Report

1st Research Coordination Meeting

on

“Nanosized Delivery of Radiopharmaceuticals”

IAEA Headquarters, Vienna, Austria

July 07-11, 2014

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FOREWORD

The currently used therapeutic agents in nuclear medicine continue to pose medical challenges mainly

due to the limited uptake of radiocompound within tumour sites. This limited accumulation accounts for the

fact that current beta-emitting therapeutic nuclear medicine agents have failed to deliver optimum

therapeutic payloads at tumour sites. Actually, no other radiolabelled therapeutic agent has been capable to

get close to the remarkably high target accumulation that was demonstrated by the long-lasting

radionuclide I-131 in thyroid cancer. This means that metastases of several different types of aggressive

cancers cannot be controlled, thus resulting in propagation of cancers to various other organs including

bone, making it difficult to treat cancer patients. Therefore, new delivery modalities that result in (i)

effective delivery of therapeutic probes with optimum payloads, site specifically at the tumour sites, with

minimal/tolerable systemic toxicity, and (ii) higher tumour retention, would bring about a clinically

measurable shift in the way cancers are diagnosed and treated. Such oncological advances in targeted

delivery of radioactive therapeutic probes would provide significant benefits to patient population

worldwide.

Nanotechnology has the potential to bring about this paradigm shift in the early detection and therapy

of various forms of human cancers because radioactive nanoparticles of optimum sizes for penetration

across tumour cell membranes can be engineered through a myriad of interdisciplinary approaches,

involving teams of experts from nuclear medicine, materials sciences, physics, chemistry, tumour biology

and oncologists. Therefore, the overall approach which encompasses application of nanoparticulate

radioactive probes in combination with polymeric nanomaterials has a realistic potential to generate the

next generation of tumour specific theranostic nanoceuticals and minimize/eliminate delivery and tumour

accumulation problems associated with the existing traditional nuclear medicine agents.

This CRP was formulated based on the conclusions and recommendations of a Consultants Meeting

(27−31 May 2013), and will utilize the knowledge and expertise in synthesizing polymer nanoparticles

using radiation technologies developed under the framework of a completed CRP “Nanoscale Radiation

Engineering of Advanced Materials for Potential Biomedical Applications”. It is expected to result in new

nanoceuticals, nanoparticles capable of forming stable bonds with diagnostic and therapeutic radioisotopes,

and with tumor specific biomolecules and proteins leading to well-defined delivery devices.

The first RCM was held on 7-11 July 2014 in Vienna, Austria. The participants reviewed the relevant work

being carried out in their respective institutions, as well as discussed and adopted the work plan for the next

period. This meeting report contains all relevant information, in a concise way in the Summary part, and

with full details in the individual reports of the participants.

The IAEA wishes to thank all participants for their valuable contributions. The IAEA officers

responsible for this publication were Agnes Safrany and Adriano Duatti of the Division of Physical and

Chemical Sciences.

1. INTRODUCTION

Nuclear medicine is an important medical specialty involving the application of radioactive

substances in the diagnosis and treatment of diseases. There are a number of diagnostic and therapeutic

radiopharmaceuticals that are FDA approved for use in human patients. These radiopharmaceuticals, once

administered to the patient, can localize to specific organs or cellular receptors. This property of

radiopharmaceuticals allows nuclear medicine the ability to image the extent of a disease process in the

body, based on the cellular function and physiology, rather than relying on physical changes in the tissue

anatomy. Nuclear medicine has the ability to identify medical problems at an earlier stage than other

diagnostic tests. Nuclear medicine is often compared to "radiology done inside out", or "endo-radiology",

because it records radiation emitting from within the body rather than radiation that is generated by external

sources such as X-rays. The therapeutic effect of radiopharmaceuticals relies on the tissue-destructive

power originating from the emission of massive, short-range ionizing radiation by some selected

radionuclides. The combined use of a pair of diagnostic and therapeutic radiopharmaceuticals for the

treatment of the same disease constitutes the basic paradigm of the field of theranostics as applied to

nuclear medicine.

Currently used diagnostic PET and SPECT agents in Nuclear Medicine utilize radiolabelled small-

molecules, proteins and peptides as targeting vectors. On the therapy front, β-emitting radionuclides

conjugated with tumor specific peptides or monoclonal antibodies, are routinely used to ablate tumours and

metastatic lesions. Only a small number of small-molecule radiolabelled compounds are routinely

employed in therapy mostly for pain palliation of metastatic bone cancer. There exist very few examples of

radionuclides administered under a simple ionic form; the most widely employed being I-131 injected as

iodide salt for the treatment of thyroid cancer. Strontium-89 is employed as Sn2+

ion for treating metastatic

bone cancer and, recently, the alpha-emitting 223

Ra as Ra2+

ion has been approved for the same indication.

1.1. Remaining Challenges

The currently used therapeutic agents in nuclear medicine continue to pose vexing medical challenges

mainly due to the limited uptake of radiocompound within tumour sites. This limited accumulation

accounts for the fact that current beta-emitting therapeutic nuclear medicine agents have failed to deliver

optimum therapeutic payloads at tumour sites. Actually, no other radiolabelled therapeutic agent has been

capable to get close to the remarkably high target accumulation that was demonstrated by the long-lasting

radionuclide I-131 in thyroid cancer. This means that metastases of several different types of aggressive

cancers cannot be controlled, thus resulting in propagation of cancers to various other organs including

bone, making it difficult to treat cancer patients. These challenges have resulted in minimal or no

improvement in the quality of life for cancer patients. 177

Lu-AuNPs conjugated to different peptides have

been proposed as a new class of theragnostic radiopharmaceuticals. These radioconjugates may function as

both diagnostic molecular imaging agents, radiotherapeutic systems and thermal-ablation nanodevices

Therefore, new delivery modalities that result in (i) effective delivery of therapeutic probes with optimum

payloads, site specifically at the tumour sites, with minimal/tolerable systemic toxicity, and (ii) higher

tumour retention, would bring about a clinically measurable shift in the way cancers are diagnosed and

treated. Such oncological advances in targeted delivery of radioactive therapeutic probes would provide

significant benefits to patient population worldwide.

1.2. Role of nanotechnology

Nanotechnology has the potential to bring about a paradigm shift in the early detection and therapy of

various forms of human cancers because radioactive nanoparticles of optimum sizes for penetration across

tumour cell membranes can be engineered through a myriad of interdisciplinary approaches, involving

teams of experts from nuclear medicine, materials sciences, physics, chemistry, tumour biology and

oncologists. In particular, intervention of nanotechnology in nuclear medicine is poised to offer tremendous

benefits in site-specific delivery of both diagnostic and therapeutic probes site specifically at tumour sites.

This is possible because gamma and beta emitting isotopes can be converted into their corresponding

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nanoparticles. The sizes of diagnostic and therapeutic probes can be engineered to be within the 10-50 nm

range so that they can penetrate tumour cells (which are 5-10 microns in size) to provide optimum

diagnostic and therapeutic payloads for efficient diagnostic imaging and therapy.

Radiation induced production of polymeric nanoparticles using both the natural and synthetic

polymers and proteins in combination with theranostic radioactive Au-198/199 (and other beta and gamma

emitting radionuclides of Re, Sm, Rh etc.) would afford nanoceuticals within the 30-50 nm range. This size

range would allow facile penetration of diagnostic and therapeutic payloads across tumour vasculature to

achieve (i) effective delivery of optimum diagnostic/therapeutic payloads accompanied by

minimal/tolerable systemic toxicity, and (ii) higher tumour retention using homogeneously dispersed

diagnostic/therapy agents. Therefore, the overall approach which encompasses application of

nanoparticulate radioactive probes in combination with polymeric nanomaterials has a realistic potential to

generate the next generation of tumour specific theranostic nanoceuticals and minimize/eliminate delivery

and tumour accumulation problems associated with the existing traditional nuclear medicine agents.

In this context, nanostructured antibodies when tagged with nanostructured diagnostic/therapeutic probes

may also offer significant benefits over conventional radio-immunotherapy, because nanoparticulates can

penetrate tumour vasculature and being retained by the tumour mass more efficiently than traditional

antibody labelled radiopharmaceuticals.

1.3. Research already in progress in Member States

Within the framework of successfully completed CRP “Nanoscale Radiation Engineering of

Advanced Materials for Potential Biomedical Applications” considerable progress has been made in the

Member States in synthesizing polymer nanoparticles using radiation technique. These new nanomaterials

include, among others, protein nanoparticles, nanogels of various physical and chemical properties, as well

as nanoparticles based on polysaccharides (Argentina, Brazil, China, Egypt, Hungary, India, Italy, Korea,

Malaysia, Poland, Serbia, Thailand and Turkey). These approaches were aimed at precise control of the

structure, size, shape and functionality of the nanoscale products. In some cases, procedures were

developed for preparing “hybrid” products, e.g., where the surfaces of nanoparticles and nanogels were

decorated with functional polymers and biomolecules. It will be very valuable to build up on this

accumulated knowledge and experience for creating new products where radiation-engineered

nanoparticles are the basis of new generation of diagnostic and therapeutic systems for use in nuclear

medicine.

This CRP was formulated based on the conclusions and recommendations of a Consultants Meeting

(27−31 May 2013), and will utilize the knowledge and expertise in synthesizing polymer nanoparticles

using radiation technologies developed under the framework of a completed CRP “Nanoscale Radiation

Engineering of Advanced Materials for Potential Biomedical Applications”. It is expected to result in new

nanoceuticals, nanoparticles capable of forming stable bonds with diagnostic and therapeutic radioisotopes,

and with tumor specific biomolecules and proteins (including monoclonal antibodies) leading to well-

defined delivery devices. The proposed CRP will provide tremendous benefits to MS because the nanosized

diagnostic and therapeutic agents planned for investigation and development might be potentially used in

alleviating pain and suffering of human patients globally.

2. CRP OVERALL OBJECTIVE

The overall objective of this CRP is to exploit the unique properties of materials at the nanometer

scale for developing nanocarriers of radioactivity capable of selectively targeting and penetrating cancerous

cells.

2.1. Specific Objectives

To synthesize polymeric (both synthetic and natural) nanoparticles capable of forming in vitro and in

vivo-stable bonds, through chelate interactions, with diagnostic and therapeutic radioisotopes—for the

creation of new generation of nanoparticle-based tumour specific radiopharmaceuticals.

To formulate tumour specific proteins (including monoclonal antibodies) and generate well-defined

nanoparticles, through radiation methods, for use in conjugation reactions with diagnostics and

therapeutic radioisotopes (Tc-99m, Re-186/188)—for the generation of nanosized tumour specific

radiopharmaceuticals.

To formulate nanoparticles derived from inherently diagnostic/ therapeutic radioisotopes (Au-198,

Au-199, Re-186/188, Rh-105) and to conjugate them with tumour specific peptide(s)—in order to

generate radioactive nanoceuticals for diagnostic imaging and therapy.

To develop a functional in vivo platform for efficient testing of various radiolabelled nanoconstructs

using animal models that mimic human tumours—all aimed at clinical translation of

diagnostic/therapeutic efficacy from animal for human applications.

2.2.Expected research outputs

Polymeric (synthetic and natural) nanoparticles for the creation of new generation of nanoparticle-

based tumour specific radiopharmaceuticals.

Protocols for conjugation reactions with diagnostics and therapeutic radioisotopes (Tc-99m, Re-

186/188)—for the generation of nanosized tumour specific radiopharmaceuticals.

Nanoparticles derived from inherently diagnostic/ therapeutic radioisotopes (Au-198, Au-199, Re-

186/188, Rh-105) and conjugated with tumour specific peptide(s).

Normalized in vivo platform for efficient testing of various radiolabelled nanoconstructs using animal

models that mimic human tumours—all aimed at clinical translation of diagnostic/therapeutic efficacy

from animal for human applications.

The first Research Coordination Meeting (RCM) was held in Vienna, on 7-11 July 2014 and was

attended by 14 participants and 3 external observers coming from thirteen different countries. During the

meeting, presentations were given by all participants showing the main results of their research activity in

the development of new nanomaterials and in their applications to the design and production of novel

diagnostic and therapeutic radiopharmaceuticals. After a throughout discussion and analysis of the status of

the field, the research program for the next coming years of the CRP was intensively discussed and final

detailed workplan was designed and approved.

3. REVIEW OF THE WORK IN INDIVIDUAL INSTITUTIONS

3.1. Argentina

More than twenty-five years ago it has been demonstrated that radiation technology can be used to

generate polymer microparticles by precipitation polymerization with a very narrow particle size

distribution. Particle diameter can be achieved, in the range 0.8 to 8 microns, by selection of the appropriate

organic solvent. However lower-diameter particles still have not been achieved by this technique.

By using a different strategy water-soluble polymers can be intramolecularly cross-linked in diluted

solutions by e-beam irradiation. Considering their aqueous environment and their rheological properties

they were called nanogels.

Proteins are also soluble macromolecules; however, most of them have a very compact and defined

three-dimensional structure instead random coil conformation, which are known as globular proteins. The

effect of ionizing irradiation onto globular proteins solutions was study for many years, mainly in features

related to the biological effect of protein degradation or agglomeration.

Globular proteins are very sensitive to microenvironment, thus the effect of the solvent during the

irradiation process could have a great effect onto the sample. Having in mind that organic polar solvents

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induce protein aggregation, without protein denaturation, it is possible to prepare protein-based

nanoparticles by irradiation of water/ethanol solutions of globular proteins with ionizing radiations.

We describe for the first time albumin nanoparticles (Alb-NP) preparation by ionizing radiation cross-

linking. Particle size can be tailored by changing the water/ethanol ratio in the protein solution, reaching

particles in the range of 20 nm to 40 nm. The conformational structure of albumins in the nanoparticles is

conserved in a high degree and preliminary toxicity analysis in Zebrafish model did not show significant

toxicity changes.

In the proposed research work it was expected to cover gold nanoparticles (Au-NP) with several

layers of albumin, in a controlled and stable manner, to create a protein-shell by using the previous

developed technique. Novel core-shell nanoparticles will be characterized by different spectroscopic

techniques, in addition the classical characterization techniques available for Au-NP. Off-target

accumulation and receptor-mediated internalization of Alb-Au-NP will be studied by in-vitro human cell

culture lines. The optimized protocol will be translated to prepare this core-shell NP using Au-198/199.

3.2. Brazil

Nanoparticle protein-bound (Nab) pharmaceuticals has been considered as a very promising route for

the delivery of chemo and radio-pharmaceutics for cancer cells. Protein-bound paclitaxel is

an injectable formulation of paclitaxel, a mitotic inhibitor drug used in the treatment of breast cancer, lung

cancer and pancreatic cancer. In this formulation, paclitaxel is bonded to albumin as a delivery vehicle. It is

sold in the United States under the trade name Abraxane. The preparation of albumin nanoparticles of

Abraxane is based on strong shear and pressure in solvent medium followed by extraction. Therefore a

number of possible shortcomings can be foreseen. A better process in a pure and mild, solvent free medium

to synthesize albumin nanoparticle and to encapsulate chemo and radio pharmaceutics should be

developed. Nanoparticles of albumin by radio-induced crosslinking was recently demonstrated and proved

by Grasselli’s group and papain nanoparticle was demonstrated and proved by our group in Brazil. Our new

process will be applied to encapsulate radiopharmaceuticals. This report details the establishment of a novel

platform for the development of protein-based nanoparticles for the delivery of chemo and

radiopharmaceutics, including an overview of the process, scale up and irradiation dose effects, as well as

provides an experimental evidence to clarify the mechanism involved in the nanoparticle formation.

3.3. Egypt

Research proposal title: Development of radio-nanoscale delivery systems for diagnosis and treatment

of cancer.

Main objective: Our aim is to formulate, synthesize, and evaluate nanosized gold-198, iodine-125/131

and technetium-99m radiopharmaceuticals as diagnostic or therapeutic agents for cancers.

This work will be conducted in Egyptian Atomic Energy Authority. It will be cooperation between 2

main research labs, Radioisotope Production Facility (RPF)-ETRR-2 complex and National Center for

Radiation Research and Technology.

We had made different studies on the preparation of nanosized polymeric materials using radiation

synthesis technique such as PVP/PAAc hydrogel to act as carrier for different drugs for medical

applications. Besides, our team is highly specialized in radiolabeling techniques using iodine-125/131 and

technetium-99m for the preparation of some diagnostic and therapeutic radiopharmaceuticals. We had

made studies on the preparation of gold-198 nanoparticles in cooperation with Dr Kattesh Katti’s labs in

USA. We are able to perform preclinical studies in mice models and induction of some diseases such as

inflammation, infection and solid tumour.

We are planning to prepare targeting nanosized radiopharmaceuticals for cancer diagnosis and

treatment. These preparations will be based upon a nano-polymer matrix, which will be produced by the

radiation synthesis technique. The control of their sizes and shapes will be investigated and characterized

using full-scale instruments. These nano-polymers will be tested to serve as carriers for radioisotopes such

as; gold-198, iodine-125/131 and technetium-99m. Decoration of theses nano-sized radiopharmaceuticals

with targeting vector like folate proteins and antibodies to act as a targeting moiety guiding selectively the

radiopharmaceuticals to cancer cells will be studied. The quality and in vitro stability of these preparations

will be evaluated. In vivo studies will be carried out in solid tumour bearing mice.

3.4. Iran

During recent years, production and quality control and marketing of various radiopharmaceuticals

based on SPECT and PET as well as therapeutic radioisotopes has been performed in Iran (including Tc-

99m, In-111, Tl-201, Ga-67, F-18, Cu-64, Cu-61, Ga-68, Y-90, Ho-166, Lu-177, Sm-153 etc.). The

development of nanoscience based research centers and groups in Universities and research institutes is

also of great interest according to national development policy and this has put Iran among the top-ranking

countries in nanoscience research in the region as well as the globe.

According to parallel advances of both radiopharmaceutical sciences as well as nanoscience, few

research projects for developing nan size radiopharmaceutical delivery systems has been initiated including

radiolabeled super paramagnetic iron oxide nanoparticles (SPION) with or without coating and decorations

including receptor binding moieties such as folate.

Recently a new program has been initiated to develop naturally based polymer nanoparticles such as

chitosan, dextran, etc. According to development of nanopharmaceutical products in Iran including

Sinadoxosome, Curcumino-some etc. for cancer therapy and prophylaxis and also few on-going clinical

trials on new products, it is expected that in case of developing any new nano-sized radiopharmaceutical,

the National Medical Ethics Committee would allow the initiation of new clinical trials as was the case for

successful clinical trials for 15 radiopharmaceuticals developed in last 5 years.

In this project according to recent advances in developing reproducible results on chitosan-based NPs

as well as possibility of creating natural chelating moieties in the structure a few possible tumour-targeting

moieties (such as folic acid/immunomolecules) can be developed and radiolabeled using available medical

radioisotopes including Tc-99m, Ga-68 etc. followed by quality control, in vitro tests, animal studies with

tumour xenografts as well as preliminary imaging in this models.

3.5. Italy (Milan)

The group in Milan can contribute to the success of this CRP with its expertise mainly focused on

radiochemistry/radiopharmacy.

In particular:

- To study and optimize the reaction conditions in order to conjugate bifunctional chelating agents (such as

DOTA) to different nanoparticles

- To perform the radiolabeling using various radionuclides available on site either for diagnosis and/or

therapy

- To optimize the quality control procedures on the radiolabeled materials

- To study the in-vitro stability of the radiolabeled particles in different medium

- To provide detailed procedures for the labelling to the participants who wants to test this radiolabeled

nanoparticles in animal models

An important expected output from this CRP is to identify and make available to the participants a

nanoparticle system, which can be efficiently radiolabeled with different isotopes for application to the

diagnosis and therapy of tumours. In particular, it will be very useful to our group if the ongoing research

on DOTA conjugated human albumin will originate a particulate compound which can be used in the

ROLL procedure.

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It is also very appealing to us the possibility to attach a targeting molecule to the surface of

nanoparticles. Our group has pioneered in Europe the therapy of neuroendocrine tumours using

radiolabeled peptides. Therefore, the possibility to obtain from this CRP efforts a nanosystem with high

affinity for somatostatin receptors and at the same time carrying a radioactivity payload will be extremely

important for us.

In this scenario the following approach is proposed. The groups in this CRP, which are expert in

nanoparticle’s preparation, should initially try to conjugate the octreotide peptide (commercially available)

to the surface of the particles. The DOTA chelator will be also attached to other reactive groups (especially

amine groups) made available on the surface of the particles. This new construct should be initially checked

in-vitro for its affinity to somatostatin receptors performing binding studies. Then, following optimization

of the radiolabeling steps, biodistribution studies will be performed in animal models bearing neuroendrine

tumours (well described in literature) in comparison with the widely used radiolabeled octreotide

derivatives.

These preliminary results may be very helpful to tune up and optimize all the conjugation procedures

either with decorating agents or with chelating agents aiming to the final goal of this CRP to provide to the

MS a nanoparticles system flexible for different in-vivo applications.

3.6. Italy (Padova)

Colorectal Cancer (CRC) is the third most common malignant neoplasm worldwide and the second

leading cause of cancer deaths (irrespective of gender). Despite advances in surgical techniques and

adjuvant therapy have significantly increased the life expectation of patients affected by primary CRC,

there has been only a modest improvement in survival for patients who present with advanced neoplasms

and metastasis. Pharmaceutical nanotechnologies offer excellent opportunities to reduce the morbidity and

mortality from CRC In particular, engineered colloidal systems obtained by physical or chemical

combination of structural and functional modules (drugs, targeting agents, microenvironmental or external

stimuli sensitive materials) have being studied for the treatment of a variety of tumours. These

supramolecular nanostructured systems can be exploited to overcome a few problems that limit the

anticancer drug efficacy such as low stability and solubility and rapid clearance. Furthermore, colloidal

systems may be designed for tissue or cell targeting thus providing for selective and controlled drug release

in the disease site, which avoids systemic toxicity. On the other hand, such systems have been poorly

exploited as carrier of diagnostic and/or therapeutic radionuclides.

The research project is aimed at designing and developing novel poly-ethylen-glycol (PEG)-based

multifunctional polymer bioconjugate therapeutic systems (smart nanoradiopharmaceuticals), for efficient

theragnostics of colorectal carcinoma (CRC) and its liver metastasis. The research group is

multidisciplinary and comprises expertise in polymer chemistry, radiochemistry, radiopharmacy, nuclear

medicine and biology. Multifunctional bioconjugate nanoradiopharmaceuticals will be obtained by

derivatization with chelators for selected radionuclides for either PET and/or SPECT imaging or therapy,

and by functionalization with the YHWYGYTPQNVI (GE11) peptide, a dodecaptide that selectively

recognises the epidermal growth factor receptor (EGFR) overexpressed by the CRC cells, to achieve the

desired active CRC tumour targeting.

The modules forming the multifunctional colloidal systems will be extensively characterized

according to various analytical techniques present in house or available through integrated collaboration:

chromatography (RP-HPLC, GPC); spectrometry (UV-Vis, fluorescence, IR, NMR, PCS, ESI-TOF,

MALDI-TOF, atom absorption, SPR); microscopy (TEM, AFM, confocal microscopy); calorimetry (MC,

DSC, TGA); Squid magnetometry, sonography.

Investigations will be carried out using cell lines that overexpress and non-overexpress the EGFR to

evaluate the cell selectivity and the cytotoxicity of the products. In this regard, biochemical and confocal

microscopy studies will allow measuring biological effects and fate of the systems within the neoplastic

cell. Moreover, ex vivo perfusion of human colon segments bearing a primary carcinoma will permit to

study the distribution and the effects of drug nanodelivery systems in a model that most reproduces in vivo

human conditions. Finally, in vivo biodistribution and therapeutic efficacy in CRC mouse models will be

analysed by PET/SPECT/CT molecular imaging approaches.

3.7. Italy (Palermo)

Major challenges of delivery devices in cancer diagnostic and therapy are the low bioavailability of

contrast agents or drugs within cancer cells and the high toxicity of these "actives" to normal organs or

tissues. Nanotechnology holds the promise to deliver more efficient diagnostics and therapeutics for

treating cancer. Among the thousands of nanomedicine formulations described in the scientific literature

over the last two to three decades, only a very few are clinically well established or accepted for clinical

evaluation. This is due to the lack of comprehensive information on nanodrugs performance, often tested

only on cell cultures or simple animal models, to the lack of confidence in their manufacturing processes,

complex and difficult to scale up, and to the several issues related to targeting and delivering to specific

sites. Theranostic nanodevices incorporating, in a single construct, a ligand for "active" targeting, contrast

agents for MRI or radionuclides for nuclear imaging, and a chemotherapeutics or tumour

radiochemotherapeutics can be particularly helpful to carry out the preclinical and clinical efficacy studies

required for translation of advanced nanomedicines to the clinical application for treating cancer patients.

The present research project is aimed to build up on the main outcomes of the research activity

carried out in the framework of a previous CRP, “Nanoscale Radiation Engineering of Advanced Materials

for Potential Biomedical Applications", that can be summarised in the following:

[1] a novel family of polymeric nanocarriers, in particular nanogels (NGs) with a Poly(N-vinyl

pyrrolidone)-base structure has been manufactured by e-beam irradiation of polymer aqueous

solutions, showing a variety of average particles size, relatively narrow particle size distributions,

generally anionic surface charge densities and several functional groups (carboxyl or amino groups)

per nanoparticle;

[2] All the NGs produced have been proved to be biocompatible and rapidly internalized by cells;

[3] The many functional groups grafted on the NGs have been used for coupling reactions with bioactive

molecules, such as small peptides, antibodies, oligonucleotides and anti-cancer drugs;

[4] The proposed synthetic approach, that makes use of industrial type accelerators and typical operating

conditions used for sterilisation purposes, has granted high yields in terms of recovered product and

high throughputs. Moreover, through a fine-tuning of the experimental parameters (both formulation

composition and irradiation parameters), NGs with different physico-chemical and functional

properties have been obtained.

In this project we will develop protocols for tailoring nanogel carriers of contrast agents for Magnetic

Resonance Imaging (MRI) and/or radionuclides for Nuclear Imaging and either chemotherapeutics or

radiotherapeutics.

The protocol development will be based on detailed knowledge about the mechanism and kinetics of

electron beam irradiation driven synthesis of nanogels in aqueous solution. Nanogels are formed upon

crosslinking of polymers in semi-dilute solutions. Hence, mechanistic and kinetic studies of this process

will be carried out as an integer part of the project. The aim is to be able to control nanoparticle size, shape

and size distribution by varying process parameters such as dose per pulse, pulse frequency, polymer

concentration and concentration and nature of chemical additives. Parallel with the development of a

protocol for nanoparticle synthesis, methods for functionalization of the nanoparticles for specific binding

of contrast agents, radionuclides and chemotherapeutics will be carried out. In particular, the possibility of

performing the functionalization as an integrated part of the radiation chemical synthesis will be

investigated.

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Functionalized nanoparticles will be manufactured for chemical characterization as nanocarriers,

including long-term stability tests under relevant conditions, and for in-vitro and in-vivo testing by other

groups within the current CRP.

3.8. Malaysia

Nanoscale therapeutic systems have emerged as novel therapeutic modalities for cancer treatment and

are expected to lead to major advances in cancer detection, diagnosis and treatment. In this proposal we

plan to explore two distinct types of nanocarriers in order to exploit the physical properties of both types of

construct. The first type is nanogel. The nanogel to be used as a platform for multifunctional delivery of

radiopharmaceuticals was previous developed under the CRP project “Radiation synthesis of nanogel for

chitosan immobilization”. Covalently cross-linked nanogel for PEG-DA using an inverse micelle system

for irradiation with electron beam was developed. Nanogel makes an ideal nanocarrier as it has a high

degree of steric stabilization and reduced non-specific interaction, properties that are beneficial for targeted

delivery. In addition nanogels can achieve a high degree of encapsulation leading to an increase in drug

loading capacities leading to a more effective therapy and image contrast.

Additionally this study will explore the use of gold nanoparticles for targeted delivery of

radiopharmaceuticals. Compared with other nanostructures, metallic nanoparticles have proven to be the

most flexible nanostructures owing to the synthetic control of their size, shape, composition, structure,

assembly and encapsulation, as well as the resulting tenability of their optical properties. To further

functionalize these systems a tumour targeting peptide, or other suitable targeting ligand, will be appended

to the nanogel and AuNP to endow it with tumour homing/targeting capability. Furthermore to track its

biodistribution in vivo, it will be radiolabeled with technetium (Tc-99m) and scan using a gamma camera to

assess its tumour accumulation.

3.8.1. Research objectives

1. Synthesis of nanogel (from polyethylene glycol diacrylate [PEGDA]) and gold nanoparticle endowed

with tumour homing peptide

2. Evaluate biological activity of targeted nanoparticles

3. In vivo imaging and targeting of murine tumours

3.8.2. Anticipated outcomes

1. Able to functionalize two types of nanoparticles with tumour targeting ligands. Targeting specifically

to cancer cells allow specific accumulation and retention which would enhance drug therapeutic

efficacy as well as image contrast.

2. Labelling with a suitable radioisotope e.g. Tc-99m allows characterization of biodistribution and PK of

the different types of nanoparticles

3.9. Mexico

3.9.1. Title of the proposed research

Theragnostic Radiopharmaceuticals Based on Gold Nanoparticles Labeled with 177

Lu and Conjugated

to Peptides by Blanca E. Ocampo-García, Guillermina Ferro-Flores, Clara L. Santos-Cuevas, Flor de María

Ramírez and Erika P. Azorín-Vega.

3.9.2. Scientific Backround.

The somatostatin receptors, which are overexpressed in a majority of neuroendocrine tumors, are

targets for radiopeptide-based imaging. Somatostatin analogues, including 99m

Tc-Tyr3-Octreotide, 99m

Tc-

Tyr3-Octreotate, 177

Lu-Tyr3-Octreotate are successfully being used for neuroendocrine tumor imaging and

targeted radiotherapy.

Multivalency is a design principle by which organized arrays amplify the strength of a binding

process, such as the binding of multimeric peptides to specific receptors located on cell surfaces. Due to

passive and active-targeting mechanisms, the radiolabeled multimeric and multivalent peptides exhibit

higher tumor uptake than does a peptide-monomer or -dimer. Radiopharmaceuticals based on nanoparticles

have other advantages as multifunctional agents. Multifunctional systems with multiple receptor targeting

and multiple therapeutic entities are potentially useful as imaging and therapeutic agents.

The overall aim of this research is to prepare two nanosized multimeric and multifunctional systems

for neuroendocrine tumor imaging and targeted therapy, 99m

Tc-PAMAM-Tyr3-Octreotide and

188Re-

PAMAM-Tyr3-Octreotide, and to evaluate their in vitro uptake in somatostatin receptor-positive AR42J

cancer cells as well as their biodistribution profile in athymic mice bearing somatostatin receptor-positive

AR42J tumors.

3.9.3. Specific objectives

1. Synthesis and chemical characterization of PAMAM-Tyr3-Octreotide

2. Radiolabeling of PAMAM-Tyr3-Octreotide with Tc-99m and evaluation of the radiochemical purity and

human serum stability.

3. Radiolabeling of PAMAM-Tyr3-Octreotide with Re-188 and evaluation of the radiochemical purity and

human serum stability.

3. Evaluation of the in vitro uptake and binding affinity of 99m

Tc-PAMAM-Tyr3-Octreotide and

188Re-

PAMAM-Tyr3-Octroetide in somatostatin receptor-positive AR42J cancer cells.

4. Evaluation of the biokinetics and dosimetry of 99m

Tc-PAMAM-Tyr3-Octreotide and

188Re-PAMAM-

Tyr3-Octreotide in athymic mice bearing somatostatin receptor-psotive AR42J tumors.

5. To verify the in vivo nanoradiopharmaceutical retention in tumors by imaging.

3.9.4. Expected outputs

To obtain two novel nanosized multimeric and multifunctional systems useful for neuroendocrine

tumor imaging and targeted therapy: 99m

Tc-PAMAM-Tyr3-Octreotide and

188Re-PAMAM-Tyr

3-

Octreotide.

To publish at least two scientific articles in International Journals (indexed) related to the preparation

and evaluation of two novel nanosized radiolabeled analogues of somatostatin for neuroendocrine

tumor Imaging and targeted therapy.

3.10. Pakistan

The over-expression of neuropeptide Y1-receptors in human breast cancer leads toa potential

application of selective neuropeptide Y (NPY) Y1-receptor ligands as a new generation of agents for cancer

diagnosis and therapy. For the first time, we showed the clear uptake of the 99mTc(core)3+-(N_His-ac)-

labelled Y1-receptor selective [Phe7, Pro34]NPY peptide in human breast cancer patients by whole body

scintimammography. In addition, peptide uptake was detected in metastases spread in other organs,

originating from the tumour. In contrast, a healthy volunteer showed no peptide uptake, especially in

breasts. Our pre-clinical studies, including different in vitro and in vivo analysis as well as our imaging

studies in human breast cancer patients confirmed the suitability of the NPY-Y1-receptor system for

selective breast tumour targeting. These excellent results prompted us to develop peptide-based gold and

silver nanoparticles, thus ultimately, resulting highly specific radiolabeled nanoconstructs decorated with

peptide molecules to act as ‘nanotheranostics’. For this purpose, we produced highly stable gold and silver

nanoparticles (NPs) using arabinoxylan (AX) from ispaghula (Plantago ovata) seed husk. The NPs were

synthesized by stirring a mixture of AX and HAuCl4·H2O or AgNO3, separately, below 100_ C for less

than an hour, where AX worked as the reducing and the stabilizing agent. The synthesized NPs were

12

characterized by surface plasmon resonance (SPR) spectroscopy, transmission electron microscopy (TEM),

atomic force microscopy (AFM), and Xray diffraction (XRD). The particle size was (for silver: 5-20 nm

and gold: 8-30 nm) found to be dependent on pH, temperature, reaction time and concentrations of AX and

the metal salts used. The NPs were poly-dispersed with a narrow range. They were stable for more than two

years time.

3.11. Poland

Based on the Consultant Meeting held in Vienna in May 2013, the team of the Institute of Applied

Radiation Chemistry (Lodz University of Technology) identified the tasks which it can undertake and

perform within this CRP. Exact formulation of these tasks will be made upon consultations with other

national teams during the current 1st RCM.

Within the first task, using its experience in radiation chemistry and sonochemistry of water-soluble

polymers, IARC will focus on developing methods and procedures for synthesizing gold nanoparticles.

These methods will utilize the fact that both in irradiation and sonication reductive species are formed

which are capable of reducing ionic gold precursors (such as chloroauric ions) to atomic gold, the latter

aggregating into gold nanoparticles. Polymers and/or oligomers present in the system will form the

protective layer around these nanoparticles to prevent their further aggregation. At the same time,

polymeric ligands may provide functions (stability, hydrophilicity, charge, diameter, specific interactions,

etc.) necessary for the stabilized particles to perform the designed role in biological systems. Gold

nanoparticles are employed in radiotherapy, in two modes – to increase local dose deposition in tissue

during radiotherapy or as a local emitter of gamma and beta rays, if the nanoparticles contain radioactive

gold isotopes.

At IARC experimental studies have been launched on radiation synthesis of gold nanoparticles

stabilized by three kinds of polymeric ligands – oligomers of poly(acrylic acid), medium-chain-length

chitosan and thiolated poly(ethylene glycol). While in all cases the nanoparticles have been obtained, much

work is still required to understand the influence of synthesis’ parameters on size and stability of the

products. As a complementary technique, sonochemical synthesis based on the same systems has also been

successfully performed. Further plans, including also nanogel-based syntheses of gold nanoparticles, will

be discussed at the RCM with the project partners.

The second task will be the radiation synthesis and modification of tailored polymer nanogels for

binding radioactive metal ions in order to form a nanoconstruct suitable for radiotherapy. Realization of

task 2 will be based on longstanding experience of IARC in radiation synthesis of nanogels by

intramolecular cross-linking of polymers using method originally developed at our laboratory.

Besides the already existing co-operation (Argentina, Brazil, Italy, Malaysia), further networking is

planned, especially in the fields of desired biomedical properties of the products and in experimental

techniques (e.g. TEM) necessary for studying the physicochemical parameters of polymer-stabilized gold

nanoparticles. IARC input to the networking can be the experience in radiation chemistry and

sonochemistry of water-soluble polymers (including kinetic and mechanistic studies by pulse radiolysis),

nanogel synthesis, polymer and nanoparticle characterization (selected methods) and also well equipped

laboratories with radiation sources (electron beam, gamma) and sonochemical reactors.

3.12. Singapore

Nanotechnology has found its presence in many industries and applications, and one such promising

application is in the biomedical arena. It has been exploited for the development of nanomedicine, for drug

delivery and bioimaging purposes. For example, drug delivery in the form of nanomedicine utilizes nano-

sized particles to transport and release pharmaceutical compound into the body, to achieve the most

desirable therapeutic outcome and in the safest possible manner. In this presentation, we will focus on how

nanotechnology can be applied for nanomedicine, i.e. drug delivery and bioimaging. The scope will be on

the use of bottom-up approaches to develop various types of particles, and how these can be modified for

targeted, controlled and sustained release of drugs in orthopedic, geriatric, and oncologic applications. We

will also review how multi-layered and hollow particles are currently developed to deliver multiple drugs

and bioimaging probes. At the same time, this presentation would also review the toxicological responses

of some nanomaterial candidates, and how this would translate to developing safer nano-materials for

biomedical applications. At the end of the presentation, we will explore how some of these particulate drug

delivery systems can be exploited for the delivery of radiopharmaceuticals.

In summary, the key project objective of the CRP would be to develop a novel nanosystem that could

be potentially used in the delivery of radiopharmaceuticals, and to investigate its integrity under the

influence of radiation and biological conditions. The secondary objective, with collaborators, would be to

develop some of these delivery systems for delivery of radiopharmaceuticals.

3.13. Thailand

3.13.1. Title of the proposed research

Green synthesis of polysaccharide and polypeptide-based nanoparticles toward the creation of stable and

functionalized radiopharmaceuticals for diagnosis and therapy, by W. Pasanphan, T. Trattanawongwiboon

and S. Wongkrongsak of the Department of Material Science, Faculty of Science, Kasetsart University,

Bangkok, Thailand.

3.13.2. Summary of the proposed research project

Current status of the research work includes a series of nanosized drug delivery system with different

formulations, i.e., (i) gold nanoparticles (AuNPs) reduced and stabilized using natural polymers, (ii)

amphiphilic core-shell polymeric NPs and (iii) nanogels (NGs) or NPs containing polychelating groups.

The proposed work (ii) is the production of water-soluble nanostructural polysaccharide (water-soluble

chitosan nanoparticles, WSCS-NPs) and polypeptide (water-soluble silk fibroin nanoparticles, WSSF-NPs)

as natural antioxidants and reducing agents for a green synthesis of AuNPs [1]. Furthermore, radiation

assisted the production of AuNPs in such water-based biopolymers is also demonstrated. The possibilities

to use the successful procedure for the creation of radioactive 198

AuNPs analogue for therapy via -rays

emission and diagnosis via -rays emission using SPECT are the strategy in progress. For series (ii), the

development of amphiphilic core-shell CS nanoparticles using chemical conjugation and radiation grafting

techniques is proposed in the last CRP. According to water-soluble limitation of the obtained polyethylene

glycol monomethacrylate-grafted-deoxycholic acid chitosan nanoparticles (PEGMA-g-DCCS-NPs, 80 nm)

from the first generation [2], further research aims to improve the solubility of this amphiphilic NPs by

using an alternative WSCS-NPs as the main biopolymer. It is expected that the next generation of the

PEGMA-g-DCWSCS-NPs would serve as a possible merit to be used not only for hydrophobic drug

encapsulation in chemotherapy but also radionuclide entrapment among the polychelating groups on the

PEGMA shells for radiodiagnosis. To achieve nanosized delivery with superior radionuclide entrapment,

new polymeric NGs or NPs containing polychelating groups will be further development as in series (iii).

Herein, poly--glutamic acid (PGA), a poly (amino acid) will be modified and fabricated as inter-

penetrating network NGs, otherwise the polypeptide SF will be modified with polyacrylic acid (PAA).

Both model contain polychelating groups of the carboxylic acid (-COOH), which would provide the sites

for strongly entrapping the radiopharmaceutical substances. The surface of all models of the nanocarriers

have amino (–NH2), hydroxyl (-OH) or –COOH functionalities, therefore they are possible to be

conjugated with the specific targeting molecules, such as monoclonal antibody, folic acid, or glucose.

Beside the synthesis and characterization, the performance of the radiopharmaceutical nanocarriers

regarding localization and uptake, in vitro biocompatibility and diagnosis and/or therapeutic efficiencies

will also be demonstrated and cooperated under this CRP.

3.13.3. References

14

[1] PASANPHANA, W., RATTANAWONGWIBOON, R., CHOOFONG, S., GÜVENC, O., KATTID,

K.K., “Irradiated chitosan nanoparticle as a water-based antioxidant and reducing agent for green

fabrication of gold nanoplatforms”, Radiat. Phys. Chem., (2014) Revised manuscript.

[2] PASANPHAN, W., RATTANAWONGWIBOON, T., RIMDUSIT, P., PIROONPAN, T., “Radiation-

induced graft copolymerization of poly (ethylene glycol) monomethacrylate onto deoxycholate-

chitosan nanoparticles as a drug carrier”, Radiat. Phys. Chem. 94 (2014) 199–204.

3.14. United States of America

3.14.1. Title of the proposed research

“Radioactive Nanoparticles In Molecular Imaging and Therapy: Production, In Vitro and In Vivo

Testing Facilities”, by Kattesh V. Katti, Curator's Professor of Radiology and Physics Director, University

of Missouri Cancer Nanotechnology Platform, University of Missouri, Columbia, Missouri 65212, USA.

In the proposed research contract agreement for CRPF22064 entitled “In vivo studies and production

of nanosized delivery systems and Radiopharmaceuticals”, the University of Missouri Cancer

Nanotechnology Platform will provide various in vitro and in vivo facilities to all the investigators. This

research contract will address the following specific aims.

Specific Aim 1: Provide clinical grade samples of radioactive Au-198/199 nanoparticles (and other

isotopes) produced at the University of Missouri Research Reactor;

Specific Aim 2: Provide services on optimization of he synthesis, gain insights into mechanism of

cellular interaction, and tumour specific interaction of nanoparticles.

Specific Aim 3: Evaluate the therapeutic efficacy of targeted 198

AuNP conjugates in tumour bearing

orthotopic mouse models and tumour bearing dogs (services include studies in mice and dogs for

prostate, pancreatic and breast tumours );

Specific Aim 4: Evaluate the toxicity and pharmacokinetics of targeted nanoconjugates in pigs.

5. SUPPORTING MATERIAL

TABLE 1. AVAILABLE NANOCONSTRUCTS

Type of nanoconstruct Size range (nm) Stage of development* Nation/laboratory

PVP-based nanogels 20-200 PR ITALY (Palermo)

Albumine NPs 20-40 PR ARGENTINA

Albumine-Au NPs 40-80 UD ARGENTINA

Chitosan-PEG-folate NPs 20-50 UD IRAN

PVP-PAAc nanogels 70-150 PR EGYPT

Au NPs 8-30 UD PAKISTAN

Ag NPs 5-20 UD PAKISTAN

PEG-DA based nanogels 70-200 UD MALAYSIA

EPOLA-based nanogels 90-200 UD MALAYSIA

AuNP@silica core-shell 200 (30) UD *initial development MALAYSIA

(AuNP core) was for industrial

application

Papain NPs 5-20 PR BRAZIL

Albumin NPs 5-20 PR BRAZIL

Albumin macroagregates 100-200 microns PR ITALY (Milano)

177Lu-AuNP-RGD 20 PR MEXICO

99mTc/177Lu-AuNP-TAT-Bn 5 PR MEXICO

99mTc-PAMAM-Tyr3-

Octreotide UD MEXICO

PEG-G11 <5 PR ITALY (Padova)

Nanogels of various synthetic

polymers 15-100 PR POLAND

CMC-based nanogels 20-80 UD POLAND

Au NP-PAA/chitosan or PEG-

SH 5-20 core-size UD POLAND

198 Au NP; 199 Au NP 15-20 core-size PR USA

109 Pd NP 5-10 PR USA

PLGA / PLLA nanoparticles 50 nm – 500 um PR NTU, Singapore

Hydroxyapatite (HA)-coated

PLGA / PLLA nanoparticles 50 nm – 500 um PR NTU, Singapore

Multi-layered polymeric

particles 1um – 500 um PR NTU, Singapore

Microencapsulation of

nanoparticles 1um – 500 um PR NTU, Singapore

Hydrophilic-hydrophobic core-

shell particles 1um – 500 um PR NTU, Singapore

HA nanoparticles 50 – 500

(different shapes) PR NTU, Singapore

WSCS-NPs (water-soluble

chitosan NPs) 15 and 70 nm PR Thailand

WSSF-NPs (water-soluble silk

fibroin NPs) 40 nm PR Thailand

AuNPs-WSCS 20 nm PR Thailand

AuNPs-WSSF 25 nm PR Thailand

PEGMA-g-CS-NPs 80 nm PR Thailand

PEGMA-g-WSCS-NPs 80-150 (not clear) UD Thailand

16

* Prototype Ready (PR); Under Development (UD)

4.1. Guidelines for particle characterization (physico chemical properties)

TABLE 2. POLYMER NANOPARTICLES AND NANOGELS

No. Property Recommended analytical method Auxiliary analytical

methods Remarks

1. Chemical composition

- general Elemental analysis, XPS NMR, Raman

2.

Chemical composition

– presence of specific

groups or functions

FT-IR and tests specific to given

function (potentiometric, fluorimetric

titration, etc.)

UV-Vis Absorbance

and/or Emission

spectrometry after

binding of a marker

3. Colloidal stability Turbidity (e.g. by UV-Vis) DLS, MALLS

Specify solvent

composition (e.g.

water + pH range,

PBS, saline, etc.)

4 Avg. mass / avg.

molecular weight Multiangle (static) laser light scattering (MALDI)

5. Molecular weight

distribution HPLC-GPC

6.

Size distribution –

avg. hydrodynamic

radius

Dynamic light scattering Laser diffraction,

viscometry

For nanogels – size in

the swollen state

7. Shape and size

distribution when dry SEM, TEM, AFM

May be problematic

for nanogels

8. Radius of gyration MALLS

9. Zeta potential Electrokinetic measurements (Zetasizer)

10. Metal ion content ICP Det. of free metal ions

4.2. Polymer nanoparticle dispersion specs for labeling

• Concentration mass/volume

• pH

• Molecular weight and hydrodynamic size

• Av Number of DOTA per nanoparticle

• Colloidal stability after incubation at 90°C for 30 min.

Crosslinked PEG-PGA

Nanogel UD (Plan) Thailand

• Presence of metal impurities

TABLE 3. GOLD NANOCONSTRUCTS

No. Property Recommended analytical

method

Auxiliary analytical

methods

Remarks

1. Chemical composition FT-IR, UV-Vis absorption,

Raman

NMR, elemental

analysis, XPS

2. Chemical composition –

presence of specific groups or

functions

FT-IR, UV-vis absorption

spectroscopy

Titration, UV-Vis

spectrofluorimetry

after binding of a

marker

3. Colloidal stability Turbidity (e.g. by UV-Vis) DLS, MALLS Specify solvent

composition (e.g.

water + pH range,

PBS, etc.)

4. Presence and location of

Plasmon resonance band

UV-Vis absorption spectroscopy

5. Shape, size distribution of the

metal core (+ presence of

aggregation)

UV-Vis absorption spectroscopy,

SEM, TEM, AFM

6. Size distribution – avg.

hydrodynamic radius of the

whole nanoparticle

Dynamic light scattering*, TEM,

Disc centrifuge (CPS)

Laser diffraction,

viscometry

*Providing the

analytical

wavelength does

not coincide with

absorption bands

7. Radius of gyration of the

whole nanoparticle

MALLS* *Providing the

analytical

wavelength does

not coincide with

absorption bands

8. Zeta potential Electrokinetic measurements

(Zetasizer)*

*Providing the

analytical

wavelength does

not coincide with

absorption bands

4.3. Guidelines for quality control and in vitro stability

Radioactive nanoparticles and various radiolabeled polymeric nanoparticles will be tested for quality

control through various radioanalytical techniques.

18

Radiochemical purity will be established through TLC, HPLC, size exclusion or gel permeation

chromatographic techniques. In vitro stability for periods of 1-7 days (and beyond) will be evaluated by

incubating at 37°C aliquots of radiolabeled nanoconstructs in saline, PBS buffer, HSA.

In vitro stability will be measured through UV spectrophotometry and the radioanalytical techniques

above described.

Additional in vitro stability may include dilution studies where radiolabeled nanoconstructs will be

diluted in aqueous media at physiological pH at concentrations to mimic cellular levels and stability

analyzed through UV spectrophotometric TLC, HPLC, size exclusion or gel permeation chromatographic

techniques.

Once the initial in vitro stability is established, further in vitro toxicological analyses will include cell

viability tests on Peripheral Blood Mononuclear Cells (PBMCs), hemolysis assays, complement activation,

platelet aggregation assays.

4.4. Binding affinity assays

The bombesin decorated constructs will be assayed in a competitive binding study with the 125

I-

bombesin analogue, prior to conjugation, using cells expressing GRP receptors.

TABLE 3. IN VITRO AND IN VIVO TESTING FACILITIES

Tumor type Animal

model/cell line

Receptor

type

In-vitro

Testing

facilities

In-vivo Testing

facilities

Label/isotope

type

Country

Prostate

cancer

Xenographt and

orthotropic

Laminin

GRP Cell culture Vet school

Au-198/199

Pd-109 USA

Breast cancer Xenographt and

orthotropic GRP Cell culture Vet school

Au-198/199

Pd-109 USA

Pancreatic

cancer

Xenographt and

orthotropic EGFR Cell culture Vet school

Au-198/199

Pd-109 USA

Breast cancer MCF-7 Y1 Cell culture SPECT/PET,

Animal house

Tc-99m, F-18, Lu-

177, Re-188, Au-

198

Pakistan

Prostate

cancer PC-3 GRP Cell culture

SPECT/PET,

Animal house

Tc-99m, F-18, Lu-

177, Re-188, Au-

198

Pakistan

Brest cancer Mice GRP

Confocal

microscopy,

cytotoxicity

test, activity

assays

SPECT/PET Fluorescent, Tc-

99m, Ga-68, I-131 Malaysia

Prostate

cancer Mice GRP

Confocal

microscopy,

cytotoxicity

test, activity

assays

SPECT/PET Fluorescent, Tc-

99m, Ga-68, I-131 Malaysia

Colon cancer Mice TGFR

Confocal

microscopy,

cytotoxicity

test, activity

assays

SPECT/PET Fluorescent, Tc-

99m, Ga-68, I-131 Malaysia

Head and

neck cancer Mice EGFR

Confocal

microscopy,

cytotoxicity

test, activity

assays

SPECT/PET Fluorescent, Tc-

99m, Ga-68, I-131 Malaysia

Pancreatic

cancer Mice SSTR

Microscopy

cell culture,

Cytotoxicity

test, activity

assays

Optical imaging Tc-99m, Lu-177,

Re-188 Mexico

Breast cancer Mice GRPR

Microscopy

cell culture,

Cytotoxicity

test, activity

assays

Optical imaging Tc-99m, Lu-177,

Re-188 Mexico

Prostate

cancer Mice GRPR

Microscopy

cell culture,

Cytotoxicity

test, activity

assays

Optical imaging Tc-99m, Lu-177,

Re-188 Mexico

Glioma Mice Integrins

Microscopy

cell culture,

Cytotoxicity

test, activity

assays

Optical imaging Tc-99m, Lu-177,

Re-188 Mexico

Prostate

cancer

Mice, TRAMP

Mice

PSMA,

PSCA,

Mesothelin

Cytometry,

Confocal

microscopy,

cytotoxicity

Flourescence and

bioluminescent

optical Imaging,

PET/SPECT/CT

Tc-99m, Re-188,

Lu-177, Cu-64, Ga-

67, F-18, In-111,

Ga-68, I-

124/125/131, Y-90

Italy-

Padova

Breast cancer Mice, HER2/neu EGFR, HER-

2

Cytometry,

Confocal

microscopy,

cytotoxicity

Flourescence and

bioluminescent

optical Imaging,

PET/SPECT/CT

Tc-99m, Re-188,

Lu-177, Cu-64, Ga-

67, F-18, In-111,

Ga-68, I-

124/125/131, Y-90

Italy-

Padova

Ovarian

cancer Mice

EGFR, HER-

2, CD44

Cytometry,

Confocal

Flourescence and

bioluminescent

Tc-99m, Re-188,

Lu-177, Cu-64, Ga-

Italy-

Padova

20

microscopy,

cytotoxicity

optical Imaging,

PET/SPECT/CT

67, F-18, In-111,

Ga-68, I-

124/125/131, Y-90

Oseophageal

cancer Mice CD44

Cytometry,

Confocal

microscopy,

cytotoxicity

Flourescence and

bioluminescent

optical Imaging,

PET/SPECT/CT

Tc-99m, Re-188,

Lu-177, Cu-64, Ga-

67, F-18, In-111,

Ga-68, I-

124/125/131, Y-90

Italy-

Padova

Gastric

cancer Mice CD44

Cytometry,

Confocal

microscopy,

cytotoxicity

Flourescence and

bioluminescent

optical Imaging,

PET/SPECT/CT

Tc-99m, Re-188,

Lu-177, Cu-64, Ga-

67, F-18, In-111,

Ga-68, I-

124/125/131, Y-90

Italy-

Padova

Mesotheliom

a Mice

CD44,

Mesothelin

Cytometry,

Confocal

microscopy,

cytotoxicity

Flourescence and

bioluminescent

optical Imaging,

PET/SPECT/CT

Tc-99m, Re-188,

Lu-177, Cu-64, Ga-

67, F-18, In-111,

Ga-68, I-

124/125/131, Y-90

Italy-

Padova

Pancreatic

cancer Mice

CD44,

Mesothelin

Cytometry,

Confocal

microscopy,

cytotoxicity

Flourescence and

bioluminescent

optical Imaging,

PET/SPECT/CT

Tc-99m, Re-188,

Lu-177, Cu-64, Ga-

67, F-18, In-111,

Ga-68, I-

124/125/131, Y-90

Italy-

Padova

SW480, Xenograft in

mice VEGF Cell culture

Animal

house/animal

SPECT

Tc-99m, In-111,

Ga-67,68, Cu-64,

Ho-166, lu-177, Y-

90

Iran

MCF-7 Xenograft in

mice Folate Cell culture

Animal

house/animal

SPECT

Tc-99m, In-111,

Ga-67,68, Cu-64,

Ho-166, lu-177, Y-

90

Iran

SKBR-3 Xenograft in

mice Her-2 Cell culture

Animal

house/animal

SPECT

Tc-99m, In-111,

Ga-67,68, Cu-64,

Ho-166, lu-177, Y-

90

Iran

Raji cells Xenograft in

mice CD-20 Cell culture

Animal

house/animal

SPECT

Tc-99m, In-111,

Ga-67,68, Cu-64,

Ho-166, lu-177, Y-

90

Iran

4T1 Syngeneic Folate Cell culture

Animal

house/animal

SPECT

Tc-99m, In-111,

Ga-67,68, Cu-64,

Ho-166, lu-177, Y-

90

Iran

Ehrlich cell

line Mice - - Animal house

Tc-99m, I-131, Au-

198 EGYPT

4.5. Collaborative research activities for the first 15 months of the CRP

1. Naked and bombesin-functionalised polymeric nanocarriers labelled with a fluorescent probe

(preferably, CY5.5, BODIPY) will be sent to ITALY-PADOVA for preliminary binding tests

(cytometry) and NPs localisation through confocal microscopy.

2. Bombesin and DOTA derivatized NPs to complex Lu-177/Ga-67 will be produced by ARGENTINA,

BRAZIL, ITALY-PALERMO, POLAND, SINGAPORE/THAILAND. Samples with Specs for

Labeling containing 2-50 mg of solid will be sent to ITALY-MILAN and IRAN for radiolabeling and

to ITALY-PADOVA, MALAYSIA, MEXICO and PAKISTAN for in-vitro/in-vivo evaluation or both.

3. Functionalised natural polymers will be provided by THAILAND to USA, EGYPT, PAKISTAN and

MALAYSIA for stabilizing radioactive Au NPs.

4. Radioactive Au NP labeling and selected in-vitro cell studies will be performed by USA on bombesin-

functionalised polymer NPs able to bind gold NPs, along with an already established protocol

developed with non-radioactive gold.

5. CONCLUSIONS AND RECOMMENDATIONS

The five days meeting involved extensive, stimulating discussions with participation from all the

scientists from participating Member States—Culminating into consensus toward the development

of nanosized radiopharmaceuticals.

The Participants to the meeting have demonstrated a variety of expertise in different and

complementary fields, from the design, synthesis and characterization of nanoconstructs to the

radiolabeling and in vitro/in vivo evaluation. The expertise and enthusiasm shown by the

participants may produce significant outcomes in the course of the CRP.

The meeting also helped to strategize various different ways on how the participants can exchange

scientific information and research samples as outlined below.

A list of nanoconstructs, available or under development is provided in Table 1. Selected

nanoconstructs will be sent to the relevant partners for preliminary biological evaluation and

labeling with radioisotopes.

Participants have agreed to decorate the nanocarriers with the selected targeting peptide

(bombesin) in order to standardize the evaluation protocols. This approach will ensure a common

base for comparison of different material platforms.

Following the preliminary testing, Participants will be engaged in the conjugation of the

nanocarriers with DOTA chelator in order to proceed with the labeling tests.

A list of minimum product specification to accompany the produced nanocarriers has been

discussed and outlined above.

A comprehensive list of characterization tools for various nanoconstructs is outlined in Table 2.I

and 2.II.

Resources available for in vitro and in vivo testing of nano-radiopharmaceuticals are summarized

in Table 3.

There are no standard protocols in terms of in vitro stability and in vivo analysis for the

development of nanosized radiopharmaceuticals. Therefore, the Members of CRP, through

collective efforts, will formulate standard protocols for the development of nanosized

radiopharmaceuticals for use in Academia and Industry involved in related research and

development activities.

It is recommended to have the next CRP meeting either in Brazil or in South Africa.

22

Radiation-induced nano-structuration of biological materials using ionizing radiation

Macarena Siri1, Silvia del Valle Alonso

1 and Mariano Grasselli

2

1 Laboratorio de BioMembranas, Dpto. de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal,

Buenos Aires, Argentina 2

Laboratorio LaMaBio, Dpto. de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos

Aires, Argentina

1. INTRODUCTION

The first attempt to organize the public research work in the nanotechnology field in Argentine was

done in the year 2004. The main action was the creation of nanotechnology networks from the existing

public research and development laboratories. Four networks, each including more than three groups were

organized.

In 2005, the Ministry of Science and Technology (MINCyT) creates the Argentine Foundation of

Nanotechnology (FAN) to promote the development of this area. During 2006, nanotechnology was

claimed as priority area by the MINCyT and opened different R+D oriented-grants to support the research

in the following years.

In 2012 there were 93 research groups working in R+D in the field (12 in the bio-nanotechnology).

Since this year, nanotechnology is considered by MINCyT as one of the three technologies of general

propose considered as priority, with applications in agro-industry, energy, health, and environment issues.

Since 1999 the Laboratorio de Materiales Biotecnológicos (LaMaBio), headed by Dr. M. Grasselli, at

the Universidad Nacional de Quilmes developed expertise in application of gamma radiation to polymers

for biotechnological applications. The goal of our research was the preparation of new adsorptive polymers

for recovery specific proteins from biological liquors. Polymer modifications were performed by radiation-

induced grafting techniques using gamma irradiation from a Cobalt-60 source. In the following years new

specific applications on biological science were added, i.e. grafting polymerization onto nuclear track-

etched membranes by remanent radical after etching process.

In 2009, LaMaBio was awarded with the first two grants to start R+D projects in the nanotechnology

field. One of them was involved with the development of functional nanochannels into track-etched

membranes. The second one was the use of radiation techniques to prepare nanostructured materials, as a

member of a Coordinate Research Project supported by IAEA.

In the following paragraphs a description of some of the main results achieved in this latter above

mentioned project, which is in connection to the present CRP.

1.1. Preparation of protein nanoparticles by radiation-induced crosslinking

More than twenty-five years ago it has been demonstrated that radiation technology can be used to

generate polymer microparticles by precipitation polymerization with a very narrow particle size

distribution [1]. In this way, methacrylate-based microspheres can be prepared by radiation-induced

polymerization of diethyleneglycol dimethacrylate (DEGDMA) and others cross-linker monomers in

appropriate organic solvent [1,2]. Tailor-made microspheres were further developed by copolymerization

of DEGDMA with reactive monomers, such as glycidyl methacrylate (GMA), or special type of monomers

to reach functional microspheres [3,4], however lower-diameter particles (<0. een

achieved by this technique.

Another strategy has been described to obtain nano-sized polymeric particles using ionizing radiation

technology. Soluble synthetic polymers can be intramolecularly cross linked in diluted solutions by

quantum-ray creating nanogels [5,6]. Changing irradiation conditions it is possible to manage inter or

intramolecular crosslinking onto soluble polymer molecules in random coil conformation. Considering

their aqueous environment and their rheological properties they were called nanogels.

Proteins are also soluble macromolecules; however, most of them have a very compact and defined three

dimensional structure instead random coil conformation, which are known as globular proteins. The effect

of gamma-ray irradiation onto globular proteins solutions was study for many years, mainly in features

related to the biological effect of protein degradation or agglomeration, especially human related proteins

such as Albumin [7, 8]. All these studies were run mainly in aqueous conditions according to the

physiological milieu.

Albumin is the most abundant protein in the mammalian plasma and it serves as a carrier of

hydrophobic biological and synthetic molecules such as anticancer drugs. Thus, preparation of Albumin

nanoparticles should improve the drug delivery properties of this natural carrier.

Globular proteins are macromolecules very sensitive to microenvironment, thus the effect of the

solvent during the irradiation process would have a great effect onto the sample. Having in mind that

organic polar solvents induce protein aggregation, without protein denaturation, therefore it would be

possible to prepare protein-based nanoparticles by irradiation of water/ethanol solutions of globular

proteins with ionizing radiations. Ethanol is the preferred solvent since it has been used for protein

precipitation for more than sixty years in plasma fractionation by Cohn process [9].

2. MATERIALS AND METHODS

Bovine serum albumin, Fraction V (BSA) was obtained from Sigma Aldrich. All other reagents were

of analytical grade and used as received.

BSA was dissolved in buffer phosphate 30 mM (BP) pH 7. Different amounts of ethanol were added drop

wise onto the protein solution, keeping the temperature at 0 ºC under constant stirring. BSA solutions were

irradiated by a 60

Co gamma-rays source (PISI CNEA-Ezeiza) at a dose rate lower than 1 kGy/h and keeping

sample temperature in the range of 5-10 ºC during irradiation. Alternative irradiation was performed with a

ELU-6 linear electron accelerator (Eksma, Russia) at 1 kGy/pulse.

After irradiation, protein samples were diluted to a suitable concentration with phosphate buffer

saline (PBS) for different experiments.

Particle size was determined by dynamic light scattering (DLS) at 25 °C in a 90Plus/Bi-MAS particle

size analyzer, with a light source of 632.8 nm and a 10-mW laser. Each data is the average of three

measurements. Samples were kept at 4 °C until analyzed, and measurements were carried out on days 1

and 30 after sample preparation.

UV-vis spectra were performed in a UV-Vis Shimadzu 160 A spectrophotometer. Circular Dicroism

(CD) measurements were carried out at 20 °C on a Jasco 810 spectropolarimeter equipped with a Peltier-

effect device for temperature control (Jasco Corporation, Japan). Six or ten spectra were recorded and

averaged. Fluorescence spectra were recorded with Spectrofluorometer FS 2 Scinco at 25 °C.

3. RESULTS AND DISCUSSION

Engineered nanoparticles are attracting interest for application in different medical fields. These

nanosized materials have different absorption properties in biological tissues and this constitutes one of the

relevant characteristics for cosmetic and medical applications [10,11].

24

For a long time, albumins have attracted the attention of the pharmaceutical industry because it is the

most abundant serum protein and it has the ability to bind a wide variety of drug molecules, and alter their

pharmacokinetic parameters. Recently, the discovery of the albumin recycling pathway opens the

possibility to produce drugs with tailored half-life properties [12].

Albumin nanoparticles can be prepared by emulsification, but this method requires an organic solvent

for removal of oil and surfactant. Desolvation process with organic solvents is another commonly used

method for protein nanoparticles preparation [13,14]. Chemical cross linking with glutaraldehyde or heat

denaturation are standard stabilization steps done to avoid particle dissolution. It is also known that

albumin oligomerization by thermal process involves protein denaturation [15].

Globular proteins are macromolecules which are very sensitive to the microenvironment and their

two most common protein precipitants —ethanol and ammonium sulfate— were considered as modifiers in

irradiated protein solutions. Both additives alter its solubility by a dewatering effect onto the solvated

protein. In the particular case of BSA, more than 60 % ethanol is required to induce its precipitation.

The addition of increasing quantities of ammonium sulfate to the protein solution did not change the

average size of the particles after irradiation. However, the addition of sub-precipitant concentrations of

ethanol reached nanoparticle formation. Using buffer/ethanol mixtures, from 20% ethanol , nanoparticles

can be found with sizes that increased in the same proportion of solvent increment in the irradiated mixture.

Other protein-compatible polar solvents, such as acetonitrile and isopropanol can also induce nanoparticle

formation.

BSA solutions were irradiated at different radiation doses in the range of 1 to 20 kGy. Irradiations

higher than 5 kGy were necessary to obtain nanoparticles (NP) from non-degased samples. Protein size of

samples irradiated without ethanol remained approximately in their original size when they were irradiated

within this dose range. These results are in agreement with data reported for molecular weight

determination by DLS of an irradiated BSA solution in an oxygen atmosphere [8]. Using a pulse linear

accelerator, only 2 kGy were enough to obtain albumin NP (BSA-NP).

The three dimensional structure of BSA included in the nanoparticles was studied by fourth-

derivative UV-visible spectroscopy (4dUVv), Circular Dichroism (CD), and Fluorescence spectroscopy

(FS). It has been established that these techniques are sensitive to detect instantaneous protein

conformational changes. Alteration of the microenvironment (polarity, hydration, hydrophobic interactions,

and packing density) of tyrosine and tryptophan amino acids can be followed by these spectroscopic

techniques (4dUVv and FS).

Fig. 1. UV-vis spectra of BSA and BSA-NP (left) and the fourth-derivative spetra of the same samples (right). No significant

diference in the Trp region has been found.

The BSA molecule has two tryptophan amino acids, one in the Domain I and the second in the

Domain II. The 4dUVv spectra of the irradiated BSA samples have the same spectrum shape and no

absorbance shifts were noticed, indicating that the microenvironments of aromatic amino acids, especially

Trp, were maintaining the same conformational features as the protein [16]. From the analysis of FS

emission spectra, no major changes in the Trp environment were observed; the shift of BSA-NP is very

subtle. We can assumed that the structure in the BSA-NP is conserved (Fig. 2), therefore not disturbance in

the Domain I and II of the BSA are found in the NP.

Fig. 2. Fluorescence spectra of BSA and BSA-NP using excitation at 295 nm

Far CD gives information about the composition of secondary structure of the proteins. According to

Fig. 3, the spectrum corresponding to BSA-NP prepared by EB irradiation at 2 kGy showed an

approximately 20% decreasing in the alpha/beta secondary structures, in comparison to the native BSA.

This change is considered a small global change assuming that a NP is made of dozens of BSA molecules.

Fig. 3. Far-Circular dicroism of the irradiated BSA samples at 0, 1 and 2 kGy (last one has NP)

26

Near CD gives information about ternary protein structure and the symmetry around aromatic amino

acids where only folded proteins have CD spectral information. Fig. 4 shows the CD characterization of

BSA samples. CD spectra of the irradiated samples showed no changes in the general spectral shape.

However, ethanol concentrations from 20% to 40% showed an increase in the CD signal. This effect could

be attributed to a more rigid conformational structure of the protein in the NP.

Fig. 4. Near-Circular dicroism of the irradiated BSA samples with different ethanol proportions.

From CD and UV-vis data, most of BSA proteins in the NP kept their native structure. Thus,

nanoparticles can be considered as formed by an aggregation of protein molecules in their native

conformation.

In order to study the nature of the linkage between protein molecules, particle size was determined by LDS

under chaotropic condition, 6 M guanidinium chloride (GdmCl), to unfold (denature) the protein structures

(Table I).

Table I. Average particle diameter of BSA and BSA-NP solutions in buffer and diluted in GdmCl 6 M 20 h

previously to be measured in DLS.

Diameter in buffer

(nm)

Diameter in denaturant

(nm)

BSA 5.9 +/- 1.3 630 +/- 500

BSA-NP 20.3 +/- 3.4 100 +/- 80

As expected, for unfolded and open protein structures particles of longer diameters are present. Data

of higher dispersion in denaturant condition can be attributed to the random coil unstructured particles.

Additionally, an inverse correlation between BSA and BSA-NP in unfolded condition is shown in Table I.

The lower diameter extension of NP can be assigned to the presence of covalent intercatenary cross linking

bounds.

In order to elucidate the mechanism of nanoparticle formation further studies using LDS were

performed. BSA and BSA-NP prepared in buffer/ethanol were diluted with its corresponding solvent or

with buffer. Under these conditions LDS measurements showed quite different results. Diameter

distribution profiles of BSA in the presence of ethanol show protein aggregation irrespective of irradiation

condition. Non-irradiated sample prepared with buffer/ethanol and further buffer diluted do not show

aggregation. Thus, the protein aggregation step it is a fast reversible process that varies dynamically

according to the ethanol concentration in the media and irradiation the sample with ionizing radiation

freeze the protein aggregates in an irreversible way.

Fig. 5. Scheme of BSA nanoparticle formation according to LDS data from different steps

4. CONCLUSION

Irradiation of globular protein solutions has been studied by many authors in the past. Most of these

studies have focused on the physicochemical changes in the protein structure. However, a globular protein,

such as the BSA, can be used as a basic unit to build nanoparticles by ionizing irradiation under specific

conditions.

By c hanging the ethanol/water ratio protein clusters could be dynamically created in solution, which can

be further stabilized by radiation crosslinking. Protein nanoparticles in the range of 20 to 40 nm were found

after gamma or electron beam irradiation of a protein dissolved in an aqueous / ethanol mixture.

Nanoparticles were characterized by DLS, UV, fluorescence, and CD spectroscopies, which showed that

protein molecules keep their overall structure.

Another important achievement is the potentiality of size tailoring of the BSA nanoparticles.

Nanoparticle size could be controlled by the ethanol-water proportion in the sample. It is important to

highlight this method can synthesize high concentration of protein nanoparticles without precipitation and

to achieve high yield without addition of any additional chemical cross-linker.

Diameter (nm)

1 10 100

Nu

mb

er

0

20

40

60

80

100

120

NanoBSA

BSA solution in buffer BSA solution in buffer+ethanol

Gamma irradiation

Diameter (nm)

1 10 100

Nu

mb

er

0

20

40

60

80

100

120

BSA

Diameter (nm)

1 10 100

Nu

mb

er

0

20

40

60

80

100

120

BSA EtOH 35%

28

This nanoparticle radiosynthesis could also be extended to others proteins by co-aggregation with BSA.

ACKNOWLEDGEMENT

M.S. thanks to CONICET for the fellowship. S.A. and M.G. are senior researchers from CONICET.

This work was partially supported by grants from Universidad Nacional de Quilmes and MINCyT. Authors

thanks to Dr. S. Kadłubowski from the Institute of Applied Radiation Chemistry, Lodz, Poland, for the e-

beam irradiation.

REFERENCES

[1] YOSHIDA, M., ASANO, M., KAETSU, I. AND MORITA, Y., Radiat. Phys. Chem. 30 (1987) 39.

[2] NAKA, Y., KAETSU, I., YAMAMOTO, Y. AND HAYASHI, K., Preparation of microspheres by

radiation-induced polymerization. I. Mechanism for the formation of monodisperse poly(diethylene

glycol dimethacrylate) microspheres. J. Polym. Sci. A: Polym. Chem. 29 (1991), 1197–1202.

[3] CHEN, C.-H. AND LEE, W.-C. Preparation of methyl methacrylate and glycidyl methacrylate

copolymerized nonporous particles. J. Polym. Sci. A: Polym. Chem. 37 (1999) 1457–1463

[4] SAFRANY, A., KANO, S., YOSHIDA, M., OMICHI, H., KATAKAI, R. AND SUZUKI, M.,

Functional polymeric microspheres synthesized by radiation polymerization Radiat. Phys. Chem. 46

(1995) 203-206

[5] KADLUBOWSKI, S., GROBELNY, J., OLEJNICZAK, W., CICHOMSKI M. AND ULANSKI, P.,

Pulses of Fast Electrons as a Tool To Synthesize Poly(acrylic acid) Nanogels. Intramolecular Cross-

Linking of Linear Polymer Chains in Additive-Free Aqueous Solution, Macromol. 36 (2003) 2484-

2492

[6] ULANSKI, P., JANIK, I, ROSIAK, J.M., Radiation formation of polymeric nanogels Radiat. Phys.

Chem, 52 (1998) 289-294.

[7] DAVIES, K. J. A. Protein damage and degradation by Oxygen Radicals. I. General Aspects, J. Biol.

Chem. 262 (1987) 9895-9901.

[8] KUME, T. AND MATSUDA, T., 1995. Changes in structural and antigenic properties of proteins by

radiation. Radiat Phys. Chem. 46 (1995) 225-231.

[9] COHN, E. J., HUGHES JR., W. L., WEARE, J. H., Preparation and Properties of Serum and Plasma

Proteins. XIII. Crystallization of Serum Albumins from Ethanol-Water Mixtures, J. Am. Chem. Soc.,

69 (1947) 1753–1761.

[10] [Pardeike, J., Hommoss, A., Muller, R.H., 2009. Lipid nanoparticles (SLN, NLC) in cosmetic and

pharmaceutical dermal products. Int. J. Pharm., 366, 170-184

[11] [11] Fortina, P., Kricka, L.J., Graves, D.J., Park, J., Hyslop, T., Tam, F., Halas, N., Surrey, S. and

Waldman, S. A., 2007. Applications of nanoparticles to diagnostics and therapeutics in colorectal

cancer Trends in Biotechnol., 25, 145-152.

[12] PLUMRIDGE, A., SLEEP, D., CAMERON, J., SANDLIE, I., ANDERSEN, J. T.,

[13] FRIIS, E. P.. Albumin variants, patent WO/2011/051489

[14] Marty, J.J., Oppenheimer, R.C., Speiser, P., 1978. Nanoparticles - a new colloidal drug delivery

system. Pharm. Acta Helv. 53, 17-23.

[15] Weber, C., Coester, C., Kreuter, J., Langer, K., 2000. Desolvation process and surface

characterisation of protein nanoparticles. Int. J. Pharm. 194, 91–102.

[16] Vaiana, S.M., Emanuele, A., Palma-Vittorelli, M.B. and Palma, M.U., 2004. Irreversible formation of

intermediate BSA oligomers requires and induces conformational changes. Proteins, 55, 1053-1062.

[17] Soto Espinoza, S. L., Sánchez, M. L., Risso, V., Smolko, E. E. and Grasselli M., Radiation Synthesis

of Seroalbumin Nanoparticles” Radiat. Phys. Chem. 81 (2012) 1417–1421.

Radio-induced crosslinking of albumin nanoparticles for radiopharmaceuticals delivery

system

Ademar B. Lugão, Gustavo H. C. Varca; PhD. José R. Rogero, MS. Sizue O. Rogero, PhD.

Luis Goulart

Abstract

Nanoparticle Protein-bound (Nab) pharmaceuticals has been considered as a very promising route for the delivery of

chemo and radio-pharmaceutics for cancer cells. Protein-bound paclitaxel is an injectable formulation of paclitaxel, a mitotic

inhibitor drug used in the treatment of breast cancer, lung cancer and pancreatic cancer. In this formulation, paclitaxel is

bonded to albumin as a delivery vehicle. It is sold in the United States under the trade name Abraxane. The preparation of

albumin nanoparticles of Abraxane is based on strong shear and pressure in solvent medium followed by extraction.

Therefore a number of possible shortcomings can be foreseen. A better process in a pure and mild, solvent free medium to

synthesize albumin nanoparticle and to encapsulate chemo and radio pharmaceutics should be developed. Nanoparticles of

albumin by radio-induced crosslinking was recently demonstrated and proved by Grasselli’s group and papain nanoparticle

was demonstrated and proved by our group in Brazil. Our new process will be applied to encapsulate radio-pharmaceuticals.

This report details the establishment of a novel platform for the development of protein-based nanoparticles for the delivery

of chemo and radiopharmaceutics, including an overview of the process, scale up and irradiation dose effects, as well as

provides an experimental evidence to clarify the mechanism involved in the nanoparticle formation.

1. INTRODUCTION

This report addresses the research project entitled: "RADIO-INDUCED CROSSLINKING OF

ALBUMIN NANOPARTICLES FOR RADIOPHARMACEUTICALS DELIVERY SYSTEM", and

contains the information related to the ongoing research progress, mainly describing the activities and goals

achieved during the involved period.

Proteins as a whole represent an important group of the therapeutic agents available nowadays for the

treatment of a wide range of disorders. Despite direct applications, these biomolecules may also be used to

functionalize, confer biopharmaceutical advantages and constitute novel drug delivery systems for

available drugs among other aspects (Banta et al, 2010; Sezaki and Hashida, 1985).

Perhaps the most relevant aspect to be taken into account with regard to globular proteins in

pharmaceutics and industrial processes is attributed to instability in unusual environments and intrinsic

limitations of such biomolecules. Thus, many approaches have been directed towards overcoming such

limitations (Polizzi et al., 2007) including nanotechnological tools (Crommelin et al., 2003), chemical

modifications (Fernandez-Lafuente et al, 1995), immobilization (Sheldon, 2007), the use of additives such

as sugars (Arakawa and Timasheff, 1982, Varca et al., 2010) among others, considering that overcoming

such problems and intrinsic limitations would allow a great expansion in the use of such compounds

(Arnold, 1993).

Particularly the use of high energy radiation is known to directly or indirectly damage or impair

biological function of macromolecules and proteins (Saha, 1995; Davies et al., 1987; Furuta, 2002) and as a

result its use is therefore limited. However, over the last decade some researchers have attempted to use

radiation (Akiyama et al., 2007; Furusawa et al., 2004) to achieve nanometer-sized particles and nanogels

based on proteins and peptides. A few years later, Soto-Espinoza et al. (2012) evaluated the use of solvents

combined with radiation to synthesize bovine serum albumin nanoparticles.

Specifically the use of ionizing radiation to achieve particle size control of enzyme nanoparticles with

preserved bioactivity for biomedical applications was recently demonstrated by our group, using papain

(Varca et al, 2014). This enzyme is a cysteine protease, widely applied as model enzyme for several studies

30

due to its renowned biotechnological relevance (Kao Hwang & A. C. Ivy 2006) as well as its defined

structure and biological properties (Kamphius et al., 1984).

The approach given in this report details the establishment of a novel platform for the development of

protein-based nanoparticles for the delivery of chemo and radiopharmaceutics, including an overview of

the process, scale up and irradiation dose effects, as well as provides an experimental evidence to clarify

the mechanism involved in the nanoparticle formation.

2. GENERAL SCOPE

To develop a cleaner process free from solvents and free from shear and high pressure for the

production of stable Albumin nanoparticles with size control based on the recently discovered radio-

induced crosslinking of proteins.

3. SPECIFIC GOALS

Development and characterization of Albumin nanoparticles;

Radio-induced crosslinking of Albumin;

Development of the capability for production of large amount of radio-induced crosslinked proteins

nanoparticles;

Development of the capability for controlling the size and size distribution of protein nanoparticles.

Encapsulation of Yttrium by albumin nanoparticles

Study of encapsulation stability;

Crosslinking of new peptides for radiopharmaceuticals encapsulation

4. EXPECTED OUTPUTS

1. Control of yield and control of size of protein nanoparticles.

2. Data on Albumin encapsulation of Yttrium.

3. Data on the possibilities of producing peptides nanoparticles and radio-pharmaceutical encapsulation.

4. Control protein nanostructure to achieve instructive scaffolds for tissue engineering.

5. Network of researchers from different areas.

5. MAIN ACHIEVEMENTS

5.1. Development of Protein-based nanocarriers

5.1.1. Experimental

5.1.1.1. Materials

Papain 30000 USP-U/mg (EC 3.4.22.2) and ethylenediaminetetraacetic acid were purchased from Merck

(Germany); L-cysteine hydrochloride monohydrate, dimetilsulfoxide, sodium chloride, ethanol, sodium

hydroxide, chloridric acid, acetic acid and heptahydrate disodium phosphate from Synth (Brazil), and Nα-

Benzoyl-DL-arginine p-nitroanilide from Sigma-Aldrich®

(USA). All reagents were of analytical grade.

5.1.1.2. Methods

Particle synthesis. Phosphate buffer (50mM), papain solution and ethanol 0-35% (v/v) were added

drop wise to glass vials on ice bath and allowed to stabilize overnight prior to the beginning of experiments

at refrigerated conditions (8oC). Samples were exposed to γ-irradiation on ice bath and dose rate of 1.2

kGy.h-1

using 60

Co as radioactive source in a gammacell 220 Irradiator. The samples were properly filtered

using 0.22µm filters and stored at ±8oC prior to analysis. Controls were prepared under the same

conditions.

5.1.1.3. Process Overview

ɣ-irradiation dose. The effect of γ-irradiation dose over particle size changes was evaluated as a

function of exposure to 2.5, 5, 7.5 and 10 kGy in presence and absence of 20% (v/v) ethanol as additive.

5.1.1.4. Scale up

The effect of papain concentration over the particle formation was evaluated in the range of 12.5-50

mg.mL-1

at 10kGy.

5.1.1.5. Particle Characterization

Particle Size. Particle size measurements were performed by Dynamic Light Scattering analysis (DLS)

on a Zetasizer Nano SZ90 device at 20 oC and 90

o scattering angle in triplicates of 3 runs of 12s each.

5.1.1.6. Bityrosine evaluation

The samples were properly diluted in buffer to reach equivalent absorbance. The readings were

performed at λ=280nm using a Cary 1E Uv-Vis Varian® spectrophotometer. The samples were then

checked for bityrosine emission on a F4500 Hitachi® Fluorescence spectrophotometer using λEx=325nm

λEm = 340-500nm, scan speed of 240nm/min, Exslit of 5nm and Emslit of 10nm.

6. RESULTS AND DISCUSSION

In order to track down the effect of γ-irradiation over papain particle size increment and allow a proper

selection of optimum irradiation dose for the synthesis of the nanoparticles the samples were submitted to

distinct irradiation doses (Figure 1). As reported in literature, the effects of irradiation exposure in proteins

preferably lead to chain scission and degradation effects (Furuta, 2002; Saha et al., 1995) and thus, the dose

range evaluated did not exceed 10kGy to preserve bioactivity and minimize protein degradation.

0,0 2,5 5,0 7,5 10,0

4,5

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

9,0

9,5

10,0

10,5 20% EtOH

0% EtOH

Siz

e (d

.nm

)

Radiation dose (kGy)

FIG. 1. Papain particle size increment as a function of irradiation dose in presence (20% v/v) and absence of ethanol.

In absence of ethanol minor changes were observed as a function of dose ranging from 0 to 10kGy by

means of particle size as observed in figure 1. This profile was distinct in presence of ethanol (20% v/v),

where papain did undergo changes at 2.5 kGy irradiation and similar effects were also observed up to

10kGy, whereas minimum changes were registered among the doses ranging from 2.5 to 7.5 kGy. Based on

these results optimized conditions for the nanoparticles synthesis were achieved at irradiation dose of

10kGy. Similar results were observed in the case of BSA (Soto-Espinoza et al., 2012), where albumin

nanoparticles presented the highest particle size increase at 10 kGy.

32

FIG. 2. Bityrosine monitoring in papain (12.5 mg.mL-1) as a function of irradiation dose in absence (a) and presence (b) of

ethanol (20% v/v).

The bityrosine evaluation of the samples is described in figure 2. The irradiation led to an increase in

bityrosine formation in both cases. Particularly in absence of ethanol (Figure 2A), the bityrosine linkages

increased as the irradiation dose increased. The same profile was observed in presence of ethanol, but in

this case (Figure 2B) the increase was smaller and corroborated the DLS measurements (Figure 1), where

minimum changes took place between 2.5-7.5 kGy and more pronounced changes at 10kGy. These data

highlighted the optimized dose established as 10 kGy, and experimentally supported the proposed

mechanism, attributed to the protein crosslinking by means of bityrosine formation.

6.1. Effect of concentration

As an attempt to understand to what extent the applications of the protein-based nanoparticles could

be applied to, scale up experiments involving distinct papain concentrations - 12.5, 20 and 50 mg.ml-1

-

irradiated at the optimized dose of 10 kGy were performed as presented in figure 3.

0% 10% 20% 30% 35%

4

6

8

10

12

14 12.5mg.ml-1

20mg.ml-1

50mg.ml-1

Siz

e (

d.n

m)

Ethanol%

FIG. 3. Scale up experiments involving different papain (12.5, 20 and 50 mg.mL-1) and ethanol (0-35% v/v) concentrations

irradiated at 10 kGy.

Under the assayed conditions the particles ranged from 5 to 13 nm with minimum deviation for all

concentrations assayed. These results revealed that the technique was effective at broad concentration

range, from 12.5 to 50mg.mL-1

, considering that similar particle size and size distribution profiles, as a

function of ethanol concentration were obtained for the samples (figure 3). This evidenced a minor

influence of the total protein concentration over particle size increment caused by the process.

To some extent, similar profiles were observed for the samples in presence of ethanol without

irradiation. However, as reported in our previous work (Varca et al., 2014) dilution tends to invert the

process. Such fact did not take place when the samples were irradiated, supporting the occurrence of novel

linkages and protein crosslinking as a consequence.

It’s relevant to mention that some deviation (±1.5-2 nm) has been observed from our previous results

(Varca et al., 2014). We support such changes considering that at this time the flaks were completely filled

in order to minimize the effects of oxygen (Davies et al, 1987; Saha et al., 1995). In this case, a little shift

was observed in terms of particle size and also a better size distribution was achieved up to 35% ethanol

concentration, with a decrease in the formation of aggregates. However, more experiments shall be

conducted in order to establish the role of the atmospheric conditions and oxygen itself over the irradiation

process.

FIG. 4. Bityrosine monitoring in papain (12.5 mg.mL-1) non-irradiated (a) and irradiated (b) at 10kGy under different

ethanol concentrations (0-35%).

In absence of irradiation, the presence of ethanol led to a minimum increase in bityrosine

fluorescence units (figure 4A). These changes were attributed to microenvironmental changes in the solvent

which holds some effects over protein and may lead to distinct values.

At defined irradiation dose of 10 kGy figure 4B, the samples containing ethanol presented considerably

higher bityrosine intensity if compared to non-irradiated papain indicating an increase in the formation of

such linkages if compared to the native form of the enzyme. At 30-35% ethanol, the values were a little bit

34

higher if compared to lower ethanol concentrations (10-20%). The same profiles were observed for

samples prepared at higher papain concentrations 20 and 50mg.mL-1

(data not shown).

The interaction of ethanol over proteins is well established. This solvent is widely applied as protein

precipitant agent, which is known to induce changes on protein structure and denaturation as well,

depending upon ethanol concentration, the protein itself and experimental conditions (Yoshikawa, 2012).

Regarding the radiation induced nanoparticle, the role of ethanol in the process was evidenced by

comparing irradiated papain in absence and presence of ethanol. This compound created a specific

environment suitable for the particle size increase, which induced the formation of bityrosine in a

controlled manner, possibly related crosslinks of intermolecular nature, which was not achieved otherwise.

To experimentally support such information (figures 1 and 4A), irradiation in absence of ethanol led to

higher levels of bityrosine crosslinks with no relevant changes in particle size whatsoever, and thus

indicated that the formation of such linkages, in this case, were at intramolecular level.

Additionally, the bityrosine experiments provided clear evidence that the mechanism involved is a

result of building of novel tyrosine linkages, possibly of intermolecular nature, and they were sufficient to

demonstrate a direct relation between the linkages and the particle formation itself. Whatsoever, more

experiments shall be performed in order to clarify the nature of such linkages.

6.2. Mechanistic approach

The results supported the proposed mechanism involved in the nanoparticle formation induced by

irradiation (figure 5) and revealed that it occurred in a similar way of the so called protein crosslinking

(Baylei, A.J, 1991; Matheis & Whitaker, 1987) and the changes caused by UV exposition (Garcia-

Castinaras, 1978), where the main mechanism involve changes – linkages at intermolecular level - in

tyrosine residues.

FIG. 5. Representative scheme of the proposed mechanism involved in the papain nanoparticle formation induced by

irradiation.

In the case of gamma irradiation, the radiation induced formation of bityrosine occurs as a result of

the interaction between the tyrosine residues, if properly accessible, and the species produced as a result of

the water radiolysis. Even though the dimerization of tyrosine residues by irradiation is not yet established

(Saha et al., 1995), there is evidence to support the crosslinking formation by C-O-C bonds induced by

interaction with OH.-radical (Karam et al., 1984, Saha et al., 1995) and in parts by the generated tyrosine

phenoxyl radicals (Casas-Finet et al., 1984).

In conclusion the radiation-induced synthesis of papain nanoparticles under optimized conditions led

to the formation of protein based nanoparticles with size ranging from 5 to 13nm depending upon ethanol

concentration and irradiation dose. The optimized irradiation dose was confirmed at 10kGy and scale up

experiments revealed that protein concentration (5-50mg.mL-1

) did not hold a major influence in the

particle formation. Therefore, these results indicated that the radiation induced technique may be carried

out under a wide protein concentration range with minimum size variation.

Bityrosine changes as a function of the process were observed and provided experimental evidence

that such linkages were directly related to the particle formation process. This effect was attributed to

irradiation in specific conditions which led to a controlled formation of bityrosine, possibly of

intermolecular nature, which is not achieved otherwise. Ethanol was capable of promoting this

microenvironment suitable for the nanoparticles synthesis. Possible applications of the technique concern

the development of protein-based nanostructured bioactive nanoparticles and/or drug carriers for distinct

biomedical applications.

7. ACHIEVEMENTS

In this report we summarized the preliminary results of the work developed as well as the initial work

plan for the project. On this account, the mains achievements are described below.

Development of the capability for controlling the size and size distribution of protein

nanoparticles: the radiation-induced synthesis of protein nanoparticles (papain) under optimized

conditions led to the formation of protein based nanoparticles with size ranging from 5 to 13nm

depending upon ethanol concentration and irradiation dose. The optimized irradiation dose was

confirmed at 10kGy.

Development of the capability for production of large amount of radio-induced crosslinked

proteins nanoparticles: scale up experiments revealed that protein concentration (5-50mg.mL-1

) did

not hold a major influence in the particle formation. Therefore, these results indicated that the

radiation induced technique may be carried out under a wide protein concentration range with

minimum size variation.

7.1. Collaborations

We have performed a collaborative work with Argentina, where Professor Mariano Grasselli visited

Brazil and Gustavo Varca visited Argentina for a period of time in order to produce papain particles, and

also to develop protein based drug carriers using radio-induced protein reticulation. As an attemp to clarify

the mechanism as well as the role of the radicals over the nanoparticle formation, Gustavo Varca visited

Poland with the help of Dr. Ulansky and Rosiak, where pulse-radiolysis experiments were carried out.

7.2. Published articles

1. Varca, G. H.C. ; Ferraz, C. C. ; Lopes, P. S.; Mathor, M. B. ; Grasselli, M. ; Lugao, A. B. . Radio-

synthesized protein-based nanoparticles for biomedical purposes. Radiation Physics and Chemistry, v. 94,

p. 181-185, 2013.

2. Varca, G. H. C. ; Perosi, G. ; Grasselli, M. ; Lugao, A. B. . Radiation synthesized protein-based

nanoparticles: a technique overview. Radiation Physics and Chemistry, 2014. DOI:

10.1016/j.radphyschem.2014.05.020

7.3. Conferences

1. Perossi, G. ; Varca, G. H. C. ; Grasselli, M. ; Mathor, M. B. ; Lugao A. B. Enzyme nanoparticles as

bioactive compounds for biotechnological applications. 2013. Xii encontro da sbpmat.

2. Varca, g. H. C. ; Grasselli, m. ; Mathor, M. B. ; Lopes, P. ; Lugao A. B.. Protein based nanocarriers for

drug delivery. 2013. XII encontro da sbpmat.

3. Varca, Gustavo; Ferraz, C; Grasselli, M.; Lugao, A. B. Radiation induced protein crosslinking - protein

nanoparticles for biomedical purposes. 2013. 17th international meeting on radiation processing - imrp 17.

4. Varca, G. ; Lugao, A.B. Radiation synthesized papain nanoparticles: a stability approach. 2013.

Nanotoday 2013.

5. Varca, G. ; Lugao, A.B.. Protein nanoparticles synthesized by gamma irradiation. 2013. Nanotoday

2013.

36

6. Varca, G. ; Lugao, A.B.. Reticulação radio-induzida de proteínas para fins biomédicos. 2012. 7o

congresso latino americano de orgãos artificiais e biomateriais - colaob.

7. Varca, G. ; Grasselli, K.; Lugao, A.B.. . Radio-synthesized papain nanoparticles for biomedical purposes.

2012. 10th meeting of the ionizing radiation and polymers symposium irap'2012.

7.3. Patents

1. Varca, G. ; Lugao, A.B.. Processo de Produção de Nanopartículas Proteicas utilizando radiação ionizante

. 2012, Brasil. Número do registro: BR1020130050342, data de depósito: 10/10/2012, título: "Processo de

Produção de Nanopartículas Proteicas utilizando radiação ionizante ", Instituição de registro:INPI -

Instituto Nacional da Propriedade Industrial.

2. Varca, G. ; Lugao, A.B.. Processo simultâneo de reticulação, Esterilização e Produção de sistema

polimérico contendo nanopartículas proteicas. 2014, Brasil. Número do registro: BR1020140047182, data

de depósito: 27/02/2014, título: "Processo simultâneo de reticulação, Esterilização e Produção de sistema

polimérico contendo nanopartículas proteicas" , Instituição de registro:INPI - Instituto Nacional da

Propriedade Industrial

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protein nanogel by quantum-ray irradiation. Colloid. Polym. Sci. 285, 801–807.

Arakawa, T., Timasheff, S.N., 1982. Stabilization of protein structure by sugars. Biochem. 21(25), 6536–

6544.

Arnold, F. H., 1993. Protein engineering for unusual environments. Curr. Opin. Biotechnol. 4(4), 450–455.

Banta, S., Wheeldon, I. R., Blenner, M. 2010. Protein Engineering in the Development of Functional

Hydrogels. Annu. Rev. Biomed. Eng. 12, 167–186.

Baylei, A.J, 1991. The chemistry of natural enzyme-induced cross-links of proteins. Amino. Acids. 1, 293–

306.

Casas-Finet, J.R., Toulme, J.J., Santus R., Butler, J., Land, E.J., Swallow, A.J., 1984. Influence of DNA

binding on the formation and reactions of tryptophan and tyrosine radicals in peptides and proteins. Int. J.

Radiat. Biol. 45, 119-132.

Cromellin, D.J.A, Storm, G., Jiskoot, W., Stenekes, R., Mastrobattista, E., Hennink W.E, 2003.

Nanotechnological approaches for the delivery of macromolecules. J. Control. Release. 87, 81–88.

Davies, K.J.A., 1987. Protein damage and degradation by oxygen radicals. I general aspects. J. Biol. Chem.

262, 9895–9901.

Fernandez-Lafuente, R., Rosell, C.M., Rodriguez, V., Guisan, J.M., 1995. Strategies for enzyme

stabilization by intramolecular crosslinking with bifunctional reagents. Enzyme. Microb. Tech. 17, 517–

523.

Furusawa, K., Terao, K., Nagasawa, N., Yoshii, F., Kubota, K., Dobashi, T., 2004. Nanometer-sized gelatin

particles prepared by means of gamma-ray irradiation. Colloid. Polym. Sci. 283, 229–233.

Furuta M., 2002. Irradiation effects of hydrases for biomedical application. Radiat. Phys. Chem. 57, 455–

457.

Garcia-Castinaras, S., Dillon, J., Spector, A., 1978. Detection of bityrosine in cataractous human lens

protein. Sci. 199, 897–899.

Hwang, K. and Ivy, A. C., 1951. A review of the literature on the potential therapeutic significance of

papain. Ann. N. Y. Acad. Sci., 54, 161–207.

Kamphuis, I., Kalk, K., Swarte, M., Drenth, J., 1984.Structure of papain refined at 1.65 angstrom

resolution. J. Mol. Biol. 179, 233–256.

Karam, L.R., Dizdaroglu, M., Simic, M.G. 1984. OH radical-induced products of tyrosine peptides. Int. J.

Radiat. Biol. 46, 715- 724.

Lal, M., 1994. Radiation induced oxidation of sulphydryl molecules in aqueous solutions. A comprehensive

review. 43 (6), 595–611.

Matheis, G., Whitaker, J.R., 1987. A review: enzymatic cross-linking of proteins applicable to foods. J.

Food Biochem. 11, 309–327.

Polizzi, K.M., Bommarius, A.S., Broering, J.M., Chaparro-Riggers, J.F., 2007. Stability of biocatalysts.

Curr. Opin. Chem. Bio. 11(2), 220–225.

Saha, A., Mandal, P.C., Bhattacharyya, S.N., 1995. Radiation-induced inactivation of enzymes–A review.

Radiat. Phys. Chem. 46(1), 123–145.

Sezaki, H., Hashida, M., 1985. Macromolecules as Drug Delivery Systems. Exp. Biol. Med. 7, 189–208.

Sheldon, R.A., 2007. Enzyme Immobilization: The Quest for Optimum Performance. Adv. Synth. Catal.

349, 1289– 1307.

Soto Espinoza, S.L., Sánches, M.L., Risso, V., Smolko, E.E., Grasselli, M., 2012. Radiation synthesis of

seroalbumin nanoparticles. Radiat. Phys. Chem. 81, 1417–1421.

Varca, G.H.C., Andreo-Filho, N., Lopes, P.S., Ferraz, H.G., 2010. Cyclodextrins: an overview of the

complexation of pharmaceutical proteins. Curr. Protein. Pept. Sc. 11, 255–263.

Varca, G.H.C., Ferraz, C.C., Lopes, C.C., Mathor, M.B., Grasselli, M., Lugao, A.B., 2014. Radio-

synthesized protein nanoparticles for biomedical purposes. Radiat. Phys. Chem. 94, 181–185.

Yoshikawa, H., Hirano A., Arakawa T., Shiraki, K., 2012. Effects of alcohol on the solubility and structure

of native and disulfide-modified bovine serum albumin. Int. J. Biol. Macromol. 50, 1286–1291.

38

Development of nanoscale delivery systems for diagnosis and treatment of cancer

Tamer M. Hafez

National Centre for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy

Authority (EAEA), Cairo, Egypt

Abstract

Development specialized polymeric nano carrier for radiopharmaceuticals like gold-198 for the diagnosis and treatment of

cancer has been investigated. The preparation and characterization of nanosized gold-198 was carried out. It was found that it

has high radiochemical purity and in vitro stability upto 7 days. Also, it had shown high retention time in the site of injection

when it is tested by intra-articualr injection in joints of guinea pigs. On the other side, the preparation of nano-polymeric

materials using radiation synthesis technique, which will serve as carriers for the radioisotopes, was investigated. Polymeric

pH-sensitive hydrogel nanoparticles (nanogels) of narrow size distributions were directly prepared by gamma radiation-

induced polymerization of acrylic acid (AAc) in an aqueous solution of polyvinylpyrrolidone (PVP) as a template polymer.

The particle sizes of the PVP/PAAc nanogels at different pH values were evaluated using dynamic light scattering (DLS) and

the morphology was assessed using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Smaller

and more stable nanogel particles can be produced by irradiating a feed solution containing about 50 to 75 mol% of AAc and

using PVP of high molecular weight. The particle size increases with the increase in feed concentration and the suitable

concentration might be from 1% to 2%. The nanogel particles prepared under air or nitrous oxide atmosphere were slightly

smaller and more stable (lower polydispersity index at high pH) than those prepared under nitrogen atmosphere. Lowering

the irradiation temperature can also decrease the particle size. Increasing the irradiation dose decreased the swelling degree of

the PVP/ PAAc nanogel particles. These nanoparticles (nanogels) are expected to be potentially useful in

radiopharmaceutical delivery due to their controllable sizes. Besides, the presence of carboxylic groups enables the particles

to adsorb radiopharmaceuticals cations

1. INTRODUCTION

Nuclear medicine is an important medical specialty involving the use of radioactive substances for the

diagnosis and treatment of diseases. There are a number of diagnostic and therapeutic radiopharmaceuticals

that are FDA approved for use in human patients. These radiopharmaceuticals, once administered to the

patient, localize to specific organs or cellular receptors based on their in vivo properties and allows for

noninvasive imaging and/or treatment at the molecylar level. Nuclear medicine procedure measures the

radiation emitted from within the body rather than radiation that is generated by external sources such as X-

rays allowing for the detection at a much earlier stage that is far more sensitive and quantitative than other

anatomical imaging techniques. The therapeutic effect of radiopharmaceuticals relies on the tissue-

destructive power originating from the emission of short-range ionizing radiation by radionuclides. The

combined use of a pair of diagnostic and therapeutic radiopharmaceuticals for the treatment of disease

constitutes the basic paradigm of the field of theranostics as applied to nuclear medicine.

Currently used diagnostic and therpeutic radiopharmaceuticals in nuclear medicine utilize

radiolabelled small-molecules, proteins and peptides as targeting vectors. On the therapy front, β-emitting

radionuclides conjugated with tumor specific peptides or monoclonal antibodies are used to ablate tumors

and metastatic lesions.

Nanosized therapeutic radioisotopes of size 50-100 nm range will facilitate their penetration across

tumor vasculature with restrictive 300-400 nm porosity. This may provide new approaches to maximize

drug uptake, enhance therapeutic efficacy and consequently minimize side effects due to nonspecific

localization in healthy cells. Gold-198 (198

Au), as a therapeutic radioisotope, provides an efficient beta

energy emission (βmax = 0.96 MeV) and half-life (2.7days) that are optimum for the destruction of tumor

cells. Its penetration range (up to 4 mm in tissue or up to 1100 cell diameters) is sufficiently long to provide

crossfire effects to destroy tumor cells/tissue, but short enough to minimize radiation exposure to adjacent

tissues. Different nanoparticle preparations based upon 198

Au have been reported such as: Gum Arabic-198

AuNP and EpiGalloCatechin Gallate-198

AuNP. The fabrication of GA-198

AuNP nanotherapeutic agent of

85nm hydrodynamic diameter are highly effective for delivering high therapeutic doses directly into tumor

cells because they possess suitable sizes to tumor vasculature and porosity. Besides, technetium-99m and

ioine-131 play very important roles in the diagnostic and therapeutic nuclear medicine, respectively.

Recently, there is a growing interest in synthesis and design of soft nanomaterials to be applied in the

biomedical field. Among these materials, hydrogel nanoparticles have gained considerable attention due to

their tunable dimensions, large surface area, stable interior network structure, and fast response to

environmental factors, such as ionic strength, pH, and temperature. These properties demonstrate the great

potential of nanogels for bio-related applications such as drug delivery systems (DDS) and bio-imaging.

Majority of methods utilized for nanogel synthesis are often performed in microemulsion in order to

prevent formation of bulk material. However, the formation of hydrogel nanoparticles without using

organic solvents and surfactants is considered a challenge. Recently, the concept of interpolymer

complexation via hydrogen bonding has been exploited for this purpose. Hydrogen- bonded interpolymer

complexes (IPCs) are formed between proton-donating poly(carboxylic acids) and proton- accepting non-

ionic polymers. An interesting example which has attracted a continuing interest as a model of biological

systems is the formation of polymer complexes between poly(ethylene oxide) and poly(acrylic acid) via H-

bonding in aqueous media. These complexes are novel individual compounds and their properties are

entirely different from the properties of their component polymers. Moreover, they are pH-sensitive since

their formation depends on the degree of ionization of the poly(carboxylic acid) and thus on the

environmental pH. Henke et al. reported an approach that involved irradiation of PVP–PAAc complexes by

pulses of fast electrons in dilute, deoxygenated solutions. The radiation treatment of IPC induces

intramolecular (intracomplex) crosslinking leading to the formation of permanent PVP–PAAc nanogels.

Practically, nanogels produced by this method are especially well suited for biomedical applications

because they are free of residual toxic chemicals, e.g. monomers, initiators or crosslinkers, and there is no

need for additional purification steps.

In our research proposal, we aim to formulate, synthesize and evaluate nanosized gold-198, iodine-

125/131 and technetium-99m radiopharmaceuticals, based upon nanohydrogel polymer, as diagnostic or

therapeutic agents for cancers.

2. EXPERIMENTAL

The gold-198 was prepared by irradiation in MURR. Res-Au-198NP was prepared successfully by

mixing resveratrol with the radioactive gold with stirring resulting a dark ruby red color is produced. The

radiochemical purity and the UV-Vis spectroscopy were performed using TLC and HPLC. Also, in vitro

stability in room temperature, saline and rat serum were studied.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene)

Preparations of PVP/PAAc hydrogel nanoparticles PVP/PAAc hydrogel nanoparticles were prepared

via gamma radiation-induced polymerization of AAc in an aqueous solution of PVP. Aqueous solutions

containing different feed compositions of PVP/AAc mixtures were introduced into glass bottles and

40

subjected to c-rays generated from a 60Co source provided with a temperature control unit at a dose rate of

3.85 kGy/h. The irradiation process was carried out at 35oC under air atmosphere; otherwise conditions will

be mentioned. In some cases, the feed solutions were purged with nitrogen or nitrous oxide for 20 min to

remove the dissolved oxygen, and then the bottles were sealed and irradiated. To calculate the yield of

nanoparticles, the colloidal nanogel suspensions were centrifuged (SORVALL_ ULTRA 80, USA) at

20,000 rpm for 30 min. at 4oC. Supernatants as well as aggregated nanogels were collected and freeze-dried

in order to determine the weight of the polymers, which formed nanogels. The production yields were

calculated from the mass ratio of polymers forming nanogel and the polymer and monomer initially

introduced in the preparation procedure. Particle sizes of PVP/PAAc nanogels were measured by the

dynamic light scattering (DLS) technique using a PSSNICOMP Zeta Potential/Particle Sizer 380ZLS (PSS-

NICOMP, Santa Barbara, CA, USA). Samples were properly diluted with freshly prepared deionized water

(filtered with a 0.2 lm syringe filter) until an intensity of 250–350 kHz was achieved. The dilutions were

also convenient to avoid. particle interactions. The pH of the solutions was adjusted by adding few drops of

NaOH or HCl. The scattered light intensity was detected at a 90 angle and measurements were run for at

least 10 min. The volume-weighted hydrodynamic mean diameters were reported to know the size

contributing the most volume. The polydispersity index (PDI) (the square of the coefficient of variation),

which is a measure of the size distribution breadth, was also calculated. FT-IR spectra of PVP, PAAc and

freeze-dried PVP/PAAc nanogel as KBr pellets were recorded by a JASCO FT/IR- 6300 spectrometer in

the range of 400–4000 cm_1. To follow the formation of PVP/PAAc complexes, solutions were analyzed

by measuring the change in transmittance at 500 nm using a JASCO UV/VIS spectrophotometer V-560.

Viscosity measurements were conducted at 30.0 Å} 0.5 _C using Ubbelohde viscometer. The pH of the

solutions was adjusted by adding few drops of NaOH or HCl. Deionized water was used as a reference

solvent. Transmission electron microscopy (TEM; JEOL JEM-100CX, Japan) and atomic force microscopy

(AFM; Agilent AFM 5500, USA) were used to observe the morphology of nanogel particles. For TEM

observations, the nanogel suspension was properly diluted and dripped onto carboncoated copper grid and

then dried at room temperature. For AFM observations, one drop of properly diluted nanogel suspension

was placed on the surface of freshly cleaved mica and then dried at room temperature.

3. RESULTS

Preparation of stable RES gold-198 NP

This following figures shows the UV absorbance of 198Au-Resveratrol (λ = 540nm), which is

typically the range of the gold NP and the radiochemical purity of 198Au-Resveratrol measure using TLC-Bioscanner radiochromatogram confirming its purity about 96.5% and of particle size around 50nm.

FIG. 1. UV-Vis of 198Au-Resveratrol

FIG. 2. Radiochemical purity of 198Au-Resveratrol as measured by TLC.

FIG. 3. TEM scan of 198Au-Resveratrol

3.1. Preparation of nanoscale carriers

The following figures confirm the successful preparation of PVP/PAAc hydrogel nanoparticles.

PVP/PAAc hydrogel nanoparticles were prepared via gamma radiation-induced polymerization of AAc in

an aqueous solution of PVP. These figures show the factors studied which affect the preparation of the

PVP/PAAc hydrogel nanoparticles such as: PVP-AAc concentration, PVP/AAc composition, Exposure

dose, PVP Mwt, Oxygen atmosphere, Temperature, Type of irradiator, PH of the Media. And shows the

characterization of this nano-polymeric materials using different instruments such as: Thermogravimetric

analysis (TGA), Scanning electron microscopy (SEM), Ultraviolet spectrophotometer (UV- Vis), Infrared

(FTIR).

42

FIG. 4. Changes of transmittance of PVP/AAc solution with irradiation dose. Feed composition: 35/65 (PVP/AAc); feed

conc.: 1.5%; PVP molecular weight (Mw) = 1,300,000. Samples were diluted 5-fold with water.

FIG. 5. Intensity (blue), volume (red) and number (green) weighted Gaussian distribution analysis for the PVP/PAAc

nanogels at pH 4 (a) and 7 (b). Feed composition: 35/65 (PVP/AAc); feed conc.: 1.5%; PVP molecular weight (Mw) =

1,300,000; 20 kGy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web

version of this article.)

Dose ( kGy)

0 1 2 3 4 5

% T

rans

mitt

ance

0

20

40

60

80

100

Scheme 1. Proposed scheme for radiation-induced formation of PVP/PAAc nanogel particles.

FIG. 6. FTIR spectra of PAAc, PVP and PVP/PAAc nanogel particles.

44

FIG. 7. Effect of PVP molecular weight (Mw) on the particle size of the prepared PVP/PAAc nanogels measured at pH 4

(main frame) and pH 7 (insert). Feed composition: 35/65 (PVP/AAc); feed conc.: 1.5%; 20 kGy. Numbers inside parentheses

represent polydispersity index (PDI).

FIG. 8. Effect of the environmental atmosphere during irradiation on the particle size of the prepared PVP/PAAc nanogels

at low and high pH. Feed composition: 35/65 (PVP/AAc); feed conc.: 1.5%; PVP molecular weight (Mw) = 1,300,000; 20

kGy. H2O2 conc.: 1% (based on feed conc.). Numbers inside parentheses represent polydispersity index (PDI).

PVP Mw

Mean

Dia

mete

r (

nm

)

0

100

200

300

400

1000

1500

2000

pH 4

pH 7

8000 58000 1300000

(0.089)

(0.035)(0.041)

(0.224)

(0.078)

Multimodal

Mea

n d

iam

eter

( n

m )

0

100

200

300

400

500

600

pH 4

pH 7 (NaOH)

Air/H2O2 Air N2O Nitrogen

Multimodal (0.239)

(0.053)

(0.064)

(0.041)

(0.078)

(0.086)

(0.045)

(0.015)

FIG. 9. Effect of H2O2 concentration on the particle size of the prepared PVP/PAAc nanogels. Feed composition: 35/65

(PVP/AAc); feed conc.: 1.5%;PVP molecular weight (Mw) = 1,300,000; 20 kGy.

FIG. 10. Effect of feed concentration on the particle size of the prepared PVP/PAAc nanogels at pH 4 and 7. Feed

composition: 35/65 (PVP/AAc); PVP 1,300,000; 20 kGy. Numbers inside parentheses represent polydispersity index (PDI).

H2O2 ( wt % )

(based on feed concentration)

0 1 2 3 4 5 6

Mea

n di

amet

er (

nm )

0

100

200

300

400

500

pH 7 (NaOH)

pH 4

Feed Concentration ( wt % )

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Me

an

Dia

me

ter

( n

m )

0

100

200

300

400

500

600

700

800

pH 4

pH7 (NaOH)

(0.048)

(0.102)

(0.078)

(0.055)

(0.112)

(0.051)(0.04) (0.041) (0.041)

(0.066)

46

FIG. 11. Effect of feed composition on the particle size of the prepared PVP/PAAc nanogels at pH 4. Insert represents

particle sizes at pH 7 for nanogels prepared from three different feed compositions. Feed conc.: 1.5%; PVP 1,300,000; 20

kGy. Numbers inside parentheses represent polydispersity index(PDI).

FIG. 12. Effect of irradiation temperature on the particle size of PVP/PAAc nanogels prepared from different feed

compositions when measured at pH 4 (a) and pH 7 (b). Feed conc.: 1.5%; PVP 1,300,000; 20 kGy. Numbers inside

parentheses represent polydispersity index (PDI).

10 20 30 40 50 60

Mea

n Di

amet

er (

nm )

60

80

100

120

140

160

180

200

220

240

Zeta

Pot

entia

l ( m

V )

-32

-30

-28

-26

-24

-22

-20

-18

-16

Mean Diameter

Zeta Potential

90 80 70 60 50 40

PVP

AAc

Feed Composition ( mol/mol % )

(0.042)

(0.049)

(0.041) (0.036) (0.089)

Feed composition (PVP/AAc mol/mol %)

Mean

dia

mete

r (n

m)

0

100

200

300

400

500

600

700

Irradiated at ~35 0 C

Irradiated at ~10 0 C

25/75 35/65 55/45

(b) pH 7

(0.120)

(0.140)

(0.209)

(0.072)

(0.078)

(0.152)

FIG. 13. Effect of irradiation dose on the particle size of the prepared PVP/PAAc nanogels. Feed composition: 35/65

(PVP/AAc); feed conc.: 1.5%; PVP 1,300,000. Numbers inside parentheses represent polydispersity index (PDI).

FIG. 14. Effect of irradiation dose on the relative viscosity of PVP/PAAc nanogels at different pH values. Samples were

diluted 5-fold with water. Feed composition: 35/65 (PVP/AAc); feed conc.: 1.5%, PVP 1,300,000.

Irradiation dose(kGy)

10 20 30 40

Me

an

dia

me

ter(

nm

)

0

200

400

600

800 pH ~ 4

pH ~ 7

(0.04) (0.041) (0.037) (0.038)

(0.117)

(0.078)

(0.048)(0.058)

pH

3 4 5 6 7 8

Re

lati

ve

vis

co

sit

y

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

20 kGy

40 kGy

48

FIG. 15. Morphology of PVP/PAAc nanogel particles investigated in deionized water at pH 4. (a) TEM image without

staining; (b) TEM image after staining with uranyl acetate; (c) AFM image (topography and three dimensional). Feed

composition: 35/65 (PVP/AAc); feed conc.: 1.5%; PVP 1,300,000; 20 kGy.

FIG. 16. TEM and 3D AFM images of PVP/PAAc nanogel particles prepared by irradiation doses of 20 (a) and 40 (b) kGy,

and deposited from an aqueous solution at pH 7 without staining. Feed composition: 35/65 (PVP/AAc); feed conc.: 1.5%;

PVP 1,300,000.

4. CONCLUSIONS

As a first step results, chemically crosslinked, pH-sensitive PVP/PAAc hydrogel nanoparticles were

successfully prepared in a high yield via gamma radiation-induced polymerization of acrylic acid in an

aqueous solution of polyvinylpyrrolidone (PVP) as a template polymer. The particle sizes of the

PVP/PAAc nanogels at different pH values were evaluated using dynamic light scattering (DLS) and the

morphology was assessed using atomic force microscopy (AFM) and transmission electron microscopy

(TEM).. These nanoparticles (nanogels) are expected to be potentially useful in drug delivery due to their

controllable sizes. Besides, the presence of carboxylic groups enables the particles to adsorb cationic drugs

or to be conjugated with some bioactive molecules.

50

REFERENCES

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nanogels prepared by γ radiation as artificial tears for dry eye treatment, submitted for publication.

2. Abd El-Rehim, H.A., Hegazy, E.A., Hamed, A.A., Swilem, A.E., Controlling the size and swellability of

stimuli-responsive polyvinylpyrrolidone–poly(acrylic acid) nanogels synthesized by gamma radiation-

induced template polymerization, Eur. Polym. J. (2013), in press.

3. Abd El-Rehim, H.A., Hegazy, E.A, Swilem, A.E., Klingner, E.A., Hamed, A.A., Developing the potential

ophthalmic applications of pilocarpine entrapped into polyvinylpyrrolidone−poly(acrylic acid) nanogel

dispersions prepared by γ radiation, Biomacromolecules (2013), in press.

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6. Chanda N, Shukla R, Zambre A, Mekapothula S, Kulkarni RR, Katti K, Bhattacharyya K, Fent GM,

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52

Report from the European Institute of Oncology (IEO), Milan, Italy

Marco Chinol

1. INTRODUCTION

Accurate localization of non-palpable breast lesion increases the probability of radical tumor excision

and decreases the mortality. The European Institute of Oncology (IEO) developed a medical procedure for

localization and surgical removal of nonpalpable mammalian lesions, which combines the use of

radioactive tracers with a probe for radioguided surgery. This methodology is called ROLL - Radioguided

Occult Lesion Localization - and involves the injection of human albumin macroaggregates (MAA)

labelled with radioactive technetium (99m

Tc) into a small clinically nonpalpable nodule during

mammography or ultrasonography. Correct inoculation is verified by superimposing the mammography

image over a scintigraphy scan of the breast acquired in the nuclear medicine facility. Both set of imaging

are available in the operating room to assist the surgeon in locating the lesion.

Then, by means of a gamma detecting probe for radioguided surgery, the lesion is localized as a hot

spot and can be removed efficiently during surgery, maintaining the breast integrity. The validity of this

methodology is demonstrated by its international diffusion (> 10,000 patients only at the IEO in the last

decade) and by the high number of scientific reports. The aspecific complex Technetium-MAA, due to the

short radioactive half-life of the 99m

Tc, does not allow planning surgical procedures on several days and,

not negligible aspect, also to carry out the localization as an outpatient exam, as well as its verifications in

the days preceding surgery. These factors limit patient accessibility to this methodology, severely affecting

hospitalization times and costs.

2. OBJECTIVES OF THE RESEARCH PROPOSAL

The main objective will be to obtain an insoluble conjugate of human albumin (HA) conjugated with

the macrocyclic chelator DOTA capable of binding with high in-vivo stability several radioisotopes for

imaging such as 111

In and for therapy such as 177

Lu with half-lifves longer than 99m

Tc.

Secondary objectives will be:

- Optimization and reproducibility of the conjugation steps

- Optimization and control of the particle size

3. EXPECTED OUTPUTS

It is expected that the new nanoceutical will possess the suitable size for the ROLL application and be

prepared in a controlled environment for further application in clinical trials and preferably in a freeze dry

kit to facilitate its use to the other CRP participants.

4. METHODS

Commercial HA, obtainable in a defined particle size, was initially purified from excipients and

mixed with p-SCN-Bn-DOTA testing different molar ratios. The mixture was first brought to pH=9.0 using

carbonate buffer and then heated at 40 oC. The solid phase thus formed underwent a series of separation,

dilution and further heating at the same temperature for a total processing time of 16 h. Evidence for the

formation of the conjugated albumin (HAC) as a new chemical entity was provided by MALDI-TOF mass

spectrometry and by IR and UV spectra. The degree of substitution was also checked by the colorimetric

arsenazo method. Radiolabelling tests using 111

In and 177

Lu were performed by heating the HAC particles

for 30 min at 90 oC. Stability studies of

177Lu-DOTA-HAC up to 6 days at 37 °C either in PBS or human

serum were also performed.

5. RESULTS

The degree of substitution (DS) was influenced by reactant molar ratio, pH, temperature and reaction

time. In the optimized conditions, DS averaged 5 nDOTA/nHA, coherent with the estimation obtained by

MALDI-TOF. Stability studies showed no evidence of radiolysis or biodegradation of the conjugate.

Radiolabelling tests using 111

In and 177

Lu showed that HAC can withstand to the high temperature required

for a high labelling yield.

6. CONCLUSIONS

A new HA conjugate with the DOTA chelator has been developed and has been successfully labelled

with 111

In and 177

Lu. However, the conjugation procedure still requires optimization and reproducibility in

order to apply this new particulate product to the ROLL procedure.

54

Cancer-fighting diagnostic and therapeutic nanogels

CLELIA DISPENZA

Dipartimento di Ingegneria Chimica, Gestionale, Informatica, Meccanica. Università degli Studi di

Palermo, Viale delle Scienze Ed. 6, 90128 Palermo; CNR - Istituto di Biofisica (Palermo Unit), Via U. La

Malfa 153, 90146 Palermo, Italy, Email: clelia.dispenza@unipa.it

1. INTRODUCTION

Nanotechnology applied to Medicine holds the promise to improve the current therapeutic and

diagnostic approaches in healthcare. However, most of the proposed nanoconstructs have shown limitations

related either to their inherent properties and in vivo performance or to the manufacturing process for the

complexity of material formulations and production steps. The research activity carried out by Dispenza

and co-workers in the framework of the previous CRP on “Nanoscale Radiation Engineering of Advanced

Materials for Potential Biomedical Applications” demonstrated the possibility of generating Poly-N-(Vinyl-

Pyrrolidone)-based nanogels with tailored physico-chemical properties in terms of particles size

distribution, surface charge density and degree of functionalization. In particular, these nanoparticles were

produced via e-beam irradiation of semi-dilute aqueous solutions of the polymer, also in the presence of a

functionalizing monomer, carrying either carboxylic or primary amino groups. High-energy irradiation

promotes crosslinking of the polymer, grafting of functional monomers and simultaneous sterilization of

the so obtained aqueous dispersion of the polymer, provided that the irradiation doses were within the

sterilization dose range (20-40 kGy). [1-5]

Different variants were obtained by varying the polymer concentration in water, the concentration of

the functionalizing monomer and the irradiation conditions, i.e. the delivered dose and dose-rate (by

changing the dose per pulse and pulse repetition rate). Purification from low molecular weight byproducts

was performed by dialysis. A detailed products analysis was carried by the combination of different

techniques, such as dynamic and static light scattering, gel permeation chromatography, photo-correlation

spectroscopy, FT-IR, Raman and solid-state NMR spectroscopies and XPS, SEM or AFM.

The main evidences from the above characterizations are:

1. All PVP-based nanogels have a globular morphology and hydrodynamic size varying from 20 to 200

nm with PDI of about 0.2-0.3.

2. All the generated nanogels are chemically stable and show new chemical functionalities with respect

to the parent PVP polymer, formed both by reaction of water radiolysis products with the polymer

and through grafting of suitable monomers to PVP. The nature of the new functional groups is the

same for the different variants produced while their relative amount mainly depends on polymer

concentration and to a less extent on dose and dose-rate.

3. The pure PVP variants are slightly anionic, the carboxyl group-modified variants are more markedly

anionic and the amino-functionalized variants are non-ionic, due to electrical charge compensation

between the anionic charge of the irradiated PVP and the cationic charge introduced with the amino

group in its protonated form.

4. Colloidal stability in water and PBS buffers with pH in the range 3 to 9 has been demonstrated for the

base and carboxyl group-grafted variants, bot at 25°C and 37°C. The amino functionalized variants

have a higher propensity to aggregation due to the suppression of electrostatic charge repulsion in the

colloidal stabilization mechanism.

5. No significant modification of hydrodynamic size has been observed by changing the pH of the

dispersing medium, thus suggesting that the ionizable moieties are mainly grafted on the surface of

the nanogels.

6. Shelf-stability of the base and carboxyl acid grafted nanogel variants up to two years has been

demonstrated.

7. Re-dispersability of the freeze-dried product has been demonstrated for the variants obtained in

correspondence to the higher doses, provided that a suitable cryoprotector is used.

8. Radiation effects on the formed nanogel particles have been investigated by imparting twice as much

the maximum dose delivered for their production: smaller particles show to be less sensitive to

irradiation in terms of molecular weight, particle size and chemical composition modification.

The PVP-based nanogels have been then tested as substrate for the assembly of tumor-targeted

“composite” nanodevices. In particular, the possibility of decorating the nanoparticle with different

“model” ligands or molecules with a biological function has been explored. Moreover, the bicompatibility

and localization pattern of the nanocarriers in cell cultures have been evaluated. For the last purpose,

fluorescent variants of the nanogels have been generated by covalent attachment of suitable probes (such as

FITC, TRITC, fluorescamine). [3, 7-9]

It has been demonstrated that:

1. All PVP-based NGs produced are non-cytotoxic and able to be internalized by cells with

preferential perinuclear localization. Preliminary studies aimed to elucidate the internalization

pathways suggest a macropinocytic mechanism.

2. Nanogels are first uptaken by cells and then released, with a maximum of uptake by cells that is at

about 6-7 h from incubation, for the bare PVP nanogels.

3. Folic acid, a single strand oligonucleotide, a variety of antibodies, and proteins such as BSA or

insulin have been covalently coupled to the nanogels. The degree of conjugation for the various

molecules has been determined. The biological activity of the attached moiety has been tested in-

vitro.

4. Polychelating DTPA-derivatized nanogels able to complex gadolinium have been produced. The

DTPA that is chemically bound to the nanogels forms quite stable complexes with Gd3+

. These

results, although preliminary, encourage to progress in the characterization of these materials to

demonstrate their utility as enhanced contrast agents for bio-imaging.

FIG. 1. Radiation engineered nanogels decorated with various functional moieties.

In conclusion, a platform to generate “as-born” sterile, noncytotoxic, multifunctional nanogel

particles, with tunable size and surface properties, amenable of conjugation with peptides, oligonucleotides,

antibodies, fluorescent molecules/enzymes is available.

56

Current research and development activities are now devoted to the following topics.

1. Establishing the mechanisms of nanogel formation.

2. Studying the interaction of the nanoparticles with plasma proteins.

3. Studying the biodistribution of the nanoparticles in vivo.

4. Developing theranostic nanodevices incorporating, in a single construct, a ligand for "active"

targeting, contrast agents for MRI or radionuclides for Nuclear Imaging, and a chemotherapeutic drug

and/or radiotherapeutic isotopes.

REFERENCES

[1] DISPENZA, C., GRIMALDI, N., SABATINO, M.A., SOROKA, I.L., JONSSON, M., Radiation-

engineered functional nanoparticles in aqueous system. Journal of Nanoscience and Nanotechnology, in

press.

[2] DISPENZA, C., SABATINO, M.A., GRIMALDI, N., BULONE, D., BONDI, M.L., CASALETTO, M.P.,

RIGOGLIUSO, S., ADAMO, G., GHERSI, G., Minimalism in Radiation Synthesis of Biomedical

Functional Nanogels. Biomacromolecules 13 (2012) 1805-1817.

[3] SABATINO, M.A., BULONE, D., VERES, M., SPINELLA, A., SPADARO, G., DISPENZA, C.,

Structure of e-beam sculptured poly(N-vinylpyrrolidone) networks across different length-scales, from

macro to nano. Polymer 54 (2013) 54-64.

[4] DISPENZA, C., GRIMALDI, N., SABATINO, M.A., TODARO, S., BULONE, D., GIACOMAZZA, D.,

PRZYBYTNIAK, G., ALESSI, S., SPADARO, G., Studies of network organization and dynamics of e-

beam crosslinked PVPs: from macro to nano. Radiat. Phys. Chem., 81 (2012) 1349-1353.

[5] DISPENZA, C., SABATINO, M.A., GRIMALDI, N., SPADARO, G., BULONE, D., BONDÌ, M.L.,

ADAMO, G., RIGOGLIUSO, S., Large-scale Radiation Manufacturing of Hierarchically Assembled

Nanogels. Chem. Eng. Transact. 27 (2012) 229-234.

[6] GRIMALDI, N., SABATINO, M.A., PRZYBYTNIAK, G., KALUSKA, I., BONDI’, M.L., BULONE, D.,

ALESSI, S., SPADARO, G., DISPENZA, C., High-energy radiation processing, a smart approach to obtain

PVP-graft-AA nanogels. Radiat. Phys. Chem. 94 (2014) 76-79.

[7] DISPENZA, C., ADAMO, G., SABATINO, M.A., GRIMALDI, N., BULONE, D., BONDI’, M.L.,

RIGOGLIUSO, S., GHERSI, G., Oligonucleotides-decorated-poly(N-vinyl pyrrolidone) nanogels for gene

delivery. J. Appl. Polym. Sci. 131 (2014) 39774-39782.

[8] DISPENZA, C., RIGOGLIUSO, S., GRIMALDI, N., SABATINO, M.A., BULONE, D., BONDI’, M.L.,

GHERSI, G., Structure and biological evaluation of amino-functionalised PVP nanogels for fast cellular

internalisation. React. Funct. Polym. 73 (2013) 1103-1113.

[9] RIGOGLIUSO, S., SABATINO, M.A., ADAMO, G., GRIMALDI, N., DISPENZA, C., GHERSI G.,

Polymeric nanogels: Nanocarriers for drug delivery application. Chem. Eng. Transact. 27 (2012) 247-252.

Radiolabeled Nanosized Delivery Systems for Theragnostics of primary and Metastatic

Colorectal Cancer

A. Rosato

Department of Surgical Sciences, Oncology and Gastroenterology, University of Padua,

Padua, Italy

1. INTRODUCTION

Dr. Rosato started is presentation with a brief outline of the project advanced in the framework of the

CRP “Nanosized Delivery Systems for Radiopharmaceuticals”. In particular, the research project is aimed

at designing and developing novel poly-ethylen-glycol (PEG)-based multifunctional polymer bioconjugate

therapeutic systems (smart nanoradiopharmaceuticals), for efficient theragnostics of colorectal carcinoma

(CRC) and its liver metastasis. Multifunctional bioconjugate nanoradiopharmaceuticals will be obtained by

derivatization with chelators for selected radionuclides for either PET and/or SPECT imaging or therapy,

and by functionalization with the YHWYGYTPQNVI (GE11) peptide, a dodecaptide that selectively

recognises the epidermal growth factor receptor (EGFR) overexpressed by the CRC cells, to achieve the

desired active CRC tumor targeting. In this regard, in fact, it must be stressed that CRC is the third most

common malignant neoplasm worldwide and the second leading cause of cancer deaths (irrespective of

gender). Despite advances in surgical techniques and adjuvant therapy have significantly increased the life

expectation of patients affected by primary CRC, there has been only a modest improvement in survival for

patients who present with advanced neoplasms and metastasis. These aspects demand prompt solutions.

Pharmaceutical nanotechnologies offer excellent opportunities to reduce the morbidity and mortality from

CRC. In particular, engineered colloidal systems obtained by physical or chemical combination of

structural and functional modules (drugs, targeting agents, microenvironmental or external stimuli sensitive

materials) have being studied for the treatment of a variety of tumours. These supramolecular

nanostructured systems can be exploited to overcome a few problems that limit the anticancer drug efficacy

such as low stability and solubility and rapid clearance. Furthermore, colloidal systems may be designed for

tissue or cell targeting thus providing for selective and controlled drug release in the disease site, which

avoids systemic toxicity. Notwithstanding, such systems have been poorly exploited as carrier of diagnostic

and/or therapeutic radionuclides.

The presentation continued with the introduction of the research group, which is multidisciplinary and

comprises competences in polymer chemistry, radiochemistry, radiopharmacy, nuclear medicine and

biology. Based on this expertise, the modules forming the multifunctional colloidal systems will be

extensively characterized according to various analytical techniques present in house or available through

integrated collaboration: chromatography (RP-HPLC, GPC); spectrometry (UV-Vis, fluorescence, IR,

NMR, PCS, ESI-TOF, MALDI-TOF, atom absorption, SPR); microscopy (TEM, AFM, confocal

microscopy); calorimetry (MC, DSC, TGA); Squid magnetometry, sonography. Moreover, investigations

will be carried out using cell lines that overexpress and non-overexpress the EGFR to evaluate the cell

selectivity and the cytotoxicity of the products. In this regard, biochemical and confocal microscopy studies

will allow to define biological effects and fate of the systems within the neoplastic cell. Moreover, ex vivo

perfusion of human colon segments bearing a primary carcinoma will permit to study the distribution and

the effects of drug nanodelivery systems in a model that most reproduces in vivo human conditions.

Finally, in vivo biodistribution and therapeutic efficacy in CRC mouse models will be analyzed by

PET/SPECT/CT molecular imaging approaches.

After such introduction, Dr. Rosato presented an outline of the facilities available for the realization

of the project. Specifically, the Department of Surgery, Oncology and Gastroenterology of Padova

University is a fully-equipped institution that offers several supports through its main core facilities that

will be available to the project if needed. In particular, the core facilities of: Genomics (TaqMan,

genotyping, a complete Affymetrix platform for microarrays, ChIP-on-chip, sequencing, RFLP), Cytomics

58

(FACS-sorter), Microscopy confocal, and fluorescence microscopy, multidimensional confocal

microscopy), Laser microdisection (LCM), Immunohistochemistry. Further, the institution has a >400 m2

Specific Pathogen Free (SPF) animal facility adapted for rodents, with a professional fully qualified staff of

veterinarians and technicians. The facility has a specific area for animal surgery, a BL3 dedicated room

with microisolators and holds several knock-out, transgenic and immunosuppressed xenograft tumor mouse

models. The animal facility is equipped with a platform for in vivo imaging, comprising the eXplore Locus

MicroCT scanner (GE Healthcare; Figure 1A and B, the instrument and a 3D reconstruction of a mouse,

respectively), an apparatus for bioluminescence Lumina II (Xenogen/Caliper, now Perkin Elmer; Figure 1C

and D, the instrument and an example of a mouse imaged after i.v. injection of tumor cells engineered to

express luciferase, respectively) and the MX2 optical imager (ART, Canada; Figure 1E and F, the

instrument and an example of a mouse bearing a s.c. prostate tumor imaged after administration of a

fluorophore-labelled anti-PSMA mAb, respectively). The MX2 is a high performance in vivo fluorescence

optical imaging system designed to characterize, quantify and visualize cellular and molecular events in

living animals using specific or non-specific fluorescent probes. The eXplore Locus MicroCT scanner is

optimised to deliver high quality volumetric images with low dosing to support the most demanding

longitudinal studies. As the two instruments shares a common animal bed to enable multi-modality image

fusion by dedicated visualization software tools, it follows that combination of the high sensitivity of MX2

with the high resolution of the eXplore Locus allows biochemical activities to be detected, quantified,

monitored and registered to a specific anatomical location. Moreover, the Department has an agreement

with the Laboratory of Radiopharmaceuticals and Molecular Imaging (LRIM) at the National Laboratories

of Legnaro (Padua), INFN (National Institutes of Nuclear Physics). This structure is fully authorized to

Nuclear Medicine practice, and represents one of the few facilities for preclinical studies present in Italy.

The LRIM is dedicated to explore new radiopharmaceuticals for imaging, diagnosis and radiotherapy, as

well as to develop new detecting systems and instrumentations. The radiochemical laboratory, part of

LRIM, is authorized to use radionuclides, such as 18

F, 99m

Tc, 111

In, 67

Ga, 68

Ga, 64

Cu, 124

I, 125

I, 131

I, 90

Y, 177

Lu and 188

Re, and is equipped with a system for low gamma-ray background measurements using a well-

shaped scintillation detector, devoted for - and ray activity measurements, an HPLC system with an in-

line radioactivity detector to analyze and characterize radiolabelled compounds, as well as Biohazard and

chemical safety hoods, CO2 incubators, and cell centrifuges. Three fully equipped systems for small-object

-ray imaging give the possibility to conduct in vivo biodistribution experiments with small animals. Two

of them are based on high-spatial resolution position-sensitive scintillator cameras employing segmented

YAP scintillators. One of the -ray imaging systems (field of view = 40x40mm; spatial resolution <1.5mm)

is designed to operate with 99m

Tc-labelled radiopharmaceuticals. The other imaging system (field of view =

50x50mm; spatial resolution < 2.7mm) is designed to operate with 188

Re-labelled radiopharmaceuticals.

The third system is a planar gamma-camera with a NaI-scintillator (field of view = 100x100mm; spatial

resolution about 2mm and high sensitivity). Most importantly, Dr. Rosato has been awarded a 1.2 million

Euro grant to acquire a preclinical PET/SPECT/CT integrated trimodal apparatus, which will be installed at

LRIM representing an exceptional reinforcement of technical equipment of the facility and that will be

absolutely instrumental to carry out the present project. In this regard, the instrument is expected to be

installed in the second semester of 2014, to be fully operative by the beginning of 2015. The personnel at

LRIM has the necessary expertise not only for gamma cameras but also for PET because a neurologic

clinical PET for preclinical research was already available in the lab and has been discharged because

obsolete.

Conventional studies aimed at analysing cancer growth and response to therapeutic agents in small

animal models have usually relied upon euthanizing cohorts of animals at multiple time points. This

experimental strategy has been the foundation for the study of cancer pathogenesis and treatment, but there

are inherent weaknesses that limit applications of this approach for testing new therapeutic strategies.

Analysing groups of mice at multiple time points after treatment precludes serial studies of disease

progression in the same animal. Indeed, data from longitudinal studies in the same animal may reveal key

information about animal-to-animal variations in therapeutic efficacy. Furthermore, conventional animal

studies require large numbers of animals to generate statistically meaningful data, which in turn

necessitates larger amounts of infused drugs for initial pre-clinical testing. In vivo imaging has emerged as

a powerful alternative to these conventional studies of cancer pathogenesis and treatment. Such innovative

approaches permit to represent, characterize and quantify biological processes at cellular and subcellular

levels within living organisms, and constitute a real improvement for promotion and follow-up of new

experimental therapies with a consequent acceleration in their transfer to clinical practice. In this context,

previous experience with scintigraphy techniques and optical imaging has led the team to gain high

expertise in tracking tumor cell growth and response to therapy in a variety of different mouse models (1,

2), in assessing trafficking and tumor homing of adoptively transferred T cells (3), and in evaluating in vivo

biodistribution of fluorochrome-labelled novel drugs, nanoparticles (4), polymers and monoclonal

antibodies (5). Starting from the illustration of some selected applications, Dr. Rosato concluded the second

part of his presentation illustrating two biological examples where molecular imaging has turned out

critically instrumental to provide meaningful insights about the use of new investigational drugs.

1.1. The MDA-MB-231 in vivo triple negative breast cancer (TNBC) mouse model

Among the many small animal imaging approaches that have been recently developed,

bioluminescence imaging (BLI) is at the forefront of technologies used to study tumors in mouse models.

BLI allows in vivo quantification of tumor replication, dissemination, and response to therapy and,

Importantly, these processes can be interrogated repetitively in the same animal over the course of hours to

weeks, overcoming many of the limitations of conventional assays. Signal-to-noise for BLI is higher than

other imaging techniques because there is only very minimal background light in the intestine from

chlorophyll or similar pigments present in most mouse chows. Furthermore, it is possible to image more

animals per hour with BLI than other small animal imaging modalities. As a result, several different

subgroups can be analyzed in the context of one experiment, thus accelerating the evaluation of the

therapeutic efficacy of different compounds. In this regard, we have set up a very robust and reliable TNBC

in vivo model represented by the MDA-MB-231 cell line (the 4T1 mouse counterpart has been also

prepared) transduced with Lentiviral vectors carrying the Firefly Luciferase. These bioluminescent TNBC

tumor cells can be injected in immunodeficient mouse strains (SCID, NOD/SCID, RAG/, NSG) either

intravenously (iv) to generate "artificial" lung metastases or can be implanted orthotopically in the fat pad;

in both cases, local tumor growth and metastatic progression can be monitored by optical imaging. In

particular, the fat pad route fairly well recapitulates the biological behaviour of the original neoplasia: upon

injection, tumor growth can be detected locally but by week 3 metastases at the homolateral axillary lymph

nodes become apparent that are followed by detectable lung signals a couple of weeks later. Such model is

highly reproducible and the cells have been engineered to obtain gain- and loss-of-function mutants to

assess the role of relevant metastasis regulator genes. Moreover, the same luciferase-expressing tumour cell

line has been instrumental to monitor the response to therapy induced by a selected set of anti-metastatic

compounds, and to assess the potentiality of antagomiRs to modulate malignant and metastatic cancer

behaviour (6-14).

Notwithstanding the MDA-MB-231 model can be envisaged to play a pivotal role in many different

steps of the research program, an important drawback is represented by the extreme paucity of TNBC cell

lines that are internationally well characterized and available. Moreover, MDA-MB-231 is used in the lab

for more than forty years and it is conceivable that its "genetic drift" from the original tumour is now quite

relevant. To overcome such limit and to provide a more physiological platform of study, we are trying to

set up a human-in-mouse model based on the in vivo stabilization of primary TNBC tumours from patient

specimens. Thus far, 33 samples have been already collected from either primary biopsies or surgery and

implanted in highly immunosuppressed mice. Six of such implants have already successfully grown and

has been propagated 3 times in vivo. Preliminary analyses conducted in histology and

immunohistochemistry on the grown tumour and subsequent passages showed a substantial maintenance of

the structural architecture and phenotype compared to the original sample from the patient. Apart the

current use of the luciferase-expressing MDA-MB-231 model that will likely represent a kind of internal

"gold standard" for any future study aimed addressing the role of emerging relevant pathways or molecules

involved and potentially interfering with the metastatic cascade, the set up of a cohort of "mouse TNBC

patients" fully reflecting the characteristics of the original primary tumors will allow to conduct

"preclinical" phase I trials to test the therapeutic concepts generated within the research program in a very

rapid manner, thus accelerating their transfer to the clinical setting.

60

1.2. Hyaluronic acid (HA) as carrier of drugs, cytokines and radionuclides

Dr. Rosato presented a brief synopsis of his long-standing experience with HA for drug delivery

applications. In particular, HA has been proposed for a) the conjugation with various chemical drugs, such

as paclitaxel, SN38 (the active metabolite of camptothecin) and doxorubicin (15-19); these conjugates have

been studied for biodistribution (Figure 2; 20-22) and therapeutic efficacy against a variety of different

tumour histotypes, proving to be very efficient, and have reached the level of clinical exploitation in phase

I/II clinical trials (23). b) The labeling with therapeutic radionuclides (- emitters) for the radiotherapy of

liver cancer (24-26). c) The linking to carboranes for Boron Neutron Capture Therapy (BNCT) of tumours

(27, 28). d) The sustained release formulation of human Interferon (INF); HA-IFN bioconjugate

retained the antiviral and antiproliferative activity, induced STAT1 phosphorylation and gene modulation

on PBMC and tumour cells similarly to the free cytokine, and exerted relevant therapeutic activity against

susceptible target cells. Upon intravenous inoculation, the bioconjugate disclosed a rapid and almost

complete accumulation in the liver, thus suggesting the possibility to realize an organ-focused therapeutic

approach especially for virus-related diseases and primary and metastatic liver tumours.

FIG. 1. The in vivo imaging platform available and selected

examples of applications (see the text).

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administration through different routes. A) The YAP gamma-camera; B) the field of view; C-F)

scintigraphy images obtained after intravenous, intraperitoneal, intravesical and oral

administration, respectively.

62

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Bicciato S, Cordenonsi M, Piccolo S (2012). SHARP1 suppresses breast cancer metastasis by promoting

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Thompson A, Mano M, Rosato A, Crook T, Scanziani E, Means AR, Lozano G, Schneider C, Del Sal G

(2011). A Pin1/mutant p53 axis promotes aggressiveness in breast cancer. CANCER CELL, vol. 20, p. 79-

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AR, Poletti A, Daidone MG, Dupont S, Basso G, Bicciato S, Piccolo S (2011). The Hippo transducer TAZ

confers cancer stem cell-related traits on breast cancer cells. CELL, vol. 147, p. 759-772.

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Spruce T, Parenti AR, Daidone MG, Bicciato S, Piccolo S (2010). A MicroRNA targeting dicer for

metastasis control. CELL, vol. 141, p. 1195-1207.

13. RUSTIGHI A, TIBERI L, SOLDANO A, NAPOLI M, NUCIFORO P, ROSATO A, KAPLAN F,

CAPOBIANCO A, PECE S, DI FIORE PP, DEL SAL G (2009). The prolyl-isomerase Pin1 is a Notch1

target that enhances Notch1 activation in cancer. NATURE CELL BIOLOGY, vol. 11, p. 133-142.

14. ADORNO M, CORDENONSI M, MONTAGNER M, DUPONT S, WONG C, HANN B, SOLARI A,

BOBISSE S, RONDINA MB, GUZZARDO E, PARENTI AR, ROSATO A, BICCIATO S, BALMAIN A,

PICCOLO S. (2009). A mutant-p53/Smad complex opposes p63 to empower TGF-beta induced metastasis.

CELL, vol. 137, p. 87-98.

15. Montagner IM, Banzato A, Zuccolotto G, Renier D, Campisi M, Bassi P, Zanovello P, Rosato A (2013).

Paclitaxel-hyaluronan hydrosoluble bioconjugate: mechanism of action in human bladder cancer cell lines.

UROLOGIC ONCOLOGY, vol. 31, p. 1261-1269.

16. D'Este M, Renier D, Pasut G, Rosato A (2010). Process for the synthesis of conjugates of

glycosaminoglycanes (GAG) with biologically active molecules, polymeric conjugates and relative uses

thereof. WO2010145821.

17. BANZATO A, BOBISSE S, RONDINA M, RENIER D, BETTELLA F, ESPOSITO G, QUINTIERI L.,

MELENDEZ-ALAFORT L, MAZZI U, ZANOVELLO P, ROSATO A (2008). A paclitaxel-hyaluronan

bioconjugate targeting ovarian cancer affords a potent in vivo therapeutic activity. CLINICAL CANCER

RESEARCH, vol. 14, p. 3598-3606.

18. ROSATO A, BANZATO A, DE LUCA G, RENIER D, BETTELLA F, PAGANO C, ESPOSITO G,

ZANOVELLO P, BASSI P (2006). HYTAD1-p20: A new paclitaxel-hyaluronic acid hydrosoluble

bioconjugate for treatment of superficial bladder cancer. UROLOGIC ONCOLOGY, vol. 24, p. 207-215.

19. Capodilupo AL, Crescenzi V, Francescangeli A, Joudioux R, Leonelli F, Marini R, Renier D, Banzato A,

Rosato A (2003). Hydrosoluble, Metabolically Fragile Bioconjugates by Coupling Tetrabutylammonium

Hyaluronan with 2′ Paclitaxel-4-Bromobutyrate: Synthesis and Antitumor Properties. In: Hyaluronan 2003.

p. 391-395.

20. BANZATO A, RONDINA M, MELENDEZ-ALAFORT L, ZANGONI E, NADALI A, RENIER D,

MOSCHINI G, MAZZI U., ZANOVELLO P, ROSATO A (2009). Biodistribution imaging of a paclitaxel-

hyaluronan bioconjugate. NUCLEAR MEDICINE AND BIOLOGY, vol. 36, p. 525-533.

21. MELENDEZ-ALAFORT L, NADALI A, ZANGONI E, BANZATO A, RONDINA M, ROSATO A,

MAZZI U (2009). Biokinetic and dosimetric studies of Re-188-hyaluronic acid: a new radiopharmaceutical

for treatment of hepatocellular carcinoma. NUCLEAR MEDICINE AND BIOLOGY, vol. 36(6), p. 693-

701.

22. MELENDEZ-ALAFORT L, RIONDATO M, NADALI A, BANZATO A, CAMPORESE D,

BOCCACCIO P, UZUNOV N, ROSATO A, MAZZI U (2006). Bioavailability of Tc-99m-Ha-paclitaxel

complex [Tc-99m-ONCOFID-P] in mice using four different administration routes. JOURNAL OF

LABELLED COMPOUNDS & RADIOPHARMACEUTICALS, vol. 49, p. 939-950.

23. Bassi PF, Volpe A, D'Agostino D, Palermo G, Renier D, Franchini S, Rosato A, Racioppi M (2011).

Paclitaxel-hyaluronic acid for intravesical therapy of Bacillus Calmette-Guerin refractory carcinoma In situ

of the bladder: results of a phase I study. THE JOURNAL OF UROLOGY, vol. 185, p. 445-449.

24. ANTOCCIA A, BANZATO A, BELLO M, BOLLINI D, DE NOTARISTEFANI F, GIRON C, MAZZI U,

ALAFORT LM, MOSCHINI G, NADALI A, NAVARRIA F, PERROTTA A, ROSATO A,

TANZARELLA C, UZUNOV N (2007). (188)Rhenium-induced cell death and apoptosis in a panel of

tumor cell lines. NUCLEAR INSTRUMENTS & METHODS IN PHYSICS RESEARCH. SECTION A,

ACCELERATORS, SPECTROMETERS, DETECTORS AND ASSOCIATED EQUIPMENT, vol. 571, p.

471-474.

25. ANTOCCIA A, BALDAZZI G, BANZATO A, BELLO M, BOCCACCIO P, BOLLINI D, DE

NOTARISTEFANI F, MAZZI U, ALAFORT LM, MOSCHINI G, NAVARRIA FL, PANI R,

PERROTTA A, ROSATO A, TANZARELLA C, UZUNOV NM (2007). A YAP camera for the

biodistribution of Re-188 conjugated with Hyaluronic-Acid in "in vivo" systems. NUCLEAR

INSTRUMENTS & METHODS IN PHYSICS RESEARCH. SECTION A, ACCELERATORS,

SPECTROMETERS, DETECTORS AND ASSOCIATED EQUIPMENT, vol. 571(1-2), p. 484-487.

26. ANTOCCIA A, BANZATO A, BELLO M, BOLLINI D, DE NOTARISTEFANI F, GIRON MC, MAZZI

U, MELENDEZ-ALAFORT L, MOSCHINI G, NADALI A, NAVARRIA F, PERROTTA A, ROSATO A,

TANZARELLA C, UZUNOV N (2006). 188Rhenium-induced cell death and apoptosis in a panel of tumor

cell lines. NUCLEAR INSTRUMENTS & METHODS IN PHYSICS RESEARCH. SECTION A,

ACCELERATORS, SPECTROMETERS, DETECTORS AND ASSOCIATED EQUIPMENT, vol. 571, p.

471-474.

27. DI MEO C, PANZA L, CAMPO F, CAPITANI D, MANNINA L, BANZATO A, RONDINA M,

ROSATO A, CRESCENZI V (2008). Novel types of carborane-carrier hyaluronan derivatives via "click

chemistry". MACROMOLECULAR BIOSCIENCE, vol. 8, p. 670-681.

28. DI MEO C, PANZA L, CAPITANI D, MANNINA L, BANZATO A, RONDINA M, RENIER D,

ROSATO A, CRESCENZI V (2007). Hyaluronan as carrier of carboranes for tumor targeting in boron

neutron capture therapy. BIOMACROMOLECULES, vol. 8, p. 552-559.

64

Nanosized Delivery Systems for Radiopharmaceuticals

Siti Najila Mohd Janib

Malaysian Nuclear Energy, Ministry of Science, Technology and Innovation, Kajang,

Selangor, Malaysia

Receptor-based radiopharmaceuticals provide a unique tool for the specific delivery of radionuclides

to diseased tissues like cancerous cells. By incorporating a targeting molecule that specifically binds to an

antigen or receptor uniquely expressed or overexpressed by the tumour, the ligand-targeted approach is

expected to selectively deliver radiopharmaceuticals/radionuclides to tumour tissues with greater

efficiency. Nanoscale (1-100nm) systems have emerged as novel therapeutic modalities for cancer

treatment and are expected to lead to major advances in cancer detection, diagnosis and treatment. The

progression of nanoparticle technology toward solving these problems is mainly a result of the properties of

these compounds, which include:

1. Their diminutive size allows preferential accumulation in the tumors via the enhanced permeability

and retention effect (EPR). In addition the sizes also allow escape from renal elimination for

increase therapeutic efficacy.

2. The ability to endow disease specific targeting agents to enhance of therapeutic delivery in a tissue-

or cell-specific manner.

3. The ability to deliver a combination of imaging and therapeutic agents for real time-monitoring of

therapeutic efficacy.

4. The high surface area to volume can allow increase loading therapeutics

5. Nanoparticle multivalency greatly enhance its ability to bind to targets with great affinity and avidity

It is for these reasons nanoparticles would make an ideal platform for the delivery of targeted

radiopharmaceuticals.

The high sensitivity of nuclear imaging techniques allows the use of nano- to picomolar

concentrations of the imaging agents to achieve acceptable signal to noise ratios. SPECT imaging is much

more widely available than PET imaging and the radionuclides used for SPECT are easier to prepare and

usually have a longer half-life than those used for PET (6 h for [99m

Tc], 67 h for [111

In], and 13.2 h for

[123

I]). However, PET is approximately ten times more sensitive than SPECT and is able to detect

picomolar concentrations of tracer. At the moment, the high sensitivity of PET is only reached by optical

imaging (OI) techniques, but not by MRI, computed tomography (CT) or ultrasound (US). Moreover, both

PET and SPECT can be quantitative, especially with regard to PET. This is an advantage over MRI and

conventional optical imaging techniques (Beer and Schwaiger, 2008).

At Nuclear Malaysia we have developed multiple nanoplatforms that would be suitable to use for the

targeted delivery of radiopharmaceuticals. We proposed to explore two distinct types of nanocarriers in

order to exploit the physical properties of both types of construct. The first type is nanogels. Oil in water

(O/W) and water in oil (W/O) based nanogels are currently under investigation. One of the nanogel

preparations was previously developed under the CRP project “Radiation synthesis of nanogel for chitosan

immobilization (Project agreement no. 15464)”. Here an inverse micelle system was used as a nanoreactor

for the polymerization of polyethylene glycol diacrylate (PEGDA) via irradiation. Inverse micelles were

formed by the addition of sodium dioctyl sulfosuccinate (AOT) in n-heptane above its critical micelle

concentration. These nano sized micelles were used to trap PEGDA in its water phase. Irradiation of the

PEGDA containing micelles lead to the formation of radicals which subsequently form crosslinked

nanogels. Addition of 10% PEGDA into 0.15M AOT in n-heptane produces micelles with a narrow size

distribution. The PEGDA nanogel size increases as the polymerization irradiation dose is increased.

FIG. 1. Reversed surfactant micelles as template for nanogel formation. Cross linking of PEG diacrylate based

nanogels was initiated by irradiation.

To complement this work, water in oil nanogel preparation was also developed. In this second study,

the radiation induced initiator method was utilized for the formation of nanoparticles. A microemulsion

system consisting of epoxidized palm oil acrylate (EPOLA) and the aqueous surfactant pluronic F-127 (PF-

127) was subjected to gamma irradiation to form the crosslinked EPOLA nanoparticles. The size of the

nanoparticle obtained is in the range of 98 to 200 nm after irradiation using a gamma irradiator.

FIG. 2 Formation of EPOLA-based nanogels. The non-polar part of EPOLA structure is trapped together with the

hydrophobic portion of the surfactant e.g. the PF127 micelle, to form the mixed surfactant and EPOLA micelle.

The development of this nanocarrier has been taken a step further by loading it with a bioactive

compound called thymoquinone. Thymoquinone (TQ), is the main bioactive component of the the black

seed (Nigella sativa), and has historically been used as an anti-oxidant, anti-inflammatory and

antineoplastic medicines. Previous studies reported that thymoquinone exhibited inhibitory effects on cell

proliferation of many types of cancer cell lines, including breast, ovarian, colorectal, pancreatic uterine and

lung carcinoma among others.

66

FIG. 3 (a) Nigella sativa seeds (black seeds). (b) The chemical structure of thymoquinone; 2-isopropyl-5-methyl-1,4-

benzoquinone (C10H12O2); MW 164.2.

TQ was successfully loaded into the EPOLA micelle and further characterization revealed that the

EPOLA micro-and nanoparticle can retain and control the release rate of the thymoquinone for up to 72

hours. Additionally cytotoxicity studies performed on the human colon adenocarcinoma cell line, HT29,

revealed anti-tumour effects.

FIG. 4 . TEM images of the (a) surfactant/EPOLA particles and (b) TQ-loaded surfactant/EPOLA particles. TQ release

profile of EPOLA/SDS and EPOLA/PF127 (c). The nanoparticles formulated from TQ/EPOLA/SDS releases TQ faster than

TQ/EPOLA/PF127 in PBS solution.

We also propose to explore the use of gold nanoparticles for the targeted delivery of

radiopharmaceuticals. At Nuclear Malaysia the 198

Au@SiO2 nanoparticles has been developed as a

radiotracer for industrial processes. However we feel that due to its many advantages such as ease of

synthesis, high stability, tunable size and shape, biocompatibility, and being amenable to surface

functionalization, makes this metallic nanoparticle an ideal nanocarrier for the purposes of this project.

FIG. 5 Procedure for preparation of gold silica core shell structure based radiotracer

The colloidal Au-SiO2 with core-shell structure currently in development was neutron activated in a

nuclear reactor to produce 198

Au (T1/2=2.7 d) emitting gamma ray of 412 keV. Using conventional citrate-

reduction method, gold nanoparticles were prepared from its corresponding metal salts in aqueous solution

then coated with uniform shells of amorphous silica via a sol-gel reaction. The citrate-reduction-based

method provides gold nanoparticles with higher concentration and narrow size distribution. The resultant

particle size and silica coatings could be varied from tens to several hundred of nanometers by controlling

the catalyser and precipitation time. The silica coating prevents the particles from aggregating and due to its

chemical inertness it is biocompatible and stable in biological environment, making it ideal for medical

applications. In addition, the silica surface offers ease of functionalization and can endow hydrophobic and

hydrophilic properties to the gold nanoparticles. Thus, by using an established system, albeit for a different

purpose that what it was originally intended for, it demonstrates the versatility of this nanoparticle.

Target specific radiopharmaceuticals is an emerging research field, which has demonstrated great

potential for improvement of healthcare in clinic, especially in clinical oncology for delivering

radiation/radionuclide to the diseased sites and reducing side effects. To date, target specific delivery of

radionuclides/radiopharmaceuticals has employed numerous targeting moieties, including small molecules,

peptides, proteins, antibody, antibody fragments and nanoparticles. Among these targeting moieties low

molecular weight small peptides demonstrate a number of distinct advantages over others. In particular

small peptides have favourable PK, increased permeability, lower toxicity, lower immunogenicity and

considerable flexibility in chemical modification.

Under this project we propose to use established targeting ligands/moiety e.g. RGD to assess the

viability of the nanoplatforms chosen for functionalization, and its potential use as a nanocarrier for

radiopharmaceuticals. For nanogels, biofunctionality is incorporated into the hydrogel network primarily

through copolymerization of peptide and/or protein monoacrylate and diacrylate macromers. Biofunctional

macromers may be synthesized by reacting acryl-PEG-NHS with peptide. Similarly the surface of the

AuNP can be readily modified with ligands containing functional groups such as thiols, phosphines, amines

and carboxyls. These functional groups are then used to anchor specific ligands. Furthermore to track its

biodistribution in vivo, it will be radiolabeled with technetium (99m

Tc) and scan using a gamma camera to

assess its tumour accumulation.

68

Theranostic Radiopharmaceuticals Based on Gold Nanoparticles Labeled with 177

Lu

and Conjugated to Peptides

Guillermina Ferro-Flores, Blanca E. Ocampo-García, Clara L. Santos-Cuevas, Flor de María

Ramírez and Erika P. Azorín-Vega

Abstract

Gold nanoparticles (AuNPs) have been proposed for a variety of medical applications such as localized heat sources

for cancer treatment and drug delivery systems. The conjugation of peptides to AuNPs produces stable multimeric systems

with target-specific molecular recognition. Lutetium-177 is a β- and γ-emitting radionuclide successfully used in peptide

radionuclide therapy.

Recently, 177Lu-AuNPs conjugated to different peptides have been proposed as a new class of theragnostic

radiopharmaceuticals. These radioconjugates may function simultaneously as molecular imaging agents, radiotherapy

systems and thermal-ablation systems.

This work covers advancements in the design, synthesis, physicochemical characterization, molecular recognition

assessment and preclinical therapeutic efficacy of gold nanoparticles radiolabeled with 177Lu and conjugated to RGD (-Arg-

Gly-Asp-), Lys3-Bombesin and Tat(49-57) peptides.

1. INTRODUCTION

Several monomeric radiopharmaceuticals based on lutetium-177-labeled peptides have successfully

been developed for target-specific radionuclide therapy. Clinical applications of different 177

Lu-octreotide

peptide derivatives have shown good results on tumor response, overall survival, and quality of life in

patients with neuroendocrine tumors [1,2].

The term nanoparticle (NP) describes a sub-classification of ultra-fine solids with dimensions from 1

to 100 nm and novel properties that distinguish them from the bulk material.7 NPs can be design as

multimeric systems to produce multivalent effects due to multiple simultaneous interactions between the

biomolecules conjugated to the nanoparticle surface and cell surface specific receptors [3-8].

Gold nanoparticles (AuNPs) have been proposed as localized heat sources for cancer treatment [9-

13]. The photo-thermal conversion effect in AuNPs is focused on taking advantage of the collective

oscillations of the electrons under optical excitation (surface plasmon resonance) which provide strong

localized heating when they are irradiated with a laser. The localized heating reaching temperatures about

700°C around AuNPs, causes irreversible thermal cellular destruction of cancer tissues [14-15]. When

AuNPs are exposed to a certain radiofrequency field, they also release enough heat to destroy malignant

cells [16, 17].

Plasmonic AuNPs can be fluorescent when they are prepared by thermal reduction procedures or

when they are designed to absorb in the near infrared region (e.g. nanoshells and nanorods), therefore

AuNPs can function as optical imaging agents [18].

177

Lu-labeled AuNPs conjugated to different peptides have been proposed as a new class of

theragnostic radiopharmaceuticals. These multifunctional systems could be useful for the identification of

malignant tumors and monitoring of treatment (SPECT/NIR fluorescence imaging), for targeted

radiotherapy (β-particle-energy delivered per unit of targeted mass) and for photothermal therapy (localized

heating).

2. DEVELOPMENT OF [177

Lu-DOTA-GGC-AuNP-cRGDfK(C)]

Radiolabeled peptides based on the Arg-Gly-Asp (RGD) sequence have been reported as

radiopharmaceuticals with high affinity and selectivity for α(ν)β(3) and α(ν)β(5) integrins. These peptides

are thus useful in the non-invasive monitoring of tumor angiogenesis by molecular imaging techniques [20-

23]. In breast cancer and gliomas, α(ν)β(3) and/or α(ν)β(5) integrins are over-expressed in both endothelial

and tumor cells [24, 25].

2.1. Design and synthesis.

To prepare gold nanoparticles conjugated to RGD a thiol derivative was synthesized. The cyclo[Arg-

Gly-Asp-Phe-Lys(Cys)] (c[RGDfK(C)]) peptide with a purity of > 98% was prepared and analyzed by

reversed-phase HPLC (RP-HPLC) and mass spectroscopy. In the c[RGDfK(C)] molecule, the sequence -

Arg-Gly-Asp- (-RGD-) acts as the active biological site, the D-Phe (f) and Lys (K) residues completed the

cyclic and pentapeptide structure, and Cys (C) was the spacer and active thiol group that interacts with the

gold nanoparticle surface. For the labeling with 177

Lu, the DOTA-Gly-Gly-Cys (DOTA-GGC) peptide was

also synthesized. DOTA was the chelator used to form a stable 177

Lu complex, -Gly-Gly- was the spacer

and Cys was used as the active thiol group to interact with the AuNP surface.

For the conjugate preparation, a solution of c[RGDfK(C)] was prepared in water and then was added

to the AuNP suspension. An average of 100-170 molecules of c[RGDfK(C)] were attached per

nanoparticle. No further purification was required since the maximum number of peptides that can be

bound to one AuNP (20 nm) is from 520 to 1701 depending of the peptide structure. The number of

peptides per nanoparticle was calculated by UV–Vis titration. To obtain 177

Lu-DOTA-GGC-AuNP-

c[RGDfK(C)], the 177

Lu-DOTA-GGC radiopeptide was first prepared and added to a solution of AuNP-

c[RGDfK(C)] to attach about 170 radiomolecules per nanoparticle [20, 21].

2.2. Physicochemical characterization

The chemical conjugation was confirmed by UV-Vis, medium and far IR, and Raman spectroscopy.

Transmission electron microscopy (TEM), Z potential measurement and size distribution by dynamic laser

were also performed and correlated with the design AuNP-peptide system.

Size-exclusion chromatography and ultrafiltration were used to evaluate the radiochemical purity of

the final radiopharmaceutical solution. When a PD-10 column and injectable water as the eluent is used, the

first radioactive and red eluted peak (3.0–4.0 mL) corresponds to radiolabeled AuNP-c[RGDfK(C)]. The

free radiolabeled peptide (177

Lu-DOTA-GGC) appears in the fraction that eluted at 5.0–7.0 mL, and 177

LuCl3 remains trapped in the column matrix. Upon ultrafiltration (30,000 MW cut off), the 177

Lu-DOTA-

GGC-AuNP-c[RGDfK(C)] remains in the filter, while free 177

Lu-DOTA-GGC and 177

LuCl3 pass through

the filter. In the radio-HPLC size exclusion system (using a column for proteins) two chromatographic

profiles can be obtained using two different detector systems, the UV Vis detector and a radiometric

detector. The UV-Vis spectrum (obtained with the photodiode array of the system) assigned to the first

peak (at 4-5 min corresponding to the molecule with higher molecular weight) and associated with the

radioactivity, exhibited the AuNP surface plasmon band at 521-522 nm.

2.3. In vitro affinity

In vitro affinity has been determined by a competitive binding assay. Results have indicated that the

concentration of c(RGDfK) required to displace 50 % of the 177

Lu-DOTA-GGC-AuNP-c[RGDfK(C)]

(IC50=10.2 nM) or 177

Lu-DOTA-E-c(RGDfK)2 (IC50=5.3 nM) from the receptor (α(v)β(3) protein) was the

same order of magnitude for both, demonstrating high in vitro affinity for the α(v)β(3) integrin for both

conjugates. However, the amount of c(RGDfK) required to displace 177

Lu-DOTA-GGC-AuNP-

c[RGDfK(C)] from the α(v)β(3) protein was twice to that necessary to displace the 177

Lu-DOTA-E-

c(RGDfK)2, which was attributed to the multimeric and multivalent effect of the AuNP system.

70

2.4. Specific internalization in cancer cells

The 177

Lu-DOTA-GGC-AuNP-c[RGDfK(C)] binding was specific for integrins in MCF7 cells,

although through α(ν)β(5) integrins considering that these breast cancer cells do not express the α(ν)β(3)

protein [47]. Fluorescence microscopy images demonstrated that 177

Lu-labeled AuNP-c[RGDfK(C)]

(20 nm) was internalized in the cytoplasm of the MCF7 cells.

2.5. In vitro studies of thermotherapy and radiotherapy

After laser irradiation (Nd:YAG laser, irradiance 0.65 W/cm2), the presence of AuNP-c[RGDfK(C)]

in MCF7 cells causes a significant increase in the temperature of the medium (ΔT = 13.5°C, compared to

ΔT = 3.3°C without AuNPs) resulting in a significant decrease in MCF7 cell viability down to 9 %. AuNP

and AuNP-c[RGDfK(C)] significantly reduced MCF7 cell viability (20 % and 9 % respectively) with

respect to the laser irradiated cells treated without nanoparticles (cell viability=90 %). The cellular

internalization is a key factor to release heat near cell organelles when laser is used as a thermal conversion

source in AuNP because of its strong localized heating mechanism. That is, during the AuNP laser

irradiation, the energy transformation process starts by the fast phase loss of the coherently excited

electrons (on femtoseconds) via electron-electron collisions leading hot electrons with temperatures as high

as 3227°C. Then the electron transfers the energy to the phonon by electro-phonon interactions on the order

of 0.5-1 ps, resulting in a hot lattice with high temperature (727°C) and transferring the generated thermal

energy to the surrounding medium [14].

After treatment with 177

Lu-DOTA-GGC-AuNP-c[RGDfK(C)], the MCF7 cell proliferation was

significantly reduced (<5%). This effect on cell proliferation was significantly higher than that obtained

with 177

Lu-DOTA-GGC-AuNP (10%) or 177

Lu-DOTA-E-c(RGDfK)2 (40%) treated with the same initial

activity.

2.6. Preclinical studies

In a first approach Luna-Gutierrez et al. [20] compared the radiation absorbed dose of 177

Lu-DOTA-

GGC-AuNP-c[RGDfK(C)] (multimeric) with that of 177

Lu-labeled monomeric and dimeric RGD peptides

to α(ν)β(3) integrin-positive U87MG tumors. Biokinetic studies were accomplished in athymic mice with

U87MG-induced tumors by intraperitoneal administration of the radiopharmaceutical. 177

Lu absorbed doses

per injected activity (per MBq administered) delivered to U87MG tumors were 0.36 Gy (multimer), 0.25

Gy (dimer) and 0.10 Gy (monomer). Doses (Gy/MBq) delivered to kidney were 0.12 (multimer), 0.15

(dimer) and 0.13 (monomer). In the spleen, radiation absorbed doses were 0.13, 0.08 and 0.02 Gy/MBq for

the multimer, dimer and monomer respectively. Therefore, the multimeric system of 177

Lu-DOTA-GGC-

AuNP-c[RGDfK(C)] showed the best properties for use in targeted radiotherapy.

2.7. Therapeutic efficacy 177

Lu-DOTA-GGC-AuNP-c[RGDfK(C)] significantly decreased glioma tumor progression in mice

by combined modalities of molecular targeting therapy and radiotherapy [26]. Specifically, the therapeutic

response of 177

Lu-DOTA-GGC-AuNP-c[RGDfK(C)] in athymic mice bearing α(v)β(3)-integrin-positive C6

gliomas was studied. The radiation absorbed dose, metabolic activity, histological characteristics and

VEGF gene expression in tumor tissues following treatment with 177

Lu-DOTA-GGC-AuNP-c[RGDfK(C)]

(177

Lu-AuNP-RGD), 177

Lu-DOTA-GGC-AuNP (177

Lu-AuNP) or 177

Lu-DOTA-E-c(RGDfK)2 (177

Lu-RGD)

were stated. Results showed that the mean tumor uptakes (percent of the injected activity: % IA) 3 h post

intratumoral administration were 68 % (177

Lu-AuNP-RGD), 48 % (177

Lu-AuNP) and 27 % (177

Lu-RGD),

with high tumor retention for the radiolabeled nanoparticles. The tumor retention of 177

Lu-AuNP-RGD at

96 h (35 % IA) was significantly higher than that of 177

Lu-AuNP (16 % IA), whereas 177

Lu-RGD exhibited

the highest tumor clearance (5.7 % IA at 96 h).

177

Lu-AuNP-RGD delivered the highest tumor radiation absorbed dose (63 Gy) compared to that of 177

Lu-AuNP (38 Gy) and 177

Lu-RGD (17 Gy) [49]. These results correlated with the observed therapeutic

response in which 177

Lu-labeled AuNP-c[RGDfK(C)] significantly (p<0.05) slowed tumor progression and

tumor metabolic activity and decreased intratumoral vessels and VEGF gene expression as a consequence

of the high tumor retention and the molecular targeting therapy (multimeric RGD system) and radiotherapy

(177

Lu) combination. The inhibition of VEGF gene expression, involved in angiogenesis, indicated a

molecular response. [18

F]fluor-deoxy-glucose-microPET/CT images after treatment showed a significant

decrease in tumor metabolic activity. There was low uptake in non-target organs, and no renal toxicity was

induced [26].

177

Lu multivalent system can also be used in combination with thermo-ablative therapy using laser

heating or an RF field. In fact, Sanchez-Hernández et al. [27] demonstrated a significant effect on cell

viability of MCF7 cells incubated with AuNP-c[RGDfK(C)] after exposure to an RF field (200 W, 100

V/cm). The presence of nanoparticles in cells caused a significant increase in the temperature of the

medium (ΔT = 29.9 ± 1.7°C) and the MCF7 cell viability was significantly reduced (down to 19 %).

3. DEVELOPMENT OF Tat(49-57) and Lys3-BOMBESIN PEPTIDES CONJUGATED TO

177Lu-labeled AuNPs

The gastrin-releasing peptide (GRP) receptor is overexpressed in prostate and breast cancer, and 99m

Tc-Lys3-bombesin has been reported as a radiopharmaceutical with specific binding to cells expressing

GRP receptors [28, 29].

Of particular interest was the development of dual-radiolabeled gold nanoparticles linked to Tat(49-

57)-Lys3-bombesin. Tat(49-57) is a peptide derived from the transactivator of the transcription protein of

the HIV-1 virus that has a membrane translocation domain and a nuclear localization peptide sequence,

therefore Tat(49-57) is a cell-penetrating peptide that reaches the DNA [30-32]. As previously mentioned, 177

Lu has successfully been used for radiopeptide therapy with an efficient cross-fire effect in cancer cells,

while 99m

Tc produces Auger energy of 0.90 keV/decay and internal conversion (IC) electron energy of

15.40 keV/decay, which represent 11.4 % of the total 99m

Tc energy release per decay [33,34]. At the single-

cell level, short-range charged particles, such as IC electrons and Auger electrons, impart a dense ionizing

energy deposition pattern associated with increased radiobiological effectiveness. However, they must be

able to penetrate to the cytoplasm and reach the nucleus.

Recently, Jimenez-Mancilla et al. [35,36] prepared a multifunctional system of 177

Lu- and 99m

Tc-

labeled gold nanoparticles (AuNPs of 5 nm) conjugated to Tat(49-57)-Lys3-bombesin (

177Lu/

99mTc-AuNP-

Tat-BN) and evaluated the in vitro potential of the radiopharmaceutical as a plasmonic photothermal

therapy and targeted radiotherapy system in PC3 prostate human cancer cells, as well as the radiation

absorbed dose to GRP receptor-positive PC3 tumors in mice.

3.1. Design and synthesis

The Tat(49-57) peptide was conjugated to Gly10

-Gly11

-Cys12

-Gly13

-Cys14

(Acm)-Gly15

-Cys16

(Acm)-

NH2 to produce the Tat(49-57)-spacer-GCGC peptide. The sequence G13

C14

G15

C16

was added for use as the

specific chelating site by the AuNPs. The Lys3-bombesin was conjugated to a maleimidopropyl moiety

through Lys3, and the maleimidopropyl group was used as the branch position to form a thioether with the

Cys12

side chain of the Tat(49-57)-spacer-GCGC peptide. High-performance liquid chromatography

(HPLC) analysis, matrix-assisted laser desorption/ionization mass spectral analysis (MALDI), amino acid

analysis and peptide content determination showed a chemical purity of more than 90 % and a molecular

weight of 3779.5 g/mol.

For the conjugate preparation, a 25 µM solution of Tat-BN was prepared using injectable-grade water.

Then, 0.025 mL of solution (6.02 × 1014

molecules/mL) was added to 1 mL of the AuNP solution (5.5 ×

1013

particles/mL) followed by stirring for 5 min. Under those conditions, an average of 7 molecules of Tat-

BN were attached per nanoparticle (5 nm, surface area = 78.5 nm2, 2310 surface Au atoms). No further

purification was required since it has been evaluated that the maximum number of peptides that can be

bound to one AuNP (5 nm) is from 52 to 106 depending on the peptide structure.

72

For the labeling with 177

Lu, the DOTA-Gly-Gly-Cys (DOTA-GGC) peptide was also synthesized (10

molecules were attached per nanoparticle) and for 99m

Tc labeling the hydrazinonicotinyl-Tyr3-Octreotide

(HYNIC-TOC) was used (8 molecules were attached per nanoparticle). 99m

Tc-HYNIC-TOC is currently

used as a conventional radiopharmaceutical in nuclear medical practice and is therefore of easy availability.

In this study, it was successfully used as a radiotracer for 99m

Tc labeling of AuNPs. The AuNP-TOC

spectra suggested interactions of the AuNPs with the thiol groups of the broken TOC cysteine disulfide

bridge (DB). DB are known to cleave to interact with Au because the Au–S bond strength approximates S–

S strength (approximately 40 kcal/mol compared with approximately 50 kcal/mol) [37, 38]. In fact, the use

of 177

Lu-octreotide is a good alternative for the labeling of AuNPs with 177

Lu.

3.2. Physicochemical characterization

The chemical conjugation was confirmed by UV-Vis, medium and far IR, and Raman spectroscopy.

Transmission electron microscopy (TEM), Z potential measurement and size distribution by dynamic laser

were also performed and correlated with the design AuNP-peptide system.

Size-exclusion chromatography and ultrafiltration were used to evaluate the radiochemical purity of the

final radiopharmaceutical solution under the same conditions mentioned for the 177

Lu-DOTA-GGC-AuNP-

c[RGDfK(C)] radiopharmaceutical. The radiochemical purity of all radio-AuNPs was 96 ± 2 %.

3.3. Specific internalization in PC3 cancer cells 99m

Tc/177

Lu-AuNP-Tat-BN had specific recognition because the GRP-r positive cell internalization

was 52.5 % higher than that of 99m

Tc/177

Lu-AuNP. The specific tumor cell internalization (internalization in

unblocked cells minus internalization in blocked cells) was 7.7 %. Internalization results and the obtained

fluorescence images of 99m

Tc/177

Lu-AuNP-Tat-BN showing the internalization in PC3 cell nuclei, also

demonstrated specific nanoparticle internalization and transfer across the cytoplasm with 5 nm particles

achieving nuclear entry.

3.4. In vitro studies of thermotherapy and radiotherapy

After laser irradiation (Nd:YAG laser, irradiance 0.65 W/cm2), the presence of gold nanoparticles

significantly increased the temperature of the medium (ΔT=9.4°C, compared to ΔT = 2.5°C without

AuNP). The AuNP-Tat-BN system caused a significant decrease in PC3 cell viability down to 1.3 % by the

end of treatment (6 min) when compared to AuNP (10.1 %).

After treatment with 99m

Tc/177

Lu-AuNP-Tat-BN the PC3 cell proliferation was significantly inhibited

(<0.5%) compared to that of 177

Lu-AuNP-Tat-BN (4%), 99m

Tc-AuNP-Tat-BN (8%), 99m

Tc/177

Lu-AuNP

(10%) and 99m

Tc-Tat-BN (40%), which was attributed to the greater PC3 cell internalization of beta

particles, IC and Auger electrons inside nuclei. Although 99m

Tc did not produce a high absorbed radiation

dose as that of 177

Lu (4 Gy/Bq and 55 Gy/Bq respectively), the significant difference between 99m

Tc/177

Lu-

AuNP-Tat-BN and 177

Lu-AuNP-Tat-BN on PC3 cell proliferation was attributed to the biological effect of

Auger and low-energy IC. Lundqvist et al. [39] have shown that it is important to distinguish between what

might be a normal increased cellular dose and the biological Auger effect. Electron energies close to the

ionization potential (<30 eV) will only have a marginal biological effect, and electrons above 5 keV will

not contribute to the local effect. For example, an Auger electron with an energy of approximately 20 keV

is ideal for full deposition within the size of a mammalian cell, but it will be far from the DNA and will not

have the local DNA impact that is usually associated with the biological Auger effect, which occurs within

cubic nanometers [39]. Therefore, it is possible that in the 99m

Tc/177

Lu-AuNP-Tat-BN radiopharmaceutical,

the positive charges of Tat(49-57) generate an electrostatic interaction with the negatively charged DNA,

producing cell toxicity from a biological Auger effect and not only from a simple increase in the

macroscopic absorbed dose.

3.5. Preclinical studies

Jiménez-Mancilla et al. [58] compared the in vivo tumor retention of 99m

Tc/177

Lu-AuNP-Tat-BN with

that of 99m

Tc/177

Lu-AuNP and 99m

Tc-Tat-BN to GRP receptor-positive PC3 tumors. Biokinetic studies were

accomplished in athymic mice with PC3-induced tumors by intratumoral administration of the

radiopharmaceutical. The tumor retention at 1 h was 58 % (99m

Tc/177

Lu-AuNP-Tat-BN), 29 % (99m

Tc/177

Lu-

AuNP) and 8 % (99m

Tc-Tat-BN) and after 24 h the retention was 44 %, 16 % and 5 % respectively.

For the 99m

Tc/177

Lu-AuNP-Tat-BN, the 177

Lu absorbed dose per intratumorally injected activity delivered to

the PC3 tumors was 7.9 Gy/MBq, and the 99m

Tc absorbed dose that was delivered to the nuclei was

estimated at 0.53 Gy/MBq.

Therefore, the 177

Lu/99m

Tc-AuNP-Tat-BN system showed properties suitable for a targeted

radionuclide therapy of tumors expressing GRP receptors due to the energy deposition from β-emissions

and the Auger and IC electron emissions near the DNA [35, 36]. This radio-therapeutic system can also be

used in combination with thermo-ablative therapy and for SPECT/CT imaging. For therapeutic applications

in humans, nanoparticles would have to be administered by an intratumoral injection or via a selective

artery to avoid high uptake by organs of the reticuloendothelial system because of the colloidal nature of

the nanoparticles. Injection of the multimeric system into an artery of the affected organ would permit high

uptake to a tumor and potentially to micrometastases or individual cancer cells.

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Lu-labeled gold nanoparticles-tat(49-57)-Lys3-bombesin

internalized in nuclei of prostate cancer cells. J. Labelled Comp. Radiopharm., 2013, 56, 663-671.

[37] Aubin-Tam, M.E.; Hamad-Schifferli, K. Structure and function of nanoparticle-protein conjugates.

Biomed. Mater., 2008, 3, 034001–0340017.

[38] Whitesides, G.M.; Laibinis, PE. Wet chemical approaches to the characterization of organic surfaces:

self-assembled monolayers, wetting, and the physical–organic chemistry of the solid–liquid interface.

Langmuir, 1990, 6, 87-96.

[39] Lundqvist, H.; Stenerlow, B.; Gedda L. The Auger effect in molecular targeting therapy. In: Stigbrand

T, Carlsson J, Adams GP, editors. Targeted radionuclide tumor therapy. Ed.; New York: Springer;

2008, pp. 195-214.

76

RADIATION- AND SONOCHEMICAL SYNTHESIS OF POLYMER-STABILIZED

GOLD NANOPARTICLES AND OF POLYMER NANOGELS FOR

RADIOTHERAPY

Renata Czechowska-Biskup, Sławomir Kadłubowski, Janusz M. Rosiak, Piotr Ulański

Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland

Abstract

This report summarizes the preliminary considerations on the planned activities of IARC team in the CRP, where the

team’s expertise in radiation chemistry, sonochemistry and polymer chemistry shall serve the needs of the

radiopharmaceutical community, focused on formulating, optimizing and testing of nanocarriers for radiopharmaceuticals.

Two general tasks have been postulated as the main input of IARC, radiation- and sonochemical synthesis of polymer-

stabilized gold nanoparticles, as well as radiation synthesis and tailoring polymer nanogels as potential radioisotope carriers.

Precise formulation of these tasks, including the need for cooperation and networking, will evolve from the discussions and

conclusions of the 1 RCM. This report contains also results of preliminary studies undertaken at IARC aimed at gaining

experience and demonstrating the possibility of synthesizing polymer-stabilized Au nanoparticles using ionizing radiation

and ultrasound.

1. Introduction

Based on the Consultant Meeting held in Vienna in May 2013, the team of the Institute of Applied

Radiation Chemistry, Lodz University of Technology (IARC) identified the tasks which it can undertake

and perform within this CRP. Exact formulation of these tasks will be made upon consultations with other

national teams during the current 1st RCM.

Using its experience in radiation chemistry and sonochemistry of water-soluble polymers, IARC will

focus on developing methods and procedures for synthesizing gold nanoparticles (task 1) and on radiation

synthesis of nanoparticles, in particular nanogels, to be used as carriers of radiopharmaceuticals (task 2). In

realization of task 1 we will utilize the fact that both in irradiation and sonication reductive species are

formed which are capable of reducing ionic gold precursors (such as chloroauric ions) to atomic gold, the

latter aggregating into gold nanoparticles. Polymers and/or oligomers present in the system will form the

protective layer around these nanoparticles to prevent their further aggregation. At the same time,

polymeric ligands may provide functions (stability, hydrophilicity, charge, diameter, specific interactions,

etc.) necessary for the stabilized particles to perform the designed role in biological systems. Gold

nanoparticles are employed in radiotherapy, in two modes – to increase local dose deposition in tissue

during radiotherapy or as a local emitter of gamma and beta rays, if the nanoparticles contain radioactive

gold isotopes. Realization of task 2 will be based on longstanding experience of IARC in radiation

synthesis of nanogels by intramolecular cross-linking of polymers, a method originally developed at our

laboratory.

2. Radiation- and sonochemical synthesis of polymer-stabilized gold nanoparticles for radiotherapy

One of the approaches in radiotherapy utilizes the idea of synthesizing gold nanoparticles, preferably

of the 10 – 20 nm size (which corresponds to several hundred thousand gold atoms) in which some fraction

are atoms of radioactive gold isotopes (Fig. 1). Such nanoparticles, being beta and gamma emitters, are

placed in the tumor, preferentially by intratumor injection, where they fulfill their function of killing the

cancer cells by the emitted ionizing radiation.

FIG. 1. Schematic representation of a gold nanoparticle containing radioactive gold isotopes [Kattesh V. Katta, Consultants

Meeting, Vienna, IAEA Headquarters, 27-31 May 2013].

In order to provide nanoparticle stability (see below), higher retention time in the cancer tissue and

possibility of functionalization, the surface of gold nanoparticles is usually grafted with organic molecules,

often oligomers or polymers (Fig. 2). Therefore the total diameter the so decorated nanoparticle, as

determined for instance by dynamic light scattering, can be significantly higher than the diameter of Au

core (which can be measured using transmission electron microscopy).

FIG. 2. Schematic representation of polymer-stabilized gold nanoparticle [Kattesh V. Katta, Consultants Meeting, Vienna,

IAEA Headquarters, 27-31 May 2013].

The typical way of anchoring an organic ligand to the surface of gold (including Au nanoparticles) is

to use a thiol, for instance a thiol-capped poly(ethylene glycol), or other sulfur-containing molecule, which

usually form spontaneously gold-sulfur bonds (Fig. 3).

78

FIG. 3. Thiolated ligands used for stabilization of gold nanoparticles in aqueous systems [Kattesh V. Katta, Consultants

Meeting, Vienna, IAEA Headquarters, 27-31 May 2013].

The idea we are planning to explore within this CRP is a one-step synthesis of Au nanoparticles

coated with a layer of polymer molecules, by irradiating aqueous solutions containing the polymer and a

ionic Au precursor, such as chloroauric acid. Irradiation of dilute aqueous solutions of these compounds

will mainly lead to radiolysis of water (reaction 1), hydrated electrons, hydrogen atoms and hydroxyl

radicals being the main reactive species formed.[1] Hydrated electrons and hydrogen atoms reduce the

gold precursor to atomic gold (a multi-step process schematically presented as reactions 2 and 3).[1] Since

hydroxyl radicals could potentially oxidize Au0 back to a higher oxidation state, it is reacted with purposely

added scavenger, such as aliphatic alcohol or formate (reactions 4 and 5), which may also react with

hydrogen atoms (reactions 6 and 7).

H2O ⇝ e-aq, H

+, H

•,

•OH, H2O2, H2 (1)

M+ + e

-aq → M

0 (2)

M+ + H

• → M

0 + H

+ (3)

(CH3)2CHOH + •OH → (CH3)2C

•OH + H2O (4)

HCOO- +

•OH →

•COO

- + H2O (5)

(CH3)2CHOH + H• → (CH3)2C

•OH + H2 (6)

HCOO- + H

• →

•COO

- + H2 (7)

Hydroxyl radicals and a fraction of hydrogen atoms which were scavenged in reactions (4)-(7) are in fact

not lost, since the alfa-hydroxyalkyl radicals and formate radicals are capable themselves of reducing the

gold precursor (reactions 8 and 9).

M+ + (CH3)2C

•OH →M0 + (CH3)2CO + H

+ (8)

M+ +

•COO

- → M

0 + CO2 (9)

Gold atoms start to coalesce (reaction 10), and it is expected that the growing gold nanoparticles can

also incorporate gold ions (reaction 11) which are likely to be formed at various steps of the reduction

process. Finally, the coalescence process (reactions 10 and 12) should be terminated if the nanoparticles

reach the desired size.

M0 + M0 → M2 (10)

M0 + M+ → M2

+ (11)

Mx+

m+x + My+

n+y → Mz+

p+z (12)

In general nanoparticles are prone to spontaneous coalescence and/or aggregation, which might

finally lead to the formation of big aggregates and to precipitation. This is in part due to their large surface-

to-volume ratio, leading to strong interactions between the surfaces. If nanoparticle preparations are to

possess good stability in time, direct contact between the surfaces should be prevented. This can be done

either by introducing steric hindrance (for instance, by a dense corona of polymer chains) or/and by

sufficiently high surface charge (zeta potential) (Fig. 4).

FIG. 4. (a) Sterical stabilization of metal nanoparticles. The barrier is formed by high local contentration of polymer chains

between the particles. (b) Electrostatic stabilization of metal nanoparticles. The barrier is formed by coulombic repulsion

between the highly charged particles [Gniewek A., Trzeciak A.M., Chemical News (in Polish: Wiadomości Chemiczne), 63,

955-981, 2009].

While the typical way in which organic molecules can be bound to gold is via the sulfur-gold link

(see above), there have been literature reports indicating that also other binding modes are possible and

sulfur-free polymers as poly(N-vinylpyrrolidone),[2-7] poly(acrylic acid)[8,9] and chitosan[10] can be

efficiently attached to gold nanoparticles. At IARC experimental studies have been launched on radiation

synthesis of gold nanoparticles stabilized by three kinds of polymeric ligands – oligomers of poly(acrylic

acid) (Mw of 1.8 kDa corresponding to ca. 25 monomer units), medium-chain-length chitosan (degree of

deacetylation 90 %, medium molecular weight 50 – 100 kDa) and thiolated poly(ethylene glycol) (Mw ca. 6

kDa). While in all cases the nanoparticles have been obtained, much work is still required to understand the

influence of synthesis’ parameters on size and stability of the products. As a complementary technique,

sonochemical synthesis based on the same systems has also been successfully performed (data not shown).

The obtained products have been analysed by UV-Vis spectroscopy, dynamic light scattering, zeta potential

measurements and transmission electron spectroscopy. Exemplary data are presented below.

Deoxygenated aqueous solutions containing 1 mM chloroauric acid as the Au nanoparticle precursor,

20 mM (in momoner units) poly(acrylic acid) as the ligand and 0.2 M isopropanol as OH radical scavenger

and a precursor of reducing alfa-hydroxyalkyl radicals have been irradiated by 6 MeV electron beam

(pulsed mode) with doses up to 2.5 kGy. Pictures of the resulting solutions, made at different time delays

after irradiation, are shown in Fig. 6.

80

FIG. 5. Deoxygenated, irradiated aqueous solutions of 1 mM chloroauric acid, 20 mM PAA, 0.2 M isopropanol, pH 2.3: (A)

before irradiation, (B) 5 min. after irradiation, (C) 1 hour after irradiation, (D) 1 day after irradiation. Absorbed dose 0.1 ÷

2.5 kGy.

It can be seen that doses lower than 1 kGy proved too low to efficiently reduce the precursor, while 1

– 2.5 kGy is sufficient to make the conversion clearly visible. This is also evidenced by UV-Vis spectra

(Fig. 6), where the absorbance characteristic for chloroauric ions disappears completely at 1 kGy, with the

parallel build-up of the plasmon resonance band at ca. 530 nm, characteristic for gold nanoparticles. An

interesting observation is that this band undergoes evolution not only with dose, but also with time after

irradiation for at least one hour.

FIG. 6. UV-Vis spectra of deoxygenated, irradiated aqueous solutions of 1 mM chloroauric acid, 20 mM PAA, 0.2 M

isopropanol, pH 2.3. Spectra recorded 30 minutes after irradiation. Absorbed dose 0.1 ÷ 2.5 kGy.

In order to gain a general idea on stability of the obtained nanoparticles, we followed time evolution

of the UV-Vis spectra for up to 35 days (Fig. 7). While changes in the shape of the spectra are very

moderate, a decrease in absorbance at 530 nm is observed. This indicates that stability is not prefect and

this issue definitely has to be addressed in further studies.

FIG. 7. UV-Vis spectra of deoxygenated, irradiated aqueous solutions of 1 mM chloroauric acid, 20 mM PAA, 0.2 M

isopropanol, pH 2.3. Absorbed dose 0.1 ÷ 2.5 kGy. Time after irradiation: 1 day (solid line), 7 days (broken line) and 35

days (dotted line).

Similar experiments have been performed using chitosan instead of poly(acrylic acid). In this case

acetic acid has been added to assure complete chitosan solubility. An important difference between the

chitosan- and poly(acrylic acid) is that in the latter no background reaction was expected nor observed,

while chitosan has been shown before to undergo a spontaneous reaction with gold ions, at least at elevated

temperature.[11] In preliminary tests we have confirmed the occurrence of this slow, spontaneous reaction

also at R.T.. However, at least at the conditions used in this study, the spontaneous reaction led to opaque

suspension, not suitable for isolation of gold nanoparticles (see Fig. 8F). Therefore we proceeded with the

radiation-induced reduction of gold precursor in the presence of chitosan as a ligand. Deoxygenated

aqueous solutions containing 1 mM HAuCl4, 20 mM of chitosan, 50 mM of acetic acid, 0.2 M of

isopropanol, pH 4.8, was irradiated by electron beam using doses up to 2.5 kGy. Results are presented in

Figs. 8-10. In contrast to the spontaneous reaction, here transparent solutions were obtained. As in the case

of PAA, both dose- and time evolution has been observed.

FIG. 8. Irradiation of deoxygenated aqueous solutions containing 1 mM HAuCl4, 20 mM of chitosan, 50 mM of acetic acid,

0.2 M of isopropanol, pH 4.8. (A) Before irradiation, (B) 5 min. after irradiation, (C) 1 day after irradiation, (D) 1 week after

82

irradiation, (E) 5 weeks after irradiation; absorbed dose 0.5 ÷ 2 kGy. Sample (F) non-irradiated solution 1 day after

preparation.

FIG. 9. UV-Vis spectra of deoxygenated, irradiated aqueous solutions containing 1 mM HAuCl4, 20 mM of chitosan, 50 mM

of acetic acid, 0.2 M of isopropanol, pH 4.8. UV-Vis spectra recorded 30 min. after irradiation.

FIG. 10. UV-Vis spectra of deoxygenated, irradiated aqueous solutions containing 1 mM HAuCl4, 20 mM of chitosan, 50 mM

of acetic acid, 0.2 M of isopropanol, pH 4.8. UV-Vis spectra recorded 24 h after irradiation.

These preliminary data show some promise towards the possibility of using ionizing radiation for a

simple and fast, one-step synthesis of polymer-stabilized gold nanoparticles. Discussion with partners and

optimization of synthesis procedures are necessary for further development of these products.

3. Radiation synthesis of polymer nanogels as potential radioisotope carriers

At IARC, there is large body of expertise in using ionizing radiation to synthesize polymer nanogels.

The team involved in this CRP in late 1990’ pioneered the approach based on intramolecular cross-linking

of single polymer chains in additive-free polymer-solvent system.[12-14] Since then this method has

been demonstrated to be quite universal, allowing to produce nanogels based not only on typical neutral

hydrophilic polymers, but also on polyelectrolytes[15] and thermosensitive polymers.[16-18] Further

progress in the synthesis involved developing two ways allowing for independent control of the product

diameter and mass, i.e., to control the inner density of the nanogel.[19] The method and products are

being explored at several labs, including Egypt, Germany,[16-18] Italy,[20-24] Turkey and United

States.[25] Significant progress on modification of chemical structure, introducing particular functional

groups, binding biomolecules, biomedical properties (including cellular uptake) and applications of

radiation-synthesized nanogels has been achieved at the laboratory of the Italian partner of this CRP in

Palermo.[20-24]

Within the current CRP it is intended to explore the possibility of using this approach to synthesize

nanogels capable of binding radioactive atoms, but at the same time fulfilling the application-driven

requirements as to their chemical composition, size, etc. The first task will be to establish testing methods

and quality control protocol(s) for nanogels. Such tools are necessary if we want to assure the

reproducibility of the future nanogel-based radiopharmaceuticals. During the RCM discussions will be held

to guide the activities of research teams working on nanogels in the sense of selecting product parameters

which show the greatest promise for the intended application.

4. Concluding remarks

It is postulated that IARC will participate in two main tasks of the current CRP, namely radiation- and

sonochemical synthesis of polymer-stabilized gold nanoparticles, as well as radiation synthesis and

tailoring polymer nanogels as potential radioisotope carriers. Precise formulation of these tasks, including

the need for cooperation and networking, will evolve from the discussions and conclusions of the 1 RCM.

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[3] TANG, X.L., JIANG, P., GE, G.L., TSUJI, M., XIE, S.S., GUO, Y.J., Poly(N-vinyl-2-pyrrolidone)

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colloidal gold nanoparticles: Synthesis and properties, Nanotechnology 21 45 (2010)

[9] DENG, Z.J., LIANG, M., TOTH, I., MONTEIRO, M.J., MINCHIN, R.F., Molecular interaction of

poly(acrylic acid) gold nanoparticles with human fibrinogen, ACS Nano 6 10 (2012) 8962.

[10] VO, K.D.N., KOWANDY, C., DUPONT, L., COQUERET, X., HIEN, N.Q., Radiation synthesis of

chitosan stabilized gold nanoparticles comparison between e-beam and gamma irradiation, Radiat.

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[13] ULANSKI, P., ROSIAK, J.M., The use of radiation technique in the synthesis of polymeric nanogels,

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[14] ULANSKI, P., ROSIAK, J.M., "Polymeric Nano/Microgels", Encyclopedia of Nanoscience and

Nanotechnology (NALWA, H.S., Eds.) American Scientific Publishers, Stevenson Ranch, CA (2004)

845-871.

[15] KADLUBOWSKI, S., GROBELNY, J., OLEJNICZAK, W., CICHOMSKI, M., ULANSKI, P., Pulses

of fast electrons as a tool to synthesize poly(acrylic acid) nanogels. Intramolecular cross-linking of

linear polymer chains in additive-free aqueous solution, Macromolecules 36 (2003) 2484.

[16] ARNDT, K.F., SCHMIDT, T., REICHELT, R., Thermo-sensitive poly(methyl vinyl ether) micro-gel

formed by high energy radiation, Polymer 42 16 (2001) 6785.

[17] QUERNER, C., SCHMIDT, T., ARNDT, K.F., Characterization of structural changes of poly(vinyl

methyl ether) gamma-irradiated in diluted aqueous solutions, Langmuir 20 7 (2004) 2883.

[18] SCHMIDT, T., JANIK, I., KADLUBOWSKI, S., ULANSKI, P., ROSIAK, J.M., REICHELT, R.,

ARNDT, K.-F., Pulsed electron beam irradiation of dilute aqueous poly(vinyl methyl ether) solutions,

Polymer 46 23 (2005) 9908.

[19] KADLUBOWSKI, S., ULANSKI, P., ROSIAK, J.M., Synthesis of tailored nanogels by means of two-

stage irradiation, Polymer 53 10 (2012) 1985.

[20] DISPENZA, C., GRIMALDI, N., SABATINO, M.A., TODARO, S., BULONE, D., GIACOMAZZA,

D., PRZYBYTNIAK, G., ALESSI, S., SPADARO, G., Studies of network organization and dynamics

of e-beam crosslinked PVPs: From macro to nano, Radiation Physics and Chemistry 81 9 (2012) 1349.

[21] DISPENZA, C., SABATINO, M.A., GRIMALDI, N., SPADARO, G., BULONE, D., BONDI, M.L.,

ADAMO, G., RIGOGLIUSO, S., Large-scale radiation manufacturing of hierarchically assembled

nanogels, Chemical Engineering Transactions 27 (2012) 229.

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[23] DISPENZA, C., SABATINO, M.A., GRIMALDI, N., BULONE, D., BONDI, M.L., CASALETTO,

M.P., RIGOGLIUSO, S., ADAMO, G., GHERSI, G., Minimalism in radiation synthesis of biomedical

functional nanogels, Biomacromolecules 13 6 (2012) 1805.

[24] SABATINO, M.A., BULONE, D., VERES, M., SPINELLA, A., SPADARO, G., DISPENZA, C.,

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macro to nano, Polymer (United Kingdom) 54 1 (2013) 54.

[25] AN, J.C., WEAVER, A., KIM, B., BARKATT, A., POSTER, D., VREELAND, W.N., SILVERMAN,

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25 (2011) 5746.

Nanosized Delivery Systems for Radiopharmaceuticals

Irfan Ullah Khan1, Fozia Iram

2, Mohammad Saeed Iqbal

3, Numair Younis

1, Abubaker

Shahid1

1Institute of Nuclear Medicine & Oncology (INMOL), Lahore, Pakistan

2Department of Chemistry, Lahore College for Women University (LCWU), Lahore, Pakistan

3Department of Chemistry, Forman Christian (FC) College, Lahore, Pakistan

Abstract

The over-expression of neuropeptide Y1-receptors in human breast cancer leads to a potential application of selective

neuropeptide Y (NPY) Y1-receptor ligands as a new generation of agents for cancer diagnosis and therapy. For the first time,

we showed the clear uptake of the 99mTc(core)3+-(NαHis-ac)-labelled Y1-receptor selective [Phe7, Pro34]NPY peptide in

human breast cancer patients by whole body scintimammography. In addition, peptide uptake was detected in metastases

spread in other organs, originating from the tumor. In contrast, a healthy volunteer showed no peptide uptake, especially in

breasts. Our pre-clinical studies, including different in vitro and in vivo analysis as well as our imaging studies in human

breast cancer patients confirmed the suitability of the NPY-Y1-receptor system for selective breast tumor targeting. These

excellent results prompted us to develop peptide-based gold and silver nanoparticles, thus ultimately, resulting highly specific

radiolabeled nanoconstructs decorated with peptide molecules to act as ‘nanotheranostics’. For this purpose, we produced

highly stable gold and silver nanoparticles (NPs) using arabinoxylan (AX) from ispaghula (Plantago ovata) seed husk. The

NPs were synthesized by stirring a mixture of AX and HAuCl4·H2O or AgNO3, separately, below 100◦C for less than an

hour, where AX worked as the reducing and the stabilizing agent. The synthesized NPs were characterized by surface

plasmon resonance (SPR) spectroscopy, transmission electron microscopy (TEM), atomic force microscopy (AFM), and X-

ray diffraction (XRD). The particle size was (for silver: 5-20 nm and gold: 8-30 nm) found to be dependent on pH,

temperature, reaction time and concentrations of AX and the metal salts used. The NPs were poly-dispersed with a narrow

range. They were stable for more than two years’ time.

1. INTRODUCTION

Cancer is still one of the most frequently occurring tumours. Severe and often therapy-limiting side

effects are a major obstacle in chemotherapy. New delivery concepts reducing systemic side effects are

needed in order to optimise anticancer therapies, and selective targeting concepts are required for early and

selective tumour diagnosis.

NPY is a 36 amino acid peptide amide of the pancreatic polypeptide family. It is one of the most

abundant neuropeptides in the brain and expressed in the peripheral and central nervous system1,2

. NPY

mediated functions are transmitted by so-called Y-receptors, named Y1, Y2 and Y5-receptors, which bind

NPY with nanomolar affinity3-11

. All Y-receptors are members of the class A of heptahelix receptors, that

signal through heterotrimeric G-proteins12

.

Reubi et al. have recently described Y-receptors expression in human breast cancer. They have shown

that > 90% of all breast tumours as well as 100% of the examined metastases express Y1-receptors13

.

Interestingly, a shift of the receptor subtype from Y2-receptors in healthy tissue to Y1-receptors during

neoplasm was found. Accordingly, we hypothesise that NPY and especially Y1-receptor selective ligands

might be dedicated tools for human cancer diagnosis and therapy. For the first time now, we show our

results of the detection of tumours and metastases by applying a 99m

Tc-labelled, Y1-receptor selective NPY

analogue in human breast cancer patients.

Based on NPY and on the previously identified Y1-receptor preferring peptide [Phe7,Pro

34] NPY

14 we

designed, synthesised and characterised two analogues for tumour labelling that vary in the position of the

chelator to conjugate 99m

Tc. Peptide 1a and 2a (Table 1) were synthesised with an Nα-histidinyl-acetyl-

chelator (NαHis-ac) chelator

15 at the N-terminus, whereas peptides 1b and 2b were modified with at the N

α-

86

side chain of lysine4. Rhenium was used as a cold surrogate for

99mTc and introduced for in vitro studies

(peptides 1c, 1d, 2c, 2d). In displacement competition assays using [3H-propionyl] NPY and increasing

concentrations of each peptide we could show that IC50 values reveal only minor reduction in receptor

binding on Y1- (SK-N-MC (human neuroblastoma) and MCF-7 (human breast adenocarcinoma)), Y2-

(SMS-KAN (human neuroblastoma)) and Y5- (HEC-1b-hY5 (human endometrial carcinoma)) receptor

expressing cells16

compared to the non modified analogues (Table 1). Peptides 1c and 1d showed similar

IC50 values at Y1- and Y2-receptor expressing cells and a slightly reduced affinity for Y5-receptors as

expected, whereas peptides 2c and 2d were characterised by a selective, nanomolar receptor binding only at

Y1-receptors. Accordingly, no significant influence of the Re(CO)3-(NαHis-ac) moiety was found for the in

vitro peptide receptor binding.

Table 1 Characterisation of the synthesised NPY analogues

Since, Nanotechnology is a rapidly growing area of interest because of its versatile applications in the

fields ranging from electronics to medicine18,27,32,34,35

, so we decided to couple our promising NPY

derivatives with Gold and silver nanoparticles (NPs). NPs find extensive use in medicine, catalysis and

electronics. Several synthetic methods have been employed for their preparation. These include physical,

chemical and biochemical techniques. Chemical methods involve the use of noxious reducing agents, like

sodium borohydride33

and hydrazine19

, and hazardous costly solvents. The particles produced by these

methods usually are unstable and as such require a stabilizing agent to be included in the process of

synthesis. With increasing awareness about environmental impact of synthetic methods, it is always

desirable to use the so-called green chemistry approach. Therefore, methods have been developed where

the additional use of reducing and stabilizing agents can be avoided24,29

. For application of NPs in medicine

it is more important that all the materials used as aids in synthesis should be biocompatible. Keeping in

view these requirements we here have used AX isolated from ispaghula seed husk as the reducing and

dispersing agent. AXs are gel-forming food components that are not hydrolyzed by enzymes of the upper

gastrointestinal tract, and coagulate at an acidic pH. Thus they can deliver the encapsulated material into

the last part of the large intestine (colon). AXs from isphagula (Plantago ovata) seed husk are abundantly

No. Peptide MWcalc. MWexpt. RTa

RTb

[Da] [Da] [min] [min]

1a (NαHis-ac)-NPY 4450.0 4450.9 20.1 n.d.

1b Lys4(N

αHis-ac)-NPY 4450.0 4451.8 20.5 n.d.

1c Re(CO)3-(NαHis-ac)-NPY 4720.0 4720.9 23.7 10.9

1d Lys4(Re(CO)3-(N

αHis-ac))-NPY 4720.0 4719.5 21.8 10.4

2a (NαHis-ac)-[Phe

7, Pro

34]NPY 4452.1 4453.2 21.8 n.d.

2b Lys4(N

αHis-ac)-[Phe

7, Pro

34]NPY 4452.1 4452.9 22.5 n.d.

2c Re(CO)3-(NαHis-ac)-[Phe

7, Pro

34]NPY 4722.0 4722.8 23.0 11.5

2d Lys4(Re(CO)3-(N

αHis-ac))-[Phe

7, Pro

34]NPY 4722.0 4722.9 23.1 11.9

No. Peptide IC50 [nM]

SK-N-MC MCF-7 SMS-KAN HEC-1b-hY5

1c Re(CO)3-(NαHis-ac)-NPY 3.9 ± 0.3 17.0 ± 6.5 3.2 ± 1.3 29.8 ± 1.9

1d Lys4(Re(CO)3-(N

αHis-ac))-NPY 10.5 ± 3.9 8.5 ± 6.5 6.1 ± 2.6 27.3 ± 5.1

2c Re(CO)3-(NαHis-ac)-[Phe

7, Pro

34]NPY 11.8 ± 2.6 26.9 ± 5.2 106.3 ± 22.2 ~1000

2d Lys4(Re(CO)3-(N

αHis-ac))-[Phe

7, Pro

34]NPY 1.3 ± 0.1 5.2 ± 1.0 97.5 ± 11.9 216 ± 3.6

available and well-characterized materials with potential use as drug carriers17,30

. The AXs are composed of

reducing sugars which can reduce gold or silver ions to particles and at the same time, having a hydrogel-

like network, can disperse the particles in themselves. They have also been proved to be non-toxic for drug

delivery22

. Therefore, we report here, for the first time, the use of AX from ispaghula for green synthesis of

gold and silver NPs having exceptional high stability, which may find their use in medicine.

2. EXPERIMENTAL

2.1. Materials

HAuCl4·3H2O, AgNO3, NaOH and CH3COOH were of analytical grade and procured from Sigma

Aldrich Co., USA. Ispaghula (P. ovata) seed husk was obtained from local market. Nanopure® water was

used in this work.

2.2. Isolation of AX Ispaghula seed husk

(5.0 g) was soaked in Nanopure® water (200 mL) for 24 h in a clean environment. The AX gel was

filtered under vacuum by use of a muslin cloth, washed several times with Nanopure® water, and the

suspension was reduced by evapora-tion under vacuum to 100 mL (AX-Sus) for use in synthesis of NPs. In

order to determine the reproducibility three batches of AX-Sus were prepared from ispaghula seed husk

obtained from three different sources. The isolated AX was characterized by sugar analysis, gel permeation

chromatography and FT-IR spectroscopy, and found to be similar to that reported previously (Saghir et al.,

2008).

2.3. Synthesis of NPs

To an appropriate amount of AX-Sus, prepared above, 10 mL of HAuCl4·3H2O solution (1.0 mM)

was added dropwise under vigorous stirring by use of a magnetic stirrer-cum-hotplate at ambient to 100◦C.

The inputs of the reagents used in the synthesis and the sample codes are given in Table 1. Reduction of

Au3+

to Au0 was evident from a color change (orange to purple), which was com-plete within 60 min time.

The experiment was repeated by using 2.0 and 3.0 mM HAuCl4·3H2O separately in order to study the

effect of concentration of the gold salt on NPs. The effect of time and pH was studied by changing time

from 10 to 180 min and pH from 2 to 7 (for gold) and 4 to 10 (for silver). The SPR spectra of the NPs were

also recorded beyond 180 min from time to time to determine the ultimate stability. The effect of

temperature (ambient to 100◦C) was studied at optimum pH and time conditions. Silver particles were

synthesized in a similar manner by replacing HAuCl4·3H2O with AgNO3. The change in color from

colorless to yellowish brown was observed in about 60 min. The NP suspensions thus obtained were

washed several times with Nanopure® water in order to remove any unreacted gold or silver salts. The

purple color remained stable for a very long time (a period of more than two years has lapsed at the time of

writing this manuscript when it was last observed).

2.3.1. SPR spectroscopy

The NP suspension was appropriately diluted with water to obtain the SPR spectrum in 200-700 nm range

by using Phar-maspec UV-1700 (Shimadzu, Japan) UV-Vis spectrophotometer, 1 cm quartz cell and AX

suspension in Nanopure® water as the reference.

2.3.2. X-ray diffraction

The XRD spectra were recorded on Bruker D8 Discover (Germany) diffractometer using

monochromatic Cu Kα radiation (λ = 1.5406˚A) operating at 40 kV and 30 mA. The data were collected

over a 10-80◦ 2θ range by using freeze-dried NPs.

2.3.3. Atomic force microscopy

The AFM images of AX film (5.0 µm × 5.0 µm) with dispersed gold NPs were obtained by using

Scanning Probe Microscope SPM-9500 J3 (Shimadzu, Japan) in the contact mode under normal

atmospheric conditions. A freshly prepared sample was deposited on a fine metal surface in dust-free

environment for this analysis.

88

2.3.4. Scanning electron microscopy

Scanning electron microscopic images of selected NPs were obtained by using SEM S-3700N

(Hitachi Japan) without sputter coating because the NPs were self-conducting.

2.3.5. Transmission electron microscopy

An ultrasonically dispersed sample of the solution of NPs (one drop) was placed on a carbon grid,

dried at room temperature in clean environment and TEM images were obtained by using JEM-1200EX

(JEOL, Japan) microscope at an accelerating voltage of 120 kV. The size distribution was determined by

measuring the diameter of about 300 particles and using Origin 7.5 software, which was further confirmed

by calculating the size from the highest intensity of XRD pattern by use of Debye-Scherrer equation (D =

0.9λ/ βcosθ), where the symbols have their usual meaning).

3. RESULTS AND DISCUSSION

3.1. Synthesis and optimization of conditions

The gold and silver NPs were successfully synthesized by reactions of gold and silver salts with the

AX isolated from ispaghula seed husk. The synthetic conditions were optimized in terms of temperature,

pH, time, and concentrations of AX and metal salts with respect to SPR spectral changes. The results are

shown in Figs. 1 and 2. The SPR absorbance is extremely sensitive to size and shape of particles, inter-

particle distance, and the surrounding media (Kelly, Coronado, Zhao, & Schatz, 2003). Thus optimization

was successfully carried out by use of SPR spectra.

FIG. 1. Effect of (a) temperature, (b) pH, (c) time and (d) amount of AX on the SPR spectrum of S5.

3.1.1. AgNPs

Fig. 1a shows the effect of temperature on the SPR spectrum of a sample (S5) at pH 11, time 60 min,

and AX-Sus 10 mL per 20 mL. There was no SPR band observed in the spectrum of the reaction carried out

at 25◦C. Beyond 25

◦C up to 100

◦C, the absorption started coming up between 409 and 445 nm. This

temperature dependent increase in the peak intensity shows that the silver ions reduction depends upon the

temperature. This effect is in line with the observations by other workers26,28

. The absorption at 412 nm,

corresponding to the size range of 5-12 nm31

, was observed at 75◦C. Similarly the optimum pH to obtain the

desired absorption at 412 nm was found to be 11. At pH 4.0 no absorption maximum was observed in the

range of 400-450 nm even after 24 h. However, an absorption band appeared at about 440 nm when pH was

raised to 5. The size of particles formed at pH 5, however, was large which decreased with increasing pH

up to 11 (Fig. 1b). The maximum absorption at 412 nm was achieved after 60 min under optimum

conditions of temperature and pH (Fig. 1c). The optimum amount of AX-Sus for 10 mL of 1.0 mM AgNO3

to obtain the peak at 412 nm was found to be 10 mL (Fig. 1d). The absorption maximum moved from 435

to 412 nm as the amount of AX-Sus increased from 2.0 to 10.0 mL per 20 mL and it shifted to 445 nm at

15 mL of AX-Sus. This effect may be attributed to restricted movement of particles in the medium with

increasing viscosity20

.

3.1.2. AuNPs

The SPR spectra of the AuNPs with varying amount of AX-Sus are shown in Fig. 2. The particles

exhibited characteristic absorption maxima at 535-560 nm. Position of the absorption maximum varied

with change in temperature, pH, reaction time and concen-trations of the AX-Sus and the gold salt. The

optimum conditions for obtaining a sharp absorption band at 535 nm, characteristic of AuNPs having size

in the range 10-25 nm25

, were determined through experimentation as described for AgNPs, and were

found to be: temperature: 80◦C, pH: 6, time: 40 min, amount of AX-Sus per 20 mL: 8 mL, and amount of

1.0 mM HAuCl4·3H2O per 20 mL: 10 mL. With the increase in temperature from 25◦C to 80

◦C, an increase

in peak sharpness was observed indicating a change in particle size.

FIG. 2. SPR spectra of AuNPs with varying amounts of AX-Sus.

3.1.3. X-ray diffraction analysis

The XRD spectra of the synthesized AgNPs and AuNPs are shown in Fig. 3. The spectra are

characteristic of face-centered cubic phase (JCPDS File No. 87-0720). The diffraction peaks can be

assigned to (1 1 1), (2 0 0), (2 2 2) and (3 1 1) planes as shown on the pictures. The sizes of the particles as

calculated by use of Debye-Scherrer equation were found to be 6-22 and 10-25 nm for silver and gold,

respectively. The Bragg reflections (2 0 0), (2 2 0) and (3 1 1) were weak and broadened relative to the

intense (1 1 1) reflection. This feature indicates that the nanocrystals are mainly oriented along (1 1 1)

plane and are of smaller size as confirmed by TEM data. There were some unidentified peaks in the spectra

which may be due to crystallization of an organic material from AX on the surface of the nanoparticles.

90

FIG. 3. XRD spectra of (a) AgNPs (S5) and (b) AuNPs (G4) dispersed in the polymer.

3.1.4. AFM and TEM analysis

Surface morphologies of AX films with and without nanoparticles are depicted in the AFM images

(Fig. 4). It can be seen that the surface morphology changed substantially after dispersion of nanoparticles

into the polymeric matrix. The TEM images of AgNPs (S5) and AuNPs (G4) along with size distribution

histograms are shown in Fig. 5. Average size of AgNPs was found to be 16 nm with the range 5-20 nm.

These results are consistent with the SPR and XRD data. Crystalline structure of the particles was

confirmed by the SAED pattern exhibiting bright circular spots corresponding to (1 1 1), (2 0 0), (2 2 0)

and (3 1 1) reflection planes. The d-spacings of the ring patterns correspond well to the XRD spectra. The

TEM image of AuNPs depicted an average particle size of 20 nm with a range of 8-30 nm. This was also

supported by the SPR and XRD data. All these measurements were replicated under optimum conditions on

the AX-Sus obtained from three different sources and thereby reproducibility was demonstrated. This study

clearly demonstrates that AX works as the reducing and stabilizing agent simultaneously. It may be

postulated that polysaccharides hydrolyze to some extent into monosaccharides which in turn exist in cyclic

and acyclic (aldehyde form) forms in equilibrium in aqueous medium21

. Thus the aldehyde group becomes

available for reduction of metal ions. However, it may be noted that AgNPs were produced in a basic and

AuNPs in an acidic environment. This observation points toward different chemistries taking place with

these elements, which needs to be investigated further. This study has identified AX as the excellent

stabilizing agent as the AgNPs and AuNPs dispersed in it exhibited unaltered particle size after the lapse of

over two years’ time. The AgNPs and AuNPs possessing exceptional high stability in AX-Sus can be used

for various biomedical and engineering applications without the risks of contamination by conventional

reducing and capping agents.

FIG. 4. AFM images of AX film (a) without and (b) with AuNPs (G4).

92

FIG. 5. TEM images and particle size distribution histograms of (a) AgNPs (S5) and (b) AuNPs (G4).

4. CONCLUSION

Since we observed excellent uptake of NPY derivatives in breast cancer subjects, so we hypothesize

that nanoconstructs developed by silver and gold NPs having particle size 5-20 and 8-30 nm respectively,

by using AX, from ispaghula seed husk, as the reducing and stabilizing agent, could prove promising

candidates. The method used in this work totally eliminates the use of noxious reducing and stabilizing

agents. The particles prepared by this method possess exceptional stability as more than two years passed

when they were last observed. As the AX used in this work is highly biocompatible, the AgNPs and AuNPs

thus prepared can be safely used for various biomedical and engineering applications.

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13. Reubi, J. C., Gugger, M., Waser, B. & Schaer, J. C. Y(1)-Mediated Effect of Neuropeptide Y in

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14. Soll, R. M. et al. Novel Analogues of Neuropeptide Y with a Preference for the Y1-Receptor. Eur J

Biochem 268, 2828-2837 (2001).

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Strategies of Peptides. Q J Nucl Med 43, 155-158 (1999).

16. Moser, C. et al. Cloning and Functional Expression of the hNPY Y5 Receptor in Human Endometrial

Cancer (HEC-1B) Cells. Can J Physiol Pharmacol 78, 134-142 (2000).

17. Akbar, J., Iqbal, M. S., Chaudhary, M. T., Yasin, T., & Massey, S. (2012). A QSPR study of drug

release from an arabinoxylan using ab initio optimization and neural networks. Carbohydrate

Polymers, 88(4), 1348-1357.

18. Du, W. L., Niu, S. S., Xu, Y. L., Xu, Z. R., & Fan, C. L. (2009). Antibacterial activity of chitosan

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Nanotoday, 2, 34-43.

Radioactive Nanomedicine – Imparting radioactivity to particulate drug delivery

systems

Say Chye Joachim Loo

Nanyang Technological University, Singapore, Singapore

Nanotechnology has found its presence in many industries and applications, and one such promising

application is in the biomedical arena. It has been exploited for the development of nanomedicine, for drug

delivery and bioimaging purposes. For example, drug delivery in the form of nanomedicine utilizes nano-

sized particles to transport and release pharmaceutical compound into the body, to achieve the most

desirable therapeutic outcome and in the safest possible manner. Here, we review some of the particulate

drug delivery systems that have been developed from the School of Materials Science and Engineering

(MSE), Nanyang Technological University (NTU), Singapore. These drug delivery systems will be

explored for their potential in delivery radiopharmaceuticals.

1. Multilayered Particulate Systems

Double-layered ternary-phase microparticles composed of a poly(D,L-lactide-co-glycolide) (50:50)

(PLGA) core and a poly(L-lactide) (PLLA) shell impregnated with poly(caprolactone) (PCL) particulates

were loaded with ibuprofen (IBU) and metoclopramide HCl (MCA) through a one-step fabrication process.

MCA and IBU were localized in the PLGA core and in the shell, respectively. The aim was to study the

drug release profiles of these double-layered ternary-phase microparticles in comparison to binary-phase

PLLA (shell) / PLGA (core) microparticles and neat microparticles (FIG. 1). The particle morphologies,

configurations and drug distributions were determined using scanning electron microscopy (SEM) and

Raman mapping. The presence of PCL in the PLLA shell gave rise to an intermediate release rate of MCA

between that of neat and binary-phase microparticles. The ternary-phase microparticles were also shown to

have better controlled release of IBU than binary-phase microparticles. The drug release rates for MCA and

IBU could be altered by changing the polymer mass ratios [1].

Ternary-phase microparticles, therefore, provide more degrees of freedom in preparing microparticles

with a variety of release profiles and kinetics. This study presents a feasible route to load

radiopharmaceuticals into such particles, and would be further explored.

96

FIG. 1. (a) Double-layered microparticle; (b) Fluorescent-labelled double-layered sub-micron particle; (c) Raman mapping

of drug-loaded double-layered microparticle; (d) Sequential release of two different drugs from double-layered

microparticle; (e) Different morophologies of double-layered microparticles. Ref: PCT Application no.

PCT/SG2010/000215; Acta Biomaterialia 2012; 8: 2271.

2. Microencapsulation of Nanoparticulates

Drug delivery is the administration of pharmaceutical agent(s) to achieve a therapeutic effect in patients.

Conventional drug-delivery strategies often require repetitive drug administration and this can lead to

strongly fluctuating drug levels that may pose undue physiological discomfort to the recipient. Fur-

thermore, certain drugs at high concentrations can sometimes trigger undesirable toxicity or immunological

responses in the patient, which can be detrimental. Proper drug administration requires a fine balance

between time, dosage, and mode of delivery, and, more importantly, should aid in improving patient

compliance and clinical outcomes. To help realize a multiple-drug delivery system, the design of a

polymeric microcapsule that can house different drug-loaded particles using a modified

solid/water/oil/water (S/W/O/W) emulsion technique was developed. A simple emulsion technique was

devised to encapsulate multiple fluorophore-loaded particles within a single polymeric microcapsule (FIG.

2(a)). The largeness in size for the microcapsules had been selected to present insights into the interior

structure of the construct as precedence to miniaturization of the system. Visual analyses (SEM and CLSM)

had confirmed the feasibility of this methodology (FIG. 2(b)). Two drugs of different hydrophilicities were

also loaded into PLLA microparticles and successfully encapsulated in the PLGA microcapsule. Drug

release studies showed that the PLGA shell provided a means to retard burst release, while controlling the

release [2-4]. The results presented offer more opportunities to further examine the system’s effectiveness

as a drug delivery platform for radiopharmaceuticals, which would be explored in future research

undertakings.

3. Hybrid Ceramic/Polymer Particulates for Bone Targeting

Using sodium dodecyl sulphate micelles as template, hollow-cored calcium phosphate nanocapsules

were produced. The surfaces of the nanocapsule were subsequently silanised by a polyethylene glycol

(PEG)-based silane with an N-hydroxysuccinimide ester end groups which permits for further attachment

with bisphosphonates (BP). Characterizations of these nanocapsules were investigated using Field

Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy, Fourier Transform

Infra-Red Spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy and Dynamic Light

Scattering. To further validate the bone-targeting potential, dentine discs were incubated with these

functionalised nanocapsules. FESEM analysis showed that these surface-modified nanocapsules would

bind strongly to dentine surfaces compared to non-functionalized nanocapsules. We envisage that

respective components would give this construct a bifunctional attribute, whereby (1) the shell of the

calcium phosphate nanocapsule would serve as biocompatible coating aiding in gradual osteoconduction,

while (2) surface BP moieties, acting as targeting ligands, would provide the bone-targeting potential of

these calcium phosphate nanocapsules [5-6]. Such nanocapsules can be considered for the delivery of Sm-

153-EDTMP for bone targeting.

(a) (b)

FIG. 2 (a) Schematic representation of simultaneously encapsulation of particles loaded with two different dyes within a

single microcapsule; (b) FESEM images of a typical microcapsule excised to disclose the cross-section region. (a–c)

Subsequent magnification had revealed the presences of smaller PLLA microparticles residing within the hollow space of

the microcapsule. Note that no microparticles were found on the outer shell region of the microcapsule. Ref: Assigned

Singapore Patent No. 190009; Advanced Healthcare Materials 2012; 1: 159.

98

FIG. 3 (a) Schematic representation of coating drug-loaded PLGA particles with calcium phosphate layer;

(b) SEM images of PLGA particles showing calcium phosphate nanoparticles coated on its surface. Ref:

USA Appl. No.: 13/885,931 and Europe Appl. No.: 11842961.2; PCT/SG2010/000214; Journal of

Materials Science: Materials in Medicine 2014 Accepted.

4. Hydrophilic-Hydrophobic Core-Shell Particles

Current focus on particulate drug delivery entails the need for increased drug loading and sustained

release of water soluble drugs. Commonly studied biodegradable polyesters, such as poly(lactide-

coglycolide) (PLGA) and poly(L-lactide) (PLLA), are lacking in terms of loading efficiency of these drugs

and a stable encapsulation environment for proteins. While hydrogels could enable higher loading of

hydrophilic drugs, they are limited in terms of controlled and sustained release. With this in mind, the aim

was to develop microparticles with a hydrophilic drug-loaded hydrogel core encapsulated within a

biodegradable polyester shell that can improve hydrophilic drug loading, while providing controlled and

sustained release. Herein, we report a single step method of fabricating microparticles via a concurrent

ionotropic gelation and solvent extraction. Microparticles fabricated possess a core–shell structure of

alginate, encapsulated in a shell constructed of either PLGA or PLLA. The cross-sectional morphology of

particles was evaluated via scanning electron microscopy, calcium alginate core dissolution, FT-IR

microscopy and Raman mapping (FIG. 4). The incorporation of alginate within PLGA or PLLA was shown

to increase encapsulation efficiency of a model hydrophilic drug metoclopramide HCl (MCA). The

findings showed that the shell served as a membrane in controlling the release of drugs [7]. Such gel-core

hydrophobic-shell microparticles thus allow for improved loading and release of water soluble drugs, and

can be considered for the loading of fluid based hydrophilic radiopharmaceuticals.

FIG. 4 Schematic diagram of alginate-PLGA/PLLA core-shell particle and SEM images of subsequent particles prepared

accordingly. Release profile from such a particle is found on the bottom right corner. Ref: PCT/SG2013/000051;

Biomaterials Science 2013; 1: 486.

5. Conclusions

Particulate drug delivery systems can be developed for the delivery of drugs. Such delivery systems

shown here include multi-layered particles, microencapsulation of nanoparticles, ceramic-polymer hybrid

particles and hydrophobic-hydrophilic core-shell particles. These particulate systems can be further

developed for the delivery of radiopharmaceuticals.

REFERENCES

[1] SCJ Loo, WL Lee. Multi-phase microparticles and method of manufacturing multi-phase microparticles. NTU

TD ref: TD/019/09 – US Provisional No. 61/184,559 filed 5 Jun 2009 (lapsed). PCT Application no.

PCT/SG2010/000215 filed 7-Jun-2010. National phase: USA.

[2] SCJ Loo, WL Lee, PX Wee. Multi-drug-loaded gastric-floating microcapsules. US Provisional

Application no. 61/683,955 filed on 16-Aug-2012. US PCT Application No: PCT/SG2013/000282. 8

July 2013.

[3] T Steele, SCJ Loo, S Venkatraman, FYC Boey. Drug loaded nanoparticles and microparticles encapsulated

into low-melting polyester wax films locally delivered by angioplasty balloons. PCT Application no.

PCT/SG2011/000457.

[4] SCJ Loo, YL Khung, WL Lee. Method for encapsulating particles. PCT Application No:

PCT/SG2011/000414. National phase: Europe (Appl. No.: 11 842 853.1), Singapore (Appl. No.: 201303178-

6)

[5] SCJ Loo, K Bastari, S Venkatraman. Method for coating particles with calcium phosphate and particles,

microparticles and nanoparticles formed thereof. NTU TD ref: TD/076/09 – US Provisional Application No.

100

61/416,973 filed 24-Nov-2010 (lapsed). PCT Application no. PCT/SG2011/000414 filed 24-Nov-2011.

National phase: USA (Appl. No.: 13/885,931), Europe (Appl. No.: 11842961.2), Singapore.

[6] S Venkatraman, SCJ Loo. Targeted drug delivery to bone. NTU TD ref: TD/024/08 – US Provisional

No. 61/184,548 filed 5 Jun 2009 (lapsed). PCT Application no. PCT/SG2010/000214 filed 7-Jun-2010.

National phase: Singapore, China, Europe, India and USA

[7] SCJ Loo, MP Lim, WL Lee. Methods of manufacturing core-shell microparticles, and microparticles

formed thereof. PCT Application No: PCT/SG2013/000051 filed 6-Feb-2013.

GREEN SYNTHESIS OF POLYSACCHARIDE AND POLYPEPTIDE-BASED

NANOPARTICLES TOWARD THE CRATION OF STABLE AND

FUNCTIONALIZED RADIOPHARMACEUTICALS FOR DIAGNOSIS AND

THERAPY

W. PASANPHAN, T.RATTANAWONGWIBOON, S.WONGKRONGSAK

Department of Materials Science, Faculty of Science, Kasetsart University,

Bangkok, Thailand

Abstract

Current status of the research work includes a series of nanosized drug delivery system with different formulations,

i.e., (i) gold nanoparticles (AuNPs) reduced and stabilized using natural polymers, (ii) amphiphilic core-shell

polymeric NPs and (iii) nanogels (NGs) or NPs containing polychelating groups. The proposed work (ii) is the

production of water-soluble nanostructural polysaccharide (water-soluble chitosan nanoparticles, WSCS-NPs) and

polypeptide (water-soluble silk fibroin nanoparticles, WSSF-NPs) as natural antioxidants and reducing agents for a

green synthesis of AuNPs. Furthermore, radiation assisted the production of AuNPs in such water-based

biopolymers is also demonstrated. The possibilities to use the successful procedure for the creation of radioactive 198AuNPs analogue for therapy via -rays emission and diagnosis via -rays emission using SPECT are the

strategy in progress. For series (ii), the development of amphiphilic core-shell CS nanoparticles using chemical

conjugation and radiation grafting techniques is proposed in the last CRP. According to water-soluble limitation of

the obtained polyethylene glycol monomethacrylate-grafted-deoxycholic acid chitosan nanoparticles (PEGMA-g-

DCCS-NPs, 80 nm) from the first generation, further research aims to improve the solubility of this amphiphilic

NPs by using an alternative WSCS-NPs as the main biopolymer. It is expected that the next generation of the

PEGMA-g-DCWSCS-NPs would serve as a possible merit to be used not only for hydrophobic drug encapsulation

in chemotherapy but also radionuclide entrapment among the polychelating groups on the PEGMA shells for

radiodiagnosis. To achieve nanosized delivery with superior radionuclide entrapment, new polymeric NGs or NPs

containing polychelating groups will be further development as in series (iii). Herein, poly--glutamic acid (PGA),

a poly (amino acid) will be modified and fabricated as inter-penetrating network NGs, otherwise the polypeptide

SF will be modified with polyacrylic acid (PAA). Both model contain polychelating groups of the carboxylic acid

(-COOH), which would provide the sites for strongly entrapping the radiopharmaceutical substances. The surface

of all models of the nanocarriers have amino (–NH2), hydroxyl (-OH) or –COOH functionalities, therefore they are

possible to be conjugated with the specific targeting molecules, such as monoclonal antibody, folic acid, or glucose.

Beside the synthesis and characterization, the performance of the radiopharmaceutical nanocarriers regarding

localization and uptake, in vitro biocompatibility and diagnosis and/or therapeutic efficiencies will also be

demonstrated and cooperated under this CRP.

1. INTRODUCTION

Biodegradable or bio-based polymeric nanomaterial is a good promising for being nanocarrier for

radiopharmaceuticals because it helps to prolong circulation time of the drug substances and it can be

degraded and eliminated via biological mechanism. There are a number of diagnostic and therapeutic

radiopharmaceuticals that are Food and Drug Administration (FDA) approved for use in human patients.

Radionuclide imaging is commonly divided into two general modalities; single photon emission computed

tomography (SPECT) and positron emission tomography (PET). These radionuclides are almost beta (β)-

emitters and alpha (α)-emitters for radiotherapy and gamma ()-emitters for diagnosis. For PET

applications, positron-emitting radionuclides of first choice are F-18, C-11, N-13, and O-15. The principal

applied radionuclide for PET is F-18 (half-life, t1/2 = 1.83 h). For diagnosis, many types of radionuclides,

such as Tc-99m and Ga-67, have been used for imaging using SPECT [1].

Nanoscience and technologies have been considered in many areas including biomedicine. For

biomedical purposes, nanosized delivery system provides several superior properties. For example, it

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improves drug solubility, prolongs drug circulation time, decreases severe toxic effect, enhances

permeation and retention effect in tumor tissue and enhances the drug’s therapeutic efficacy.

Drug delivery and targeting systems are well known for diagnosis and therapy of cancer. Due to their

targeting ligands, the drug targeting can selectively and effectively carry several molecules, such as genes,

chemotherapeutic drugs, contrast agents, radioimaging agents, etc. into the targeted tumour cells. The first

generation of nanocarriers is known for its rapid clearance from the blood stream and high uptake by the

macrophages of the reticuloendothelial system (RES) due to the opsinisation processes occurring at the

surface of these carriers following an intravenous administration. In the second generation, the nanocarriers

surface properties have been modified by covalently binding hydrophilic poly (ethylene glycol) (PEG) to

reduce their toxicity and overcome the RES capture and prolong their blood circulation time [i]. For further

development in the third generation on the nanocarriers, the nanocarrier surface was bound with the

specific recognition ligands, e.g., monoclonal antibodies [ii], folic acid [iii] and D-glucose [iv]. This allows

an active rather than passive targeting of the cancerous cells. Several types of nanocarriers, such as

liposome, micro/nanoparticle, dendrimer and hydrogel, have been proposed for the development of drug

delivery for radiopharmaceuticals (Fig. 1).

Liposomes: Various types of chelating molecules, e.g., deferoxamine or nitrilotriacetic acid labelled

with Ga-67, In-111 and Tc-99m and loaded into the liposomes were reported. The particle with the size as

small as 80 nm exhibited a good tumor accumulation and higher intrinsic tumor compared to that of free

Ga-67, In-111 and Tc-99m [v]. Surface modification of the hydrophilic molecules induces a prolongation

in the circulatory half-life of the drug-loaded liposome including other types of nanostructural carriers.

Modification of liposome and loaded with 111

In-DTPA into the PEGylated could enhance the liposome

circulation time and increase tumor uptake [vi]. To approach an active targeting, the liposomes labeled

radionuclide, such as Ga-67 and linked to a specific antibody have been carried out.

FIG 1. Examples of nanosized delivery system for radiopharmaceuticals [1].

Micro/nanoparticles: For microparticles (MPs) and nanoparticles (NPs), glass MPs, albumin MPs,

solid-lipid NPs including polymeric MPs and NPs were developed for loading Y-90, Ho-166, Ga-68, Tc-

99m and In-111. For example, Mumper et al. developed the poly (L-lactic acid, PLLA) MPs labeled with

Ho-166 microsphere as a potential agent for the internal radiation therapy of hepatic tumors [vii]. Recently,

gold NPs (AuNPs) have been recognized in clinical applications. It provides many advantages in clinical

applications over other metallic particles due to biocompatibility and non-cytotoxicity [viii]. They have

long been considered as possible drug delivery vehicles applicable for therapy. Owing to their excellent

surface functionalization chemistry, the AuNPs are attractive for a number of reasons. For example,

different shape and size of AuNPs provide special and specific biomedical applications. In addition, the

invention of AuNPs have preclinical studied and reported their radiosensitizing effect [ix]. In the recent

year, a group of researcher developed a radioactive analogue AuNPs (Au-198 NPs) for the possible use as

both diagnosis and therapy for treating a postage cancer. The radioactive analogue AuNPs was prepared via

green synthesis using gum arabic glycoprotein (GA) [x]. The GA-Au-198 NPs (85 nm) exhibited effective

control in the growth of prostate tumors over 30 days.

Polymeric micelle: Polymeric micelles are spherical, nanosized (10–100 nm) supramolecular

constructs with a core–shell structure resulting from the self-assembly of amphiphilic block copolymers in

aqueous environments. Generally, the hydrophobic core provides the loading space for water-insoluble

anticancer drug, whereas the hydrophilic shells hinder plasma protein adsorption and prolongation of the

circulation time. Both anticancer drug and contrast agents (gadolinium-diethylene triamine penta acetic

acid, Gd-DTPA) have been incorporated into the polymeric micelles. The polymeric core-shell has been

applied by incorporation of the anticancer drugs (e.g., doxorubicin, paclitaxel), incorporation of

paramagnetic Gd complexes for MRI, incorporation of In-111 or Tc-99m for gamma scintigraphy and

incorporation of iodine for x-ray computed tomography.

To incorporate a chelated radionuclide in polymeric micelles for diagnosis, the amphiphilic chelating

agents are basically used. For example, micelles created from DTPA-phosphatidylethanolamine and

DTPA-stearylamine were reported for labeling with In-111 and attached with 2C5 antibody [xi]. To further

improve high loading of radionuclide into the polymeric micelles, the polychelating amphiphilplic

polymers (PAP), which contain multiple chelating groups bring about the increase of loading efficiency and

a suitable means for incorporation into liposomes, particles and micelles. The use of PAP allows for

increasing the number of chelated radionuclide metal atoms attached to a single anchor and it is able to be

incoperated into the micelle core resulting in better image intensity even a lower micelle doses [xii]. The

polymeric backbone providing the sites for attachment of the multiple chelating moieties should possess a

sufficient number of reactive groups, such as amino (–NH2), carboxy (–COOH), aldehyde (–CHO), thiol (–

SH) and ether (–C–O–C–). For example, DTPA-derivative poly-L-lysine (PLL) containing multiple free –

NH2 groups, polyacrylic acid containing -COOH side groups and PEG containing ether –O–C–O– between

the ethylene oxide repeating units – (CH2CH2–O) –.

Hydrogel: Hydrogel are also good promising radiopharmaceutical delivery systems because they

have ability to become locally gelatinized after a lag of time in order to be retained in the tissue. Several

polymers and biopolymers, i.e., chitosan, fibrin and alginate have been studied and proposed as good

examples of these systems. The Ho-166 was incubuated in chitosan solution to from a complex of Ho-166-

chitosan. The complex hydrogel could reduce C6 glioma cell growth rate by 97% and 2 times longer

survival period up to 59 days than that of the control (22.8 days) [xiii]. For preparing the stable covalent

hydrogel, glutaraldehyde was used to create chemical crosslink of chitosan [xiv]. The I-131 was

incorporated into crosslinked chitosan hydrogel. It was found that 80% of the incorporated 131

I-chitosan

hydrogel was released within a month after implantation. Besides, fibrin glue [xv] and alginate [xvi]

hydrogel were also prepared for entrapment of Re-188 for treating the breast cancer cells. The Re-188

alginate hydrogel could delay in tumor growth and it localized in the tumor site up to 48 h post-injection.

According to the previous literatures, several carrier formulations have been studied for

radiopharmaceutical delivery system. It can be summarized that to archive the effective carrier for delivery

system, the materials should provide (i) multiple chelating groups for enhancing the radionuclide

entrapment, (ii) nanoscaled size as small as 80 nm for increasing radionuclide permeation and accumulation

in the tumor cell, (iii) surface modification with hydrophilic polymers (e.g., PEG) for enhancing the

stability and prolonging the circulation time, (iv) surface modification with targeting molecule (e.g.,

monoclonal antibody or glucose, folic acid) for improving the specific targeting to tumor site. Although

there are a number of research has proposed the preparation of nanosized carrier and its abilities to use for

radiopharmaceutical delivery applications, the production of nanosized and multifunctional biopolymer-

based nanocarrier, i.e., AuNPs, core-shell amphiphilic NPs and multiple chelating nanogel fabricated from

polysaccharide (i.e., chitosan) and poly (amino acid) or polypeptide (i.e, silk fibroin, poly-gamma-glutamic

acid) using a green irradiation synthesis have not yet been reported spanning in a wide range of this clinical

area.

In the present report, the current status of our research works related to nanopolymer production and

its possible use as nanocarrier in the radiopharmaceutical delivery system is proposed. Herein, a series of

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the biopolymer-based nanoparticles have been mainly focusing and produced using irradiation technology

accompanying with the common chemical modification process. Future development of the successful

nanostructural materials based on the previous challenge would serve as a good promising nanosized

delivery system for radiopharmaceuticals for diagnosis and therapy applications.

2. GREEN SYNTHEIS OF GOLD NANOPARTICLE AND ITS RADIOACTIVE ANALOUGE USING

WATER-SOLUBLE POLYSACCHARIDE OR POLYPEPTIDE BIOPOLYMERS AND RADIOLYSIS

METHOD

Synthesis of gold nanoparticles (AuNPs) is one of the expanding research areas in nanotechnology.

With their novel properties, they have been applied in many applications particularly biomedicine and other

bio-related applications, such as drug delivery [xvii], photothermal therapy [xviii], diagnosis [xix]

including radiotherapy and diagnosis [x]. In term of radiopharmaceuticals, the radioactive properties of

198Au with its beta (Eβ = 0.96 MeV) and gamma (Eγ= 0.411 MeV) energy emission make them valuable

candidates for dual therapy and single-photon emission computed tomography (SPECT) imaging

applications [x]. In addition, the surface of AuNPs can be decorated with the tumor specific peptides (and

proteins) or monoclonal antibody to develop a new generation of nanotheranostic agents capable of

performing effective singular nanoceuticals containing diagnosis, therapy and targeting properties.

Several methods, particularly chemical reduction [xx], have been developed to synthesize metal NPs

including AuNPs. In the past decade, with some hazardous chemicals remaining in the products and it is

concerned in the clinical applications, the alternative green synthesis has been emerged. The green

synthesis of AuNPs has been performed using natural reducing and stabilizing agents.

Biomolecules from plant extracts have been widely proposed as green synthesis of AuNPs [xxi] as a

result of their antioxidant and reducing properties. However, small molecular natural products still lack of

the stability including chelating and stabilizing efficiencies. Biomacromolecules, e.g., starch, chitosan,

gelatin and alginate, have been the most widely used as capping agents for the synthesis of metal NPs [xxii,

xxiii, xxiv]. Unlike the small molecular reducing agents, the biopolymers themselves exhibit low reactivity

to reduce metal ion to metal atom because of their inherent high molecular weight, low solubility or lack of

reducing power function.

2.1 Preparation of water-soluble chitosan and silk fibroin nanoparticles as green antioxidant and reducing

agents

Chitosan (CS): CS, a deacetylated form of chitin, makes up the second-most naturally abundant

copolysaccharides next to cellulose. It dictates numerous advantage properties, such as biodegradability,

biocompatibility, bioactivity, and non-toxicity [xxv]. The amino (-NH2) and hydroxyl (-OH) groups of CS

at C-2 and C-6 positions not only provide reactive sites for functionalization but also exhibit strong metal

ion chelation [xxvi], oil absorption [xxvii], antimicrobial [xxviii], and antioxidant activities [xxix]. It is

known that the most common chemical species, which act as antioxidant or reducing agents are hydroxyl, -

OH (e.g. phenolics), sulfhydryl, -SH (e.g. cysteine and glutathione) and amino, -NH2 groups (e.g. uric acid,

spermine and proteins) [xxx].

Therefore, CS has attracted much attention to develop as natural antioxidant and reducing agent since

it has plenty of –OH and –NH2 groups as H-atom donation. It has been concluded that the –OH group in the

saccharide units can react with free radicals by the H-abstraction reaction and the –NH2 group of CS can

react with free radicals to form additional stable macroradicals [xxxi]. Although the -OH and

FIG. 2. Chemical structures, physical appearances and particle morphologies of (A) WSCS-NPs [32] (B) WSSF-NPs.

-NH2 groups of CS exhibit many unique properties including antioxidant and reducing power, CS has

its inherent drawback of non-reactivity and water-insolubility. This is a vastest limitation to use CS in

aqueous solution at neutral pH to apply in a wide range of applications particularly biological media.

To improve water-solubility and nanosized formation of CS, irradiation of the CS was carried out

[xxxii]. The water-soluble chitosan nanoparticles (WSCSNPs) were prepared by simply irradiation with

the dose of 80 kGy accompanying with appropriate separation. The WSCS-NPs product (Fig. 2A) was well

characterized to confirm it remaining chemical structure after irradiation. The changes in chain packing

structure, thermal stability and morphology of the obtained WSCSNPs reflect the improvement of its

solubility, nanoscaled structure and reactivity. TEM image confirmed the successful preparation of

nanoscaled WSCS without derivatization. The WSCSNPs exhibited more excellent antioxidant activity

with the EC50 (0.115 mg/mL). The WSCSNPs are non-toxic and more biocompatible to human skin

fibroblasts. This WSCS-NPs is expected to use as a green reducing and capping agent for the production of

the radioactive 198

AuNPs for diagnosis and therapy.

Silk fibroin (SF): SF is a polypeptide consisting of repetitive protein with layers of antiparallel beta

sheets. SF extracted from Bombyx mori silkworms have been discovered to use as biomaterials according to

its specific properties. It has impressive biocompatibility, biodegradability, aqueous processibilty and

antioxidant properties [xxxiii]. Its primary structure mainly consists of amino acid sequence of glycine,

alanine and serine [(Gly-Ala-Gly-Ala-GlySer)]. Although the chemical structure of SF is similar to

collagen, SF exhibits less inflammatory response than the collagen including poly (lactic acid) [xxxiv].

Generally, SF exhibited high molecular weight (250-350 kDa), self-assemble into an antiparallel β-

sheet structure resulting in high crystalline and mechanical properties. Although, SF provide satisfied

properties for some application particularly tissue engineering, its hydrophobic and water-insoluble, soluble

in hot acid and alkaline solution, and less reactive may limited its application as a water-soluble antioxidant

and reducing agent to create AuNPs and as a main biopolymer for the production of nanosized delivery for

radiopharmaceuticals.

Our current study has been focused on production of water-soluble nanosized silk fibroin (WSSF-

NPs) using electron-beam irradiation. The effects of irradiation doses on molecular weight reduction,

antioxidant activity, reducing power and nano structural morphology of nano-SF were clarified. The

samples were irradiated in a water system with electron beam dose ranging from 1 to 30 kGy. The

antioxidant and reducing power were increased by irradiation. The antioxidant activity of WSSF-NPs was

increased up to 89.31%. The obtained WSSF-NPs showed spherical shape with the size as small as 40 nm,

which was observed by TEM (Fig 2(B)).

2.2 Green synthesis of AuNPs using WSCS-NPs and WSSF-NPs including radiolytic method.

The obtained WSCS-NPs and WSSF-NPs assist the production of monodispersed AuNPs in one-pot

reaction. Fig. 3 showed the physical appearances and particle morphologies of AuNPs produced in

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FIG. 3. Green synthesis of AuNPs using 0.1 mM AuHCl4 in different concentrations of (A) WSCS-NPs and (B) WSSF-NPs for

24 h.

WSCS-NPs (Fig. 3A) and WSSF-NPs (Fig. 3B). The color of the solution changed particularly in

WSCS-NPs indicated the successful preparation of AuNPs. The particle morphology of the obtained

AuNPs was spherical. The particle size of the AuNPs obtained from WSCS-NPs and WSSF-NPs were 20

and 15 nm.

To assist the production of the AuNPs in the WSCS-NPs and WSSF-NPs, irradiation process is a

strategy in our research. Compared with non-irradiated sample, the color of the irradiated samples changed

as seen in Fig. 4. The absorbent at a maximum wavelength also increased when the irradiation dose

increased from 1-10 kGy. The results demonstrate the successful production of AuNPs under electron-

beam irradiation.

FIG. 4. Green synthesis of AuNPs using 0.05 mM of (A) WSCS-NPs and (B) WSSF-NPs accompanying with electron-beam

irradiation with the doses from 1-30 kGy.

2.3 From current status of AuNPs production to the future prospects in nanosized delivery of

radiopharmaceuticals

The successful productions of WSCS-NPs and WSSF-NPs are expected to be novel, practical, natural

water-soluble polysaccharide and polypeptide antioxidants or reducing agents as well as capping agents for

green synthesis of stable AuNPs. This simple and rapid green synthesis of AuNPs using WSCS-NPs and

WSSF-NPs can be applied in biomedical areas, such as drug carrier, therapy and diagnostic applications. In

addition, the increase of AuNPs amount can be performed by irradiation process.

A B

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

400 450 500 550 600 650 700

Wavelength (nm)

Ab

sorb

ance

0 kGy1 kGy5 kGy10 kGy30 kGy

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

400 450 500 550 600 650 700

Wavelength (nm)

Ab

sorb

ance

0 kGy1 kGy5 kGy10 kGy30 kGy

A

B

FIG. 5. (A) Preparation of radioactive 198

AuNPs using green synthesis as observed in section 2.3 and

(B) Diagnosis and therapeutic efficiencies of 198

AuNPs in tumour-bearing animal models that mimic

human cancers.

The effects of all related parameters, i.e., AuHCl4, WSCS-NPs and WSSF-NPs concentrations and

irradiation doses on the AuNPs formation will be carefully demonstrated and characterized.

The appropriate protocol of AuNPs production will be further used for the creation of the radioactive

AuNPs analogue (i.e., 198

AuNPs). The 198

AuNPs will be produced by neutron (1n0) activation. We will also

extend our work in the future to a study of the 198

AuNPs stabilized WSCS-NPs and WSSF-NPs efficiencies

for diagnosis and therapy [x]. Localization and uptake of nonradioactive 197

AuNPs and in vitro

biocompatibility will also be demonstrated.

3. AMPHIPHILIC CORE-SHELL NANOPARTICLES FOR HYDROPHOBIC DRUG AND

RADIOPHARMACEUTICAL ENCAPSULATION

3.1 The first generation of an amphiphilic core-shell PEGMA-g-DCCS NPs [xxxv]

Poly (ethylene glycol) monomethacrylate-grafted-deoxycholate chitosan nanoparticles (PEGMA-g-

DCCSNPs) were successfully prepared by radiation-induced graft copolymerization. The hydrophilic

PEGMA was grafted onto DCCS in an aqueous system. The radiation absorbed dose is an important

parameter on degree of grafting, shell thickness and particle size of PEGMA-g-DCCSNPs. Owing to their

amphiphilic architecture, PEGMA-g-DCCSNPs self-assembled into spherical core–shell nanoparticles in

aqueous media (Fig. 6A). The PEGMA-g-DCCSNPs encapsulated a model anticancer

FIG. 6. Morphologies of (B) PEGMA-g-DCCS NPs, (C) PXT-loaded PEGMA-g-DCCS NPs and (D) in vitro PTX releasing

feature from the PEGMA-g-DCCS NPs in the PBS solution at pH 7.4 and 37C after 10 days.

drug, i.e., paclitaxel (PTX) of around 30% (Fig. 6B) and slow released around 20-30% in PBS (pH 7.4) at

37C over a period of 30 days. The PEGMA-g-DCCSNPs evidently showed its efficiencies in

A B

302 ± 15 nm

281 ± 19 nm

200 nm

79 ± 12 nm

A B C

PEGMA-g-DCCS

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nanoparticulate formation, drug encapsulation and controlled release; however, the particles were

agglomerated within an hour and rarely soluble in water even containing the grafted PEGMA shells.

3.2 The next generation of amphiphilic core-shell PEGMA-g-DCWSCS-NPs

Because of limitation in water-solubility of the PEGMA-g-DCCS-NPs obtained from the first

generation, the next generation of the amphiphilic core-shell nanoparticles has been synthesized using

WSCS-NPs as main polymers instead of CS. Similarly, the DC core was fabricated by chemical

conjugation onto WSCS-NPs, whereas the PEGMA shell was eventually decorated onto DCWSCS-NPs

using radiation-induced grafting. The final step of grafting would be very mind process and suitable for

clinical purpose because it was carried out in pure water system without any chemical initiators. The

obtained PEGMA-g-WSCS-NPs exhibited spherical shape and better water-solubility (Fig. 7). The particle

sizes were around 80-100 nm.

According to the chemical structure of these core-shell PEGMA-g-WSCS NPs nanoparticle, it is

expected that they would provide not only the anticancer drug for chemotherapy but also the multiple

chelating groups owing to the ether group (-C-O-C-) on the PEGMA shell. Thus PEGMA shell is expected

to increase water-solubility, prolong the circulation time including chelate radionuclide ions, such as Tc-

99m or Ga-67, for SPECT/CT imaging. For further improve the targeting efficiency, the monoclonal

antibody can be conjugated on the particle surface via the hydroxyl group (-OH) of PEGMA and the

carboxylic acid group (-COOH) of the antibody.

FIG. 7. Possible functionalities of the PEGMA-g-DCWSCS NPs for both radiopharmaceutical diagnosis and chemotherapy.

For material development, the parameters affecting particle size and solubility including drug

encapsulation and radionuclide entrapment will be carefully studied and characterized. We will extend our

study on the ability of PEGMA-g-DCWSCS-NPs for the possible use as nanosized delivery for

radiopharmaceuticals in term of localization and uptake and in vitro biocompatibility including imaging if

applicable.

4. POLYMERIC NANOGEL/NANOPARTICLE CONTAINING MULTICHELATING GROUPS FOR

RADIOPHAMACEUTICAL ENTRAPMENT

To improve the chelating efficiency of the nanocarrier, two models of polymeric nanogel or

nanoparticle containing poly-chelating groups are proposed as seen in Fig. 8. Poly (glutamic acid) (PGA) is

poly amino acid present in nature. The amide linkages in the poly amino acids involve side chain function,

such gamma-carboxylic (Fig. 8A), whereas amide bonds in protein or polypeptide (i.e., SF) (Fig. 8B) are

only from between alpha-amino and alpha carboxylic groups (alpha-amide linkage). PGA is an anionic

biopolymer containing –COOH groups which is able to chelate metal ions. Like CS and SF, PGA is

biodegradable, edible and non-toxic towards human and environment, therefore it has been suggested to be

a good candidate for sustained release materials, drug carrier, highly water absorbable hydrogel and heavy

metal absorbers [xxxvi].

O

H

CH3

H

C O

CH3

OH

OH

H H

OO

OHNH

2

OO

OH

OH

NHCOCH3

O

O

C

CH2

C

CH2

O

H

O

CH2

CH3

m

400

FIG. 8. Molecular design of (A) PEGMA-grafted-PGA and (B) PAA-grafted-SF the polychelating nanogel/nanoparticles.

In the present work, the new molecular designs of crosslinked PEGMA-grafted-PGA nanogel (Fig.

8A) and the polyacrylic acid (PAA)-grafted-SF nanoparticle are proposed as polymeric nanocarrier

containing multi-chelating groups for superior radiopharmaceutical entrapment. Irradiation procedure will

be focused for the synthesis of these two structural nanocarriers. Synthesis, characterization and

performance of these new synthesis and novel materials for radiopharmaceutical delivery will be carefully

demonstrated as previous described.

5. CONCLUSIONS

Three main different types of nanostructural polymers as nanosized delivery system for

radiopharmaceuticals are presented. For the first type, AuNPs produced from WSCS-NPs and SFCS-NPs

including radiolysis method would be a good candidate for the creation of radioactive 198

AuNPs for

imaging and therapy. For the second type, the next generation of the water-soluble amphiphilic core-shell

PEGMA-g-DCWSCS-NPs not only provide drug loading core but also radionuclide chelating shell. Thus it

would be serve as a good choice for chemotherapy accompanying with radio imaging if applicable. For the

last type, biopolymeric nanogel or nanoparticle containing multi-chelating groups will be synthesized and

characterized. The localization and uptake, in vitro biocompatibility and the radiopharmaceutical diagnosis

and/or therapeutic efficiencies will be demonstrated and cooperated in this CRP.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge IAEA’s Coordinated Research Project (CRP) in Nanosized

delivery system for radiopharmaceuticals (No.18316), International Atomic Energy Agency (IAEA)

for supports. Appreciation is expressed to Office of Atoms for Peace (OAP) and Thailand Institution

of Nuclear Technology (TINT), Thailand for providing irradiation facilities.

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