Report
1st Research Coordination Meeting
on
“Nanosized Delivery of Radiopharmaceuticals”
IAEA Headquarters, Vienna, Austria
July 07-11, 2014
2
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
4
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.
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[3] CHEN, C.-H. AND LEE, W.-C. Preparation of methyl methacrylate and glycidyl methacrylate
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[5] KADLUBOWSKI, S., GROBELNY, J., OLEJNICZAK, W., CICHOMSKI M. AND ULANSKI, P.,
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Linking of Linear Polymer Chains in Additive-Free Aqueous Solution, Macromol. 36 (2003) 2484-
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[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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binding on the formation and reactions of tryptophan and tyrosine radicals in peptides and proteins. Int. J.
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Furusawa, K., Terao, K., Nagasawa, N., Yoshii, F., Kubota, K., Dobashi, T., 2004. Nanometer-sized gelatin
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papain. Ann. N. Y. Acad. Sci., 54, 161–207.
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resolution. J. Mol. Biol. 179, 233–256.
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Radiat. Biol. 46, 715- 724.
Lal, M., 1994. Radiation induced oxidation of sulphydryl molecules in aqueous solutions. A comprehensive
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Food Biochem. 11, 309–327.
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Curr. Opin. Chem. Bio. 11(2), 220–225.
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Sheldon, R.A., 2007. Enzyme Immobilization: The Quest for Optimum Performance. Adv. Synth. Catal.
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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
1. Abd El-Rehim HA, Swilem AE, El-Shazly, A, Hegazy EA, Hamed AA., Use of poly(acrylic acid)-based
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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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: [email protected]
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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(2011). A Pin1/mutant p53 axis promotes aggressiveness in breast cancer. CANCER CELL, vol. 20, p. 79-
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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,
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BOBISSE S, RONDINA MB, GUZZARDO E, PARENTI AR, ROSATO A, BICCIATO S, BALMAIN A,
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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.
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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
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Hyaluronan with 2′ Paclitaxel-4-Bromobutyrate: Synthesis and Antitumor Properties. In: Hyaluronan 2003.
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20. BANZATO A, RONDINA M, MELENDEZ-ALAFORT L, ZANGONI E, NADALI A, RENIER D,
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hyaluronan bioconjugate. NUCLEAR MEDICINE AND BIOLOGY, vol. 36, p. 525-533.
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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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[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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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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Strategies of Peptides. Q J Nucl Med 43, 155-158 (1999).
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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
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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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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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