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I NANOMATERIALI AL SERVIZIO DELLA SALUTE UMANA
Agnese Molinari
Dipartimento di Tecnologie e salute
Salute ed ambiente in Italia Istituto Superiore di Sanità
5 - 6 dicembre 2011
Attività svolte nel Dipartimento di Tecnologie e salute
Nanomateriali al servizio della salute umana: “needs and challenges”
- Physico-chemical characteristic of the drugs (low water solubility: paclitaxel 0,0015 mg/ml; dexamethasone 0,1 mg/ml);
- Biodistribution (1/10,000-1/100,000 drug molecules reach target site, high doses needed);
- Narrow therapeutic window.
LIMITATIONS HINDERING DRUG CLINICAL TRANSLATIONS AND SUCCESS
THUS THERE IS THE NEED OF:
- Increasing drug stability;
- Increasing drug solubility;
- Targeting the drug to the site of action.
Nanotechnology allows creation of platforms with:
• superior drug carrier capabilities • selective responsiveness to the environment • unique contrast enhancement profiles • improved accumulation at the disease site.
Imaging Diagnosis
Therapy
! Nano-sized drug crystals ! Cross-linked nanogels ! Carbon nanoparticles ! Gold nanoparticles ! Nanoshell ! Albumin nanoparticles ! Polymer. drug conjugates ! Liposomes ! Polymer micelles ! Nanoshelles ! Denrimers ! Immunopolymers, immunotoxins
! Superparamagnetic agents ! Metal nanoparticles ! Synthetic carbon-based nanoparticles ! Others: (Liposomes, Dendrimers, ! Polymer conjugates, bacteriophage, etc.)
! Nanoporous silica chips ! Smart hydrogel particles ! Nano-biosensors: ! Nanowires ! Micro- and nano-cantilever systems
Regenerative medicine
! Nanofibrous scaffolds ! Amphiphilic peptides ! Nanoparticles (spheres, capsules, liposomes, micelles, densrimers) ! Biodegradable polymers (PLA, PEG, PGA)
Nano-scaled materials and devices
1-100 nm
IMAGING Nanoparticle-based contrast agents. I
Magnetic Resonance Imaging
Superparamagnetic agents
Metal nanoparticles
Synthetic carbon-based nanoparticles
Others:
liposomes, dendrimers, polymer conjugates
Positron emission tomography Single-photon emission computed tomography
Advantages:
! high contrast ! tunable size and shape ! surface properties ! multiple functionalities ! long circulation times
Composition Contrast source Target Indication
Poly-L-lysine coated IO IO Mammalian cells Tracking of transplanted cells
Antibody-targeted IO IO Her-2 Breast cancer
Peptide/protein-targeted SPIO IO Clotted plasma proteins, MMP-2 Various tumors
Radiolabeled antibody-targeted SPIO 111In, IO, IRDye 800CW Membrane glycoproteins, EGFR-2 Various cancer
Aptamer-doxorubicin SPIO conjugate IO, doxorubicin PSMA Prostate cancer
Peptide-targeted USPIO IO αvβ3, E-selectin Various tumors, inflammation
Antibody-targeted USPIO IO CD20 antigen, E-selectin Non-Hodkin’s lynphoma, inflammation
Baculovirus-targeted USPIO IO, LacZ Mammalian cells Gene therapy
Micelle-encapsulated MnSPIO IO Macrophages Liver lesions
Antibody-targeted MnMEIO IO Her-2 Breast cancer
Radiolabeled passive-targeted MnMEIO 124I, IO Lymph nodes Lymph node mapping
Fluorescent CLIO IO, Cy5.5 Macrophages Macrophage infiltration
Radiolabeled fluorescent CLIO 64Cu, IO, Cy5.5 Macrophages Macrophage infiltration
Fluorescent peptide-targeted CLIO IO, Cy5.5/FITC Proteases, bombesin receptor, plectin, uMUC-1, hepsin, αvβ3, H-2Kd, VCAM-1, phosphatidylserine
Various tumors, autoreactive T-cells, inflammation, apoptosis
Fluorescent siRNA-CLIO conjugate IO, Cy5.5 Birc5 gene Various cancer
IMAGING Nanoparticle-based contrast agents. III (1) Preclinical development Superparamagnetic metal nanoparticles
Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89
Composition Contrast source Target Indication Polymer-coated gold nanoshells Au Tumor accumulation Solid tumors
Fluorescent passive-targeted gold Au, Hilyte 647 Tumor accumulation Solid Tumors
Fluorescent antibody-targeted gold Au, ICG EGFR Epithelial cancer
Antibody-targeted QD QD Her-2, PSMA, VEGFR Various tumors
Growth factor-targeted QD QD EGFR Epithelial cancers
Radiolabeled peptide-targeted QD 64Cu, QD Αvβ3, VEGFR Various cancer
Protein-targeted paramagnetic QD Gd, QD Phosphatidylserine Apoptosis
(2) Preclinical development other metal nanoparticles
IMAGING Nanoparticle-based contrast agents. IV
Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89
(3) Preclinical development liposome-based nanoparticles
Composition Contrast source Target Indication Radiolabeled peptide-targeted liposomes 18F Macrophages Inflammation
Antibody-targeted paramagnetic liposomes Gd, Texas red ICAM-1 Inflammation and neuroinflammatory
disease
Radiolabeled, dye-filled liposomes 99mTc, blue dye Lymph nodes Lymph node identification,
inflammation
Fluorescent protein-targeted paramagnetic liposomes
Gd, AF680 Transferrin receptor, E-selectin
Various cancers
Electron dense liposomes Gd, AF680 - Blood pooling
(4) Preclinical development synthetic carbon-based nanoparticles
Composition Contrast source Target Indication Peptide-targeted SWNT SWNT Integrin αvβ3 Various cancers
Gd-filled fullerenes, fullerenols, and SWNT
Gd Macrophages Macrophage infiltration, blood pooling
Radiolabeled antibody-targeted SWNT 111In, SWNT CD20 Lymphoma
Radiolabeled peptide-targeted SWNT 64Cu, 111In, SWNT Integrin αvβ3, EGFR Various cancers
Radiolabeled MWNT 99mTc, 125I - TBD
Nanoparticle-based contrast agents in preclinical development (4): other platforms
IMAGING - Nanoparticle-based contrast agents. V
Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89
Composition Contrast source Target Indication Bismuth sulfide polyvinylpyrrolidone nanoparticles
Bi - Blood pooling
Radiolabeled hormone-targeted bacteriophage
111In MC-1 receptor Melanoma
Ioxilan carbonate particles Iodine Macrophages Liver lesions
Antibody-targeted paramagnetic perfluorocarbon emulsions
Gd, 19F Fibrin, Integrin αvβ3, collagen III
Atheroslerosis
Radiolabeled amphiphillic block copolymers
64C Folate receptor Various cancer
Iodinated amphiphillic block copolymers Iodine Macrophages Lymph lesions
Fluorescent paramagnetic dendrimers Gd, Cy5.5 - Sentinal lymph node identification
IMAGING Nanoparticle-based contrast agents. II
Composition Trade name Company Indication Administration Destran coated SPION (ferumoxides)
Feridex I.V./Endorem Bayer Healthcare Pharmaceuticals, Inc.
Detection and evaluation of liver lesions
i.v.
Carboxydextran-coated SPION (ferucarbotran)
Resovist/Cliavist (EU, AUS, JPN only)
Bayer Schering Pharma AG
Detection and evaluation of liver lesions
i.v.
Silicon-coated SPION (ferumoxsil)
GastroMARK Covidien, Ltd. Bowel marking Oral
Composition Trade name Company Indication Administration
Dextran-coated USPIO (ferumoxtran-10)
Combidex/Sinerem AMAG Pharmaceuticals, Inc. Differentiation of cancerous from noncancerous lymph nodes
i.v.
Carboxy dextran-coated USPIO (ferucarbotran)
Supravist Bayer Schering Pharm AG Detection of blood pooling using MRA
i.v.
Polyglucose sorbitol carboxymethyl ether-coated SPIO(ferumoxytol)
- AMAG Pharmaceuticals, Inc. Nervous system disease, brain neoplasms, peripheral artery disease
i.v.
Citrate-coated very small SPIO
VSOP-C184 Charité-Universitätsmedizin Berlin
Detection of blood pooling using MRA
i.v.
Radiolabeled-Her-2-Affibody®
ABY-025 Affibody Holding AB Breast cancer i.v.
Clinically approved nanoparticle-based contrast agents
Nanoparticle-based contrast agents in clinical trials
Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89
Theranostic SPIONs
SuperParamagnetic Iron Oxide Nanoparticles
Surface Engineering of Iron Oxide Nanoparticles for Targeted Cancer Therapy"FORREST M. KIEVIT AND MIQIN ZHANG*"
Biological barriers. I
Biological barriers. II
ACCOUNTS OF CHEMICAL RESEARCH Vol. 44, No. 10 ’ 2011 ’
Enhanced permeability and retention effect EPR
ACCOUNTS OF CHEMICAL RESEARCH Vol. 44, No. 10 ’ 2011 ’
Nano-based Injectable drug-delivery devices
First generation: passive mechanisms (e.g. liposomes - EPR mechanism)
Second generation: active mechanisms (e.g. m-Ab conjugated liposomes - magnetic liposomes)
Third generation: active mechanisms (multistage delivery system)
Sakamoto et al. 2010
Functional taxonomy
Multistage Nanovectors: From Concept to Novel Imaging Contrast Agents and Therapeutics Vol. 44, No. 10 ’ 2011 ’ 979–989 ’ ACCOUNTS OF CHEMICAL RESEARCH
MULTISTAGE NANOVECTORS (MSVs). I
Multistage Nanovectors: From Concept to Novel Imaging Contrast Agents and Therapeutics Vol. 44, No. 10 ’ 2011 ’ 979–989 ’ ACCOUNTS OF CHEMICAL RESEARCH
MULTISTAGE NANOVECTORS (MSVs). II
Multistage Nanovectors: From Concept to Novel Imaging Contrast Agents and Therapeutics Vol. 44, No. 10 ’ 2011 ’ 979–989 ’ ACCOUNTS OF CHEMICAL RESEARCH
Sistemi di drug delivery attualmente approvati
Manomaterial Trade name Company Indication Current Status
Pegylated Liposomes
Caelyx (doxorubicin) anticancer
Janssen Pharmaceutica Metastatic breast cancer Ovarian cancer Multiple myeloma AIDS-related Kaposis
Commercialized"
Pegylated Liposomes Mepact (mifamurtide) immunomodulator
Mitsubishi Pharmaceutical, Japan
High grade non metastatic osteosarcoma
Commercialized"
Pegylated Liposomes Myocet (doxorubicin) anticancer
Cephalon Europe Metastatic breast cancer
Commercialized"
Nano-scale particles of the active substance
Abraxane (paclitaxel)
Celgene Europe Limited Metastatic breast cancer
Commercialized"
Nano-scale particles of the active substance
Emend (aprepitant) Anti-emetic
Merck Sharp & Dome Ltd Cancer Commercialized"
Nano-scale particles of the active substance
Rapamune (sirolimus)
Wyeth Lederle Rejection of transplanted kidney
Commercialized"
Nanomaterials for Drug Delivery EMEA approved
FROM THE BENCH TO THE BED
Preclinical studies: • in vitro studies • in vivo studies
PHASE 1: Healthy subjects: Effects on body functions, dose definition, pharmacokinetics
PHASE 2: Selected patients Effect on disease:
Safety efficacy dose pharmacokinetics
PHASE 2: Patient groups: Comparison with shandon therapy
PHASE 3: Approval from FDA or EMEA
General use Long-term benefit-risk evaluation
Design, characterization, production
In vitro testing
• Cytoxicity
• Haematocompatibility
• Drug release
• Intracellular fate
• Therapeutic efficacy
In vivo testing
• Body distribution
• Organ specific toxicity
• Immunogenicity
• Pharmacological activity
NEEDS FOR SAFETY AND EFFICACY DEFINITION. I
GENERALLY, IMPROVEMENT OF NON-CLINICAL METHODOLOGY TO AID DEFINITION OF LIKELY CLINICAL EFFICACY AND TOXICITY IS NEEDED
Models should be developed to more closely correlates with the appropriate pathophysiology scenario present in the specifc, target clinical situation. Important considerations must be:
• disease localization • disease progression • likely access to target tissues and cells • impact of angiogenesis (vascular permeability) • immune status
(Gaspar and Duncan, Adv Drug Del Rev. 61: 1220-1231 - 2009)
NEEDS FOR SAFETY AND EFFICACY DEFINITION. II
SELECTION OF OPTIMAL PATIENT POPULATION TO ENTER CLINICAL TRIALS
There is a need to identify those patients who are most likely to benefit from a novel therapy and to select the optimal patient population to enter clinical trials.
INDIVIDUALIZED NANO-THERAPY
Patient-specific molecular profiling allow the individuation of specific biomarkers useful to identify: • target specific site of disease • follow up pharmacological response • identify potential adverse reactions.
(Gaspar and Duncan, Adv Drug Del Rev. 61: 1220-1231 - 2009)
CONGRESS TOPIC DEVELOPING A NANOPARTICLE
!Design, !Modelling, !Characterization
FROM THE ADMINISTRATION TO THE TARGET SITE !Administration routes !Biological barrier !Immunological respons
CLINICAL TRANSLATION OF NANODRUGS !Laboratory optimization !Pre-clinical safety evaluation !I/II/III phase clinical studies !Clinical successes !Industry reports
Chairperson: Dr.ssa Giovanna Mancni CNR, Roma
Dr.ssa Agnese Molinari Istituto Superiore di Sanità
PROSSIMO EVENTO"
Department of Technology and Health
UNITS involved in Nanomaterials and Human Health Research
Ultrastructural Infectious Pathology
Ultrastructural Methods for Innovative Anticancer Therapies
Biomaterials and Contaminants
Risk Assessment Studies
Effect of pre- and post-exposure to nanoparticles on viral and bacterial infections
CTR! ZnO 5 µg/cm2!
ZnO 10 µg/cm2! TiO2 5 µg/cm2!
Nanoparticles toxicity on cultured cells
Relation between nanoparticles cytotoxicity and their physico-chemical characteristics
Department of Technology and Health
Biomedical applications
Nanoparticles-plasma membrane interaction: uptake and transport
Employment of Nanoparticles as selective drug carriers at the site of disease
Possible use of Nanoparticles as antimicrobial agents
LIPOSOME!
PROTOPLASMIC FRACTURE FACE!
CYTOPLASM!
LIPOSOMES!
EXTRACELLULAR SPACE!
Studies on implantable devices made or covered with nanomaterials: characterization of biomechanical alterations and potential surface performance
Department of Technology and Health
Impiego di nanomateriali per terapie innovative antitumorali
Dipartimento di Tecnologie e Salute
INNOVATIVE ANTICANCER THERAPIES
Collaborations
Tumor markers Natural products
Nanotechnology
2 µm!
Scanning electron microscopy
Laser scanning confocal microscopy
BAF Device!
Transmission electron microscopy
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
Impiego di liposomi cationici per la terapia fotodinamica del glioblastoma
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
MECCANISMO DI AZIONE DELLA PDT SUI TUMORI
INFIAMMAZIONE
NECROSI E APOPTOSI
CHIUSURA DEL MICROCIRCOLO
F
F *
STATO FONDAMENTALE DI SINGOLETTO
*
STATO ECCITATO
SINGOLETTO
STATO ECCITATO
TRIPLETTO
F
1O2
3O2
*
REAZIONE DI TIPO II
SUBSTRATO Radicali liberi
STATO FONDAMENTALE
DI TRIPLETTO
STATO ECCITATO
DI SINGOLETTO
REAZIONE DI TIPO I
N H
N
N
N H
OH
OH
OH
OH
H H
H H
m-THPC, Foscan® (650-700 nm)
Meso-tetraidrossifenilclorina
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
100 nm
L’EFFICACIA DELLA PTD PUO’ ESSERE MIGLIORATA USANDO DELLE FORMULAZIONI LIPOSOMICHE :
" Migliorano l’accumulo del fotosensibilizzante nei tumori
" Limitano la formazione di aggregati in soluzione acquosa dei fotosensibilizzanti idrofobici, aumentando la popolazione fotoattiva
" Possono influenzare in modo positivo la farmacocinetica ed il destino subcellulare del fotosensibilizzante
I LIPOSOMI SONO VESCICOLE CHIUSE COSTITUITE DA UNO O PIU’ DOPPI STRATI DI FOSFOLIPIDI
SEPARATI DA COMPARTIMENTI ACQUOSI.
LA MICROGRAFIA ELETTRONICA RAPPRESENTA LIPOSOMI UNILAMELLARI OOSSERVATI MEDIANTE LA TECNICA DEL FREEZE-FRACTURING. Dipartimento Tecnologie e salute - Istituto Superiore di Sanità - Roma
Impiego di liposomi cationici per la terapia fotodinamica del glioblastoma Dipartimento di Tecnologie e Salute
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
DMPC/G1 m-THPC/DMPC m-THPC/DMPC/G1
Formulazioni!(DMPC + G1 =12,5 mM)!
DMPC!(%mol)!
G1!(%mol)!
m-THPC!
DMPC/G1!m-THPC/DMPC!m-THPC/DMPC/G1(8:2)!m-THPC/DMPC/G1(7:3)!m-THPC/DMPC/G1(6:4)!
60!100!80!70!60!
40!---!20!30!40!
---!50 µM!50 µM!50 µM!50 µM!
m-THPC/DMPC Foscan m-THPC/DMPC/G1 7:3
DMPC/G1! 4.85 ± 0.5 5.61 ± 0.6 4.79 ± 0.41
m-THPC/DMPC! 4.04 ± 0.66 4.01 ± 0.25 6.54 ± 0.67
Foscan! 3.21 ± 0.95 5.79 ± 0.39 14.29 ± 3.25
m-THPC/DMPC/G1 8:2! 35.66 ± 3.9 42.27 ± 4.53 76.98 ± 6.38
m-THPC/DMPC/G1 7:3! 42.44 ± 3.87 58.24 ± 3.11 80.82 ± 3.47
m-THPC/DMPC/G1 6:4! 73.32 ± 4.83 90.13 ± 3.91! 105.53 ± 5.56
30 min! 1 h! 4 h!
DMPC/G1 6:4
m-THPC/DMPC
Foscan
m-THPC/DMPC/G1 8:2
m-THPC/DMPC/G1 7:3
m-THPC/DMPC/G1 6:4
120
0
20
40
60
80
100
30 min!1 h!
4 h!
Can
ale
med
io d
i flu
ores
cenz
a
ACCUMULO DI m-THPC CELLULE DI GLIOBLASTOMA DI RATTO (C6): confronto con il FOSCAN®
0!
10!
20!
30!
40!
50!
60!
70!
80!
90!100!
LN229+drugs+laser! 100! 100 ± 0.0 73.66 ± 7.2 43.33 ± 9.6 3.66 ± 1.18 0.02 ± 0.02 0.34 ± 0.5
LN229+drugs! 100! 100 ± 10 100 ± 20 100 ± 1.21 100 ± 12 100 ± 3 94.0 ± 4.6
CTR! DMPC/G1!FOSCAN! m-THPC/DMPC! 8:2! 7:3! 6:4!
CTR
m-THPC/DMPC/G1 8:2 m-THPC/DMPC/G1 7:3
Foscan m-THPC/DMPC
m-THPC/DMPC/G1 6:4
DMPC/G1 6:4 LN229
CITOTOSSICITA’ confronto con il FOSCAN®
Test di clonogenicità
Frazi
on
e d
i so
pra
vviv
en
za
%
Liposomi+ LASER INTERSTIZIALE
FOSCAN + LASER
INTERSTIZIALE
LIPOSOMI/m-THPC+ LASER
INTERSTIZIALE
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
Lysozyme Microbubbles for magnetic resonance imaging
and ultrasound triggered drug delivery "
Ferritin Coating
In vitro cellular uptake studies in breast cancer cells (4 hrs)
In vitro cytotoxicity test (MTT)
SKB3
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
Partecipanti all’attività Nanomateriali
Dipartimento di Tecnologie e salute
Ing. Velio Macellari
Rossella Bedini Giuseppina Bozzuto Annarica Calcabrini Marisa Colone Maria Condello Giuseppe Formisano Magda Marchetti Stefania Meschini Agnese Molinari Annarita Stringaro Fabiana Superti Laura Toccacieli
Collaboratori esterni
Istituto di Neurochirurgia, Università Cattolica,
Roma Giulio Maira
Annunziato Mangiola Stefano Mannino
Istituto di Metodologie Chimiche, CNR, Roma
Cecilia Bombelli Paola Luciani
Giovanna Mancini!