I NANOMATERIALI AL SERVIZIO DELLA SALUTE UMANA Salute ed ambiente in Italia

Salute ed ambiente in Italia
Istituto Superiore di Sanità
5 - 6 dicembre 2011
I NANOMATERIALI AL SERVIZIO DELLA SALUTE UMANA
Agnese Molinari
Dipartimento di Tecnologie e salute
Nanomateriali al servizio della salute umana:
“needs and challenges”
Attività svolte nel Dipartimento di Tecnologie e salute
LIMITATIONS HINDERING DRUG CLINICAL TRANSLATIONS AND
SUCCESS
-  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.
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.
Nano-scaled materials and devices
Imaging
!  Nanoporous silica chips
!  Smart hydrogel particles
Diagnosis
!  Nano-biosensors:
!  Nanowires
!  Micro- and nano-cantilever systems
!  Superparamagnetic agents
!  Metal nanoparticles
!  Synthetic carbon-based nanoparticles
!  Others: (Liposomes, Dendrimers,
!  Polymer conjugates, bacteriophage, etc.)
Therapy
1-100 nm
Regenerative medicine
!  Nanofibrous scaffolds
!  Amphiphilic peptides
!  Nanoparticles (spheres, capsules, liposomes, micelles,
densrimers)
!  Biodegradable polymers (PLA, PEG, PGA)
!  Nano-sized drug crystals
!  Cross-linked nanogels
!  Carbon nanoparticles
!  Gold nanoparticles
!  Nanoshell
!  Albumin nanoparticles
!  Polymer. drug conjugates
!  Liposomes
!  Polymer micelles
!  Nanoshelles
!  Denrimers
!  Immunopolymers, immunotoxins
IMAGING
Nanoparticle-based contrast agents. I
Superparamagnetic agents
Advantages:
Metal nanoparticles
!  high contrast
!  tunable size and shape
!  surface properties
!  multiple functionalities
!  long circulation times
Synthetic carbon-based nanoparticles
Others:
liposomes, dendrimers, polymer conjugates
Magnetic Resonance Imaging
Positron emission tomography
Single-photon emission computed
tomography
IMAGING Nanoparticle-based contrast agents. III
(1) Preclinical development
Superparamagnetic metal nanoparticles
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,
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,
Lymph nodes
Lymph node mapping
Fluorescent CLIO
IO, Cy5.5
Macrophages
Macrophage infiltration
Radiolabeled fluorescent CLIO
64Cu,
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 Tcells, inflammation, apoptosis
Fluorescent siRNA-CLIO conjugate
IO, Cy5.5
Birc5 gene
Various cancer
IO, IRDye 800CW
IO
IO, Cy5.5
Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89
IMAGING Nanoparticle-based contrast agents. IV
other metal nanoparticles
(2) Preclinical development
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,
Αvβ3, VEGFR
Various cancer
Protein-targeted paramagnetic QD
Gd, QD
Phosphatidylserine
Apoptosis
QD
liposome-based nanoparticles
(3) Preclinical development
Composition
Contrast source
Target
Indication
Radiolabeled peptide-targeted liposomes
18F
Macrophages
Inflammation
Gd, Texas red
ICAM-1
Inflammation and neuroinflammatory
disease
99mTc,
Lymph nodes
Lymph node identification,
inflammation
Antibody-targeted paramagnetic liposomes
Radiolabeled, dye-filled liposomes
blue dye
Fluorescent protein-targeted paramagnetic
liposomes
Gd, AF680
Transferrin receptor, Eselectin
Various cancers
Electron dense liposomes
Gd, AF680
-
Blood pooling
Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89
IMAGING - Nanoparticle-based contrast agents. V
(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,
CD20
Lymphoma
Radiolabeled peptide-targeted SWNT
64Cu, 111In,
Integrin αvβ3, EGFR
Various cancers
Radiolabeled MWNT
99mTc, 125I
-
TBD
SWNT
SWNT
Nanoparticle-based contrast agents in preclinical development (4): other platforms
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
Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89
IMAGING Nanoparticle-based contrast agents. II
Clinically approved nanoparticle-based contrast agents
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
Nanoparticle-based contrast agents in clinical trials
Trade name
Company
Indication
Administration
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 ethercoated 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-2Affibody®
ABY-025
Affibody Holding AB
Breast cancer
i.v.
Composition
Dextran-coated USPIO
(ferumoxtran-10)
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
Functional taxonomy
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
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
Nanomaterials for Drug Delivery EMEA approved
Manomaterial
Trade name
Company
Indication
Current Status
Caelyx
Janssen Pharmaceutica
Metastatic breast
cancer
Commercialized"
(doxorubicin)
Pegylated Liposomes
Ovarian cancer
Multiple myeloma
anticancer
AIDS-related Kaposis
Mepact
Pegylated Liposomes
(mifamurtide)
Mitsubishi
Pharmaceutical, Japan
immunomodulator
Pegylated Liposomes
High grade non
metastatic
osteosarcoma
Commercialized"
Myocet
Cephalon Europe
Metastatic breast
Commercialized"
Sistemi
di
drug
delivery
attualmente
approvati
cancer
(doxorubicin)
anticancer
Nano-scale particles
of the active
substance
Abraxane
Nano-scale particles
of the active
substance
Emend
Nano-scale particles
of the active
substance
Rapamune
Celgene Europe Limited
Metastatic breast
cancer
Commercialized"
Merck Sharp & Dome Ltd
Cancer
Commercialized"
Wyeth Lederle
Rejection of
transplanted kidney
Commercialized"
(paclitaxel)
(aprepitant)
Anti-emetic
(sirolimus)
FROM THE BENCH TO THE BED
Design,
characterization,
production
PHASE 1:
Preclinical
studies:
Healthy subjects:
Effects on body
functions, dose
definition,
pharmacokinetics
•  in vitro studies
•  in vivo studies
In vitro testing
In vivo testing
• Cytoxicity
• Haematocompatibility
• Drug release
• Therapeutic efficacy
Approval from FDA or
EMEA
General use
Long-term benefit-risk
evaluation
• Organ specific toxicity
• Immunogenicity
• Intracellular fate
PHASE 3:
• Body distribution
PHASE 2:
Selected patients
• Pharmacological activityEffect on disease:
PHASE 2:
Patient groups:
Comparison with
shandon therapy
Safety efficacy dose
pharmacokinetics
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
Biomaterials and
Contaminants
Ultrastructural
Infectious Pathology
Ultrastructural Methods for Innovative
Anticancer Therapies
Risk Assessment Studies
Department of Technology and Health
CTR!
ZnO 10 µg/cm2!
ZnO 5 µg/cm2!
TiO2 5 µg/cm2!
Nanoparticles
cultured cells
toxicity
on
Relation between nanoparticles
cytotoxicity and their physicochemical characteristics
Effect of pre- and post-exposure
to nanoparticles on viral and
bacterial infections
Biomedical applications
Department of Technology and Health
Nanoparticles-plasma membrane
interaction: uptake and transport
EXTRACELLULAR SPACE!
LIPOSOME!
Employment of Nanoparticles as
selective drug carriers at the site
of disease
PROTOPLASMIC FRACTURE FACE!
LIPOSOMES!
CYTOPLASM!
Possible use of Nanoparticles as
antimicrobial agents
Studies on implantable devices
made
or
covered
with
nanomaterials: characterization of
biomechanical alterations and
potential surface performance
Impiego di nanomateriali per terapie innovative
antitumorali
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
Dipartimento di Tecnologie e Salute
Tumor markers
Natural
products
!
BAF Device
INNOVATIVE
ANTICANCER THERAPIES
Nanotechnology
Collaborations
Transmission
electron
microscopy
2
µ
m
!
Laser scanning
confocal microscopy
Scanning
electron
microscopy
Impiego di liposomi cationici per la terapia
fotodinamica del glioblastoma
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
MECCANISMO DI AZIONE DELLA PDT SUI TUMORI
REAZIONE DI TIPO I
OH
OH
REAZIONE DI TIPO II
*
F
STATO
ECCITATO
SINGOLETTO
F
H
H
N
H
N
N
H
Radicali liberi
SUBSTRATO
N
OH
HH
OH
Meso-tetraidrossifenilclorina
m-THPC, Foscan®
(650-700 nm)
*
STATO
FONDAMENTALE
DI TRIPLETTO
STATO
ECCITATO
TRIPLETTO
3O
2
NECROSI E APOPTOSI
STATO
FONDAMENTALE
DI SINGOLETTO
F
STATO
ECCITATO
DI SINGOLETTO
*
1O
2
CHIUSURA DEL
MICROCIRCOLO
INFIAMMAZIONE
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
Impiego di liposomi cationici per
la terapia fotodinamica del
glioblastoma
Dipartimento di Tecnologie e Salute
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
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
aumentando la popolazione fotoattiva
fotosensibilizzanti
idrofobici,
"  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
100 nm
MEDIANTE LA TECNICA DEL FREEZE-FRACTURING.
Dipartimento Tecnologie e salute - Istituto Superiore di Sanità - Roma
DMPC/G1
m-THPC/DMPC
m-THPC/DMPC/G1
Formulazioni!
DMPC!
G1!
(DMPC + G1 =12,5 mM)!
(%mol)!
(%mol)!
DMPC/G1!
60!
40!
---!
m-THPC/DMPC!
100!
---!
50 µM!
m-THPC/DMPC/G1(8:2)!
80!
20!
50 µM!
m-THPC/DMPC/G1(7:3)!
70!
30!
50 µM!
m-THPC/DMPC/G1(6:4)!
60!
40!
50 µM!
m-THPC!
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
ACCUMULO DI m-THPC CELLULE DI GLIOBLASTOMA DI RATTO (C6):
confronto con il FOSCAN®
120
Canale medio di fluorescenza
100
80
DMPC/G1 6:4
m-THPC/DMPC
Foscan
60
m-THPC/DMPC/G1 8:2
m-THPC/DMPC/G1 7:3
40
m-THPC/DMPC/G1 6:4
20
0
4 h!
1 h!
30 min!
30 min!
1 h!
4 h!
DMPC/G1!
4.85 ± 0.5
5.61 ± 0.6
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
76.98 ± 6.38
4.79 ± 0.41
m-THPC/DMPC/G1 8:2!
35.66 ± 3.9
42.27 ± 4.53
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
Foscan
m-THPC/DMPC
m-THPC/DMPC/G1 7:3
Liposomi+
LASER INTERSTIZIALE
CITOTOSSICITA’
confronto con il FOSCAN®
Test di clonogenicità
CTR
DMPC/G1 6:4
Foscan
m-THPC/DMPC
m-THPC/DMPC/G1 8:2
m-THPC/DMPC/G1 7:3
LN229
%
100!
90!
80!
m-THPC/DMPC/G1 6:4
70!
Frazione di
sopravvivenza
60!
50!
40!
FOSCAN +
LASER
INTERSTIZIALE
30!
20!
10!
0!
CTR!
DMPC/G1!FOSCAN! m-THPC/DMPC! 8:2!
7:3!
6:4!
LN229+drugs+laser! 100! 100 ± 0.0 73.66 ± 7.243.33 ± 9.63.66 ± 1.180.02 ± 0.020.34 ± 0.5
LN229+drugs!
100! 100 ± 10
100 ± 20 100 ± 1.21 100 ± 12
100 ± 3
94.0 ± 4.6
LIPOSOMI/m-THPC+
LASER
INTERSTIZIALE
Lysozyme Microbubbles for magnetic resonance imaging
and ultrasound triggered drug delivery
"
Reparto Metodi ultrastrutturali per terapie innovative antitumorali
Ferritin Coating
In vitro cytotoxicity test (MTT)
In vitro cellular
uptake studies in
breast cancer cells
(4 hrs)
SKB3
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!
`