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!