Nanotechnology Advances in Targeted Drug Delivery Systems

Nanotechnology Advances in Targeted Drug Delivery Systems Professor Costas Kiparissides Department of Chemical Engineering, Aristotle University of Thessaloniki & Center for Research and Technology Hellas (CERTH)

INNOVATION, RECHERCHE ET ENTREPRENARIAT ENERGIE, BIOTECHNOLOGIE, ENVIRONMENT, SANTE October 9, Thessaloniki, Greece CPERI/AUT

Outline ¾ Nanomedicine ¾ Controlled Drug Delivery Systems ¾ Development of Novel Nanocarriers ¾ Respiratory Delivery ¾ Future Challenges

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Nanomedicine The term Nanomedicine refers to the application of nanotechnology to diagnosis and treatment of diseases. 9 It deals with the interactions of nanomaterials (surfaces, particles, etc.) or analytical nanodevices with “living” human material (cells, tissue, body fluids).

9 It is an extremely large field ranging from in vivo and in vitro diagnostics to therapy including targeted delivery and regenerative medicine.

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Drug Delivery Systems The concept of “Clever” drug targeting system includes the coordinating behavior of three components: the targeting moiety, the carrier and the therapeutic drug.

• • •

The first one recognizes and binds the target. The second one carries the drug The third one provides a therapeutic action to the specific site

Targeting moieties:

• • • •

antibodies oligonucleotides

Antibodies Proteins Lipoproteins Hormones

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• Charged molecules • Polysaccharides • Low-molecular-weight ligands

receptors

carbohydrates

peptides

Interactions Between Biological Systems and Nanostructures The potential of targeted delivery will only be realized with a much better understanding of how such structures interact with the body and its components – in vitro and in vivo.

9 Interaction of nanostructures with plasma proteins and relation between protein adsorption and removal of nanostructures from the circulation by the reticulo-endothelial system.

9 Adsorption of nanostructures to cells (in relation

fuse absorption

to the surface chemical characteristics, size and shape of the nanostructures).

9 Uptake and recycling, trans-endocytosis and endosomal escape of nanostructures.

9 Safety evaluation: In vitro/in vivo cytotoxicity, haemocompatibility, genotoxicity testing.

immunogenicity

and

9 In vivo carrier biodistribution and degradation. CPERI/AUT

phagocytosis

endocytosis

Nanocarriers as DDS ¾ The potential of nanocarriers as Drug Delivery Systems 9 Exhibit higher intracellular uptake 9 Can penetrate the submucosal layers while the microcarriers are predominantly localized on the epithelial lining.

9 Can be administered into systemic circulation without the problems of particle aggregation or blockage of fine blood capillaries.

9 The development of targeted delivery is firmly built on extensive experience in pharmaco-chemistry, pharmacology, toxicology, and nowadays is being pursued as a multi- and interdisciplinary effort.

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Our Mission ¾ “NanoBioPharmaceutics”

aims at breakthrough advances in novel biopharmaceutics delivery systems for the treatment of diabetes, cancer, AIDS, Alzheimer’s disease, and other neurodegenerative diseases.

¾ Nanocarrier-based

protein/peptide (P/P) delivery systems for respiratory and oral delivery and blood brain barrier (BBB) crossing applications are developed within this project allowing a targeted and controlled release of the drugs.

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Polymer- P/P Drug Complexes ¾ Covalent attachment of P/P Drugs to polymer chains via specific linkers.

linker

targeting ligand

polymeric carrier

P/P

• P/P • Linker • Targeting ligand • Polymer carrier CPERI/AUT

: peptide / protein : enzymatically or pH sensitive : peptide / saccharide : Hydrophilic polymers, polyelectrolytes

PEGylated TNF–alpha (PEG-TNF) PEG-TNF for Cancer Therapy Cys-SH

protein Cys-analogs for sitespecific pegylation

n at ylatio he G E P p of t the ti e r t r im 3 x 10 kDa 3 x 20 kDa PEG y the b lation at ase o f t r im e r opp the o sit to th e tip e

+

•A

O

mPEG-(CH2)3-NHCO-(CH2)2 O

•B

protein

O

Cys-S N O

Goals: 9 Prolonged half-life (30 min Æ 5 – 10 hrs) 9 Reduced toxicity 9 Better protection to degradation 9 Improved antitumor activity

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N

(CH2)2-CONH-(CH2)3-mPEG

Dendritic Polymers ¾ Multifunctional dendrimers and hyperbranched polymers as DDS. 9 Cell specificity via attachment of targeting ligands. 9 Decreased toxicity, biocompatibility, stability, and protection in the biological milieu via functionalization with PEG.

FITC-labeled PEGylated biodegradable hyperbranched polyester as a carrier for ADNF peptide BH40-PEG-FITC OH O

O

HO O

O

O OH

O

O

O

O

O

O

O O

OH

O

~

O

O

O O

O

O

O

O

O

O

O

O

~

OH O

O

HO

O

O

O

18

C

* 26

O

Confocal microscopy on A549 cells revealed preferential uptake of BH40-PEG in cells nuclei

TBTL HO

18 H N O C S

24h, rt

OH 26

O

O

O O

OH

OH

PEGylated BOLTORN H40 H40 PEGylated BOLTORN

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*

OH

O

N

O

18

OH O

O

OH

O

O O

O

O

O

O

OH

S

O

OH

O O

* OH

OH 25

Block Copolymer Micelles Poly(glycidol)-block-poly(lactide) NPs loaded with ovalbumin (OVA) and diphosphoryl lipid A (DPLA)

9 9 9 9 9

Mean size: ~30nm Zeta potential: -19.1 ± 16.8 mV OVA loading: up to 10%wt DPLA loading: up to 5%wt Labelling:1,1’–dioctadecyl-3,3,3’3’, tetramethylindocarbocyanine perchlorate (Dil)

HO O

O O

O x

H y

9 NPs are stable following 7 days incubation in water Dil

N

ClO4

16

N 16

Caco-2 cells treated with NPs labelled with Dil CPERI/AUT

Nanogels ¾ Three-dimensional, hydrophilic, stimuli-responsive polymeric networks: exhibit dramatic changes in network structure or swelling behavior in response to various external stimuli.

9 Thermosensitive: NIPAAM-

9

Aam, NIPAAM-DMAM, DEAM-DMAM pH sensitive: 2-hydroxyethyl methacrylate, acrylic acid Adhesion SH

H2N

Cysteamine hidrocloride

Surface modification

Fluorescence H2N HO3S

O

O

C NH(CH2)5NH2 CH3 H2 O

Alexa fluor 350 cadaverine

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Thiomer Nanogels ¾ Biodegradable nanogels by crossliniking thiol functionalized starPEG or poly(glycidols) in the inverse miniemulsion via oxidation or Michael addition with diacrylates.

9 Synthesis of hydrophilic oligomers via radical polymerization with acrylosuccinimide.

cysteamine-modified

starPEG nanogels

N-

9 Crosslinking of hydrophilic polymers possessing hydroxyl groups with disulfide crosslinker.

Poly(glicydols) nanogels

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Chitosan ¾ Synthesis of chitosan-6 mercaptonicotinic acid (CS-6-MNA) via carbodiimide mediated reaction.

a

¾ Preparation of NPs with CS-6-MNA and unmodified CS by ionic gelation.

Structure of CS-6-MNA Mucoadhesion (NP1, NP2, NP3: 30, 55, 255 μmol thiols/g polymer)

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9 Size: 250nm 9 Zeta potential: 10-20mV 9 Very high mucoadhesion (> 70 fold improvement over non thiolated polymer) 9 Very strong and rapid in situ gelling properties

Particle stability

Nasal Vaccination ¾ Vaccination is the most effective way of fighting infectious diseases like HIV, malaria, influenza, etc.

¾ Among the potential needle-free routes, nasal vaccination attractive.

is

particularly

¾ The nose is easily accessible (i.e., administration via drops or sprays) and the nasal cavity is equipped with a high density of dendritic cells (DC) that can mediate strong systemic and local immune responses against pathogens that invade the human body through the respiratory tract.

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Sagittal section of human nasal cavity

A Roadmap to Successful Nasal Vaccine Delivery

1. Prolonging the nasal residence time (mucoadhesion).

2. M-cell targeting (antigen uptake by M-cell transport).

3. Delivery

to and subsequent activation/maturation of dendritic cells (DC).

4. Induction

of cytotoxic Tlymphocyte immune responses.

Three major elements should constitute the nanostructure-based vaccines: the carrier, the antigen and the adjuvant (e.g., MPLA, CpG, etc.) CPERI/AUT

Particle Synthesis Synthesis of PLGA NPs by the Double Emulsion Method Aq. sln of antigen

Polymer dissolved in volatile solvent

Sonication

W/O EMULSION

PVA aq. sln

Sonication

W/O/W EMULSION Solvent Evaporation

POLYMER PRECIPITATION / NANOPARTICLE FORMATION Washing

RECOVERY OF NANOPARTICLES

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PLGA: Resomer RG752H Antigen: Profos AG EndoGradeTM Ovalbumin,