Lezione 9 12 Maggio 2010 - Università degli Studi di Pisa

Lezione 9
12 Maggio 2010

Rilascio Controllato e Mirato di Farmaci
“Magic Bullet”
Paul Erlich (1906)
A Drug will be able to perform its therapeutic action if present
at the site of action for an adeguate period of time and in an
optimal concentration.
concentration

NANOMEDICINA
Applicazione medica/clinica delle nanotecnologie

Nanotechnology-Based Drug Delivery Systems
Veicolazione controllata e mirata di farmaci
Diagnostica ed imaging

Liposomi
I liposomi sono vescicole a base di fosfolipidi di dimensioni variabili tra I 20 nm e 1 µm. Sono
generalmente costituti da uno o piu’ doppi strati lipidici. Il nucleo spesso contiene una soluzione
acquosa. Tipicamente sono utilizzati per veicolare farmaci idrofili che non potrebbero passare le
membrane cellulari. Farmaci lipofili possono essere dispersi nel doppio strato lipidico. Sono stati
utilizzati come modello di membrana cellulare. Si ottengono da dispersioni in acqua di fosfolipi.

Esempio: Liposomi con catene di PEG recanti
all’estremita’ molecole di transferrina.
Trattamento dei gliomi.
I liposomi possono essere modificati per impartire
proprieta’ antiopsonizzanti (PEG) e/o con molecole che
direzionano I vettori su cellule/organi specifici

Doxorubicina: Chemioterapico idrosolubile per il
trattamento di tumori solidi (es. Seno)

Micelle
Le micelle sono aggregati di molecole anfifiliche (testa polare,
coda idrofoba) che in ambiente acquoso si organizzano in modo
da esporre le teste polari verso l’acqua e le code idrofobe verso
l’interno. La parte interna e’ quindi formata da lunghe catene
alchiliche non polari.
Le micelle si formano solo quando la concentrazione della
molecola anfifilica in acqua raggiunge una determinata
concentrazione chiamata concentrazione micellare critica
(CMC)
La micellizzazione dipende dal bilancio di due effetti principali: la tendenza delle code idrocarburiche ad evitare il
contatto con l’acqua, e la repulsione tra le teste cariche, un effetto destabilizzante sul processo di aggregazione.
Le catene idrocarburiche evitano il contatto col solvente puntando verso l’interno dell’aggregato, privo di acqua,
mentre la repulsione tra le teste cariche sulla superficie della micella è attenuata dalla presenza di ioni di carica
opposta (controioni). L’associazione favorevole delle code apolari all’interno della micella avviene attraverso
l’interazione idrofobica, che è l’effetto dominante nella formazione di questi grandi aggregati di molecole.

Micelle Inverse
Oltre che in acqua, le molecole anfifiliche possono formare micelle anche in solventi
organici non polari. In questi casi, gli aggregati micellari prendono il nome di micelle inverse
perché la situazione è "capovolta" rispetto all’acqua. Infatti, le code idrocarburiche sono
esposte al solvente non polare, mentre le teste polari sono rivolte all’interno dell’aggregato
per evitare il contatto con il solvente.
Le micelle inverse sono capaci di trattenere quantità relativamente grandi di acqua all’interno
della struttura, verso cui puntano le teste polari. In questo modo si crea una "tasca" ideale per
sciogliere e trasportare soluti polari attraverso un solvente apolare.

Applicazioni Micelle per Drug Delivery

Peg-PCL micelles loaded with magnetic nanoparticles
Polymer: Polycaprolactone-block-Polyethylene Glycol (PCL 2000 /PEG 2000 ) 75/25 (Mw 25000)
Preparation method: Co-precipitation/Solvent Evaporation; Dialysis
Magnetic Core: Cobalt Ferrite or Magnetite (Colorita)
DLS
Organic NPs
148±41 nm
Nanoparticle Suspensions
Hybrid
CoFe
Organic
Hybrid/
Fe 3 O 4

Micro-Nanoparticelle Polimeriche per il Rilascio Controllato e Mirato di Farmaci
➼ Dimension ≤ 1 µm (nanoDDS 100nm)
➼ Capability to incorporate high dose of
therapeutics
➼ Able to cross biological barriers
➼ Possibility to biofunctionalize surfaces for a
targeted release
➼ Suited for hydrophobic or proteic drugs
➼ Biodegradable/Bioeliminable
Control of Pharmacokinetics & Biodistribution - Enhance Drug Physical and Chemical Stability

Metodi di Preparazione di Micro-Nanoparticelle
Micro-Nanoparticelle
The site of action and therapeutic regimes will guide in the
individuation of the best type of nanoparticulate system and in the
choice of the best suited polymer matrix. These aspects, combined
with drug features, are the leading factors for the definition of the
proper preparation and purification procedures.
Le metodologie di preparazione si distinguono in:
1. Polimerizzazione in situ
2. Preparazione da polimeri preformati

In-situ Polymerization Procedures
Direct polymerization of the low molar mass building blocks,
in presence of the active agent
Pros: High encapsulation efficiency
Cons: Use of organic solvent which may alter the loaded drug
Contamination by residual unreacted monomers
Time consuming for the purification process
Most Common Methods
¬ Emulsion Polymerization
(organic or acqueous)
¬ Dispersion Polymerization
¬ Interfacial Polymerization

Emulsion Polymerization
Most applied and fast
Aqueous or Organic Continuous Phase
¬ Emulsion or inverse emulsion of the monomer
and drug in the continuous phase
¬ Addition of surfactants to prevent aggregation
(generally over the critical micellar concentration)
¬ Water soluble initiators or γ/UV/VIS radiation
Formation of Solid Nanoparticles
Nanoparticelle di polialchilcianoacrilati: preparate per polimerizzazione anionica di monomeri di cianoacrilato dispersi in una
fase acquosa acida

Dispersion Polymerization
Single Step Technique
¬ The monomer is dissolved in a proper solvent
containing drug and stabilizer
¬ Radical polymerization of the monomer is
generally performed
¬ The oligomers will start to precipitate at a critical
chain length, adsorbing or entrapping the drug
Formation of Solid Nanoparticles
Nanoparticelle di poli(butilcianoacrilato) preparate per polimerizzazione in dispersione del monomero n-butyl cianoacrilato

Interfacial Polymerization
Polymerization, Polycondensation or Polyaddition
¬ Preparation of a w/o or o/w emulsion with two
monomers (one for each phase), drug and stabilizers
(generally in the inner phase for osmotic stability)
¬ The reaction starts and proceeds at the interface
Formation of Nanocapsules with
water or oil core

Preparation from Preformed Polymers
Most widely applied, compared to in-situ polymerization methods
Basically, the nanoparticles are formed due to
1. Polymer Aggregation or Precipitation for Solvation Loss
Emulsion-Solvent Evaporation
Phase Separation
Solvent Displacement (nanoprecipitation, co-precipitation,
dialysis)
Spray Drying
2. Formation of Insoluble Complexes
Self-Assembly (polyplexes, micelles)
3. Cross-linking
Chemical or Physical Crosslinking
• The loading of the drug may occur simultaneously with particle
formation or on preformed particles
• The drug can be physically entrapped or adsorbed

Emulsion-Solvent Evaporation 1
Single Emulsion (o/w) Hydrophobic Drugs
¬Polymer organic solution in which the drug is
dissolved/dispersed (sonication)
¬Emulsification in water (addition of surfactants)
¬Removal of the organic solvent by evaporation or extraction
¬Formation of Solid Nanoparticles

Emulsion-Solvent Evaporation 2
Double Emulsion (w/o/w) Hydrophilic Drugs
¬Emulsification of the water solution of the drug in the organic
solution containing the polymer (primary emulsion)
¬Transfer of the primary emulsion into an excess of water
solution containing a surfactant (vigorous stirring)
¬Removal of the organic solvent by evaporation or extraction
I Emulsion
Evaporation/
Extraction
II Emulsion

Phase Separation
¬ Dissolution or dispersion of the drug in the polymer
organic solution (PLA, PLGA, PVC…)
¬ Addition of an organic non solvent (silicone oil, vegetable
oils..) under continuous vigorous stirring until complete
extraction of the first solvent and formation of a soft
coacervate
¬ Hardening of the coacervate with exposure to another
non solvent (hexane, diethyl ether)
Formation of Solid Nanoparticles
Drawbacks: formation of aggregates and use of several organic solvents

Nanoprecipitation
Hydrophobic Drugs dissolved
in the polymer solution
Solvent Displacement
¬ The polymer is dissolved in an organic solution
¬ The solution is added dropwise to a non solvent
(other organic solvent or water), miscible with the
former one, kept under stirring
¬ Microphase separation of the polymer with
concurrent interaction with drug and stabilizers
Co-precipitation
Hydrophilic Drugs dissolved
in the water solution
Formation of Solid Nanoparticles

Nanoprecipitazione
- Il polimero, il farmaco e stabilizzanti vengono sciolti nello stesso solvente (es. Acetone)
- La soluzione polimerica viene fatta gocciolare in un non-solvente miscibile con quello
utilizzato per sciogliere il polimero
- Indicata per il caricamento di composti idrofobici
PLGA
4mg/ml
Elicene GA007
in DMSO (25_g/ml)
Pluronic F-127
0,1 % w/v
H 2 O

PLGA
PHB

Co-precipitation
- Original procedure - Avoid use of aggressive organic solvents
- Straightforward and reproducible - Tunable for different polymeric materials
+
HO
HO
HO
O
O
HO
HO
O
O OH
OH
O OH
HO
O
OH
HO
O
O
OH
OH
OH
HO
O
O
HO
O
OH
O
OH
O
O
EtOH/H 2 O
OH
OH
OH
H 2 O

¬ Dissolution of polymer, drug and
surfactants in the same solvent
(organic).
¬ Dialysis of the solution vs. a non
solvent, miscible with the former
one (water).
¬ Progressive aggregation of polymer,
drug, and surfactant.
¬ Formation of solid nanoparticles
Dialysis
Drawbacks: formation of aggregates and interaction with the dialysis membrane.

PLGA Nanoparticles
PLGA/RA Nanoparticles

Spray Drying
Mild conditions, good reproducibility
¬ The polymer is dissolved in a volatile solvent
¬ The drug is either dispersed or dissolved in the polymer solution
¬ The solution/dispersion is then sprayed against a stream of cold
air (-60°C)
¬ The cold droplets are then dried in a cold essicator
¬ Formation of Solid Nanoparticles

Self Assembly
Self Assembly of oppositely charged polyions
¬Generally applied for DNA plasmids, Antisense
Oligodeoxynucletides (ODNs) and siRNA (negatively charged)
¬Addition of an aqueous solution of the active agent to the
aqueous solution of water soluble cationic polymers (PEI, PLL,
etc.). Mixed mixed under stirring at room temperature.
Formation of Polyion Complexes
(Polyplexes)

¬ Sizing: Dynamic Light Scattering
Characterisations
¬ Morphology: Microscopy (SEM, TEM, AFM)
¬ Surface: Charge (Zeta Potential), Hydrophobicity (HIC), Plasma Protein
Adsorption (2-DE), ESCA
¬ Encapsulation Efficiency (EE%):
¬ Loading (%):
¬ Encapsulated Activity (EA%):
¬ Drug Release Kinetics
mg Loaded Agent
•100
mg Dried NPs
¬ In-vitro and In-vivo Characterizations
¬ Shelf-Life and Stability of the Dosage Form
mg Loaded Agent
•100
mg Agent in the Formulation
UI
Agent Activity
•100
mg Lyophilised
NPs

Targeting strategies
⎫ The therapeutic agent can be directly administered in the site of action by
means of cannulas or catheters, as well as release from an implant.
⎫ Passive drug targeting: usually based on the relation between the size of
the drug carrier and tissue characteristics (e.g. EPR effect, respiratory tract)
⎫ Physical targeting involves endogenous or exogenous physical factors that
can mediate targeted drug delivery (e.g. pH changes in gastrointestinal tract,
external magnetic field).
⎫ Active targeting: epitope-paratope recognition system

Effetto EPR (Enhanced Permeation and Retention)
R.Solaro, F. Chiellini, A. Battisti
Materials, 2010, 3 1928-1980

ACTIVE TARGETING IN CANCER THERAPY
ANGIOGENESIS-ASSOCIATE TARGETING:
Vascular Endothelial Growth Factor receptor
α vβ 3 Integrin (endothelial cell receptor for ECM)
Vascular Cell Adhesion Molecule-1 (VCAM-1)
Membrane Type 1- Matrix metalloproteinase (MT1-MMP)

ACTIVE TARGETING IN CANCER THERAPY
Uncontrolled Cell Proliferation Targeting:
Human Epidermal Receptor-2 (HER-2)
Epidermal Growth Factor Receptor
Transferrin Receptors
Folate Receptors

PEG
Brushes
Drug
Polymer Matrix
Targeting
Moieties

❒ Targeted &Controlled Controlled Drug Delivery System:
A Case Study
EC Funded Project TATLYS
“A New Biocompatible Nanoparticle Delivery System for Targeted
Release of Fibrinolytic Drugs”
• University of Pisa (I)
• Kedrion SpA (I)
• Polymer Laboratories Ltd (UK)
• Novetide Ltd (IL)
G5RD-CT-2000-00294
• Vulm a.s. (SK)
• Polish Academy of Science (PL)
• Czech Academy of Science (CZ)
• Slovak Academy of Science (SK)

Formation of intracoronary thrombus (95 % of cases)
Vascular
injury
❒ Acute Myocardial Infarction (AMI)
Protease
activation
Fibrin Clot
Plasminogen t-PA
u-PA
Plasmin
Prothrombin Thrombin Fibrinogen
Polymerization
Factor XIIIa
Fibrin
(monomer)
Soft clot
Hard Clot
Clot
Lyses

❒ Available Treatments for AMI
❍ Percutaneous Transluminal Coronary Angioplastic (PTCA)
❍ Fibrinolysis Standard Treatment
● Thrombolytic drugs:
- Urokinase (Uk);
- Recombinant Tissue Plasminogen Activator (rt-PA)
● Side effects: Fatal Hemorrhage Risks
Improvement of Fibrinolytic Therapy:
Targeted Selected Activation of Plasminogen in Thrombus
Site specific delivery of Urokinase

❒ Fibrin as Targeting Site
Selection of sequences exposed on the surface of Fibrin but not on Fibrinogen molecule
Selected epitope on γ chain

❒ Biofunctionalized Bioerodible Polymers
Grafting of 5% PEG 2000 Da to achieve stealth
polymer matrices & relative nanoparticles
Fab fragment against fibrin γ epitope covalently linked
to pegilated bioerodible polymer as targeting moiety
F.Chiellini, A.M. Piras, M. Gazzari, C. Bartoli, M. Ferri, L. Paolini, MACROMOLECULAR BIOSCIENCE 2008

❒ Nanoparticles Preparation by Co-Precipitation
Co Precipitation
Technique
- Avoid use of aggressive organic solvents
- Straightforward and reproducible
- Tunable for different polymeric materials
Organic bioerodible/bioeliminable polymer solution
Water solution of Human Serum Albumin (HSA)+
Protein Drug (Urokinase) + Stabilizer (Cyclodextrin)
E.E. Chiellini, F. Chiellini, R. Solaro, J. Nanosci. Nanotechnol. 2006, 6, 3310-20

❒ Bioerodible Nanoparticles Characterization
VAM41
Size: 120-150 nm
VAM41-Peg-Fab
Size: 90-120 nm
VAM41-Peg-Fab
Urokinase
Size: 90-150 nm
- Reproducible Results
- Biofunctionalization of Polymer Matrices and Introduction of Protein Drug do not alter
Nanoparticles Formation
A.M. Piras, F. Chiellini, C. Fiumi, C. Bartoli, E. Chiellini, INT. J. PHARM. 2008

❒ Bioerodible Nanoparticles Zeta Potential
Sample Grafted Z Pot
Molecule (mV± SD)
Vam41-G - -18.5 ± 1.1
Vam41-1%Fab Fab-SH -18.1± 0.9 √
Vam41-Fab Fab-SH -20.1± 1.1 √
Vam41-PEG PEG -7.7 ± 1.2
Vam41-PEG PL PEG -7.8 ± 0.5
Vam41-PEG-NHM PEG, NHM -7.8 ± 0.8
Vam41-PEG-1%Fab PEG, Fab-SH -8.1 ± 1.5
Vam41-PEG-Fab PEG, Fab-SH -10.6 ± 1.2
Measurements carried out in 0.9%NaCl at pH 5.5
•
•
•
å
å
A.M. Piras, F. Chiellini, C. Fiumi, C. Bartoli, E. Chiellini, INT. J. PHARM. 2008
Reference Material
Fab-SH Reduce Z Pot
PEG Increase Z Pot
Core-Corona Particles
Elude RES

❒ In Vivo Evaluation of Polymers Cytotoxicity
Acute Toxicity in mice after single i.v. dose polymers and stabilisers
Polymer LD50 (mg/Kg) Mw (kDa)
VAM41-1 0.6 320
VAM41-B 1.2 150
VAM41-C 1.5 130
VAM41-D 1.7 105
VAM41-F 2.5 41
VAM41-5%PEG 7.5 57
GIG-βCD 1500 -
Acute Oral Toxicity in mice
Polymer LD50 (mg/Kg) Mw (kDa)
VAM41-F ≥ 3000 41
VAM41-G ≥ 3000 38

❒ Evaluation of Urokinase Release Kinetics
Dialysis was performed at 37 °C in PBS 5X pH 7.4 containing 0.2% EDTA
using a regenerated cellulose membrane with a MWCO of 60000 Da
Protein release Activity of the released enzyme

❒
In Vivo Activity Activity
of Urokinase Loaded Nanoparticle
Artherial rtherial thrombosis in rats
Time-course of mean blood flow in a carotid artery after
i.v. application

❒ Stability Studies
Stressed Stability Conditions for Lyophilized Nanoparticles:
3 months at 35 ± 2 °C, 75 ± 5% relative humidity
Stressed Stability Conditions for Nanoparticles Suspensions:
16 months at 4 ± 2 °C, 75 ± 5% relative humidity