Lezione 9 12 Maggio 2010 - Università degli Studi di Pisa
Lezione 9 12 Maggio 2010 - Università degli Studi di Pisa
Lezione 9 12 Maggio 2010 - Università degli Studi di Pisa
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<strong>Lezione</strong> 9<br />
<strong>12</strong> <strong>Maggio</strong> <strong>2010</strong>
Rilascio Controllato e Mirato <strong>di</strong> Farmaci<br />
“Magic Bullet”<br />
Paul Erlich (1906)<br />
A Drug will be able to perform its therapeutic action if present<br />
at the site of action for an adeguate period of time and in an<br />
optimal concentration.<br />
concentration
NANOMEDICINA<br />
Applicazione me<strong>di</strong>ca/clinica delle nanotecnologie
Nanotechnology-Based Drug Delivery Systems<br />
Veicolazione controllata e mirata <strong>di</strong> farmaci<br />
Diagnostica ed imaging
Liposomi<br />
I liposomi sono vescicole a base <strong>di</strong> fosfolipi<strong>di</strong> <strong>di</strong> <strong>di</strong>mensioni variabili tra I 20 nm e 1 µm. Sono<br />
generalmente costituti da uno o piu’ doppi strati lipi<strong>di</strong>ci. Il nucleo spesso contiene una soluzione<br />
acquosa. Tipicamente sono utilizzati per veicolare farmaci idrofili che non potrebbero passare le<br />
membrane cellulari. Farmaci lipofili possono essere <strong>di</strong>spersi nel doppio strato lipi<strong>di</strong>co. Sono stati<br />
utilizzati come modello <strong>di</strong> membrana cellulare. Si ottengono da <strong>di</strong>spersioni in acqua <strong>di</strong> fosfolipi.
Esempio: Liposomi con catene <strong>di</strong> PEG recanti<br />
all’estremita’ molecole <strong>di</strong> transferrina.<br />
Trattamento dei gliomi.<br />
I liposomi possono essere mo<strong>di</strong>ficati per impartire<br />
proprieta’ antiopsonizzanti (PEG) e/o con molecole che<br />
<strong>di</strong>rezionano I vettori su cellule/organi specifici
Doxorubicina: Chemioterapico idrosolubile per il<br />
trattamento <strong>di</strong> tumori soli<strong>di</strong> (es. Seno)
Micelle<br />
Le micelle sono aggregati <strong>di</strong> molecole anfifiliche (testa polare,<br />
coda idrofoba) che in ambiente acquoso si organizzano in modo<br />
da esporre le teste polari verso l’acqua e le code idrofobe verso<br />
l’interno. La parte interna e’ quin<strong>di</strong> formata da lunghe catene<br />
alchiliche non polari.<br />
Le micelle si formano solo quando la concentrazione della<br />
molecola anfifilica in acqua raggiunge una determinata<br />
concentrazione chiamata concentrazione micellare critica<br />
(CMC)<br />
La micellizzazione <strong>di</strong>pende dal bilancio <strong>di</strong> due effetti principali: la tendenza delle code idrocarburiche ad evitare il<br />
contatto con l’acqua, e la repulsione tra le teste cariche, un effetto destabilizzante sul processo <strong>di</strong> aggregazione.<br />
Le catene idrocarburiche evitano il contatto col solvente puntando verso l’interno dell’aggregato, privo <strong>di</strong> acqua,<br />
mentre la repulsione tra le teste cariche sulla superficie della micella è attenuata dalla presenza <strong>di</strong> ioni <strong>di</strong> carica<br />
opposta (controioni). L’associazione favorevole delle code apolari all’interno della micella avviene attraverso<br />
l’interazione idrofobica, che è l’effetto dominante nella formazione <strong>di</strong> questi gran<strong>di</strong> aggregati <strong>di</strong> molecole.
Micelle Inverse<br />
Oltre che in acqua, le molecole anfifiliche possono formare micelle anche in solventi<br />
organici non polari. In questi casi, gli aggregati micellari prendono il nome <strong>di</strong> micelle inverse<br />
perché la situazione è "capovolta" rispetto all’acqua. Infatti, le code idrocarburiche sono<br />
esposte al solvente non polare, mentre le teste polari sono rivolte all’interno dell’aggregato<br />
per evitare il contatto con il solvente.<br />
Le micelle inverse sono capaci <strong>di</strong> trattenere quantità relativamente gran<strong>di</strong> <strong>di</strong> acqua all’interno<br />
della struttura, verso cui puntano le teste polari. In questo modo si crea una "tasca" ideale per<br />
sciogliere e trasportare soluti polari attraverso un solvente apolare.
Applicazioni Micelle per Drug Delivery
Peg-PCL micelles loaded with magnetic nanoparticles<br />
Polymer: Polycaprolactone-block-Polyethylene Glycol (PCL 2000 /PEG 2000 ) 75/25 (Mw 25000)<br />
Preparation method: Co-precipitation/Solvent Evaporation; Dialysis<br />
Magnetic Core: Cobalt Ferrite or Magnetite (Colorita)<br />
DLS<br />
Organic NPs<br />
148±41 nm<br />
Nanoparticle Suspensions<br />
Hybrid<br />
CoFe<br />
Organic<br />
Hybrid/<br />
Fe 3 O 4
Micro-Nanoparticelle Polimeriche per il Rilascio Controllato e Mirato <strong>di</strong> Farmaci<br />
➼ Dimension ≤ 1 µm (nanoDDS 100nm)<br />
➼ Capability to incorporate high dose of<br />
therapeutics<br />
➼ Able to cross biological barriers<br />
➼ Possibility to biofunctionalize surfaces for a<br />
targeted release<br />
➼ Suited for hydrophobic or proteic drugs<br />
➼ Biodegradable/Bioeliminable<br />
Control of Pharmacokinetics & Bio<strong>di</strong>stribution - Enhance Drug Physical and Chemical Stability
Meto<strong>di</strong> <strong>di</strong> Preparazione <strong>di</strong> Micro-Nanoparticelle<br />
Micro-Nanoparticelle<br />
The site of action and therapeutic regimes will guide in the<br />
in<strong>di</strong>viduation of the best type of nanoparticulate system and in the<br />
choice of the best suited polymer matrix. These aspects, combined<br />
with drug features, are the lea<strong>di</strong>ng factors for the definition of the<br />
proper preparation and purification procedures.<br />
Le metodologie <strong>di</strong> preparazione si <strong>di</strong>stinguono in:<br />
1. Polimerizzazione in situ<br />
2. Preparazione da polimeri preformati
In-situ Polymerization Procedures<br />
Direct polymerization of the low molar mass buil<strong>di</strong>ng blocks,<br />
in presence of the active agent<br />
Pros: High encapsulation efficiency<br />
Cons: Use of organic solvent which may alter the loaded drug<br />
Contamination by residual unreacted monomers<br />
Time consuming for the purification process<br />
Most Common Methods<br />
¬ Emulsion Polymerization<br />
(organic or acqueous)<br />
¬ Dispersion Polymerization<br />
¬ Interfacial Polymerization
Emulsion Polymerization<br />
Most applied and fast<br />
Aqueous or Organic Continuous Phase<br />
¬ Emulsion or inverse emulsion of the monomer<br />
and drug in the continuous phase<br />
¬ Ad<strong>di</strong>tion of surfactants to prevent aggregation<br />
(generally over the critical micellar concentration)<br />
¬ Water soluble initiators or γ/UV/VIS ra<strong>di</strong>ation<br />
Formation of Solid Nanoparticles<br />
Nanoparticelle <strong>di</strong> polialchilcianoacrilati: preparate per polimerizzazione anionica <strong>di</strong> monomeri <strong>di</strong> cianoacrilato <strong>di</strong>spersi in una<br />
fase acquosa acida
Dispersion Polymerization<br />
Single Step Technique<br />
¬ The monomer is <strong>di</strong>ssolved in a proper solvent<br />
containing drug and stabilizer<br />
¬ Ra<strong>di</strong>cal polymerization of the monomer is<br />
generally performed<br />
¬ The oligomers will start to precipitate at a critical<br />
chain length, adsorbing or entrapping the drug<br />
Formation of Solid Nanoparticles<br />
Nanoparticelle <strong>di</strong> poli(butilcianoacrilato) preparate per polimerizzazione in <strong>di</strong>spersione del monomero n-butyl cianoacrilato
Interfacial Polymerization<br />
Polymerization, Polycondensation or Polyad<strong>di</strong>tion<br />
¬ Preparation of a w/o or o/w emulsion with two<br />
monomers (one for each phase), drug and stabilizers<br />
(generally in the inner phase for osmotic stability)<br />
¬ The reaction starts and proceeds at the interface<br />
Formation of Nanocapsules with<br />
water or oil core
Preparation from Preformed Polymers<br />
Most widely applied, compared to in-situ polymerization methods<br />
Basically, the nanoparticles are formed due to<br />
1. Polymer Aggregation or Precipitation for Solvation Loss<br />
Emulsion-Solvent Evaporation<br />
Phase Separation<br />
Solvent Displacement (nanoprecipitation, co-precipitation,<br />
<strong>di</strong>alysis)<br />
Spray Drying<br />
2. Formation of Insoluble Complexes<br />
Self-Assembly (polyplexes, micelles)<br />
3. Cross-linking<br />
Chemical or Physical Crosslinking<br />
• The loa<strong>di</strong>ng of the drug may occur simultaneously with particle<br />
formation or on preformed particles<br />
• The drug can be physically entrapped or adsorbed
Emulsion-Solvent Evaporation 1<br />
Single Emulsion (o/w) Hydrophobic Drugs<br />
¬Polymer organic solution in which the drug is<br />
<strong>di</strong>ssolved/<strong>di</strong>spersed (sonication)<br />
¬Emulsification in water (ad<strong>di</strong>tion of surfactants)<br />
¬Removal of the organic solvent by evaporation or extraction<br />
¬Formation of Solid Nanoparticles
Emulsion-Solvent Evaporation 2<br />
Double Emulsion (w/o/w) Hydrophilic Drugs<br />
¬Emulsification of the water solution of the drug in the organic<br />
solution containing the polymer (primary emulsion)<br />
¬Transfer of the primary emulsion into an excess of water<br />
solution containing a surfactant (vigorous stirring)<br />
¬Removal of the organic solvent by evaporation or extraction<br />
I Emulsion<br />
Evaporation/<br />
Extraction<br />
II Emulsion
Phase Separation<br />
¬ Dissolution or <strong>di</strong>spersion of the drug in the polymer<br />
organic solution (PLA, PLGA, PVC…)<br />
¬ Ad<strong>di</strong>tion of an organic non solvent (silicone oil, vegetable<br />
oils..) under continuous vigorous stirring until complete<br />
extraction of the first solvent and formation of a soft<br />
coacervate<br />
¬ Hardening of the coacervate with exposure to another<br />
non solvent (hexane, <strong>di</strong>ethyl ether)<br />
Formation of Solid Nanoparticles<br />
Drawbacks: formation of aggregates and use of several organic solvents
Nanoprecipitation<br />
Hydrophobic Drugs <strong>di</strong>ssolved<br />
in the polymer solution<br />
Solvent Displacement<br />
¬ The polymer is <strong>di</strong>ssolved in an organic solution<br />
¬ The solution is added dropwise to a non solvent<br />
(other organic solvent or water), miscible with the<br />
former one, kept under stirring<br />
¬ Microphase separation of the polymer with<br />
concurrent interaction with drug and stabilizers<br />
Co-precipitation<br />
Hydrophilic Drugs <strong>di</strong>ssolved<br />
in the water solution<br />
Formation of Solid Nanoparticles
Nanoprecipitazione<br />
- Il polimero, il farmaco e stabilizzanti vengono sciolti nello stesso solvente (es. Acetone)<br />
- La soluzione polimerica viene fatta gocciolare in un non-solvente miscibile con quello<br />
utilizzato per sciogliere il polimero<br />
- In<strong>di</strong>cata per il caricamento <strong>di</strong> composti idrofobici<br />
PLGA<br />
4mg/ml<br />
Elicene GA007<br />
in DMSO (25_g/ml)<br />
Pluronic F-<strong>12</strong>7<br />
0,1 % w/v<br />
H 2 O
PLGA<br />
PHB
Co-precipitation<br />
- Original procedure - Avoid use of aggressive organic solvents<br />
- Straightforward and reproducible - Tunable for <strong>di</strong>fferent polymeric materials<br />
+<br />
HO<br />
HO<br />
HO<br />
O<br />
O<br />
HO<br />
HO<br />
O<br />
O OH<br />
OH<br />
O OH<br />
HO<br />
O<br />
OH<br />
HO<br />
O<br />
O<br />
OH<br />
OH<br />
OH<br />
HO<br />
O<br />
O<br />
HO<br />
O<br />
OH<br />
O<br />
OH<br />
O<br />
O<br />
EtOH/H 2 O<br />
OH<br />
OH<br />
OH<br />
H 2 O
¬ Dissolution of polymer, drug and<br />
surfactants in the same solvent<br />
(organic).<br />
¬ Dialysis of the solution vs. a non<br />
solvent, miscible with the former<br />
one (water).<br />
¬ Progressive aggregation of polymer,<br />
drug, and surfactant.<br />
¬ Formation of solid nanoparticles<br />
Dialysis<br />
Drawbacks: formation of aggregates and interaction with the <strong>di</strong>alysis membrane.
PLGA Nanoparticles<br />
PLGA/RA Nanoparticles
Spray Drying<br />
Mild con<strong>di</strong>tions, good reproducibility<br />
¬ The polymer is <strong>di</strong>ssolved in a volatile solvent<br />
¬ The drug is either <strong>di</strong>spersed or <strong>di</strong>ssolved in the polymer solution<br />
¬ The solution/<strong>di</strong>spersion is then sprayed against a stream of cold<br />
air (-60°C)<br />
¬ The cold droplets are then dried in a cold essicator<br />
¬ Formation of Solid Nanoparticles
Self Assembly<br />
Self Assembly of oppositely charged polyions<br />
¬Generally applied for DNA plasmids, Antisense<br />
Oligodeoxynucletides (ODNs) and siRNA (negatively charged)<br />
¬Ad<strong>di</strong>tion of an aqueous solution of the active agent to the<br />
aqueous solution of water soluble cationic polymers (PEI, PLL,<br />
etc.). Mixed mixed under stirring at room temperature.<br />
Formation of Polyion Complexes<br />
(Polyplexes)
¬ Sizing: Dynamic Light Scattering<br />
Characterisations<br />
¬ Morphology: Microscopy (SEM, TEM, AFM)<br />
¬ Surface: Charge (Zeta Potential), Hydrophobicity (HIC), Plasma Protein<br />
Adsorption (2-DE), ESCA<br />
¬ Encapsulation Efficiency (EE%):<br />
¬ Loa<strong>di</strong>ng (%):<br />
¬ Encapsulated Activity (EA%):<br />
¬ Drug Release Kinetics<br />
mg Loaded Agent<br />
•100<br />
mg Dried NPs<br />
¬ In-vitro and In-vivo Characterizations<br />
¬ Shelf-Life and Stability of the Dosage Form<br />
mg Loaded Agent<br />
•100<br />
mg Agent in the Formulation<br />
UI<br />
Agent Activity<br />
•100<br />
mg Lyophilised<br />
NPs
Targeting strategies<br />
⎫ The therapeutic agent can be <strong>di</strong>rectly administered in the site of action by<br />
means of cannulas or catheters, as well as release from an implant.<br />
⎫ Passive drug targeting: usually based on the relation between the size of<br />
the drug carrier and tissue characteristics (e.g. EPR effect, respiratory tract)<br />
⎫ Physical targeting involves endogenous or exogenous physical factors that<br />
can me<strong>di</strong>ate targeted drug delivery (e.g. pH changes in gastrointestinal tract,<br />
external magnetic field).<br />
⎫ Active targeting: epitope-paratope recognition system
Effetto EPR (Enhanced Permeation and Retention)<br />
R.Solaro, F. Chiellini, A. Battisti<br />
Materials, <strong>2010</strong>, 3 1928-1980
ACTIVE TARGETING IN CANCER THERAPY<br />
ANGIOGENESIS-ASSOCIATE TARGETING:<br />
Vascular Endothelial Growth Factor receptor<br />
α vβ 3 Integrin (endothelial cell receptor for ECM)<br />
Vascular Cell Adhesion Molecule-1 (VCAM-1)<br />
Membrane Type 1- Matrix metalloproteinase (MT1-MMP)
ACTIVE TARGETING IN CANCER THERAPY<br />
Uncontrolled Cell Proliferation Targeting:<br />
Human Epidermal Receptor-2 (HER-2)<br />
Epidermal Growth Factor Receptor<br />
Transferrin Receptors<br />
Folate Receptors
PEG<br />
Brushes<br />
Drug<br />
Polymer Matrix<br />
Targeting<br />
Moieties
❒ Targeted &Controlled Controlled Drug Delivery System:<br />
A Case Study<br />
EC Funded Project TATLYS<br />
“A New Biocompatible Nanoparticle Delivery System for Targeted<br />
Release of Fibrinolytic Drugs”<br />
• University of <strong>Pisa</strong> (I)<br />
• Kedrion SpA (I)<br />
• Polymer Laboratories Ltd (UK)<br />
• Novetide Ltd (IL)<br />
G5RD-CT-2000-00294<br />
• Vulm a.s. (SK)<br />
• Polish Academy of Science (PL)<br />
• Czech Academy of Science (CZ)<br />
• Slovak Academy of Science (SK)
Formation of intracoronary thrombus (95 % of cases)<br />
Vascular<br />
injury<br />
❒ Acute Myocar<strong>di</strong>al Infarction (AMI)<br />
Protease<br />
activation<br />
Fibrin Clot<br />
Plasminogen t-PA<br />
u-PA<br />
Plasmin<br />
Prothrombin Thrombin Fibrinogen<br />
Polymerization<br />
Factor XIIIa<br />
Fibrin<br />
(monomer)<br />
Soft clot<br />
Hard Clot<br />
Clot<br />
Lyses
❒ Available Treatments for AMI<br />
❍ Percutaneous Transluminal Coronary Angioplastic (PTCA)<br />
❍ Fibrinolysis Standard Treatment<br />
● Thrombolytic drugs:<br />
- Urokinase (Uk);<br />
- Recombinant Tissue Plasminogen Activator (rt-PA)<br />
● Side effects: Fatal Hemorrhage Risks<br />
Improvement of Fibrinolytic Therapy:<br />
Targeted Selected Activation of Plasminogen in Thrombus<br />
Site specific delivery of Urokinase
❒ Fibrin as Targeting Site<br />
Selection of sequences exposed on the surface of Fibrin but not on Fibrinogen molecule<br />
Selected epitope on γ chain
❒ Biofunctionalized Bioero<strong>di</strong>ble Polymers<br />
Grafting of 5% PEG 2000 Da to achieve stealth<br />
polymer matrices & relative nanoparticles<br />
Fab fragment against fibrin γ epitope covalently linked<br />
to pegilated bioero<strong>di</strong>ble polymer as targeting moiety<br />
F.Chiellini, A.M. Piras, M. Gazzari, C. Bartoli, M. Ferri, L. Paolini, MACROMOLECULAR BIOSCIENCE 2008
❒ Nanoparticles Preparation by Co-Precipitation<br />
Co Precipitation<br />
Technique<br />
- Avoid use of aggressive organic solvents<br />
- Straightforward and reproducible<br />
- Tunable for <strong>di</strong>fferent polymeric materials<br />
Organic bioero<strong>di</strong>ble/bioeliminable polymer solution<br />
Water solution of Human Serum Albumin (HSA)+<br />
Protein Drug (Urokinase) + Stabilizer (Cyclodextrin)<br />
E.E. Chiellini, F. Chiellini, R. Solaro, J. Nanosci. Nanotechnol. 2006, 6, 3310-20
❒ Bioero<strong>di</strong>ble Nanoparticles Characterization<br />
VAM41<br />
Size: <strong>12</strong>0-150 nm<br />
VAM41-Peg-Fab<br />
Size: 90-<strong>12</strong>0 nm<br />
VAM41-Peg-Fab<br />
Urokinase<br />
Size: 90-150 nm<br />
- Reproducible Results<br />
- Biofunctionalization of Polymer Matrices and Introduction of Protein Drug do not alter<br />
Nanoparticles Formation<br />
A.M. Piras, F. Chiellini, C. Fiumi, C. Bartoli, E. Chiellini, INT. J. PHARM. 2008
❒ Bioero<strong>di</strong>ble Nanoparticles Zeta Potential<br />
Sample Grafted Z Pot<br />
Molecule (mV± SD)<br />
Vam41-G - -18.5 ± 1.1<br />
Vam41-1%Fab Fab-SH -18.1± 0.9 √<br />
Vam41-Fab Fab-SH -20.1± 1.1 √<br />
Vam41-PEG PEG -7.7 ± 1.2<br />
Vam41-PEG PL PEG -7.8 ± 0.5<br />
Vam41-PEG-NHM PEG, NHM -7.8 ± 0.8<br />
Vam41-PEG-1%Fab PEG, Fab-SH -8.1 ± 1.5<br />
Vam41-PEG-Fab PEG, Fab-SH -10.6 ± 1.2<br />
Measurements carried out in 0.9%NaCl at pH 5.5<br />
•<br />
•<br />
•<br />
å<br />
å<br />
A.M. Piras, F. Chiellini, C. Fiumi, C. Bartoli, E. Chiellini, INT. J. PHARM. 2008<br />
Reference Material<br />
Fab-SH Reduce Z Pot<br />
PEG Increase Z Pot<br />
Core-Corona Particles<br />
Elude RES
❒ In Vivo Evaluation of Polymers Cytotoxicity<br />
Acute Toxicity in mice after single i.v. dose polymers and stabilisers<br />
Polymer LD50 (mg/Kg) Mw (kDa)<br />
VAM41-1 0.6 320<br />
VAM41-B 1.2 150<br />
VAM41-C 1.5 130<br />
VAM41-D 1.7 105<br />
VAM41-F 2.5 41<br />
VAM41-5%PEG 7.5 57<br />
GIG-βCD 1500 -<br />
Acute Oral Toxicity in mice<br />
Polymer LD50 (mg/Kg) Mw (kDa)<br />
VAM41-F ≥ 3000 41<br />
VAM41-G ≥ 3000 38
❒ Evaluation of Urokinase Release Kinetics<br />
Dialysis was performed at 37 °C in PBS 5X pH 7.4 containing 0.2% EDTA<br />
using a regenerated cellulose membrane with a MWCO of 60000 Da<br />
Protein release Activity of the released enzyme
❒<br />
In Vivo Activity Activity<br />
of Urokinase Loaded Nanoparticle<br />
Artherial rtherial thrombosis in rats<br />
Time-course of mean blood flow in a carotid artery after<br />
i.v. application
❒ Stability <strong>Stu<strong>di</strong></strong>es<br />
Stressed Stability Con<strong>di</strong>tions for Lyophilized Nanoparticles:<br />
3 months at 35 ± 2 °C, 75 ± 5% relative humi<strong>di</strong>ty<br />
Stressed Stability Con<strong>di</strong>tions for Nanoparticles Suspensions:<br />
16 months at 4 ± 2 °C, 75 ± 5% relative humi<strong>di</strong>ty
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