Nanoparticles for Drug Delivery

Nanoparticles for Drug Delivery

• Introduction – Systemic Drug Delivery

• Nanoparticles

• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting

• Examples :

Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery

Methods of Drug Delivery Advantages of Nanoparticles for Drug Delivery

• Oral Delivery

• Inhalation

• Transdermal

• Implantation

• Injection

University of Illinois, Urbana-Champagne

Examples of Nanoparticles for Drug Delivery

Liposomal Amphoterin, sold by Gilead (Ambisome) and Enzon (Abelcet)

~$200 Million in Ambisome sales in 2003.

Examples of Nanoparticles for Drug Delivery

Applications: Non-resistant cancers

Gilead Sciences

• Amphotericin treats fungal and parasite infection

• Most commonly used in patients with depressed white blood cell count (cancer and chemotherapy patients, HIV-infected patients, elderly patients).

• Liposomal formulation is preferred because of decreased side effects

and prolonged drug exposure (due to slow release)

•In hepatic metastases model, the reduction in number of metastases was greater with Dox-loaded nanospheres than free dox. (Why hepatic model?) •No special affinity for tumor tissue detected. Most nanospheres located within Kupffer cells. See proposed mechanism of action •Note that this approach reduces side effects and toxicity! Small molecules are distributed throughout the body.

Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)

Examples of Nanoparticles for Drug Delivery Nanoparticles for Drug Delivery

Applications: Intracellular Infections

• Introduction – Systemic Drug Delivery

Macrophages infected with Salmonella incubated with ampicillin-loaded PACA nanospheres

Antibiotic-loaded nanospheres Resistance of many microorganisms to

antibiotics is often related to low uptake of antibiotics or reduced activity in acidic pH of lysosomes.

1. Ampicillin-loaded nanospheres for Listeria

treatment. Dramatic improvement over free drug; bacterial counts in liver reduced at least 20-fold.

2. Ampicillin-loaded nanospheres for Salmonella treatment. Drug alone – required 32 mg per mouse; with nanoparticle, only 0.8 mg ensured survival.

Vauthier, et al. Adv. Drug Del Rev v55:519-548

(2003)

• Nanoparticles

• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting

• Examples :

Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery

Drug Carrier Systems

from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu

Nanoparticles for Drug Delivery

• Metal-based nanoparticles • Lipid-based nanoparticles • Polymer-based nanoparticles • Biological nanoparticles

Metal Nanoparticles for drug delivery Metal Nanoparticles

Generation

Size Definition Examples

First

> 1 µm

Able to release a drug at the target site but needing a particular type of administrat ion

Microspheres and microcapsules for chemoembolization

Second

< 1 µm

Carriers that can be given by a general route able to

transport a drug to the target site

Liposomes, nanoparticles, polymer drug carriers

Third

< 1 µm

Carriers able to recognize a specific target

Monoclonal antibodies; second-generation carriers with targeted antibodies or

other ligands

Metal Nanoparticles Lipid-based nanoparticles for Drug Delivery

DOPE

DSPC

DOTAP

Note: direct tissue injection

Lipid-based nanoparticles for Drug Delivery

Deliverables:

-Small molecules (Amphotericin B, Daunorubicin, Dox orubicin – all approved and marketed drug formulations) Gilead, A lza

-Viruses and bacteria (as vaccines) – in developmen t

-Nucleic acids – in development

Lipid-based nanoparticles for Drug Delivery Many formulation methods -- 1. Mixing lipids together

in organic solution. 2. Remove solvent

by evaporation 3. Hydration with aqueous

solvent containing drug to form multilamellar vesicles

4. Sonication or extrusion are common methods to reduce the size of the liposomes

Avanti Polar Lipids

Lipid-based nanoparticles for Drug Delivery

Drug properties and Liposome association

Hydrophilic Retained in aqueous interior Slowly rel eased over

**may be difficult to get several hours-several day s high loading

Hydrophobic Inserted into hydrophobic Excellent ret ention

interior of the liposome bilayer **can disrupt liposome at high concentrations

Intermediate Rapidly partition between Rapid releas e from liposomes

lipid bilayer and aqueous but pH manipulation or phase formation of molecular

complexes can result in good retention

Polymer-based nanoparticles for Drug Delivery • Poly(alkylcyanoacrylates) used extensively for tissue

adhesives for skin wounds and surgical glue (late 1960’s)

• Application as drug nanoparticulate carriers (1980s)

• For detailed review, see:

Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)

Polymer-based nanoparticles for Drug Delivery Polymer-based nanoparticles for Drug Delivery

Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)

Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)

Polymer-based nanoparticles for Drug Delivery Polymer-based nanoparticles for Drug Delivery

DEGRADATION OF NANOPARTICLES

Hydrolysis of ester bond; degradation products (alkylalcohol and poly(cyanoacrylic acid) are eliminated by kidney filtration.

Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)

Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)

Biological nanoparticles --Viruses Biological nanoparticles --Viruses

Stratagene, Inc.

From an excellent review by Thomas et al. (2003) Nature v4:346

Biological nanoparticles --Viruses Nanoparticles for Drug Delivery

• Introduction – Systemic Drug Delivery

• Nanoparticles

• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting

• Examples :

Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery

Thomas et al. (2003) Nature v4:346

Stability of Colloids in Physiological Environments

1. Attractive van der Waals forces and random Br ownian motion

cause particle flocculation

2. Stabilization of colloids by electrostatic st abilization (DLVO theory): -- Charged particles have a counter-ion layer. The charged surface

and counter-ion layer is called the “electrostatic double layer”. For homogenous colloid suspensions, this electrostatic double layer acts as a repulsive force between particles.

--The sum of the van der Waals force and the double layer repulsion force gives the DVLO interaction potentia l:

Stability of Colloids in Physiological Environments

Aggregation of Colloids

A

-higher ionic strengths collapse this boundary layer

water 150 mM salt

M. Nikolaides

-at high salt concentration, no stable region -physiologic salt concentration ~150 nm

-- in intravenous delivery, this can be a major cau se of toxicity

Stability of Colloids in Physiological Environments

3. Stabilization of colloids by steric stabilizat ion: -- Add polymers to the surface of particles to prev ent the particles from

coming in close proximity to each other. At these distances, there is not enough attractive force for flocculation to occ ur.

--Note that this phenomena is solvent dependent (af fects the structure and interaction of the surface polymers)

Steric Stabilization

Steric Stabilization Dispersion Stabilization Choi, et al. J Disp Sci Tech (2003) v24:415

Steric Stabilization Steric Stabilization

Choi, et al. J Disp Sci Tech (2003) v24:415 Ogris et al. (1999) Gene Therapy 6:595 (1999).

Liposome Stability Liposome Stability Optimization

•Chemical stability

•Reducing oxidation – addition of antioxidants, stor age at low temperatures and pH 6.5 •Removal of water – spray drying or lyophilization ( but both have to occur under controlled and optimized conditions)

•Physical stability

•Electrostatic stabilization •Steric stabilization •Cream or hydrogel incorporation

Nanoparticles for Drug Delivery

• Introduction – Systemic Drug Delivery

• Nanoparticles

• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting

• Examples :

Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery

Mechanisms of Removal from Circulation • Fast removal from circulation

-binding to cells, membranes, or plasma proteins -uptake by phagocytes (macrophages) -trapping in capillary bed (lungs)

• Renal clearance

-size restriction for kidney glomerulus is ~30-35 kDa for polymers (~20-30 nm)

• Extravasation

-depends on the permeability of blood vessels -capillaries are thought to be more permissive to extravasation -note: for cancer applications this works to our advantage: EPR!

from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu

Drug Carrier Systems: Influence of Physicochemical Properties

• Molecular weight -macromolecules smaller than renal threshold are rapidly eliminated -for larger, non-degradable molecules, excretion is useful to decrease toxicity.

• Charge

-positively-charged macromolecules will interact with cells and membranes (remember the negatively charged proteoglycans?) -negatively charged macromolecules are picked up by macrophages such as Kupffer cells, that contain polyanion scavenger receptors on their surface.

• PEGylation

-may increase circulation by reducing non-specific protein binding.

from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu

Drug Carrier Systems: Influence of Physicochemical Properties

Kupffer Cells

http://education.vetmed.vt.edu/curriculum/VM8054/La bs/Lab20/Examples/exkupff.htm

Drug Carrier Systems: Influence of Physicochemical Properties Biodistribution of PEGylated Polymer-based Nanoparticles

1000 nm

6000 nm

500 nm

200 nm

10,000 nm

“+”-charged

“-”-charged

neutral

PEGylated

Non- PEGylated

Ogris et al. (1999) Gene Therapy 6:595 (1999).

Biodistribution of Lipid-based Nanoparticles PEGylation

Non-PEGylated liposomes

Why is there a dose-dependence?

PEGylated liposomes

Nanoparticles for Drug Delivery • Introduction – Systemic Drug Delivery

• Nanoparticles

• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting

• Examples

Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery

Allen, TM and Stuart, DD. “Liposome Pharmacokineti cs” In Liposomes Ed. Janoff, AS (2000)

Non-specific targeting: EPR Effect

• Tumors generally can’t grow beyond 2 mm in size without becoming angiogenic (attracting new capillaries) because difficulty in obtaining oxygen and nutrients.

• Tumors produce angiogenic factors to form new capillary structures.

• Tumors also need to recruit macromolecules from the blood stream

to form a new extracellular matrix.

• Permeability-enhancing factors such as VEGF (vascular endothelial growth factor) are secreted to increase the permeability of the tumor blood vessels.

• This effect is called the “enhanced permeability and retention

effect” (EPR)

from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu

Non-specific Targeting

Targeting Ligands Targeting

• Small Molecules

– Galactose/Glucose/Mannose

– Folate

• Peptides

– RGD

• Proteins

– Transferrin

– Antibodies – LDLs

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endocytosis.html Choi, et al. J Disp Sci Tech (2003) v24:415

Targeting Nanoparticles for Drug Delivery • Introduction – Systemic Drug Delivery

• Nanoparticles

• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting

• Examples :

Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery

Choi, et al. J Disp Sci Tech (2003) v24:415

Examples of Nanoparticles for Drug Delivery

DOXIL Efficacy

Doxil (n = 118) Topotecan (n =

119) Time to progression

18.4 wks 18.3 wks

ORR CR 4* 5* PR 16* 12* TTP Plat Res 12.3 wks 6.5 wks TTP Plat Sens 28.4 wks 28.8 wks OSPlat Res 33.4 wks 37.3 wks OSPlat Sens 86.1 wks** 63.3 wks *Expressed as percentage.

**p = .01. TTP Plat Res = time-to-progression platinum resistant; TTP Plat Sens = time-to-progression platinum sensitive; OS = overall survival.

Examples of Nanoparticles for Drug Delivery Examples of Nanoparticles for Drug Delivery

DOXIL DOXIL Proposed Mechanism of Action

Formulation

1. Doxorubicin-containing

core

2. Lipid Bilayer membrane

3. PEG-coated surface

4. <100 nm

Examples of Nanoparticles for Drug Delivery

DOXIL Pharmacokinetics

Rats Dogs

Gabizon, et al. Pharm Res v10:703 (1993)

Examples of Nanoparticles for Drug Delivery

DOXIL Toxicity

•Mild white blood cell depression •Skin toxicity (unique to Doxil) with full recovery. (Long distribution time?) •Cardiac toxicity – insignificant up to 1500 mg/m 2; Much lower toxicity than doxorubicin (lower peak plasma level; decrease availability to cardiac muscle) •Hair loss – rare; only seen in ~6% of patients •Mucositis – ulceration of oral mucosa. Dose-limiti ng toxicity

Examples of Nanoparticles for Drug Delivery

DOXIL Future improvements?

H

HO

Rel

ativ

e L

ight

Uni

ts

Bac

kgro

und

Po

ly-L

-Lys

ine

Su

per

fect

(Den

dri

mer

)

Lip

ofe

ctam

ine

CD

-po

lym

er

4 P

EI

HO

HO

O

IDEAL SYSTEMIC DELIVERY VECTOR CYCLODEXTRIN POLYMER

• Non-toxic vehicle OH

O

OH O

O OH

HO O O OH

O

• Non-immunogenic O O

HO OH HO

OH O

OH OH

O

O HO

HO O

OH OH HO

HO O

OH

OH HO S

N O O

NH2

• Condensation of DNA OH HO

S O HO

O

(CH2)6 N

S NH2 2

OH HO O

OH HO

S + NH +

O HO OH HO

O O OH

O OH HO X O

HO

• Intracellular delivery • Targeting ligand

H2N O O

O OH

HO

• Stabilization of particles

• Ave. MW 5.8 kDa, polydispersity index Mw/Mn = 1.12 • Degree of polymerization ~ 5 (10 charges/chain)

verified by end group analysis and light scattering

Gonzalez et al. Bioconjugate Chemistry (1999) v10:1068-1074

PARTICLE ASSEMBLY TRANSFECTION COMPARISON

DNA

(a)

1.00E+08

1.00E+07

BHK-21

CHO-K1

1.00E+06

1.00E+05

1.00E+04

1.00E+03

1.00E+02

1.00E+01

(b)

1.00E+00

200 nm

Gonzalez et al. Bioconjugate Chemistry (1999) v10:1 068-1074

TOXICITY COMPARISON TO BHK-21 CELLS INCLUSION COMPLEX FORMATION

Polymer PEI IC50=23µM

Lipid, Lipofectamine IC50=6.4 µM

+

Polycation Superfect

IC50=11 µM

Cyclodextrin polymer IC50=320 µM

IC50 values reported as charge concentrations in the presence of DNA

Association Constant is 104–105

L

L

NEW METHOD OF SURFACE MODIFICATION TEM IMAGES OF PARTICLES

Stabilized Particle

L

L L

L L Pre-formed Particle

L L L

L L

L L L

A B A. Polyplexes in water B. PEGylated polyplexes

in water

C. Polyplexes in 50 mM

NaCl

D. PEGylated polyplexes

C D in 50 mM NaCl

Space bar is 100 nm

except in C where it

is 1000 nm

Targeted, Stabilized Particle Pun and Davis Bioconjugate Chemistry (2002 ) v13:6 30

Pun and Davis Bioconjugate Chemistry (2002 ) v13:6 30

Targeted Particles Biodistribution

Nucleic acid delivery Nanoparticle delivery

Pun, et al. Canc Biol Ther (in press) Pun, et al. Canc Biol Ther (in press)

Tumor uptake

Nucleic acid delivery Nanoparticle delivery

Pun, et al. Canc Biol Ther (in press)