nanoparticles for drug · pdf filenanoparticles for drug delivery suzie pun dept of...

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Nanoparticles for Drug Delivery

Suzie PunDept of Bioengineering, University of Washington

Frontiers in NanotechnologyMay 18, 2004


• Introduction – Systemic Drug Delivery

• Nanoparticles

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

• Examples : Liposomes for chemotherapeutic deliveryCyclodextrin particles for gene delivery

Methods of Drug Delivery

• Oral Delivery

• Inhalation

• Transdermal

• Implantation

• Injection

Advantages of Nanoparticles for Drug Delivery

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.

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)

Applications: Non-resistant cancers

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

•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.


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Applications: Intracellular Infections

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 Listeriatreatment. 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.

Examples of Nanoparticles for Drug Delivery Nanoparticles for Drug Delivery


• Nanoparticles



Drug Carrier Systems

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

Monoclonal antibodies; second-generation carriers with targeted antibodies or other

ligandsCarriers able to recognize a specific target< 1 µmThird

Liposomes, nanoparticles, polymer drug carriers

Carriers that can be given by a general route able to transport a drug to the target

site< 1 µmSecond

Microspheres and microcapsules for chemoembolization

Able to release a drug at the target site but needing a particular type of administration> 1 µmFirst

ExamplesDefinitionSizeGeneration


• Metal-based nanoparticles

• Lipid-based nanoparticles

• Polymer-based nanoparticles

• Biological nanoparticles

Metal Nanoparticles for drug delivery Metal Nanoparticles

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Metal Nanoparticles

Note: direct tissue injection

Lipid-based nanoparticles for Drug Delivery

DOPE

DSPC

DOTAP


Deliverables:

-Small molecules (Amphotericin B, Daunorubicin, Doxorubicin – all approved and marketed drug formulations) Gilead, Alza

-Viruses and bacteria (as vaccines) – in development

-Nucleic acids – in development


Many formulation methods --1. Mixing lipids together in

organic solution. 2. Remove solvent by

evaporation3. 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


Drug properties and Liposome association

Hydrophilic Retained in aqueous interior Slowly released over **may be difficult to get several hours-several dayshigh loading

Hydrophobic Inserted into hydrophobic Excellent retention interior of the liposome bilayer**can disrupt liposome at high concentrations

Intermediate Rapidly partition between Rapid release from liposomeslipid bilayer and aqueous but pH manipulation orphase formation of molecular

complexes can result in good retention

• 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:


Polymer-based nanoparticles for Drug Delivery

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DEGRADATION OF NANOPARTICLESHydrolysis of ester bond; degradation products

(alkylalcohol and poly(cyanoacrylic acid) are eliminated by kidney filtration.



Biological nanoparticles --Viruses

Scientific AmericanStratagene, Inc.

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

Biological nanoparticles --Viruses

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Thomas et al. (2003) Nature v4:346

Biological nanoparticles --Viruses Nanoparticles for Drug Delivery


• Nanoparticles



Stability of Colloids in Physiological Environments

1. Attractive van der Waals forces and random Brownian motion cause particle flocculation

2. Stabilization of colloids by electrostatic stabilization (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 potential:

M. Nikolaides

-higher ionic strengths collapse this boundary layer

-at high salt concentration, no stable region

-physiologic salt concentration ~150 nm

Aggregation of Colloids

C

A


water 150 mM salt

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


3. Stabilization of colloids by steric stabilization:-- Add polymers to the surface of particles to prevent the particles from

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

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

Steric Stabilization Dispersion Stabilization

Steric Stabilization

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

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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, storage 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



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!

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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.



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

Kupffer Cells


1000 nm

6000 nm500 nm

200 nm

10,000 nm

“+”-charged

“-”-charged

neutral

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

PEGylated

Non-PEGylated

Biodistribution of PEGylated Polymer-based Nanoparticles

Biodistribution of Lipid-based NanoparticlesPEGylation

Non-PEGylatedliposomes

PEGylatedliposomes

Why is there a dose-dependence?

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



• Nanoparticles


• Examples Liposomes for chemotherapeutic deliveryCyclodextrin particles for gene delivery

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Non-specific targeting: EPR Effect

• Tumors generally can’t grow beyond 2 mm in size without becomingangiogenic (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)


Non-specific Targeting

• Small Molecules– Galactose/Glucose/Mannose– Folate

• Peptides– RGD

• Proteins– Transferrin– Antibodies– LDLs

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endocytosis.html

Targeting Ligands Targeting


Targeting




• Nanoparticles



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DOXIL

Formulation

1. Doxorubicin-containing core

2. Lipid Bilayer membrane

3. PEG-coated surface

4. <100 nm


DOXIL Proposed Mechanism of Action


DOXIL Pharmacokinetics

Rats Dogs

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


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/m2; 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-limiting toxicity


DOXIL Efficacy

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

**p = .01.

*Expressed as percentage.

63.3 wks86.1 wks**OSPlat Sens

37.3 wks33.4 wksOSPlat Res

28.8 wks28.4 wksTTP Plat Sens

6.5 wks12.3 wksTTP Plat Res

12*16*PR

5*4*CR

ORR

18.3 wks18.4 wksTime to progression

Topotecan (n = 119)

Doxil (n = 118)


DOXIL Future improvements?

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• Non-toxic vehicle• Non-immunogenic• Condensation of DNA• Intracellular delivery• Targeting ligand• Stabilization of

particles

IDEAL SYSTEMIC DELIVERY VECTOR

• 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

CYCLODEXTRIN POLYMER

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

OHO

OHO

OHO

OHO

O

HO

HO

OO

OHHOO

OOH

OHO

O

OH

OOH

O

HOOOH

OH

OH

S

OH

HO

HO

SHN H

N

NH2+ NH2

+S

X

OHO

OHO

OHO

OHO

O

HO

HO

OO

OHHOO

OOH

OHO

O

OH

OOH

O

OH

OOH

OH

OH

OH

HO

HO

SNH2

(CH2)6

H2N

PARTICLE ASSEMBLYDNA

(a)

(b)

200 nm

OHO

OHO

OHO

OHO

O

HO

HO

OO

OHHOO

OOH

OHO

O

OH

OOH

O

HOOOH

OH

OH

S

OH

HO

HO

SHN H

N

NH2+ NH2

+S

X

OHO

OHO

OHO

OHO

O

HO

HO

OO

OHHOO

OOH

OHO

O

OH

OOH

O

OH

OOH

OH

OH

OH

HO

HO

SNH2

(CH2)6

H2N

TRANSFECTION COMPARISON

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

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

Back

grou

nd

Poly

-L-L

ysin

e

Supe

rfect

(Den

drim

er)

Lipo

fect

amin

e

CD

-pol

ymer

4 PEI

Rel

ativ

e Li

ght U

nits

BHK-21CHO-K1

Lipid,LipofectamineIC50=6.4 µM

PolycationSuperfect

IC50=11 µM

CyclodextrinpolymerIC50=320 µM

Polymer PEIIC50=23µM

IC50 values reported as charge concentrations in the presence of DNA

TOXICITY COMPARISON TO BHK-21 CELLS

Association Constant is 104–105

INCLUSION COMPLEX FORMATION

+

11

LLL

LL

L

L

LL

LL

L

L

L

L

L

L

LLLLLLL

LL

L

L

LL

LL

L

L

L

L

L

L

LL

L

L

LL

LL

L

L

L

L

L

L

LL

L

L

LL

LL

L

L

L

L

L

LPre-formed Particle

Stabilized Particle

Targeted, Stabilized Particle

PEG-Ad

Ligand-PEG-Ad

NEW METHOD OF SURFACE MODIFICATION

Pun and Davis Bioconjugate Chemistry (2002 ) v13:630

C D

BA A. Polyplexes in waterB. PEGylated polyplexes

in waterC. Polyplexes in 50 mM

NaClD. PEGylated polyplexes

in 50 mM NaCl

Space bar is 100 nm except in C where itis 1000 nm

TEM IMAGES OF PARTICLES

Pun and Davis Bioconjugate Chemistry (2002 ) v13:630

Targeted Particles

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

Biodistribution

Nucleic acid delivery Nanoparticle delivery


Tumor uptake


Nucleic acid delivery Nanoparticle delivery

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