Nanoparticles for Targeted Drug Delivery

Joseph ZasadzinskiGuohui Wu, Cecile Boyer, Ryan van Zanten,

Chris Steinbeck and Brad ChmelkaDepartment of Chemical EngineeringUniversity of California Santa Barbara

Drug Carrier Motivation: Efficacy vs Toxicity

Delivery vehicles increase Therapeutic IndexCirculating reservoirs of drug, localization at the diseased site

100%

50%

%response

Log(Dose)TD50

toxicresponse

ED50

effectiveresponse

Therapeutic Index:

TI = TD50 / ED50

TI

• Lowers the effective dose: ED50• Lowers drug exposure to healthy tissue: TD50

Drug Carrying Liposomes - Prokaryotic Cells

Liposomes in Drug Delivery:• Variety of membrane, interior compositions• Increased dose-limiting toxicities as compared to directly injected drug• Drug retention reduces exposure to healthy tissue / toxic side effects• 24 hour circulation time → passive accumulation in leaky tumors• 30 years of scientific evaluation as drug carriers• FDA approved (Doxil and Daunosome), but few, if any others - why?

Single compartment

Single Bilayer

Controlled transport across the boundary

Prokaryotic Cell MimicsUnilamellar Vesicles

Technology Limiting Problem: Liposomes Leak

Most small molecule drugs are released too quickly in a physiological environment for best efficacy

No new liposome carriers approved for past decade

• Vincristine: Chemotherapy agent

Liposome retention > 8 weeks in saline, < 1 hour in serumImproved biodistribution in rats, but efficacy limited by premature release

(Webb et al., Cancer Chemotherapy and Pharmacology 42, 461 (1998))

• Ciprofloxacin: Antibiotic

Liposome retention > 4 weeks in saline, < 30 minutes in serumImproved efficacy against pneumonia in rat model, but efficacy

limited by premature release(Bakker-Woudenberg et al., Antimicrobial Agents, 45, 1487 (2001))

Eukaryotic Cell Mimic – the Vesosome

Multiple encapsulation approach:• Each membrane can be of a different composition• Each membrane can be optimized for a particular set of functions• Improved and independent control over permeation and degradation• Macromolecules / Colloids can be encapsulated and protected• Co-encapsulation of different drugs, imaging agents, triggers…• Self-assembly and phase behavior is key to formation

Multi compartment structure

Higher levels of functionality

Nature’s approach to better retention

and division of laborEukaryotic Cell The Vesosome

+ EthanolHeat toT > Tm

T<Tm (Lβ’ phase) T>Tm (Lα phase)Interdigitated

Interdigitated Phase of Saturated Lipids

High curvature stress→ Formation of stiff, open bilayer sheets

0.50 µm

< 45 C

Above Tm, the bilayers become more flexible and close to form complete shells

1.00 µm

> 45 C

HEATT > Tm

Vesosome Self-Assembly Process

Washing

Interdigitationfusion+ EtOH

Encapsulation

Separation

Testing Drug Release

T>Tm

Drug Loaded50nm vesicles

or other nanoparticles

The vesosome self-assembly processcan be used to encapsulate any

colloidal particle in a suspension within a bilayer

MIX

US Patent 6,565,889, (2003)

Interior Nanoparticles

Exterior Membrane

Phospholipase A2 – induced dye release

PLA2 does not reach the inside liposomes!effective protection from PLA2 degradation

Liposomes 10ng/mL

Liposomes 5ng/mL

Vesosomes0ng/mL

Vesosomes5ng/mL

Vesosomes10ng/mL

0%

20%

40%

60%

80%

100%

0 4 8 12 16 20 24

% C

F re

leas

e

Time (hours)

Serum – induced dye release

The external membrane extends the 1/2 life for dye release from ~ 15 minutes to > 15 hours in serum

0%

20%

40%

60%

80%

100%

0 2 4 6 8 10 12Hours

75% Serum

50% Serum

25% Serum

Buffer

Unilamellar Liposomes

Whatever in serum is enhancing release,it is depleted and release slows

1/2 life: 15 min

0%

20%

40%

60%

80%

100%

0 2 4 6 8 10 12Hours

75% Serum50% Serum25%SerumBuffer

Vesosomes

Ciprofloxacin, pH Loading and NMR DetectionpH ~ 7

OUT

Lipid bilayer

INAcidic pH

Protonation into cationic, impermeable form

Accumulation into acidic compartment

Easy detection with 19F NMR

• Ammonium Sulfate gradient pH gradientNH+

+

COOH

NH+

Neutral

COO-

NH+

+

COOH

NH+

Neutral

COO-Cullis P.R., et al., BBA-Reviews on Biomembranes 1331, 187 (2000)

The fluorine peak shifts with total ciproconcentration to measure initial loading

Internal and external cipro are monitored by 19F NMR during release via shifts due to diffusion and pH

9mMCipro

45mMCipro

121 119120120.5 119.5 ppm

126 116121 118119 117120122123124125

Ciproin

~ 64.7%

Ciproout

ppm

Serum – Induced Cipro Release

Time (hours)

% C

ipro

rele

ase

(from

inte

rnal

pea

k de

crea

se)

Drug Release Time extended by factor of 100!Literature Hypothesis The neutral / permeable form of Cipro is

released via permeation as the pH gradient is depleted

-2

-1

0

3.5 4.5 5.5 6.5 7.5 8.5

pKa(Cipro)=6.2

pH

Encapsulated, pH 5 Free, pH 7

0 -219F Chemical Shift (kHz)

Che

mic

al s

hift

(kH

z)

Incr

easi

ng ti

me

Cipro can be used as a pH meter!

We observe only pH 5 or pH 7 environments in serum!

In liposomes, release is by a failure of carrier rather than by a diffusive equilibration of pH

followed by fast permeation of neutral species!

No

cipr

oat

inte

rmed

iate

pH

!

The Cipro chemical shift depends on the solution pH!

We can monitor the local environmentthe Cipro encounters during release

Some insights on the release mechanism via NMR

Chemical Shift Data on unilamellar liposomes in serumevolves with time to show changes in various environments

Choline peaks in serum increase with time

Net removal of lipids from vesicles

Unsaturated Lipid and high density lipoproteins (HDL)

decreaseInsertion into bilayer

In the bloodstream, release from liposomes is by degradation of the carrier (protein insertion, enzymatic digestion), not by permeation. This explains the dramatic decrease in drug half-life relative to saline exposure

In the Vesosome, the exterior bilayer acts as a barrier to enzymes, lipases, etc to prevent attack on the interior carriers. The bilayers have to be compromised in sequence, to release their contents. Release occurs over the same time scale as circulation lifetime.

The Vesosome structure provides the differenceOptimizing membrane composition is next goal

Conclusions and Acknowledgements• Drug release from liposomes is via carrier failure• Vesosomes can be constructed via simple self-assembly

and knowledge of lipid phase behavior• Structure of Vesosome provides 100 fold increase in

drug retention in serum/blood• Vesosome drug retention is similar to best carrier

circulation lifetime, potential new platform carrier• NMR allows for non-invasive, no label monitoring

of drug release profiles in serum/blood

Support for this project came from a Program of ExcellenceIn Nanotechnology Grant from the NIH and the Institute for Collaborative Biotechnology.