Post on 03-Feb-2022
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.