Modified Liposomes with Biomaterials Delivery...

Modified Liposomes with Biomaterials Delivery Applications

Dr. Rahau Shirazi

AOCS Meeting - Newcastle, Australia

November 8th, 2013

Outline

Motivation and Background Focus Future Direction

Efficient Vector:

Encapsulate, Deliver, Release Non-viral vs. viral vectors:

+ Ease of production + Unlimited Encapsulation Capacity + Safety - Efficiency

Liposomes – Delivery Vehicles

Modified Liposomes Tailored to enhance efficiency Encapsulate (Cationic – pH Sensitivity) Protect (e.g. Oxidative Damage, Enzymatic Degradation, Aggregation in Serum) Deliver (Enhance Circulation, Controlled Release/Environmentally responsive,

Increase Bioavailability)

5 µm

Cationic Lipid DNA

++

++++

++

++

++- -

- -- -

--

- -

- -- -

+++

- -- -

- - - --

-

++ +++++

+++++++

Positively Charged

Liposomes

50-200 nm

+

CL–DNA

ComplexesCounterion Release is Driving

Force of Formation

+-

+-

+

-

+-

+

-

Dry Lipid Film

Sonication Extrusion

Hydration Incubation

Agitation

Liposomes – Development

Non-viral Gene Delivery Vectors

Membrane Contain Mixtures of Lipids (Charged, Neutral, Bioactive,..)

Cationic Liposome-Nucleic acid self-assembly

Electrostatic interactions

DNA Complexes with CL

+

+ + + + + + +

+ + + + + +

DNA

- - Lip

id B

ilaye

r

nt

CLCL

N

nNnN

N

N

PEGylation: + Delivery in vivo + Stability of Liposomes + Circulation lifetime - Reduce membrane interactions Modified Liposomes Control Release / disassembly

Poly (Ethylene Glycol)-Cationic Liposomes Encapsulation

A pH-sensitive linker, (acylhydrazone-based PEG2000-lipid): Stable at neutral pH (blood) triggered at pH 5 to 4, (late endosomes)

Chan, C.-L.; Majzoub, R. N.; Shirazi, R. S; Ewert, K. K.; Chen, Y.-J.; Liang, K. S.; Safinya, C. R.: Biomaterials 2012

O

O

O

N

H

N

H

N

O

OO

OO

OH

n

OO

O

n

n

O

O

O

N

H

N

H

NH2

O

O

O

O

OH NH3

+O

OCl-

O

O

O

N O

O

H

+

+

TBTU, DIEA

N2H4 hydrate

( 20 x excess)

CH2Cl2/MeOH

mol. sievespH 5

N

O

PhI(OAc)2,

cat.

DOB--Ala-hydrazide ("Tail") mPEG2000-CHO ("PEG")

acid-labile HPEG2K-lipid ("PL")

Stimuli Responsive Cationic Lipids

Ref. Chan, C.-L.; Majzoub, R. N.; Shirazi, R. S; Ewert, K. K.; Chen, Y.-J.; Liang, K. S.; Safinya, C. R.: Biomaterials 2012

Mature endosome acidifies its contents cleaves the PEG chains closer contact between the membranes of complex and endosomes

Live-cell imaging of transfection by PEGylated CL–DNA complexes: DIC and fluorescence micrographs of mouse L-cells incubated with doubly labeled PEGylated CL–DNA complexes. (A, B) Images for complexes prepared with the acid-stable PEG2K-lipid. (C, D) Images for complexes prepared with the acid-labile HPEG2K-lipid.

Ref.: Chan, C.-L.; Majzoub, R.; Shirazi, R. S; Ewert, K.; Chen, Y.; Liang S.; Safinya, C. Biomaterials 2012

Stimuli Responsive Cationic Lipids

Outline Motivation and Background

Focus Future Direction

+

+ + + + + + +

+ + + + +

+

+ +

+ + + + +

Hydrophobic: Protected in lipid bilayers Hydrophilic: Sandwiched between lipid bilayers Amphiphilic: Orient accordingly Liposome-Encapsulated Biomaterials Characterization: Microscopy – Structural Characterisation (Optical, ESEM, STEM) Fluorescent Assays, Nanodrop, UV-Vis – Encapsulation Ability Bioassays – Efficiency and toxicity - Gene Delivery in vivo

Biomaterials for Nano-Delivery:

Natural :

CAEP (ADM12) Olympic Krill oil

(SD238) PGC 80 ICF8

Liposomes : Synthetic and Natural Sources Synthetic:

Cationic – DOTAP, DC-Cholesterol pH Sensitive – DODAP, CHEMs Neutral – DOPE, DOPC PEGylation – PEG2000-PE (18:0, 14:0)

18:1 (Δ9-Cis) PE (DOPE)

18:1 TAP (DOTAP)

DC-Cholesterol∙HCl

18:1 DAP (DODAP)

Cholesteryl hemisuccinate CHEMS

18:1 (Δ9-Cis) PC (DOPC)

Development of Modified Liposomes

CAEP (ADM12)

Agitation (top) vs Extrusion (below)

ESEM – Development Matters

ESEM - Natural State Olympic Krill oil

(SD238)

746 nm

PGC 80 ICF8

DC-Chol:DOPE:PEG2000PE 18:0

ESEM - Natural State

Pegylated Cationic Liposomes DOTAP-DOPE:PEG2000PE14:0

Liposomes STEM – Negative Staining

Liposomes STEM – Negative Staining

DC-Chol:DOPE:PEG2000PE 18:0

Liposomes as Nano-Protectors

Unprotected Astaxanthin Liposome Encapsulated Astaxanthin

STEM – Negative Staining

Liposomes Target Gene Delivery

Unique Cationic Stealth Liposomes

Encapsulation/protect CpG- Oligodeoxynucleotides (CpG-ODN) - Nuclease degradation Enhance Bioactivity - Unique Phosphatidylglyceroylalkylamine (PGAA) based lipids isolated from thermophilic bacteria Meiothermus silvanus

Specific CpG-ODN sequences with Immunostimulatory adjuvant activity mediated through Toll-Like Receptor 9

Synergistic Effects of Strong Encapsulation Ability Combined with Specific Bioactivity of

PGAA

Outline Motivation and Background Focus and Achievements

Conclusion

- Liposomes encapsulate hydrophobic nano-material within their hydrophobic bilayer, and hydrophilic guests in the larger interior.

- 'stealth‘/PEGylated liposomes developed for drug-delivery applications can carry specific peptides, polymers, bioactive materials.

- Cationic liposome–NA complexes: onion-like structure with NA sandwiched between cationic membranes.

- Stimuli responsive liposomes aid control release of biomaterials in stimulus media (e.g. HPEG2000)

Cyrus Safinya et al. Nature 489, 372–374, 2012

Qu

est

ion

s?

Thank you

Lipid Team

IBT Group

Bradley Williams

Gavin Painter

Karen Johnston

Mallaghan Institute of Medical

Research

Carbohydrate Chemistry

Group

AOCS Meeting