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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