modified liposomes with biomaterials delivery...
TRANSCRIPT
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Modified Liposomes with Biomaterials Delivery Applications
Dr. Rahau Shirazi
AOCS Meeting - Newcastle, Australia
November 8th, 2013
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Outline
Motivation and Background Focus Future Direction
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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)
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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
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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
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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
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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
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Outline Motivation and Background
Focus Future Direction
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+
+ + + + + + +
+ + + + +
+
+ +
+ + + + +
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:
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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
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CAEP (ADM12)
Agitation (top) vs Extrusion (below)
ESEM – Development Matters
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ESEM - Natural State Olympic Krill oil
(SD238)
746 nm
PGC 80 ICF8
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DC-Chol:DOPE:PEG2000PE 18:0
ESEM - Natural State
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Pegylated Cationic Liposomes DOTAP-DOPE:PEG2000PE14:0
Liposomes STEM – Negative Staining
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Liposomes STEM – Negative Staining
DC-Chol:DOPE:PEG2000PE 18:0
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Liposomes as Nano-Protectors
Unprotected Astaxanthin Liposome Encapsulated Astaxanthin
STEM – Negative Staining
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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
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Outline Motivation and Background Focus and Achievements
Conclusion
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- 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)
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Cyrus Safinya et al. Nature 489, 372–374, 2012
Qu
est
ion
s?
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Thank you
Lipid Team
IBT Group
Bradley Williams
Gavin Painter
Karen Johnston
Mallaghan Institute of Medical
Research
Carbohydrate Chemistry
Group
AOCS Meeting