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pH-Sensitive Liposomes for Effective Release of Therapeutics
By: Savita RamlallMentor: Professor Sofou & Shrirang Karve
Polytechnic University
Laboratory of Drug Delivery Systems
What are Liposomes?A liposomes is a spherical vesicle with a membrane composed of a phospholipid bi-layer used to deliver drugs or genetic material into a cell.
When using liposomes as drug carriers the main aim is to reduce toxic side affects in sensitive organs such as the heart and brain and to target specific tissues such as cancer cells.
The Purpose of This Project
It involves lipid separation which leads to imperfections in membrane. This leads to leakage of drugs.
Materials
Prepared liposomes
Pipettes
Fluorescence Spectrometer
ProceduresTake Lipids in (CHCl3 solution) in a round
bottom flask dried under N2 for 5 minutesAnneal it with PBS solution for 2 hoursExtrude it through 100 nm membraneMeasure the intensities for excimer and
monomer for pH 7.4; 5.5; 4.0
Leakage mechanism: lipids with pyrene-labeled fatty acid chains to detect membrane asymmetry and destabilization
ex: 344 nm/ em: 396 nm ex: 344 nm/ em: 470 nm
time (minutes)10 100 1000
% c
onte
nt r
eten
tion
-60
-40
-20
0
20
40
60
80
100
pH = 7.4 pH = 5.5pH = 4.0
day 2day 3
Release of contents from rigid PEGylated liposomes vs pH (in PBS)
• pH dependent content release
• size of ‘lipid-packing’ discontinuities (3kDa dextrans are released similarly, 10kDa dextrans are retained at all pH values)
Release of contents from rigid PEGylated liposomes vs pH (in media with 10% serum)
time (minutes)10 100 1000
% c
ont
ent
ret
ent
ion
-60
-40
-20
0
20
40
60
80
100
day 2 day 3
pH = 7.4 pH = 5.5pH = 4.0
• Instantaneous content release at pH 4• significant release at pH 5.5 • stable content retention at pH 7.4 the first hours
Monomer-to-eximer ratio
day 1 day 2 day 3
pH = 7.4 0.64 ± 0.05 0.56 ± 0.08 0.57 ± 0.05
pH = 5.5 0.62 ± 0.06 0.52 ± 0.08 0.45 ± 0.06
pH = 4.0 0.58 ± 0.05 0.40 ± 0.05 0.42 ± 0.11
monomer-to-eximer ratio raft formation
Result & DiscussionContent release at different pH in PBS & media (10% serum) studied using calcein leakage.Higher content release at lower pHMembrane asymmetry at different pH was studied using pyrene monomer – excimer ratio.Asymmetry increases with lower values of pH.
Further StudiesDuring the remaining summer I would like to see the effect of temperature on the raft formation of the liposomal membranes and compare it with the effect of pH levels.
References
http://biophysics.org/discussions/2004/london.pdf
http://www.ajhp.org/cgi/content/abstract/46/8/1576
http://explantion-guide.info/meaning/liposome.html
Ulrich, A Biophysical Aspects of Using Liposomes as Delivery Vehicles vol. 22, NO. 2 (2002) 129-146
Torchilin, V Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate. (2004) 96-107
Lasic, D Noel applications of liposomes vol. 16 (1998) 307-319
AcknowledgementsProfessor Sofou
Shrirang Karve
Polytechnic University
Everyone in the Laboratory of Drug Delivery Systems
Dr. Sat
Harlem Children Society
The Audience
Special Thanks
LAB