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Wenting XuDepartment of Pharmaceutical Engineering
2011.6.2
A study on the development of mucoadhesive targeting liposomes
Aims of the study
o Development of folate-tethered mucoadhesive liposomes containing polymeric drug
o To improve gastrointestinal absorption of hydrophilic polymer drug
Ideas of the studyo Approach 1: Increase of the rigidity of liposomal
membrane DSPC Vs EPC liposomes
o Approach 2: Increase of mucoadhesion of liposomes by coating with chitosan
o Approach 3: Enhanced intestinal absoption of liposomes through folate transport system(Folate-PEG)
Introduction
What is a Liposome?
o In drug delivery system:
• Bilayer: Hydrophobic drugs
• Inside of liposomes:Hydrophilic drugs
o Vesicles made of lipid bilayer
Basic composition of liposomes
o Phospholipid
o Cholesterol
Phospholipids
DSPC (Distearoyl phosphatidyl choline)
EPC(Egg phosphatidylcholine)
Fatty acid chain16:0-18:1
Fatty acid chain18:0-18:0
EPC &DSPC
EPC DSPC
Chemical formula C42H82NO8P C44H88NO8P
Phase transition temperature −10 ± 5 ◦ C 55 ◦ C
Liposomal state at RT Liquid Gel
Fluidty of Liposomal membranes at RT High Low
Cholesterol
Fluidity reduction
Hydrophilic
Hydrophobic
Liposome/Cell Interaction
Adsoption Fusion
EndocytosisLipid transfer
Modification of liposomal surface
o Protection of drug
o Avoidance of RES ( reticuloendothelial system )
o Extended release
o Targeting
Mucoadhesion
Mucoadhesion
o Attachment to a biological substrateMucous gel layer
o Increasing residence time of liposomes
o Mucin
Chitosan
o Nontoxic
o Mucin recognitive molecules
o Chitosan-coating liposome Liposome ( - ) : A Chitosan ( + ): B
Folate-conjugated liposomes
Intestinal absorption enhancement :
? Through folate transport system in brush border membrane
FITC-dextran 3k
As a model drug mimicking peptide drug such as calcitonin
o High sensitivityConcentrations down to 1ng/ml can be detected in tissue fluids
Experiments & Results
Preparation of liposomes
Composition of liposomes
Lips EPC DSPC DCP Cholestrol PEG2000PE
DSPEPEG2000FOLATE
FFITC-Dextran 3k
EPC-PEG 8 2 1 0.1 0.1
EPC-PEG-FOLATE 8 2 1 0.1 0.1
DSPC-PEG 8 2 1 0.1 0.1
DSPC-PEG-FOLATE 8 2 1 0.1 0.1
Preparation of Liposomes
Liposome suspension
Preparation of Liposomes
Extrusion through 0.4um and then 0.2um membrane
Removal of unencapsulated dextran- 3K
Dialysis
Encapsulation efficiency
Encapsulation efficiency
Encapsulation efficiency =
Drug-to-phosphate ratio of loadingDrug-to-phosphate ratio measured after dialysis ×100%
Encapsulation efficiency
EPC-PEG Lips EPC-PEG-Folate Lips DSPC-PEG Lips DSPC-PEG-
Folate Lips
Encapsulation efficiency 29.53+6.66% 38.16+16.1% 30.1+4.96% 34.1+4.49%
Result:
Uptake by Caco2 cell
Uptake by Caco2 cell
The Caco-2 cell line
o Derived from colon cancer cell
o Known to have similar characteristics with the small intestinalepithelial cells
o Can be used as HTSS for studing the drug uptake in instestine
Hamilton test simulation system
Uptake by Caco2 cell
o Folate-free RPMI 1640 medium Folate receptor overexpressed (2 weeks)·
o Folate medium RPMI 1640 medium
To study folate receptor mediated transport
Cell culture
Procedure of Caco2 cell uptake experiment
Cell seeding: 50000 cells/well
in 24-well overnight
Remove old medium
Add serum free RPMI 1640
Incubate 30minsIncubated with Liposomes for
2hours
Wash three times with cold
PBS
Add Triton X -100
Fluorescence(485nm/535nm)
Result of Caco2 cell uptake experiment
No treat EPC-PEG Lips EPC-PEG-FOLATE Lips DSPC-PEG Lips DSPC-PEG-FOLATE Lips0.0
50.0
100.0
150.0
200.0
250.0
Folate mediumNo folate medium
%Fluorescent
Mucoadhesion studies
&
Evaluation of the absorption of liposomes
Male SD rats( fasted for at least 24h)Oral administration of liposomes
Collection of blood( 2hours)Sacrifice of rats
Removal of intestines and collection of segments(duodenum, jejunum and ileum)
Homogenization of intestion in PBS
Mesurement of fluorescence after centrifugation
In vivo experiment
Result of mucoadhesion studies
0
50
100
150
200
250 DuodenumJejunumIleum
%Fl
uore
scen
ce
Blanck
EPC-PEG
Lips
EPC-PEG
-CS Lips
EPC-PEG
-FOLA
TE Lip
s
EPC-PEG
-FOLA
TE-CS
DSPC-PEG
Lips
DSPC-PEG
-CS-Lips
DSPC-PEG
-FOLA
TE Lip
s
DSPC-PEG
-FOLA
TE-CS L
ips0
50
100
150
200
250
Jejunum
%Fl
uore
scen
ce
Blood dextran 3k concentration
Blanck
EPC-PEG
Lips
EPC-PEG
-CS Lips
EPC-PEG
-FOLA
TE Lip
s
EPC-PEG
-FOLA
TE-CS
DSPC-PEG
Lips
DSPC-PEG
-CS-Lips
DSPC-PEG
-FOLA
TE Lip
s
DSPC-PEG
-FOLA
TE-CS L
ips0
200
400
600
800
1000
1200
%Bl
ood
dext
ran
conc
entr
ation
Conclusion
o Approach 1: Increase of the rigidity of liposomal membrane(DSPC) DSPC significantly increased the drug retention in mucosal layer of
intestine and blood drug concentration
o Approach 2: Increase of the mucoadhesion of liposomes(Chitosan) Modification with chitosan could not increase the mucoadhesion of
liposomes (might be due to the bigger size and breaking the liposomes)
o Approach 3: Enhanced intestinal absoption of liposomesthrough folate transport system(Folate-PEG)
Modification with folate could not increase the intestinal absoption of liposomes
Thank you !
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