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 !