Materials and Method

Curcumin as the API is extracted from turmeric. The liposome is prepared using thinlayer homogenization method. The liposom is created using three different materials, S100PC,
CHOL, and mPEG2000-DSPE. S100PC (Soybean phosphatidylcholine), Cholesterol (CHOL),
andmPEG2000-DSPE(1,2-distearoyl-sn-glycero-3-phosphoethanolamine[methoxy(polyethyleneglycol)-2000]) will be use in a 90:5:5 ratio. Organic solution (Methanol and Chloroform)
will be use to disolve the materials before the formation of liposome. For the antigen, we will
use antigen OX26 mAb.
The first step of this experiment is to extract curcumin from turmeric. The maserasi
extraction method will be used to extract API from turmeric. The steps are:
1. Drying and grinding the dried turmeric using blender grinder and separated through a
sieve tray.
2. Extraction of turmeric using ethanol and water by adding the grinded turmeric with
ethanol 96% with the ratio of 1:3.
3. Stirred the solution every 3 hours to better extract the curcumin.
In order to synthesis an immunoliposome, first we need to encapsulate API into the
liposomes and created a liposome. The preparation step in the formation of (PEG)ylatedliposomes are:
1. Disolving 20 mg of lipid into a mixture of organic solution containing methanol and
chloroform with the ratio of 1:9 v/v until the concentration of 10 mg/ml achieved.
2. Evaporating the organic solution using rotary evaporator or using nitrogen stream.
3. A thin layer of lipid will be deposite at the flask wall.
4. Disolving the API (Curcumin) into PBS (Phosphat Buffer Saline) to encapsulate it
into the lipid layer.
5. Forming the liposome by hydrating lipid layer with PBS-API solution until the lipid
concentration is 40 mg/ml and sonicate the solution.
6. Adding more PBS until a concentration of 4 mg/ml is achieved, than sonicate it to
reduce it size to make it unilamelar (SUV).
After we prepared the liposomes and API, we formed an antigen linkage onto the
liposomes, forming an immunoliposomes. The steps are:
1. Harvesting the antigen used for the formation of immunoliposomes. In this
experiment, if rat was used as test subject, then the antigen will be anti-transferrin
receptor OX26 mAb.
2. Purifying the antigen using G Sepharose affinity chromatigraphy
3. Reacting OX26 mAb with iodine (125I) and chloramine to label the antigen
4. Reacting the labelled antigen with 2-iminothiolane in 0,15 M Na-borate buffer/0,1
mM EDTA for 60 minutes.

5. Conjugating the thiolated antigen with maleimide derivatized PEG 2000-DSPE for 24
hours at 4oC forming antigen-PEG2000-DSPE.
6. Incubating the antigen-PEG2000-DSPE with (PEG)ylated liposome at 4oC for 24 hours
to form immunoliposomes.
7. Separating the immunoliposomes formed using sepharose chromatography and
purifying the immunoliposomes using freeze dryer.
To evaluate the performance of our liposomes, we design a release experiment, both
in vitro and in vivo to see the performance of our drugs. The in vitro release of curcumin will
be evaluated using the dialysis method. To perform this test, an aliquot of liposomal solution
(0,1 mL) was placed in a dialysis tube after reconstituting the purified immunoliposome in
PBS. The tube is immeresed in a release medium (such as PBS) containing 0,1% Tween to
maintain sink condition. The release profile than analyse by taking samples in certain
intervals for 24 hours. The curcumin concentration can be determine using HPLC.
As for the in vivo test we will use cellular uptake experiments using rats as test
subject. Rats was first anesthetized using ketamine and/or xylazine. To see the performance
of our drug, we inject immunoliposomes via femoral vein. Then, we collect the data from
blood samples at predetermined intervals for 1 hours. After 24 hours from immunoliposomes
injection, rats will be decapitated for removal of the brain. The pharmacokinetic parameters
then can be calculated by fitting plasma radioactivity (from the labelled immunoliposome)
data to a biexponential equation.