I have been trying to encapsulate VEGF (a kind of protein) using PLGA w/o/w. The EE%, that I determined, was quite low: 10%. Here is the protocol I used:
1. 100 ug VEGF was reconstituted in 1ml DI water and 1ml Human Albumin Serum (HSA) 5%.
2. Dissolve 20mg PLGA in 2ml DCM.
3. Add 40ul VEGF solution into PLGA solution.
4. Ultrasonicate first emulsion in 10 sec under condition: 750W, and 40% amplitude.
5. Pipetting slowly the first emulsion into PVA 1.5% and Tween80 2%.
6. Ultrasonicate second emulsion in 30sec under condition: 750W, and 40% amplitude.
7. Waiting 3hr to allow DCM evaporate.
8. Free-dry overnight and then collect particle
After that, I determine amount of VEGF in particle by using VEGF ELISA kit.
Dear Huy Nguyen,
Can you describe the method to determine the EE% of VEGF before you use the ELISA kit? I mean, is there any pre-treatment procedure before hand?
Hi Raditya Iswandana ,
Here is the procedure for the pre-treatment of determination EE%:
1. 2mg VEGF-loaded PLGA particle was dissolve in 200ul DMSO in 10 minute.
2. Dilute solution with ratio 1:10 by DI water.
3. Continue dilute solution with Standard Dilute Buffer from the ELISA kit with ratio 1:10, 1:20, 1:40 and 1:80.
4. Do ELISA for quantifying VEGF concentration.
Hi Huy Nguyen,
I don't know exactly if the step 1 in your procedure is able to dissolve all VEGF content in your PLGA particle. Can you make sure that by following this step all VEGF content will be extracted?
Hi Raditya Iswandana
After adding DMSO to VEGF-loaded PLGA particles, the solution was looked transparent, which means PLGA was dissolved completely.
On the other hand, I didn't extract VEGF. I directly diluted the VEGF in DMSO with DI water (DMSO also can be dissolved in water). So, I don't think the VEGF was lost during pre-treatment stage.
Hi Huy Nguyen,
Have you ever read these articles, perhaps it will help you to solve your problem:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106449/
https://www.sciencedirect.com/science/article/pii/S0168365912000752
https://www.ncbi.nlm.nih.gov/m/pubmed/26004370/
https://www.sciencedirect.com/science/article/pii/S1549963415001495?via%3Dihub
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