I have encapsulated the hydrophobic drug in liposomes. I analyzed the encapsulation efficiency by HPLC for both the supernatant and the pellet.
First, I centrifuged the liposomal solution, took the supernatant, lysed the pellet with triton 2%, and analyzed them separately. However, there is a big difference between the results. The supernatant showed 40-50 % EE and the lysed pellet showed less than 3%. I used these formulas:
For supernatant: EE%=((total drug-loss drug)/ total drug)*100
For lysed pellet: EE%=((entrapped drug/total drug)*100
To find out the %EE which method is accurate?
Hi Zahra Kariminezhad ,
A good part of the answer depends on where (supernatant vs pellet) you are expecting to find your drug-loaded liposomes. A bit more clarification of how the loss drug is determined and how this centrifugation fits in your methodology would be good for a more accurate answer.
Since you mentioned "lysed" pellet, I am assuming that the centrifugation is for pellet-recovering purposes and that you are expecting to find your drug-loaded liposomes in the pellet.
If that is the case, the %EE is the value you obtain with the pellet. The %EE is only associate with the liposomal component of your preparation. So, if you are not expecting to find the liposomes in the supernatant, there is no need calculate %EE for it. Also, the drug amount you obtain from the pellet doesn't have to match what you have in the supernatant. However, the addition of both %drug (compared to total drug) will need to be close to 100% which is not the case here. This discrepancy can either stem from inaccurate total drug quantification or if supernatant volume is not accounted for or poor lysis of the pellet.
Perhaps, another look at what you define as the "loss drug" is worthwhile. The supernatant would be the immediate liquid environment of your liposomes and could contain the loss drug (if the drug is expected to be soluble in the supernatant). A couple of considerations regarding this is that 1) if the supernatant is aqueous, it is likely that some loss drug would be in the pellet as hydrophobic drugs tend to precipitate and centrifugation would facilitate this. 2) Depending on the speed of the centrifugation, you could still have liposomes in the supernatant...
This answer is based on the assumption that that the liposomes are in the pellet.
Check out his thread about a similar topic
https://www.researchgate.net/post/How-can-I-determine-the-encapsulation-efficiency#:~:text=The%20EE%25%20was%20calculated%20as,detected%20only%20in%20the%20supernatant.
This article about a liposomal formulation with a hydrophobic drug may also help with the calculation of %EE (Page 3652).
Article Liposomal Encapsulated Curcumin Effectively Attenuates Neuro...
Good luck!
Dear Akpedje Serena Dossou,
Thank you very much for your valuable explanation and the article.
The centrifuge condition that I used was 36000 rpm, for 1h, at 4 C.
You're welcome Zahra Kariminezhad !
From the centrifugation info, it looks like you are expecting the drug in the drug-loaded liposomes in the pellet. So, you will only need to calculate the EE% in the lysed pellet :).
Question: This is the only centrifugation involved in the methodology? If not, is there an initial gentle centrifugation step that you perform on your preparation to remove the unbound/non-incorporated drug? This info could further help in providing a more accurate answer (if you still need an answer).
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