Hi everyone,
I am aiming to load a hydrophilic drug inside liposomes using thin film hydration method but the issue is i can't assure the final concentration inside the liposomes is achieved because some evaporation occur during the final thin film hydration step causing the concentration to increase (for example if i added 10mM drug solution the final concentration of the liposomal suspension will be 12mM due to evaporation of some volume).
Hi Mohammed
The method I generally use is as follows:
- Following formation of your drug encapsulated liposomes purify a sample using centrifuge spin columns with a MWCO appropriate for your drug (I use Amicon spin columns). This will separate out any uncapsulated drug from your liposomes
- I would then disrupt the liposomes using 0.1 % Triton X to liberate the encapsulated drug. You can now analyse the drug via HPLC and a standard curve of your drug. You will have to optimise your HPLC conditions specific to your drug however.
- To ensure this method is accurate, I would also use a control of the same concentration of unfiltered drug encapsulated liposome (assuming no drug is lost in formulation, this is your 100% drug reading in order to get % encapsulated). It would also be advisable to analyse a drug free liposome sample in order to normalise any background reading.
I hope this helps!
Alan
Thanks for your reply alan, from what you said i understand the procedure you mentioned is to assess the loading efficiency of the process and assess the final concentration.
my issue is how to avoid evaporation of the drug solution and ensure that the final drug concentration is acheived as i always get higher concentration,
For instance if i am aiming to prepare 10 ml of liposomal solution with concentration of 10mM i end with 8 ml with higher concentration of 12.5 mM due to evaporation.
How can i overcome this issue.
Thanks,
Mohamed hafiz.
Ah ok, apologies for the confusion. Seeing as though it is a hydrophilic drug, I assume you are forming your liposomes in PBS or dH2O? It is surprising you are getting such a high level of evaporation in this case. How long do you let your liposomes hydrate and at what temperature? I would suggest sealing your container with parafilm and storing at 2-8C. If you are not using PBS or dH2O, I would suggest switching to a less volatile solvent and reducing the air circulation as you make your formulation.
Generally, I work off % w/w loading of drug/liposome components so I’ve always just determined my final products µg/ml of drug and diluted appropriately if it was too concentrated for any subsequent experiment.
All the best,
Alan
I use acetate buffer , i always prepare 2 ml or 1ml only so the evaporation will have some impact, i hydrate the lipid film at 35oC for 1 hour at the rotavapor at speed of about 80 rpm which is sealed.
I think if we go for higher volumes this effect will be reduced although no matter what evaporation will occur and concentration will be affected.
can you suggest a less time/ rotation speed.
regards,
Mohammed
Hi Mohammed Hafiz Alshafie Ahmed Mohammed ,
Did you end up overcoming this challenge? I am experiencing evaporation, too. However, my method is through extrusion rather than rotary evaporation.
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