Hello, I need to help about my thesis. I syhnthesize catechin loaded plga nanoparticle. I use indirect analysis method for calculate encapsulation efficiency and I get negative encapsulation efficiency results.
For example; I load 20 mg molecule into nanaoparticle. I calculate 24mg molecule in upper phase.
I use pva as a emulsifier. I think, It is interesting with pva (polyvinyl alcohol).
What do you think about this problem?
Thank you for your answers.
Dear Burcu,
Please forgive me in advance if I ask you some questions that may make it seem like I am doubting your experimental protocol. I am just trying to help you potentially resolve your problem.
Let us assume there are no biases in your catechin assay method.
- So you observe a certain amount of grams catechin per milliliter of serum phase surrounding your nanoparticle by perhaps UV-Vis or by HPLC.
- You then have to take the known total volume of the serum phase to convert that grams catechin/milliliter value you observed into a total mass of catechin in the serum phase.
- You then compare that total mass of found catechin to the formulation amount of catechin mass you added at the beginning of the encapsulation process.
A critical step in the above is making sure that you know you continuous phase, or serum volume. If the nanoparticles occupy 20% of the volume of the sample, then the continuous phase occupies the remaining 80% of volume. If no catechin was actually encapsulated, then all the catechin would be in that 80% volume of the serum phase. This would make the grams catechin per ml of serum phase look enriched versus the grams of catechin per total ml of sample.
Is it perhaps something like this that is causing your problem?
Best regards,
Mike Bender
Dear Burcu,
There are some few things you should do first which I think it will help,
You need to revise your analysis protocol for catechin. You are measuring the conc. of the free catechin (un-encapsulated) in the external PVA phase. Here we should know how you did your standard curve which you used to detect the free drug conc. I mean did you used different catechin conc. in PVA solution for your standard curve or you dissolved catechin in your mobile phase in case of HPLC or other solvents in case of UV.
Have you tried to measure entrapped catechin by using direct method after extracting it from your loaded NPs. I think this will help you o confirm if there is any encapsulation occurred or you have zero encapsulation as you measured by the indirect method. Here, you will have a good chance to compare between the two results of the indirect and direct methods.
PVA external aqueous will improve the diffusion of your drug (hydrophilic drug) to it but will not affect its concentration. You might need to optimize your formulation in order to improve your encapsulation efficiency.
Hopefully, this will help.
Regards
I came across the same problem. As above, it is really critical to determine the volume of the supernatant! In the end, I just used the indirect method. You will have to hydrolyse the purified NPs prior to analysis, for example in 1 M KOH and stirring at RT for some hours. But make sure your drug is still intact under these conditions :) good luck!
I faced a similar problem, and eventually I found out that the problem was in the separation of the nanoparticles. On moderate speeds on centrifuge (20,000 rpm), Nps are not separated efficiently and consequently the tiny ones still in the supernatant affect the UV absorbency; it raises the baseline. Try ultra centrifuge (40,000 rpm), otherwise you will have to use the direct method ( degradation of NPs).
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