Dear fellow researcher,
I am working with gelatin nanoparticles for gene delivery. I prepared cationic gelatin by adding cholamine as a cationic agent. The size and zeta are all good, however, I find difficulty in transfection. The gelatin are not able to transfect the DNA. I tried after one, two, and three days but all did not work. Any help or advice.
Thanks
Dear Osama,
As far I understood, you coat your cationic nanoparticles with oligonucleotides based on electrostatic interactions. I believe you see the shift from positive to negative zeta potential (or a decrease) since you mentioned the zeta potential was good. Have you checked the loading efficiency? The pH can dramatically affect the loading. Are you using pH 7.4? Another reason might be the internalization of the particles by the cells. If everything is ok, but, somehow, your cells don't take up particles, you might electroporate them to see if this will change your results. I hope this info helps.
Dear Sezin,
Thank you for your answer. Yes when I said zeta is good, I mean the charge is positive for DNA and gelatin togethe. I added the treatment to the media containing 10% FBS. I agree with you, the cells might need electroporation but I have not done this kind of technique previously.
Can you tell me how to do it?
You can check the supplementary info from our paper to see our conditions for electroporation: Data 2014 PLGA NPs Supplementary
However, I would suggest to make an optimization test for the best condition (cuvette size, field strength, electroporation buffer, etc.) for your cells and this paper along with many others can help: Article Optimizing Electroporation Conditions in Primary and Other D...
Also, you need to keep in mind that smaller cells require higher voltages. Do you know whether your particles aggregate in cell culture medium? If yes, you might also try to improve this. Good luck with your experiments!
Thank you Sezin!
This is really helpful. I will take your suggestions into consideration, hopefully it works.
Hi
The following paper may be useful for you
Its higly recommended to use PEI as a positive control.
http://nmj.mums.ac.ir/article_8047.html
Mohsen
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