I am trying to make LNP for mRNA delivery using Dlin MC3-DMA,DSPC,cholesterol and DMG-PEG2000 (50:10:38.5:1.5 molar ratio) by rapid mixing method, but I am unable to successfully transfect the LNPs in Huh7 cells. Can some one please suggest if LNPs can be made by rapid mixing method without using microfluidic/T mixer equipment? If yes, then please suggest a suitable protocol for this as I am unable to find one.
b. Also, regarding the N/P ratio, I am using N/P ratio 6 following the paper (doi:10.3390/pharmaceutics3020125) where N/P ratio= number of moles of cationic lipid x number of ionizable nitrogen/ number of moles of siRNA x number of negatively charged phosphates. Please suggest if any other formula may be used for calculating N/P ratio which worked in your experiment.
Thanks in advance.
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