I am basically wondering how I can determine how much siRNA has been loaded in nanoparticles after formulation. Can anyone suggest the best or simplest method?
Please suggest. Thank you.
For UV assay, I think the answer is yes. In general the background of the same drug-free solution should be subtracted. first get absorbance of blank NP supernatant (A1) and of drug loaded NP supernatant (A2), then get the absorbance of a siRNA solution that has the same amount loaded in NP (A4), and finally absorbance of the same vehicle in which siRNA is dissolved (A3). The EE% should be 100 - [(A2-A1) x 100 / (A4 - A3)]. Or get A2-A1 and calculate the siRNA conc using a standard curve. It works better if you have a nanodrop machine. Or even using fluorescent siRNA.
You can use the fluorescent-labeled siRNA. Once you prepare the nanoparticles you can centrifuge them down and measure the fluorescence of the supernatant, and also measure the fluorescence of the unencapsulated labeled siRNA. You can obtain the loading efficiency by the indirect method. Alternatively, you can also measure the UV absorbance of the supernatant similarly. you can check this reference out
10.1016/j.biomaterials.2010.12.057
Thank you Mr. Youssef Wahib. It really helped much! Kind regards
Rakesh
You can also do electrophoresis gel. It's gonna be less quantitative but works good if you have problems to separate the nanoparticles from supernatant.
Thanks Dr. Curcio, I think you both gave one of best solutions. SO much thanks-God bless.
Sir, I would suggest Centrifugation followed by UV/fluorescence estimation as the BEST method as it would give you the exact quantitation of siRNA loading. And if you are having NanoDrop instrument, it would even work with lowest of quantities of samples.
For UV assay, I think the answer is yes. In general the background of the same drug-free solution should be subtracted. first get absorbance of blank NP supernatant (A1) and of drug loaded NP supernatant (A2), then get the absorbance of a siRNA solution that has the same amount loaded in NP (A4), and finally absorbance of the same vehicle in which siRNA is dissolved (A3). The EE% should be 100 - [(A2-A1) x 100 / (A4 - A3)]. Or get A2-A1 and calculate the siRNA conc using a standard curve. It works better if you have a nanodrop machine. Or even using fluorescent siRNA.
Thanks Dr. Youseffa nd Dr. Amira. I appreciate collective efforts of all researchers for making progress in the field and inquired question.
Wishes
Rakesh
Both by direct and indirect method. Indirect method is simple as you asked. After centrifugation of formulation collect the supernatant, using calibration curve method calculate free drug present in it and using formula find the drug loaded.
functional readout is always the best, just transfect siRNAs and measure the effect (eg knockdown of the corresponding mRNA target). despite many folks traditionally keep measuring it - encapsulation efficiency wont tell you much...
Thank you very much Prof. Alexander Vlassov for your answer and worthy suggestion.
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