I use liposomes as a vehicle for gene silencing....so after encapsulation of my siRNA-protein polyplexes I measure what is left outside the liposomes using Quantiflour RNA kit (promega).
I have a dilemma. After loading the siRNAs-protein complexes in the liposomes I use trypsin to break all proteins and set the siRNA free to be able to measure it. Before filtration and washing of liposomes I measure the left RNA it is substantial and so by subtracting it from the original starting amount I get an encapsulation efficiency of 60-70%. But, if I take the same mixture and filter on Amicon filters (100kd) the amount of RNA drops to only traces and the encapsulation efficiency star rockets to 95%. what happened ? where did the RNA go? Amicon filters are used to concentrate proteins and Nucleic acids....help and advice is much appreciated
Perhaps I am misunderstanding your question, but are you not filtering out your siRNA? siRNA are about 13 kDa, so if you wash the particles with a 100 kDa Amicon filter, you are washing out all of the unencapsulated siRNA. So you will get almost 0 unencapsulated siRNA left with the particles on your filter. If you measure the RNA quantity in the solution that got filtered through and multiply by the centrifuge dilution factor, you will probably see the 30-40% unencapsulated siRNA there.
So instead of quantifying siRNA loading efficiency by substracting what didn't get loaded from the original loading stock, I would suggest that you instead directly measure the siRNA that was loaded. After washing your particles with the Amicon filter, use Triton-X-100 to lyse your liposomes to release all of the encapsulated siRNA, trypsin for protein dissociation, and perform the Quant-iT assay. You would need to do a RNA standard curve in Triton-X-100 and trypsin at the same concentration as your particles for this.
Hi,
Check out these 2019 articles on siRNA encapsulation employing liposomes / polyplexes, you may find precise answers to your questions there:
Effectiveness of sirna delivery via arginine-rich pei-based polyplex in metastatic and doxorubicin resistant breast cancer cells
S Lu, VB Morris, V Labhasetwar - Journal of Pharmacology and …, 2019 - ASPET
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pH/Redox Dual-Responsive Polyplex with Effective Endosomal Escape for Co-Delivery of siRNA and Doxorubicin against Drug-Resistant Cancer Cells
Y Gao, L Jia, Q Wang, H Hu, X Zhao… - … applied materials & …, 2019 - ACS Publications
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Effectiveness of Small Interfering RNA Delivery via Arginine-Rich Polyethylenimine-Based Polyplex in Metastatic and Doxorubicin-Resistant Breast Cancer Cells
S Lu, VB Morris, V Labhasetwar - Journal of Pharmacology and …, 2019 - ASPET
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Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery
J Hendershot, AE Smith, TA Werfel - JoVE (Journal of Visualized …, 2019 - jove.com
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