Biochemistry
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Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device
Chapters
Summary March 22nd, 2022
Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.
Transcript
Lipid nanoparticle size affects the biodistribution, anti-tumor effect, and gene silencing performance. Therefore, the LNP size control method is an effective technique for producing nano medicines, including RNA delivery systems. This original microfluidic device, named the iLiNP device, was able to control the LNP size ranging from 20 to 100 nanometers at 10 nanometers intervals.
The iLiNP device can produce not only siRNA loaded LNPs, but all also mRNA loaded LNPS, and ribonucleoprotein loaded LNPs. To begin, take the lipid ethanol solutions and produce the siRNA loaded LNPs by mixing DOTAP, DSPC, cholesterol and DMG-PEG2k solutions at a molar ratio of 50 to 10 to 38.5 to 1.5. Adjust the total lipid concentration to eight millimolar.
Then take the aqueous solutions of 154 millimolar saline and 25 millimolar acetate buffer at pH 4 and filter them through 0.2 micrometer membrane filters or syringe filters. Prepare the siRNA buffer solution by dissolving 70 micrograms of siGL4 into one milliliter of 25 millimolar acetate buffer. Fill one milliliter glass syringes with lipid and aqueous solutions, respectively.
Connect the glass syringes to the peak capillaris using syringe connectors, then set the flow rate of the lipid and aqueous solutions and introduce the lipid and aqueous solutions separately into the iLiNP device using syringe pumps. Collect the LNP suspensions in a microtube from the outlet of the iLiNP device. Dialyze the LNP suspension using a dialysis membrane of 12 to 14 kiloDalton molecular weight cutoffs at four degree is Celsius overnight against saline or DPBS for POPC LNPs and siRNA loaded LNPs, respectively.
Collect the dialyzed LNP suspensions in microtubes and then pipet 20 to 30 microliters of the LNP suspension to a micro quartz cell. Finally, measure the LNP size, LNP size distribution and poly dispersity index by dynamic light scattering. POPC LNP size distribution produced at different flow conditions, such as the total flow rate and the flow rate ratios are shown here.
Precise LNP sizes ranging from 20 to 100 nanometers can be controlled using this iLiNP device. Small sized LNPs are formed at high total flow rate conditions. In addition, the LNP sizes that are formed at the FRR 5 are smaller than those of the FRR 3, regardless of the total flow rate.
Size distribution of siRNA loaded LNPs is shown here. siRNAs are encapsulated into the LNPs by electrostatic interaction between the cationic lipid DOTAP and the negatively charged siRNAs. The iLiNP device produced 90 nanometer siRNA loaded cationic LNPs with a narrow distribution.
The siRNA encapsulation efficiency was 95%because of the electrostatic interaction between the cationic lipid and the negatively charged siRNAs. Cytotoxicity and the gene silencing activity of the 19 nanometer siRNA loaded LNPs were evaluated. siRNA loaded LNPs showed cytotoxicity at a dose of 10 to 100 nanomolar siRNA.
The expression level of luciferase was decreased depending on siRNA concentration. The siRNA loaded LNPs suppressed 80%luciferase expression at a dose of 100 nanomolar siRNA. Optimizations of the flow conditions are the most important steps in the protocol to obtain the desired sized LNPs.
The microfluidic based LNP production method including the iLiNP device does not require any complicated procedure and is expected to be employed as the standard LNP production method.
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