Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

摘要

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

摘要

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

引言

Drug carriers are used to protect and deliver a therapeutic with typical favorable properties including low cytotoxicity, increased bioavailability, and improved stability^1,2,3. Polymeric nanoparticles, micelles, and lipid-based particles have previously been explored for nucleic acid encapsulation and delivery^4,5,6,7. Lipids have been used in different types of nanocarrier systems, including liposomes, and lipid nanoparticles, as they are biocompat....

方案

A schematic of the overall process is provided in Figure 1.

1. Preparation of buffers

NOTE: Sterile filtering of the buffers is highly suggested here to remove any particulates which may impact the nucleic acid and LNP quality.

1. Phosphate Buffered Saline (PBS)
   1. Prepare 1x PBS using 8 mM Na₂HPO₄, 2 mM KH₂PO_4, 137 mM NaCl, and 2.7 mM KCl in nuclease free water and adjust the pH to 7.4.
   2. Sterilize by vacuum filtration using a 0.22 µm pore-size filter.
2. Citrate Buffer
   1. Prepare ci....

结果

Multiple batches of LNPs with the same lipid formulation and N/P ratio of 6 were developed on separate days to demonstrate reproducibility of the technique. Batch 1 and 2 resulted in overlapping size distributions with similar polydispersity (Figure 2A) No significant difference was observed in the size or encapsulation efficiency between the two different batches (Figure 2B). The encapsulation efficiency was high for each batch .......

讨论

Reproducibility, speed, and low volume screening are significant advantages of using microfluidic mixing to form LNPs compared to other existing methods (e.g., lipid film hydration and ethanol injection). We have demonstrated the reproducibility of this method with no impact on encapsulation efficiency or particle size observed with different LNP batches. This is an essential criterion for any therapeutic, including LNPs, to become clinically available.

The technique described here employs sta.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇-2000 （C-14 PEG）	Avanti 极性脂质	880151P
10 µl 渐变滤镜提示 （无 RNase、DNase、DNA）	美国科学	1121-3810
1000 µl 刻度过滤头（无 RNase-，DNase-， DNA）	USA Scientific	1111-2831
20 µl 斜面滤芯吸头（不含 RNase-，DNase-， DNA）	USA Scientific	1120-1810
200 µl 刻度滤芯吸头（无RNase-，DNase-，DNA）	美国科学公司	1120-8810
3&β;-羟基-5-胆甾烯，5-胆甾-3&β;-醇（胆固醇）	Sigma-Aldrich	C8667
BD滑头无菌注射器（1毫升注射器）	Thermo Fisher Scientific	14-823-434
BD滑头无菌注射器（3ml注射器）	Thermo Fisher Scientific	14-823-436
BD Vacutainer 通用注射器针头（BD 钝填充针 18G）	Thermo Fisher Scientific	23-021-020
台式离心机	Beckman Coulter
黑色 96 孔板	Thermo Fisher Scientific	14-245-177
BrandTech 品牌 BIO-CERT 无 RNase、DNase、DNA 微量离心管 （1.5mL）	Thermo Fisher Scientific	14-380-813
柠檬酸	Fisher Scientific	02-002-611
康宁 500 毫升真空过滤器/储存瓶系统，0.22 um 孔径	康宁	430769
一次性折叠毛细管细胞	马尔文	DTS1070
乙醇，纯 200 标准	Sigma-Aldrich	459844
Fisher 品牌半微量比色皿	Thermo FisherScientific	14955127
Invitrogen 锥形管 （15 mL）（不含 DNase-RNase）	Thermo Fisher Scientific	AM12500
MilliporeSigma Amicon Ultra 离心过滤装置	Thermo Fisher Scientific	UFC901024
NanoAssemblr 台式	精密 Nanyosystems
无核酸酶水	Fisher Scientific	AM9930
磷酸盐缓冲盐水 （PBS）	Thermo Fisher Scientific	AM9624
Quant-iT PicoGreen dsDNA 检测试剂盒	Thermo Fisher Scientific	P7589
Quant-iT RiboGreen RNA 检测试剂盒	Thermo Fisher Scientific	R11490
氯化钠	Fisher Scientific	02-004-036
柠檬酸钠，二水合物，颗粒	状 Fisher Scientific	02-004-056
SpectraMax i3x	Molecular Devices
Zetasizer Nano	Malvern