测试通过微流控混合配制的mRNA-脂质纳米颗粒的 体外 和 体内 效率
Summary
在这里，提出了一种用于配制脂质纳米颗粒（LNP）的方案，该脂质纳米颗粒封装编码萤火虫荧光素酶的mRNA。这些 LNP 在体外 HepG2 细胞和体内 C57BL/6 小鼠中测试 了它们的效力。
Abstract
最近，随着 Moderna 和辉瑞/BioNTech 成功开发 COVID-19 mRNA 疫苗，脂质纳米颗粒 （LNP） 引起了广泛关注。这些疫苗证明了mRNA-LNP疗法的疗效，并为未来的临床应用打开了大门。在 mRNA-LNP 系统中，LNP 作为递送平台，保护 mRNA 货物免受核酸酶降解并介导其细胞内递送。LNP 通常由四种组分组成：可电离脂质、磷脂、胆固醇和脂质锚定聚乙二醇 （PEG） 偶联物 （lipid-PEG）。在这里，通过微流控混合含有LNP脂质成分的有机相和含有mRNA的水相来制备封装编码萤火虫荧光素酶的mRNA的LNP。然后使用基于生物发光板的测定法在体外测试这些 mRNA-LNP，以评估它们在 HepG2 细胞中的转染效率。此外，在通过侧尾静脉静脉注射后，在C57BL / 6小鼠中在体内评估mRNA-LNP。全身生物发光成像是通过使用体内成像系统进行的。显示了 mRNA-LNP 特性、它们在 HepG2 细胞中的转染效率以及 C57BL/6 小鼠中的总发光通量的代表性结果。
Introduction
近年来，脂质纳米颗粒（LNPs）在非病毒基因治疗领域展现出巨大的前景。2018 年，美国食品和药物管理局 （FDA） 批准了有史以来第一个 RNA 干扰 （RNAi） 疗法 Onpattro by Alnylam，用于治疗遗传性转甲状腺素蛋白淀粉样变性 1,2,3,4。这是脂质纳米颗粒和基于RNA的疗法向前迈出的重要一步。最近，Moderna 和辉瑞/BioNTech 的 mRNA-LNP 疫苗获得了 FDA 批准，用于对抗 SARS-CoV-2 4,5。在每一种基于 LNP 的核酸疗法中，LNP 都有助于保护其货物免受核酸酶降解，并促进有效的细胞内递送 6,7。虽然 LNP 在 RNAi 疗法和疫苗应用中取得了成功，但 mRNA-LNP 也被探索用于蛋白质替代疗法8 以及 Cas9 mRNA 的共同递送和引导 RNA 的递送，用于递送用于基因编辑的 CRISPR-Cas9 系统9。然而，没有一种特定的制剂适合所有应用，LNP制剂参数的细微变化会极大地影响体内的效力和生物分布8,10,11。因此，必须开发和评估单个 mRNA-LNP，以确定每种基于 LNP 的疗法的最佳配方。
LNP 通常由四种脂质成分配制而成：可电离脂质、磷脂、胆固醇和脂质锚定聚乙二醇 （PEG） 偶联物 （lipid-PEG）11,12,13。LNP促进的有效细胞内递送部分依赖于可电离脂质成分12。该成分在生理pH值下呈中性，但在内体11的酸性环境中带正电。离子电荷的这种变化被认为是内体逃逸的关键因素12,14,15。除了可电离脂质外，磷脂（辅助脂质）成分还改善了货物的包封并有助于内体逃逸，胆固醇提供稳定性并增强膜融合，脂质-PEG 最大限度地减少循环中 LNP 聚集和调理作用10,11,14,16.为了配制LNP，将这些脂质成分结合在有机相（通常是乙醇）中，并与含有核酸货物的水相混合。LNP配方工艺用途广泛，因为它允许以不同的摩尔比轻松替换和组合不同的组分，以配制许多具有多种物理化学性质的LNP配方10,17。然而，在探索种类繁多的LNP时，至关重要的是，必须使用标准化程序对每种配方进行评估，以准确测量表征和性能的差异。
本文概述了mRNA-LNP的配方及其在细胞和动物中性能评估的完整工作流程。
Protocol
注意：在配制 mRNA-LNP 时，始终保持无 RNase 条件，方法是用 RNase 和 DNA 的表面去污剂擦拭表面和设备。仅使用不含 RNase 的吸头和试剂。 所有动物程序均按照宾夕法尼亚大学的《实验动物护理和使用指南》和宾夕法尼亚大学机构动物护理和使用委员会（IACUC）批准的协议进行。 1.制剂前准备 用 200-300 mL 新鲜的 10x 磷酸盐缓冲盐水 （PBS） …
Representative Results
mRNA-LNPs采用微流控仪器配制，其平均流体动力学直径为76.16 nm，多分散指数为0.098。通过进行 TNS 测定，发现 mRNA-LNP 的 pKa 为 5.7518。使用改良荧光测定法和 方程式 4.4 计算出这些 mRNA-LNP 的包封效率为 92.3%。用于细胞处理和动物给药的总RNA浓度为40.24 ng/μL。该值是从改良的荧光测定19中获得的，特别是通过将用1%Triton X-100和1x TE缓冲液稀?…
Discussion
通过该工作流程，可以配制和测试各种mRNA-LNP的体外和体内效率。可电离脂质和赋形剂可以以不同的摩尔比和不同的可电离脂质与mRNA重量比进行交换和组合，以产生具有不同物理化学性质的mRNA-LNPs22。在这里，我们配制了摩尔比为 35/16/46.5/2.5（可电离脂质：辅助脂质：胆固醇：脂质-PEG）的 C12-200 mRNA-LNP，可电离脂质与 mRNA 重量比为 10：1。这些 LNP 在体外 HepG2 ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
M.J.M. 感谢美国国立卫生研究院 （NIH） 主任新创新者奖 （DP2 TR002776）、科学界面 （CASI） 的 Burroughs Wellcome 基金职业奖、美国国家科学基金会职业奖 （CBET-2145491） 以及美国国立卫生研究院（NCI R01 CA241661、NCI R37 CA244911 和 NIDDK R01 DK123049）的额外资助。
Materials
| 0.1 M Hydrochloric Acid | Sigma | 7647-01-0 | |
| 0.22 μm Syringe Filters | Genesee | 25-243 | |
| 1 mL BD Slip Tip Syringe | BD | 309659 | |
| 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000) | Avanti Polar Lipids | 880150P | |
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) | Avanti Polar Lipids | 850725P | |
| 1.5 mL Eppendorf Tubes | Fisher Scientific | 05-408-129 | |
| 15 mL Conical Tubes | Fisher Scientific | 14-959-70C | |
| 200 proof Ethanol | Decon Labs | 2716 | |
| 23G Needles | Fisher Scientific | 14-826-6C | |
| 3 mL BD Disposable Syringes with Luer-Lok tips | Fisher Scientific | 14-823-435 | |
| 3 mL Dialysis Cassettes | Thermo Scientific | A52976 | |
| 96 Well Black Wall Black Bottom Plate | Fisher Scientific | 07-000-135 | |
| 96 Well White/Clear Bottom Plate, TC Surface | Thermo Scientific | 165306 | |
| Ammonium Acetate, 1 Kilogram | Research Products International | 631-61-8 | |
| Ammonium Citrate dibasic | SIgma | 3012-65-5 | |
| BD Luer-Lok Syringe sterile, single use, 5 mL | BD | 309646 | |
| C12-200 Ionizable Lipid | Cayman Chemical | 36699 | |
| C57BL/6 Mice | Jackson Laboratory | 000664 | |
| Cholesterol | Sigma | 57-88-5 | |
| CleanCap FLuc mRNA (5moU) | TriLink Biotechnologies | L-7202 | |
| Disposable cuvettes | Fisher Scientific | 14955129 | |
| D-Luciferin, Potassium Salt | Thermo Scientific | L2916 | |
| DMEM, high glucose | Thermofisher Scientific | 11965-084 | |
| Exel Insulin Syringes – 0.5 mL | Fisher Scientific | 1484132 | |
| Fetal Bovine Serum | Corning | 35-010-CV | |
| Hep G2 [HEPG2] | ATCC | HB-8065 | |
| HyPure Molecular Biology Grade Water | Cytiva | SH30538.03 | |
| Infinite 200 PRO Plate Reader | Tecan | N/A | |
| IVIS Spectrum In Vivo Imaging System | Perkin Elmer | N/A | |
| Large Kimwipes | Fisher Scientific | 06-666-11D | |
| Luciferase Assay Kit | Promega | E4550 | |
| NanoAssemblr Ignite Cartridges – Classic – 100 Pack | Precision Nanosystems | NIN0065 | |
| NanoAssemblr Ignite Instrument | Precision Nanosystems | NIN0001 | |
| PBS – Phosphate-Buffered Saline (10x) pH 7.4, RNase-free | Thermo Scientific | AM9624 | |
| Penicillin-Streptomycin | Thermofisher Scientific | 15140122 | |
| QB Citrate Buffer, (Citrate 100 mM) pH 3.0 | Teknova | Q2442 | |
| Quant-it RiboGreen RNA Assay Kit | Thermo Scientific | R11490 | |
| Reporter Lysis 5x Buffer | Promega | E3971 | |
| RNase Away Surface Decontaminant | Thermofisher Scientific | 7000TS1 | |
| Sodium Chloride | Sigma | 7647-14-5 | |
| Sodium Hydroxide | Sigma | 1310-73-2 | |
| Sodium Phosphate | Sigma | 7601-54-9 | |
| TNS reagent (6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt) | Sigma | T9792 | |
| Triton X-100 | Sigma | 9036-19-5 | |
| Zetasizer | Malvern Panalytical | NanoZS |
References
- Cheng, Q., et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nature Nanotechnology. 15 (4), 313-320 (2020).
- Wood, H. FDA approves patisiran to treat hereditary transthyretin amyloidosis. Nature Reviews Neurology. 14 (9), 509 (2018).
- Zhang, X., Goel, V., Robbie, G. J. Pharmacokinetics of patisiran, the first approved RNA interference therapy in patients With hereditary transthyretin-mediated amyloidosis. Journal of Clinical Pharmacology. 60 (5), 573-585 (2019).
- Shepherd, S. J., et al. Scalable mRNA and siRNA lipid nanoparticle production using a parallelized microfluidic device. Nano Letters. 21 (13), 5671-5680 (2021).
- Barbier, A. J., Jiang, A. Y., Zhang, P., Wooster, R., Anderson, D. G. The clinical progress of mRNA vaccines and immunotherapies. Nature Biotechnology. 40 (6), 840-854 (2022).
- Mukalel, A. J., Riley, R. S., Zhang, R., Mitchell, M. J. Nanoparticles for nucleic acid delivery: Applications in cancer immunotherapy. Cancer Letters. 458, 102-112 (2019).
- Akhtar, S. Oral delivery of siRNA and antisense oligonucleotides. Journal of Drug Targeting. 17 (7), 491-495 (2009).
- Guimaraes, P. P. G., et al. Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening. Journal of Controlled Release. 316, 404-417 (2019).
- Qiu, M., et al. Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3. Proceedings of the National Academy of Sciences of the United States of America. 118 (10), 2020401118 (2021).
- Zhang, R., et al. Helper lipid structure influences protein adsorption and delivery of lipid nanoparticles to spleen and liver. Biomaterials Science. 9 (4), 1449-1463 (2021).
- El-Mayta, R., et al. A nanoparticle platform for accelerated in vivo oral delivery screening of nucleic acids. Advanced Therapeutics. 4 (1), 2000111 (2021).
- Patel, S., et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA. Nature Communications. 11, 983 (2020).
- Kulkarni, J. A., et al. Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA. Nanomedicine: Nanotechnology, Biology, and Medicine. 13 (4), 1377-1387 (2017).
- Cheng, X., Lee, R. J. The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery. Advanced Drug Delivery Reviews. 99, 129-137 (2016).
- Varkouhi, A. K., Scholte, M., Storm, G., Haisma, H. J. Endosomal escape pathways for delivery of biologicals. Journal of Controlled Release. 151 (3), 220-228 (2011).
- Granot, Y., Peer, D. Delivering the right message: Challenges and opportunities in lipid nanoparticles-mediated modified mRNA therapeutics-An innate immune system standpoint. Seminars in Immunology. 34, 68-77 (2017).
- Gan, Z., et al. Nanoparticles containing constrained phospholipids deliver mRNA to liver immune cells in vivo without targeting ligands. Bioengineering and Translational Medicine. 5 (3), 10161 (2020).
- Patel, S. K., et al. Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells. Journal of Controlled Release. 347, 521-532 (2022).
- Robinson, E., et al. Lipid nanoparticle-delivered chemically modified mRNA restores chloride secretion in cystic fibrosis. Molecular Therapy. 26 (8), 2034-2046 (2018).
- Love, K. T., et al. Lipid-like materials for low-dose, in vivo gene silencing. Proceedings of the National Academy of Sciences of the United States of America. 107 (5), 1864-1869 (2010).
- Kauffman, K. J., et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Letters. 15 (11), 7300-7306 (2015).
- Billingsley, M. M., et al. Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering. Nano Letters. 20 (3), 1578-1589 (2020).
- Ramaswamy, S., et al. Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proceedings of the National Academy of Sciences of the United States of America. 114 (10), 1941-1950 (2017).
- Leung, A. K. K., et al. Lipid nanoparticles containing siRNA synthesized by microfluidic mixing exhibit an electron-dense nanostructured core. Journal of Physical Chemistry C. 116 (34), 18440-18450 (2012).
- Billingsley, M. M., et al. Orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of CAR T cells. Nano Letters. 22 (1), 533-542 (2022).
- Khalil, A. A., et al. Subcutaneous administration of D-luciferin is an effective alternative to intraperitoneal injection in bioluminescence imaging of xenograft tumors in nude mice. ISRN Molecular Imaging. 2013, 689279 (2013).
- Qin, J., et al. RGD peptide-based lipids for targeted mRNA delivery and gene editing applications. RSC Advances. 12 (39), 25397-25404 (2022).
- Pardi, N., et al. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. Journal of Controlled Release. 217, 345-351 (2015).
- Finn, J. D., et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Reports. 22 (9), 2227-2235 (2018).
- Truong, B., et al. Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency. Proceedings of the National Academy of Sciences of the United States of America. 116 (42), 21150-21159 (2019).
- Cheng, Q., et al. Dendrimer-based lipid nanoparticles deliver therapeutic FAH mRNA to normalize liver function and extend survival in a mouse model of hepatorenal tyrosinemia type I. Advanced Materials. 30 (52), 1805308 (2018).
- Sedic, M., et al. Safety evaluation of lipid nanoparticle-formulated modified mRNA in the Sprague-Dawley rat and cynomolgus monkey. Veterinary Pathology. 55 (2), 341-354 (2018).
- Veiga, N., et al. Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes. Nature Communications. 9 (1), 4493 (2018).
- Pattipeiluhu, R., et al. Anionic lipid nanoparticles preferentially deliver mRNA to the hepatic reticuloendothelial system. Advanced Materials. 34 (16), 2201095 (2022).
- Rosenblum, D., et al. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Science Advances. 6 (47), (2020).
- Fenton, O. S., et al. Bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mRNA delivery. Advanced Materials. 28 (15), 2939-2943 (2016).
- Kauffman, K. J., et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Letters. 15 (11), 7300-7306 (2015).
- Tombácz, I., et al. Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs. Molecular Therapy. 29 (11), 3293-3304 (2021).
- Kim, J., et al. Engineering lipid nanoparticles for enhanced intracellular delivery of mRNA through inhalation. Nano. 9 (9), 14792-14806 (2022).
- Bevers, S., et al. mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells. Molecular Therapy. 30 (9), 3078-3094 (2022).
Cite This Article
El-Mayta, R., Padilla, M. S., Billingsley, M. M., Han, X., Mitchell, M. J. Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing. J. Vis. Exp. (191), e64810, doi:10.3791/64810 (2023).