Effect of using amino acids in the freeze‑drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection

Tang,Min; Hattori,Yoshiyuki

doi:10.3892/br.2021.1448

Effect of using amino acids in the freeze‑drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection

Authors:
- Min Tang
- Yoshiyuki Hattori
View Affiliations

Affiliations: Department of Molecular Pharmaceutics, Hoshi University, Tokyo 142‑8501, Japan
Published online on: July 12, 2021 https://doi.org/10.3892/br.2021.1448
Article Number: 72

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Recently, small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) have become a crucial research tool for studying gene function. Easy and reliable siRNA transfection with a large set of siRNAs is required for the successful screening of gene function. Reverse (Rev)‑transfection with freeze‑dried siRNA lipoplexes is validated for siRNA transfection with a large set of siRNAs in a multi‑well plate. In our previous study, it was shown that Rev‑transfection with siRNA lipoplexes freeze‑dried in disaccharides or trisaccharides resulted in long‑term stability with a high level of gene‑knockdown activity. In the present study, the effects of amino acids used as cryoprotectants in the freeze‑drying of siRNA lipoplexes on gene knockdown via Rev‑transfection were assessed. A total of 15 types of amino acids were used to prepare freeze‑dried siRNA lipoplexes, and it was found that the freeze‑drying of siRNA lipoplexes with amino acid concentrations >100 mM strongly suppressed targeted gene expression regardless of the amino acid type; however, some amino acids strongly upregulated or downregulated gene expression in the cells transfected with negative control siRNA. Amongst the amino acids tested, the presence of asparagine showed specific gene‑knockdown activity, forming large cakes after freeze‑drying and retaining a favorable siRNA lipoplex size after rehydration. These findings provide valuable information regarding amino acids as cryoprotectants for Rev‑transfection using freeze‑dried siRNA lipoplexes for the efficient delivery of siRNA into cells.

View Figures

View References

1	Wilson RC and Doudna JA: Molecular mechanisms of RNA interference. Annu Rev Biophys. 42:217–239. 2013.PubMed/NCBI View Article : Google Scholar
2	Zhang MM, Bahal R, Rasmussen TP, Manautou JE and Zhong XB: The growth of siRNA-based therapeutics: Updated clinical studies. Biochem Pharmacol. 189(114432)2021.PubMed/NCBI View Article : Google Scholar
3	Thapa B, Remant KC and Uludağ H: siRNA library screening to identify complementary therapeutic pairs in triple-negative breast cancer cells. Methods Mol Biol. 1974:1–19. 2019.PubMed/NCBI View Article : Google Scholar
4	Erfle H, Neumann B, Liebel U, Rogers P, Held M, Walter T, Ellenberg J and Pepperkok R: Reverse transfection on cell arrays for high content screening microscopy. Nat Protoc. 2:392–399. 2007.PubMed/NCBI View Article : Google Scholar
5	Zhang S, Zhi D and Huang L: Lipid-based vectors for siRNA delivery. J Drug Target. 20:724–735. 2012.PubMed/NCBI View Article : Google Scholar
6	Yadava P, Gibbs M, Castro C and Hughes JA: Effect of lyophilization and freeze-thawing on the stability of siRNA-liposome complexes. AAPS PharmSciTech. 9:335–341. 2008.PubMed/NCBI View Article : Google Scholar
7	Hattori Y, Hu S and Onishi H: Effects of cationic lipids in cationic liposomes and disaccharides in the freeze-drying of siRNA lipoplexes on gene silencing in cells by reverse transfection. J Liposome Res. 30:235–245. 2020.PubMed/NCBI View Article : Google Scholar
8	Tang M, Hu S and Hattori Y: Effect of pre freezing and saccharide types in freeze drying of siRNA lipoplexes on gene silencing effects in the cells by reverse transfection. Mol Med Rep. 22:3233–3244. 2020.PubMed/NCBI View Article : Google Scholar
9	Franzé S, Selmin F, Samaritani E, Minghetti P and Cilurzo F: Lyophilization of liposomal formulations: Still necessary, still challenging. Pharmaceutics. 10(10)2018.PubMed/NCBI View Article : Google Scholar
10	Abdelwahed W, Degobert G, Stainmesse S and Fessi H: Freeze-drying of nanoparticles: Formulation, process and storage considerations. Adv Drug Deliv Rev. 58:1688–1713. 2006.PubMed/NCBI View Article : Google Scholar
11	Ball RL, Bajaj P and Whitehead KA: Achieving long-term stability of lipid nanoparticles: Examining the effect of pH, temperature, and lyophilization. Int J Nanomedicine. 12:305–315. 2016.PubMed/NCBI View Article : Google Scholar
12	Kundu AK, Chandra PK, Hazari S, Ledet G, Pramar YV, Dash S and Mandal TK: Stability of lyophilized siRNA nanosome formulations. Int J Pharm. 423:525–534. 2012.PubMed/NCBI View Article : Google Scholar
13	Andersen MØ, Howard KA, Paludan SR, Besenbacher F and Kjems J: Delivery of siRNA from lyophilized polymeric surfaces. Biomaterials. 29:506–512. 2008.PubMed/NCBI View Article : Google Scholar
14	Mohammed AR, Coombes AG and Perrie Y: Amino acids as cryoprotectants for liposomal delivery systems. Eur J Pharm Sci. 30:406–413. 2007.PubMed/NCBI View Article : Google Scholar
15	Crowe JH and Crowe LM: Factors affecting the stability of dry liposomes. Biochim Biophys Acta. 939:327–334. 1988.PubMed/NCBI View Article : Google Scholar
16	Koster KL, Webb MS, Bryant G and Lynch DV: Interactions between soluble sugars and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) during dehydration: Vitrification of sugars alters the phase behavior of the phospholipid. Biochim Biophys Acta. 1193:143–150. 1994.PubMed/NCBI View Article : Google Scholar
17	Stärtzel P: Arginine as an excipient for protein freeze-drying: A mini review. J Pharm Sci. 107:960–967. 2018.PubMed/NCBI View Article : Google Scholar
18	Paik SH, Kim YJ, Han SK, Kim JM, Huh JW and Park YI: Mixture of three amino acids as stabilizers replacing albumin in lyophilization of new third generation recombinant factor VIII GreenGene F. Biotechnol Prog. 28:1517–1525. 2012.PubMed/NCBI View Article : Google Scholar
19	Hattori Y, Nakamura T, Ohno H, Fujii N and Maitani Y: siRNA delivery into tumor cells by lipid-based nanoparticles composed of hydroxyethylated cholesteryl triamine. Int J Pharm. 443:221–229. 2013.PubMed/NCBI View Article : Google Scholar
20	Hattori Y, Hara E, Shingu Y, Minamiguchi D, Nakamura A, Arai S, Ohno H, Kawano K, Fujii N and Yonemochi E: siRNA delivery into tumor cells by cationic cholesterol derivative-based nanoparticles and liposomes. Biol Pharm Bull. 38:30–38. 2015.PubMed/NCBI View Article : Google Scholar
21	Hattori Y, Nakamura M, Takeuchi N, Tamaki K, Ozaki K and Onishi H: Effect of cationic lipid type in pegylated liposomes on siRNA delivery following the intravenous injection of siRNA lipoplexes. World Acad Sci. 1:74–85. 2019.PubMed/NCBI View Article : Google Scholar
22	Varga J, Li L, Mauviel A, Jeffrey J and Jimenez SA: L-Tryptophan in supraphysiologic concentrations stimulates collagenase gene expression in human skin fibroblasts. Lab Invest. 70:183–191. 1994.PubMed/NCBI
23	Vary TC, Jefferson LS and Kimball SR: Amino acid-induced stimulation of translation initiation in rat skeletal muscle. Am J Physiol. 277:E1077–E1086. 1999.PubMed/NCBI View Article : Google Scholar
24	Forney-Stevens KM, Bogner RH and Pikal MJ: Addition of amino acids to further stabilize lyophilized sucrose-based protein formulations: I. Screening of 15 amino acids in two model proteins. J Pharm Sci. 105:697–704. 2016.PubMed/NCBI View Article : Google Scholar