Effective mRNA transfection of tumor cells using cationic triacyl lipid‑based mRNA lipoplexes

Hattori,Yoshiyuki; Shimizu,Ryohei

doi:10.3892/br.2024.1903

Effective mRNA transfection of tumor cells using cationic triacyl lipid‑based mRNA lipoplexes

Authors:
- Yoshiyuki Hattori
- Ryohei Shimizu
View Affiliations

Affiliations: Department of Molecular Pharmaceutics, Hoshi University, Shinagawa, Tokyo 142‑8501, Japan
Published online on: December 4, 2024 https://doi.org/10.3892/br.2024.1903
Article Number: 25
Copyright: © Hattori et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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

Previously, it was reported that mRNA/cationic liposome complexes (mRNA lipoplexes) composed of the cationic triacyl lipid, 11‑((1,3‑bis(dodecanoyloxy)‑2‑((dodecanoyloxy)methyl)propan‑2‑yl)amino)‑N,N,N‑ trimethyl‑11‑oxoundecan‑1‑aminium bromide (TC‑1‑12), with 1,2‑dioleoyl‑sn-glycero‑3‑phosphoethanolamine and poly(ethylene glycol) cholesteryl ether, induce high protein expression in human cervical carcinoma HeLa cells. In the present study, the authors aimed to optimize mRNA transfection using TC‑1‑12‑based mRNA lipoplexes. mRNA lipoplexes were prepared at various charge ratios (+:‑) using modified ethanol injection (MEI) and thin‑film hydration (TFH) methods and compared the protein expression efficiency after transfection of HeLa cells with the developed mRNA lipoplexes. Firefly luciferase (FLuc) and enhanced green fluorescent protein (EGFP) mRNA lipoplexes prepared using the MEI method exhibited higher Luc and EGFP expression levels in cells than those prepared using the TFH method. Moreover, FLuc mRNA lipoplexes prepared using the MEI and TFH methods at charge ratios of 3:1 and 4:1, respectively, exhibited the highest Luc expression in cells. However, transfection with mRNA lipoplexes using the MEI and TFH methods induced moderate cytotoxicity in HeLa cells (46 and 57% cell viability, respectively). Furthermore, Cy5‑labeled mRNA lipoplexes, which were prepared using the MEI method, showed higher cellular uptake of mRNA than those prepared using the TFH method. In the transfection of FLuc mRNA lipoplexes prepared using the MEI method, the storage of the lipid‑ethanol solution at 37˚C for 4 months did not decrease Luc expression in HeLa cells. Additionally, FLuc mRNA lipoplexes prepared using the MEI method, induced relatively high Luc expression in human prostate carcinoma PC‑3 and human liver cancer HepG2 cells with low cytotoxicity (103 and 81% cell viability, respectively). Overall, the results highlighted the potential of TC‑1‑12‑based mRNA lipoplexes prepared using the MEI method for efficient mRNA delivery to cells.

View Figures

View References

1	Sahin U, Karikó K and Türeci Ö: mRNA-based therapeutics-developing a new class of drugs. Nat Rev Drug Discov. 13:759–780. 2014.PubMed/NCBI View Article : Google Scholar
2	Liu T, Liang Y and Huang L: Development and delivery systems of mRNA vaccines. Front Bioeng Biotechnol. 9(718753)2021.PubMed/NCBI View Article : Google Scholar
3	Barbier AJ, Jiang AY, Zhang P, Wooster R and Anderson DG: The clinical progress of mRNA vaccines and immunotherapies. Nat Biotechnol. 40:840–854. 2022.PubMed/NCBI View Article : Google Scholar
4	Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, et al: Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 7:618–630. 2010.PubMed/NCBI View Article : Google Scholar
5	Ramachandran S, Satapathy SR and Dutta T: Delivery strategies for mRNA vaccines. Pharmaceut Med. 36:11–20. 2022.PubMed/NCBI View Article : Google Scholar
6	Yen A, Cheng Y, Sylvestre M, Gustafson HH, Puri S and Pun SH: Serum nuclease susceptibility of mRNA cargo in condensed polyplexes. Mol Pharm. 15:2268–2276. 2018.PubMed/NCBI View Article : Google Scholar
7	Chen H, Ren X, Xu S, Zhang D and Han T: Optimization of lipid nanoformulations for effective mRNA delivery. Int J Nanomedicine. 17:2893–2905. 2022.PubMed/NCBI View Article : Google Scholar
8	Malone RW, Felgner PL and Verma IM: Cationic liposome- mediated RNA transfection. Proc Natl Acad Sci USA. 86:6077–6081. 1989.PubMed/NCBI View Article : Google Scholar
9	Tang M, Sagawa A, Inoue N, Torii S, Tomita K and Hattori Y: Efficient mRNA delivery with mRNA lipoplexes prepared using a modified ethanol injection method. Pharmaceutics. 15(1141)2023.PubMed/NCBI View Article : Google Scholar
10	Bangham AD, Standish MM and Watkins JC: Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol. 13:238–252. 1965.PubMed/NCBI View Article : Google Scholar
11	Hattori Y, Nakamura M, Takeuchi N, Tamaki K, Shimizu S, Yoshiike Y, Taguchi M, Ohno H, Ozaki KI and Onishi H: Effect of cationic lipid in cationic liposomes on siRNA delivery into the lung by intravenous injection of cationic lipoplex. J Drug Target. 27:217–227. 2019.PubMed/NCBI View Article : Google Scholar
12	Barichello JM, Ishida T and Kiwada H: Complexation of siRNA and pDNA with cationic liposomes: The important aspects in lipoplex preparation. Methods Mol Biol. 605:461–472. 2010.PubMed/NCBI View Article : Google Scholar
13	Hattori Y, Saito H, Nakamura K, Yamanaka A, Tang M and Ozaki KI: In vitro and in vivo transfections using siRNA lipoplexes prepared by mixing siRNAs with a lipid-ethanol solution. J Drug Deliv Sci Technol. 75(103635)2022.
14	Hattori Y, Tamaki K, Sakasai S, Ozaki KI and Onishi H: Effects of PEG anchors in pegylated siRNA lipoplexes on in vitro genesilencing effects and siRNA biodistribution in mice. Mol Med Rep. 22:4183–4196. 2020.PubMed/NCBI View Article : Google Scholar
15	Smith MC, Crist RM, Clogston JD and McNeil SE: Zeta potential: A case study of cationic, anionic, and neutral liposomes. Anal Bioanal Chem. 409:5779–5787. 2017.PubMed/NCBI View Article : Google Scholar
16	Jarzebska NT, Frei J, Lauchli S, French LE, Guenova E, Gouttefangeas C, Kündig TM, Mellett M and Pascolo S: Lipofection with synthetic mRNA as a simple method for T-cell immunomonitoring. Viruses. 13(1232)2021.PubMed/NCBI View Article : Google Scholar
17	Wei X, Shao B, He Z, Ye T, Luo M, Sang Y, Liang X, Wang W, Luo S, Yang S, et al: Cationic nanocarriers induce cell necrosis through impairment of Na(+)/K(+)-ATPase and cause subsequent inflammatory response. Cell Res. 25:237–253. 2015.PubMed/NCBI View Article : Google Scholar
18	Opsomer L, Jana S, Mertens I, Cui X, Hoogenboom R and Sanders NN: Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers. J Mater Chem B. 12:3927–3946. 2024.PubMed/NCBI View Article : Google Scholar
19	Ahlemeyer B, Vogt JF, Michel V, Hahn-Kohlberger P and Baumgart-Vogt E: Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: Evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency. Histochem Cell Biol. 142:577–591. 2014.PubMed/NCBI View Article : Google Scholar
20	Hattori Y, Yoshizawa T, Koga K and Maitani Y: NaCl induced high cationic hydroxyethylated cholesterol-based nanoparticle-mediated synthetic small interfering RNA transfer into prostate carcinoma PC-3 cells. Biol Pharm Bull. 31:2294–2301. 2008.PubMed/NCBI View Article : Google Scholar
21	Hattori Y, Hagiwara A, Ding W and Maitani Y: NaCl improves siRNA delivery mediated by nanoparticles of hydroxyethylated cationic cholesterol with amido-linker. Bioorg Med Chem Lett. 18:5228–5232. 2008.PubMed/NCBI View Article : Google Scholar
22	Hattori Y, Ding WX and Maitani Y: Highly efficient cationic hydroxyethylated cholesterol-based nanoparticle-mediated gene transfer in vivo and in vitro in prostate carcinoma PC-3 cells. J Control Release. 120:122–130. 2007.PubMed/NCBI View Article : Google Scholar
23	Koulov AV, Vares L, Jain M and Smith BD: Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues. Biochim Biophys Acta. 1564:459–465. 2002.PubMed/NCBI View Article : Google Scholar
24	Ma X, Wu F, Peng C, Chen H, Zhang D and Han T: Exploration of mRNA nanoparticles based on DOTAP through optimization of the helper lipids. Biotechnol J. 18(e2300123)2023.PubMed/NCBI View Article : Google Scholar
25	Anderluzzi G, Lou G, Gallorini S, Brazzoli M, Johnson R, O'Hagan DT, Baudner BC and Perrie Y: Investigating the impact of delivery system design on the efficacy of self-amplifying RNA vaccines. Vaccines (Basel). 8(212)2020.PubMed/NCBI View Article : Google Scholar
26	Hattori Y and Tang M: Effect of cationic and neutral lipids in cationic liposomes on antibody production induced by systemic administration of mRNA lipoplexes into mice. J Drug Deliv Sci Technol. 100(106034)2024.