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Guanidinium-functionalized Block Copolyelectrolyte Micelleplexes for Safe and Efficient siRNA Delivery

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Abstract

RNAi-based therapeutics utilizing small interfering RNAs (siRNAs) are of significance in the clinic as it serves great potentials for gene-based treatment of human diseases. Currently, siRNA-based RNAi efficiency has been limited by facile degradation, poor cell membrane penetration, and short half-life time of siRNA. In this study, block copolyelectrolytes containing a poly(ethylene oxide) (PEO) neutral block and a cationic block were synthesized by anionic polymerization and post-polymerization modification. In the cationic block, guanidinium and ammonium groups were randomly incorporated with various fractions to achieve micelleplexes for safe and efficient siRNA delivery. Compared to traditional polyethylenimine-based polyplexes, all micelleplexes exhibited enhanced cellular internalization and better gene silencing efficiency with higher stability. As the fraction of guanidinium groups increased, the uptake level and siRNA transfection were enhanced due to stronger binding of guanidinium groups with siRNA. However, the trade-off between cellular internalization and toxicity was inevitable with increasing guanidinium fraction. The fraction of guanidinium group in block copolyelectrolytes was optimized by the systemic evaluation of cytotoxicity and gene silencing efficiency of the micelleplexes.

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References

  1. Dowdy, S. F. (2017) Overcoming cellular barriers for RNA therapeutics. Nat. Biotechnol. 35: 222–229.

    Article  CAS  Google Scholar 

  2. Jasinski, D., F. Haque, D. W. Binzel, and P. Guo (2017) Advancement of the emerging field of RNA nanotechnology. ACS Nano 11: 1142–1164.

    Article  CAS  Google Scholar 

  3. Kim, B., J. H. Park, and M. J. Sailor (2019) Rekindling RNAi therapy: materials design requirements for in vivo siRNA delivery. Adv. Mater. 31: e1903637.

    Article  Google Scholar 

  4. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498.

    Article  CAS  Google Scholar 

  5. Guan, X., Z. Guo, L. Lin, J. Chen, H. Tian, and X. Chen (2016) Ultrasensitive pH triggered charge/size dual-rebound gene delivery system. Nano Lett. 16: 6823–6831.

    Article  CAS  Google Scholar 

  6. Guo, Y., Z. Wu, S. Shen, R. Guo, J. Wang, W. Wang, K. Zhao, M. Kuang, and X. Shuai (2018) Nanomedicines reveal how PBOV1 promotes hepatocellular carcinoma for effective gene therapy. Nat. Commun. 9: 3430.

    Article  Google Scholar 

  7. Hu, B., L. Zhong, Y. Weng, L. Peng, Y. Huang, Y. Zhao, and X. J. Liang (2020) Therapeutic siRNA: state of the art. Signal Transduct. Target. Ther. 5: 101.

    Article  CAS  Google Scholar 

  8. Soutschek, J., A. Akinc, B. Bramlage, K. Charisse, R. Constien, M. Donoghue, S. Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John, V. Kesavan, G. Lavine, R. K. Pandey, T. Racie, K. G. Rajeev, I. Röhl, I. Toudjarska, G. Wang, S. Wuschko, D. Bumcrot, V. Koteliansky, S. Limmer, M. Manoharan, and H. P. Vornlocher (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432: 173–178.

    Article  CAS  Google Scholar 

  9. Toudjarska, I. and A. de Fougerolles (2009) Silencing prostate cancer. Nat. Biotechnol. 27: 821–823.

    Article  CAS  Google Scholar 

  10. Kumar, P., H. Wu, J. L. McBride, K. E. Jung, M. H. Kim, B. L. Davidson, S. K. Lee, P. Shankar, and N. Manjunath (2007) Transvascular delivery of small interfering RNA to the central nervous system. Nature 448: 39–43.

    Article  CAS  Google Scholar 

  11. Nair, J. K., J. L. S. Willoughby, A. Chan, K. Charisse, M. R. Alam, Q. Wang, M. Hoekstra, P. Kandasamy, A. V. Kel’in, S. Milstein, N. Taneja, J. O’Shea, S. Shaikh, L. Zhang, R. J. van der Sluis, M. E. Jung, A. Akinc, R. Hutabarat, S. Kuchimanchi, K. Fitzgerald, T. Zimmermann, T. J. C. van Berkel, M. A. Maier, K. G. Rajeev, and M. Manoharan (2014) Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 136: 16958–16961.

    Article  CAS  Google Scholar 

  12. Ramishetti, S., I. Hazan-Halevy, R. Palakuri, S. Chatterjee, S. N. Gonna, N. Dammes, I. Freilich, L. K. Shmuel, D. Danino, and D. Peer (2020) A combinatorial library of lipid nanoparticles for RNA delivery to leukocytes. Adv. Mater. 32: e1906128.

    Article  Google Scholar 

  13. Yang, J., Q. Zhang, H. Chang, and Y. Cheng (2015) Surface-engineered dendrimers in gene delivery. Chem. Rev. 115: 5274–5300.

    Article  CAS  Google Scholar 

  14. Liu, C., N. Shao, Y. Wang, and Y. Cheng (2016) Clustering small dendrimers into nanoaggregates for efficient DNA and siRNA delivery with minimal toxicity. Adv. Healthc. Mater. 5: 584–592.

    Article  CAS  Google Scholar 

  15. Endoh, T. and T. Ohtsuki (2009) Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape. Adv. Drug Deliv. Rev. 61: 704–709.

    Article  CAS  Google Scholar 

  16. Nothisen, M., M. Kotera, E. Voirin, J. S. Remy, and J. P. Behr (2009) Cationic siRNAs provide carrier-free gene silencing in animal cells. J. Am. Chem. Soc. 131: 17730–17731.

    Article  CAS  Google Scholar 

  17. Averick, S. E., E. Paredes, S. K. Dey, K. M. Snyder, N. Tapinos, K. Matyjaszewski, and S. R. Das (2013) Autotransfecting short interfering RNA through facile covalent polymer escorts. J. Am. Chem. Soc. 135: 12508–12511.

    Article  CAS  Google Scholar 

  18. Fang, H., Z. Guo, L. Lin, J. Chen, P. Sun, J. Wu, C. Xu, H. Tian, and X. Chen (2018) Molecular strings significantly improved the gene transfection efficiency of polycations. J. Am. Chem. Soc. 140: 11992–12000.

    Article  CAS  Google Scholar 

  19. Xu, C., Y. Zhang, K. Xu, J. J. Nie, B. Yu, S. Li, G. Cheng, Y. Li, J. Du, and F. J. Xu (2019) Multifunctional cationic nanosystems for nucleic acid therapy of thoracic aortic dissection. Nat. Commun. 10: 3184.

    Article  Google Scholar 

  20. Luten, J., C. F. van Nostrum, S. C. De Smedt, and W. E. Hennink (2008) Biodegradable polymers as non-viral carriers for plasmid DNA delivery. J. Control. Release 126: 97–110.

    Article  CAS  Google Scholar 

  21. van Waarde, A., A. A. Rybczynska, N. K. Ramakrishnan, K. Ishiwata, P. H. Elsinga, and R. A. J. O. Dierckx (2015) Potential applications for sigma receptor ligands in cancer diagnosis and therapy. Biochim. Biophys. Acta 1848: 2703–2714.

    Article  CAS  Google Scholar 

  22. Vargas, J. R., E. G. Stanzl, N. N. H. Teng, and P. A. Wender (2014) Cell-penetrating, guanidinium-rich molecular transporters for overcoming efflux-mediated multidrug resistance. Mol. Pharm. 11: 2553–2565.

    Article  CAS  Google Scholar 

  23. Priegue, J. M., D. N. Crisan, J. Martínez-Costas, J. R. Granja, F. Fernandez-Trillo, and J. Montenegro (2016) In situ functionalized polymers for siRNA delivery. Angew. Chem. Int. Ed. Engl. 55: 7492–7495.

    Article  CAS  Google Scholar 

  24. Yu, C., E. Tan, Y. Xu, J. Lv, and Y. Cheng (2019) A guanidinium-rich polymer for efficient cytosolic delivery of native proteins. Bioconjug. Chem. 30: 413–417.

    Article  CAS  Google Scholar 

  25. Chang, H., J. Zhang, H. Wang, J. Lv, and Y. Cheng (2017) A combination of guanidyl and phenyl groups on a dendrimer enables efficient siRNA and DNA delivery. Biomacromolecules 18: 2371–2378.

    Article  CAS  Google Scholar 

  26. Lee, Y. and K. Kataoka (2012) Delivery of nucleic acid drugs. pp. 95–134. In: A. Murakami (ed.). Nucleic Acid Drugs. Springer, Berlin, Germany.

    Google Scholar 

  27. Liu, X.-Q., C.-Y. Sun, X.-Z. Yang, and J. Wang (2013) Polymeric-micelle-based nanomedicine for siRNA delivery. Part. Part. Syst. Charact. 30: 211–228.

    Article  CAS  Google Scholar 

  28. Cabral, H., K. Miyata, K. Osada, and K. Kataoka (2018) Block copolymer micelles in nanomedicine applications. Chem. Rev. 118: 6844–6892.

    Article  CAS  Google Scholar 

  29. Kumar, R., C. F. Santa Chalarca, M. R. Bockman, C. V. Bruggen, C. J. Grimme, R. J. Dalal, M. G. Hanson, J. K. Hexum, and T. M. Reineke (2021) Polymeric delivery of therapeutic nucleic acids. Chem. Rev. 121: 11527–11652.

    Article  CAS  Google Scholar 

  30. Kataoka, K., H. Togawa, A. Harada, K. Yasugi, T. Matsumoto, and S. Katayose (1996) Spontaneous formation of polyion complex micelles with narrow distribution from antisense oligonucleotide and cationic block copolymer in physiological saline. Macromolecules 29: 8556–8557.

    Article  CAS  Google Scholar 

  31. Katayose, S. and K. Kataoka (1997) Water-soluble polyion complex associates of DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer. Bioconjug. Chem. 8: 702–707.

    Article  CAS  Google Scholar 

  32. Itaka, K., K. Yamauchi, A. Harada, K. Nakamura, H. Kawaguchi, and K. Kataoka (2003) Polyion complex micelles from plasmid DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer as serum-tolerable polyplex system: physicochemical properties of micelles relevant to gene transfection efficiency. Biomaterials 24: 4495–4506.

    Article  CAS  Google Scholar 

  33. Tockary, T. A., K. Osada, Y. Motoda, S. Hiki, Q. Chen, K. M. Takeda, A. Dirisala, S. Osawa, and K. Kataoka (2016) Rod-to-globule transition of pDNA/PEG-poly(l-lysine) polyplex micelles induced by a collapsed balance between DNA rigidity and PEG crowdedness. Small 12: 1193–1200.

    Article  CAS  Google Scholar 

  34. Christie, R. J., Y. Matsumoto, K. Miyata, T. Nomoto, S. Fukushima, K. Osada, J. Halnaut, F. Pittella, H. J. Kim, N. Nishiyama, and K. Kataoka (2012) Targeted polymeric micelles for siRNA treatment of experimental cancer by intravenous injection. ACS Nano 6: 5174–5189.

    Article  CAS  Google Scholar 

  35. Christie, R. J., K. Miyata, Y. Matsumoto, T. Nomoto, D. Menasco, T. C. Lai, M. Pennisi, K. Osada, S. Fukushima, N. Nishiyama, Y. Yamasaki, and K. Kataoka (2011) Effect of polymer structure on micelles formed between siRNA and cationic block copolymer comprising thiols and amidines. Biomacromolecules 12: 3174–3185.

    Article  CAS  Google Scholar 

  36. Miyata, K., Y. Kakizawa, N. Nishiyama, A. Harada, Y. Yamasaki, H. Koyama, and K. Kataoka (2004) Block catiomer polyplexes with regulated densities of charge and disulfide cross-linking directed to enhance gene expression. J. Am. Chem. Soc. 126: 2355–2361.

    Article  CAS  Google Scholar 

  37. Nishida, H., Y. Matsumoto, K. Kawana, R. J. Christie, M. Naito, B. S. Kim, K. Toh, H. S. Min, Y. Yi, Y. Matsumoto, H. J. Kim, K. Miyata, A. Taguchi, K. Tomio, A. Yamashita, T. Inoue, H. Nakamura, A. Fujimoto, M. Sato, M. Yoshida, K. Adachi, T. Arimoto, O. Wada-Hiraike, K. Oda, T. Nagamatsu, N. Nishiyama, K. Kataoka, Y. Osuga, and T. Fujii (2016) Systemic delivery of siRNA by actively targeted polyion complex micelles for silencing the E6 and E7 human papillomavirus oncogenes. J. Control. Release 231: 29–37.

    Article  CAS  Google Scholar 

  38. Tabujew, I., C. Freidel, B. Krieg, M. Helm, K. Koynov, K. Müllen, and K. Peneva (2014) The guanidinium group as a key part of water-soluble polymer carriers for siRNA complexation and protection against degradation. Macromol. Rapid Commun. 35: 1191–1197.

    Article  CAS  Google Scholar 

  39. Hori, M., H. Cabral, K. Toh, A. Kishimura, and K. Kataoka (2018) Robust polyion complex vesicles (PICsomes) under physiological conditions reinforced by multiple hydrogen bond formation derived by guanidinium groups. Biomacromolecules 19: 4113–4121.

    Article  CAS  Google Scholar 

  40. Kim, B. S., M. Naito, H. Chaya, M. Hori, K. Hayashi, H. S. Min, Y. Yi, H. J. Kim, T. Nagata, Y. Anraku, A. Kishimura, K. Kataoka, and K. Miyata (2020) Noncovalent stabilization of vesicular polyion complexes with chemically modified/single-stranded oligonucleotides and PEG-b-guanidinylated polypeptides for intracavity encapsulation of effector enzymes aimed at cooperative gene knockdown. Biomacromolecules 21: 4365–4376.

    Article  CAS  Google Scholar 

  41. Miyazaki, T., S. Uchida, Y. Miyahara, A. Matsumoto, and H. Cabral (2021) Development of flexible polycation-based mRNA delivery systems for in vivo applications. Mater. Proc. 4: 5.

    Google Scholar 

  42. Truong Hoang, Q., T.-Y. Heo, D. G. Choi, S.-H. Choi, and M. S. Shim (2020) Guanidinium-incorporated micelleplexes for low toxic and efficient siRNA delivery. Macromol. Res. 28: 1160–1165.

    Article  Google Scholar 

  43. Shim, M. S. and Y. J. Kwon (2009) Controlled cytoplasmic and nuclear localization of plasmid DNA and siRNA by differentially tailored polyethylenimine. J. Control. Release 133: 206–213.

    Article  CAS  Google Scholar 

  44. Calnan, B. J., B. Tidor, S. Biancalana, D. Hudson, and A. D. Frankel (1991) Arginine-mediated RNA recognition: the arginine fork. Science 252: 1167–1171. (Erratum published 1992, Science 255: 665)

    Article  CAS  Google Scholar 

  45. Shen, W., R. Wang, Q. Fan, Y. Li, and Y. Cheng (2020) Natural polyphenol assisted delivery of single-strand oligonucleotides by cationic polymers. Gene Ther. 27: 383–391.

    Article  CAS  Google Scholar 

  46. ISO (2009) Biological Evaluation of Medical Devices — Part 5: Tests for In Vitro Cytotoxicity. 3rd ed. International Organization for Standardization, Geneva, Switzerland.

    Google Scholar 

  47. Whitehead, K. A., R. Langer, and D. G. Anderson (2009) Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8: 129–138. (Erratum published 2009, Nat. Rev. Drug Discov. 8: 516)

    Article  CAS  Google Scholar 

  48. Hong, C. A. and Y. S. Nam (2014) Functional nanostructures for effective delivery of small interfering RNA therapeutics. Theranostics 4: 1211–1232.

    Article  Google Scholar 

  49. Bhang, S. H., K. Kim, W. J. Rhee, and M. S. Shim (2018) Bioreducible polyspermine-based gene carriers for efficient siRNA delivery: effects of PEG conjugation on gene silencing efficiency. Macromol. Res. 26: 1135–1142.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by Research Assistance Program (2019) in the Incheon National University and 2022 Hongik University Innovation Support Program Fund.

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  1. Tae-Young Heo and Quan Truong Hoang contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical Engineering, Hongik University, Seoul, 04066, Korea

    Tae-Young Heo, Seung-Hwan Oh, Moon-Chul Ryu & Soo-Hyung Choi

  2. Department of Nano-Bioengineering, Incheon National University, Incheon, 22012, Korea

    Quan Truong Hoang, Thuy Giang Nguyen Cao & Min Suk Shim

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  1. Tae-Young Heo
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Heo, TY., Hoang, Q.T., Cao, T.G.N. et al. Guanidinium-functionalized Block Copolyelectrolyte Micelleplexes for Safe and Efficient siRNA Delivery. Biotechnol Bioproc E 27, 1004–1013 (2022). https://doi.org/10.1007/s12257-022-0222-6

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