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Double-grafted chitosans as siRNA nanocarriers: effects of diisopropylethylamine substitution and labile-PEG coating

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Abstract

The preparation of safe and efficient siRNA carriers remains a challenge that has limited the therapeutic applications of siRNA. In this study, the design of a new small interfering RNA (siRNA) carrier based on diisopropylaminoethyl-chitosan was devised for application in non-viral gene therapy. Polycations having varied proportions (11–32%) of diisopropylethylamine groups (DIPEA) and grafted with polyethylene glycol (1–3%) were synthesized and characterized. The physicochemical and biological properties of the polymers and their nanoparticles were evaluated at pH 6.3 and pH 7.4. The degrees of ionization at pH 7.4 were precisely controlled by the composition and increased from 13% for chitosan to 47% for the more substituted derivative. Nanoparticles with very low toxicities and sizes in the range of 100–200 nm, remained stable up to 24 h after their preparation in both the evaluated pHs under plasma osmolality. As probed by scanning electron and confocal microscopies, an efficient cell uptake of spherical nanoparticles mediated a TNFα knockdown of almost 60% in RAW 264.7 macrophages, and mRNA silence levels higher than the Lipofectamine (up to 90%) in HeLa cells. Overall, the results showed that these derivatives are promising vectors for in vivo studies under physiological conditions.

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Acknowledgements

This research was supported by a grant from the São Paulo Research Foundation, FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) grant n. 2017/10331-5. A. M. Martinez-Junior acknowledge the support of the National Council for the Improvement of Higher Education, CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) grant n. 2017/10331-5. A. M. Martinez-Junior and M. S. Petrônio acknowledge the support of FAPESP (grants 2019/27801-0 and 2015/05148-1). J. Fernandes and M. Benderour would like to thank the Ministère de l’Économie, de la Science et de l’Innovation du Québec, (PSR-SIIRI-960) and the Chaire de Recherche en Orthopédie de l’Université de Montréal à l’Hôpital du Sacré-Cœur de Montréal). The authors would also like to thank: the CMIB-UNESP (EMU-FAPESP project nº 2009/53989-4) for instrumentation access and Dr. F. R. de Moraes for his help with 13C NMR analysis; Dr. S. C. M. Agustinho and A. L. Tognon (MSc) for help with 1H NMR analysis (IQSC-USP); the LMA-IQ (UNESP) laboratory for providing SEM facilities and Dr. D. Tita for his assistance with Scanning Electron Microscopy; Dr. M. F. Lima (LQBOA Group-UNESP/IBILCE), Dr. M. P. S Cabrera (Peptides Research Group-UNESP/IBILCE) and Dr. C. R. B. Domingos (LHGDH Group-UNESP/IBILCE) for access to facilities and instrumentation.

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  1. Department of Chemistry and Environmental Sciences, IBILCE, São Paulo State University-UNESP, R. Cristóvão Colombo, São José do Rio Preto, SP, 2265, 15054-000, Brazil

    André Miguel Martinez Junior, Maicon Segalla Petrônio, Grazieli Olinda Martins, Vera Aparecida Oliveira de Tiera & Marcio José Tiera

  2. Department of Physics, IBILCE, São Paulo State University-UNESP, São José do Rio Preto, Brazil

    Ricchard Hallan Felix Viegas de Souza

  3. Orthopedic Research Laboratory, Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Montréal, Canada

    Júlio Cesar Fernandes & Mohamed Benderdour

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  1. André Miguel Martinez Junior
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MJT, JCF, AMMJ, RHFVS and MB have filed for a patent on the nanoparticles described in this study. The authors report no other conflicts of interest in this study.

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Supplementary file1 1H and 13C NMR spectra, dynamic light scattering measurements, cytotoxicity in RAW 264.7 cells, 3D confocal images, GPC chromatograms, and titrations data can be found in the supplementary information of the manuscript. (DOCX 5337 KB)

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Martinez Junior, A.M., de Souza, R.H.F.V., Petrônio, M.S. et al. Double-grafted chitosans as siRNA nanocarriers: effects of diisopropylethylamine substitution and labile-PEG coating. J Nanostruct Chem 13, 605–624 (2023). https://doi.org/10.1007/s40097-022-00487-0

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