Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application

Abstract

Purpose

Nanodiamonds (NDs) are emerging as an attractive tool for gene therapeutics. To reach their full potential for biological application, NDs should maintain their colloidal stability in biological milieu. This study describes the behavior of lysine-functionalized ND (lys-ND) in various dispersion media, with an aim to limit aggregation and improve the colloidal stability of ND-gene complexes called diamoplexes. Furthermore, cellular and macromolecular interactions of lys-NDs are also analyzed in vitro to establish the understanding of ND-mediated gene transfer in cells.

Methods

lys-NDs were synthesized earlier through covalent conjugation of lysine amino acid to carboxylated NDs surface generated through re-oxidation in strong oxidizing acids. In this study, dispersions of lys-NDs were prepared in various media, and the degree of sedimentation was monitored for 72 hours. Particle size distributions and zeta potential measurements were performed for a period of 25 days to characterize the physicochemical stability of lys-NDs in the medium. The interaction profile of lys-NDs with fetal bovine serum showed formation of a protein corona, which was evaluated by size and charge distribution measurements. Uptake of lys-NDs in cervical cancer cells was analyzed by scanning transmission X-ray microscopy, flow cytometry, and confocal microscopy. Cellular uptake of diamoplexes (complex of lys-NDs with small interfering RNA) was also analyzed using flow cytometry.

Results

Aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Size distributions showed good stability, remaining under 100 nm throughout the testing period. A positive zeta potential of >+20 mV indicated a preservation of surface charges. Size distribution and zeta potential changed for lys-NDs after incubation with blood serum, suggesting an interaction with biomolecules, mainly proteins, and a possible formation of a protein corona. Cellular internalization of lys-NDs was confirmed by various techniques such as confocal microscopy, soft X-ray spectroscopy, and flow cytometry.

Conclusion

This study establishes that dispersion of lys-NDs in aqueous medium maintains long-term stability and also provides evidence that lysine functionalization enables NDs to interact effectively with the biological system to be used for RNAi therapeutics.

Keywords:

• colloidal stability
• particle size
• zeta potential
• protein corona
• diamoplex
• microscopy
• flow cytometry

Acknowledgments

The authors acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Saskatchewan for funding this project. Saniya Alwani would also like to thank the College of Pharmacy and Nutrition, the College of Graduate Studies and Research at the University of Saskatchewan for supporting her graduate studies. The authors thank Tosoh Corporation, USA, for the kind donation of the YTZ grinding media used in these studies. The scanning transmission X-ray microscopy data described in this paper were collected at the Soft X-ray Spectromicroscopy beamline of the Canadian Light Source (CLS), which is supported by NSERC, the National Research Council Canada (NRC), the Canadian Institutes of Health Research (CIHR), the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan.

Disclosure

The authors report no conflicts of interest in this work.