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.

Surface Modified Multifunctional and Stimuli Responsive Nanoparticles for Drug Targeting: Current Status and Uses

“…However, for most biomedical applications including drug delivery, the precise control of their surfaces via functionalization is necessary [ 83 , 84 , 85 , 86 , 87 ]. It has been pointed out that NDs are not used neat but are conjugated with biocompatible polymers and drugs, similarly to previously reported cases with CNTs and GOs focusing on cancer treatment [ 84 , 88 , 89 , 90 , 91 , 92 ].…”

Biomedical applications of nanodiamond (Review)

“…Perhaps, the most studied application of ND for gene delivery is based on the non-covalent integration of polycationic molecules onto the ND surface followed by association with negatively charged nucleic acids [69,87,152,[178][179][180][181][182][183] (inset, figure 6). For example, pEGFPLuc plasmids encoding Luciferase and green fluorescent protein (GFP) have been successfully delivered in cytoplasm by means of aa ND-PEI vector [178].…”

Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein

“…DLS measurements of proanthocyanidins were performed at 0.1, 1, 5, 10, and 50 μM concentrations in DMEM + 10% FBS (constant 0.5% final DMSO across all samples) using a Brookhaven 90 Plus particle size analyzer. Measurements were conducted after incubating samples for 30 minutes at room temperature (covered to protect from dust particles) using a viscosity of 0.94 cP ( Fröhlich et al, 2013 ), refractive index of 1.345 ( Alwani et al ), wavelength of 658 nm and scattering angle of 90°.…”