title: Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Saniya
authors.initials: S
authors.lastname: Alwani
authors.affiliation: Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Randeep
authors.initials: R
authors.lastname: Kaur
authors.affiliation: Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Deborah
authors.initials: D
authors.lastname: Michel
authors.affiliation: Department of Chemical & Biological Engineering, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Jackson M
authors.initials: JM
authors.lastname: Chitanda
authors.affiliation: Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Verrall
authors.affiliation: Canadian Light Source, Saskatoon, SK, Canada.
authors.forename: Chithra
authors.initials: C
authors.lastname: Karunakaran
authors.affiliation: Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Ildiko
authors.initials: I
authors.lastname: Badea
mesh_terms: D000818:Animals; D002417:Cattle; D003102:Colloids / Q000737:chemistry*; D004337:Drug Carriers; D016503:Drug Delivery Systems; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D018613:Microscopy, Confocal; D058612:Nanodiamonds / Q000737:chemistry*; D010587:Phagocytosis; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics*
chemical_list: D003102:Colloids; D004337:Drug Carriers; D058612:Nanodiamonds; D034741:RNA, Small Interfering; D008239:Lysine
keywords: colloidal stability; diamoplex; flow cytometry; microscopy; particle size; protein corona; zeta potential