Repository logo
 

Amino acid functionalized nanodiamonds as nucleic acid delivery vectors: synthesis and optimization of histidine-modified nanodiamonds, biological interaction studies and utilization for nucleic acid therapeutics

Date

2023-03-13

Journal Title

Journal ISSN

Volume Title

Publisher

ORCID

0000-0002-4506-9269

Type

Thesis

Degree Level

Doctoral

Abstract

Utilization of siRNA as therapeutic agents requires sophisticated carriers capable of packing, protecting, transporting, and delivering them to the target cells in an intact form. Among many non-viral nucleic acid delivery vectors, nanodiamonds (NDs) have shown promise to serve this purpose. Our group have covalently functionalized the surface of NDs with basic amino acids to impart a net positive charge facilitating complexation with negatively charged nucleic acids. The prototype lysine-NDs (K-NDs) exhibited excellent dispersion stability and cellular internalization of the siRNA, however, the transfection efficiency of K-NDs diamoplexes was sub-optimal due to endo-lysosomal entrapment in the cells. Therefore, current work focused on introducing histidine on the surface to modulate pH sensitivity of the NDs and facilitate endo-lysosomal escape of diamoplexes. Histidine was incorporated in the functionalization scheme in two configurations: (1) lysyl-histidine-NDs (HK-NDs) and (2) lysine/lysyl-histidine-NDs (H50K50-NDs) where histidine was attached to either 100% or 50% of the lysine residues on the surface. Upon encounter with the acidic medium of the endosomes, histidine undergoes protonation leading to a cascade of counter ion and water influx in the organelle, that leads to endosomal swelling and rupturing of the membrane, i.e., proton sponge effect. Timely release of diamoplexes from the endosomes prevents degradation of the siRNA and thus improves the overall transfection efficiency of the carrier. Functionalization of NDs with lysine and lysyl-histidine amino acid conjugates was maintained with desired physicochemical properties i.e., particle size < 200 nm and zeta potential higher than +20 mV. HK-NDs exhibited a surface loading of 14.49 mmoles/g. H50K50-NDs with two distinct functional moieties on the surface, lysine and lysyl-histidine, showed a surface loading of 2.73 and 1.74 mmoles/g respectively. The nucleic acid binding capacity, cellular uptake, intracellular fate, biocompatibility and transfection efficiency of fNDs varied with surface functionalization. HK-NDs complexed the siRNA at a ratio of 50:1 (ND: siRNA), 2.5 times higher than the prototype K-NDs, while H50K50-NDs showed improved nucleic acid complexation reducing the mass ratio to 30:1 (ND: siRNA), i.e., comparable to the K-NDs. Exposure of HK-NDs affected cell viability, showing more than 25% cell death even at therapeutic concentrations. H50K50-NDs successfully restored the innate biocompatible nature of NDs maintaining a cell viability above 85% up to 500 µg/mL. The ranking of cellular uptake was: H50K50-NDs (~49%) >> K-NDs (~25%) > HK-NDs (~21%). All fNDs showed exit after 5 days, leaving less than 10% cells with diamonds. Unlike the K-NDs, histidine-modified fNDs showed successful endosomal escape process driven by histidine-mediated proton sponge effect. Finally, we established that fNDs are feasible as siRNA delivery vectors to target oncogenes overexpressed in hepatocellular carcinoma. Both HK-NDs and H50K50-NDs diamoplexes transfected the siRNA against anti-myeloid cell leukemia-1 (anti-MCL-1) i.e., the target oncogene overexpressed in hepatocellular carcinoma, exhibiting a decline in relative MCL-1 expression with respect to untreated cells by 45% and 61% respectively. H50K50-NDs performed ~ 1.3 times better than the gold standard Lipofectamine control. Furthermore, downregulation of MCL-1 oncogene increased the sensitivity of the cells by 1.8 times towards an apoptosis inducing drug melphalan. Overall, my work demonstrated that histidine-mediated pH modulation renders high efficiency to diamoplexes.

Description

Keywords

nanodiamonds, nucleic acid delivery, amino acid functionalization, lysine, histidine, diamoplexes, MCL-1 oncogene

Citation

Degree

Doctor of Philosophy (Ph.D.)

Department

Pharmacy and Nutrition

Program

Pharmacy

Part Of

item.page.relation.ispartofseries

DOI

item.page.identifier.pmid

item.page.identifier.pmcid