ENGINEERING FUNCTIONALIZED DIAMOND NANOPARTICLES FOR GENE DELIVERY: BIODISTRIBUTION STUDIES

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Date
2020-02-18Author
Rai, Raj 1990-
Type
ThesisDegree Level
MastersMetadata
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Purpose: To understand the biodistribution of lysine and lysyl-histidine functionalized NDs (collectively ‘fND’) employed as gene carriers. MicroPET imaging and biodistribution studies will elucidate their fate at the organ level.
Method: lysine and lysine histidine fNDs were synthesized via covalent conjugation using a 3-carbon chain linker. 1HNMR was used to confirm synthesis of amino acid conjugates after every step. Method development was carried out to optimize a synthetic approach for designing radiolabeled fNDs. A chelating agent desferoxamine (DFO) was conjugated to fNDs to allow labeling with a radionuclide, Zirconium 89 (89Zr). Thermograms of fNDs were used to quantify the percentage of DFO conjugation on ND surface. DFO was conjugated at 8%, 6%, 3% and 1% and characterized to maintain size and positive surface without compromising optimum radiolabeling efficiency. Pharmacokinetic and biodistribution studies of 89Zr-labeled fNDs were performed in naive Balb/c mice using microPET/CT imaging and ex vivo biodistribution
Results: Among all conjugation ratios, 3% coverage of DFO to fNDs maintained the optimal size of under 200 nm and positive surface charge of +19.4±4.1 mV. The conjugates formed diamoplexes (DFO conjugated fNDs/siRNA complexes) at biocompatible mass ratios. Radiolabeling method was developed and optimized with respect time and temperature achieving more than 90% radiochemical yield (RCY). The 89Zr ND complexes were stable in phosphate buffer saline and mouse blood serum for over 96 h (97 ± 2%). Biodistribution assays revealed high accumulation of fNDs in liver after 6 h (6.11 ± 4.06) and 72 h (2.32 ± 2.09) followed by spleen (0.17 ± 2.71 at 6 h and 1.66 ± 1.31 at 72 h). PET images further confirmed the finding of biodistribution assays.
Conclusion: This study establishes an understanding of in vivo behavior of fNDs for future design, optimization and application of these novel carriers for targeted gene therapy.
Degree
Master of Science (M.Sc.)Department
Pharmacy and NutritionProgram
PharmacySupervisor
Badea, Ildiko; Fonge, HumphreyCommittee
Krol, Ed; Yang, Jian; Dadachova, Ekaterina; Taghibiglou, ChangizCopyright Date
December 2019Subject
Nanodiamonds, Micro PET, delivery system, biodistribution