Date of Award
Doctor of Philosophy
Metallurgical and Materials Engineering
Leishmaniasis, a neglected tropical disease (NTD), manifests through protozoan parasites belonging to the genus Leishmania. Treatments for cutaneous leishmaniasis (CL), the most distributed form of the disease, have controversial effects and are high in cost leading to an urgent need for novel therapies. Î±,Î² unsaturated ketones are promising agents with effective, anti-parasitic properties hindered primarily by their hydrophobic nature. In its nascence, this work sought to improve the solubility of previously reported anti-leishmanial compound NC2459 using nanoparticles (NPs) made from gelatin type A. However, the conditions for NP formation were found incompatible to NC2459, re-directing our efforts towards analogue molecule, 1-acetyl-3,5-dibenzylidene-4-piperidone (AO1-Ac). In addition, we included bovine serum albumin (BSA) as an alternate protein base for our delivery system. The aims of this work were three-fold: 1) optimize NP formulations to properly encapsulate AO1-Ac, 2) determine the toxicity of loaded and unloaded NPs, and 3) validate the cellular uptake of the aforementioned NP variations. Prioritizing stability, size, and loading capacity, particles were prepared using variations of the desolvation method. The fabrication of NPs was first examined using gelatin in PBS at a pH range concordant with the buffer and isoelectric point (pI) of gelatin type A (7-9). For pH 5.5, 7.4, and 9, the average sizes for GNPs in PBS were 151 Â± 42 nm, 121 Â± 29 nm, and 191 Â± 15 nm. Though the resultant sizes were considered acceptable, protein NPs produced in PBS displayed a larger distribution and thus a higher size heterogeneity compared to protein particles developed in DI water. In an aqueous solution of pH 4, the size, polydispersity index (PDI), and zeta potential for GNPs containing AO1-Ac were found to be 177 Â± 16 nm, 0.34 Â± 0.08, and 12 Â± 0.4 mV, respectively. Under alkaline conditions, pH 9, the values for BSA nanoparticles (BSA-NPs) were 187 Â± 10 nm, 0.18 Â± 0.02, and -13 Â± 0.5 mV. Release studies using an agitation method in 60% ethanol determined the encapsulation efficiency (EE%) of AO1-Ac within the PBNPs to be 7.8 Â± 3.2% and 40.8 Â± 16.5% for BSA-NPs and GNPs, respectively. For L. major promastigotes, the EC50 values of AO1-Ac in PBNPs was 3.75 ÂµM for BSA-NPs and 7.5 ÂµM for GNPs. Further in vitro viability studies of PBNPs containing 0.4 ÂµM of AO1-Ac showed a significant decrease in promastigote proliferation compared to the control group in both NP variants (p < 0.01). To test for cytotoxicity, blank PBNPs and PBNPs containing 10 ÂµM of AO1-Ac were examined in human foreskin fibroblasts which revealed minimal cell death for all variations. PBNP uptake into cells was observed in mouse monocytes using Atto 565-conjugated BSA-NPs and GNPs containing rhodamine B. Confocal imaging displayed NP aggregation on the cell surface and within the cell membrane validating internalization for all NP types. SEM analysis determined that all PBNPs had smooth surfaces, were uniform in size, and had spherical appearance. Collectively, we demonstrate an effective method for encapsulating a hydrophobic treatment molecule enhancing its capacity for drug delivery. Furthermore, the evidence provided supports the application of PBNPs as promising delivery vehicles for other non-polar therapeutic agents.
Received from ProQuest
Carlos Serna III
Serna Iii, Carlos, "Formulation Of Protein-Based Nanoparticles As Vehicles For Drug Delivery" (2020). Open Access Theses & Dissertations. 3037.