الفهرس 
General introductionOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma (HCC) is a debilitating highly prevalent tumor that is usually managed via systemic chemotherapy. However, the currently available treatments fall short due to the lack of selectivity to tumor tissues, which in turn leads to low availability of the drug at the active site begetting poor efficacy and increased distribution all over the body resulting in intolerable side effects.
Resveratrol (Res) is a natural polyphenol that exhibits multiple health benefits being antioxidant, anti-inflammatory and anticancer. The anti-proliferative activity of Res against HCC while showing mild adverse events raised hopes that synthetic harsh cytotoxic agents could be replaced with green phytochemicals. On the other hand, Res has some limitations regarding its delivery to the tumor site, where Res has low water solubility, photo-degradability, proneness to alkaline degradation, poor oral bioavailability, short half-life and high plasma protein binding. Thus, a drug delivery system (DDS) capable of mitigating these challenges is sought after to improve the anticancer potential of Res.
The era of nanomedicine came with great promise to enhance the delivery of challenging drugs. Zein is a plant protein extracted from maize. It was approved by the FDA as generally regarded as safe as it is biocompatible and biodegradable. Its low water solubility, capability to get nanonized into small-sized nanoparticles (NPs) which can carry high payload, and sustain the drug release and the flexibility of surface engineering encourage further exploitation of zein nanoparticles (ZNPs).
Res-loaded ZNPs were prepared by the anti-solvent precipitation method (ASP). The effect of the factors affecting Res-ZNPs characteristics namely; ethanol concentration, the concentration of zein and the percentage of the anti-solvent phase were investigated and optimized utilizing Box-Behnken design (BBD). Twelve formulae were prepared and characterized where they showed high entrapment efficiency (EE%) of Res (>53%) with a monomodal distribution of small particle size (<200 nm). The optimized formulation (R4) had a particle size (PS) 158±2.81 nm, polydispersity index (PDI) 0.092±0.023, zeta potential (ZP) 35.1±0.6 mV and EE% 79.42%±1.5%.
In an attempt to increase the efficacy of Res-ZNPs, their surface was engineered to create PEGylated and ligand-conjugated Res-ZNPs to achieve long-circulating NPs that can evade the mononuclear phagocytic system (MPS) and hepato-selective NPs that can be more efficiently internalized specifically by hepatocytes, respectively. The PEGylation was carried out via 2 strategies. One strategy was applied by adding polyethylene glycol 4000 (PEG 4000) during the NPs fabrication and the other was applied by adding it after the NPs fabrication creating a hybrid PEG-R4 and PEG-coated-R4, respectively. Successful PEGylation and insights on the location of PEG were deduced from the dynamic light scattering data (DLS). In hybrid-R4, the PEG was partially embedded in the core and partially protruding on the surface, whereas in coated-R4, PEG was located exclusively on the surface. Both PEGylated formulae with PEG 4000 concentration 2.5% w/v had a small PS (172.3 and 168.5 nm) with good homogeneity, but they differed in the ZP (31.2 and 25.3 mV) and the EE% (70.76% and 38.85%) for the hybrid and the coated formulae, respectively.
Glycyrrhetinic acid (GA) and lactobionic acid (LBA) are natural ligands that can bind with high affinity to the asialoglycoprotein receptor (ASGPR) that is found preferentially at the surface of hepatocytes. These ligands were chemically conjugated to zein via EDC/NHS coupling protocol and the degree of conjugation was verified and quantified using 1H-NMR spectroscopy. The mass ratio of GA to zein was 128.93 mg/g and that of LBA to zein was 117.43 mg/g. The colloidal properties of GA and LBA-conjugated R4 were as follows: PS (166.1 and 169.4 nm), PDI (0.084 and 0.097), ZP (23.5 and 24.2 mV) and EE% (80.27 and 79.56%) for GA-and LBA-R4, respectively.
All the modified Res-ZNPs had a smooth spherical geometry displaying a core-shell feature as were evident in the transmission electron microscope (TEM) micrographs. Native and modified Res-ZNPs were able to release Res in a sustained manner over 96 h unlike free Res that was completely released in 3 h. Coated-R4 showed the slowest rate and the least amount released of Res while GA-R4 showed the highest amount released after 96 h.
The anti-proliferative activity of Res against HepG2 cell line was improved via the native and the ligand-conjugated formulae where the IC50 was in the order of GA-R4 < LBA-R4 < R4 < Res. Both of the PEGylated formulae did not cause cytotoxicity to HepG2 cells owing to their poor cellular uptake due to the steric hindrance of the PEG shell, a phenomenon known as” The PEG dilemma”. A competitive binding assay was performed where the ASGPR was saturated with free ligands before incubation with the ligand-conjugated formulae. This had the effect of decreasing the cytotoxicity of GA-R4 and LBA-R4 as clarified by the increased IC50, which is a testimonial on the role of receptor-mediated endocytosis in the NPs uptake.
A computational study was undergone to compare the affinity of GA and LBA to ASGPR via molecular docking. The study concluded that GA had a higher binding affinity than LBA suggesting better uptake. This result backed up the cytotoxicity results that showed that GA-R4 had a lower IC50 than LBA-R4.
The storage stability of the 5 formulae at 4 ℃ was tracked over 6 months where the colloidal properties and the EE% were measured monthly. Hyb-R42.5% and GA-R4 were the most stable with no change in the PS or the EE% over the whole 6 months. R4 had good storage stability showing only slight increment in the PS while maintaining the EE%. LBA-R4 suffered some enlargement in its PS but the EE% was intact. Coated-R42.5% was the least stable formulation where the PS increased over 5 months till some aggregates formed at the 6th month. The EE% decreased sharply after 1 month.