الفهرس 
Scope of workOnly 14 pages are availabe for public view
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Abstract
Excessive free radicals’ generation and cellular oxidative stress are implicated as a causative or an adjuvant of various pathologies, such as hepatic fibrosis, pulmonary inflammation, cardiomyopathy, diabetic complications, renal disease, brain aging and neurodegenerative conditions. Several chemotherapeutic agents are a potential source of free radicals and reactive species, which impede their therapeutic use.
Doxorubicin (DOX) is a highly effective chemotherapeutic agent. DOX metabolization, on the other hand, generates reactive oxygen species (ROS) and hydroxyl radicals (OH). Excessive ROS generation triggers the lipid peroxidation and the depletion of endogenous antioxidant enzymes resulting in mitochondrial dysfunction, cellular injury, and multi-organ toxicity. Hence, searching for a protective agent remains a challenge.
Recently, there is a great interest towards the combination of natural antioxidants with chemotherapeutics to avoid the harmful non-targeted effects, which can be toxic to healthy cells. However, most natural antioxidant agents suffer from their low bioavailability and cellular uptake.
Encapsulating antioxidant agents in biodegradable and biocompatible nanoparticles might enable to overcome these issues. Such albumin nanoparticles can be internalized and engulfed by endocytosis, might provide sustained drug release and enhanced cellular uptake. Besides, albumin nanoparticles (ANPs) play a crucial role in passive targeting and accumulate in solid tumors and inflamed tissues because of their enhanced permeability and retention. Furthermore, albumin has endogenous direct and indirect antioxidant capabilities that may enhance the efficacy of the loaded antioxidant agent.
Sesamol (SML), a natural phenolic antioxidant agent, has a potent antioxidant, anti-inflammatory, and free radical scavenging activity. However, SML has poor intracellular bioavailability (Fic). SML also suffers from rapid clearance that is associated with the appearance of its metabolites such as; sesamol sulphate/glucuronide within four hours.
In this study, sesamol-loaded albumin nanoparticles were fabricated and assessed for the first time as a potential nanoplatform to overcome the limited bioavailability and improve the pharmacokinetics profile.
As a result, the work in this thesis was separated into two main chapters:
• Chapter I: Preparation, optimization and characterization of sesamol-loaded albumin nanoparticles
The desolvation technique allowed fabrication of a homogenous SML-ANPs with a drug loading capacity ranging from 14.8 to 116.7 µg/mg HSA. All the prepared formulations showed particle diameters ranging from 111.5 to 367.1 nm with acceptable PDI < 0.3, and zeta potential.
The concentration of HSA, pH of albumin-drug solution and the type of the desolvating agent were treated as three independent variables affecting the SML-ANPs formulation. Three levels for each of the investigated factors were chosen to generate an experimental design, namely, the D-optimal design. The design matrices comprised 18 experimental runs. The optimum formulation according to the predetermined parameters in the design and data analysis was successfully fabricated at pH 8 using HSA with a weight ratio of 5:1 of HSA to SML ethanol acetone mixture and GA as a desolvating agent and a cross-linker, respectively. Formulation no. 14 was significantly in agreement with their corresponding predicted values gained by the design expert software. So, it was chosen for further examination.
Mean particle size and PDI were determined by dynamic light scattering technique where, the selected SML-ANPs had an average particle size of 127.24 ±2.12 nm and a PDI of 0.07±0.014. SEM imaging of SML-ANPs showed that the particles had a regular spherical shape with a smooth surface and a mean particle size of 120.7917.69 nm (n = 10), which is consistent with the findings obtained from DLS study. The surface charge of nanoparticle dispersions was determined by measuring the ZP. All the fabricated nanoparticles had overall negative Zeta-potential values of -26.2±1.53 mV, and that value rose primarily with increasing the pH value.
Sesamol HPLC procedure revealed good linearity of the calibration line and reproducibility of the test in the concentration range of 10 to 100 g/ml. Where, the unbounded HSA concentration in the supernatant was determined spectrophotometrically based on the method of Bradford. The encapsulation efficiencies and SML payloads of SML-ANPs measured by HPLC optimized procedure were of 87.8±61.0% and 96.8±92.4 g/mg, respectively, where drug EE% rose when the HSA ratio was raised.
FT-IR analysis revealed an interaction between HSA and SML, as well as incorporating SML into albumin nanoparticles, supporting the hypothesis that the hydroxyl group in phenolic compounds such as sesamol can interact with the C=O and C–N groups in protein polypeptide chains besides the aromatic hydrophobic interaction and had a significant influence on the modifications in the secondary structure of the protein.
Differential scanning calorimetry (DSC) analysis was performed to detect any change in the physical state of SML loaded in albumin nanoparticles. The thermogram of SML/HSA 4-h incubated mixture after lyophilization showed a change and broadening in the melting peak of sesamol, thus indicating an interaction between albumin and sesamol. Moreover, SML-ANPs thermogram did not show the characteristic endothermic peaks of SML at 65.3 °C indicating SML crystallization was inhibited by albumin during nanoparticles formation, thus the drug is no longer existing in the crystalline form.
The tested nanoparticle formulations exhibited a biphasic release pattern, comprising an initial burst release within the first 2 h, followed by a controlled release pattern, with a total percentage of SML released over 72 h around 62.3 %. The first rapid release can be because of SML desorption and diffusion from the outer surface of ANPs, while the slow release is mainly because of the slow diffusion of SML through the albumin matrix. The release data of the nanoparticle’s formulation were fitted to several kinetic models, with a satisfying match to the Higuchi model and the Korsmeyer-Peppas model showing that SML release from albumin nanoparticles was governed by a Fickian diffusion mechanism.
The freeze-drying process was adopted to maintain the suspension of SML-ANPs and enhance their long-term efficacy. The absence of cryoprotectants led to an enormous increase in particle size to 386 nm and a lack of homogeneity (PDI, 0.52). After reconstitution of the freeze dried SML-ANPs with different types of cryoprotectants, trehalose was able to maintain the properties of SML-ANPs and showed a long-term stability up to six months with a non-significant difference (p≥0.05).
As a result, SML-ANPs may be regarded as a potential delivery strategy for extending SML half-life and overcoming limited bioavailability. Thus, it was chosen for additional in vitro and in vivo antioxidant activity tests.
• Chapter II: Antioxidant activity of sesamol-loaded albumin nanoparticles in vitro and in vivo.
In vitro and in vivo assessments were carried out in animal models of oxidative stress induced by DOX. The drug cellular protection of SML-ANPs was tested on rats’ hepatocytes pretreated with 1 µM doxorubicin and showed 1.2-fold higher protective activity than the free sesamol. The co-treatment with both free and encapsulated sesamol had a protecting effect. However, higher hepato-protectivity of the used antioxidant drug was observed when it was encapsulated in albumin nanoparticles compared to the free drugs. This can be explained by the efficient internalization, localization and endocytic uptake of albumin nanoparticles via different pathways.
The pharmacokinetic study showed that the average half-life, mean retention time and AUC were significantly higher (p ˂ 0.05) than that of SML-treated group, whereas the clearance rate was lower (p ˂ 0.05). The above results indicates that SML-ANPs increased the systemic circulation time leading to a higher amount of SML available for cellular uptake.
Furthermore, in vivo efficacy and biochemical assessment of lipid peroxidation, cardiac biomarkers and liver enzymes were significantly ameliorated after administration of the sesamol-loaded albumin nanoparticles. The sesamol-loaded albumin nanoparticles considerably improved the in vivo effectiveness and biochemical assessment of lipid peroxidation, cardiac biomarkers, and liver enzymes. By cumulative i.p. injection of DOX, serum levels of CK, LDH, AST, and ALT were significantly elevated to 2.7, 4, 3.3, and 3-fold above the corresponding control values, respectively. However, co-treatment with SML-ANPs significantly reduced the elevated serum CK and LDH by 59.3 and 64.5% respectively, while AST and ALT were reduced by 60.5 and 59% in comparison to the corresponding values of free SML treated group were 35.6, 47.2, 49.9 and 41.2%, respectively. The in vivo results were accompanied by a significant increase in the MDA levels above the control values by 21.6, 9.4, 114, 7.5 and 8.8-folds in serum, cardiac, liver, kidney and testis samples, respectively. The protection of doxorubicin -treated rats with SML-ANPs succeeded in normalizing MDA levels. Where, MDA activity was significantly reduced in serum, cardiac, liver, kidney and testis by 88.2, 77.3, 80, 72.3 and 81.2% respectively in comparison to the corresponding values of free SML-treated group were 61.9, 55.9, 59, 41.7 and 86.5%, respectively.
The biochemical assessments were also corroborated with the histopathological examination data. Sesamol-loaded albumin nanoparticles, prepared under controlled conditions, might provide an enhanced protective effect against non-targeted doxorubicin toxicity. In histopathological study, DOX treated group showed remarkable degenerative changes at different grades in the cardiac, liver, kidney and testis tissues in the form of cellular atrophy, cytoplasmic vacuolization, lymphoid cell aggregation, coagulative necrosis, edema and hemorrhages. SML counteracted the damaging effects of oxidation. However, SML-HAS-NPs treatment markedly ameliorated the DOX-induced pathological changes and maintained the normal histological picture.
In animal experiments, although there was no significant difference in the animals’ body weight between SML-treated or SML-ANP-treated animals during the first two weeks, the difference after the third week was highly significant (p value < 0.001). Consequently, these findings pointed to the enhanced protective effect of SML-ANPs at the tested dose. Besides, during the study period, there was no mortality in both control and SML-ANP-treated groups. However, throughout the experiment, three animals died; two animals in DOX-treated GP and only one animal in SML-treated GP.
Finally, according to these findings, sesamol-loaded albumin nanoparticles enhanced and sustained sesamol antioxidant efficacy against doxorubicin-induced oxidative stress. As a result, sesamol-loaded albumin nanoparticles may be a promising nano-platform to give improved protection against non-targeted doxorubicin toxicity.