الفهرس 
General IntroductionOnly 14 pages are availabe for public view
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Abstract
Evaluation of sperm countExcessive 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 nontargeted
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
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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
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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
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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
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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
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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,
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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 SMLtreated
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 DOXinduced
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 SMLANP-
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 DOXtreated
GP and only one animal in SML-treated GP.
Finally, according to these findings, sesamol-loaded albumin
nanoparticles enhanced and sustained sesamol antioxidant efficacy
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against doxorubicin-induced oxidative stress. As a result, sesamolloaded
albumin nanoparticles may be a promising nano-platform to
give improved protection against non-targeted doxorubicin toxicity.