الفهرس 
General Introduction
MethodologyOnly 14 pages are availabe for public view
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Abstract
Title of thesis:
Lipid Polymer Hybrid Nanoparticles as an Efficient Hepatic Drug Delivery
System
Summary
In spite of the great development in the field of hepatology, CHB, is a
major public health problem worldwide. Unfortunately, CHB is a major public
health challenge in Egypt where 1.4% of the total population were diagnosed with
HBV and millions are undiagnosed or at risk. E, a nucleoside reverse
transcriptase inhibitor, is one of the first line potent antiviral therapies for HBV
which could reduce the risk of virus progression to liver cirrhosis and
hepatocellular carcinoma. Although its proved efficacy, E is accompanied with
some drawbacks as high risk of resistance and serious adverse effects as
neuropathy, skeletal and cardiac myopathy, nephrotoxicity, pancreatitis and
lactic acidosis. Moreover, the high rate of non-compliance in patients with
chronic diseases and long-life treatment are serious healthcare concerns that pose
a great challenge to the successful delivery of pharmaceuticals.
To tackle these problems, intramuscular administration of E has been
attempted. To provide a suitable delivery system, we hypothesized that LPH will
be able to control the hydrophilic drug release in a sustained manner.
Accordingly, the ultimate goal of this study was to control E release and
improve its macrophage uptake. A special focus will be given to the effects of E
loading technique, the tuning of the lipid corona as well as vitamin E coating on
its release and uptake.
To fulfill the above goal, the work in this thesis was divided into three
chapters:
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I. Preparation and characterization of Entecavir Lipid Polymer Hybrid
Nanoparticles.
II. Preparation and characterization of Entecavir-Hyaluronate conjugate
Lipid Polymer Hybrid Nanoparticles.
III. Biological Studies on Different Entecavir Lipid polymer Hybrid
Nanoparticles.
Chapter I
Preparation and characterization of Entecavir Lipid Polymer Hybrid
Nanoparticles.
In this chapter we aimed to prepare LPH with high drug EE% and PS <
200 nm suitable for the controlled delivery of E, to the liver. Four studies were
performed: study I: A preliminary study to set the surfactant concentration and
time of stirring which deemed to be of crucial importance during
nanoprecipitation. Study II, where optimization of different critical process
parameters on E LPH was done to achieve the quality target product profile.
Thirdly the release of the optimized LPH, with the highest EE% and minimum
PS<200 nm, was modified using different shell lipid with the evaluation of their
different physicochemical characteristics. Finally, the effect of vitamin E coating
on the characteristics of the optimally lipid modified LPH was investigated.
PS and EE % could be manipulated by changing the polymer, lipid and/ or
E amount as well as the stirring speed. The optimized formula were selected
depending on the maximum EE % and PS < 200 nm with the following
composition:
F1-LEC: 5 mg PLGA, 2.85 mg lecithin and 13.33 mg E prepared at 900
rpm.
F2-LEC: 15 mg PLGA, 1 mg lecithin and 9.45 mg E prepared at 900 rpm.
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F3-LEC: 5 mg PLGA, 2.93 mg lecithin and 9.45 mg E prepared at 1000
rpm.
All the prepared formulae showed a negative zeta potential ranged from
-14.51±1.85to -25.51±2.62 depending on E amount and lecithin content. The in
vitro release study showed biphasic drug release with T100% achieved in 12-18 h.
The in vitro release depended directly on the drug amount and inversely polymer
to lecithin ratio.
In our study, using lecithin as the sole component in the lipid shell
prolonged the release for less than 24 h. Hence, it deemed necessary to modify
the lipid shell in order to, provide more control over the E release from the
proposed LPH. Different lipid mixtures were tried to control drug release namely;
lecithin in combination with cholesterol or glyceryl monostearate in different
ratios (1:1, 1:1.5, 1:2) while keeping the total lipid amount constant during the
fabrication. All the tried lipid mixture ratios resulted in significant increase in PS.
However and as previously highlighted, a PS below 200 nm is crucial for hepatic
passive targeting. Therefore, E LPH prepared using lecithin to cholesterol ratio
(1:1.5) and lecithin to GMS (1:1) were elected for further studies, viz. zeta
potential, drug loading efficiency and in vitro drug release. Moreover, the
prepared lipid shell modified LPH showed a significant increase in zeta potential.
Both cholesterol based and GMS based LPH extended E release for 36-48 h and
72-120 h respectively. A further coat with vitamin E extended the release for one
week, while keeping the PS ≤ 200 nm. conclude that the modification of lipid
shell and vitamin E coating of E loaded LPH have been provided a lucrative
option to control the E release over a period of one week. Such a control release
is efficient to achieve patient compliance and would be suitable for patients with
renal impairment. In the next chapter attempts were made for to prolong drug
release for liver patients without any accompanied diseases.
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Chapter II
Preparation and characterization of Entecavir-Hyaluronate conjugate
Lipid Polymer Hybrid Nanoparticles.
Moreover, the impact of drug incorporation technique in the LPH on E
release was studied. In this chapter, the preparation of vitamin E coated E-HA
LPH was exploited in order to achieve a controlled release of E. The drug was
first conjugated with HA and the resultant compound was characterized. An indepth
study of all structural alterations occurred during conjugation process was
assessed by UV spectrophotometric analysis, 1H NMR, 13C NMR and Mass
spectroscopy. The synthetized conjugate was further incorporated in LPH using
the optimized lipid shell composition and vitamin E coating based on the results
of chapter I. The primary purpose of using a drug-polymer conjugate for
nanocarriers’ drug delivery is the modulation of drug’s release and kinetics
towards controlled one and ideally, zero order release kinetics. Therefore, the
effect of using E-HA conjugate in vitamin E coated LPH fabrication was studied
by drug association, PS and zeta potential. AFM and TEM were used to give an
insight on the morphological characteristics of the proposed system. Moreover,
the feasibility of controlling the release of Ewas tested by in vitro drug release
study and comparing the results to vitamin E coated LPH with passively loaded;
unconjugated E. In this study, the grafting of E with HA was based on the
carbodiimide chemistry with conjugation efficiency up to ≈ 30%. The prepared
E-HA conjugate was successfully used to fabricate LPH with also the same
QTPP (PS < 200 nm and maximum association efficiency). The obtained results
showed that using E-HA conjugate led to controlled release over a period of one
month. The conjugation of E to HA prior to fabrication the LPH as well as the
customizable lipid shell could provide an efficient way to control E release. The
proposed system showed good stability in the presence of serum as well as shelf
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life stability. Further work will be conducted in the next chapter to evaluate the
feasibility of E-HA to improve drug cellular internalization.
Chapter III
Biological Studies on Different Entecavir Lipid polymer Hybrid
Nanoparticles
In this chapter, the cytocompatibility of the developed LPHs was evaluated
by testing its hemolysis effect on red blood cells and cytotoxicity on macrophage
J774 cells. The feasibility of vitamin E coating to improve the macrophage uptake
of different E LPHs was evaluated. The in vivo biodistribution of the
intramuscularly injected DiR labelled EE-HA LPH mice were assessed.
Furthermore, the body weight change, vital organ weight and histopathological
examination of liver, spleen, lung, kidney and intestine tissues were evaluated as
toxicity markers. The results of the toxicity studies were compared to those of
PBS injected mice.
The biocompatibility and cytocompatibility of either E LPH or E- HA LPH
were proven by low haemolytic effect on rats’ RBCs (< 5%) and negligible effect
on macrophage J774 cells viability. Moreover, the negative charge of the
proposed systems was able to evade the adsorption of plasma proteins and
subsequently kept the system stable under physiological conditions.
The second target of this study focused on the assessment of the LPH
ability on cellular retention in J774 macrophages cells. The cellular uptake of E
LPH was confirmed qualitatively and quantifiably by confocal laser microscope
and flow cytometry respectively. The presence of vitamin E coat on both types
of fabricated LPH significantly improved the macrophage uptake in comparison
to uncoated ones. Long muscular residence and high liver uptake were confirmed
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by in vivo imaging of the prepared E-HA based LPH which is positively
correlated with the in vitro release results.