الفهرس 
general introductionOnly 14 pages are availabe for public view
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Abstract
Summary
For centuries, crude CUR was used as food spice and dietary supplement, in
addition to traditional Asian medicine. Commercial turmeric extract contains
structurally-related curcuminoids; 75% diferuloylmethane/curcumin (curcumin
I), 20% demethoxycurcumin (curcumin II), 5% bisdemethoxycurcumin BDMC
(curcumin III). CUR gained immense popularity in the last decade with around
8400 hits on PubMed on usage of “curcumin” as the search string; possibly as a
‘next-generation multipurpose drug’ due to its widespread applicability in
prophylaxis and treatment of various pro-inflammatory chronic diseases,
including neurodegenerative diseases, cardiovascular, pulmonary, metabolic and
many other diseases.
Moreover, recently CUR anti-cancer activity has been extensively investigated
and supporting evidences were found for its potential use in chemoprevention
and treatment of a wide variety of tumors; including head and neck, lung,
colorectal, breast, GIT, genitourinary, melanoma, neurological and sarcoma.
Curcumin exerts its anti-cancer effect by various mechanisms; including:
activating apoptosis signaling and inhibiting cell proliferation as it inhibits Bcl2
and activates Caspase 9 to induce apoptosis, also it inhibits many signaling
pathways for tumour cell proliferation such as MAP kinase pathway, AKT
pathway and mTOR pathways. Moreover, it inhibits inflammatory cytokines
(TNFα, Interleukins & multiple protein kinases). CUR delivery faces some
challenges owing to their poor aqueous solubility, photodegradation, chemical
instability, poor bioavailability and rapid metabolism in vivo.
Interestingly, curcumin III (Bisdemethoxycurcumin) (BDMC) which is the
bisdemethoxylated derivative of curcumin was found to possess enhanced anti-
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Summary
cancer potency yet more hydrophobicity. Accordingly, its main delivery
problem lies in its extreme poor aqueous solubility. Currently, BDMC is under
extensive research for enhancing its targeted delivery to cancer cells and
improving its poor solubility.
Nanotechnology is considered the cutting edge of biomaterials research and the
science of future. Nano-systems gained widespread popularity because of their
potential to enhance the biological effect of incorporated drugs possibly by
protecting drugs from enzymatic degradation, providing controlled release,
altering their pharmacokinetics and prolonging their residence in plasma,
decreasing their toxicity and limiting nonspecific uptake to undesirable tissues.
Additionally, among the various applications of nanotechnology is enhancement
of aqueous solubility of lipophilic drugs like CUR and maximization of its
bioavailability and tissue biodistribution in drug delivery systems.
These nano-systems take the advantage of the unique properties of malignant
tissues to deliver chemotherapy specifically towards solid tumors and spare
normal cells with maximized therapeutic efficiency and minimized side effects.
This gold standard in Passive cancer targeting identified as EPR (Enhanced
Permeability and Retention) coined by Maeda and Matsumura back in 1986,
depends on the characteristic properties of tumour vasculature which are: (1)
Highly defective architecture of blood vessels with elevated levels of
permeability factors, to afford sufficient nutrients and oxygen for rapid tumour
growth (2) Manifested large endothelial cell-cell gap openings (3) Highly
impaired lymphatic drainage from tumor tissue. All these factors support
leakage of macromolecules (above 40 kDa) outside vessels and their selective
accumulation in tumour cells while having limited access to normal organs
because of their tightly-knitted endothelial lining.
Therefore, the aim of this thesis was to formulate and prepare BDMC-loaded
nanosystems, mixed micelles and polymeric nanoparticles, dedicated to improve
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Summary
its aqueous solubility, passive targeting and reduced untoward side effects for
cancer therapy.
Hence the work in this thesis was divided into two chapters:
1- Chapter I: Preparation and evaluation of BDMC-loaded Pluronic mixed
micelles.
2- Chapter II: Preparation and evaluation of BDMC-loaded PLGA
nanoparticles.
Chapter I: Preparation and evaluation of BDMC-loaded Pluronic mixed
micelles
In this chapter, BDMC-loaded Pluronic mixed micelles were prepared adopting
thin-film hydration technique. The mixed micelles were prepared according to a
full factorial design to study the effect of two independent variables, namely the
ratio of Pluronics® used (F68%:F127%) at 3 levels: 15:85, 20:80 and 25:75,
and ratio of organic solvent utilized in micellar thin film preparation
(Acetone%: Acetonitrile %) at 3 levels (100:0, 50:50, 0:100) leading to
preparation of 9 (32) formulations. Whereas, the dependent variables
(responses) were the particle size (PS), polydispersity index (PDI), zeta
potential (ZP), and entrapment efficiency (EE %).
The values of critical micelle concentration (CMC) of the used Pluronics were
determined via pyrene fluorescence method, as 0.25 mg/ml (2.994 10-5 M) and
0.1 mg/ml (7.93 10-6 M) for F68 and F127, respectively. The calculated CMC
for mixed micellar formulations were around 1 10-5 M, according to the
simplified equation of Markov chain model of mixed surfactant systems which
confirms high thermodynamic stability of the prepared micelles, as well as
expected in-vivo longevity even with many fold dilution within plasma.
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Summary
The compatibility of BDMC and the used polymers was confirmed via
Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD) studies,
indicating reduction of drug crystallinity and the incorporation of drug within
micelles in amorphous form which enhances its aqueous solubility. Differential
light scattering (DLS) study indicated that the particle size of prepared mixed
micelles were in the range of 25-38 nm. This small size allows extravasation of
the particles from the leaky tumour vasculature, preventing their uptake by RES
cells, enhancing their accumulation inside the solid tumour via EPR effect and
passive tumor targeting. DLS showed single peaks for the prepared micelles
with average PDI value of 0.3, indicating a fair monodisperse pattern of particle
size. Smaller particle sizes (P.S) and polydispersity indices (PDI) were obtained
with acetone: acetonitrile 50%:50% organic solvent mixture, than either solvent
when used alone.
The observed ζ-potential was in the range of -3.4 to -4.6 mV with reasonable
stability, confirmed by their 6-months storage stability, possibly due to
electrostatic repulsion and steric and hydrophilic interactions between the
hydrophilic chains in micellar corona; hence preventing aggregation of micelles
and providing colloidal stability.
Polymeric mixed micelles flaunt with enhanced entrapment efficiency (EE %)
specially for hydrophobic drugs as BDMC, with average EE% = 84.22 %
(w/w). Such high encapsulation may provide a solution for the dose-related
toxicity and allow smaller doses with enhanced therapeutic effect. Significant
rise in the encapsulation efficiency (EE %) was observed with acetone as sole
solvent. Moreover, approaching the ratio 20:80 (F68%:F127%) resulted in
optimum particle size, PDI, zeta-potential and entrapment efficiency.
TEM images of selected formulations confirmed the homogenous, nonaggregated
spherical shape of the mixed micelles in the size range 30-70 nm.
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Summary
In-vitro release profile showed biphasic release pattern with rapid initial release
for about 50% in the first 4 hrs, followed by slower sustained yet complete
release during the rest of the 24 hours. Different release kinetic models were
obtained as the formulations demonstrated zero, first and Higuchi release
kinetics for the prepared samples. This may be attributed to various processes
controlling drug release from the prepared polymeric mixed micelles.
The effect of ageing on mixed micelles was studied for long term stability at
37ºC in shaking water bath for six months. No statistically significant change in
particle size or PDI of the prepared mixed micelles was observed on storage for
6 months; confirming their stability and ability to withstand storage for long
periods. It is worth noting, that the zeta potential and the encapsulation
efficiency may show slight decrease on long term storage possibly due to drug
release from micellar preparation, a case that may lead to charge neutralization
of the utilized Pluronic® micelles.
In-vitro cytotoxicity study was conducted on hepatocellular carcinoma (Hep G-
2) cells using crystal violet method. It revealed higher cytotoxic effect of
BDMC loaded within Pluronic® mixed micelles rather than free BDMC, with
obtained IC50 value of 0.74 μg/ml.
Augmentation of the anticancer activity of BDMC via Pluronics®, could be
attributed to several factors including enhanced cellular uptake into malignant
cells by membrane fluidization, inhibition of several drug resistance
mechanisms (P-gp efflux, drug sequestration within cytoplasmic vesicles) and
ATP depletion in MDR cancer cells.
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Summary
Chapter II: Preparation and evaluation of BDMC-loaded PLGA
nanoparticles
BDMC-loaded PLGA nanoparticles were prepared adopting the
nanoprecipitation technique according to a full factorial study design to study
the effect of three independent variables each at two levels, namely; polymer
type (PLGA 50/50 or PLGA 75/25), polymer concentration (5 mg/ml and 10
mg/ml) and PVA concentration (1% and 2%) according to 23 = 8 formulations.
The studied responses were particle size (PS), polydispersity index (PDI), zeta
potential and BDMC entrapment efficiency (EE %).
The prepared nanoparticles were characterized via XRD to study the
compatibility of the used polymers with the encapsulated BDMC. The obtained
results revealed decreased crystallinity of drug peaks and indicated amorphous
dispersion within PLGA matrix structure, which suggests higher solubility of
the hydrophobic drug.
Drug loading analysis showed increased drug entrapment and loading using
PLGA 75/25 with higher polymer concentration, owing to the higher lactate
ratio and polymer amount which provide better higher ability to accommodate
the hydrophobic drug, reaching the highest observed entrapment efficiency of
97.84 %.
Particle size analysis showed a relatively small particle size in the range of 171-
197 nm, which is relatively small enough (below 200 nm) rendering them
amenable to extravasation and retention inside solid tumors via the EPR effect.
Significant small PDI values exhibited a highly monodisperse pattern of
nanoparticulate size. TEM results confirmed the spherical morphology of the
prepared nanoparticles.
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Summary
The obtained results of zeta potential were in the range of (-9 to -13 mV)
indicating high stability of the colloidal dispersion and hence less liability to
aggregation.
In-vitro drug release showed biphasic release pattern for the prepared
nanoparticles, with initial burst effect during the first 8 hours, followed by a
sustained effect up to 1 week (F8), mostly due to slow erosion of the
hydrophobic PLGA75/25, as well as the higher PVA used concentration which
may hinder drug release and extend the release profile.
A selected formulation (F8) was subjected to further coating using various
coating moieties, namely PEG, Tween 80 and F68, in order to impart a
hydrophilic stealth character to the surface. The surface hydrophobicity was
assessed using Rose Bengal test; where the obtained results emphasized higher
adsorption of hydrophobic dye onto uncoated NPs compared to the hydrophilic
coated counterparts.
The entrapment efficiency of coated NPs using various coating moieties was
almost unchanged. No significant increase in particle size was recorded for NPs
coated with Tween 80. On the other hand, slight increase in particle size was
obtained upon coating with PEG or F68. Whereas, a significant DROP in zeta
potential of coated NPs occurred indicating successful complete surface coating.
TEM results showed a spherical morphology with smoother surface due to
sufficient full circumference coating with coating moieties. In-vitro release from
the selected coated formulation (F8) showed sustained release profile for 1 week
as well.
In-vitro cytotoxicity test was carried out using HepG-2 cells adopting the crystal
violet method. Coated NPs showed the highest inhibition of malignant cells
viability, than uncoated NPs and free BDMC. IC50 of Pluronic-F68 coated NPs
was 0.5437 μg/ml, due to augmented effect against malignant cells due to better
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Summary
internalization within malignant cells and augmented anticancer effect thanks to
Pluronic incorporated in coating.
In conclusion, the prepared nano-systems; the polymeric mixed micelles and the
PLGA nanoparticles exhibited high entrapment efficiency, suitable size for
passive tumor targeting, and acceptable stability. They were successful in the
delivery of Bisdemethoxycurcumin (curcumin III), sustaining its release and in
augmenting its effect on HepG-2 cancerous cells fulfilling the aims and scope of
this thesis.
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