الفهرس 
MethodologyOnly 14 pages are availabe for public view
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Abstract
Oxaliplatin loaded albumin nanoparticles for cancer immunotherapy
Summary
Cancer management strategies include surgery, radiotherapy and
chemotherapy. Recently, gene therapy, hormonal therapy and immunotherapy
are progressively investigated in cancer management. Chemotherapeutics as
oxaliplatin, stimulate cancer cells to induce immunogenic cell death (ICD). ICD
is an apoptosis process that amplifies an inflammatory immune response.
Interestingly, previous reports demonstrated that the implantation of tumour cell
lines previously treated with ICD inducers acts to vaccinate mice from rechallenge
with the same tumour cells. In addition, tumour local treatment with
ICD inducers can lead to diminution of distal tumour proving the conception of
systemic immune recognition.
The main physiological characteristics of ICD are cell surface expression
of calreticulin, release of different Damage-associated molecular patterns
(DAMPs) as heat shock proteins (HSPs) and Adenosine triphosphate (ATP) as
well as high mobility group box 1 (HMGB1). Calreticulin is an “eat me signal”
that combines with low-density lipoprotein receptor-related protein 1 (CD91) on
phagocytes, DAMPs release provides a ‘find me’ signal and HMGB1 maturates
antigen-presenting cells (APCs). Consequently, these factors lead to stimulation
of host immune system. Calreticulin expression is considered the crucial factor
in ICD bona fide.
Oxaliplatin is a platinum-based alkylating cytotoxic agent that acts via
impeding DNA replication. Oxaliplatin has a wide range of activity against
colorectal, pancreatic, gastric, ovarian, bladder, breast, small- and non-small-cell
lung, and head and neck cancers. Oxaliplatin suffers from various side effects as
neuropathy, low blood count and dysesthesias. These harmful side effects could
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be attributed to the nonspecific biodistribution of oxaliplatin into the healthy
tissues. As a consequence, the development of a suitable carrier with an enhanced
tumour internalization is crucial.
In addition, CD44 is a transmembrane glycoprotein with different isoforms
intricate in cell-cell and cell-matrix interactions. Moreover, CD44 is highly
expressed on Cancer Stem Cells and has a key role in tumor growth, metastasis
and chemotherapeutic resistance. In colon cancer, CD44 isoforms are reported to
trigger tumour invasion, impede apoptosis and patient survival. Downregulation
of CD44 could be accomplished via different strategies as antibodies, aptamer,
siRNA, miRNA and is usually associated with improved therapeutic efficacy.
Zinc oxide nanoparticles (nZnO) are FDA approved biodegradable,
biocompatible nanocarriers with proved anticancer activity as well as
antibacterial and antiviral activity. In addition, nZnO are reported to have
immunomodulatory effect.
Nanotechnology is considered as the most noticeable tool in the
development of new drug carrier systems, providing versatile clinical
applications and scale-up for industrial production. Polymeric nanoparticles and
lipid nanocarriers are the mainly investigated types of nanoparticles which had
been approved by FDA for clinical applications. Commonly, drug targeting is
divided into passive and active targeting strategies. Cancer passive targeting of
nanoparticles relies on the discontinued fenestrations in the endothelial layer in
the tumor microenvironment that ranges from 300 to 4700 nm in diameter.
Moreover, poor lymphatic drainage is encountered in the tumors due to the
dysfunctional lymph angiogenesis and compression of lymphatic vessels by
proliferating cancer cells. This phenomena of nanomedicine accumulation in
tumors is referred to as enhanced permeability and retention effect.
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Accordingly, the ultimate goal of this study was to combine oxaliplatin and
nZnO into albumin NPS to improve cellular uptake, cytotoxicity and apoptosis.
The effect of RBCs coating on macrophage uptake in vitro and in vivo was also
assessed. A special focus was given to the effects of the proposed platform on
immunological stimulation against colon cancer cells.
To fulfill the above goal, the work in this thesis was divided into three
chapters:
I. Preparation and characterization of zinc oxide nanoparticles.
II. Preparation and characterization of RBCs Coated Oxaliplatin-Zinc
Oxide Loaded Albumin Nanoparticles
III.Immunological and Biological Studies on RBCs Coated Oxaliplatin-
Zinc Oxide Loaded Albumin Nanoparticles.
Chapter I
Preparation and characterization of zinc oxide nanoparticles
In this chapter, we aimed to synthesize nZnO with minimum crystallite
size as a possible adjuvant in cancer immunotherapy. Briefly, two studies were
performed: a preliminary study to set the growth time and temperature for
efficient fabrication of nZnO. The second study involved the optimization of
different CPPs including precursor concentration, pH and stirring speed affecting
the nZnO crystallite size to achieve the QTPP.
nZnO was successfully prepared by chemical precipitation technique,
where the preparation process optimized by QbD by response surface
methodology using BBD.The influence of different CPPs on crystallite size was
statistically fitted to the quadratic model. Increasing zinc precursor concentration
and/ or stirring speed decreased crystallite size. Contrarily, nZnO NPs crystallite
size was directly proportional to the reaction pH .The optimum formula was
selected based on the criteria of attaining minimum crystallite size. This formula
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was prepared using 500 mMZn(NO3)2.6H2O at pH 10 and 1000 rpm stirring
speed.
The XRD data revealed that the obtained nZnO had hexagonal wurtzite
P63mc crystal structure .The optimized nZnO was also characterized by FT-IR
spectroscopy where the main stretching band of ZnO at 430 cm-1 infers the
successful fabrication of nZnO. The optimized nZnO was characterized in terms
of UV spectroscopy with a characteristic λmax of 364 nm corresponding to the
pure nZnO .The optimized nZnO showed a reversal surface charge ranged from
+25.32 ±3.21 to -18.78± 1.58 depending on the dispersant pH. The morphological
architecture of the nZnO demonstrated heterogeneous hexagonal and spherical
nanostructure with crystallite size in good agreement with that determined by
applying Scherrer’s equation on the data derived by XRD technique .The in vitro
Zn2+ release from nZnO was pH dependent due to the limited solubility of Zn2+
at physiological pH. In the next chapter, attempts were made to co-load nZnO
with oxaliplatin into long circulating RBCs coated albumin NPs.
Chapter II
Preparation and characterization of RBCs Coated Oxaliplatin-Zinc
Oxide Loaded Albumin Nanoparticles.
Bovine serum albumin nanoparticles (albumin NPs) have proven success
in the clinical arena as a platform for cancer treatment, with an existing privation
for multifunctional particulates nurturing combinatory therapies. Targeting
multiple pharmacological bits are thought to improve cancer’s therapeutic
outcomes. Interestingly, FDA had approved, nZnO as a multifunctional platform
capable of combating various types of cancer, while achieving multiple tasks.
The multi-target anticancer activity of nZnO could be attributed to its ability to
provoke reactive oxygen species (ROS), macrophage polarization promoting
cancer apoptosis, as well as, down regulation of CD44 expression hampering
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both cancer adhesion and migration. Currently, the use of natural membrane coat
derived from RBCs would add several merits as biocompatibility, nonimmunogenicity,
improved cellular interactions and targeting efficiency.
In this chapter, RBCs coated albumin NPs co-loaded with oxaliplatin and
nZnOwere prepared and evaluated using four studies: 1- setting the ratio of desolvent
to solvent phases and stirring time for oxaliplatin albumin NPs
formulation; 2- optimization of different CPPs for drug albumin NPs fabrication;
3- co-loading of different concentrations of nZnO with the optimized drug
albumin NPs and 4- RBCs coating of the chosen co-loaded albumin NPs,
followed by its in-depth characterization.
Briefly, oxaliplatin albumin NPs were successfully prepared by
desolvation technique, where the preparation process optimized by QbD by
response surface methodology using BBD. The influence of different CPPs on
particle size and EE % was statistically fitted to the 2FI and quadratic models
respectively. Increasing BSA and oxaliplatin concentrations increased both
particle size and oxaliplatin EE%, while increasing the stirring speed decreased
both responses. All the obtained oxaliplatin albumin NPs had a PDI value less
than 0.2 indicating the fabrication of homogenously dispersed systems. The
optimum formula was selected on the criteria of attaining a minimum particle
size and maximum oxaliplatin EE%. The optimum formula (Alboxa) was
composed of BSA (5 mg/mL) and oxaliplatin (3 mg/mL) and prepared at 1000
rpm.
Oxaliplatin albumin NPs were co-loaded with nZnO to improve the
expected antitumor activity of the proposed system. The complexation between
BSA and nZnO was confirmed by UV spectroscopy, fluorescence spectroscopy
and FT-IR spectroscopy. The co-loading of nZnO into oxaliplatin albumin NPs
significantly increased particle size. The presence of nZnO in a concentration of
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30% w/w and above respective to BSA amount significantly decreased
oxaliplatin EE%. The selected nZnO concentration loaded into Alboxa was 20%
w/w. Alboxaco-loaded with nZnO (Alboxa-nZnO) had a particle size of 131± 3.61nm,
PDI of 0.184 ± 0.012 and oxaliplatin EE% equals 81.65± 1.01%.
The presence of nZnO during the desolvation preparation of Alboxa did not
interfere with the percent yield. The conformational changes in the native BSA
structure during the preparation of NPs with and without oxaliplatin and nZnO
were investigated by FT-IR spectroscopy. The morphological architecture of the
proposed Alboxaand Alboxa-nZnO were visualized by SEM showing almost spherical
and doughnut-shaped nanostructures respectively. The presence of platinum and
zinc elements were confirmed by EDX analysis.
RBCs coated Alboxa-nZnO NPs were successfully prepared using freshly
prepared mice blood. The RBCs derived membrane obtained by hypotonic
treatment of erythrocytes was fused with Alboxa-nZnO. The coating of Alboxa-nZnO
significantly increased particle size with a neglected effect on oxaliplatin and
nZnO EE%. All the prepared formulae showed a negative zeta potential ranged
from -17.62± 2.34 to -29.38± 1.95 mV depending on the presence of nZnO and
RBCs coat. The presence of nZnO with and without RBCs coat decreased
oxaliplatin LE% from 7.99 ± 1.15% to 4.85± 0.26%.
The proper orientation of RBCs coat on the Alboxa-nZnO was confirmed by
quantification of %glycoprotein, sialic acid content and the extent of surface
CD47 protein expression. The successful coating of Alboxa-nZnO with RBCs was
also confirmed with TEM, where a non-aggregated spherical core-shell
nanostructures were observed. Incubation of RBCs coated Alboxa-nZnO with FBS
(10% and 50% v/v) for 48 h had an insignificant effect on particle size, PDI and
zeta potential indicating the colloidal stability of the proposed system.
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The in vitro oxaliplatin release from RBCs coated Alboxa-nZnO had a more
prolonged oxaliplatin released than Alboxaand Alboxa-nZnO in PBS pH 7.4 (in
presence of 50% FBS) and acetate buffer pH 5.5 (in presence of glutathione 10
mM). The in vitro Zn+2 release from Alboxa-nZnO with and without RBCs coat
showed a pH dependent behavior due to the inherent solubility of Zn in acidic
conditions. Stability studies of RBCs coated Alboxa-nZnO after storage for 21days
at 4 °C revealed nonsignificant changes in the measured physicochemical
characteristics. The aforementioned results gave a rational for immunological and
biological assessment of the prepared formulation.
Chapter III
Immunological and Biological Studies on RBCs Coated Oxaliplatin-
Zinc Oxide Loaded Albumin Nanoparticles.
In this chapter, the efficacy of the developed C-AlbOxa-nZnO was evaluated
by testing its cytotoxicity and cellular uptake on CT26 colon cancer cells. The
effect of RBCs coating on the macrophage uptake was also assessed. The
consequent in vitro immune stimulation of the proposed system was tested by
assessing calreticulin CT26 cell surface expression, ATP and HMGB1 secretion,
as well as, the expression of CD80, CD86 and MHC II on macrophage cells. The
in vitro phagocytosis efficiency of macrophage against pre-treated cancer cells
with the combinatory systems was investigated. The effect of C-AlbOxa-nZnOon
CD44 expression and consequently cell adhesion and migration ability was
investigated on CT26 cells. The in vivo biodistribution of fluorescence labelled
C-AlbOxa-nZnOin tumour bearing mice was also assessed.
The prepared C-AlbOxa-nZnO (composed of 5 mg BSA, 3 mg oxaliplatin and
1 mg nZnO and coated with RBCs) showed ≈ 2 fold increase in cytotoxicity
compared to that of free drug solution. C-AlbOxa-nZnO significantly induced CT26
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apoptosis markers namely; caspase 3 and 7 over untreated cells or those treated
with either C-AlbOxaor C-AlbnZnO. The macrophage uptake of both coated and
uncoated AlbOxa-nZnO was assessed qualitatively and quantitatively by confocal
laser microscope and flow cytometry respectively.
RBcs coating was able to diminish macrophage internalization of the
proposed system. The uptake of the fluorescence labelled C-AlbOxa-nZnO by CT26
cells was investigated also qualitatively and quantitatively by confocal laser
microscope and flow cytometry respectively. The confocal microscope graphs
revealed the uptake of C-AlbOxa-nZnO intracellularly adjacent to the nucleus.CT26
cellular capturing expressed as MFI, was improved in time- and concentration
dependent manners. Tracking different endocytic pathway indicated that clathrinmediated
endocytosis is the endocytic pathway involved in the uptake of the
prepared C-AlbOxa-nZnO.
The incorporation of nZnO into the proposed platform was responsible for
downregulation of surface CD44 and consequently hampering both cell adhesion
and migration. C-AlbOxa-nZnO was able to induce macrophage polarization towards
pro-inflammatory M1phenotype in terms of upregulation of IL-6, TNF-α, CD80,
CD86 and MHC II expressions on J774 macrophage cells. C-AlbOxa-nZnOwas able
to induce ICD hall marks on CT26 cells namely; surface calreticulin expression,
ATP and HMGB1 release over untreated cells or those treated with either
monotherapy. C-AlbOxa-nZnO was able to stimulate macrophage recognition and
phagocytosis of the treated CT26 cells over untreated cells or those treated with
either monotherapy.
Lower RES deposition, yet higher tumour preferential accumulation were
noticed following IV administration of C-AlbOxa-nZnO compared to the uncoated
counterpart. Accordingly, C-AlbOxa-nZnO can be considered a potentially
promising nanosystem triggering the anti-tumour immune responses and thus,
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could be utilized as an injectable delivery system with long-standing action,
controlled release of oxaliplatin and preferential tumour deposition. To support
the outcomes of this study, it seems to be essential to carry out further antitumor
efficacy and re-challenge studies.