الفهرس 
Preparation and characterization of QT Loaded PLGA Polymeric NanocapsulesOnly 14 pages are availabe for public view
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Abstract
Quercetin [3,3‘,4‘, 5, 7-pentahydroxyflavone] (QT) is a polyphenolic flavonoid
that has been recently extensively investigated for its biological activities, such as
anti-inflammatory, antioxidant, hepatoprotective activities, antitumor and
antiproliferative effects on a wide range of human cancer cell lines. Its antitumor
activity was found to be through inhibiting glycolysis and macromolecules
synthesis in addition to some enzymes such as matrix metalloproteinases (MMPs),
NaıKıATPase, protein kinase C, tyrosine kinases, and pp60ı kinase. It is
considered a promising candidate for clinical trials but its extreme water
insolubility hinders its introduction in clinical trials. It is usually administered in
dimethyl sulfoxide (DMSO) as a vehicle which carries risks of vasoconstriction
and neurological, liver and kidney toxicity.
Therefore, the work in this thesis aimed at formulating QT in injectable polymeric
nanocapsules with an oil core in which the drug is soluble which would result in
high encapsulation of the drug hence overcoming its extreme water insolubility
and allowing its i.v. administration in a stable nanoform. Polymeric nanocapsules
offers the advantages of the possibility of loading high amount of water insoluble
drug molecules into the oil core, their physicochemical stability, and protection
against enzymatic degradation due to the presence of the polymeric wall.
Moreover, their subcellular size allows relatively higher intracellular uptake.
Furthermore, attaching a targeting moiety (passive or active) to these nanocapsules
would enable targeted delivery of the drug enhancing the efficiency of treatment
through concentrating the drug at the tumor site and hence decreasing its systemic
side effects which was tried in this thesis as well. In the first chapter, QT loaded PLGA polymeric nanocapsules (PNCs) were
prepared and characterized. To determine the oil to be used in preparation of the
nanocapsules, the saturated solubility of QT in different oils namely; castor oil,
ethyl oleate, soybean oil, mygliol, labrafac propylene glycol (Labrafac PG), oleic
acid, olive oil and sesame oil was determined at 25°C. QT polymeric nanocapsules
(QT-PNCs) were then prepared using the nanoprecipitation technique utilizing the
oil selected from solubility study. A preliminary study was done on some factors
to select the suitable conditions to formulate QT-PNCs of appropriate size range.
The studied factors were; the lipophilic emulsifier type and organic solvent
composition, oil and drug concentration, Soybean Lecithin Type and PLGA Type.
For further optimization of particle size (PS), a full factorial design experiment
was built up to study the effect of three factors namely; Tween 80 concentration,
polymer concentration and soybean lecithin concentration using the PS as
response and performing check point analysis. The selected formulations with
optimum PS were characterized through studying their zeta potential, EE%, invitro
drug release and physical stability. Results showed that castor oil dissolved
the highest amount of QT so it was selected as the oil core for preparation of QTPNCs.
Most suitable formulation conditions among the studied ones to produce
QT-PNCs of PS lower than 200 nm were found to be: Epikuron E145V soybean
lecithin as an emulsifier with acetone: ethanol 6:4, 3% w/v castor oil, 5 mg QT
and PLGA 7502 A as a polymer. The results of factorial design experiment
showed that increasing both Tween 80 and E145V concentration significantly
decreased PS of QT-PNCs (p˂0.0001) while polymer concentration didn‘t have a
significant effect on PS in the studied range. Moreover, a significant two way
interaction was found between Tween 80 and soybean lecithin concentrations
(p˂0.0001). QT-PNCs carried a highly negative charge due to soybean lecithin
and carboxylic groups of PLGA which resulted in high stability of PNCs. The EE% of QT in PNCs was between 94 -98% which provides the advantage of
formulating QT in a nanoparticulate system allowing its parenteral administration
in the required dose. In-vitro release studies in 70:30 ethanol: water at 37oC
showed a biphasic pattern of QT release from PNCs; an initial burst release, 50%
in 24 h, which was still lower than that obtained from other studies, followed by
sustainment of release over a long period (3 days). Non-significant change in PS
and zeta potential of selected formulations was observed over 6 months indicating
its suitable stability.
In the second chapter, the composition (3% castor oil, 0.5% soybean lecithin,
0.5% PLGA 7502 A polymer and 0.2% Tween 80) was selected for modification
by pegylation and conjugation of folic acid (FA) moiety for passive and active
targeting, respectively. For this aim, PLGA conjugates were synthesized; PLGAPEG
conjugate for passive targeting and PLGA-PEG-FA for active targeting.
PLGA-PEG-FA was synthesized using two synthetic methods which were
compared. The synthesized conjugates were characterized by FTIR and H1-NMR,
furthermore, polyethylene glycol (PEG) and FA content were quantified using
colorimetric and spectrophotometric assay, respectively. QT-PNCs were then
prepared using the synthesized conjugates and characterized by determination of
their PS, zeta potential, EE%, in-vitro release study and physical stability.
Morphological examination was done by Transmission Electron Microscopy
(TEM), Atomic Force Microscopy (AFM) and Cryo-Transmission Electron
Microscopy (Cryo-TEM). The chemical conjugation and formation of PLGA-PEG
and PLGA-PEG-FA conjugates was confirmed by FTIR and 1H-NMR. All
conjugates showed high PEG content of 14-16%. Moreover, the FA content of
PLGA-PEG-FA synthesized by both tried synthetic methods was found to be
similar. PNCs formulated using PLGA-PEG showed significantly smaller size and
lower zeta potential than non pegylated ones. PLGA-PEG-FA NCs size and zeta potential were close to that with PLGA only. No difference was found in EE%
among the PNCs formulated with the three different polymers. AFM and TEM
confirmed the spherical shape of PNCs and confirmed the presence of polymer
shell around the oil core. The release profile of the three formulations, NC1
(PLGA), NC3 (PLGA-PEG), NC5 (PLGA-PEG-FA) in phosphate buffer saline
(PBS) was similar with a biphasic pattern of burst release followed by sustained
one. In presence of serum, NC3 and NC5 showed higher stability than NC1 due to
presence of PEG that provides more protection from destabilization by serum
proteins. No significant change in size and zeta potential of particles were
observed after 90 days showing high shelf life stability. Therefore, NC1, NC3 and
NC5 were selected for further cytotoxicity and in-vivo studies.
In the third chapter, In-vitro cytotoxicity and cellular uptake of QT loaded PNCs
was divided into two sections; cytotoxicity studies and cellular uptake ones. The
cytotoxicity studies were done using the MTT assay. First, the cytotoxicity of free
QT in DMSO solution was determined on four different cell lines, namely; C6,
B16F10, CT26 and HeLa, to determine those of highest sensitivity to QT. CT26
and HeLa cells showed the highest sensitivity to QT followed by B16F10 with C6
glioma cell line showing least sensitivity.Therefore, CT26 and HeLa cells were
selected for further studies. The next step was to test the cytotoxicity of QT versus
QT loaded PNCs, NC 1, NC 3 & NC 5, on the selected cell lines, CT26 and HeLa,
to determine the effect of drug encapsulation on its cytotoxicty. To test selectivity
and active targeting of PNCs, cytotoxicity studies were done on folate expressing
HeLa cells in folate free and folate enriched media (200 μm). For further
confirmation, similar experiment was done on non-folate expressing CT26 cells. It
was found that encapsulation of QT in PNCs didn‘t compromise its in-vitro
cytotoxicity on both CT26 and HeLa cell lines. FA-targeted PNCs, NC 5, showed
highest cytotoxicity, 56.63%, on Hela cells compared to QT, NC 1 & NC 3 at 10 μM after 24 h in folate free medium due to their uptake by folate recpetors,
whereas, in the presence of excess FA, it showed equal cytotoxicity to other
formulations due to saturation of folate receptors by free folic acid in the medium.
No difference in the cytotoxicity of the targeted and non-targeted PNCs was seen
on CT26 in both folate free and folate enriched medium further confirming the
results. In the second part of this chapter, the uptake of PNCsin HeLa cells was
studied by fluorescence labeling of PNCs through incorporation of DiI in the oil
core of PNCs. The uptake of fluorescently labeled PNCs in HeLa cells, at 10
ug/ml after 1 & 4 h in folate free and folate enriched medium, was quantified by
spectrofluorimetry and examined by Confocal Laser Scanning Microscopy
(CLSM). NC5 was found to have higher uptake than NC 1& NC 3 in folate free
medium after 1h and 4 h incubation. Higher uptake was found after 4h than 1h. In
presence of excess FA in the medium, all PNCs formulations showed similar
uptake. Similar results were shown by CLSM. Therefore, both cytotoxicity and
cellular uptake studies proved superiority and active targeting of FA-targeted
PNCs, NC 5 than non-FA ones.
In the fourth chapter, the three PNCs formulations, NC1, NC 3 & NC 5, were
selected for in vivo uptake studies which were done using two different cell lines.
The first cell line was the CT26 which was selected with the aim of studying and
proving the passive targeting of PNCs in case of non folate expressing tumors.
This was done utilizing a radiolabeling technique. In this study PLGA-PEG-DTPA
conjugate was synthesized for incorporation in the polymeric shell of PNCs and
then chelation with radiotracer, In111. Its chemical conjugation and formation was
confirmed by FTIR and H1-NMR. It was then incorporated in the polymeric shell
of PNCs in different ratios with the polymer (PLGA or PLGA-PEG or PLGAPEG-
FA) forming the coat (1-50%) to find the optimum ratio to be used for
efficient radiolabeling without affecting the properties of the PNCs optimized previously. The efficiency and stability of radio-labeling was tested using thin
layer chromatography (TLC) technique. 5% w/w PLGA-PEG-DTPA was found to
be efficient for labeling. Furthermore, labeled PNCs showed stability after
incubation with PBS (pH 7.4) and serum for 24 h. No significant change on the PS
or zeta potential of PNCs (NC 1, NC 3 & NC 5) was observed after incorporation
of the 5% PLGA-PEG-DTPA. After proving the efficiency and stability of labeled
PNCs, they were injected in CT26 tumor bearing mice for studying tumor uptake
by live animal SPECT/CT imaging and tissue biodistribution was determined
quantitatively by Gamma Scintigraphy. In the SPECT/ CT, animals were imaged
at 3 time points, 0.5, 4 & 24 h. Similar organ biodistribution was observed in case
of NC 3 and NC 5 formulations with obvious prolonged blood circulation profile
than NC 1 which showed fast accumulation in the liver and spleen (~90% ID)
even within the first 30 min confirming the stealth characteristics of the PEGylated
PNCs (NC 3& NC 5). Therefore, NC 1 was excluded from further studies. In the
quantitative biodistribution studies, NC 5 showed lower blood % and lower %
injected dose/ g (ID/g) of CT26 tumor than NC 3 by gamma counting due to
uptake of particles by folate receptors in the liver. The % ID/g of tumor accounted
for 3.9% in case of pegylated PNCs, NC 3, which proves passive targeting of these
particles and high accumulation in tumors. For further confirmation an in vivo
tumor growth delay study was carried out on CT26 tumor bearing BALB/cs.
When the tumor reached appropriate size (200 mm 3), PNCs, NC 3 & NC 5, were
injected i.v. for a total of 4 doses over 12 days; 50 mg/ kg QT every 3 days, and
tumor size was measured using a vernier caliper. NC 3 showed significant tumor
size reduction starting 3rd day after the start of treatment. This proves the
efficiency of the pegylated NCs, NC 3, in targeting QT and increasing its uptake to
the tumors, hence increasing its therapeutic effect. The second part of the work
was done on folate expressing tumors, HeLa and IGROV1, to prove the active targeting of NC 5. This was done through using fluorescence
labeling of PNCs, NC 3 & NC 5, through incorporation of DiR fluorescent dye in
oil core of PNCs. HeLa and IGROV-1 bearing animals were kept on folate free
diet one week before and during the period of the experiment. PNCs were injected
in animals and they were imaged at different time points, 1, 4 and 24 h, using
IVIS® Lumina Series III in Vivo Imaging System. Organs and tumor uptake was
imaged and uptake quantified using Living Image® software. Tumors were frozen
and sections were cut by a cryostat and examined by CLSM. Both NC 3 and NC 5
showed similar tumor accumulation (%ID per gram of tumor) in HeLa and
IGROV-1 folate expressing tumor models due to their uptake by EPR effect
although uptake of NC 5 may occur by folate receptors in liver as mentioned
before. Moreover, the selectivity of uptake of folate bearing PNCs, NC 5, was
further shown by altered intra-tumoral distribution and better association with
cancer cells thus confirming the in vitro results.
Therefore from the work in this thesis, a stable intravenously administered
nanopaticulate system of QT of optimum size for tumor targeting (˂200μm) was
successfully prepared. Conjugation of PEG and PEG-FA moieties for both passive
and active targeting was achieved. The efficiency of the prepared particles for both
passive and active targeting of QT was proved both in-vitro and in-vivo.