الفهرس 
Preparation??and??characterization??of??chitosan-coated??doxorubicinloaded ??nanodiamonds??for??intravesical??deliveryOnly 14 pages are availabe for public view
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Abstract
Nanodiamonds (NDs) have been extensively evaluated for their applications in the field of drug delivery due to many of their superior properties such as biocompatibility, ease of production, large surface area and tunable surface chemistry. NDs-mediated anticancer drug delivery was especially under spot after the discovery of the NDs potential to enhance the chemosensitivity of antineoplastic drugs against resistant tumors.
Doxorubicin is a popular chemotherapeutic agent that is administered among the first-line treatment protocols of multiple types of tumors. Doxorubicin is clinically approved for administration to humans by both intravenous and intravesical routes. This latter is a special route of administration that aims to deliver therapeutics locally to the bladder to treat local disorders such as superficial bladder carcinomas and interstitial cystitis.
The objective of this thesis was to optimize the NDs-doxorubicin system for intravesical administration. Intravesical delivery of chemotherapeutics is challenged by two main problems: the short residence of the administered therapeutics before it is washed by the urine, in addition to resistance being developed by many mechanisms; the most popular of them is the efflux pumps.
Accordingly, the work in this thesis was divided into three chapters:
Chapter 1: Preparation and characterization of chitosan-coated doxorubicin-loaded nanodiamonds for intravesical delivery.
Chapter 2: Stabilization of chitosan-coated doxorubicin-loaded nanodiamonds.
Chapter 3: Cytotoxicity, cellular uptake and mucoadhesion of chitosan-coated doxorubicin-loaded nanodiamonds.
Chapter 1: Preparation and characterization of chitosan-coated doxorubicin-loaded nanodiamonds for intravesical delivery
This chapter aimed at developing NDs-doxorubicin nanoparticles coated with chitosan to allow the nanoparticles to adhere to the mucosa of the urinary bladder.
The results of this chapter could be summarized in the following points:
The particle size and zeta potential of native NDs as measured by dynamic light scattering were 81.0±0.7 nm and -37.4±1.33 mV respectively. The colloidal stability of NDs was conditioned by neutral or alkaline pH and low ionic strength (<10 mM NaCl).
NDs could successfully load doxorubicin in a weight ratio of 5:1 with a loading efficiency close to 100% yielding doxorubicin-loaded NDs (NDX). The optimized NPs scored a particle size and zeta potential of 87.1±0.2 nm and -26.7±0.3 mV, respectively. However, the colloidal stability of NDs-doxorubicin nanoparticles was very poor in presence of electrolytes (>10 mM NaCl) or low pH (<7).
Chitosan could successfully coat the NDX nanoparticles, as confirmed by the complete inversion of the zeta potential of nanoparticles from -26.7±0.3 mV to more than +20 mV along with increase in particle size. FT-IR and transmission electron microscopy provided further evidence of the success of chitosan coating.
A full factorial (32) study was carried out to investigate the effect of chitosan molecular weight and pH on the particle size, zeta potential and loading efficiency of the nanoparticles. The three dependent variables were affected by the two factors under investigation as well their interactions and a quadratic model of high significance was suggested for each dependent variable. Particle size of the nanoparticles ranged from 136.47 to 219.07 nm, zeta potential from +23.70 to +34.13 mV while loading efficiency exceeded 84.99 % in all formulae. The values of the dependent variables were statistically optimized to achieve smallest particle size, highest loading efficiency, and highest absolute zeta potential. The formulation with highest desirability factor had a particle size of 136.47±5.36 nm, zeta potential of 29.60±0.82 mV and loading efficiency of 89.71±0.70%.
Both NDX and chitosan coated ND-doxorubicin (Chi-NDX) nanoparticles exhibited pH-dependent release with more drug liberated in acidic media. Chi-NDX showed enhanced release, in comparison with uncoated ones, in acidic media probably due to their enhanced colloidal stability.
Despite the high stability of Chi-NDX in acetate buffer in which it was prepared, upon mixing with pH neutral media such as phosphate buffered saline or RPMI 1640 (both supplemented with 10% FBS), rapid aggregation occurred followed by precipitate formation indicating physical incompatibility with cell culture media and physiological media.
Chapter 2: Stabilization of chitosan-coated doxorubicin-loaded nanodiamonds
The objective of this chapter was to overcome the physical instability problem of Chi-NDX in cell culture media and physiological media in order to be able to proceed to the in vitro testing of the delivery system on the cellular level.
Two strategies were investigated to face this challenge: first, to add an extra layer of dextran and second, to crosslink the chitosan present in the NPs by the addition of TPP. Dextran sulphate was incorporated using two mode of mixing: the first consisted of adding a low Mw chitosan solution to a mixture of NDX and dextran sulphate and the second involved addition of dextran sulphate solution to optimized Chi-NDX. In each mode, the effect of different concentrations of components and pH were investigated. Dextran modified chitosan coated nanodiamonds-doxorubicin complexes (Dex-Chi-NDX E1), prepared using low Mw chitosan, dextran at a concentration of 0.03% and at a final pH of 5.0 was selected for further characterization. This formula scored a particle size of 224.0±6.1 nm, zeta potential of −34.0±1.9 mV and loading efficiency of 94.2±0.8%.
TPP was incorporated by adding different concentrations of TPP to a fixed volume of Chi-NDX prepared either from high or low Mw chitosan followed by evaluating the particle size and surface charge of the resultant NPs. Aggregation and precipitation of the formulae occurred upon addition of TPP in concentrations higher than 20 µg/ml for Chi-NDX prepared from high Mw or higher than 10 µg/ml for those prepared with low Mw chitosan. The particle size and zeta potential of Chi-NDX prepared from high molecular weight chitosan significantly decreased by the addition of TPP, while those prepared from low molecular weight chitosan were unaffected. TPP modified chitosan coated nanodiamonds-doxorubicin complexes (TPP-Chi-NDX L10), prepared using low Mw chitosan, TPP at a concentration of 10µg/ml and at a pH of 4.75±0.05, recording particle size, zeta potential and drug loading efficiency of 134.7±2.0 nm, 23.8±0.5mV and 95.8%, respectively.
Selected dextran and TPP modified Chi-NDX prepared from low Mw chitosan were found to be stable in PBS and RPMI 1640 (both supplemented with 10% fetal bovine serum). Drug release in case of dextran-coated Chi-NDX, interestingly, exhibited pH dependent release pattern opposite to that encountered with Chi-NDX, displaying enhanced release in alkaline medium. Furthermore, the formulae were found to be stable following storage at 4ºC for a period of six months.
Chapter 3: Cytotoxicity, cellular uptake and mucoadhesion of chitosan-coated doxorubicin-loaded nanodiamonds.
The aim of this chapter was to evaluate the cytotoxicity and cellular uptake of selected formulations as well as ex vivo testing of the potential of selected formulations to enhance the drug retention in excised bovine bladder.
Upon testing the cytotoxicity of selected formulae on a bladder cancer cell line, selected TPP-stabilized Chi-NDX had an IC50 value of 0.13±0.002 µg/ml, which is 3.38 less than free drug (doxorubicin) and 1.64 folds less than chitosan-uncoated NDX (p<0.001). IC50 value of Dextan-coated Chi-NDX was ~11 times that of free doxorubicin and ~7 times that of uncoated NDX. Flow cytometry analysis confirmed the cellular uptake of NDs as well free drug and all doxorubicin-loaded formulae.
The potential of Chi-NDX-TPP to enhance the drug retention in bladder was investigated by applying different formulae to ex-vivo bovine bladder and analyzing the amount of drug retained in the bladder wall. The % doxorubicin retained in the bladder wall significantly increased from 26.0%±0.03 in case of NDX to 46.3%±2.47 for TPP-stabilized Chi-NDX formula.
To conclude, TPP-stabilized Chi-NDX proved to be promising to help overcome the two main challenges of intravesical therapy; it could enhance the cytotoxicity of doxorubicin against bladder cancer cell line as well enhancing its retention in the bladder wall.
Although dextran-coated NDX failed to enhance the cytotoxicity of the anticancer drug, its drug release profile can be exploited for other applications such as delayed release orally administered delivery systems to minimize drug release in the stomach and deliver the drug cargo specifically to the intestine.