Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713139
Title: Delivery of antioxidants to the lens using nanoparticles
Author: Al-Kinani, Ali Athab Tahar
Awarding Body: Kingston University
Current Institution: Kingston University
Date of Award: 2016
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Abstract:
Introduction: Cloudiness of the eye-lens -cataract- is part of the eye lens aging process. Cataract is known to be the leading cause of blindness worldwide as it contributes towards 51% of all blindness cases. A cararact formation has been associated with reduced levels of glutathione (GSH) in the eye. Recent work showed that cystine-glutamine exchangers are expressed in the lens nucleus which suggests that these exchangers could be targeted to increase the cysteine (GSH precursor) that plays a vital role in the GSH lenticular biosynthesis. L-2-oxothiazolidine-4-carboxylic acid (OTZ) is a cysteine pro-drug that is believed to increase intracellular GSH levels. Hypothesis: Ocular supplementation of cysteine or OTZ (cysteine prodrug) would increase the lenticular level of GSH, this is likely to prevent or delay the formation of cataract. Experimental: Preformulation studies were conducted to assess OTZ aqueous solubility and permeation through excised bovine cornea and sclera. A new analytical method based on hydrophilic interaction liquid chromatography (HILIC) was developed and validated following FDA guidelines to separate and quantify OTZ in different biological matrices. Four different nanoparticle delivery systems, namely chitosan, modified chitosan, poly (lactic-co-glycolic acid) -PLGA- and cerium oxide nanoparticles were formulated. The formation of chitosan nanoparticles was optimised using experimentally design chitosan nanoparticles. Considered responses include particle size, polydispersity index, [Zeta] potential, entrapment efficiency and release profile. Optimised chitosan nanoparticles loaded with OTZ were formulated using an ionotropic gelation method. A short alkyl glycerol group was added ti the chitosan backbone at the N-position to ofm N-acyl glycerol chitosan, formation of N-modified chitosan was investigated using several analytical and spectroscopic tecniques. N-modified chitosan nanoparticles were fabricated by ionic gelation. A double emulsion solvent diffusion (DES-D) method was used to formulate OTZ-loaded PLGA nanoparticles. HPMC-coated cerium oxide nanoparticles were fabricated in two steps: first, double emulsion formation to entrap OTZ into HPMC; second, homogenous precipitation to formulate cerium oxide nanoparticles. Particle size, [Zeta] potential, entrapment efficiency, particle morphology and in vitro release were measured for all the formulated nanoparticle systems. Further, permeation studies for all the formulated nanoparticles were performed on excised bovine sclera and cornea. Ocular tolerability was assessed using bovine corneal opacity and permeability followed by histological examination, Red Blodd Cell hemolysis and Hen's Eggs Test Chorioallantoic Membrane test. Different concentrations of the four nanoparticulate systems were used to assess the cytotoxicity to a lens epithelium cell line using the neutral red uptake (NRU) and suforhodamine B colorimetric (SRB) assays. The effect of those systems on the lens epithelium cell line was monitored using real time imaging techniques; this allows monitoring of the cell morphology and shape in addition to calculating the cell growth in real time. The effect of cystine on the ceulluar level of GSH was invetigated. Monochlorobimane (MCB) dye was used to quantify the GSH levels of cysteine treated HLEC-B3 cells. Results: The HILIC developed method was found to accurate and precise wiht LLOD and LOOQ on 100 and 200 ng/mL respectively. The optimisation process revelaed that the two most important variables in the formulation of chitosan nanoparticles were chitosan and TPP concentration. These optimised conditions were chosen to produce the chitosan nanoparticles. The optimised nanoparticles had a size and [Zeta] potential of 133.0 nm and 28 mV repectively. Using chitosan nanoparticles, the permeation of OTZ thriugh excised bovine cornea and sclera was higher than the OTZ simple solution. This increase may be attributed to the permeation enhancing properties of chitosan. The analytical and spectroscopic techniques confirmed the success of the N-acyle glycerol modification on the chitosan backbone. Both NMR and IR showed the modification peaks, gel permeation chromatography (GPC) showed an increase in the molecular weight of the modified chitsan (197.8 kDa) compared to the unmodified (151.4 kDa). The modified chitosan nanoparticles had a size of 176.7 nm and [Zeta] potential of 24.2 mV. Although the entrapment efficiency of the modified chitosan nanoparticles was less compared to the unmodified form (73.3 and 86.9% respectively), the permeation of OTZ from modified chitosan nanoparticles was comparable to the unmodified one, this indicates that the modification had improved the ocular permeation of OTZ from the nanoparticles. The formulated PLGA nanoparticles had a particle size, and [Zeta] potential of 92.9nm, and -8.6mV respectively, with an entrapment efficiencey of 68.4%. The mechanical properties of the formulated PLGA nanoparticles were investigated using atomic force microscopy (AFM), the results showed uniform and non-porous nanoparticles. Trans-scleral permeation of OTZ was 6 times that through the cornea using PLGA nanoparticles. Nanoceria were formulated and coated with hydroxypropyl methylcellulose (HPMA). The X-Ray diffractograms of coated nanoceris confirmed the formation of an HPMC coat. FT-IR spectra further confirmed the presence of polymer coatung on the surface of the nanoceria by showing peaks representing hydroxypropyl and epoxy groups of HPMC. Particle size and zeta potential of HPMC coated nanoceria were 15 [plus or minus] 1.19 nm and -1.67 [plus or minus] 0.2 mV respectively. Scanning electron microscopy and transmission electron microscopy revealed the formation of uniform well dispersed spherical nanoparticles. Furthermore, certain oxide nanotparticles showed [zeta] potential of -1.67 [plus or minus] 2.07 mV with HPMC and -34.67 [plus or minus] 2.33 mV without HPMC coating. This reduction in [Zeta] potential after coating which is attributed to the ability of the HPMC coat to mask the negative charge of cerium oxide. The ex-vivo permeation studies revealed that the scleral permeation of OTZ is 10 times higher than the corneal permeation. OTZ is a hydrophilic molecule, and as such is expected to show better permeation through the sclera over the cornea for all the permeation studies. The results obtained from the BCOP, HET-CAM and RBC haemolysis assays alongside with the histological examination illustrated the, the formulated nanoparticulate systems were wll endured by the ocular surface and dveoid of any irritation effect on the ocular surfaces. Additionally, NRU and SRB cytoxic assays showed no cytoxic effect from modified chitosan, PLGA nanoparticles and HPMC coated nanoceria on the HLEC-B3 cell line. However, these assays revealed that chitosan nanoparticles were mild irritantsto the HLEC-B3 cell line at a higher concetration. Real time imaging was used to study and evaluate the cell morpholology and growht before, during and after exposure to the nanoparticulate systems. The imaging technology confirmed that those cells were grown normally without any abnormality. HLEC-B3 cells treated with cyctine for 24 hours showed an increase in GSH levels. Cystenine is a GSH precursor and it is the rate limiting step in GSH biosynthesis. As such, it would be expected that, supplementing the cell with cyctine will enhance the GSH level. Conclusion: Formulation of OTZ (cysteine prodrug) has been fine-tuned through incorporation into various nanoparticulate systems. These nanoparticulate systems have the potential to prolong OTZ residence time, control the release and ocular permeation of OTZ. Cysteine has been shown to increase the GSH cellular levels in HLEC-B3 cell line. These findings warrant further studies on the possible use of these nanoparticulate systems for cataract prophylaxis as an alternative to surgery.
Supervisor: Not available Sponsor: Kingston University
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.713139  DOI: Not available
Keywords: Chemistry ; Pharmacy
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