Theses and dissertations (Applied Sciences)
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Item The in vitro delivery of Doxorubicin using biosynthesized versus chemically synthesized stealth site-specific Bimetallic Selenium-silver nanoparticles(2023) Malinga, Thoko Winnie; Kudanga, Tukayi; Mbatha, Londiwe SimphiweProblems related to the limitations of chemotherapeutic treatments compel the pressing necessity to develop a drug-delivery system that will specifically target tumor cells and have minimal or no harmful effects on normal/healthy cells. This study aimed to comparatively evaluate the ability of chemically versus biologically synthesized site-specific selenium-silver bimetallic stabilized folic targeted nanoparticles (SeAgChPEGFA NPs) to efficiently deliver doxorubicin (DOX) in cervical cancer cells (HeLa). The NPs were synthesized using a coreduction method chemically using sodium borohydride and polyvinylpyrrolidone, and biologically using fenugreek seed extract. Moreover, the NPs from both methods of synthesis were stabilized and functionalized using carbodiimide and adsorptionreaction procedures. The drug/DOX-loaded nanocomplexes (NCs) were prepared via anadsorption and amide bonding reaction process between the co-polymer stabilized NPs and DOX. The bimetallic NPs and their DOX-loaded NCs were characterized using ultraviolet- visible (UV-vis) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and zeta Sizer. The drug release, loading, and encapsulation capabilities were evaluated in an in vitro environment. The effects of the synthesized NCs on cell viability and programmed cell death analysis were evaluated by means of the 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and a dual staining technique in selected human non-cancer and cancer cell lines (HEK293 and HeLa cells), respectively. The NPs and their drug-loaded NCs were successfully formulated and characterized. The successful synthesis of the NPs was initially validated by the reaction mixtures’ change in colour from cloudy to red-orange and subsequently from yellow-gold to orange-brown, signifying the formation of SeAg NPs and F-SeAg NPs, respectively. The UV- spectroscopy revealed that the SeAg NPs absorbance peaks were between 260 nm and 320 nm, while the FTIR verified the stabilization and functionalization of the NPs by revealing the presence of carbodiimide and amide bonds. All the resultant NPs and their drug-loaded NCs were shown asspherical withthe NPs appearing predominantly monodispersed and the NCs as groups. The sizes of the chemically and biosynthesized NPs ranged between 103.5 nm and 138.8 nm and that of the DOX-loaded NCs ranged between 154.9 nm and 158.7 nm respectively. The DOXloaded chemically- and biologically-synthesized NCs showed good stability with zeta measurements of 53.1 ± 2.3 mV and 57.4 ± 1.9 mV, respectively. The encapsulation efficiency (EE%) and drug loading (DL%) percentages of the chemically synthesized NCs were calculated to be 84% and 26%, respectively, while the percentages of the biosynthesized NCs were 87% and 22%, respectively. The cytotoxicity and anticancer activities of the BMNPs/NCsfrom both methods of synthesis were cell-specific and concentration-dependent. Overall, the encapsulation of DOX to the eco-friendly formulated BNPs enhanced the biocompatibility, bioavailability, and therapeutic effects ofthe drug in tumor cells, with limited harm to the healthy cells, thus showing promise as alternative delivery systems for targeted cancer treatment. The findings indicated that both the chemically synthesized and biosynthesized NPs showed great potential as anticancer drug-delivery modalities, with the biosynthesized SeAgChPEGFA@DOX NCs showing superior optical, surface charge stability, and drug encapsulation properties than the chemically synthesized SeAgChPEGFA@DOX NCs and free DOX. Moreover, among the studied synthesis methods, biosynthesis is reported to be eco-friendly and as a result the more ideal anticancer drug-delivery system with favourable features forfuture in vivo applications. Thus, future studies can encompass in vivo assessment of this eco-friendly system to further evaluate at a broader scale the bimetallic system’s efficacy and safety before using these NPs clinically.Item Enzymatic modification of Bambara groudnut protein for the production of hydrogels(2021) Ruzengwe, Faith Matiza; Kudanga, Tukayi; Amonsou, Eric OscarNatural polymer-based, especially plant protein hydrogels have recently been gaining attention because of their biodegradability and biocompatibility. Bambara groundnut is a potential source of protein in hydrogel production. However, the use of Bambara groundnut protein in such applications is limited because it is associated with the formation of inadequate crosslinks between polymer chains. Enzymatic treatment can potentially be used for improving the strength of Bambara groundnut protein hydrogel. In this study, the effect of laccase and transglutaminase (separately and in combination) on the microstructural, structural, rheological and mechanical properties of Bambara groundnut protein hydrogels was investigated for potential application in encapsulation and release of bioactive compounds. In the first part of this study, the effect of pH and NaCl concentration on the rheological and microstructural properties of Bambara groundnut protein gels were optimised using response surface methodology (RSM) to determine ideal starting conditions before enzymatic treatment. The effect of using crosslinking enzymes (transglutaminase and laccase) on the textural, rheological, structural and microstructural properties of Bambara groundnut protein hydrogels were then investigated. Since the effectiveness of enzymatic processes may be limited by using single enzymes, the use of a combination of enzymes was also investigated for the first time in gelation and optimised using RSM. Subsequently, encapsulation efficiency and release properties of the enzymatically crosslinked Bambara groundnut protein hydrogel were investigated using riboflavin as a model bioactive compound. The heat induced Bambara groundnut protein isolate (BPI) gels optimised for pH and NaCl prior to enzymatic treatment showed G’> G’’ over a frequency range of 0-100 rad s-1 . Although BPI gels displayed the characteristics of weak gels, slightly acidic conditions (pH 6) coupled with low NaCI concentration (0.5 M) promoted the formation of more rigid gels. These gels had the lowest water holding capacity and thiol content, suggesting the participation of disulphide linkages during network formation. Their microscopy images showed that the network was composed of porous homogeneous aggregates. Amino acid analysis showed that Bambara groundnut protein contains substantial amounts of amino acids including lysine, glutamic acid, cysteine and tyrosine with potential active sites for transglutaminase and laccase action. Laccase modification of Bambara groundnut protein caused a decrease in the gelation point temperature from approximately 85°C in the absence of laccase to 29°C at an activity of 3 U/g protein. Laccase treated samples showed a sharp increase in the G’ and G” values during the heating ramp as well as a wider gap between the moduli suggesting the formation of a more established network structure. The difference between G’ and G” increased to approximately 1 log and the dependency on angular frequency reduced suggesting improvement in the strength of the formed gels. Bambara groundnut protein crosslinking by laccase, was demonstrated by the decrease in thiol and phenolic content and crosslinking of amino acids (glutathione, cysteine and lysine) in model reactions. Microscopy images of the gel showed an increase in homogeneity and compactness of the lath sheet-like structure with increase in laccase activity up to 2 U/g protein. Transglutaminase crosslinking at 15 U/g protein resulted in the formation of hydrogels with well-organised network structures and small pores. Gel strength improved as observed from the highest G’ (6947 Pa) and hardness (5.60 N) recorded upon use of this activity. Transglutaminase-mediated crosslinking of BPI hydrogel was demonstrated by the reduction in amine and thiol groups and the formation of a new protein band (56 kDa) in crosslinked hydrogels. The combined use of transglutaminase and laccase showed a G’ > 10G” over a frequency range of 0 – 100 rad/s suggesting the dominance of the elastic behaviour. BPI hydrogel with the highest hardness (15.96 N) and encapsulation efficiency (98.8%) was formed at 15 and 0.5 U/g protein of transglutaminase and laccase activities, respectively. The lowest swelling capacity recorded in this hydrogel contributed to the lowest release kinetic constants in both simulated gastric fluid (0.51) and simulated intestinal fluid (0.73) in the presence of digestive enzymes which indicated that riboflavin release was due to diffusion and swelling. Overall, modification of Bambara groundnut protein using a combination of crosslinking enzymes increased the crosslinking density and promoted the formation of strong hydrogels. The hydrogels effectively encapsulated and prevented the early release of a heat sensitive compound (riboflavin) in the stomach while making it available in the small intestines. Therefore, the optimised enzyme combination of laccase and transglutaminase is a potential strategy for application in Bambara groundnut protein gelation.Item Microencapsulation of Bifidobacterium animalis and Lactobacillus casei using resistant starch from Vigna unguiculata(2020) Rengadu, Danielle; Mellem, John JasonThe use of functional foods is increasing globally with individuals aiming to maintain a healthy gut causing an increasing trend associated with probiotics in the health sector. Probiotics are live microorganisms that aid in improving the digestive system and gut health, however, the main problem associated with probiotics are ensuring a safe delivery through transition to the colon in harsh gastrointestinal conditions. For probiotics to be considered effective to the host a growth of Log 7 is essential in the colon, thus the need for microencapsulation. Therefore, this study was aimed at analysing resistant starch isolated from cowpea as an encapsulation material for Lactobacillus casei and Bifidobacterium animalis, for beverage application. Five different cultivars of cowpea (Bechuana white, Fahari, PAN 311, TVU 11424 and DT129-4) were analysed to determine the amount of resistant starch yielded as well as structural and physicochemical properties to determine the most suitable cultivar for the encapsulation process. The resistant starch percentage obtained was found in the range of 9.42-13.74%, with DT129-4 yielding the most resistant starch. The structural and physicochemical results obtained showed that the resistant starch isolated from cowpea has the potential for microencapsulation with cultivar DT129-4 exhibiting the most favourable results. Resistant starch was used as an encapsulating medium for Lactobacillus casei (RSL), Bifidobacterium animalis (RSB) and for a combination of the two probiotic microorganisms (RSLB) at a ratio of 1:1. The encapsulation yield after freeze drying were between 81.55-88.78% with the viability of the microcapsules under simulated gastrointestinal conditions also observed. The microcapsules were added to apple juice and the viability and stability of the microcapsules examined over 28 d. The final viability for microcapsules in the juice at the end of 28 d for RSL, RSB and RSLB were 7.53, 6.98 and 7.46 Log CFU/mL. This study shows that that resistant starch from cowpea has great potential as an encapsulating membrane within the nutraceutical beverage manufacturing industry.Item Production of oligosaccharides from lignocellulosic biomass(2020) Arumugam, Nanthakumar; Pillai, Santosh Kumar Kuttan; Singh, SurenLignocellulosic biomass is the most abundant plant material present on earth which is primarily composed of cellulose, hemicellulose and lignin. The composition of lignocellulosic biomass varies depending on the type of plant material and the conditions at which the plant grow. Exploration of lignocellulose for the production of value-added compounds including all types of platform chemicals, biofuels and bioactive compounds is gaining momentum. However, extensive research needs to the carried out to minimize the cost of production to make the processing of this biomass more viable. In the last two decades, several agricultural biomass types have been studied to facilitate the production of biochemicals and biofuels at a low cost. Biomass such as peanut shells, bambara, cowpea and sorghum are some of the indigenous crops of South Africa that are yet to be explored for value addition. Therefore, this study was designed to characterize the underutilized agro-residues such as peanut shell, bambara, cowpea and sorghum biomass for the enzymatic production of prebiotic xylooligosaccharides (XOS) and their application.