Design, synthesis, manufacture, characterization and evaluation of lipid nanocapsules in chitosan-iota-carrageenan based hydrogel scaffold as a potential anti-Covid-19 drug delivery system
- Authors: Mukubwa, Grady Kathondo
- Date: 2022-10-14
- Subjects: Nanocapsules Design , Hydrogel , COVID-19 (Disease) , Characterization , Drug delivery systems
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/364955 , vital:65665
- Description: Covid-19 is a deadly viral disease that has been rampant around the world since 2019. Although the successful introduction of the vaccine has reduced the spread of covid-19, new cases and deaths are still being recorded. To date, no specific curative antiviral treatment has been approved for covid-19. However, many existing antiviral drugs have been and are still being studied against covid-19 and some of them, such as Remdesivir, have shown promise and could be repurposed to treat this infection. Unfortunately, antiviral drugs are prone to resistance as most of them have poor biopharmaceutical properties, including low solubility, permeability and bioavailability, which could hinder any clinical success. Recent advances in nanotechnology-based delivery systems have made it possible to improve the biopharmaceutical properties of many drugs, especially those of poorly water-soluble drugs, by formulating them as lipid nanoparticles (LNP). Thus, in order to contribute to the fight against covid-19, this work aimed to develop Lipid Nanocapsules (LNC), based on some natural raw materials, which could improve the biopharmaceutical properties of antiviral drugs. In addition, since covid-19 infection is mainly respiratory, this work also aimed to fabricate a targeted delivery system based on a hydrogel capable of entrapping LNC and ensuring their efficient deposition and release in the lungs. The LNC consisted of a mixture of medium-chain triglycerides oil (MCT oil), crude soy lecithin, tween 80, NaCl and water, while the hydrogel consisted of a chitosan-grafted-iota carrageenan-grafted-poly (acrylamide-co-acrylic acid) system (CS-iCar-p (AAm-Co-AA)). Efavirenz (EFV), a drug with very low water solubility that has recently been demonstrated to have the potential to influence sars-cov-2 life cycle through different targets (3CLP, RdRp, Hellicase, 3’to5’exonuclease, 2’-O-ribose methyltransferase and EndoRNAse), was chosen as the model drug to evaluate the developed delivery system. The combination of LNP and hydrogel results in a delivery system known as the LNP-hydrogel composite, an emerging area of research in the field of drug delivery. To date, no research has reported the design and fabrication of an LNC-CS-iCar-p (AAm-Co-AA) hydrogel composite that could effectively deliver an antiviral drug to the lungs in addition to its advantages in terms of biological activities. Prior to the design of experiment, EFV solubility was assessed in water, labrafac lipophile 1349 and MCT oil. After that, the Design Expert Software version 13 was used to design the different experiments performed in this work. The I-optimal mixture design of experiments was performed for both LNC preparation and CS-iCar-p (AAm-Co-AA) hydrogel synthesis to study the impact of raw materials on the characteristics of these delivery systems. LNC were prepared using the phase inversion method while the free radical precipitation graft copolymerization method was used to synthesize hydrogel. In order to build polynomial models that could predict the amount of drug both LNC and CS-iCar-p (AAm-Co-AA) hydrogel can entrap, a D-optimal (custom) randomized design was performed. Moreover, various characterization techniques were used to investigate the physicochemical properties of the developed delivery systems. Thereafter, drug release studies were performed using a 1% sodium lauryl sulfate solution adjusted to either pH 4 or 7. Solubility studies revealed that EFV was more soluble in labrafac lipophile 1349 and in MCT oil than in water; therefore, given its affordability, MCT oil was used for the LNC formulation. The design of experiment carried out allowed the construction of polynomial models that could predict, on the one hand, the droplet size, the polydispersity index and the Zeta potential of LNC, which were respectively around 50nm, below 0.2 and below -33. On the other hand, the model could predict the swelling capacity of the synthesized hydrogel, which was optimised to about 30,000% (300 g of water to 1 g of hydrogel). This turned out to be influenced by the proportion of polymers, the ratio of monomers as well as the concentration of the cross-linking agent. In addition, the characterization techniques further supported the improvement of EFV solubility by highlighting its conversion into its amorphous state after encapsulation in LNC. They also confirmed successful synthesis of CS-iCar-p (AAm-co-AA) hydrogel. LNC were able to encapsulate about 87% of EFV while the synthesized CS-iCar-p (AAm-co-AA) hydrogel entrapped around 53% of EFV encapsulated in LNC. While LNC were able to release 42% and 27% of EFV after 74 hours in a 1% sodium lauryl sulfate solution (SLS) at pH 7 and pH 4 respectively, the LNC-CS-iCar-p (AAm-co-AA) hydrogel composite released about 50% and 40% of the drug after 9 days in the same release medium. Interestingly, the chemical integrity of the drug was preserved throughout the manufacturing process up to after its release, suggesting that the developed LNC-CS-iCar-p (AAm-co-AA) hydrogel composite could be used as a novel potential anticovid-19 drugs delivery system. , Thesis (MSc) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mukubwa, Grady Kathondo
- Date: 2022-10-14
- Subjects: Nanocapsules Design , Hydrogel , COVID-19 (Disease) , Characterization , Drug delivery systems
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/364955 , vital:65665
- Description: Covid-19 is a deadly viral disease that has been rampant around the world since 2019. Although the successful introduction of the vaccine has reduced the spread of covid-19, new cases and deaths are still being recorded. To date, no specific curative antiviral treatment has been approved for covid-19. However, many existing antiviral drugs have been and are still being studied against covid-19 and some of them, such as Remdesivir, have shown promise and could be repurposed to treat this infection. Unfortunately, antiviral drugs are prone to resistance as most of them have poor biopharmaceutical properties, including low solubility, permeability and bioavailability, which could hinder any clinical success. Recent advances in nanotechnology-based delivery systems have made it possible to improve the biopharmaceutical properties of many drugs, especially those of poorly water-soluble drugs, by formulating them as lipid nanoparticles (LNP). Thus, in order to contribute to the fight against covid-19, this work aimed to develop Lipid Nanocapsules (LNC), based on some natural raw materials, which could improve the biopharmaceutical properties of antiviral drugs. In addition, since covid-19 infection is mainly respiratory, this work also aimed to fabricate a targeted delivery system based on a hydrogel capable of entrapping LNC and ensuring their efficient deposition and release in the lungs. The LNC consisted of a mixture of medium-chain triglycerides oil (MCT oil), crude soy lecithin, tween 80, NaCl and water, while the hydrogel consisted of a chitosan-grafted-iota carrageenan-grafted-poly (acrylamide-co-acrylic acid) system (CS-iCar-p (AAm-Co-AA)). Efavirenz (EFV), a drug with very low water solubility that has recently been demonstrated to have the potential to influence sars-cov-2 life cycle through different targets (3CLP, RdRp, Hellicase, 3’to5’exonuclease, 2’-O-ribose methyltransferase and EndoRNAse), was chosen as the model drug to evaluate the developed delivery system. The combination of LNP and hydrogel results in a delivery system known as the LNP-hydrogel composite, an emerging area of research in the field of drug delivery. To date, no research has reported the design and fabrication of an LNC-CS-iCar-p (AAm-Co-AA) hydrogel composite that could effectively deliver an antiviral drug to the lungs in addition to its advantages in terms of biological activities. Prior to the design of experiment, EFV solubility was assessed in water, labrafac lipophile 1349 and MCT oil. After that, the Design Expert Software version 13 was used to design the different experiments performed in this work. The I-optimal mixture design of experiments was performed for both LNC preparation and CS-iCar-p (AAm-Co-AA) hydrogel synthesis to study the impact of raw materials on the characteristics of these delivery systems. LNC were prepared using the phase inversion method while the free radical precipitation graft copolymerization method was used to synthesize hydrogel. In order to build polynomial models that could predict the amount of drug both LNC and CS-iCar-p (AAm-Co-AA) hydrogel can entrap, a D-optimal (custom) randomized design was performed. Moreover, various characterization techniques were used to investigate the physicochemical properties of the developed delivery systems. Thereafter, drug release studies were performed using a 1% sodium lauryl sulfate solution adjusted to either pH 4 or 7. Solubility studies revealed that EFV was more soluble in labrafac lipophile 1349 and in MCT oil than in water; therefore, given its affordability, MCT oil was used for the LNC formulation. The design of experiment carried out allowed the construction of polynomial models that could predict, on the one hand, the droplet size, the polydispersity index and the Zeta potential of LNC, which were respectively around 50nm, below 0.2 and below -33. On the other hand, the model could predict the swelling capacity of the synthesized hydrogel, which was optimised to about 30,000% (300 g of water to 1 g of hydrogel). This turned out to be influenced by the proportion of polymers, the ratio of monomers as well as the concentration of the cross-linking agent. In addition, the characterization techniques further supported the improvement of EFV solubility by highlighting its conversion into its amorphous state after encapsulation in LNC. They also confirmed successful synthesis of CS-iCar-p (AAm-co-AA) hydrogel. LNC were able to encapsulate about 87% of EFV while the synthesized CS-iCar-p (AAm-co-AA) hydrogel entrapped around 53% of EFV encapsulated in LNC. While LNC were able to release 42% and 27% of EFV after 74 hours in a 1% sodium lauryl sulfate solution (SLS) at pH 7 and pH 4 respectively, the LNC-CS-iCar-p (AAm-co-AA) hydrogel composite released about 50% and 40% of the drug after 9 days in the same release medium. Interestingly, the chemical integrity of the drug was preserved throughout the manufacturing process up to after its release, suggesting that the developed LNC-CS-iCar-p (AAm-co-AA) hydrogel composite could be used as a novel potential anticovid-19 drugs delivery system. , Thesis (MSc) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-10-14
Production, purification and characterization of a multifunctional, thermostable and acido/alkaline stable putative xylanase from the psychrotrophic bacterium, Sphingomonas aerolata
- Authors: Mathibe, Brian Nkanyiso
- Date: 2020
- Subjects: Sphingomonas , Sphingomonas aerolata , Psychrotrophic bacteria , Xylanases Purification , Thermodynamics , Characterization
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164478 , vital:41122
- Description: Enzymes are biological catalysts produced by living organisms in order to increase the rate of chemical reactions that are essential for life. Enzymes are now applied as catalysts in a variety of fields in response to a worldwide shift towards sustainable industrial processes, with enzymatic processes having been found to be specific, fast and to result in savings in energy and raw materials, all while generating less hazardous wastes when compared to conventional chemical processes. Microorganisms, specifically fungi and bacteria, are the most common sources for the production of enzymes because they grow rapidly meaning the production of enzymes is also rapid and can be easily manipulated using genetic engineering to obtain enzymes with desired characteristics. Due to the harsh conditions required in most industrial processes, there have been growing interests in enzymes that can withstand such harsh conditions. It has been found that extremophiles, microorganisms that can survive and grow in extreme conditions that would otherwise be detrimental to other life forms, are a source of extremozymes, enzymes that can withstand harsh conditions and therefore be utilised industrially where production processes are often harsh. Psychrophiles are examples of extremophiles, having the ability to be prolific in extremely low-temperature environments, and have been found to produce cold-active enzymes that have high specific activity at low temperatures. Cold-active enzymes from psychrophiles are of industrial and biotechnological significance as they could potentially result in energy savings when applied in industries that do not require high temperatures such as the transformation of heat-sensitive substrates at low temperatures. In this study, a psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated in both solid and liquid media and found to grow optimally at 28°C on media composed of (w/v): peptone, 1%; NaCl, 0.5%; yeast extract, 1%, and hydrolysed potato starch, 1%; with 1.5% agar incorporated to the solid medium formulation. This bacterium was found to produce an extracellular xylan degrading enzyme, which was purified to homogeneity using ultrafiltration with Amicon molecular weight cut-off filtration units. This enzyme was purified to a fold purification of 4.2 with percentage yield of 14.29%. A single band corresponding to approximately 36 kDa was observed using SDS-PAGE analysis of the purified putative xylanase sample. The xylanolytic activity of the purified sample was confirmed by a band of xylan hydrolysis using non-denaturing native activity-PAGE analysis, with 0.1% (w/v) beechwood xylan incorporated into the resolving gel. However, biochemical characterization of the enzyme revealed it to be also active on Avicel cellulose (22.64 U/mg), hydrolysed potato starch (19.11 U/mg), arabinogalactan (16.82 U/mg) as well as xylan substrates, beechwood xylan (23.69 U/mg) and wheat arabinoxylan (20.15 U/mg), making it a multifunctional glycoside hydrolase enzyme. Substrate competition assays between beechwood xylan and Avicel, and between beechwood xylan and hydrolysed potato starch, revealed that the enzyme uses the same active site to hydrolyse all three substrates, confirming the cross-specificity, or substrate promiscuity, of the enzyme. The enzyme, referred to as the putative xylanase in this study, was found to be cold-active, maintaining over 25% relative activity on beechwood xylan at 4˚C, consistent with other cold-active xylanases that have been reported in literature. The putative xylanase showed optimal xylanase activity at 40˚C and pH 6.0, and was found to be relatively thermostable, maintaining at least 78% relative activity on beechwood xylan after incubation for 24 hours at 50˚C. The enzyme was also found to be acido/alkaline stable, maintaining over 60% relative activity between pH 2.0 and 11.0, and also stable in the presence of various divalent cations, ethylenediaminetetraacetic (EDTA) and β-mercaptoethanol. The kinetic parameters of the putative xylanase on different substrates were determined using Hanes-Woolf plots. The Vmax values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 24.63, 27.40, 12.20, and 19.34 U/mg, respectively. The KM values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 3.03, 4.09, 4.49 and 2.21 mg/ml, respectively. In conclusion, the psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated under laboratory conditions and found to produce an extracellular putative xylanase that was successfully purified and characterized, and determined to possess a number of novel features such as the ability to hydrolyse different substrates using one active site, activity at low temperatures coupled with high thermostability, and stability at acidic and alkaline pH conditions. This novel enzyme could be very useful as a biocatalyst in different fields due to its novel features, and further study into its structure especially around the active site that has been shown to be possibly fluid enough to maintain reaction rates at low temperatures while also stable enough to maintain high reaction rates after long incubation periods at high temperatures, can bind and hydrolyse different substrates and can maintain high reaction rates across acidic, moderate and alkaline conditions, could further expand on the existing knowledge on biocatalysts and their properties. , Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Mathibe, Brian Nkanyiso
- Date: 2020
- Subjects: Sphingomonas , Sphingomonas aerolata , Psychrotrophic bacteria , Xylanases Purification , Thermodynamics , Characterization
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164478 , vital:41122
- Description: Enzymes are biological catalysts produced by living organisms in order to increase the rate of chemical reactions that are essential for life. Enzymes are now applied as catalysts in a variety of fields in response to a worldwide shift towards sustainable industrial processes, with enzymatic processes having been found to be specific, fast and to result in savings in energy and raw materials, all while generating less hazardous wastes when compared to conventional chemical processes. Microorganisms, specifically fungi and bacteria, are the most common sources for the production of enzymes because they grow rapidly meaning the production of enzymes is also rapid and can be easily manipulated using genetic engineering to obtain enzymes with desired characteristics. Due to the harsh conditions required in most industrial processes, there have been growing interests in enzymes that can withstand such harsh conditions. It has been found that extremophiles, microorganisms that can survive and grow in extreme conditions that would otherwise be detrimental to other life forms, are a source of extremozymes, enzymes that can withstand harsh conditions and therefore be utilised industrially where production processes are often harsh. Psychrophiles are examples of extremophiles, having the ability to be prolific in extremely low-temperature environments, and have been found to produce cold-active enzymes that have high specific activity at low temperatures. Cold-active enzymes from psychrophiles are of industrial and biotechnological significance as they could potentially result in energy savings when applied in industries that do not require high temperatures such as the transformation of heat-sensitive substrates at low temperatures. In this study, a psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated in both solid and liquid media and found to grow optimally at 28°C on media composed of (w/v): peptone, 1%; NaCl, 0.5%; yeast extract, 1%, and hydrolysed potato starch, 1%; with 1.5% agar incorporated to the solid medium formulation. This bacterium was found to produce an extracellular xylan degrading enzyme, which was purified to homogeneity using ultrafiltration with Amicon molecular weight cut-off filtration units. This enzyme was purified to a fold purification of 4.2 with percentage yield of 14.29%. A single band corresponding to approximately 36 kDa was observed using SDS-PAGE analysis of the purified putative xylanase sample. The xylanolytic activity of the purified sample was confirmed by a band of xylan hydrolysis using non-denaturing native activity-PAGE analysis, with 0.1% (w/v) beechwood xylan incorporated into the resolving gel. However, biochemical characterization of the enzyme revealed it to be also active on Avicel cellulose (22.64 U/mg), hydrolysed potato starch (19.11 U/mg), arabinogalactan (16.82 U/mg) as well as xylan substrates, beechwood xylan (23.69 U/mg) and wheat arabinoxylan (20.15 U/mg), making it a multifunctional glycoside hydrolase enzyme. Substrate competition assays between beechwood xylan and Avicel, and between beechwood xylan and hydrolysed potato starch, revealed that the enzyme uses the same active site to hydrolyse all three substrates, confirming the cross-specificity, or substrate promiscuity, of the enzyme. The enzyme, referred to as the putative xylanase in this study, was found to be cold-active, maintaining over 25% relative activity on beechwood xylan at 4˚C, consistent with other cold-active xylanases that have been reported in literature. The putative xylanase showed optimal xylanase activity at 40˚C and pH 6.0, and was found to be relatively thermostable, maintaining at least 78% relative activity on beechwood xylan after incubation for 24 hours at 50˚C. The enzyme was also found to be acido/alkaline stable, maintaining over 60% relative activity between pH 2.0 and 11.0, and also stable in the presence of various divalent cations, ethylenediaminetetraacetic (EDTA) and β-mercaptoethanol. The kinetic parameters of the putative xylanase on different substrates were determined using Hanes-Woolf plots. The Vmax values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 24.63, 27.40, 12.20, and 19.34 U/mg, respectively. The KM values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 3.03, 4.09, 4.49 and 2.21 mg/ml, respectively. In conclusion, the psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated under laboratory conditions and found to produce an extracellular putative xylanase that was successfully purified and characterized, and determined to possess a number of novel features such as the ability to hydrolyse different substrates using one active site, activity at low temperatures coupled with high thermostability, and stability at acidic and alkaline pH conditions. This novel enzyme could be very useful as a biocatalyst in different fields due to its novel features, and further study into its structure especially around the active site that has been shown to be possibly fluid enough to maintain reaction rates at low temperatures while also stable enough to maintain high reaction rates after long incubation periods at high temperatures, can bind and hydrolyse different substrates and can maintain high reaction rates across acidic, moderate and alkaline conditions, could further expand on the existing knowledge on biocatalysts and their properties. , Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Africa‘s Heritage No. 4: Bantu humour
- Authors: Tracey, Hugh
- Subjects: Humour , Nuance , Laughter , Tragedy , Nyasaland , Valley of Surprises , A place where you have to pray quickly , Christian , Heathen , Writing , Marimba , The Talkative Woman , Tumbuka , Drum , The Drum is Broken , Onomatopaeia , Bird sounds , Aural observation , Clowning , Miming , Sticks , Train , Engine whistle , Mbira , Hand piano , Church of Sinners , Translations , Fooding , Drinking , Tonga , Cunning , Indignation , Bravado , Story telling , Characterization , Bicycles , Horses , Taxes , Policemen , Magistrate , Courts , Drums , Jonathan Swift , Mukazi Wamulomo , Chinooma Chakubaruka , Shangaan , Kwaya , Ndau , Sitima , Music for the Horse , Music for the Bicycle , Conversation between Politician and Small Girl , Dialogue Whistle and Dogs
- Language: English
- Type: Sound , Radio broadcast , Music
- Identifier: vital:15100 , http://hdl.handle.net/10962/d1008528 , Reel number: BC129
- Description: 4th programme in the ‘Africa‘s Heritage‘ Series, about Bantu humour, broadcast by the South African Broadcasting Corporation , For further details refer to the ILAM Document Collection: Hugh Tracey Broadcasts
- Full Text: false
- Authors: Tracey, Hugh
- Subjects: Humour , Nuance , Laughter , Tragedy , Nyasaland , Valley of Surprises , A place where you have to pray quickly , Christian , Heathen , Writing , Marimba , The Talkative Woman , Tumbuka , Drum , The Drum is Broken , Onomatopaeia , Bird sounds , Aural observation , Clowning , Miming , Sticks , Train , Engine whistle , Mbira , Hand piano , Church of Sinners , Translations , Fooding , Drinking , Tonga , Cunning , Indignation , Bravado , Story telling , Characterization , Bicycles , Horses , Taxes , Policemen , Magistrate , Courts , Drums , Jonathan Swift , Mukazi Wamulomo , Chinooma Chakubaruka , Shangaan , Kwaya , Ndau , Sitima , Music for the Horse , Music for the Bicycle , Conversation between Politician and Small Girl , Dialogue Whistle and Dogs
- Language: English
- Type: Sound , Radio broadcast , Music
- Identifier: vital:15100 , http://hdl.handle.net/10962/d1008528 , Reel number: BC129
- Description: 4th programme in the ‘Africa‘s Heritage‘ Series, about Bantu humour, broadcast by the South African Broadcasting Corporation , For further details refer to the ILAM Document Collection: Hugh Tracey Broadcasts
- Full Text: false
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