Browsing by Author "Omolo, Calvin Andeve."

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Design of advanced materials and nano delivery approaches for enhancing activity against Methicillin resistant Staphylococcus aureus.
(2018) Omolo, Calvin Andeve.; Govender, Thirumala.; Mocktar, Chunderika.
Infectious diseases, including bacterial infections, continue to be a significant cause of morbidity and mortality globally, antimicrobial resistance has further made them fatal. Limitations of conventional dosage forms have been found to be one of the contributing factors to antimicrobial resistance. Novel nano delivery systems are showing potential to combat antimicrobial resistance. The search for novel materials for efficient delivery of antibiotics is an active research area. The aim of the study was to design and synthesize advanced materials and explore nano-based strategies for preparations of novel drug delivery systems to treat SA and MRSA infections. In this study two novel materials; a linear polymer dendrimer hybrid star polymer (3-mPEA) comprising of a generation one poly (ester-amine) dendrimer (G1-PEA) and copolymer of methoxy poly (ethylene glycol)-b-poly(ε-caprolactone) (mPEG-b-PCL) and oleic acid based quaternary lipid (QL) were synthesized and characterized and Poloxamer 188 (P188) material available in the market were employed to formulate three nano drug delivery systems for efficient and targeted delivery of antibiotics. The synthesized materials and the drug delivery system were found to be biosafe after exhibiting cell viability above 75% in all the cell lines tested on using MTT assay. The formulated nano based systems were evaluated for sizes, polydispersity indices (PDI), zeta potential (ZP), surface morphology, drug release, in vitro and in vivo antibacterial activity. Nanovesicles were formulated from 3-mPEA and they had sizes, PDI, ZP and entrapment efficiency of 52.48 ± 2.6 nm, 0.103 ± 0.047, -7.3 ± 1.3 mV and 76.49 ± 2.4%. respectively. QL lipid was employed to formulate vancomycin (VCM) loaded liposomes with Oleic acid based ‘On’ and ‘Off’” pH responsive switches for infection site and intracellular bacteria targeting. They were found to have the size of 98.88 ± 01.92 at pH 7.4. and exhibited surface charge switching from negative at pH 7.4 to positive charge accompanied by faster drug release at pH 6.0. Fusidic acid nanosuspension (FA-NS) with size, PDI and ZP of 265 ± 2.25 nm, 0.158 ± 0.026 and -16.9 ± 0.794 mV respectively was formulated from P188. The drug release profile from both the nanovesicles and liposomes was found to have sustained release. In vitro antibacterial activity for the nanovesicles, FA-NS and liposomes showed 8, 6 and 4-fold better activity at pH 7.4, while the liposome being a pH responsive antibacterial system at pH 6 showed 8- and 16- fold better activity against both Methicillin susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) respectively when compared with the bare drugs. An in vivo BALB/c mice, skin infection model revealed that treatment with VCM-loaded nanovesicles, liposomes and FA-Ns significantly reduced the MRSA burden compared to bare drugs and untreated groups. There was a 20, 6.33 and 76-fold reduction in the MRSA load in mice skin treated with nanovesicles, liposomes and FA-NS respectively compared to those treated with bare VCM and fusidic acid. In summary, synthesized material showed to be biosafe and potential for the development of nano-based drug delivery systems of antibiotics against bacterial infections. The data from this study has resulted in one book chapter and 3 first authored and 3 co-authored research publications.
Design of advanced multifunctional biomaterial-based biomimetic and pH-responsive hybrid nanocarriers for antibiotic delivery against bacterial infections and sepsis.
(2023) Elhassan, Eman Hussain Elmubarak.; Govender, Thirumala.; Omolo, Calvin Andeve.
Despite the notable improvements in the management of bacterial infections and sepsis, the mounting threat of antibiotic resistance on a global scale is leading towards a post-antibiotic era. Nano-drug delivery systems have improved the delivery and efficacy of various antibiotics. Biomimicry and stimuli-responsiveness have recently been used to improve the targetability of these nanocarriers, and enhance their localization at infected sites, thus improving overall therapeutic outcomes and reducing toxicity. Strategies such as targeting bacterial biofilms and efflux pumps can further enhance the delivery and effectiveness of antibiotics. Developing smart biomaterials with multifunctional properties to confer biomimetic, stimuli-responsive and antivirulence properties to antibiotic nanocarriers is the focus of ongoing research. Therefore, the general aim of this study was to investigate the potential of various novel multifunctional biomaterial-based hybrid nanocarriers (HNs), including biomimetic and/or pH-responsive HNs in enhancing the targeted delivery of antibiotics and modulating the proinflammatory response against bacterial infections and sepsis. In this study, two biomaterials with multifunctional activities, hyaluronic acid-lysine conjugate (HA-Lys) and tannic acid (TA), were employed to design, formulate, and extensively characterize innovative biomimetic and pH-responsive HNs for efficient and targeted delivery of antibiotics. The novel HA-Lys was synthesized and fully characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy and Fourier-transform infrared spectroscopy (FT-IR). Then it was successfully employed with tocopherol succinate (TS) and Oleylamine (OLA) to fabricate biomimetic pH-responsive vancomycin-loaded hybrid nanostructured lipid carriers (VCM-HNLCs). The prepared VCM-HNLCs were spherical and had average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 110.77  1.69 nm, 0.11  0.02, -2.92  0.21 mV, 76.27  1.20 % and 8.36  0.25 %, respectively. Both HA-Lys conjugate and its respective nanoformulations had excellent biosafety profiles (>70 % cell viability and ˂ 1 % hemolytic effect). Possible VCM-HNLCs competitive inhibition activity to toll-like receptors 2 and 4 (TLR2 and TLR4) was demonstrated via microscale thermophoresis (MST) analysis, which showed a 5-times and 16-times lower Kd values than their natural substrates peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively. VCM-HNLCs exhibited a pH-responsive drug release profile under acidic conditions, higher bacterial killing kinetics, enhanced antibacterial, anti-biofilm, and efflux pump inhibition activities over bare VCM. Also, they showed an improved activity in neutralizing reactive oxygen species (ROS) and modulating the inflammatory response induced by LPS. On the other hand, tannic acid (TA) and Oleylamine (OLA) were successfully employed to formulate biomimetic ciprofloxacin-loaded tannic acid hybrid nanoparticles (CIP-loaded TAH-NPs) to enhance the efficacy of CIP against bacterial infections and sepsis. The prepared HNs had onion-shaped morphology, with average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 85.65 ± 0.89 nm, 0.126 ± 0.01, +16.3 ± 0.23 mV, 68.73 ± 0.54 % and 6.86 ± 0.09 %, respectively. The hemolysis and MTT assays confirmed the biosafety and non-hemolytic activity of CIP-loaded TAH-NPs formulations (>70 % cell viability and ˂ 1 % hemolytic effect). The results of MST investigations and in-silico simulations demonstrated that TA and its nanoformulation (CIP-loaded TAH-NPs) competitively inhibited TLR4 compared to its natural substrate LPS. CIP-loaded TAH-NPs showed a diffusion-based sustained release profile at physiological pH 7.4. Also, in comparison to bare CIP, the hybrid nanovesicles demonstrated improved antibacterial, anti-biofilm and efflux pump inhibition properties, as well as faster bacterial killing kinetics. Moreover, they showed a significant neutralization of ROS and the ability to control the inflammatory responses brought on by LPS. In summary, VCM-HNLCs and CIP-loaded TAH-NPs were successfully formulated and showed significant improvement in antibiotics efficacy and overall therapeutic outcomes. This study confirmed the potential of biomimetic stimuli-responsive antibiotic hybrid nanocarriers for enhancing antibiotic efficacy against bacterial sepsis and addressing the antimicrobial resistance crisis. The data from this study has resulted in one first-authored review article, two first-authored research publications and one co-authored review article.
Formulation of nanostructured lipid carriers using eugenol and D-α-tocopherol succinate for the inhibition of bacterial efflux pumps to enhance delivery of ciprofloxacin.
(2023) Dlamini, Sbongumusa.; Govender, Thirumala.; Omolo, Calvin Andeve.
The continuously growing antibacterial resistance crisis is decreasing the availability of the antibiotics to treat bacterial infections. Bacteria develop additional mechanisms to survive lethal concentrations of antibiotics such as efflux pumps and biofilms. Novel drug delivery systems are urgently required to enhance antibiotic efficacy and overcome resistance. Furthermore, natural derivatives from medicinal plants have shown great potential as bacterial adjuvants to inhibit these mechanisms. Therefore, employing these compounds in the formulation of nanocarriers could restore antibiotic efficacy and overcome antibiotic resistance. Aim: The aim of this study was to explore the potential of ciprofloxacin-loaded nanostructured lipid carriers (CIP-NLCs) designed using D-α-tocopherol succinate (TS) and eugenol for enhancing antimicrobial activity and overcoming resistance mechanisms against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa). Methods: CIP-NLCs were prepared using hot homogenization/ultrasonication method. The particle size, polydispersity index (PDI) and zeta potential (ZP) of CIP-NLCs were determined using the dynamic light scattering technique. Transmission electron microscopy analysis was conducted to confirm particle size and visualize the morphology of CIP-NLCs. The entrapment efficiency (EE %) of CIP-NLCs was determined using the ultrafiltration method and was quantified using High-Performance Liquid Chromatography (HPLC). In vitro drug release of CIP-NLCs were conducted using the dialysis bag technique and CIP released was quantified using HPLC. Drug release kinetics were analysed using the DDSolver program. Haemocompatibility of CIP-NLCs was performed using sheep blood. The in vitro antibacterial activity of CIP-NLCs were determined using micro broth assay against Staphylococcus aureus (SA), Escherichia coli (E. coli), MRSA, and P. aeruginosa. Bacterial killing kinetics were performed against MRSA and P. aeruginosa using the plate colony counting method. MRSA and P. aeruginosa biofilm inhibition of CIP-NLCs was evaluated using microtiter method. DPPH scavenging was used to study the antioxidant activity of CIP-NLCs. In vivo antibacterial activity of MRSA was studied using the systemic MRSA infection on BALB/c mice. Results: CIP-NLCs had a particle size, PDI, ZP, and EE % of 147.4 ± 0.59 nm, 0.219 ± 0.009, -9.64 ± 2.22 mV, and 82.8 ± 0.39 %, respectively. The in vitro biosafety evaluation revealed CIP-NLCs as non- haemolytic. The in vitro drug release study showed a biphasic release of CIP from the CIP-LNCs for 48 hours at physiological pH (7.4). The in vitro antibacterial activity of CIP-NLCs (SA:0.195 μg/mL and MRSA:12.5 μg/mL) showed 2-fold lower minimum inhibitory concentration (MIC) values over bare ciprofloxacin (SA: 0.39 μg/mL and MRSA: 25 μg/mL) against MRSA, SA, whereas the MIC values of CIP-NLCs (E. coli: 0.048 μg/mL and P. aeruginosa: 0.097 μg/mL) were 4-fold lower CIP ( E. coli: 0.195 μg/mL and P. aeruginosa: 0.39 μg/mL) against E. coli, and P. aeruginosa. The bacterial-killing kinetic test showed 100% elimination of MRSA and P. aeruginosa within eight and one hour(s) of treatment with CIP-NLCs, respectively. Conversely, 100% elimination of MRSA and P. aeruginosa was shown within 24 and 12 hours of treatment with bare ciprofloxacin, respectively. CIP-NLCs eliminated 3-fold MRSA biofilm compared to bare ciprofloxacin, whereas 1.25-fold P. aeruginosa biofilms were eliminated. The efflux pump inhibition potential of CIP-NLCs was confirmed using cartwheel assay, which showed high fluorescence intensity on bacteria treated with CIP-NLCs. The DPPH scavenging assay of CIP-NLCs proved antioxidant activity equivalent to Vitamin C (ascorbic acid), which is reported to be a potent antioxidant. The in vivo systematic infection in BALB/c mice reduced MRSA infection in kidney, liver, and blood by 12.27-fold, 4.47-fold, and 1613-fold, respectively. Conclusion: CIP-NLCs designed using TS and eugenol could enhance the antimicrobial activity against MRSA and P. aeruginosa infections and inhibit efflux pumps associated with these bacteria. Therefore, the CIP-NLCs may serve as a promising tool for enhanced delivery of ciprofloxacin and treatment of bacterial infections.
Formulation of pH-responsive lipid-polymer hybrid nanoparticles for co-delivery and enhanced antibacterial activity of 18β-glycyrrhetinic acid and vancomycin against MRSA.
(2020) Jaglal, Yajna.; Govender, Thirumala.; Omolo, Calvin Andeve.
Background: Due to the rise in antimicrobial resistance and the challenges accompanied by conventional antibiotic dosage forms, there is a need for developing drug delivery systems that enhance, protect and potentiate the current antibiotics in the market. Furthermore, natural derivatives from plants have proven to be potent antimicrobial agents. Therefore, their combination with antibiotics could be effective in overcoming antimicrobial resistance. Aim: The aim of this study was to co-deliver vancomycin and 18β-glycyrrhetinic acid via pH-responsive lipid-polymer hybrid nanoparticles (VCM-GAPAH-LPHNPs) formulated from polyallylamine and oleic acid (OA) and to explore its potential for enhanced activity and targeted delivery. Methods: Molecular dynamics and stability studies were used to determine the stability of the oil and water phases independently as well as VCM-GAPAH-LPHNPs as a complex. VCM-GAPAH-LPHNPs were prepared using the micro-emulsion technique. The size, polydispersity index and zeta potential of VCM-GAPAH-LPHNPs were determined using the dynamic light scattering technique. Transmission electron microscopy analysis was conducted to determine the morphology of VCM-GAPAH-LPHNPs. The entrapment efficiency and drug loading were determined using the ultrafiltration method. Differential scanning calorimetry was used to determine the thermal profiles of VCM-GAPAH-LPHNPs and its components. In vitro drug release studies were performed using the dialysis bag technique. Drug release kinetics were analysed using the DDSolver program. Cytotoxicity of VCM-GAPAH-LPHNPs were determined using the MTT assay. Haemolysis of VCM-GAPAH-LPHNPs were performed at different concentrations using sheep blood. In vitro antibacterial activity of VCM-GAPAH-LPHNPs were determined against SA and methicillin-resistant Staphylococcus aureus (MRSA) at pH 6 and 7.4. Time killing assay was performed using the plate colony count method. MRSA biofilm study was performed using the crystal violet assay. Results: Molecular dynamics indicated VCM-GAPAH-LPHNPs to be stable. VCM-GAPAH-LPHNPs were successfully prepared using the micro-emulsion technique. VCM-GAPAH-LPHNPs size, polydispersity index, zeta potential and encapsulation efficiency were found to be 198.4 ± 0.302 nm, 0.255 ± 0.003, - 3.8 ± 0.335 mV and 69.46 ± 2.52 % respectively. Thermal profiles of lyophilized VCM-GAPAH-LPHNPs showed transformation from crystallization to amorphous form. In vitro drug release studies revealed that VCM-GAPAH-LPHNPs released 60% of VCM after 24 h whereas bare VCM released 90% of VCM after 24 h hence VCM-GAPAH-LPHNPs showed sustained drug release compared to bare VCM. At pH 6 VCM-GAPAH-LPHNPs released 82% of VCM after 24 h whereas at pH 7.4 VCM-GAPAH-LPHNPs released 60% of VCM after 24 h indicating VCM-GAPAH-LPHNPs had a faster drug release at pH 6 compared to pH 7.4. The Weibull model was considered the best fit model for VCM-GAPAH-LPHNPs. The MTT assay revealed 75% > cell viability which indicated VCM-GAPAH-LPHNPs to be non-cytotoxic. At 0.5 mg/ml VCM-GAPAH-LPHNPs showed < 1% haemolysis. Stability studies at 4 °C and room temperature indicated VCM-GAPAH-LPHNPs to be stable. In vitro antibacterial activity against MRSA treated with VCM-GAPAH-LPHNPs demonstrated a 16-fold lower minimum inhibitory concentration than bare VCM at acidic conditions. The time-killing assay study at 12 h revealed that VCM-GAPAH-LPHNPs eliminated 100% of MRSA cells whereas bare VCM eliminated 55% of MRSA cells. The crystal violet assay analysis revealed VCM-GAPAH-LPHNPs ability to eliminate MRSA biofilms. Conclusion: VCM-GAPAH-LPHNPs could effectively treat MRSA infections at a faster rate as compared to bare VCM. Therefore, this novel pH-responsive LPHNPs may serve as a promising nanocarrier for enhancing antibiotic delivery and antibacterial activity.
pH-responsive gelatin nanoparticles for targeted delivery of ciprofloxacin against bacterial infections.
(2024) Hlabisa, Minenhle.; Govender, Thirumala.; Omolo, Calvin Andeve.
Background: Given the rise in antimicrobial resistance and challenges associated with traditional antibiotic dosage forms, there is an urgency to develop drug delivery systems that improve, safeguard, and augment the present antibiotics on the market. Further research is required to maximize extended and targeted drug release, which can be accomplished via stimuli-responsive approaches such as pH-responsive nano-drug delivery systems. Furthermore, these pH-responsive nanosystems may be employed as carriers for antimicrobial drugs, which could be beneficial against antimicrobial resistance. Aim: The aim of this study was to prepare novel pH-responsive gelatin nanoparticles to function as delivery agents of Ciprofloxacin (CIP) to enhance their antibacterial effectiveness against methicillin-resistant Staphylococcus aureus (MRSA). Methods: CIP-GNPs were prepared using a two-step desolvation method. The particle size, polydispersity index (PDI) and zeta potential (ZP) of CIP-GNPs were determined using the dynamic light scattering technique. Transmission electron microscopy analysis was conducted to confirm particle size and visualize the morphology of CIP-GNPs. The entrapment efficiency (EE %) of CIP-GNPs was determined using the ultrafiltration method and was quantified using High-Performance Liquid Chromatography (HPLC). In vitro drug release of CIP-GNPs was conducted using the dialysis bag technique and CIP released was quantified using HPLC. Drug release dissolution factors were analysed using the DDSolver program. Hemocompatibility of CIP-GNPs was performed using sheep blood. In vitro antibacterial activity of CIP-GNPs was determined using micro broth assay against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), MRSA, and P. aeruginosa. Bacterial killing kinetics were performed against MRSA and P. aeruginosa using the plate colony counting method. MRSA and P. aeruginosa biofilm inhibition of CIP-GNPs was evaluated using the microtiter method. Results: CIP-GNPs had a particle size, polydispersity index, zeta potential, and entrapment efficiency of 212.3 ± 1.739, 0.259 ± 0.023, +4.58 ± 0.148 mV and 38.1 ± 3.85%, respectively. In vitro, biosafety testing identified CIP-GNPs as non-hemolytic. The CIP-GNPs demonstrated pH responsiveness with an increase in particle size from 204.1 ± 0.100 to 226.4 ± 0.451 nm and a charge switch on the zeta potential from -3.59 ± 0.428 to 1.06 ± 0.271 mV, followed by a significantly faster release of CIP at pH 6.0 compared to 7.4. The in vitro antibacterial activity of CIP-GNPs showed 2-fold lower minimum inhibitory concentration values compared to bare ciprofloxacin against Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). Moreover, the bacterial-killing kinetic test showed 100% elimination of MRSA and P. aeruginosa within eight and one hour(s) of treatment with CIP-GNPs, respectively. In contrast, 100% elimination of MRSA and P. aeruginosa was observed within 24 and 12 hours of treatment with bare ciprofloxacin, respectively. CIP-GNPs eliminated 3,75-fold MRSA biofilm compared to bare ciprofloxacin, whereas 1.4-fold Pseudomonas aeruginosa biofilms were eliminated. Conclusion: CIP-GNPs could effectively treat MRSA infections at a faster rate as compared to bare CIP. Therefore, this novel pH-responsive CIP-GNPs may serve as a promising nanocarrier for enhancing antibiotic delivery and antibacterial activity