Browsing by Author "Machumele, Khanani Peggy"

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    Synthesis and characterization of novel short antimicrobial peptides with wound healing properties
    (University of the Witwatersrand, Johannesburg, 2019) Machumele, Khanani Peggy; Makatini, Maya Mellisa
    In recent years, there has been an increasing health crisis due to multidrug-resistant microbes. These pathogens are strains of bacteria that have become resistant to antibiotic drugs. In the year 2016, the World Health Organisation (WHO) had appealed to the members of states in the USA to create a priority list of other bacteria that are resistant to antibiotics in order to support research and development of effective drugs. According to literature, antimicrobial peptides have the potential to be potent agents against pathogens that have multidrug-resistant properties. Despite these studies, there are still substantial limitations (toxicity and susceptibility to proteases) that have affected their clinical and commercial development. In this study, the focus was on bacteria that infect wounds. The lack of potent chronic wound treatment has resulted in an enormous financial and physical burden on patients and the health care system. The stress of multi-resistant microbes heighten the challenges plagued on a patient due to untreatable infected wounds. Peptides which are able to kill bacteria and promote the wound healing process would greatly benefit patients. For example, patients with diabetic foot ulcers are prone to chronic wounds because of their condition, which may lead to amputation. Wound healing antimicrobial peptides are able to kill bacteria in the wound and induce the formation of collagen which will result in fewer amputations. The aim of this proposed research is to develop novel wound healing and antimicrobial compounds by derivatizing bioactive peptides into selective and protease-stable peptidomimetics. Tigerinin RC1 is an antimicrobial peptide with wound healing properties. It was chosen as a starting point for the design of analogues with drug-like properties and it was also conjugated to silver nanoparticle (AgNPs) to improve its bactericidal activity. In this study, 16 Tigerinin RC1 peptide analogues were successfully synthesized using the solid phase peptide synthesis strategy. Peptides were purified using the semi prep-HPLC however, the desired purity of > 90% was only achieved after two or more purification runs. Thus only 4 of the peptide analogues had a purity great than 90% which were KM-PEP-carb, KM-PEP-cyc-amide, KM-PEP-ada and KM-PEP-CT. These peptides were tested for antimicrobial activity and KM-PEP-cyc-amide peptide showed promising results with the minimum inhibitory concentration of 128 μg/ml against P. aeruginosa. Cytotoxicity studies also revealed that conjugation of KM-PEP-carb to AgNPs improved cytotoxicity because when 25 μg/ml of KM-PEP-carb was tested against human T cells the cell viability was -1.48% and when conjugated to AgNPs the cell viability increased to 35.17.
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    Synthesis of carbon nanodots-peptide conjugates decorated with germanium for bioimaging
    (University of the Witwatersrand, Johannesburg, 2023-10) Machumele, Khanani Peggy; Makatini, Maya Mellisa; Maubane-Nkadimkeng, Manoko
    The World Health Organization Global Cancer Observatory estimates that cancer caused 9.96 million deaths worldwide in 2020, making early detection crucial for diagnosis and treatment. Accurate identification of cancer plays a crucial role in the diagnosis and treatment process. It allows for customized and efficient therapies, minimizes unnecessary procedures and adverse effects, and improves the prognostic insights for patients and healthcare providers alike. The challenges in diagnosis include overdiagnosis, false positives/negative outcomes, and limited sensitivity. Advanced technologies are needed for better imaging accuracy and minimizing harm. This study aims to fabricate carbon dot-peptide conjugates to enhance bio-imaging capacity and selectivity. The peptides used are derived from the GKPILFF cell-penetrating peptide sequence and the RLRLRIGRR peptide, which is selective to cancerous cells. The Carbon dots were used to provide the photoluminescent properties required for bio-imaging of cancerous cells. Carbon dots (CDs) were synthesized using iso-ascorbic acid as the source of carbon using a microwave-assisted method. The nitrogen and germanium-modified carbon dots (Iso-N-Ge-CDs) demonstrated the highest photoluminescent properties compared to the unmodified CDs (Iso-CDs) and those with either N (Iso-N-CDs) or Ge (Iso-Ge-CDs). Photoluminescence emissions of longer wavelengths suitable for cell imaging were observed for the CDs, and the Iso-N-Ge-CDs demonstrated excitation-dependent emission wavelength behavior, pH sensitivity, and Fe3+ sensitivity. The 13 peptides derived from the peptide accelerating sequence GKPILFF and the cancer-selective peptide RLRLRIGRR were successfully synthesized. The peptides were characterized using Liquid Chromatography Mass Spectrometry (LCMS) and purified using preparative High-Pressure Liquid Chromatography (prep-HPLC). The secondary structure of the L-GKPILFF penetration acceleration peptide sequence (Pas) adopted a helical secondary structure. The D-GKPILFF derivative was found to adopt a random coil structure. These were confirmed using Nuclear Magnetic Resonance (NMR) techniques such as Total Correlation Spectroscopy (TOCSY) and Rotating Frame Overhauser Enhancement Spectroscopy (ROESY) NMR. The CDs-peptide conjugates were successfully synthesized, and the confirmation of conjugation involved multiple methods, including UV-Vis and PL techniques. To the best of our knowledge, the thesis incorporates the first study to demonstrate long-range interactions through ROESY NMR. The NMR analysis indicated that the helical structure of the peptide could be affected after conjugation, leading to notable peak shifts. Since the helical structure is crucial for the peptide's bioactivity and stability enhancement, NMR spectra with fewer structural changes in the peptide region may improve its biological properties. The research contained valuable information for scientists aiming to design and characterize Carbon dot-peptide conjugates with enhanced permeability and selectivity that can effectively deliver materials into cytosolic space.