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Design and synthesis of novel pH-responsive fatty acid-based lipids for the development of nano-delivery systems to enhance Vancomycin activity against Methicillin-resistant Staphylococcus aureus (MRSA).

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The ability of antimicrobials to prevent and treat infections caused by a range of microorganisms, including bacteria, is threatened by the emergence of drug-resistant microorganisms that is associated with high mortality rates globally. Novel nano-drug delivery systems, including lipidbased drug delivery systems, represent an alternative therapeutic approach to combat antimicrobial resistance resulting from conventional dosage forms. Since bacteria are associated with an acidic environment and the bacterial envelope is made up of lipid bilayer, the application of pHresponsive lipid-based nanomaterials for targeted antibiotic delivery is recognized as an active area of research. The aim of this study was to design and synthesize fatty acid-based pH-responsive lipids ( FAL, OLA-SPDA and DMGSAD-lipid) and explore their potential for the preparation of pH-responsive nano-based vancomycin (VCM) delivery systems to treat infectious diseases caused by methicillin-resistant Staphylococcus aureus (MRSA) infections. All the lipids were synthesized, and its structures were confirmed by FTIR, 1H NMR, 13C NMR and HR-MS. The nontoxic nature of the synthesized lipids was demonstrated by cell viability results above 75% on all tested mammalian cell lines using the MTT assay. After the synthesis and characterization, the novel fatty acid-based lipids were employed to formulate three pH-responsive lipid-based nanodrug delivery systems (liposomes, micelles and lipid polymer hybrid nanoparticles) for efficient and targeted delivery of VCM for the treatment S. aureus and MRSA infections. These systems were characterised for their physicochemical properties (Zetasizer), in vitro drug release (dialysis bag), morphology (HR-TEM), in vitro cell viability studies (flow cytometry), in vitro cytotoxicity (MTT assay), in vitro antibacterial activity (broth dilution method) and in vivo antibacterial activity (mice skin infection model). The four formulated pH-responsive liposomes had a mean size ranging from 86.28 ± 11.76 to 282 ± 31.58 nm, with their respective PDI’s ranging from 0.151 ± 0.016 to 0.204 ± 0.014 at pH 7.4 and 6.0 respectively. The ZP values were negative at physiological pH (7.4) and shifted towards positivity with a decrease in pH (6.0). The encapsulation efficiency (%EE) and loading capacity were in the range of 29.86 ± 4.5% and 44.27 ± 9.2%, The drug release profiles of all formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. Enhanced in vitro antibacterial activity at pH 6.0 was observed for the DOAPA-VAN-Liposome and DLAPA-VANLiposome formulations. Flow cytometry studies indicated a high killing rate of MRSA cells using DOAPA-VAN-Lipo (71.98%) and DLAPA-VAN-Lipo (73.32%) using the MIC of 1.59 µg/ml. In vivo studies showed reduced MRSA recovery from mice treated with liposome formulations (DOAPA-VAN-Lipo and DLAPA-VAN-Lipo) by 4- and 2-folds compared to bare VCM-treated mice respectively. The pH-responsive oleic acid-based dendritic lipid amphiphile self-assembled into stable micelles with particle size, PDI, ZP and %EE of 84.16 ± 0.184 nm, 0.199 ± 0.011 and -42.6 ± 1.98 mV and 78.80 ± 3.26%, respectively. The micelles demonstrated pH-responsiveness with an increase in particle size to 141.1 ± 0.070 nm at pH 6.0. The drug release profiles of formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. The in vitro antibacterial efficacy of VCM-OLA-SPDA-micelle against MRSA was 8-fold better when compared to bare VCM, and the formulation was 4-fold better at pH 6.0 when compared to the formulation’s MIC at pH 7.4. The MRSA viability assay showed that the micelles had a high percentage killing of 93.39% when compared to bare VCM (58.21%) at the same MIC (0.98 µg/ml). The in vivo mice skin infection model also demonstrated an enhanced antibacterial effect, showing 8-fold reduction in MRSA burden on skin treated with VCM-OLA-SPDA-micelles when compared with the skin sample treated with bare VCM. The optimized pH responsive lipid polymer hybrid nanoparticles (LPHNPs) formulations, RH40_VCM_LPHNPs had a particle size, PDI and ZP of 64.05 ± 0.64 nm, 0.277 ± 0.057 and 0.55 ± 0.14Vm, respectively, whereas SH15_VCM_LPHNPs displayed a size of 73.41 ± 0.468 nm, PDI of 0.487 ± 0.001 and ZP of -1.55 ± 0.184 Vm at pH 7.4. There was a significant change in particle size and ZP to 113.6 ± 0.20 nm and 9.44 ± 0.33 Vm for RH40_VCM_LPHNPs, respectively, whereas for SH15_VCM_LPHNPs, there was no change in is size but a significant change in surface charge switch to 9.83 ± 0.52 Vm at pH 6.0. The drug release profiles of formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. The VCM release profile, together with release kinetic study on LPHNPs, demonstrated the influence of pH on the high rate of VCM release at pH 6.0 as compared to pH 7.4. The LPHNPs a had better antibacterial activity against S. aureus and MRSA at both pH conditions when compared to bare VCM. Furthermore, the MIC of LPHNPs against MRSA was better by 8-fold at pH 6.0 than at 7.4. In summary, synthesized novel lipid materials showed superior biosafety profiles and potential in the development of lipid-based pH-responsive nanoantibiotic delivery systems against bacterial infections and other disease types characterized by low pH. The data from this study has resulted in three first-authored research publications, one co-authored research publication and one coauthored review article.


Doctoral Degree. University of KwaZulu-Natal, Durban.