RESUMO
Uropathogenic Escherichia coli express hairlike proteinaceous surface projections, known as chaperone-usher pathway (CUP) pili. Type 1 pili are CUP pili with well-established pathogenic properties. The FimH adhesin subunit of type 1 pili plays a key role in the pathogenesis of urinary tract infections (UTIs) as it mediates the adhesion of the bacteria to urothelial cells of the bladder. In this study, two breast cancer cell lines, MDA-MB-231 and MCF-7, were used to demonstrate the cytotoxic activities of type 1 piliated uropathogenic E. coli UTI89 on breast cancer cells in a type 1 pili and FimH-mediated manner. E. coli were grown in static and shaking conditions to induce or inhibit optimal type 1 pili biogenesis, respectively. Deletion constructs of UTI89 ΔfimH and a complemented strain (UTI89 ΔfimH/pfimH) were further utilized to genetically assess the effect of type 1 pili and FimH on cancer cell viability. After incubation with the different strains, cytotoxicity was measured using trypan blue exclusion assays. UTI89 grown statically caused significant cytotoxicity in both breast cancer cell lines whereas cytotoxicity was reduced when the cells were incubated with bacteria grown under shaking conditions. The incubation of both MDA-MB-231 and MCF-7 with UTI89 Δfim operon or ΔfimH showed a significant reduction in cytotoxicity exerted by the bacterial strains, revealing that type 1 pili expression was necessary for cytotoxicity. Complementing the ΔfimH strain with pfimH reversed the phenotype, leading to a significant increase in cytotoxicity. Incubating type 1 pili expressing bacteria with the competitive FimH inhibitor D-mannose before cancer cell treatment also led to a significant reduction in cytotoxicity on both MDA-MB-231 and MCF-7 cancer cells, compared to vehicle control or D-mannose alone, indicating the requirement for functional FimH for cytotoxicity. Overall, our results reveal that, as opposed to UTI89 lacking type 1 pili, type 1 piliated UTI89 causes significant cancer cell mortality in a FimH-mediated manner, that is decreased with D-mannose.
RESUMO
AIM: To synthesise cytarabine-loaded SLNs modified with the RGD peptide as a ligand, suitable for effective cancer therapy. METHODS: SLNs were synthesised by the high shear, hot homogenisation technique. A 2 level 3 factor analysis was used in optimisation. Particle size, zeta potential, poly-dispersion index and surface morphology were measured. Drug encapsulation, drug release, release kinetics, nanoparticle stability and chemical structure were determined. LIVE/DEAD® Fluorescence Assay was used to qualify cytotoxicity and Tryphan Blue assay to quantify. RESULTS: Cyt-SLNs exhibited a size of 161 ± 2.25 nm, a PDI of 0.49 ± 0.15 and a zeta potential of -19.8 mV. Entrapment fell at 88.87 ± 0.02% and release at 83.5 ± 0.95%. The in vitro release kinetics pointed towards a diffusion-based drug release mechanism. SLNs remained stable for 60 d. Cytotoxicity studies revealed that conjugation of the ligand with the RDG peptide resulted in a significant decrease in cell viability in both cell lines. CONCLUSION: Overall, the study suggests that RGD-SLN-cyt can be used for effective cancer therapy.
