Fig 1.
Schematic of nanoparticle-mediated TFD delivery and inhibition of S. aureus replication.
A) Normal transcription process whereby (i) the transcription factor protein (s) bind their relevant promoter region which results in (ii) transcription and mRNA production which is then (iii) translated to functional protein. B) (i) Following administration of TFD-nanoparticles, (ii) these traverse the bacterial cell wall and membrane and TFD is decomplexed (iii) TFDs competitively inhibit the transcription factor binding to DNA promotor region in the bacterial chromosome and therefore (iv) considerably reduce or even inhibit transcription and (v) translation of crucial cell wall proteins (image made in BioRender—biorender.com).
Fig 2.
Composition of nanoparticles tested for TFD delivery.
A) cationic nanostructured lipid carriers and B) chitosan based nanocarrier (CS-NCs) and C) Structure and properties of the WalR TFD (redrawn from [17, 22]).
Fig 3.
Stability analysis of cNLC-TFD nanocomplexes at N/P = 32 over a 72-hour timeframe in a variety of biological buffers.
A) Size and polydispersity analysis B) zeta potential analysis C) agarose gel electrophoresis analysis of nanocomplex integrity following incubation at 37°C for 72 hours.
Fig 4.
Agarose gel electrophoresis to quantify the TFD entrapped in chitosan nanocapsules.
A) Addition of 50 μg TFD. Lane 1, empty CS-NCs; lanes 2–7, free TFD calibration; lane 8, empty well; lanes 9–12, TFD-CS-NCs diluted 1:2; lane 13, filtered TFD-CS-NCs. B) Addition of 100 μg TFD. Lanes 1–4, free TFD calibration; lanes 5–6, empty CS-NCs; lanes 7–9, TFD-CS-NCs diluted 1:5; lanes 10–12, TFD-CS-NCs diluted 1:10; lane 13, filtered TFD-CS-NCs. C) Addition of 200 μg TFD. Lane 1, empty CS-NCs; lanes 2–7, free TFD calibration; lanes 8–11, TFD-CS-NCs diluted 1:8; lane 12, filtered TFD-CS-NCs.
Table 1.
Drug loading and entrapment efficiency of TFD-CS-NCs.
Fig 5.
Stability analysis of TFD-CS-NC nanocarriers over a 72-hour timeframe in a variety of storage and biological buffers.
A) Size analysis B) zeta potential analysis and C) polydispersity index.
Table 2.
MIC values of vancomycin and either cNLC-TFD or CH-NC-TFD nanocomplexes as well as relevant controls (n = 6) against S. aureus CECT794 strain using reduced sample assay.
Fig 6.
Synergy assay demonstrating enhanced antimicrobial effect against MRSA strain CECT 5190 when read at 570 nm.
From left, untreated MRSA controls, MRSA treated with sub-MIC dose of 0.6 μg/ml vancomycin, MRSA treated with cNLC-TFD nanocomplexes at a TFD concentration of 125 nM and MRSA treated with dual therapy of 0.6 μg/ml free vancomycin and cNLC-125nM TFD nanocomplexes (n = 3 ±SEM, *P<0.05, **P<0.01, one-way ANOVA).
Fig 7.
WST-1 assay analysis of cell viability following cNLC-TFD nanocomplex administration in A549 and HUVEC cells.
Viability was assessed as percentage change against untreated negative control cells including cyclosporine positive control samples (PC). (n = 3 ±SEM).
Fig 8.
Percentage of haemolysis from whole human blood following incubation with cNLC-TFD nanocomplexes.
Samples were incubated for 90 minutes at 37°C (n = 6 ±SEM, one-way ANOVA).