# Optimizing CNT Loading in Antimicrobial Composites for Urinary Tract Application
## 3 Results and Discussion
### AntibioticMins Assays  
To evaluate the influence of CNT-O loading in the antib biofilm activity of CNT-O/PDMS composites, a dynamic biofilm formation assay was performed with _E. coli_ CECT 434 GFP. The results of culturable and total cells quantification after 24 h of incubation with 0.1, 1, 2, 3, 4 and 5 wt% CNT-O/PDMS composites are presented in Figure 4.  
Despite the notorious increase in the percentage of culturable cells (Figure 4a) for surfaces with CNT loadings between 0.1 and 2 wt%, a decrease in those values was obtained for surfaces with higher CNT-O content. A total reduction of 39, 28 and 22% were achieved for 3, 4 and 5 wt% CNT-O/PDMS surfaces, respectively. However, only the surface with 3 wt% CNT-O loading presented a statistically significant reduction of cell culturability (\(p<0.001\)), being the most promising surface for the inhibition of _E. coli_ biofilms. The lower antimicrobial activity of 4 and 5 wt% CNT-O/PDMS surfaces may be justified by the decrease of CNTs dispersion degree imposed by increased loading, once the CNTs antimicrobial activity depends on their dispersion state [44]. Since no major differences were observed in the total cell number (Figure 4b), it is possible to assume that this decrease in cell culturability is a consequence of the antimicrobial activity of CNT-O/PDMS composites (an anti-adhesive activity would lead to a higher decrease in the total cell number). Indeed, an optimal CNT-O loading increases the availability of CNTs to interact with microbial cells on the matrix, without decreasing their dispersion degree, allowing CNTs to directly damage microbial membranes, with consequent cell death [22]. Figure 4: Percentage of _E. coli_ CECT 434 GFP (**a**) culturable and (**b**) total cells after 24 h of biofilm formation on PDMS (control) and CNT/PDMS composite surfaces with different CNT-O loading (ranging from 0.1 to 5 wt%). The means \(\pm\) standard error (SE) for three independent experiments are illustrated. Significant differences were considered for \(p\)-values \(<0.05\) (\(**\)\(p<0.001\)). These results were corroborated by the contact angle measurements, where the introduction of distinct CNT-O concentrations only gave rise to slight changes in surface hydrophobicity. After the initial optimization of the CNT-O loading, where the antimicrobial activity of 3 wt% CNT-O/PDMS composites against _E. coli_ CECT 434 GFP stood out from the remaining surfaces, a new assay was performed in order to assess the influence of the CNTs dispersion pattern on the antimicrobial activity of the composites. In this particular case, PDMS matrices filled with 1, 2, 3 and 4 h ball-milled CNTs (at 3 wt%) were prepared. The results obtained for culturable and total cell counts are shown in Figure 5. After 24 h of biofilm growth, no significant reductions in the total cell number were observed. However, the number of culturable cells shows a statistically significant reduction in comparison to PDMS (_p_ < 0.001). An average reduction of about 44% was obtained, indicating, once again, an antimicrobial effect against _E. coli_ biofilms. A higher decrease in _E. coli_ biofilm formation was achieved for the surfaces whose CNTs were ball-milled during 3 and 4 h (with a decrease of 54 and 53%, respectively, compared to PDMS). Results show a further decrease in the number of culturable cells for the ball-milled samples of around 24% when compared to the same loading (3 wt%) of CNT-O. This result suggests that BM treatment increases CNT's ability to disperse in the PDMS matrix, inducing morphological changes in the CNT/PDMS composites [45], which may increase the CNT surface contact with the microbial cells, leading to cell death [44]. Moreover, the effect of ball-milled CNTs on the reduction of _E. coli_ adhesion and its association with the degree of dispersion of CNTs were already demonstrated [24]. Representative CLSM images of the _E.