# Rifampicin-Loaded PLGA/Alginate-Grafted pNVCL-Based Nanoparticles for Wound Healing
## 3 Results and Discussions
### Cell Scratch Assay (Wound Healing)  
A cell scratch assay was used to assess the wound-healing potential of the nanoparticles-based formulations. Lower concentrations than the MTT assay were tested to avoid cytotoxic effects. During a period of 30 h, wound closure was monitored at 0-, 12-, 24- and 30-h intervals. A relative wound area (%) was determined. Figure 7 describes the development of scratches in the presence of tested materials. As shown within Figure 7, the microscope images were taken at 0, 12, 24, 30 h and % wound area was determined by using Image J software, a Java-based image processing program developed at the National Institutes of Health and the Laboratory for Optical and Computational Instrumentation. The percentage of wound area data were normalized via proportioning according to hour 0. The statistical significance of the difference between different groups at the same hours of the data showing % wound area against time were shown via two-way ANOVA test and the statistical significance of the difference between different hours of the same groups was shown via one-way ANOVA test. The ANOVA test was used to investigate whether the difference between the groups in % wound healing calculated according to the formula specified in the procedure was statistically significant. [PERSON]'s multiple comparisons test was used to determine between which groups there was a significant difference. Figure 8 described the closure activity in time and the wound area was determined after each time interval. As observed, the activity of the rifampicin loaded within the nanoparticles was more obvious after 24 h from the application of the nanoparticle's solution containing RF. Figure 7: The microscopical observations of the closures over a period of up to 30 h.  
When the closure activity is compared between the tested samples (Figure 9) it can be seen that the activities of the distinct solutions of nanoparticles and rifampicin are much higher than the complex formulation of Rf loaded inside the nanoparticles. A total closure effect of 60% was determined in the case of the RF-loaded nanoparticles while separate solutions of rifampicin and nanoparticles showed around 80% (Figure 10). Previously reported results [28] demonstrated the capacity to proliferate the cells when hydrogels composed of the similar polymeric carrier were loaded with rifampicin. The scratch assay was performed to test the migration and proliferation of Normal Human Dermal Fibroblasts (NHDF) within a maximum time frame of 72 h. The RF-loaded hydrogels demonstrated a synergetic effect between polymeric matrix and drug, the wound closure activity being more evident after 24 h from exposure, while RF itself showed a decreased activity after 24 h. This outcome may be explained by the advantage that RF was trapped inside the polymeric matrix and the closure activity had a sustained characteristic. However, many studies found a poor closure activity of rifampicin-based polymeric systems. Most of the studies associating the activity of rifampicin with an antibacterial one [36] in topical treatment of surgical wounds and rifampicin-based systems may be recommended for wound site irrigation [37] as an antiseptic agent rather than purely for wound healing. As reported, wound irrigation supposes the usage of the material as an antiseptic solution to sanitize the postoperative wound and to remove the elements that affect the healing process. When performed correctly, it helps removing any extra cellular debris, surface bacteria, wound exudate, dressing residue, and residual topical agents [38]. Figure 8: Wound areas of 3T3-L1 cells incubated with control (DMEM + 1%FBS), rifampicin, nanoparticles, rifampicin + nanoparticles until 30 th h. \(p<0.05\).