# Rifampicin-Loaded PLGA/Alginate-Grafted pNVCL-Based Nanoparticles for Wound Healing
## 3 Results and Discussions
### Cytotoxicity Studies  
Cytotoxicity is a crucial factor in wound dressing fabrication. More importantly, it can support skin tissue restoration. The first step of the assessment of the cytotoxicity of rifampicin was to calculate the IC50 values. The cytotoxicity data about rifampicin were limited. In a study by [PERSON] et al., the IC50 value of rifampicin was found to be 87 ug/mL. For the assessment, human monocyte cells (THP-1) were used; the cells were incubated with rifampicin for 72 h and then measured for their cytotoxicity with Alamar blue [33]. Within the present study, the IC50 value for rifampicin was found to be 208.8 ug/mL for 3T3-L1 cell line. Cells were incubated with rifampicin for 24 h and cytotoxicity was measured via MTT assay. The differences between the two tests were primarily that the cell type and then also incubation time obviously resulted in different IC50 values. As the proposed formulations are intended to be used for external use on wounds, the most relevant cell type to be tested was decided to be 3T3-L1. Figure 6 describes the cytotoxicity results and the IC50 values calculated for all studied samples.  
IC (inhibitory concentration) curves are dose/response curves used to determine the specific drug concentration required to reduce the viable cell population by a certain percentage compared to cells grown without drug exposure. The IC50, or inhibitory concentration at 50%, represents the concentration of a compound needed to inhibit a biological process by half. IC50 values are crucial in cytotoxicity assessments as they indicate the amount of a drug required to achieve this inhibition. The IC curves demonstrate changes in the population due to increased cell death or decreased cell proliferation [34]. Determining the IC50 value has significant implications beyond merely inhibiting cell growth or increasing cell death. In cancer treatment, using certain drugs at the IC50 concentration can reduce tumor growth by half. If IC50 is identified at a lower concentration, it indicates that the drug will be effective at lower doses, thereby reducing systemic toxicity in patients. Utilizing the IC50 concentration can effectively kill cancer cells and halt their growth while minimizing the toxic effects on healthy cells in the body. The values found for the prepared formulations were 192.1 ug/mL for the nanoparticle, 208.8 ug/mL for pure rifampicin, and the nanoparticle loaded with rifampicin had a IC50 value of 718 ug/mL. As observed, the IC50 value of the conjugate (i.e., nanoparticle of AgA-g-pNVCL/PLGA loaded with RIF) had a higher value than its constituents itself. Within the literature this phenomenon is defined as \"mixture effect\" [35]. From a toxicological point of view, the study of the combined effects of substances is crucial because organisms are often exposed  
Figure 5: In vitro release profile of rifampicin from the polymeric micro/nanoparticles of AgA-g-pNVCL/PLGA.  
to complex mixtures of chemicals rather than single substances. When the combined effect of chemicals is greater than the sum of their individual effects, a synergetic effect of the mixture is reported. It seems that it is also the case of the present formulation; the loaded nanoparticle with rifampicin presented a value of IC50 three times higher than its constituents. The reason behind this phenomenon needs to be investigated in further investigations up to the concentration dose and time of exposure.  
Figure 6: Cell viability test’s results for the studied samples: (**a**) pure rifampicin; (**b**) synthesized nanoparticles (AgA-g-PNVCL/PLGA); and (**c**) rifampicin-loaded nanoparticles. Within the caption, the IC50 values were determined by applying the sigmoidal mathematical model of the program GRAPHPAD.