# Rifampicin-Loaded PLGA/Alginate-Grafted pNVCL-Based Nanoparticles for Wound Healing
## 3 Results and Discussions
### In Vitro Release Profile  
Prior to evaluating the in vitro release behavior of rifampicin, the amount of drug trapped within the polymeric chains was determined via HPLC assay, and it was found out that 20 mg polymeric micro/nanoparticles consisted of 160 mg/mL of rifampicin as was reported within the previous study [4]. This outcome is useful for further studies such as cytotoxicity assessment, in vitro release, and wound closure capacities. Figure 5 describes the in vitro release profile of rifampicin within 7 h from the beginning of the experiment. As shown in Figure 5, in the first 60 min from the administration of the formulation, there was an amount of about 1% of the total loaded rifampicin. As observed from the in vitro release profile, over the time interval release, the release capacity was studied and the low amount of rifampicin was determined via the HPLC method. More specifically, within the first hour from the administration, approximately 1% was released, followed by 1% more being further determined after another interval of 2 h. The outcome regarding the low release of RF from various matrices was previously reported. [PERSON] et al. [28] found out an amount of released RF within 4 h of 5.98 ppm while [PERSON] et al. [29] reported a delivered amount of RF of 10% within 10 h in the case of encapsulated RF within liquid-crystalline folate nanoparticles. Interestingly, they reported a self-assembly behavior to control the loading capacity of rifampicin and additionally its delivery profile. They found  
Figure 3: DSC thermograms of polymeric nanoparticles with and without RF. out that by decreasing the particle size of the particles encapsulating rifampicin, the loaded amount was lower. However, the low amount of rifampicin detected within the matrix did not show significant differences concerning its cytotoxicity activity or release profile. An additional factor reported which seemed to influence the release behavior was that rifampicin, when encapsulated within nanoparticles, was bound via its functional groups creating internal crosslinks which may have slowed down the release. Within the present study, rifampicin was encapsulated within the matrix of grafted alginate with poly(N-vinylcaprolactam) and protected by a shell of PLGA. The size of formed particles was determined as being around 100 without RF and 200 nm when RF was loaded, as presented within Figure 2. The explanation on the low efficacy regarding the loading amount but also the capacity of the nanoparticle to release the trapped RF was found to be on the base of created crosslinks between RF and polymeric matrix. Another reason was also the drug's low solubility. [PERSON] et al. [30] reported the low solubility of rifampicin in water and buffer system up to the content of the amorphous moiety from the raw drug and the careful selection of the drug as a raw material is needed. The rifampicin used within the study was provided from a local medicine company and according to the DSC analysis (thermogram from Figure 3) a form II of rifampicin was determined so the polymorphism could influence the solubility in aqueous phases [31]. The drug release mechanism was assessed via the theoretical kinetic evaluation by applying the semi-empiric equation (1) of [PERSON] et al. [20] of the release profile within the first hour, which showed an abnormal non-Fickian mechanism (case II) and a matrix-dependent mechanism considering the values of the release rate of 0.958 min\({}^{-1}\) (R\({}^{2}\) = 0.9911) and n value of 0.7502 indicating that the release mechanism was case II which involved swelling and erosion of the matrix structure.