# Investigation of Gentamicin Release from
## 3 Results and Discussion
### In Vitro Drug Release from G-PDA NPs  
The pH-responsive drug release for gentamicin was investigated for two batches viz. ; G-PDA NPs 1:1 and G-PDA NPs 0:6:1 against three pH values, i.e., 7.4, 5 and 3. The release profile of gentamicin was determined from the LC-MS. The concentrations of the drug release were obtained through a calibration curve of standard gentamicin, as shown in Figure S1 (Supplementary Materials). The standard gentamicin peaks were also obtained from all three of the isomers C1a, C2/C2a and C1, respectively, at their corresponding m/z values as shown in Figures S2-S4 (Supplementary Materials). An ideal drug-loaded nanocarrier normally shows a release-inhibition phenomenon at physiological pH and becomes free to release the bound drug only when and where bacterial multiplication takes place, thus decreasing the local pH in the vicinity. Based on this concept, three pH conditions were studied for the drug release from the G-PDA NPs. Notably, the PDA's zwitterionic nature allows it to substitute surface charge at variable pH \begin{table}
\begin{tabular}{c c} \hline \hline
**Elements of LC-MS Used** & **Experimental Conditions** \\ \hline Instrument & Xevo TQS micro with Acquity UPLC 100 \\ \hline Column & Waters Ethylene Bridged Hybrid (BEH) C18 \\ \hline Column size & 2.1 mm ID \(\times\) 50 mm length \\ \hline Particle size & 1.7 \(\upmu\)m \\ \hline Eluent & 20 mM Heptafluorobutyric acid (HFBA) in 70:30 Water: Acetonitrile \\ \hline Flow rate & 0.5 mL/min \\ \hline Injection volume & 0.5 \(\upmu\)L \\ \hline Detection & MRM in positive mode \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Elements and experimental conditions of LC–MS. conditions [28]. Therefore, PDA should block the release of aminoglycosides in normal physiological conditions because of its negatively charged surface at alkaline pH. During bacterial colonization on the surface, the local acidification induced by bacterial metabolism should change the structural properties of PDA, due to the protonation of the amine groups, hence accelerating the release of the gentamicin from the G-PDA NPs. To investigate the pH-dependent drug release of G-PDA 1:1 and 0.6:1, the in vitro drug release experiment was carried out at three different pH values, 7.4, 5 and 3. No initial burst release of gentamicin was observed in both NPs, as shown in Figures 1 and 2. The cumulative release of the gentamicin from the G-PDA NPs 1:1 at pH 5 and 3 was approx. 40% and 55%, even after 168 h, and expected to release slowly with the passage of time by maintaining its release rate, thus exhibiting a long-term release profile. On the contrary, a very slow drug-release profile (11.4%) was achieved at pH 7.4. This finding suggests the pH-dependent behavior of PDA in terms of its zwitterionic property. Figure 1: (**A–C**) are representing non-cumulative gentamicin release at pH 7.4, 5 and 3, respectively; and (**D**) is representing cumulative percent release of gentamicin at all pH conditions from G-PDA. 1:1. Data were mean \(\pm\) SD (\(n\) = 3). Hence, the mechanism of drug release could be explained depending on the behavior of the PDA functional groups in response to pH. At an acidic pH, both the PDA NPs and gentamicin are positively charged because of the protonation of the amine groups. Thereby, the gentamicin molecules easily escape from PDA, because of the repulsive forces. Moreover, hydrogen bonding between PDA and gentamicin is also disturbed at an acidic pH, due to the interchangeable active sites of PDA resulting in gentamicin's release from the PDA NPs. Although the G-PDA NPs 1:1 was best considered in terms of the highest possible loading efficiency as discussed in previous work, the drug release behavior for another batch of drug-loaded particles with a comparatively lower quantity of the loaded drug, i.e., G-PDA NPs 0.6:1, was studied as well. This study was performed intentionally in order to determine the effect of the loading percentage on drug release profile.