## 3 Results and Discussion
### Drug Release  
The next step in the development of our nanosystem was to characterize the release of DOX. This was done using a diffusion bag technique, where each MSN-based- system was loaded into a dialysis bag and the release media collected at varying time points. For this experiment a positive control was employed, where pure DOX was loaded into the dialysis bag and the releasing media consisted of either the pH 7.4 or pH 5 buffer systems. The media collected was then analysed by HPLC, in which the characteristic peak of pure DOX was obtained at 4.3 min, while the peak eluted before three minutes were the solvent (buffers) peak as per previous literature [33]. The first systems to be evaluated were those synthesized by the pre-surface grafting loading method (Figure 2a). When the DOX@MSNs from the pre-grafting loading method were evaluated for release, DOX was detected in both the release mediums of pH 7.4 and 5 (Figure 4). This indicated that encapsulated DOX was liberated into the releasing media. In addition to this, the peak of DOX from DOX@MSN at pH 7.4 has a lower AUC compared to the peak of DOX from DOX@MSN at pH 5, indicating a higher release of DOX from native MSNs under acidic conditions. This is as expected as DOX is known to be more soluble at acidic pH [31; 32]. Unfortunately, when the release of the functionalized particles DOX@MSN-PLH-TAM (synthesized by pre-surface grafting method, Figure 2a) was evaluated using the same release procedure, no DOX was detected in the release media. It was hypothesized that there was insufficient amount of DOX left within the mesopores after multiple surface grafting steps and that what remained was well below the detection limit of the instrument. This is consistent with the similar FT-IR spectrum of the DOX loaded systems vs. unloaded systems, implying a low loading of DOX (Figure 3; Figures S1 and S2). To investigate the in vitro release pattern of DOX@MSN-PLH and DOX@MSN-PLH-TAM (synthesized by post-surface grafting loading method, Figure 2b), the release of DOX from native MSNs was used as a control for comparison. The release kinetic of DOX@MSNs, DOX@MSN-PLH and DOX@MSN-PLH-TAM were demonstrated in Figure 5 below. Figure 3: **(a)** Photos of MSN-PLH-TAM vs. DOX@MSN-PLH-TAM, highlighting the colour change upon DOX loading. **(b)** FT-IR spectra of two MSN-based systems. No additional signals were observed following the loading of DOX into the MSN-PLH-TAM. More DOX was released at acidic pH in all three systems; native MSNs, MSN-PLH and MSN-PLH-TAM. This reaffirmed the knowledge that DOX molecules are more soluble in acidic environments [31; 32]. For native MSNs, 8.7% more DOX was released at pH 5 than at pH 7.4. It was observed that the release of DOX@MSNs reached a plateau, with an overall release after 72 h of 33.6% for DOX at pH 7.4; and 42.3% at pH 5. However, the total amount of DOX released from the native MSNs was less than typically expected from this  
Figure 4: Chromatography of demonstrating the in vitro release kinetics of DOX@MSNs at pH 7.4 and 5. Figure 5: In vitro cumulative release profile of DOX@MSN, DOX@MSN-PLH and DOX@MSN-PLH-TAM over 72 h.  
type of material, where a release of over 80% after 24 h is reported [30; 37]. This incomplete release from native MSN may be attributed to absorption of DOX molecules inside the deep channels of our materials, requiring the need for releasing media to penetrate inside and solubilize the drug, hence, prolonging the releasing time. For functionalized MSNs, it was observed that the cumulative amount of DOX released from MSN-PLH and MSN-PLH-TAM was less than that of native MSNs. The maximal cumulated release of DOX molecules from DOX@MSN-PLH after 72 h was \(\approx\)18% at pH 7.4 and \(\approx\)23% at pH 5, respectively. Whereas approximately 5% of DOX was released from DOX@MSN-PLH-TAM at pH 7.4 and 9% released at pH 5 over 72 h (Figure 5).