br The synthesis route is shown in Scheme A
The synthesis route is shown in Scheme 2A. We used CTAC as template for preparation of MSN by hydrolysis of TEOS under basic condition. Fig. 1A shows TEM image of the MSN. The highly ordered porous structure of the MSN could be clearly seen from Fig. 1A. The MSN was then reacted with 3-mercaptopropyltrimethoxysilane to ob-tain MSN-SH. As represented in Fig. 2B, the characteristic peak of MSN-SH at 2550 cm−1 indicates presence of thiol group (−SH) on the MSN-SH. Meanwhile, stretching vibration absorbance peaks of SieO and −CH2- appear at 1100 cm−1 and 2900 cm −1, respectively, further confirming that the MSN-SH was synthesized successfully. Afterwards, MSN-SH was reacted with S-(2-aminoethylthio)-2-thiopyridine hydro-chloride and propargyl bromide step by step, to generate MSN-SS-al-kyne. The characteristic peak at 2120 cm−1 of alkyne groups shown in Fig. 2C proves the existence of alkyne groups on the surface of MSN-SS-alkyne. Subsequently, to connect KLA onto the MSN, KLA was synthe-sized manually employing a standard Fmoc chemistry through the solid phase peptide synthesis and terminated with azide groups. (The detail of synthesis of KLA-N3 is provided in the supporting information.) The structure of KLA is shown in Scheme 2B. The structure of KLA-N3 was confirmed by ESI-MS and provided as Fig. S4. By reaction of MSN-SS-alkyne and KLA-N3 via "click chemistry", redox-responsive MSN-SS-KLA was obtained. As shown in Fig. 2D, the characteristic peaks at 1650 cm−1 and 1550 cm−1 demonstrate presence of amide bonds in the MSN-SS-KLA, suggesting the successful modification of apoptotic pep-tides on the MSN.
Finally, MSN-SS-KLA was coated with BSA to improve its Lycopene in biological media . In previous studies, BSA functionalization has been reported to enhance water solubility and biocompatibility of na-noparticles . As shown in Fig. 1A and B, TEM images demonstrate that average size of the MSN is 65 nm whereas that of the [email protected]/BSA is about 80 nm. Compared with the MSN, outermost of the [email protected]/BSA became more obscure, for KLA and BSA coating. Meanwhile, size distributions of the MSN and the [email protected]/BSA were determined by DLS. DLS results show that the average sizes of MSN and [email protected]/BSA are 85.32 nm and 104.48 nm, respectively. Since BSA coating on the surface of [email protected] MSN-SS-KLA/BSA could combine much more water molecules, the hydrodynamic size of the particles become bigger than the bare MSN. Also, it needs to be noted that the particle size measured by DLS is slightly larger than that measured by TEM. Because the molecules on surface of nanoparticles are more stretched when the samples are dis-persed in water whereas TEM characterizes actual size of the nano-particles under dry state.
3.2. Redox-induced drug release
Since apoptotic peptide KLA is attached to the surface of MSN through disulfide bonds, it is expected to be released by responding to glutathione inside tumor cells. In order to understand the release ki-netics of KLA from MSN-SS-KLA under reductive conditions, KLA was labeled by FITC. In this study, we chose reductive agent DTT as an alternative to glutathione, to mimic reductive environment inside cells. It can be seen from the profiles that within a few hours at the beginning (Fig. 3), the release rate of KLA from the MSN-SS-KLA with DTT is significantly faster than that without DTT, and the drug release rate increases as the concentration of DTT increases. This is due to the cleavage of the disulfide bonds between KLA and MSN under reductive condition. After 60 h, the total amount of cumulative drug release at 0 mM DTT, 0.5 mM DTT and 5 mM DTT were 9.3%, 27.6% and 69.0%,
Scheme 1. Schematic illustration of the func-tional MSNs for highly controllable drug re-lease and synergistic anticancer therapy. (a) The DOX-loaded MSNs target cancer cells and penetrate cell membrane. (b) Endocytosis by specific tumor cell. (c) Trypsin triggered BSA degradation. (d) The highly concentrated GSH in cytoplasm induces the cleavage of disulfide bonds, leading to a burst release of DOX. (e) The released KLA and DOX synergistically en-hance therapy eﬀect.