br Fig Release profiles of DOX from DOX
Fig. 4. Release profiles of DOX from [email protected]/BSA under diﬀerent conditions.
investigate the influence of reductive agent on DOX release, the Hela Lovastatin were treated with 1 mL DMEM containing 1 mM DTT for 4 h. Here, DTT was chosen as an analog to imitate the concentrated glutathione in the intracellular matrix of tumor cells . As shown in Fig. 6, with the addition of DTT, the red fluorescence inside the cells is much stronger than that without addition of DTT, indicating that more DOX can be released intracellularly by stimuli of reductive agent with higher con-centration. The results are consistent with those shown in Fig. 4 and Colloids and Surfaces B: Biointerfaces 175 (2019) 65–72
imply that the newly fabricated [email protected]/BSA in this study shows great potential as a highly controllable DDS for cancer therapy.
3.5. In vitro cytotoxicity
Furthermore, to verify the inhibition eﬀect of [email protected]/ BSA on tumor cells, in vitro cytotoxicity experiments were done by MTT assay on HeLa cells. As displayed in Fig. 7A, MSN/BSA showed little cytotoxicity to Hela cells, suggesting that MSN/BSA is biocompatible. When the concentration of MSN/BSA is 40 μg/mL, the cell viability is about 74%. In contrast, the cell viability after co-incubation with MSN-SS-KLA/BSA (40 μg/mL) was about 37%. Once the MSN-SS-KLA/BSA enters the cells, KLA can be released via the cleavage of disulfide bonds. Then the apoptotic peptide KLA would selectively distort the matrix of the mitochondrial phospholipid physically and prompt the mitochon-dria-dependent apoptosis [40,46–48]. That’s why the MSN-SS-KLA/BSA exhibits obvious cytotoxicity compared with MSN/BSA. For [email protected] MSN-SS-KLA/BSA (40 μg/mL), the cell viability decreases dramatically to ∼8%, due to the dual responsive release of DOX and KLA as men-tioned above. Moreover, [email protected]/BSA shows more cyto-toxicity to Hela cells than Free DOX at the same DOX concentration. These results strongly indicate that co-delivery of DOX and KLA can achieve a synergetic eﬀect.
Then, we incubated the cells with DTT ahead of co-incubation of cells with the samples to verify the influence of reductive agent con-centration on the viabilities of the cells. As shown in Fig. 7B, [email protected] MSN-SS-KLA/BSA cultured with DTT exhibits more obvious inhibition eﬀect on Hela cells compared with that without DTT at the same con-centration. According to Figs. 4 and 6, more DOX is released with the increase of DTT concentration, thus the viabilities of Hela decrease with the addition of DTT.
Due to the poor hydrophilicity of [email protected], we used BSA to improve the stability of the system [41,42]. When designing a drug nanocarrier for in vivo application, surface adsorption of blood com-ponents should be considered. So we investigated the stability of [email protected] MSN-SS-KLA/BSA in aqueous solution and 100% serum, respectively. As shown in Fig. 8A, within 24 h, [email protected]/BSA shows no significant change of particle size in aqueous solution. Also, the particle size of [email protected]/BSA did not change obviously in 100% serum within 24 h according to Fig. 8B. On the contrary, as shown in Fig. 8C and D, after 24 h, [email protected] without BSA shows sig-nificant change of particle size in aqueous solution. Also, the particle size of [email protected] changed obviously in 100% serum within 3 h according to Fig. 8D. The results indicate that the hydrophilic BSA
Fig. 6. Fluorescent inverted microscopy micrographs of Hela cells co-cultured with [email protected]/BSA for 4 h.
Fig. 7. (A) Viability of HeLa cells incubated with MSN/BSA, MSN-SS-KLA/BSA, [email protected]/BSA, free DOX for 48 h; (B) Viability of HeLa cells incubated with [email protected]/BSA, [email protected]/BSA with DTT for 48 h.