Preparation of MPCT@Li-R NPs
The synthesis process of MPCT@Li-R is illustrated in Scheme 1. MSN-based drug vehicles were first synthesized and then modified with -NH2 to effectively chelate Pt ions while improving water stability, then Pt ions were reduced by NaBH4, so as to achieve the goal of in-situ growth of Pt NPs in the nanochannels. After the adsorption of photosensitizer Ce6 and MTH1 inhibitor TH588 through electrostatic action, the RGD-functionalized liposome shell was introduced to the periphery of the NPs in order to further realize the controllable release and precise targeting of Ce6 and TH588.
Characterization of MPCT@Li-R NPs
The synthesis process of the core-shell MPCT@Li-R was monitored by TEM. As shown in Figure. 1a, b, MSN was spherical, with uniform particle size (about 95nm by TEM measurement) and possessed homogeneous mesoporous structure, in which the black part is the skeleton of MSN and the bright area is the mesoporous channel. After the reduction of NaBH4, it can be observed that the bright black dot structure with an average diameter of 3.4 nm is evenly distributed in the mesoporous structure of MSN, indicating the successful grafting of Pt NPs. The morphology of MPCT@Li-R was monitored by TEM, in which the thickness of the liposome shell was about 7nm, and the particle size increased to nearly 105nm compared with MSN-Pt, indicating the successful aggregation of lipid bilayer on the surface of the NPs (Figure. 1c, d). SEM was used to further observe the uniform spherical structure of MPCT@Li-R and the corresponding element distribution (Figure. S1). The polydispersity index (PDI), as an evaluation of the inhomogeneity of size distribution, showed that the PDI of the final product in deionized water was 0.265, which proved its good dispersion stability. As shown in the Figure. S2, both the intermediate product and the final product MPCT@Li-R exhibited good colloidal stability in aqueous solution. In addition, the particle sizes of MSN and MPCT@Li-R were measured by dynamic light scattering (DLS), and the results were similar to those measured by TEM (Figure. 1e, f). Studies have shown that the cell uptake efficiency of MSN is negatively correlated with particle size, and smaller particle size means higher uptake[39]. Fang et al. reported that MSN with a particle size of around 100nm exhibited the best intracellular uptake rate and endosomal escape efficiency, which perfectly matched our results. In addition, energy dispersive spectrometer (EDS, OXFORD, Xplore 30) was used to analyze the composition of different elements in MPCT@Li-R, in which Si, O, Pt, C and other elements can be clearly observed, further indicating the successful preparation of the composite material (Figure. 1g-i).
For effectively induce the growth of Pt NPs in mesoporous of MSN, the MSN was modified with -NH2, resulting in the formation of positively charged nanochannels which further promotes the electrostatic bonding of negatively charged PtCl62- with -NH2. Zeta potential is shown in Figure. 2a: in the aqueous solution, the surface of MSN was covered by hydroxyl groups (-OH) due to the presence of SiO2 molecule and exhibited negative potential. After APTS grafting, the potential of MSN was reversed and exhibited positive potential, indicating its successful amination. Due to the positive charge of Pt NPs, the potential of MSN-Pt was further increased after the successful reduction of Pt ions in the nanochannels. Then, photosensitizer Ce6 and MTH1 inhibitor TH588 were successively loaded into the synthesized MSN-Pt through electrostatic adsorption to form MPCT NPs, which were further coated with RGD functionalized liposome shell. According to the UV-vis absorption spectra analysis, as shown in Figure. 2b, there are two obvious characteristic absorption peaks at 400nm and 660nm for Ce6. Meanwhile, MPC and MPCT@Li-R also exhibited similar characteristic absorption peaks, demonstrating the successful loading of Ce6.
Drug loading and TH588/Ce6 pH responsive release in vitro
Firstly, ultraviolet absorption curves of Ce6 and TH588 with different concentrations were measured by ultraviolet spectrophotometer (Figure. S3), and the concentration-fluorescence intensity standard curve was drawn according to the results, as shown in the Figure. 2c, d. The drug loading capacity and encapsulation rate of TH588 were 8.67% and 94.93%, and that of Ce6 were 9.08% and 99.85%, respectively.
The pH-responsive release of TH588 and Ce6 is an inevitable requirement for PDT-CHT combination therapy after the targeted arrival of NPs to tumor cells. Different pH values (pH 7.4, 5.0) were selected to simulate the pH values of normal physiological environment and tumor acidic environment. As shown in Figure. 2e, explosive drug release of MPCT NPs was observed in PBS solutions at all pH values, the release proportion of TH588 and Ce6 from MPCT was nearly 90% and 85% at 10h, respectively. In contrast, MPCT@Li-R has a completely different TH588 and Ce6 release profile. As shown in Figure. 2f, MPCT@Li-R is pH-dependent on the release of TH588 and Ce6. For TH588, only 30% was released at pH 7.4 within 30 h, in contrast to more than 75% at pH 5.0. The release behavior of Ce6 was similar to that of TH588, with only 17% release at pH 7.4 and more than 60% release at pH 5.0 within 36 hours. In general, the gatekeeper liposomes can be cleaved in acidic environments and unlock mesoporous channels, eventually leading to the release of TH588 and Ce6. This property provides a strong guarantee for the continuous drug release of NPs in acidic medium after entering tumor cells.
Evaluation of O2 and ROS generation in vitro
As shown in Figure. 2g, obvious transparent bubbles were attached to the tube wall after MPCT and MPCT@Li-R were co-incubated with H2O2 solution for 30min. The catalytic performance of Pt NPs was found to be excellent by dissolved oxygen meter, and Ce6, TH588, even liposome shell had a negligible influence on its performance. In addition, the catalase activity of the final product was further investigated. As shown in Figure. 2h, in the absence of H2O2, the content of O2 in the solution hardly changes. With the increase of H2O2 concentration, the content of dissolved O2 also increased gradually, indicating that MPCT@Li-R had excellent catalase-like activity. Time-dependent detection of H2O2 concentration showed that more than half of the H2O2 was decomposed within 30 minutes (Figure. S4). After repeated addition of H2O2 for several times, its catalytic activity was still excellent, indicating its good catalytic stability (Figure. 2i).
ROS can ablate tumors by destroying nucleic acids and proteins in tumor cells[40]. In order to investigate whether MPCT@Li-R can produce ROS under the irradiation of 660nm laser, Ce6, TH588, MPC@Li-R and MPCT@Li-R were co-incubated with HOS cells and DCFH-DA probe was used to detect ROS generation. As shown in the Figure. 3a, negligible fluorescence was observed in the control group, while relatively obvious green fluorescence was observed in the other 5 treatment groups, indicating that CHT, PDT or NPs + laser can effectively produce ROS in tumor cells. Compared with Ce6 + laser, MPC@Li-R + laser exhibited a stronger green fluorescence, which may be attributed to the catalase properties of Pt NPs or the targeting ability of RGD peptide. In all treatment groups, MPCT@Li-R + laser (660nm, 500mW cm-2, 5min) exhibited the highest green fluorescence intensity, which demonstrated the unique advantage of CHT-PDT combination therapy with dual amplifying effect in ablation of tumor cells.
Since MTH1 inhibitor TH588 can inhibit the purification of 8-oxo-dGTP, resulting in DNA damage. Therefore, immunofluorescence staining was utilized to observe the content of 8-oxo-dGTP in different treatment groups. There is no doubt that the dissipation of MTH1 protein can lead to the accumulation of 8-oxo-dGTP in DNA, as demonstrated by pink fluorescence. In addition, the single PDT or MSN-Pt based in situ oxygen-generation promoting PDT process, and the reciprocal effect of TH588 and O2-facilitated PDT could significantly increase the intracellular content of 8-oxo-dGTP, resulting in oxidative damage to DNA (Figure. 3b).
Cancer cellular uptake of NPs
To evaluate the uptake capacity of HOS cells to MPCT@Li-R, different formulations (free Ce6, MPCT@Li, MPCT@Li-R) were co-incubated with HOS cells for 12h and fluorescein imaging was performed using a fluorescence microscope. As shown in Figure. 3c, after being co-incubated with free Ce6, although the blue fluorescence of the nucleus was clearly visible, only a small amount of red fluorescence was observed in the cytoplasm, which may be due to the poor solubility of Ce6 and its inability to enter HOS cells effectively. In contrast, there was a strong red fluorescence signal in the cytoplasm of MPCT@Li-R group, indicating that RGD peptides enhanced the endocytosis of NPs by interacting with integrin receptors.
Antitumor efficacy of NPs in vitro
Excellent biocompatibility is the prerequisite for composite to play the therapeutic role. Hemoglobin can be released from the broken red blood cells, generating the red supernatant and resulting in enhanced absorbance at 570nm. In the hemolysis experiment, composites with a series of gradient concentration were co-incubated with red blood cells in PBS solution, as shown in Figure. 4a, the hemolytic activity of MPCT@Li-R was less than 5% even at concentrations up to 400 μg/ml.
To evaluate the cytotoxicity of NPs, CCK8 assay was utilized to analyze the viability of HOS cells. As shown in Figure. S5, the cellular viability of HOS cells remained above 90% even after 48h co-incubation with 200 μg/ml MP@Li-R, which proves their excellent biocompatibility. Subsequently, we further explored the cytotoxicity of different treatment methods. As shown in Figure. 4b, the TH588 group and the Ce6 + laser group showed relatively low cell viability compared with the control group, indicating the killing effect of CHT or PDT on tumor cells. The cellular viability of MPC@Li-R + laser group was significantly lower than that of PDT group, which may be attribute to the Pt-related O2-enhanced PDT effect or RGD targeting effect. Undoubtedly, the cellular viability of MPCT@Li-R + laser with dual amplification effect is the lowest, indicating that it has the best therapeutic effect. Based on the above findings, in order to explore whether the inhibition of the proliferation of tumor cells by MPCT@Li-R + laser is caused by inducing their apoptosis, we conducted a live (green)/dead (red) staining analysis on the cells. As shown in Figure. 4c, compared with the TH588 group and the Ce6 + laser group, the MPCT@Li-R + laser group exhibited stronger red fluorescence signal, which confirmed its unique advantages in killing tumor cells as a multifunctional nanotherapy platform.
Next, Western blot was used to assess the expression of some key marker proteins during combination therapy. As shown in Figure. 4d, e, MTH1 expression can be inhibited by TH588 or MPCT@Li-R. Moreover, MTH1 inhibitor can trigger p53-mediated apoptosis of cancer cells by inducing DNA damage. The expression of p53 protein was upregulated in all treatment groups, especially in the MPCT@Li-R group (Figure. S6). Bcl-2 can prevent the release of cytochrome c from mitochondria and has an anti-apoptotic effect, while BAX can interact with voltage-dependent ion channels on mitochondria to mediate the release of cytochrome c and have an apoptotic effect. As can be seen from Figure. S6, BAX and Bcl-2 exhibited an opposite trend. In the MPCT@Li-R + laser treatment group, the expression level of BAX was the highest, while the Bcl-2 was the lowest, which confirmed the efficacy of CHT-PDT combination therapy in mediating mitochondrial injury to kill tumor cells.
Fluorescence imaging and biodistribution of NPs in mouse
RGD peptide confers tumor targeting ability to MPCT@Li-R NPs through integrin receptor mediated endocytosis[41]. To further explore the appropriate irradiation time after NPs treatment, HOS tumor-bearing mice models were further utilized to investigate the tumor accumulation and biological distribution of MPCT@Li-R. Fluorescence images were collected at different time points (1h, 3h, 6h, 12h, 24h) after tail vein injection. As shown in Figure. 5a, a strong fluorescence signal appeared in the liver region 1h after injection. With the EPR effect and the active targeting effect of RGD, fluorescence signals began to appear in the tumor region 3 h post injection, and reached the peak intensity 6 h post injection. It is noteworthy that we can still detect residual fluorescent signals in the tumor area 24h after injection. Moreover, mice were sacrificed 24h post injection, tumor tissues and organs were harvested. Quantitative analysis of fluorescence signals in tumors and major organs showed that the fluorescence intensity in tumor tissues was significantly higher than that in other major organs, confirming the excellent targeting ability, high uptake and retention ability of MPCT@Li-R NPs (Figure. 5b, c).
Antitumor efficacy of NPs in vivo
Encouraged by the excellent targeting, in situ O2 generation facilitated PDT effect and CHT effect of MPCT@Li-R, antitumor efficacy was further observed in HOS tumor xenograft mouse model. Tumor-bearing mice were randomly divided into five groups (n=3 each group) and received different prescriptions after tumor volume reached about 80-100mm3. Based on the results of fluorescence imaging and biological distribution in vivo, 6h after intravenous injection was determined as the optimal time window for laser irradiation (660nm, 1 W cm-2, 5 min). As shown in Figure. 5d, Tumors in the control group showed a faster growth trend and no inhibition trend was observed. TH588 and Ce6 + laser group had certain inhibitory effect on tumor growth. However, in the Ce6 + laser group, tumor growth volume began to accelerate after day 8, demonstrating that hypoxia within the larger solid tumors may impair the efficacy of PDT. Encouragingly, ROS generation based on Pt NPs catalytic properties can effectively inhibit tumor growth, while the addition of MTH1 inhibitor can bring out the best effect of this dual-amplification therapy model. Figure. 5e shows the general image of mice after 14 days of treatment, in which the change of tumor volume is consistent with the trend of Figure. 5d. Moreover, the tumor tissue images and weight further confirmed the changing trend of tumor volume (Figure. 5f, h).
Biosafety assessment
To assess the biosafety of MPCT@Li-R, body weight changes of mice were recorded every other day during each treatment period. Compared with the PBS treatment group, the other five treatment groups showed no significant weight fluctuations (Figure. 5g). Finally, tumor tissues and major organs of the mice were collected. No obvious inflammatory lesions or histological abnormalities were observed in the organs, which fully confirmed the excellent biosafety of MPCT@Li-R (Figure. S7). In contrast, TUNEL, Ki67 analysis and H&E staining showed that MPCT@Li-R + laser had a definite anti-tumor effect compared to the other five treatment groups. (Figure. 5i)