Cu2+-Chelating Mesoporous Silica Nanoparticles for Synergistic Chemotherapy/Chemodynamic Therapy

In this study, a pH-responsive controlled-release mesoporous silica nanoparticle (MSN) formulation was developed. The MSNs were functionalized with a histidine (His)-tagged targeting peptide (B3int) through an amide bond, and loaded with an anticancer drug (cisplatin (CP)) and a lysosomal destabilization mediator (chloroquine (CQ)). Cu2+ was then used to seal the pores of the MSNs via chelation with the His-tag. The resultant nanoparticles showed pH-responsive drug release, and could effectively target tumor cells via the targeting effect of B3int. The presence of CP and Cu2+ permits reactive oxygen species to be generated inside cells; thus, the chemotherapeutic effect of CP is augmented by chemodynamic therapy. In vitro and in vivo experiments showed that the nanoparticles are able to effectively kill tumor cells. An in vivo cancer model revealed that the nanoparticles increase apoptosis in tumor cells, and thereby diminish the tumor volume. No off-target toxicity was noted. It thus appears that the functionalized MSNs developed in this work have great potential for targeted, synergistic anticancer therapies.


Preparation of MSN and MSN−NH 2
CTAB (2 g) and TEA (150 µL) were sequentially dispersed in H 2 O (20 mL) and stirred vigorously at 95 • C. After 1 h, TEOS (1.5 mL) was added to the solution dropwise, and the Pharmaceutics 2022, 14, 1200 4 of 18 mixture was stirred for another 1 h. After recovery by centrifugation, the solid product was washed with ethanol several times and dried under vacuum at 60 • C. MSNs were obtained via calcination in a muffle furnace at 550 • C for 3 h.
The prepared MSNs (50 mg) were next dispersed in ethanol (50 mL). APTES (3 mL) was added, and the mixture was stirred at room temperature for 24 h. After recovery by centrifugation, the solid product was washed with ethanol several times and dried under vacuum to obtain MSN−NH 2 NPs.

Preparation of MSN−HtB
HtB peptide (20 mg) was dissolved in PBS (pH = 7.4, 10 mL) containing EDC (60 mg) and NHS (40 mg), and stirred for 2 h to activate the peptide. Then, the MSN−NH 2 NPs (25 mg) were dispersed in the HtB solution, and the mixture was stirred at room temperature for 24 h. The resultant suspension was dialyzed with deionized water for 48 h (MWCO: 3500 Da) and then lyophilized.

Preparation of CP/CQ@MSN−HtB/Cu 2+
CQ (8 mg) and CP (8 mg) were dissolved in an aqueous saline solution (0.9%, w/v, 10 mL), and then added to a dispersion of MSN−HtB NPs (1 mg·mL −1 ) in PBS (10 mL). After stirring for 12 h at room temperature, CuCl 2 (0.5 g) was added, and the reaction mixture was stirred for a further 24 h. After this, the solid product was dialyzed with deionized water for 48 h (MWCO: 3500 Da) and then lyophilized, resulting in CP/CQ@MSN−HtB/Cu 2+ NPs. Control nanoparticles were prepared, in which drug-free nanoparticles were sealed with Cu 2+ ; these were termed MSN−HtB/Cu 2+ NPs, and were generated by directly chelating Cu 2+ to the MSN−HtB NPs without first drug-loading them. CP@MSN-HtB/Cu 2+ NPs were prepared as above but with the omission of CQ from the reaction mixture.

Materials' Characterization
The morphologies of the nanoparticles were studied by transmission electron microscopy (TEM) on a JEM-2100UHR instrument (JEOL, Tokyo, Japan). Particle size and zeta potential data were obtained on a Zetasizer Nano ZS90 instrument (Malvern Instruments, Malvern, UK). Infrared absorption spectra were collected on a Nicolet Nexus 870 spectrometer (Thermo Fisher, Waltham, MA, USA). UV-Vis absorption spectra were collected from 200 to 1000 nm using a UV 3600 spectrophotometer (Shimadzu, Kyoto, Japan) at ambient temperature. Thermogravimetric analysis (TGA) was performed on a TGA-101 analyzer (JB, Shanghai, China). Pore size and specific surface area were quantified on a TriStar 3000 automatic nitrogen adsorption specific surface area analyzer (Micromeritics Instruments Corporation, Atlanta, GA, USA). X-ray photoelectron spectroscopy (XPS) was used to probe the surface chemical composition, with experiments carried out using an ESCALAB 250Xi X-ray photoelectron spectrometer (Thermo Fisher, Waltham, MA, USA). Elemental composition was determined on a 710-ES inductively coupled plasma-optical emission spectrometer (ICP-OES; Varian, Salt Lake City, UT, USA). For the latter, the samples were digested in HNO 3 and then diluted into the calibration range with deionized water.
To determine the percentage encapsulation efficiency (EE) and drug loading (DL) of CP and CQ in CP/CQ@MSN−HtB at the end of the CP/CQ loading process, the NP dispersion in PBS was centrifuged to precipitate any solid material, and the amount of unencapsulated drug in the supernatant was determined by UV-Vis spectroscopy. The CP and CQ concentrations were quantified with reference to a predetermined calibration curve. In the case of CP, the supernatant was first mixed with an equal volume of o-phenylenediamine (OPD), and heated at 95 • C for 10 min prior to UV-Vis measurements [47]. The EE and DL were calculated as follows [48][49][50]:

In Vitro Release Experiments
In vitro release of CP or CQ from the CP/CQ@MSN−HtB/Cu 2+ NPs was studied by dispersing CP/CQ@MSN−HtB/Cu 2+ NPs (5 mg) in 2 mL of two different buffer solutions (PBS at pH 7.4 or 5.0) placed in a dialysis bag (MWCO: 3500 Da). This was then dialyzed against PBS (20 mL) at the appropriate pH at 37 • C, under stirring. At specific time intervals, the dialysate (2 mL) was removed and replaced with the same amount of preheated PBS. The amount of released CQ was determined by UV-Vis spectroscopy at λ = 343 nm. CP quantification was performed by mixing the release aliquot with OPD (1.2 mg·mL −1 , 2 mL) in a 9% w/v aqueous NaCl solution at 95 • C. This mixture was heated for 10 min to form a complex, and analyzed with a UV-Vis spectrophotometer at λ = 705 nm.

Evaluation of Extracellular ·OH Production Capacity
Methylene blue (MB) is a dye that can be degraded by ·OH, and can thus be used as an indicator for the generation of ·OH [52]. Experiments were set up at 37 • C under stirring, as follows:

Cell Culture
B16 cells were cultured in RPMI 1640 medium containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin solution. L-929 cells were cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin solution. All of the cells were cultured in an atmosphere of 5% CO 2 at 37 • C. Negative controls comprised cells treated with an equal volume of PBS (pH 7.4). Three independent experiments were performed, with five replicate wells per experiment.

Cytotoxicity Studies
The cytotoxicity of the nanoparticles to tumor cells was analyzed by the MTT assay. B16 cells were seeded in 96-well plates (1 × 10 5 cells·mL −1 , 100 µL) and incubated for 24 h at 37 • C before the media was aspirated. Subsequently, the cells were incubated with different concentrations of CP, CQ, and CP/CQ@MSN−HtB/Cu 2+ NPs, and cultured in complete 1640 medium for another 12 h. Next, the MTT reagent (5 mg·mL −1 , 100 µL) was added to each well, and the plate was incubated for 4 h. The media was aspirated, and DMSO (150 µL) added to each well of the plate, which was then incubated for another 20 min to dissolve the formazan crystals. Finally, the absorbance at 570 nm was measured by a microplate reader (Thermo Scientific Multiskan FC, Waltham, MA, USA).
The cytotoxicity of the nanoparticles to L-929 cells was also tested by MTT assay. The experimental method was the same as above, except that the cells were treated with different concentrations of MSN−HtB NPs and CP/CQ@MSN−HtB/Cu 2+ NPs.
Calcein AM/PI double-labeling was employed to visualize the effects of the NPs on cells. B16 cells were seeded in 6-well plates (1 × 10 5 cells·mL −1 , 2 mL) and in-Pharmaceutics 2022, 14, 1200 6 of 18 cubated for 24 h at 37 • C. The medium was then removed and replaced with fresh medium containing the test formulations. The cells were divided into 6 groups-(1) control; (2) free CP; (3) free CQ; (4) MSN−HtB NPs; (5) CP@MSN−HtB/Cu 2+ NPs; and (6) CP/CQ@MSN−HtB/Cu 2+ NPs-and incubated at an equivalent concentration of Cu 2+ (10 µg·mL −1 ) or CP (30 µg·mL −1 ) for another 12 h. The cells were then washed with PBS three times and cultured in RPMI for a further 24 h. The cells were subsequently stained with a mixture of calcein-AM/PI solution for 10 min. Living cells (green) and apoptotic cells (red) were observed with an inverted fluorescent microscope (Nikon TE-2000U, Tokyo, Japan).
The annexin V-FITC and PI double-staining method was used to quantify cell death. B16 cells were seeded in 6-well plates (1 × 10 5 cells·mL −1 , 2 mL) and incubated for 24 h at 37 • C. The medium was removed and replaced with fresh medium containing the test formulations. The cells were divided into 6 groups-(1) control; (2) free CP; (3) free CQ; (4) MSN−HtB NPs; (5) CP@MSN−HtB/Cu 2+ NPs; and (6) CP/CQ@MSN−HtB/Cu 2+ NPs-and incubated for 12 h. Next, the cells were trypsinized (0.25% trypsin without EDTA), collected, and rinsed three times with PBS (pH 7.4). They were re-suspended with binding buffer (500 µL) at a concentration of 1 × 10 6 cells·mL −1 . An annexin V-FITC/PI cell apoptosis analysis kit was used to stain the cells according to the manufacturer's protocol, and the samples were quantified by flow cytometry (BD FACS Calibur, Franklin Lakes, NJ, USA).

Cellular Uptake
In order to study the cellular uptake of NPs, fluorescent DOX was loaded in the MSNs instead of CP and CQ, following the same protocol as detailed above. To check the influence of the His-tag on uptake, control nanoparticles in which B3int was conjugated to the NP surface were also prepared. These were produced by replacing HtB with B3int in the MSN synthesis procedure, and then loading with DOX. The cellular uptake of DOX@MSN−HtB/Cu 2+ NPs was evaluated by confocal laser scanning microscopy (CLSM, Nikon C1-si, Tokyo, Japan). B16 and L-929 cells were separately plated on glass-bottomed culture dishes (1 × 10 5 cells·mL −1 , 2 mL) for 24 h at 37 • C. The media was aspirated, and then replaced with fresh media containing the test formulations. The cells were divided into 4 groups-(1) control; (2) free DOX (0.5 µg·mL −1 ); (3) DOX@MSN−B3int NPs (DOX: 0.5 µg·mL −1 ); and (4) DOX@MSN−HtB/Cu 2+ NPs (DOX: 0.5 µg·mL −1 )-and incubated in complete medium for another 4 h. After removal of the medium and washing with PBS, the cells were stained with a lysosome staining kit for 45 min, fixed with aqueous formaldehyde (4%, v/v) at room temperature, stained with DAPI for 3 min, and then imaged by CLSM.

Intracellular ROS Generation
Intracellular total ROS generation in B16 cells was detected using a DCFH-DA ROS assay kit. Cells were seeded in 6-well plates (1 × 10 5 cells·mL −1 , 2 mL) and incubated for 24 h at 37 • C. After removing the medium and replenishing it with fresh medium containing the test formulations, five groups of experiments were performed as follows: (1) control; (2) CuCl 2 ; (3) MSN−HtB-Cu 2+ NPs; (4) CP/CQ@MSN−HtB NPs; and (5) CP/CQ@MSN−HtB/ Cu 2+ NPs. The formulations were added to give equivalent concentrations of Cu 2+ (10 µg·mL −1 ) or CP (30 µg·mL −1 ), and the cells were incubated for another 12 h. Next, the culture medium was replaced with fresh RPMI containing DCFH-DA (10 µM) for another 30 min of incubation. Afterwards, the cells were washed three times with PBS. Finally, the intensity of green fluorescence was observed using an inverted fluorescence microscope.

In Vivo Biodistribution
All animal experiments were undertaken following review and approval by the Experimental Animal Committee of Donghua University (SYXK(SH)2020-0018). C57BL/6 black mice (specific pathogen-free grade, 18-20 g) were acquired from Shanghai Jiesijie Experimental Animal Co., Ltd. (Shanghai, China). Tumors were implanted by subcutaneous Pharmaceutics 2022, 14, 1200 7 of 18 injection of 2 × 10 6 B16 cells in 100 µL of PBS (pH 7.2-7.4) into the right flank area of each mouse. The mice were randomized into two groups (5 mice each) when the tumor volume (determined as length × width 2 /2) reached ca. 100 mm 3 .
ICP-OES was used to determine the content of Pt and Cu, and to investigate in vivo biodistribution. CP/CQ@MSN−HtB/Cu 2+ NPs or free CP in PBS solution (CP concentration: 1 mg·mL −1 , 200 µL) were injected into the tail vein. The mice were euthanatized after 24 h, and the tumors and major organs (i.e., heart, liver, spleen, lungs, and kidneys) were collected and weighed before being digested with aqua regia. After diluting the digestion solution 10 times with water, the contents of Pt and Cu were determined by ICP-OES.

In Vivo Antitumor Efficacy and Biosafety
Tumor-bearing mice with tumor volumes of ca. 100 mm 3 were randomly divided into six groups (n = 5), and injected intravenously through the tail vein with the formulations under test (200 µL). The following treatments were investigated: (1) PBS, (2) free CP, (3) free CQ, (4) MSN−HtB/Cu 2+ NPs, (5) CP/CQ@MSN−HtB NPs, and (6) CP/CQ@MSN−HtB/Cu 2+ NPs. The amount of CP in each treatment was set at 300 µg·kg −1 . During the experiment, the tumor volume and body weight were measured every three days.

Histological Analysis
After 12 days, mice were euthanatized by cervical dislocation. The tumor tissues and major organs (i.e., the heart, lungs, spleen, liver, and kidneys) were excised from the mice and fixed in 4% aqueous formaldehyde. They were then embedded in paraffin and sliced into 5 µm sections. These sections were stained with hematoxylin and eosin (H&E) and studied under an optical microscope. In addition, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining was used to analyze apoptosis.

Kaplan-Meier Analysis
Additional tumor-bearing mice with tumor volumes of 100 mm 3 were employed for Kaplan-Meier analysis. These mice were divided into the abovementioned 6 groups (n = 10), and injected intravenously through the tail vein with the formulations under test (200 µL). The survival status was recorded every day for 20 days.

Statistical Analysis
Statistical analyses were performed using either Student's t-test or one-way ANOVA analysis, followed by a Newman-Keuls test; p-values < 0.05 were set as statistically significant findings, and the data were marked with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001.

Synthesis and Characterization of CP/CQ@MSN−HtB/Cu 2+ NPs
The MSN morphology was first investigated by TEM (Figure 1a). The nanoparticles were spherical, and about 160 nm in size. The mesopores were dense and distributed evenly throughout the nanoparticles. N 2 adsorption-desorption isotherms confirmed the mesoporous structure of the MSNs, and showed that the pore size was ca. 2.6 nm (Supplementary Materials, Figure S1). mesoporous structure of the MSNs, and showed that the pore size was ca. 2.6 nm (Supplementary Materials, Figure S1). Next, the surface of the nanoparticles was modified. The MSNs were first functionalized via co-condensation to obtain MSN−NH2 [53]. Amidation between MSN−NH2 and the carboxyl group in HtB peptides then allowed the generation of MSN−HtB. The FTIR spectra of MSN−NH2 and MSN−HtB (Figure 1c) showed the bending vibration peak of N-H bonds at 1574 cm −1 and the stretching vibration peak of C=O bonds at 1655 cm −1 , respectively, confirming the successful incorporation of amino groups and HtB peptides onto the surface. Changes in zeta potential also validated the functionalization ( Figure S2). The zeta potential of the initial MSNs was −28.4 ± 1.5 mV, but this increased to +39.4 ± 1.0 mV after modification with APTES, and changed again to 24.1 ± 1.1 mV after connecting the HtB peptide.
To determine the HtB peptide content on the MSN surface, thermogravimetric analysis (TGA) was performed to analyze the weight loss (Figure 1d). The decrease in mass below 100 °C arose from the evaporation of H2O. The mass of MSN−NH2 and MSN−HtB was reduced by 7.2% and 16.5%, respectively, compared with MSN between 100 °C and 600 °C, indicating that the content of grafted HtB peptide was 9.3% w/w. CP and CQ were next loaded into the MSN−HtB NPs, and Cu 2+ was chelated onto the surface to seal the pores. UV spectra are shown in Figure S3. Comparing the spectra of CP, CQ, and CP/CQ@MSN−HtB, we can see that MSN−HtB was successfully loaded with CQ, as the characteristic peaks of CQ (e.g., at 343 nm) can be seen. The CP peaks are hard to distinguish from those of CQ. After Cu capping, the CQ peaks are less distinct, likely owing to absorption by the Cu-HtB complex. This indicates that the drugs were successfully sealed in the pores. After drug loading and sealing, the pores of the NPs remained intact (Figure 1b), and the particle size increased from 160 to 200 nm (Figure 1e). Figure 1f depicts XPS data. The peaks of Cu, Pt, and Cl are all visible in the XPS spectrum, confirming the presence of these elements in the NPs. The presence of Pt in the formulation confirms CP loading. ICP analysis revealed that the content of Cu was 10.5 ± 0.8 mg·g −1 and the content of Pt was 2.2 ± 0.4 mg·g −1 (Figure 1f). UV analysis shows that the DL of CP is Next, the surface of the nanoparticles was modified. The MSNs were first functionalized via co-condensation to obtain MSN−NH 2 [53]. Amidation between MSN−NH 2 and the carboxyl group in HtB peptides then allowed the generation of MSN−HtB. The FTIR spectra of MSN−NH 2 and MSN−HtB (Figure 1c) showed the bending vibration peak of N-H bonds at 1574 cm −1 and the stretching vibration peak of C=O bonds at 1655 cm −1 , respectively, confirming the successful incorporation of amino groups and HtB peptides onto the surface. Changes in zeta potential also validated the functionalization ( Figure S2). The zeta potential of the initial MSNs was −28.4 ± 1.5 mV, but this increased to +39.4 ± 1.0 mV after modification with APTES, and changed again to 24.1 ± 1.1 mV after connecting the HtB peptide.
To determine the HtB peptide content on the MSN surface, thermogravimetric analysis (TGA) was performed to analyze the weight loss (Figure 1d). The decrease in mass below 100 • C arose from the evaporation of H 2 O. The mass of MSN−NH 2 and MSN−HtB was reduced by 7.2% and 16.5%, respectively, compared with MSN between 100 • C and 600 • C, indicating that the content of grafted HtB peptide was 9.3% w/w. CP and CQ were next loaded into the MSN−HtB NPs, and Cu 2+ was chelated onto the surface to seal the pores. UV spectra are shown in Figure S3. Comparing the spectra of CP, CQ, and CP/CQ@MSN−HtB, we can see that MSN−HtB was successfully loaded with CQ, as the characteristic peaks of CQ (e.g., at 343 nm) can be seen. The CP peaks are hard to distinguish from those of CQ. After Cu capping, the CQ peaks are less distinct, likely owing to absorption by the Cu-HtB complex. This indicates that the drugs were successfully sealed in the pores. After drug loading and sealing, the pores of the NPs remained intact (Figure 1b), and the particle size increased from 160 to 200 nm (Figure 1e). Figure 1f depicts XPS data. The peaks of Cu, Pt, and Cl are all visible in the XPS spectrum, confirming the presence of these elements in the NPs. The presence of Pt in the formulation confirms CP loading. ICP analysis revealed that the content of Cu was 10.5 ± 0.8 mg·g −1 and the content of Pt was 2.2 ± 0.4 mg·g −1 (Figure 1f). UV analysis shows that the DL of CP is 381 ± 13 µg·mg −1 and the EE is 84.1 ± 5.8 %. The DL of CQ is 178.5 ± 9.4 µg·mg −1 , and the EE is 75.9 ± 7.2 %. The chelation of Cu 2+ with the His-tag was probed using a PV assay. After adding Cu 2+ to HtB peptides and MSN−HtB NPs, insoluble flocculent precipitates were generated ( Figure S4a). In the corresponding UV spectra, the redshift of the phenolic absorbance peak in the mixture from 430 nm to 655 nm indicates the production of oligomers ( Figure S4b). This clearly evidences successful chelation of Cu 2+ by the HtB on the NPs.

In Vitro Drug Release
The complex of His-tag and Cu 2+ chelates present in alkaline conditions (pH = 7-8) collapses in acidic environments (pH = 4-6) [54]. Drug release data are presented in Figure 2a,b. Approximately 20-30% of the CP and CQ loading was released at pH 7.4. CP and CQ were released to a greater extent at pH 5.0, indicating that pore sealing by Cu 2+ is reversed in an acidic environment. Thus, the formulations have pH-responsive drug release, and are likely to free more of their cargo in the TME than under normal physiological conditions. Pharmaceutics 2022, 14, x FOR PEER REVIEW 9 of 17 381 ± 13 μg·mg −1 and the EE is 84.1 ± 5.8 %. The DL of CQ is 178.5 ± 9.4 μg·mg −1 , and the EE is 75.9 ± 7.2 %. The chelation of Cu 2+ with the His-tag was probed using a PV assay. After adding Cu 2+ to HtB peptides and MSN−HtB NPs, insoluble flocculent precipitates were generated ( Figure S4a). In the corresponding UV spectra, the redshift of the phenolic absorbance peak in the mixture from 430 nm to 655 nm indicates the production of oligomers ( Figure  S4b). This clearly evidences successful chelation of Cu 2+ by the HtB on the NPs.

In Vitro Drug Release
The complex of His-tag and Cu 2+ chelates present in alkaline conditions (pH = 7-8) collapses in acidic environments (pH = 4-6) [54]. Drug release data are presented in Figure  2a,b. Approximately 20-30% of the CP and CQ loading was released at pH 7.4. CP and CQ were released to a greater extent at pH 5.0, indicating that pore sealing by Cu 2+ is reversed in an acidic environment. Thus, the formulations have pH-responsive drug release, and are likely to free more of their cargo in the TME than under normal physiological conditions.

Evaluation of Extracellular ·OH Production
Methylene blue (MB) was used as an indicator to explore the effect of CP/CQ@MSN−HtB/Cu 2+ on the production of ·OH in vitro. ·OH has the ability to destroy the conjugated structure of organic dyes and, thus, cause color fading. Therefore, the production of ·OH can be detected by measuring the change in the absorbance of MB [55,56].

Evaluation of Extracellular ·OH Production
Methylene blue (MB) was used as an indicator to explore the effect of CP/CQ@MSN−HtB/ Cu 2+ on the production of ·OH in vitro. ·OH has the ability to destroy the conjugated structure of organic dyes and, thus, cause color fading. Therefore, the production of ·OH can be detected by measuring the change in the absorbance of MB [55,56]. After adding H 2 O 2 for 24 h, the MB solution did not fade, indicating that MB was stable at this concentration of H 2 O 2 [57]. As demonstrated in Figure 2c, at pH = 5, the color of an MB solution containing H 2 O 2 almost completely faded after adding CP/CQ@MSN−HtB/Cu 2+ NPs. This was because Cu 2+ released in the acidic environment can react with H 2 O 2 to produce ·OH. In contrast, at pH 7.4, the MB solution faded to a notably lesser degree. This may be due to much of the Cu 2+ remaining chelated, with a reduced amount being released into solution. The UV spectra confirm the visual observations (Figure 2d). The MB absorption peak at 665 nm disappeared at pH 5.0 in the presence of the NPs and H 2 O 2 , indicating that in an acidic environment the nanoparticles can effectively release Cu 2+ . This effect was reduced at pH 7.4.

Intracellular Uptake
CP and CQ cannot easily be seen by fluorescent microscopy. Hence, the nanoparticles were loaded with the fluorescent drug DOX to detect cellular uptake. The ability of DOX@MSN−HtB NPs to target cancer cells was evaluated in B16 and L-929 cells by CLSM. The resultant images (Figure 3a,b) revealed that both cell lines showed a certain amount of free DOX uptake (as evidenced by red intracellular fluorescence). In addition, it was observed that the intracellular DOX fluorescence of cells exposed to DOX@MSN−HtB NPs was stronger than that of those receiving free DOX, and was similar to that of cells given DOX@MSN−B3int NPs. The results show that DOX@MSN−HtB NPs can target B16 cells, and that the targeting ability of the B3int peptide is not affected by His-tag. The red DOX overlaps with the green fluorescence of the lysosome, suggesting that the NPs could be located within the lysosomes. In contrast, there was almost no fluorescence with L-929 cells treated with DOX@MSN−B3int and DOX@MSN−HtB (Figure 3b). This is because there is no overexpressed integrin β3 on the surface of L-929 cells and, thus, no active targeting.
After adding H2O2 for 24 h, the MB solution did not fade, indicating that MB was stable at this concentration of H2O2 [57]. As demonstrated in Figure 2c, at pH = 5, the color of an MB solution containing H2O2 almost completely faded after adding CP/CQ@MSN−HtB/Cu 2+ NPs. This was because Cu 2+ released in the acidic environment can react with H2O2 to produce ·OH. In contrast, at pH 7.4, the MB solution faded to a notably lesser degree. This may be due to much of the Cu 2+ remaining chelated, with a reduced amount being released into solution. The UV spectra confirm the visual observations (Figure 2d). The MB absorption peak at 665 nm disappeared at pH 5.0 in the presence of the NPs and H2O2, indicating that in an acidic environment the nanoparticles can effectively release Cu 2+ . This effect was reduced at pH 7.4.

Intracellular Uptake
CP and CQ cannot easily be seen by fluorescent microscopy. Hence, the nanoparticles were loaded with the fluorescent drug DOX to detect cellular uptake. The ability of DOX@MSN−HtB NPs to target cancer cells was evaluated in B16 and L-929 cells by CLSM. The resultant images (Figure 3a,b) revealed that both cell lines showed a certain amount of free DOX uptake (as evidenced by red intracellular fluorescence). In addition, it was observed that the intracellular DOX fluorescence of cells exposed to DOX@MSN−HtB NPs was stronger than that of those receiving free DOX, and was similar to that of cells given DOX@MSN−B3int NPs. The results show that DOX@MSN−HtB NPs can target B16 cells, and that the targeting ability of the B3int peptide is not affected by His-tag. The red DOX overlaps with the green fluorescence of the lysosome, suggesting that the NPs could be located within the lysosomes. In contrast, there was almost no fluorescence with L-929 cells treated with DOX@MSN−B3int and DOX@MSN−HtB (Figure 3b). This is because there is no overexpressed integrin β3 on the surface of L-929 cells and, thus, no active targeting.

Intracellular ·OH Detection
Since the DCFH-DA probe can be oxidized to DCF-a green fluorescent molecule-under the action of ROS, it can be used to detect ROS produced in the cell. Figure 3c reveals that in the control group and CP/CQ@MSN−HtB group there was almost no fluorescence in the cells, due to the absence of Cu 2+ . Cells treated with CuCl 2 or MSN−HtB/Cu 2+ produced relatively weak fluorescence, due to some ROS being generated by the Cu 2+ present. The CP/CQ@MSN−HtB/Cu 2+ group gave markedly enhanced fluorescence, indicating that the NPs successfully released CP and Cu 2+ in the acidic environment of the lysosome. The presence of CP and Cu 2+ aids Fenton-reaction-mediated production of ROS [58].

Cytocompatibility and Cytotoxicity
B16 cells were used to evaluate the cytotoxicity of the CP/CQ@MSN−HtB/Cu 2+ NPs (Figure 4a). Compared with free CP, CP/CQ@MSN−HtB/Cu 2+ NPs showed greater toxicity to B16 cells after 24 h of incubation. Similarly, the cytotoxicity of CP/CQ@MSN−HtB/Cu 2+ NPs was also higher than that of free CQ. These results can be explained by dint of the CP/CQ@MSN−HtB/Cu 2+ NPs releasing their drug cargo and providing synergistic therapy. Healthy L-929 cells were used to assess the biocompatibility of the nanoparticles. As shown in Figure 4b, CP/CQ@MSN−HtB/Cu 2+ NPs induced negligible cytotoxicity to L-929 cells even when the material concentration was as high as 5 µg·mL −1 , indicating that the CP/CQ@MSN−HtB/Cu 2+ NPs have good biocompatibility.

Intracellular ·OH Detection
Since the DCFH-DA probe can be oxidized to DCF-a green fluorescent moleculeunder the action of ROS, it can be used to detect ROS produced in the cell. Figure 3c reveals that in the control group and CP/CQ@MSN−HtB group there was almost no fluorescence in the cells, due to the absence of Cu 2+ . Cells treated with CuCl2 or MSN−HtB/Cu 2+ produced relatively weak fluorescence, due to some ROS being generated by the Cu 2+ present. The CP/CQ@MSN−HtB/Cu 2+ group gave markedly enhanced fluorescence, indicating that the NPs successfully released CP and Cu 2+ in the acidic environment of the lysosome. The presence of CP and Cu 2+ aids Fenton-reaction-mediated production of ROS [58].

Cytocompatibility and Cytotoxicity
B16 cells were used to evaluate the cytotoxicity of the CP/CQ@MSN−HtB/Cu 2+ NPs (Figure 4a). Compared with free CP, CP/CQ@MSN−HtB/Cu 2+ NPs showed greater toxicity to B16 cells after 24 h of incubation. Similarly, the cytotoxicity of CP/CQ@MSN−HtB/Cu 2+ NPs was also higher than that of free CQ. These results can be explained by dint of the CP/CQ@MSN−HtB/Cu 2+ NPs releasing their drug cargo and providing synergistic therapy. Healthy L-929 cells were used to assess the biocompatibility of the nanoparticles. As shown in Figure 4b, CP/CQ@MSN−HtB/Cu 2+ NPs induced negligible cytotoxicity to L-929 cells even when the material concentration was as high as 5 μg·mL −1 , indicating that the CP/CQ@MSN−HtB/Cu 2+ NPs have good biocompatibility.  Calcein AM/PI double-staining was used to evaluate the apoptosis of B16 cells (Figure 4c). Compared with free CP and CQ, the cells treated with CP/CQ@MSN−HtB/ Cu 2+ NPs showed strong red fluorescence (apoptotic cells), confirming that the NPs can effectively transport CP and CQ to the cells and cause cell apoptosis. The cells treated with CP/CQ@MSN−HtB NPs showed stronger green fluorescence (living cells) than those exposed to CP/CQ@MSN−HtB/Cu 2+ NPs, indicating that Cu 2+ can augment the chemotherapy provided by CP and CQ, proving synergistic chemotherapy/chemodynamic therapy. In addition, the MSN−HtB group had negligible red fluorescence, indicating that MSN−HtB has almost no biological toxicity.
Flow cytometry analysis was used to confirm the results of these assays (Figure 4d,e). In the PBS-treated group, 97.4% of the cells survived. By contrast, the apoptotic rates of the free CP and CQ groups were 22.9% and 32.0%, respectively, while the equivalent value for the CP/CQ@MSN−HtB group was 35.0%; this indicates that CP and CQ can effectively be transported into the cell via the NP carrier and cause apoptosis. In the CP/CQ@MSN−HtB/Cu 2+ group, 46.5% of the cells were apoptotic. This increased percentage is attributable to the CDT effects induced by Cu 2+ . The cell survival rate after treatment with MSN−HtB was 88.4%, consistent with the results above, and showing the NPs also have only slight cytotoxicity.

In Vivo Biodistribution
Free CP or CP/CQ@MSN−HtB/Cu 2+ NPs were injected into mice through the tail vein. After 24 h, the contents of Pt and Cu in the heart, liver, spleen, lungs, kidneys, and tumor cells were measured by ICP-OES. Both Pt and Cu could be seen to be concentrated in the tumor (Figures 5 and S5). Compared with an injection of an equivalent amount of free CP, the content of Pt in the tumor site increased markedly when using CP/CQ@MSN−HtB/Cu 2+ NPs ( Figure 5), indicating that the nanoparticles were enriched in the tumor site more effectively than the free drug. This can be attributed to the targeting ability of the NPs. The levels of Cu and Pt in the liver and kidneys were also relatively high. The former was due to the uptake of CP/CQ@MSN−HtB/Cu 2+ NPs by the mononuclear phagocyte system, while the latter resulted from renal excretion [59]. Calcein AM/PI double-staining was used to evaluate the apoptosis of B16 cells (Figure 4c). Compared with free CP and CQ, the cells treated with CP/CQ@MSN−HtB/Cu 2+ NPs showed strong red fluorescence (apoptotic cells), confirming that the NPs can effectively transport CP and CQ to the cells and cause cell apoptosis. The cells treated with CP/CQ@MSN−HtB NPs showed stronger green fluorescence (living cells) than those exposed to CP/CQ@MSN−HtB/Cu 2+ NPs, indicating that Cu 2+ can augment the chemotherapy provided by CP and CQ, proving synergistic chemotherapy/chemodynamic therapy. In addition, the MSN−HtB group had negligible red fluorescence, indicating that MSN−HtB has almost no biological toxicity.
Flow cytometry analysis was used to confirm the results of these assays (Figure 4d,e). In the PBS-treated group, 97.4% of the cells survived. By contrast, the apoptotic rates of the free CP and CQ groups were 22.9% and 32.0%, respectively, while the equivalent value for the CP/CQ@MSN−HtB group was 35.0%; this indicates that CP and CQ can effectively be transported into the cell via the NP carrier and cause apoptosis. In the CP/CQ@MSN−HtB/Cu 2+ group, 46.5% of the cells were apoptotic. This increased percentage is attributable to the CDT effects induced by Cu 2+ . The cell survival rate after treatment with MSN−HtB was 88.4%, consistent with the results above, and showing the NPs also have only slight cytotoxicity.

In Vivo Biodistribution
Free CP or CP/CQ@MSN−HtB/Cu 2+ NPs were injected into mice through the tail vein. After 24 h, the contents of Pt and Cu in the heart, liver, spleen, lungs, kidneys, and tumor cells were measured by ICP-OES. Both Pt and Cu could be seen to be concentrated in the tumor (Figures 5 and S5). Compared with an injection of an equivalent amount of free CP, the content of Pt in the tumor site increased markedly when using CP/CQ@MSN−HtB/Cu 2+ NPs ( Figure 5), indicating that the nanoparticles were enriched in the tumor site more effectively than the free drug. This can be attributed to the targeting ability of the NPs. The levels of Cu and Pt in the liver and kidneys were also relatively high. The former was due to the uptake of CP/CQ@MSN−HtB/Cu 2+ NPs by the mononuclear phagocyte system, while the latter resulted from renal excretion [59].

In Vivo Antitumor Efficacy and Biosafety
We investigated the therapeutic performance of CP/CQ@MSN−HtB/Cu 2+ NPs using B16 melanoma tumor-bearing mice. After the tumors had grown to ca. 100 mm 3 in volume, mice were injected with PBS, free CQ, free CP, MSN−HtB/Cu 2+ , CP/CQ@MSN−HtB, and CP/CQ@MSN−HtB/Cu 2+ NPs. Tumor volume data (Figure 6a) show that the tumor growth of mice treated with CP/CQ@MSN−HtB/Cu 2+ was significantly inhibited. The CP/CQ@MSN−HtB treatment group also displayed a certain degree of tumor growth inhibition but, due to the lack of Cu 2+ -mediated CDT, this was less marked than with CP/CQ@MSN−HtB/Cu 2+ .

In Vivo Antitumor Efficacy and Biosafety
We investigated the therapeutic performance of CP/CQ@MSN−HtB/Cu 2+ NPs using B16 melanoma tumor-bearing mice. After the tumors had grown to ca. 100 mm 3 in volume, mice were injected with PBS, free CQ, free CP, MSN−HtB/Cu 2+ , CP/CQ@MSN−HtB, and CP/CQ@MSN−HtB/Cu 2+ NPs. Tumor volume data (Figure 6a) show that the tumor growth of mice treated with CP/CQ@MSN−HtB/Cu 2+ was significantly inhibited. The CP/CQ@MSN−HtB treatment group also displayed a certain degree of tumor growth inhibition but, due to the lack of Cu 2+ -mediated CDT, this was less marked than with CP/CQ@MSN−HtB/Cu 2+ .  The mice were euthanatized and the tumors were excised at day 12 ( Figure 6b). The photographs revealed that the tumor shrank much more after treatment with CP/CQ@MSN−HtB/ Cu 2+ compared with the other groups. When these tumors were weighed ( Figure S6), mice treated with CP/CQ@MSN−HtB/Cu 2+ had an average tumor mass of ca. 2.74 g-notably less than with the free CP (5.79 g) or free CQ treatment groups (6.03 g).
Body weight data are given in Figure 6c. There was no significant weight loss in any of the treatment groups, indicating that the CP/CQ@MSN−HtB/Cu 2+ NPs have good biocompatibility and are well tolerated, with minimal off-target side effects. In addition, the survival time of tumor-bearing mice was evaluated using the Kaplan-Meier test (Figure 6d). The survival curves show that 80% of the mice treated with nanoparticles survived the 20-day experimental period. All of the mice treated with PBS, free CP, and free CQ died within 15 days. The survival time of mice treated with CP/CQ@MSN−HtB/Cu 2+ nanoparticles was notably higher than that of other groups.

Histological Analysis
The tumors were processed for histological analysis, including H&E and TUNEL staining (Figure 6e). In H&E staining, the tumor tissue of the CP/CQ@MSN−HtB/Cu 2+ treatment group showed a large area of tissue necrosis, indicated by pink/red-stained unstructured areas, and the cell nuclei (blue) were barely visible. There was no significant tissue necrosis in the free CP and free CQ treatment groups, which was attributed to their being distributed throughout the body rather than being localized in the tumor. The necrotic area of the tumor tissue increased greatly after the addition of Cu 2+ . TUNEL staining (Figure 6e) revealed that the number of apoptotic cells (green) in the tumor tissue after the CP/CQ@MSN−HtB/Cu 2+ treatment was greater than that in the CP/CQ@MSN−HtB treatment group, while the TUNEL positive signal in the other groups was markedly lower still. This confirms the potent synergistic chemotherapy/CDT properties of CP/CQ@MSN−HtB/Cu 2+ .
Tissue sections of the heart, liver, spleen, lungs, and kidneys were additionally subjected to H&E staining ( Figure S7). The results showed no obvious pathological abnormalities, indicating that the NP formulations had good selectivity and histocompatibility.
In this work, we designed and prepared a Cu 2+ -chelating mesoporous silica drug delivery system and evaluated its antitumor efficacy. Compared with other mesoporous silicon-based drug delivery systems, this has a higher cisplatin drug-loading and entrapment efficiency [60,61]. Cell experiments show that the formulation has good biocompatibility. In contrast, previously reported peptide-sealed mesoporous silica NPs showed a degree of cytotoxicity [21]. The results of in vivo experiments confirm that the CP/CQ@MSN−HtB/Cu 2+ NPs provide effective cisplatin/Cu 2+ synergistic therapy against cancer, which is consistent with other results in the literature [62].

Conclusions
In this work, we designed pH-responsive drug-loaded nanoparticles allowing synergistic chemotherapy/chemodynamic therapy of tumors. These were based on mesoporous silica nanoparticles (NPs) loaded with cisplatin and chloroquine. The NPs were functionalized with the targeting ligand His-tag-B3int, and the NP pores were sealed via the chelation of Cu 2+ with the His-tag. Drug release was found to be accelerated in slightly acidic pH, where the Cu 2+ was freed into solution and the NP pores opened. The NPs were shown to be able to generate ROS, and to be selectively taken up by cancer cells in vitro, where they appeared to be localized in the lysosomes. The presence of chloroquine then caused lysosomal membrane permeability, and released cisplatin and Cu 2+ into the cytoplasm for synergistic chemotherapy/chemodynamic therapy. In vivo results showed the therapeutic potential of this approach. The NP platform developed is selectively trafficked to the tumor, where it leads to marked increases in cell apoptosis and a reduction in tumor volume over time. No evident off-target toxicity was noted. Thus, this work offers a new strategy to