pH-Responsive and Biodegradable ZnO-Capped Mesoporous Silica Composite Nanoparticles for Drug Delivery

As a drug delivery system (DDS), traditional mesoporous silica nanoparticles (MSNs) suffer from bioaccumulation in vivo and premature drug release in systemic circulation due to low degradation rate and lack of protective gatekeeper. Herein, we developed a safe and intelligent DDS with characteristics of pH-responsive biodegradation and controlled drug release based on mesoporous silica composite nanoparticles (MSCNs) capped with ZnO quantum dots (ZnO QDs). Acidic degradable MSCNs were successfully synthesized by doping Ca2+ and PO43− into the MSNs’ framework. The in vitro doxorubicin hydrochloride (DOX) release was inhibited at neutral pH 7.4 but triggered significantly at pH 5.0 due to the dissociation of ZnO caps. The internalization behavior and cytotoxicity of 4T1 cells indicated MSCNs-ZnO could efficiently deliver DOX into the cells with significant antitumor activity. Such a DDS with pH-responsive biodegradation and controlled drug release has promising potential for cancer therapy.


Introduction
Mesoporous silica nanoparticles (MSNs) are widely employed in biomedical fields such as bioimaging [1], drug delivery [2], cancer diagnosis and therapy [3,4]. With huge specific surface area and pore volume, tailorable pore aperture and easy surface modification by well-known chemistries, MSNs show great potential as drug delivery carrier [5][6][7]. It has been reported that chemotherapeutic therapy for current cancer could lead to severe side effects on normal tissues during the systemic administration of toxic drugs [8]. MSNs with the encapsulated drugs inside can effectively avoid premature leakage as well as significantly improve drugs' efficacy [9][10][11]. However, it is well-known that pure MSNs have a very slow degradation rate in the physiological environment due to their stable framework. The majority of MSNs would accumulate in the reticuloendothelial system (RES) generating long-term tissue toxicity and restricting their clinical application [12,13]. Thus, MSNs with degradability in vivo could be a promising drug carrier in clinical application.
Organic [14,15] or inorganic entities [16,17] were doped into the MSNs' framework to transform structural features, which enabled composite nanoparticles more sensitive to the tumor microenvironment Scheme 1. Schematic illustration of ZnO-capped mesoporous silica composite nanoparticles (MSCNs) drug delivery system (DDS) and its procedure of pH-responsive degradation and drug release. DOX: doxorubicin hydrochloride.

Synthesis of ZnO QDs
Zinc acetate, 1.32 g, was refluxed in 40 mL ethanol at 78 °C for 3 h under vigorous stirring (denoted as solution A). Lithium hydroxide, 0.3024 g, was dissolved in 20 mL ethanol (denoted as solution B). Solution A was cooled to 20 °C followed by adding solution B with vigorous stirring for 20 min to obtain ZnO QDs [39]. The ZnO QDs solution was then dispersed in hexane till the color turned white. Afterwards, the ZnO QDs were washed with ethanol twice and centrifuged at 10,000 r/min for 5 min followed by vacuum drying. To the anchored amino group with ZnO QDs (ZnO-NH2), the as-prepared ZnO QDs were well-ultrasonically dispersed in 20 mL DMF followed by addition of 100 μL APTES, then the mixture was stirred for 20 min at 120 °C. The ZnO-NH2 QDs were obtained by washing with DMF twice and centrifuging at 10,000 r/min for 5 min. Scheme 1. Schematic illustration of ZnO-capped mesoporous silica composite nanoparticles (MSCNs) drug delivery system (DDS) and its procedure of pH-responsive degradation and drug release. DOX: doxorubicin hydrochloride.

Synthesis of ZnO QDs
Zinc acetate, 1.32 g, was refluxed in 40 mL ethanol at 78 • C for 3 h under vigorous stirring (denoted as solution A). Lithium hydroxide, 0.3024 g, was dissolved in 20 mL ethanol (denoted as solution B). Solution A was cooled to 20 • C followed by adding solution B with vigorous stirring for 20 min to obtain ZnO QDs [39]. The ZnO QDs solution was then dispersed in hexane till the color turned white. Afterwards, the ZnO QDs were washed with ethanol twice and centrifuged at 10,000 r/min for 5 min followed by vacuum drying. To the anchored amino group with ZnO QDs (ZnO-NH 2 ), the as-prepared ZnO QDs were well-ultrasonically dispersed in 20 mL DMF followed by addition of 100 µL APTES, then the mixture was stirred for 20 min at 120 • C. The ZnO-NH 2 QDs were obtained by washing with DMF twice and centrifuging at 10,000 r/min for 5 min.

Synthesis of MSCNs
MSCNs were prepared by doping CaCl 2 and Na 2 HPO 4 during the synthetic process of MSNs based on the Stöber approach [40]. CTAB, 0.20 g, and 0.18 g Na 2 HPO 4 ·12H 2 O were dissolved in 70 mL deionized water followed by adding 20 mL ethanol and 0.27 mL ammonia. The mixture was stirred magnetically for 30 min at 40 • C. Then 1 mL TEOS was dropwise added into the solution with vigorously stirring. Aqueous solution, 1 mL, containing 0.083 g CaCl 2 was immediately introduced into the solution and stirred for 24 h. The resulted product MSCNs-CTAB were isolated by centrifugation at 8000 r/min for 10 min and washed three times with deionized water and ethanol alternately. To remove surfactant CTAB, the final MSCNs were calcined at 550 • C for 6 h with a heating rate of 1 • C/min.

Amino Functionalization of MSCNs
For the synthesis of amine groups modified MSCNs (MSCNs-NH 2 ), 100 mg MSCNs were dispersed in 20 mL ethanol containing 1 mL APTES. The mixture was continually reacted at room temperature by stirring overnight. MSCNs-NH 2 were collected by centrifuging at 8000 r/min for 10 min and washing with deionized water and ethanol thrice alternately.

Carboxyl Functionalization of MSCNs
The carboxyl group modified-MSCNs (MSCNs-COOH) were synthesized based on the previous report with slight modification [41]. MSCNs-NH 2 were dispersed in 10 mL DMF including 86 mg succinic anhydride, which was vigorously stirred at room temperature for 24 h under nitrogen protection. Then the as-prepared MSCNs-COOH were washed with deionized water and ethanol thrice respectively.

Biodegradation of MSCNs in PBS Solution and in Cells
MSCNs, 20 mg, were suspended in 20 mL phosphate-buffered saline (PBS) with pH value of 5.0 and 7.4. The mixture was continuously shaken at 37 • C for days. At 1, 3 and 7 d, the nanoparticles in the pH 5.0 and 7.4 were collected for TEM and SEM observation. Simultaneously, the supernatant was collected at fixed times (1 d, 3 d, 5 d, 7 d) to detect the cumulative dissolved rate of Ca 2+ by inductively coupled plasma-optical emission spectrometer (ICP-OES).
The intracellular biodegradation behavior of the MSCNs was evaluated by bio-TEM observation. Typically, 100 µg/mL MSCNs dispersed in DMEM were co-cultured with 4T1 cells for 1, 3 and 7 d. The cells were harvested by centrifugation at 1000 r/min for 5 min and fixed with 2.5% glutaraldehyde. Then ultrathin cell sections were cut for bio-TEM characterization.

Synthesis of DOX@MSCNs
DOX, 20 mg, was dispersed in 40 mL of pH 7.4 PBS followed by adding 80 mg MSCNs-COOH. The mixture was shaken at 25 • C for 24 h. The DOX-loaded MSCNs (DOX@MSCNs) were collected by centrifugation at 8000 r/min for 5 min and dried under vacuum. The absorbance of the supernatant was measured by UV-vis spectrophotometer at 480 nm, the loading amount (mg/g) and efficiency (%) of DOX were calculated by the following Equations (1) and (2): DOX loading amount (mg/g) = Mass of DOX loading into particles (mg) Mass of particles (g)

ZnO-Capped DOX@MSCNs
The above DOX@MSCNS were dispersed in 10 mL aqueous solution containing 50 mg ZnO-NH 2 QDs and 60 mg EDC. After stirring at room temperature for 5 h, the precipitate was centrifuged (5000 r/min, 5 min) and washed several times with deionized water until the supernatant became transparent. The ZnO-capped drug loaded particles (DOX@MSCNs-ZnO) were collected after freeze-drying. The ZnO-capped MSCNs (MSCNs-ZnO) were prepared using the same synthetic approach as DOX@MSCNs-ZnO.

In Vitro pH-Responsive Drug Release of DOX@MSCNs-ZnO
To investigate the release behavior of DOX from DOX@MSCNs-ZnO, 4 mg of DOX@MSCNs-ZnO were dispersed into 4 mL pH 5.0 and 7.4 PBS, respectively and placed in a 37 • C constant temperature shaker to release. The supernatant was gathered at 1 h, 4 h, 8 h, 12 h, 24 h, 36 h and 48 h, respectively by centrifugation at 8000 r/min for 5 min. Subsequently, the supernatant was supplemented to 4 mL for releasing again.
As control, the release behavior of DOX from DOX@MSCNs (without ZnO capping) was also performed with the same experimental process as that of DOX@MSCNs-ZnO. The cumulative release rate (%) of DOX was calculated by the absorbance at 480 nm of the supernatant according to the following Equation (3).

Cell Culture
4T1 cells (mouse breast cancer cell line) were kindly presented by Jiangsu Center for the collaboration and innovation of cancer biotherapy from the cell bank of the Chinese Academy of Sciences (Shanghai, China), which were incubated with DMEM containing 10% FBS and 1% penicillin-streptomycin under a humidified 5% CO 2 atmosphere at 37 • C.

Intracellular Endocytosis of DOX@MSCNs-ZnO
The 4T1 cells were seeded into a 24-well plate at the concentration of 1 × 10 4 per well, and were treated with 100 µg/mL of DOX@MSCNs-ZnO in DMEM. At different intervals (0.5, 1, 4 and 8 h), after discarding the medium, the cells were washed with pH 7.4 PBS three times and then fixed with 4% (g/mL) paraformaldehyde for 30 min. Subsequently, after removal of paraformaldehyde and washing with pH 7.4 PBS, the nuclei were stained by DAPI (blue) for 10 min. With the cells sealed with a prolong antifade kit, cellular uptake behavior was observed by confocal laser scanning microscopy (CLSM).

In Vitro Cytotoxicity Assays
In vitro cell viability of the 4T1 cells was measured by CCK-8 assay. The cells were seeded into a 96-well plate with 3.0 × 10 4 cells per well and incubated overnight at 37 • C. The samples were divided into two groups involving the first group of ZnO and Zn 2+ (ZnCl 2 ) with concentrations 0.1, 0.5, 2.5, 5, 25, 50 and 100 µg/mL, and the second group of MSCNs, MSCNs-ZnO, DOX@MSCNs-ZnO, DOX@MSCNs with concentrations 1, 5, 10, 25, 50, 100 and 200 µg/mL and free DOX with the corresponding concentrations: 0.188, 0.94, 1.88, 4.7, 9.4, 18.8 and 37.6 µg/mL. The 4T1 cells were treated with the above samples for 24 h. Afterwards, 10 µL CCK-8 was added into the medium per well followed by incubation for another 2 h. The optical density (OD) of the samples was measured using a microplate spectrophotometer at 450 nm. The cell viability was calculated based on the following Equation (4): where ODe is the absorbance value of experimental well (DMEM with cells, CCK-8 and samples), ODc is the absorbance value of control well (DMEM with cells, CCK-8, and without samples) and ODb is the absorbance value of blank well (DMEM without cells and CCK-8). Data statistics were calculated based on six independent results.

Characterization of Nanoparticles
Scanning electron microscope (SEM) images were taken with SU8220 microscope (Hitachi, Ibaraki, Japan) at an accelerating voltage of 10 Kv. SEM elements mapping images were obtained on an energy dispersive spectrometer (EDS) with XFlash Detector 6|30 (Bruker Nano GmbH, Karlsruhe, Germany). Transmission electron microscopy (TEM) was carried out at Tecnai G2 F20 microscope (FEI, Hillsboro, OR, USA) at an exciting voltage of 200 kV. The powder X-ray diffraction (XRD) patterns were recorded on a D8 ADVANCE (Bruker, Karlsruhe, Germany) X-ray diffractometer with Cu-Kα radiation (λ = 1.5418 Å) at the scanning step of 0.02 • . The specific surface area, pore volume and pore size of the particles were measured at ASAP 2020 instrument (Micromeritics, Norcross, GA, USA), which was calculated applying the Brunauer-Emmett-Teller (BET) and the Barrett-Joyner-Halenda (BJH) method. The hydrodynamic diameter of the particles was detected by NanoBrook Omni instruments (Brookhaven Instruments Corporation, Holtsville, NY, USA). The element component of nanoparticles was carried out on ESCALAB250Xi X-ray photoelectron spectroscopy (XPS). The drug absorbance was examined by Evolution 60 ultraviolet-visible (UV-vis) spectrophotometer (Thermo, Walsham, MA, USA). An inductively coupled plasma-optical emission spectrometer (ICP-OES, Optima8000, PerkinElmer, Waltham, MA, USA) was used to analyze the amount of dissolved ion. The confocal laser scanning microscopy (CLSM) was carried out on ZEISS LSM880 (Jena, Germany) to observe the intracellular endocytosis of the drug-loaded particles with a blue fluorescence excitation wavelength of 405 nm and a red fluorescence excitation wavelength of 561 nm. The cell sections were observed by a JEM1010 bio-TEM (JEOL, Tokyo, Japan) to evaluate the intracellular biodegradation of nanoparticles.

Statistical Analysis
Data were statistically analyzed by OriginPro (version 8.0, OriginLab, Northampton, MA, USA) and expressed as mean ± standard deviation (SD) at least three times.

Morphology and Particle Size of ZnO QDs
The morphology and particle size were observed by TEM. It can be seen from Figure 1a

Elemental and Structural Analysis of ZnO QDs
Elemental composition of ZnO QDs was analyzed by EDS. As shown in Figure 2a, copper (Cu), carbon (C), zinc (Zn) and oxygen (O) elements were detected. Cu and C elements were mainly from the network material Cu mesh and carbon film while the Zn and O elements were derived from ZnO QDs. The wide-angle x-ray diffraction (WAXRD) of ZnO QDs is shown in Figure 2b, which could match well with the standard card Joint Committee on Powder Diffraction Standards (JCPDS) 36-1451 of the hexagonal wurtzite structure.

Elemental and Structural Analysis of ZnO QDs
Elemental composition of ZnO QDs was analyzed by EDS. As shown in Figure 2a, copper (Cu), carbon (C), zinc (Zn) and oxygen (O) elements were detected. Cu and C elements were mainly from the network material Cu mesh and carbon film while the Zn and O elements were derived from ZnO QDs. The wide-angle x-ray diffraction (WAXRD) of ZnO QDs is shown in Figure 2b, which could match well with the standard card Joint Committee on Powder Diffraction Standards (JCPDS) 36-1451 of the hexagonal wurtzite structure.

Acidic Degradation Behavior of ZnO QDs
As shown in Figure 3, the percentage of dissolved Zn 2+ was detected by ICP-OES after dispersing ZnO QDs in PBS at pH 5.0 for 30 min and pH 7.4 for 7 d. It was calculated that 98.7% ± 3.6 of Zn 2+ was found in pH 5.0 PBS after 30 min immersion, which was ascribed to the rapid decomposition of the ZnO QDs in the acidic environment. On the contrary, when immersed in pH 7.4 PBS for a relatively long time (7 d), only 15.7% ± 1.8 of Zn 2+ dissolved out, indicating ZnO QDs were more stable in the neutral environment. This was further verified by the visualized yellow-green fluorescence in pH 7.4 PBS and no fluorescence in pH 5.0 PBS under 365 nm UV irradiation (as shown in the inset images in Figure 3). These acidic-triggered, decomposed ZnO QDs could be developed as the pH-responsive gate to seal a drug in the pores of MSCNs and release the entrapped drug in the acidic compartment of the cancer cells.

Acidic Degradation Behavior of ZnO QDs
As shown in Figure 3, the percentage of dissolved Zn 2+ was detected by ICP-OES after dispersing ZnO QDs in PBS at pH 5.0 for 30 min and pH 7.4 for 7 d. It was calculated that 98.7% ± 3.6 of Zn 2+ was found in pH 5.0 PBS after 30 min immersion, which was ascribed to the rapid decomposition of the ZnO QDs in the acidic environment. On the contrary, when immersed in pH 7.4 PBS for a relatively long time (7 d), only 15.7% ± 1.8 of Zn 2+ dissolved out, indicating ZnO QDs were more stable in the neutral environment. This was further verified by the visualized yellow-green fluorescence in pH 7.4 PBS and no fluorescence in pH 5.0 PBS under 365 nm UV irradiation (as shown in the inset images in Figure 3). These acidic-triggered, decomposed ZnO QDs could be developed as the pH-responsive gate to seal a drug in the pores of MSCNs and release the entrapped drug in the acidic compartment of the cancer cells.

Morphology and Particle Size of MSCNs
The degradable MSCNs were prepared by adding CaCl2 and Na2HPO4 during the synthesis process of MSNs. The doping of Ca 2+ and PO4 3− into the framework of MSNs was expected to transform the original stable structure and make it possible to decompose MSCNs in an acidic environment. As shown in TEM images (Figure 4a,b), the as-prepared MSCNs were spherical in shape and about 100 nm in average diameter with good dispersion and well-ordered porous structure. The average size distribution with diameter of 100 nm was confirmed by dynamic light scattering (DLS) technique (Figure 4c).

Morphology and Particle Size of MSCNs
The degradable MSCNs were prepared by adding CaCl 2 and Na 2 HPO 4 during the synthesis process of MSNs. The doping of Ca 2+ and PO 4 3− into the framework of MSNs was expected to transform the original stable structure and make it possible to decompose MSCNs in an acidic environment. As shown in TEM images (Figure 4a,b), the as-prepared MSCNs were spherical in shape and about 100 nm in average diameter with good dispersion and well-ordered porous structure. The average size distribution with diameter of 100 nm was confirmed by dynamic light scattering (DLS) technique ( Figure 4c).

Morphology and Particle Size of MSCNs
The degradable MSCNs were prepared by adding CaCl2 and Na2HPO4 during the synthesis process of MSNs. The doping of Ca 2+ and PO4 3− into the framework of MSNs was expected to transform the original stable structure and make it possible to decompose MSCNs in an acidic environment. As shown in TEM images (Figure 4a,b), the as-prepared MSCNs were spherical in shape and about 100 nm in average diameter with good dispersion and well-ordered porous structure. The average size distribution with diameter of 100 nm was confirmed by dynamic light scattering (DLS) technique (Figure 4c). In Figure 5b WAXRD pattern, the 2θ of 22° was the amorphous characteristic peak of silica. The other occurring peaks could be attributed to hydroxyapatite consisting of Ca 2+ and PO4 3− , which was in agreement with the standard card JCPDS 09-0432 of hexagonal hydroxyapatite, indicating the successful incorporation of Ca3(PO4)2 into the pore walls of the MSNs. Under an acidic environment, Ca3(PO4)2 was prone to be hydrolyzed into Ca 2+ and PO4 3− , which would lay the foundation for MSCNs to achieve acidic degradation. The nitrogen adsorption desorption curve of MSCNs (Figure 5c) was a type IV curve, further implying the synthesized MSCNs have a mesoporous structure. The calculated BET specific surface area of the MSCNs was 973.3 m 2 /g, and the pore volume was 0.83 cm 3 /g with the mean pore diameter of 3.7 nm (Figure 5d). MSCNs doped with Ca 2+ and PO4 3− still maintained the excellent features (ordered mesoporous structure, high specific surface area and pore volume, suitable pore diameter) and had high drug-loading capacities as pure MSNs.

Elemental Analysis of MSCNs
SEM elemental mapping and an EDS curve were applied to investigate the elemental distribution of the MSCNs. Calcium (Ca) and phosphorus (P) signals were detected as uniformly distributed in MSCNs, indicating the successful incorporation (Figure 6a). The elemental components and chemical structure of the MSCNs were further investigated by XPS (Figure 6b).

Degradation of MSCNs in PBS Solution
Since the as-prepared MSCNs were expected to be sensitive to the acidic environment, the in vitro degradation behavior was evaluated in PBS solution at different pH values (5.0 and 7.4). MSCNs were immersed in pH 5.0 and 7.4 PBS for 1, 3 and 7 d. The changes of the particles' morphology were tracked by SEM and TEM images. The MSCNs were spherical with a smooth surface and highly ordered mesoporous structure before degradation (Figure 7a). In pH 5.0 PBS solution, the MSCNs underwent significant time-dependent degradation (Figure 7b). After 1 d immersion (Figure 7b1,b2), there was partial structural breakdown of some nanoparticles with morphology changing from spherical to blurred. With time, more considerably biodegraded nanoparticles were found, which exhibited severe structural damages after 3 d (Figure 7b3,b4). When immersion time extended to 7 d, the spherical morphology of the particles was completely invisible (Figure 7b5) and also no mesoporous structure was observed (Figure 7b6), indicating that a complete collapse had occurred. Notably, some of them even disintegrated into smaller fragments as shown in Figure 7b7, which provided a prerequisite for metabolizing particles out of the body in a relatively short time. In contrast, no evident morphological changes were found in pH 7.4 PBS solution (Figure 7c). Though after 7 d immersion, MSCNs still maintained the spherical morphology and intact structure, indicating that the particles were stable in the neutral environment. Moreover, the acid-sensitivity characteristic of the MSCNs was further corroborated by the Ca 2+ dissolution amount from the framework in pH 5.0 and 7.4 PBS by ICP-OES detection (Figure 7d). The final cumulative dissolved rate of Ca 2+ was calculated to be 55% ± 1.0 in pH 5.0 and less than 5% ± 0.9 Ca 2+ in pH 7.4, which further demonstrated the obvious acid-sensitivity degradation characteristic of MSCNs.

Degradation of MSCNs in PBS Solution
Since the as-prepared MSCNs were expected to be sensitive to the acidic environment, the in vitro degradation behavior was evaluated in PBS solution at different pH values (5.0 and 7.4). MSCNs were immersed in pH 5.0 and 7.4 PBS for 1, 3 and 7 d. The changes of the particles' morphology were tracked by SEM and TEM images. The MSCNs were spherical with a smooth surface and highly ordered mesoporous structure before degradation (Figure 7a). In pH 5.0 PBS solution, the MSCNs underwent significant time-dependent degradation (Figure 7b). After 1 d immersion (Figure 7b1,b2), there was partial structural breakdown of some nanoparticles with morphology changing from spherical to blurred. With time, more considerably biodegraded nanoparticles were found, which exhibited severe structural damages after 3 d (Figure 7b3,b4). When immersion time extended to 7 d, the spherical morphology of the particles was completely invisible (Figure 7b5) and also no mesoporous structure was observed (Figure 7b6), indicating that a complete collapse had occurred. Notably, some of them even disintegrated into smaller fragments as shown in Figure 7b7, which provided a prerequisite for metabolizing particles out of the body in a relatively short time. In contrast, no evident morphological changes were found in pH 7.4 PBS solution (Figure 7c). Though after 7 d immersion, MSCNs still maintained the spherical morphology and intact structure, indicating that the particles were stable in the neutral environment. Moreover, the acid-sensitivity characteristic of the MSCNs was further corroborated by the Ca 2+ dissolution amount from the framework in pH 5.0 and 7.4 PBS by ICP-OES detection (Figure 7d). The final cumulative dissolved rate of Ca 2+ was calculated to be 55% ± 1.0 in pH 5.0 and less than 5% ± 0.9 Ca 2+ in pH 7.4, which further demonstrated the obvious acid-sensitivity degradation characteristic of MSCNs.  a1, b1, b3, b5, c1, c2, c3) and TEM (a2, b2, b4, b6, b7

Degradation of MSCNs in 4T1 Cells
Furthermore, the biodegradation behavior of MSCNs intracellularly was observed by Bio-TEM after co-cultivation with 4T1 cells for 1, 3 and 7 d. As shown in Figure 8, the MSCNs could be efficiently internalized into cells and the evident time-dependent biodegradation was found in the intracellular microenvironment, which was consistent with the above in vitro degradation at pH 5.0. After 1 d incubation, the endocytosed nanoparticles (shown by red arrow) were evenly dispersed in cells and kept their intact structure with a spherical shape and clear edge. When co-cultivation time extended to 3 d, the spherical morphology of MSCNs became blurred with structural collapse indicating the endocytosed MSCNs gradually underwent degradation in the acidic compartments. After 7 d co-cultivation, it was difficult to find nondegraded particles inside the cells, suggesting MSCNs were almost completely degraded and excreted out of the cells. The endocytosed MSCNs experienced this significant degradation behavior at a cellular level. As known, the pH value in the endosome or lysosome was low (pH 5.0), which would be the useful factor to prompt the pH-sensitive MSCNs' biodegradation [34] in cells.   a1, b1, b3, b5, c1, c2, c3) and TEM (a2, b2, b4, b6, b7)

Degradation of MSCNs in 4T1 Cells
Furthermore, the biodegradation behavior of MSCNs intracellularly was observed by Bio-TEM after co-cultivation with 4T1 cells for 1, 3 and 7 d. As shown in Figure 8, the MSCNs could be efficiently internalized into cells and the evident time-dependent biodegradation was found in the intracellular microenvironment, which was consistent with the above in vitro degradation at pH 5.0. After 1 d incubation, the endocytosed nanoparticles (shown by red arrow) were evenly dispersed in cells and kept their intact structure with a spherical shape and clear edge. When co-cultivation time extended to 3 d, the spherical morphology of MSCNs became blurred with structural collapse indicating the endocytosed MSCNs gradually underwent degradation in the acidic compartments. After 7 d co-cultivation, it was difficult to find nondegraded particles inside the cells, suggesting MSCNs were almost completely degraded and excreted out of the cells. The endocytosed MSCNs experienced this significant degradation behavior at a cellular level. As known, the pH value in the endosome or lysosome was low (pH 5.0), which would be the useful factor to prompt the pH-sensitive MSCNs' biodegradation [34] in cells.  a1, b1, b3, b5, c1, c2, c3) and TEM (a2, b2, b4, b6, b7

Degradation of MSCNs in 4T1 Cells
Furthermore, the biodegradation behavior of MSCNs intracellularly was observed by Bio-TEM after co-cultivation with 4T1 cells for 1, 3 and 7 d. As shown in Figure 8, the MSCNs could be efficiently internalized into cells and the evident time-dependent biodegradation was found in the intracellular microenvironment, which was consistent with the above in vitro degradation at pH 5.0. After 1 d incubation, the endocytosed nanoparticles (shown by red arrow) were evenly dispersed in cells and kept their intact structure with a spherical shape and clear edge. When co-cultivation time extended to 3 d, the spherical morphology of MSCNs became blurred with structural collapse indicating the endocytosed MSCNs gradually underwent degradation in the acidic compartments. After 7 d co-cultivation, it was difficult to find nondegraded particles inside the cells, suggesting MSCNs were almost completely degraded and excreted out of the cells. The endocytosed MSCNs experienced this significant degradation behavior at a cellular level. As known, the pH value in the endosome or lysosome was low (pH 5.0), which would be the useful factor to prompt the pH-sensitive MSCNs' biodegradation [34] in cells.

ZnO-Capped DOX@MSCNs
To prevent drug from premature release, the as-prepared pH-sensitive ZnO QDs were used to block MSCNs pores. The amino-modified ZnO QDs were tethered to the outlet of the MSCNs-COOH nanopores by amidation coupling reaction. The morphology of the MSCNs-ZnO was observed by TEM images (Figure 9a), which evidently displayed that ZnO QDs were distributed on the surface of the MSCNs, realizing the purpose of plugging nanopores. Interestingly, after capping, MSCNs-ZnO exhibited the same yellow-green fluorescence behavior as the pure ZnO QDs, which further verified the presence of ZnO QDs on the surface of the nanoparticles (Figure 9b). Figure 9c displays the SAXRD of MSCNs and MSCNs-ZnO. Since ZnO QDs blocked the pores of MSCNs, the diffraction peak intensity of MSCNs-ZnO was lower than that of the bare MSCNs. WAXRD pattern of MSCNs-ZnO showed the peak overlay of ZnO and MSCNs, attributing to the successfully modified ZnO on the surface of MSCNs (Figure 9d). After the pores were capped with ZnO, the BET-specific area of MSCNs-ZnO reduced to 185 m 2 /g from 973.3 m 2 /g of MSCNs (Figure 9e). Together, a series of significant changes between bare MSCNs and MSCNs-ZnO demonstrated the successful capping of ZnO on MSCNs.

ZnO-Capped DOX@MSCNs
To prevent drug from premature release, the as-prepared pH-sensitive ZnO QDs were used to block MSCNs pores. The amino-modified ZnO QDs were tethered to the outlet of the MSCNs-COOH nanopores by amidation coupling reaction. The morphology of the MSCNs-ZnO was observed by TEM images (Figure 9a), which evidently displayed that ZnO QDs were distributed on the surface of the MSCNs, realizing the purpose of plugging nanopores. Interestingly, after capping, MSCNs-ZnO exhibited the same yellow-green fluorescence behavior as the pure ZnO QDs, which further verified the presence of ZnO QDs on the surface of the nanoparticles (Figure 9b). Figure 9c displays the SAXRD of MSCNs and MSCNs-ZnO. Since ZnO QDs blocked the pores of MSCNs, the diffraction peak intensity of MSCNs-ZnO was lower than that of the bare MSCNs. WAXRD pattern of MSCNs-ZnO showed the peak overlay of ZnO and MSCNs, attributing to the successfully modified ZnO on the surface of MSCNs (Figure 9d). After the pores were capped with ZnO, the BET-specific area of MSCNs-ZnO reduced to 185 m 2 /g from 973.3 m 2 /g of MSCNs (Figure 9e). Together, a series of significant changes between bare MSCNs and MSCNs-ZnO demonstrated the successful capping of ZnO on MSCNs.

In Vitro pH-Responsive Drug Release of DOX@MSCNs-ZnO
To verify the pH-responsive drug release of MSCNs-ZnO, DOX as the anticancer model drug was loaded into the pores of MSCNs-COOH and subsequently plugged with ZnO QDs. The loading amount and efficiency of DOX were calculated as 231.3 mg/g and 18.8%, respectively. Since DOX was loaded into the interior of pores and the "nanovalves" ZnO sealed the outlet of pores, the BET-specific surface area of DOX@MSCNs-ZnO reduced to 104.4 m 2 /g compared to bare MSCNs (973.3 m 2 /g) (Figure 10a).
To validate the effect of pH-responsive uncapping on drug release, the release behavior of DOX@MSCNs-ZnO was studied in acidic and neutral environments (pH 5.0 and 7.4 PBS solution) as shown in Figure 10b. It can be seen that negligible DOX was released from MSCNs-ZnO in pH 7.4 due to the capped pores with ZnO. However, in the pH 5.0 environment, an initial burst release was

In Vitro pH-Responsive Drug Release of DOX@MSCNs-ZnO
To verify the pH-responsive drug release of MSCNs-ZnO, DOX as the anticancer model drug was loaded into the pores of MSCNs-COOH and subsequently plugged with ZnO QDs. The loading amount and efficiency of DOX were calculated as 231.3 mg/g and 18.8%, respectively. Since DOX was loaded into the interior of pores and the "nanovalves" ZnO sealed the outlet of pores, the BET-specific surface area of DOX@MSCNs-ZnO reduced to 104.4 m 2 /g compared to bare MSCNs (973.3 m 2 /g) (Figure 10a). DOX@MSCNs-ZnO. It revealed that the ZnO nanovalves could plug drugs into the pores in pH 7.4, preventing the premature drug release into systemic circulation in neutral conditions. The final cumulative drug release amount in pH 5.0 was nearly the same as DOX@MSCNs-ZnO, suggesting that the existence of ZnO nanovalves would not affect the drug release. Thus, considering the above results, the ZnO QDs were excellent candidates as the pH-responsive "on-off" for the drug controlled release system.

Endocytosis of DOX@MSCNs-ZnO
The cellular uptake behavior of DOX@MSCNs-ZnO was observed by CLSM after culturing with 4T1 cells for 30 min, 1 h, 4 h and 8 h. As shown in Figure 11, DOX was released over time as MSCNs-ZnO was gradually degraded in the acidic endocytic vesicles. After 30 min and 1 h, part of DOX had begun to be released into the cytoplasm, which was attributed to uncapped pores caused by the rapid dissolution of ZnO. After continuous incubation for 4 h and 8 h, more-and-more DOX was released and distributed evenly throughout the cells. When internalized into endosome or lysosome with lower pH value [34], ZnO nanovalves were rapidly decomposed and the DOX was released from the nanopores intracellularly. Meanwhile, as depicted in the intracellular biodegradation experiment, MSCNs could be degraded into fragments in cells, which further enhanced the drug release. The "DOX@MSCNs-ZnO" DDS could efficiently transport drugs into cancer cells and release drugs into cells in a sustained and pH-stimulated controlled way. To validate the effect of pH-responsive uncapping on drug release, the release behavior of DOX@MSCNs-ZnO was studied in acidic and neutral environments (pH 5.0 and 7.4 PBS solution) as shown in Figure 10b. It can be seen that negligible DOX was released from MSCNs-ZnO in pH 7.4 due to the capped pores with ZnO. However, in the pH 5.0 environment, an initial burst release was found with a cumulative release rate of 27% ± 1.7 at 1 h, which was attributed to the rapid dissolution of ZnO QDs. Furthermore, release tended to reach a saturation at 12 h and the final cumulative release reached 45% ± 2.0 at 48 h. This result was also confirmed by the color change of DOX@MSCNs-ZnO soaked in pH 7.4 and 5.0 for 1 h. As shown in the inset images of Figure 10b, after immersion in pH 7.4, the supernatant remained unchanged, indicating a negligible release of DOX. On the contrary, when immersed in pH 5.0, the solution was gradually transformed from the supernatant liquid to red, suggesting the continuous release of DOX in acidic environment. The lesser released amount in pH 7.4 but significantly increased release in pH 5.0 indicated the acid-responsive release capacity of the ZnO nanovalves. Figure 10c presents the cumulative release of DOX from DOX@MSCNs without capping with ZnO under the same situation. Apparently, without ZnO nanovalves, the final cumulative release of DOX from DOX@MSCNs reached 27% ± 1.4 in pH 7.4, which was higher than that from DOX@MSCNs-ZnO. It revealed that the ZnO nanovalves could plug drugs into the pores in pH 7.4, preventing the premature drug release into systemic circulation in neutral conditions. The final cumulative drug release amount in pH 5.0 was nearly the same as DOX@MSCNs-ZnO, suggesting that the existence of ZnO nanovalves would not affect the drug release. Thus, considering the above results, the ZnO QDs were excellent candidates as the pH-responsive "on-off" for the drug controlled release system.

Endocytosis of DOX@MSCNs-ZnO
The cellular uptake behavior of DOX@MSCNs-ZnO was observed by CLSM after culturing with 4T1 cells for 30 min, 1 h, 4 h and 8 h. As shown in Figure 11, DOX was released over time as MSCNs-ZnO was gradually degraded in the acidic endocytic vesicles. After 30 min and 1 h, part of DOX had begun to be released into the cytoplasm, which was attributed to uncapped pores caused by the rapid dissolution of ZnO. After continuous incubation for 4 h and 8 h, more-and-more DOX was released and distributed evenly throughout the cells. When internalized into endosome or lysosome with lower pH value [34], ZnO nanovalves were rapidly decomposed and the DOX was released from the nanopores intracellularly. Meanwhile, as depicted in the intracellular biodegradation experiment, MSCNs could be degraded into fragments in cells, which further enhanced the drug release. The "DOX@MSCNs-ZnO" DDS could efficiently transport drugs into cancer cells and release drugs into cells in a sustained and pH-stimulated controlled way.

In Vitro Cell Cytotoxicity
CCK-8 assay was employed to investigate the cytotoxicity of different samples cultured with 4T1 cells for 24 h. Firstly, the cell viability of ZnO and Zn 2+ (as ZnCl2) with different concentrations is shown in Figure 12a. It is clearly seen that both ZnO and Zn 2+ exhibited similar concentrationdependent tendency. At the concentration of less than 25 μg/mL, both had almost no toxic effect on 4T1 cells. Interestingly, when the concentration surpassed 25 μg/mL, the cell viability declined sharply to 50% ± 5.0 for ZnO and 26% ± 1.2 for Zn 2+ . ZnO QDs dissolved into Zn 2+ in the acidic environment of cellular compartments. According to previous reports [44][45][46], the accumulation of Zn 2+ inside cells induced the production of reactive oxygen species (ROS) followed by peroxidation of lipid and damage of DNA. As a result, ZnO QDs had a therapeutic effect on cancer cells. Figure 12b shows the viability of 4T1 cells in the presence of MSCNs, DOX@MSCNs-ZnO, DOX@MSCNs and free DOX with different concentration. It is clearly seen that as the drug carrier itself, the bare MSCNs had no obvious cytotoxicity even at the concentration of 200 μg/mL, suggesting that MSCNs have excellent biocompatibility and can act as the safe drug vehicle. MSCNs-ZnO exhibited concentration-dependent cytotoxicity, which was mainly caused by ZnO attached on MSCNs. Free DOX, DOX@MSCNs and DOX@MSCNs-ZnO all exhibited significant toxicity with the increased concentration. However, among these, the half-maximal inhibitory concentration (IC50) of DOX@MSCNs-ZnO was calculated to be 4.3 μg/mL of equivalent DOX concentration, less than that of DOX@MSCNs (7.3 μg/mL) and free DOX (17.6 μg/mL). At the same DOX concentration, DOX@MSCNs-ZnO can effectively inhibit more cancer cell growth than DOX@MSCNs and free DOX, which was the result of the synergistic anticancer therapeutic effect of the released DOX and ZnO from DOX@MSCNs-ZnO.

In Vitro Cell Cytotoxicity
CCK-8 assay was employed to investigate the cytotoxicity of different samples cultured with 4T1 cells for 24 h. Firstly, the cell viability of ZnO and Zn 2+ (as ZnCl 2 ) with different concentrations is shown in Figure 12a. It is clearly seen that both ZnO and Zn 2+ exhibited similar concentration-dependent tendency. At the concentration of less than 25 µg/mL, both had almost no toxic effect on 4T1 cells. Interestingly, when the concentration surpassed 25 µg/mL, the cell viability declined sharply to 50% ± 5.0 for ZnO and 26% ± 1.2 for Zn 2+ . ZnO QDs dissolved into Zn 2+ in the acidic environment of cellular compartments. According to previous reports [44][45][46], the accumulation of Zn 2+ inside cells induced the production of reactive oxygen species (ROS) followed by peroxidation of lipid and damage of DNA. As a result, ZnO QDs had a therapeutic effect on cancer cells.

Conclusions
In summary, we developed a safe and intelligent DDS "DOX@MSCNs-ZnO" based on "MSCNs" capping with "ZnO QDs" nanovalves, which possessed the traits of pH-responsive biodegradability and pH-responsive drug release, aiming at overcoming the crucial drawback of bioaccumulation of traditional MSNs and developing the on-demand drug release mechanism. Both ZnO QDs and MSCNs exhibited improved biodegradability in the acidic environment, endowing the DDS with pHresponsive degradation behavior in the acidic TME. Furthermore, the acid degradable ZnO QDs successfully prevented DOX in the pores of MSCNs from premature leakage in neutral environment but effectively triggered the release of DOX in an acidic environment. Besides, MSCNs-ZnO could efficiently transport DOX into cancer cells and showed enhanced cancer cells inhibition. Thus, this DDS with the excellent features of pH-responsive biodegradability, pH controlled drug release, avoiding premature drug leakage and enhancing therapeutic efficiency is expected to have a promising potential in clinical application for cancer therapy.   Figure 12b shows the viability of 4T1 cells in the presence of MSCNs, DOX@MSCNs-ZnO, DOX@MSCNs and free DOX with different concentration. It is clearly seen that as the drug carrier itself, the bare MSCNs had no obvious cytotoxicity even at the concentration of 200 µg/mL, suggesting that MSCNs have excellent biocompatibility and can act as the safe drug vehicle. MSCNs-ZnO exhibited concentration-dependent cytotoxicity, which was mainly caused by ZnO attached on MSCNs. Free DOX, DOX@MSCNs and DOX@MSCNs-ZnO all exhibited significant toxicity with the increased concentration. However, among these, the half-maximal inhibitory concentration (IC 50 ) of DOX@MSCNs-ZnO was calculated to be 4.3 µg/mL of equivalent DOX concentration, less than that of DOX@MSCNs (7.3 µg/mL) and free DOX (17.6 µg/mL). At the same DOX concentration, DOX@MSCNs-ZnO can effectively inhibit more cancer cell growth than DOX@MSCNs and free DOX, which was the result of the synergistic anticancer therapeutic effect of the released DOX and ZnO from DOX@MSCNs-ZnO.

Conclusions
In summary, we developed a safe and intelligent DDS "DOX@MSCNs-ZnO" based on "MSCNs" capping with "ZnO QDs" nanovalves, which possessed the traits of pH-responsive biodegradability and pH-responsive drug release, aiming at overcoming the crucial drawback of bioaccumulation of traditional MSNs and developing the on-demand drug release mechanism. Both ZnO QDs and MSCNs exhibited improved biodegradability in the acidic environment, endowing the DDS with pH-responsive degradation behavior in the acidic TME. Furthermore, the acid degradable ZnO QDs successfully prevented DOX in the pores of MSCNs from premature leakage in neutral environment but effectively triggered the release of DOX in an acidic environment. Besides, MSCNs-ZnO could efficiently transport DOX into cancer cells and showed enhanced cancer cells inhibition. Thus, this DDS with the excellent features of pH-responsive biodegradability, pH controlled drug release, avoiding premature drug leakage and enhancing therapeutic efficiency is expected to have a promising potential in clinical application for cancer therapy.

Conflicts of Interest:
The authors declare no conflict of interest.