“Block and attack” strategy for tumor therapy through ZnO2/siRNA/NIR‐mediating Zn2+‐overload and amplified oxidative stress

Intracellular zinc ion (Zn2+) accumulation disrupts the Zn2+ homeostasis, providing an ion‐overloading anticancer strategy with great potential. The self‐adaptation of tumor cells to ion concentration, however, puts forward higher requirements for the design of ion‐overloading strategy. Herein, “block and attack” antitumor strategy was applied through a composite nanomaterials (UHSsPZH NPs). The strategy demonstrated powerful ion interference ability through both “blocking” the efflux of excess Zn2+ via gene silencing and “attacking” tumor cells via target delivery of ZnO2. After cellular internalization, ZnO2 was degraded to Zn2+ and hydrogen peroxide (H2O2), and the gene expression of zinc transporter 1 (ZnT1) was silenced by targeting of released siRNA, which together caused intracellular Zn2+‐overload. Disorder of Zn2+ further interfered with intracellular Ca2+ homeostasis, inhibited the electron transport chain and promoted the production of endogenous reactive oxygen species (ROS), which assisted the “attack” to tumor cells together with the exogenous ROS generated by UHSsPZH NPs under 980 nm laser irradiation. In summary, this work supplies a “block and attack” strategy for the application of ion homeostasis interference in tumor therapy.


INTRODUCTION
Ion interference therapy, as a potential cancer treatment strategy, changes the physical and chemical properties and biological functions of cancer cells by accelerating the imbalance of osmotic pressure, pH or redox, further inducing functional protein inactivation as well as initiation of apoptotic pathways. [1][2][3][4] However, when ion distribution is abnormal, tumor cells can regulate intracellular in the distribution of ions and exceed the threshold of self-regulation. [9,10] So far, a variety of ion interference methods, including Ca 2+ , Cl − , Fe 2+ /Fe 3+ and Mn 2+ , have been successfully applied for anti-tumor therapy through combining interference of channel inhibitors and ion donors. [11][12][13][14] Zinc ions (Zn 2+ ) is a key auxiliary component in key metabolic regions of several enzyme molecules involved in protein synthesis, such as ribose nucleic acid (RNA) polymerase. [15] Excess Zn 2+ could block over-active calcium signal, thus selectively inhibiting the growth of cancer cells without affecting normal cells. [16] In addition, excess Zn 2+ is potentially toxic for tumor cells through promoting production of endogenous reactive oxygen species (ROS) and destroying mitochondrial membrane potential. [17][18][19] Therefore, the dyshomeostasis of Zn 2+ could be adopted as an important anticancer strategy. Zinc peroxide (ZnO 2 ) NPs have been proven to cause zinc uptake and overloading of tumor cells by endocytosis, resulting in a series of cell signal changes and apoptosis of tumor cells, which cannot be achieved by other forms of Zinc such as ZnCl 2 . [20] ZnO 2 could be decomposed to be hydrogen peroxide (H 2 O 2 ) and Zn 2+ under tumor microenvironment (TME), which induces Zn 2+ dyshomeostasis and promotes the production of endogenous ROS simultaneously, further resulting in oxidative stress. [20][21][22] In summary, ZnO 2 shows great potential in Zn 2+ interference strategy of tumors and provides exogenous H 2 O 2 to promote the generation of endogenous ROS induced by mitochondrial oxidative stress. The specific mechanism of tumor death associated with the accumulation and overloading of Zn 2+ , however, still needs further discussion and experimental verification. [23][24][25] Tumor cells show high adaptability to "illegal invasion" of ions through the regulation of channel proteins on the cell membrane. [26] Specifically, intracellular zinc levels are tightly regulated by two families of zinc transporters: Zrt/Irtrelated protein (ZIP) and zinc transporter (ZnT). [27] ZIP introduces Zn 2+ into the cytoplasm from the extracellular environment or organelle lumen. [28,29] And ZnT1, as the only Zn 2+ efflux transporter on the plasma membrane, plays an important role in maintaining zinc homeostasis. [30,31] When the intracellular Zn 2+ increases, tumor cells will accelerate zinc ion efflux through upregulation of ZnT1 channel. [32] The expression inhibition of ZnT1 could significantly promote the intracellular accumulation and overloading of Zn 2+ . Small interfering ribose nucleic acid (siRNA), showing high specificity and low toxicity, could silence targeting gene precisely after cell delivery, providing great potential for gene therapy of various diseases including cancer. [33][34][35][36][37] Inhibition of the expression of targeted proteins by gene silence of siRNA has become a promising technology for the treatment of cancer and other diseases. [38,39] Therefore, the development of siRNA transport vectors to effectively inhibit ZnT1 expression supplies an effective means to induce intracellular zinc accumulation of tumors.
In this study, a multifunctional composite upconversion nanoparticles-hypericin@mesoporous silica@polyminesmall interfering ribonucleic acid@zinc peroxide@ hyaluronic acid (UCNPs-Hy@mSiO 2 @PEI-siRNA@ ZnO 2 @HA, UHSsPZH nanoparticles [NPs]) were prepared for "block and attack" strategy of tumor treatment (Scheme 1). Hypericin as a probe of fluorescence imaging to provide accurate and effective guidance for tumor treatment [40,41] (3-isocyanatopropyl)triethoxysilane (TESPIC) premodified hypericin (Hy) were combined with mesoporous silica (mSiO 2 ) that coated upconversion NPs (UCNPs) to achieve photodynamic therapy (PDT) simultaneously. [42][43][44] In addition, the siRNAs were more conducive to be adsorbed after modification of polymine (PEI). The coating of ZnO 2 blocked the leakage of siRNA and provided Zn 2+ and H 2 O 2 after disruption under acidic condition (pH = 6.5). Hyaluronic acid (HA) not only enabled the nano-platform to target highly-expressed CD44 receptor in tumor cells but also achieved charge reversal to allow the NPs easier to get into tumor cells. [45][46][47] After ZnO 2 was disrupted, siRNA was released into tumor cytoplasm to silence the gene expression of ZnT1 protein and further interfere with the homeostasis of Zn 2+ in tumor cells. It is noteworthy that over-loaded Zn 2+ can increase mitochondrial superoxide anion (O 2⋅− ) and H 2 O 2 production by inhibiting the electron transport chain, which further produce effective oxidative stress-induced cell damage during tumor therapy. In summary, the fluorescence imaged UHSsPZH NPs supplied a typical "block" and "attack" model material for anticancer strategy through highly efficient Zn 2+ homeostasis destruction and gene silence of ZnT1 protein, which are expected to be applied for other ion interference methods to achieve biomedical applications.

Characterization of multifunctional composite
The preparation process of UHSsPZH NPs are displayed in Scheme 1A, and the UCNPs were synthesized according to the method previously reported. [48] Transmission electron microscope (TEM) in Figure S1 proved the successful preparation of the uniformly dispersed UCNPs with an average particle size of 22.78 ± 0.07 nm. Uniform silica layer of coreshell UCNPs-Hy@mSiO 2 NPs (UHS NPs) was about 14 nm thickness ( Figure 1A and Figure S2). The significant changes of morphology and size between UHSsPZ NPs (60.67 ± 8.57 nm) and UHS NPs suggested the successful coating of ZnO 2 ( Figure 1B and Figure S3). The lattice spacing in the high-resolution TEM image ( Figure 1C) of UHSPsZ NPs was 0.265 and 0.3038 nm, which represented the crystal diffraction peak of ZnO 2 (221) and (210), respectively. The TEM image of UHSsPZH NPs ( Figure 1D) and element mappings of Na, F, Er, Y, Yb, Si, O, C, and Zn in UHSsPZH NPs ( Figure 1E) further revealed the element composition and core-shell structure of UHSsPZH NPs. Moreover, the successful coating of SiO 2 and ZnO 2 on the UHSsPZH NPs was directly demonstrated by the energy dispersive spectroscopy spectrum ( Figure S5). In the X-ray diffraction pattern of UCNPs@mSiO 2 @ZnO 2 NPs (USZ NPs) ( Figure S4), the representative diffraction peaks of UCNPs were identified with β-NaYF 4 (JCPDS: 28-1129) standard card, which also confirmed the successful synthesis of UCNPs. [49,50] In addition, the characteristic peaks at 2θ = 30 • and 2θ = 32 • indicated the synthesis of SiO 2 and ZnO 2 respectively. [21] The strong red and green emission peaks at 980-nm excitation in the up-conversion luminescence spectra of UCNPs ( Figure 1F) were due to the transitions of 4 F 9/2 → 4 I 15/2 and 2 H 11/2 / 4 S 3/2 → 4 I 15/2 on Er 3+ . [ in the TME to "attack" cells but also "block" efflux of excess Zn 2+ by tumor cells through gene silencing showed certain absorption effect in the wavelength range of 550 nm, which was combined with peak attenuation ( Figure S6). The changes of surface potential obtained by zeta potentiometer also reflected the preparation process of UHSsPZH NPs ( Figure 1G). Positively charged and mesoporous UCNPs-Hy@mSiO 2 -PEI NPs (UHSP NPs) was conducive for the encapsulation of negatively charged siRNA [53] . Moreover, negatively charged HA was not only beneficial for transportation of UHSsPZH NPs in the blood but also helps the NPs achieve potential reversal and thus enhances the endocytosis of tumor cells when the NPs are targeted to the surface of tumor cell membrane [45][46] . The N 2 adsorption/desorption isotherms of UCNPs@mSiO 2 NPs (US NPs) ( Figure S7) exhibited a typical type IV isotherm, further demonstrating the mesoporous structure of US. The specific surface area was calculated to be 123.104 m 2 ⋅g −1 , which could ensure sufficient space for drug adsorption. In addition, thermogravimetric analysis ( Figure 1H) indicated that the drug loading rate of Hy in UHS was 38%.

Dissociation and ROS generation behavior of UHSsPZH NPs
The pH-dependent dissociation behavior of ZnO 2 at the acidic environment (pH 6.5) of TME was investigated in this study. In particular, ZnO 2 can react with H + to form H 2 O 2 and Zn 2+ in acid environment (Figure 2A and 2B), and UHSsPZH NPs can release more H 2 O 2 and Zn 2+ in higher speed with the increase of acid concentration. In addition, the stability of UHSsPZH NPs in phosphate buffer solution (PBS) at different pH values (pH = 6.5 or 7.4) was used to investigate the safety of NPs during blood transport ( Figure  S8). UHSsPZH NPs showed satisfactory stability even after dispersion in PBS (pH = 7.4) for 12 h, while the ZnO 2 coating was broken after dispersion in PBS (pH = 6.5) for 12 h. Therefore, the accumulation of Zn 2+ from ZnO 2 NPs solution was due to the responsive degradation of NPs under acid condition ( Figure 2C). According to electron spin resonance (ESR) analysis, the characteristic signals of ⋅OH (1:2:2:1) and 1 O 2 (1:1:1) of UHSsPZH NPs under 980 nm irradiation (1.0 W⋅cm −2 ) were observed ( Figure 2D and 2E), demonstrating the simultaneous contribution of hypericin and ZnO 2 to generation of free radical. The fluorescence spectra of single oxygen sensor green (SOSG) showed that with the extension of laser irradiation (980 nm, 1.0 W⋅cm −2 ), more and more 1 O 2 could be produced by UHSsPZH NPs through PDT type II pathway ( Figure 2F).

Determination of gene silencing and the concentration of Zn 2+
The siRNA-ZnT1 I/II/III with transfection rate over 80% were respectively designed to inhibit the expression of ZnT1  Figure S9). The western blotting bands in the siRNA-ZnT1 III group were obviously the weakest ( Figure 3A), suggesting the best inhibitory effect of siRNA III on ZnT1, and ZnT1 inhibition rate of siRNA III reached to be 71% ( Figure 3B). This was further confirmed by real-time quantitative polymerase chain reaction, which showed the most obvious inhibition effect of siRNA-ZnT1 III on ZnT1 gene ( Figure 3C). Therefore, the siRNA-ZnT1 III (sense(5′-3′): CGCCAACAGUUAGCAUUUCUUTT, antisense(5′-3′): AAGAAAUGCUAACUGUUGGCGTT) was utilized for preparation of UHSsPZH NPs. Strong green fluorescence signal of UHSsPZH NPs with siRNA-FITC could be observed in 4T1 cells after endocytosis, indicating that siRNA of UHSsPZH NPs were effectively transmitted into tumor cells ( Figure 3D). Calcein acetoxymethyl ester/propidium iodide (Calcein AM/PI) staining of 4T1 cells proved that no signifi-cant lethal effect was achieved when tumor cells were treated with only ZnO 2 NPs even under 50 µg⋅mL −1 ( Figure 3E), suggesting excellent self-regulate ability of 4T1 cells. With the assistance of siRNA, however, 10 µg⋅mL −1 ZnO 2 had induced severe damage of 4T1 cells ( Figure 3E). The damaged cells were then collected and lysed, and inductively coupled plasma (ICP, Figure 3F) showed that the intracellular concentration of Zn 2+ reached 5.35 µmol⋅L −1 under the simultaneous treatment of ZnO 2 and siRNA. Therefore, combined with the results of immunofluorescence histochemistry and biotransmission electron microscopy (Bio-TEM), it could be inferred that obvious apoptosis of tumor cells and change of cell permeability occurred when the cells were treated with 10 µg⋅mL −1 ZnO 2 and siRNA, which promoted the intracellular concentration of Zn 2+ to be higher than 5.35 µmol⋅L −1 . Zinquin was used to detect the content of intracellular Zn 2+ , and blue fluorescence intensity was positively correlated with the concentration of Zn 2+ ( Figure 4A, Figures S10 and S11). There was no significant change of intracellular Zn 2+ concentration between USsPH group and control group, suggesting no significant change of intracellular Zn 2+ concentration when the cells were treated by siRNA alone. Moreover, the blue fluorescence in USsPZH group and UHSsPZH group was obviously enhanced, proving obvious increase of the intracellular Zn 2+ concentration, which could be further promoted through the responsive release of ZnO 2 and the gene silencing of siRNA-ZnT1 synergistically. 2′, 7′-dichlorodihy drofluorescein diacetate (DCFH-DA) was coincubated with 4T1 cells to verify intracellular ROS levels ( Figure 4B, Figures S12 and S13). The promotion of the release of ROS by PDT could be confirmed by strong green fluorescence intensity of UHSH + Laser group ( Figure 4B). The green fluorescence intensity of UHSsPZH + Laser group was strongest, proving that the participation of ZnO 2 could further promote ROS release. After treatment by group III, VI and IV, siRNA showed significant inhibitory effect on ZnT1 protein ( Figure 4C), which would lead to series of intracellular metabolic disturbance triggered by zinc disruption. Orai1, an overexpressed Ca 2+ entry channel in tumor tissue, could promote Ca 2+ oscillation and cell proliferation of tumor tissue. According to Figure 4D, the expression of Oria1 in the USsPZH group and UHSsPZH + Laser group was down-regulated significantly, indicating obvious distruction of zinc overload on calcium homeostasis. Caspase-dependent apoptosis pathway was also significantly activated, which demonstrated that the apoptotic process was initiated by treatment of UHSsPZH + Laser ( Figure 4E).

Intracellular Zn 2+ -overload and production of endogenous ROS
Cystine residues play important roles in proteins, including binding to various metal ions. [54][55][56] In particular, cysteine residues have a high affinity for Zn 2+ , and the Zn 2+ -cysteine complexes play a key role in mediation of protein structure, catalysis and regulation ( Figure 4F). [57,58] Mutations in cysteine may distort the conformational structure of Oria1, leading to down-regulation of Oria1 expression ( Figure 4G). L-Cystine metabolites were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS), and the down-regulation of free cysteine proved that Zn 2+ had prominent chelating effect on cysteine, and it was inferred that Zn 2+ could directly interfere with the normal physiological functions of proteins rich in cysteine residues ( Figure 4H). Meanwhile, during the differential metabolite screening analysis of intracellular amino acids and organic acids with their derivatives, it was showed that the metabolites with fold change (FC) ≥ 2 and FC ≤ 0.5 were significantly different. Interestingly, out of 94 groups of metabolites, 42 groups of amino acids, organic acids and their derivatives showed significant differential expression (Table S1, Figures S14-S18). Amino acids, such as ornithine, citrulline, high arginine, histidine glycine, glutamate, alanine, leucine, lysine, tryptophan, threonine, serine and proline, were all down-regulated. In addition, the overall expression of this pathway (biosynthesis of amino acids, biosynthesis of aminoyl-tRNA, biosynthesis of cofactors, protein digestion and absorption and central carbon metabolism in cancer, etc.) tend to be down-regulated ( Figure S19). Therefore, it was inferred that Zn 2+ -overload may further interfere with the metabolic pathway of amino acids and induce the down-regulation of amino acid expression, thus realizing the starvation of tumor cells. In addition, the expression of ZnT1 protein in the cells co-incubated with UHSsPZH NPs with 980-nm laser at different times (0, 3, 6, 9, 12, 24 h) was monitored by the western bloting ( Figures  S20A and S20B). The ZnT1 protein in the cells co-incubated with UHSsPZH NPs was slightly silenced at 9 h. Nevertheless, the concentration of Zn 2+ could reach the maximum at 9 h in the cells ( Figure S20C). On the contrary, the silencing effect of ZnT1 protein was excellent at 24 h, but the intracellular concentration of Zn 2+ decreased at this time, which was due to increased membrane permeability and Zn 2+ outflow of the 4T1 cells. In summary, UHSsPZH NPs not only supplied enhanced PDT to release ROS under 980 nm laser with H 2 O 2 produced by ZnO 2 but also inhibited ZnT1 expression by gene silencing, thereby inducing intracellular Zn 2+ -overload and further leading to mitochondrial membrane damage and production of endogenous ROS. Finally, tumor apoptosis was rapidly accelerated by Zn 2+ -overload, and the attack toward tumor cells could be promoted through PDT oxidative stress amplification and Ca 2+ homeostasis destruction ( Figure 4I).

Evaluation of cytotoxicity and biosafety
Bio-TEM was used to observe the endocytosis and exocytosis of UHSsPZH NPs as well as the resulting morphological and structural changes of organelles ( Figure 5A and 5B). Without treatment of NPs, organelles of the 4T1 cells in the control group were intact and clearly defined ( Figure 5A), while vacuolation of mitochondria and fragmentation of organelles were observed in the UHSsPZH group. In addition, the structure change of UHSsPZH NPs co-incubated with the physiological environment of the tumor site could be observed through Bio-TEM ( Figure S21), which further proved that UHSsPZH NPs could target into 4T1 cells and respond to the TME environment. Mitochondrial membrane potential change and mitochondrial damage were further analyzed through double labeling of mitochondria and nucleus by Mito Tracker Red CMXRos and Hoechst33341 ( Figure 5C). The red fluorescence intensity was positively correlated to the mitochondrial membrane potential. Compared with the control group, the red fluorescence intensity of USsPZH group and UHSsPZH + Laser group was significantly weaker and the membrane potential decreased obviously. While there was no obvious change detected in UHSH + Laser group, suggesting that decrease of membrane potential was mainly caused by Zn 2+ -overload. Survival rate of human umbilical vein endothelial cells (HUVECs) treated with different concentration of UHSsPZH NPs (125 µg⋅mL −1 , 250 µg⋅mL −1 , 500 µg⋅mL −1 , 1 mg⋅mL −1 , 2 mg⋅mL −1 ) was investigated ( Figure 5D) to evaluate the biosafety of UHSsPZH nano-platform. When the concentration of NPs was lower than 2 mg⋅mL −1 , survival rate of HUVEC cells was more than 70%, demonstrating low toxicity and good biosafety of UHSsPZH NPs in normal cells without laser irradiation. Furthermore, the cytotoxicity of USZH, USsPH, USsPZH, UHSH + Laser, and UHSsPZH + Laser on the 4T1 cells was dose-dependent ( Figure 5E). Compared with control group (PBS + Laser, 1.0 W⋅cm −2 , 10 min), the viability of 4T1 cells in the UHSH + Laser group was significantly reduced, which was mainly due to the PDT effect of UHSH NPs. The viability of 4T1 cells treated with 1.0 mg⋅mL −1 and 2.0 mg⋅mL −1 UHSsPZH reached respectively to be 33% and 17.0% under 980 nm irradiation, which was the combining results of the combination of oxidative stress and Zn 2+ overload. Therefore, taking into account of both biosafety and antitumor efficacy, 1 mg⋅mL −1 was chosen as the optimal concentration of UHSsPZH NPs to evaluate in vivo therapy. Calcein AM/PI staining of live/dead 4T1 cells with different treatment groups exhibited the killing effect of different NPs ( Figure 5F). Compared with UHSH + Laser group, red fluorescence intensity in UHSsPZH + Laser group were significantly increased, confirming satisfactory anti-tumor effect of UHSsPZH NPs.

Evaluation of oxidative stress
Under oxidative stress, phospholipids, and cholesterol esters containing polyunsaturated fatty acids in cell membranes and lipoproteins can be easily oxidized and corresponding oxidation products formed through lipid peroxidation (LPO) induced by free radicals. 4T1 cells showed high activity and low permeability of cell membrane under treatment of 200 µg⋅mL −1 UHSsPZH NPs ( Figure S22). When under 1 mg⋅mL −1 , however, PI probe could pass through the disordered cell membrane with increased permeability, presenting a strong red fluorescence of nucleus. Significant up-regulation of LPO in 4T1 cells after treatment of UHSsPZH NPs ( Figure 6A) resulted in further increased oxidative stress. Downstream metabolites of oxidized lipids, including arachidonic acid, linoleic acid (LA), A-linolenic acid (ALA), docose hexaenoic acid, eicosapentaenoic acid, and dohomo-γ-linolenic acid, were analyzed through LC-MS/MS platform. Among the 141 kinds of metabolites detected, the contents of differential metabolites of 72 kinds (Table S2) were significantly downregulated ( Figure 6B). The correlation network diagram of the differential metabolites ( Figure 6C) showed that the size of the point was correlated with the degree of connection, and red indicated positive correlation. Z-Score map, which was used to normalize the differential metabolites of different samples, showed significant downregulation of oxidized lipids with the treatment of the UHSsPZH NPs under 980 nm laser ( Figure 6D). All the differences of metabolic pathways abundance and chart are shown in Figure 6E, in which downregulation of metabolites expression could be identified when the differential abundance score was −1. Therefore, under the treatment of UHSsPZH NPs, the increase of cell membrane permeability further induced disorder of the cell membrane, disruption of cellular structure as well as serious outflow of intracellular metabolites.

Evaluation of antitumor effect of "attack and block" strategy in vivo
4T1 tumor model was established to study the in vivo antitumor effect of UHSsPZH NPs ( Figure 7A). When the tumor diameter reached about to be 12 mm, 150 tumor-bearing mice were randomly divided into six groups for treatment. Before treatment, UHSsPZH NPs were intravenously injected into 4T1 tumor-bearing Balb/c mice for detection of fluorescence imaging of hypericin ( Figure 7B), which reached the peak at 6 h-9 h after intravenous injection, providing guidance for specific treatment options. Except for tumor, UHSsPZH NPs mainly accumulated in liver and spleen due to the clearance of reticuloendothelial system (RES). ICP mass spectrometry (ICP-MS) was applied to analyze the concentration of Y element (the characteristic element of UHSsPZH NPs) in the homogenate of organ tissues (kidney, liver, spleen, and tumor), which provided reliable evidence about the metabolic pathway of UHSsPZH NPs ( Figure S23). In addition, the total amount of Y element decreased gradually from 24 to 72 h after injection and the concentration of Y in the liver increased, proving that the UHSsPZH NPs could be gradually metabolized and removed, and the main metabolic pathway maybe through the liver. Representative images of 4T1 tumor-bearing mice in different groups on the 6th and 15th day ( Figure 7C and Figure S24) showed that significant elimination of the tumor could be observed on the 15th day through treatment of UHSsPZH NPs under 980-nm irradiation (1.0 W⋅cm −2 , 10 min), which was consistent with evaluation of tumor mass and relative tumor volume ( Figure  7D and 7E). In addition, there was no significant change of body weight in different groups, and UHSsPZH + Laser treatment significantly extended survival time ( Figure 7F and 7G). Hematoxylin and eosin staining assay as well as terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick  Figure S25). No pathological changes of the heart, liver, spleen, lung, and kidney were observed ( Figure S26) in UHSsPZH + Laser group. Blood biochemical and blood routine analysis further proved good biosafety of UHSsPZH NPs ( Figures S27, S28), which was also confirmed by HE staining of organ tissues on 15th days after treatment of UHSsPZH + Laser ( Figure S29). Therefore, good tumor targeting and in vivo therapeutic effect could be achieved by the UHSsPZH nano-platform under the premise of low cytotoxicity, which is important for the clinical application of Zn 2+ -interference therapy.
To verify Zn interference of UHSsPZH NPs and subsequent tumor apoptosis during in vivo therapy, the expression of ZnT1, Orai1 and caspase-3 in tumor tissues were detected. The expression of ZnT1 in tumor tissues was obviously inhib-ited by USsPH, USsPZH and UHSsPZH + Laser ( Figure 8A and 8D), which was mainly due to the gene silence of siRNA. Zn 2+ -overload caused by ZnO 2 supply and inhibition of ZnT1 expression could significantly up-regulate the expression of Orai1, confirming the inhibition of Zn 2+overload by UHSsPZH NPs (Figure 8B and 8E). Consistent with the in vitro results, UHSsPZH could further promoted the up-regulation of caspase-3 ( Figure 8C and 8F), demonstrating the initiation of the tumor apoptosis. In addition, western blotting and quantitative analysis of ZnT1, Orai1, and Caspase-3 in tumor tissues was consistent with the results of fluorescence immunohistochemistry ( Figure 8G-L). The above experimental results indicated that UHSsPZH could effectively promote Zn 2+ overload through "attack" and "block" pathways in both in vitro and in vivo treatment, and finally achieved the resulting tumor metabolic disorder and cell apoptosis.

CONCLUSION
In this study, a composite UHSsPZH nanoplatform with both offensive and defensive capabilities was designed by promoting zinc overload and inhibiting zinc outflow. The efficient release of ZnO 2 , hypericin and siRNA at the tumor site can be realized through the response of ZnO 2 to TME by NPs, which is not only conducive to tumor targeting but also effectively avoids drug leakage during blood transport of NPs. ZnO 2 not only promotes zinc overload but also further enhances the photodynamic effect of hypericin through H 2 O 2 , which together achieves "attack" on tumor cells. The inhibition of zinc ion efflux was successfully achieved by the participation of siRNA targeting ZnT1 inhibition. In conclusion, the development of "attack and block" strategy significantly improves the efficacy of ion interference therapy for tumor and provides a new idea and clinical application paradigm for effective ion interference therapy. Laboratory Animals. The 4T1 murine breast cancer cells line was purchased from Procell Life Scientific and Technology Co., Ltd., China (number: CL-0007).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in wiley at https://doi.org/10.1002/agt2.321.

E T H I C S S TAT E M E N T
This study involved 150 healthy female Kunming mice. All animal experiments were carried out following the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory Animals (NIH publications number: 8023, revised 1978). During biopsy, anesthesia was used, and animals were decapitated when necessary. All efforts were made to minimize animal suffering. All animal experiments and the research project hereby were approved by the Ethics Committee of Qingdao Agricultural University (approval number: 20220068).