Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Open AccessCCS ChemistryRESEARCH ARTICLE29 Mar 2022Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics Zhiyi Wang†, Shuren Wang†, Xiaoguang Zhang, Ziyuan Li, Zeeshan Ali, Donghai Yu, Hongtao Zhang, Fugeng Sheng, Song Gao and Yanglong Hou Zhiyi Wang† Beijing Key Laboratory for Magnetoelectric Materials and Devices, School of Materials Science and Engineering, Peking University, Beijing 100871 Institute of Spin-X Science and Technology, South China University of Technology, Guangzhou 510641 , Shuren Wang† Beijing Key Laboratory for Magnetoelectric Materials and Devices, School of Materials Science and Engineering, Peking University, Beijing 100871 , Xiaoguang Zhang Beijing Key Laboratory for Magnetoelectric Materials and Devices, School of Materials Science and Engineering, Peking University, Beijing 100871 , Ziyuan Li Beijing Key Laboratory for Magnetoelectric Materials and Devices, School of Materials Science and Engineering, Peking University, Beijing 100871 , Zeeshan Ali School of Chemical and Materials Engineering, National University of Sciences & Technology, Islamabad 44000 , Donghai Yu Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200032 , Hongtao Zhang Fifth Medical Center of Chinese PLA General Hospital, Beijing 100071 , Fugeng Sheng Fifth Medical Center of Chinese PLA General Hospital, Beijing 100071 , Song Gao Institute of Spin-X Science and Technology, South China University of Technology, Guangzhou 510641 and Yanglong Hou *Corresponding author: E-mail Address: [email protected] Beijing Key Laboratory for Magnetoelectric Materials and Devices, School of Materials Science and Engineering, Peking University, Beijing 100871 https://doi.org/10.31635/ccschem.022.202201805 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The development of smart drug delivery systems (SDDSs) based on engineered nanomaterials is important for clinical applications. Nevertheless, controllable administration of chemotherapeutic drugs for deep tumors and the avoidance of side effects caused by off-targeting during delivery remain a great challenge. Herein, a stimulus-responsive system of mesoporous nanospheres (composed of [email protected]2[email protected]2) with good magnetothermal effect is introduced into the tumor microenvironment. This system plays an important role in image-guided controllable targeted drug delivery that is independent of tumor depth. Aggregation-induced emission luminogen-based fluorescence imaging and magnetic resonance imaging were utilized since these techniques visualize the delivery process in real time. In addition, the degraded nanocarriers showed high catalytic activity for Fenton and Fenton-like reactions, upregulating the level of hydroxyl radicals (•OH) in cancer cells to realize chemodynamic therapy. The induced •OH led to the overexpression of pho-STAT3, activating the STAT3 signaling pathway, eventually inducing cancer cell apoptosis. Through metabolic monitoring, this SDDS is removed from the body after its degradation in vivo. The synergistically enhanced therapeutic effect was obtained in the chemo-chemodynamic therapy of 4T1 tumor-bearing mice, offering a platform for efficient cancer therapy with a personalized theranostic strategy. Download figure Download PowerPoint Introduction Pharmacokinetic obstacles have been a major underlying reason for the failure to treat solid tumors, according to cumulative scientific evidence in recent decades.1 It has been generally accepted that pharmacokinetic disorders can be divided into three sublevels: (1) systemic pharmacokinetic disorders, (2) intratumoral pharmacokinetic disorders, and (3) cellular pharmacokinetic disorders. As the intratumoral pharmacokinetics disorders represent a core obstacle for drug delivery,2 to achieve the optimal anticancer effect, anticancer drugs need to be delivered not only to every single micromilieu of solid tumor microregions, but also at cytotoxic concentrations. Tumor cells placed in anticancer drugs with subcytotoxic concentrations of anticancer drug have met with tumor resistance, not just treatment failure.3,4 In other words, the challenge of drug delivery within the solid tumor micromilieu is mainly attributed to the complicated biology of the intratumoral microenvironment.5 Hence, to resolve this issue, a series of stimulus-responsive nanocarrier-mediated drug delivery systems (DDSs), including endogenous stimuli-responsive nanomaterials (NMs) (such as pH, enzyme, and redox),6 exogenous stimuli-responsive NMs,7–10 and multistimuli -esponsive NMs (such as temperature, light, ultrasound, and magnetic field),11,12 have been developed over the past few decades. These nanocarrier-mediated SDDSs have shown several obvious advantages, including greater accumulation of drugs at the diseased or pathological sites, enhanced cellular uptake, prolonged circulation time, high systemic stability, and reduction of toxic effects of the encapsulated compounds on healthy tissues.13–15 All these aspects of DDSs greatly promote the controllability and efficiency of drug delivery for cancer therapy. Nevertheless, the development of DDSs is still restricted by the problems with controllable drug delivery in the deepest tissues and off-targeting in the process of drug delivery. In addition to the drug delivery efficiency mentioned above, the pharmacological effect of drugs will also affect the therapeutic effect in the treatment of major diseases. Hence, some catalytic reactions such as Fenton and Fenton-like reactions have been employed to improve that efficacy. This strategy is considered as the classical chemical reaction in the field of environmental science,16 while the core reaction is to employ Fe2+, Cu+, and other variable valence transition metal ions to catalyze hydrogen peroxide (H2O2) to produce hydroxyl radical (•OH) with strong oxidation under the acidic conditions of the tumor microenvironment (TME).17,18 This strategy, defined as chemodynamic therapy (CDT), is a new treatment strategy with great potential for clinical transformation and development.17 At present, CDT has become a leading subject of cutting-edge research for the interdisciplinary scientific community while the distinctive characteristics of Fenton-like chemistry are the core in the field of chemical biology.19 Considering the fact that the catalyst plays a decisive role in kinetics and efficiency of Fenton and Fenton-like reactions, the selective design of CDT nanocatalysts will prove to be of great significance in improving its therapeutic effect on cancer, and will be given priority to combine with other treatments to achieve a synergistic enhancement effect. As a proof of concept, we designed a special kind of SDDS with degradable mesoporous magnetic nanospheres (NSs) for personalized imaging-guided cancer therapy (Figure 1). Primarily, [email protected]2[email protected]2 NSs with rich mesoporous structures were designed and synthesized as nanocarriers. Then the doxorubicin (DOX) and synthesized aggregation-induced emission luminogen (AIEgen) of TPE-based schiff base (TPE-SB) were filled into the mesopores of the [email protected]2[email protected]2 NSs and encapsulated by phase-change materials (PCMs) (i.e., lauric acid/stearic acid (LA/SA)) to avoid off-targeting during delivery. The combination of TPE-SB-based fluorescence imaging and magnetic resonance imaging (MRI) was used to visualizethe delivery process of SDDSs. Afterward, nucleolin-specific aptamer AS1411 was used to improve the targeting of 4T1 cancer through acylation reaction on the surface of SDDSs as depicted in Figure 1a. These SDDSs showed high catalytic activity for Fenton and Fenton-like reactions, which could upregulate the level of •OH in 4T1 cells, and led to the controlled overexpression of pho-STAT3 to activate the STAT3 signaling pathway so as to induce apoptosis of cancer cells. Hence, such nanocarriers are strongly reccomneded for use in CDT (Figure 1b). Controlled release of DOX was achieved for the SDDSs in mild alternating magnetic fields (AMF). In addition, synergistic enhancement of the cancer therapeutic effect was finally achieved by chemo-chemodynamic therapy in 4T1 tumor-bearing mice. In summary, our study provides a platform for personalized therapy of cancer with smartly engineered design, high-efficiency, targeted and controlled drug delivery, and enhanced therapeutic effect. Figure 1 | (a) Fabrication of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411. (b) Schematic representation of magnetothermal effect-induced smart drug delivery system for precision tumor theranostics. Download figure Download PowerPoint Experimental Methods Synthesis of TPE-SB TPE-SB was synthesized by the aldimine condensation between 1,2,4,5-Benzenetetramine and 4-(1,2,2-triphenylvinyl)benzaldehyde (TPE-CHO). First, the hydrochloric acid in amine hydrochloride (1,2,4,5-Benzenetetramine tetrahydrochloride) was removed by extraction operation under alkaline condition; then, the above obtained 1,2,4,5-Benzenetetramine and TPE-CHO were mixed in chloroform solvent for 12 h. Finally the reaction product TPE-SB was isolated and purified by recrystallization. Synthesis of [email protected]2C nanoparticles [email protected]2C nanoparticles (NPs) were synthesized by a thermal decomposition method in oil phase. Typically, copper(II) acetylacetonate (1 mmol), 1-octadecene (ODE) (15 mL), OAm (5 mL) and NH4Br (0.1 mmol) were mixed under a gentle N2 flow for 10 min in a four-necked flask. Then the solution was heated to 120 °C for 30 min to remove the organic impurities. Fe(CO)5 (5 mmol) was injected into the reaction system when the temperature reached 180 °C for 10 min, and the system was raised up to 265 °C for another 30 min. After the system cooled down to room temperature, 27 mL of acetone was added to the system. After centrifugation, the product was washed by ethanol and hexane. Finally, the [email protected]2C NPs were dispersed in hexane. Synthesis of [email protected]2[email protected]2 NSs [email protected]2[email protected]2 NSs were synthesized by coating mesoporous silicon on the surface of [email protected]2C NPs. Typically, [email protected]2C NPs (20 mg NPs in 1 mL hexane) and triethylamine (2 mmol) were dissolved in a mixed solution of cetyltrimethylammonium chloride (CTAC) solution (25 wt %, 24 mL) and deionized water (36 mL). Then the resulting solution was heated to 60 °C for 4 h to remove the hexane. After 4 h, tetraethoxysilane (TEOS) (1 mL, dispersed in 20 mL hexane) was added dropwise into the above mixed solution and continued to react for 48 h. After the reaction, when the system reached room temperature, 50 mL of absolute ethanol was added to the mixture and then centrifuged and collected. The collected product was washed with absolute ethanol three times, and the product was finally dispersed in deionized water for storage. Synthesis of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411 [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411 was synthesized by the amidation between [email protected]2[email protected]2-LA/SA/DOX/TPE-SB and AS1411. Typically, [email protected]2[email protected]2-LA/SA/DOX/TPE-SB (50.0 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (0.12 mmol), and N-hydroxysuccinimide (NHS) (0.15 mmol) were dissolved in 10 mL of N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer (pH 7.2) with stirring at room temperature. After 4 h, AS1411 (5 OD) was added in the above mixture, and it continued to react for 24 h. Finally, the reaction product was isolated and purified by dialysis. In addition, the synthesis of [email protected]2[email protected]2-LA/SA/TPE-SB-AS1411 was consistent with the above synthesis process. Degradation experiment in vitro Degradation of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB in vitro was directly observed in the time-dependent structural evolution of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB in different pH conditions by transmission electron microscope (TEM) during the degradation evaluation. Typically, [email protected]2[email protected]2-LA/SA/DOX/TPE-SB was added into phosphate-buffered saline (PBS) buffer. To investigate the pH influence on biodegradation, the PBS buffer solution with different pH conditions was adopted (pH 7.4, 6.5, and 5.4). All the evaluations were based on the concentration of 10 mg mL−1 [email protected]2[email protected]2-LA/SA/DOX/TPE-SB. The testing solution was put into a water bath (at 37 °C) under slow magnetic stirring (i.e., at 300 rpm) for 9 days. Samples were taken at different time points (0, 1, 2, 3, 4, and 9 days), and the morphological changes of the samples were observed by TEM. Magnetothermal effect of [email protected]2[email protected]2-LA/SA/DOX 1.5 mL of [email protected]2[email protected]2-LA/SA/DOX dispersions with different concentrations (of 0, 20, 40, 60, 80, and 100 mg L−1) were tested by AMF (heating current: 9A, oscillation frequency: 45–50 kHz) for 5 min, and their temperature in solution was recorded by an online-type thermocouple thermometer. Cell culture NIH3T3 and 4T1 cell lines were obtained from the Cancer Institute and Hospital of the Chinese Academy of Medical Science. All cell-culture-related reagents were purchased from Invitrogen. RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin was used to culture cells at 37 °C under 5% CO2 with 100% humidity. Colocalization of lysosomes and NSs assay The as-prepared sample of [email protected]2[email protected]2 NSs was conjugated with. 4T1 cells (5 × 104 cells per well) which was then seeded into a glass-bottom cell culture dish (20 mm). When the cell density reached 80–90%, the cells were then incubated with [email protected]2[email protected]2 NSs for 6 h. Subsequently, 4T1 cells were counterstained with Lyso-Tracker Green (L7526, Life TechnologiesTM, United States) and 4′,6-diamidino-2-phenylindole (DAPI) (C0060, Solaribio, Beijing, China) for 15 min. Finally, the cells were washed with PBS three times for NIR-II fluorescence imaging observation through multidimensional confocal microfluorescence imaging system (FLIM+confocal+AFM, Q2, ISS-USA). Double staining of living/dead cells assay Double staining of the living/dead cells assay was measured by Calcein-AM/PI Double Stain Kit (40747ES76, Yeasen, Shanghai, China). 4T1 cells were seeded into a 24-well culture plate (2 × 104 cells per well). When the cell density reached 70%, the cells were then incubated with [email protected]2[email protected]2 NSs for 24 h. After washing out the free NPs with PBS, fresh culture medium was added. AMF (oscillation frequency: 45–50 kHz, heating current: 9 A) was then used to cover the cells for 5 min. The staining was carried out as per given instructions. The cells were finally visualized using an inverted microscope (Olympus IX71). Fluorescence imaging of reactive oxygen species generation in 4T1 cells The reactive oxygen species (ROS)-generating capabilities of control group (saline) and [email protected]2[email protected]2 NSs were assessed by dihydrorhodamine 123 (DHR123) respectively. DHR123 staining was carried out as follows: cells were incubated with saline or [email protected]2[email protected]2 NSs (60 mg L−1) for 24 h. Then, 1 μg of DHR123 was added to cell media under laser irradiation (808 nm, 0.3 W cm−2) for 1 min. Confocal laser scanning microscopy and flow cytometry were used to observe the fluorescence intensity of DHR123 (λex/em = 488 nm/520 nm, n = 3). Intracellular ROS assay Intracellular ROS assay was measured by ROS Assay Kit (S0033, Beyotime, China). 4T1 cells were seeded into a 24-well culture plate (2 × 104 cells per well). When the cell density reached 70%, the cells were then incubated with [email protected]2[email protected]2 NSs for 24 h. After washing out the free NPs with PBS, fresh culture medium was added. AMF (oscillation frequency: 45–50 kHz, heating current: 9 A) was then used to cover the cells for 5 min. The staining was carried out as per given instructions. The cells were finally visualized using an inverted microscope (Olympus IX71) or detected through flow cytometry using BD Fortessa flow cytometer (BD Biosciences). Animals and tumor model Balb/c mice four to five weeks old with an average weight of 20 g were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). An specific pathogen-free (SPF) animal house was provided to mice under a 12 h light and 12 h darkness cycle, and they were fed a standard laboratory diet and tap water ad libitum. For the subcutaneous tumor model, 4T1 cells (2 × 106 cells in 0.1 mL of saline) were injected subcutaneously into these mice. Fluorescence imaging in vivo The 4T1 triple-negative breast cancer (TNBC)-bearing mice were intravenously injected with [email protected]2[email protected]2-LA/SA/TPE-SB and [email protected]2[email protected]2-LA/SA/TPE-SB-AS1411 (20 mg kg−1, 200 μL) for fluorescence imaging in vivo. The fluorescence signal was recorded by the CRi Maestro ex in vivo imaging system (United States) at 0, 3, 6, 12, 24, and 48 h after the injection. To confirm the in vivo distribution of [email protected]2[email protected]2-LA/SA/TPE-SB and [email protected]2[email protected]2-LA/SA/TPE-SB-AS1411, mice were sacrificed 48 h post-injection. The main organs (liver, heart, lung, spleen, tumor, and kidneys) were collected for imaging and semiquantitative biodistribution analysis (n = 6). MRI in vivo The 4T1 TNBC-bearing mice were intravenously injected with [email protected]2[email protected]2-LA/SA/TPE-SB and [email protected]2[email protected]2-LA/SA/TPE-SB-AS1411 (20 mg kg−1, 200 μL) for MRI in vivo. After the injection, T2 images were obtained at 0, 6, 12, 24, and 48 h by a clinical 3.0 T MRI scanner (Philips, TR = 1200 ms, TE = 30.2 ms, slice thickness = 2.5 mm). The intensity of the MRI signal before injection was used as the control (n = 6). In vivo antitumor efficiency evaluation Mice bearing 200 mm3 4T1 TNBC were randomly divided into six groups: (1) control (only saline); (2) AMF; (3) [email protected]2[email protected]2-LA/SA/TPE-SB; (4) [email protected]2[email protected]2-LA/SA/DOX/TPE-SB; (5) [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411; and (6) [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411+AMF. Each group contained six mice. After 200 mL of saline or NPs (20 mg kg−1) were intravenously injected into 4T1 tumor-bearing mice for 1, 4, and 7 days, the mice were exposed to AMF (heating current: 9 A, oscillation frequency: 45–50 kHz) for 5 min three times. The changes in body weight and tumor volume during the 20 days of the treatment period were recorded. Histological evaluation Mice from each group were euthanized, and major organs and tumors were recovered, followed by fixing them with 10% neutral buffered formalin after 18 days of treatment. The organs were embedded in paraffin and sectioned at 5 mm. Hematoxylin and eosin (H&E) or Prussian blue staining was performed for histological examination. The slides were observed under an optical microscope (n = 6). Statistical analysis Statistical analysis was carried out by Tukey's post-hoc test with statistical significance assigned at **P < 0.01 (moderately significant), ***P < 0.001 (highly significant), and ****P < 0.0001 (highly significant). Results and Discussion Characterization of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411 The procedure for the synthesis of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411 is presented in Figure 1a while Figure 1b shows the tumor theranostic precision mechanism of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411. Initially, monodispersed core-shell structure [email protected]2C NPs were synthesized by the thermal decomposition method in the oil phase. TEM images clearly show that Cu cores are surrounded by the Fe2C domains with a thickness of nearly 3 nm (Figure 2a). The high-resolution TEM (HRTEM) analysis presented in Figure 2b evidences the lattice of spacing 2.08 Å between two (111) adjacent planes in the Cu region while the lattice spacing is 2.09Å in the Fe2C region, corresponding to the (101) planes of the hexagonal phase. Furthermore, energy dispersive X-ray (EDX) mapping of [email protected]2C NPs (presented in Supporting Information Figure S1), has also confirmed the composition and core-shell structure of [email protected]2C NPs. Subsequently, TEM and scanning electron microscopy (SEM) images [email protected]2[email protected]2 NSs after mSiO2 coating are presented in Figure 2c and Supporting Information Figure S2a. From these images, the estimated total size of [email protected]2[email protected]2 NSs is ∼65 nm. TEM images depicted in Supporting Information Figures S2b and S2c show characteristic lattice spacing of [email protected]2C NPs, which again reassures us that the NPs coated with mSiO2 are [email protected] NPs. EDX mapping and energy dispersive spectroscopy (EDS) line scan of [email protected]2[email protected]2 NSs are shown in Figures 2d and 2e, respectively, which has confirmed the composition and core-shell structure of [email protected]2[email protected]2 NSs. It is important to note that the X-ray diffraction (XRD) results presented in Figure 2f are consistent with TEM results. The X-ray photoelectron spectroscopy (XPS) of Cu 2p and Fe 2p (Figures 2g and 2h and Supporting Information Figures S3 and S4) has confirmed the existence of Cu0 and Fe0 in [email protected]2C NPs and [email protected]2[email protected]2 NSs.20,21 The specific surface area of [email protected]2[email protected]2 NSs is 424.156 m2 g−1 ( Supporting Information Figure S5a) while the average pore size distribution of [email protected]@mSiO2 NSs is ∼5.625 nm after simulation calculation and analysis ( Supporting Information Figure S5b). We also synthesized the PCMs LA/SA before loading the drug into the mesopores of [email protected]2[email protected]2 NSs. As shown in Supporting Information Figure S6, when the mass ratio of LA to SA reaches 88:12, the onset melting point of the PCMs LA/SA is 37.9 °C, which is very favorable for the controllable release of drugs in vivo by inducing the magnetothermal effect of nanosystems through mild AMF. In addition, we amidated 1,2,4,5-tetraaminobenzene with TPE-CHO by organic synthesis to obtain TPE-SB ( Supporting Information Figure S7). This organic synthetic product was characterized and verified by 1H NMR ( Supporting Information Figure S8) and Fourier transform infrared (FT-IR; Supporting Information Figures S9 and S10). Before and after the formation of the amide bond, the position of the carbon-based stretching vibration absorption peak shifted significantly in the direction of a high wave number ( Supporting Information Figure S10a). This result confirmed that the above reactants were amidated, and the TPE-SB were prepared. Later, an appropriate amount of TPE-SB and DOX were evenly mixed at a high temperature (50 °C) with PCMs LA/SA and then filled into the mesopores onto the surface of [email protected]2[email protected]2 NSs by vacuum impregnation to form [email protected]2[email protected]2-LA/SA/DOX/TPE-SB. In addition, we covalently grafted AS1411 on the surface of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB through amidation reaction to realize the active targeting ability of [email protected]2[email protected]2-LA/SA/DOX/TPE-SB-AS1411. It should be noted that the AS1411 has been developed and well known for treating nucleolin-overexpressed metastatic renal cell carcinoma in phase II trials.22,23 Moreover, as a small-molecule drug transporting agent, it has been widely utilized in targeted cancer therapy.24–27 The realization of covalent grafting was determined by the high wavenumber movement of the stretching vibration peak of the carbonyl of amide bond in the FT-IR spectrum ( Supporting Information Figure S10b). Figure 2 | Characterization of [email protected]2[email protected]2 NSs. (a) TEM image of [email protected]2C NPs. (b) HRTEM image of [email protected]2C NPs. (c) TEM image of [email protected]2[email protected]2. (d) EDS-mapping of [email protected]2[email protected]2 NSs. (e) EDS line scan of [email protected]2[email protected]2 NSs. (f) XRD patterns of [email protected]2C NPs and [email protected]2[email protected]2 NSs. (g) High-resolution XPS of Cu 2p for [email protected]2[email protected]2 NSs. (h) High-resolution XPS of Fe 2p for [email protected]2[email protected]2 NSs. Download figure Download PowerPoint Biodegradation of [email protected]2[email protected]2-LA/SA in vitro The biodegradation ability of [email protected]2[email protected]2-LA/SA was evaluated by pH-dependent (pH 7.4, 6.5, and 5.4) TEM imaging after 9 days (Figure 3a and Supporting Information Figure S11). There was no visible degradation of [email protected]2[email protected]2-LA/SA under these conditions till day 1. But, after 2 days, with the prolonged dispersion of [email protected]2[email protected]2-LA/SA in the PBS buffer (pH 5.4), more obvious degradation was evident than in the other two groups (pH 7.4 and 6.5). We also measured the hydrodynamic diameter of [email protected]2[email protected]2-LA/SA under three groups of pH conditions for different times by dynamic light scattering (DLS) (Figure 3b), which was consistent with the above results of TEM. These conforming results prove that [email protected]2[email protected]2-LA/SA can degrade in a certain time under acidic conditions. The biodegradation behavior of [email protected]2[email protected]2-LA/SA was further evaluated in 4T1 cells. After 48 h of intracellular coincubation, [email protected]2[email protected]2-LA/SA was almost completely degraded into ultrasmall NPs in the lysosomes of 4T1 cells. These results are shown in the bio-TEM images in Figure 3c. It is understood that the pH of the tumor extracellular microenvironment is about 7.2–6.5 depending upon the type and stage of tumor while pH of intracellular early endosome and lysosome reaches 6.2–5.0. The TEM observations and variation curve of dynamic particle size for [email protected]2[email protected]2-LA/SA provide direct evidence that the acidic condition promotes fast biodegradation of –Si–O–Si– bonds. PCMs LA/SA in mesoporous silica channels can finally be degraded into water and carbon dioxide through lysosomes. The core [email protected]2C NPs also degrade to a certain extent under acidic conditions to achieve ionization. In conclusion, the biodegradability of the [email protected]2[email protected]2-LA/SA has undoubtedly been validated through these experiments. Based on the above experimental results, the degradation process of [email protected]2[email protected]2-LA/SA is illustrated in Figure 3d. First, the heat generated by the magnetothermal effect of the [email protected]2[email protected]2 NSs is used to drive the LA/SA in the mesopores from the solid phase to the liquid phase so as to separate from the NSs; Then the mesoporous part of the NSs disintegrates slowly under the condition of weak acid. Finally, the degraded NSs are reduced in the presence of glutathione (GSH) in the TME, resulting in the release of Cu+ and Fe2+. Figure 3 | Degradability evaluation of [email protected]2[email protected]2-LA/SA in vitro. (a) TEM observations of [email protected]2[email protected]2-LA/SA under three different pH conditions (pH 7.4, 6.5, and 5.4) over 9 days. (b) Variation curve of dynamic particle size for [email protected]2[email protected]2-LA/SA which measured by DLS. (c) Bio-TEM imaging of [email protected]2[email protected]2-LA/SA in 4T1 cell. (d) Degradation process diagram of [email protected]2[email protected]2-LA/SA. Download figure Download PowerPoint Biological performance evaluation at cellular level The above results reveal unique degradation characteristics of the NSs under weak acid conditions. After the degradation of nano-microspheres, the low valence Cu and Fe (mainly including Fe0 and Cu0) in [email protected]2[email protected]2 NSs may get exposed. The particular reaction equations of corresponding Fenton and Fenton-like reaction processes can be written as: Fe 0 → Oxide Fe 3 + → [ GSH ] Fe 2 + (1) Cu 0 →