In Situ Autophagy Disruption Generator for Cancer Theranostics | ACS

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Biological and Medical Applications of Materials and Interfaces

An in Situ Autophagy Disruption Generator for Cancer Theranostics Huijuan Zhang, Yanping Ren, Fang Cao, Jianjiao Chen, Chengqun Chen, Junbiao Chang, Lin Hou, and Zhenzhong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10578 • Publication Date (Web): 31 Jul 2019 Downloaded from pubs.acs.org on August 1, 2019

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ACS Applied Materials & Interfaces

An in Situ Autophagy Disruption Generator for Cancer Theranostics Huijuan Zhang a,b,c, Yanping Rena, Fang Caoa, Jianjiao Chena, Chengqun Chend, Junbiao Chang c, Lin Hou *,a,b,c and Zhenzhong Zhang*,a,b,c

a

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China

b

Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province

c

Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou,

China d

Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University

* Corresponding Author: Email address: [email protected] (Zhenzhong Zhang), [email protected] (Hou Lin); Tel.: +86 371 6778 1910. Fax: +86 371 6778 1908. Mailing address: No.100, Kexue Road, Zhengzhou 450001, P. R. China

ABSTRACT: Cancer remains a serious clinical disease awaiting new effective treatment strategies. Autophagy modulation has emerged as a novel and promising pharmacologic target critical to future drug development and anti-cancer therapy applications. Herein, we constructed an in-situ autophagy disruption generator to break the balance of autophagy flow for tumor targeting therapy. Hollow mesoporous manganese trioxide (Mn2O3) nanoparticles (NPs) were synthesized and conjugated with hyaluronic acid (HA) to form tumor targeting drug carriers. Then traditional autophagy inhibitor hydroxychloroquine (HCQ) was loaded into the hollow core of HA-Mn2O3, to form a multifunctional theranostics platform (HA-Mn2O3/HCQ). This nanoplatform displayed specific localization and retention in lysosomes after entering tumor cells. The synchronous release of HCQ and manganese ion (Mn2+) induced lysosomal alkalization and osmotic pressure elevation. 1

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Significantly greater lysosomal deacidification and autophagy blockade effect emerged after treatment by this nanoplatform, with in vitro tumor inhibition rate of 92.2%. Imaging experiment proved that it could selectively deliver HCQ to tumor sites and further degraded to realize simultaneous release of Mn2+ and HCQ. Micromorphological and immunofluorescence analysis demonstrated that in situ high concentrations of these two substances would achieve effective autophagy blockade. Pharmacodynamics test showed this nanogenerator displaying the best therapeutic efficacy with 5.08-folds tumor inhibition ratio compared with HCQ group. Moreover, generated Mn2+ can be used as T1 contrast agent for visualizing tumor lesions and monitoring therapeutic effects. Overall, the as-made multifunctional drug delivery system might provide a promising platform for cancer theranostics upon in situ autophagy disruption. KEYWORD: autophagy blockade, in situ, cancer theranostics, HCQ, 4T1 cells, lysosomal deacidification

1. Introduction Autophagy is a lysosome-dependent catabolic cellular salvage pathway which exists extensively in eukaryotic cells, which is an important mechanism for the survival of cells1, 2. This pathway allows cells to degrade a portion of abnormal organelles or biological macromolecules to reduce self damage and maintain the cellular homoeostasis required for normal growth, development and adaption to stress3. Degradation of the sequestered material generates nucleotides, amino acids, and free fatty acids that are recycled for macromolecular synthesis and ATP generation4. Both healthy and malignant cells can utilize autophagy. In rapidly dividing tumor cells, autophagy is additionally enhanced to meet cellular survival needs under intratumoral nutrient starvation and hypoxic microenvironment5. Moreover, tumor cells rely on autophagy to develop 2


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resistance to various targeted agents and traditional treatment process (such as chemotherapy and radiotherapy)6, 7. Therefore, inhibiting autophagy and alternative cell death pathways has arisen as a new effective treatment strategy for refractory tumors8, 9. Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) have been used as classic antimalarial drugs for a long time due to their favorable high biological safety, efficacy, and powerful immune-modulating properties. Intriguingly, most recent studies have highlighted on their promising anticancer repurposing efforts in various tumor types, such as glioblastoma, hepatocellular carcinoma, breast cancer, prostate cancer, pancreatic cancer and esophageal carcinoma10, 11. They can inhibit autophagy by alkalizing lysosomes and subsequently blocking the fusion of autophagosomes-lysosomes, to damage the autolysosomes at the last step of autophagy12. Some other studies have shown that their antitumor effect is ascribed to direct targeting of tumor cells and/or stromal endothelial cells mechanistically13, 14. In fact, CQ is currently the subject of several clinical trials as part of combination tumor therapy15. Nevertheless, it has pointed out that the clinical usefulness of CQ is limited because high doses are required to compensate for its nonselective distribution in vivo

15.

Researches have reported that HCQ can not accumulate to

sufficient levels in many solid tumors to adequately inhibit autophagy for effective tumor treatment in vivo16. Nanotechnology provides an effective way to solve this limitation12,17. Nanotherapeutics could accumulate around disorganized tumorous vasculatures via the enhanced permeability and retention effect, and thus have been used as delivery tools for nonselective drugs to tumors

15.

Due to the

good biocompatibility and specific responsiveness to internal or external stimuli, such as pH, temperature and redox potential, manganese oxide nanomaterials have been developed in a variety of fields, such as drug targeted delivery, biosensing and molecular imaging18-20. The integration of 3



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diagnosis and treatment of tumors is conducive to mastering the best treatment time, monitoring the treatment effect in real time and even adjusting the treatment plan. Among all manganese oxide nanomaterials, manganese dioxide (MnO2) nanosheet is the most common used and researched. It can rapidly degrade to manganese ions in tumor-specific microenvironment (H+ and glutathione (GSH)) for payloads intelligent release and tumor enhanced magnetic resonance imaging (MRI) 22.

21,

The latest researches suggest that this class of material has many other outstanding features for

cancer therapy. It is effective in improving tumor oxygenation, as well as reducing hydrogen peroxide (H2O2) and acidity to overcome tumor microenvironment (TME) factors23, enhance photodynamic therapy for cancer treatment24, or trigger a series of anti-tumor immune responses25. Compared with two-dimensional MnO2 nanosheets, hollow MxOy nanoparticles with interior void spaces are the most attractive candidates owing to their large water-accessible surface areas, which are able to carry high payloads of MR-active magnetic centers. Meanwhile, they can load a large amount of therapeutic drug within their interior void26. Hollow MxOy nanostructures with large cavities (such as hollow mesoporous MnO2 and Mn3O4) have demonstrated to be ideal to realize the most effective drug loading as well as precisely controlled release of therapeutic agents25-28. However, as smart drug delivery system (DDS), hollow Mn2O3 nanoparticles DDS have not yet been reported to our best knowledge. In this study, we for the first time report a novel and facile synthesis of hollow mesoporous Mn2O3 nanoparticles and their potential application as a kind of intelligent and multifunctional theranostic platform. In this system, we firstly synthesized hollow mesoporous Mn2O3 using carbon spheres as a template. This material owned a large surface area and excellent drug loading capacities. In order to reduce drug leakage in the body circulation, natural anionic polysaccharide hyaluronic acid (HA) was grafted onto the shell of Mn2O3 nanoparticles. In addition, Mn2O3 nanoparticles were endowed with good water dispersibility, great 4


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physiological stability and improved tumor-targeted ability by HA modification. HA has served as a tumor targeting ligand because it can recognize and bind to CD44 receptor overexpressing on numerous kinds of tumor cells29-30, such as 4T1 breast cancer and A549 lung cancer31-32. Furthermore, HA can be specifically degraded by hyaluronidase (HAase), highly being expressed in tumor tissue, in the body30-32. Thus, HA-based nanomaterials have been employed not only as a safe carrier but also as a stimuli-responsive reservoir susceptible to HAase for the targeted delivery of drugs and imaging agents33. As Fig 1 shown, after this nano-platform enter the tumor it will achieve TME-responsive rapid release of HCQ and Mn2+, for simultaneous enhanced MR imaging and improved synergistic autophagy inhibition. Our research demonstrated this smart drug delivery system could effectively inhibit the proliferation of 4T1 tumors.

Figure 1. A: Formation of HA-Mn2O3/HCQ theranostic platform and its tumor microenvironment (TME) responsive MR imaging 5



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and drug release; B: Schematic illustration of HA-Mn2O3/HCQ nanoparticles for anti-tumor proliferation in 4T1-tumor bearing mice via inhibiting the autophagy-dependent tumor nutrition supply.

2. Experimental section 2.1. Materials Hydroxychloroquine sulphate (HCQ, >98.0%) was purchased from Shanghai Dibai Biotechnology Limited Company (Shanghai, China). Sodium hyaluronate (HA, MW≈12000, >98%) was bought from Bloomage Freda Biopharm Co. Ltd. (Jinan, Shandong, China). Manganese nitrate solution (Mn(NO3)2, 50%) was got from Nanjing Chemical Reagent Co., Ltd. (Nanjing, Jiangsu, China). Sucrose was got from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). N-(3-dimethylamino propyl-N′-ethylcarbodiimide) hydrochloride (EDC•HCl, 98.5%), N-Hydroxysuccinimide (NHS, 99%), glutathione (GSH, 99%), hyaluronidase (HAase, 99%) and sulforhodamine B (SRB, 99%) were purchased from Sigma-Aldrich (St Louis, MO, USA). The LC3-II and LC3-I ELISA kits were got from Shanghai Enzyme-linked Biotechnology Co., Ltd (Shanghai, China).

2.2. Synthesis of hollow mesoporous Mn2O3 nanoparticles Briefly, 16g of sucrose was dissolved in 80mL of water to get a clear solution. This solution was then heated at 190°C for 4h in muffle furnace. After above solution was cooled to room temperature, the produced carbon spheres template was collected by centrifugation (12000r/min, 10min), washed four times with water and ethanol alternately and dried at 40°C for further use. 1g of as-prepared carbon spheres template was dispersed into 40mL of Mn(NO3)2 solution (50%). This mixture was sonicated for 15min to get a uniform dispersion, followed by 24h of stirring and 48h of impregnation. Subsequently, the mixture solution was separated by centrifugation (12000r/min, 10min). After washed four times with water and ethanol alternately, the precipitation was put in crucible and roasted at 450°C for 6h to obtain the final hollow mesoporous Mn2O3 nanoparticles.

2.3. Synthesis of HA-modified Mn2O3 nanoparticles Mn2O3 nanoparticles (20mg) were added into PEI solution (MW=10000, 1.5mg/mL). After stirring at room temperature for 24h in the dark, the resulting mixture was dialyzed by a dialysis bag (MW cutoff =14kDa) for 12h to remove free PEI. Finally, NH2-Mn2O3 was obtained by filtering and washing. 50mg of as-prepared NH2-Mn2O3 was dispersed in 10mL of formamide. Add 173mg of EDC and 103mg of NHS into the above dispersion and stir overnight at room temperature. After that, 50mg of HA was dissolved in this mixture dispersion and keep stirring for 24h. Then add 3 to 4 times the amount of pre-cooled acetone for precipitation. After that, the product was taken out to dialyze (MW cutoff =14kDa) for 48h to get rid of the excessive acetone, EDC, NHS and unreacted HA. Finally, HA-Mn2O3 was freeze-dried for further use.

2.4. Particle characterizations The morphology of Mn2O3 and HA-Mn2O3 nanoparticles were examined by transmission electron microscopy (TEM). The size distribution and zeta potential of nanoparticles were determined by the laser particle size distribution analyzer (DLS). Elemental composition and atomic valence states of Mn2O3 nanoparticles were estimated by HAADF-STEM and X-ray photoelectron spectroscopy (XPS) analysis, respectively. The crystal structure identification of Mn2O3 nanoparticles was demonstrated by X-ray diffraction (XRD). The surface modification of Mn2O3 with HA was analyzed using FT-IR spectra recorded on a Nicolet iS10 spectrometer. Specific surface areas and pore diameter of Mn2O3 nanoparticles were obtained according to Brunauer−Emmett−Teller (BET) and Barrett−Joyner−Halenda (BJH) analyses using a gas adsorption instrument (Nova-Touch TM, USA). The modification amount of HA was measured based on the weight loss of HA-Mn2O3 nanoparticles by a thermogravimetric analyzer (TGA, STA 409 PC, Germany). 6


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2.5. Evaluation of drug loading ability and TME-responsive drug release property For HCQ loading, free HCQ (2mg/mL) aqueous solution was mixed with HA-Mn2O3 nanoparticles (1mg/mL) in PBS. Then place the mixture in the ultrasound system for 48h at room temperature. Subsequently, the nanosuspension was dialyzed by a dialysis bag (MW cutoff = 3.5kDa) for 24 h to remove unloaded free HCQ. The HCQ amount loaded in HA-Mn2O3 nanoparticles was determined as follows. Firstly, HCQ loaded in HA-Mn2O3/HCQ nanosuspension was extracted by using 10-fold volume of methanol. After ultra-sonicated using an ultrasonic cell disruption system (300W, 20times) under ice-bath condition, the supernatant containing HCQ was collected by high-speed centrifugation. Finally, the concentration of loaded HCQ was measured by UV-visible spectrophotometer at 343nm. The entrapment efficiency and loading efficiency were calculated using formula 1 and 2, respectively. Entrapment efficiency (%) = W Loaded HCQ / W Totally added HCQ × 100% (1) Loading efficiency (%) = W Loaded HCQ / (W Loaded HCQ + W HA-Mn2O3) × 100% (2) The release profiles of HCQ from HA-Mn2O3/HCQ were evaluated by a dialysis method. In brief, HA-Mn2O3/HCQ nanosuspensions were sealed into dialysis bags (MW cutoff = 3.5kDa). Then, they were immersed in 100mL of PBS buffer solution containing different TME-responsive substances ( ① pH=7.4; ② 10μg/mL of HAase; ③ pH=5.5; ④ 5mM GSH; ⑤ pH=5.5, 5mM GSH and 10μg/mL of HAase) and gently shaken at an incubator shaker (37°C, 100 rpm). The release samples (2mL) were drawn at various time points, and the medium was replaced by the same volume of PBS solution. The concentration of HCQ released from HA-Mn2O3/HCQ was quantified by UV-visible spectrophotometer at 343nm.

2.6. TME-responsive structural changes and MR imaging of Mn2O3 nanoparticles 1mL of Mn2O3 or HA-Mn2O3 (1mg/mL) aqueous solution was diluted with 10mL of PBS buffer solution containing different TME-responsive substances ( ①pH=7.4; ②pH=7.4 and 5mM GSH; ③pH=5.5; ④pH=5.5 and 5mM GSH) and gently shaken at an incubator shaker (37°C, 100rpm). The dispersion solutions were drawn at 4h. Structural changes were demonstrated by TEM. The Mn2+ concentration in samples was quantified using inductively coupled plasma absorption emission spectroscopy (ICP-AES). Moreover, we prepared two 0.5mM of Mn2O3 dispersion solutions with different medium ( ①pH=7.4; ②pH=5.5 and 5mM GSH), respectively. After standing for 4h, the above solutions were diluted to 0.25mM, 0.125mM, 0.0625mM, 0.0312mM and 0.0156mM to determine their MR imaging ability.

2.7. Cellular uptake The quantitative targeting efficiency evaluation of this drug delivery system was carried out on CD44-positive 4T1 cells and A549 cells by flow cytometry analysis. 4T1 cells or A549 cells in logarithmic growth phase were seeded in 6-well plates at a density of 3×105 cells per well. After culturing for 24h, cells were treated with FITC labeled HA-Mn2O3 or Mn2O3 (10μg/mL) for 2 and 4h, respectively. Thereafter, cells were washed with PBS and collected for quantitative analysis. The excitation and emission wavelengths for flow cytometry determination were 488 and 525nm, respectively. Herein, NIH 3T3 cell line was chose as CD44-negative cell control.

2.8. Lysosomal localization 4T1 cells in logarithmic growth phase were seeded in 6-well plates at a density of 3×105 cells per well. After incubation for 24 h, cells were treated with FITC labeled HA-Mn2O3 (10μg/mL) for 4h. Thereafter, cells were stained with Lyso-Tracker Red (25nM) for 20min and examined by a Fluorescence Microscope (Zeiss LSM 510). Moreover, CD44-positive A549 cell line was also selected to validate the results.

2.9. Intracellular pH determination 7



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BCECF-AM fluorescent indicator was used for intracellular pH determination. 4T1 cells were seeded in 6-well plates at a density of 3×105 cells per well. Then cells were incubated with 10μg/mL of HA-Mn2O3, 20μg/mL of HCQ or HA-Mn2O3/HCQ for 6h, respectively. Thereafter, cells were washed with PBS and stained by BCECF-AM (2μM) for 30min at 37°C. Wash the cell samples twice with fresh culture medium and record the images under a Fluorescence Microscope. The excitation and emission wavelengths were 488 and 535nm, respectively. Moreover, this experiment was also carried out using CD44-positive A549 cell line to validate the results.

2.10. Lysosome deacidifying effect 4T1 cells in logarithmic growth phase were seeded in 6-well plates at a density of 3×105 cells per well. After incubation for 24 h, cells were treated with HCQ and HA-Mn2O3/HCQ (20, 50μg/mL) or HA-Mn2O3 (10μg/mL) for 6h, respectively. Afterwards, cells were fixed with 4% paraformaldehyde and stained with LysoTracker Red (25nM) for 20min. Laser confocal microscope was used to record the results. The excitation and emission wavelengths for determination were 577 and 590nm, respectively. Moreover, this experiment was also carried out using CD44-positive A549 cell line to validate the results.

2.11. Autophagy inhibition 4T1 cells were seeded in 24-well plates at a density of 5×104 cells per well. After incubation for 24 h, cells were treated with HCQ or HA-Mn2O3/HCQ for 6h at 20μg/mL. After fixing with 4% paraformaldehyde and 0.2% Triton X-100 for 15min, LC3B rabbit polyclonal antibody was added and incubated for 2h at room temperature. Cells were washed three times with PBS and then incubated with FITC-labeled IgG for 5h in the dark. Finally, autophagosome was recorded under a Laser confocal microscope. Furthermore, TEM was used to observe intracellular autophagosome more clearly. After incubated with HCQ or HA-Mn2O3/HCQ (20μg/mL) for 3 and 6h, cells were fixed with 4% glutaraldehyde and then post-fixed with 2% osmiumtetroxide in 0.1M sodium phosphate buffer for 30min. Cell slices were observed using TEM. In addition, LC3-II and LC3-I protein contents were evaluated by using the mouse LC3-II ELISA kit and LC3-I ELISA kit, respectively. After treated with HCQ or HA-Mn2O3/HCQ for 6h at 20μg/mL, cells were washed and collected. The cell suspension was diluted to 1×106 cells/mL with PBS (pH 7.4). After repeated freezing and thawing, the cells were destroyed to release intracellular proteins. The supernatant was collected by centrifugation for 20 min (3000 rpm), and tested according to the manufacturer’s protocol. Thereafter, LC3-II: LC3-I value can be calculated to evidence autophagosome accumulation effect caused by HA-Mn2O3/HCQ. In addition, the autophagy inhibition effect of HA-Mn2O3/HCQ in presence of autophagy inducer was also investigated via LC3 immunohistochemistry method. Earle’s balanced salts solution (EBSS) containing no amino acids and no serum component was used to induce autophagy. Briefly, 4T1 cells pre-treated with EBSS for 24 h were further treated with HCQ or HA-Mn2O3/HCQ for 3 and 6h at 20μg/mile Thereafter, the autophagy behavior was observed by immunohistochemistry (IHC). Moreover, autophagy inhibition effect of HA-Mn2O3/HCQ was also carried out using CD44-positive A549 cell line to validate the results.

2.12. Cytotoxicity 4T1 cells were plated in 96-well plates at a density of 5×103 cells per well and incubated for 24h. After that, cells were treated with HCQ or HA-Mn2O3/HCQ, respectively. The concentrations of HCQ in above formulations were 2, 5, 20 and 40μg/mL.After incubation for 24h or 48h, SRB assay was used to determine cell viability. In addition, after 4T1cells were treated with HCQ or HA-Mn2O3/HCQ (20μg/mL) for 24 or 48h under the aforementioned conditions, cells were stained with calcein acetoxymethyl ester (calcein AM, 1μM) and propidium iodide (PI, 1μM) to distinguish the live cells from dead cells through a fluorescence microscope (Zeiss LSM 510). Moreover, this experiment was also carried out using CD44-positive A549 cell line to validate the results. 8


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2.13. Pharmacokinetics study 0.6 mL of blood was drawn from eyes of healthy BALB/c mice after treatment with HCQ, Mn2O3/HCQ or HA-Mn2O3/HCQ (HCQ: 50mg/kg; Mn2O3: 12.5 mg/kg) for 0.083、0.25、0.5、1、2、4、6、8 and 12 h. After centrifugation (4000 r/min, 10 min), 0.2 mL of supernatant was placed into the centrifuge tubes. 0.8 mL of acetonitrile was added to the above tubes, followed by vortex for 3 min. After centrifugation (4000 r/min, 10min), the supernatant was pipetted and dried under nitrogen at 35 °C. And then the residue was dissolved in 200μL of mobile phase. Finally, 10μL of each sample was injected to high performance liquid chromatography (HPLC) to determine HCQ concentration under the following chromatographic conditions. The chromatographic conditions: column model: Waters Symmetry® C18 (150 × 4.6 mm, 5 μm); detector type: UV detector; detection wavelength: λ = 254 nm; mobile phase composition: 0.6% formic acid in water (pH = 3.2) : methanol = 80:20; flow rate: 1.0 mL/min; column temperature: 25 ° C; injection volume: 10 μL. The PKSolver software was used to calculate the main pharmacokinetic parameters.

2.14. 4T1 tumor bearing mouse models Female BALB/c mice at the age of 4-6 weeks were purchased from Henan laboratory animal center, and then implemented in strict accordance with protocols approved by Henan laboratory animal center. 4T1 cells (3×106) suspended in 100μL of PBS were subcutaneously injected into the right shoulder of mouse. The mouse models were used for in vivo experiments once the volume of tumors reached about ~80mm3.

2.15. In vivo distribution and intratumoral degradation of HA-Mn2O3 In order to visually observe the biodistribution of the carrier in the body, a near-infrared (NIR) dye IR783 was loaded into HA-Mn2O3 (HA-Mn2O3/IR783) with the same method of loading HCQ. Mice were intravenously injected with free IR783, Mn2O3/IR783 and HA-Mn2O3/IR783 via tail vein, respectively, with the same dosage of IR783 (0.8 mg/kg). Then a noninvasive optical imaging system FX PRO (Kodak, USA) was utilized to record and analyze in vivo distribution images at different times (excitation: 700 nm, emission: 830 nm). At 12h post-injection, the mice were sacrificed. Then the main organs (heart, liver, spleen, lung, and kidney) and tumors were excised for ex vivo fluorescence imaging. The intratumoral degradation behavior of the carrier was monitored by MR imaging with a 3.0 T clinical MRI scanner (Philips, Netherlands). Firstly, 4T1 tumor bearing mice were anesthetized with 5% chloral hydrate (i.p., 200μL/20g). And then mice were intravenously injected with saline, Mn2O3 and HA-Mn2O3 via tail vein, respectively, with the same dosage of Mn2O3 (4mg/kg). In vivo T1-Weighted MRI was performed after 1, 2, 4 and 8h post-injection using a fast spin three echo sequence with the following parameters: TR/TE = 5310/99 ms, 256 × 256 matrices, repetition time 1. Finally, the MR signal intensities of tumors were obtained by the histogram equalization method at the region of interest (ROI) with the same diameter placed at the tumor site in the same slice on T1-weighted images.

2.16. In vivo anti-tumor effect and autophagy in tumor tissues For in vivo pharmacodynamic evaluation, tumor bearing mice were divided into six groups (n = 10): saline, Mn2O3, HA-Mn2O3, HCQ, Mn2O3/HCQ and HA-Mn2O3/HCQ (HCQ: 50mg/kg; Mn2O3: 12.5 mg/kg). Formulations were intravenously injected into mice via tail vein every other day for seven times. The body weights and tumor volumes were monitored before injection every time. The tumor volume was calculated according to the following formula (A× B2)/2, where A represents the larger diameter and B represents the smaller one. At the end of the experiment, two mice in each group were sacrificed and their tumor tissues were taken out for hematoxylin and eosin (H&E) staining to evaluate the histopathological changes. Moreover, in order to observe the autophagy level at the tumor site, two mice in each group were sacrificed 9



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and tumor tissues were taken out for TEM slices and immunofluorescence staining. To prepare TEM samples, excised tumor tissues were fixed with 2.5% glutaraldehyde and then dehydrated and embedded in Agar 100 resin. After being cut into nanometre sections, the autophagosome can be observed under a TEM. In addition, the other tumor tissue was soaked in 10% formalin solution and embedded with paraffin for immunofluorescence staining using fluorescein-labeled LC3 and p62, respectively.

2.17. In vivo safety evaluation The systemic toxicity for different formulations was assessed by monitoring the weight changes of tumor-bearing mice during treatment. At the end of the experiment, two mice in each group were sacrificed and the major organs (heart, liver, spleen, lung, and kidney) were excised for H&E staining to evaluate the histopathological changes. The remaining 6 mice in each group continued to raise to record their survival rate. Moreover, the toxicity of HA-Mn2O3/HCQ drug delivery system was also evaluated on health mice. Briefly, HA-Mn2O3/HCQ NPs were intravenously administrated every 2 days for 6 times, with the dose of 50 mg/kg. At the 12th day, the toxicity was evaluated by the serum biochemistry assay.

2.18. Statistical analysis All data analyzed by using GraphPad Prism6 software (GraphPad Software, Diego, USA). One-way ANOVA was used for both multiple comparisons and a two-group comparison in multi-groups. The level of significance was set at probabilities of *P < 0.05 and **P < 0.01.

3. Results and discussion 3.1. Synthesis and characterization of Mn2O3 and HA-Mn2O3 nanoparticles The procedure for the synthesis of HA-Mn2O3/HCQ was illustrated in Fig 1A. Firstly, hollow mesoporous Mn2O3 nanoparticles were synthesized via a carbon spheres template method. A uniform layer of manganese oxide was grown on the surface of as-made carbon spheres by simply mixing them with 50% Mn(NO3)2 solution. The hollow mesoporous manganese oxide nanoparticles were obtained after roasting at 450 °C for 6 h to remove carbon core. TEM images (Fig 2A and 2B) clearly demonstrated the typical hollow interiors and smooth surface (indicating by red arrows) of spherical Mn2O3 NPs. DLS results showed the particle size and zeta potential of these nanoparticles were 314 nm and -28.4 mV, respectively (Fig 2C and 2D). Furthermore, high-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping further confirmed there were only two elements (Mn and O) distributing on our product besides the supporting carbon film composed of C element (Fig 2E). Next, the sample was further characterized by XPS spectra and 10


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XRD pattern to confirm the crystal form of this manganese oxide. The characteristic signals of MnO2 are 642.0 eV and 653.8 eV for Mn 2p3/2 and Mn 2p1/2, respectively. For the samples of Mn2O3 and Mn3O4, the binding energies for Mn 2p3/2 and Mn 2p1/2 show a continuous downward trend, with the decrease of Mn valence state28. The characteristic signals of Mn2O3 are 641.4 eV and 653.4 eV for Mn 2p3/2 and Mn 2p1/2, respectively. The characteristic signals of Mn3O4 are 641.0 eV and 653.1 eV for Mn 2p3/2 and Mn 2p1/2, respectively28. As shown in Fig 2F, the signals at 641.42 eV for Mn 2p3/2 and 653.42 eV for Mn 2p1/2 of the sample were consistent with Mn2O3 reported in the study28. Moreover, we compared the XRD pattern of the sample and standard cards of different manganese oxide nanoparticles (MnO2, Mn2O3 and Mn3O4). The results can be seen in Fig 2G and Fig S1. All the reflections in the XRD pattern of the sample (Fig 2G) agreed well with the reported Mn2O3 phase (JCPDS 41-1442). The sharp reflections indicated the good crystallinity of Mn2O3 nanoparticles28, 34. The surface area and average pore diameter of hollow mesoporous Mn2O3 were determined to be 107m2g-1 and ~2.5nm, respectively (Fig S2 and S3). These parameters suggested Mn2O3 carrier with large hollow interiors and proper mesoporous shells was ideal for efficient drug loading. Free small molecule drugs with a particle size of less than 2 nm, such as HCQ, can enter the hollow cavity by free diffusion through the mesopores on the surface of Mn2O3 nanoparticles. To improve their water solubility and physiological stability, Mn2O3 carriers were modified with HA polysaccharide via the amide bond. As Fig 2H shown, HA with carboxy group grafting onto aminated Mn2O3 (PEI modification firstly) was confirmed by the typical C=O stretching vibration absorption at 1734.61 cm-1. The peaks at 668.58 and 618.26 cm-1 belong to Mn-O vibration absorption. TEM image showed the uniform particle size of HA-Mn2O3 NPs (Fig 2I). Fig. 2J further demonstrated obvious light gray surface coating (indicating by arrows) on Mn2O3 particles which accounted for the HA grafting. The elemental mapping of HA-Mn2O3 (Fig. 2K) showed 11



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HA-Mn2O3 NPs were composed of Mn, O and C elements. And the C element mainly distributed on the surface of nanoparticles, which further confirmed the HA surface coating. The grafting amount of HA was estimated about 11.4% calculated via TGA method (Fig S4). After HA modification, the zeta potential of HA-Mn2O3 changed from -28.4 mV to -23.7 mV (Fig S5). The size stability of Mn2O3 and HA-Mn2O3 NPs in the cell medium had been tested. The size and PDI changes can be seen in Fig S6, suggesting that HA modified Mn2O3 NPs can be stable for one week in physiological solution.

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Figure 2. Characterizations of hollow mesoporous Mn2O3 nanoparticles and HA modified Mn2O3 nanoparticles (HA-Mn2O3). A and B: TEM images of Mn2O3. C: Size distribution of Mn2O3. D: Zeta Potential of Mn2O3. E: The HAADF-STEM image of Mn2O3, elemental mapping showed Mn2O3 NPs are composed of only Mn and O elements. F: XPS survey scan of Mn2O3. G: The XRD pattern of samples (Red) and standard cards of Mn2O3 (Black, JCPDS 41-1442). H: FTIR spectra of Mn2O3, HA and HA-Mn2O3. I and J: TEM images of HA-Mn2O3. K: The elemental mapping of HA-Mn2O3 showed HA-Mn2O3 NPs are composed of Mn, O and C elements. Furthermore, the C element mainly distributed on the surface of nanoparticles, which accounted for the HA surface coating.

3.2. Evaluation of drug loading ability and release profile To employ hollow mesoporous Mn2O3 for tumorous autophagy regulation application, autophagy inhibitor HCQ was chosen as the model drug. After incubating HA-Mn2O3 nanoparticles 13



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with different amounts of HCQ for 48 h, drug loading efficiency and entrapment efficiency were determined. As Fig 3A shown, at the feeding weight ratio (HCQ: HA-Mn2O3) of 2:1, drug loading efficiency and entrapment efficiency both demonstrated high ratios of 61.8% and 81.0%, respectively, suggesting Mn2O3 with hollow mesoporous structure can be employed as an excellent cargo for HCQ loading. Moreover, the stability of HA-Mn2O3/HCQ NPs was studied in the cell culture medium. As Fig S7 shown, HA-Mn2O3/HCQ NPs can be stable for one week in physiological solution. Nowadays, “activable” drug delivery system is increasingly valued35. In this nanoplatform, HA was grafted onto the surface of Mn2O3 to prevent drug leakage. After HA grafting, HCQ leakage rates decreased from 24.5% to 5.8% in a week (Fig 3B). This indicated HA modification can significantly reduce the leakage of drugs, thereby reducing the ineffective and harmful release of drugs during in vivo circulation. Once HA-Mn2O3/HCQ reached targeted site, HA shell degraded in HAase-rich tumor microenvironment. Fig 3C showed in the presence of HAase, the cumulative release percentages of HCQ increased from 18.3% to 53.4% within 24 h, suggesting its excellent gating effect. Furthermore, acidity and high GSH level are distinct characteristics of solid tumors36. More comprehensive drug release data indicated the release speeds of HCQ were found to be faster in mild acidic solution at pH 5.5 and much faster in the presence of GSH (5mM). In mild acidic solution simultaneously containing HAase and GSH, the cumulative release percentage of HCQ even achieved 92.1% within 12 h. This result demonstrated HA-Mn2O3/HCQ could realize tumor-specifically responsive drug release. 3.3. TME-responsive degradation property for Mn2O3 nanoparticles Manganese oxide is known to be unstable in acidic and reductive environment. Acidity and high GSH level are distinct characteristics of solid tumor microenvironment (TME)36. So next we

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explored structural changes of Mn2O3 in presence of different TME-specific factors (①pH=7.4; ② pH=7.4 and 5 mM GSH; ③ pH=5.5; ④ pH=5.5 and 5 mM GSH). After gently shaking at an incubator shaker (37 °C, 100 rpm) for 4h, four Mn2O3 dispersions with the same initial concentration presented different colors, due to different degradation degrees of Mn2O3 (Fig S8). Then the structural changes of Mn2O3 nanoparticles in different groups were recorded using TEM images. As Fig 3D shown, the morphology of Mn2O3 nanoparticles showed no significant change in pH 7.4 solution after 4 h, indicating that Mn2O3 nanoparticles could maintain structural integrity in neutral environment. However, in mild acidic solution at pH 5.5 for 4 h, Mn2O3 nanoparticles exhibited slight degradation. While in GSH-containing solutions, Mn2O3 nanoparticles showed significant structural damage. Especially in the GSH-containing acidic solution, there were no visible nanoparticles can be seen in view due to the decomposition of Mn2O3 into Mn2+ ions. Moreover, the TME-responsive degradation properties of HA-modified Mn2O3 had also been tested. As shown in Fig. S9, the TME-responsive structure change trend of HA-Mn2O3 was consistent with that of Mn2O3 (Fig 3D), suggesting that modification with HA did not affect the TME response characteristics of Mn2O3 nanoparticles. The concentrations of Mn2+ in pH 7.4, pH 5.5, pH 7.4+5 mM GSH and pH 5.5+5 mM GSH groups were determined to be 0.74, 3.21, 12.67 and 38.45 μg/mL, respectively (Fig 3E). All these results suggested that acid may accelerate the degradation of Mn2O3 to be Mn2+ ions by GSH. As we know, Mn2+ ions have recently been reported as potent T1 MRI contrast agents37. So next, we evaluated the effectiveness of hollow mesoporous Mn2O3 nanoparticles as MRI agents in simulating tumor microenvironment in vitro. As shown in Fig 3F, Mn2O3 dispersed in GSH-containing acidic solution (pH 5.5+5 mM GSH) exhibited much stronger enhancement in T1-weighted MRI than that dispersed in the neutral solution (pH 7.4) at the same Mn2O3 concentration. The specific relaxivity, obtained by measuring the relaxation rate as a 15



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function of the concentration of the contrast agent, were found to be markedly increased in pH 5.5+5 mM GSH group as compared to the pH 7.4 group. The r1 values of hollow mesoporous Mn2O3 nanoparticles in pH5.5+5mM GSH and pH7.4 dispersions were 6.49 and 0.17, respectively (Fig 3G). These data indicated Mn2O3 nanocarriers owned excellent MRI capability after degradation in simulated TME in vitro.

Figure 3. A: HCQ loading efficiency and entrapment efficiency in HA-Mn2O3 at different feeding HCQ: HA-Mn2O3 weight ratios (n=3). B: Leakage rates of HA-Mn2O3/HCQ and Mn2O3/HCQ in a week (n=3). C: The release curves of HA-Mn2O3/HCQ in different 16


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conditions (n=3). The concentrations of GSH and HAase were 5mM and 10μg/mL, respectively. D: The structure changes of Mn2O3 in PBS buffer dispersion containing different TME-responsive substances. E: The concentration of Mn2+ determined by ICP in different Mn2O3 dispersion containing various TME-responsive substances (n=3). F: T1-weighted MR pseudo color images of Mn2O3 nanoparticles recorded using 3T MR scanner in different aqueous environments (pH7.4 and pH 5.5+5mM GSH). The NMR signals were enhanced obviously (from blue to red) with increasing concentration of Mn2O3 in pH 5.5+5mM GSH group. G: The transverse relativities were 0.17 and 6.49 mM−1 s−1 for Mn2O3 at pH7.4 and pH 5.5+5mM GSH aqueous environments, respectively. （*P < 0.05 and **P < 0.01）

3.4. Lysosomotropic activity of HA-Mn2O3/HCQ How to improve intracellular drug concentration is the key factor to impact the autophagy inhibition of HCQ15, 16. We firstly tested the targeting efficiency of this drug delivery system. In this study, Mn2O3 was further conjugated tumor-targeting HA to endow the nanocarriers with additional ability to selectively bind tumor cells. 4T1 cells were selected as the model because their overexpressed CD44 on the cell membrane and NIH 3T3 cells were employed as the CD44-negative control. After incubation with FITC-labeled Mn2O3 and HA-Mn2O3 for 4 h, the fluorescent intensities within the 4T1 cells were 31.2% and 71.4%, respectively (Fig 4A). This significant difference suggested a specific and rapid internalization of HA-Mn2O3 by CD44-positive 4T1 cells. However, similar result was not found on NIH 3T3 cells (Fig S10). On account that HCQ is a lysosomal targeted drug which is widely used to neutralise lysosomal pH and block lysosomal degradation4, next we explored the intracellular localization of HA-Mn2O3/HCQ. Lysosomes were stained with Lyso-Tracker Red probes and cells were incubated with FITC-labeled HA-Mn2O3. After incubation for 4 h, HA-Mn2O3 displayed an ultimate localization and retention in lysosomes (Fig S11). After substantial accumulation in lysosomes, we compared the lysosomotropic capacity and functional effects between HCQ and HA-Mn2O3/HCQ. After incubation for 6 h, there were much fewer Lyso-Tracker dye–stained red flecks observed in HA-Mn2O3/HCQ-treated cells than HCQ-treated cells at both 20 μg/mL and 50 μg/mL. As shown in Fig 4B, there were about 28.9 red 17



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flecks per cell in blank cells group. Because HA-Mn2O3 could consume H+ and GSH to generate Mn2+, the Lyso-Tracker positive flecks decreased to 22.2 per cell in HA-Mn2O group. After treatment with 20 μg/mL of HCQ and HA-Mn2O3/HCQ, the number of intracellular red flecks decreased to 13.1 and 2.03 per cell, respectively. The reduction amount of red flecks in HA-Mn2O3/HCQ group was 6.45-fold than that in HCQ group. Moreover, when drug concentration was increased to 50 μg/mL, there were almost no Lyso-Tracker-positive red flecks observed in HA-Mn2O3/HCQ-treated cells. Moreover, some of the treated cells showed apoptotic features, such as nuclear shrinkage and lysis. These results showed that HA-Mn2O3/HCQ owned much greater lysosomotropic activity than free HCQ. Because the principle of Lyso-Tracker Red probes is based on the acidity of lysosome, this result also suggested HA-Mn2O3/HCQ could induce an increase in lysosomal pH (deacidifying effect) and lysosomal dysfunction, such as lysosomal membrane permeability changes and inactivation of lysosomal enzymes38. There were three main reasons to explain higher lysosomotropic activity of HA-Mn2O3/HCQ. Firstly, as a kind of highly water-soluble drug, the capacity of permeating the lipid membranes of cells for hydroxychloroquine sulphate was limited. HA-Mn2O3 could deliver more HCQ into tumor cells, as Fig 4A shown. Secondly, HA-Mn2O3 displayed a specific localization and retention in lysosomes after entering tumor cells. Therefore HA-Mn2O3/HCQ could achieve lysosome targeting and transfer HCQ to their targeted organelle directly. Thirdly, HA-Mn2O3/HCQ had HAase, acidity and GSH responsive Mn2+ and drug release property. Once accumulation in lysosomes, HA shell will be degraded and removed, and then Mn2O3 nanoparticles disintegrate rapidly to realize HCQ and Mn2+ synchronous release. Lysosomal alkalization and elevated osmotic pressure synergistically lead to lysosomal destruction. Fig 4C showed 6 h after administration, HA-Mn2O3/HCQ could also elevate intracellular pH significantly. This may be due to drug overflow into cytoplasm after lysosomal 18


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destruction. The amines in HCQ act as proton acceptors to neutralize pH in living tumor cells. In order to verify the lysosomotropic property of HA-Mn2O3/HCQ nanoparticles, another CD44-positive A549 cancer cell line was selected to repeat these experiments. As Fig S12 shown, HA-Mn2O3/HCQ also demonstrated outstanding deacidifying effect and lysosomal dysfunction on A549 cells.

Figure 4. A: Intracellular uptake amount of FITC-labeled Mn2O3 and HA-Mn2O3 nanoparticles after incubating with CD44-positive 4T1 cells for 2 and 4h. B: Confocal imaging of 4T1 cells treated with different formulations for 6h and stained with Lyso-Tracker Red (blue: DAPI staining nuclear). Yellow arrows: Lyso-Tracker positive flecks. The number of red flecks per cell was scored which can be seen in the column chart. The data presented are the mean ± SD. （*P < 0.05 and **P < 0.01）. C: Intracellular pH changes 19



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indicated by BCECF-AM fluorescent indicator after treating with HA-Mn2O3, HCQ or HA-Mn2O3/HCQ for 6h. The fluorescence intensity of BCECF is pH-dependent. The fluorescence intensity enhances with increasing pH.

3.5. Inhibition of autophagy and cytotoxicity of HA-Mn2O3/HCQ Lysosomal function is required for autophagy to proceed. The lysosome deacidifying effect can lead to further effective autophagy inhibition during the digestive stage of autophagic flux17. To demonstrate autophagosome accumulation effect induced by HA-Mn2O3/HCQ, green fluorescence labeled autophagy-related protein LC3B was used to survey autophagic flux in cells. 4T1 cells were treated with either 20 μg/mL of HCQ or HA-Mn2O3/HCQ. 6 h after administration, all cells in the field of view treated with HCQ demonstrated obviously green LC3B-positive flecks, indicating suppression of terminal degradation of intracellular autophagosomes. While cells treated with HA-Mn2O3/HCQ of the same concentration showed highly dense green flecks (Fig 5A), suggesting the distinct accumulation of autophagosomes. The number of flecks (green) per cell was scored which can be seen in the column chart (Fig 5A). The number of autophagosomes in HCQ-treated 4T1 cells showed a significant 4.06-fold increase compared with that in untreated blank cells. While HA-Mn2O3/HCQ-treated 4T1 cells exhibited a more striking 4.03-fold increase in LC3B-positive flecks compared to HCQ treatment. Overall, quantitative analysis demonstrated after treatment with 20 μg/mL of HA-Mn2O3/HCQ for 6 h, a 16.36-fold increase in LC3B-positive autophagosomes was observed. This high level of intracellular autophagy may cause significant cytotoxicity. TEM images were adopted to further visually observe the level of autophagy in cells. As Fig 5B shown, after treatment for 3h, 4T1 cells treated with 20 μg/mL of HCQ and HA-Mn2O3/HCQ exhibited significant differences in the size and number of autophagic vesicles. 6 h after administration, there were many autophagic vesicles appeared in HA-Mn2O3/HCQ-treated cells, whereas there were fewer in untreated blank cells. In addition, the LC3-II: LC3-I value was also provided to evidence autophagosome accumulation effect caused by HA-Mn2O3/HCQ. As Fig S13 shown, after 20


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incubation with 20 μg/mL of HCQ, Mn2O3/HCQ and HA- Mn2O3/HCQ for 6 h, LC3-II: LC3-I value of 4T1 cells increased from 3.29 to 4.22, 4.80 and 5.67, respectively. Moreover, the autophagy inhibition performance of HA-Mn2O3/HCQ in presence of autophagy inducer was also studied. Earle’s balanced salts solution (EBSS) containing no amino acids is often used to induce autophagy in vitro. Therefore, we next investigated the autophagy inhibition effect of HA-Mn2O3/HCQ on EBSS pre-treated 4T1 cells with LC3 immunohistochemistry. Fig 5C showed that compared with cells cultured with complete medium (RPMI-1640 group), there were obvious brown LC3-positive autophagosomes present in EBSS group, suggesting successfully induced autophagy. After HCQ and HA-Mn2O3/HCQ treatment, accumulated intracellular LC3 protein increased significantly. Especially in HA-Mn2O3/HCQ 6h group, a sharp increase in LC3 positive autophagosomes and nuclear shrinkage appeared. All these results indicated HA-Mn2O3/HCQ can be used as a kind of autophagy inhibition nanogenerator for cancer therapy. This may be related to the excellent lysosome deacidification effect induced by the intracellular synchronous release of HCQ and Mn2+. The remarkable enhanced autophagy blockade property of HA-Mn2O3/HCQ prompted us to study its cytotoxicity in vitro. Firstly, the cytotoxicity of Mn2O3 and HA-Mn2O3 nanocarriers on 4T1 cells was tested. As Fig S14 shown, the cell inhibition rate was lower than 10% in the concentration range of 1-40 μg/mL for the HA-Mn2O3 NPs, suggesting that HA-Mn2O3 was a safe biomaterial for drug delivery. While HCQ and HA-Mn2O3/HCQ both exhibited an obvious concentration- and time-dependent cytotoxicity on 4T1 cells, as Fig 5D shown. Furthermore, for the four concentrations tested, HA-Mn2O3/HCQ-treated groups all exhibited a significant increase in the tumor cell inhibition rate than the free HCQ. After treatment with 40μg/mL of HA-Mn2O3/HCQ for 48h, the 4T1 cell inhibition rate could even reach 92.2%. Likewise, live/dead staining 21



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experiment also exhibited the effective anti-tumor efficacy of HA-Mn2O3/HCQ (Fig 5E), in accordance with the results of cytotoxicity assay. In addition, we selected another cancer cell line A549 with high CD44 expression for validation. As shown in Fig S15A and B, there was significant autophagic bubble accumulation in the HA-Mn2O3/HCQ group. This because the lysosome deacidifying effect can lead to further effective autophagy inhibition during the digestive stage of autophagic flux. As a result, live/dead cell staining result exhibited the best anti-tumor efficacy of HA-Mn2O3/HCQ (Fig S15C). Based on above results, as a kind of enhanced autophagy inhibition nanogenerator, HA-Mn2O3/HCQ offered a new strategy for autophagy blockade mediated tumor therapy.

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Figure 5. A: The accumulation of autophagosomes (green, LC3B marker) assessed by confocal microscopy. Representative images of HCQ-treated and HA-Mn2O3/HCQ-treated 4T1 cells after incubation for 6h. The concentration of HCQ and HA-Mn2O3/HCQ were both 20μg/mL. Red arrows: LC3B-positive flecks. The number of flecks (green) per cell was scored which can be seen in the column chart. The data presented are the mean ± SD（*P < 0.05 and **P < 0.01）. B: Representative TEM images of HCQ-treated 23



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and HA-Mn2O3/HCQ-treated 4T1 cells after incubation for 3 and 6h. The concentration of HCQ and HA-Mn2O3/HCQ were both 20μg/mL. Black arrows: autophagic vesicles. C: The immunohistochemistry result of LC3 in 4T1 cells treated with different drugs. D: Cytotoxicity of HCQ and HA-Mn2O3/HCQ on 4T1 cells. The concentration of HCQ was indicated in X-axis. The data presented are the mean ± SD (n=6). E: Fluorescent images of Calcein AM and PI co-stained 4T1 cells with different treatments, indicating live cells (green) and dead cells (red), respectively.

3.6. In vivo behavior and intratumoral degradation of HA-Mn2O3/HCQ Adequate drug concentration in tumors is a key factor in determining the in vivo antitumor effect of HCQ16. This requires HA-Mn2O3 to specifically deliver HCQ to the tumor target site and achieve a fixed-point rapid release. To validate the above capabilities of HA-Mn2O3, noninvasive NIR optical imaging and clinical T1-weighted MR imaging techniques were applied. The real-time images at different time points were recorded in Fig 6. As Fig 6A shown, IR783 was lack of tumor targeting ability. On the contrary, IR783 labeled Mn2O3 and HA-Mn2O3 could accumulate in tumor region at 2h post injection, and signals were still strong up to 24h. In addition, tumor-targeted accumulation amount of HA-Mn2O3/IR783 was significantly more than that of Mn2O3/IR783 at each time point. After 24h, mice were sacrificed and major organs and tumors were harvested for ex vivo imaging. As shown in Fig 6B, HA-Mn2O3/IR783 displayed significantly enhanced fluorescence intensity in tumor site, while free IR783 preferred to accumulate in liver. This notable tumor targeting capacity could be elucidated by Mn2O3-mediated EPR effect based on nanoscale size as well as CD44 receptor-mediated endocytosis. Once HA-Mn2O3 carrier reaches the targeted tumor site, can it be degraded to release the drug quickly? Fig.3F demonstrated that Mn2O3 nanocarriers owned excellent MR imaging capability after degradation into Mn2+ in simulated tumor microenvironments in vitro. So next, MR imaging technique was applied to monitor the tumor-specific degradation behavior of HA-Mn2O3 in vivo. As presented in Fig 6C and 6D, the T1-weighted MR images of tumors treated with Mn2O3 and HA-Mn2O3 presented a bright signal enhancement in a time-dependent manner, suggesting its 24


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strong positive contrast. This could be elucidated by Mn2+-mediated MR enhancement effect. This result testified the degradation of Mn2O3 and the production of Mn2+ in tumor site after treatment with Mn2O3 or HA-Mn2O3, which was consistent with the results of in vitro TME-responsive degradation property for Mn2O3 nanoparticles. Moreover, tumor treated with HA-Mn2O3 presented 1.31-, 1.24-, 1.40- and 1.47-folds higher MR signal intensity than Mn2O3 group at 1, 2, 4 and 8h. This might be attributed to active tumor targeting caused by HA. Taken together, all above results suggested HA-Mn2O3 nanocarriers could selectively deliver drugs to tumor sites. And then they were further degraded in acidic and reductive (high GHS level) tumor microenvironment, to realize tumor responsive simultaneous release of Mn2+ and HCQ. This behavior was conducive to an effective HCQ accumulation level at the target site, to adequately inhibit autophagy in vivo. In addition, compared with saline control group, more distinct tumor boundaries could be seen in mice treated with Mn2O3 or HA-Mn2O3, suggested their excellent MR diagnostic performance. Due to their outstanding MR imaging capability plus efficient drug delivery ability, HA-Mn2O3 nanoparticles could serve as a multifunctional nanocarrier for visualizing tumor lesions using MRI with concomitantly delivering therapeutic agents specifically to the tumors and monitoring their therapeutic effects, thereby allowing more precise diagnosis and treatment of cancers. Moreover, to investigate the pharmacokinetics feature, the concentration of HCQ in blood samples of BALB/c mice after injection of HCQ, Mn2O3/HCQ and HA-Mn2O3/HCQ were determined by HPLC, according to the standard curve shown in Fig S16. The main pharmacokinetic parameters were listed in Tab S1. The order of drug elimination rate in mice was: HCQ group > Mn2O3/HCQ group > HA-Mn2O3/HCQ group, indicating that HA-Mn2O3/HCQ delivery system can prolong the retention time of HCQ in vivo and increase the bioavailability of HCQ, thereby 25



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improving the therapeutic effect of the drug.

Figure 6. A: In vivo NIR imaging of 4T1 tumor-bearing mice after the intravenous injection of free IR783, IR783-loaded Mn2O3 or HA-Mn2O3 carriers at 2, 8, 12 and 24h post injection. Tumor tissues are indicated by yellow arrows B: NIR imaging of various major tissues in different groups at 24h post injection. C: In vivo MR images of mice bearing implanted 4T1 tumor after the intravenous injection of saline, Mn2O3 or HA-Mn2O3 nanoparticles at 1, 2, 4, and 8h. The areas in green box represent tumors. D: Quantification of the tumor MR signals intensity enhancement in 4T1 tumor -bearing mice according to MR images obtained at 1, 2, 4, and 8h post injection. The data presented are the mean ± SD. （n=3, *P < 0.05 and **P < 0.01）

3.7. In vivo antitumor effect The promising antitumor effect displayed by HA-Mn2O3/HCQ in vitro led us to ask whether it would have activity on 4T1 tumor-bearing mice in vivo. During two weeks of treatment, changes of tumor volume for each group were summarized in Fig 7A. As Fig 7A and Fig S17 shown, HCQ, 26


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Mn2O3/HCQ and HA-Mn2O3/HCQ all showed obvious tumor growth inhibition compared to saline and nanocarriers groups. The mean tumor volume after 6 times administration (endpoint) was 471.7 mm3 for saline group, 448.7 mm3 for Mn2O3 group, 431.3 mm3 for HA-Mn2O3 group, 276.6 mm3 for HCQ group, 111.4 mm3 for Mn2O3/HCQ group and 54.5 mm3 for HA-Mn2O3/HCQ group. HA-Mn2O3/HCQ displayed the best therapeutic efficacy with 5.08-folds tumor inhibition ratio than HCQ group. What’s more, the histological changes of tumor tissues were tested through H&E (Fig 7B) and TUNEL staining (Fig S18). It could be observed a compact cell arrangement in saline group, without apoptotic cells being present. whereas small amounts of apoptotic cells (brown granules) and cell shrinkage emerged in HCQ group. Furthermore, HA-Mn2O3/HCQ group displayed apparent cell apoptosis and lysis, implying the greatest antitumor activity. 3.8. Autophagy inhibition in tumor tissue In order to explore the mechanism resulting in tumor inhibition by HA-Mn2O3/HCQ, micro-morphological study and immunofluorescence analysis were adopted. After two weeks of treatment, tumors were excised from mice for TEM and immunologic tests. As Fig 7C shown, compared to saline and nanocarriers groups, there was significantly increased accumulation of autophagic vesicles in HA-Mn2O3/HCQ treated tumors (indicated by red arrow), suggesting blocked autophagy process. It is well known that autophagosome elongation steps become stalled in the presence of lysosomotropic agents, like HCQ39. Lipidation of the ubiquitin-like protein, referred to here as LC3, is an important step in autophagosome elongation40. As a result of autophagy disruption, a kind of specified substrates to the autophagosome, referred to cargo adaptor protein p62, will accumulate in tumor cells. Herein, the expression levels of LC3 and p62 proteins in tumor tissues were additionally determined by immunofluorescence analysis to survey autophagic flux in tumors treated with HA-Mn2O3/HCQ. As Fig 7D-7G shown, compared with saline control group, 27



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LC3 and p62 proteins were both up-regulated in HCQ and HA-Mn2O3/HCQ groups. Moreover, tumor treated with HA-Mn2O3/HCQ resulted in significantly higher expression levels of LC3 as well as p62, indicating blocked downstream autophagy and autophagolysosome degradation pathway. Above results suggested HA-Mn2O3/HCQ could be as a kind of in situ nano-generators to realize enhanced autophagy inhibition and tumor therapy. After HA-Mn2O3 selectively delivered HCQ to tumor sites, the nanocarriers further degraded in specific tumor microenvironment, to realize simultaneous release of Mn2+ and HCQ. In situ high concentrations of these two substances would achieve effective autophagy blockade attributable to lysosomal dysfunction. This behavior may induce tumor cell death through lysosome-mediated cell death pathways 17, as well as cutting off autophagolysosome-mediated natural nutrients and energy sources reused of waste sources in the tumors 3,4, so as to realize efficient tumor suppression.

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Figure 7. A: Tumor volume changes of mice treated with different formulations within two weeks. B: H&E stained tumor tissues harvested from mice after two weeks of treatment with different formulations. Typical pathological changes are indicated in red section. C: TEM images of excised tumors that harvested from mice after two weeks of treatment with saline, HA-Mn2O3, HCQ or HA-Mn2O3/HCQ. Typical autophagic vesicles are indicated by red arrows. D: Immunofluorescence images to detect LC3 in the tumors that harvested from mice after two weeks of treatment with saline, HA-Mn2O3, HCQ or HA-Mn2O3/HCQ (200x; red: LC3; blue: nucleus). E: The quantitative fluorescence intensity of LC3 can be seen in histogram. F: Immunofluorescence images to detect p62 in the tumors that harvested from mice after two weeks of treatment with different formulations (400x; red: p62; blue: nucleus). G: The quantitative fluorescence intensity of p62 can be seen in histogram.

3.9. In vivo safety The toxicity of different formulations was further examined. The changes in the body weights of 29



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tumor-bearing mice were recorded as an indication of safety. As shown in Fig 8A, there was not significantly different of body weights in all groups during the treatment, suggesting the low toxicity of our drug delivery system. Fig 8B showed the survival curves of mice treated with various formulations. In saline treatment group, all mice died after 35d of treatment. The group treated with HCQ showed a higher survival rate (50% at 35d) than saline group. Remarkably, mice treated with HA-Mn2O3/HCQ showed a 100% survival rate over 40d. This result further demonstrated the effectiveness and biocompatibility of HA-Mn2O3/HCQ. The toxicity of main organs was also evaluated by H&E staining (Fig 8C). No pathological changes in the heart, liver, spleen, lung, kidney and brain were observed as compared to control group, further demonstrating the biosafety of this nanoplatform. Finally, the potential toxicity of HA-Mn2O3/HCQ in vivo was further evaluated by the serum biochemistry assay on health mice. As shown in Tab S2, after administration at the therapy dose, there were no significant differences in all biochemical parameters. These results demonstrated that HA-Mn2O3/HCQ could be used as a safe nanoplatform for tumor treatment in vivo.

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Figure 8. A: Body weight changes of mice in each group during two weeks treatment. B: Survival rate of mice-bearing 4T1 tumors after various treatments as indicated. C: H&E staining of the major organs (heart, liver, spleen, lung, kidney and brain) that harvested from mice after two weeks of treatment with different formulations

4. Conclusion In summary, HA-Mn2O3/HCQ was successfully synthesized as a novel in situ autophagy disruption generator and potential 4T1 tumor-targeted T1 contrast agent. Pharmaceutical studies showed HA-Mn2O3 could degrade in tumor microenvironment, to achieve HCQ and Mn2+ 31



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simultaneous release. CD44-positive 4T1 cell line was chose as tumor model. Results in vitro demonstrated HA-Mn2O3 displayed a specific localization and retention in lysosomes after entering tumor cells. Then intracellular Mn2O3 disintegrate rapidly to release HCQ and Mn2+, leading to representative lysosome deacidification and autophagy blockade effect. In vivo results proved HA-Mn2O3/HCQ was conducive to achieve an effective HCQ accumulation level at the target site. It displayed the best therapeutic efficacy with 5.08-folds tumor inhibition ratio than HCQ group based on autophagy blockade pathway. Additionally, in vivo MR imaging demonstrated that HA-Mn2O3 exhibited an enhanced contrast effect in the tiny 4T1 tumor region. Therefore, HA-Mn2O3 nanoparticles could serve as multifunctional carriers for visualizing tumor lesions using MRI with concomitantly delivering drug specifically to the tumors and monitoring their therapeutic effects, thereby allowing more precise diagnosis and treatment of cancers.

Supporting Information Figure S1: A: The XRD pattern of samples; Figure S2: N2 adsorption and desorption curves; Figure S3: The pore diameter distribution; Figure S4: TGA curves; Figure S5: Zeta Potential; Figure S6: The size change of Mn2O3 and HA-Mn2O3 NPs in the cell medium; Figure S7: The size change of HA-Mn2O3/HCQ NPs in the cell medium; Figure S8: The changes of Mn2O3 dispersions; Figure S9: The structure changes of HA-Mn2O3; Figure S10: Intracellular uptake of nanoparticles; Figure S11: Lysosomal localization; Figure S12: Lysosomal destruction results of A549 cells; Figure S13: LC3-II: LC3-I value of 4T1 cells; Figure S14: Cytotoxicity of Mn2O3 and HA-Mn2O3; Figure S15: The accumulation of autophagosomes in A549 cells; Figure S16: Linear relationship of HCQ; Table S1: The main pharmacokinetic parameters; Figure S17: The images of tumors; Figure S18: TUNEL immumohistochemical results; Table S2: In vivo toxicity evaluation.

ORCID Zhenzhong Zhang: 0000-0002-8704-0974

ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 81573364), Henan Postdoctoral Research Grant (No.1902003) and Key Program for Science and Technology Research in Henan Province (No.192102310153).

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CONFLICT OF INTEREST The authors declare no conflict of interest.

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TOC Figure:

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