Cancer Cell Membrane-Biomimetic Nanoplatform for Enhanced

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Biological and Medical Applications of Materials and Interfaces

Cancer Cell Membrane-Biomimetic Nanoplatform for Enhanced Sonodynamic Therapy on Breast Cancer via Autophagy Regulation Strategy Qianhua Feng, Xuemei Yang, Yutong Hao, Ning Wang, Xuebing Feng, Lin Hou, and Zhenzhong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10948 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Cancer Cell Membrane-Biomimetic Nanoplatform for Enhanced Sonodynamic Therapy on Breast Cancer via Autophagy Regulation Strategy Qianhua Feng,†,‡,§ Xuemei Yang,† Yutong Hao,† Ning Wang,† Xuebing Feng,¶ Lin Hou,*,†,‡,§ Zhenzhong Zhang*,†,‡,§

†School

of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou

450001, China ‡Collaborative

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

Zhengzhou 450001, China §Key

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

Zhengzhou 450001, China ¶School

of Stomatology, Shandong university, Shandong Province, Jinan 250012, China

* Corresponding author. Tel: 86-371-67781910; Fax: 86-371-67781908. Email: [email protected] (L. Hou), [email protected] (Z. Zhang).

ABSTRACT Autophagy was considered as a double-edged sword that might cooperate, aggravate or antagonize apoptosis. We found that the sonodynamic therapy (SDT) in low dosage induced autophagy might function as a survival pathway for breast cancer and exhibited resistance to SDT mediated apoptosis. In this sense, it was highly desired to enhance SDT via autophagy regulation strategy. Herein, we reported a biomimetic nanoplatform based on hollow mesoporous titanium dioxide nanoparticles (HMTNPs) by autophagy inhibitor (hydroxychloroquine sulphate, HCQ) loading and cancer cell membrane (CCM) coating. Owing to the biomimetic surface functionalization, the CCM-HMTNPs/HCQ could escape from macrophage phagocytosis, actively recognize and “home” to tumor by homologous targeting ability. Afterwards, the released HCQ in response to ultrasound stimulus was capable of blocking autophagic flux and cutting off the nutrients supply derived from the damaged organelles, which was anticipated to abrogate the cells’ resistance to SDT. Meanwhile, the vessel normalization effect of HCQ alleviated the tumor hypoxia, ACS Paragon Plus Environment

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which was bound to enhance the oxygen-dependent HMTNPs mediated SDT treatment. Based on the above findings, it was undoubtedly logical that CCM-HMTNPs/HCQ would sensitize breast cancer cells to SDT via autophagy regulation strategy, which held a great promise in cancer treatment. KEYWORDS: sonodynamic therapy; hollow mesoporous titanium dioxide; autophagy regulation; biomimetic; homologous targeting

1. INTRODUCTION In the past few years, reactive oxygen species (ROS)-mediated sonodynamic therapy (SDT) has emerged as an alternative to the traditional treatments of cancer, benefiting from its deep penetration and noninvasive features.1,2 In comparison with sonosensitizer molecules, nanosensitizers such as TiO2 and MnWOx nanoparticles have drawn a widespread attention for their potential as drug carriers.3 Interestingly, the hollow mesoporous titanium dioxide nanoparticles (HMTNPs) possessed high drug loading efficiacy due to their large surface area and great pore volume.4,5 Classically, the ultrasound (US) activated ROS generation from HMTNPs primarily targeted and destroyed the organelles such as mitochondria in the SDT cases, further activating mitochondria apoptosis signaling pathway.6 Despite of these advantages, several resistance mechanisms were involved in the SDT cases. Accumulated data has proposed the existence of the cross-talk between apoptosis and autophagy,7 since autophagy was a lysosome-dependent process for eliminating impaired proteins and organelles (mitochondria) resulted from SDT.8 However, the role of autophagy for acting as a tumor suppressor or promoter in cell fate was hotly contested. Autophagy was considered as a double-edged sword that might cooperate, aggravate or antagonize apoptosis, which depended on the particular tumor stage, cell line, specific SDT dosage and the types of sonosensitizers.9 Recent studies had investigated the selective modulation of autophagy at different stages of tumour progression.10 It had reported that autophagy mainly acted as a tumor suppressor preventing carcinogenesis in early stage cancer, but a tumor promotor providing nutrients in late stage cancer.11 In the treatment of late stage cancer, low SDT dosage induced autophagy was not enough to induce

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cell death, while it might function as a survival pathway to cells through clearing the damaged organelles and recycling nutrients for energy supply, leading to a resistance to SDT mediated apoptosis. To tackle these issues, it was necessary to introduce autophagic modulators into the system. It has been suggested that autophagy modulation was proposed as a strategy to sensitize cells to chemotherapy or other traditional treatments.12,13 Typically, a well-known autophagy inhibitor, hydroxychloroquine sulphate (HCQ), could deacidify the lysosome and inhibit the autophagosome fusion with lysosomes, thus terminally blocking the autophagic flux.14 Such effect would cut off the nutrients supply derived from the damaged mitochondria, which was anticipated to abrogate the cells’ resistance to SDT. More importantly, HCQ could interfere with intracellular Notch1 trafficking and signaling to exhibit the vessel normalization effect, as reported in literature.15 Therefore, HCQ alleviated the tumor hypoxia by improving tumor perfusion and oxygenation, which was bound to enhance the oxygen-dependent SDT treatment. From this viewpoint, it was more than critical to obtain a promising platform based on HMTNPs loaded with HCQ for elevating the SDT sensitivity of MCF-7 cells. For naked HMTNPs, the premature drug release and lack of tumor targeting ability should be settled before their application. Hence, it was imperative to modify HMTNPs with functional groups. Excitingly, the biomimetic drug delivery system with natural cell membranes coating has drawn wide attentions.16,17 Such a top-down approach bypassed the laborious group-modified engineering, benefiting from the absolute duplication of surface antigentic diversity. Of several different types of membranes reported to date,18,196 cancer cell membrane (CCM) bestowed the homologous targeting effect and immune evasive ability on nanoparticles, which relied on the surface antigens such as cellular adhension molecules and “marker-of-self” protein.20-22 In addition, the tight coverage of HMTNPs by CCM could minimize leakage of HCQ in circulation and ensure ultrasound responsive drug release in tumor site due to the oxidation and dissociation of CCM by SDT. Inspired by these advantages, the CCM biomimetic surface functionalization on HMTNPs was expected to enhance the antitumor effect and reduce the potential side effect. In this study, we developed a CCM biomimetic nanoplatform based on HMTNPs loaded with ACS Paragon Plus Environment


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HCQ. Herein, the MCF-7 human breast cancer cell was selected as a model cell line. As illustrated in Scheme 1, the cancer cell membrane-biomimetic nanoplatform (CCM-HMTNPs/HCQ) could escape from the macrophage phagocytosis, actively recognize and “home” to MCF-7 tumor due to the biomimetic surface mediated homotypic adhesive interactions. On the one hand, the vessel normalization effect of HCQ alleviated the tumor hypoxia by improving tumor perfusion and oxygenation, which was bound to enhance the oxygen-dependent SDT treatment. On the other hand, we found that the low SDT dosage (20 mg kg-1 HMTNPs, 1 W cm-2) induced autophagy in MCF-7 cells functioned as a survival pathway to provide energy and nutrients, which exhibited resistance to SDT mediated apoptosis. While the released HCQ in response to US stimulus was capable of blocking autophagic flux to abrogate the MCF-7 cells’ resistance to SDT. Reasonably, it was undoubtedly logical that CCM-HMTNPs/HCQ would enhance SDT on breast cancer via autophagy regulation strategy, which held a great promise in cancer treatment.

Scheme 1 Schematic of the cancer cell membrane biomimetic nanoplatform. A) Formulation of CCM-HMTNPs/HCQ. B) The vessel normalization effect of HCQ for enhancing the oxygen-dependent SDT treatment. C) Schematic mechanism of CCM-HMTNPs/HCQ for enhanced


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SDT on breast cancer via autophagy regulation strategy.

2. RESULTS AND DISCUSSION The TEM image (Figure 1A, a) of the synthesized HMTNPs clearly showed their hollow interiors with a size of ~100 nm. And the high-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping further demonstrated the distribution of two elements (Ti, O) on HMTNPs. According to the N2 adsorption-desorption isotherm (Figure S1), the average pore diameter was about 3.9 nm and the surface area of HMTNPs was calculated as 169.6 m2 g-1, allowing for HCQ loading inside the pores in doses sufficient for high therapeutic efficacy. To fabricate the CCM-biomimetic nanoparticles, CCM were collected from MCF-7 human breast cancer cell as a model cell line, and displayed the irregular hollow vesicles and thickness of ~10 nm in Figure 1A, b. Then CCM-HMTNPs/HCQ was prepared via the co-extrusion method after coating CCM on the surface of HMTNPs/HCQ. The optimum CCM-to-HMTNPs weight ratio was 2:1, with which CCM-HMTNPs/HCQ exhibited excellent dispersibility after 10 days storage in PBS (Figure S2). The hydrodynamic diameter of the resulted CCM-HMTNPs/HCQ was 131.7 ± 1.6 nm (Figure 1B), slightly larger than that of HMTNPs/HCQ (110.4 ± 2.3 nm). While the zeta potential of CCM-HMTNPs/HCQ was -23.8 ± 0.9 mV, close to that of CCM (-21.6 ± 1.3 mV). These results demonstrated the coating CCM was present in the right-side-out orientation. Moreover, the morphology of CCM-HMTNPs/HCQ was also characterized by TEM (Figure 1A, c and Figure S3), which showed the core-shell structure with the CCM shell thickness of ~9.1 nm, very close to the measurement of CCM, verifying the successful coverage of HMTNPs/HCQ by CCM. In addition, the CCM-HMTNPs/HCQ exhibited good stability and there was no visible precipitation in PBS at different pH and culture medium containing 10% fetal bovine serum (Figure S4). To obtain more insights into the formulation, the optical property of CCM-HMTNPs/HCQ was examined (Figure S5). It displayed the absorption peak of HCQ at 221 nm and 343 nm,23,24 indicating the successful HCQ loading. And the loading efficiency of HCQ was calculated as 46.4%. Keeping the HMTNPs as the sonosensitizers in mind, it was highly desired to assess the ultrasound activated ROS (especially 1O2) generation for CCM-HCQ or CCM-HMTNPs by using SOSG as an



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indicator, which could emit fluorescence at 525 nm after capturing 1O2 (Figure 1C).25 Figure S6 showed that the SOSG emitted low fluorescence intensity under US irradiation in CCM-HCQ group. By contrast, it was found that the 1O2 production from CCM-HMTNPs showed ultrasonic time-dependent features, exhibiting an excellent SDT effect. This phenomenon could be explained by the fact that the HMTNPs as the sonosensitizer would transfer energy to oxygen molecules,26 resulting in the generation of 1O2. Based on the HMTNPs mediated ROS generation, the CCM integrity of CCM-HMTNPs/HCQ under US irradiation was assessed (Figure 1A, d). Surprisingly, it showed that the gray CCM outer shell was destroyed into fragments after US irradiation, which might be probably due to that the US activated ROS generation from HMTNPs induced the oxidation and dissociation of CCM. Hence, it was interesting to explore the drug release behavior by applying US stimulation (Figure 1D). With the excellent coating efficiency and stability, CCM-HMTNPs/HCQ displayed a slow and sustained drug release pattern compared to the HMTNPs/HCQ group. Then the US stimulation was conducted experimentally at the beginning. As expected, it was not surprised to observe that the HCQ release from CCM-HMTNPs/HCQ increased by 54.2%, indicating a ultrasound responsive drug release profile owing to the US induced CCM damage. In this sense, it was reasonable to infer that CCM-HMTNPs/HCQ should open up new possibilities for a more sophisticated on-demand cargo delivery and HCQ mediated autophagy regulation-associated sonodynamic therapy.

Figure 1. Characterization of CCM-HMTNPs/HCQ. A) a) TEM image (left) and the correspondent ACS Paragon Plus Environment

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HAADF-STEM image (right) of HMTNPs, elemental mapping showed the distribution of Ti (green) and O (yellow). b and d) TEM images of CCM vesicles (b), CCM-HMTNPs/HCQ (c) and CCM-HMTNPs/HCQ under US irradiation (1 W cm-2, 1 min) (d), respectively. B) Size and zeta potential of CCM, HMTNPs/HCQ and CCM-HMTNPs/HCQ. C) US irradiation time-dependent 1O generation of CCM-HMTNPs (20 μg mL-1) determined using singlet oxygen sensor green 2 (SOSG) as indicator. D) Release profiles of HCQ from CCM-HMTNPs/HCQ under US irradiation (1 W cm-2, 30 s). The data points represent mean ± S.D. (n = 3).

According to several published protocols, the membrane surface antigens played crucial roles in the homologous adhesion feature of tumor cells.27 Then the western blotting analysis of the various proteins on the biomimetic nanoplatform was carried out systematically. Figure 2A showed that the CCM-HMTNPs/HCQ retained CD44 (MCF-7 cells surface antigen marker), as well as CD47, which acted as the molecular “marker-of-self” to prevent the macrophages phagocytosis in reticular endothelial system.28 Notably, there was a considerable enrichment of cellular adhension molecules (CD176, galectin-3, E-cadherin) on CCM-HMTNPs/HCQ, suggesting the successful transfer of the marker proteins to the shell of the nanoparticles. The retainment of these cellular adhension molecules with the adherence capabilities was responsible for the specific self-recognition to hypotypic cancer cells via homologous-binding mechanism. To prove our assumption, the cellular uptake of HMTNPs and CCM-HMTNPs was evaluated in five different cell lines. As illustrated in Figure 2B and Figure S7, in the case of the CCM coating nanoparticles, the fluorescence intensity of nile red (NR) as the fluorescence dye was far superior in the corresponding source cell (MCF-7 cell) over those in heterotypic cells. These results indicated that the biomimetic CCM-HMTNPs possessed a higher affinity to homologous MCF-7 cells due to the adhesion molecules mediated homotypic adhesive interactions, as observed in other reports.29 After exposed to US irradiation, brighter red fluorescence was observed in CCM-HMTNPs/NR treated cells (Figure S8), which was consistent with the ultrasound responsive drug release property as aforementioned. Taken together, CCM-HMTNPs developed here displayed highly tumor-selective targeting of the homotypic tumor cells. Much evidences have shown that the tumor cells possessed the immune evasive ability due to the tumor-associated antigens on the surface membrane.30 In light of this, the Raw264.7 mouse macrophage cells were selected to evaluate the immunocompatibility of the CCM-HMTNPs/NR. ACS Paragon Plus Environment


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The quantitative flow cytometric analysis in Figure 2C verified the lower uptake of CCM-HMTNPs/NR (8.2%) than that of the non-membrane coated nanoparticles (76.4%). And the CLSM images also showed the similar results (Figure 2D, Figure S7). These results implied that CCM-HMTNPs suppressed the macrophage engulfment and exhibited the favorable immune evasive efficacy, which might be ascribed to the “marker-of-self” CD47 capable of inhibiting phagocytosis through interactions with signal regulatory protein-α expressed on macrophages.28

Figure 2. Evaluation of the homologous targeting and immune escape effect of CCM-HMTNPs/HCQ. A) Western blotting analysis of cancer cell lysate (a), cancer cell membrane vesicles (b) and CCM-HMTNPs/HCQ (c) for cell membrane protein markers. B) Cellular uptake of HMTNPs/NR and CCM-HMTNPs/NR in MCF-7 (Human breast cancer cell from Michigan Cancer Foundation), MDA-MB-231 (Human breast cancer cell), Hs538Bst (Normal human breast cell), HepG2 (Human hepatocellular carcinoma cell) and NIH3T3 (Mouse fibroblasts) cells. C and D) Flow cytometric assay (C) and CLSM images (D) of Raw264.7 cells after incubation with HMTNPs/NR and CCM-HMTNPs/NR, respectively. Nuclei were stained with DAPI.

Encouraged by the ultrasound activated ROS generation of CCM-HMTNPs in cell-free experiments, we moved on to investigate its feasibility on MCF-7 cells by using DCFH-DA fluorescent probe. Quantitative flow cytometric analysis indicated the large amount of ROS production of CCM-HMTNPs + US and CCM-HMTNPs/HCQ + US with a positive cell percentage ACS Paragon Plus Environment

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of 57.3% and 61.4%, respectively (Figure 3A). Generally, mitochondria were considered as the primary targets in the SDT cases.6 Then the mitochondrial damage induced by CCM-HMTNPs was detected by using JC-1 mitochondrial membrane potential (MMP) assay kit. Figure 3B,C showed the CCM-HMTNPs group with US irradiation brought out a high JC-1 monomer (green) /aggregate (red) ratio, suggesting the enormous loss of MMP and mitochondrial damage. Classically, the destruction of mitochondria structure would activate mitochondria apoptosis signaling pathway, and further result in the SDT effect.

Figure 3. A) Flow cytometric assay of ROS with different treatments. B) Mitochondrial membrane potential detection via JC-1 staining. JC-1 aggregate (red) and monomer (green) formation were recorded. C) The bar graph indicated the JC-1-monomer/JC-1-aggregate formation ratio. *P

Cancer Cell Membrane-Biomimetic Nanoplatform for Enhanced

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