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Platelet Membrane Camouflaged Black Phosphorus Quantum Dots Enhance Anticancer Effect Mediated by Apoptosis and Autophagy Yinghui Shang, Qinghai Wang, Bin Wu, Qiangqiang Zhao, Jian Li, Xueyuan Huang, Wansong Chen, and Rong Gui ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04735 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Applied Materials & Interfaces

Platelet Membrane Camouflaged Black Phosphorus Quantum Dots Enhance Anticancer Effect Mediated by Apoptosis and Autophagy

Yinghui Shang a, Qinghai Wang b, Bin Wu c, Qiangqiang Zhao a, Jian Li d, Xueyuan Huang a, Wansong Chen e and Rong Gui a a

Department of Blood Transfusion, the Third Xiangya Hospital, Central South

University, Changsha 410013, P. R. China b

Department of Cardiology, the Second Hospital of Shandong University, Jinan

250000, P. R. China c

Department of Transfusion Medicine, Wuhan Hospital of Traditional Chinese and

Western Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, P. R. China d

Clinical Laboratory of the Third Xiangya Hospital, Central South University,

Changsha 410013, P. R. China e

College of Chemistry and Chemical Engineering, Central South University,

Changsha 410083, P. R. China



Corresponding author. Phone/Fax: +86-731-8861 8513.

E-mail address: [email protected] (R. Gui)

ACS Paragon Plus Environment

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ABSTRACT: Hederagenin (HED) has poor anticancer activity, whose mechanism remains unclear and unsystematic. Free drugs for cancer treatment exhibit disadvantages such as poor targeting and efficacy. To address this problem, we constructed a nanoplatform of black phosphorus quantum dots (BPQDs) camouflaged with a platelet membrane (PLTm) carrying HED, termed PLT@BPQDs-HED. PLTm vesicles serve as a shell to encapsulate multiple high-efficiency drug-loaded nanocores, which can target tumor sites and significantly improve antitumor activity. Compared with free HED, this platform significantly reduced tumor cell viability and the mitochondrial membrane potential (MMP), while increasing the production of intracellular reactive oxygen species (ROS). The platform also significantly increased the

amounts

of

nick-end-labeling

terminal

deoxyribonucleotide

(TUNEL)-positive

cells

and

transferase-mediated decreased

the

dUTP

number

of

Ki-67-positive cells. In addition, the platform upregulated proapoptotic factor Bax, downregulated the anti-apoptotic molecule Bcl-2, activated Caspase-9 and Caspase-3, and stimulated Cytochrome C release. Moreover, the platform promoted the formation of autophagosomes, upregulated Beclin-1, and promoted LC3-I conversion into LC3-II. This study demonstrated that the above platform significantly enhances tumor targeting and promotes mitochondria-mediated cell apoptosis and autophagy in tumor cells. KEYWORDS: Platelet membrane, black phosphorus quantum dots, apoptosis, autophagy, hederagenin


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1. INTRODUCTION Natural plants and their extracts are valuable resources for antineoplastic agent research and development 1. Identifying potent antineoplastic agents with low toxicity from natural plants is a promising research direction for anticancer treatment 2. China represents a rich source of medicinal natural plant species, and the Chinese medicine has a long history of tumor treatment 3. Triterpenoid saponins demonstrate antitumor activities against a wide variety of carcinomas 4. Hederagenin (HED) is a pentacyclic triterpenoid compound with diverse pharmacological features. It has been reported that HED analogs have potential anticancer activities. H6, derived from HED, may improve the antitumor action of paclitaxel 5. HED saponins (such as macranthoside B), triterpene saponin compound 8, and Pulsatilla saponin D analog (compound 10i) exhibit antitumor effects 5-7. It was indicated that the sugar structures (i.e., α-hederin) of triterpenoid saponins are closely related to their anticancer properties 8. HED and analogs have potential anticancer effects; however, the efficiency of HED is poor, with the underpinning anticancer mechanism remaining unclear. The main issues of anticancer drugs are poor targeting and low efficacy. In the past 30 years, research assessing nanomaterials has indicated that a variety of nanomaterials can be used as drug carriers to enhance the antitumor activities of drugs in vivo

9.

A systematic review revealed that nanomaterials loaded with

chemotherapeutic drugs have therapeutic effects on tumors of the digestive system 10. The antitumor effects of nanomaterials are related not only to their internal



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characteristics, such as antioxidant activity, but also to external stimuli, including high heat caused by infrared rays or magnetic fields 11. Due to its unique physical structure, good optical properties

12,

and other characteristics such as high surface area and

drug-loading rate 13, black phosphorus (BP) has become an outstanding nanomaterial. Indeed, BP quantum dots (BPQDs) show good biological imaging functionality and tissue penetration, with the characteristics of photothermal/photodynamic synergistic antitumor treatments 11. Phosphorus, as the main element of BPQDs, represents one of the essential elements of an organism's life activities; thus, BPQDs are almost nontoxic, with good properties such as biocompatibility, degradability, and therapeutic potential

14.

In addition, BPQDs with particle sizes of 10-20 nm are not

easily engulfed by macrophages; thus, they have a long cyclic half-life, and are more likely distributed in tumor tissues by an enhanced permeability and retention (EPR) activity

9, 15-18,

enabling the loaded antitumor drugs to accumulate at the tumor site.

Besides, BP degradation is increased under acidic conditions 19, thereby accelerating the release of antitumor agents in acidic tumor microenvironment. These strengths make it possible to develop nontoxic and highly effective treatments based on BP nanomaterials. According to the abovementioned characteristics, BPQDs are expected to be excellent carriers of chemotherapeutic drugs for the treatment of tumors. Studies have shown that platelets (PLTs) bind to tumor cells through P-selectin and the CD44 receptor

20-21

as well as their structure-based capture

22.

Based on the

close relationship between PLTs and tumors, bionic strategies for delivering drugs to tumor tissues have emerged. Purified PLTs are readily available and show weak


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antigenicity and immunogenicity. Furthermore, compared with drug-loaded nanoparticles alone, drug-loaded nanoparticles coated with a platelet membrane (PLTm) can reduce uptake by macrophages and avoid the activation of complements in autogenous plasma, thus increasing the retention time of the drug in the body. In addition, compared with bare nanoparticles, hydrophilic surface glycans of the plasma membrane contribute to the stabilizing effect of PLTm-camouflaged nanoparticles, thereby improving their colloidal stability

23.

damaged blood vessels and enhances binding

Meanwhile, the PLTm also adheres to 20

so that the drug can be targeted for

delivery to the tumor tissue. Therefore, application of the PLTm with these features has become a biomimetic strategy for the targeted delivery of antitumor drugs. Since the PLTm-camouflaged BPQDs drug-loading system possesses high drug-loading efficiency, excellent biological safety, good compatibility, and good targetability, we designed a platform of PLTm-coated BPQDs loaded with HED and explored its antitumor effect (Figure 1). This study showed that this platform can enhance antitumor activity, providing a new tool for tumor treatment, which has certain potential in the field of cancer therapy.



Figure 1. Schematic diagram of PLT@BPQDs-HED construction and targeted therapeutic mechanisms. 2. MATERIALS AND METHODS 2.1. Materials. BPQDs Dispersion was purchased from XFNANO Materials Technology (China). HED, Cy5, rhodamine B (RhB), rhodamine 123 (Rh123), Hoechst 33342, and distearoyl phosphatidyl ethanolamine-fluorescein isothiocyanate (DSPE-FITC) were provided by Yeasen Biotechnology (China). Dialysis membranes


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(2 kD) were purchased from Solarbio (China). Annexin V-FITC/PI Apoptosis Detection and ROS Assay Kits were provided by KeyGEN BioTECH (China). Cell Counting Kit-8 (CCK-8) was manufactured by Dojindo Laboratories (Japan). Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM) (high glucose), RPMI-1640 and trypsin were purchased from Life Technologies (USA). A terminal deoxyribonucleotide transferase (TdT) In Situ Apoptotic Kit (DAB) (R&D Systems, Shanghai, China), Ki-67 Detection Kit (DAB) (R&D Systems), and hematoxylin and eosin (H&E) were purchased from the Servicebio Technology (China). Primary antibodies targeting Bax, Bcl-2, Caspase-3, Caspase-9, Cytochrome C, Beclin-1, LC3, and β-actin, as well as horseradish peroxidase (HRP) goat anti-rabbit secondary antibodies were from Proteintech (USA); Cy3-linked goat anti-IgG, FITC-conjugated goat anti-IgG, and DAPI were manufactured by Servicebio Technology (China). Polycarbonate porous membrane syringe filters (200 nm) were provided by Whatman (USA). 2.2. Cells and mice. Human breast cancer MCF-7 cells were kindly provided by the Cancer Research Institute, Central South University, and maintained in DMEM (high glucose) supplemented with 10% FBS. RAW264.7 cell culture was performed in RPMI-1640 supplemented with 10% FBS. Cell culture was carried out for both lines at 37°C in presence of 5% CO2. Female BALB/c nude mice (6 weeks) were provided by the Hunan SJA Laboratory Animal Co., Ltd. 2.3. Preparation of PLTm vesicles. Whole blood samples collected in tubes containing heparin anticoagulant from female BALB/c nude mice were used. PLTs



were separated from whole blood by centrifugation and washing. The PLTm was extracted by repeated freezing and thawing, and suspended in phosphate-buffered saline (PBS), followed by ultrasound treatment (2 min, 42 kHz, 100 W) to yield PLTm vesicles. 2.4. BPQDs-HED preparation. BPQDs (5 mg) and HED (2.8 mg) in PBS (2 mL) were mixed (24 h at ambient). Then, free HED was removed by passing through a 2 kD dialysis membrane. The dialyzed sample was obtained for HED concentration assessment. Encapsulation efficiency (EE) was calculated as follows: EE% = mass of HED loaded on BPQDs / mass of HED added × 100%. Loading efficiency (LE) was calculated as follows: LE% = mass of HED loaded on BPQDs / mass of BPQDs × 100%. 2.5. Construction of PLT@BPQDs-HED. PLTm vesicles underwent fusion with an equal volume of BPQDs-HED by ultrasound (5 min, 42 kHz, 100 W), and the resulting specimens were filtered 20 times using porous syringe filters with a membrane pore size of 200 nm. After centrifugation (2500 rpm, 10 min), excess PLTm was removed, and PLT@BPQDs-HED was obtained. 2.6. Characterization of PLT@BPQDs-HED. PLT@BPQDs were morphologically assessed by transmission electron microscope (TEM) with a Tecnai G2 Spirit TEM (FEI, USA), detecting PLTm encapsulation of nanoparticles and determining the size of nanoparticles. A Zetasizer Nano ZS (Malvern Nano series, Malvern, UK) was


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employed for surface charge assessment. PLT@BPQDs-HED absorbance was obtained by UV/vis spectrometry (ScanDrop, Analytik Jena, Germany). 2.7. PLT@BPQDs-HED release properties for HED. In vitro drug release assays were carried out at pH 7.4 and pH 5.4 to determine whether HED could be released in a pH-dependent manner. To this end, 1 mL of PLT@BPQDs-HED was dialyzed at 37 ℃ in 20 mL of PBS solutions at pH 7.4 and pH 5.4, respectively. HED absorbance values in dialysates were determined at 405 nm. The cumulative amounts of HED released were based on a standard curve. 2.8. PLT@BPQDs biocompatibility. PLT@BPQDs biocompatibility was assessed according to hemolytic rates. PLT@BPQDs specimens of different concentrations (0.25-2.0 mg/mL) were mixed with 5% red blood cell suspensions from female BALB/c nude mice, incubated at 37 ℃ (2 h) and submitted to centrifugation at a speed of 3500 rpm (5 min). Absorbance at 545 nm was obtained in the resulting supernatants, with ultra-pure water and PBS used as positive and negative controls, respectively. The following formula was employed for hemolytic rate assessment: hemolytic ratio% = (absorbance of experimental sample - absorbance of negative control) / (absorbance of positive control - absorbance of negative control) × 100%. To determine the capacity of PLT@BPQDs to perform immune evasion, RAW264.7 cells were seeded in 6-well plates (approximately 3×105 cells/well), followed by addition of PLT@BPQDs-RhB for 24 h and staining with Hoechst 33342. PLT@BPQDs phagocytosis by macrophages and fluorescence signals were obtained



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under a laser confocal fluorescence microscope (LCFM) (TCS SP8 CARS, Leica, Germany). 2.9. Evaluation of the antitumor effects of PLT@BPQDs-HED in vitro. 2.9.1 Evaluation of the targeting capability in vitro. MCF-7 cells (1×106/well) were inoculated into a culture dish, stained with Hoechst 33342 after 24 h, and co-cultured with DSPE-FITC-labeled PLTm vesicles for 8 h. After three PBS washes, observation was carried out under a LCFM, and images were acquired. 2.9.2 Cytotoxic effects of PLT@BPQDs-HED in MCF-7 cells assessed by CCK-8. MCF-7 cells seeded in 96-well plates at 2×103/well were incubated for 24 h and administered

PBS,

BPQDs,

PLT@BPQDs,

HED,

BPQDs-HED,

and

PLT@BPQDs-HED, respectively. HED at 2.0 μg/mL was included when applicable. Upon 48 h of incubation, CCK-8 solution (10 μL) was added per well for another 3 h followed by absorbance reading at 450 nm. 2.9.3 Apoptosis assessment by flow cytometry. To further assess in vitro antitumor effects of PLT@BPQDs-HED, Annexin V-FITC/PI Apoptosis Detection Kit was applied for assessing MCF-7 cell apoptosis. Briefly, cells seeded in small culture vessels at 106/flask were treated as described above for 48 h. Apoptosis detection was performed flow-cytometrically (FACS CantoTM II; BD, USA). 2.9.4 Reactive oxygen species (ROS) assessment by flow cytometry. ROS Assay Kit was employed for detecting ROS amounts in MCF-7 cells. Briefly, 1×106 cells were inoculated into each culture dish and administered the above treatments for 48 h.


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Then, all cells were collected, and submitted to PBS washes, staining (according to the kit’s protocol) and detection by flow-cytometry (FACS CantoTM II). 2.9.5 Mitochondrial membrane potential (MMP) evaluation by Rh123 staining. A total of 1×106 cells were inoculated into each culture dish and administered the above treatments. After 48 h, all cells were washed, resuspended in PBS and stained with Rh123 for flow-cytometry detection on a FACS CantoTM II. 2.9.6 Ultrastructure observation under TEM. A total of 1×106 cells were inoculated into each culture dish and administered the above treatments. After 48 h, all cells were washed, resuspended in PBS, and centrifuged (1000 rpm for 5 min). Precipitated cell clumps (approximately 1-2 mm3) were fixed with 3% glutaraldehyde at 4 ℃ for 2 h, washed with PBS, mounted with 1% osmic acid for 2 h, dehydrated in ethanol and acetone, and Epon821 embedded. After polymerization, the samples were sliced using an ultrathin slicing machine, counterstained with oil and citric lead acetate and observed under TEM. 2.10. PLT@BPQDs-HED distribution assessment in vivo. To assess the targeting ability of PLT@BPQDs-HED in vivo, Cy5-labeled PLT@BPQDs were compared with BPQDs labeled with Cy5. Then, MCF-7 tumor-bearing mice were administered Cy5-labeled PLT@BPQDs by tail vein injection. The dose of Cy5 in the composite was 3 μg/kg. A Xenogen IVIS Lumina XR imaging system (Caliper Life Sciences, USA) was employed to assess fluorescence signals at 6, 24, and 48 h post-administration. The animals were then sacrificed, and tumor tissues and visceral



organs were collected for further imaging. Finally, tumor tissues were sliced into frozen sections, and observed under a LCFM. 2.11. PLT@BPQDs-HED treatment in breast cancer-bearing mice. When the tumor tissues exceeded 100 mm3 in volume, the animals were randomized into four groups (n = 3) and intravenously (tail vein injection) administered 100 μL of PBS, BPQDs, PLT@BPQDs, HED, BPQDs-HED, or PLT@BPQDs-HED, once daily, for 4 consecutive days. HED was included at 70 mg/kg/d when applicable. Then, tumor sizes and body weights in the animals were assessed at four-day intervals. Animal euthanasia was carried out at 20 days, and blood specimens, tumors, and internal organs (heart, liver, spleen, lung, and kidney) were collected. Whole blood was collected in tubes containing EDTA for anticoagulation, for blood cell count on a 5-part differential hematology analyzer (BC-5390; Mindray, China). Serum enzyme levels were assessed on a 7100 automatic biochemical analyzer (HITACHI, Japan) and a Cobas 6000 e601 immunology analyzer (ROCHE, USA), respectively, after centrifugation (3000 rpm, 10 min). The sampled organs and tumors underwent fixation with 4% formalin and were frozen at -80°C. The frozen tumor tissues were sliced into frozen sections for immunofluorescent staining, and assessed by Western blotting for Bax, Bcl-2, Caspase-3, Caspase-9, Beclin-1, Cytochrome C, LC3, and β-actin protein amounts. The fixed tissues were embedded in paraffin and sliced into sections. This step was followed by H&E staining, immunofluorescent staining, and immunohistochemical staining.


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2.12. Immunohistochemical staining and immunofluorescence. 2.12.1 TUNEL and Ki-67 assays. Paraffin-embedded tissue samples underwent deparaffinization and antigen retrieval. Apoptotic nuclei were examined by TdT In Situ Apoptotic Kit as directed by the manufacturer. Ki-67 Detection Kit use was based on the protocol provided by the manufacturer. Cells were observed under light microscopy, and images were acquired. 2.12.2 MMP assessment and ROS analysis. MMP assessment and ROS analysis were performed by immunofluorescent staining of JC-1 and DCFH-DA based on standard protocols. Counterstaining was carried out with DAPI. A LCFM was employed for analysis, acquiring images. 2.13. Immunoblot. Total protein extraction from cells was carried out with RIPA buffer, and quantitated with the BCA protein assay kit. Then, Bax, Bcl-2, Caspase-3, Caspase-9, Beclin-1, Cytochrome C, LC3, and β-actin protein amounts in MCF-7 cells were assessed by immunoblot based on standard procedures. 2.14. Statistical analysis. Data were assessed by SPSS 20.0 and expressed as mean ± SD. Intergroup differences were assessed by one-way ANOVA, followed by Tukey’s posttest (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). 3. RESULTS AND DISCUSSION 3.1. Preparation and characterization of the PLT@BPQDs-HED nanocomposite. To prepare the PLT@BPQDs-HED nanocomposite, HED was first loaded into BPQDs to form BPQDs-HED, which underwent encapsulation into PLTm



nanovesicles by ultrasound (Figure 1). BPQDs were monodispersed with diameters averaging approximately 15 nm (Figure 2Aa). For PLT@BPQDs, several BPQDs encapsulated into one PLTm nanovesicle can be clearly observed (Figure 2Ac). The SDS-PAGE results (Figure 2B) revealed that almost all PLTm proteins were retained in the PLT@BPQDs nanocomposites. Based on dynamic light scattering (DLS) data (Figure 2C), the obtained PLT@BPQDs nanocomposites averaged 140 nm in size (slightly smaller than PLTm nanovesicles, whose size was approximately 150 nm). BPQDs had zeta potential values of -40.5±3.2 mV; upon encapsulation, PLT@BPQDs showed lower values of -30.5±2.5 mV, which are close to those of PLTm vesicles (-27.8±2.4 mV) (Figure 2D). UV-vis spectrometry (Figure 2E), revealed absorption peaks at 210 nm, 405 nm, and 200 nm, respectively, for PLT@BPQDs-HED, consistent with those of BPQDs, HED, and PLTm vesicles assessed alone. The above finding further confirmed that PLT@BPQDs-HED was successfully assembled.


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Figure 2. Characterization of PLT@BPQDs-HED. (A) TEM images of a) BPQDs, b) PLTm vesicles, and c) PLTm vesicle-camouflaged BPQDs. Scale bar: 100 nm. (B) SDS-PAGE protein assessment. M) Marker, a) BPQDs, b) PLTm vesicles, and c) PLTm vesicle-camouflaged BPQDs. (C) The particle sizes and (D) zeta potential values of BPQDs, PLTm vesicles, and PLTm vesicle-camouflaged BPQDs. a) BPQDs, b) PLTm vesicles, and c) PLTm vesicle-camouflaged BPQDs. Data are mean ± SD (n = 3). (E) UV-Vis spectra of HED, BPQDs, PLTm vesicles, and PLT@BPQDs-HED. 3.2. Drug LE and release rate. Similar to BP nanosheets, BPQDs with abundant phosphate groups on the surface might interact with drugs via hydrogen bonds or electrostatic forces 13; hence, BPQDs may be an important delivery platform in tumor therapy. Using BPQDs as drug trappers, the HED EE and LE of the PLT@BPQDs-HED nanocomposites were 89.5±3.5% and 74.3±2.8%, respectively (Figure 3A). These values were apparently higher than those of many typical nanoparticle-based nanodelivery platforms whose drug LEs range from 10% to 30% 24.

PLT@BPQDs-HED nanocomposites were stably dispersed in PBS (pH 7.4) with



average size around 140 nm (Figure S1). Then, the drug release profiles of the PLT@BPQDs-HED nanocomposites and BPQDs-HED were evaluated (Figure 3B). At pH 7.4, only 61.2±5.2% and 64.0±4.4% of HED came out of PLT@BPQDs-HED and BPQDs-HED, respectively, within 48 h. However, at pH 5.4, within 48 h, 95.4±3.9% and 97.3±2.8% of HED came out of PLT@BPQDs-HED and BPQDs-HED, respectively. Increased cumulative amounts of the released drug were obtained for PLT@BPQDs-HED and BPQDs-HED at pH 5.4. This result is due to accelerated degradation of BP in acidic conditions 19. Thus, HED is released faster at pH 5.4 than at pH 7.4. Since the tumor environment is weakly acidic, the pH-responsive drug release profile of the PLT@BPQDs-HED nanocomposites is favorable for tumor therapy. Overall, in vitro drug loading and release experiments suggested that BPQDs are powerful drug carriers, and the acidic environment facilitates HED release from PLT@BPQDs-HED.

Figure 3. Drug LE of BPQDs and release rate of PLT@BPQDs-HED. (A) EE and LE of BPQDs. (B) Cumulative release rates of HED from PLT@BPQDs-HED or BPQDs-HED at different pH values (5.4 and 7.4). Data are mean ± SD (n = 3).


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PLT@BPQDs-HED (pH 7.4) group vs PLT@BPQDs-HED (pH 5.4) group, 0.05; BPQDs-HED (pH 7.4) group vs BPQDs-HED (pH 5.4) group, * p < 0.05.

*

p

Platelet-Membrane-Camouflaged Black ... - ACS Publications

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