Erythrocyte Membrane-Camouflaged IR780 and DTX Coloading

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Article Cite This: Mol. Pharmaceutics 2019, 16, 3208−3220

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Erythrocyte Membrane-Camouflaged IR780 and DTX Coloading Polymeric Nanoparticles for Imaging-Guided Cancer Photo−Chemo Combination Therapy Qian Yang,*,† Yao Xiao,‡ Yanlong Yin,† Gaoyin Li,† and Jinrong Peng*,‡ †

School of Pharmacy, Chengdu Medical College, No. 783, Xindu Avenue, Xindu District, Chengdu 610500, Sichuan, P. R. China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan 610041, P. R. China

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ABSTRACT: Conventional systemic chemotherapy leads to poor therapeutic outcomes at moments in cancer therapy because the nontargeting anticancer drug release results in adverse effects and consequently drug resistance. The combination therapeutic strategy provides an alternative way to solve the conundrums. Herein, drug delivery systems with a rational design and tumor-targeting abilities become the ideal carriers for combinatorial therapy. IR780 iodide possesses near-infrared fluorescence intensity for fluorescence imaging (FI) and photothermal conversion for photoacoustic imaging (PAI), which also can be employed for tumor phototherapy (including photothermal therapy and photodynamic therapy). However, hydrophobicity and rapid elimination in vivo limit its biomedical applications. Furthermore, the hydrophobicity and high crystallization of IR780 result in poor drug-loading capacity and low stability. In this study, the high-pressure homogenization method was utilized for hydrophobic molecular IR780 and DTX coloading to construct IR780/DTX-PCEC nanoparticles which exhibit narrow size distribution and satisfactory drug-loading capacity. With further erythrocyte membrane [red blood cell (RBC)] camouflaging, the obtained IR780/DTX-PCEC@RBC nanoparticles present desired stability and prolonged circulation time in vivo. Additionally, the IR780/DTX-PCEC@RBC nanoparticles not only can be employed as a FI/PAI dual model imaging probe but also exhibit the property for phototherapy and chemotherapy of tumors. Based on the therapeutic outcome of combination therapy, the IR780/DTX-PCEC@RBC nanoparticles can serve as promising FI- and PAI-guided photo−chemo combination therapy agents for the future treatment of breast cancer. KEYWORDS: dual drug-loaded nanoparticles, long circulation, dual model imaging, combination therapy

1. INTRODUCTION

a single and simple therapeutic strategy will not obtain efficient tumor growth inhibition. Combination therapy may provide an alternative strategy to further enhance the activity of therapeutic strategies.4 In preclinical research, radiotherapy/chemotherapy, radiotherapy/immunotherapy, or chemotherapy/immunotherapy has been combined for enhancing the tumor growth inhibition.5−7 Some preclinical results indicated that the combination of

Heterogeneity is a ubiquitous feature in human cancer. The intratumoral heterogeneity, resulting from genomic instability or cellular differentiation, indeed makes the tumor microenvironment more complicated, and as a feedback, the tumor microenvironment further aggregates the tumor heterogeneity to further promote the progression of tumor.1−3 This feature may cause low therapeutic outcome of targeting therapeutics in cancer therapy because of the persistent variation in the intratumoral microenvironment and the tumor heterogeneity, and for the worst, the heterogeneity even promotes the formation of drug resistance. Therefore, it can be expected that © 2019 American Chemical Society

Received: Revised: Accepted: Published: 3208

April 16, 2019 May 27, 2019 May 30, 2019 May 30, 2019 DOI: 10.1021/acs.molpharmaceut.9b00413 Mol. Pharmaceutics 2019, 16, 3208−3220

Article

Molecular Pharmaceutics

circulation, for example, CD47.30 By a simple extracting procedure, the membrane of erythrocyte with retained membrane proteins can be obtained.31 In addition, with further coextrusion with NPs, erythrocyte membrane-coated NPs can also be obtained.32,33 We have previously confirmed that the introduction of an erythrocyte membrane indeed prolongs the circulation time of Prussian blue/manganese dioxide hybrid NPs.34 Therefore, the erythrocyte membrane will also be extracted and co-infused with the NIR dye NPs in this study. Meanwhile, we also have used a block copolymer-formed micellar structure to load NIR dye. However, because of the low rigidity and small size (hydrodynamic diameter < 50 nm) of the micelles which are formed by the block copolymer with low molecular weight (Mn = 3000−4000 kDa), it is hard to coinfuse the micelles with the erythrocyte membrane. The NPs formed by high molecular weight block copolymer via highpressure homogenization may be a promising candidate for the construction of the erythrocyte membrane-coated drug-loaded NPs.35,36 Therefore, in this study, we plan to use poly(caprolactone) (PCL)−PEG−PCL (PCEC) triblock copolymer to coload IR780 and DTX to obtain NPs with narrow distribution. Then, the obtained NPs were co-infused with a pre-extracted erythrocyte membrane to impart the NPs with long circulation. The synergistic therapeutic effects of the obtained IR780/DTX-PCEC@RBC NPs in vivo and in vitro have been evaluated in detail. In addition, the results demonstrated the IR780/DTX-PCEC@RBC NPs exhibiting great potential for cancer combination therapy.

different therapeutic mechanisms, such as ERK and autophagy inhibition, may be an effective strategy for the treatments for pancreatic ductal adenocarcinoma.8 In our previous study, we have introduced a voltage-gated Na+ channel inhibitor lidocaine to combine with cisplatin for breast cancer therapy, which could even effectively inhibit the tumor growth and alleviate metastasis.9 It can be concluded that the combination therapy has combinatorial approaches that integrate the advantages of the multiple therapeutic strategies to target not only the subsets of predominant tumor cells but also the drugtolerant cells after treatment, which results in inducing longterm durable responses.3 With the development of nanobiomaterials and nanotechnology, it has been proved that the nano drug delivery systems have great potential in combination therapy.10 By introducing the nanocarriers, the hydrophobicity of some therapeutics, which results in low bioavailability in vivo of the drugs, can be improved. Among the numerous combination strategies, photo−chemo combination therapy has been highlighted because of the improved chemotherapeutic activity induced by phototherapy. Different kinds of agents have been developed for phototherapy, including metal-based nanoparticles (NPs), carbon-based nanostructures or dyes, and so forth.11−14 The dyes, especially the dye that has strong adsorption in the NIR region, exhibit some unique characters while comparing with the other types of agents for phototherapy because of their photoadsorption capacity and ability to fluoresce in fluorescent imaging.15,16 Some NIR dyes have been invented, such as ICG, IR820, IR780, and so forth. ICG, a soluble NIR dye, is used as an indicator for evaluating the metabolic performance of the liver. However, in another aspect, it indicates that the soluble form of NIR dyes may encounter a short half-life in vivo, which may result in a weak enrichment in the tumor site while being used as an agent for photothermal therapy (PTT). Conversely, the hydrophobic forms of NIR dyes, such as IR780, have limited applications in vivo because of their low bioavailability. Combining with nanoencapsulation may favor the resolving of this controversy dilemma because of the ability of nanocarriers in improving the solubility of hydrophobic drugs.17−19 Besides, it has been proved that PTT alone cannot obtain satisfactory therapeutic outcome in cancer therapy.20,21 By introducing with conventional chemodrugs, the growth of primary tumor as well as the distal tumor could be effectively inhibited, and the tumor metastasis also can be alleviated, which may be ascribed to the synergistic effect triggering of the immunotherapy in vivo.20,22 It demonstrates the potentials of PTT combined with chemotherapy in cancer therapy. Moreover, the tumor-targeting of the nanoformulation is critically important in enhancing the therapeutic activity of both the NIR dye and chemodrugs.23 In addition, the tumortargeting strategies still need to be invented and developed to realize satisfactory targeting efficiency.24 In recent years, it has been revealed that the enrichment of NPs in the tumor region is mainly dependent on the enhanced permeability and retention (EPR) effect.25,26 In addition, prolonging the circulating time of the NPs can further strengthen the accumulation of NPs into tumor tissues. It spurs the invention and development of numerous strategies for achieving long circulation.27 Co-infusing with the membrane of erythrocyte (red blood cells, RBCs) is a promising one.28,29 Erythrocyte has a half-life more than 120 h during circulation because of the existence of kinds of proteins which favor the long

2. MATERIALS AND METHODS 2.1. Materials. Polyethylene glycol (PEG, average Mn 4000), ε-caprolactone (ε-CL), stannous octoate (Sn(Oct)2), F127 and IR-780 iodide, and Coumarin-6 dyes were purchased from Sigma-Aldrich (St. Louis, MO, USA). DTX was obtained from Dalian Meilun Biotechnology; dichloromethane (DCM), acetone, ethanol, ethyl acetate (EA), and petroleum ether were provided by Kelong Chemical Company (Chengdu, China). PEG was vacuum-dried at 60 °C before use, and other materials in this study were of analytic reagent grade. 2.2. Composition of IR780/DTX−PCEC@RBC NPs. 2.2.1. Synthesis of PCEC Copolymer. PCEC copolymers were synthesized with ε-CL initiated by PEG via ring-opening polymerization, which was reported in our previous study. In the topical syntheses, the calculated amount of ε-CL and PEG were introduced into a dry glass vessel with nitrogen atmosphere, and a specific amount of Sn(Oct) 2 was subsequently added into the reaction mixture with mild agitation. The reaction was kept at 135 °C lasting for 10 h. After cooling to room temperature, the reaction mixture was first dissolved in DCM, then precipitated in excess cold petroleum ether, and then filtrated. The obtained copolymers were vacuum-dried and kept in desiccators for further use. 1 H NMR (Bruker 400 spectrometer, German) and GPC (HLC-8320GPC, EcoSEC, TOSOH, Japan) were introduced to identify the chemical structure of the obtained PCEC triblock polymers. 2.2.2. Preparation of IR780/DTX Co-Loaded NPs. PCEC NPs were prepared by the method of high-speed homogenization.37 PCEC (500 mg) was dissolved in the mixed cosolvent (acetate/acetone/DCM, 7:1:2, v/v/v). The mixed organic phase was added dropwise into 50 mL of aqueous phase containing 0.05% (w/v) poloxamer as the surfactant and 3209

DOI: 10.1021/acs.molpharmaceut.9b00413 Mol. Pharmaceutics 2019, 16, 3208−3220

Article

Molecular Pharmaceutics then emulsified for six cycles (2 min for a cycle with 30 s interval) with a high-speed homogenizer (AH-Basic-I, Jiangsu, China). The organic solvent in the o/w emulsion solution was removed by a rotary evaporator (RV10 digital S96, KIA, Germany). Finally, the obtained dispersion of NPs was filtered through a 0.22 μm membrane filter and stored at 4 °C. To formulate NPs loaded with IR780 or/and DTX, the drugs with different ratios were dissolved in ethanol and introduced in the initial step of NP formation, followed by the same procedure described above. The basic recipes for the preparation of PCEC NPs are listed in Table 2. 2.2.3. RBC Membrane-Coated IR780/DTX-PCEC NPs. The RBC membrane was obtained and purified via an osmotic pressure adjusting and centrifugation procedure. In brief, after the whole blood was obtained from the mice, it was diluted by multiple times of 1× PBS. Centrifugation (800g, 5 min per time) was introduced to collect RBCs and to eliminate the other cell types in the blood. The obtained RBCs which have been washed by 1× PBS several times were redispersed in 0.25× PBS to undergo a hemolysis-like process. The RBCs dispersed in 0.25× PBS were placed in an ice bath and maintained for more than 20 min. Then, the RBC membrane was collected by centrifugation and purified by further washing with 1× PBS several times. Then, the collected membrane was extruded though a 400 nm filter to obtain RBC membrane vesicles. To prepare RBC membrane-camouflaged IR780/DTXPCEC NPs, the IR780/DTX-PCEC NPs were mixed with the RBC membrane vesicle, and the mixture was then sonicated in a bath sonicator for 2 min. Then, the sonicated mixture was extruded by an Avatar extruder by pressing the mixture though the 400 nm filter and 200 nm filter orderly. In addition, the obtained RBC-camouflaged IR780/DTX-PCEC NPs were stored at 4 °C for following applications. 2.3. Characterization of NPs. The average particle sizes and particle size distributions of obtained NPs were measured by a particle size analyzer which is based on the mechanism of dynamic light scattering (Nano-ZS 90, Malvern Instruments, UK). The temperature of the analytic environment was settled at 25 °C. Particle size distributions were further confirmed by NP tracking analysis (Particle Metrix, Diessen, Germany). The morphologies of the different drug-loaded NPs were examined by a transmission electron microscope (TEM, JEM100S, Japan). The absorbance spectrum of obtained NPs was acquired by a UV/vis/NIR spectrometer (UV-2600, Shimadzu, Japan). 2.3.1. Drug Loading and Releasing Behavior. IR780 concentration in different formulations was detected by the absorbance of 790 nm using a UV/vis/NIR spectrometer. The content of DTX was quantified by HPLC at the detection wavelength of 254 nm (HPLC 1260, Agilent, U.S., with a C18 column, 4.6 mm × 150 mm, 5 mm). The mobile phase was composed of acetonitrile and 0.6% (w/v) acetic acid (60:40, v/ v) with a flow rate of 1.0 mL/min. Drug-loading (DL) content and encapsulation efficiency (EE) were obtained by the following equations

In vitro drug release experiments were performed at physiological condition. The IR780/DTX-PCEC and IR780/ DTX-PCEC@RBC desperations corresponding to 1 mg/mL DTX were placed in a dialysis bag with a molecular weight cutoff of 8−14 kDa and then submerged in releasing buffer. The releasing buffer was PBS (0.2 M, pH = 7.4) containing 0.5% (w/w) Tween-80. At a definite time point, the drugcontaining medium was collected and fresh buffer was replaced. The amount of DTX in the release medium was quantified by HPLC, and water−acetonitrile (45/55, v/v) was chosen as the mobile phase. Each sample for quantitation was performed in triplicate, and the data were indicated as mean ± SD. 2.4. Photothermal Convention Investigation. IR780PCEC dispersion was diluted in saline with the IR780 concentration of 10, 25, and 50 μg/mL, and a 808 nm NIR laser was introduced for irradiation subsequently (1.5 W/cm2, 5 min). Meanwhile, IR780/DTX-PCEC and IR780/DTXPCEC@RBC were diluted with the IR780 concentration of 50 μg/mL and then irradiated with the same procedure, and the saline solution was used as the control. At definite time intervals, temperature variation of each dispersion was recorded by the infrared imaging camera (Fluke, T32, USA). 2.5. Cellular Experiments. MCF-7 breast cancer cell line was cultured with a RAMI 1640 medium (supplemented with 10% of fetal bovine serum and 1% of penicillin), which was consistent with our previous culture condition.38 The cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD). 2.5.1. Cellular Uptake of IR780/DTX-PCEC@RBC. The investigation of intracellular uptake was conducted by different Cou-6-loaded NPs using our previous protocol.39 Free Cou-6, Cou-6-PCEC, or Cou-6-PCEC@RBC (Cou-6 as the fluorescent probe with a concentration of 100 ng/mL for each sample) was incubated with MCF-7 cells in a 6-well plate. After being coincubated for 2 h at 37 °C, the cells were washed and fixed consequently. The captures of Cou-6-labeled NPs were visualized by a fluorescence microscope (Zeiss OBSERVER D1/AX10 cam HRC). Further semiquantification of the uptake was performed by flow cytometry. After being coincubated with various NPs described above, a certain number of cells were collected for flow cytometric analysis (NovoCyteTM Flow Cytometer, ACEA Bioscience. Inc., USA). 2.5.2. Cell Growth Inhibition. The MTT assay was conducted to evaluate the cytotoxicity of different DTX formulations. The MCF-7 cells were seeded in 96-well plates (5 × 103 cells/well) and preincubated at 37 °C for 24 h. The culture medium was replaced with 200 μL of a fresh medium containing different concentrations of IR780-PCEC, DTXPCEC, IR780/DTX-PCEC, and IR780/DTX-PCEC@RBC, and some wells were treated with laser 4 h after incubation. Finally, the MTT assay was performed to detect the cell survival. 2.5.3. Cell Apoptosis Analysis. MCF-7 cells were seeded in 6-well plates and preincubated for 24 h. Sequentially, cells were treated with different formulations (final concentration of IR780 and DTX in various formulations were fixed to 34 and 10 μg/mL, respectively), and untreated cells were used as control. Some wells for phototherapy study, were irradiated by the 808 nm NIR laser (1.5 W/cm2, 5 min) after 4 h of incubation, whereas others without irradiation were just used for chemotherapy. After another 12 h of culture, all cells were

DL % = (weight of drugs in obtained NPs) /(weight of obtained NPs) × 100% EE % = (weight of drugs in obtained NPs) /(weight of drugs used) × 100% 3210

DOI: 10.1021/acs.molpharmaceut.9b00413 Mol. Pharmaceutics 2019, 16, 3208−3220

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Molecular Pharmaceutics collected, and the cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI stain according to our previous protocol.9 2.5.4. Evaluation of IR780-DTX-PCEC@RBC-Induced ROS Production in Cancer Cells. To investigate the IR780-DTXPCEC@RBC-mediated reactive oxygen species (ROS) production in cancer cells, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was chosen as the fluorescent probe. Briefly, MCF-7 cancer cells were placed in a 12-well plate and cultured for 24 h. Then, the cancer cells were co-cultured with IR780/DTX-PCEC@RBC and saline, respectively, with the presence of H 2 DCFDA. The final concentrations of H2DCFDA and IR780 in IR780/DTX-PCEC@RBC-treated groups were 40 μM and 20 μg/mL, respectively. After culturing for another 4 h, the cancer cells were washed with PBS and then irradiated by a 808 nm laser for 5 min (1.5 W/ cm2). The fluorescent images of the treated cancer cells were immediately captured by a fluorescence microscope (Zeiss Observer D1/AX10 cam HRC). 2.6. In vivo MCF-7 Tumor Targeting Efficiency Study. Balb/c-nu mice were used as the model animal to evaluate the pharmacokinetics and biodistribution of IR780/DTX-PCEC@ RBC NPs. Balb/c-nu mice were purchased from the Vital Laboratory Animal Center (Beijing, China). In addition, all of the animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals of Chengdu Medical College and Sichuan University, China. 2.6.1. Pharmacokinetics Investigation. The tumor-free BALB/c-nu mice were utilized for the pharmacokinetics investigation of erythrocyte membrane-camouflaged NPs, and IR780 was used as fluorescent dye to visualize the IR780/ DTX-PCEC@RBC optical imaging. After the IR780/DTXPCEC or IR780/DTX-PCEC@RBC NPs were i.v. injected (at approximately 1.6 mg/kg of IR780), respectively, 50 μL of blood was collected at 0, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 48, and 60 h post-injection for each mouse. The serum samples were separated and consequently diluted with 0.01 M PBS in 96-well plates, and the fluorescence intensity was measured in a 96-well plate using the IVIS Lumina XR system (PerkinElmer, USA; with the excitation of 740 nm and emission of 790 nm for the fluorescent probe). 2.6.2. Tumor-Targeting Evaluation of IR780/DTX-PCEC@ RBC NPs in Vivo by FI. BALB/c-nu mice were subcutaneously injected with 1 × 107 MCF-7 cells into the right flank. After the MCF-7 tumor model was established (the tumor volume reached 150 mm3), IR780/DTX-PCEC or IR780/DTXPCEC@RBC was i.v. administered (at approximately 1.6 mg/kg of IR780). Living NIR imaging was carried out at predetermined time points with the IVIS Lumina XR system by using the same parameters described above. At last, the organs and tumors of each group were harvested and subjected to ex vivo fluorescent imaging, when all mice were sacrificed 24 h post-injection. 2.6.3. Biodistribution of DTX. Mice were sacrificed 24 h post-injection, and the samples including plasma, tumor, heart, spleen, liver, lung, and kidney were also collected for the detection of DTX concentrations. The concentration of DTX in the organs was measured by a UPLC/MS/MS system. In brief, the weighted samples were cut into pieces in 250 μL of saline and homogenized by a homogenizer (ultra-turraxT10 basic, IKA, Germany). Then, 300 μL of EA−hexane (1:1, v/v) was added into each sample to extract the DTX. After vortex and centrifuge, the organic

layer was separated from the water layer and dried by nitrogen gas at 40 °C. Methanol (50 μL) was further added into each tube to dissolve the residue in each tube; then, the solutions were centrifuged to isolate the undissolved substances (16 000 rpm, 5 min). In addition, 5 μL of the supernatant was sampled for UPLC/MS/MS detection. The detail method was as follows: QSight Triple Quad LC/MS/MS System (PerkinElmer, USA), with a C18 column (4.6 × 100 mm, 2.6 μm). The mobile phase used was 0.1% formic acid−acetonitrile at a flow rate of 0.3 mL/min where the percentage of acetonitrile was changed as follows: 0 min, 30%; 0.5 min, 30%; 1.5 min, 90%; 5 min, 90%; 5.5 min, 30%; and 7 min, 30%. Electrospray ionization was performed in the positive ion mode. The ion pairs were 830.1/549.0 and 830.1/304.0, electrospray voltage was 5.5 kV, source temperature was 500 °C, and nebulizer and collision gas flows were 600 and 25 L/h, respectively. 2.6.4. Tumor-Targeting Evaluation of IR780/DTX-PCEC@ RBC NPs by PAI in Vivo. Because of the introduction of IR780, the obtained IR780/DTX-PCEC@RBC NPs can generate PA signals. Therefore, we used photoacoustic imaging (PAI) to further evaluate the tumor-targeting of IR780/DTX-PCEC@ RBC NPs in vivo. PAI was performed by the Multiple-Spectral Optoacoustic Tomography Imaging system (MSOT inVision 128, iThera Medical, Germany). The MCF-7 tumor-bearing mice were treated with saline, IR780/DTX-PCEC NPs, and IR780/DTX-PCEC@RBC NPs. The PA signals of the mice treated with different NPs were detected according to methods we used in our previous study with the change of the laser wavelengths: 700, 715, 730, 760, 800, 820, and 850 nm.11 2.7. In Vivo Therapeutic Efficacy Evaluation. BALB/cnu mice were subcutaneously injected with 1 × 107 MCF-7 cells into the right flank. When the tumor volumes of each mouse reached approximately 100−150 mm3, mice were randomly sorted for different treatments with saline, free DTX, IR780/DTX-PCEC, IR780/DTX-PCEC@RBC, IR780/DTXPCEC + laser, and IR780/DTX-PCEC@RBC + laser. Each formulation was administered daily for 3 days (10 mg DTX/kg, 1.67 mg IR780/kg). For the laser-treated groups, 808 nm laser irradiation was introduced 24 h after the first treatment (laser power: 1.5 W/cm2, 5 min). A fluke infrared imaging camera was introduced to monitor the tumor surface temperature during laser irradiation. The tumor volumes and body weights were measured and recorded every other day in a 42 days’ experiment period. 2.8. Histological Analysis. The tumors were harvested and fixed with formalin before being sectioned. The sliced tissues were stained for immunohistochemistry assay and observed by a Zeiss microscope. 2.8.1. Immunohistochemistry. The apoptosis of the cancer cells was identified by immunohistochemically approaches, which is similar to our previous study. 9 A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling kit (TUNEL, Promega, Madison, WI) was introduced. These histological sections of tumors were also stained with antibodies against proliferating cell nuclear antigen Ki67 to measure proliferation. Antibodies used for immunohistochemistry was purchased from Abcam (USA). The percentage of the proliferation of the cancer cells in tumor tissues were also calculated according to the method we used in previous study.11 All of the captures were obtained via a fluorescence microscope (400× magnification). 2.8.2. Statistical Analysis. SPSS 15.0 software (IBM Corporation, Armonk, NY) was used for the statistical analysis. 3211

DOI: 10.1021/acs.molpharmaceut.9b00413 Mol. Pharmaceutics 2019, 16, 3208−3220

Article

Molecular Pharmaceutics

Scheme 1. (A) Construction of IR780/DTX-PCEC@RBC NPs; (B) Drug-Loaded NPs (IR780/DTX-PCEC) by Surface RBC Membrane Camouflaging Can Serve as FI- and PAI-Guided Combination Therapy Agent for the Treatment of Breast Cancer

Figure 1. Characterization of NPs. (A) TEM imaging of DTX-PCEC, IR780-PCEC, IR780/DTX-PCEC, extracted RBC membrane, and IR780/ DTX-PCEC@RBC (the scale bar = 100 nm); (B) CD47 analyzed by electrophoresis with Coomassie brilliant blue staining; (C) expression levels of CD47; (D) size distribution of corresponding samples; (E) in vitro drug release profiles of corresponding samples.

The results were all expressed as means ± standard derivations. Multigroup comparisons were performed using one-way analysis of variance (ANOVA), and p values of