pH-triggered biomimetic red blood cell membranes camouflaged

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Biological and Medical Applications of Materials and Interfaces

Light/pH-triggered biomimetic red blood cell membranes camouflaged small molecular drug assemblies for imagingguided combinational chemo-photothermal therapy Shefang Ye, Fanfan Wang, Zhongxiong Fan, Qixin Zhu, Haina Tian, Yubing Zhang, Beili Jiang, Zhenqing Hou, Yang Li, and Guanghao Su ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00897 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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ACS Applied Materials & Interfaces

Light/pH-triggered Biomimetic Red Blood Cell Membranes Camouflaged Small Molecular Drug Assemblies for Imaging-guided Combinational Chemo-photothermal Therapy Shefang Ye

1#,

Fanfan Wang1#, Zhongxiong Fan1, Qixin Zhu3, Haina Tian1, Yubing

Zhang1, Beili Jiang1, Zhenqing Hou1*, Yang Li2,5*, Guanghao Su4* 1

Department of Biomaterials, College of Materials, Research Center of Biomedical

Engineering of Xiamen & Key Laboratory of Biomedical Engineering of Fujian Province & Fujian Provincial Key Laboratory for Soft Functional Materials Research, Xiamen University, Xiamen 361005, China 2

CAS Key Laboratory of Design and Assembly of Functional Nanostructures, and

Fujian Provincial Key Laboratory of Nanomaterials, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, China 3

School of Pharmaceutical Science, Fujian Provincial Key Laboratory of Innovative

Drug Target Research, Xiamen University, Xiamen 361005, China 4 Children’s 5

Hospital of Soochow University, Suzhou 215025, China

Department of Translational Medicine, Xiamen Institute of Rare Earth Materials,

Chinese Academy of Sciences, Xiamen 361024, P. R. China.

ABSTRACT Nanoparticles camouflaged by red blood cell (RBC) membranes have attracted considerable attention owing to reservation of structure of membrane and surface proteins, endowing prominent cell-specific function including biocompatibility, prolonged circulation lifetime, and reduced RES uptake ability. Considering the drawbacks of carrier-free nanomedicine including the serious drug burst release, poor stability, and lacking of immune escape function, herein we developed and fabricated a novel RBC membranes biomimetic combinational therapeutic system by enveloping the small molecular drug co-assemblies of 10-hydroxycamptothecin (10-HCPT) and indocyanine green (ICG) in the RBC membranes for prolonged circulation, controlled 1 ACS Paragon Plus Environment

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drug

release,

and

synergistic

chemo-photothermal

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therapy

(PTT).

The

self-reorganized RBCs@ICG-HCPT nanoparticles (NPs) exhibited a diameter of ∼150 nm with core-shell structure, high drug payload (∼92 wt%), and reduced RES uptake function. Taking advantage of the stealth functionality of RBC membranes, RBCs@ICG-HCPT NPs remarkably enhanced the accumulation at the tumor sites by passive targeting followed by cellular endocytosis. Upon the stimuli of near-infrared (NIR) laser followed by acidic stimulation, RBCs@ICG-HCPT NPs showed exceptional instability by heat-mediated membrane disruption and pH change, thereby triggering the rapid disassembly and accelerated drug release. Consequently, compared with individual treatment, RBCs@ICG-HCPT NPs under dual-stimuli accomplished highly efficient apoptosis in cancer cells and remarkable ablation of tumors by chemo-PTT. This biomimetic nanoplatform based on carrier-free, small molecular drug co-assemblies integrating imaging capacity as a promising theranostic system provides potential for cancer diagnosis and combinational therapy.

Keywords: small molecular drug co-assemblies, biomimetic red blood cell membranes, light/pH-response, on-demand drug release, chemo-PTT therapy

1. INTRODUCTION As a horrible disease that seriously threatens the safety of human life, cancer kills 2 million people every year in China alone.

1

Therefore, the treatment of cancer has

become one of the major concerns for researchers in medicine,2 chemistry,3 and materials science.4 Considerable efforts have been made in cancer therapy, including operative treatment, chemotherapy,5 and radiotherapy.6 Carrier-free small-molecule self-assembly provides a novel and simplified strategy for drug delivery and cancer treatment,7-8 which is a hopeful strategy that could realize both nanoscale advantages and high drug payload while avoiding the significant safety concerns from carrier materials. Nevertheless, these nanosystems still suffer from many shortcomings existed in a variety of complex biological barriers in vivo, simply summarized as poor 2 ACS Paragon Plus Environment

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stability, serious drug burst release,9 short blood circulation, and lacking of immune escape function.10-11 Recently, combining naturally occurring bio-membranes with synthesized nanoparticles (NPs) by simple extrusion process to obtain nanosystems with biomimetic properties has achieved tremendous attention.12-15 Taking advantage of the complex properties and biological stability formed naturally during the process of evolution, the translocation process of the membranes could be simply and effectively translocated from organism to composite. To date, a series of cell membranes such as platelet membrane,13 bacterial cell membrane,16 and macrophage membrane17 have been chosen to prepare and develop membrane-coated nanosystems, in which RBC membranes engineering has been proposed as a biomimetic strategy used in drug delivery and cancer therapy due to the advantages that it could gain low immunogenicity and good biocompatibility to prolong the circulation time in blood stream while avoiding the rapid macrophage uptake and elimination from blood.18-21 In addition, although chemotherapy is a primary treatment means for tumor therapy in clinic, it frequently suffers from many drawbacks, including serious side effects on normal cells and high probability of tumor recurrence and metastasis,22 which significantly reduce the effect of chemotherapy. Thus, the unsatisfactory therapeutic effect still remains a challenge in clinical translation of nanomedicine. To address

these

issues,

newly-developing

therapeutic

modality

integrating

photosensitizers appeared.23-24 As a representative modality and a noninvasive approach for tumor therapy, photothermal therapy (PTT) integrating photothermal conversion agent could convert absorbed light into thermal energy to destroy localized cancer cells while reduce the side effects on normal cells. Furthermore, combinational therapy possesses a huge superiority in clinical use since it could take advantages of both therapeutic modalities while overcoming the disadvantages of each therapeutic modality.25 Particularly, chemo-PTT is one of the most preferable and effective modality, which could achieve the cooperative anticancer efficacy and better clinical outcome by synergistically augmenting the chemo-cytotoxicity. 3 ACS Paragon Plus Environment


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Indocyanine green (ICG) as a cyanine near-infrared (NIR) dye is an FDA-approved medical diagnostic agent. ICG molecule contains two polycyclic indole skeletons with two sulfonate groups, which imparts amphiphilic character. The NIR spectral absorption peak of ICG is approximately 820 nm, which could penetrate tissues deeply, making ICG not only used for NIR fluorescence (NIRF) imaging,26 but also used for PTT.27 Nevertheless, the theranostic use of ICG is still restricted by the defects of photo-/thermal-/aqueous-instability, non-specific binding to plasma proteins,

rapid

blood

clearance,

and

lacking

of

targeting

capability.28

10-Hydroxycamptothecin (HCPT) is an FDA-approved clinically used chemo-drug and exhibits great anticancer activity against a broad variety of malignant tumors by binding with nuclear DNA enzyme topoisomerase I and disrupting DNA structure and function.29 However, the clinical application of HCPT is still restricted greatly due to the extremely low water solubility, rapid opsonization, non-specific biodistribution, and serious systemic toxicity.30 In light of the electron-rich π-conjugated structure of ICG/HCPT, amphiphilicity of ICG, and hydrophobicity of HCPT, we envisaged that ICG and HCPT could form nanoscale co-assembled particles in the aqueous solution by non-covalent interactions including electrostatic, π–π stacking, and/or hydrophobic interactions. Besides, with an aim of achieving highly efficient tumor accumulation and activated intracellular drug release, we combined PTT and chemotherapy to fabricate NIR laser-triggered biomimetic small molecular drug co-assemblies cloaked with RBC membranes (RBCs) for significantly enhancing therapeutic potential. As illustrated in Figure 1A, a highly hydrophobic crystalline chemo-drug (HCPT) and an amphiphilic photothermal conversion agent (ICG) were co-assembled within aqueous media into carrier-free nanodrug (designed as ICG-HCPT NPs). Then the RBCs derived from rat blood were cloaked onto the ICG-HCPT NPs’ surface using a multiple-step extrusion procedure (designed as RBCs@ICG-HCPT NPs). It is expected that the RBCs@ICG-HCPT NPs would have an exceptionally high drug payload with the protection of HCPT and ICG by RBCs from premature release/leakage during blood 4 ACS Paragon Plus Environment

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circulation. Furthermore, the RBCs-cloaked nanodrug could improve the stability of ICG-HCPT NPs, moreover, and protect HCPT and ICG from premature drug leakage and serious burst release during circulation in the blood stream. Once self-delivered into tumor sites through enhanced permeability and retention (EPR) effect,31 the RBCs could be thermally disrupted upon the irradiation of NIR laser which triggered the accelerated release of co-loaded HCPT/ICG, thereby boosting the drug concentration within tumor regions and inside tumor cells, and eventually enhancing the chemo-PTT efficacy (Figure 1B). In consideration of the simplicity of co-self-assembly, cooperative modalities, and RBCs engineering, the bioinspired strategy might provide a new universal simplified approach in the design and development of clinically translatable drug assemblies to realize highly effective chemo-PTT and tumor ablation. The reorganized RBCs@ICG-HCPT NPs could also be used for NIRF imaging upon irradiation.

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Figure 1. (A) Schematic illustration of preparation procedure of RBCs@ICG-HCPT NPs. (B) Schematic illustration of NIRF imaging-guided chemo-PTT combinational therapy through intravenous injection.

2. RESULTS AND DISCUSSION 2.1. Preparation and Characterization of RBCs@ICG-HCPT NPs. A wide range of small molecular drugs could be assembled into NPs with various size and morphology. In addition, supramolecular self-assembly has aroused great interest in the field of both biomedicine and nanotechnology.

32-33

Herein, a highly

hydrophobic crystalline chemo-drug (HCPT) and an amphiphilic photothermal conversion agent (ICG) were initially co-assembled within aqueous media into carrier-free nanodrug (designed as ICG-HCPT NPs) (Figure 1A). To simplify the 6 ACS Paragon Plus Environment

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study, the molar ratio between ICG and HCPT was first optimized via dynamic light scattering (DLS) assay and transmission electron microscopy (TEM). Interestingly, the data from DLS measurements certified the mean hydrodynamic diameter (Dh) of ICG-HCPT nanosystems could be regulated from nanometer to micrometer via changing the molar ratio of ICG to HCPT (Table S1). Notably, the results were in accordance with the TEM and AFM experimental results (Figure 2A and 2B). Meanwhile, it was found that mixing of ICG and HCPT at the molar ratio of 1: 2 resulted in the generation of NPs with a mean Dh of ∼110 nm (Table S1), and the Dh and polydisperse index (PDI) was smaller compared to the other groups. Therefore, the molar ratio of ICG to HCPT (1: 2) was very suitable for the enhanced penetration and retention (EPR) effect. To precisely track and investigate the formation process of ICG-HCPT NPs, they were characterized by TEM (Figure 3A). As the reaction time elapsed, the Dh of ICG-HCPT NPs gradually largen and their shape became uniform. This result could be attributed to intermolecular interactions of ICG with HCPT, including hydrogen bonding, electrostatic, and π−π stacking interactions. As mentioned above, the molar ratio of ICG to HCPT was selected to be 1: 2 and the reaction time was 6 h in the following experiment. According to this molar ratio, the morphology, Dh, and PDI of ICG-HCPT NPs were characterized with TEM, SEM, CLSM, and DLS, respectively. Figure 3C, and Figure 3F exhibited that the Dh of ICG-HCPT NPs was ∼110 nm with uniform spherical morphology. It was also found that the sphere-shaped ICG-HCPT NPs was completely different from the needle shaped HCPT (Figure 3B) and the unregularly ICG (Figure S1A). As shown in Figure S2, the absorption bands of ICG-HCPT NPs exhibited an increased peak width and a slight red shift (∼4 nm) in the UV-vis-NIR absorption spectra compared to the absorption bands of HCPT and ICG. These results indicated that ICG-HCPT NPs have been successfully fabricated.



Figure 2. (A) TEM images of ICG-HCPT NPs with different molar ratio of ICG/HCPT (4: 1, 2: 1, 1: 1, 1: 2, and 1: 4). (B) AFM image of ICG-HCPT NPs with optimized molar ratio (1: 2).

In order to make ICG-HCPT NPs have excellent biocompatibility, long circulation time, and excellent reduced RES uptake ability, the biomimetic RBCs derived from rat blood were cloaked onto the ICG-HCPT NPs’ surface using a multiple-step extrusion procedure (designed as RBCs@ICG-HCPT NPs) (Figure 1A). After RBCs coating, the RBCs@ICG-HCPT NPs showed a typical core-shell structure with an outer lipid shell of ∼20 nm in thickness (Figure 3D and Figure 3E), which was different from free RBC membranes in water (Figure S1B). The increase of 20 nm was about the coverage of three layers of RBC-membranes lipid bilayers which was known as about 8 nm34. Besides, the Dh, PDI, and zeta potential of RBCs@HCPT-ICG NPs was ∼150 nm, 0.216, and ∼28 mV, respectively (Figure 3F and Figure 3G). The change of surface zeta potential was attributed to the charge 8 ACS Paragon Plus Environment

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screening effect of the membrane coating. Moreover, UV-vis-NIR absorption spectra exhibited that RBCs@ICG-HCPT NPs have gained an additional absorption peak around 405 nm after the extrusion process, which was consistent with the characteristic absorption peak of RBC membranes-cloaked vesicles (Figure 3H). To better demonstrate the transfer of the red blood cell membranes protein to the ICG-HCPT NPs, gel electrophoresis and western blotting were conducted. Obviously, as shown in Figure S3, RBCs@ICG-HCPT NPs displayed almost identical protein components to RBCs, indicating that the membrane proteins in RBCs were efficiently retained. Last but not least, based on the western blotting analysis, it was found that key membrane protein (CD47) was retained on RBCs@ICG-HCPT NPs (Figure 7B). The result further confirmed the reservation of membrane protein within RBCs. The drug payload content of ICG and HCPT was calculated to be ∼30.6% and ∼68.4% respectively. More than 83.1% of ICG and 90.5% of HCPT were encapsulated into RBCs@ICG-HCPT NPs as measured by UV-vis-NIR absorption spectra. Therefore, we successfully speculated biomimetic RBC membranes camouflaged small molecular nanodrugs with a small diameter, negative charge, high drug payload, and excellent stability in biological condition might be beneficial for EPR effect of RBCs@ICG-HCPT NPs, immune escape function, and reduced reticular endothelial system (RES) uptake, guaranteeing the passive tumor-targeting with prolonged circulation lifetime during the circulation.



Figure 3. Characterization of ICG-HCPT NPs and RBCs@ICG-HCPT NPs. (A) TEM images and Dh distribution. The co-assembly process of ICG-HCPT NPs was monitored at different reaction time (0.5, 2, and 6 h). (B) TEM, SEM, and CLSM images of free HCPT. (C) TEM, SEM, and CLSM images of ICG-HCPT NPs. (D) and (E) TEM image of RBCs@ICG-HCPT NPs. (F) Dh distribution (Inset: Tyndall effect of free ICG (left) and RBCs@ICG-HCPT NPs (right)) and (G) Zeta potential of ICG-HCPT NPs and RBCs@ICG-HCPT NPs. (H) UV-vis-NIR spectra of RBCs, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs.

2.2. In Vitro Stability Study of RBCs@ICG-HCPT NPs. The physical stability of nanodrug is very important for efficient drug delivery. 10 ACS Paragon Plus Environment

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The variation in particle size of nanodrug was a pivotal exhibition of their stability in PBS or FBS. Thus, to verify the stability of RBCs@ICG-HCPT NPs, the Dh was recorded at predetermined times. It was found that Dh of RBCs@ICG-HCPT NPs was not obviously changed after the storage of RBCs@ICG-HCPT NPs dispersion in PBS at 25°C for 120 h (Figure 4A) compared to ICG-HCPT NPs (Figure S4). In addition, no significant precipitation in RBCs@ICG-HCPT NPs was found at pH 7.4. On the contrary, too much yellow precipitation was shown in ICG-HCPT NPs at pH 5.5 after standing for 24 h. This phenomenon was very similar to HCPT solution (Figure 4G). This could be illustrated that the interaction between ICG and HCPT was broken in a weakly acidic environment. To better explain this result, the fluorescence spectra, TEM, and DLS were performed. It was found that ICG-HCPT NPs had significant fluorescence switch effect (Figure 4B). Besides, the fluorescence intensity of ICG-HCPT NPs was greatly weakened at 380 nm (excitation wavelength of HCPT) comparing to free HCPT. Interestingly, the fluorescent signal is restored to the origination after ICG-HCPT NPs incubated with PBS at pH 5.5. Notably, an obvious shift from 820 nm to 836 nm was appeared at 740 nm excitation and the fluorescence intensity was significantly enhanced. Meanwhile, optical photos photographed at UV light (365 nm) also explained the result (Figure 4C). This result might be attributed to the migration of excitation energy among π-stacked ICG/HCPT molecules as well as the change of molecule conformation in the co-self-assembly process. ICG-HCPT NPs incubated with PBS at pH 7.4 for 24 h could still maintain shaped sphere well. However, ICG-HCPT NPs incubated with PBS at pH 5.5 for 6 h was disassembled and then drugs were sustainably released (Figure 4D). Next, the photo-stability of RBCs@ICG-HCPT NPs in aqueous solutions was also evaluated via UV-vis-NIR and fluorescence analysis at predetermined times (Figure 4E and Figure 4F). Obviously, ∼90% of UV absorption and ∼80% of fluorescence intensity of free ICG was respectively declined. Notably, the UV absorption and fluorescence intensity of RBCs@ICG-HCPT NPs still maintained for more than 50% and 60% of the initial intensity respectively, which was better than ICG-HCPT NPs 11 ACS Paragon Plus Environment


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(Figure S5). These results implied that the photo-stability of ICG encapsulated in RBCs@ICG-HCPT NPs was greatly improved. The phenomenon could be ascribed to the double bonds of ICG were protected after it was enveloped into RBCs@ICG-HCPT NPs. 2.3. In Vitro Hemolysis Study of RBCs@ICG-HCPT NPs. To assess the hemolysis of RBCs@ICG-HCPT NPs, an equivalent amount of red blood cells was added into solution of H2O, PBS, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs, respectively. It was found that all of the red blood cells were completely ruptured and hemoglobin dissolved in water. However, the ICG-HCPT NPs and RBCs@ICG-HCPT NPs were no significant different compared to the PBS-treated group (Figure 4H). To quantitatively evaluate the of the hemolysis of RBCs@ICG-HCPT NPs, the absorption at 541 nm of H2O, PBS, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs was measured by a microplate reader. The hemolysis rates of ICG-HCPT NPs and RBCs@ICG-HCPT NPs were 0.87% and 1.49% (international

standard:

5%),

respectively.

These

results

indicated

that

RBCs@ICG-HCPT NPs have excellent biocompatibility and can be used for intravenous injection.


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Figure 4. In vitro stability, assembly/disassembly, and hemolysis study. (A) Dh of RBCs@ICG-HCPT NPs in water, PBS, and 10% of FBS aqueous solution for 120 h. (B) Fluorescence spectra of HCPT (ex: 380 nm), ICG (ex: 740 nm), ICG-HCPT NPs, and ICG-HCPT NPs at pH 5.5. (C) Photographs of HCPT, ICG, and ICG-HCPT NPs at pH 7.4 and pH 5.5 upon UV light at 365 nm. (D) TEM images and DLS analysis of ICG-HCPT NPs at pH 7.4 and pH 5.5. (E) UV-vis-NIR absorption of free ICG and RBCs@ICG-HCPT NPs in aqueous solution stored for 96 h. (F) Fluorescence spectra of free ICG and RBCs@ICG-HCPT NPs in aqueous solution stored for 96 h. (G) Photographs of HCPT, ICG, and ICG-HCPT NPs at pH 5.5 and pH 7.4 after standing for 24 h. (H) Hemolysis percentage of water, PBS, ICG-HCPT NPs, and 13 ACS Paragon Plus Environment


RBCs@ICG-HCPT NPs. Inset: photographs of water, PBS, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs dispersed in red blood cells.

2.4. In Vitro Photothermal Effect of RBCs@ICG-HCPT NPs. Next, to assess the photothermal effect of RBCs@ICG-HCPT NPs, the photothermal performance of different formulation dispersions was conducted upon the irradiation of NIR laser. It was found that the maximum temperature of RBCs@ICG-HCPT NPs could reached to ∼80oC after irradiated by NIR laser (808 nm, 1 W/cm2) for 5 min. This result implied that RBCs@ICG-HCPT nanosystems still remained the originally photothermal response compared to free ICG aqueous solution (Figure 5A and Figure 5B), whereas the temperature increases of saline solution was negligible at the same condition. In addition, as shown in Figure 5C∼5F, ICG-HCPT NPs and RBCs@ICG-HCPT NPs significantly displayed concentration (50∼200 μg/mL)/power (0.25∼1 W/cm2)-dependent photothermal effect. These results illustrated that this nanodrug system was suitable for further biological applications in photothermal therapy. 2.5. In Vitro Drug Release of RBCs@ICG-HCPT NPs. In vitro release profile of HCPT from RBCs@ICG-HCPT NPs was measured using a dialysis membrane diffusion technique in the simulated physiological (pH 7.4) and acidic (pH 5.5) condition at 37 °C for 48 h. As shown in Figure 5G and Figure 5H, RBCs@ICG-HCPT NPs obviously exhibited NIR/pH-responsive release behaviors of HCPT. Notably, approximately 80% of HCPT was released from ICG-HCPT NPs and RBCs@ICG-HCPT NPs (with laser irradiation treatment) at the weak acidic pH 5.5. The HCPT was released ∼50% within 8 h. Subsequently, HCPT was continuously released as the time collapsed. This release behavior made sure the HCPT continually kill tumor cells after tumor implantation and further played a long-acting chemotherapy effect. On the contrary, only about 12% and 7% of HCPT were released from ICG-HCPT NPs and RBCs@ICG-HCPT NPs (without laser irradiation treatment) at pH 7.4. These results could be attributed to weak interaction 14 ACS Paragon Plus Environment

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between ICG and HCPT might be broken at pH 5.5. In addition, as shown in Figure 6B and Figure S6, morphological analysis of RBCs@ICG-HCPT NPs irradiated by NIR laser (808 nm, 1 W/cm2, 5 min) also proved exceptional instability by heat-mediated membrane disruption process.

Figure 5. In vitro photothermal effect of RBCs@ICG-HCPT NPs. (A) Infrared thermographic images and (B) corresponding photothermic effect curves of saline, free ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs. (C-E) Photothermic effect curves of free ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs at different ICG concentrations. (F) Temperature change curves of RBCs@ICG-HCPT NPs under different power intensities. (G) Cumulative release of HCPT from ICG-HCPT NPs and RBCs@ICG-HCPT NPs at pH 7.4 and 5.5. (H) Cumulative release of HCPT from ICG-HCPT NPs and RBCs@ICG-HCPT NPs at pH 7.4 and 5.5. (with laser). Data were presented as mean ± s. d. (n= 3). 15 ACS Paragon Plus Environment


2.6. In Vitro Cellular Uptake of Self-assembled NPs. Previous studies have proved that some organic small molecules had the auto-fluorescence characteristics,35-36 which made them used as fluorescence probes for cell imaging37. Herein, the auto-fluorescence of ICG and HCPT made RBCs@ICG-HCPT NPs suitable for cell imaging. To qualitatively analyze the cellular uptake of RBCs@ICG-HCPT NPs, the process was traced by CLSM. The free HCPT and ICG were used as controls. HeLa cells were incubated with ICG, HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs for 1 h and 4 h. As displayed in Figure 6A, the red fluorescence of ICG was weak in HeLa cells treated with free ICG. On the contrary, the red fluorescence of HeLa cells treated with ICG-HCPT NPs and RBCs@ICG-HCPT NPs was much stronger. These results indicated the ICG amounts of RBCs@ICG-HCPT NPs uptaken by HeLa cells were higher than those of free ICG. Meanwhile, it was also found that some needle-shaped fluorescence signals were obviously diffused into cells. The phenomenon could be investigated that HCPT was self-assembled into micrometer club-like shaped HCPT nanocrystals in the medium of cell.38 The fluorescence switch effect was found in cellular uptake process of ICG-HCPT NPs and RBCs@ICG-HCPT NPs. In detail, the fluorescence signal of HCPT was not found after incubation with ICG-HCPT NPs and RBCs@ICG-HCPT NPs for 1 h. This phenomenon could be illustrated that ICG-HCPT NPs and RBCs@ICG-HCPT NPs was not dis-assembled. This indirectly stated that ICG-HCPT NPs and RBCs@ICG-HCPT NPs could be intact in a relatively long time in blood and cells. As the incubation time was extended from 1 h to 4 h, the appearance of the strong fluorescence signals illustrated that ICG-HCPT NPs was dis-assembled and then ICG and HCPT was continuously released. Meanwhile, as incubation time was extended from 1 h to 4 h and treated with NIR laser, the appearance of the strong fluorescence signals illustrated that RBCs@ICG-HCPT NPs was dis-assembled. As is well-known, the cellular uptake of nanodrug is directly related to the 16 ACS Paragon Plus Environment

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cell-killing effect39. Therefore, it was very necessary to quantitatively evaluate the cellular uptake/internalization of RBCs@ICG-HCPT nanosystems. As shown in Figure 6C and 6D measured by flow cytometer, the fluorescence signals of ICG from ICG-HCPT NPs and RBCs@ICG-HCPT NPs group were much higher than those of free ICG group. However, the HCPT fluorescence signals of each group at 1 h and 4 h were well consistent with the analysis results of CLSM. For further quantitative analysis of cellular uptake of RBCs@ICG-HCPT NPs, we also conducted the fluorescence measurements. As displayed in Figure 6E, the average ICG fluorescence intensity of RBCs@ICG-HCPT NPs group was much higher compared to that of free ICG group. Similarly, the HCPT fluorescence intensity of each groups at 1 h and 4 h were also in agreement with the analysis results by using CLSM and flow cytometer. We speculated that the enhanced cellular uptake of RBCs@ICG-HCPT NP (discussed as below) could result in the high cell-killing efficiency. 2.7. In Vitro chemo-PTT therapy of RBCs@ICG-HCPT NPs. To further the in vitro synergistical chemo-PTT of RBCs@ICG-HCPT NPs, the cytotoxicity of different formulations was assessed using MTT assay. In brief, HeLa cells were incubated with free ICG, free HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs without or with treatment of NIR laser irradiation (5 min, 1 W/cm2) for 24 h. As shown in Figure 6F, the chemotherapeutic effect of RBCs@ICG-HCPT NPs was much more effective than free ICG, free HCPT, and ICG-HCPT NPs at the same drug concentration. In detail, when treated with ICG-HCPT NPs and RBCs@ICG-HCPT NPs (2 μg/mL of HCPT) for 24 h incubation, the inhibition in cell viability of HeLa cells was up to ∼50% and ∼41%, respectively. In contrast, the cell-killing ability of HeLa cells treated with free ICG and free HCPT was reduced to ∼8% and ∼44%, respectively. These

phenomena

could

be

investigated

that

the

enhanced

cellular

uptake/internalization of RBCs@ICG-HCPT nanosystems led to the highly effective cell-killing effect. Besides, the free ICG served as a kind of photosensitizer presented certain biosecurity and ~100% of HeLa were still alive even at concentration of 20 17 ACS Paragon Plus Environment


μg/mL. Next, the photothermal performance of various formulations was assessed upon the irradiation of NIR laser (Figure 6G). After laser irradiation, as compared to the free HCPT, free ICG groups, the RBCs@ICG-HCPT NPs group exhibited significantly higher cancer cell-killing ability after 24 h incubation. When the concentration of ICG reached to 20 μg/mL, only 11.8% of cells survived. Thus, the incomplete eradication of cancer cells by chemotherapy alone could well be settled by the combinational therapy. In addition, to measure the apoptosis capability of RBCs@ICG-HCPT NPs, the Annexin V-FITC/PI apoptosis detection was conducted. The total apoptotic ratio of HeLa cells was measured after incubated with free ICG (with or without laser), free HCPT, ICG-HCPT NPs (with or without laser), and RBCs@ICG-HCPT NPs (with or without laser). As shown in Figure 7A, the sum of both the early apoptotic ratio and the late apoptotic ratio of RBCs@ICG-HCPT NPs was ∼67.1%. This value was obviously higher than any other treatment group. Besides, the groups treated by laser irradiation showed a higher apoptotic rate than the groups without laser irradiation, which illustrating superior PTT effect of ICG. To further confirm these results, the hallmark of cell apoptosis induction, the cleaved caspase-3, pro-caspase-3, and cleaved PARP analyzed. As shown in Figure 7C, caspase-3 protein and cleaved PARP expression of group treated by RBCs@ICG-HCPT NPs with laser was up-regulated slightly compared to other groups, while the expression of substrate pro-caspase-3 was significantly decreased. Based on these results, it could be inferred that RBCs@ICG-HCPT NPs had the ability to synergistically induce apoptosis in HeLa cancer cells.


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Figure 6. Cellular uptake assays and cytotoxicity test of RBCs@ICG-HCPT NPs. (A) Confocal laser scanning microscopy images of free HCPT, free ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs for HeLa cells. (B) TEM image of RBCs@ICG-HCPT NPs after NIR laser irradiation (808 nm, 1 W/cm2, 5 min). (C) Flow cytometry detection of free ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs for HeLa cells. (D)

Flow

cytometry

detection

of

free

HCPT,

ICG-HCPT

NPs,

and

RBCs@ICG-HCPT NPs with and without NIR laser irradiation for HeLa cells. (E) Quantification analysis of mean fluorescence intensity. (F, G) Cell viability of HeLa cells incubated with different concentrations of free ICG, HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (F) with or (G) without NIR laser irradiation (808 nm, 1 W/cm2, 5 min), respectively. Data were presented as mean ± s. d. (n= 5). 19 ACS Paragon Plus Environment


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2.8. In Vivo NIRF Imaging of RBCs@ICG-HCPT NPs. To gain insight into the passive tumor-targeting capability of RBCs@ICG-HCPT NPs in the tumor tissues and other organs (liver, lung, spleen, kidney, and heart), in vivo NIR fluorescence imaging was conducted using HeLa tumor-bearing nude mice at different time points after intravenous administration. The in vivo fluorescence imaging was performed. As displayed in Figure 7D, the intense fluorescence signals of RBCs@ICG-HCPT NPs could be observed at the tumor tissue at 24 h post-injection. In contrast, it was found that the weak fluorescence signal was shown at the tumor tissue of mice with ICG-HCPT NPs and free ICG. These phenomena implied that the RBCs@ICG-HCPT nanosystems could efficiently accumulate in the tumor regions via the passive tumor-targeting-mediated by EPR effect. Additionally, to

quantificationally

analyze

the

passive

tumor-targeting

capability

of

RBCs@ICG-HCPT nanosystems, we also performed the fluorescence measurements (Figure 7E). The result was well in agreement with the above phenomenon. These results also demonstrated that RBCs@ICG-HCPT NPs were more likely to escape the RES and prolong the circulation time under the shielding effect of RBCs. 2.9. In Vivo Photothermal Treatment of RBCs@ICG-HCPT NPs. Previous studies have reported that tumor cells would be effectively destroyed when the surrounding temperature was over 50°C.40-41 Considering the enhanced tumor accumulation of RBC@ICG-HCPT NPs in NIRF imaging experiments in vivo, 24 h post-injection as a time point was chosen to conduct the in vivo photothermal therapy. After the intravenous injection of saline, ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs into HeLa tumor-bearing nude mice, the tumor regions were exposed to laser irradiation (808 nm, 8 min) at 24 h post-injection and photographed using infrared (IR) camera. As shown in Figure 7G, when the mice were injected with saline and ICG upon the irradiation of laser, the temperature only reached to 43.6°C and 48.6°C, respectively. This could be attributed to low enrichment of free ICG in tumor tissues. Besides, the data regarding thermal imaging also exhibited that the temperature around tumor tissues of RBCs@ICG-HCPT NPs-treated mice rapidly 20 ACS Paragon Plus Environment

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rose to 56 °C and stably kept within 8 min (Figure 7F). The increase of significant temperature in RBCs@ICG-HCPT NPs-treated groups further proved the superior PTT effect of RBCs@ICG-HCPT NPs in vivo after NIR laser irradiation, while demonstrating the efficiently passive tumor accumulation and certain reduced RES uptake ability.

Figure 7. Cell apoptosis and in vivo NIRF imaging-guided photothermal treatment of RBCs@ICG-HCPT NPs. (A) Flow cytometry analysis for apoptosis of HeLa cells apoptosis induced by ICG, HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (with or without laser) for 24 h. (B) Expression levels of CD47. Group 1 was ICG-HCPT NPs, group 2 was RBCs, and group 3 was RBCs@ICG-HCPT NPs. (C) Expression levels of caspase-3 in HeLa cells after treated for 24 h and analyzed by 21 ACS Paragon Plus Environment


Western blotting. The β-actin was used as the loading control. (D) In vivo NIRF imaging and (E) average NIRF intensity in the tumor areas of HeLa tumor-bearing nude mice treated with free ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs at 1, 3, 6, 12, and 24 h post-injection. (F) Temperature changes and (G) infrared thermal images in the tumor regions over time of HeLa tumor-bearing nude mice at 0, 1, 2, 3, 4, 5, and 8 min treated with saline, ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs under NIR laser irradiation (808 nm, 1 W/cm2). Data were presented as mean ± s. d. (n= 6).

2.10. Pharmacokinetics and Distribution In Vivo. To further investigate whether the RBCs@ICG-HCPT NPs retained the prolonged blood circulation and immune escape function from natural RBC membranes, the in vivo pharmacokinetics and biodistribution experiments were performed. After intravenous injection, venous blood was collected at a specified time and then the concentration of NPs was measured. As depicted in Figure 8B, the blood concentration of RBCs@ICG-HCPT NPs group was about 16.3% ID/g at 24 h post-injection. In sharp contrast, only about 2.0% and 7.5% ID/g of free ICG and ICG-HCPT NPs was retained in blood circulation system, respectively. Therefore, RBCs@ICG-HCPT NPs showed the prolonged blood circulation due to the camouflage of RBC membranes on the surface of ICG-HCPT NPs. Inspired by the pharmacokinetics results, the in vivo biodistribution experiment of different NPs was further conducted. After 24 h post-injection, tumors and major organs of each group were collected and measured. As shown in Figure S7, the biodistribution results exhibited that free ICG and ICG-HCPT NPs were highly accumulated in the spleen and liver. The result might be attributed to their high RES uptake, which is a universal phenomenon for nanodrug. The distribution of RBC@ICG-HCPT NPs in the spleen and liver was lower compared to ICG and ICG-HCPT NPs. It was also found that the accumulation of RBCs@ICG-HCPT NPs at tumor site was higher than that of other groups. Accumulating evidence has suggested that a transmembrane glycoprotein 22 ACS Paragon Plus Environment

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CD47 can be expressed on the surface of red blood cells. CD47 protein can form a signal complex with SIRPα, then produce an inhibitory regulatory signal, inhibit the phagocytosis of red blood cells by immune cells, and thus maintain prolonged circulation of red blood cells in the body42. Herein, ICG-HCPT NPs, RBCs, and RBCs@ICG-HCPT NPs were assayed in parallel by using western blotting. The result unequivocally

revealed

the

presence

of

CD47

on

the

surface

of

the

RBCs@ICG-HCPT NPs (Figure 7B). These results further illustrated that the RBC membranes-camouflaged NPs had stealth ability to escape RES clearance. 2.11. In Vivo Antitumor Effect Study of RBCs@ICG-HCPT NPs. In order to evaluate the in vivo antitumor effect of ICG-HCPT nanosystems, HeLa tumor-bearing nude mice were injected with saline, ICG, HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs without and with laser irradiation. During the treatment, the photographs were obtained on the 1st, 7th, and 20th days, which clearly exhibited the destruction pattern of tumor (Figure 8A). The tumor volume change curves and tumor weight were shown in Figure 8C and Figure 8D, respectively. As compared to the saline group, the ICG, ICG, HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs group without the treatment of laser irradiation showed some degree of suppression in the tumor growth. Moreover, the tumor growth was largely suppressed when mice were treated with ICG-HCPT NPs/RBCs@ICG-HCPT NPs and laser irradiation. Particularly in the case of RBCs@ICG-HCPT NPs, the tumor growth inhibition ability was relatively stronger compared to that of ICG-HCPT NPs. The phenomenon could be attributed to the better passive tumor-targeting mediated by EPR effect and immune escape ability of RBCs@ICG-HCPT NPs. Additionally, no significant changes regarding body weight were observed in all formulations group, indicating no obvious systemic toxicity in the course of treatment (Figure 8E). Thus, compared with individual treatment, chemotherapy or PTT, RBCs@ICG-HCPT NPs under photo/pH-dual-stimulate accomplished remarkable ablation of tumors by chemo-PTT. Additionally, histological analysis of tumor sections was conducted to assess the antitumor efficacy. The RBCs@ICG-HCPT NPs-treated group showed the much 23 ACS Paragon Plus Environment


more irregular shape in tumor cells with obvious shrinking nucleus. However, in the control groups, the tumor cells showed the densest and regular morphology with normal nucleus (Figure 8F). Besides, no significant differences were observed between saline-treated group and RBCs@ICG-HCPT NPs-treated group. The result indicated that the improved nanodrug administration exhibited low toxicity and did not induce off-target damage to liver or other tissues injury compared other groups. Based on these results, it may be concluded that RBCs@ICG-HCPT NPs holds great potential for the treatment of cancer utilizing this treatment modality of chemo-PTT.


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Figure 8. In vivo antitumor effect of RBCs@ICG-HCPT NPs. (A) Representative photographs of HeLa tumor-bearing mice in different groups during the treatment. (B) Pharmacokinetic curves of ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs. Data were presented as mean ± s. d. (n= 4). (C) Tumor volume of mice in different groups after treatment. (D) Mean weight of tumors separated from mice in different groups after treatment. (E) Body weight of mice in different groups during the treatment. (F) H&E staining histological images of different groups obtained from tissues including heart, liver, spleen, lung, kidney, and tumor of HeLa tumor-bearing mice. Data were presented as mean ± s. d. (n= 6).

3. CONCLUSIONS In conclusion, we successfully developed a biomimetic nanosystem of RBC membranes-cloaked two small molecular drugs (HCPT and ICG) co-self-assembling core for imaging-guided synergistic chemo-PTT. In this RBCs@ICG-HCPT nanosystem, ICG was used as photothermal agent and near-infrared fluorescence (NIRF) imaging agent while HCPT was used as chemotherapeutic agent. Because of the effective cloaking of RBC membranes, RBCs@ICG-HCPT NPs showed the significantly improved tumor accumulation by EPR effect-mediated passive tumor targeting capabilities, prolonged circulation lifetime, and reduced RES uptake ability. Upon the irradiation of NIR laser, the RBC membranes were thermally disrupted to trigger the accelerated release of small molecular drugs. Then the released HCPT was internalized inside tumor cells by disrupting the structure of nuclei to inhibit the cell replication. Meanwhile, the released ICG was able to produce heat by irradiation to induce cell apoptosis. Moreover, the in vitro and in vivo results demonstrated that RBCs@ICG-HCPT NPs showed the higher anticancer efficacy by combined effect of chemo-PTT, leading to effective tumor ablation. This work suggested that this cooperative assembly of therapeutic and imaging agents and subsequent cloaking of RBC membrane NPs provided potential in imaging, targeted drug delivery and combination cancer therapy. 25 ACS Paragon Plus Environment


4. EXPERIMENTAL SECTION 4.1. Materials. Indocyanine green was purchased from Sigma-Aldrich Co. Ltd (America), 10-Hydroxycamptothecin was obtained from Huangshi Pharmaceutical Co. Ltd (China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Propidium Iodide (PI) were obtained from Sigma-Aldrich Co. Ltd (America). Dimethyl sulfoxide (DMSO) was received from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). And all reagents were of analytical grade without purification. Dulbecco’s modified Eagle media (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone (USA). 4.2. Preparation of ICG-HCPT NPs. The carrier-free nanodrug systems were achieved in a green and simple self-assembly approach between two small molecules drugs (ICG and HCPT). In brief, 200 μL of a solution of ICG in ethanol (1 mg/mL) and 200 μL of a solution of HCPT in ethanol (1 mg/mL) were injected into 2 mL of pure water simultaneously and stirred at room temperature for 6 h in dark environment. Subsequently, the solution was dialyzed for 12 h (MWCO= 3500 Da) using deionized water, during which the water was renewed every 4 h. Finally, the ICG-HCPT NPs were prepared and stored at 4°C environment for future use. 4.3. Preparation of RBC Membranes-Derived Vesicles. Biomimetic RBC membranes-derived vesicles were achieved in the light of a well-established procedure with a few modifications. In brief, the fresh blood (∼3 mL) was collected from Sprague Dawley (SD) rats (male, 200 ± 5 g) and then suspended in 8 mL of phosphate saline buffer (1×PBS) solution. In order to collect the erythrocytes, the solution was centrifuged at 3500 rpm for 5 min at 4°C. Afterwards, in order to remove plasma and other unwanted cellular components, RBC membranes were washed three times with cold PBS (1 × ) solution and recollected by centrifugation. For hemolysis process to remove the hemoglobin, the resulting RBC 26 ACS Paragon Plus Environment

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membranes solution were then subjected to the hypotonic solution (1/4 × PBS) and centrifuged at 12000 rpm for three times to hypotonic treatment. Then, the collected RBCs ghosts were extruded serially through 1 μm, 0.45 μm and then 0.2 μm polycarbonate porous membranes (Avanti Polar Lipids Inc). For further use, the resulting pink RBCs vesicles were stored at -80°C after lyophilization. 4.4. Preparation of RBCs@ICG-HCPT NPs. The ICG-HCPT NPs were prepared as mentioned above, then 200 μL of RBCs solution ghosts in PBS was injected into the above solution slowly. After sonicated for 3 min in water bath at 200 Hz and extruded serially through 1 μm, 0.45 μm, and 0.2 μm polycarbonate porous membranes. The reaction solution was centrifuged at 12000 rpm for 20 min at 4°C and diluted with deionized water for further use. The encapsulation efficiency (EE) and loading efficiency (LE) of HCPT and ICG were determined using dialysis method (MWCO= 3500 Da). After 12 h of dialysis, UV–vis-NIR absorption spectra of the solutions of ICG-HCPT NPs and RBCs@ICG-HCPT NPs were measured. EE and LE were inferred by the following equations: EE (%) = LE (%) =

𝑊𝑒 𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑁𝑃𝑠 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑁𝑃𝑠 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑁𝑃𝑠

× 100% (1) × 100% (2)

4.5. Evaluation of Size and Morphology of Nanostructures. The morphology of HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs was observed by a transmission electron microscope (TEM, JEOL-1400, Japan) operating at 100 kV and a scanning electron microscope (SEM, SU-70, Japan) operating at 10 kV. In addition, Leica TCS SP5 confocal laser scanning microscopy (CLSM, Leica Microsystems, Germany) was also used for morphology observation. Moreover, the size distribution and surface potential change of NPs were detected by dynamic light scattering (DLS) with a Malvern Zeta-sizer Nano-ZS (Malvern Instruments, UK). 4.6. Stability Evaluation. The physical stability of ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs in 27 ACS Paragon Plus Environment


aqueous solution was evaluated by depositing samples (containing 15 μg/mL of ICG) in dark environment at room temperature for 96 h. During this period, UV–vis-NIR absorption spectra were recorded on Shimadzu UV-2550 spectrophotometer and fluorescence emission spectra were recorded by using a fluorescence spectrometer (a FluoroMax-4 Spectrofluorometer HORIBA Jobin Yvon, USA) at 740 nm excitation wavelength at regular intervals. Besides, the size distribution of nanodrugs in water, PBS, and 10% FBS was measured respectively within certain time intervals to evaluate the physical stability. 4.7. Hemolysis Experiment. Hemolysis experiments were used to evaluate the irritancy of nanodrug to red blood cells and biocompatibility. Blood samples (3 mL) were collected from the plexus venous in the eyeground of SD mice. Red blood cells were collected by centrifugation and washed three times with cold PBS (1×) at 3500 rpm for 15 min at 4°C until the supernatant was colorless. Then 200 μL of cell suspension was diluted to 10% (v/v0) cell suspension. 1 mL of PBS and water were added into 200 μL of cell suspension respectively, acting as positive control and negative control. Meanwhile, l mL of ICG-HCPT NPs and RBCs@ICG-HCPT NPs were added as experimental groups. After that, all of experimental groups were shaken at 100 rpm for 8 h at 37°C. Then experimental groups were centrifuged at 3500 rpm for 10 min at 4°C to collect the supernatant and measured its absorption at 541 nm using a microplate reader (Biotek, USA). HP (hemolysis percentage) was calculated by the following equations: HP (%) =

𝐴𝑠 ― 𝐴𝑠(0) ― 𝐴𝑐( ― ) 𝐴𝑐( + ) ― 𝐴𝑐( ― )

× 100% (3)

In the above equations, As, As(0), Ac(+), Ac(-) represented the absorption values of the experimental groups containing RBC, the experimental group without RBC, the positive control group and the negative control group, respectively. 4.8. In Vitro Photothermal Response Study. 200 μL of saline, free ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs solution with determined concentration of ICG were respectively added into orifice plates and irradiated with continuous 808 nm NIR laser at 1 W/cm2 for 5 minutes and used a 28 ACS Paragon Plus Environment

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FLIR A × 5 thermal imager (FLIR Systems Inc., USA) to record temperature changes simultaneously. Besides, different power of 808 nm laser (1 W/cm2, 0.75 W/cm2, 0.5 W/cm2, 0.25 W/cm2) were used at the same method to measure the temperature changes. 4.9. In Vitro Drug Release. The release efficiency of HCPT from free HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (with or without 808 nm laser) were conducted by using a dialysis method. Samples with the same concentration of HCPT were injected into dialysis bags (MWCO= 3500 Da) and suspended into PBS buffer solutions with different pH (pH 7.4 and 5.5). Then the dialysis bags gently shaking at 120 rpm and 37°C. At regular intervals, 3 mL of PBS buffer solution was removed and then fresh PBS buffer solution replaced. The cumulative released HCPT was determined by UV-vis-NIR spectrophotometer according to standard curve of HCPT. In the illumination groups, the 808 nm laser was irradiated onto the dialysis bags at regular intervals. 4.10. Cell Lines and Mice. HeLa cells (human cervical carcinoma cell line) were chosen from American Type Culture Collection. Medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin was used for cell culture. Cells were cultivated at 37°C with 5% CO2 atmosphere. Male BALB/c nude mice (20± 2 g) were obtained from Xiamen University Laboratory Animal Center. All animal experiments were operated according to Institutional Animal Care and Use Committee. 4.11. In Vitro Cellular Uptake. Cellular uptake was cunducted in HeLa cells by CLSM. In brief, HeLa cells were seeded in 6-well plates with slides at a density of 2× 105 per well. After incubated for 24 h at 37°C and 5% CO2, the culture medium was removed and replaced by fresh DMEM medium including different samples (ICG, HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs) containing 5 μg/mL of ICG or HCPT without FBS. Then all of cells were incubated for 1 or 4 h. After incubated predetermined times later, the 29 ACS Paragon Plus Environment


cells were washed with PBS. Then 4% paraformaldehyde were used to fix the cells on the slides and dipped with propidium iodide solution for 20 min. Finally, all slides were stained with CLSM to observe cellular internalization. Flow cytometry was adopted for further quantification. In brief, HeLa cells (2 × 105 cells per well) were seeded in 6-well plates and grown 24 h in the fresh medium containing 10% FBS. After that, all of the previous medium was removed. The complete DMEM containing free ICG, free HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (10 μg/mL of ICG or HCPT) was added into each well. At predetermined times (1 h and 4 h), the cells were collected and suspended into cold PBS and analyzed by flow cytometry (Thermo, USA) immediately. 4.12. In Vitro Cytotoxicity Assay. The in vitro cytotoxicity experiments were conducted by the MTT assay. In brief, HeLa cells were precultured in 96-well plates at the density of 1×104 cells per well for 24 h. After that, previous medium was replaced with DMEM contained various concentrations of free ICG, free HCPT, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (0.5, 1, 2, 5, 10 μg/mL of ICG or HCPT) and incubated 24 h at 37°C in the dark. After that, 10 μL of MTT (5 mg/mL) solution was added into each well for another 4 h to test succinate dehydrogenase in living cell mitochondria. The MTT solution was replaced by 150 μL of DMSO and the plate was agitated for 10 min in shaking table to adequately dissolve the formazan crystals. The absorbance at 490 nm was measured in each well using a microplate reader (Biotek, USA). To compare the photocytotoxicity effect, HeLa cells were cltivated with the same samples as described above for 24 h. After incubation, the cells were irradiated with 808 nm laser at 1 W/cm2 for 5 min. The subsequent experimental steps were the same as above. Each experimental group contains three parallel groups. 4.13. Cellular Apoptosis Study. Cellular apoptosis was conducted by using Annexin V-FITC/PI fluorescent stain. HeLa cells were precultured in 6-well plates at the density of 5 × 105 cells per well 30 ACS Paragon Plus Environment

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for 24 h. Then the culture medium was replaced with fresh DMEM containing same drug concentration (2 μg/mL) and incubated for 24 h. The laser irradiation groups were irradiated with 808 nm laser for 5 min at 1 W/cm2. HeLa cells without any treatment were set as a control group. Subsequently, all suspended and adherent cells were acquired and washed three times with cold PBS. Annexin V-FITC binding solution was used to resuspend 1 × 105 cells, and stained with Annexin V-FITC and PI. These samples were measured by flow cytometry. Additionally, western blotting analysis was conducted to further detected the cell apoptosis. HeLa cells were precultured in 6-well plates at the density of 5 × 105 cells per well for 24 h. Then the culture medium was replaced with fresh DMEM containing same drug concentration (2 μg/mL) and incubated for 24 h. The laser irradiation groups were irradiated with 808 nm laser for 5 min at 1 W/cm2. HeLa cells without any treatment were set as a control group. Subsequently, Laemmli buffer was used to extracted the cellular proteins. Then 20 μL of samples were added to a 10% SDS-PAGE and electrotransferred to 0.22 μm PVDF membranes, which were then blocked and probed with antibodies against β-actin and caspase-3 followed by horseradish peroxidase (HRP) conjugated anti-rabbit immunoglobulin-G. Protein bands were identified by chemiluminescence using the ECL Western blotting substrate (Themo Scientific, USA). Finally, the imaging system (Bio-Rad, USA) were used to analyze. 4.14. In Vivo Tumor Imaging and Tumor-Targeting Efficiency Study. The same procedure as previously described was conducted to generate HeLa tumors. When the tumors volume reach to ≈ 80 mm3, 200 μL of saline, ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (100 μg/mL of ICG) were intravenously injected into HeLa tumor-bearing nude mice through tail vein. Fluorescence imaging was conducted at different time points (1, 3, 6, 12, 24 h) after injection (SI Imaging Amix, USA.) using 740 nm excitation wavelength and 790 nm emission wavelength. The free ICG-treated mice were used for comparison. The relative fluorescence intensity was obtained using Living Image software. 31 ACS Paragon Plus Environment


4.15. In Vivo Photothermal Therapy. Male BALB/c nude mice injected with HeLa cells at the right legs were used as the animal model. When the tumors volume reach to ≈ 80 mm3, 200 μL of saline, ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (100 μg/mL of ICG or HCPT) were intravenously injected into HeLa tumor-bearing nude mice via the tail vein. After that, each mouse was treated with 8 min laser irradiation (808 nm, 1 W/cm2). During the process, the real-time temperature at tumor sites was recorded. At the same time, the temperature curve profiles were also drawn at the same time using AnalyzIR software. 4.16. Pharmacokinetics and Distribution In Vivo. BALB/c mice-bearing HeLa tumor were divided into three groups when the tumor volume reach to approximately 100 mm3 (n= 4). After intravenous injection of ICG, ICG-HCPT NPs, and RBCs@ICG-HCPT NPs (200 μL of ICG (100 μg/mL)), respectively, the venous blood of those mice was collected at different time points. Subsequently, the supernatant was collected after centrifuged at 1000 rpm for 10 min and then measured by a fluorescence spectrometer at 740 nm excitation wavelength. All mice were sacrificed 24 h later and major organs (heart, liver, spleen, lung, kidney, and tumors) were harvested and then homogenized in lysis buffer (Beyotime Biotech, China). The supernatant was collected after centrifuged at 1000 rpm for 10 min to measure the fluorescence intensity. The concentration was expressed as percentage of injected dose per gram of tissue (% ID/g). 4.17. In Vivo Antitumor Study and Histological Analysis. HeLa cells at logarithmic growth phase were dispersed in PBS with a density of 5 ×106/mL and injected into the right leg of male BALB/c nude mice. After 15 days of feeding, all mice were divided into eight groups (n= 6). The mice were injected with saline, ICG (with or without laser), HCPT, ICG-HCPT NPs (with or without laser) and RBCs@ICG-HCPT NPs (with or without irradiation, 1 W/cm2, 5 min) (The ICG and HCPT doses were 2 mg/kg), respectively. After 24 h later, the tumor size and body weight were recorded every three days for 24 d. V = L × S2/2 was used to 32 ACS Paragon Plus Environment

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calculate tumor volume, in which L was the largest diameter and S was the smallest diameter. 24 days later, all mice were killed and main organs were collected and fixed in 4% paraformaldehyde solution, and then embedded in paraffin and stained with hematoxylin and eosin (H&E). 4.18. Statistical Analysis. Statistical significance was performed by using the t-test and one-way and two-way analyses of variance, in which P value < 0.05 (*) was considered significant, P < 0.01 (**) was very significant, and P＜0.001 (***) was highly significant.

Author information Corresponding Authors *E-mail: [email protected]; [email protected]; [email protected] Acknowledgements The work is supported by the National Natural Science Foundation of China (31271071), the research projects of the Health Commission of Jiangsu Province of China (H2018070), and the “Double-First Class” Foundation of Materials and Intelligent Manufacturing Discipline of Xiamen University. References (1) Chen, W.; Zheng, R.; Baade, P. D.; Zhang, S.; Zeng, H.; Bray, F.; Jemal, A.; Yu, X. Q.; He, J. Cancer Statistics in China, 2015. CA: A Cancer Journal for Clinicians 2016, 66 (2), 115-132. (2) Lim, S. H.; Sun, J. M.; Lee, S. H.; Jin, S. A.; Park, K.; Ahn, M. J. Pembrolizumab for The Treatment of Non-small Cell Lung Cancer. New England Journal of Medicine 2015, 372 (21), 2018-2028. (3) Murray, B. S.; Babak, M. V.; Hartinger, C. G.; Dyson, P. J. The Development of RAPTA Compounds for The Treatment of Tumors. Coordination Chemistry Reviews 2016, 306, 86-114. (4) Lei, P.; An, R.; Zhang, P.; Yao, S.; Song, S.; Dong, L.; Xu, X.; Du, K.; Feng, J.; Zhang, H. Ultrafast Synthesis of Ultrasmall Poly(Vinylpyrrolidone) ‐ Protected Bismuth Nanodots as a Multifunctional Theranostic Agent for In Vivo Dual ‐ Modal CT/Photothermal ‐ Imaging ‐ Guided Photothermal Therapy. Advanced Functional Materials 2017, 27 (35), 1702018-1702028. (5) Spyratos, F., .; Briffod, M., .; Tubiana-Hulin, M., .; Andrieu, C., .; Mayras, C., .; 33 ACS Paragon Plus Environment


Pallud, C., .; Lasry, S., .; Rou?Ssé, J., . Sequential Cytopunctures During Preoperative Chemotherapy for Primary Breast Carcinoma. II. DNA Flow Cytometry Changes During Chemotherapy, Tumor Regression, and Short-term Follow-up. Cancer 2015, 69 (2), 470-475. (6) Kunjachan, S.; Detappe, A.; Kumar, R.; Ireland, T.; Cameron, L.; Biancur, D. E.; Motto-Ros, V.; Sancey, L.; Sridhar, S.; Makrigiorgos, G. M. Nanoparticle Mediated Tumor Vascular Disruption: A Novel Strategy in Radiation Therapy. Nano Letters 2016, 96 (2), S97-S97. (7) Huang, P.; Wang, D.; Su, Y.; Huang, W.; Zhou, Y.; Cui, D.; Zhu, X.; Yan, D. Combination of Small Molecule Prodrug and Nanodrug Delivery: Amphiphilic Drug– Drug Conjugate for Cancer Therapy. Journal of the American Chemical Society 2014, 136 (33), 11748-11756. (8) Shamay, Y.; Shah, J.; Işık, M.; Mizrachi, A.; Leibold, J.; Tschaharganeh, D. F.; Roxbury, D.; Budhathoki-Uprety, J.; Nawaly, K.; Sugarman, J. L.; Baut, E.; Neiman, M. R.; Dacek, M.; Ganesh, K. S.; Johnson, D. C.; Sridharan, R.; Chu, K. L.; Rajasekhar, V. K.; Lowe, S. W.; Chodera, J. D.; Heller, D. A. Quantitative Self-assembly Prediction Yields Targeted Nanomedicines. Nature Materials 2018, 17 (4), 361-368. (9) Singh, R.; Jr.ab, J. W. L. Nanoparticle-based Targeted Drug Delivery. Experimental and Molecular Pathology 2009, 86 (3), 215-223. (10)Omid C, F.; Robert, L. Impact of Nanotechnology on Drug Delivery. Acs Nano 2009, 3 (1), 16-20. (11)Dobrovolskaia, M. A.; Aggarwal, P.; Hall, J. B.; Mcneil, S. E. Preclinical Studies To Understand Nanoparticle Interaction with The Immune System and Its Potential Effects on Nanoparticle Biodistribution. Mol Pharm 2008, 5 (4), 487-495. (12)Ding, H. M.; Ma, Y. Q. Theoretical and Computational Investigations of Nanoparticle-biomembrane Interactions in Cellular Delivery. Small 2015, 11 (9-10), 1055-1071. (13)Li, J.; Angsantikul, P.; Liu, W.; Esteban-Fernández, d. Á. B.; Chang, X.; Sandraz, E.; Liang, Y.; Zhu, S.; Zhang, Y.; Chen, C. Biomimetic Platelet-Camouflaged Nanorobots for Binding and Isolation of Biological Threats. Advanced Materials 2017, 30 (2), 1704800-1704808. (14)Hu, C. M. J.; Fang, R. H.; Wang, K. C.; Luk, B. T.; Thamphiwatana, S.; Dehaini, D.; Nguyen, P.; Angsantikul, P.; Wen, C. H.; Kroll, A. V. Nanoparticle Biointerfacing via Platelet Membrane Cloaking. Nature 2015, 526 (7571), 118-121. (15)Wei, X.; Ying, M.; Dehaini, D.; Su, Y.; Kroll, A. V.; Zhou, J.; Gao, W.; Fang, R. H.; Chien, S.; Zhang, L. Nanoparticle Functionalization with Platelet Membrane Enables Multifactored Biological Targeting and Detection of Atherosclerosis. Acs Nano 2017, 12 (1), 109-116. (16)Weiwei, G.; Fang, R. H.; Soracha, T.; Luk, B. T.; Jieming, L.; Pavimol, A.; Qiangzhe, Z.; Hu, C. M. J.; Liangfang, Z. Modulating Antibacterial Immunity via Bacterial Membrane-coated Nanoparticles. Nano Letters 2015, 15 (2), 1403. (17)Minjun, X.; Jingxin, S.; Luru, D.; Qiang, H.; Junbai, L. Nanocapsules: Macrophage Cell Membrane Camouflaged Mesoporous Silica Nanocapsules for In 34 ACS Paragon Plus Environment

Page 34 of 37

Page 35 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Vivo Cancer Therapy (Adv. Healthcare Mater. 11/2015). Advanced Healthcare Materials 2015, 4 (11), 1578-1578. (18)Luk, B. T.; Zhang, L. Cell Membrane-camouflaged Nanoparticles for Drug Delivery. Journal of Controlled Release Official Journal of the Controlled Release Society 2015, 220 (Pt B), 600-607. (19)Lang, R.; Lin-Lin, B.; Jun-Hua, X.; Bo, C.; Guang-Tao, Y.; Xiaolei, Y.; Zhaobo, H.; Qinqin, H.; Andrew, L.; Shi-Shang, G. Red Blood Cell Membrane as a Biomimetic Nanocoating for Prolonged Circulation Time and Reduced Accelerated Blood Clearance. Small 2016, 11 (46), 6225-6236. (20)Wu, Z.; Li, T.; Gao, W.; Xu, T.; Jurado ‐ S á nchez, B.; Li, J.; Gao, W.; He, Q.; Zhang, L.; Wang, J. Cell-Membrane-Coated Synthetic Nanomotors for Effective Biodetoxification. Advanced Functional Materials 2015, 25 (25), 3881-3887. (21)Wan, G.; Chen, B.; Li, L.; Wang, D.; Shi, S.; Zhang, T.; Wang, Y.; Zhang, L.; Wang, Y. Nanoscaled Red Blood Cells Facilitate Breast Cancer Treatment by Combining Photothermal/Photodynamic Therapy and Chemotherapy. Biomaterials 2018, 161, 25-40. (22)Ding, Y.; Su, S.; Zhang, R.; Shao, L.; Zhang, Y.; Wang, B.; Li, Y.; Chen, L.; Yu, Q.; Wu, Y. Precision Combination Therapy for Triple Negative Breast Cancer via Biomimetic Polydopamine Polymer Core-shell Nanostructures. Biomaterials 2017, 113, 243-252. (23)Yue, H.; Yamin, Y.; Hongjun, W.; Henry, D. Synergistic Integration of Layer-by-Layer Assembly of Photosensitizer and Gold Nanorings for Enhanced Photodynamic Therapy in the Near Infrared. Acs Nano 2015, 9 (9), 8744-8754. (24)Xu, D.; You, Y.; Zeng, F.; Wang, Y.; Liang, C.; Feng, H.; Ma, X. Disassembly of Hydrophobic Photosensitizer by Bio-degradable Zeolitic Imidazolate Framework-8 for Photodynamic Cancer Therapy. Acs Applied Materials & Interfaces 2018, 10 (18), 15517–15523. (25)Yang, L.; Liu, G.; Ma, J.; Lin, J.; Lin, H.; Su, G.; Chen, D.; Ye, S.; Chen, X.; Xuan, Z. Chemotherapeutic Drug-photothermal Agent Co-self-assembling Nanoparticles for Near-infrared Fluorescence and Photoacoustic Dual-modal Imaging-guided Chemo-photothermal Synergistic Therapy. Journal of Controlled Release 2017, 258, 95-107. (26)Tan, X.; Wang, J.; Pang, X.; Liu, L.; Sun, Q.; You, Q.; Tan, F.; Li, N. Indocyanine Green-Loaded Silver Nanoparticle@Polyaniline Core/Shell Theranostic Nanocomposites for Photoacoustic/Near-Infrared Fluorescence Imaging-Guided and Single-Light-Triggered Photothermal and Photodynamic Therapy. Acs Applied Materials & Interfaces 2016, 8 (51), 34991–35003. (27)Hu, D.; Zhang, J.; Gao, G.; Sheng, Z.; Cui, H.; Cai, L. Indocyanine Green-Loaded Polydopamine-Reduced Graphene Oxide Nanocomposites with Amplifying Photoacoustic and Photothermal Effects for Cancer Theranostics. Theranostics 2016, 6 (7), 1043-1052. (28)Cheng, Y.; Tan, X.; Wang, J.; Wang, Y.; Song, Y.; You, Q.; Sun, Q.; Liu, L.; Wang, S.; Tan, F. Polymer-based Gadolinium Oxide Nanocomposites for FL/MR/PA 35 ACS Paragon Plus Environment


Imaging Guided and Photothermal/Photodynamic Combined Anti-tumor Therapy. Journal of Controlled Release 2018, 277, 77-88. (29)Zhao, Y.; Chen, F.; Pan, Y.; Li, Z.; Xue, X.; Okeke, C. I.; Wang, Y.; Li, C.; Peng, L.; Wang, P. C. Nanodrug Formed by Coassembly of Dual Anticancer Drugs to Inhibit Cancer Cell Drug Resistance. Acs Applied Materials & Interfaces 2015, 7 (34), 19295-19305. (30)Yang, J.; Ni, B.; Liu, J.; Zhu, L.; Zhou, W. Application of Liposome-encapsulated Hydroxycamptothecin in The Prevention of Epidural Scar Formation in New Zealand White Rabbits. Spine Journal Official Journal of the North American Spine Society 2011, 11 (3), 218-223. (31)Denise, B.; Pierre, C.; Céline, F.; Marie-Laure, V.; Fran?Ois, G.; Muriel, B. H. Nanoparticles as Vehicles for Delivery of Photodynamic Therapy Agents. Trends in Biotechnology 2008, 26 (11), 612-621. (32)Wei, S.; Xi, Z.; Min, L.; Lian, L.; Jiaju, Z.; Wei, S.; Zhirong, Z.; Yuan, H. Overcoming The Diffusion Barrier of Mucus and Absorption Barrier of Epithelium by Self-assembled Nanoparticles for Oral Delivery of Insulin. Acs Nano 2015, 9 (3), 2345. (33)Ping, H.; Dali, W.; Yue, S.; Wei, H.; Yongfeng, Z.; Daxiang, C.; Xinyuan, Z.; Deyue, Y. Combination of Small Molecule Prodrug and Nanodrug Delivery: Amphiphilic Drug-drug Conjugate for Cancer Therapy. Journal of the American Chemical Society 2014, 136 (33), 11748-11756. (34)Hochmuth, R. M.; Evans, C. A.; Wiles, H. C.; Mccown, J. T. Mechanical Measurement of Red Cell Membrane Thickness. Science 1983, 220 (4592), 101-102. (35) Choi, K. Y.; Chung, H.; Min, K. H.; Hong, Y. Y.; Kim, K.; Park, J. H.; Kwon, I. C.; Jeong, S. Y. Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010, 31 (1), 106-114. (36)Yahia-Ammar, A.; Sierra, D.; Mérola, F.; Hildebrandt, N.; Le, G. X. Self-Assembled Gold Nanoclusters for Bright Fluorescence Imaging and Enhanced Drug Delivery. Acs Nano 2016, 10 (2), 2591-2599. (37)Hee, L. M.; Jong Seung, K.; Sessler, J. L. Small Molecule-based Ratiometric Fluorescence Probes for Cations, Anions, and Biomolecules. Chemical Society Reviews 2015, 44 (13), 4185-4191. (38)Yang, L.; Jinyan, L.; Yu, H.; Yanxiu, L.; Xiangrui, Y.; Hongjie, W.; Shichao, W.; Liya, X.; Lizong, D.; Zhenqing, H. Self-Targeted, Shape-Assisted, and Controlled-Release Self-Delivery Nanodrug for Synergistic Targeting/Anticancer Effect of Cytoplasm and Nucleus of Cancer Cells. Acs Applied Materials & Interfaces 2015, 7 (46), 25553-25559. (39)Qian, C.; Chao, L.; Chao, W.; Zhuang, L. An Imagable and Photothermal "Abraxane-like" Nanodrug for Combination Cancer Therapy to Treat Subcutaneous and Metastatic Breast Tumors. Advanced Materials 2015, 27 (5), 903-910. (40)Siperstein, A. E.; Rogers, S. J.; Hansen, P. D.; Gitomirsky, A. Laparoscopic Thermal Ablation of Hepatic Neuroendocrine Tumor Metastases. Surgery 1997, 122 (6), 1147-1155. (41)Ju, E.; Dong, K.; Liu, Z.; Pu, F.; Ren, J.; Qu, X. Tumor Microenvironment 36 ACS Paragon Plus Environment

Page 36 of 37

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Activated Photothermal Strategy for Precisely Controlled Ablation of Solid Tumors upon NIR Irradiation. Advanced Functional Materials 2015, 25 (10), 1574-1580. (42)Ayi, K.; Lu, Z.; Serghides, L.; Ho, J. M.; Finney, C.; Wang, J. C.; Liles, W. C.; Kain, K. C. CD47-SIRPα Interactions Regulate Macrophage Uptake of Plasmodium falciparum-Infected Erythrocytes and Clearance of Malaria In Vivo. Infection & Immunity 2016, 84 (7), 2002-2010.

a Table of Contents graphic


pH-triggered biomimetic red blood cell membranes camouflaged

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