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Erythrocyte Membrane-Coated Upconversion Nanoparticles with Minimal Protein Adsorption for Enhanced Tumor Imaging Lang Rao, Qianfang Meng, Lin-Lin Bu, Bo Cai, Qin qin Huang, Zhi-Jun Sun, WenFeng Zhang, Andrew Li, Shi-Shang Guo, Wei Liu, Tza-Huei Wang, and Xing-Zhong Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14450 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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ACS Applied Materials & Interfaces

Erythrocyte Membrane-Coated Upconversion Nanoparticles with Minimal Protein Adsorption for Enhanced Tumor Imaging Lang Rao,† Qian-Fang Meng,† Lin-Lin Bu,‡ Bo Cai,† Qinqin Huang,† Zhi-Jun Sun,‡ Wen-Feng Zhang,‡ Andrew Li,§ Shi-Shang Guo,† Wei Liu,*,†,§,║ Tza-Huei Wang§,║ and Xing-Zhong Zhao† †

Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School

of Physics and Technology, Wuhan University, Wuhan, Hubei 430072, China ‡

Department of Oral Maxillofacial Head Neck Oncology, School and Hospital of

Stomatology, Wuhan University, Wuhan, Hubei 430072, China §

Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore,

Maryland 21205, United States ║

Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland

21218, United States * Corresponding author E-mail: [email protected]

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ABSTRACT: Upconversion nanoparticles (UCNPs) with superior optical and chemical features have been broadly employed for in vivo cancer imaging. Generally, UCNPs are surface modified with ligands for cancer active targeting. However, nanoparticles in biological fluids are known to form a long-lived "protein corona", which covers the targeting ligands on nanoparticle surface and dramatically reduces the nanoparticle targeting capabilities. Here, for the first time, we demonstrated that by coating UCNPs with red blood cell (RBC) membranes, the resulting cell membrane-capped nanoparticles (RBC-UCNPs) adsorb virtually no proteins when exposed to human plasma. We further observed in various scenarios that the cancer targeting ability of folic acid (FA)-functionalized nanoparticles (FARBC-UCNPs) is rescued by the cell membrane coating. Next, the FA-RBC-UCNPs were successfully utilized for enhanced in vivo tumor imaging. Finally, blood parameters and histology analysis suggest that no significant systematic toxicity was induced by the injection of biomimetic nanoparticles. Our method provides a new angle on the design of targeted nanoparticles for biomedical applications.

KEYWORDS: diagnosis and therapy, drug delivery, protein corona, cancer targeting, red blood cell membrane, upconversion nanoparticle


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1. INTRODUCTION Versatile and accurate tumor imaging holds great importance in cancer early prevention and advanced metastasis management.1,

2

Amongst the growing amount of in vivo imaging

techniques including magnetic resonance imaging (MRI), computer tomography (CT), positron emission tomography (PET), and photoacoustic tomography (PAT), fluorescence imaging is one of the most powerful modalities.3, 4 Upconversion nanoparticles (UCNPs), which can convert light from the near-infrared (NIR) range to the visible range, are a promising agents for in vivo fluorescence imaging.5-10 Compared with conventional downconversion fluorescent nanoprobes, such as organic dyes and quantum dots, UCNPs own superior chemical and optical properties, such as narrow emission peaks, low toxicity, and good photo-stability.11-16 For in vivo tumor imaging, UCNPs typically require specific tumor targeting capabilities, which enables the specific delivery of UCNPs to the tumor sites, improving the practicality of imaging.17, 18 However, when these nanoparticles enter into biological fluids (e.g., serum and plasma), proteins and other biomolecules aggregate around the nanoparticles.19-21 This in situ generation of protein coronas covers the targeting ligands and dramatically weakens the nanoparticle targeting capabilities.22-24 One classical approach used to prevent the protein corona formation is surface functionalization of nanoparticles with polymer materials (e.g., poly(ethylene glycol) (PEG), poly(vinyl pyrrolidone) (PVP) and poly(carboxybetaine) (PCB)).25-33 However, these materials either still interact with certain biomolecules or induce the activation of immune responses at different degrees.34, 35 Thus, there still exists great concerns in the validity and bio-safety of using synthetic polymers to prevent the formation of protein coronas.36, 37 The cell is the basic unit of organism structure and function that continually inspires passion and provides insight for life science researchers. Unlike nanoparticles, cells have a 3 ACS Paragon Plus Environment


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cell membrane that regulates the adsorption of biological components.38 Thus it is conceivable to exploit the natural properties of cell membranes to prevent the protein corona formation and thus maintain the nanoparticle targeting capabilities in a complex biological environment. In this work, cell membranes were donated from red blood cells (RBCs), reconstructed into vesicles (RBC-vesicles), and then coated onto UCNPs. We demonstrated that the obtained RBC membrane-capped nanoparticles (RBC-UCNPs) adsorb virtually no proteins after being exposed to 100% human plasma. Subsequently, cancer-targeting molecules were modified onto the surface of cell membrane-coated nanoparticles to investigate the fidelity of our nanoparticle targeting. Benefiting from the prevention of protein adsorption, the targeting efficiency of cell membrane-coated nanoparticles was significantly improved. Given the superior optical and chemical performances of UCNPs, this biomimetic nanoplatform was further employed for efficient tumor imaging. Finally, the in vivo toxicity evaluation results point to superior biocompatibility of our biomimetic nanoparticles. Our cell membrane-coated nanoplatform embodies a bio-inspired strategy to solve the long-standing challenges in nano-bio interactions.

2. EXPERIMENTAL SECTION 2.1. Materials. Phosphate buffer solution (PBS) was obtained from Thermo-Fisher (USA). Bicinchoninic acid (BCA) assay kit, folate receptor β (FRβ), paraformaldehyde (PFA), 4’,6diamidino-2-phenylindole (DAPI), and uranyl acetate were purchased from Sigma-Aldrich (USA). Sodium dodecyl sulfate (SDS)-polyacrylamide gel and SDS-buffer were purchased from Beyotime (China) and Invitrogen (USA). Phospholipids with various functionalized head-groups

including

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[folate(polyethylene (DSPE-PEG-FA),

and

glycol)-2000]

(ammonium

salt)

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Cy5(poly4 ACS Paragon Plus Environment

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ethylene glycol)-2000] (ammonium salt) (DSPE-PEG-Cy5) were obtained from Nanocs (USA) and Avanti Polar Lipids (USA). All the aqueous solutions were prepared by using deionized (DI) water purified on a experimental water purification system (Direct-Q3, Millipore, USA). And the other solvents used in the present work were obtained from Aladdin-Reagent (China) and Sinopharm Chemical Reagent (China). 2.2. Preparation and Characterization of RBC-UCNPs. To encapsulate UCNPs (i.e., DSPE-PEG-functionalized UCNPs) into RBC-vesicles, RBC-vesicles derived from 10 µL mice blood were firstly mixed with 50 µg UCNPs in 1 mL PBS. Subsequently, the mixture was repeatedly extruded through a 200-nm nuclepore polyester membrane on a mini-extruder, and then centrifuged at 1,000 g for 10 min to eliminate excess RBC-vesicles.39 Finally, the resulting RBC-UCNPs were stored in PBS at 4 °C for the following experiments. 50 µg UCNPs or RBC-UCNPs suspended in 1 mL PBS were used to detecting the mean diameter and zeta potential at room temperature by using a dynamic light scatter (DLS; Nano-Zen 3600, Malvern Instruments, UK). The morphology of UCNPs and RBC-UCNPs were also observed by using a transmission electron microscope (TEM; JEM-2010 ES500W, Japan). Before the TEM characterization, the samples were prepared by contacting the suspension droplet containing UCNPs or RBC-UCNPs with copper grids for 60 s and then negatively stained with uranyl acetate for 30 s. The cell membrane proteins were further characterized by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).40 The RBC lysate, RBC-vesicles, and purified RBC-UCNPs were loaded into SDS buffer as measured by the BCA assay kit. Then the samples were heated at 95 °C for 5 min and 20 µg of each sample was added into each well in a 10% SDS-polyacrylamide gel. Samples were run at 120 V for 2 h and the obtained gel was stained with Commassie blue, washed with DI water and imaged. 2.3. Preparation and Characterization of Nanoparticle-Protein Complexes. 1 mg 5 ACS Paragon Plus Environment


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UCNPs or RBC-UCNPs was incubated in 1 mL 100% human plasma on ice for 4 h. To obtain hard protein corona complexes, the resulting mixture was centrifuged at 1,000 g for 10 min to obtain the particle-protein complexes. The final products were washed with PBS for three times before being re-suspensed in PBS. This treatment allows us to remove the proteins with low affinity (i.e., soft protein corona) from the nanoparticles.27 The size of UCNPs and RBC-UCNPs before and after incubation in 100% human plasma was measured by DLS. The nanoparticle-protein complexes were also analyzed by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Before the LC-MS/MS analysis, the protein content of hard protein coronas was determined by the BCA kit. Then all samples were individually re-suspended in 25 µL Sequencing Grade Trypsin (ammonium bicarbonate, 12.5 ng/µL), and digested by using a digestor (CEM Discover Microwave, USA) for 20 min at 60 °C. And then the digestion was stopped by adding 200 µL of acetonitrile/water/formic acid (50 : 45 : 5, v/v/v) solution to the samples. The solvents were removed by using a SpeedVac (Thermo Scientific, USA), and the residue was dissolved in 13 µL of 5% aqueous acetonitrile containing 0.1% formic acid. 10 µL samples were analyzed by LC-MS/MS performed on a mass spectrometer (Q-ToF, Waters, USA) connected to an ultra performance LC (UPLC) (nano Acquity, Waters, USA). LC was performed on a C-18 column (Waters Atlantis, USA) at a flow rate of 250 nL/min. Peptides were eluted by using 0-60% aqueous acetonitrile containing 0.1% formic acid. The mass spectrometer was set at data dependent acquisition mode, and MS/MS was performed on the most abundant four peaks detected at any time. Spectrum was searched by using BioworksBrowser 3.3.1 SP1 software (Thermo Scientific, USA) with Sequest Uniprot/Swiss-Prot database. The normalized percentage of spectral counts detected by LC-MS/MS for protein k (NSpCk) was calculated according to following equation:24


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NSpCk =

SpC/(M w ) k

n

∑ (SpC/(M ) ) i =1

× 100%

w i

2.4. Preparation and Characterization of FA-RBC-UCNPs. First, empty RBCs were obtained from 1 mL blood as described previously and then incubated with 50 µg DSPEPEG-FA for 30 min to form FA-inserted empty RBCs.41 All samples were centrifuged at 1,000 g for 10 min and then washed with PBS for three times before further use. To ensure the successful FA insertion, DSPE-PEG-Cy5 was used instead of DSPE-PEG-FA. The empty RBCs modified without or with DSPE-PEG-Cy5 were analyzed on a flow cytometer (FACScaliber, Becton Dickinson, USA) and data was analyzed by using FlowJo software (Tree Star, USA). The Cy5-modified empty RBCs were also observed by employing a confocal laser scanning microscope (CLSM; IX81, Olympus, Japan). In order to obtain cancer-targeting FA-RBC-UCNPs, the FA-modified RBC membranes were re-constructed into vesicles and then coated onto UCNPs as described previously. To confirm coating of DSPE-PEG-FA onto the oleic acid-capped UCNPs, DSPE-PEG-Cy5 was used instead of DSPE-PEG-FA and the resulting Cy5-RBC-UCNPs were observed by CLSM under an external 980-nm laser device (Hi-Tech Optoelectronics, China). DLS and SDS-PAGE were also employed to analyze the differences before and after surface modification in hydrodynamic size, zeta potential and proteins of RBC-UCNPs. 2.5. Evaluation of Nanoparticle Targeting to FRs and MCF-7 Cells. UCNPs, RBC-UCNPs, FA-UCNPs (i.e., DESPE-PEG-FA-functionalized UCNPs) and FA-RBC-UCNPs were individually incubated in 100% human plasma for 4 h and then washed with PBS for three times to remove proteins and soft protein coronas. Subsequently, 5 mg UCNPs, RBC-UCNPs, FA-UCNPs and FA-RBC-UCNPs with or without incubation in human plasma were individually mixed with 5 µg FRβ for 1 h. Then all samples were centrifuged at 1,000 g for 10 min at 4 °C and then washed with PBS for three times before further use. Both the pellet



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and suspension were collected separately and the amount of FRβ in the suspension was determined by BCA kit. MCF-7 human breast carcinoma cells were seeded in confocal dishes and cultured for 24 h. Before the test, the cell medium was discarded. The cells were washed with PBS for three times before adding cell medium or medium containing 50 µg/mL various Cy5-labelled nanoparticles. The cells grown without the addition of any nanoparticles were used as a control. Then the cells were orderly incubated for 6 h, washed with PBS, stained with DAPI, washed again, fixed with 4% PFA and observed by using CLSM under the 980nm irradiation. Further, MCF-7 cells were seeded in 6-well culture plates, grown for 24 h, incubated with 50 µg/mL of the Cy5-labelled nanoparticles for 6 h. The cells grown without the addition of nanoparticles were used as a control. Single cell suspensions were prepared by trypsinization, washed with PBS for three times and then analyzed by flow cytometry as described previously.42, 43 To quantify the targeting of nanoparticles to MCF-7 cells, HNO3 was added to dissolve the cells that were incubated with Cy5-labelled nanoparticles for 6 h. The mixture samples were stored at room temperature for 12 h, then heated at 70 °C to eliminate the acids, cooled down to room temperature and re-suspended in DI water for the Y3+ determination by using an inductively coupled plasma-atomic emission spectrometry (ICP-AES; IRIS Intrepid II XSP, Thermo Elemental, USA).7 2.6. In Vivo Tumor Imaging and Biodistribution Evaluation. For conducting in vivo fluorescence imaging, 30 BALB/c nude mice bearing MCF-7 human breast tumor xenografts (n = 6) were intravenously (i.v.) injected with 200 µL PBS or PBS containing 5 mg/mL various nanoparticles. At 48 h after the injection, all mice were anesthetized intraperitoneally (i.p.) by injection of 80 µL 10% chloral hydrate solution, and then the in vivo imaging was conducted by a modified small animal imaging system (Xenogen IVIS Spectrum, Caliper, USA) equipped with a fluorescent filter set (excitation/emission = 980/535 nm). The view field was 12.5 cm in diameter and the photos were acquired for 0.5 s and further analyzed by 8 ACS Paragon Plus Environment

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using Living Image software (PerkinElmer, USA).44 All mice were euthanized, and their tumors were extracted to observe the ex vivo fluorecence signals by using the small animal imaging system and the corresponding software. Further, the biodistribution of various nanoparticles in the mice were detected and quantified. The tumors, blood samples and major organs of the euthanized mice were collected for the Y3+ quantification with ICP-AES as described previously. 2.7. In Vivo Toxicity Evaluation. For conducting systematic toxicity evaluation, 12 ICR mice (n = 6) were i.v. injected with 200 µL PBS or PBS containing 5 mg/mL RBC-UCNPs. In order to evaluate the general status of mice, veterinarian examined the mice daily and measured the body weights of mice every three day. All mice were euthanized on the 15th d after the initial injection and their blood samples and major organs were harvested for blood test and histology analysis. The blood parameters from the control and treated mice were detected by using a blood biochemical auto-analyzer (7080, HITACHI, Japan). The major organs were fixed in 4% neutral formalin buffer, paraffin embedded, sectioned at 4 µm, stained with hematoxylin and eosin (H&E), and finally imaged by using a typical optical microscope (BX51, Olympus, Japan). 2.8. Statistical Analysis. Experimental data was analyzed by using one-way ANOVA followed by the post-Tukey comparison tests with GraphPad Prism 5.0 software. P < 0.05 indicates statistical difference.

3. RESULTS AND DISCUSSION 3.1. Preparation of RBC-UCNPs. The production of biomimetic RBC-UCNPs consists of three steps: i) preparation of RBC-vesicles, ii) synthesis of hydrophilic UCNPs, and iii) fusion of RBC-vesicles onto UCNPs (Figure 1a). First, RBCs harvested from the fresh mice blood were permeablized, resulting in empty RBCs (Figure S1, Supporting Information).



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Empty RBCs were then sonicated and extruded through a 400-nm membrane on an extruder to obtain the RBC-vesicles (Figure S2).39, 45, 46 Concurrently, hexagonal β-NaYF4:Er3+,Yb3+ UCNPs were prepared as described in our previous papers,44, 47 and characterized (Figure S3). UCNPs were then surface functionalized with DSPE-PEG to improve the water solubility (Note: UCNPs used in this work were all modified DSPE-PEG).48 DSPE-PEG-Cy5 was used to replace DSPE-PEG to validate the successful surface modification (Figure S4). Finally, the DSPE-PEG-functionalized UCNPs were mixed with RBC-vesicles and the mixture was repeatedly extruded through 200-nm pores. The mechanical force induced by the extrusion process promoted the synthesis of bio-inspired RBC-UCNPs.45 DLS was employed to characterize changes in the nanoparticles before and after the cell membrane coating. The hydrodynamic diameter of nanoparticles increased ~20 nm (Figure 1b and Figure S5a) and the zeta potential changed approximately to that of the level of RBCvesicles (Figure 1c and Figure S5b), indicating successful cell membrane coating. Furthermore, TEM photos undoubtedly showed a hexagonal nanoparticle core of ~80 nm and a cell membrane shell of ~10 nm (Figure 1d-f). SDS-PAGE protein analysis further demonstrated that RBC membrane proteins were successfully transferred to UCNPs (Figure 1g). We further tried to optimize the membrane coating and found that the lowest core-toshell ratio at which RBC-UCNPs possessed a steady size was approximately 1 mg UCNPs per 0.2 mL blood (Figure S6). At this ratio, the obtained RBC-UCNPs possessed good stability in 1 × PBS and 100% human plasma over 15 d (Figure S7). In addition, under 980nm NIR irradiation, hydrophilic UCNPs showed strong green upconversion luminescence (UCL) and the emission peaks were agreed with the typical 543 and 655 nm.47 It is also noteworthy that the biomimetic RBC membrane coating had virtually no influence on the UCL emission (Figure S8), ensuring the quality of further in vivo tumor imaging experiments. 3.2. Evaluation of Nanoparticle-Protein Interactions. The formation of protein coronas, 10 ACS Paragon Plus Environment

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which involves both reversible and irreversible adsorption of proteins on the nanoparticle surface (i.e., soft and hard protein coronas),40,

49

changes the interface characteristics of

nanoparticles (Figure 2a) and plays a key role in the fate of nanoparticles after entering a complex physiological environment.22, 50 After confirming the cell membrane coating, we hypothesized that they can efficiently prevent the formation of protein coronas (Figure 2b). To test this, UCNPs and RBC-UCNPs were individually incubated in 100% human plasma for 4 h and then centrifuged and washed with PBS to eliminate excessive proteins and soft coronas. DLS was employed to characterize the uncoated and cell membrane-coated nanoparticles. After the incubation, the hydrodynamic diameter of UCNPs was markedly increased (Figure 2c and Figure S9a), which can be attributed to the formation of hard protein coronas around nanoparticles. However, there was no obvious changes in size of RBCUCNPs (Figure 2d and Figure S9b), suggesting that the RBC membranes can effectively prevent protein adsorption. The nanoparticle-protein interactions were further analyzed by using LC-MS/MS. After the incubation in 100% human plasma, certain proteins were adsorbed onto the uncoated UCNPs (Figure 2e, Table S1 and S2). In contrast, the protein components on the RBC-UCNPs stayed essentially the same before and after the incubation, demonstrating that the proteins were inherited from RBC membranes and not adsorbed from biological fluids, and further indicating that the RBC membrane coating could successfully reduce the protein aggregation around nanoparticles. 3.3. Preparation of Cancer-Targeting FA-RBC-UCNPs. FA molecules have been widely used for cancer targeting because their receptors (i.e., folate receptors (FRs)) are a class of highly selective tumor markers overexpressed in various cancers.51 After confirming that the cell membrane-coated nanoparticles are basically free of protein coronas, FA molecules were modified onto the surface of nanoparticles to test the targeting efficacy of nanoparticles (Figure 3a). Owing to the fluidity of lipid bilayered membranes, we were able to obtain FA11 ACS Paragon Plus Environment


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modified RBC membranes by physically mixing empty RBCs and DSPE-PEG-FA.41 Fluorescent DSPE-PEG-Cy5 was used to demonstrate the successful functionalization. After mixing DSPE-PEG-Cy5 and empty RBCs, cell membranes that were originally nonluminous exhibited bright Cy5 fluorescence (Figure 3b,c). It is should be point out that, once the DSPE-PEG with various functionalized head-groups were bonded to the empty RBCs, they would not be shared across other empty RBCs (Figure S10), suggesting good stability of the interaction of DSPE-PEG and empty RBCs. Afterwards, the Cy5- or FA-inserted RBC membranes were coated onto UCNPs by the same process used to produce RBC-UCNPs (Figure S11). Unmodified and FA-modified RBC-UCNPs showed similar physicochemical properties (Figure 3d-f and Figure S12,S13), suggesting that the surface modification of DSPE-PEG-FA had minor influences on the properties of nanoparticles. It is also noteworthy that the physical extrusion of cell membranes onto UCNPs did not cause loss of the desirable features of cell membranes.45 Thus, the process for preparing these cancer-targeting cell membrane-coated nanoparticles ensures good biocompatibility and the potential of using these biomimetic nanoparticles for further applications. Lastly, as a non-membrane-coated control, cancer-targeting FA-UCNPs were prepared by the same procedures, but with DSPEPEG-FA instead of DSPE-PEG. After generating two types of cancer-targeting nanoparticles, the changes in hydrodynamic size of nanoparticles before and after being incubated in 100% human plasma were monitored. We found that the modification of FA molecules did not hurt the anti protein adsorption properties of the cell membrane-coated nanoparticles (Figure S14). 3.4. Evaluation of Nanoparticle Targeting. When FA-UCNPs were exposed to biological fluids, the in situ formation of protein coronas concealed the surface targeting FA molecules, resulting in low efficiency of FA-UCNPs targeting to FR-overexpressed cancer cells (Figure 4a).52 In contrast, after being coated with cell membranes, the targeting efficiency of nanoparticles was significantly improved, which may be attributed to the lack of protein 12 ACS Paragon Plus Environment

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coronas (Figure 4b). FRβ, a member of the functional FRs, was mixed with nanoparticles to test the binding efficiency between FA-functionalized nanoparticles and FRs before and after plasma exposure. After exposing nanoparticles to 100% human plasma, the amount of FRβ bound to FA-UCNPs was markedly reduced, whereas for FA-RBC-UCNPs, the amount of bound FRβ maintained prior levels (Figure 4c and Figure S15a). This indicates that the cell membrane coating can help FA-functionalized nanoparticles preserving their targeting capabilities to FRs even in complex biological fluids. It is also worth noting that FA-UCNPs and FA-RBC-UCNPs exhibited similar FRs binding efficiencies in PBS (Figure S15b), suggesting that the cancer targeting abilities of two nanoparticles are essentially equivalent in PBS. To evaluate the nanoparticle targeting in vitro, FR-overexpressing MCF-7 human breast carcinoma cells were used. We first performed a cell viability assay and found that the potential cytotoxic effects of nanoparticles were negligible (Figure S16), ensuring the fidelity of further in vitro experiments. MCF-7 cells were incubated with equal amounts of various Cy5-labelled nanoparticles, observed by using CLSM under a NIR laser device, and further analyzed by flow cytometry and ICP-AES. The cells treated with FA-UCNPs and FA-RBCUCNPs exhibited bright UCL, and the amount of FA-RBC-UCNPs bound to the cells was significant higher than other nanoparticles (Figure 4d,e and Figure S17). These results indicate that the cancer targeting capability of FA-UCNPs is quite poor when exposed to cell culture medium and can be dramatically enhanced with the use of cell membrane coating. 3.5. In Vivo Tumor UCL Imaging. For tumor UCL imaging with nanoparticles, BALB/c nude mice bearing MCF-7 human breast tumor xenografts were i.v. injected with equal amounts of various nanoparticles. At 48 h after the injection, in vivo tumor imaging was conducted by using a modified small animal imaging system.44 All mice were then euthanized and their tumors were harvested and used for the ex vivo imaging. The mice 13 ACS Paragon Plus Environment


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injected with FA-RBC-UCNPs showed the brightest UCL at the tumor site both in vivo and ex vivo (Figure 5a,b), suggesting good targeting ability of FA-RBC-UCNPs to MCF-7 tumor xenografts. In addition, the nanoparticle contents in the tumor sites, major organs, and blood samples were quantitatively analyzed by ICP-AES. When compared to other nanoparticles, FA-RBC-UCNPs demonstrated higher tumor accumulation (Figure 5c), suggesting that RBC membrane-coated UCNPs retain good tumor targeting capability even in a complex in vivo environment. It should also be noted that RBC-UCNPs had low accumulation in the liver and spleen (Figure 5d), supporting the notion that the nanoparticles acquire the immune evasion capabilities after being coated with RBC membranes.39, 45 It is believed that the interaction between CD47 and signal regulatory protein-alpha (SIRP-α) helps nanoparticles to evade the reticuloendothelial system (RES) uptake.53 3.6. In Vivo Toxicity Evaluation. When antibodies are generated against a person's own RBCs, causing them to lyse, autoimmune hematolytic anemia occurs.54 We used ICR mice to evaluate systematic toxicity of the RBC-mimicking nanoparticles. The mice were i.v. injected with PBS or PBS containing RBC-UCNPs and then euthanized on the 15th d after the injection. The systematic toxicity of RBC-UCNPs was investigated by blood test and histology analysis. The body weight undulation can be translated to an index for assessment of in vivo toxicity of nanoparticles. In the work presented here, neither mice death nor obvious weight difference between the control and treated group was observed over 15 d after the injection (Figure S18), demonstrating that RBC-UCNPs have no significant side effects on mice in general. Three liver function indicators (i.e., AST: aspartate aminotransferase, ALP: alkaline phosphatase, and ALT: alanine aminotransferase) and two kidney function indicators (i.e., CRE: creatinine and BUN: blood urea nitrogen) were also measured and the results suggest no distinct hepatic and renal toxicity was induced by these bio-inspired RBCUCNPs (Figure 6a-c). Complete blood parameters (i.e., RBC: red blood cell, PLT: platelets, 14 ACS Paragon Plus Environment

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WBC: white blood cell, HGB: hemoglobin, MCHC: mean corpuscular hemoglobin concentration, MCH: mean corpuscular hemoglobin, MCV: mean corpuscular volume, and HCT: hematocrit) suggested no noticeable molecule-level toxicity (Figure 6d-k). Furthermore, no obvious organ damage was observed from the hematoxylin and eosin (H&E)-stained sections (Figure 6l). All systematic toxicity results point to good biocompatibility of our biomimetic RBC-UCNPs.

4. CONCLUSIONS In summary, we reconstructed RBC membranes into vesicles and encapsulated them onto the fluorescent UCNPs. By using the resulting RBC-UCNPs, we demonstrated that cell membrane coating could efficiently prevent the protein corona formation around nanoparticles. Next, cancer-targeting RBC-UCNPs were obtained by inserting targeted FA molecules onto the cell membranes and were used to test the targeting efficiency of nanoparticles in biological fluids. Molecular, and cellular level responses demonstrate that, after being coated with cell membranes, the targeting efficiency of nanoparticles in a biological environment was rescued. Next, the RBC membrane-coated fluorescent nanoparticles were employed demonstrating superior tumor UCL imaging. Finally, in vivo toxicity evaluation results demonstrated that no significant systematic toxicity was induced by the RBC-UCNPs injection. The current design uses UCNPs as the core and RBC membranes as the shell of nanoparticles. However, we can easily envision our method for maintaining nanoparticle targeting in biological fluids being used with a wide array of cell membranes and nanoparticles. By applying a cell membrane coating, the nanoparticles acquire not only greater targeting capabilities, but can also inherit other beneficial characteristics. Examples of these traits include immune escape and the ability to absorb pore-forming toxins from erythrocytes,45, 55 communication with endothelial cells from leukocytes,56 homologous cell 15 ACS Paragon Plus Environment


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binding from cancer cells,44, 57 and adhesion to damaged vasculatures from platelets.58 Our method overcomes the problem of unwarranted protein coronas, but also creates a desired "protein corona" in the form of cell membranes, providing benefits such as a stealth coating and a biological identity to nanoparticles.30, 59, 60

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Supplementary experimental details; photos of RBCs and empty RBCs; hydrodynamic size curves and zeta potential distribution of various nanoparticles; XRD spectrum and SEM photo of UCNPs; CLSM photos of various nanoparticles; size stability and optimization of RBC-UCNPs; emission spectrum of various nanoparticles; LC-MS/MS list of proteins on UCNPs and RBC-UCNPs; the contents of FRs that bound to various nanoparticles; cell cytotoxicity; the contents of various nanoparticles that bound to cancer cells; mice body weight change curves (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We would like to thank Mrs. Jinwen Yang and Dr. Yaoyao Ren (Center for Electron Microscopy, Wuhan University) for their kind help in TEM characterization. This work was 16 ACS Paragon Plus Environment

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supported by National Research and Development Program for Major Research Instruments (Grant No. 81527801) and National Natural Science Foundation of China (Grant Nos. 81272443, 61474084, and 51272184).

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Figure 1. Preparation of RBC-UCNPs. (a) Schematic of preparation of RBC-UCNPs. (b) Size intensity and (c) zeta potential of UCNPs, RBC-vesicles and RBC-UCNPs. Error bars: standard deviations (n = 3). TEM photos of (d) uncoated UCNPs and (e,f) RBC-UCNPs. Scale bars: 25 nm. TEM samples were negatively stained with uranyl acetate before characterization. (g) SDS-PAGE protein identification photo of RBC lysate, RBC-vesicles and RBC-UCNPs.



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Figure 2. Evaluation of nanoparticle-protein interactions. Illustrations of certain proteins that (a) bind to UCNPs and (b) do not bind to RBC-UCNPs. Size intensity of (c) UCNPs and (d) RBC-UCNPs before and after being incubated in 100% human plasma for 4 h. (e) Heatmap of the most abundant proteins on UCNPs and RBC-UCNPs before and after being incubated in 100% human plasma, respectively. Only those proteins that constitute at least 0.5% of the total proteins on UCNPs and RBC-UCNPs are shown.


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Figure 3. Preparation of cancer-targeting FA-RBC-UCNPs. (a) Schematic of preparation of FA-RBC-UCNPs. (b) Flow cytometry analysis of unmodified and modified empty RBCs with DSPE-PEG-Cy5. (c) CLSM photo of empty RBCs modified with DSPE-PEG-Cy5. Scale bar: 20 µm. (d) Size intensity, (e) zeta potential, and (f) SDS-PAGE protein identification photo of unmodified and modified RBC-UCNPs with DSPE-PEG-FA. Error bars: standard deviations (n = 3).



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Figure 4. Evaluation of nanoparticle targeting. Illustrations of (a) uncoated UCNPs that are surrounded by protein coronas losing their ability to target cancer cells, and (b) cell membrane-coated nanoparticles retaining its cancer targeting capabilities in a biological environment. (c) Normalized content of FRs that bound to various nanoparticles. Nanoparticles were first treated with or without incubation in 100% human plasma for 4 h. Error bars: standard deviations (n = 4). NS, * and ***: no statistical difference, P < 0.05 and P < 0.001, respectively. (d) CLSM photos and (e) flow cytometry analysis of MCF-7 cancer cells after being incubated with various Cy5-labelled nanoparticles. Cells grown without the addition of any nanoparticles were used as a control. Scale bar: 20 µm.


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Figure 5. In vivo tumor UCL imaging. (a) In vivo UCL photos of the tumor-bearing nude mice at 48 h after being i.v. injected with PBS or PBS containing various nanoparticles. Red circles: tumor sites. (b) Ex vivo UCL photo of tumors from the euthanized mice at 48 h after the injection. Nanoparticle content in the (c) tumor sites, and (d) major organs and blood samples at 48 h after the injection. Error bars: standard deviations (n = 6). ** and ***: P < 0.01 and P < 0.001.



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Figure 6. In vivo toxicity evaluation. Blood biochemistry test: (a) AST, ALP, and ALT, (b) CRE, and (c) BUN. Complete blood panel analysis: (d) RBC, (e) PLT, (f) WBC, (g) HGB, (h) MCHC, (i) MCH, (j) MCV, and (k) HCT. Error bars: standard deviations (n = 6). (l) H&Estained tissue slice photos of major organs from the euthanized mice on the 15th d after being i.v. injected with PBS or PBS containing RBC-UCNPs. Scale bar: 50 µm.


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