Biodegradable zwitterionic polymer membrane coating endowing nanoparticles with ultra-long circulation and enhanced tumor photothermal therapy

Journal Pre-proof Biodegradable zwitterionic polymer membrane coating endowing nanoparticles with ultra-long circulation and enhanced tumor photothermal therapy Shaojun Peng, Boshu Ouyang, Yongzhi Men, Yang Du, Yongbin Cao, Ruihong Xie, Zhiqing Pang, Shun Shen, Wuli Yang PII:

S0142-9612(19)30779-3

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119680

Reference:

JBMT 119680

To appear in:

Biomaterials

Received Date: 10 September 2019 Revised Date:

10 December 2019

Accepted Date: 11 December 2019

Please cite this article as: Peng S, Ouyang B, Men Y, Du Y, Cao Y, Xie R, Pang Z, Shen S, Yang W, Biodegradable zwitterionic polymer membrane coating endowing nanoparticles with ultra-long circulation and enhanced tumor photothermal therapy, Biomaterials (2020), doi: https://doi.org/10.1016/ j.biomaterials.2019.119680. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Biodegradable Zwitterionic Polymer Membrane Coating Endowing Nanoparticles with Ultra-Long Circulation and Enhanced Tumor Photothermal Therapy Shaojun Penga, Boshu Ouyangb,c, Yongzhi Mend, Yang Dub,c, Yongbin Caoa, Ruihong Xiea, Zhiqing Pange, *, Shun Shenb, *, Wuli Yanga,*

a

State Key Laboratory of Molecular Engineering of Polymers & Department of

Macromolecular Science, Fudan University, Shanghai, 200433, PR China b

The Institute for Translational Nanomedicine, Shanghai East Hospital, Tongji University

School of Medicine, Shanghai 200120, PR China c

Central Laboratory, First Affiliated Hospital, Dalian Medical University, Dalian 116021, PR

China d

Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai,

200080, PR China e

Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of

Pharmaceutics, School of Pharmacy, Fudan University, Shanghai,201203, PR China

*

Corresponding author

E-mail:

[email protected]

(Z.

Pang),

[email protected] (W. Yang).

1

[email protected]

(S.

Shen)

and

ABSTRACT Long blood circulation is the basic requirement of advanced drug delivery systems for tumor treatment, which leads to enhanced tumor therapeutic efficiency and reduced side effects. However, the pharmacokinetics of the current nanoparticles in vivo is still unsatisfactory, which leads to limited success to translate nanoparticles into clinical applications. Inspired by the natural cell membrane-coating strategy, a series of zwitterionic polymer membranes are firstly developed and coated onto different kinds of nanoparticles in this work. Intriguingly, the zwitterionic polymer membrane shows stronger protein adsorption resistance and reduced macrophage uptake compared with the corresponding zwitterionic polymer brush or the red blood cell (RBC) membrane, which results in longer blood circulation time and higher tumor accumulation of the coated nanoparticles. Combined with the photothermal effect of model nanoparticles, Fe3O4, zwitterionic polymer membrane-coated Fe3O4 shows enhanced photothermal therapy (PTT) efficacy on A549 tumors compared with the corresponding zwitterionic polymer brush or RBC membrane-coated Fe3O4. Notably, Fe3O4 coated by carboxybetaine-based biomimic membranes exhibits the ultra-long blood circulation (t1/2=96.0 h) and strongest PTT efficacy compared with those coated by phosphorylcholinebased or sulfobetaine-based biomimic membranes. In addition, the zwitterionic biomimic membrane exhibits rapid glutathione-triggered degradation with the products of low molecular weight (<2000 g mol-1). Therefore, the biodegradable zwitterionic biomimic membrane coating offers an universal platform for the design and application of longcirculating biomedical nanoparticles, which may pave the way for the clinical applications of biomedical nanoparticles in tumor therapy. Keywords: zwitterionic polymer membrane, long circulation, drug delivery, protein adsorption, photothermal therapy

2

1. Introduction Biomedical nanoparticles have made impressive progress in the field of cancer diagnosis and therapy during the past two decades [1,2]. Owing to the enhanced permeation and retention (EPR) effect, nanoparticles prefer to accumulate in tumor tissues, which achieves better therapeutic efficacy and reduced side effects compared with free drugs [3]. Nevertheless, the pharmacokinetics of nanoparticles in vivo is still unsatisfactory, which leads to limited success to translate nanoparticles into clinical applications [4]. One of the important reasons is that the body evolves the sophisticated immune system to recognize the non-self materials as invaders and eliminate them by the reticuloendothelial system (RES) quickly [5,6].Therefore, the delivery efficiency of nanoparticles to target sites is relatively low with a large part of them cleared by the RES in blood circulation [7,8]. Hence, it is desirable to develop facile and suitable coatings to endow nanoparticles with stealthy property to escape the monitor of immune system and clearance by RES, thereby reaching the desired target sites ultimately. In recent years, cell membrane coating has provided a novel top-down approach for nanoparticle camouflage, which endows nanoparticles with surface antigenic diversity and biological benefits [9-14]. Especially, red blood cell (RBC) membranes with abundant selfmarkers, such as CD47 proteins, peptides, glycans, acidic sialyl moieties, can help the nanoparticles escape the recognition by immune systems [15]. Therefore, RBC membrane coating strategy has been proved as a powerful method to offer the inner nanoparticles extended blood circulation time, superior biocompatibility, and reduced immune response [16,17]. For instance, Zhang et al. have made a series of innovative work to demonstrate that the RBC membrane coating can greatly prolong the blood half-life of polymer nanoparticles from several hours to approximately 40 h [18]. Sun et al. have proved that RBC membranecoated Fe3O4 showed longer blood circulation time and negligible accelerated blood clearance compared with bare Fe3O4 [19]. Our group also uncovered that RBC membrane-coated 3

melanin or polypyrrole nanoparticles showed extended blood circulation time and higher tumor accumulation, which leaded to enhanced photothermal therapeutic effects [20,21]. Despite great progress in the RBC membrane coating strategy, the necessity to match the blood type and difficulty in large-scale production may be hurdles to translate this advanced strategy into clinical applications. Zwitterionic polymers which contain equal cationic and anionic groups have aroused increasing attention in biomedical field [22-24]. Due to the strong electrostatically induced hydration and neutral charge, zwitterionic polymers exhibit outstanding resistance to nonspecific protein adsorption [25,26]. Nanoparticles coated by zwitterionic polymers show superior biocompatibility, prolonged blood circulation time, and negligible immune response [27-29]. Therefore, many types of zwitterionic polymers have been developed and explored in biomedical fields, among which phosphorylcholine, carboxybetaine, and sulfobetaine polymers are the most widely used [30-32]. For instance, Ishihara et al. have demonstrated that nanoparticles covered with phosphorylcholine polymers were proved effective in preventing protein adsorption at the surface [33]. Jiang et al. revealed that carboxybetaine polymers showed superior blood circulation time and lower immune response than polyethylene glycol (PEG) [34]. Our group also found that sulfobetaine-based nanogels exhibited longer blood circulation time and higher tumor accumulation than PEGylated nanogels, thereby achieving better tumor therapeutic effects [35]. Therefore, zwitterionic polymers may be ideal coating to endow nanoparticles with long blood circulation time and reduced immune response. However, the current zwitterionic polymer coatings are mainly concentrated on linear zwitterionic polymer chains just like the polymer brushes, which may be not as dense as cell membranes to mask nanoparticle surface completely and conceal all “danger signals” from inner nanoparticles sufficiently. Therefore, developing a zwitterionic polymer membrane coating strategy which mimics cell membrane coating may not only endow the inner core with superior stealthy ability compared with the zwitterionic polymer 4

brush coating but also bridge the gap between the zwitterionic polymer coating and the cell membrane coating.

Fig. 1. Schematic showing the fabrication, protein adsorption resistance, and tumor delivery of the zwitterionic polymer membrane, zwitterionic polymer brush or red blood cell (RBC) membrane-coated nanoparticles. (a) Phosphorylcholine, sulfobetaine or carboxybetaine polymers are coated onto nanoparticles in the forms of zwitterionic polymer membranes or zwitterionic polymer brushes; RBC membranes are also coated onto nanoparticles. (b) The protein adsorption resistance behavior of bare nanoparticles or nanoparticles coated by the zwitterionic polymer membrane, zwitterionic polymer brush, and RBC membrane, respectively. (c) The blood circulation, tumor tissue accumulation, and photothermal therapy of nanoparticles coated by the zwitterionic polymer membrane, zwitterionic polymer brush or RBC membrane. MPC, 2-methacryloyloxyethyl phosphorylcholine; SBMA, sulfobetaine methacrylate; CBMA, carboxybetaine methacrylate; NP, nanoparticle.

5

In this work, three kinds of zwitterionic polymer membranes similar to cell membranes are facilely coated onto nanoparticles (Fig. 1a). The zwitterionic polymer membrane-coated nanoparticles show superior protein adsorption resistance compared with zwitterionic polymer brush- or RBC membrane-coated nanoparticles, which leads to reduced macrophage uptake, longer blood circulation time, and higher tumor accumulation of nanoparticles (Fig. 1b and c). Using Fe3O4 nanoparticles with inherent photothermal conversion ability as the model nanoparticles, zwitterionic polymer membrane-coated Fe3O4 shows a higher tumor temperature and stronger PTT efficacy in vivo than the corresponding zwitterionic polymer brush or RBC membrane-coated Fe3O4 [36]. Moreover, nanoparticles coated by the carboxybetaine-based polymer membrane exhibits the longest blood circulation time among three typical zwitterionic polymer membranes, which leads to superior PTT therapeutic efficacy. Lastly, the zwitterionic polymer membrane displays prominent degradability in reduction environment due to the incorporation of crosslinkers containing disulfide-bonds, which could reduce the accumulation risk of zwitterionic polymers in human body [37]. Thus, our work offers a favorable biomimic membrane-coating strategy endowing nanoparticles with ultra-long blood circulation time, which could promote the application of biomedical nanoparticles in vivo. 2. Materials and methods 2.1.Materials 2-methacryloyloxyethyl phosphorylcholine (MPC) was purchased from Joy-Nature company in Nanjing. Sulfobetaine methacrylate (SBMA), bis(acryloyl)cystamine (BAC), 3methacryloxypropyltrimethoxysilane

(MPS),

3-aminopropyltriethoxysilane

(APS),

2-

bromoisobutyryl bromide (BB), triethylamine, cuprous bromide, 2,2'-bipyridine and glutathione (GSH), 1-Ethyl-3-[3-dimethylaminopropyl]-1 carbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide (NHS) were purchased from Alfa Aesar. Sodium citrate 6

dehydrate, ferric chloride hexahydrate, sodium acetate, ethylene glycol, tetraethoxysilane, 3hydroxytyramine hydrochloride, aqueous ammonia solution (28-30%), zinc nitrate hexahydrate,

dimethylmidazole,

methacrylate

(DMAEMA),

tert-Butyl

trifluoroacetic

bromoacetate, acid

(TFA),

2-(dimethylamino)ethyl triethyl

silane,

2,2-

azobisisobutyronitrile (AIBN), hydroquinone monomethyl ether (MEHQ), acetonitrile, dichloromethane (DCM), diethyl ether, dimethyl formamide (DMF), dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich. Human lung adenocarcinoma cell line (A549 cells), macrophage cell RAW264.7 were purchased from Shanghai Meixuan Biotechnology CO., Ltd. (China). Sulfo-Cyanine5 amine (Cy5-NH2) was purchased from Shanghai Suofei Biotechnology CO., Ltd. (China). All the cell culture-related chemicals were from Aladdin reagent. All the chemicals were used as received unless otherwise mentioned. 2.2. Synthesis of nanoparticles and monomers Fe3O4 nanoparticles are prepared by the solvothermal reaction reported previously [38]. The obtained Fe3O4 nanoparticles are dispersed in 10 mL of ethanol for the further use. SiO2 nanoparticles are prepared by the method reported previously [39]. The as-prepared SiO2 nanospheres are acquired by centrifugation and washed three times with deionized water. Polydopamine (PDA) nanospheres are prepared by the oxidation and self-polymerization of dopamine [40]. The as-prepared PDA nanospheres are dispersed in 10 mL of water for the further use. Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles are prepared by the method reported previously [41]. The as-prepared ZIF-8 nanoparticles are dispersed in 10 mL of methanol for the further use. The carboxybetaine methacrylate (CBMA) monomer is prepared by the method reported previously [42]. The white powder is collected by suction filtration and washed with diethyl ether for several times. Finally, the CBMA is obtained by vacuum drying at 25 oC. 2.3.Coating Fe3O4 nanoparticles with zwitterionic polymer brush 7

Briefly, 200 mg of Fe3O4 is mixed with 1.5 mL of aqueous ammonia solution (28-30%), 10 mL of deionized water and 30 mL of ethanol. The mixture is vigorously stirred at 60 oC for 30 min and 300 mg of APS is slowly injected into the above mixtures and the stirring is continued at 60 oC for 12 h. Finally, the APS-modified Fe3O4 (Fe3O4-APS) is acquired by centrifugation and washed three times with ethanol and freeze-dried for the further use. 100 mg of Fe3O4-APS is dispersed into 5 mL of anhydrous DMF and 185.5 µL of triethylamine is added with vigorous stirring at 4 oC. Then, 82.3 µL of 2-bromoisobutyryl bromide is dropwise added into the above mixture. The reaction is continued for 2 h at 4 oC and another 12 h at room temperature. Finally, the 2-bromoisobutyryl bromide-modified Fe3O4 (Fe3O4-APS-BB) nanoparticles are separated and washed with deionized water for several times and freeze-dried for further use. 100 mg of Fe3O4-APS-BB, 1.1 g of monomer, namely MPC, SBMA or carboxybetaine-1methacrylate tert-butyl ester (CBMA-1-tBu) synthesized according to the previous literature [42] and 5.8 mg of 2,2'-bipyridine are dispersed into 10 mL of anhydrous DMSO and the mixture is vigorously stirred at 90 oC for 30 min in N2 atmosphere. Then, 2.7 mg of cuprous bromide in 20 µL of anhydrous DMSO is injected into the above solution and the reaction is continued for 12 h. Finally, the poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) or poly(sulfobetaine methacrylate) (PSBMA) brush-modified Fe3O4 (Fe3O4-PMPC, Fe3O4PSBMA) are separated and washed with deionized water for several times and dispersed in 10 mL of ethanol for the further use. The obtained poly(carboxybetaine-1-methacrylate tert-butyl ester) (PCBMA-1-tBu) brush-modified Fe3O4 is dispersed into DCM, and TFA and triethyl silane are added to remove the tert-butyl ester. Finally, poly(carboxybetaine methacrylate) (PCBMA) brush-modified Fe3O4 (Fe3O4-PCBMA) is separated and washed with deionized water for several times and dispersed in 10 mL of water for the further use. 2.4. Coating Fe3O4 nanoparticles with RBC membrane 8

RBC membrane-derived vesicles are prepared by following the published protocol [18]. 200 µL RBC membrane-derived vesicles are mixed with 800 µL of Fe3O4 dispersion at a final Fe concentration of 0.1 mg mL-1 and sonicated for 30 s (53 kHz, 100 W). The mixture is then extruded 20 times through 200 nm porous membranes using the Avanti mini extruder. Excess vesicles are removed by centrifuging at 3500 rpm for 5 min at 4 oC, and the resulting Fe3O4@RBC is collected and redispersed for the future use. 2.5.Coating nanoparticles with zwitterionic polymer membranes and fluorescent dye modification. Briefly, 200 mg of nanoparticles (Fe3O4, SiO2, PDA, and ZIF-8, respectively) are mixed with 1.5 mL of aqueous ammonia solution (28-30%), 10 mL of deionized water and 30 mL of ethanol. The mixture is vigorously stirred at 60 oC for 30 min and 300 mg of MPS is slowly injected into the above mixture and the stirring is continued at 60 oC for 12 h. Finally, the MPS-modified nanoparticles are acquired by centrifugation and washed three times with ethanol and dispersed in 20 mL of acetonitrile for the further use. Briefly, 25 mg of MPS-modified nanoparticles are mixed with 4 mg of AIBN, 5 mg of BAC, 95 mg of zwitterionic monomers (MPC, SBMA and CBMA, respectively), and 40 mL of acetonitrile. The mixture is ultrasound for 5 min and vigorously stirred at 100 oC for 1 h under the atmosphere of N2. Finally, the zwitterionic polymer membrane-coated nanoparticles (Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA) are acquired by centrifugation, washed three times with water, and dispersed in 10 mL of water for the further use. For fluorescent dye modification, 25 mg of Fe3O4@PCBMA is dispersed in 10 mL of deionized water. Then the dispersion is added with 30 mg of EDC and 5 mg of NHS, stirred at 25 oC for 0.5 h, and collected by centifugation. After wash two times with deionized water, Fe3O4@PCBMA is re-dispersed in 10 mL of deionized water, followed by adding 1 mg of

9

Cy5-NH2 and stirring for 3 h at 25 oC. The obtained nanoparticles are collected by centifugation and washed three times with deionized water for further use. 2.6.Material characterizations The morphology of the nanoparticles is tested by transmission electron microscope (TEM) performed on a JEOL 1230 instrument. The average size and zeta potential of the nanoparticles are determined by using a dynamic light scattering (DLS) (Malvern Nano-ZS90) at a scattering angle of 90°. The average molecular weight of degraded polymer chains is estimated by using a gel permeation chromatography (GPC) system (HP Agilent series 1100). Fe content is measured using inductively coupled plasma atomic emission spectroscopy (ICPAES) (Z-5000, Hitachi, Japan). Fourier transform infrared (FT-IR) spectra are recorded using KBr-pressed plates on a Nicolet 6700 FTIR spectroscope. The thermogravimetric analysis (TGA) is conducted on a Pyris 1 TGA instrument under an N2 environment at a heating rate of 20 °C min−1. X-ray photoelectron spectra (XPS) measurements are analyzed using an ESCA-Lab-200i-XL spectrometer with monochromatic Al Kα radiation (1486.6 eV). The scanning electron microscopy energy dispersive spectroscopy (SEM-EDS) pattern is obtained with Ultra 55. 808 nm laser (PSU-H-LED, Changchun New Industries Optoelectronics Technology Co., Changchun, China) irradiation at a power density of 3 W cm-2 for different amount of time. The temperature is measured using a near-infrared (NIR) camera (VarioCAM HR, InfraTec, Germany). The magnetic resonance imaging (MRI) capability of the nanoparticles are evaluated using a 7.0 T Bruker Pharmascan animal instrument (Fudan University Shanghai Cancer Center, Shanghai, China). 2.7. Protein adsorption studies Protein adsorption is quantified using the following method. Phosphate buffer saline (PBS) (1

mL,

negative

control),

Fe3O4,

Fe3O4-PMPC,

Fe3O4-PSBMA,

Fe3O4-PCBMA,

Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA, or Fe3O4@RBC (1 mL, 1 mg of Fe per 10

mL) are mixed with 3 mL of mouse whole blood and incubated at 37 °C for 30 min. After incubation, samples are separated by magnetic field and washed 3 times with PBS to remove unabsorbed proteins. Finally, the amount of protein adsorbed is determined by bicinchoninic acid (BCA) assays. The protein amount of Fe3O4@RBC before incubation is also evaluated by BCA assay to eliminate the proteins derived from RBC membranes. 2.8. Macrophage uptake and penetration in tumor spheroids Approximately 2×106 RAW264.7 cells are seeded into 6-well plates in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. After 24 h, cells are washed by warm PBS and incubated with Fe3O4, Fe3O4-PMPC, Fe3O4-PSBMA, Fe3O4-PCBMA, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA, or Fe3O4@RBC at a Fe concentration of 50 µg·mL-1 in the cell culture medium for 1 or 4 h. Then, cells are gently washed with PBS three times to remove free nanoparticles. Cells are digested by trypsin and counted. Then, 0.2 mL of perchloric acid and 0.8 mL of hydrochloric acid are added into each sample to break down cells and dissolve nanoparticles into iron ions. The acid solutions are placed at room temperature for 2 days, heated to 60 oC, and kept at this temperature for another 12 h. The samples are then diluted by deionized water to a certain volume and Fe content is measured by ICP-AES. Cells without nanoparticle treatment are treated with the same procedure in parallel and measured for Fe content as controls. A549 cells are seeded in ultra-low-attchment 96-well plates at a density of 2.0×104 cells per well and incubated in DMEM at 37 °C for five days to form 3D tumor spheroids. Then, Cy5modified Fe3O4-PCBMA with the concentration of 100.0 µg mL−1 are added into the wells and incubated for different times (0.5, 1, 2, 4 h, and 6 h, respectively). Afterward, 3D tumor spheroids were washed by PBS for three times and observed by confocal laser scanning microscopy. 2.9. In vivo pharmacokinetics studies 11

Forty male ICR mice (6-7 week) are obtained from Fudan University Experimental Animal Center (Zhangjiang Campus, Shanghai, China) and are handled under the protocols approved by the ethics committee of Fudan University. Mice are randomly assigned to eight groups (n=5) and were administrated with Fe3O4, Fe3O4-PMPC, Fe3O4-PSBMA, Fe3O4-PCBMA, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA, and Fe3O4@RBC (2.5 mg of Fe per kg) via tail vein injection, respectively. At different time points (0.02, 0.5, 1, 2, 4, 8,12, 24, 48 h), 50 µL of blood is collected through orbital sinus. The Fe concentration is determined as the above described with ICP-AES. 2.10. In vivo immunogenic response studies The immunogenic response of nanoparticles is explored by measuring the immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in blood from the mice injected with the corresponding nanoparticles. Briefly, male BALB/c mice weighed 20 ± 4 g are randomly divided into nine groups. For the Fe3O4, Fe3O4-PMPC, Fe3O4-PSBMA, Fe3O4-PCBMA, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA and Fe3O4@RBC groups, mice received an injection of nanoparticles at a dose of 2.5 mg/kg Fe, respectively. Mice in the Control group receive an injection of an equal volume of saline. Five days after injection, 1 mL of blood is collected from each mouse through orbital sinus (n = 5 per group) and the obtained serum is used for IgM measurement according to the enzyme-linked immunosorbent assay (ELISA) protocol. Seven days after the first injection, all mice in the nine groups receives the second injection of the nanoparticles (2.5 mg/kg Fe) or an equal volume of saline. Five days after the second injection, 1 mL of blood is collected from each mouse for the IgG measurement (n = 5 per group). 2.11. In vivo biodistribution studies Nude mice bearing A549 xenograft tumors are used to evaluate biodistribution of the obtained nanoparticles. Sixty female nude mice aged 4 weeks are purchased from Fudan 12

University Experimental Animal Center. Approximately 2×105 A549 cells in 100 µL of PBS are injected subcutaneously into each mouse to form tumors. After the tumor volume reached 150~200 mm3, mice are randomly divided into 6 groups, and injected with Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA or Fe3O4-PCBMA (2.5 mg Fe kg-1) respectively via tail vein. At 4 or 24 h post the injection, five mice from each group are sacrificed. The heart, liver, spleen, lung, kidney and tumor from each mouse are collected after myocardial perfusion, and homogenized with PBS. Acid mixture is added into each sample and Fe content in the samples is determined as the above described with ICP-AES. 2.12. In vivo MRI studies Tumor-bearing

mice

injected

with

Fe3O4,

Fe3O4@PMPC,

Fe3O4@PSBMA,

Fe3O4@PCBMA or Fe3O4-PCBMA (2.5 mg/kg Fe) are used to test the MRI capability of asprepared nanomaterials in vivo. Mice are scanned for T2-weighted MR images at different time points before or after the nanoparticle injection by Bruker Pharmascan 7.0 T. 2.13. In virtro and in vivo photothermal therapy To test the biocompatibility and photothermal effect of the nanoparticles in vitro, human lung adenocarcinoma cell line A549 was selected as a representative cell line. The cytotoxicity of all the nanoparticles was measured applying Alamar Blue cell proliferation kit (KeyGEN bioTECH, Nanjing, China). Briefly, 2×104 A549 cells were seeded in black 96well plates in Dulbecco's modified Eagle's medium (DMEM) with 10 % fetal bovine serum and 1 % penicillin and streptomycin. After cultured with 5 % CO2 at 37 oC overnight, cells were incubated with all of the nanoparticles at different indicated Fe concentrations for 48 h. Then, Alamar Blue reagent was added into each well, incubated for 5 h. Afterward, the 96well plates were directly measured for fluorescence intensity at the emission wavelength of 590 nm with the excitation wavelength of 530 nm using a Synergy 2 microplate reader (Biotek Instruments Inc., USA). The photothermal effect of all the nanoparticles on A549 13

cells was explored by the same Alamar Blue kit mentioned above. Briefly, after 2×104 A549 cells were seeded and attached into 96-well plates, all nanoparticles were added into each well to achieve a final Fe concentration of 100 µg mL-1 and were incubated with the cells for one hour. Subsequently, an 808 nm laser was applied to each single well at 3 W cm-2 for varying amount of time. After the irradiation by the 808 nm laser, cells were incubated for another 12 h and relative cell viabilities were measured by Alamar Blue cell proliferation kit. To directly visualize the ablating and killing capacity of nanoparticles, A549 cells after 808 nm laser irradiation were co-stained with Calcein-AM and PI accordingly and were observed via a laser scanning fluorescence confocal microscopy (Carl Zeiss LSM710, Zeiss, Germany). A549 xenograft-bearing nude mice are used as animal models for in vivo PTT treatment. Thirty-five female mice are subcutaneously injected with A549 cells to generate tumor as described above. After the tumor volume reaches approximately ~100 mm3, mice are randomly divided into 7 groups (n=5), and are i.v. injected with PBS, Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA and Fe3O4-PCBMA (2.5 mg Fe kg-1), respectivey. 4 h after the administration, mice are anesthetized with 5% chloral hydrate and their tumors are irradiated using an 808-nm laser for 5 min (3 W cm-2). The spot size of the laser is 5 mm × 8 mm which is suitable to cover the whole tumor regions. Meanwhile, the local temperature of the tumor area under laser irradiation is recorded using the NIR camera. After the PTT treatment, the tumor size and mouse body weight are measured every other day. All mice are euthanized, and tumors are harvested and weighed at day 14. The terminaldeoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay is performed to detect apoptotic cells in the tumor slices. 2.14. H&E-stained histology

14

For the hematoxylin and eosin (H&E)-stained histological evaluation, major organs (heart, liver, spleen, lung and kidney) and tumors are harvested 14 days after PTT treatment, fixed in a 4% polyoxymethylene solution, and then embedded in paraffin for the H&E staining. 2.15. Statistical analysis The data were showed as mean ± standard deviation. The differences within groups and between groups were applied by one-way ANOVA with Fisher's LSD and unpaired student's t tests, respectively. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Fabrication of zwitterionic polymer membrane-coated nanoparticles Four typical nanoparticles including Fe3O4, SiO2, polydopamine (PDA), and ZIF-8 (one of the mental-organic frameworks) are selected for the poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) membrane-coating. In addition, poly(sulfobetaine methacrylate) (PSBMA) membranes and poly(carboxybetaine methacrylate) (PCBMA) membranes are also coated onto Fe3O4. Carboxybetaine methacrylate (CBMA) is synthesized based on the previous report (Fig. S1) [29]. Carbon-carbon double bonds are firstly modified to the nanoparticles, which is proved by fourier transform infrared spectroscopy (FTIR) and showed by transmission electron microscope (TEM) images after the modification (Fig S2 and S3). Next, zwitterionic polymer membranes are coated onto the nanoparticles by reflux precipitation polymerization with the advantages of surfactant-free and time-saving [43]. Fig. 2a-d shows the clear morphology of the bare nanoparticles with the particle size ranging from 80 to 120 nm. After the zwitterionic polymer membrane coating, contiguous membranes appear on the surface of the composite nanoparticles, generating the obvious core-shell structure, which are denoted as Fe3O4@PMPC, SiO2@PMPC, PDA@PMPC, ZIF-8@PMPC, Fe3O4@PSBMA, and Fe3O4@PCBMA, respectively (Fig. 2e-j). The thickness of the coated zwitterionic polymer membranes varies from 5 to 15 nm according to the coated nanoparticles, 15

which is close to that of RBC membrane with the lipid bilayer [44]. Furthermore, the thickness of PMPC, PSBMA and PCBMA membranes on Fe3O4 are similar, which is conductive to compare the property difference among zwitterionic polymer membranes. Meanwhile, the hydrodynamic size of zwitterionic polymer membrane-coated nanoparticles increases by 20 to 50 nm compared with non-coated nanoparticles, which indicates zwitterionic polymer membranes are highly swollen in the aqueous medium (Fig. 2k). Furthermore, the zeta potential of all nanoparticles dropped to nearly zero after polymer membrane coating due to the equal positive and negative charges of zwitterionic polymers (Fig. 2l). X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy energy dispersive spectroscopy (SEM-EDS) also jointly prove the successful coating of zwitterionic polymer membrane onto the nanoparticles (Fig. S3-S7). Therefore, it could be concluded that not only the zwitterionic polymer membrane could coat a variety of nanoparticles (Fe3O4, SiO2, PDA and ZIF-8) but also the nanoparticles could be coated by different types of zwitterionic polymers (PMPC, PSBMA and PCBMA), which exhibits great universality and versatility as a biomimic membrane coating platform.

16

Fig 2. Morphology and colloidal properties of the nanoparticles. TEM images of (a) Fe3O4, (b) SiO2, (c) PDA, (d) ZIF-8, (e) Fe3O4@PMPC, (f) SiO2@PMPC, (g) PDA@PMPC, (h) ZIF8@PMPC, (i) Fe3O4@PSBMA, and (j) Fe3O4@PCBMA (Scale bar: 100 nm), amplified images of the nanoparticles are listed at the top right corner (Scale bar: 30 nm). Hydrodynamic size (k) and zeta potential (l) of the nanoparticles. The biodegradability of the zwitterionic polymer membrane is investigated by incubating the zwitterionic polymer membrane-coated nanoparticles in a reduction environment. Glutathione (GSH), which is ubiquitous in mammal, is known to be highly expressed in tumor cells with its concentration ranging from 2 to 10 mM [45]. Therefore, zwitterionic polymer membrane-coated Fe3O4 is incubated in 10 mM GSH and it displays that the hydrodynamic sizes of Fe3O4@PMPC, Fe3O4@PSBMA or Fe3O4@PCBMA all decrease significantly after incubation for 30 min (Fig. 3a), and meanwhile the zeta potentials change from nearly neutral to negatively charged (Fig. 3b). TEM images show that the zwitterionic polymer membranes on Fe3O4 disappear completely after incubation for 1 h (Fig. 3c-e). These results reveal that 17

zwitterionic polymer membranes on Fe3O4 could be successfully disintegrated owing to the breakage of disulfide bonds in the reduction environment. Notably, the molecular weights of the degraded zwitterionic polymer membranes are relatively low (<2000 g mol-1) with a narrow molecular weight polydispersity index (Fig. 3f-h). The products of the degraded zwitterionic membranes with such a low molecular weight could be eliminated by kidney easily, thereby reducing the accumulation risk of zwitterionic polymers in the body [46].

Fig. 3. The biodegradability of zwitterionic polymer membranes. The hydrodynamic size (a) and zeta potential (b) change of Fe3O4@PMPC, Fe3O4@PSBMA and Fe3O4@PCBMA; TEM photograph of Fe3O4@PMPC (c), Fe3O4@PSBMA (d) and Fe3O4@PCBMA (e); Molecular weights of the degraded products of Fe3O4@PMPC (f),

Fe3O4@PSBMA (g) and

Fe3O4@PCBMA (h) after incubation in 10 mM GSH for 1 h. The scale bar was 100 nm. Different structures of polymers on nanoparticles display different properties, and nanoparticles with dense and compact hydrophilic polymer shell show enhanced colloid 18

stability and bio-inertness in the physiological medium [47,48]. Given that the polymer membranes may exhibit more compact structure than polymer brushes, we wonder whether zwitterionic polymer membrane-coated nanoparticles could bring superior bio-inert property in vitro and in vivo compared with zwitterionic polymer brush-coated analogues. To demonstrate this, we prepare zwitterionic polymer brush-coated Fe3O4 (named as Fe3O4PMPC, Fe3O4-PSBMA, and Fe3O4-PCBMA) by grafting well-controlled linear zwitterionic polymers onto Fe3O4 with surface initiated atom transfer radical polymerization [49]. The zwitterionic polymer brush-coated Fe3O4 nanoparticles are controlled with the hydrodynamic size and zeta potential similar to those of the zwitterionic polymer membrane-coated Fe3O4, thereby avoiding the possible influence of hydrodynamic size and zeta potential on biological behaviors of the nanoparticles (Fig. S11a-b, S13a-b and S15a-b). TEM images reveal that the zwitterionic polymer brush-coated Fe3O4 nanoparticles do not exhibit obvious core-shell structure, which might be attributed to the collapse of the soft zwitterionic polymers in dehydrated state (Fig. S11c, S13c and S15c). Furthermore, the molecular weights of the grafted zwitterionic polymer chains are measured by dissolving the inner Fe3O4 in an acid environment, which are 39000, 42000, and 37000 g mol-1 for phosphorylcholine-based, sulfobetaine-based, and carboxybetaine-based polymer brushes, respectively (Fig. S11d, S13d and S15d). In addition, FTIR, XPS, and SEM-EDS also demonstrate that zwitterionic polymer brushes are successfully coated onto Fe3O4 (Fig. S11e-f, S12, S13e-f, S14, S15e-f and S16). To make a better comparison between zwitterionic polymer membranes and RBC membranes, RBC membrane-coated Fe3O4 nanoparticles (Fe3O4@RBC) are also prepared according to the previous work [50]. The TEM image shows that the contiguous outer membrane shell is coated onto the rough surface of Fe3O4, generating the final Fe3O4@RBC with a slightly increased size (Fig. S17a). In consistency, DLS results demonstrate that RBC membrane-coating onto Fe3O4 leads to an increase in the hydrodynamic size from 181 to 215 nm and a sharp increase in the zeta potential from -40.5 to -12.4 mV (Fig. S17b and S17c). 19

Moreover, ultraviolet and visible (UV-vis) absorption spectra also display that Fe3O4@RBC obtains an absorption peak around 400 nm, which is identical with the characteristic absorption peak of RBC membranes but absent in the curve of the bare Fe3O4 (Fig. S17d) [51,52]. The above results demonstrate the successful coating of RBC membranes onto Fe3O4. 3.2. Biological behaviors of the zwitterionic polymer membrane-coated Fe3O4 Before exploring the biological behaviors of the coated nanoparticles, thermogravimetric analysis is applied to investigate the density difference between zwitterionic polymer membranes and zwitterionic polymer brushes. It shows that the weight loss of zwitterionic polymer membrane-coated Fe3O4 are significantly higher than that of zwitterionic polymer brush-coated Fe3O4 (Fig. S18). For instance, the weight ratio of PCBMA membranes on Fe3O4@PCBMA is 2.3-fold higher than PCBMA brushes on Fe3O4-PCBMA (Fig. 4a). It suggests that zwitterionic polymer membranes on nanoparticles exhibit denser and more compact state than zwitterionic polymer brushes on nanoparticles with similar hydrodynamic size and zeta potential. Next, the protein adsorption resistance of the bare Fe3O4 and coated Fe3O4 are performed in the mouse blood. It shows that bare Fe3O4 adsorbs proteins significantly, whereas zwitterionic polymer membranes, zwitterionic polymer brushes or RBC membranes on Fe3O4 greatly suppress the protein adsorption. Furthermore, zwitterionic polymer membrane-coated Fe3O4 displays superior protein adsorption resistance compared with the corresponding zwitterionic polymer brush-coated Fe3O4 (e.g. Fe3O4@PMPC to Fe3O4-PMPC) (Fig. 4b). With denser and more compact structure, zwitterionic polymer membranes might generate enhanced steric effect compared with zwitterionic polymer brushes and prevent proteins from overcoming the osmotic barriers and physically interacting with Fe3O4 cores. In addition, carboxybetainebased polymer membranes (Fe3O4@PCBMA) exhibit the lowest protein adsorption among the three zwitterionic polymer membranes system, due to the less hydrophobic methylene 20

groups between the anion group and cation group in the carboxybetaine group compared with the phosphorylcholine and sulfobetaine groups [53]. Intriguingly, PCBMA membranes also show reduced protein adsorption compared with RBC membranes, which indicates the stronger anti-fouling ability of PCBMA membranes than RBC membranes.

Fig. 4. The density and biological behaviors of zwitterionic polymer membrane-coated Fe3O4 nanoparticles. (a) The weight ratio of zwitterionic polymer membranes and zwitterionic polymer brushes on Fe3O4. (b) Protein adsorption of the nanoparticles after incubation in the mouse blood for 30 min. (c) Cellular uptake of the nanoparticles by RAW264.7 macrophages after co-incubation for 1 h and 4 h. (d) Pharmacokinetics of the nanoparticles after intravenous injection. (e) Blood circulation half-life of the nanoparticles. (f) Blood retention of the nanoparticles 24 h after intravenous injection. (g) IgM level at the fifth day after the first intravenous injection of nanoparticles and (h) IgG levels at the fifth day after the second 21

intravenous injection of nanoparticles. *p< 0.1, **p < 0.01, ***p < 0.001, and n.s. represents no significance. Data are means ± s.d. N=5. The adsorption of plasma proteins often leads to the opsonization of nanoparticles, which induces the accelerated recognition, phagocytosis, and clearance by RES [54]. To explore the interactions of RES with bare Fe3O4 and the coated Fe3O4, RAW 264.7 murine macrophage cells are selected to explore the anti-phagocytosis capacity of the nanoparticles. As shown in Fig. 4c, the zwitterionic polymer membrane, zwitterionic polymer brush or RBC membranecoated Fe3O4 all exhibit much lower uptake by RAW 264.7 compared with bare Fe3O4, which indicates the anti-phagocytosis of zwitterionic polymers and RBC membranes. Furthermore, the macrophage uptake of Fe3O4 coated by zwitterionic polymer membranes is significantly lower than that of Fe3O4 coated by the corresponding zwitterionic polymer brushes. For example, the macrophage uptake of Fe3O4-PCBMA is 2.0-fold higher than Fe3O4@PCBMA after incubation for 4 h. The macrophage uptake of Fe3O4@PCBMA is also significantly lower than that of Fe3O4@PMPC or Fe3O4@PSBMA, which agrees well with the protein adsorption results. In addition, PCBMA membrane-coated Fe3O4 displays lower macrophage uptake compared with RBC membrane-coated Fe3O4, demonstrating the superior antiphagocytosis ability of PCBMA membranes. The outstanding protein adsorption resistance and anti-phagocytosis ability encourage us to further explore the pharmacokinetics behaviors of polymer membrane-coated nanoparticles. Fig. 4d-f shows that the bare Fe3O4 is rapidly cleared from the blood circulation after intravenous injection with the elimination half-life (t1/2) of 8.9 h. The blood circulation time of the coated Fe3O4 is significantly extended compared with that of the bare Fe3O4 (Table S1). It is found that the zwitterionic polymer membrane-coated Fe3O4 displays long blood circulation superior to the corresponding zwitterionic polymer brush-coated Fe3O4. For instance, the t1/2 of Fe3O4@PCBMA is 96.0 h which is longer than Fe3O4-PCBMA (t1/2=74.9 h) and the blood retention of Fe3O4@PCBMA is 1.4-fold than Fe3O4-PCBMA at 24 h post intravenous 22

injection.

Furthermore,

Fe3O4@PCBMA

displays

longer

blood

circulation

than

Fe3O4@PSBMA and Fe3O4@PMPC, which is consistent well with the protein adsorption and macrophage uptake results. In addition, Fe3O4@PCBMA shows 2.6-fold blood retention than Fe3O4@RBC at 24 h post intravenous injection, which indicates the advantages of zwitterionic polymer membranes as a long-circulating biomimic membrane coating. The immune responses of the bare Fe3O4 and coated Fe3O4 are explored by detecting the serum immunoglobulin M (IgM) level 5 days after the first nanoparticle injection and the serum immunoglobulin G (IgG) level 5 days after the second nanoparticle injection (Fig. 4gh). The results show that the bare Fe3O4 significantly induces the rise of both IgM and IgG levels in mice, whereas Fe3O4 coated by zwitterionic polymer membranes, zwitterionic polymer brushes or RBC membranes give rise to negligible immune response. Therefore, the zwitterionic polymer membrane, zwitterionic polymer brush or RBC membrane all show preferable immunocompatibility, which is beneficial for the in vivo applications. 3.3 Tumor accumulation and phothermal properties of the zwitterionic polymer membranecoated Fe3O4. Although nanoparticles prefer to accumulate in tumor tissue because of the EPR effect, the tumor accumulation of most biomedical nanoparticles still remains unsatisfactory due to the undesirable blood circulation. Encouraged by the superior blood circulation of zwitterionic polymer membrane-coated Fe3O4, we further explore the tumor imaging and biodistribution of these nanoparticles. Firstly, in order to explore the magnetic resonance imaging (MRI) functionality of the coated nanoparticles and determine the best time window for the following PTT, an in vivo T2-weighted MRI study is performed on mouse models. Nude mice bearing A549 xenograft tumors are intravenously injected with bare Fe3O4 or the coated Fe3O4 nanoparticles and are imaged at different time points post nanoparticle injection. It shows that significant darkening effects are observed for all mice after nanoparticle injection, which 23

indicates the imaging abilities of the nanoparticles (Fig. 5a). In the meantime, darker tumor areas and longer lasting of the contrast signals could be observed in tumor regions of mice injected with Fe3O4@PCBMA compared with other groups. The lowest intensity ratio is observed at 4 h post nanoparticle injection among all the images, which suggests the most abundant tumor accumulation of nanoparticles is achieved at 4 h post nanoparticle injection (Fig. S19). Altogether, the in vivo MRI data have well demonstrated that 4 h after nanoparticle injection could be a good timing for laser intervention. Furthermore, the biodistribution of the bare Fe3O4 and coated Fe3O4 is evaluated by detecting the Fe concentration in the heart, liver, spleen, lung, kidney and tumor. It shows that the bare Fe3O4 prefers to accumulate in liver and spleen and only 1.3 %ID/g of Fe3O4 accumulate in tumor tissues 4 h post intravenous injection (Fig. 5b). In contrast, Fe3O4 nanoparticles coated by zwitterionic polymer membranes, zwitterionic polymer brushes or RBC membranes show significantly increased tumor accumulation and reduced nonspecific distribution in normal organs. Notably, Fe3O4@PCBMA shows the tumor accumulation of 13.2 %ID/g which is significantly higher than Fe3O4-PCBMA (9.9 %ID/g). Meanwhile, Fe3O4@PCBMA also shows higher tumor accumulation superior to Fe3O4@PSBMA, Fe3O4@PMPC or Fe3O4@RBC, which could be attributed to the longer blood circulation of nanoparticles endowed by PCBMA membranes than that endowed by other membranes. Furthermore, Fe3O4@PCBMA remains high tumor accumulation at 24 h post intravenous injection, indicating that nanoparticles with prolonged blood circulation could accumulate in tumor tissues for an extended time (Fig. 5c). To further explore the penetration ability of PCBMA membrane-coated Fe3O4 in tumor tissues, A549 tumor spheroids are cultured with Cy5modified Fe3O4@PCBMA. It is found that the fluorescence intensity of tumor spheroids increases with the incubation time and the whole tumor spheroid is full of red fluorescence signals after incubation for 6 h, demonstrating the favorable tumor penetration capability of Fe3O4@PCBMA (Fig. S20). 24

Fig. 5. The MR imaging and biodistribution of the nanoparticles. (a) T2-weighted MR images of A549 tumor-bearing mice injected with the bare Fe3O4 and coated Fe3O4 nanoparticles (2.5 mg Fe kg-1) taken at different time points after intravenous injection. White circles highlight the tumor site. The biodistribution of the bare Fe3O4 and coated Fe3O4 nanoparticles at 4 h (b) and 24 h (c) after intravenous injection. *p< 0.1, **p < 0.01, ***p < 0.001, and n.s. representing no significance. Data are means ±s.d. N=5. As a benign photothermal conversion reagent with biocompatibility, Fe3O4 has been applied for MRI-guided PTT, which is extensively studied in recent years [38,50,55]. UV-vis adsorption spectra show that the absorbance of all nanoparticles at 808 nm are similar and the temperature of all nanoparticle dispersions could rise from 25 oC to around 60 oC irradiated by a near-infrared (NIR) laser, which indicates that the photothermal conversion ability of Fe3O4 is not impaired after different coatings (Fig. S21a and S21b ). Photothermal effect of all nanoparticles for cell ablation is further confirmed by CLSM. Calcein-AM is used to stain live cells whereas propidium iodide (PI) is used to stain dead cells. FCM images reveals that A549 cells incubated with all kinds of nanoparticles show strong red fluorescence after laser treatment, which indicates that severe cell death is induced by the photothermal effect of nanoparticles (Fig. 6a). Furthermore, the cell viability experiments reveal the superior biocompatibility of different Fe3O4, whereas A549 cells could be effectively killed after NIR irradiation for 5 min without significant differences among all groups (Fig. 6b and 6c). 25

Therefore, the photothermal effects of all nanoparticles in vitro remain identical, which is beneficial to compare the influence of different coatings on the PTT effects of the coated Fe3O4 in vivo.

Fig 6. In vitro photothermal effect of the nanoparticles. (a) Fluorescence confocal microscopy images of A549 cells stained with PI and Calcein-AM after incubation with PBS and Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA and Fe3O4-PCBMA after NIR laser irradiation (Incubation time=4 h, 808 nm, irradiation time=300 s). Red channel images were obtained from PI (λex/λem, 535/617 nm) while green channel images were obtained from Calcein-AM (λex/λem, 495/515 nm). (b) Cell viability of A549 cells incubated with Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA and Fe3O4PCBMA of various concentrations for 48 h, respectively. (c) Cell viabilities of A549 Cells

26

treated with PBS, Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA and Fe3O4-PCBMA (100 µg Fe mL-1) after 808 nm laser irradiation for 0, 1, 2, 3 and 5 min. Inspired by the MRI data and PTT effects of these nanoparticles in vitro, we further explore the influence of different coatings of Fe3O4 on the tumor temperature in vivo. The thermal images of mice from different treatment groups under laser irradiation are shown in Fig. 7. The tumor local temperature rises by 5.4, 8.8, 13.7, 15.3, 21.3, 25.3, and 22.2 oC for the PBS, Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA, and Fe3O4-PCBMA groups, respectively (Fig. S22). Given that the PTT effects of the nanoparticles are similar in vitro, the differences of tumor temperature rise are probably due to the different blood circulation behaviors and tumor accumulations of nanoparticles.

Fig. 7. Infrared thermal images of the bare Fe3O4 and coated Fe3O4 nanoparticles under 808 nm NIR laser irradiation for 300 s with the injection dose of 2.5 mg Fe kg-1. White circles highlight the tumor site. 3.4 PTT efficacy of the zwitterionic polymer membrane-coated Fe3O4.

27

Encouraged by the excellent PTT effects achieved in vitro, PTT experiments in vivo are then performed on A549 tumor models. Seven groups are included: PBS, Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA, and Fe3O4-PCBMA (n = 5 per group). Seven groups of A549-bearing mice are i.v. injected with 0.1 mL of PBS or nanoparticle dispersions (0.5 mg of Fe per mL) when the tumor volume is around 100 mm3. The mice are then irradiated by the 808 nm NIR laser for 300 s at 4 h after injection. The tumor growth curves show that the tumors in the PBS and Fe3O4 groups grow rapidly, indicating that Fe3O4 without stealthy coating cannot inhibit the tumor growth via PTT effect probably due to its unfavorable blood retention and tumor accumulation (Fig. 8b). Among all the groups, the Fe3O4@PCBMA group exerts the strongest tumor regression over time and four of five tumors are ablated completely without recurrence after a single treatment (Fig. 8c). The calculated tumor inhibition rates based on the tumor weight (Fig. 8d) are 38.4%, 63.0%, 79.5%, 84.9%, 98.6%, and 89.0% for the Fe3O4, Fe3O4@RBC, Fe3O4@PMPC, Fe3O4@PSBMA, Fe3O4@PCBMA and Fe3O4-PCBMA groups, respectively. Meanwhile, in situ terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) analysis demonstrates that the most cell apoptosis is observed in the Fe3O4@PCBMA group, which is also supportive of the best antitumor efficacy of Fe3O4@PCBMA among all the treatment groups. To verify the safety of the coated Fe3O4, mice injected with different nanoparticles are sacrificed 14 d after PTT treatment, with major organs (heart, liver, spleen, lung and kidney) collected and sliced for histology analysis. No obvious tissue damages or adverse effects to the main organs are observed for each group, which indicates the favorable biocompatibility of all nanoparticles (Fig. S23). Meanwhile, neither obvious body weight loss (Fig. 8a) nor abnormal behavior are observed for all the treatment groups, demonstrating the bio-safely of these nanoparticles. Therefore, the results suggest that zwitterionic polymer membrane-coated Fe3O4 possesses no obvious toxicity in mice. 28

Fig 8. In vivo photothermal therapy. (a) Body weight curves of mice in each group observed for 14 days. (b) Tumor volume curves of A549 tumor-bearing mice with 808 nm laser irradiation for 5 min. (c) Photographs of tumors from different groups and (d) tumor weights of each group after laser treatment. (e) H&E images of tumor slices after PTT treatment. The scale bar is 200 µm. (f) TUNEL staining of tumor tissues 14 days after PTT treatment. Nuclei and apoptotic cells were stained blue and green, respectively. Scale bars indicate 50 µm. *p< 0.1, **p < 0.01, ***p < 0.001, and n.s. representing no significance. Data are means ±s.d. N=5. 29

4. Conclusions In this work, a facile biodegradable zwitterionic polymer membrane coating platform is developed, by which different kinds of nanoparticles could be coated by different types of zwitterionic polymers. The coated zwitterionic polymer membranes shows stronger protein adsorption resistance than the corresponding zwitterionic polymer brushes or RBC membranes, leading to superior anti-phagocytosis ability and longer blood circulation of the coated Fe3O4. With preferable blood circulation, the zwitterionic polymer membrane-coated Fe3O4 shows higher tumor accumulation and stronger MRI signal than the corresponding zwitterionic polymer brush or RBC membrane-coated Fe3O4, leading to superior PTT effects on tumors. Furthermore, the carboxybetaine-based polymer membrane-coated nanoparticles exhibit ultra long blood circulation and stronger PTT effects than the phosphorylcholinebased polymer membrane, sulfobetaine-based polymer membrane or RBC membrane-coated nanoparticles. Therefore, the fabrication of zwitterionic polymer membranes mimicking cell membranes endows the inner core with preferable blood circulation and desirable therapeutic benefits compared with the current zwitterionic polymer brushes or RBC membranes, which may inspire the design of biomimic nanoparticles for biomedical applications.

Notes The authors declare no competing financial interest.

Acknowledgements This work is financially supported by the National Key R&D Program of China (Grant No. 2016YFC1100300) and the National Natural Science Foundation of China (Grant Nos. 51873041, 51933002, 81773283 and 81671815).

Data availability statement 30

The data are available from the corresponding author ([email protected]) on reasonable request.

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Biodegradable Zwitterionic Polymer Membrane Coating Endowing Nanoparticles with Ultra-Long Circulation and Enhanced Tumor Photothermal Therapy Shaojun Peng, Boshu Ouyang, Yongzhi Men, Yang Du, Yongbin Cao, Ruihong Xie, Zhiqing Pang, Shun Shen, Wuli Yang

Competing Interests: The authors declare no competing interests.