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Aug 2, 2016 - Zwitterionic Phosphorylcholine−TPE Conjugate for pH-Responsive. Drug Delivery and AIE Active Imaging. Yangjun Chen,. †. Haijie Han,...
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Zwitterionic Phosphorylcholine-TPE Conjugate for pHResponsive Drug Delivery and AIE Active Imaging Yangjun Chen, Haijie Han, Hongxin Tong, Tingting Chen, Haibo Wang, Jian Ji, and Qiao Jin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06071 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 4, 2016

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Zwitterionic Phosphorylcholine-TPE Conjugate for pH-Responsive Drug Delivery and AIE Active Imaging Yangjun Chen†, Haijie Han†, Hongxin Tong†, Tingting Chen†, Haibo Wang‡, Jian Ji*†, Qiao Jin*†

† MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, China. ‡ Textile Institute, College of Light Industry, Textile and Food Engineering, Sichuan University, Chengdu, 610065, China.

Keywords: theranostics, zwitterionic polymers, pH-responsive, aggregation induced emission (AIE), drug delivery, bioimaging

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ABSTRACT: Polymeric micelles have emerged as promising nano-platform for cancer theranostics. Herein, we developed doxorubicin (DOX) encapsulated pH-responsive polymeric micelles for combined aggregation induced emission (AIE) imaging and chemotherapy. The novel

zwitterionic

copolymer

poly(2-methacryloyloxyethyl

phosphorylcholine-co-2-(4-

formylphenoxy)ethyl methacrylate) (poly(MPC-co-FPEMA)) was synthesized via RAFT polymerization and further converted to PMPC-hyd-TPE after conjugation of tetraphenylethene (TPE, a typical AIE chromophore) via acid-cleavable hydrazone bonds. The AIE activatable copolymer PMPC-hyd-TPE could self-assemble into spherical PC-hyd-TPE micelles and DOX could be loaded through hydrophobic interactions. The zwitterionic micelles exhibited excellent physiological stability and low protein adsorption due to the stealthy phosphorylcholine (PC) shell. In addition, the cleavage of hydrophobic TPE molecules under acidic condition could induce swelling of micelles, which was verified by size changes with time at pH 5.0. The in vitro DOX release profile also exhibited accelerated release rate with pH value decreasing from 7.4 to 5.0. Fluorescent microscopy and flow cytometry studies further demonstrated fast internalization and accumulation of drug loaded PC-hyd-TPE-DOX micelles in HepG2 cells, resulting in considerable time/dose-dependent cytotoxicity. Meanwhile, high-quality AIE imaging of PChyd-TPE micelles was confirmed in HepG2 cells. Notably, ex vivo imaging study exhibited efficient accumulation and drug release of PC-hyd-TPE-DOX micelles in the tumor tissue. Consequently, the multifunctional micelles with combined nonfouling surface, AIE active imaging and pH-responsive drug delivery showed great potential as novel nano-platforms for new generation of cancer theranostics.

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1. Introduction Theranostic nanomedicine1-4 is a relatively new and popular field that integrates both therapeutic (e.g. chemotherapy, photo-thermal/dynamic therapy) and diagnostic functions into one single system. Nanoscale drug delivery systems (DDSs) with simultaneous fluorescent imaging module have attracted tremendous interest. Nevertheless, traditional fluorescent dyes could suffer from aggregation caused quenching (ACQ) when incorporated in nanoparticles at high loadings,5 which greatly impedes the imaging performance. In 2001, Tang’s group introduced a novel category of fluorogens which instead possessed high fluorescent efficiency in aggregated state because of the inhibition of intramolecular rotation.6 The special aggregation induced emission (AIE) dyes thus provided an efficient approach to overcome the ACQ problem.5,7 In recent years, numerous AIE based fluorescent nanoparticles have been designed for efficient bioimaging,5,8,9 with AIE dyes either physically encapsulated10-12 or covalently conjugated13-15. Moreover, theranostic systems with combined drug delivery and AIE fluorescent imaging modules have recently been explored.16-22 Therefore, developing AIE active nanosystems (e.g., polymeric micelles) as simultaneous drug carriers is now an attractive and significant issue for the development of nanomedicine. The passively targeting accumulation of nanoparticles in tumor tissue could be notably improved by prolonging blood circulation time via the enhanced permeability and retention (EPR) effect.23,24 Hence, DDSs are usually modified with hydrophilic polyethylene glycol (PEGylation) to reduce non-selective protein absorption and avoid the subsequent rapid clearance by the reticuloendothelial system. However, the susceptibility to oxidation damage and the recent discovery of anti-PEG immunological response25,26 have triggered a new perspective of PEGylation and great efforts have been made to investigate alternative non-fouling materials.27,28

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Zwitterionic phosphorylcholine (PC)29 is a major component of cell membranes and possesses superior non-fouling property to PEG because of the electrostatically induced hydration formation.30 2-Methacryloyloxyethyl phosphorylcholine (MPC) based polymers31,32 have been widely synthesized to mimic the cell membranes and have been considered as stealthy materials. In our previous studies, metal nanoparticles33,34 and polymeric micelles35-37 with bioinspired PC shells exhibited great biostability and biocompatibility, which ensured and even improved their functions as therapeutic or imaging agents. Additionally, AIE active polymeric nanoparticles based on copolymerization of MPC and AIE monomers were developed by Wei’s group38,39 as biocompatible imaging agents. Yet they only focused on the single imaging application and rational design of AIE activatable MPC-based polymers for controlled drug delivery will be very promising. The unique tumor microenvironment or intracellular physiological conditions,40 such as acidic pH gradient,41 higher glutathione concentration42 and overexpressed level of enzymes43, allow for tailored release of drugs. Herein,

we

successfully

methacryloyloxyethyl

synthesized

a

novel

zwitterionic

copolymer

phosphorylcholine-co-2-(4-formylphenoxy)ethyl

poly(2-

methacrylate)

(poly(MPC-co-FPEMA)) with pedant benzaldehyde groups for further conjugation of tetraphenylethene (TPE, a typical AIE chromophore) via pH-cleavable hydrazone bonds44,45. It is anticipated that the resultant AIE active copolymer (PMPC-hyd-TPE) could self-assemble into micelles in which the hydrophobic TPE core serves for cell imaging and zwitterionic PC shell endows them with excellent stability and biocompatibility (Scheme 1). Doxorubicin (DOX) was further encapsulated through hydrophobic interactions to form theranostic PC-hyd-TPE-DOX micelles. Under endo/lysosomal acidic conditions, TPE was expected to be cleaved from the polymer, inducing the disassembly of micelles and the following drug release. The in vitro AIE

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imaging, cellular uptake and cytotoxicity against cancer cells as well as ex vivo DOX biodistribution were further evaluated in detail.

Scheme 1. Schematic Illustration of Phosphorylcholine-TPE Conjugate as a Novel Cancer Theranostic Nano-Platform for Combined AIE Active Imaging and pH-Responsive Drug Delivery

2. Experimental Section Materials.

4-Cyanopentanoic

acid

dithiobenzoate

(CTA),46

2-methacryloyloxyethyl

phosphorylcholine (MPC),47 2-(4-formylphenoxy)ethyl methacrylate (FPEMA)48 and the tetraphenylethene derivative with hydrazine group (TPE-hyd)45 were synthesized following published reports. Doxorubicin hydrochloride (DOX·HCl) was bought from Beijing Zhongshuo Pharmaceutical Technology Development Co., Ltd.

4-Nitrophenyl chloroformate 3-(4, 5-

dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) was purchased from SigmaAldrich. Dimethyl formamide (DMF), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

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Characterization. Fourier transform infrared (FT-IR) analysis of poly(MPC-co-FPEMA) was recorded with a Bruker Vector 2 spectrometer. 1H NMR spectra of polymers were recorded on a Bruker DMX500 instrument. The polydispersity index of poly(MPC-co-FPEMA) was measured by aqueous gel permeation chromatography (GPC) equipped with Shimadzu RI and UV-vis detection. Monodisperse PEG was used as standard and an aqueous solution containing 0.2 M guanidinium chloride and 0.2 M NaNO3 was used as the eluting phase at a flow rate of 0.45 mL/min.49 The morphology of micelles was measured via transmission electron microscopy (TEM, HT7700, Hitachi). The size distribution and size change of zwitterionic micelles were determined by dynamic light scattering (DLS) using Zetasizer Nano-ZS (Malvern Instruments). UV-vis spectra of TPE and DOX were recorded by a Shimadzu UV-2550 UV-vis spectrometer. Fluorometric measurements were performed by a Shimadzu RF-530 spectrometer.42 Synthesis of poly(MPC-co-FPEMA). The novel zwitterionic copolymer with pendant benzaldehyde groups poly(MPC-co-FPEMA) was synthesized via RAFT polymerization. Briefly, MPC (0.354 g, 1.2 mmol), FPEMA (0.281 g, 1.2 mmol), CTA (8.4 mg, 30 µmol) and azobisisobutyronitrile (1.6 mg, 10 µmol) were added into a flask together with mixed solvent of 4 mL methanol and 4 mL DMF. The mixture was degassed using argon (g) for 30 min and reacted at 70 °C. After 24 h, the flask was exposed to air to terminate the polymerization and the copolymer was obtained by dropwise precipitation into excess amount of cold THF. The product was further dialyzed against deionized water for 1 day and lyophilized to get poly(MPC-coFPEMA) as light pink solid (70% yield).35 Synthesis of PMPC-hyd-TPE. TPE molecules were grafted onto poly(MPC-co-FPEMA) via pH-responsive hydrazone bonds. Briefly, poly(MPC-co-FPEMA) (0.1 g, 0.19 mmol for aldehyde) and TPE-hyd (0.16 g, 0.38 mmol) were dissolved in a mixture of methanol (5 mL) and

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DMSO (7 mL). Then a small amount of trifluoroacetic acid was added as catalyst and the mixture was kept reaction under N2 atmosphere.45 After 24 h, the solution was dialyzed against DMSO 6 times to remove unreacted TPE-hyd, then deionized water for 48 h to remove DMSO. The final solution was filtered through a 0.45 µm pore-sized microporous membrane and lyophilized to obtain PMPC-hyd-TPE (58% yield). Preparation of Zwitterionic Micelles and Drug Loading. PC-hyd-TPE-DOX and PC-hydTPE micelles were prepared by the dialysis method. Typically, PMPC-hyd-TPE (10 mg) and DOX (1 mg) were dissolved in a mixture of methanol (2 mL) and DMSO (2 mL). Then, 4 mL of distilled water was dropwise added under violent stirring. After stirring for 3 h. the mixture was dialyzed against deionized water for 2 days to remove any residual solvent and free DOX. Afterwards, the solution was filtered through 0.45 µm pore-sized microporous membrane to obtain DOX loaded PC-hyd-TPE-DOX micelles. The blank PC-hyd-TPE micelles were prepared similarly in the absence of DOX. The drug loading efficiency (DLE) and drug loading content (DLC) were calculated following published reports.50 Colloidal Stability and Protein Adsorption of Micelles. Micelles with zwitterionic PC shell were expected to be stable and stealthy. Therefore, the micellar stability of PC-hyd-TPE in PBS was monitored by DLS over 1 month. In addition, the serum stability of PC-hyd-TPE micelles was further examined. PC-hyd-TPE micelles and DMEM culture medium (10% fetal bovine serum, FBS) were mixed in 1:1 volume ratio and size changes were recorded by DLS.51 Fibrinogen (FBG) and bovine serum albumin (BSA) were adopted as models of plasma proteins to evaluate the protein adsorption of micelles according to published reports.51,52 The zwitterionic micelles (0.3 mg/mL) and protein solutions (0.5 mg/mL) were mixed in 1:1 volume ratio. After incubation for 3 and 24 h, the mixed solution was centrifuged at 12 000 g for 15 min

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to precipitate protein-micelle aggregates. Subsequently, Brandford assay was used to determine the adsorbed protein content. pH-Responsive Destabilization and Drug Release. The acid-sensitive cleavage of TPE molecules could induce destabilization of micelles, resulting in controlled DOX release. Firstly, pH-responsive destabilization of PC-hyd-TPE-DOX micelles was determined by DLS measurement. The micellar solution were incubated under different pH values (7.4 and 5.0) at 37 °C and size changes at different time intervals were monitored. Subsequently, in vitro DOX release profile of PC-hyd-TPE-DOX micelles was studied by dialysis method at pH 7.4 and 5.0, respectively. Briefly, 2 mL of PC-hyd-TPE-DOX micelle were transferred into a dialysis tube (MWCO 3.5 kDa) that was immersed into 10 mL of PBS with different pH values. The dialysis tubes were incubated in an air bath with constant shaking at 37 °C. At desired time intervals, 2 mL of release media was withdrawn to calculate the released DOX content by measuring fluorescence intensity. The same amount of corresponding fresh media was replenished immediately. Cell Line and Culture. The human hepatic carcinoma cells (HepG2) were cultured in complete DMEM medium. The human umbilical vein endothelial cells (HUVECs) and pancreatic cancer cells (BxPC-3) were cultured in complete RAMI 1640 medium. All the cells were incubated at 37 °C in a balanced air humidified incubator with an atmosphere of 5% CO2.35 Cell Imaging of PC-hyd-TPE Micelles. To investigate the AIE active imaging of PC-hydTPE micelles, HepG2 cells were planted in glass bottom dishes with 1×105 cells per dish. After incubation for 24 h, PC-hyd-TPE micelles was added with a final polymer concentration of 100 µg/mL and cells were allowed to be treated for 3 h. Afterwards, cells were washed with PBS, fixed with 4% paraformaldehyde and imaged by fluorescence microscope.45

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Cellular Uptake and Intracellular Drug Release of PC-hyd-TPE-DOX Micelles. The cellular uptake behaviour was studied by flow cytometry analysis. HepG2 cells were planted into 24 well plates at 2×105 cells per well. Then PC-hyd-TPE-DOX micelles and free DOX were added with the same final drug concentration of 5 mg/L. After incubation for different time periods (1 and 5 h), cells were washed with PBS, harvested and analyzed using a FACScan flow cytometer. The intracellular DOX release was with fluorescence microscopy. HepG2 cells were planted into glass bottom dishes at 1×105 cells per dish in DMEM medium for 24 h, then incubated with PC-hyd-TPE-DOX micelles and free DOX with the same final DOX concentration of 5 mg/L. After incubation for different time periods (1 and 5 h), cells were washed with PBS, fixed with 4% paraformaldehyde and imaged by fluorescence microscopy.45 Cytotoxicity Studies by MTT Assay. The in vitro cytotoxicity of PC-hyd-TPE-DOX micelles and empty PC-hyd-TPE micelles was determined with MTT assay. For PC-hyd-TPE-DOX micelles, HepG2 cells were seeded into 96 well plates (5000 cells per well) using 200 µL culture medium and incubated for 1 day. Afterwards, fresh culture medium containing free DOX or PChyd-TPE-DOX micelles was added with a final drug concentration gradient from 0.05 to 7.5 µg/mL. After treatment for 48 or 72 h, cell cytotoxicity was determined via standard MTT assay. The cytotoxicity of blank PC-hyd-TPE micelles was evaluated similarly using HUVECs and HepG2 cells with a final polymer concentration gradient from 5 to 250 µg/mL. Ex Vivo DOX Distribution and Tumor Accumulation. BALB/c nude mice (male, 3–4 weeks) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. The xenograft model was generated by subcutaneous injection of pancreatic cells (BxPC-3, 1 × 107 for each mouse) in the right flank of mice. Free DOX or PC-hyd-TPE-DOX micelles (5mg DOX equiv./kg) were

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intravenously administrated when the tumors reached about 400 mm3 in volume. After 6 h, mice were sacrificed and major organs including heart, liver, spleen, lung, kidney and tumor were harvested. Fluorescence images of DOX were acquired with the Maestro in vivo fluorescence imaging system.42 All handling of animals was carried out under protocols approved by Institutional Animal Care and Use Committee.35

3. Results and Discussion Synthesis and Characterization of poly(MPC-co-FPEMA) and PC-hyd-TPE. As shown in Scheme 2, the zwitterionic copolymer poly(MPC-co-FPEMA) was synthesized via RAFT polymerization, then AIE activatable TPE molecules were grafted onto the copolymer with pHcleavable hydrazone bonds.

Scheme 2. Synthetic Route of poly(MPC-co-FPEMA) and PMPC-hyd-TPE

The chemical structure and composition of poly(MPC-co-FPEMA) were measured by FT-IR, 1

H NMR and aqueous GPC. As shown in Figure S1, all characteristic peaks of both MPC and

FPEMA units were visible in the FT-IR spectrum. We could see absorption bands of ester and aldehyde carbonyl group at 1728 and 1689 cm-1, respectively. The typical peaks for asymmetric

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O=P−O− stretching (1253 cm−1), symmetric O=P−O− stretching (1087 cm−1) and −N+(CH3)3 (964 cm−1) of the MPC units could also be seen clearly. Figure 1A showed the 1H NMR spectrum of poly(MPC-co-FPEMA) copolymer and all characteristic signals of CTA, MPC and FPEMA could be observed clearly. By comparing the typical peak integrals of MPC (methylene, δ 3.74) and FPEMA (phenyl, δ 7.15 and 7.87) with that of CTA (phenyl, δ 7.45, 7.53 and 7.68), the polymerization degrees of MPC and FPEMA were calculated to be 35 and 39, respectively. Thus, the molecular weight of poly(MPC-co-FPEMA) was calculated to be 19.7 kDa. We also measured the molecular weight distribution of poly(MPC-co-FPEMA) using aqueous GPC. The GPC trace of poly(MPC-co-FPEMA) in Figure S2 exhibited a unimodal peak with a narrow polydispersity index (Mw/Mn) of 1.24, which confirmed the well-controlled nature of RAFT polymerization. TPE molecules were further grafted onto poly(MPC-co-FPEMA) via reaction between aldehyde and hydrazine. Figure 1B showed the 1H NMR spectrum of PMPC-hyd-TPE. Compared with poly(MPC-co-FPEMA), the characteristic peaks of aldehyde group (δ 9.88) in the FPEMA units completely disappeared and new peaks corresponding to TPE (δ 6.5-7.2) were observed, indicating that poly(MPC-co-FPEMA) was successfully converted to PMPC-hyd-TPE with 100% conversion efficiency.

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Figure 1. 1H NMR spectra of (A) poly(MPC-co-FPEMA) and (B) PMPC-hyd-TPE.

Preparation and Characterization of Micelles. The amphiphilic PMPC-hyd-TPE copolymer could self-assemble into nanoscale micelles with hydrophobic TPE core and zwitterionic PC shell by dialysis method. The size distribution and morphology of the PC-hyd-TPE micelles were studied by DLS and TEM, respectively. As shown in Figure 2A, the intensity-mean hydrodynamic diameter of PC-hyd-TPE micelles was about 195.5 nm with a polydispersity index (PDI) of 0.19. The inserted TEM image demonstrated that PC-hyd-TPE micelles dispersed very well with regular spherical shape and had an average diameter of around 93 nm. After

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encapsulation of DOX, the size of PC-hyd-TPE-DOX micelles became smaller than that of PChyd-TPE micelles (Figure S3). We think that here DOX worked as “physical crosslinker” of the randomly distributed TPE molecules via hydrophobic-hydrophobic interactions, resulting in the more compact micellar core of drug loaded micelles than blank micelles as previously reported.50,53 Hydrophobic TPE moieties were trapped into the micellar core during the micellar formation and the restricted TPE would excite the AIE behaviour. As shown in Figure 2B, the maximum UV absorption of TPE located at around 310 nm. Under the same polymer concentration and excitation condition, PC-hyd-TPE micelles exhibited strong fluorescence (FL) intensity whereas the FL intensity of PMPC-hyd-TPE in DMSO/methanol mixture was negligible. This indicated that PC-hyd-TPE micelles could be suitable for the potential bioimaging application.

Figure 2. (A) Size distribution and TEM image of PC-hyd-TPE micelles. (B) UV-vis and fluorescence (FL) spectra of PC-hyd-TPE micelles.

Colloid Stability and Protein Adsorption of Micelles. PC-hyd-TPE micelles with zwitterionic PC shell were expected to possess excellent physiological stability. The size changes of PC-hyd-TPE micelles in pH 7.4 PBS and DMEM culture medium with 10% FBS were studied by DLS. As shown in Figure 3A, PC-hyd-TPE micelles were fairly stable in PBS for 5 days.

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Even after 1 month, only a little change in size was observed. Additionally, Figure 3B showed that PC-hyd-TPE micelles were quite stable in serum contained culture medium for 48 h. We also evaluated the interaction of micelles with proteins, using FBG and BSA as model plasma proteins. As displayed in Figure S4, the micelles exhibited low protein adsorption even after 24 h incubation, which could be ascribed to the superhydrophilic and nonfouling property of zwitterionic PC moiety. These results indicated that the theranostic micelles with stealthy PC shell may possess prolonged circulation time and enhanced tumor accumulation in vivo.

Figure 3. Colloid stability of PC-hyd-TPE micelles (A) at pH 7.4 and (B) in DMEM with 10% FBS.

pH-Responsive Micelle Swelling and Drug Release. The hydrophobic TPE molecules were conjugated with hydrazone bonds and could be cleaved under acid condition, leading to micellar swelling and subsequent drug release. The size changes at pH 7.4 and 5.0 were monitored by DLS. As shown in Figure 4A, after incubation at pH 7.4 for 24 h, no obvious size change of the drug loaded PC-hyd-TPE-DOX micelles was observed. However, there was a significant size increase in 6 h when the pH decreased to 5.0. Moreover, the micelles further expanded to hundreds of nanometers in 72 h, indicating remarkable destruction of the micellar structure.

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DOX was loaded via hydrophobic interaction and could be released after hydrophobic TPE molecules were cleaved and micelles were disassembled. The DLC and DLE of PC-hyd-TPEDOX micelles were calculated to be 5.2% and 57.2%, respectively. The release profile of PChyd-TPE-DOX micelles was studied at physiological pH 7.4 and intracellular acidic pH 5.0. As shown in Figure 4B, the release of DOX exhibited a pH-dependent behaviour. At pH 7.4, less than 20% of the total DOX was released in 72 h. In comparison, the DOX release rate strongly increased at pH 5.0. It was observed that about 70% of the total DOX was released from PC-hydTPE-DOX micelles in the first 24 h. The remarkable increase of DOX release under intracellular pH condition could be attributed to acid-triggered cleavage of TPE and the following disassembly of micelles, as demonstrated above. These results demonstrated that the micelles exhibited great potential to reduce premature drug release in the bloodstream while augment drug release under endo/lysosomal acidic condition in tumor cells.

Figure 4. (A) pH-Triggered disassembly monitored by DLS and (B) in vitro DOX release of PChyd-TPE-DOX micelles.

Cell Imaging of PC-hyd-TPE Micelles. The AIE active cell imaging of PC-hyd-TPE micelles was studied by fluorescence microscopy. HepG2 cells were co-cultured with PC-hyd-TPE micelles for 3 h. Subsequently, we could observe clear blue fluorescence of TPE which mainly

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located in the cytoplasm region (Figure 5), indicating efficient internalization of PC-hyd-TPE micelles through the endocytosis process. Hence, we could expect that the cell membrane mimicking PC-hyd-TPE micelles might serve as promising candidate for cell imaging.

Figure 5. AIE imaging of HepG2 cells after co-culture with PC-hyd-TPE micelles for 3 h.

Cellular Uptake and Intracellular Drug Release. Efficient internalization and rapid intracellular drug release were crucial to achieve ideal therapeutic effect. The internalization behaviour was studied by flow cytometry, which was demonstrated by quantitative fluorometry of internalized DOX from incubated cells. As shown in Figure 6, stronger DOX fluorescence intensity at 5 h than that at 1 h was observed, indicating that the uptake of micelles was timedependent. Moreover, the intracellular drug release and distribution were evaluated by fluorescence microscopy. As shown in Figure 7, red fluorescence of DOX and blue fluorescence of TPE could be observed after 1 h incubation and mainly located in the cytoplasm. The fluorescence intensity of both TPE and DOX was significantly enhanced with longer incubation time of 5 h, which was consistent with the result of flow cytometry. Meanwhile, certain DOX fluorescence appeared in the cell nuclei, implying successful intracellular DOX release and further delivery into cell nuclei. Faster internalization of free DOX was confirmed with most DOX fluorescence emerged in the cell nuclei. This phenomenon could be explained by the fact that free DOX could permeate cellular and nuclear membranes quickly via passive diffusion.50

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Figure 6. Cytometer assay of HepG2 cells incubated with free DOX and PC-hyd-TPE-DOX micelles.

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Figure 7. Fluorescence microscopy images of HepG2 cells co-cultured with PC-hyd-TPE-DOX micelles or free DOX for 1 and 5 h. From left to right: AIE (blue), DOX (red), and overlays of two images.

In Vitro Cytotoxicity Study. The biocompatibility of empty PC-hyd-TPE micelles was determined using HUVECs and HepG2 cells. More than 80% cell viability was achieved with a tested concentration up to 250 µg/mL (Figure S5), indicating excellent biocompatibility of the cell membrane bioinspired polymer. By contrast, the drug loaded PC-hyd-TPE-DOX micelles and free DOX showed remarkable cytotoxicity against HepG2 cells. As shown in Figure 8, PChyd-TPE-DOX micelles exhibited time/dose-dependent cytotoxicity and the half maximal inhibitory concentration (IC50) values were 4.37 and 0.96 µg/mL in 48 and 72 h, respectively. With regard to free DOX, the IC50 values were 0.39 and 0.21 µg/mL correspondingly. Free DOX induced higher cytotoxicity than PC-hyd-TPE-DOX micelles, which coincided well with the results of cellular uptake studies. The possible reason may be that PC-hyd-TPE-DOX micelles should undergo complex internalization and subsequent drug release process to induce inhibition of cell proliferation.

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Figure 8. Cytotoxicity of free DOX and PC-hyd-TPE-DOX micelles against HepG2 cells after treatment for (A) 48 and (B) 72 h, respectively.

Ex Vivo DOX Biodistribution and Tumor Accumulation. As demonstrated above, zwitterionic PC-hyd-TPE-DOX micelles with excellent colloidal stability and high protein resistance were promising to have long blood circulating time and improved tumor accumulation via EPR effect. Ex vivo fluorescence images of isolated tissues were visualized at 6 h postinjection of PC-hyd-TPE-DOX micelles and free DOX (Figure 9). The zwitterionic PC-hyd-TPEDOX micelles exhibited much higher DOX fluorescence intensity in tumor tissue than in major organs. By contrast, strong DOX fluorescence was observed in the liver, lung and kidney for free DOX. Additionally, PC-hyd-TPE-DOX micelles possessed higher tumor accumulation than free DOX. These results indicated that the zwitterionic PC-shelled micelles could effectively accumulate in pancreatic tumor xenografts via EPR effect and release DOX in the tumor cells.

Figure 9. Fluorescence images of excised tissues at 6 h post-injection of PC-hyd-TPE-DOX micelles and free DOX.

4. Conclusions Herein, we developed zwitterionic copolymer PMPC-hyd-TPE bearing AIE active TPE molecules for combined drug delivery and fluorescent imaging. The zwitterionic copolymer

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could self-assemble into spherical micelles with excellent physiological stability and high protein resistance due to the stealthy PC moiety. The DOX loaded PC-hyd-TPE-DOX micelles could undergo swelling at pH 5.0 due to the cleavage of TPE and showed remarkable pH-regulated drug release behaviour. In vitro results revealed that PC-hyd-TPE-DOX micelles could be efficiently internalized and induce remarkable cytotoxicity to HepG2 cells with IC50 of 4.37 and 0.96 µg/mL after 48 and 72 h, respectively. Meanwhile, high-quality AIE imaging of PC-hydTPE micelles was confirmed in HepG2 cells. Further ex vivo fluorescence imaging results demonstrated enhanced accumulation and DOX delivery of PC-hyd-TPE-DOX micelles in tumor tissue. Therefore, the cell membrane mimicking copolymer bearing AIE active molecules showed great potential as novel nano-platforms for cancer theranostics.

ASSOCIATED CONTENT Supporting Information. FT-IR spectrum and aqueous GPC trace of zwitterionic copolymer poly(MPC-co-FPEMA), size distribution of PC-hyd-TPE-DOX micelles by DLS, protein adsorption of micelles and MTT results of blank PC-hyd-TPE micelles. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Qiao Jin*: E-mail: [email protected]; Jian Ji*: E-mail: [email protected] Fax/Tel: +86-571-87953729

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The authors declare no competing financial interest.

Acknowledgments Financial support from the Key Science Technology Innovation Team of Zhejiang Province (no. 2013TD02), the National Natural Science Foundation of China (51303154, 51573160, 21574114), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry ([2015]311) are gratefully acknowledged.

References (1) Choi, K. Y.; Liu, G.; Lee, S.; Chen, X. Theranostic Nanoplatforms for Simultaneous Cancer Imaging and Therapy: Current Approaches and Future Perspectives Nanoscale 2012, 4, 330-342. (2) Luk, B. T.; Fang, R. H.; Zhang, L. Lipid- and Polymer-Based Nanostructures for Cancer Theranostics Theranostics 2012, 2, 1117-1126. (3) Elsabahy, M.; Heo, G. S.; Lim, S. M.; Sun, G.; Wooley, K. L. Polymeric Nanostructures for Imaging and Therapy Chem. Rev. 2015, 115, 10967-11011. (4) Chen, Q.; Ke, H.; Dai, Z.; Liu, Z. Nanoscale Theranostics for Physical Stimulus-Responsive Cancer Therapies Biomaterials 2015, 73, 214-230. (5) Mei, J.; Leung, N. L.; Kwok, R. T.; Lam, J. W.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718-11940.

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(6) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z. Aggregation-Induced Emission of 1-Methyl-1,2,3,4,5-pentaphenylsilole Chem. Commun. 2001, 1740-1741. (7) Liang, J.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobes Based on AIEgen Conjugates Chem. Soc. Rev. 2015, 44, 2798-2811. (8) Zhang, X.; Wang, K.; Liu, M.; Zhang, X.; Tao, L.; Chen, Y.; Wei, Y. Polymeric AIE-Based Nanoprobes for Biomedical Applications: Recent Advances and Perspectives Nanoscale 2015, 7, 11486-11508. (9) Yan, L.; Zhang, Y.; Xu, B.; Tian, W. Fluorescent Nanoparticles Based on AIE Fluorogens for Bioimaging Nanoscale 2016, 8, 2471-2487. (10) Ding, D.; Li, K.; Qin, W.; Zhan, R.; Hu, Y.; Liu, J.; Tang, B. Z.; Liu, B. Conjugated Polymer Amplified Far-Red/Near-Infrared Fluorescence from Nanoparticles with AggregationInduced Emission Characteristics for Targeted in Vivo Imaging Adv. Healthcare Mater. 2013, 2, 500-507. (11) Li, Y.; Shao, A.; Wang, Y.; Mei, J.; Niu, D.; Gu, J.; Shi, P.; Zhu, W.; Tian, H.; Shi, J. Morphology-Tailoring of a Red AIEgen from Microsized Rods to Nanospheres for TumorTargeted Bioimaging Adv. Mater. 2016, 28, 3187-3193. (12) Wang, X.; Song, P.; Peng, L.; Tong, A.; Xiang, Y. Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging ACS Appl. Mater. Interfaces 2016, 8, 609-616. (13) Wang, Z.; Chen, S.; Lam, J. W.; Qin, W.; Kwok, R. T.; Xie, N.; Hu, Q.; Tang, B. Z. LongTerm Fluorescent Cellular Tracing by the Aggregates of AIE Bioconjugates J. Am. Chem. Soc. 2013, 135, 8238-8245.

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Page 23 of 29

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(14) Wan, Q.; Wang, K.; He, C.; Liu, M.; Zeng, G.; Huang, H.; Deng, F.; Zhang, X.; Wei, Y. Stimulus Responsive Cross-Linked AIE-Active Polymeric Nanoprobes: Fabrication and Biological Imaging Application Polym. Chem. 2015, 6, 8214-8221. (15) Ma, H.; Qi, C.; Cheng, C.; Yang, Z.; Cao, H.; Yang, Z.; Tong, J.; Yao, X.; Lei, Z. AIEActive Tetraphenylethylene Cross-Linked N-Isopropylacrylamide Polymer: A Long-Term Fluorescent Cellular Tracker ACS Appl. Mater. Interfaces 2016, 8, 8341-8348. (16) Yuan, Y.; Kwok, R. T.; Tang, B. Z.; Liu, B. Targeted Theranostic Platinum(IV) Prodrug with a Built-In Aggregation-Induced Emission Light-Up Apoptosis Sensor for Noninvasive Early Evaluation of Its Therapeutic Responses in Situ J. Am. Chem. Soc. 2014, 136, 2546-2554. (17) Yuan, Y.; Kwok, R. T.; Zhang, R.; Tang, B. Z.; Liu, B. Targeted Theranostic Prodrugs Based on an Aggregation-Induced Emission (AIE) Luminogen for Real-Time Dual-Drug Tracking Chem. Commun. 2014, 50, 11465-11468. (18) Chen, J. I.; Wu, W. C. Fluorescent Polymeric Micelles with Aggregation-Induced Emission Properties for Monitoring the Encapsulation of Doxorubicin Macromol. Biosci. 2013, 13, 623632. (19) Han, H.; Jin, Q.; Wang, Y.; Chen, Y.; Ji, J. The Rational Design of a Gemcitabine Prodrug with AIE-Based Intracellular Light-Up Characteristics for Selective Suppression of Pancreatic Cancer Cells Chem. Commun. 2015, 51, 17435-17438. (20) Wang, H.; Liu, G.; Gao, H.; Wang, Y. A pH-Responsive Drug Delivery System with an Aggregation-Induced Emission Feature for Cell Imaging and Intracellular Drug Delivery Polym. Chem. 2015, 6, 4715-4718.

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Page 24 of 29

(21) Zhang, C.; Jin, S.; Li, S.; Xue, X.; Liu, J.; Huang, Y.; Jiang, Y.; Chen, W. Q.; Zou, G.; Liang, X. J. Imaging Intracellular Anticancer Drug Delivery By Self-Assembly Micelles With Aggregation-Induced Emission (AIE Micelles) ACS Appl. Mater. Interfaces 2014, 6, 5212-5220. (22) Xue, X.; Jin, S.; Zhang, C.; Yang, K.; Huo, S.; Chen, F.; Zou, G.; Liang, X.J. Probe-Inspired Nano-Prodrug with Dual-Color Fluorogenic Property Reveals Spatiotemporal Drug Release in Living Cells ACS Nano 2015, 9, 2729-2739. (23) Liu, X.; Li, H.; Jin, Q.; Ji, J. Surface Tailoring of Nanoparticles via Mixed-Charge Monolayers and Their Biomedical Applications Small 2014, 10, 4230-4242. (24) Maeda, H. Macromolecular Therapeutics in Cancer Treatment: The EPR Effect and Beyond J. Controlled Release 2012, 164, 138-144. (25) Yang, W.; Liu, S.; Bai, T.; Keefe, A. J.; Zhang, L.; Ella-Menye, J.-R.; Li, Y.; Jiang, S. Poly(carboxybetaine) Nanomaterials Enable Long Circulation and Prevent Polymer-Specific Antibody Production Nano Today 2014, 9, 10-16. (26) Liang, S.; Liu, Y.; Jin, X.; Liu, G.; Wen, J.; Zhang, L.; Li, J.; Yuan, X.; Chen, I. S. Y.; Chen, W.; Wang, H.; Shi, L.; Zhu, X.; Lu, Y. Phosphorylcholine Polymer Nanocapsules Prolong the Circulation Time and Reduce the Immunogenicity of Therapeutic Proteins Nano Res. 2016, 9, 1022-1031. (27) Schlenoff, J. B. Zwitteration: Coating Surfaces with Zwitterionic Functionality to Reduce Nonspecific Adsorption Langmuir 2014, 30, 9625-9636. (28) Venault, A.; Zheng, Y. S.; Chinnathambi, A.; Alharbi, S. A.; Ho, H. T.; Chang, Y.; Chang, Y. Stimuli-Responsive and Hemocompatible Pseudozwitterionic Interfaces Langmuir 2015, 31, 2861-2869.

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(29) Matsuno, R.; Ishihara, K. Integrated Functional Nanocolloids Covered with Artificial Cell Membranes for Biomedical Applications Nano Today 2011, 6, 61-74. (30) Chen, S.; Zheng, J.; Li, L.; Jiang, S. Strong Resistance of Phosphorylcholine SelfAssembled Monolayers to Protein Adsorption:  Insights into Nonfouling Properties of Zwitterionic Materials J. Am. Chem. Soc. 2005, 127, 14473-14478. (31) Iwasaki, Y.; Ishihara, K. Cell Membrane-Inspired Phospholipid Polymers for Developing Medical Devices with Excellent Biointerfaces Sci. Technol. Adv. Mater. 2012, 13, 64101-64114. (32) Jin, Q.; Chen, Y.; Wang, Y.; Ji, J. Zwitterionic Drug Nanocarriers: A Biomimetic Strategy for Drug Delivery Colloids Surf., B 2014, 124, 80-86. (33) Zhou, W.; Shao, J.; Jin, Q.; Wei, Q.; Tang, J.; Ji, J. Zwitterionic Phosphorylcholine as a Better Ligand for Gold Nanorods Cell Uptake and Selective Photothermal Ablation of Cancer Cells Chem. Commun. 2010, 46, 1479-1481. (34) Liu, X.; Zhu, H.; Jin, Q.; Zhou, W.; Colvin, V. L.; Ji, J. Small and Stable Phosphorylcholine Zwitterionic Quantum Dots for Weak Nonspecific Phagocytosis and Effective Tat Peptide Functionalization Adv. Healthcare Mater. 2013, 2, 352-360. (35) Chen, Y.; Li, Z.; Wang, H.; Wang, Y.; Han, H.; Jin, Q.; Ji, J. IR-780 Loaded Phospholipid Mimicking Homopolymeric Micelles for Near-IR Imaging and Photothermal Therapy of Pancreatic Cancer ACS Appl. Mater. Interfaces 2016, 8, 6852–6858. (36) Wang, H.; Wang, Y.; Chen, Y.; Jin, Q.; Ji, J. A Biomimic pH-Sensitive Polymeric Prodrug Based on Polycarbonate for Intracellular Drug Delivery Polym. Chem. 2014, 5, 854-861. (37) Wang, Y.; Wang, H.; Chen, Y.; Liu, X.; Jin, Q.; Ji, J. Biomimetic Pseudopolyrotaxane Prodrug Micelles with High Drug Content for Intracellular Drug Delivery Chem. Commun. 2013, 49, 7123-7125.

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(38) Liu, M.; Zhang, X.; Yang, B.; Deng, F.; Huang, Z.; Yang, Y.; Li, Z.; Zhang, X.; Wei, Y. Ultrabright and Biocompatible AIE Dye Based Zwitterionic Polymeric Nanoparticles for Biological Imaging RSC Adv. 2014, 4, 35137. (39) Li, H.; Zhang, X.; Zhang, X.; Yang, B.; Yang, Y.; Huang, Z.; Wei, Y. Zwitterionic Red Fluorescent Polymeric Nanoparticles for Cell Imaging Macromol. Biosci. 2014, 14, 1361-1367. (40) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery Nat. Mater. 2013, 12, 991-1003. (41) Wang, Y.; Xiao, H.; Fang, J.; Yu, X.; Su, Z.; Cheng, D.; Shuai, X. Construction of Negatively Charged and Environment-Sensitive Nanomedicine for Tumor-Targeted Efficient siRNA Delivery Chem. Commun. 2016, 52, 1194-1197. (42) Han, H.; Wang, H.; Chen, Y.; Li, Z.; Wang, Y.; Jin, Q.; Ji, J. Theranostic ReductionSensitive Gemcitabine Prodrug Micelles for Near-Infrared Imaging and Pancreatic Cancer Therapy Nanoscale 2016, 8, 283-291. (43) Chen, W. H.; Luo, G. F.; Lei, Q.; Jia, H. Z.; Hong, S.; Wang, Q. R.; Zhuo, R. X.; Zhang, X. Z. MMP-2 Responsive Polymeric Micelles for Cancer-Targeted Intracellular Drug Delivery Chem. Commun. 2015, 51, 465-468. (44) Cai, T.; Chen, Y.; Wang, Y.; Wang, H.; Liu, X.; Jin, Q.; Agarwal, S.; Ji, J. Functional 2Methylene-1,3-dioxepane Terpolymer: A Versatile Platform to Construct Biodegradable Polymeric Prodrugs for Intracellular Drug Delivery Polym. Chem. 2014, 5, 4061-4068. (45) Wang, H.; Liu, G.; Dong, S.; Xiong, J.; Du, Z.; Cheng, X. A pH-Responsive AIE Nanoprobe as a Drug Delivery System for Bioimaging and Cancer Therapy J. Mater. Chem. B 2015, 3, 7401-7407.

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(46) Chen, X.; Lawrence, J.; Parelkar, S.; Emrick, T. Novel Zwitterionic Copolymers with Dihydrolipoic Acid: Synthesis and Preparation of Nonfouling Nanorods Macromolecules 2013, 46, 119-127. (47) Wang, H.; Xu, F.; Li, D.; Liu, X.; Jin, Q.; Ji, J. Bioinspired Phospholipid Polymer Prodrug as a pH-Responsive Drug Delivery System for Cancer Therapy Polym. Chem. 2013, 4, 20042010. (48) Hu, X.; Li, H.; Luo, S.; Liu, T.; Jiang, Y.; Liu, S. Thiol and pH Dual-Responsive Dynamic Covalent Shell Cross-Linked Micelles for Triggered Release of Chemotherapeutic Drugs Polym. Chem. 2013, 4, 695-706. (49) Wang, Z.; Ma, G.; Zhang, J.; Yuan, Z.; Wang, L.; Bernards, M.; Chen, S. Surface Protonation/Deprotonation Controlled Instant Affinity Switch of Nano Drug Vehicle (NDV) for pH Triggered Tumor Cell Targeting. Biomaterials 2015, 62, 116-127. (50) Huang, P.; Wang, W.; Zhou, J.; Zhao, F.; Zhang, Y.; Liu, J.; Liu, J.; Dong, A.; Kong, D.; Zhang, J. Amphiphilic Polyelectrolyte/Prodrug Nanoparticles Constructed by Synergetic Electrostatic and Hydrophobic Interactions with Cooperative pH-Sensitivity for Controlled Doxorubicin Delivery ACS Appl. Mater. Interfaces 2015, 7, 6340-6350. (51) Huang, P.; Liu, J.; Wang, W.; Li, C.; Zhou, J.; Wang, X.; Deng, L.; Kong, D.; Liu, J.; Dong, A. Zwitterionic Nanoparticles Constructed with Well-Defined Reduction-Responsive Shell and pH-Sensitive Core for "Spatiotemporally Pinpointed" Drug Delivery. ACS Appl. Mater. Interfaces 2014, 6, 14631-14643. (52) Yuan, Y. Y.; Mao, C. Q.; Du, X. J.; Du, J. Z.; Wang, F.; Wang, J. Surface Charge Switchable Nanoparticles Based on Zwitterionic Polymer for Enhanced Drug Delivery to Tumor Adv. Mater. 2012, 24, 5476–5480.

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(53) Zhao, J.; Wang, H.; Liu, J.; Deng, L.; Liu, J.; Dong, A.; Zhang, J. Comb-like Amphiphilic Copolymers Bearing Acetal-Functionalized Backbones with the Ability of Acid-Triggered Hydrophobic-to-Hydrophilic Transition as Effective Nanocarriers for Intracellular Release of Curcumin Biomacromolecules 2013, 14, 3973-3984.

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