An Asymmetrical Polymer Vesicle Strategy for Significantly Improving

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An Asymmetrical Polymer Vesicle Strategy for Significantly Improving T1 MRI Sensitivity and Cancer-Targeted Drug Delivery Qiuming Liu, Shuai Chen, Jing Chen, and Jianzhong Du* School of Materials Science and Engineering, Key Laboratory of Advanced Civil Engineering Materials of Ministry of Education, Tongji University, 4800 Caoan Road, Shanghai 201804, China S Supporting Information *

ABSTRACT: Traditional T1 magnetic resonance imaging (MRI) contrast agents such as diethylenetriaminepentacetatic acid (DTPA) chelated gadolinium [Gd(III)] have poor sensitivity, leading to a risk of accumulated toxicity in vivo. To significantly improve the sensitivity of a T1 MRI contrast agent and to enhance the efficacy of cancer chemotherapy, herein for the first time we report a noncytotoxic asymmetrical cancer targeting polymer vesicle based on R-poly(L-glutamic acid)-block-poly(ε-caprolactone) [R is folic acid (FA) or DTPA]. Such asymmetrical vesicles have a cancer-targeting outer corona and a Gd(III)-chelating and drug-loading-enhancing inner corona, exhibiting an extremely high T1 relaxivity (42.39 mM−1 s−1, 8fold better than DTPA-Gd) and anticancer drug loading efficiency (52.6% for doxorubicin hydrochloride, DOX·HCl). Moreover, the DOX-loaded vesicles exhibited excellent antitumor activity (2-fold better than free DOX). This “chelating-just-inside” strategy for synthesizing asymmetrical polymer vesicles demonstrated promising potential theranostic applications in magnetic resonance imaging and cancer-targeted drug delivery.

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INTRODUCTION Theranostic nanoparticles which can simultaneously deliver diagnostic and therapeutic agents have been developed for more accurate diagnosis and more efficient cancer chemotherapy.1−3 Typical theranostic nanoparticles include polymer conjugates,4−6 micelles,7 vesicles,8,9 and inorganic nanoparticles.10,11 Those nanoparticles may physically encapsulate or chemically conjugate anticancer drugs, diagnostic agents, and targeting ligands to achieve co-incorporation of both theranostics and site-selected delivery.12 Magnetic resonance imaging (MRI) has been widely used in clinical disease diagnosis due to its superb spatiotemporal resolution, deep penetration into soft tissues, and facile operation.13−15 Usually, the MRI detection sensitivity depends on the local proton relaxation of water molecules in the normal and lesion sites, which can be enhanced with the administration of contrast agents.16 At present, predominant commercial contrast agents include T1-type gadolinium [Gd(III)] chelate complexes [e.g., diethylenetriaminepentacetatic acid (DTPA)Gd and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-Gd]17−20 and T2-type ones (e.g., Feridex and MnFe2O4 nanoparticles).9,21−24 In clinic, T1-type contrast agents are more popular than T2type ones because the former favorably offers positive imaging contrast enhancement. Usually, the efficiency of the contrast enhancement of an MRI contrast agent can be evaluated by its relaxivity (r1).25 According to the Solomon−Bloembergen− Morgan theory, the optimal relaxivity can be attained by optimizing two key features: mean residence lifetime (τM, the inverse of the water-exchange rate, kex) and rotational correlation time (τR).26 Small Gd(III) chelates generally have © XXXX American Chemical Society

low r1 due to fast tumbling of the molecules in water (a.k.a. low τR).27 Therefore, Gd(III) chelates had been conjugated to macromolecular nano-objects to slow down the tumbling rate.28−31 For example, Liu et al. reported diblock copolymer micelles32 and star copolymers micelles33 with covalently conjugated DOTA-Gd moieties exhibited T1 relaxivities of 26.29 and 11.4 mM−1 s−1, respectively. Davis and co-workers found that the precise molecular location of gadolinium atoms has a significant influence on the efficacy of nanoparticulate MRI positive contrast agents.34 However, to minimize its dosage in clinic, further improvement of the T1 relaxivity value of an MRI contrast agent is still an important challenge. Therefore, multifunctional polymer vesicles35−39 with an inside water pool and bigger size than polymer micelles may be a next generation of nanocarrier of T1 contrast agent for further MRI contrast enhancement. As an attempt, we recently reported a theranostic vesicle based on poly(ethylene glycol)-block-poly(L-lactic-co-glycolic acid) and bovine serum albumin−gadolinium (BSA-Gd) complexes.8 However, the T1 relaxivity value has not been improved because the simple physical encapsulation of BSA-Gd inside the polymer vesicle only provides a weak chelation of BSA with Gd. In addition, those vesicles did not have an active targeting unit to cancer cells. Recently, we reported that polymer vesicles with asymmetrical outer and inner coronas exhibited much faster endocytosis rate and endosomal escape ability than polymer vesicles with symmetrical coronas.40 This suggested besides Received: November 7, 2014 Revised: December 30, 2014

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DOI: 10.1021/ma502255s Macromolecules XXXX, XXX, XXX−XXX

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vesicles in aqueous solution. The membrane of the asymmetrical vesicle is composed of biocompatible and biodegradable PCL. Biodegradable hydrophilic PGA with a longer chain length and an FA terminal group is designed as the outer corona of the polymer vesicle, while the short PGA with DTPA chelating agent is the inner corona. Furthermore, the PGA chains can enhance the loading and stabilization of the anticancer drug DOX·HCl via electrostatic interactions between −COO− and −NH3+ groups. The terminal group of the inner corona, micromolecular DTPA, is designed to chelate a T1 contrast agent, Gd(III), to significantly improve the MRI contrast effect and to decrease the toxicity of Gd(III).

biocompatibility and biodegradability the following issues need to be considered in the design of highly effective nextgeneration theranostic polymer vesicles. First, compared with simple physical entrapment of contrast agents, the covalent conjugation of toxic T1 contrast agents only in the hollow cavity of polymer vesicles may provide many advantages such as high T1 contrast and low cytotoxicity. Second, polymer vesicles with active cancer targeting coronas may greatly improve the anticancer efficiency and decrease the cytotoxicity in vivo.23 Finally, the loading efficacies of hydrophilic anticancer drugs such as doxorubicin could be further improved [at present, the drug loading efficiency (DLE) values of most polymer vesicles are usually between 20% and 30%].41,42 On the basis of the above considerations, herein we report the design and synthesis of a new generation of noncytotoxic biocompatible and biodegradable theranostic polymer vesicle for ultrasensitive MRI and efficient intracellular anticancer drug delivery (Figure 1). This folic acid (FA) targeting pH-sensitive

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Figure 1. Illustration of multifunctional asymmetrical polymer vesicles for ultrasensitive T1 MRI and effective cancer targeted drug delivery. Blue: biodegradable hydrophilic PGA chains with different chain lengths. Red: biocompatible and biodegradable PCL. Orange: tumor targeting group, folic acid (FA). Green: chelating agent, diethylenetriaminepentacetatic acid (DTPA). Pink: doxorubicin hydrochloride (DOX·HCl). Black: T1 contrast agent Gd3+.

asymmetrical polymer vesicle has been synthesized based on two kinds of diblock copolymers with the same length of hydrophobic PCL block but different lengths of hydrophilic chains and different terminal ligands. The copolymers are folic acid−poly(L-glutamic acid)-block-poly(ε-caprolactone) (FAPGA75-b-PCL30, polymer 4 in Table 1) and DTPA−poly(Lglutamic acid)-block-poly(ε-caprolactone) (DTPA-PGA22-bPCL30, polymer 5 in Table 1). As shown in Figure 1, the mixture of both polymers can self-assemble into asymmetrical Table 1. Properties of Synthesized Polymers polymera

compositionb

Mn,NMRb

Mn,GPCc

Mw/Mnc

1 2 3 4 5

PCL30-NH2 PBLG75-b-PCL30 PBLG22-b-PCL30 FA-PGA75-b-PCL30 DTPA-PGA22-b-PCL30

3500 20100 8000 13800 6800

1600 20200 9600

1.02 1.49 1.36

EXPERIMENTAL SECTION

Synthesis of PCL-NH-Boc. A flask charged with a magnetic flea, εcaprolactone (30.58 g, 265.2 mmol), and dry toluene (60 mL) was placed into an oil bath at 144 °C. Toluene was removed by azeotropic distillation under argon so as to remove traces of water from the flask. The reaction temperature was set at 110 °C, and the solution was further degassed using an argon sparge for 30 min. N-(tertButoxycarbonyl)-2-aminoethanol (1.500 g, 8.800 mmol) was then added via syringe. Sn(Oct)2 (0.0188 g, 0.0442 mmol) was added by a micropipet under a positive argon pressure. Then the polymerization was carried out at 110 °C for 48 h under an argon atmosphere with stirring. The resulting product was dissolved in 10 mL of DCM and poured dropwise into 200 mL of cold methanol under vigorous stirring at room temperature. This purification protocol was repeated at least three times. The precipitate was harvested and dried in vacuum at 40 °C. Yield: ∼92%. Synthesis of PCL-NH2. PCL30-NH-Boc (18.00 g) was dissolved in 20 mL of anhydrous DCM under argon, and then 20 mL of anhydrous TFA was added. The reaction solution was stirred at room temperature for 4 h. The solvents were then removed under vacuum. Then the polymer was redissolved in DCM and washed sequentially with 5% NaHCO3 aqueous solution and deionized water and then dried over MgSO4. After filtration and evaporation of the solvent, the resulting NH2-terminated aminoethyl-PCL30 was dried in vacuum. Yield: ∼78%. Synthesis of Bz-Glu NCA Monomer. γ-Benzyl-L-glutamate (10.00 g, 42.10 mmol) and α-pinene (29.25 g, 210.7 mmol) were suspended in 150 mL of anhydrous ethyl acetate in a reaction flask fitted with a reflux condenser. After heating to reflux, triphosgene (9.476 g, 31.60 mmol) was gradually added through an addition funnel, and the reaction was allowed refluxing under argon for 4−5 h. The clear reaction solution was allowed to cool to room temperature, and the crude NCA monomer was precipitated three times in hexane to yield Bz-Glu NCA monomer. Yield: ∼86%. Synthesis of NH2-PBLG-b-PCL Diblock Copolymers with Different Lengths. Polymerizations of Bz-Glu-NCA were performed by mixing a solution of NH2-PCL30 in anhydrous dimethylformamide (DMF) with a solution with different contents of freshly prepared BzGlu-NCA in anhydrous DMF. The reaction mixture was stirred under argon at room temperature for 48 h. The reaction mixture was then poured into a large excess of diethyl ether. The precipitates were collected by centrifugation, washed with diethyl ether, and dried in a vacuum. Then two kinds of diblock copolymers NH2-PBLG75-b-PCL30 and NH2-PBLG22-b-PCL30 were obtained. Yield: ∼69%. Synthesis of FA/DTPA-PBLG-b-PCL Diblock Copolymers. The mixture of diblock copolymer NH2-PBLG22-b-PCL30 (500.0 mg, 0.06274 mmol), micromolecular diethylenetriaminepentacetatic acid (DTPA; 148.1 mg, 0.3764 mmol), and o-(7-azabenzotriazole-1-yl)N,N,N,N′-tetramethyluronium hexafluorophosphate (HATU; 47.71 mg, 0.1255 mmol) in dry DMF was stirred for 1 h. Diisopropylethylamine (DIPEA; 16.22 mg, 0.1255 mmol) was added to the stirring solution. The resulting reaction mixture was stirred an additional 24 h. The solvent was removed under vacuum, and the byproducts and impurities were removed by precipitation in diethyl ether three times. The product was dried under vacuum for 24 h. The diblock copolymer

f PGAd

0.68 0.38

a

Polymers 1, 4, and 5 are synthesized from the precursor polymers PCL30-NH-Boc, 2, and 3, respectively. bThe compositions of copolymers are determined by 1H NMR in CDCl3 or DMSO-d6. c Determined by DMF GPC calibrated with near-monodisperse PEO standards. dThe calculation of the relative volume fraction of hydrophilic PGA blocks is shown in the Supporting Information. See Figures S1−S9 for details. B

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In Vitro Drug Release. The controlled release of anticancer drug was achieved according to the following protocol. The DOX-loaded polymer vesicles after removing free drug was divided into six parts and immediately transferred into a new dialysis tube (cutoff Mn = 8000−14 000). The final drug release process was carried out by dialyzing 3.0 mL of DOX-loaded vesicles in the dialysis tube against 80 mL of tris buffer (0.01 M; pH 7.4 or pH 5.0) in a beaker (100 mL), at 37 °C and 190 rpm of stirring rate. The volume of the liquid in the beaker (outside the dialysis tube) was ensured around 80 mL during the measurement. At desired time intervals, 3.0 mL of release media was withdrawn for fluorescence spectroscopy (excitation at 461 nm and emission at 591 nm), and the cumulative release curve of DOX was obtained. The calibration curve was shown in the reported literature.44 The cumulative DOX release was calculated according to the formula

FA-PBLG75-b-PCL30 was also prepared from the polymer NH2PBLG75-b-PCL30 and micromolecular folic acid by amide reaction in the same way. Yield: ∼85%. Synthesis of FA/DTPA-PGA-b-PCL Diblock Copolymers. The diblock copolymers PCL-b-PBLG-FA/DTPA were dissolved in 33 wt % HBr/CH3COOH solution. After stirring for 4 h, the byproducts and impurities were removed by precipitation in diethyl ether three times. The product was dried under vacuum for 24 h. For further purification, the desired copolymer was dissolved in DMF, transferred into a dialysis tube, and dialyzed against deionized water for 2 days to remove traces of residual HBr/CH3COOH solution. White (DTPA-PGA22-bPCL30) and yellow (FA-PGA75-b-PCL30) powders were obtained after lyophilization. Yield: ∼78%. Preparation of Polymer Vesicles. Polymer vesicles were prepared by a solvent switching method at initial concentrations of 0.75, 1.7, 2.7, and 5.0 mg/mL. The DTPA-PGA22-b-PCL30 and FAPGA75-b-PCL30 copolymers were dissolved in DMF, and water was added dropwise into the polymer solution with continuous stirring. Then the solution was transferred into a dialysis tube to dialyze against aqueous solution for 2 days by changing dialysis medium three times each day to remove DMF. Preparation of Gd(III)-Chelated Polymer Vesicles. The Gd(III)-chelated polymer vesicles were prepared by a solvent switching method. DTPA-PGA22-b-PCL30 and FA-PGA75-b-PCL30 copolymers were dissolved in DMF, and the aqueous GdCl3 solution was added dropwise into the polymer solution with continuous stirring. Then the solution was transferred into a dialysis tube to dialyze against tris buffer (0.01 M; pH 7.4) for 2 days by changing dialysis medium three times each day to remove DMF. After that, the self-assembled vesicle solution was dialyzed in 1.0 mM ethylenediaminetetraacetic acid (EDTA) solution to further remove free Gd. In Vitro Stability of Vesicles in PBS and FBS by DLS. The hydrodynamic diameters and the size distribution of the Gd(III)chelated polymer vesicles incubated in phosphate buffered saline (PBS; 0.01 M at pH 7.4) and fetal bovine serum (FBS) at different concentrations for 7 days were evaluated by dynamic light scattering (DLS).40 Preparation of DOX-Loaded Polymer Vesicles. The DOXloaded polymer vesicles were prepared according to a similar method for preparing the Gd(III) chelated polymer. DTPA-PGA22-b-PCL30 (5.0 mg) and FA-PGA75-b-PCL30 (5.0 mg) copolymers were dissolved in 4.0 mL of DMF. Then 8.0 mL of 0.25 mg/mL aqueous DOX·HCl solution was added dropwise into the mixed polymer solution with continuous stirring. The unloaded free drug and DMF solvent were removed by dialysis using a dialysis tube (cutoff Mn = 8000−14 000) according to a reported procedure.43 The dialysis tube was immersed in 1000 mL of tris buffer (0.01 M; pH 7.4) and dialyzed at 25 °C with stirring in a 1000 mL beaker at a rate of 300 rpm. Fresh tris buffer (0.01 M; pH 7.4) was renewed for 6 times in 3.0 h (0.5 h each). The DLE in the dialyzed vesicle solution was determined using a UV−vis spectrophotometer (UV-759S, Q/YXL270) to compare the absorbance of this solution at 483 nm with a calibration curve of aqueous DOX solutions with known concentration. The drug loading content (DLC) and DLE were calculated according to the equations

DLC (%) =

weight of drug encapsulated in vesicles × 100% weight of polymer

DLE (%) =

weight of drug encapsulated in vesicles × 100% weight of drug in feed

cumulative DOX release (%) = M t /M 0 × 100% where Mt is the total amount of DOX released from vesicles at time t and M0 is the amount of DOX initially loaded into the vesicles. A control solution without any polymers was prepared by simply adding 1.0 mg of DOX to 3.0 mL of water in the dialysis tubing. This sample was not dialyzed prior to elution. The elution experiments were carried out immediately after the DOX−vesicle samples were dialyzed. The DOX solution was added to the dialysis tubing and dialyzed against 80 mL of 0.01 M tris buffer at pH 7.4 and 37 °C. After suitable time intervals, 3.0 mL of tris buffer solution was periodically removed to determine the DOX concentration by fluorescence spectroscopy. Cytotoxicity Test. The cytotoxicity of the polymer vesicles without and with Gd and the DOX-loaded vesicles against human normal liver cells (L02) and human live cancer cells (SMMC-7721) were evaluated by measuring the inhibition of cell growth using the Cell Counting Kit-8 (CCK-8) assay, whereby dehydrogenase activities were determined via the reduction of WST-8 (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt) to a yellow product (formazan). The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. L02 and SMMC7721 cells were treated with polymer vesicles at various concentrations from 62.5 to 1000 μg/mL, respectively. First, L02 or SMMC-7721 cells were seeded with equal density in each well of 96-well plates (4000 cells/well) in 100 μL of Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% FBS for 24 h at 37 °C in a humidified 5% CO2-containing atmosphere. Then 20 μL of polymer vesicle and DOX-loaded vesicle solutions with different concentrations were added and incubated with cells for another 48 h. Untreated cells served as a control group. At the end of the treatment, CCK-8 dye was added to each well, and the plates were incubated for another 1 h at 37 °C. Subsequently, the absorbance was measured by dual wavelength spectrophotometry at 450 and 630 nm using a microplate reader. Each treatment was repeated five times. The relative cell viability (%) was determined by comparing the absorbance at 450 nm with control wells containing only cell culture medium. In Vitro Enzymatic Degradation. Biodegradation of the polymer vesicles was in situ conducted in the DLS sample cells at 37 °C. The vesicle solution with lipase or trypsin was degraded on a shaking platform at 37 °C, and then the derived count rates of the vesicle solutions were monitored by DLS at different time intervals.40,41 T1 Relaxivity Measurement. The longitudinal and transverse relaxation times of Gd(III)-chelated polymer vesicles and micromolecule contrast agent DTPA-Gd were measured using a 1.41 T minispec mq 60 NMR analyzer (Bruker, Germany) at 37 °C. Relaxivity values were calculated via linear least-squares fitting of 1/ relaxation time (s−1) vs the Gd3+ concentration (mM). In Vitro MR Imaging. The T1 weights of the polymer vesicle solution with Gd and micromolecule DTPA-Gd at various concentrations were measured with a GE Discovery MR750 3.0 T clinical MRI instrument at room temperature. The longitudinal T1 measurements were acquired using a multiple spin−echo 2D imaging

As aqueous polymer vesicle solution also absorbs UV radiation at 485 nm, the UV absorbance of pure DOX loaded in the vesicles could be obtained by subtracting the UV absorbance of the vesicle solution from the UV absorbance of the DOX-loaded vesicle solution. Then, the concentration of DOX was obtained by calibration curve of DOX· HCl in distilled water (y = 0.01630x + 0.08706, R2 = 0.9998, where y is the UV absorption intensity at 485 nm and x is the concentration of DOX with the unit of μg/mL). C

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Figure 2. Synthetic route to FA or DTPA terminated PGA-b-PCL diblock copolymers with various chain lengths of PGA. Zeta Potential Studies. Zeta potential studies of aqueous polymer vesicle solutions were determined using a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK). No background electrolyte was added. Each reported measurement was conducted for three runs. Transmission Electron Microscopy. TEM images were obtained using a JEOL JEM-2100F instrument at 200 kV equipped with a Gatan 894 Ultrascan 1 k CCD camera. To prepare TEM samples, 5 μL of diluted vesicles suspension was dropped onto a carbon-coated copper grid, and the vesicles without Gd were negatively stained with 1% phosphotungstic acid (pH = 7). The water droplet was allowed to evaporate slowly under ambient conditions before measurement.37,38 Atomic Force Microscopy. AFM was employed to verify the hollow structure of the vesicles. The sample solutions in water at a concentration of 0.03 mg/mL were dropped (10 μL) onto the substrate and dried at room temperature for 12 h. The fresh silicon wafer was used as the sample substrate, which was washed with acetone for 4 times before sample preparation. The observation was conducted on a Seiko (SPA-300HV) instrument operating in tapping mode at 200−400 kHz drive frequency. UV−vis Studies. UV−vis studies were conducted using a UV−vis spectrophotometer (UV-759S, Q/YXL270, Shanghai Precision & Scientific Instrument Co., Ltd.) with a scan speed of 300 nm min−1. The absorbance and transmittance spectra of the DOX-loaded vesicles were recorded in the range of 300−650 nm. Fluorescence Measurements. Fluorescent experiments were carried out to study the cumulative DOX release of DOX-loaded polymer vesicles (excitation at 461 nm and emission at 591 nm) via a Lumina fluorescence spectrometer (Thermo Fisher). Inductively Coupled Plasma Atomic Emission Spectrometry. The total Gd concentrations of the Gd(III)-chelated polymer vesicles and micromolecular DTPA-Gd were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, JY/T 015-1996) after degrading the Gd-chelated vesicles in boiling HCl (35%).

sequence [TR = 1000 ms, TE = 9 ms (5 echoes), SL = 4 mm, FOV = 17 × 17 cm, MA = 256 × 256]. In Vivo MR Imaging. For in vivo MR imaging investigation, male nude mice (∼25 g) bearing liver tumor were employed. Contrast enhanced images of rats were obtained on a GE Discovery MR750 3.0 T clinical MRI instrument. The imaging parameters were TR = 1000 ms, TE = 9 ms (6 echoes), FOV = 12 × 12 mm, MA = 256 × 256, 60° flip angle, and 0.5 mm coronal slice thickness. The T1-weighted contrast agents and the Gd(III)-chelated polymer vesicle solution were injected into the nude mice at a dose of 0.013 mmol/kg via the tail vein. Images were acquired at 15 and 60 min after injection of the Gd(III)-chelated polymer vesicle solution. Images collected at preinjection were also obtained as a control. Characterization. DMF GPC. The molecular weights of the polymers were assessed by gel permeation chromatography (GPC) which were carried out with a Waters Breeze 1525 GPC analysis system with two PL mix-D columns using poly(ethylene oxide) (PEO, purchased from TOSOH) as standard. The mobile phase was DMF with 0.5 M LiBr at a flow rate of 1.0 mL min−1 and 40 °C. 1 H NMR Spectra. Proton nuclear magnetic resonance (1H NMR) spectra were recorded using Bruker AV 400 MHz spectrometers using CDCl3 or DMSO-d6/CH3COOD as solvent and TMS as standard at room temperature. For polymers containing PGA block after lyophilization, two drops of CD3COOD were added to break the hydrogen bond. DLS Studies. DLS studies of aqueous polymer vesicle solutions were determined using a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK) at a fixed scattering angle of 90°. Each reported measurement was conducted for three runs. All the aqueous solutions were analyzed using disposable cuvettes. The data were processed by cumulative analysis of the experimental correlation function, and the particle diameters were calculated from the computed diffusion coefficients using the Stokes−Einstein equation. SLS Studies. The statistic light scattering (SLS) was used to determine the radius of gyration (Rg) and was conducted using ALV/ 5000E laser light scattering (LLS). The data were analyzed using the Zimm plot method to determine Rg.45 D

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RESULTS AND DISCUSSION Syntheses of Diblock Copolymers. Two kinds of amphiphilic diblock copolymers (FA-PGA75-b-PCL30 and DTPA-PGA22-b-PCL30) containing different hydrophilic chain lengths and functional terminal groups were synthesized by sequential ring-opening polymerization (ROP) and amidation reaction (Figure 2). First, the PCL block (polymer 1) was synthesized through the ROP of ε-caprolactone at 110 °C in toluene for 48 h, followed by transforming into a macroinitiator via the deprotection of the Boc end group in TFA/DCM (see 1 H NMR spectra in Figures S1 and S2 of the Supporting Information). Second, ROP of the Bz-Glu-NCA monomer (Figure S3) afforded PBLG75-b-PCL30 (polymer 2; Figure S4) and PBLG22-b-PCL30 (polymer 3; Figure S5). Third, the copolymer with a long PBLG chain (Figure S6) was functionalized with a FA targeting end group by amidation reaction, while the copolymer with a short PBLG chain (Figure S7) was functionalized with a chelating agent, DTPA. Finally, the benzyl ester protecting group in the PBLG side chain was removed by 33% HBr/CH3COOH to afford FA-PGA75-bPCL30 (polymer 4; Figure S8) and DTPA-PGA22-b-PCL30 (polymer 5; Figure S9). The FA and DTPA end-group fidelities were ∼94.5% and 98.6%, respectively, according to the 1 H NMR results in Figures S6 and S7. The detailed calculation procedures of the degrees of polymerization of PCL and PBLG and the fidelities of FA and DTPA groups are shown in the Supporting Information. The properties of all the polymers are summarized in Table 1. The GPC results showed that the polydispersities of all the copolymers are less than 1.5. Nonetheless, the molecular weight of PCL30-NH2 determined by GPC was less than half of that of Boc-NH-PCL30 (Mn = 4506; PDI = 1.13) after deprotection. This is because the adherence of −NH2 groups with the GPC column. Based on the previous theoretical and experimental studies on asymmetrical vesicles,40,46−49 the asymmetrical polymer vesicle showed rapid endocytosis rate and much faster endosomal escape ability compared with the symmetrical vesicle. In this work, the self-assembly of FA-PGA75-b-PCL30 and DTPA-PGA22-b-PCL30 diblock copolymers with different lengths of hydrophilic PGA chains and different functional terminal groups should form an asymmetrical corona. The hydrophobic PCL forms the vesicle membrane. The longer PGA chains with FA terminal groups should form the outer coronas. The shorter PGA chains with DTPA chelating agents should form the inner coronas. The FA groups are designed to be located in the end of the outer coronas of the asymmetrical vesicle for the specific binding with the FA receptors on the surface of cancer cells. The DTPA agents are designed in the inner coronas of the asymmetrical vesicle to chelate T1 contrast agent Gd(III) to increase its contrast effect and to decrease the toxicity. If the FA groups are designed on the both sides of traditional symmetrical vesicles, the inside coronas will be not fully used as the targeting groups, resulting in lower effective in cell uptake. The size of polymer vesicles can be easily controlled by the initial copolymer concentration. As demonstrated in Figure 3, dynamic laser light scattering (DLS) studies revealed that the mean-intensity-averaged hydrodynamic diameter (Dh) of the polymer vesicles increased with the initial copolymer concentration (corresponding to various final copolymer concentrations in pure water), from 128 nm at 0.15 mg/mL,

Figure 3. Control of the size of polymer vesicles by initial copolymer concentration. The vesicles were prepared from diblock copolymers FA-PGA75-b-PCL30 and DTPA-PGA22-b-PCL30 at initial concentrations of 0.75, 1.7, 2.7, and 5.0 mg/mL, corresponding to final concentrations of 0.15, 0.30, 0.50, and 1.0 mg/mL at different pH.

223 nm at 0.30 mg/mL, 289 nm at 0.50 mg/mL to 318 nm at 1.0 mg/mL, while the zeta potentials (ξ) were −35.6 mV to −32.4, −30.2, and −29.4 mV, respectively, indicating excellent stability of vesicles in water.50 The PGA vesicle coronas are pH-sensitive, and its pKa is ca. 5.0. At pH 6.25, the −COOH group in the PGA chains is deprotonated, resulting in a large number of negative charges on the surface of the polymer vesicles (the zeta potential was −32.4 mV). The electrostatic interactions in polyelectrolyte nanoparticles can create an electrostatic field, which fluctuates with the Brownian motion of the vesicles and reversely influences their motion dynamics by slowing it down.51 This results in so-called “slow mode” in the distribution of diffusion coefficients. As a consequence, the measured apparent hydrodynamic diameter (D h) is always much larger than the true value for polyelectrolyte nanoparticles.52,53 Furthermore, the DLS studies of vesicles were performed at a fixed pH value of 7.0. As shown in Figure S10 of the Supporting Information, the same behavior was observed that the Dh of the polymer vesicles increased with the initial copolymer concentration, from 337 nm at 0.15 mg/mL, 372 nm at 0.30 mg/mL, 404 nm at 0.50 mg/mL to 417 nm at 1.0 mg/mL, while the zeta potentials (ξ) were −43.3 mV to −42.6, −40.5, and −39.8 mV, respectively. Compared with the polymer vesicles before adjusting the pH, the Dh’s of the vesicles are larger after increasing the solution pH to 7.0. This is because the higher zeta potential at pH 7.0 leads to stronger electrostatic repulsion, resulting in the stretch of the hydrophilic PGA chains. Furthermore, the higher zeta potential at pH 7.0 aggravates the “slow mode” in the distribution of diffusion coefficients in DLS measurement. The transmission electron microscopy (TEM) studies in Figure 4A and Figure S11 revealed the phase-segregated membrane of polymer vesicles. The dark and white patterns on the vesicles were related to the uneven distribution of phosphotungstic acid (PTA) stains on the vesicles at pH 7.4. The number-averaged diameter of vesicles (∼150 nm by Nano Measurer software) in the TEM images was reasonably smaller than that by DLS analysis (∼223 nm, Figure 3). To accurately evaluate the membrane thickness of vesicles through TEM images according to our recent protocol,37 some stack-up vesicles with a ring-like image on the TEM grid were E

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Figure 4. TEM analysis of polymer vesicles. (A) TEM images of polymer vesicles at a concentration of 0.30 mg/mL. The ring-like regions indicated by black arrows in (A) confirm the hollow structure of stack-up vesicles. The stack-up vesicle magnified in (B) and indicated by the white square was selected for the determination of the membrane thickness. (C) Schematic diagram of stack-up vesicles. (D) The electron transmittance simulation of a rigid hollow sphere, where d is the real membrane thickness. (E) The enlarged electron transmittance simulation chart. (F) The electron transmittance chart relates to the red scan-line in (B), suggesting that the actual membrane thickness is the width (7 nm) between the first inflection point and the peak point.

selected (Figure 4B). The width between the first inflection point and the peak point in the electron transmittance diagram (corresponding to the red scan line) refers to the membrane thickness (ca. 7.0 nm). To further verify the vesicle structure of the self-assemblies, the radius of gyration (Rg) of the polymer vesicles at a concentration of 0.15 mg/mL (the first sample in Figure 3) was measured by static light scattering (SLS). Usually, the Rg/Rh value can predict the particle morphology. For example, a solid sphere has an Rg/Rh of 0.774, while a thin-layer hollow sphere of 1.00.45,54 From the SLS studies shown in Figure 5, the Rg of the polymer vesicles at 0.15 mg/mL was 68 nm. Therefore, the Rg/Rh value is 1.06, indicating a vesicular structure with a hollow cavity. Moreover, no noteworthy change in both hydrodynamic diameter and polydispersity index was observed (Figure S12), implying that the dilution process scarcely affects the morphology of the polymer vesicles.

Preparation of Gd(III)-Chelated Polymer Vesicles. The chelation of Gd(III) to the DTPA-terminated inner coronas of polymer vesicle was accomplished in the process of selfassembly of the polymer vesicles. To completely remove all the redundant Gd3+ ions from the vesicles solution, a dilute EDTA solution was used as a receiving medium during the dialysis process. The ICP-AES analysis revealed that the content of Gd(III) in the vesicle solution was 5.28 μg/mL, which was very close to the theoretical value (5.41 μg/mL) but high enough for MR imaging.55 Zeta-potential studies also revealed the chelation of Gd(III) to the vesicle. Compared with polymer vesicles without Gd(III), the ξ potential of the Gd(III)-chelated vesicles decreased from −29.4 to −26.0 mV due to the electrostatic interaction between Gd(III) ions and the −COO− groups in the PGA chains of inner vesicle coronas. The Dh of Gd(III)chelated vesicles was 318 nm (Figure 6C), which was the same as the vesicles before Gd chelation. Furthermore, the strong contrast in the TEM images of polymer vesicles without any staining clearly confirmed the chelation of Gd3+ on the collapsed vesicles with a hollow structure (Figure 6B and Figure S13). Otherwise, it is not possible to see the regular structure by this conventional TEM without staining of samples. The corresponding schematic representation of vesicles indicated by a red square in the TEM image in Figure 6A demonstrated the collapse of Gd(III)chelated polymer vesicle. Moreover, atomic force microscopy (AFM) study further confirmed the formation of the Gd(III)-chelated vesicle (Figure S14). The marked area had a width of nearly 300 nm, which was much higher than the height of ∼35 nm. This is due to the incomplete collapse of those soft and deformable vesicles on the silicon substrate.56 In Vitro Stability of Gd(III)-Chelated Polymer Vesicles in PBS and FBS. To evaluate the physiological stability of

Figure 5. SLS study of polymer vesicles: Zimm plot and Rg determination. The Rg/Rh value of 1.06 indicates the hollow structure of vesicles. F

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Figure 6. TEM and DLS studies of Gd(III)-chelated polymer vesicles. (A) Schematic representation of a collapsed Gd-chelated polymer vesicle in the TEM image in (B). The yellow dotted cycle in (B) corresponds to the double vesicle membrane in (A). (C) DLS study of Gd(III)-chelated polymer vesicles at a polymer concentration of 1.0 mg/mL and pH 7.4. The vesicles in (B) were not stained before TEM analysis.

Figure 7. (A) Schematic representation of enhanced loading of DOX by electrostatic interaction with vesicles. (B) UV−vis spectra of vesicles without DOX (a) and DOX-loaded vesicles (b) at 0.6 mg/mL.

Figure 8. Cytotoxicity, drug release, and antitumor activities studies. Cytotoxicity of polymer vesicles and Gd-chelated polymer vesicles against normal liver L02 cells (A) and cancer liver SMMC-7721 cells (B) at various concentrations. L02 cells and SMMC-7721 cells were incubated with polymer vesicles for 48 h. The relative cell viabilities were determined by CCK-8 assays (n = 5). (C) Cumulative release profiles of DOX in the absence and in the presence of polymer vesiclesat 37 °C: (a) DOX in the absence of vesicles at pH 7.4; (b) DOX-loaded vesicles at pH 5.0; (c) DOX-loaded vesicles at pH 7.4. (D) Antitumor activities and IC50 values of (a) free DOX and (b) DOX-loaded vesicles against SMMC-7721 cells. The relative cell viabilities were determined by CCK-8 assays (n = 5).

Gd(III)-chelated polymer vesicles were placed in 0.01 M PBS at pH 7.4, there was no obvious size change even after 80 h. In Figure S15B, compared with the vesicle size distribution in pure

Gd(III)-chelated polymer vesicles, the vesicles in PBS and different FBS concentrations were monitored by DLS in vitro for more than 80 h. As shown in Figure S15A, when the G

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Figure 9. In vitro MRI studies revealed high r1 relaxivity of Gd(III)-chelated polymer vesicles. (A) T1-weighted spin-echo MR images recorded for aqueous solutions of Gd(III)-chelated polymer vesicles and commercial DTPA-Gd at various concentrations. The T1 relaxation rate (1/T1, black) and the T2 relaxation rate (1/T2, red) as a function of Gd concentration are recorded in (B) Gd(III)-chelated polymer vesicles and (C) DTPA-Gd.

according to the calibration curve of DOX in aqueous solution (y = 35.1035x + 11.7306, R2 = 0.9996, where y is the fluorescence intensity and x is the concentration of DOX with the unit of 0.01 μg/mL).44 Such DLE value was too low for a vesicle with a water pool. This is because the fluorescence quenching of the large amount of DOX adsorbed on the vesicle corona via electrostatic interactions. Therefore, the UV−vis spectroscopy was used for more accurate evaluation than fluorescence spectroscopy, giving a DLE of 52.6%. The in vitro kinetic release profiles of free DOX (without vesicles) at pH 7.4 and DOX-loaded polymer vesicle solutions at pH 7.4 and pH 5.0 were carried out in 0.01 M tris buffer at 37 °C, respectively. As shown in profiles b and c in Figure 8C, after 25 h, the DOX release content at pH 5.0 was 15% higher compared with the same DOX-loaded vesicles but kept at pH 7.4. This was because the protonation of the −COO− groups in the PGA chains at pH 5.0 accelerated the DOX release from the vesicle corona. The control experiment utilizing an aqueous solution of 0.33 mg/mL DOX in the absence of any vesicles indicated rapid drug elution (profile a in Figure 8C), and the DOX release percentage was about 70% after 1.5 h, as expected. The release rates under those conditions indicated significantly retarded release of the drug at pH 7.4 due to its entrapment within the vesicles (see profiles a and c), suggesting that pH-triggered disassembly of vesicles was the dominant drug release mechanism (see profiles b and c).

H2O, except for the appearance of two peaks for FBS protein size at around 5−70 nm, no obvious size change for vesicles (320 nm) was observed upon exposure to PBS and different concentrations of FBS from 5% to 10%. This phenomenon demonstrated the high stability of the Gd(III)-chelated polymer vesicles in PBS and FBS. Loading and in Vitro Release of DOX with Polymer Vesicles. DOX loading and in vitro release experiments were performed to evaluate the potential applications of polymer vesicles as a drug delivery vehicle. As shown in Figure 7, the polymer vesicles with PGA coronas may improve the DLE of DOX·HCl due to the electrostatic interactions between the −COO− groups of PGA and the −NH3+ of DOX·HCl [pKa,DOX·HCl = 8.25].42 The zeta potential of the DOX-loaded polymer vesicles was −20.5 mV, which was nearly 9.0 mV lower than the empty vesicles (−29.4 mV) due to the consumption of −COO− groups. To evaluate the DOX loading efficiency in the polymer vesicles, the DOX-loaded vesicle solution was investigated by monitoring the absorption peak of the solution at 485 nm by UV−vis spectroscopy, as shown in Figure 7. The DLE was ca. 52.6%, which was much higher than that of most reported drug delivery systems including vesicles. First, fluorescence experiments were carried out to calculate the DLE of DOX-loaded polymer vesicles. The fluorescence intensity of DOX-loaded polymer vesicles (excitation at 461 nm and emission at 591 nm) was recorded via a Lumina fluorescence spectrometer. However, the DLE was only 8.7% H

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Macromolecules Cytotoxicity Tests of Polymer Vesicles. The cytotoxicities of the polymer vesicles and Gd-chelated polymer vesicles against human normal liver cells (L02) and human liver cancer cells (SMMC-7721) were evaluated by using a sensitive colorimetric CCK-8 (Cell Counting Kit-8) assay over 48 h. The cell viabilities were calculated using the ratio of the numbers of L02 and SMMC-7721 cells of the treated group over the untreated control (Figure 8). As expected, the polymer vesicles (without Gd) and the Gd-chelated polymer vesicles treated with L02 and SMMC-7721 cells remained high metabolic activities at various concentrations (≤1000 μg/mL) after 48 h, suggesting low cytotoxicities of both the polymer vesicles and Gd-chelated polymer vesicles. Antitumor Activities of DOX-Loaded Polymer Vesicles. The cytotoxicity tests confirmed that the polymer vesicles without and with Gd had no cytotoxicity against human L02 normal cells and SMMC-7721 liver cancer cells when the concentration was less than 1000 μg/mL (Figure 8). The antitumor activities of DOX-loaded polymer vesicles were investigated in SMMC-7721 cells by CCK-8 assay with free DOX as control. The proper concentration of DOX to realize the ideal therapeutic efficacy (cell viability of SMMC-7721 cells is less than 20%) should be more than 0.5 μg/mL (Figure 8). As shown in Figure 8D, the DOX-loaded vesicles retained extremely high drug efficacy, and the IC50 (inhibitory concentration to produce 50% cell death; calculated by SPSS) of DOX·HCl from the DOX-loaded polymer vesicles was 0.0686 μg/mL (equiv 0.1304 μg/mL DOX-loaded polymer vesicles), which was only 1/3 of the value from the free DOX without vesicles (IC50 = 0.2215 μg/mL). The high antitumor activity of DOX-loaded polymer vesicles indicates that the DOX·HCl has been delivered and released into the nucleus of SMMC-7721 cells with a high efficiency. This is due to the specific binding of FA groups on the surface of the polymer vesicles with the FA receptors on the cancer cell membrane, resulting in DOX·HCl rapidly delivered and released into the nucleus of SMMC-7721 cells with a high efficiency. Therefore, these DOX-loaded polymer vesicles have great potential in biomedical applications. In Vitro MR Imaging of Gd(III)-Chelated Polymer Vesicles. Gd(III)-based T1-type MRI contrast agent can considerably improve the diagnostic sensitivity toward malignant tissues over normal ones. 57−59 It has been demonstrated that micromolecular contrast agents of Gd(III) complex often suffer from limited sensitivity and unsatisfactory further development in clinical applications.60,61 In an effort to improve the contrast enhancement performances, several previous literature reports have documented that when Gd(III) complexes are covalently conjugated onto polymers and polymer assemblies, the positive MR imaging contrast enhancement can be effectively improved.62 In the current study, we fabricated a novel kind of polymer vesicles with FA targeted groups on the outside corona and DTPA chelating groups on the inside corona as a nanoplatform to increase the T1 relaxivity of MR imaging contrast agents. Typical T1 weighted spin-echo MR images recorded for Gd(III)-chelated polymer vesicles at various Gd(III) concentrations in aqueous solution are shown in Figure 9A. The performance of a micromolecular DTPA−Gd complex was measured as a control. Upon gradually increasing the concentration of DTPA-Gd, a lightly positive contrast enhancement of MR signals can be observed by the reinforcement of the spot brightness.

To determine the relaxivity value of the Gd(III)-chelated polymer vesicles and the DTPA−Gd complex in aqueous solution, longitudinal (T1) and transverse proton relaxation times (T 2 ) were measured as a function of Gd(III) concentration at 1.41 T and 37 °C. As shown in Figure 9B, the Gd(III)-chelated polymer vesicles exhibited extremely high r1 and r2 values of 42.93 and 78.24 mM−1 s−1, respectively. The r2/r1 value of 1.82 indicates that the Gd(III)-chelated polymer vesicles has a significant advantages as a positive contrast agents.63 It is noteworthy that the r1 value of this Gd(III)-chelated polymer vesicle is much higher than the reported Gd-based nanoparticles, reaching nearly 8-fold enhancement compared with micromolecular DTPA-Gd complex (4.568 mM−1 s−1, shown in Figure 9C). The transverse relaxivity (r2 = 78.24 mM−1 s−1) of this Gd(III)-chelated polymer vesicle is also significantly higher than that of the micromolecular DTPA−Gd complex in solution (r2 = 4.936 mM−1 s−1). Such high relaxivity of the Gd(III)-chelated polymer vesicles is particularly useful in dosage reduction, which is desirable for patients with weak kidney function. In Vivo MR Imaging of Gd(III)-Chelated Polymer Vesicles. Based on the above results, the in vivo MR imaging effect of Gd(III)-chelated polymer vesicles in nude mice bearing liver tumor was evaluated, as indicated by red circles in Figure 10. Before and after the intravenous injection of the

Figure 10. In vivo MRI studies of Gd(III)-chelated polymer vesicles revealed dramatic positive contrast enhancement in the tumor sites with prominent accumulation and positive enhancement primarily in the liver, bladder, and kidney after 1 h. Representative image of nude mouse bearing liver tumor (red circle indicated, A) and T1-weighted MR images recorded for the mouse acquired preinjection (B), 15 min (C) and 60 min (D) after intravenous injection via the tail vein of 2.0 mL of 3.0 mg/mL Gd(III)-chelated polymer vesicles.

resulting vesicle solution, the MR imaging of the mice was acquired at specific time intervals. The MR signal intensity in the blood vessel directly reflects the intravenous injection of the suspension via the tail vein after 15 min (Figure 10C). Also, some lightened areas of T1-weighted MR images were found around the liver tumor site, rather than inside the tumor. This is because the blood supply in the tumor is not as abundant as the surroundings. Within 60 min after injection, an obvious enhancement of the signal intensities around the tumor site was observed, while it gradually disappeared in the blood vessel. Meanwhile, the liver, kidney, and bladder clearly showed positive contrast enhancement, indicating the concentration of Gd-chelated polymer vesicles in these organs and excretion of the contrast agents via glomerular filtration. Ultrasensitive T1 MRI and Efficient Cancer Targeted Delivery. The efficacy of a T1-weighted contrast agent is defined by its relaxivity, r1, i.e., the increment of the total paramagnetic relaxation rate enhancement of the free water protons (R1p) against the concentration of paramagnet (mM−1 I

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Macromolecules s−1) (eq 1). The r1 value of the Gd(III)-chelated polymer vesicles (42.93 mM−1 s−1) is ca. 8-fold higher than that of the control group (4.568 mM−1 s−1). The reason for the high r1 value is because of the slow tumbling of the polymer vesicles in water (a.k.a. long rotational correlation time, high τR). The T1 relaxivity depends on the inner sphere (R1pIS) and outer sphere (R1pOS) relaxation mechanisms (eq 2). Usually through chemical methods, we have greatest control over the larger inner-sphere contribution which consists of interactions between Gd and directly bound water molecules, rather than the outer sphere that consists of interactions with second sphere and closely diffusing water molecules. The parameters contributed to R1pIS are shown in eq 3, where C is the molar concentration of paramagnetic compound, q is the number of bound water molecules, T1M is the longitudinal relaxation time of the bound water protons, and τm is the mean residence lifetime in coordination sites. These parameters can be finetuned to give a more efficient contrast agent.26

r1 = R1P/[Gd]

(1)

IS OS R1P = R1P + R1P

(2)

IS = R1P

Cq 1 55.6 T1M + τm

enzymatic degradation of the hydrophobic PCL block by lipase (a family of enzymes showing a high activity for ester chain scission) and hydrophilic PGA block by trypsin (a family of enzymes showing a high activity for amide chain scission). At a shaking rate of 100 rpm and 37 °C, the biodegradation process can be monitored by measuring the decrease in the derived count rate of DLS with time, which is proportional to both the molecular weight and the concentration of vesicles. As shown in Figure S16, in the presence of 0.80 mg/mL of trypsin, about 95% of polymer vesicles degraded within 70 h, nearly 30% more than that with 0.70 mg/mL of lipase. This is because of the degradation of PGA corona, resulting in instability and aggregation even precipitation of polymer vesicles. Finally, the polymer vesicles were degraded into short chains and micromolecules after several days.

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CONCLUSIONS In summary, we have successfully synthesized a cancer targeted noncytotoxic asymmetrical polymer vesicle based on two kinds of diblock copolymers FA-PGA75-b-PCL30 and DTPA-PGA22-bPCL30. TEM, DLS, and SLS confirmed the formation of polymer vesicles. The pH-sensitive PGA vesicle coronas can significantly enhance the loading and stabilization of the anticancer drug DOX·HCl via electrostatic interactions (DLE ∼ 52.6%). The DOX-loaded vesicles exhibited slow release at neutral conditions but fast release at acidic conditions. Furthermore, antitumor experiments confirmed that the FA terminal groups in the outer coronas can greatly improve the cancer targeting and anticancer effect. Moreover, the DTPA in the inner coronas of vesicles can effectively chelate T1 MRI contrast agent Gd(III), affording 8-fold enhancement in the T1 relaxivity. This “chelating-just-inside” strategy also significantly minimized the toxicity of Gd(III). Finally, in vivo MR imaging investigation in nude mice revealed dramatic positive contrast enhancement in the tumor sites. Overall, such multifunctional asymmetrical polymer vesicle provided effective cancer targeting, high drug loading efficiency, noncytoxicity, and ultrahigh T1 MRI sensitivity on a single platform, suggesting endless potential applications for theranostics and nanomedicine.

(3)

At the magnetic field strengths used in MRI scans, the longitudinal relaxation time of the bound water protons T1M is dominated by the rotational correlation time τR. The slower the Gd(III) complex tumbles, the longer the correlation time. As a result, this leads to faster relaxation rates (i.e., shorter longitudinal relaxation time, lower T1M) and hence higher relaxivities. A common approach to decrease the tumbling rate and to lengthen the τR is to attach the Gd(III) complex to slowly tumbling polymers5,8,28,64,65 and polymer self-assemblies.31,32 The rotational correlation time τR can be estimated in a number of ways. For spherical structures, e.g., polymer micelles and vesicles, eq 4 can be used to calculate the τR where a is the radius of the sphere and η is the viscosity.18 τR = 4πa3η /3kT

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(4)

In our work, the radius (a) of the Gd-chelated polymer vesicle is estimated more than 150 nm from the DLS studies (Figure 6C), which is much higher than the reported nanoparticles31,32 and the micromolecular DTPA-Gd. Therefore, according to eqs 1−3, the longitudinal relaxation time of the bound water protons T1M of this kind of contrast agent with a fast relaxation rate is extremely short due to the long rotational correlation time τR, resulting in a significant enhancement of the relaxivity, r1. In the in vivo MRI measurement (Figure 10), the fast appearance of the high positive contrast and the increasing contrast enhancement around the tumor site strongly suggests the tumor target of the FA-decorated polymer vesicles. Consequently, the corresponding polymer vesicles-based contrast agents in the same Gd(III) concentration range exhibit substantially enhanced MR signal contrast, confirming that attaching multiple micromolecule Gd(III) complexes into polymer vesicles can significantly improve the contrast effect in the tumor site. In Vitro Enzymatic Degradation of Polymer Vesicles. The polymer vesicles prepared from diblock copolymers FAPGA75-b-PCL30 and DTPA-PGA22-b-PCL30 were subjected to

ASSOCIATED CONTENT

S Supporting Information *

Materials and additional characterizations. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Fax +86-21-6958 4723; Ph +8621-6958 0239 (J.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.D. is supported by Shanghai 1000 Plan, Eastern Scholar Professorship, NSFC (21174107 and 21374080), Ph.D. Program Foundation of Ministry of Education (20110072110048), and Fok Ying Tong Education Foundation (132018).

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DOI: 10.1021/ma502255s Macromolecules XXXX, XXX, XXX−XXX