Theranostic Vesicles Based on Bovine Serum Albumin and Poly

Apr 1, 2014 - Poly(ethylene glycol)-block-poly(L‑lactic-co-glycolic acid) for ... University, 4800 Caoan Road, Shanghai 201804, People,s Republic of...
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Theranostic Vesicles Based on Bovine Serum Albumin and Poly(ethylene glycol)-block-poly(L‑lactic-co-glycolic acid) for Magnetic Resonance Imaging and Anticancer Drug Delivery Qiuming Liu,†,§ Hongshi Zhu,†,§ Jingya Qin,† Haiqing Dong,*,‡ and Jianzhong Du*,† †

School of Materials Science and Engineering, Tongji University, 4800 Caoan Road, Shanghai 201804, People’s Republic of China Shanghai East Hospital, The Institute for Biomedical Engineering and Nano Science, Tongji University School of Medicine, No. 150 Jimo Road, Shanghai 200120, People’s Republic of China

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ABSTRACT: Presented in this article is the preparation of a new theranostic vesicle which exhibits excellent in vitro and in vivo T1 magnetic resonance (MR) imaging contrast effect and good anticancer drug delivery ability. The theranostic vesicle has been easily prepared based on an amphiphilic biocompatible and biodegradable dibock copolymer, poly(ethylene glycol)-block-poly(L-lactic-co-glycolic acid) (PEG-b-PLGA) and bovine serum albumin-gadolinium (BSA-Gd) complexes. Dynamic light scattering (DLS), transmission electron microscopy (TEM), UV−vis spectroscopy, and inductively coupled plasma atomic emission spectroscopy (ICP-AES) measurements confirmed the formation and physiological stability of BSA-Gd@PEG-b-PLGA vesicles. Furthermore, the in vitro and in vivo MR imaging experiments revealed their excellent T1-weighted MR imaging function. Red blood cell hemolysis and cytotoxicity experiments confirmed their good blood compatibility and low cytotoxicity. Doxorubicin (DOX) loading and release experiments indicated a more retarded release rate of DOX in those theranostic vesicles than sole PEG-b-PLGA nanoparticles without BSA. Overall, this new biocompatible and biodegradable vesicle shows promising potential in theranostic applications.

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containing nanohybrids, as shown in Scheme 1. As Gd3+ was hardly incorporated to polymers without abundant ligand such as PEG-b-PLGA, bovine serum albumin (BSA), an important

INTRODUCTION Theranostics, a term derived from therapy and diagnostics to link the fields of diagnostics and therapeutics, is expected to improve patient outcomes and safety through a more personalized approach to medicine.1 Therefore, the field of theranostic systems has gained much attention during past decades.2−4 For example, a series of delivery vehicles for theranostics have been developed with a special interest in combining therapeutic molecules with imaging agents in one entity by physical entrapment or chemical conjugation.2,5−19 Poly(ethylene glycol)-block-poly(L-lactic-co-glycolic acid) (PEG-b-PLGA) has been widely used in biomedicine because both PEG and PLGA segments are approved by FDA for clinical use.20−23 Unfortunately, this copolymer was largely used as conventional drug carriers due to its limited functional groups.23−25 However, as an important T1 magnetic resonance imaging (MRI) agent, gadolinium (Gd) has many advantages over T2 contrast agents. For example, T1 MRI can distinguish the void from the contrast agent from other signal voids,26 with a wider dynamic range than T2 MRI.27−31 However, Gd3+ also shows disadvantages such as high cytotoxicity, nonspecificity, rapid excretion in vivo, etc. Therefore, it will be of great interest to develop new T1 MRI contrast agents with high efficiency and low cytoxicity for biomedical applications. Considering that metal-based agents such as Gd-containing nanoparticles have excellent MR imaging effect,32−35 we propose herein a new theranostic system by combining the advantages of PEG-b-PLGA block copolymer and Gd© 2014 American Chemical Society

Scheme 1. Preparation of Theranostic DOX-Loaded BSAGd@PEG-b-PLGA Vesicles

Received: December 8, 2013 Published: April 1, 2014 1586

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loading efficiency (DLE) and the drug loading content (DLC) were calculated according to the following formulas:

blood protein composed of hundreds of amino acid residues as well as many active functional groups such as carboxyl groups and thiol groups,36 was employed to chelate with Gd3+ to form biomineralized nanohybrids, which were then encapsulated in PEG-b-PLGA nanoparticles via a physical entrapment. Doxorubicin (DOX), a hydrophobic anticancer drug, was also encapsulated in the PEG-b-PLGA vesicles. The resulted theranostic BSA-Gd@PEG-b-PLGA vesicles showed good biocompatibility and excellent T1-weighted MR imaging effect, as well as effective cancer cell inhibition ability.

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DLC(%) = mass of drug encapsulated in nanoparticle /mass of polymer × 100%

DLE(%) = mass of drug encapsulated in nanoparticle /mass of fed drug × 100% The DLC and DLE for BSA-Gd@PEG-b-PLGA vesicles are 3.14% and 20.9%, respectively. In a control experiment, the DLC and DLE for PEG-b-PLGA nanoparticles without BSA-Gd nanohybrids were 3.47% and 23.1%, respectively, which were evaluated by using the aqueous solution of DOX instead of BSA-Gd solution with DOX. Drug Release Behavior of DOX-Loaded BSA-Gd@PEG-b-PLGA Vesicles. The suspension of the DOX-loaded BSA-Gd@PEG-b-PLGA vesicles after removing the free drug was divided into three parts, which were put into three new dialysis tubes immediately to evaluate the drug release behavior in parallel. The drug release process was carried out by dialyzing 5.0 mL of DOX-loaded nanoparticles in the dialysis tube against 150 mL of tris buffer (0.01 M; pH 7.4) in a 250 mL beaker at 37 °C and stirring at the rate of 190 r/min. The volume of the buffer solution in the beaker outside of the dialysis tube was maintained around 150 mL during the measurement. At different time intervals, the liquid in the beaker was measured with a fluorescence spectroscopy (excitation at 461 nm and emission at 591 nm) and the cumulative release curve of DOX was obtained. In Vitro and in Vivo MR Imaging. For in vitro MRI of BSA-Gd@ PEG-b-PLGA vesicles at various concentrations, their T1 relative signal intensity was detected with a 1.5 T medical superconducting MRI system (T1 weighted imaging, SE sequence; TR, 600 ms; TE, 12 ms). The relaxivity values were calculated via linear least-squares fitting of 1/relaxation time (s−1) vs the Gd3+ concentration (mM), which were determined by MR scanner and ICP-AES, respectively. As control groups, the PEG-b-PLGA nanoparticles without BAS and aqueous BSA solution were also subjected to MRI test. For in vivo MRI evaluation, female nude mice (∼20 g) bearing tumor were used. The BSA-Gd@PEG-b-PLGA nanoparticles were injected into the mice via the tail vein. Images were collected at different time intervals postinjection of vesicles. Images collected at preinjection were also obtained as a control. The relative signal intensities at preinjection and postinjection in the region of interest (ROI) were recorded for analysis. Hemolytic Test. The blood compatibility of the BSA-Gd@PEG-bPLGA nanoparticles was determined by testing hemoglobin release from the fresh blood of mice. Various concentrations of vesicles were made by dilution with tris buffer (pH 7.4). A total of 1.0 mL of diluted vesicles suspension was added into 1.0 mL of mouse blood. The mixture was then put in a shaker at 37 °C for 60 min. Subsequently, the mixture was subjected to centrifugation at a rate of 4000 r/min in a centrifugal machine for 5 min. The reference liquid was the supernatant made by the blood of mice only at the same conditions. The transmissions of the supernatant were measured by a UV−vis spectrometer at 540 nm deducting the absorbance of reference liquid.39−42 Cytotoxicity Test. Cytotoxicity of BSA-Gd@PEG-b-PLGA vesicles against HeLa cells was determined by a standard MTT assay. Hela cells were seeded in a 96-well cell-culture plate at 5 × 103 cells per well using DMEM medium supplemented with 10% FBS and incubated at 37 °C for 24 h under 5% CO2 humid conditions. The medium in each well was then replaced with DMEM containing BSA-Gd@PEG-bPLGA(100 μL per well, containing 1% HEPES) at concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.813, and 3.906 μg/mL. After incubation for 24 h, 15 μL of MTT (5 mg/mL) was added to each well and incubated for another 4 h. Then the medium was removed and washed with PBS 2 times before addition of dimethyl sulfoxide (DMSO, 150 mL per well); the assay plate was allowed to shake at room temperature in dark for 10 min. The optical density (OD) was

EXPERIMENTAL METHODS

Materials. Methoxy poly(ethylene glycol)-block-poly(lactic-coglycolic acid) (PEG5k-b-PLGA5k) was purchased from Jinan Daigang Biomaterial Co., Ltd. All chemicals were purchased from SigmaAldrich and used as-received. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin−streptomycin, trypsin, Dulbecco’s phosphate buffered saline (DPBS), and 3-(4, 5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Gibco Invitrogen Corp. Tris buffer (0.01 M; pH 7.4), doxorubicin hydrochloride (DOX·HCl), and the dialysis tubing (molecular weight cutoff 6000−8000 Da) were purchased from Aladdin. Animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee of Tongji University. Methods. Synthesis of BSA-Gd Nanohybrids. In a typical experiment, aqueous gadolinium chloride solution (20 mL, 1.0 mM) was slowly added to a BSA solution (20 mL, 5.0 mg/mL) under vigorous stirring. NaOH solution (2.0 mL, 0.01 M) was introduced 2.0 min later. The reaction was allowed to proceed under vigorous stirring at room temperature. After 12 h, the solution was dialyzed against deionized water with refreshing water timely to remove excess Gd3+. Preparation of Theranostic BSA-Gd@PEG-b-PLGA Vesicles by PEG-b-PLGA Diblock Copolymer and BSA-Gd Nanohybrids. In a typical procedure, PEG-b-PLGA copolymer (15.0 or 7.5 mg) was dissolved into 5.0 mL THF. Then BSA-Gd solution (10 mL, 1.0 mg/ mL; pH 7.9) was tuned by adding aqueous HCl solution to pH 7.4, then added to the copolymer solution at a rate of 1 drop/10 s under vigorous stirring. After 2 h stirring at room temperature, the solution was dialyzed against water with a dialysis tube (MWCO 6000−8000). The concentration of Gd3+ inside the vesicles was measured with inductively coupled plasma atomic emission spectroscopy (ICP-AES). Fifty microliters of vesicle solution was diluted to 10.0 mL with a 3% w/v nitrate solution. The resulting solution was used for ICP-AES measurement. In Vitro Stability of BSA-Gd@PEG-b-PLGA Vesicles in PBS and FBS. The hydrodynamic diameters and size distribution of BSA-Gd@ PEG-b-PLGA vesicles incubated in PBS (0.01 M at pH 7.4) and FBS at different concentrations for 80 h were evaluated by dynamic light scattering (DLS). Loading Anticancer Drug into BSA-Gd@PEG-b-PLGA Vesicles. The loading of anticancer drug (DOX) was achieved according to the following protocol. Ten milligrams of PEG-b-PLGA copolymer was dissolved in 5.0 mL of THF, then 19.5 mL of BSA-Gd solution (1.0 mg/mL) with 0.5 mL of DOX solution (ca. 3.0 mg/mL) was placed into the above polymer solution under vigorous stirring. The obtained mixture was adjusted to pH 7.4 by using aqueous NaOH solutions. After stirring at room temperature for 12 h, the mixture was subjected to dialysis to remove the unloaded free drug according to the reported procedures.37,38 The dialysis tube was immersed in 500 mL of tris buffer (0.01 M; pH 7.4) and dialyzed at 37 °C with 300 r/min of stirring. The same fresh tris buffer was renewed for 5 times in 2.5 h (0.5 h each). The amount of DOX before and after dialysis was calculated from the fluorescent intensity of the mixture solution at an emission wavelength of 591 nm excited at 461 nm. The concentration of DOX (c) in tris buffer (0.01 M; pH 7.4) was obtained from the calibration curve: c (μg/mL) = (I − 11.7306)/35.1035, where I is the fluorescent intensity at an emission wavelength of 591 nm. The drug 1587

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measured at 570 nm with a Tecan Infinite M200 monochromatorbased multifunction microplate reader. The following formula was used to calculate the viability of cell growth: relative cell viability (%) = (ODtreated /ODcontrol) × 100, where OD control was obtained in the absence of BSA-Gd@PEG-b-PLGA. The incubation of cells with DOX-loaded BSA-Gd@PEG-b-PLGA vesicles as well as controls such as PEG-b-PLGA nanoparticles and free DOX were also carried out using MTT assay. Characterization. 1H NMR spectra were recorded using a Bruker AV 400 MHz spectrometer at ambient temperature using CDCl3 or D2O as solvents. Transmission electron microscopy (TEM) images were obtained using a JEM-2100 electron microscope equipped with a Gatan 1K × 1K digital camera operating at an acceleration voltage of 200 kV. To prepare a TEM sample of BSA-Gd@PEG-b-PLGA vesicle, 5 μL of the diluted aqueous sample suspension was placed on a carbon-coated copper grid. The water droplet was allowed to evaporate at 20 °C. DLS studies were conducted using a Zetasizer Nano ZS90 instrument (Malvern Instruments) equipped with a multipurpose autotitrator (MPT-2) at a fixed scattering angle of 90°. The data were processed by cumulants analysis of the experimental correlation function, and particle diameters were calculated from the computed diffusion coefficients using the Stokes−Einstein equation (Dh = kT/ (3πηD)). Each reported measurement was conducted for three runs. The UV−vis absorption spectra of samples were acquired using a U3010 spectrophotometer (HITACHI).

Figure 2. Size distributions of PEG-b-PLGA nanoparticles using different organic cosolvents at 0.5 mg/mL determined by DLS at 25 °C.

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RESULTS AND DISCUSSION Synthesis of BSA-Gd Nanohybrids. The synthetic route of BSA-Gd solution is described in Scheme 1. Briefly, the

Figure 3. Size distributions of BSA-Gd@PEG-b-PLGA vesicles prepared at 0.5 mg/mL of PEG-b-PLGA at pH 7.4 (determined by DLS at 25 °C).

Figure 1. Size distributions of pure BSA and BSA-Gd nanohybrids at 2.0 mg/mL in water determined by DLS at 25 °C. Figure 4. TEM images (left) and DLS study (right) of as-prepared BSA-Gd@PEG-b-PLGA vesicles prepared at 0.5 mg/mL of PEG-bPLGA block copolymer.

aqueous gadolinium chloride solution is added to the BSA solution slowly under vigorous stirring. The BSA-Gd mixture solution appears cloudy and opaque before the addition of NaOH, indicating the interaction between the BSA and the metal ion Gd3+. After adding NaOH, the solution becomes optically transparent. The BSA protein then undergoes structural changes and transforms into its unfolded or tertiary configuration at alkaline environment.43 Such unfolded or denatured BSA is much efficient in controlling the crystal growth. After 12 h, the solution is dialyzed against deionized water to remove excess Gd3+. The nanohybrids suspension with BSA and Gd(OH)3 and Gd2O3 were obtained. In contrast, only large precipitation was observed in the absence of BSA, indicating that BSA is necessary for stabilizing BSA-Gd nanohybrids. The sizes of the pure BSA and BSA-Gd nanohybrids were evaluated by DLS (Figure 1), which suggested that the average

size of the BSA-Gd nanohybrids was slightly smaller than pure BSA, as a result of the complex interaction between BSA and Gd3+. Synthesis and Characterization of BSA-Gd@PEG-bPLGA Vesicles. The organic cosolvents usually have an impact on the morphology of self-assemblies because they are related to the self-assembly behavior of core-forming block and corona repulsions.44 The size of the micelle core can be determined by the solubility parameter of the hydrophobic block and the property of the solution, while the dielectric constant of the solvent controls the repulsion of the corona among polymer chains.45 Therefore, the choice of solvent is quite important in controlling the size of micelles.39,44 In our research, different 1588

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Figure 5. (A) Effect of the time on the size of BSA-Gd@PEG-b-PLGA vesicles in 0.01 M PBS at pH 7.4. (B) Size distribution of BSA-Gd@PEG-bPLGA vesicles in FBS, PBS, and H2O.

Figure 6. UV absorbance of BSA (c) and BSA-Gd@PEG-b-PLGA suspension prepared from different concentration of PEG-b-PLGA copolymer: (a) 1.0 and (b) 0.5 mg/mL, respectively.

solvents such as DMF and THF, which are miscible with water, were used to study the self-assembly of amphiphilic PEG5k-bPLGA5k copolymer at neutral pH. Nanoparticles with different sizes were obtained using two different solvents, as shown in Figure 2. In this article, the theranostic BSA-Gd@PEG-b-PLGA vesicles were prepared in THF/water mixture. The DLS measurements were performed to analyze the diameters of selfassemblies, as shown in Figure 3. At pH 7.4, the mean hydrodynamic diameter (Dh) was about 280 nm at 0.5 mg/mL of PEG-b-PLGA diblock copolymer. Transmission electron microscopy (TEM) revealed the vesicular morphology of BSA-Gd@PEG-b-PLGA vesicles, as indicated by the dotted circles in Figure 4. The small particles in the TEM image are the broken vesicles with fragmented BSA-Gd nanohybrids during the preparation of TEM samples. In order to evaluate the physiological stability of BSA-Gd@ PEG-b-PLGA vesicles, the vesicles in PBS and different FBS concentrations were monitored by DLS in vitro for more than 80 h. As shown in Figure 5A, when BSA-Gd@PEG-b-PLGA vesicles were placed in 0.01 M PBS at pH 7.4, there was no obvious size change even after 80 h. In Figure 5B, compared with vesicle size distribution in pure H2O, except for the appearance of two peaks for FBS protein size at around 5−20 nm, no obvious size change for vesicles (280 nm) was observed upon exposure to PBS and different concentrations of FBS from 5% to 10%. This phenomenon demonstrated the high

Figure 7. (A) In vitro MR imaging of (from left to right) PEG-bPLGA (1.0 mg/mL), BSA (1.0 mg/mL), BSA-Gd@PEG-b-PLGA (1.0 mg/mL), BSA-Gd@PEG-b-PLGA (0.5 mg/mL), and H2O. (B) The r1 relaxivity curve is obtained from the BSA-Gd@PEG-b-PLGA vesicles at various concentrations.

Figure 8. Assigned 1H NMR spectra of PEG-b-PLGA diblock copolymer in CDCl3 and D2O. 1589

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Figure 9. T1-weighted MR images of the nude mice acquired (a) prior to and (b,c) postinjection of BSA-Gd@PEG-b-PLGA vesicle suspension at different times. The white arrows denote xenograft tumors.

Figure 10. Cumulative release profile of (a) pure DOX, (b) DOXloaded PEG-b-PLGA nanoparticles, and (c) DOX-loaded BSA-Gd@ PEG-b-PLGA vesicles at 37 °C in buffer saline of pH 7.4.

Figure 12. (A) Cell proliferation of HeLa cells incubated with PEG-bPLGA nanoparticles and BSA-Gd@PEG-b-PLGA vesicles at different concentrations. (B) Cell proliferation of HeLa cells incubated with pure DOX and DOX-loaded BSA-Gd@PEG-b-PLGA vesicle solutions containing an equal concentration of DOX at various concentrations.

spectroscopy, as shown in Figure 6. The absorption peak of the solution at higher concentration was higher than that at lower concentration due to the higher encapsulation amount of BSAGd in the former one. According to the calibration curve of BSA at 280 nm in PBS buffer (A = 0.6103C + 0.0547, R2 = 0.9997, where A is the UV absorption intensity at 280 nm and C is the concentration of BSA with the unit of mg/mL), we can calculate that there is around 120 μg/mL of BSA in the vesicles prepared from the PEG-b-PLGA at 1.0 mg/mL. Furthermore, the ICP-AES analysis revealed that the content of Gd3+ in the BSA-Gd@PEG-b-PLGA solution was 3.1 μg/mL, which was high enough for MR imaging.32 In Vitro and in Vivo MR Imaging. Using a 1.5 T medical superconducting MRI system, the T1 relative signal intensity of the BSA-Gd@PEG-b-PLGA vesicle suspension was evaluated, as well as four controls (Figure 7A). Its intensity was significantly higher than that of the other four control groups. Also, the T1 relative signal intensity is related to the concentration of BSA-Gd@PEG-b-PLGA vesicles. Higher concentration leads to brighter signals (Figure 7A). As shown in Figure 7B, the BSA-Gd@PEG-b-PLGA vesicles exhibited the r1 value of 2.77 s−1 per mM of Gd3+.

Figure 11. Hemolysis of BSA-Gd@PEG-b-PLGA vesicles after incubation with red blood cells for 60 min at 37 °C. The initial concentration of the aqueous BSA-Gd@PEG-b-PLGA solution in PBS was 2.0 mg/mL. A series of concentrations of solutions were obtained by 2-fold dilutions. Experiments were performed in triplicate.

stability of the BSA-Gd@PEG-b-PLGA vesicles in PBS and FBS. To further confirm the encapsulation of BSA-Gd nanohybrids into PEG-b-PLGA block copolymer nanoparticles, the aqueous copolymer solution at neutral pH after centrifugation was investigated by monitoring the absorption peak of the solution at different concentrations at 280 nm by a UV−vis 1590

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Partial Hydration of Polymer Vesicle Membrane. Since the MR effect of gadolinium is based on its interaction with water molecules, the MR signal of BAS-Gd nanohybrids may be seriously suppressed if they are located in the hydrophobic domain. Anyway, most of them have been encapsulated in the water pool of vesicles. Furthermore, the vesicle membrane is not fully hydrophobic, but partially hydrated, which was proved by the 1H NMR studies. As shown in Figure 8, The PEG-bPLGA diblock copolymers are molecularly dissolved in CDCl3. In D2O, PEG signals are still visible while PLGA signals partially disappeared. Signals from ca. 58% of PLA (peaks c and d) and ca. 93% of PGA (peak f) still appeared by comparing with PEG (peak b). This indicates that the PLGA vesicle membrane is partially hydrated, consequently facilitating the MR signal. The in vivo MR imaging data of the BSA-Gd@PEG-b-PLGA vesicles are shown in Figure 9. HeLa cell line was subcutaneously injected into the middle back of mice to prepare tumor-bearing mice. Following the intravenous injection of the resulting vesicles suspension, the MR imaging of the mice was acquired at specific time intervals. The tumor site was indicated by white arrows. After injection of the suspension, some lightened areas on the T1-weighted MR images were found within the tumor site. An enhancement of the signal intensities was observed with time (Figure 9). Specifically, at 15 min postinjection, it presents relatively lower relative signal intensity. Afterward, the gradually enhanced signal intensity appears. After 45 min, the relative signal intensity within the tumor site was much higher. The quick enhancement of the signal intensity indicates the excellent MR imaging function of the BSA-Gd@PEG-b-PLGA vesicles solution within short time, which is important for an efficient MR contrast agent with fewer side effects. Drug Loading/Release Profiles of BSA-Gd@PEG-bPLGA Vesicles. DOX loading and release experiments were performed to evaluate the potential of BSA-Gd@PEG-b-PLGA vesicle for drug delivery application. The hydrophobic interaction between DOX and the hydrophobic membrane of vesicles drives the loading process, with the drug loading content of 23.1 and 20.9 wt % for pure PEG-b-PLGA nanoparticles (without BAS-Gd nanohybrids) and BSA-Gd@ PEG-b-PLGA vesicles, respectively, indicating that the introduction of BSA-Gd into the PEG-b-PLGA vesicle has slight effects on the drug loading content of the polymer. As shown in Figure 10, the in vitro DOX release behaviors in PBS (pH 7.4) at 37 °C were investigated for only DOX (curve a), DOX-loaded PEG-b-PLGA nanoparticles (curve b), and BSA-Gd@PEG-b-PLGA vesicles (curve c). The BSA-Gd@ PEG-b-PLGA vesicles displayed an obviously slower drug release behavior than the other two controls, possibly due to the electrostatic and hydrophobic interaction between the DOX and BSA.46,47 Hemolytic Test. Red blood cell hemolysis experiment was performed to test the blood compatibility of BSA-Gd@PEG-bPLGA vesicles. As shown in Figure 11, even at 1.0 mg/mL, they showed good blood compatibility, as a result of low cytotoxicity of both PEG48 and PLGA blocks,49,50 as well as PEG-b-PLGA nanoparticles acting as a retarding space to entrap BSA-Gd nanohybrids. Cytotoxicity Test. The cytotoxicity tests were carried out by the conventional MTT assay using a HeLa cell line. HeLa cells were incubated with different materials including BSAGd@PEG-b-PLGA vesicles, PEG-b-PLGA nanoparticles, free

DOX, and DOX-loaded BSA-Gd@PEG-b-PLGA vesicles at various concentrations for 24 h. As shown in Figure 12A, the BSA-Gd@PEG-b-PLGA vesicles and the PEG-b-PLGA nanoparticles did not obviously affect the HeLa cell viability even at a high concentration of 1000 μg/mL, suggesting effective encapsulation of Gd3+ into the BSA-Gd@ PEG-b-PLGA vesicles. In contrast, higher cell viability was observed for DOX-loaded BSA-Gd@PEG-b-PLGA vesicles than for free DOX (Figure 12B), as a result of less free DOX released from DOX-loaded BSA-Gd@PEG-b-PLGA vesicles within 24 h. It is noteworthy that the cell viability of DOX-loaded BSAGd@PEG-b-PLGA vesicles is much lower than that incubated with the BSA-Gd@PEG-b-PLGA vesicles without loading DOX, indicating an obvious inhibition effect of DOX-loaded BSA-Gd@PEG-b-PLGA to HeLa cells. Therefore, these DOXloaded BSA-Gd@PEG-b-PLGA vesicles have great potential in biomedical applications.

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CONCLUSIONS In summary, a novel theranostic DOX-loaded BSA-Gd@PEGb-PLGA vesicle has been successfully synthesized based on PEG-b-PLGA diblock copolymer and BSA-Gd nanohybrids in THF/water solution at room temperature. The vesicles showed excellent T1-weighted MR imaging function both in vitro and in vivo. Furthermore, it has good blood compatibility and significantly retarded the release rate of DOX. Moreover, this vesicle combined two important functions together: excellent MR imaging function and good drug delivery ability, suggesting its great potential for theranostics applications.

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AUTHOR INFORMATION

Corresponding Authors

*(H.D.) E-mail: [email protected]. *(J.D.) E-mail: [email protected]. Tel: +86-21-6958-0239. Fax: +86-21-6958-4723. Author Contributions

§ These authors (Q.L. and H.Z.) contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21074095, 21104059, 21174107, and 21374080), Eastern Scholar Professorship, Shanghai 1000 Plan, New Century Excellent Talents in Universities of MOE (NCET-10-0627), Ph.D. Program Foundation of MOE (20110072110048), Fok Ying Tong Education Foundation (132018), and the fundamental research funds for the central universities.

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REFERENCES

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dx.doi.org/10.1021/bm500438x | Biomacromolecules 2014, 15, 1586−1592