Multifunctional and Redox-Responsive Self-Assembled Magnetic

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Multifunctional and Redox-Responsive Self-Assembled Magnetic Nanovectors for Protein Delivery and Dual-Modal Imaging Hong Yu Yang, Moon-Sun Jang, Yi Li, Jung Hee Lee, and Doo Sung Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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ACS Applied Materials & Interfaces

Multifunctional

and

Redox-Responsive

Self-Assembled Magnetic Nanovectors for Protein Delivery and Dual-Modal Imaging Hong Yu Yang §, Moon-Sun Jang #, Yi Li §, Jung Hee Lee #,* and Doo Sung Lee §,* §

Theranostic Macromolecules Research Center, School of Chemical Engineering,

Sungkyunkwan University, Suwon 16419, Republic of Korea. #

Department of Radiology, Samsung Medical Center, Sungkyunkwan University

School of Medicine and Center for Molecular and Cellular Imaging, Samsung Biomedical Research Institute, Seoul 06351, Republic of Korea.

Keyword: Redox-reponsive, Protein delivery system, Self-assembled magnetic nanovectors, MR imaging, Near-infrared fluorescence (NIRF) imaging, HSA-Cy5.5.

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Abstract Nanoparticle (NP)-based model carriers present an emerging strategy for protein delivery. However, constructing a multifunctional nanocarrier with high loading capacity, diagnostic imaging capacity and controlled release capability is a tremendous challenge for protein delivery systems. Thus, we herein report on the fabrication of redox-responsive magnetic nanovectors (termed RMNs) through selfassembly of Fe3O4 NPs and redox-responsive polymer ligands, which could effectively transport protein and trigger intracellular protein release. These RMNs also exhibited

low

toxicity,

high

stability,

biocompatibility

and

T2-weighted

contrast-enhancement properties. In addition, they presented a quantized positively charged surface that had the capacity to load cyanine 5.5 (Cy5.5)-labeled human serum albumin (HSA) with high loading efficiency (~84%) via electrostatic interactions and which favored cellular uptake. Notably, studies of the in vitro protein release showed that HSA-Cy5.5-loaded RMNs (RMNs-HSA-Cy5.5) presented minimal cumulative release behavior under physiological conditions but release was rapidly enhanced under high glutathione concentration conditions. Confocal microscopy further revealed that protein was delivered and localized at the perinuclear region of tumor cells. Moreover, the in vivo imaging results confirmed that RMNs-HSA-Cy5.5 could serve as a dual-modal probe for simultaneous near-infrared fluorescence (NIRF) imaging and magnetic resonance (MR) imaging, which can be used for breast cancer diagnosis, and verified higher tumor accumulation of transported protein in a living body. Overall, we believe that these

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multifunctional RMNs exhibit great promise for protein delivery, cancer diagnosis and therapy, and multi-modal imaging, as well as clinical applications.

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INTRODUCTION Protein therapeutics have attracted great interest for treating a variety of human diseases including malignant tumors and autoimmune diseases.1-3 Although the development of protein therapeutics has made significant gains in the last several decades4-5, their use still suffers from several challenges, such as in vitro and in vivo instability, poor membrane permeability, enzymatic degradation and a relatively short half-life.6-8 As a result, intelligent nanoparticles such as polymeric micelles, polymer nanogels, liposomes and polymersomes have been widely investigated for protein delivery. Because they can respond to a variety of specific stimuli including pH, temperature, redox, light-irradiation and electromagnetic field to achieve “on demand” accurate release of encapsulated therapeutic agents and exhibit both also low side-effect and high stability.9-11 In addition to the aforementioned advantages, smart nanoparticles also suffer some challenges for the incorporation of magnetic nanoparticles or fluorescent organic dye into protein nanocarriers to provide protein-tracking and diagnostic imaging capability.12-13 Superparamagnetic iron oxide nanoparticles (SPIONs) have been extensively exploited for in vivo and in vitro biomedical applications, including diagnostic imaging14-17, labeling18-19 and drug delivery20-21, because of their magnetic property, biocompatibility, low cytotoxicity and surface-modification propeties.22-23 However, SPIONs have limited stability and solubility and exhibit nonspecific adsorption of plasma proteins.24-25 Therefore, the development of self-assembled iron oxide nanocarriers with surface-engineered polymers has attracted considerable attention as 4


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an excellent drug delivery system because of the long-term stability, biocompatibility and high delivery efficiency that they exhibit.

15, 26-27

Although these self-assembled

magnetic nanocarriers can effectively resolve individual hurdles in the delivery process, they still have not addressed the challenge of constructing a multifunctional magnetic nanocarrier system through the combination of multiple features to overcome these obstacles and successfully achieve a safe and efficient drug delivery system. For example, for intracellular triggered release of therapeutic agents, a bio-reducible linker based on disulfide linkages can be introduced into magnetic nanocarriers. These disulfide bridges can be cleaved under the reductive conditions in the intracellular cytoplasm, in the presence of high concentrations of glutathione (GSH)28-29, but they remain stable in the extracellular environment due to significant differences in the GSH levels between normal extracellular tissues (2-10 µM) and the tumor microenvironment (1-10 mM)

30-31

. For instance, Zhang et al. constructed a

redox-sensitive magnetic nanoparticle by surface capping with a chitosan-PEG copolymer containing disulfide crosslinking, which was applied to control the localized release of O6-benzylguanine (BG) under reductive intracellular conditions.32 Liu et al. constructed redox-responsive magnetic nanoreservoirs based on disulfide-linked β-cyclodextrin-graft-polyethylenimine polymer encapsulation of magnetic nanoparticles for efficient control of intracellular small molecule anticancer drug delivery and MR imaging diagnostics33, and Yuan et al. developed redox and temperature dual-responsive self-assembled iron oxide nanoparticles by surface modification with functionalized PCL-SS-PDMAEMA copolymer containing 5



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disulfide bonds, which could load doxorubicin (DOX) and exhibited excellent properties for controlled release by redox stimuli.34 However, these types of smart nanocarriers are rarely explored in small molecule drug delivery while limiting the delivery of larger proteins because they need to meet higher requirements, including high loading capacity, a controlled protein release profile and effective resistance of non-specific adsorption to biomolecules. To tackle the above-mentioned challenges, we developed a novel redox-responsive magnetic nanovector (RMN), which consisted of self-assembled SPIONs and multifunctional polymer ligands, for redox-triggered targeted protein delivery and diagnostic imaging. We first synthesized a multifunctional polymer ligand based on disulfide-linked

methoxy-poly(ethylene

glycol)-block-poly[dopamine-

diethylenetriamine-L-glutamate] [mPEG-SS-P(Dopa-Deta)LG] ligands, which were employed to facilitate self-assembly and provide both reduction-sensitivity and high stability. In addition, fluorescent dye (Cy 5.5)-labeled human serum albumin (HSA-Cy5.5) was selected as a model protein to complex with RMNs via electrostatic interaction. The resulting RMNs-HSA-Cy5.5 promoted rapid protein release inside HeLa cells and acted as T2 contrast agents for MR imaging. Simultaneously, the fluorescence not only can be applied to monitor cell internalization and tumor accumulation of protein but can also serve as a fluorescent imaging probe for diagnostic imaging of breast cancer (Scheme 1). Notably, RMNs-HSA-Cy5.5 also exhibits outstanding stability in various biological media. Therefore, we believe that

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combining multiple functions into a single protein nanovector system overcame many of the obstacles in protein delivery and cancer diagnostic imaging.

EXPERIMENTAL SECTION Materials. All reagents and solvents were received and used without further purification. Human serum albumin (HSA), 4-nitrophenyl chloroformate, iron (III) acetylacetonate (Fe(acac)3), dopamine hydrochloride, cystamine, phosphate-buffered saline (PBS), RPMI-1640 medium, triethylamine (TEA), tetrahydrofuran (THF), diethyl ether, phenyl ether, dimethyl sulfoxide (DMSO), chloroform (CH3CL3), N,N-dimethylformamide

(DMF),

dimethyl

sulfoxide-d6

(DMSO-d6)

and

chloroform-d6 (CDCl3) were obtained from Sigma-Aldrich Co. (USA). Triphosgene, oleic acid (OA), diethylenetriamine (Deta), oleylamine and 1,2-hexadecanediol were purchased from TCI Co (Japan). Hexane, ethanol sodium chloride (NaCl) and hydrochloric acid (HCl) were purchased from Samchun. Co (Korea). Methoxy poly(ethylene glycol) (mPEG, Mn = 5000) was obtained from SunBio. Co (Korea). Cyanine 5.5 NHS ester (Cy5.5-NHS, 95%) was obtained from Lumiprobe (USA). γ-Benzyl-L-glutamine-N-carboxyanhydride (BLG-NCA) was prepared based on our previously described method35 and purified by recrystallization thrice in a mixture of diethyl ether and hexane (1:4, v/v). mPEG-SS-NH2, as a macroinitiator was synthesized with a similar procedure reported by our group36. Synthesis

of

mpoly(ethylene

glycol)-SS-poly(benzyl-L-glutamate)

(mPEG-SS-PBLG) Diblock Copolymers. The mPEG-SS-PBLG copolymers as a 7



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platform ligand were prepared by ring-opening polymerization (ROP). Briefly, 1.0 g of mPEG-SS-NH2 and 3.16 g of BLG-NCA were fully dissolved in 40 mL of CH3CL3 under the protection of nitrogen (N2) at room temperature (RT) and the solution was stirred for 3 days. Then, the crude products were precipitated in an excess of ether two times and finally dried in a vacuum at RT for 48 h. We obtained mPEG-SS-PBLG with the desired PBLG segments lengths by controlling the molar ratio of monomers to the initiator. Synthesis

of

mPEG-SS-P(Dopa-Deta)LG

Polymer

Ligand.

The

mPEG-SS-P(Dopa-Deta)LG polymer ligands were synthesized by substitution of the benzyl groups in platform ligand with dopamine (Dopa) and diethylenetriamine (Deta) through a one-step aminolysis reaction. Briefly, mPEG-SS-PBLG (1 g, 0.055 mmol) was fully dissolved in 10 mL of DMF, and subsequently, dopamine (2.5 g, 0.013 mol) and triethylamine (1.34 g, 0.013 mol) were added. The solution was stirred for 8 h under N2 at 55 °C, and then, diethylenetriamine (3.41 g, 0.033 mol) was added and continued to react for 24 h. After the reaction, the solution was diluted with 10 ml of 0.1 N HCl, and the finally product was dialyzed against deionized water with 0.01 N HCl for three times (MWCO: 1,000 Da) followed by lyophilization. Yield: 73%. Fabrication of Redox-sensitive Magnetic Nanovectors (RMNs). The self-assembly process was triggered using the dual solvent-exchange method.37 Briefly, 3 mL of SPIONs (7 mg/mL) in chloroform was added drop by drop into 7 mL of DMSO containing 200 mg of mPEG-SS-P(Dopa-Deta)LG copolymer. The mixture was sonicated for 30 min (setting: 20% amplitude, 4 s on and 2 s off, 20 °C). The 8


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chloroform was then fully evaporated under vacuum, and the DMSO was completely replaced with excess deionized water using a dialysis membrane (MWCO: 3500 Da). The residual copolymers were removed by centrifugation at 10000 rpm for 15 min and washed thrice with a centrifugal filter (Millipore, MWCO: 30,000 Da). The newly formed RMNs were re-dispersed in water for further assays. Characterization. 1H nuclear magnetic resonance (1H-NMR) spectra were measured with a Varian Unity Inova 500NB 500 NMR spectrometer. High-resolution transmission electron microscopy (HR-TEM) measurements were conducted on a JEM-ARM 200F at 120 kV HR-TEM microscope. UV-vis absorbance was measured on a V-630 Bio UV−vis spectrophotometer. Fluorescence measurements were conducted on an FP-6200 spectrofluorometer (AMINCO, Bowman, Series 2) with 5 nm slit widths for both excitation and emission. We estimated Fe concentration using inductively coupled plasma mass spectrometry (ICP-MS) with an Agilent-7500i spectrometer. The magnetic property of RMNs was measured using a superconducting quantum interference device (SQUID) at 300 K. Hydrodynamic size and zeta potential of the nanovector were characterized via dynamic light scattering (DLS) using a Zetasizer-ZS90 (Malvern Instrument, UK) with a helium laser (633 nm) at a scattering angle of 90°. Cyanine 5.5 Labeling of Human Serum Albumin (HSA-Cy5.5). Briefly, 5 mg of Cy5.5-NHS ester was dissolved in 20 mL of PBS solution (pH 8.0) containing 500 mg of HSA and stirred at room temperature in the dark for 8 h. After the reaction, excess Cy5.5-NHS dye was remove by dialysis against 10 mM PBS solution using a dialysis 9



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membrane (MWCO: 3,500 Da) followed by free-dying. The obtained Cy5.5-labeled HSA was re-dispersed in buffer solution to calculate the degree of Cy5.5 labeling of HSA using a UV−vis spectrophotometer at an absorption wavelength of 685 nm. Encapsulation and Release of HSA-Cy5.5. To obtain HSA-Cy5.5-loaded RMNs (RMNs-Cy5.5-HSA), a certain amount of HSA-Cy5.5 was incubated in a solution of RMNs in 10 mM PBS at pH 7.4, and then the solution was shaken for 1 h at RT in the dark. Mass ratios of HSA-Cy5.5 to RMNs were discretely varied among samples from 0 to 0.4. Excessive HSA-Cy5.5 was removed by dialyzing (MWCO 300 kDa) against PBS (10 mM, pH 7.4, RT) for 8 h, and medium was refreshed four times. Lyophilized RMNs-Cy5.5-HSA were re-dispersed in PBS (0.1µM GSH, pH 7.4) and diluted 10 times to determine protein loading efficiency (PLE) and protein loading content (PLC) by comparing absorbance values of the samples at 685 nm to a calibration curve of assigned HSA-Cy5.5 concentrations using a UV-vis spectrometer. PLC and PLE were estimated using the following formula =

amount of loaded protein × 100% total amount of polymer and loaded protein amount of loaded protein = × 100% amount of protein in feed

Studies of in vitro protein release from the RMNs were conducted at 37 °C in pH 7.4 in PBS or PBS containing 10 mM GSH using a dialysis (MWCO: 300 kDa). Then, 5 mL of medium was withdrawn at predetermined time points and replaced by an equal volume of new medium. The mass of HSA-Cy5.5 was calculated by measuring the absorbance at 685 nm using a UV−vis spectrophotometer. In Vitro Physiological Stability of RMNs-HSA-Cy5.5. The hydrodynamic particle 10


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size stability and the fluorescence emission stability of RMNs-Cy5.5-HSA after incubation for 24 h in different media, including H2O, PBS, 0.5 M NaCl solution, pH 5.5, RPMI 1640 and fetal bovine serum (FBS) were monitored with DLS and a fluorescence spectrometer, respectively, at different time intervals (0 h, 4 h, 8 h, 12 h and 24 h). In Vitro MR Relaxivity Measurement. To test the possibility of using RMNs-HSA-Cy5.5 as MRI contrast agents. The longitudinal (r1) and transverse (r2) relaxivities of RMNs-HSA-Cy5.5 at different concentrations were measured with a Philips Medical Systems 3.0 T MR scanner (Philips, Achieva ver. 1.2, Netherlands). The r1 and r2 relaxivity values of RMNs-HSA-Cy5.5 were computed through linear regression analysis (Microsoft EXCEL) to fit relaxation rates and Fe concentration (mM). Cell Culture. HeLa cells were obtained from the Korea Cell Line Bank (KCLB, Seoul, Korea). HeLa cells were grown on 96-well plates at a density of 1 × 104 and incubated for 24 h (5% CO2, 37 °C and 95% humidity ) in cell growth medium (Gibco, Grand Island, NY) containing 10% FBS and 1% penicillin–streptomycin. Cellular Internalization Observed with Confocal Laser Scanning Microscopy (CLSM). HeLa cells were exposed to RMNs-HSA-Cy5.5 or HSA-Cy5.5 at 37 °C. After incubation for the predetermined times (1 h and 4 h), cells were fixed with 4% formaldehyde for 1 h and washed with PBS thrice. Then, cells were stained with HOECHST 33342 for 10 min, and the slides were rinsed three times with PBS. Finally, cellular internalization of RMNs-HSA-Cy5.5 was observed using confocal 11



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laser scanning microscopy (CLSM, LSM780, Germany). Fluorescent signals from Hoechst stain were monitored by setting excitation at 405 nm and emission at 410-450 nm. Cy 5.5 fluorescence was detected at an excitation wavelength of 633 nm and an emission wavelength at 707-715 nm. Scale bar = 20 µm. Cytotoxicity Test. The in vitro cytotoxicity of RMNs was examined with an MTT assays using HeLa cells. Briefly, cells were incubated with RMNs at varying concentrations from 10 to 200 µg/mL. After 48 h of incubation, 20 µL of MTT stock solution was added to each well, and cells were further cultured for 4 h. Finally, the medium in each well was completely replaced with 0.15 mL of DMSO. The absorbance at 490 nm was recorded using Cell Counting Kit-8 for determination of the viability of the cells. Animals and Tumor models. Balb/c nude mice (19 ± 2 g, female) aged 6 weeks were purchased from Seoul Oriental Bio center and used in accordance with the “Guide for the Care and Use of Laboratory Animals” provided by of the Institute of Samsung Biomedical Research. The tumor model was initiated by subcutaneous injection of MDA-MB231 mouse breast cancer cells in the right flank of Balb/c nude mice. In Vivo NIRF Imaging and Quantitative Analysis. For in vivo NIRF imaging, RMNs-HSA-Cy5.5 (dose: 5 mg/kg, 200 µL; mass ratio of HSA-Cy5.5 to RMNs is 0.4) were injected intravenously into the MDA-MB-231 mice via the tail vein. In vivo NIR fluorescence imaging was monitored at predetermined time points (pre, 4 h and 24 h) with an IVIS 200 Imaging system (Xenogen-Caliper, MA, USA) using a self-fixed excitation at a wavelength of 650 nm and a self-fixed emission filter at 720 nm. At 24 12


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h post-injection, the mice were humanely killed, and the tumor and the organs were excised for ex vivo fluorescence imaging. The fluorescence intensities were recorded using Living Image 2.5 software (Caliper Life Science) and quantitated as average radiance (p/s/cm2/sr). In Vivo MR Imaging. In vivo MR imaging was measured using the same mouse-treated with the RMNs-HSA-Cy5.5 for in vivo NIRF imaging. The T2-weighted MR images were obtained with a 7.0 T MR scanner (Bruker BioSpin, Fallanden, Switzerland) at different time intervals (pre, 4 h and 24 h), and the fast spin-echo (FSE) T2-weighted MRI sequence was set to TR = 2000 ms, TE = 45 ms, slice thickness (SL) = 1 mm, echo train length (ETL) = 6, and field of view (FOV) = 3.56 × 2.56 × 2.56 cm3. Histological analysis. For histological analysis, the tumor tissue was isolated immediately after 24 h of MR scanning, and fixed in 10% neutral buffered formalin (NBF) for 24 h and then embedded in paraffin. The embedded tumor tissues were cut into 5-µm sections and then sliced in accordance with MR images. The sectioned slices were stained with Prussian blue for detection of SPINOs and nuclear fest red as a counterstain. The most highly stained areas in the sections were visualized at 100 magnification using an Olympus DP70 Digital Microscope Camera. Statistical Analysis. Quantitative data were analyzed and reporeted as the mean ± SD from several separate experiments. Differences were assessed via Student’s t-test and were considered statistically significant if *P < 0.05 or **P < 0.01.

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RESULTS AND DISCUSSION Synthesis of SPIONs and Multifunctional Polymer Ligand To construct magnetic nanovectors, small and homogeneous spherical SPIONs (~4 nm) were prepared using a simple organic-phase synthesis method38-39, and the morphology and size were determined with HR-TEM and DLS, respectively (Figure 1a and b). mPEG-SS-PBLG acted as platform ligand and were first synthesized through ROP of BLG-NCA in CH3Cl3 at RT using mPEG-SS-NH2 as a macroinitiator (Scheme 2a). The length of the platform ligand was adjusted by changing the initiator/monomer molar ratio. As shown in Figure S1,

1

H NMR spectra clearly displayed the

characteristic peaks of PEG (δ ∼3.64), the proton peaks of the methylene groups adjacent to a disulfide bond (δ ∼2.89 and∼3.51) and signals corresponding to the benzyl groups in the platform ligand at δ ∼5.1 and ∼7.4. The polymerization degree of the mPEG-SS-PBLG was calculated from the integral ratio of the benzyl proton peaks (2H, δ ∼ 5.10 ppm) to the PEG proton peaks (4H, δ ∼ 3.64 ppm) (Table 1). Subsequently, multifunctional redox-sensitive mPEG-SS-P(Dopa-Deta)LG polymer ligand was prepared by substitution of flanking benzyl groups in platform ligand with an excessive amount of amine-terminated dopamine (Dopa) and diethylenetriamine (Deta) groups through a sequential aminolysis reaction in the presence of the bifunctional catalyst, 2-hydroxypyridine that can effectively maintain the integrity of the polymer ligand backbone. (Scheme 2b) Compared to the 1H NMR spectra of platform ligand, the 1H NMR spectrum of mPEG-SS-P(Dopa-Deta)LG (Figure 2) showed new proton peaks at ~6.6, ~6.7 and ~6.76 ppm, which represented the 14


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dopamine groups of the polymer ligand, and peaks ranging from ~3.1 to ~3.4 ppm, which belonged to the diethylenetriamine groups in the newly formed polymer ligand. The degree of grafting of Dopa and Deta of mPEG-SS-P(Dopa-Deta)LG was estimated by comparing the integral areas of the Dopa peak (1H, δ ∼6.6) and the characteristic peak of PEG (4H, δ ∼ 3.64), and the results are shown in Table 1. For the final mPEG-SS-P(Dopa-Deta)LG polymer ligand, the dopamine group serves as a robust anchor to facilitate self-assembly through high-affinity binding of SPIONs and incorporating the diethylenetriamine provides a high protein loading capacity. Construction and Characterization of Redox-Sensitive Magnetic Nanovectors (RMNs) Hydrophilic and biocompatible redox-sensitive magnetic nanovectors were fabricated through self-assembly of SPIONs and mPEG-SS-P(Dopa-Deta)LG polymer ligand using the dual solvent-exchange approach as schematically described in Figure S2. The periphery of the small SPIONs could be densely wrapped with polymer ligand containing the catechol anchor, facilitating the self-assembly of SPIONs into magneto-core/shell structures, where the magnetic core is composed of the SPIONs and the PEG segment of the polymer ligand serves as a hydrophilic shell (Scheme 1). The HR-TEM image in Figure 3a clearly displays the morphology of RMNs, and the particle size of the RMNs was approximately 60-70 nm which is consistent with the results of the DLS measurements (Figure 3b). The superparamagnetic property of RMNs was investigated. The magnetization curve of the RMNs exhibited excellent superparamagnetism owing to the absence of a 15



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hysteresis loop and the saturation magnetization (Figure 3c). The superparamagnetic property of RMNs was further assessed by observing the dispersed/accumulated state of RMNs dispersed in aqueous solution with/without an applied magnetic field. As shown in Figure S3, the RMNs were homogeneously dispersed in an aqueous solution in the absence of an external magnetic field, but they rapidly accumulated near the magnet and the solution becomes clear as a magnetic field is applied for 1 h. The contrast-enhanced MRI capability of the RMNs is shown in Figure 3d. The transverse relaxivity (r2) of RMNs was estimated to be 73.16 mM-1·s-1, while the longitudinal relaxivity (r1) of RMNs is estimated to be 0.6 mM-1·s-1, indicating that they can serve as potential T2-weighted MRI contrast-enhanced agents. Moreover, an in vitro T2-weighted MRI assessment further confirmed that RMNs exhibited superior T2 contrast-enhancement ability with increasing the concentration of Fe, and dark images appeared gradually deepened (Figure 3e). As a potential protein delivery carrier and imaging probe for biological applications, cytotoxicity evaluation of the RMNs is important. As shown in Figure 3f, the viability of HeLa cells incubated with the RMNs not show significant toxicity at concentration variations from 10 to 200 µg/mL after 48 h of incubation, and the viability of cells remained higher than 90% according to MTT assays. These results demonstrated that RMNs displayed low cytotoxicity and good biocompatibility. Loading and Release of Protein Human serum albumin (HSA) was chosen as a model protein and was labeled with the dye cyanine 5.5 (HSA-Cy5.5), resulting in the NIRF emission (Figure S4) that 16


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was used to investigate the in vitro release, cellular internalization and tumor accumulation abilities of protein-loaded RMNs as well as their utility for cancer diagnostic imaging. HSA-Cy5.5 could be easily loaded into RMNs (RMNs-HSA-Cy5.5) based on strong electrostatic interaction between HSA-Cy5.5 and the RMNs. This interaction can be explained by the zeta potential measurements shown in Figure 4. The results indicated that the self-assembled SPIONs with mPEG-SS-P(Dopa-Deta)LG polymer ligand provided a quantized positive surface charge of around +19 mV, which facilitated the loading of the negatively charged HSA-Cy5.5, and the zeta potential value of the RMN surface decreased from around +10.2 mV to around +3.8 mV with an increase in the mass ratio of HSA-Cy5.5 to RMNs from 0.1 to 0.4. The protein loading efficiency of RMNs ranged from 84.1% to 86.2% at theoretical loading content of approximately 25.8 wt%, and the size of RMNs increased from approximately 68 nm to approximately 89 nm with increasing mass ratio of HSA-Cy5.5 to RMNs from 0.1 to 0.4 according to the DLS measurement (data not shown). The cumulative release curve of HSA release from the RMNs shown in Figure 5 revealed that only ~17% of the HSA was released within 24 h in PBS solution without GSH (pH 7.4). However, the quantity of HSA was released from RMNs quickly increased up to ~75% in 24 h under a high concentration of GSH (10 mM) because of the reduction-triggered breaking of the disulfide bridges in the RMNs, leading to rapid shedding of the hydrophilic shell and destabilization of the RMNs. This result 17



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indicates that the release of protein can be regulated by GSH in the reductive environment of the cytoplasm. In Vitro Physiological Stability of RMNs-HSA-Cy5.5 Studying

the

influences

of

counter-ions

in

physiological

media

on

RMNs-HSA-Cy5.5 is important for cellular uptake and protein delivery. The particle size of the RMNs-HSA-Cy5.5 changes when incubated in various biological media, such as buffer solution (PBS, pH 7.4), high ionic strength solutions (NaCl, 0.5 M), low pH deionized water (pH 5.5), 100% cell growth media (RPMI-1640) and FBS , and these changes were evaluated with DLS, which was used to investigate the in vitro physiological stability. Figure 6a reveals that the size of RMNs-HSA-Cy5.5 remained

almost

constant,

without

significant

differences,

indicating

that

RMNs-HSA-Cy5.5 exhibited outstanding stability in various types of biological media for 24 h. The Cy5.5 fluorescence intensity of RMNs-HSA-Cy5.5 was further utilized to measure the in vitro stability by incubating RMNs-HSA-Cy5.5 in the same biological conditions. The results, shown in Figure 6b, confirmed that the fluorescence intensity of the RMNs-HSA-Cy5.5 did not obviously change for 24 h, indicating that the proteins were firmly sealed in the RMNs-HSA-Cy5.5 system. The in vitro physiological stability of the RMNs-HSA-Cy5.5 may be attributed to the presence of the compact PEG outer shell and structural integrity of the protein delivery system, which could effectively inhibit non-specific adsorption of proteins in high ionic strength solution.40-41 The intracellular release behavior of protein 18


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HeLa cells were applied to explore the cellular internalization and intracellular protein release behavior of RMNs-HSA-Cy5.5 by CLSM. The confocal images in Figure 7 show obvious red fluorescence at the perinuclear region of cancer cells after 1 h treatment and stronger fluorescence intensity was observed after 4 h of incubation. The results indicated that effective cellular uptake of the RMNs-HSA-Cy5.5 accelerated transport of protein and the cleavable disulfide bonds boosted intracellular protein release. As expected, almost no fluorescence was present in the cells after 4 h of incubation with free HSA-Cy5.5, likely due to a lack of cellular uptake capacity. It is evident, therefore, that redox-responsive RMNs can act as a promising platform for intracellular protein delivery. In Vivo Fluorescence/ MR Dual-modal Imaging In vivo NIRF imaging analysis was performed in breast cancer tumor-bearing nude mice to track the RMNs-HSA-Cy5.5 after being injected using a sensitive IVIS Imaging System. NIRF images in Figure 8a clearly show that RMNs-HSA-Cy5.5 enable high resolution NIRF imaging of tumors in living mice with increasing injection time. Subsequently, the tumor tissue and other major organs (e.g., liver, heart, lung, kidney and spleen) were isolated for ex vivo fluorescence imaging analysis (Figure 8b). Quantitative studies indicated that the fluorescence intensity of Cy5.5 in tumor tissue was significantly higher than in normal organ tissue (Figure 8c). The above results support the notion that RMNs-HSA-Cy5.5 not only can be used as a tumor-targeting protein delivery system for effective transport of proteins to reach tumor sites by EPR effects42 but can also serve as an advanced imaging probe for 19



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detection of solid tumors. T2-weighted MR images were collected before and after the RMNs-HSA-Cy5.5 injection. As shown in Figure 8d, the T2 enhancement effect was clearly displayed at the tumor site of the mice 24 h after injection. To further verify the tumor-targeting capacity of RMNs-HSA-Cy5.5, Fe staining of the tumor tissues was implemented and images

of

the

Prussian

Blue

staining

(Figure

8e)

confirmed

that

the

RMNs-HSA-Cy5.5 preferentially localized at the tumor site in mice, which is consistent with the results of the in vivo fluorescence imaging. Thus, the results of in vivo NIRF imaging and in vivo MR imaging not only confirmed that RMNs-HSA-Cy5.5 could act as a dual-modal imaging probes for breast cancer tumor diagnosis but also verified that they effectively accumulated at tumor sites. CONCLUSION We

designed

stable,

biocompatible and hydrophilic

SPIONs

through

self-assembly assisted by multifunctional polymer ligands. The resulting RMNs exhibited

redox-responsive

properties;

superparamagnetic,

T2-weighted

contrast-enhanced capacity and low cell cytotoxicity; and they presented a quantized positively charged surface that was used to load Cy5.5-labeled HSA through a highaffinity interaction. On the basis of redox-sensitive disassembly behavior, accelerated protein release in the intracellular environment, which contains high levels of glutathione was achieved. Moreover, the RMNs-HSA-Cy5.5 showed outstanding stability and were effective against non-specific adsorption of biomolecules in surplus high-strength ions and protein biological media. In addition, both in vivo NIRF/MR 20


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imaging not only revealed that the RMNs-HSA-Cy5.5 could act as a promising protein carrier for targeted-tumor protein delivery but also indicated that it could serve as a dual-modal probe, which can provide more accurate diagnostic imaging information in tumor staging. Overall, this versatile RMNs-HSA-Cy5.5 delivery system may open exciting opportunities for multimodal imaging, cancer diagnosis and simultaneous protein delivery even in clinical applications. ASSOCIATED CONTENT Supporting Information 1

H NMR spectrum of mPEG-b-PBLG polymer ligand backbone. Schemaic illustration of self-assembly process of Fe3O4 and mPEG-SS-P(Dopa-Deta)LG (RMNs). Photographs of the RMNs dispersed in aqueous solution with applied magnetic field at 0 h and 1h. UV−vis absorption and fluorescence spectrum of the RMNs-HSA-Cy5.5. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J.H.L). *E-mail: [email protected] (D.S.L). Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through a National Research Foundation of Korea grant funded by the Korean Government (MEST) (20100027955) and the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2012M3A9B6055205).

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Scheme 1. Schematic diagram of the RMNs-HSA-Cy5.5 as the multifunctional nanovector for intracellular protein delivery and dual-model NIRF imaging and MR imaging

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Scheme 2. Synthesis process of mPEG-SS-PBLG platform ligand (a) and multifunctional mPEG-SS-P(Dopa-Deta)LG polymer ligand (b).

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Figure 1. (a) HR-TEM images and (b) size distribution of SPIONs (average particle size, ~4.2 nm).

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Figure 2. 1H NMR spectra of mPEG-SS-P(Dopa-Deta)LG polymer ligand (500MHz, D2O)

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Figure 3. (a) HR-TEM image of the RMNs. (insert) Enlarged image. (b) Hydrodynamic particle size and particle size distribution of the RMNs. (c) SQUID magnetization curve of the RMNs at 300 K. (d) Relaxation rate r (1/T, s−1 ) as a function of the iron concentration (mM) and (e) the corresponding T2-weighted MRI images of RMNs. (f) Cell viability evaluation of the RMNs by an MTT assay after 48 h treatment.

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Figure 4. Zeta potential of HSA-Cy5.5, RMNs and the different mass ratio of HSA-Cy5.5/RMNs.

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Figure 5. In vitro release of HSA-Cy5.5 from redox-sensitive RMNs in PBS (pH 7.4, 10 mM GSH) and PBS (pH 7.4) at 37oC.

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Figure 6. (a) In vitro particle size stability of the RMNs-HSA-Cy5.5 in PBS, H2O, 0.5 M NaCl, RPMI-1640, pH 5.5 and FBS at 37 oC. (b) Cy5.5 fluorescence stability of the RMNs-HSA-Cy5.5 in PBS, H2O, 0.5 M NaCl, RPMI-1640, pH 5.5 and FBS. Data were presented as mean ± SD (n = 3)( mass ratio of HSA-Cy5.5 to RMNs is 0.4).

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Figure 7. Confocal images of HeLa cells incubated with RMNs-HSA-Cy5.5 at 1 h, 4 h and free HSA-Cy5.5 at 4 h, respectively. (Mass ratio of HSA-Cy5.5 to RMNs is 0.4). Scale bar = 20 µm.

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Figure 8. In vivo NIRF imaging and MR imaging of the RMNs-HSA-Cy5.5. (a) In vivo NIRF imaging of breast cancer tumor-bearing mice after intravenous injection of the RMNs-HSA-Cy5.5 at before injection, 4 and 24 h post injection. Arrows indicated the sites of tumors. (b) Ex vivo NIRF imaging of breast cancer tumor-bearing mice after intravenous injection of the RMNs-HSA-Cy5.5 at 24 h post injection (including: tumor, heart, lung, kidney, spleen and liver). (c) Average fluorescence signal intensities of tumors and normal major excised from the breast cancer tumor tumor-bearing mice at 24 h post injection. A quantification of the ex vivo tissues and tumor was recorded as fluorescence intensity (p/s/cm2/sr). All data are represented as mean SD (n = 3). ∗∗ is represented as significant (P < 0.01). (d) In vivo T2-weighted MR imaging of breast cancer tumor tumor-bearing mice after intravenous administration of the RMNs-HSA-Cy5.5 at before injection, 4 and 24 h post injection. Arrows indicated the enlarge sites of tumor. (e) Prussian blue staining images of the tumor tissues treated by RMNs-HSA-Cy5.5 at 24 h post injection. Red imaginary line is enlarged tissues images of tumor.

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Table 1 the characteristics of the prepared mPEG-SS-P(Dopa-Deta)LG polymer ligand Ligand

Molar ratio of

Degree of

Grafting ratio

Grafting ratio

Initiator/monomer

polymerizationa

of Dopaa

of Detaa

----~11%

----~89%

1/60 -----

mPEG-SS-PBLG mPEG-SS-P(Dopa-Deta)LG a)

58 58

Calculated from 1H NMR

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The Table of Contents Entry

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Multifunctional and Redox-Responsive Self-Assembled Magnetic

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