Photoregulated Cross-Linking of Superparamagnetic Iron Oxide

Article pubs.acs.org/Macromolecules

Photoregulated Cross-Linking of Superparamagnetic Iron Oxide Nanoparticle (SPION) Loaded Hybrid Nanovectors with Synergistic Drug Release and Magnetic Resonance (MR) Imaging Enhancement Kangning Zhu, Zhengyu Deng, Guhuan Liu,* Jinming Hu,* and Shiyong Liu* CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, iChem (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *

ABSTRACT: The development of stimuli-responsive magnetic resonance imaging (MRI) contrast agents that can selectively enhance imaging contrasts at pathological sites is of potential use in clinical diagnosis. Herein, a T2-type MRI contrast agent with synergistically photoregulated enhanced MRI contrast and drug release was achieved by coassembly of superparamagnetic iron oxide nanoparticles (SPIONs) and doxorubicin (DOX) with amphiphilic block copolymer assemblies. Photosensitive amphiphilic diblock copolymers, poly(ethylene oxide)-b-poly(2-((((2-nitrobenzyl)oxy)carbonyl)amino)ethyl methacrylate) (PEO-b-PNBOC), were synthesized through reversible addition−fragmentation chain transfer (RAFT) polymerizations. The resulting block copolymers were coassembled with hydrophobic oleic acid (OA)-stabilized SPIONs and DOX via an oil-in-water (O/W) emulsion and a subsequent solvent evaporation procedure, resulting in the formation of DOX/SPION coloaded hybrid nanovectors. The asassembled hybrid nanovectors exhibited retarded DOX release and weak T2 relaxivity (r2) prior to UV-irradiation. However, upon UV-irradiation, the hybrid nanovectors underwent cross-linking and a hydrophobic-to-hydrophilic transition within the cores, thereby selectively triggering DOX release and elevating T2 relaxivities. In vitro DOX release results revealed approximately 85% of DOX was released within 10 h under 20 min UV-irradiation, and this was in sharp contrast with less than 5% of DOX release without UV-irradiation. The selective DOX release under UV-irradiation showed significantly increased cytotoxicity toward HepG2 cells. Meanwhile, the r2 of UV-irradiated nanovectors exhibited 4.5- and 1.9-fold increases as compared to cetyltrimethylammonium bromide (CTAB)-stabilized monodispersed SPIONs and nonirradiated hybrid nanovectors. Moreover, there was a linear correlation between the r2 changes and cumulative DOX release extents, enabling instantaneously visualizing the DOX release by the MRI technique. Further, we demonstrated that the cellular internalization efficiency of the coloaded hybrid nanovectors increased by 2.7-fold in the presence of an external magnet. The magnetically guided cellular uptake, triggered release profile, and enhanced MRI contrast characteristics may presage potential applications as a new generation of theranostic platform.

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INTRODUCTION Magnetic resonance imaging (MRI) has been widely applied in clinical diagnosis due to its nonradiation, noninvasiveness, high lateral and depth resolution in soft tissues, and the capability to provide excellent anatomical details.1−4 To date, a number of MRI contrast agents have been developed based on either T1type (e.g., gadolinium chelates)5−8 or T2-type contrast agents (e.g., superparamagnetic iron oxide nanoparticles, SPIONs),9−17 exhibiting positive (brightening) and negative (darkening) contrasts, respectively. In comparison with T1-type contrast agents, SPION-based T2-type contrast agents could be delivered in a magnetically guided fashion18−20 and be used for hyperthermia treatment.21,22 However, monodispersed SPIONs with small sizes cannot be readily manipulated by an external magnet and are subjected to insufficient imaging sensitivity.23 Therefore, much effort has been devoted to increasing the relaxivity and magnetically targeted property. To resolve this © XXXX American Chemical Society

issue, SPIONs are commonly encapsulated into polymeric matrices, in which the loaded SPIONs closely packed and formed clustering structures, exhibiting much higher T2 relaxivities than for dispersed SPIONs at the same concentration.24−38 As for practical application concerns, administrated MRI contrast agents are expected to selectively switch on imaging signals only at desired sites (e.g., pathologic areas) to increase the contrasts between normal and pathological tissues.39−51 However, although the encapsulation of SPIONs into polymeric matrices can efficiently enhance MRI relaxivities, in many cases, the MRI signals are always “on” and the relaxivities are not tunable.24,27,28 Note that this is apparently unfavorable Received: October 4, 2016 Revised: December 26, 2016

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DOI: 10.1021/acs.macromol.6b02162 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

Scheme 1. Schematics of the Fabrication of DOX/SPION Coloaded Hybrid PEO-b-PNBOC Nanovectors via an O/W Emulsion and a Subsequent Solvent Evaporation Procedurea

a

For hybrid nanovectors dispersions, photoirradiation triggers cascade decaging reactions of PNBOC side chains and generation of primary amine moieties; extensive amidation reactions then occur, leading to concomitant cross-linking and a hydrophobic-to-hydrophilic transition within the cores of DOX/SPION coloaded hybrid nanovectors. This photoactuated transition results in synergistic DOX release and MRI enhancement, which could also be accomplished within living cells.

simultaneously increased r2 and improved stability upon specific stimuli. Previously, we incorporated T1-type MRI contrast agent (e.g., gadolinium complex) into a pH-responsive core crosslinked (CCL) micelle and demonstrated that the MRI signals could be switched on under mildly acidic pH as a result of protonation of tertiary amine moieties.61 The increased water accessibility within CCL micelles led to enhanced MRI signals. Note that water accessibility had a crucial impact on the MRI signals.62 For instance, Park and co-workers demonstrated that water accessibility toward SPION loaded polymeric aggregates dramatically affected T2 relaxivities.30 We envisioned that if the polymeric matrices of SPION loaded hybrid vectors could be concurrently rendered hydrophilic and cross-linked rather than disassembled upon external stimuli, stimuli-responsive MRI contrast agents with increased relaxivities would probably be achieved. Note that hydrophilic microenvironment would ensure water accessibility while the formation of cross-linked networks should ban the release of encapsulated SPIONs. To screen a platform that can fulfill the above criteria, we reasoned that our recently developed traceless cross-linking strategy could be an eligible candidate.63 Our previous results demonstrated that the permeability and stability of polymersomes fabricated from tracelessly cross-linkable polymers could be concurrently increased. The formation of reactive primary

for differentiating pathological regions from normal tissues due to the high background noise as a result of nonspecific distributions of MRI contrast agents. To this end, pathological tissue-specific microenvironments such as acidic pH and enzyme were applied as a triggering event to regulate MRI signals.52−56 For example, Lee et al.54,55 incorporated hydrophobic SPIONs into pH-responsive polymers. The SPION payloads were released and were gradually accumulated upon arriving at the acidic condition of pathological areas (e.g., tumor tissues). Although the MRI signals from accumulated SPIONs could indicate pathological regions, there was no direct comparison between the r2 values before and after pH-triggered disassembly of micellar nanoparticles. Thus, whether the pHtriggered release of SPIONs from micellar nanoparticles led to an increase in r2 was unclear. Later on, Almutairi et al.56 demonstrated that SPIONs could be embedded into pHresponsive poly-β-aminoester ketal-2 polymers, and it was found the released SPIONs precipitated out the solution under acidic conditions, resulting in decreased T2 relaxivity. On the other hand, these SPION loaded hybrid vectors may be subjected to spontaneous disassembly and SPION release after intravenous injection due to high dilution in blood after administration, leading to premature leakage and unwanted accumulation of SPIONs.57−60 Therefore, it remains challengeable to fabricate a T 2 -type MRI contrast agent with B


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Macromolecules

standards were employed for calibration. The degrees of polymerization, DP, of synthesized polymers were determined by 1H NMR analysis. UV/vis absorbance was carried out on a TU-1910 doublebeam UV−vis spectrophotometer (Puxi General Instrumental Company, China). Fluorescence experiments were performed on an F-4600 (Hitachi) fluorospectrometer. The slit widths for both excitation and emission monochromators were set to 5 nm. Dynamic laser light scattering (DLS) measurements were conducted on a Zetasizer Nano ZS (Malvern). Scattered light was collected at a fixed angle of 173° for a total duration of 5 min. Thermogravimetric analysis (TGA) was performed in air using a PerkinElmer Diamond TG/DTA Instruments at a heating rate of 10 °C/min. Inductively coupled plasma atomic emission spectrometry (ICP-AES) (PerkinElmer Corporation Optima 7300 DV) was used for Fe(III) content analysis. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB-MK II instrument (VG, UK) equipped with a monochromatic Al Kα X-ray source. Transmission electron microscopy (TEM) measurements were conducted on a JEOL 2010 electron microscope. The samples for TEM observations were prepared by dropping 20 μL of aqueous dispersions of hybrid nanovectors onto copper grids successively coated with thin films of Formvar and carbon. Atomic force microscopy (AFM) measurements were performed on a Bruker (Digital Instruments) Multimode Nanoscope IIID operating in the tapping mode under ambient conditions. A silicon cantilever (RFESP) with a resonance frequency of ∼80 kHz and a spring constant of ∼3 N/m was used. The set-point amplitude ratio was maintained at 0.7 to minimize sample deformation induced by the tip. The samples were prepared by dropping 20 μL of an aqueous dispersion of hybrid nanoparticle onto freshly cleaved mica surface. Confocal laser scanning microscopy (CLSM) images were acquired on a Leica TCS SP5 microscope. Synthesis of Amphiphilic Diblock Copolymers PEO45-b-PNBOCn (BP1−BP4). PEO45-b-PNBOCn diblock copolymers were prepared according to our previous report.63 Typical procedures employed for the RAFT synthesis of PEO45-b-PNBOCn are as follows. Using the preparation of BP1 as an example, NBOC (0.5 g, 1.60 mmol, 40.0 equiv), PEO-CTA (92 mg, 0.04 mmol, 1.0 equiv), and AIBN (1.3 mg, 0.008 mmol, 0.20 equiv) were dissolved in 1,4-dioxane (1.6 mL) with a magnetic stirring bar. The tube was carefully degassed by three freeze− pump−thaw cycles and then sealed under vacuum. After being thermostated at 70 °C in an oil bath and stirred for 24 h, the reaction tube was quenched into liquid nitrogen and opened; the reaction mixture was then precipitated into an excess of diethyl ether. The above dissolution−precipitation cycle was repeated three times. PEO45-b-PNBOCn was obtained as a pale red solid (0.3 g, yield: 60%). The DP of PNBOC block was determined to be 37 by 1H NMR spectroscopy (Figure S2), and the resulting block copolymer was thus denoted as PEO45 -b-PNBOC37 (BP1). According to similar procedures, PEO45-b-PNBOC54 (BP2), PEO45-b-PNBOC70 (BP3), and PEO45-b-PNBOC80 (BP4) were also synthesized by increasing the molar ratios of NBOC monomer to PEO macroRAFT agent. Fabrication of SPION Loaded Hybrid PEO-b-PNBOCn Nanovectors and DOX/SPION Coloaded Nanovectors. SPION loaded hybrid nanovectors were fabricated from amphiphilic PEO-b-PNBOCn block copolymers and hydrophobic OA-stabilized SPIONs via an O/ W emulsion and a subsequent solvent evaporation procedure. Briefly, PEO-b-PNBOCn (5 mg) copolymers were first dissolved in 100 μL of chloroform (CHCl3), and 40 μL of chloroform dispersion of Fe3O4 (25 mg/mL) was added. The mixture solution was then injected into 10 mL of deionized water and was treated with ultrasonication in a continuous or pulse-pause manner for varying durations. After removal of residual CHCl3 by rotatory evaporation, SPION loaded hybrid nanovectors were achieved. For the preparation of DOX/SPION coloaded nanovectors, a similar procedure was applied except additional DOX (1 mg) being added in the CHCl3 mixture containing block copolymers and SPIONs. Determination of SPION and DOX Loading Contents within Hybrid BP3 Nanovectors. SPIONs and DOX loading contents (defined as the weight percentage of SPIONs in the SPION loaded hybrid BP3 nanovectors and DOX in the DOX/SPION coloaded

amine groups from decaged carbamate linkages underwent spontaneous amidation reactions and allowed for permeabilizing the bilayer membranes without disintegrating vehicle integrity.63,64 We hypothesized that if SPIONs could be coassembled with tracelessly cross-linkable polymers, the formation of cross-linked networks within nanoparticles could well confine loaded SPIONs without leakage and the hydrophobic-to-hydrophilic transition should enable water accessibility into nanovectors, thereby elevating T2 relaxivity. Note that this transition could also be associated with drug release process, enabling the fabrication of stimuli-responsive theranostic vectors with triggered release profiles and enhanced imaging capability. In this work, we extend our tracelessly cross-linkable platforms to fabricate a photoreactive theranostic nanovectors with synergistic drug release and MRI enhancement performance. Photoreactive amphiphilic diblock copolymers, poly(ethylene oxide)-b-poly(2-((((2-nitrobenzyl)oxy)carbonyl)amino)ethyl methacrylate) (PEO-b-PNBOC), and hydrophobic SPIONs and chemotherapeutic drug doxorubicin (DOX) were coassembled into micellar nanoparticles stabilized by PEO coronas. The resulting hybrid nanovectors exhibited retarded DOX release and relatively low T2 relaxivity. However, upon photoirradiation-induced cross-linking and a hydrophobic-to-hydrophilic transition within the cores of nanovectors, embedded DOX was triggered release while SPIONs were restricted within hydrophilic cross-linked networks, resulting in synergistic drug release and increased r2. This design provides a possibility instantaneously image the therapeutic efficiency of theranostic nanovectors by taking advantage of synergistically increased MRI signals (Scheme 1).

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EXPERIMENTAL SECTION

Materials. Poly(ethylene oxide) monomethyl ether (PEO45-OH, Mn = 2.0 kDa, Mw/Mn = 1.06) was purchased from Aldrich and used as received. 2-Isocyanatoethyl methacrylate (stabilized with BHT) was purchased from TCI. 2-Nitrobenzyl alcohol and dibutyltin dilaurate (DBTL) were purchased from Shanghai Haiqu Chemical Co., Ltd. Doxorubicin (DOX) was purchased from Iffect Chemphar Co., Ltd. Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from GIBCO and used as received. LysoTracker Green and DAPI were purchased from Molecular Probes. 2,2-Azoisobutyronitrile (AIBN) was purified by recrystallization from 95% ethanol prior to use. Water was deionized with a Milli-Q SP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ·cm. All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., and used as received unless otherwise noted. The photoreactive monomer, 2-((((2-nitrobenzyl)oxy)carbonyl)amino)ethyl methacrylate (NBOC),63 PEO45-based macroRAFT agent (PEO45-CTA),65 and oleic acid (OA)-stabilized SPIONs with an average diameter of 7 nm were synthesized according to literature procedures (Figure S1a).66 Cetyltrimethylammonium bromide (CTAB)-coated SPIONs were synthesized from OAstabilized SPIONs via an established phase transfer procedure (Figure S1b).67 Characterization. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV300 NMR spectrometer (resonance frequency of 300 MHz for 1H and 75 MHz for 13C) operated in the Fourier transform mode. Fourier transform infrared (FT-IR) spectra were recorded on a Bruker VECTOR-22 IR spectrometer. Molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC) equipped with a Waters 1515 pump and a Waters 2414 differential refractive index detector (set at 45 °C). It used a series of two linear Styragel columns (HR2 and HR4) at an oven temperature of 45 °C. The eluent was DMF at a flow rate of 1.0 mL/min. A series of low polydispersity polystyrene C


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Table 1. Molecular Parameters of PEO-b-PNBOC BCPs and Corresponding SPION Loaded Hybrid Nanovectors before and after UV-Irradiation before UV entry

samples

BP1 BP2 BP3 BP4

PEO45-b-PNBOC37 PEO45-b-PNBOC54 PEO45-b-PNBOC70 PEO45-b-PNBOC80

Mna

(kDa)

13.6 18.8 23.8 26.9

Mn (kDa)

Mw/Mnb

⟨Dh⟩ (nm)

16.6 25.8 28.8 35.1

1.20 1.22 1.30 1.35

244 365 196 435

b

c

after UV μ2/Γ

2c

0.11 0.13 0.09 0.14

⟨Dh⟩ (nm)

μ2/Γ2 c

248 394 199 442

0.13 0.12 0.11 0.12

c

Determined by 1H NMR analysis in CDCl3. bObtained from GPC analysis using DMF as eluent (1 mL/min). cHydrodynamic diameters, ⟨Dh⟩, and polydispersity index, μ2/Γ2, of SPION loaded hybrid nanovectors determined by DLS measurements.

a

hybrid BP3 nanovectors) were quantified by TGA measurements and UV−vis spectroscopy, respectively. SPION loading content was determined to be 15.8 wt % by TGA measurement (Figure S4). The loading content of DOX was determined to be 10 wt % using absorbance spectroscopy at 480 nm based on a standard calibration curve. In Vitro DOX Release Profiles of DOX/SPION Coloaded BP3 Nanovectors. Typically, 4 mL of DOX loaded hybrid BP3 nanovectors was equally divided into four batches. Each batch contained 1 mL of hybrid nanoparticle dispersions and was placed in a dialysis tube (molecular weight cutoff: 3500 Da). After UVirradiation with predetermined times, the dialysis tubes were immersed in 10 mL of PBS medium under gentle stirring at 37 °C. Periodically, 10 mL of external buffer solutions was extracted and replaced with equal volumes of fresh PBS buffer medium. Upon each sampling, 1 mL of PBS buffer solution was diluted with 9 mL of DMSO. DOX concentration was quantified by measuring the UV−vis absorbance at 480 nm against a standard curve. In Vitro MRI Relaxivity Measurement. For in vitro MRI tests of SPION loaded hybrid nanovectors, T2 was acquired at room temperature using a GE Signa Horizon 1.5 T MR scanner equipped with a human shoulder coil. The T2-weighted images were acquired with a conventional spin-echo acquisition (TR = 4000 ms) with TE values ranging from 22 to 99 ms. The T2 relaxivity value, r2, was calculated via the least-squares curve fitting of 1/T2 (s−1) versus Fe concentration plots (mM). In Vitro MR Imaging of HepG2 Cells. HepG2 cells were first cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 °C in a CO2−air (5:95) incubator for 2 days. Then HepG2 cells were seeded in a 12-well plate at an initial density of ca. 100 000 cells/well in 1.0 mL of complete DMEM medium. After incubation for 24 h, the cells were treated with the SPION loaded hybrid nanovectors. After 8 h, the culture medium was removed, and cells were washed, irradiated with or without UV 365 nm light for 10 min, trypsinized, and neutralized. The cells without adding hybrid nanovectors were used as a control. After centrifugation at 1000 rpm for 5 min, cells were resuspended in 0.6 mL of PBS and 0.6 mL of 2% paraformaldehyde, followed by incubation at 4 °C for 1 h. After that, cells were washed with PBS to remove paraformaldehyde and were centrifuged at 1000 rpm for 5 min. A mixture of fresh PBS buffer (0.5 mL) and 2% agarose solution (0.5 mL) was then added and was transferred into a 1.5 mL centrifuge tube. Cells were kept at 4 °C overnight before MRI scan. The T2-weighted images were obtained using a GE Signa Horizon 1.5 T MR scanner equipped with a human shoulder coil as detailed above. In Vitro Cytotoxicity Assay of DOX/SPION Coloaded BP3 Nanovectors. HepG2 cells were first cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 °C in a CO2−air (5:95) incubator for 2 days. For the cytotoxicity assay, HepG2 cells were seeded in a 96-well plate at an initial density of ca. 5000 cells/well in 100 μL of complete DMEM medium. After incubation for 24 h, DMEM was replaced with fresh medium, and the cells were treated with DOX/SPION coloaded or DOX-free SPION loaded hybrid nanovector dispersions at varying concentrations for 2 h. Afterward, the cells were rinsed with PBS buffer, and 180 μL of fresh culture medium was added. The cells were

or were not irradiated with UV 365 nm light for 10 min and were incubated in a humidified environment with 5% CO2 at 37 °C for another 24 h. MTT reagent (in 20 μL PBS, 5.0 g L−1) was added to each well, and the cells were further incubated at 37 °C for an additional 4 h. After that, the medium in each well was then removed and replaced with 150 μL of DMSO. The plate was gently agitated for 15 min before the absorbance at 570 nm was recorded by a microplate reader (Thermo Fisher). Each experiment was conducted in quadruple, and the date were shown as the mean plus a standard deviation (±SD). Magnetically Guided Cell Uptake of DOX/SPION Coloaded Hybrid BP3 Nanovectors. HepG2 cells were plated onto glass-bottom Petri dishes at a density of 80 000 cells per dish and then cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL) for 24 h at 37 °C in CO2−air (5:95). UV-irradiated DOX/SPION coloaded hybrid BP3 nanovectors were added, and the equivalent DOX concentration was finally adjusted to 5 μg/mL. The cells were incubated in the presence and absence of an external magnet for 1 h. After that, cells were thoroughly rinsed with PBS buffer. The fluorescence of DOX was excited with a 543 nm laser, and the emission channel was collected between 570 and 650 nm. The uptake efficiencies of DOX/SPION coloaded hybrid BP3 nanovectors by HepG2 cells were roughly evaluated by the DOX emission intensities using an Imaging-Pro Plus software. Cellular Internalization and Intracellular Trafficking of DOX/ SPION Coloaded Hybrid BP3 Nanovectors Observed with Confocal Laser Scanning Microscopy (CLSM). HepG2 cells were plated onto glass-bottom Petri dishes at a density of 80 000 cells per dish and then cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL) for 24 h at 37 °C in CO2−air (5:95). DOX/SPION coloaded hybrid BP3 nanovectors were added, and the equivalent DOX concentration was finally adjusted to 5 μg/mL. The cells were incubated for 2 h, irradiated with UV 365 nm light for 10 min, and then incubated for another 2, 6, 10, and 22 h. Afterward, cell nuclei were stained with DAPI and endolysosomes were stained with LysoTracker green. After rinsing with PBS buffer, fluorescence images were taken using a confocal laser scanning microscopy. DOX was excited by a 543 nm laser with the emission channel was set to be 570−650 nm. DAPI and LysoTracker green were excited by 405 and 488 nm lasers, and emission channels were set to be 420−480 and 500−550 nm, respectively. The cells incubated with DOX/SPION coloaded hybrid nanovectors without UV-irradiation were employed as a control. Live/Dead Assay of HepG2 Cells. A live/dead assay was performed for the analysis of cell viabilities after cellular uptake of DOX/SPION coloaded hybrid BP3 nanovectors without or with 365 nm light irradiation. DOX/SPION coloaded hybrid BP3 nanovectors (200 μg/ mL, equivalent to 20 μg/mL DOX) were added to HepG2 cells and incubated for 2 h. The cells were rinsed with PBS and 180 μL of fresh medium was added, which was or was not subjected to 365 nm light irradiation for 10 min. After irradiation, the cells were incubated for another 24 h and were stained with fluorescein AM and propidium iodide (Molecular Probes) for 30 min to visualize the populations of live and dead cells. After rinsing with PBS buffer, the cells were imaged by confocal microscopy using 488 and 543 nm lasers as the excitation D


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Macromolecules wavelengths, and the emission detection channels were set to 500−530 and 560−700 nm, respectively.

fabricated from BP2 and BP4 (Figure 1b,d). Further, DLS analysis confirmed the TEM observations and manifested that the hybrid nanovectors fabricated from BP3 exhibited a relatively narrow polydispersity, μ2/Γ2, of 0.09 and a hydrodynamic diameter, ⟨Dh⟩, of 196 nm (Table 1). However, for other three BCPs, the resulting ⟨Dh⟩ were much larger than 200 nm, and the polydispersities ranged from 0.11 to 0.14 (Table 1). Given that, nanovectors with a ⟨Dh⟩ larger than 200 nm may be unfavorable for their potential application in a truly physiological condition. Therefore, hybrid nanovectors constructed from BP3 were employed for further studies. It is worth noting that the distributions of SPIONs within the polymeric matrices were significantly affected by the ultrasonication procedures and durations. For instance, nanoparticles with multiple SPION clusters were achieved if a pulse-pause ultrasonication was applied for 90 s, while an extended continuous ultrasonication treatment for 300 s led to the formation of colloidal nanoparticles with quite dispersed ⟨Dh⟩ and multiple SPION domains (Figure S3). From TGA analysis, the weight fraction of SPIONs in the hybrid BP3 nanovectors was calculated to be 15.8 wt %, which was very close to the initial feed ratio of SPIONs to BP3 copolymer (1/ 5, w/w) (Figure S4b). This suggested that almost all the SPIONs were encapsulated into hybrid nanovectors, in accordance with the TEM observation that no free SPIONs were observed outside the hybrid nanovectors (Figure 1c). UV-Irradiation-Induced Traceless Cross-Linking of SPION Loaded Hybrid Nanovectors. Previously, we demonstrated that PEO-b-PNBOC diblock copolymers can self-assemble into vesicles and the vesicular assemblies underwent traceless cross-linking and a hydrophobic-to-hydrophilic transition within the bilayer membranes under UVirradiation.63 Further, we found that the traceless cross-linking approach can be extended to other stimuli-responsive systems. For example, the photolabile triggering motifs can be switched to biorelevant hydrogen peroxide (H2O2)64 and bacteriasecreted enzymes.68 In the design of traceless cross-linking systems, a general strategy is to incorporate caged carbamate residues into polymerizable units through self-immolative linkages. The removal of triggering events under specific stimuli releases primary amines that subsequently elicit spontaneous amidation reactions, thereby concurrently crosslinking the assemblies and rendering them hydrophilic. For SPION loaded hybrid nanovector dispersions, to monitor the photoirradiation-triggered cross-linking and the hydrophobic-to-hydrophilic transition within the cores of nanovectors, DLS was first used to characterize the hydrodynamic diameters, ⟨Dh⟩, of the hybrid nanovectors before and after UV-irradiation, revealing a minor increase in ⟨Dh⟩ from 196 to 199 nm after UV-irradiation for 30 min (Table 1 and Figure 2a). A more detailed examination with a total irradiation duration of 30 min revealed an approximately 16.4% decrease in scattering intensities (Figure 2b). Further, UV−vis spectroscopy revealed a constant absorbance decrease at the wavelength of 277 nm and a concomitantly increased absorbance centered at 244 nm within the first 15 min irradiation and then leveled off (Figure 2c), suggesting that the photolysis reaction of NBOC moieties could be finalized within 15 min. To uncover the chemical transitions under UV-irradiation, XPS core-level N 1s spectra were recorded for the hybrid nanovectors before and after UV-irradiation (Figure 2d). Prior to UV light irradiation, the signals of N atoms of carbamate and o-nitrobenzyl moieties centered at 399.9 and 406.1 eV were clearly discerned.

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RESULTS AND DISCUSSION Fabrication of SPION Loaded Hybrid Nanovectors. Amphiphilic diblock copolymers (BCPs), PEG45-b-PNBOCn, composed of hydrophilic PEO and photoreactive hydrophobic PNBOC blocks were synthesized via RAFT polymerizations. Overall, four different diblock copolymers (BP1−BP4, Table 1) with varying PNBOC block lengths were prepared using the same PEO-based macroRAFT agent, and their structural parameters are summarized in Table 1. Oleic acid (OA)stabilized SPIONs was synthesized according to the reported procedures,66 and TEM was used to characterize the morphology and size of SPIONs. Spherical SPIONs with a diameter of 7 nm was successfully synthesized (Figure S1). To fabricate SPION loaded hybrid nanovectors, our initial attempt was to coassemble the hydrophobic SPIONs and BP1−BP4 copolymers via a cosolvent approach, which was proved to be inefficient and the loading contents of SPIONs were relatively low and nonuniform. Alternatively, an O/W emulsion and a subsequent solvent evaporation procedure was applied. Specifically, the amphiphilic block copolymers and SPIONs were dispersed in a cosolvent, chloroform (CHCl3). The mixtures were injected into aqueous solutions, followed by either continuous or pulse-pause sonication to form oil-in-water (O/W) emulsions. The residual CHCl3 was then removed under reduced pressure with the formation of water-dispersed SPION loaded hybrid nanovectors. Using a continuous ultrasonication approach for 90 s, all four block copolymers (BP1−BP4) can form stable colloidal nanoparticles in aqueous solutions. The formation of SPION loaded hybrid nanovectors was confirmed by TEM observations, revealing that the resultant hybrid colloidal nanoparticles were all in spherical morphologies (Figure 1). Remarkably, SPION loaded hybrid nanovectors with relatively narrow dispersity and uniform size were only achieved from BP3. In particular, SPIONs distributed throughout the colloidal nanoparticles constructed from BP1 (Figure 1a) while multiple domains of SPIONs were observed within the nanoparticles

Figure 1. TEM images recorded for the OA-stabilized SPION loaded hybrid nanovectors fabricated from (a) BP1, (b) BP2, (c) BP3, and (d) BP4. Hybrid nanovectors were fabricated through continuous ultrasonication for 90 s, followed by rotary evaporation to remove the organic solvents (i.e., CHCl3). The average diameter of SPIONs is 7 nm. E


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encapsulated Nile red (NR), a polarity-sensitive dye, into the cores of the hybrid nanovectors. No appreciable fluorescence changes were observed for the NR loaded hybrid nanoparticle dispersions stored in the dark (Figure S6), demonstrating that there was no polarity changes in the hybrid BP3 nanovectors. However, under UV-irradiation, the evolution of fluorescence emission spectra of NR revealed a steady decrease in fluorescence emission at 628 nm (Figure 2f). Notably, the gradually decreased fluorescence intensities were accompanied by a red-shift of the emission spectroscopy, clearly suggesting the formation of a hydrophilic microenvironment subjected to UV light irradiation (Figure 2f). To clarify whether the photoirradiation process cross-linked the hybrid nanovectors, we examined the DLS results of UVirradiated nanovectors in THF, a good solvent for PEO-bPNBOC block copolymers and OA-stabilized SPIONs. In comparison with the disassembly of nonirradiated nanovectors with a ⟨Dh⟩ of 12 nm, the UV-irradiated BP3 hybrid nanovectors had a ⟨Dh⟩ of 220 nm, slightly larger than that of spherical assemblies in aqueous solution (i.e., 199 nm, Table 1). This result clearly confirmed that the irradiated nanovectors were cross-linked (Figure S7). Further, we used TEM and AFM to monitor the morphologies of the SPION loaded hybrid BP3 nanovectors before and after UV-irradiation (Figure 3). After

Figure 2. Characterization of the SPION loaded hybrid BP3 nanovectors upon UV 365 nm light irradiation (∼1.0 mW/cm2). (a) Dh distributions of aqueous dispersions of hybrid nanovectors before and after UV-irradiation. Irradiation duration-dependent evolution of (b) ⟨Dh⟩ and scattered light intensities and (c) UV/vis absorbance spectra of SPION loaded hybrid nanovectors. The inset in (c) shows optical absorbance changes at 244 nm upon UV 365 nm irradiation. (d) XPS N 1s core level and (e) FT-IR spectra of SPION loaded hybrid nanovectors before and after UV-irradiation; the samples were obtained via lyophilization of hybrid nanovectors dispersions. (f) Irradiation-duration-dependent evolution of fluorescence emission spectra (λex = 550 nm) of Nile red loaded hybrid nanovectors upon UV-irradiation. The inset in (f) shows emission intensity changes at 628 nm under 365 nm UV-irradiation.

However, after UV-irradiation, a new peak at 405.5 eV corresponding to N atoms of protonated primary amine moieties appeared. Meanwhile, the N 1s signal at 399.9 eV slightly shifted to lower binding energy at 399.5 eV, indicative of the formation of amide derivatives. A quantitative analysis revealed that the N species of protonated primary amines accounted for 12% in the UV-irradiated hybrid nanovectors (Figure 2d), in accordance with a reverse transition of zetapotentials from −9 to +1.5 mV (Figure S5). Note that the final zeta-potential value after UV-irradiation was lower than that in the absence of SPIONs (∼+18 mV);63 we inferred that the negatively charged nature of SPIONs may partially neutralize the generated positive charges of protonated primary amine moieties. Further, the formation of amide bonds was evidenced by the FT-IR spectra (Figure 2e). Specifically, the absorbance characteristic peak of ester carbonyl moieties (1730 cm−1) considerably decreased, accompanied by an increase of amide carbonyl absorbance peak at 1640 cm−1. The absorbance characteristic peaks of nitro groups (1354 and 1520 cm−1) pronouncedly dropped after UV-irradiation, in agreement with the photocleavage of the o-nitrobenzyl groups (Figure 2e). We reasoned that the photoirradiation-actuated amidation reactions with the formation of amide bonds and protonated primary amine derivatives may lead to a significant shift in the polarity of nanovectors within the cores. To confirm this, we

Figure 3. (a−d) TEM images recorded for hybrid BP3 nanovectors (a, c) without and (b, d) with UV-irradiation for 20 min (a, b) in aqueous and (c, d) THF solutions. (e−h) AFM height images recorded for aqueous solutions of hybrid BP3 nanovectors (e) without and (g) with UV-irradiation for 20 min. (f, h) Cross-sectional profiles.

UV-irradiation, the spherical morphology of hybrid nanovectors was retained in aqueous solutions. Note that SPIONs were still encapsulated within the nanovectors after UV-irradiation, although OA-stabilized SPIONs were inherently hydrophobic (Figure 3a,b). This result could be explained by the formation of cross-linked networks after UV-irradiation that remarkably restricted the free migration of SPIONs. On the other hand, the TEM images recorded for the THF solutions of nonirradiated nanovectors showed that only dispersed SPIONs were visible. By contrast, spherical nanostructures loaded with SPIONs within BP3 matrices were retained for the UV-irradiated F


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Figure 4. (A) T2-weighted MR images obtained for CTAB-stabilized SPIONs and SPION loaded hybrid BP3 nanovectors without and with UVirradiation for 5, 10, and 20 min. (B) Iron concentration-dependent water proton transverse relaxation rates (1/T2) of aqueous dispersions (25 °C) of (a) CTAB-stabilized SPIONs and SPION loaded hybrid BP3 nanovectors (b) without and (c−e) with UV-irradiation for (c) 5, (d) 10, and (e) 20 min. (C) MR images recorded for untreated HepG2 cells (control) and HepG2 cells treated with 0.2 g/L SPION loaded hybrid BP3 nanovectors without and with 365 nm UV light irradiation for 10 min.

nanovectors, although some SPIONs were released from the irradiated nanovectors (Figure 3c,d). Therefore, we can conclude that UV-irradiation process of SPION loaded hybrid nanovectors not only led to a hydrophobic-to-hydrophilic transition within the cores but also resulted in cross-linking of the nanovectors. The formation of cross-linked nanostructures could also be supported by AFM measurements. The height images of UV-irradiated nanovectors increased to 146 nm from 71 nm of untreated hybrid nanovectors (Figure 3e−h). This result was likely attributed to the formation of more rigid crosslinked nanostructure subjected to UV-irradiation.63 To further elucidate the cross-linking mechanism, we monitored the release of 2-hydroxyethylamine (HEA) from the irradiated nanovectors under UV-irradiation. Note that the formation of HEA can only be from intrachain/interchain aminolysis reactions, and the latter will contribute to the formation of cross-linked nanostructures (Scheme 1). The generation of HEA side products was evidently confirmed by fluorescamine (FA) probes, which can specifically react with primary amine functionalities with turn-on fluorescence emission. Based on a standard calibration curve (Figure S8b,c), the generated HEA amount was determined to be 121.7 μM, corresponding to ∼15.7% of total primary amines if we assumed that all the NBOC moieties were transformed to primary amines. This result strongly suggested that the amidation reactions did occur under UV-irradiation, and the amidation reaction finally led to the formation of cross-linked nanostructures. Unfortunately, we failed to precisely determine the cross-linking degree of irradiated nanovectors since both interchain and intrachain amidation reactions would result in the release of HEA whereas only interchain amidation reactions could contribute to valid cross-linking. Therefore, the final cross-linking degree of irradiated hybrid nanovectors should be lower than 15.7% and such a low cross-linking degree would be quite favorable for the diffusion and transportation of small molecules (i.e., drug and water molecules). Considering the release of HEA small molecules under UVirradiation, it was not surprising that there was an evident increase in the weight retention of irradiated nanovectors as compared to untreated BP3 nanovectors, as evidenced by TGA result (Figure S4c). Theoretically, the weight retention of

irradiated hybrid BP3 nanovectors can be calculated according to the equation wFe3O4 ,UV wretention,UV = × 100% wFe3O4 ,UV + wOA + wBP3,UV (1) where wretention,UV is the weight retention of irradiated BP3 nanovectors, wFe3O4,UV is the weight of encapsulated SPIONs within the irradiated nanovectors, WOA is the weight of OA small molecules coated on the SPIONs, and WBP3,UV is the weight of irradiated BP3 copolymers within the nanovectors that can be further calculated from the formula wBP3,UV = wBP3 − wNSBA − wCO2 − wHEA (2) where WBP3 is the original weight of BP3 prior to UVirradiation, wNSBA and wCO2 are the weights of released onitrosobenzaldehyde and CO2 upon UV-irradiation, and wHEA is the weight of released 2-hydroxyethylamine (HEA) during the amidation reactions after UV-irradiation. If we assumed that the OA small molecules on the surfaces of SPIONs did not change during the formation of hybrid nanovectors, according to the TGA measurements (Figure S4) and the released HEA amounts quantified from the fluorogenic reaction (Figure S8), the weight ratio of BP3 to encapsulated W SPIONs ( BP3 ) was calculated to be 5.15 based on eqs 1 and WFe3O4,UV

2 while this ratio (

WBP3 ) WFe3O4

was determined to be 5.21 prior to

UV-irradiation from TGA result (Figure S4b). Note that the irradiated hybrid BP3 nanovectors were lyophilized and exhaustively washed with cyclohexane to remove any free OA-stabilized SPIONs before TGA measurement. Therefore, we can conclude that almost all encapsulated SPIONs were retained within the irradiated hybrid BP3 nanovectors without evident leakage and release. This was quite unique and drastically different from previously developed T2-type MRI contrast agents containing SPION clusters that the loaded SPIONs were released upon external stimuli, resulting in decreased relaxivities.54,55 In Vitro MR Imaging Relaxivity Measurements. SPIONs have been widely used for T2-type MRI contrast agents in clinical diagnosis, and there are several commercially G


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spin−spin relaxation rates (1/T2) increased from 3.7 to 6.5 s−1 for untreated HepG2 cells and cells incubated with hybrid nanovectors without UV-irradiation, whereas a further increase in 1/T2 was observed for HepG2 cells incubated with irradiated hybrid nanovectors (Figure 4C). The enhanced MRI signals in cells may suggest that the photoreactive hybrid nanovectors could be of potential use in practical diagnosis applications. Photoregulated DOX Release from DOX/SPION Coloaded Hybrid Nanovectors. It is worth noting that the UV-irradiation process rendered the cores of hybrid nanovectors hydrophilic and cross-linked, which could also be employed for controlled release of hydrophobic therapeutic payloads (e.g., doxorubicin; DOX). As a proof-of-example, DOX was coloaded into the hybrid nanovectors during the selfassembly process and photoirradiation-triggered DOX release was monitored by UV−vis spectroscopy. Specifically, less than 5% of DOX was released without UV-irradiation within 10 h incubation duration. However, DOX release extents dramatically increased upon UV-irradiation. For example, approximately 21% of DOX was released after 5 min UV-irradiation, and it was further increased to 52% and 85% after 10 and 20 min UV-irradiation (Figure 5a). Interestingly, there was a linear

available products such as Feridex, Resovist, and Clariscan, giving darkening contrasts in MRI images.69,70 To elevate the relaxivities of SPION-based contrast agents, SPIONs were encapsulated into diverse polymeric matrices with the formation of SPION clusters, exhibiting enhanced relaxivities (r2) as compared to monodispersed SPIONs.24,26 Also, previous studies suggested that the accessibility of surrounding water molecules to SPIONs in the hydrophobic cores of hybrid nanocarriers remarkably affected r2.30 Note that the present photoreactive SPION loaded hybrid nanovectors could simultaneously undergo a hydrophobic-to-hydrophilic transition and cross-linking reactions under UV-irradiation, which is expected to increase the water accessibility. However, the local concentration of encapsulated SPIONs within irradiated nanovectors remained unchanged. Therefore, we hypothesized that the photoirradiation process with the formation of crosslinked nanovectors should remarkably elevate the r2, exhibiting better imaging contrast. Subsequently, T2-weighted spin-echo MR images of nonirradiated SPION loaded hybrid nanovectors and UV-irradiated nanovectors for 5, 10, and 20 min were recorded using a 1.5 T MRI scanner. CTAB-stabilized SPIONs fabricated from OAstabilized SPIONs through a previously reported phase transfer procedure with an average diameter of 7 nm were used as a reference (Figure 4A).67 Upon increasing the Fe concentrations from 0 to 0.256 mM, gradually enhanced negative MRI contrasts were observed (Figure 4A). Quantitative analysis of the spin−spin relaxivities (r2) revealed that CTAB-stabilized SPIONs had the lowest r2 of 62.2 s−1 mM−1, whereas it was increased to 149.1 s−1 mM−1 after encapsulated into BP3 matrices without UV-irradiation, consistent with previous literature reports.24,26 Interestingly, the r2 was further increased to 283.0 s−1 mM−1 after 20 min UV treatment (Figure 4B). Note that previous studies suggested that the formation of nanocrystal clusters of SPIONs led to increased magnetic moment that drastically elevated the r2 relaxivities.71−73 However, in the current study, SPIONs were encapsulated into amphiphilic diblock copolymers (BP1−BP4) through hydrophobic association. Although closely packed nanostructures were formed within the cores of nanoparticles, a number of studies revealed that the magnetic moment of the embedded SPIONs did not significantly change compared to that of individual SPION.24,28,29 Given the fact that the hydrophobic cores were cross-linked and were turned hydrophilic and there was no remarkable change in the local concentration of loaded SPIONs upon UV-irradiation process, we tentatively attributed the elevated r2 to the increased water accessibility.74,75 Compared to previously developed pH-responsive polymerbased T2-type MRI contrast agents with decreased r2 after responding to acidic pH,56 we demonstrated for the first time that the relaxivity of T2-type contrast agents could be elevated through the formation of unique cross-linked nanostructures that efficiently inhibited the release of encapsulated SPIONs. Notably, the enhanced rather than decreased MRI signals should be beneficial for practical diagnosis. To demonstrate the potential application of the switchable MRI contrast agent, T2-weighted MRI images of HepG2 cells incubated with SPION loaded hybrid BP3 nanovectors with or without UV-irradiation were recorded on a clinical 1.5 T MRI scanner. The increased MRI contrasts can be readily observed by the darkening contrasts after UV-irradiation as compared to the control cells (hybrid BP3 nanovectors free) and cells without UV-irradiation. Quantitative analysis revealed the

Figure 5. (a) In vitro release profiles of doxorubicin (DOX) from DOX/SPION coloaded hybrid BP3 nanovectors without and with UV-irradiation for 5, 10, and 20 min. (b) Irradiation durationdependent evolution of T2 relaxivity, r2, and cumulative DOX release of DOX/SPION coloaded hybrid BP3 nanovectors. (c) Linear correlation of cumulative DOX release extents with corresponding r2 relaxivities without and with UV-irradiation for 5, 10, and 20 min. (d) MTT assay against HepG2 cells after incubation with nonirradiated and UV-irradiated SPION loaded hybrid BP3 nanovectors and DOX/ SPION coloaded hybrid BP3 nanovectors without and with UVirradiation for 10 min. The DOX loading content was 10 wt %.

correlation between the irradiation duration-dependent enhancement of r2 and the cumulative release amounts of DOX (Figure 5b,c), allowing for instantaneously monitoring the release behavior and reporting the therapeutic efficacy of DOX in vivo by the MRI technique. Note that this would be rather useful especially when the emission of DOX could not be readily detected in situ because of low penetration of the fluorescence technique. Next, HepG2 cells were employed to evaluate the cytotoxicity of DOX/SPION coloaded hybrid nanovectors. As shown in Figure 5d, both DOX-free hybrid nanovectors with or without UV-irradiation exhibited negligible cytotoxicity, and H


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Figure 6. (a) Representative CLSM images recorded for HepG2 cells after incubation with DOX loaded hybrid BP3 nanovectors for 2 h, followed by rinsing with PBS buffer three times, irradiation with 365 nm UV light for 10 min, and additional incubation for 2, 6, 10, and 22 h. Late endosomes/lysosomes and cell nuclei were stained with LysoTracker green (green channel) and DAPI (blue channel). (b) Relative fluorescence intensities of DOX within nuclei and (c) colocalization ratios of red channel (DOX) and green channel (LysoTracker green) fluorescence were quantified from CLSM observations. Error bars represent mean ± SD, n = 4.

more than 90% of cells survived even at a concentration of 200 μg/mL, suggesting that both the BP3 copolymers and SPIONs were inherently nontoxic to cells. However, the cell viability drastically decreased to 38% at the same concentration for DOX/SPION coloaded hybrid nanovectors in the absence of UV-irradiation. The evidently increased cytotoxicity likely arose from the free diffusion of embedded DOX. However, less than 10% cells were survived after irradiation the hybrid nanovectors with UV-irradiation for 10 min, although no significant cytotoxicity was observed for the SPION loaded hybrid nanovectors without DOX loading (Figure 5d). Therefore, the increased cytotoxicity was unlikely due to a short duration of UV-irradiation (i.e., 10 min), and we attributed the increased cytotoxicity of irradiated DOX/SPION coloaded nanovectors to the triggered release of encapsulated DOX, which in turn inhibited the proliferation of cancer cells. In the next phase of work, the cellular internalization and intracellular trafficking of DOX/SPION loaded hybrid nanoparticles were observed with CLSM. HepG2 cells were first incubated with DOX/SPION coloaded hybrid nanovectors for 2 h, followed by UV-irradiation for 10 min; the cells were then incubated for an additional 2, 6, 10, and 22 h. As shown in Figure 6a, within the first 6 h incubation, the red fluorescence of hybrid nanovectors overlapped quite well with the green channel of LysoTracker green, while negligible red fluorescence was observed within cell nuclei. Upon extending the incubation time, a gradual increase of red fluorescence within cell nuclei was discerned. Specifically, there was a cumulative 6.3-fold increase of red emission from DOX within nuclei after 22 h incubation compared to that after 2 h incubation (Figure 6b). Meanwhile, quantitative analysis of the colocalization ratio between the red channel of DOX and the green channel of LysoTracker green revealed a monotonous drop from 91% to 49% for an additional 2 and 22 h incubation, respectively, suggesting the gradual release of loaded DOX from internalized

hybrid nanovectors after UV-irradiation. However, neither the fluorescence intensity of DOX within cell nuclei nor the colocalization ratio between the red channel and green channel exhibited pronounced changes without UV-irradiation (Figure 6b,c and Figure S9). These results concurred quite well with the higher cytotoxicity of DOX/SPION coloaded hybrid nanovectors after UV-irradiation as compared to that without UV-irradiation (Figure 5d). After DOX/SPION coloaded hybrid nanovector treatment, the cells were further analyzed by a Live/Dead assay. Again, we found that 10 min of UVirradiation had negligible adverse effects on the viability of HepG2 cells, and intense green emission was observed in the absence of DOX/SPION coloaded nanovectors (Figure S10a,b), consistent with the MTT assay (Figure 5d). However, the cell viabilities dramatically dropped after incubation with DOX/SPION coloaded nanovectors and a 10 min of UVirradiation led to a further decrease in cell viability, as evidenced by the overwhelming red emission of propidium iodide that can only penetrate the cell membranes of dead cells. Although the release of DOX was retarded without UV-irradiation and no apparent accumulation was observed within cell nuclei (Figure S9), the internalized DOX/SPION coloaded nanovectors may exert adverse impact on cell viability, and a decreased cell viability was thus observed (Figure 5d). To further explore the potential applications of the SPION/ DOX loaded hybrid BP3 nanovectors, we examined the colloidal stability of hybrid nanovectors in different mediums by DLS measurements (Figure S11).76,77 Upon incubation in 10 mM PBS buffer for 24 h, the ⟨Dh⟩ and polydispersities (μ2/ Γ2) of untreated BP3 nanovectors exhibited appreciable increases while UV-irradiated nanovectors showed negligible changes in ⟨Dh⟩ and polydispersities, indicative of increased colloidal stability after cross-linking. Moreover, the increased stabilities were further verified by the presence of either bovine serum albumin (BSA, 45 g/L) or a high content of fetal bovine I


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external magnetic field was evaluated. With applying an external magnetic field, the red fluorescence of DOX in the cells was much more intense as compared to that without an external magnetic field after 1 h incubation, and the quantitative data in Figure 7e revealed that the DOX intensity exhibited a 2.7-fold enhancement, clearly demonstrating that the DOX/SPION coloaded hybrid nanovectors could be of potential use for magnetically targeted drug delivery.

serum (FBS, 90% v/v). In both cases, untreated BP3 nanovectors formed large aggregates with pronouncedly increased ⟨Dh⟩ upon extending the incubation time, whereas the cross-linking process subjected to UV-irradiation can efficiently attenuate this adverse effect and significantly decreased agglomeration (Figure S11b,c). As such, the traceless cross-linking strategy with enhanced colloidal stability of SPION/DOX loaded nanovectors presages promising application in a truly physiological condition. Magnetically Guided Delivery of DOX/SPION Coloaded Hybrid Nanovectors. The incorporation of SPIONs into polymeric matrices results in the formation of magnetic targeted carriers that renders it possible to target pathological sites following administration. In the next step, we appraised the potential of DOX/SPION coloaded nanocarriers as a magnetically targeted carrier other than photoreactive theranostic nanovectors. First, DOX/SPION coloaded nanovectors with or without UV-irradiation were more susceptible to externally magnetic fields than for CTAB-stabilized monodispersed nanoparticles, which could be more easily captured in the presence of a magnet (Figure 7a−c). This result

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CONCLUSIONS In summary, a new T2-type MRI contrast agent composed of photoresponsive PEG-b-PNBOC diblock copolymers and OAstabilized SPIONs with enhanced relaxivities under UVirradiation was reported. The SPION loaded hybrid nanovectors underwent photoinduced cross-linking and a hydrophobic-to-hydrophilic transition within the initially hydrophobic cores as a result of spontaneous amidation reactions of decaged carbamate moieties. This concurrent transition can not only exclude unwanted leakage of SPIONs from the crosslinked networks but also increase the water accessibility, thereby increasing T2 relaxivity under UV-irradiation. Moreover, therapeutic drugs could also be embedded into the nanovectors, and photoirradiation triggered release of DOX was achieved, allowing for instantaneously monitoring the release profiles and reporting therapeutic efficiency. Further, we demonstrated that the hybrid nanovectors could be potentially magnetically delivered to pathological sites, exhibiting enhanced cellular internalization performance. The combination of tunable MRI signals, triggered drug release, and magnetically guided delivery is expected to enhance the theranostic efficacy of nanovectors in practical applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b02162. Additional TEM, 1H NMR spectrum, TGA, fluorescence spectra, zeta-potential, DLS, and CLSM images (PDF)

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Figure 7. Macroscopic photographs of aqueous dispersions of (a) CTAB-stabilized SPIONs, (b) untreated SPION loaded hybrid BP3 nanovectors, and (c) UV-irradiated SPION loaded hybrid BP3 nanovectors in the absence and presence of an external permanent magnet. (d) Representative CLSM images recorded for HepG2 cells after incubation with DOX/SPION coloaded hybrid BP3 nanovectors for 1 h in the absence or presence of an external magnetic field. (e) Normalized fluorescence intensities (red channel) of HepG2 cells were quantified from CLSM observations.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.L.). *E-mail: [email protected] (J.H.). *E-mail: [email protected] (G.L.). ORCID

Zhengyu Deng: 0000-0002-7186-7491 Shiyong Liu: 0000-0002-9789-6282

preliminarily demonstrated that the DOX/SPION coloaded hybrid nanovectors could be magnetically delivered to the lesions under the assistance of an external magnetic field. Remarkably, after UV-irradiation, the cross-linked nanovectors can still be driven by an external magnet, in good agreement with the retention of SPIONs within irradiated nanovectors. Note that this result was unparalleled, and the embedded SPIONs were commonly released upon external stimuli and the resulting nanocarriers cannot be manipulated by external magnetic field anymore in previous literature.54,55 To demonstrate the magnetic targeting drug delivery in cancer cells, the uptake of the DOX/SPION coloaded hybrid nanovectors by HepG2 cells in the absence or presence of an

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support from the National Natural Scientific Foundation of China (NNSFC) Project (51690150, 51690154, 21674103, and 21274137), and the Startup Fund of USTC (KY2060000068) is gratefully acknowledged.

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