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Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 12544−12552

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Polydopamine-Derivated Hierarchical Nanoplatforms for Efficient Dual-Modal Imaging-Guided Combination in Vivo Cancer Therapy Lijiao Ao,†,§ Chunlei Wu,† Ke Liu,† Wei Wang,† Lijing Fang,† Liang Huang,*,‡ and Wu Su*,† †

Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China ‡ College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China § Shenzhen College of Advanced Technology, University of Chinese Academy of Sciences, Shenzhen 518055, P. R. China S Supporting Information *

ABSTRACT: Exploring multifunctional nanomaterials from biocompatible constituents, with integrated imaging and targeted combination therapeutic modalities of tumors in vivo, provides great prospects for clinical cancer theranostic applications. Here, we report a combination strategy for functionalization of polydopamine (PDA) nanohosts with magnetic response and stimuli-controlled drug release capabilities for in vivo cancer theranostic. The high processability of PDA as nanotemplates and surface coating layers as well as its natural affinity to metals facilitated the sandwich of a compact iron oxide nanoparticle layer into the PDA matrix, realizing enhanced near-infrared (NIR) photothermal conversion and strong superparamagnetic responsiveness. Additionally, the high reactivity of the PDA surface allowed facile linkage with reduction-responsive prodrugs and polyethylene glycol chains for in vivo chemotherapy of cancer. Under the magnetic resonance imaging/photoacoustic imaging dual-modal tumor imaging and active magnetic tumor targeting of the nanoagents in vivo, the effective tumor eradication was achieved via synergetic NIR photothermal ablation and anticancer drug delivery. KEYWORDS: polydopamine, iron oxide, drug delivery, photothermal therapy, theranostic

1. INTRODUCTION

and meticulous localization of tumor tissues via photoacoustic imaging (PAI).15 Superparamagnetic iron oxide (IO) nanoparticles (refer to as IOs) as a class of biocompatible nanomaterials have drawn great research interest in clinical imaging diagnostic and therapeutic applications.16−19 The strong chemical affinity of a catechol group to ferrite surfaces has been successfully employed for creating highly stable hydrophilic IOs using dopamine-derivated ligands.20−24 Inspired by this feature, the incorporation of an IOs layer into the PDA matrix to produce magnetic-responsive PDA composites seems attractive. The PDA-templated magnetic structure shows superiority in the control of particle size and size distribution, compared with previous single or clustered IO-cored PDA nanocomposites.12,25,26 This strategy may promote new theranostic modalities such as multimodal imaging diagnosis using both magnetic resonance imaging (MRI) and PAI as well as magnetic field-guided PTT for targeted tumor ablation. Meanwhile, the intrinsic chemical reactivity of PDA facilitates

The bioinspiration from the strong adhesion feature of invertebrate mussels realized the emergence of polydopamine (PDA) as a robust, facile, and universal surface coating material on diverse substrates,1−3 which led to an important breakthrough in materials surface functionalization. For nanoscience and nanotechnology, the striking character of PDA as a novel polymer lies in its advance for multifunctional nanostructure engineering.4−6 From the view point of synthetic scheme, the preparation of colloidal PDA via a Stöber-like process is rather facile and well-controlled for both nanosubstrates (templates) and encapsulating layers on various nanoscale objects including metals, semiconductors, oxides, and polymers, bridging discrete functionalities from different components.7−11 Moreover, as a major pigment of naturally occurring melanin,5 PDA exhibits superior biocompatibility for potential clinical theranostic applications. Noticeably, the intense and broad absorption spectrum across visible to near-infrared (NIR) region enables PDA to be an excellent photothermal agent to induce highly localized heat for cancer photothermal therapy (PTT).12−14 Simultaneously, the introduction of a pulsed NIR laser onto PDA would allow the detection of ultrasonic waves for sensitive © 2018 American Chemical Society

Received: February 19, 2018 Accepted: March 23, 2018 Published: March 23, 2018 12544

DOI: 10.1021/acsami.8b02973 ACS Appl. Mater. Interfaces 2018, 10, 12544−12552

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of the synthetic procedure and application of a PDA-based theranostic nanoplatform.

a variety of reactions for functional uses.1,5 For instance, the introduction of therapeutic drugs onto a PDA-based nanoplatform through cleavable cross-linkers may allow stimuli-response drug release at the tumor site,27−29 which could wellcompensate the limitations of PTT for synergetic in vivo cancer therapy. In this paper, a theranostic nanoplatform derived from biocompatible PDA colloids was designed for multimodal imaging and magnetic field-guided combination cancer therapy. As illustrated in Figure 1, the PDA nanospheres were employed as metal-affinity templates for in situ deposition of a compact IOs layer via high-temperature thermal decomposition to form PDA@IOs nanospheres. A thin PDA shell was encapsulated onto the IOs surface to form sandwich-type PDA@IOs@PDA (PIP) nanocomposites with simultaneous magnetic response, NIR photothermal conversion, and high surface reactivity. A redox-sensitive cross-linker containing a disulfide bond was employed to bridge the chemotherapy drug (doxorubicin, DOX) and the polyethylene glycol (PEG) spacer, which was covalently bonded to the PDA surface. With reductionresponsive drug release and PTT therapeutic capabilities of the PIP−DOX nanospheres, the in vivo combination therapy was realized under the MRI/PAI dual-modal imaging and magnetically active tumor targeting.

solution (1 mL) was added successively. The mixed solution was appropriately ultrasonicated and maintained at 70 °C until ethanol was completely evaporated. After being filled with nitrogen, the flask was kept stirring at 210 °C for 2 h. The solution was then heated to 290 °C and kept stirring for another 1 h. After being cooled to room temperature, the above solution was first diluted with acetone, and the PDA@IOs nanocomposites were purified with ethanol several times with centrifugation or magnetic separation. 2.4. Synthesis of PIP Nanospheres. The dopamine solution was prepared by dissolving dopamine hydrochloride (8 mg) in 20 mL of 10 mM Tris-HCl buffer (pH ≈ 8.5). The PIP nanospheres were prepared by dispersing the PDA@IO nanospheres (20 mg) into the above dopamine solution and stirred for 24 h. The PIP nanospheres were then purified with water several times. 2.5. Synthesis of DOX−SS−PEG-NH2 Prodrug. Dithiodipropionic anhydride (DTDPA) was first synthesized as follows: DTDP (5 g) was dissolved in acetyl chloride (15 mL) and then refluxed for 2 h at 65 °C. Acetyl chloride was preliminarily distilled by rotary evaporation. The remaining solution was added into excess ethyl ether, and the precipitate was collected and dried under vacuum to obtain DTDPA. To 3 mL of anhydrous dimethylformamide (DMF), DOX·HCl (15 mg), triethylamine (12 μL), and DTDPA (5.4 mg) were added successively. The above solution was allowed to react for 12 h in the dark at room temperature, for the preparation of DOX− DTDPA derivative. The above solution was subsequently added with 19.8 mg of EDC and 11.9 mg of NHS and stirred at room temperature for 4 h. To obtain the DOX−SS−PEG-NH2 prodrug, the above DOX−DTDPA solution was added into DMF (8 mL) containing NH2-PEG-NH2 (104 mg). After being stirred for 12 h in the dark, the reaction solution was subsequently dialyzed in ultrapure water for purification 3 days. The yields for DTDPA, DOX−DTDPA, and DOX−SS−PEG-NH2 were measured to be 40, 71, and 76%, respectively. 2.6. Synthesis of PIP−DOX Nanospheres. The obtained DOX− SS−PEG-NH2 solution was added dropwise into 10 mL of Tris-HCl buffer (pH ≈ 8.0) containing PIP nanospheres (2 mg mL−1). After stirring for 12 h, the solution was mixed with 4 mL of Tris-HCl buffer containing 52 mg of mPEG-NH2 and stirred for another 12 h. The final product was retrieved by centrifugation and washed with ultrapure water several times. 2.7. Characterization. Transmission electron microscopy (TEM) was performed on an FEI-F20 electron microscope with an accelerating voltage of 200 kV. Dynamic light scattering (DLS) was measured using a Malvern Zetasizer Instrument. The magnetic hysteresis curve was determined by the MPMS-7 (Quantum Design) superconducting quantum interference device magnetometer. The 1H nuclear magnetic resonance (1H NMR) spectra were acquired with an AVANCE 400 spectrometer at 400 MHz using CF3COOD as the solvent. The T2 relaxivity and MRI were obtained using a Siemens 3.0T clinical magnetic resonance (MR) scanner at room temperature. The PAI was performed on a photoacoustic computerized tomography scanner (Endra Nexus 128, Ann Arbor, MI). The UV−vis absorption spectra were measured with a PerkinElmer spectrometer (LAMBDA 750). The Fe content was analyzed on an inductively coupled plasma optical emission spectrometer (PerkinElmer/OPTIMA 7000DV). The

2. EXPERIMENTAL SECTION 2.1. Materials. Ferric acetylacetonate (Fe(acac)3), dopamine hydrochloride, triethylene glycol (TEG), 3,3′-dithiodipropionic acid (DTDP), N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Amine PEG amine (NH2-PEG-NH2, MW ≈ 5000) and methoxyl PEG amine (mPEG-NH2, MW ≈ 5000) were purchased from Nanocs Inc. Ethanol and ammonia solution (25− 28%) were purchased from Sinopharm Chemical Reagent Co., Ltd. DOX was purchased from Melone Pharmaceutical Co., Ltd. Hoechst, calcein-AM and propidium iodide (PI) were purchased from Thermo Fisher. Dulbecco’s modified Eagle’s medium (DMEM) medium, fetal bovine serum (FBS), trypsin, ethylenediaminetetraacetic acid (EDTA), penicillin and streptomycin were purchased from Hyclone. The ultrapure water with a conductivity of 18 MΩ·cm was used throughout the experiments. 2.2. Synthesis of PDA Nanospheres. For preparing the PDA nanospheres with an average diameter of 120 nm, the ethanol (40 mL), ultrapure water (90 mL), and aqueous ammonia solution (2.2 mL) was mixed together. Ten milliliters of water containing 0.5 g of dopamine hydrochloride was swiftly added into the above solution, which was subsequently stirred for 30 h. The PDA nanospheres were centrifuged and cleaned with water several times. 2.3. Synthesis of PDA@IOs Nanocomposites. In a typical procedure, a wet precipitate of PDA nanospheres (corresponding to 40 mg of dry powder) was dispersed in 1 mL of ethanol. To a 50 mL flask, the Fe(acac)3 (240 mg), TEG (20 mL), and PDA ethanol 12545


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ACS Applied Materials & Interfaces drug-loading content (DLC) was measured by fluorescence spectroscopy (480 nm excitation) and calculated as follows: DLC (wt %) = [weight of loaded drug/(weight of loaded drug + weight of nanoparticle)] × 100%. 2.8. In Vitro Drug Release. The drug release process was evaluated as follows: PIP−DOX (0.5 mL) was suspended in 10 mL of phosphate-buffered saline (PBS) (pH ≈ 7.4) or PBS with different concentrated glutathione (GSH). At desired time intervals, 0.8 mL of the incubation solution was taken and centrifuged, and the release amount of DOX was determined by testing the fluorescence intensity of the supernatant using a fluorescence spectrometer (LS55, PerkinElmer) with an excitation wavelength of 480 nm and an emission wavelength of 556 nm. 2.9. Cellular Experiments. 2.9.1. Cellular Uptake. The murine breast cancer cells (4T1) were supplied by Shanghai Cell Bank and cultured regularly in a DMEM culture medium with 10% heatinactivated FBS and 1% penicillin and streptomycin under 37 °C within 5% CO2. For cell uptake, 2 × 104 cells were seeded in an eightwell chambered plate in 200 μL medium. After culturing for 24 h, the medium was removed by a new medium containing free DOX, PIP− DOX, or PIP−DOX with a magnet at the bottom of the plate (all DOX were equivalent to 5 μg/mL). After 2 h incubation, the medium was discarded, and the cells were washed with PBS three times and fixed with 10% formalin for 10 min (0.5 mL/well). After washing the cells with PBS, Hoechst was added to stain the nuclei for 10 min. The cells after being washed with PBS were applied for fluorescent imaging with a confocal microscope. The cell uptake was further quantitatively evaluated by flow cytometry. The 4T1 cells were seeded into a 24-well plate in 0.5 mL of medium with 1 × 105 cells per well. After culturing for 24 h, the medium was replaced by a new medium with free DOX, PIP−DOX, or PIP−DOX with a magnet at the bottom of the plate (all DOX were equivalent to 5 μg/mL). After 2 h incubation, the medium was discarded, and the cells were washed with PBS and harvested by 0.05% trypsin EDTA. The fluorescence was determined by Accuri C6 flow cytometry. 2.9.2. In Vitro Therapeutic Treatments. The in vitro cytotoxicity of the drug and nanoagents was evaluated by the 3-[4, 5-dimethylthiazol2-yl]-2, 5 diphenyltetrazolium bromide (MTT) viability assay. The 4T1 cells with a seeding density of 5 × 103 cell/well were cultured in 96-well plates in 100 μL DMEM medium supplemented with 10% FBS at 37 °C for 24 h. After discarding the medium, the cells were treated with a fresh medium with an 808 nm laser, free DOX, PIP−DOX, or PIP−DOX with an 808 nm laser at various DOX concentrations. The laser irradiation (1 W/cm2, 5 min) was conducted after 2 h of cell treatments. After being incubated for 48 h, the cells in each well were treated with 10 μL of the MTT solution (5 mg/mL in PBS) and further incubated for 4 h. After replacing the medium and the addition of dimethyl sulfoxide (100 μL), the optical absorbance of each well at 490 nm was determined by a microplate reader. 2.10. In Vivo Experiments. Balb/c mice were used under protocols approved by Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences Animal Care and Use Committee. A suspension of 4T1 cells (2 × 106) in 100 μL of PBS was injected subcutaneously onto the right flank of the mouse. The mice were applied for experiments after 10 days of cell injection. To determine the biodistribution of the nanoagents, PIP−DOX (200 μL, 1 mg/mL) was injected intravenously into the tumor-bearing mice (n = 5). The mice were sacrificed after 24 h with the heart, liver, spleen, lung, kidney, and tumor collected. After weighting and digesting the organs, the Fe contents were determined by inductively coupled plasma optical emission spectroscopy (ICP−OES) with the background Fe signals from the organs of controlled mice subtracted. For an in vivo imaging study, the PIP−DOX solution (100 μL, 1 mg/mL) was injected intravenously into the 4T1 tumor mice (three per group), and the tumors were treated with or without a small magnet attachment. The MRI of the mice was acquired at 0 and 24 h after PIP−DOX injection on the MR scanner equipped with a special coil designed for small animal imaging. The PAI of the mice was performed at 0, 6, 12, and 24 h after PIP−DOX injection using a photoacoustic computerized tomography scanner with 810 nm excitation. The 4T1

tumor mice that were divided into four groups (five per group) were injected with 100 μL of PBS or PIP−DOX (1 mg/mL in PBS) intravenously, for an in vivo combination therapy study. The tumors were treated with or without a small magnet attachment. The tumors were treated with or without an 808 nm laser (1 W/cm2) for 5 min at 24 h post-injection. The tumor volume was calculated as A × B2/2, where A and B correspond to the maximum and minor axes of the tumor. For histology examinations, the tumors from the mice (three per group) were collected at 12 h after therapeutic treatments, fixed with 10% formalin and sectioned into 8 μm slices. The controlled mice and PIP−DOX (100 μL, 1 mg/mL) injected mice (three per group) were sacrificed at 30 d post-administration. The organs including heart, liver, spleen, lung, and kidney were collected, fixed with 10% formalin and sectioned at 8 μm. After washing, the sections were stained with hematoxylin for 3 min, washed with water, and stained with eosin for another 1 min. The observation was performed on a Nikon Eclipse 90i microscope.

3. RESULTS AND DISCUSSION The PDA nanospheres were synthesized in alkaline solution by the self-polymerization of a dopamine monomer via a facile Stöber-like process.30 The TEM image in Figure 2a shows that

Figure 2. TEM images of the PDA (a), PDA@IOs (b,c), and PIP (d) nanospheres. The inset shows the enlarged image of a PIP nanosphere.

these PDA spheres were almost monodisperse with an average diameter approaching 120 nm. By the assistance of PDA as the metal-affinity template,31 a dense layer of IOs was deposited around the PDA spheres (Figure 2b), through thermal decomposition of ferric acetylacetonate in polyalcohol. As revealed in the high-resolution TEM image (Figure 2c), the IOs with an average particle size of 7 nm and good crystallinity could be observed. The superior binding ability of iron atoms to catechol groups leads to an ultrahigh coverage of IOs on the PDA surface compared with previous reports using other conventional nanotemplates,32,33 which may greatly benefit the magnetic response and MRI contrast effect. Furthermore, to enhance the colloidal stability and endow convenient surface functionalization to the nanocomposites, a successive layer of PDA was encapsulated around PDA@IOs to form a sandwichtype PIP architecture (Figure 2d), allowing their facile covalent 12546


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Figure 3. (a) Absorption spectra of IOs, PDA, PDA@IOs, and PIP nanospheres. (b) Photographs of the water dispersions of IOs, PDA, PDA@IOs, and PIP nanospheres. (c) Temperature changes of water and aqueous dispersions of PIP with 808 nm laser irradiation (1 W/cm2). (d) Magnetic hysteresis curves of PDA@IOs and PIP nanospheres at 300 K and photographs of PIP aqueous dispersion treated without and with a magnet (inset). (e,f) Release profiles of DOX from PIP−DOX in physiological buffer (e) and with different concentrated GSH (f) at 37 °C.

conjugation with amino-terminated molecules.1,2 Compared with our previously established gold nanorod-incorporated magnetic nanocomposite,24 the current PDA-based magnetic structure shows superiorities of improved biocompatibility and biodegradability of the organic component, easy functionalization such as direct deposition of inorganic particles, as well as the natural surface reactivity for further derivation.5 The absorption spectra of the IOs and PDA-based nanocomposites are depicted in Figure 3a. The IOs without a PDA template were observed in nanoparticles or nanoparticle cluster forms (Figure S1) and showed absorption mainly across the visible region. While the PDA spheres exhibited broad and intense absorption from visible to NIR spectrum.13 After the growth of IOs, the PDA@IO nanocomposites displayed a remarkably enhanced NIR absorption, which may be attributed to the combination of a clustered IO layer with the PDA host.34,35 Moreover, the outer PDA layer also favored the enhancement of NIR absorption, giving rise to distinguished PAI and PTT performances of the final nanocomposites. The photographs of the nanospheres during the processing also confirm the darkening of the aqueous dispersions as indicated in Figure 3b. The PIP water dispersions with different concentrations were subsequently irradiated with an 808 nm NIR laser (1 W/cm2) and monitored with the temperature change. As expected, PIP exhibited a remarkable concentrationdependent photothermal conversion effect and reached final temperatures of 40.9, 47.5, 55, and 60.2 °C (corresponding to 25, 50, 100, and 250 μg/mL, respectively) within 5 min (Figure 3c), allowing sufficient photothermal ablation of malignant cells. The photothermal conversion efficiency of PIP at 808 nm was calculated to be 33.4% according to the previous method,36 which is higher than that of 21% for gold nanorods, as effective PTT agents for cancers. Meanwhile, the successive laser irradiation of six circles all induced a similar heating up of the PIP dispersion, with temperatures rising up to around 55 °C (Figure S2). Accordingly, the PIP nanospheres showed no obvious change in dimension or morphology after successive laser irradiation (Figure S3), illustrating the high photothermal stability of current nanoagents in an aqueous dispersion state. The room-temperature magnetic hysteresis curve (Figure 3d)

indicates the zero remanence/coercivity of both PDA@IOs and PIP nanospheres, probably owing to the formation of secondary nanostructures from individual small IO nanocrystals, which is similar to the previously reported magnetic supraparticles.37,38 The encapsulation of a thin PDA shell around the IO layer caused minor influence on the superparamagnetism, and the final saturation magnetization value reaches 32.5 emu/g, enabling rapid and complete separation of PIP from water dispersion. This superparamagnetic feature with strong magnetic response promotes both good dispersion state of single nanospheres and efficient magnetic-guided accumulation of theranostic agents at tumor sites.17,39 The combination of photothermal ablation with chemotherapy holds great promise in sufficient eradication of cancers in vivo.40,41 The facile amine-catechol adduct formation on the PDA surface was employed for the introduction of a proanticancer drug, which contains DOX, a reduction-responsive disulfide linker, and an amino-terminated PEG chain (Figure 1). The prodrug (DOX−SS−PEG-NH2) was synthesized by coupling both DOX and amino-terminated homo-bifunctional PEG to DTDPA as the reduction-responsive linker.42−44 The as-synthesized DOX−DTDPA and DOX−SS−PEG-NH2 were characterized by 1H NMR. As indicated in Figure S4, DOX− DTDPA exhibits characteristic peaks at 7.34−7.88 and 2.57− 2.89 ppm, which could be attributed to the protons of a benzene ring from DOX and methylene protons from DTDPA moieties, respectively, as previously reported.43,44 While DOX− SS−PEG-NH2 shows a new peak at 3.10−4.30 ppm, which was ascribed to the protons from repeated units (−OCH2CH2−) of the PEG chain, thus certifying the formation of the prodrug. As revealed in Figure S5, the PIP nanospheres exhibited a hydrodynamic diameter (HD) of 238 nm, with a zeta potential of −41.3 mV, which may result from the deprotonation of catechol groups on the particle surface.7 After being grafted with DOX−SS−PEG-NH2 and further blocked with the aminoterminated PEG monomethyl ether, the PIP−DOX nanospheres displayed an increased HD of 267 nm and a partially neutralized zeta potential of −15.6 mV (Figure S5), which could be attributed to the introduction of the prodrugs as well as PEG chains to the PDA surface. The DLC of DOX in PIP 12547


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considerable cellular uptake of the PIP−DOX nanocarriers after 2 h of incubation. While the fluorescent intensity of the cells incubated with PIP−DOX showed a remarkable increment of more than 50% with magnetic attraction, approaching that of cells treated with DOX of the same concentration. These results illustrated the effective cell internalization of the magnetic-responsible nanocarriers for guided drug delivery applications. To access the biocompatibility of the nanocarriers on a cellular level, the PEG-grafted PIP nanospheres without DOX loading were incubated with 4T1 cells. As indicated in Figure S8, the cells incubated with the nanospheres (from 10 to 500 μg/mL) all exhibited a viability approaching 90%, confirming the satisfactory biocompatibility of the plain nanocarriers consisted of ferrite and PDA as theranostic agents. Nevertheless, the incorporation of an anticancer drug into the PIP nanospheres induced a typical concentration-dependent cytotoxicity on malignant cells, which was similar with the trend for free DOX-treated cells (Figure 5), revealing their

was calculated to be 2.2%. The colloidal stabilities of the asprepared PIP−DOX nanospheres in PBS and DMEM were evaluated by DLS (Figure S6). The nanoagents exhibited relatively stable HDs in both mediums during 24 h of incubation, verifying the robustness of these PEG-grafted nanocomposites in the biological system. The stimuliresponsive drug release capability of the drug carriers was further investigated. PIP−DOX after incubation at 37 °C in physiological buffer (PBS, pH ≈ 7.4) revealed a minor DOX release of 16% over a period as long as 30 h (Figure 3e), suggesting these nanocarriers were fairly stable under physiological conditions. Whereas in the presence of 5 mM GSH, a prompt release of DOX exceeding 50% was observed immediately (within 90 min), as shown in Figure 3f. In addition, the accumulative drug release reached 84% under free GSH concentration up to 10 mM, probably owing to the breakage of disulfide bonds to release DOX molecules. Such a reduction-responsive release may largely benefit the reduction environment-specific chemotherapy in the tumor,29 while minimizing the undesired drugs’ leakage in vascular delivery. The cellular uptake of the PIP−DOX nanoagents was visualized by confocal laser scanning microscopy (CLSM) as depicted in Figure 4. After 2 h of incubation, the PIP−DOX-

Figure 5. 4T1 cells’ viabilities under different treatments for 48 h as indicated.

prospects for drug delivery applications. Noticeably, the NIR laser irradiation on the PIP−DOX-treated 4T1 cell caused a remarkable increment in the killing efficiency of malignant cells, exceeding that of free DOX with the same drug concentrations, thus confirming the synergism of chemotherapy combined with PTT on the cellular level. The costaining of cells using calceinAM and PI was further performed to visualize the therapeutic effect (Figure S9). Compared with the controlled live cells with only NIR laser irradiation, the PIP−DOX-induced chemotherapy leads to extensive but still incomplete cell death. While the combination of drug delivery with PTT resulted in the thorough elimination of cancer cells reflected by the homogeneous red fluorescence (Figure S9c), which implied the potential of the synergism with individual therapeutic modalities. The MCF-10A cells were subsequently employed for evaluating the cytotoxicity of the PIP−DOX nanoagents against normal cells. As illustrated in Figure S10, the MCF-10A cells after incubation with PIP−DOX nanospheres with various DOX concentrations all exhibited visibly higher viabilities than those treated with the same concentrated free DOX. The preliminary results revealed the effectiveness of PIP−DOX nanocarriers for reduction-responsive drug delivery against malignant cells while reducing the cytotoxicity to normal cells. For elucidating the feasibility of current nanoagents for in vivo theranostic applications, the biodistribution of PIP−DOX

Figure 4. CLSM images of 4T1 cells after incubation with free DOX (a), PIP−DOX (b), and PIP−DOX with magnetic field (c) for 2 h.

treated 4T1 cells revealed obvious red fluorescence in the cytoplasm, which could be attributed to the endocytosis of the nanoparticles by cells, compared with the free DOX that was mainly observed in the cellular nucleus probably dominated by the passive diffusion of small molecules. Notably, when subjected to the magnetic field, the cells exhibited a remarkably enhanced fluorescence (Figure 4c). This could be attributed to the high magnetic responsiveness of single PIP nanospheres for enhanced cellular uptake, enabling efficient active drug delivery under magnetic guidance. The flow cytometric analysis was performed to further confirm the cellular uptake of PIP−DOX nanocarriers as indicated in Figure S7, which reveals a 12548


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Figure 6. (a) T2-weighted MR images of PIP−DOX (upper) and linear relationship of inverse transverse relaxation times with Fe concentrations. (b) PA images of different concentrated PIP−DOX dispersions (upper) and plot of corresponding PA intensities vs PIP−DOX concentrations. (c,d) In vivo T2-weighted MR images of 4T1 tumor mice with various time durations after PIP−DOX injection without (c) and with (d) magnetic targeting at the tumor site. Red circles indicate the tumor locations. (e,f) In vivo PA images of the tumor sites with various time durations after PIP− DOX injection without (e) and with (f) magnetic tumor targeting.

Figure 7. (a) Infrared thermal images of 4T1 tumor-bearing mice under various treatments after irradiated with an 808 nm laser (1 W/cm2) for different times. (b) Photographs of 4T1 tumor-bearing mice before and 20 days after different treatments. (c) Tumor growth curves from the mice after various treatments. The data are shown as mean ± SD of 5 mice. *p < 0.05, **p < 0.01.

Fe concentration, giving a T2 relaxation rate (r2) of 128 mM−1 s−1, confirming PIP−DOX as a potential T2-weighted MRI contrast agent. The PAI capacity of the PDA-based nanospheres was evaluated by the photoacoustic tomography scanner. As revealed in Figure 6b, with the excitation of an 810 nm pulsed laser, the PA signal intensity enhanced linearly with the PIP−DOX concentration increment, illustrating a satisfactory PA contrast effect of the nanoagents based on the photothermal conversion feature of the PDA constituent. PIP− DOX was subsequently injected intravenously into 4T1 tumorbearing mice for evaluating the in vivo dual-modal imaging ability. As indicated in Figure 6c, the apparent negative MRI contrast effect at the tumor region could be attributed to the nanoagents accumulated at the tumor site probably originating from the EPR effect. After magnetic targeting at the tumor site for 24 h, a remarkably enhanced darkening effect was observed, owing to the good magnetic response of the nanoagents

nanospheres in 4T1 tumor-bearing mice was investigated by ICP−OES after intravenous administration. As indicated in Figure S11, the majority of the nanospheres were found to distribute in liver and spleen. This could be explained by the clearance of external particles by these reticuloendothelial organs.17 Simultaneously, a considerable amount of the nanospheres (approaching 5% ID/g) was observed in the tumor at 24 h post-administration. This accumulation could be attributed to the enhanced permeability and retention (EPR) effect of PIP−DOX.45 The integration of the IO layer into the PDA host facilitates the dual-modal imaging capability using both MRI and PAI for complementary diagnostic information. The potential of current nanospheres in MRI was investigated using a 3T clinical MR scanner (Figure 6a). The signal intensity of the MR decreased proportionally with the increase of Fe concentration. The T2 relaxation rate (1/T2) could be fitted linearly with the 12549


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Figure 8. H&E stained tissue sections of the (a) tumors from control mice and those injected with PIP−DOX after 12 h of the therapeutic treatment and (b) major organs from control mice and those injected with PIP−DOX after 30 d of treatment.

synergism of complementary therapeutic modalities. The major organs from the mice at 30 days post-injection of PIP−DOX were collected and examined by H&E staining. As illustrated in Figure 8b, the tissue images showed neither appreciable damage nor inflammation after PIP−DOX injection compared with the controlled ones, indicating that the nanoagents were not noticeably toxic preliminarily.

(Figure 6d). The PAI of the PIP−DOX-treated mice revealed a gradually increased PA signal at the tumor site at various time points post-injection, compared with the weak PA background signal in tumor blood vessels by endogenous hemoglobin before injection (Figure 6e).15,46 With magnetic field targeting for different time durations (Figure 6f), considerable PA signal enhancement was observed at the tumor site, reflecting the effective tumor active targeting under exogenous stimulus against the passive accumulation of the nanoagents. Such dualmodal tumor imaging using PIP−DOX nanoagents provided the opportunity for comparative in vivo cancer diagnosis using complementary imaging information, for guiding subsequent precise tumor targeting and therapy. The photothermal−chemo combination in vivo cancer therapy was conducted on 4T1 tumor-bearing mice intravenously injected with PIP−DOX nanospheres. The thermal imaging on mice was acquired to demonstrate the temperature rise at tumor sites induced by NIR laser irradiation after 24 h of the nanoagent injection. As revealed in Figure 7a, the PBStreated mice showed only a slight temperature rise (within 4 °C) under laser irradiation (808 nm, 1 W/cm2) up to 5 min. Yet, the PIP−DOX injection generated a considerable heating effect on the tumor region, reaching a final temperature of 46 °C upon NIR laser irradiation, implying the passive accumulation of the nanoagents in tumors. Encouragingly, the magnetic attraction remarkably increased the local temperature at tumor up to 54 °C which was sufficient to kill the malignant cells by thermal ablation,40 confirming the excellent magnetic response of these nanoagents in vivo. For investigating the anticancer efficiency of PIP−DOX in the tumor-bearing mice, the tumor growth was evaluated every 3 days after different treatments. Both the mice photographs and the tumor growth curves (Figure 7b,c) reflected a rapid tumor size increment for the controlled (PBS treated with NIR irradiation) group. In addition, the PIP−DOX-treated mice showed partially suppressed but still rapid tumor occurrence during the whole observation. When NIR laser was used after accumulation of the nanoagents for 24 h, the tumor growth was prominently restrained over the course of 18 d, probably benefited from the combination of PTT with anticancer drug delivery. Impressively, the combination therapy with magnetic guidance induced a complete eradication of the tumor at much earlier stage (a few days after NIR irradiation) without reoccurrence. The hematoxylin and eosin (H&E) stained tumor slice (Figure 8a) indicated a significant cell damage induced by PIP−DOX with magnetic targeting and laser irradiation, compared with the normal cell morphology in a controlled tumor tissue. This may be attributed to the enhanced accumulation of the nanoagents by active tumor targeting and the highly efficient

4. CONCLUSIONS In summary, the biocompatible colloidal PDA nanospheres were successfully employed as processable and versatile nanohosts for developing novel cancer theranostic nanoagents with complementary imaging and therapeutic modalities in vivo. Favored by the facile controlling over the self-polymerization of dopamine in a Stöber-like system as well as the natural affinity of catechol groups to iron atoms, a sandwich structure incorporating a compact IO layer in a PDA matrix was established, via thermal decomposition and surface encapsulation with a thin PDA shell. The strong magnetic responsiveness and photothermal conversion of these multifunctional nanocomposites enable MRI/PAI dual-modal tumor imaging and magnetic-targeted PTT. Moreover, the high reactivity of PDA allowed a facile linkage of surface catechol groups with an amino-terminated prodrug and PEG chain for the reductionresponsive anticancer drug release. The current theranostic nanoplatform reveals great prospects in precise in vivo cancer therapy, based on multi-modal tumor imaging diagnosis, magnetically guided active tumor targeting, and combination of PTT with chemotherapy for highly efficient tumor eradication.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b02973. TEM and HRTEM images of IOs without PDA nanospheres as templates; temperature changes of PIP aqueous dispersion; TEM images of PIP nanospheres; H NMR spectra of DOX−DTDPA and DOX−SS−PEGNH2; hydrodynamic diameter distributions and zeta potentials of PIP and PIP−DOX nanospheres, respectively; hydrodynamic diameters of PIP−DOX nanospheres in PBS and DMEM; flow cytometric plots and mean fluorescence intensities of control 4T1 cells, 4T1 cells treated with free DOX, PIP−DOX, and PIP−DOX with magnetic field; viabilities of 4T1 cells after incubation with different concentrated PIP nanospheres; fluorescent microscopic images of calcein-AM and PI stained 4T1 cells; viabilities of MCF-10A cells after free 12550


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DOX and PIP−DOX treatments; and biodistribution of PIP−DOX nanospheres in various organs of 4T1 tumorbearing mice (PDF)

AUTHOR INFORMATION

Corresponding Authors

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

Wu Su: 0000-0001-9958-3434 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (21501191 and 21672254), Shenzhen Sciences & Technology Innovation Council (JCYJ20150630114942307), and SIAT Innovation Program for Excellent Young Researchers (201513).

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