porous silicon hybrid nanocomposites as drug carriers for combined chemo-photothermal therapy of cancer

Acta Biomaterialia 51 (2017) 197–208

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Photothermal and biodegradable polyaniline/porous silicon hybrid nanocomposites as drug carriers for combined chemo-photothermal therapy of cancer Bing Xia a,b,⇑, Bin Wang b, Jisen Shi a, Yu Zhang c, Qi Zhang b, Zhenyu Chen a, Jiachen Li b a

Key Laboratory of Forest Genetics & Biotechnology (Ministry of Education of China), Nanjing Forestry University, Nanjing 210037, PR China Advanced Analysis & Testing Center, College of Science, Nanjing Forestry University, Nanjing 210037, PR China State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering & Collaborative Innovation Center for Suzhou Nano Science and Technology, Southeast University, Nanjing 210096, PR China b c

a r t i c l e

i n f o

Article history: Received 3 September 2016 Received in revised form 9 December 2016 Accepted 5 January 2017 Available online 6 January 2017 Keywords: Porous silicon nanoparticles Polyaniline Biodegradability Photothermal effect Drug delivery Combination therapy

a b s t r a c t To develop photothermal and biodegradable nanocarriers for combined chemo-photothermal therapy of cancer, polyaniline/porous silicon hybrid nanocomposites had been successfully fabricated via surface initiated polymerization of aniline onto porous silicon nanoparticles in our experiments. As-prepared polyaniline/porous silicon nanocomposites could be well dispersed in aqueous solution without any extra hydrophilic surface coatings, and showed a robust photothermal effect under near-infrared (NIR) laser irradiation. Especially, after an intravenous injection into mice, these biodegradable porous siliconbased nanocomposites as non-toxic agents could be completely cleared in body. Moreover, these polyaniline/porous silicon nanocomposites as drug carriers also exhibited an efficient loading and dual pH/NIR light-triggered release of doxorubicin hydrochloride (DOX, a model anticancer drug). Most importantly, assisted with NIR laser irradiation, polyaniline/PSiNPs nanocomposites with loading DOX showed a remarkable synergistic anticancer effect combining chemotherapy with photothermal therapy, whether in vitro or in vivo. Therefore, based on biodegradable PSiNPs-based nanocomposites, this combination approach of chemo-photothermal therapy would have enormous potential on clinical cancer treatments in the future. Statement of Significance Considering the non-biodegradable nature and potential long-term toxicity concerns of photothermal nanoagents, it is of great interest and importance to develop biodegradable and photothermal nanoparticles with an excellent biocompatibility for their future clinical applications. In our experiments, we fabricated porous silicon-based hybrid nanocomposites via surface initiated polymerization of aniline, which showed an excellent photothermal effect, aqueous dispersibility, biodegradability and biocompatibility. Furthermore, after an efficient loading of DOX molecules, polyaniline/porous silicon nanocomposites exhibited the remarkable synergistic anticancer effect, whether in vitro and in vivo. Ó 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction With the cancer death rate dropping by
20% since 1991, a great progress on cancer therapy had been made in the past decades [1]. However,
14.1 million new global cancer cases and

⇑ Corresponding author at: Key Laboratory of Forest Genetics & Biotechnology (Ministry of Education of China), Nanjing Forestry University, Nanjing 210037, PR China. E-mail address: [email protected] (B. Xia). http://dx.doi.org/10.1016/j.actbio.2017.01.015 1742-7061/Ó 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.


8.2 million cancer deaths occurred in 2012, thus there is still a great demand for the development of better cancer treatments today [2]. Recently, a novel technology of photothermal therapy based on a large variety of near-infrared (NIR) absorbing inorganic and organic nanomaterials have aroused a tremendously increasing interest, including various gold nanostructures, palladium nanosheets, lanthanide-doped upconversion nanoparticles, copper sulfide nanoparticles, carbon-based nanomaterials, organic nanoagents containing NIR dyes, or conjugated polymeric nanoparticles [3–12]. These above-mentioned photothermal nanomaterials can

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efficiently absorb light, preferably NIR light with superior tissue penetration ability to generate heat, resulting in the death of cancer cells and the subsequent tumor ablation. Except for the thermal ablation of tumor, photothermal therapy is also helpful to trigger and promote other therapeutic approaches (e.g., chemotherapy or radiotherapy) for the synergistic anticancer effect, which is called as ‘‘combination therapy” [13–15]. In many pre-clinical animal models, the ideal efficacy of the combination therapy based on inorganic nanomaterials has been reported [3–9], but their potential long-term toxicity concerns hold them back from clinical trials, resulted from their non-biodegradable nature, poor renal clearance, or heavy metal components foreign to the human body [16–18]. As a potential replacement for inorganic nanomaterials, organic nanoagents containing NIR dyes or conjugated polymeric nanoparticles for photothermal therapy have attracted much attention in the past few years, because of their better biocompatibility [10–12]. Especially, conjugated polymeric nanoparticles with extended p-electrons (e.g., polyaniline, polypyrrole, or poly( 3,4-ethylenedioxythiophene):poly(4-styrenesulfonate)) exhibited a strong and stable hyperthermia effect under repeated NIR laser irradiation [19–22]. However, some limitations from these conjugated polymeric nanoparticles such as poor water-dispersibility and low loading amount of anticancer drugs prevented them from their further applications on the combination therapy of cancer. Besides, their metabolism, biodegradability, and long-term toxicology profiles in vivo still need be explored and improved [11]. To overcome these above-mentioned drawbacks, it is of great interest and importance to develop novel biodegradable and photothermal nanocarriers with better biocompatibility for combination cancer therapy, aiming at next clinical translations [23–26]. As a multifunctional nanoplatform, porous silicon nanoparticles (PSiNPs) have showed an excellent performance on cancer diagnosis and therapy [27–31]. PSiNPs have a versatile loading capability of various exogenous organic molecules, biomacromolecules, or even nanoparticles etc., attributed to their tunable pore size and porosity, large surface area, and tailored surface functionalization [32–34]. Compared with other inorganic nanomaterials, PSiNPs with an excellent biocompatibility and biodegradability have greater potential on their future clinical translation. They can be efficiently excreted from the body through the urine and alleviate the long-term reticuloendothelial systems accumulation concerns, via self-degrading into non-toxic orthosilicic acid (Si(OH)4) in vivo [30,35–43]. Recently, to develop novel drug delivery systems with an ideal biodegradability and photothermal effect, we adopted PSiNPs as nanocarriers to coload NIR dyes and anticancer drugs via electrostatics interactions [44]. However, we found that NIR dyes molecules were easily released from PSiNPs and fast decomposed in the aqueous solution under NIR laser irradiation, limiting their future applications in vivo. Herein, to fabricate biodegradable nanocarriers with a strong and stable photothermal effect, polyaniline was chosen to be covalently grafted onto PSiNPs via surface initiated polymerization (shown in Fig. 1a), due to their robust photothermal effect [19,45–48], and excellent biocompatibility [49]. In order to analyze chemical components, size, morphology, and photothermal effect of as-prepared polyaniline/PSiNPs hybrid nanocomposites (PANi-PSiNPs), multi-characterizations including X-ray photoelectron spectra (XPS), transmission Fouriertransform infrared spectroscopy (FTIR), scanning electron microscope (SEM), transmission electron microscope (TEM), and thermal imaging had been performed in our experiments. Furthermore, their loading and dual pH/NIR light-triggered release of anticancer drugs was also assessed in different biological environments. Finally, their biocompatibility, biodegradability, and performances on chemo-photothermal therapy were systemically explored, whether in vitro or in vivo.

2. Materials and methods 2.1. Preparation of PSiNPs-based nanocomposites Single side polished, (1 0 0) oriented, and boron-doped p+-type silicon wafers with 8–10 X cm resistivity (Hefei Kejing Materials Technology Co. Ltd., China.) were boiled in 3:1 (v/v) concentrated H2SO4/30% H2O2 for 30 min and rinsed copiously with deionized (DI) water (P18 MX cm resistivity, Millipore). The porous silicon samples were prepared by electrochemically etching in a 40% HF/ ethanol electrolyte (1:1, v/v) at the current intensity of 100 mA/ cm2 for 15 min. As-prepared porous silicon samples were sonicated in N-methyl-2-pyrrolidinone (NMP) solution containing 4vinylaniline (3%, v/v) (Sigma-Aldrich Chemicals, USA), and then incubated under microwave heating at 100 °C for 1 h. Using 30 min centrifugation at 1.2  105 rpm, the PSiNPs samples were washed by NMP to prepare vinylaniline-terminated PSiNPs (VANi-PSiNPs, shown in Fig. 1a). The oxidative polymerization grafting of aniline (Sigma-Aldrich Chemicals, USA) onto VANiPSiNPs was carried out in 1 mol/L aqueous solution of HCl, containing 1 mg/mL VANi-PSiNPs, 10 mmol/L aniline, and the corresponding amount of (NH4)2S2O8 oxidant with a molar ratio of 1:1 (monomer/oxidant). With stirring in ice-bath, the polymerization reaction was need to proceed for 12 h. The surface-modified PSiNPs was subsequently washed by DI water, NMP, and ethanol to prepare polyaniline-terminated PSiNPs (PANi-PSiNPs, shown in Fig. 1a). In our experiments, others chemicals were bought from Sinopharm Chemical Reagent Co. Ltd. in China. 2.2. Characterization of PSiNPs-based nanocomposites Size and zeta potential of PSiNPs-based nanocomposites were analyzed by Zetasizer Nano ZS dynamic-light-scattering (DLS) measurements (Malvern Instruments, UK) at 25 °C. To monitor the elemental components and atomic concentrations of PSiNPsbased nanocomposites, XPS were recorded using Kratos AXIS Ultra DLD system (UK) with a monochromatic Al Ka X-ray beam (1486.6 eV) at 150 W in a residual vacuum of <4  109 Pa. The chemical composition and functional groups of PSiNPs-based nanocomposites were also detected using FTIR (Vertex 80, Bruker, USA). Their morphology was observed by SEM (JEOL JSM-7600F, Japan) with the accelerating voltage of 15 kV and TEM (JEOL JEM1400, Japan) with the accelerating voltage of 120 kV, respectively. 2.3. Degradation studies in vitro A serial of bare PSiNPs and PANi-PSiNPs samples with the concentration of 50 lg/mL was incubated in 3 mL PBS buffer (pH = 7.4) at 37 °C. An aliquot (0.45 mL) of the above degradation solutions was taken at different time points, and then ultracentrifugated (1  106 rpm, 15 min) to remove non-degraded particles from the degradation solutions. An aliquot (0.4 mL) of the resultant supernatant was diluted with 4.6 mL HNO3 (2%, v/v), and then measured by inductively coupled plasma optical emission spectrometry (ICP-OES, LEEMAN LABS Prodigy, USA) to analyze silicon elemental amount. Meanwhile, an aliquot (0.05 mL) of the resultant supernatant was also diluted with 0.95 mL DI water, and then subjected to the analysis of silicon elemental amount using a molybdenum blue colorimetric method, which was described in detail elsewhere [50]. 2.4. Biodistribution, biodegradation, and toxicity analysis in vivo All animal work was carried out under protocols approved by Laboratory Animal Center of Simcere Pharmaceutical Group in

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Fig. 1. (a) Schematic route of the fabrication of VANi-PSiNPs and PANi-PSiNPs, and their corresponding photos shown in the inset, (b) XPS, (c) FTIR spectra, (d) SEM and TEM images, and (e) the table of corresponding atomic concentrations of PSiNPs-based nanocomposites.

Nanjing. 200 lL of PANi-PSiNPs solutions (dose = 20 mg/kg, body mass) was intravenously injected into BALB/c mice, and PBS buffer with the same volume was also intravenously injected as a control. For biodegradation and biodistribution studies, the mice were sacrificed on the 1st day, 7th day and 28th day after injection, and then their brain, heart, kidney, liver, lung, and spleen tissues were harvested, respectively. These tissue samples were further weighed and digested with 2–5 mL HNO3 (65%, w/w) overnight, and then heated at 100 °C until they were completely dissolved. Subsequently, 0.5 mL HClO4 (70%, w/w) was added into the above solution, and heated at 140 °C until the residue became white. Finally, the white residue was dissolved with 10 mL HCl (7%, v/v) for the analysis of silicon elemental amount using ICP-OES. And the percentage of injected silicon dose per gram of tissue (%ID/g) was calculated. For the toxicity studies, the mass of each mouse was monitored during 28 days after injection, compared with that of control mice. Besides, after injection, heart, kidney, liver, lung, spleen, and brain samples were collected from the mice on the 1st day, 7th day, and 28th day. These organ samples were treated with hematoxylin-eosin (H&E) staining, and then examined by a pathologist.

(0.125, 0.25, 0.5, 1, 2, and 4 mg/mL) for 12 h, and then washed three times by DI water using the ultra-centrifugation (1  106 rpm, 15 min), respectively. UV–vis–NIR spectra of DOX solution before and after the co-incubation with PANi-PSiNPs were measured using Lambda 950 spectrophotometer. And the amount of DOX loading could be quantitatively calculated according to the standard curve method, described in detail elsewhere [44]. For DOX release tests, DOX@PANi-PSiNPs with the concentration of 1 mg/mL was immersed in 3 mL PBS buffer at pH 5.0 or 7.4, respectively. At different time point, DOX@PANi-PSiNPs solution was ultra-centrifuged at the speed of 1  106 rpm for 15 min, and then the absorbance intensity of the supernatant at 490 nm was detected by UV–vis–NIR spectra. For NIR light-triggered release tests, DOX@PANi-PSiNPs solution was irradiated under NIR laser with the power intensity of 1.6 W/cm2 for 6 min every hour. And the absorbance intensity of DOX in the supernatant was all detected at the beginning (Laser ON) and end (Laser OFF) of NIR laser irradiation, respectively. The release kinetics of DOX from DOX@PANi-PSiNPs could be plotted, according to the absorbance intensity of the supernatant at 490 nm. 2.7. Cell culture and cytotoxicity assays

2.5. Photothermal effect tests An optical-fiber-coupled power-tunable diode laser with the wavelength of 808 nm (maximal power = 2 W, Hi-Tech Optoelectronics, China) was used as NIR light source. Under NIR laser irradiation (1.6 W/cm2, 20 min), the thermal imaging and temperature elevation of DI water, bare PSiNPs, and PANi-PSiNPs solutions with the volume of 1 mL were observed by a IR thermal camera (Fluke, FLK-Ti32, USA), respectively. Moreover, under NIR laser irradiation with different power intensity (0.4, 0.8, 1.2, and 1.6 W/cm2) for 20 min, the thermal imaging and temperature elevation of PANiPSiNPs solution with different concentration (25, 50, 100, 200, and 400 lg/mL) were also observed by a IR thermal camera, respectively.

First, murine breast cancer 4T1 cells or human umbilical vein endothelial (HUVEC) cells were cultured in DMEM cell culture medium (KenGEN, China) containing 10% (v/v) fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin at 37 °C and 5% CO2 in a humidified atmosphere incubator. 3-(4,5-Dimethyl-2-thiazolyl)2,5-diphenyl-2-H-tetrazolium bromide (MTT) staining was adopted to investigate their cellular viabilities. After three times washing with PBS buffer, 100 lL of the new culture medium containing 10 lL MTT (KenGEN, China) with the concentration of 5 mg/mL was added, and then incubated for 4 h. After removing the culture medium and the adding of 150 lL DMSO, the absorbance intensity of cell samples was measured at 570 nm using Filter Max F5 microplate photometer (Molecular Devices, USA). Cell viability values were determined, according to the following Eq. (1):

2.6. Drug loading and release tests

cell viability ð%Þ ¼ the absorbance of experimental group

To prepare DOX@PANi-PSiNPs, 500 lg PANi-PSiNPs was incubated in 1 mL DOX solution with the different concentration

=the absorbance of blank control group  100%:

ð1Þ

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For the cytotoxicity analysis of PANi-PSiNPs in vitro, 4T1 cells or HUVEC cells (
3  105 cells/mL) were dispersed within 96-well plates to a total volume of 100 lL/well containing 12.5, 25, 50, 100, and 200 lg/mL PANi-PSiNPs for 24 h or 48 h, respectively. And then their cellular viabilities were evaluated with MTT staining method. To assess anticancer effect of DOX@PANi-PSiNPs in vitro, 4T1 cells (
3  105 cells/mL) were dispersed within 96well plates to a total volume of 100 lL/well containing 12.5, 25, 50, 100, and 200 lg/mL DOX@PANi-PSiNPs for 2, 4, 10, or 20 h, respectively. And then their cellular viabilities were evaluated with MTT staining. To investigate synergistic anticancer effect of DOX@PANi-PSiNPs plus NIR laser irradiation in vitro, 4T1 cells (
3  105 cells/mL) were dispersed within 96-well plates to a total volume of 100 lL/well containing 50 lg/mL DOX@PANi-PSiNPs, 50 lg/mL PANi-PSiNPs, or 7 lg/mL free DOX (equivalent to DOX loading amount of 50 lg/mL DOX@PANi-PSiNPs), respectively. Subsequently cell samples were immediately treated with or without NIR laser irradiation with power intensity of 1.6 W/cm2 for 20 min, and then cultured for 2, 4, 10, or 20 h. And then their cellular viabilities were also evaluated with MTT staining. To evaluate the synergistic interaction (additive interaction of two treatments might also result in a significant difference), the following Eq. (2):

DOX@PANi-PSiNPs + NIR laser. After the size of the tumors reached 50–70 mm3, all agents including PBS, free DOX, PANi-PSiNPs, or DOX@PANi-PSiNPs solutions were administrated via an intratumoral injection (dose = 1 mg/kg free DOX (equivalent to DOX loading amount of 10 mg/kg DOX@PANi-PSiNPs), 10 mg/kg PANiPSiNPs, or 10 mg/kg DOX@PANi-PSiNPs), respectively. When the tumor regions were irradiated by NIR laser with the power density of 1.4 W/cm2 for 10 min, an IR thermal camera was adopted to monitor the temperature changes of these mice. During the next 14 days, the tumor size of every mouse in our experiments was measured by a calliper every other day. Moreover, to accurately evaluate the growth inhibition of tumors, the mice were sacrificed after 14 days, and then their tumors were collected, photographed, and weighed. Besides, the sections of tumor, heart, kidney, liver, lung, and spleen tissues of different groups harvested on the 14th day were observed using H&E staining, and then examined by a pathologist. The tumor size was calculated as the volume = 0.5  (tumor length)  (tumor width)2. The inhibition efficiency of tumor growth was calculated according to the following Eq. (3):

inhibition efficiency ð%Þ ¼ ð1  ðthe weight of experimental group =the weight of control groupÞÞ  100%: ð3Þ

a ¼ SFa  SFb=SFc had been adopted in our experiments ½51; ð2Þ where SFa and SFb represented cell viability after PANi-PSiNPS + NIR laser and DOX@PANi-PSiNPS treatments, respectively, and SFc was the cell viability after DOX@PANi-PSiNPS + NIR laser treatment. 2.8. Confocal imaging First, 4T1 cells with the concentration of
3  105 cell/mL were plated onto cell culture coverslips with the area of
1 cm2 for 24 h, and then the cell culture medium was replaced by the new culture medium containing 50 lg/mL DOX@PANi-PSiNPs. At different coincubation time points (2, 4, 10, or 20 h), cell samples were washed three times with PBS buffer, and stained with 40 ,6-diamidino-2-phe nylindole (DAPI, KenGEN, China) according to the kit description for subsequent cell observation using laser scanning confocal microscopy (LSCM, LSM710 NLO, Zeiss, Germany), respectively. For NIR light-triggered release experiments, 4T1 cells with the concentration of
3  105 cell/mL were plated onto cell culture coverslips for 24 h, and then the cell culture medium was replaced by the new medium containing DOX@PANi-PSiNPs or free DOX containing 7 lg/mL DOX. After the treatment of NIR laser irradiation with the power intensity of 1.6 W/cm2 for 20 min, cell samples were cultured for 2 h, washed with three times with PBS buffer, and stained with DAPI for cell observation using LSCM. During confocal imaging, the fluorescence of DOX molecules with the emission range from 500 to 700 nm was excited at 488 nm. Mean fluorescence intensity (MFI) of cell samples were analyzed according to the statistics results of
100 different cells with the software of Zen 2012 (blue edition, copyrightÓ Carl Zeiss Microscopy, Gmbh, 2011). 2.9. Chemo-photothermal therapy in vivo To develop the tumor model, 1  106 4T1 cells were subcutaneously injected into the right back of hind leg region of every Balb/C mouse. And then five groups of 4T1-tumor-bearing mice with five mice per group were randomly chosen in our experiment: (1) PBS + NIR laser (as a control), (2) free DOX + NIR laser, (3) PANi-PSiNPs + NIR laser, (4) only DOX@PANi-PSiNPs, and (5)

2.10. Statistical analysis Statistical analysis was performed with the SPSS statistics software. All data were reported as mean ± standard deviation unless specified otherwise. Statistically significant differences (***p < 0.001, **p < 0.01, or NS (non-significant difference, p > 0.05)) were determined by ANOVA with Tukey’s post-test. 3. Results and discussion 3.1. Fabrication of photothermal PANi-PSiNPs nanocomposites As shown in Fig. 1a, to prepare VANi-PSiNPs, freshly-prepared porous silicon samples were first sonicated in 4-vinylaniline solution, and subjected to microwave-induced grafting of 4vinylaniline molecules onto the surfaces of PSiNPs. Subsequently, aniline moieties from surface-immobilized 4-vinylaniline molecules were then used as active sites for the oxidative polymerization of aniline, leading to the grafting of polyaniline onto PSiNPs (PANi-PSiNPs). Meanwhile, to remove physically adsorbed polyaniline copolymer, as-prepared PANi-PSiNPs were further washed by repeated extraction with NMP solvent, which was monitored by UV–vis-NIR spectra with the absorbance of polyaniline at 640 nm (shown in Fig. S1a). The results of UV–vis-NIR spectra showed that after the 7th washing, physically adsorbed polyaniline copolymer could be thoroughly washed from PANi-PSiNPs. In addition, when the concentration of aniline monomer was >10 mmol/L, the amount of unbounded polyaniline copolymer in reaction solution became too large, which made it difficult to thoroughly wash them from PANi-PSiNPs with NMP solvent. Therefore, the optimal concentration of aniline monomer selected in our experiments was 10 mmol/L. From Fig. 1a, we also found that after aniline polymerization, the color of PSiNPs solution changed from turbid yellow to black green, and their corresponding zeta potential also increased from 2.7 ± 0.5 mV to 29.7 ± 5.6 mV. Besides, PANi-PSiNPs could be well dispersed in an aqueous solution, without any extra hydrophilic surface modifications (e.g., polyethylene glycol or polyvinyl alcohol coating). These above results showed that polyaniline could be covalently attached onto PSiNPs via surface initiated poly-

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merization to fabricate PANi-PSiNPs with a good waterdispersibility. Next, XPS and FTIR were both used to stepwisely characterize atomic concentrations, elemental components, and functional groups of PSiNPs-based nanocomposites. The survey of XPS spectra for bare PSiNPs, VANi-PSiNPs, and PANi-PSiNPs were displayed in Fig. 1b, and the corresponding atomic concentrations of these samples were calculated in Fig. 1e. The signals of C 1s, N 1s, O 1s, Si 2p, Si 2s, and F 1s were all detected, consistent with the elemental components of 4-vinylaniline and polyaniline molecules attached onto PSiNPs. Among these elements, the elemental concentration of nitrogen provided important information to monitor the grafting of 4-vinylaniline and polyaniline. With the consecutively grafting of 4-vinylaniline and polyaniline, nitrogen concentration changed as 0.0% (bare PSiNPs) ? 1.7% (VANi-PSiNPs) ? 2.8% (PANiPSiNPs). The intensity of N 1s signal gradually increased, due to the contributions of nitrogen atoms of surface-immobilized aniline moieties. Meanwhile, with increasing the thickness of grafted polymers and decreasing the depth of silicon element detected by Xray, silicon concentration evolved as 29.2% (bare PSiNPs) ? 6.5% (VANi-PSiNPs) ? 4.5% (PANi-PSiNPs). Based on the concentration changes of nitrogen and silicon element, the overall grafting amount of 4-vinylaniline or polyaniline could be simply determined with [N]/[Si] ratio, which was shown in Fig. S1b. For VANi-PSiNPs, [N]/[Si] ratio (0.26) was much higher than that (0.05–0.06) of 4-vinylaniline monolayers on silicon surfaces [52], similar with that (0.1–0.25) of 4-vinylaniline polymer films [53]. Thus we suggested that in our experiments, the grafting of 4vinylaniline induced by microwave heating was in the form of polymerization, not simple monolayers (shown in Fig. 1a). After the grafting of aniline via oxidative polymerization, the [N]/[Si] ratio increased from 0.48 to 0.68 with reaction time increasing from 2 to 24 h. According to the relationship of [N]/[Si] ratio and polymerization time shown in Fig. S1b, the optimal reaction time selected in our experiments was 12 h. Furthermore, N 1s corelevel spectra of VANi-PSiNPs and PANi-PSiNPs were also measured to obtain the detail information of their chemical bonds. Fig. S1e comprised of a major peak component at the binding energy of 401.2 eV, attributable to amine (ANH2) species of 4-vinylaniline molecules. After aniline polymerization, the line width of N 1s core-level spectrum (in Fig. S1f) substantially increased with the appearance of a new peak component at 399.3 eV, due to imine (@NA) units of polyaniline chains. These results demonstrated that the polyaniline layer in the form of the neutral emeraldine base (EB) had been attached onto the VANi-PSiNPs after the deprotonation with equilibrating in successive amounts of NMP solvent and DI water [53]. Besides, the imine to amine ratio ([@NA]/[ANHA], 0.17) was less than the theoretical value 1 from polyaniline (EB). Here, we suggested that the excess amount of amine moieties resulted from the residue of initially grafted 4-vinylaniline polymer after aniline copolymerization. Additionally, Si 2p core-level spectra were also recorded in Fig. S1d, which exhibited one peak at 103.4 eV, assigned to SiAO, and the other peak at 99.5 eV, assigned to SiASi(C). Compared with bare PSiNPs, the SiOx amount of VANi-PSiNPs and PANi-PSiNPs was significantly enlarged, because of microwave heating and oxidative polymerization. According to the previous studies [30], the oxidation of PSiNPs could efficiently promote the water-dispersibility of PANi-PSiNPs hybrid nanocomposites without any extra hydrophilic surface coatings. Moreover, FTIR spectra of these PSiNPs-based nanocomposites could be also seen in Fig. 1c. Compared with bare PSiNPs and VANi-PSiNPs, two new bands at 1584 and 1504 cm1 of PANiPSiNPs were assigned to the C@N and C@C stretching vibration of the quinoid and benzenoid rings of polyaniline. The band at 2983 cm1 was attributed to the stretching vibration of CAH in

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the 1,4-disubstituted benzene ring, and its corresponding out-ofplane bending was reflected in the peak at 803 cm1. These two peaks at 2983 and 803 cm1 also gradually increased with the consecutively grafted polymerization of 4-vinylaniline and aniline. Meanwhile, the band at 1302 cm1 due to the CAN stretching mode for the secondary aromatic amine gradually increased, which also supported the grafted polymerization of 4-vinylaniline and aniline onto PSiNPs. Besides, the bands at 1645 and 1080 cm1 attributed to SiOAH stretching vibration, SiAOH bending vibration, and SiAOASi stretching vibration, and two bands at 962 and 874 cm1 attributed to SiAOH stretching vibration significantly became strong. The bands at 2100, 908, 670, and 616 cm1 belong to SiAHx stretching and bending vibration disappeared. These changes indicated that PSiNPs had been heavily oxidized after oxidative polymerization of aniline, in good agreement with XPS results. Furthermore, SEM and TEM were also utilized to observe the size and morphology of bare PSiNPs and PANi-PSiNPs, recorded in Figs. 1d and S1g. From SEM images, we found that whether bare PSiNPs or PANi-PSiNPs, these particles with the size of 100–200 nm were the aggregation connection of smaller silicon nanoparticle domains with the size of
10 nm, similar with our previous results [36,38,44]. For a better comparison, high-resolution TEM was used to observe their morphologies. In contrast to the dark inner core of PSiNPs consisting of silicon element, it could be observed that the outer polymer layer consisting of carbon element exhibited an irregular brightness with the thickness of 2–10 nm, marked with red arrow in Fig. 1d. Finally, to investigate the hyperthermia potential of PANiPSiNPs, an IR camera with thermal imaging was used to monitor their temperature changes in aqueous solution upon the exposure of NIR laser (shown in Fig. 2). As the exposure time of NIR laser with the power intensity of 1.6 W/cm2 increased from 0 to 20 min, the thermal imaging of 400 lg/mL PANi-PSiNPs solution continuously changed from blue (low temperature) to deep red (high temperature), revealing a significant temperature raise from 20.6 to 39.1 °C (shown in Fig. 2a and S2a). In contrast, DI water (DT = 2.2 °C) and bare PSiNPs solution (DT = 5.4 °C) showed little change under the same NIR laser irradiation. These results indicated that polyaniline layers grafted onto PSiNPs could efficiently absorb and convert NIR light to a large amount of thermal energy, compared with bare PSiNPs. In addition, the photothermal effect of PANi-PSiNPs solution with different concentration (25, 50, 100, 200, and 400 lg/mL) under NIR laser irradiation with different power intensity (0.4, 0.8, 1.2, and 1.6 W/cm2) for 20 min was also measured in Fig. 2b and c, which showed that the NIR light-toheat conversion of PANi-PSiNPs was dependent on their concentration and the power intensity of NIR laser irradiation. To investigate the photothermal stability, 400 lg/mL PANi-PSiNPs solution was further irradiated under NIR laser with the power intensity of 1.6 W/cm2 for 20 min, followed by naturally cooling to room temperature without laser irradiation, regarded as ‘‘one cycle of Laser ON/OFF”. According to Fig. 2d, the temperature elevation of 18.1, 17.5, 18.2, and 19.7 °C was achieved after sequential cycles, respectively, which indicated that PANi-PSiNPs solution had an excellent stability of photothermal effect upon repeated NIR laser irradiation. Besides, the photothermal effect PANi-PSiNPs solution at different pH value was also observed in Fig. S2b. Here, two typical pH values, such as 7.4 (physiological environments) and 5.0 (intracellular endosome/lysosome or micro-environments of tumors) were chosen. Comparing pH 7.4 (DT = 18.5 °C) with 5.0 (DT = 18.9 °C), the photothermal effect of PANi-PSiNPs was still stable in physiological environments with different pH value. Overall, polyaniline had been successfully grafted onto PSiNPs via in-situ polymerization. These resultant PANi-PSiNPs nanocomposites could be well dispersed in aqueous solution without any

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Fig. 2. T–t curves of (a) DI water, bare PSiNPs (400 lg/mL), and PANi-PSiNPs (400 lg/mL) in aqueous solution under NIR laser irradiation with the power intensity of 1.6 W/ cm2 for 20 min, (b) PANi-PSiNPs solution with different concentration (20, 50, 100, 200, and 400 lg/mL) under NIR laser irradiation with the power intensity of 1.6 W/cm2 for 20 min, (c) 400 lg/mL PANi-PSiNPs solution under NIR laser irradiation with different power intensity (0.4, 0.8, 1.2, and 1.6 W/cm2) for 20 min, and (d) photothermal stability of 400 lg/mL PANi-PSiNPs solution under repeated NIR laser irradiation with the power intensity of 1.6 W/cm2. Their corresponding thermal imagings were shown in the inset, and error bar were based on standard errors of the mean (n = 3).

extra hydrophilic coatings, and also exhibited a significant and stable photothermal effect, whether under repeated NIR laser irradiation or different physiological conditions.

3.2. Biodegradability and biocompatibility of PANi-PSiNPs in vitro and in vivo In our experiments, a molybdenum blue colorimetric method was adopted to monitor the degradation of PANi-PSiNPs and bare PSiNPs in PBS buffer (pH = 7.4) at 37 °C. Dissolved SiO2 as the degradation products of PSiNPs-based nanomaterials could be sensitively transformed into the blue-colored molybdenum complex (shown in Fig. 3a), and their corresponding concentrations could be semi-quantitatively evaluated with UV–vis-NIR spectrophotometric measurement at 815 nm (shown in Fig. 3b bottom). Besides, the concentration of silicon element in degradation solution could also be quantitatively analyzed by ICP-OES, recorded in Fig. 3b top. The degradation profiles over 48 h between PANi-PSiNPs and bare PSiNPs indicated that their degradation rate both sharply increased within the first hour, and then slowed down. In contrast to bare PSiNPs, the degradation rate of PANi-PSiNPs decreased by
50%, due to the protection of grafted polymer films. Besides, the particle size of PANi-PSiNPs and bare PSiPNs was also monitored by DLS measurements in the process of their degradation, shown in Fig. S1h. The results showed that the size of bare PSiNPs quickly decreased to zero after 1 h, however, the size of PANi-PSiNPs gradually decreased to zero after 24 h. Combined with the analysis of molybdenum blue colorimetric method and ICP-OES measurement, DLS results also confirmed that the degradation rate of

PANi-PSiNPs was slower than that of bare PSiNPs, however they still remained biodegradable under physiological conditions. Next, biodegradability and biocompatibility of PANi-PSiNPs in vitro and in vivo were further evaluated. First, cancerous 4T1 cells and noncancerous HUVEC cells were incubated with PANiPSiNPs in the concentration range (0–200 lg/mL) for 24 h or 48 h, respectively, and then their viabilities were quantitatively analyzed using MTT assay, as shown in Fig. 3c. Even after 48 h incubation with 200 lg/mL PANi-PSiNPs, the viability of 4T1 cells or HUVEC cells could still reach 85.2% or 83.4%, respectively. This result indicated that PANi-PSiNPs were relatively non-toxic to cells in vitro. For in vivo studies, 200 lL PANi-PSiNPs solution at the dose of 20 mg/kg was administrated into Balb/C mice via an intravenous injection, with 200 lL PBS buffer injected as a control. As seen in Fig. 3d, over a period of 28 days, the body weight of the mice injected with PANi-PSiNPs slightly increased in a similar pattern to that of the control mice, which indicated that the mice injected with PANi-PSiNPs could still continue to mature without any obvious toxic effects. Moreover, the biodistribution of PANi-PSiNPs in different tissues (i.e., heart, liver, spleen, lung, kidney, and brain) of the injected mice after 1 day, 7 days, or 28 days was also assessed in Fig. 3e, respectively. The injected PANi-PSiNPs accumulated mainly in the mononuclear phagocyte system-related organs such as the liver (19.8 ± 1.0% ID/g) and the spleen (20.4 ± 3.6% ID/g) after 1 day. However, the PANi-PSiNPs accumulated in the organs could be noticeably degraded within a period of 7 days (liver 11.6 ± 1.7% ID/g, or spleen 12.8 ± 3.3% ID/g) and completely cleared from the body in 28 days (liver 3.7 ± 0.8% ID/g or spleen 4.1 ± 1.1% ID/g). The mechanism of such time-dependent clearance was attributed to the degradation of porous silicon-based nanomateri-

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Fig. 3. (a) Photos of the degradation of 50 lg/mL PANi-PSiNPs and 50 lg/mL bare PSiNPs in PBS buffer (pH = 7.4) at 37 °C, stained with a molybdenum blue colorimetric method, (b) the corresponding silicon elemental amount appeared in solution monitored by ICP-OES (top) and UV–vis–NIR spectra (bottom), (c) relative viability of 4T1 cells and HUVEC cells co-incubated with PANi-PSiNPs, (d) the changes in body mass of mice injected with 200 ll PANi-PSiNPs at the dose of 20 mg/kg, compared with 200 ll PBS injected as a control, (e) in vivo biodistribution and biodegradation of PANi-PSiNPs over a period of 28 days in mice, and (f) histology of brain, heart, liver, spleen, lung, and kidney tissues collected from mice on the 1st day, 7th day and 28th day after an intravenous injection of PANi-PSiNPs, compared with that from mice 28 days after an intravenous injection of PBS as a control (scale bar = 20 lm). Error bar are based on standard errors of the mean (n = 3 or 5).

als into soluble silicic acid followed by excretion in body [30]. During the degradation of PANi-PSiNPs in vivo, they were highly localized in the some organs of the body and might induce some subsequent damages. Thus we also examined in vivo toxicity of PANi-PSiNPs in heart, liver, spleen, lung, and kidney tissues by H&E staining method, as seen in Fig. 3f. Compared with the mice injected with PBS buffer, the slight degeneration appeared in hepatocytes of liver samples, but there were no inflammatory infiltrates and sinusoids after PANi-PSiNPs injection. Spleen samples showed no significant changes in morphology of the lymphoid follicles or in the size of the red pulp, except for the slight enlargement of splenic corpuscles. For lung samples, there were also no remarkable changes, except for the slight degeneration of bronchial epithelial cells. In addition, others organ samples including heart, kidney, and brain tissues all showed no significant changes in the morphology. Overall, PANi-PSiNPs had a great promise as non-toxic and biodegradable hybrid nanomaterials for the future clinical translation. 3.3. Efficient loading and dual pH/NIR light-controlled release of DOX As a model anticancer drug, DOX was chosen to be loaded into PANi-PSiNPs for next combination cancer therapy in vitro or in vivo. First, DOX loading of PANi-PSiNPs was monitored by UV–vis-NIR spectra, which was shown in Fig. 4a. Comparing DOX@PANiPSiNPs with PANi-PSiNPs, an obvious absorption peak at 498 nm

attributed to DOX encapsulation was observed, and the solution color changed from black green to reddish brown. The amount of DOX loading could be quantitatively analyzed according to the standard curve method, which was shown in Fig. S1c. The results indicated that the optimal amount of DOX loading was calculated 14.4 ± 0.9% (w/w) when the mass ratio of PANi-PSiNPs and DOX was 1:1. DOX molecules could be efficiently encapsulated into PANi-PSiNPs, due to the strong p-p stacking between the aromatic nature of anthracyclines from DOX molecules and aniline moieties of PANi-PSiNPs [54]. Moreover, in Fig. 4c, DLS measurements showed that the mean hydrodynamic size (
165 nm) of DOX@PANi-PSiNPs in aqueous solution was slightly larger than that of PANi-PSiNPs (
156 nm), due to the encapsulation of DOX molecules. And we also found that DOX@PANi-PSiNPs could be well dispersed in aqueous solution for next biological tests. As seen in Fig. 4b, DOX@PANi-PSiNPs still retained similar photothermal effect (DT = 20.1 °C), compared with PANi-PSiNPs (DT = 18.5 °C). And NIR light-triggered release behavior of DOX from DOX@PANi-PSiNPs was further investigated, which was recorded in Fig. 4d. Here, two typical pH values such as 7.4 and 5.0 were chosen to evaluate the DOX release behavior with the treatment of NIR laser irradiation, respectively. From Fig. 4d, after 6 h incubation in PBS buffer at pH 7.4, we found that there was only 6.1% DOX released in solution, while the DOX release reached 65.7% when pH 5.0. This significant pH-responsive behavior was attributed to the strong electrostatic repulsion, resulted from the

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Fig. 4. (a) UV–vis–NIR spectra of PANi-PSiNPs and DOX@PANi-PSiNPs solution with the same concentration (400 lg/mL), and their corresponding photos shown in the inset, (b) T–t curves of 400 lg/mL DOX@PANi-PSiNPs and DI water, and their corresponding thermal imagings were showed in the inset, (c) DLS measurements of DOX@PANiPSiNPs dispersed in an aqueous solution, and (d) DOX release behavior from DOX@PANi-PSiNPs at different pH value with or without continuous NIR laser irradiation (1.6 W/ cm2, 6 min) after 1 h. Error bar are based on standard errors of the mean (n = 3).

protonation of DOX molecules and polyaniline in acidic environments. In addition, NIR laser irradiation could also efficiently promote DOX release from DOX@PANi-PSiNPs, whether pH 7.4 or 5.0. For example, followed by 6 min exposure of 1.6 W/cm2 NIR laser after 1 h incubation, the amount of DOX release increased by 17.7% at pH 5.0, and increased by 7.4% at pH 7.4. After 6 h incubation added with 6 min NIR laser exposure every hour, the cumulative amount of DOX release increased and reached 70.1% at pH 5.0 and 32.7% at pH 7.4, respectively, compared with 65.2% at pH 5.0 and 24.4% at pH 7.4 without NIR laser irradiation. The combination of NIR laser irradiation and acidic biological conditions could trigger and accelerate DOX release from DOX@PANi-PSiNPs, called as ‘‘dual pH/NIR light-controlled release”. With the help of NIR laser irradiation, this smart release behavior based on DOX@PANiPSiNPs would be of great benefit to localized release of DOX molecules in intracellular lysosomes and endosomes or the acidic microenvironments of tumor regions during cancer therapy. 3.4. Cytotoxicity assay of DOX@PANi-PSiNPs in vitro Here, MTT assays were adopted to quantitatively evaluate the viability of 4T1 cells treated with DOX@PANi-PSiNPs in the concentration range (0–200 lg/mL) for different time (2, 4, 10, or 20 h), respectively. As seen in Fig. 5b, the cytotoxicity of DOX@PANiPSiNPs was mainly dependent on their co-incubation time, not their concentration. Especially, a remarkable inhibition effect on the growth of 4T1 cells incubated with DOX@PANi-PSiNPs had been found after 10 h. In contrast to the viability of 4T1 cells trea-

ted with PANi-PSiNPs (200 lg/mL, 48 h), the cellular viability treated with DOX@PANi-PSiNPs (200 lg/mL, 20 h) noticeably decreased by 40.4%. Loading DOX of PANi-PSiNPs could efficiently improve their chemotherapeutic efficiency in vitro, because of the broad-spectrum anticancer ability of DOX molecules. Furthermore, LSCM was also utilized to observe DOX distribution inside cells. 4T1 cells were co-incubated with 50 lg/mL DOX@PANi-PSiNPs at 37 °C for different time (2, 4, 10, or 20 h), washed with PBS buffer, and then observed by LSCM at 488 nm excitation, respectively. As shown in Fig. S3a, as the co-incubation time was prolonged from 0 to 10 h, a red fluorescence signal localized in intracellular nuclei (labelled with DAPI) gradually became stronger (MFI 0.0 ? 0.6 ? 7.3 ? 26.7), which was caused by DOX release from DOX@PANiPSiNPs and then their sustained diffusion into intracellular nuclei. When 20 h, DOX fluorescence signal (MFI = 6.1) became weak, accompanied with the significant inhibition of 4T1 cells growth. The results of LSCM observations further confirmed that DOX molecules with high affinity towards DNA could efficiently accumulate in intracellular nuclei, and intercalate DNA as a cytostatic and apoptotic agent against the proliferation of cancer cells. Furthermore, to investigate the combination therapy of DOX@PANi-PSiNPs in vitro, we also assessed the cytotoxicity of free DOX, PANi-PSiNPs, or DOX@PANi-PSiNPs with NIR laser irradiation (1.6 W/cm2, 20 min), and then cultured for different time (2, 4, 10, or 20 h), respectively, which was shown in Fig. 5c. First, the results of statistical analysis showed NIR laser irradiation had a significant enhancement (***p < 0.001) of killing 4T1 cells incubated with PANi-PSiNPs or DOX@PANi-PSiNPs. For example, the viability of

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Fig. 5. (a) Typical LSCM images of 4T1 cells treated with free DOX or DOX@PANi-PSiNPs containing 7 lg/mL DOX, with or without NIR laser irradiation (1.6 W/cm2, 20 min), and then co-incubated for 2 h, respectively (scale bar = 20 lm), (b) relative viability of 4T1 cells treated with DOX@PANi-PSiNPs with different concentration for different coincubation time, and (c) relative viability of 4T1 cells alone as a control, free DOX (7 lg/mL), PANi-PSiNPs (50 lg/mL), or DOX@PANi-PSiNPs (50 lg/mL) with or without the NIR laser irradiation (1.6 W/cm2, 20 min), and then co-incubated for different time, respectively. Error bar are based on standard errors of the mean (n = 5, ***p < 0.001, or NS (non-significant difference, p > 0.05), by ANOVA with Tukey’s post-test).

4T1 cells treated with 50 lg/mL PANi-PSiNPs plus NIR laser irradiation showed a continuous reduction (94.1% ? 89.4% ? 80.5% ? 62.2%) from 0 to 20 h, in contrast to that without NIR laser irradiation (94.8% ? 94.4% ? 93.7% ? 92.6%). However, non-significant difference (NS) appeared under the same NIR laser irradiation, for 4T1 cells alone (as a control) or that incubated with only free

DOX. These results demonstrated that the mild hyperthermia induced by PANi-PSiNPs under NIR laser irradiation could directly kill cancer cells via the denaturation and aggregation of protein, or induce the programmed death (such as apoptosis) of cancer cells under the proteotoxic stress [9,55,56]. Secondly, compared with others treatments including control + NIR laser, free DOX + NIR

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laser, PANi-PSiNPs + NIR laser, and DOX@PANi-PSiNPs without NIR laser irradiation, a significant enhancement of the anticancer effect based on DOX@PANi-PSiNPs + NIR laser could be observed. For example, the combination therapy of 50 lg/mL DOX@PANiPSiNPs plus the same NIR laser irradiation appeared to more effective (84.9% ? 80.6% ? 49.5% ? 23.1%), in contrast to that without NIR laser irradiation (84.1% ? 81.9% ? 67.9% ? 42.9%). It was also found that the anticancer efficiency (83.3% ? 81.9% ? 61.2% ? 37.1%) of the only chemotherapy using 7 lg/mL free DOX could not reach the anticancer effect of the combination therapy. According to the Eq. (2), the value of a based on the treatment of DOX@PANi-PSiNPS + NIR laser, PANi-PSiNPs + NIR laser, and DOX@PANi-PSiNPs was calculated as 1.1 or 1.2, after 10 h or 20 h co-incubation, respectively. According to the reference [51], when a > 1, a synergistic effect of chemo-photothermal therapy (DOX@PANi-PSiNPS + NIR laser) appeared, in contrast to chemotherapy (DOX@PANi-PSiNPs) or photothermal therapy alone (PANi-PSiNPs + NIR laser). Similar results about the synergistic performance of chemo-photothermal therapy in vitro had been also reported using silica nanoparticles conjugated with NIR dyes [57]. Furthermore, after 4T1 cells were incubated with free DOX

and DOX@PANi-PSiNPs containing 7 lg/mL DOX at 37 °C, they were immediately treated with NIR laser irradiation of 1.6 W/cm2 for 20 min, cultured for 2 h, and then observed by LSCM, respectively (shown in Fig. 5a). After being irradiated with NIR laser, DOX fluorescence signals (MFI = 16.9) inside 4T1 cells incubated with DOX@PANi-PSiNPs could be remarkably enhanced, compared with that of others cell samples treated with free DOX (MFI = 2.2), free DOX plus NIR laser (MFI = 2.3), or only DOX@PANi-PSiNPs (MFI = 1.8). The results of LSCM observations demonstrated that NIR laser-induced heating based on photothermal DOX@PANiPSiNPs could facilitate DOX release from DOX@PANi-PSiNPs in culture medium, and simultaneously promote cellular endocytosis of DOX molecules via increasing the permeability and fluidity of cell membrane upon temperature increasing. Besides, cancer cells treated with hyperthermia induced by NIR laser could be more susceptible to the damage caused by chemotherapy [58–60]. Therefore, for DOX@PANi-PSiNPs, NIR laser-assisted hyperthermia could damage cancer cells, and was also helpful for accelerating the accumulation of DOX in intracellular nuclei and enhancing the efficiency of chemotherapy, which resulted in the synergistic effect of killing cancer cells, called as ‘‘combination therapy”.

Fig. 6. Five groups of 4T1-tumor-bearing mice with five mice per group were randomly chosen in our experiment: (1) PBS + NIR laser, (2) free DOX + NIR laser, (3) PANiPSiNPs + NIR laser, (4) only DOX@PANi-PSiNPs, and (5) DOX@PANi-PSiNPs + NIR laser. (a) Thermal imaging of different groups of tumor-bearing mice under NIR laser irradiation, (b) the changes in body mass of mice in different groups after the same treatments, (c) the weight of the tumors harvested from different groups of mice after 14 day, and (d) H&E-stained tumor slices collected from mice at the end of treatments (14th day) (scale bar = 20 lm). Error bar are based on standard errors of the mean (n = 5, ***p < 0.001, or **p < 0.01, by ANOVA with Tukey’s post-test).

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3.5. Chemo-photothermal combination therapy in vivo To investigate the synergistic antitumor effect of the combination therapy in vivo, animal experiments were designed and carried out using DOX@PANi-PSiNPs plus NIR laser irradiation. Five groups of 4T1-tumor-bearing mice with five mice per group were randomly chosen in our experiments: (1) PBS + NIR laser, (2) free DOX + NIR laser, (3) PANi-PSiNPs + NIR laser, (4) DOX@PANiPSiNPs, and (5) DOX@PANi-PSiNPs + NIR laser. All agents including PBS, free DOX, PANi-PSiNPs, or DOX@PANi-PSiNPs solutions were administrated into mice with an intratumoral injection (dose = 1 mg/kg DOX, 10 mg/kg PANi-PSiNPs, or 10 mg/kg DOX@PANi-PSiNPs), respectively. When the tumor regions were irradiated by NIR laser with a moderate power density 1.4 W/ cm2 for 10 min, an IR thermal camera was used to monitor the temperature changes of these mice, recorded in Fig. 6a. After the exposure of NIR laser irradiation for 10 min, the mice injected with PANi-PSiNPs or DOX@PANi-PSiNPs exhibited significant localized heating (44–45 °C) in the tumor region, while the temperature of the surrounding region near the tumor only reached 30–31 °C. In comparison, the tumor temperature of mice treated with free DOX or PBS showed no remarkable increasing (<37 °C) under the same experimental conditions. These results indicated that PANiPSiNPs or DOX@PANi-PSiNPs still obtained an excellent photothermal effect in body after an intratumoral injection. During the next 14 days, the tumor size of every mouse in our experiments was measured by a calliper every other day, recorded in Fig. S4b. Moreover, to accurately evaluate the growth inhibition of tumors, the mice were sacrificed after 14 days, and the tumors were collected, photographed, and weighed, which was recorded in Figs. 6c and S4c. These results showed that mice treated with DOX@PANiPSiNPs + NIR laser had the smallest tumor volumes and weight with a remarkable inhibition (90.6%) of tumor growth, when the mice in group (1) treated with PBS + NIR laser were chosen as a control. In addition, tumors on mice treated with (2) free DOX + NIR laser, (3) PANi-PSiNPs + NIR laser, and (4) DOX@PANiPSiNPs without laser irradiation exhibited 62.1%, 42.7%, and 39.4% growth inhibition, respectively. In addition, the results of statistical analysis also demonstrated that compared with the others groups, the significant synergistic anticancer effect of chemo-photothermal therapy in vivo based on DOX@PANiPSiNPs + NIR laser could be found, as demonstrated in abovedescribed experiments in vitro. Furthermore, the histology of tumor slices collected from five groups was shown in Fig. 6d. The results exhibited that cells in group (1), (3), and (4) largely retained their normal morphology with distinctive membrane and nuclear structures. In the group (2) receiving both free DOX and NIR laser irradiation, a part of tumor cells nuclei significantly shrunk, not broke. However, most tumor cells severely destroyed with their nuclei broken into pieces in the group (5) receiving both DOX@PANi-PSiNPs plus NIR laser irradiation. The histology results further confirmed that the combination therapy based on DOX@PANi-PSiNPs + NIR laser had the highest inhibition effect of tumor growth in vivo. Toxic side effects have been one of the largest obstacles for the clinic applications of engineered nanomaterials. Therefore, in our experiments, the body weights of mice was observed after receiving different treatments, which was shown in Fig. 6b. Compared with the control group, no obvious change was found for all experimental groups. Furthermore, as seen in Fig. S4a, H&E staining results of major organs from the 14th day after DOX@PANi-PSiNPs injection and NIR laser treatment indicated no obvious abnormality or damage of these tissues, which indicated that DOX@PANi-PSiNPs had negligible side effects on other tissues including heart, liver, spleen, lung, and kidney in body. Therefore, combined with NIR laser irradiation, non-toxic

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DOX@PANi-PSiNPs with an excellent antitumor effect had great potential on the combination cancer treatments in the future. 4. Conclusions In summary, the fabrication of PANi-PSiNPs nanocomposites with a robust photothermal effect via surface initiated polymerization had been reported. These resultant PANi-PSiNPs exhibited an excellent biodegradability and biocompatibility in vitro or in vivo. Except for an efficient loading of DOX, DOX release from DOX@PANi-PSiNPs was controlled with pH and NIR laser irradiation. Furthermore, the combined chemo-photothermal therapy based on DOX@PANi-PSiNPs showed a synergistic inhibition effect on the proliferation of cancer cells in vitro, and the growth of tumors in vivo, respectively. Overall, these biodegradable and photothermal DOX@PANi-PSiNPs hybrid nanocomposites would have much favor for combination cancer treatments in future clinical translation. Acknowledgements This work is funded by the National Natural Science Foundation of China (No. 30930077 and No. 31000164), Natural Science Foundation of Jiangsu Province (No. BK20130964), and bilateral Chinese-Croatian scientific project (No. 6-5). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2017.01. 015. References [1] R.L. Siegal, K.D. Miler, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66 (2016) 7–30. [2] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA Cancer J. Clin. 65 (2015) 87–108. [3] L. Zou, H. Wang, B. He, L. Zeng, T. Tan, H. Cao, X. He, Z. Zhang, X. Guo, Y. Li, Current approaches of photothermal therapy in treating cancer metastasis with nanotherapeutics, Theranostics 6 (2016) 762–772. [4] E.K. Lim, T. Kim, S. Paik, S. Haam, Y.M. Huh, K. Lee, Nanomaterials for theranostics: recent advances and future challenges, Chem. Rev. 115 (2015) 327–394. [5] Q. Chen, H. Ke, Z. Dai, Z. Liu, Nanoscale theranostics for physical stimulusresponsive cancer therapies, Biomaterials 73 (2015) 214–230. [6] G. Tian, X. Zhang, Z. Gu, Y. Zhao, Recent advances in upconversion nanoparticles-based multifunctional nanocomposites for combined cancer therapy, Adv. Mater. 27 (2015) 7692–7712. [7] L. Cheng, C. Wang, L. Feng, K. Yang, Z. Liu, Functional nanomaterials for phototherapies of cancer, Chem. Rev. 114 (2014) 10869–10939. [8] T. Sun, Y.S. Zhang, B. Pang, D.C. Hyun, M. Yang, Y. Xia, Engineered nanoparticles for drug delivery in cancer therapy, Angew. Chem. Int. Ed. 53 (2014) 12320– 12364. [9] D. Jaque, L.M. Maestro, B. Rosal, P. Haro-Gonzalez, A. Benayas, J.L. Plaza, E.M. Rodríguez, J.G. Solé, Nanoparticles for photothermal therapies, Nanoscale 6 (2014) 9494–9530. [10] X. Song, Q. Chen, Z. Liu, Recent advances in the development of organic photothermal nano-agents, Nano Res. 8 (2015) 340–354. [11] L. Xu, L. Cheng, C. Wang, R. Peng, Z. Liu, Conjugated polymers for photothermal therapy of cancer, Polym. Chem. 5 (2014) 1573–1580. [12] Z. Sheng, D. Hu, M. Xue, M. He, P. Gong, L. Cai, Indocyanine green nanoparticles for theranostic applications, Nano Micro Lett. 5 (2013) 145–150. [13] V. Shanmugam, S. Selvakumar, C.S. Yeh, Near-infrared light-responsive nanomaterials in cancer therapeutics, Chem. Soc. Rev. 43 (2014) 6254–6287. [14] Z. Zhang, J. Wang, C. Chen, Near-infrared light-mediated nanoplatforms for cancer thermo-chemotherapy and optical imaging, Adv. Mater. 25 (2013) 3869–3880. [15] M.P. Melancon, M. Zhou, C. Li, Cancer theranostics with near-infrared lightactivatable multimodal nanoparticles, Acc. Chem. Res. 44 (2011) 947–956. [16] S. Sharifi, S. Behzadi, S. Laurent, M.L. Forrest, P. Stroeve, M. Mahmoudi, Toxicity of nanomaterials, Chem. Soc. Rev. 41 (2012) 2323–2343. [17] K.L. Aillon, Y. Xie, N. El-Gendy, C.J. Berkland, M.L. Forrest, Effects of nanomaterial physicochemical properties on in vivo toxicity, Adv. Drug Delivery Rev. 61 (2009) 457–466.

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