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One-pot synthesis of polypyrrole nanoparticles with tunable photothermal conversion and drug loading capacity
Accepted Manuscript Title: One-pot synthesis of polypyrrole nanoparticles with tunable photothermal conversion and drug loading capacity Authors: Bingqian Guo, Jiulong Zhao, Chenyao Wu, Yuting Zheng, Changqing Ye, Mingxian Huang, Shige Wang PII: DOI: Reference:
S0927-7765(19)30090-6 https://doi.org/10.1016/j.colsurfb.2019.02.016 COLSUB 10007
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
10 December 2018 29 January 2019 7 February 2019
Please cite this article as: Guo B, Zhao J, Wu C, Zheng Y, Ye C, Huang M, Wang S, One-pot synthesis of polypyrrole nanoparticles with tunable photothermal conversion and drug loading capacity, Colloids and Surfaces B: Biointerfaces (2019), https://doi.org/10.1016/j.colsurfb.2019.02.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
One-pot synthesis of polypyrrole nanoparticles with tunable photothermal conversion and drug loading capacity
Bingqian Guo,a,1 Jiulong Zhao,b,1 Chenyao Wu,a Yuting Zheng,a Changqing Ye,a Mingxian Huang,a
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and Shige Wanga
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a College of Science, University of Shanghai for Science and Technology, No. 334 Jungong Road, Shanghai 200093 (China) b
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai
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200433 (China)
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*To whom correspondence should be addressed email: [email protected] (Dr. Shige Wang).
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Theses authors contributed equally to this work.
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Graphical abstract
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Highlights One-pot synthesis of surface polyvinyl pyrrolidone modified polypyrrole.
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The relationship between polypyrrole structure and physicochemical property.
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Polypyrrole exhibits combined tumor photothermal-chemotherapy ability.
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Abstract: With an excellent near-infrared (NIR) light-responsive property, polypyrrole (PPy)
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nanoparticle has emerged as a promising NIR photothermal transducing agent for tumor photothermal therapy (PTT). However, the physicochemical characteristics of PPy can be affected by
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its morphology. Therefore, it is important to seek optimal processing parameter to optimize the
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application property of PPy-PVP NPs. Herein, we reported the PVP mediated one-pot synthesis of
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colloidal stable and biocompatible PPy nanoparticles (PPy-PVP NPs) for combined tumor
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photothermal-chemotherapy. The influence of molecular weight and PVP concentration on the
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spectroscopic characteristic, photothermal feature, drug loading performance, and antitumor efficiency of the resultant PPy-PVP NPs was systematically studied. By choosing PVP with a
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molecular weight of 360 kDa (concentration of 5 mg/mL) as the template and surface modifier
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during the synthesis, PPy-PVP NPs with spectroscopic characteristic, photothermal feature, drug loading performance, and antitumor efficiency. Findings in this study are anticipated to provide an in-depth understanding of the important character of surface engineering in the rational design and
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biomedical applications of PPy NPs.
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Keywords: polypyrrole, one-pot, polyvinyl pyrrolidone, tumor therapy 1. Introduction Photothermal therapy (PTT) with high therapeutic efficacy has received special attention for the ablation of cancer cells [1-3]. Compared to traditional cancer treatments, PTT is a minimally
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invasive method characterized with simple procedures and mitigated side effects [4]. Selection of a suitable photo-absorbing agent (PTA) with prominent photothermal conversion efficiency is of great
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significance for the efficient tumor PTT [5]. Due to their splendid physical and chemical properties, inorganic materials such as two dimensional transition metal sulfides [6-8] and metal nanoparticles
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(NPs) [9] have been used as PTAs in vitro and in vivo. However, these inorganic PTAs are suffering
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from critical issues that limit their further application. For example, CuS NPs are not readily
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metabolized in organism, which raises a long-term biosafety issue [10]. Due to the “melting effect”,
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the morphology of Au based nanomaterials will be changed under laser irradiation, leading to a weak
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photothermal durability [11].
In contrast, organic compounds, typically polymeric materials such as PPy [12-16],
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polydopamine [17] and polyaniline [18, 19] have become innovative PTAs due to their good
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biocompatibility and excellent photothermal conversion performance [20-22]. Nevertheless, there remain many problems among organic PTAs. For example, although indocyanine green has been frequently studied, its applications in tumor PTT remain restricted owing to serious photobleaching
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and short circulation time in the bloodstream. Polyaniline nanoparticles have also been confronted with many obstacles including low photothermal conversion efficiency and complicated synthesis processes [23]. Thus, the design of novel biocompatibile and photothermal stable organic materials remains a challenge. 3
Nanomaterials have generated considerable research interest in biomedical fields in recent years [24-26]. Preparation of PPy-based nanomaterials has become a research hotspot in recent years attributing to their convenient synthesis approach and good biocompatibility [27-29]. PPy possesses an excellent optical adsorption in the NIR region, and moreover, it can convert the absorbed NIR
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light into heat energy. It has been reported that the application performance of PPy can be directly affected by its microscopic morphology [30]. Besides, bare PPy NPs have a poor colloidal stability.
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In order to promote their tumor PTT application, it is urgent to seek the optimized operational parameter to realize the morphology and properties-controllable preparation of PPy NPs. Pyrrole
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monomer has the same polymerization ability at α and β sites, as a consequence, to obtain PPy NPs
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with specific morphology, there must be a constraint to enable the guided polymerization in a certain
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dimension. Previous study proposed that certain dopants can guide the organized growth and
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formation of PPy with special microstructure (fibrous, tubular and microspheres, etc) [31-33]. In
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most cases, the dopants can be surfactants which are able to form a special molecular structure to confine the three-dimensional growth of PPy NPs. For example, using polyvinyl alcohol (PVA) as a
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stabilizer, researchers synthesized uniform PPy nanoparticles via a facile one-step aqueous
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dispersion polymerization [34, 35]. As a kind of nonionic surfactant with a long-standing and safe record in biomedicine, PVP has been frequently used as a template to confine the growth of nanomaterials [36-39]. Moreover, PVP can enhance the circulatory half-life of drug in plasma when
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used as a drug carrier [40]. In another research, Woo et al. found that the shape and size of PPy NPs can be controlled by changing the chain length of PVP [41]. However, the relationship between the chain length (molecular weight) and concentration of PVP and drug loading, tumor PTT and combined chemotherapy application potentials of PPy NPs has not been reported. 4
In this research, PPy NPs with synchronous surface PVP modification, high colloidal stability and good biocompatibility were synthesized via a one-step aqueous dispersion polymerization using iron (III) chloride hexahydrate (FeCl3·6H2O) as an oxidant and PVP as a template and surface modifier (Fig. 1a). Specifically, the relationship between the surface modified PVP and the
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physicochemical properties of PPy-PVP NPs were studied. Results showed that PVP can not only guide the growth of PPy NPs but also synchronously decorate the surface of PPy NPs. Moreover, the
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drug loading and tumor PTT efficiency can be tuned via reducing the chain length and concentration of PVP. We showed that PVP with a molecular weight of 360kDa and a concentration of 5 mg/mL is
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the experimental PVP chain length and concentration range.
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the optimized option to guarantee the highly efficient drug loading and tumor therapy application in
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2. Experimental section
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2.1 Materials
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Pyrrole was purchased from Adamas Chemical Reagent Co., Ltd. (Shanghai, China). FeCl3·6H2O was provided by Aladdin Reagent Co., Ltd (China). PVP (with molecular weights of 40 kDa, 360
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kDa and 1300 kDa) was obtained from Sigma-Aldrich (USA). Mice fibroblast cells (L929),
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pancreatic acinar cell line (266-6) and human colorectal carcinoma (HT29) cells were obtained from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin
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and phosphate buffer (PBS) were purchased from Gibco (Shanghai, China). The CCK-8 (cell counting kit-8) was purchased from Dojindo Laboratories (Japan). Experimental Balb/c nude mice and Kunming (KM) mice with body weight of ~20 g were bought from Shanghai Slac Laboratory Animal Center (Shanghai, China). Animals were raised according to protocols and policies of the 5
National Ministry of Health. Distilled water with a resistivity higher than 18.2 MΩ·cm used in this study was produced using the Pall Cascada laboratory water system. All chemicals were directly used as received. 2.2 Synthesis and Characterization of PPy-PVP NPs
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Three types (sample I-III) of PPy-PVP NPs were synthesized by a redox reaction using FeCl3·6H2O as an oxidant. Briefly, 1 g PVP with molecular weight of 360 kDa (sample I), or 1300
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kDa (sample II), or 0.2 g PVP with molecular weight of 360 kDa (sample III) was separately dissolved into 40 mL distilled water in a vial and magnetically stirred to obtain a homogenous
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solution at room temperature. Then, FeCl3·6H2O (2.48 g) was added in each vial and stirred to get a
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homogenous solution. After cooling the mixture solution to 4 °C using ice, pyrrole monomer (140 µL)
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was added in each vial and stirred for 4 h. The produced PPy-PVP NPs were centrifuged (13000 rpm,
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6 min) and rinsed with water for 3 times. The as-prepared PPy-PVP NPs can be re-dispersed in water
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and saline by an ultrasonic dispersing technology using the ultrasonic cell grinder (5 min, 400 w, Scientz, Ningbo, China). The size and morphology of PPy-PVP NPs were observed by the scanning
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electron microscopy (SEM, FEI Magellan 400). Before the observation, 10 µL aqueous solution
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(about 500 µg/mL) of each sample was dropped onto a mica sheet and air-dried. The hydrodynamic sizes of the PPy-PVP NPs were measured by using dynamic light scattering (DLS, Nano ZS 90, Malvern). FTIR (Nicolet Nexus 670) was used for the structural analysis of PPy-PVP NPs, the data
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were collected with 16 scans/min by transmission mode ranging from 4000 to 400 cm-1. Image J 1.40 G software were used to measure the diameter of PPy-PVP NPs. At least 50 individual NPs for each sample were randomly measured.
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2.3 Photothermal conversion performance The spectroscopic characteristic of PPy-PVP NPs in the wavelength range of 200-900 nm was measured by UV-Vis-NIR spectroscopy with a medium scanning speed (Lambda 25, PerkinElmer, USA). The as-synthesized PPy-PVP NPs (sample I-III) with concentration of 100 μg/mL or 50
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μg/mL were dispersed in holes of a 96-well plate and continuously irradiated with NIR laser (0.8 W/cm2, 808 nm, 5 min) using a high power multimode pump laser (Shanghai Connet Fiber Optics
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Company). Water was set as control. The temperature increase (△T) of PPy-PVP dispersion and water with irradiation time were measured by the FLIRTM E60 camera (FLIR, USA). To study the
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photothermal conversion efficiency, PPy-PVP NPs (100 μg/mL, sample I-III) were continuously
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irradiated with NIR laser (0.3 W/cm2, 808 nm, 5 min) and then naturally cooled to room temperature.
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The photothermal conversion efficiency was calculated according to literature [42]. To study the
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photo-stability, PPy-PVP NPs (sample III) with a concentration of 100 μg/mL were irradiated with
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NIR laser for 8 cycles (laser on: 5 min, 2 W/cm2; laser off: 5 min). The solution temperature was measured by the FLIRTM E60 camera (FLIR, USA).
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2.4 In vitro cyto-compatibility assay
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L929, HT29, and 266-6 cells were used as models to test the in vitro cyto-compatibility of PPy-PVP NPs by means of CCK-8 assay, cell morphological observation and cell staining according to previous studies [6, 8]. Briefly, cells were seeded in 96-well plates (density of 8000 cells/well) and
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cultured in a CO2 incubator for 24 h. After that, sample I-III dispersed in DMEM were added to the holes (100 μg/mL, 100 µL) and cultured for another 24 h. Cells treated with pure DMEM were set as control. The viability of treated cells was tested using CCK-8 kit according to the instruction. For cell morphological observation, cells incubated with sample I-III after 24 h were washed with PBS 7
and observed by phase contrast microscope (Leica DMIL LED). For the cell staining, cells after the morphological observation were stained with dead/live kit for 15 min. Then, cells were washed with PBS and intuitively observed by phase contrast microscope (Leica DMIL LED). 2.5 In vitro hemo-compatibility
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For in vitro hemo-compatibility assay, KM mice were anesthetized and their blood was collected by cardiac puncturing. Then, mice red blood cells (mRBCs) were separated by centrifugation and
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washed with PBS for 3 times. The mRBCs were dispersed into PBS for the hemo-compatibility assay. In this assay, 1.2 mL sample I - III (the final sample concentration: 100 μg/mL) were mixed with 0.3
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mL mRBCs and cultured at 37 oC for 2 h. 0.3 mL mRBCs treated with 1.2 mL distilled water and 1.2
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mL PBS was set as the positive and negative control, respectively. After incubation and
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centrifugation, the absorption at 570 nm of supernatant was measured by UV-Vis-NIR spectroscopy
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2.6 Drug loading and release
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(Lambda 25, Perkin Elmer, USA) to calculate the hemolysis percentage (HP).
For the in vitro drug loading evaluation, 1 mL of DOX aqueous solution was mixed with sample
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I-III (final DOX concentration: 100 μg/mL; final PPy NPs concentration: 500 μg/mL) and stirred for
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24 h. Then, the mixture solution was centrifuged to separate the drug-loaded PPy-PVP NPs. The unloaded DOX in the supernatants was measured using high performance liquid chromatography (HPLC, Ichrom 5100) to calculate the DOX loading capacity of sample I-III. The HPLC operation
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parameters were as follows: mobile phase A: acetonitrile solution (70%); mobile phase B: phosphoric acid solution of pH = 3.4 (30%); detection wavelength: 425 nm, flow rate: 0.8 mL/min. For the in vitro drug release, the drug loading PPy-PVP NPs (sample III, 3 mg) were packed with the dialysis bag (MWCO = 10000 kDa) and transferred into a sample vial that was filled with 4 mL 8
PBS (pH = 7.4) or sodium acetate-acetic buffer solution (pH = 5.4). Subsequently, the vials were incubated in a vapor-bathing vibrator at 37 oC or 50 oC. At predetermined time points, 1 mL buffer solution was sucked out from each vial using a pipettor and equal volume of fresh buffer solution was supplemented. Finally, the released DOX concentration was determined via measuring the
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solution absorbance at 480 nm using UV-Vis-NIR spectrophotometer (Lambda 25, PerkinElmer, USA).
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2.7 In vitro PTT and combined tumor therapy
HT29 cells were seeded and cultured at a density of 8000 cells/well in a 96-well plate and
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cultured in a CO2 incubator for 24 h. Then, sample III with different concentrations (25, 50, 75, 100
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and 200 μg/mL, in DMEM) were added to the cell culture holes (0.1 mL/hole). After incubated for 6
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h, cells were continuously irradiated with 808 nm NIR laser (1 W/cm 2) for 5 min and further
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incubated for 24 h. For the in vitro combined tumor therapy, sample III with a concentration of 200
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μg/mL, DOX loaded PPy-PVP NPs (PPy-PVP concentration: 200 μg/mL; DOX concentration: 10 μg/mL, according to the drug loading efficiency, pure DOX (10 μg/mL) were added to the cell
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culture holes. After 12 h incubation, cells treated with sample III and DOX loaded sample III were
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continuously irradiated with 808 nm NIR laser (1 W/cm2) for 5 min and further incubated for 24 h. Finally, CCK-8 and dead/live staining were used to evaluate the in vitro PTT effect. 2.8 In vivo biocompatibility
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In vivo biosafety of PPy-PVP NPs were evaluated from views of in vivo hemo and
histocompatibility. In vivo hemo-compatibility was studied via routine blood test and serum biochemistry assay using the Sysmex XS-800i automated hematology analyzer and DxC 800 automatic biochemical analyzer, respectively. The healthy KM mice that were intravenously (I. V.) 9
injected with sample III (200 μL, 1000 μg/mL) were anesthetized on 1, 7 and 14 days for cardiac puncturing to collect blood. KM mice treated with PBS were tested as control. Major organs including heart, liver, spleen, lung and kidney of the mice after the blood collecting were soaked in polyformaldehyde to perform hematoxylin and eosin (H&E) staining to evaluate the in vivo
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histocompatibility according to previous report [7]. 2.9 In vivo tumor PTT and combined chemotherapy
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For the construction of tumor model, ~107 HT29 cells in 0.1 mL serum-free DMEM were subcutaneously injected into the back of each Balb/c nude mouse. After 2 weeks feeding, a tumor
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nodule with diameter of 0.5 cm was formed. Then, tumor bearing Balb/c nude mice were randomly
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assigned to 6 groups including control group (I. V. injected with PBS) and experimental groups (5
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groups, group I-V). Then, mice in the first three experimental groups were separately injected (I. V.)
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with 200 μL of sample I, sample II, and sample III that were dispersed in PBS (material
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concentration: 1000 μg/mL). Mice in group IV and group V were I. V. and intratumorally (I. T.) injected with drug loaded PPy-PVP (sample III) (group IV: 200 μL, group V: 20 μL, PPy NPs
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concentration: 1000 μg/mL, respectively). Note that the I. V. or I. T. injected materials will be diluted
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by the mice blood (total volume: 1.5-2.0 mL) and the final concentration will not exceed 100 μg/mL. Finally, mice in the above 6 groups were continuously irradiated with NIR laser (1 W/cm2, 808 nm, 5 min). The temperature elevation and thermal images of mice were measured and recorded by the
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FLIRTM E60 camera (FLIR, USA). Mice after laser irradiation were continually fed to compare the in vivo tumor therapeutic efficiency. 2.10 Statistical analysis One way ANOVA statistical analysis was used to calculate the significant difference of data. 10
0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001.
3. Results and discussion
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3.1 Synthesis and characterization of PPy-PVP NPs Via selecting PVP as a template and modifier, and FeCl3·6H2O as an oxidant, PVP-modified PPy
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NPs were synthesized from the chemical oxidative polymerization of pyrrole at 4 oC. To search a process parameter that can endow PPy NPs with optimum photothermal performance, we
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investigated the influence of PVP molecular weight and concentration on the PPy morphology and
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application property. As show in Fig. 1b-c and Fig. 2, well-defined PPy NPs with spherical shape
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were obtained in all tests. The mean particle size of sample I, sample II and sample III was counted
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as 74.55 nm, 68.98 nm and 83.79 nm, respectively (Fig. 2a-d, 1b-c). Specifically, small-size
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PPy-PVP NPs were synthesized when PVP with a higher molecular weight and concentration was selected. As an ampholytic surfactant, PVP can form a polymer micelle to act as an emulsifying
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agent to increase the solubility of pyrrole monomer. When water-soluble oxidant (FeCl3•6H2O) were
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added into the aqueous solution, they diffused into the monomer containing micelle and initiate the polymerization of pyrrole monomer to form the PPy NPs. PVP with a higher molecular weight and concentration may form polymer micelles with small-size. Besides, PVP with higher molecular
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weight and concentration may act as a sticky template and exert a higher resistance to the NPs growth and thereby induce the formation of NPs with smaller size. In order to verify that PVP molecules were synchronously modified on the surface of PPy NPs during the formation, FTIR characterizations of PPy-PVP NPs (sample III was selected as the 11
representative) and pure PVP molecules (molecule weight: 360 kDa) were performed (Fig. S1). Peaks located at 1553 cm-1, 1473 cm-1, 1285 cm-1 and 1042 cm-1 corresponding to the characteristic C-C conjugation, C-N stretching vibration, C-H in-plane bend and C-H ring deformation of PPy respectively were detected from the spectrum of PPy-PVP NPs. Vibration signals located at 2950
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cm-1, 1646 cm-1, 1490-1450 cm-1 and 1291 cm-1 can be ascribed to the C-H stretching/bending vibrations, C=O stretching vibration, C-N stretching vibration and -CH2 deformation vibration of
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PVP respectively, clearly indicating that PVP has been successfully attached to the surface of PPy NPs. The peak located at 3370 cm-1 can be ascribed to characteristic peak of bounded water.
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Owing to the steric hindrance and hydrophilicity of these surface modified PVP molecules,
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PPy-PVP NPs showed a good colloidal stability. The DLS analysis revealed that all of the three kinds
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of PPy-PVP NPs were readily dispersed in water (Fig. S2a-c), PBS (Fig. 3a-c) and DMEM (Fig. 3d-f)
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and displayed the typical Tyndall effect (Fig. S2d and Fig. 3g). Moreover, the hydrodynamic size of
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three kinds of PPy-PVP NPs in different medium did not show obvious change over 48 h, laying a foundation for the evaluation of photothermal conversion and in vivo tumor killing capacity.
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3.2 Photothermal performance of PPy-PVP NPs
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Before moving to the tumor PTT assessment, the spectroscopic characteristics of three kinds of PPy-PVP NPs were studied using UV-Vis-NIR spectroscopy. As show in Fig. S3a, PPy-PVP NPs (sample I-III) showed an obvious absorption in the wavelength range of 200 - 900 nm. Notably,
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similar to the NPs size order, their light absorption level followed the order of sample II < sample I < sample III at the same PPy-NPs concentration. Providing that the surface modified PVP molecules has no contribution to the light absorption, the pure PPy contents of sample I-III were thus found to follow the same order with NPs size (sample II < sample I < sample III), while the surface modifed 12
PVP molecules follow the order of sample III < sample I < sample II, according to the Lambert-Beer's
Law.
Additionally,
the
light
absorption
of
the
PPy-PVP
NPs
was
concentration-dependent (Fig. S3b). The photothermal property of PPy-PVP NPs and their correlation with the surface modified PVP
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were investigated. At the concentration of 100 μg/mL and laser power density of 0.8 W/cm2, all kinds of PPy-PVP NPs induced a swift solution temperature increase (Fig. 4a,b). Due to the NIR laser
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inertness of PVP, the in vitro photothermal outcome of three kinds of PPy-PVP NPs was closely related to their PPy abundance. Over 5 min of laser irradiation, the solution temperature increased by
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38.1 and 32.4 oC for the solution of sample I or sample II, respectively; sample III generated the
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highest solution temperature elevation of 44.7 oC. Comparatively, the △T of distilled water was
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negligible. Besides, the photothermal performance of PPy-PVP NPs was concentration-dependent.
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When samples were diluted to 50 μg/mL, the △T of sample I, sample II and sample III was
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quantified as 28.3, 26.8, 22.9 and 41.2 oC respectively (Fig. 4c, d). The photothermal conversion efficiency of different PPy-PVP NPs was further compared to
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explore their correlation with the surface modified PVP. Similarly, the photothermal conversion
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efficiency of PPy-PVP NPs also increased with the PPy abundance and the η value of sample I, sample II and sample III was calculated to be 39.87%, 35.60% and 51.61%, respectively (Fig. 4e, f). The photothermal stability of PPy-PVP NPs was studied by selecting sample III as a representative,
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since it possesses the optimal photothermal conversion efficiency. It was found that the cycling △T of sample III kept almost similar during 8 consecutive laser on/off cycles, clearly suggesting the high tolerance of PPy-PVP NPs to NIR laser irradiation (Fig. 4g). The above results clearly suggest that the photothermal conversion ability of PPy-PVP NPs can be optimized by adjusting the molecular 13
weight and concentration of the PVP template. 3.3 Drug loading and release Beyond the spectroscopic characteristic and photothermal property, it was found that the surface modified PVP also affected the drug loading performance of PPy-PVP NPs. Similar with the pure
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PPy contents of sample I-III, the drug loading efficiency also follows the order (sample II < sample I < sample III). As show in Fig. 5a, when the concentration of DOX was 100 μg/mL, the drug loading
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efficiency of sample I, sample II and sample III was calculated as 13.5%, 11.9% and 26.4%, respectively. The difference in drug loading efficiency of sample I-III indicates that the surface
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modified PVP affected the physical adsorption of DOX: sample III has the lowest surface modified
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PPy contents and thereby the best DOX loading-efficiency. Typically, the encapsulation of
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chemotherapeutics within nanocarriers can alter their way into cancer cells from passive drug
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permeation to active endocytosis, therefore, the drug utilization can be enhanced while its
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side-effects to normal cells can be reduced [43].
The controlled DOX release was studied by choosing sample III as a representative. It was found
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that the cumulative release amount of DOX from sample III within 48 h was 20.3%, 23.7% and 25.0%
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at pH = 7.4/T = 37 oC, pH = 5.4/T = 37 oC and pH = 5.4/T = 50 oC, respectively, proving that the DOX release was increased with the rise of temperature and the decrease of pH value of surrounding solution (Fig. 5b). The increased DOX release rate is supposedly because of the enhanced DOX
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solubility in acidic environment and under a higher temperature. Such a pH and heat responsive drug release is beneficial for the enhancement of tumor therapeutic efficiency since the slightly acidic environment and the accumulated heat in tumor will locally trigger the DOX release.
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3.4 In vitro cytotoxicity and hemo-compatibility Ahead of the tumor photothermal study, the in vitro and in vivo biocompatibility of PPy-PVP NPs were analyzed. To assess the in vitro cytotoxicity, the standard CCK-8 assay, cell morphological observation and trypan blue staining were performed. It was found that the percentage of viable L929
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cells after incubated with different PPy-PVP NPs with a concentration of 100 μg/mL for 24 h were about 84.0 ± 1.3 % (sample I), 100 ± 4.1 % (sample II) and 99.8 ± 3.3 % (sample III, Fig. 6a).
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Similar with cells incubated with PBS (control), the L929 cell morphology and structural integrity were not destroyed (Fig. 6c). The cytocompatibility of PPy-PVP NPs was further studied using HT29
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and 266-6 cells as models. The percentage of viable HT29 and 266-6 cells after incubated with
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different PPy-PVP NPs with a concentration of 100 μg/mL for 24 h were about 99.14 % and 99.47%
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(sample I), 92.88 % and 95.55% (sample II), 97.09 % and 95.95% (sample III) (Fig. S4a,b).
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Dead/live staining which can stain the live and dead cells into green and red respectively indicated
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that almost all of the treated cells was green stained (Fig. 6c,d), evidences that PPy-PVP NPs are biocompatible in the experimental dosage.
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We next studied the in vitro hemo-compatibility by monitoring the HP of mRBCs that
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wereincubated with PPy-PVP NPs (Fig. 7a). Compare with saline, the HP of sample I, sample II and sample III was determined as 4.8%, 1.0% and 3.6% respectively, indicating that a small portion of mRBCs was destroyed by the PPy-PVP NPs. This is likely owing to the nonspecific interaction
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between mRBCs and PPy-PVP NPs. However, the HP of mRBCs treated with each kind of PPy-PVP NPs was lower than 5%, proving that the PPy-PVP NPs possess an outstanding in vitro hemo-compatibility in the experimental dosage [44]. Oppositely, mRBCs treated with water were completely damaged (insert of Fig. 7a). 15
3.5 In vitro tumor therapy Encouraged by the photothermal performance and in vitro biocompatibility of PPy-PVP NPs, we then explored the in vitro tumor PTT ability using sample III as a representative since it showed the highest photothermal conversion efficiency. The cell viability after PTT was evaluated using the
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standard CCK-8 assay and dead/live staining (Fig. S5a, b). After continuously irradiated with NIR laser, the HT29 cell viability gradually decreased with the concentration of PPy-PVP NPs and the
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viability decreased to less than 20% when the concentration of NPs was no less than 100 μg/mL (100 μg/mL: 16.7 %; 200 μg/mL: 12.7%; Fig. S5a). Consisted with the CCK-8 results, dead/live staining
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pictures displayed that the portion of red stained cells increased with materials concentration and
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almost all HT29 cells were red stained when the concentration was higher than 100 μg/mL (Fig. S5b),
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clearly suggesting the excellent in vitro cancer cell inhibition efficiency of PPy-PVP NPs.
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Since PPy-PVP NPs possess a tunable DOX loading and releasing ability, the in vitro combined
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tumor therapy of DOX-loaded PPy-PVP NPs was studied. As show in Fig. 6b, cells treated with saline kept healthy, while the viability of cells treated with pure DOX and DOX loaded PPy-PVP
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NPs (sample III) was significantly reduced (DOX: 79.39%, p < 0.05, versus control; sample III +
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DOX: 78.31%, p < 0.05, versus control) resulting from the cell killing effect of DOX. Meanwhile, proliferation of sample III treated cells was significantly suppressed after NIR irradiation (14.50%, p < 0.001, versus control), implying the excellent in vitro photothermal therapeutic efficiency of
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PPy-PVP NPs (sample III). Moreover, due to the NIR triggered photothermal ablation and DOX induced DNA damage, cancer cells treated with sample III + DOX + NIR presented nearly complete cell killing with a viability of 10.09% (p < 0.001, versus control), significantly lower than cells treated with sample III + DOX (p < 0.05), pure DOX (p < 0.01) and sample III + NIR (p < 0.01). In 16
accordance with CCK-8 results, dead/live staining pictures further demonstrated the synergistic photothermal and chemotherapy efficiency of DOX-loaded PPy-PVP NPs (Fig. 6d). 3.6 In vivo biocompatibility The long-term in vivo bio-safety of PPy-PVP NPs (sample III) was assessed via performing the
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routine blood test, serum biochemistry analysis and H&E staining. As show in Fig. S6 and Fig. 7b, the difference of all routine blood and serum biochemistry parameters between control and
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experimental group was meaningless, inferring that PPy-PVP NPs have an admirable in vivo hemo-compatibility. The in vivo biocompatible nature of PPy-PVP NPs was further explored by
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recording the body weight and the tissue lesion of major organs of KM mice that received the NPs
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injection. As illustrated in Fig. 7c, the weight fluctuation between the treatment group and the control
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group over the feeding period is insignificant. H&E staining images further confirmed that PPy-PVP
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NPs exerted no significant in vivo negative effect and pathological toxicity to the experimental
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animals (Fig. 7d). This fine biocompatibility together with the excellent photothermal conversion
tumor PTT.
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efficiency indicated the feasibility of employing the PPy-PVP NPs as an efficient platform for in vivo
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3.7 In vivo tumor therapy
The topic of the present study is the one-pot synthesis of PPy-PVP NPs with tunable photothermal conversion, drug loading capacity and antitumor efficiency. Therefore, the in vivo
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tumor PTT effectiveness of the PPy-PVP NPs was examined on tumor bearing Balb/c mice. Interestingly, the in vivo PTT performance of PPy-PVP NPs was related to their surface properties as well. The tumor temperature of mice that were injected with sample I and sample II increased by 9.3 o
C and 6.5 oC, respectively after 5 min of NIR irradiation (Fig. 8a and b). Followed the similar 17
tendency as the in vitro spectroscopic characteristic and photothermal property, sample III showed the most evident in vivo photothermal conversion performance with a △T of 14.2 oC. As a simple administration method, I. T. injection has been frequently used in laboratory investigations to directly demonstrate the in vivo therapeutic effects of nanomaterials [45]. As show in Fig. 8c, the in vivo
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PTT performance of mice I. T. injected with DOX-loaded sample III was most encouraging, with the temperature swiftly increased by 30 oC under laser irradiation for 5 min. Comparatively, the tumor
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temperature variation of mice with I. V. PBS injection was insignificant.
The in vivo tumor therapeutic performance was monitored by quantifying the tumor volume and
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recording the tumor appearance. After 14 days feeding, the tumor volume of mice in control group
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expanded to 6.8 times than initial. The tumor growth of the mice that were I. V. injected with sample
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I, sample II and sample III was partially inhibited with the final volume expanded about 5.3, 5.7 and
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4.5 times respectively than initial after 14 days feeding. Particularly, the PPy-PVP NPs can act as a
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promising synergistic anticancer platform after DOX loading. The tumor volume in mice injected with drug-loaded PPy-PVP NPs (sample III) was further inhibited than group I-III (Fig. 8c and d).
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More strikingly, ascribed to the multiple killing effects of PPy-PVP NPs and DOX, the tumor
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increase of mice that were I. T. injected with DOX-loaded PPy-PVP NPs was completely suppressed. It should be mentioned that the tumor cell killing effect was solely related to the PTT capacity of PPy NPs and chemotherapy effects of DOX, since pure PPy NPs have no influence on the viability of
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HT29 cells within the experimental concentration range (Fig. S4a, c). 4. Conclusions In general, we reported the structure and physicochemical property controlled synthesis of PPy-PVP NPs for highly efficient tumor PTT and chemotherapy. Different kinds (sample I-III) of 18
biocompatible PPy-PVP NPs were synthesized via a one-step aqueous dispersion polymerization using PVP as a template and surface modifier, and FeCl3·6H2O as an oxidant. The spectroscopic characteristic, photothermal property, drug loading performance, in vitro and in vivo antitumor therapy efficiency of the designed PPy-PVP NPs can be adjusted by changing the molecular weight
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and concentration of PVP. It was found that PVP with molecular weight of 360 kDa and concentration of 5 mg/mL is the most revealing option to facilitate the combined tumor photothermal
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and chemotherapy of PPy-PVP NPs. Findings in this study are anticipated to provide new ideas for the design of conjugated polymer NPs for tumor hyperthermia therapy and other clinical
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applications.
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Authors have no conflicts of interest to declare.
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Conflicts of interest
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Acknowledgements
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This work was supported by China National Natural Science Foundation of China (Grant No. 51702214), by Shanghai Sailing Program (17YF1412600) supported by the Shanghai Committee of
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Science and Technology.
References
[1] Q. Chen, J. Wen, H. Li, Y. Xu, F. Liu, S. Sun, Recent advances in different modal imaging-guided
A
photothermal therapy, Biomaterials. 106 (2016) 144-166. [2] B. Du, C. Ma, G. Ding, X. Han, D. Li, E. Wang, J. Wang, Cooperative strategies for enhancing performance of photothermal therapy (PTT) agent: optimizing its photothermal conversion and cell Internalization ability, Small. 13 (2017) 1603275. 19
[3] W. Wang, L. Wang, Y. Li, S. Liu, Z. Xie, X. Jing, Nanoscale polymermetal-organic framework hybrids for effective photothermal therapy of colon cancers, Adv. Mater. 28 (2016) 9320-9325. [4] J. Shen, X. Sheng, Z. Chang, Q. Wu, S. Wang, Z. Xuan, D. Li, Y. Wu, Y. Shang, X. Kong, L. Yu, L. Li, K. Ruan, H. Hu, Y. Huang, L. Hui, D. Xie, F. Wang, R. Hu, Iron Metabolism regulates p53
IP T
signaling through direct heme-p53 interaction and modulation of p53 localization, stability, and function, Cell Rep. 7 (2014) 180-193.
SC R
[5] X. Wang, Y. Ma, S. Xing, Y. Wang, H. Xu, Ultrathin polypyrrole nanosheets via space-confined synthesis for efficient photothermal therapy in the second near-infrared window, Nano Lett. 18 (2018)
U
2217-2225.
N
[6] H. Yang, J. Zhao, C. Wu, C. Ye, D. Zou, S. Wang, Chem. Facile synthesis of colloidal stable
A
MoS2 nanoparticles for combined tumor therapy, Chem. Eng. J. 351 (2018) 548-558.
M
[7] S. Wang, J. Zhao, H. Yang, C. Wu, F. Hu, H. Chang, G. Li, D. Ma, D. Zou, M. Huang, Bottom-up
ED
synthesis of WS2 nanosheets with synchronous surface modification for imaging guided tumor regression, Acta Biomater. 58 (2017) 442-454.
PT
[8] J. Zhao, P. Xie, C. Ye, C. Wu, W. Han, M. Huang, S. Wang, H. Chen, Outside-in synthesis of
CC E
mesoporous silica/molybdenum disulfide nanoparticles for antitumor application, Chem. Eng. J. 351 (2018) 157-168.
[9] H. Ke, J. Wang, Z. Dai, Y. Jin, E. Qu, Z. Xing, C. Guo, X. Yue, J. Liu, Ed Gold-nanoshelled
A
microcapsules: a theranostic agent for ultrasound contrast imaging and photothermal therapy, Angew. Chem., Int. , 123 (2011) 3017-3021. [10] J. Mou, P. Li, C. Liu, H. Xu, L. Song, J. Wang, K. Zhang, Y. Chen, J. Shi, H. Chen,
ltrasmall
Cu2-xS nanodots for highly efficient photoacoustic imaging-guided photothermal therapy, Small. 11 20
(2015) 2275. [11] B. Nikoobakht, J. Wang, M. A. El-Sayed, Surface-enhanced raman scattering of molecules adsorbed on gold nanorods: off-surface plasmon resonance condition, Chem. Phys. Lett. 366 (2002) 17-23.
IP T
[12] J. Y. Hong, H. Yoon, J. Jang, Kinetic study of the formation of polypyrrole nanoparticles in water-soluble polymer/metal cation systems: a light-scattering analysis, Small. 6 (2010) 679-686.
SC R
[13] X. Song, H. Gong, S. Yin, L. Cheng, C. Wang, Z. Li, Y. Li, X. Wang, G. Liu, Z. Liu, Ultra-small iron oxide doped polypyrrole nanoparticles for in vivo multimodal imaging guided photothermal
U
therapy, Adv. Funct. Mater. 24 (2014)1194-1201.
N
[14] Q. Wang, J. Wang, G. Lv, F. Wang, X. Zhou, J. Hu, Q. Wang, Facile synthesis of hydrophilic
A
polypyrrole nanoparticles for photothermal cancer therapy, J. Mater. Sci. 49 (2014) 3484-3490.
M
[15] Y. Tian, J. Zhang, S. Tang, L. Zhou, W. Yang, Polypyrrole composite nanoparticles with
ED
morphology-dependent photothermal effect and immunological responses, Small. 12 (2016) 721-726.
PT
[16] X. Song, C. Liang, L. Feng, K. Yang, Z. Liu, Iodine-131-labeled, transferrin-capped polypyrrole
CC E
nanoparticles for tumor-targeted synergistic photothermal-radioisotope therapy, Biomater. Sci. 5 (2017) 1828-1835.
[17] H.C. Zhang, X.D. Wang, P.Y. Wang, R. Liu, X.M. Hou, One-pot synthesis of biodegradable
A
polydopamine-doped mesoporous silica nanocomposites (PMSNs) as pH-sensitive targetingdrug nanocarriers for synergistic ch emo-photothermal therapy, RSC Adv. 8 (2018) 37433-37440. [18] J. Zhou, Z. Lu, X. Zhu, X. Wang, Y. Liao, Z. Ma, F. Li, NIR photothermal therapy using polyaniline nanoparticles, Biomaterials. 34 (2013) 9584-9592. 21
[19] B. P. Jiang, L. Zhang, Y. Zhu, X. C. Shen, S. C. Ji, X. Y. Tan, L. Cheng, H. Liang, Water-soluble hyaluronic acid–hybridized polyaniline nanoparticles for effectively targeted photothermal therapy, J. Mater. Chem. B. 3 (2015) 3767-3776. [20] Y. Shi, M. Liu, F. Deng, G. Zeng, Q. Wan, X. Zhang, Y. Wei, Recent progress and development
IP T
on polymeric nanomaterials for photothermal therapy: A brief overview, J. Mater. Chem. B. 5 (2016) 194-206.
SC R
[21] Y. Zhang, C. Y. Ang, Y. Zhao, Polymeric nanocarriers incorporating near-infrared absorbing agents for potent photothermal therapy of cancer, Polym. J. 48 (2015) 589-603.
U
[22] W. Zhou, L. Lu, D. Chen, Z. Wang, J. Zhai, R. Wang, G. Tan, J. Mao, P. Yu, C. Ning,
N
Construction of high surface potential polypyrrole nanorods with enhanced antibacterial properties, J.
A
Mater. Chem. B. 6 (2018) 3128-3135.
M
[23] J. F. Lovell, C. S. Jin, E. Huynh, H. Jin, C. Kim, J. L. Rubinstein, W. C. W. Chan, W. Cao, L. V.
ED
Wang, G. Zheng, Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents, Nat. Mater. 10 (2011) 324-332.
PT
[24] P. Ji, B. Zhou, Y. Zhan, Y. Wang, Y. Zhang, Y. Li, P. He, Multi-stimulative nanogels with
CC E
enhanced thermosensitivity for intracellular therapeutic delivery, ACS Appl. Mater. Interfaces. 5 (2017) 39143–39151.
[25] Y. Li, Y. Xiao, C. Liu, The Horizon of Materiobiology: A Perspective on Material-Guided Cell
A
Behaviors and Tissue Engineering, Chemical Reviews. 5 (2017) 4376−4421. [26] Y. Li, J. Rodrigues, H. Tomás, Injectable and Biodegradable Hydrogels: Gelation, Biodegradation and Biomedical Applications, Chem. Soc. Rev. 6 (2012) 2193-2221. [27] Z. Yang, W. He, H. Zheng, J. Wei, P. Liu, W. Zhu, L. Lin, L. Zhang, C. Yi, Z. Xu, One-pot 22
synthesis of albumin-gadolinium stabilized polypyrrole nanotheranostic agent for magnetic resonance imaging guided photothermal therapy, Biomaterials. 161 (2018) 1-10. [28] X. Luo, X. Liu, Y. Pei, Y. Ling, P. Wu, C. Cai, Leakage-free polypyrrole-Au nanostructures for combined raman detection and photothermal cancer therapy, J. Mater. Chem. B. 5 (2017) 7449-7962.
IP T
[29] T. T. V. Phan, N. Q. Bui and M. S. Moorthy, Synthesis and in vitro performance of polypyrrole-coated iron-platinum nanoparticles for photothermal therapy and photoacoustic imaging,
SC R
Nanoscale Res. Lett. , 12 (2017) 570.
[30] H. Liu, W. Li, Y. Cao, Y. Guo, Y. Kang, Theranostic nanoplatform based on polypyrrole
U
nanoparticles for photoacoustic imaging and photothermal therapy, J. Nanopart. Res. 20 (2018) 57.
using
carrageenan
as
a
polypyrrole/multi-walled
carbon
nanotube
M
nanocomposites, Polymers. 10 (2018) 632.
dopant:
A
polypyrrole
N
[31] M. Rahaman, A. Aldalbahi, M. Almoiqli, S. Alzahly, Chemical and electrochemical synthesis of
ED
[32] F. Khadem, M. Pishvaei, M. Salami-Kalajahi, F. Najafi, Morphology control of conducting polypyrrole nanostructures via operational conditions in the emulsion polymerization, J. Appl. Polym.
PT
Sci. 134 (2017) 44697.
CC E
[33] A. T. Mane, S. D. Sartale, V. B. Patil, Dodecyl benzene sulfonic acid (DBSA) doped polypyrrole (PPy) films: Synthesis, structural, morphological, gas sensing and impedance study, J. Mater. Sci.: Mater. Electron. 26 (2015) 1-10.
A
[34] K. Yang, H. Xu, L. Chng, C. Sun, J. Wang, Z. Liu, In vitro and in vivo near-infrared photothermal therapy of cancer using polypyrrole organic nanoparticles, Adv. Mater. 734 (2013) 5586-5592. [35] Z. Zha, X. Yue, Q. Ren, Z. Dai, Uniform polypyrrole nanoparticles with high photothermal 23
conversion efficiency for photothermal ablation of cancer cells, Adv. Mater. 5 (2013) 777-782. [36] W. Zhang, M. Tan, P. Zhang, L. Zhang, W. Dong, Q. Wang, J. Ma, E. Dong, S. Xu, G. Wang, One pot synthesis of Sb2S3 nanocrystalline films through a PVP-assisted hydrothermal process, Appl. Surf. Sci. 455 (2018) 1063-1069.
IP T
[37] S. M. Lam, M. W. Kee, J. C. Sin, Influence of PVP surfactant on the morphology and properties of ZnO micro/nanoflowers for dye mixtures and textile wastewater degradation, Mater. Chem. Phys.
SC R
212 (2018) 35-43.
[38] S. Bensaadi, O. Arous, H. Kerdjoudj and M. Amara, Evaluating molecular weight of PVP on
U
characteristics of CTA membrane dialysis, J. Environ. Chem. Eng. 4 (2016) 1545-1554.
N
[39] M. J. A. Oliveira, O. S. Estefânia, M. A. B. Lúcia, M. Regina, V. S. Amato, A. B. Lugão, D. F.
A
Parra, Influence of chitosan/clay in drug delivery of glucantime from PVP membranes, Radiat. Phys.
M
Chem. 94 (2014 )194-198.
ED
[40] D. Liu, B. Yu, G. Jiang, W. Yu, Y. Zhang, B. Xu, Fabrication of composite microneedles integrated with insulin-loaded CaCO3 microparticles and PVP for transdermal delivery in diabetic
PT
rats, Mater. Sci. Eng., C. 90 (2018) 180-188.
CC E
[41] H. Y. Woo, W. G. Jung, D. W. Ihm, J. Y. Kim, Synthesis and dispersion of polypyrrole nanoparticles in polyvinylpyrrolidone emulsion, Synth. Met. 160 (2010) 588-591. [42] S. Wang, Y. Wu, R. Guo, Y. Huang, S. Wen, M. Shen, J. Wang, X. Shi, Laponite nanodisks as an
A
efficient platform for doxorubicin delivery to cancer cells. Langmuir, 29 (2013) 5030-5036. [43] G. Yu, A. Liu, H. Jin, Y. Chen, D. Yin, R. Huo, S. Wang, J. Wang, Urchin-shaped Bi2S3/Cu2S/Cu3BiS3 composites with enhanced photothermal and CT imaging performance, J. Phys. Chem. C. 122 (2018) 3794-380. 24
[44] S. Wang, J. Zhao, F. Hu, X. Li, X, An, S. Zhou, Y. Chen, M. Huang, Phase-changeable and bubble-releasing implants for highly efficient HIFU-responsive tumor surgery and chemotherapy, J. Mater. Chem. B. 4 (2016) 7368-7378. [45] S. Wang, X. Li, Y. Chen, X. Cai, H. Yao, W. Gao, Y. Zheng, X. An, J. Shi, H. Chen, A Facile
A
CC E
PT
ED
M
A
N
U
SC R
Multi-Modality Tumor Imaging and Therapy, Adv. Mater. 27 (2015) 2775-2782.
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One-Pot Synthesis of a Two-Dimensional MoS2/Bi2S3 Composite Theranostic Nanosystem for
25
A
N
U
SC R
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Figures and Captions
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Fig. 1. (a) Schematic illustration of PVP guided synthesis of PPy-PVP NPs; (b, c) SEM micrograph
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and diameter distribution histogram of PPy-PVP NPs (sample III).
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Fig. 2. SEM micrographs and diameter distribution histograms of (a, b) sample I; (c, d) sample II.
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Fig. 3. (a-f) Time-dependent DLS profiles of sample I, sample II, sample III dispersed in PBS (a:
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sample I, b: sample II, c: sample III) and DMEM (d: sample I, e: sample II, f: sample III); (g)
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photographic images of Tyndall effect of sample I, sample II, sample III dispersed in saline (1: sample
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I, 3: sample II, 5: sample III) and DMEM (2: sample I, 4: sample II, 6: sample III). Left: after 24 h
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storage; right: after 48 h storage.
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Fig. 4. (a, c) Temperature curves of PPy-PVP NPs aqueous solutions (a: 100 μg/mL; c: 50 μg/mL)
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under laser irradiation of 808 nm laser at the power density of 0.8 W/cm2; (b, d) thermal images of
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PPy-PVP NPs aqueous solution corresponding to (a) and (c), respectively; (e) the η value of PPy-PVP NPs; (f) time constant for heat transfer of the sample III; (g) temperature curve of PPy-PVP
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NPs aqueous solution during 8 irradiation and cooling cycles.
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Fig. 5. (a) Drug loading efficiency (Mt/Mo × 100% (Mt and Mo represents the amount of loaded
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DOX and the total mass of DOX used for drug loading, respectively) of PPy-PVP NPs; (b) drug
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release of DOX loaded sample III. Data are presented as mean ± SD (n = 3).
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Fig. 6. (a) Viability of PPy-PVP NPs treated L929 cells; (b) in vitro combined tumor photothermal therapy of sample III; (c) morphology of PPy-PVP NPs treated L929 cells corresponding to (a); (d)
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morphology of dead/live staining of HT29 cells corresponding to (b). Data in panel (a) and panel (b)
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are presented as mean ± SD (n = 3).
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Fig. 7. (a) Hemolysis percentage (HP) of mRBCs co-incubated with PPy-PVP NPs solutions (100
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μg/mL, n = 3); (b) Body weight evolution of KM mice treated with PBS and sample III (1000
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μg/mL); (c) blood biochemistry parameters of KM mice that were I. V. injected with PBS or aqueous solution of sample III; (d) H&E staining images of heart, liver, spleen, lung, and kidney of KM mice.
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Data in pane (b) and panel (c) are presented as mean ± SD (n = 6).
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Fig. 8. (a) Temperature profiles of mice treated with PPy-PVP NPs and PBS (control) under NIR laser irradiation; (b) in vivo thermal images of mice under continuous irradiation for varied durations
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corresponding to (a); (c) time-dependent tumor growth profile of mice after various treatments as
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noted; (d) representative pictures of HT29 tumor bearing mice that received different treatments at
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day 1 and day 14. Data in panel (c) are presented as mean ± SD (n = 6).
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S0927-7765(19)30090-6 https://doi.org/10.1016/j.colsurfb.2019.02.016 COLSUB 10007
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
10 December 2018 29 January 2019 7 February 2019
Please cite this article as: Guo B, Zhao J, Wu C, Zheng Y, Ye C, Huang M, Wang S, One-pot synthesis of polypyrrole nanoparticles with tunable photothermal conversion and drug loading capacity, Colloids and Surfaces B: Biointerfaces (2019), https://doi.org/10.1016/j.colsurfb.2019.02.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
One-pot synthesis of polypyrrole nanoparticles with tunable photothermal conversion and drug loading capacity
Bingqian Guo,a,1 Jiulong Zhao,b,1 Chenyao Wu,a Yuting Zheng,a Changqing Ye,a Mingxian Huang,a
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and Shige Wanga
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a College of Science, University of Shanghai for Science and Technology, No. 334 Jungong Road, Shanghai 200093 (China) b
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai
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200433 (China)
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*To whom correspondence should be addressed email: [email protected] (Dr. Shige Wang).
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Theses authors contributed equally to this work.
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Graphical abstract
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Highlights One-pot synthesis of surface polyvinyl pyrrolidone modified polypyrrole.
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The relationship between polypyrrole structure and physicochemical property.
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Polypyrrole exhibits combined tumor photothermal-chemotherapy ability.
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Abstract: With an excellent near-infrared (NIR) light-responsive property, polypyrrole (PPy)
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nanoparticle has emerged as a promising NIR photothermal transducing agent for tumor photothermal therapy (PTT). However, the physicochemical characteristics of PPy can be affected by
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its morphology. Therefore, it is important to seek optimal processing parameter to optimize the
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application property of PPy-PVP NPs. Herein, we reported the PVP mediated one-pot synthesis of
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colloidal stable and biocompatible PPy nanoparticles (PPy-PVP NPs) for combined tumor
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photothermal-chemotherapy. The influence of molecular weight and PVP concentration on the
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spectroscopic characteristic, photothermal feature, drug loading performance, and antitumor efficiency of the resultant PPy-PVP NPs was systematically studied. By choosing PVP with a
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molecular weight of 360 kDa (concentration of 5 mg/mL) as the template and surface modifier
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during the synthesis, PPy-PVP NPs with spectroscopic characteristic, photothermal feature, drug loading performance, and antitumor efficiency. Findings in this study are anticipated to provide an in-depth understanding of the important character of surface engineering in the rational design and
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biomedical applications of PPy NPs.
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Keywords: polypyrrole, one-pot, polyvinyl pyrrolidone, tumor therapy 1. Introduction Photothermal therapy (PTT) with high therapeutic efficacy has received special attention for the ablation of cancer cells [1-3]. Compared to traditional cancer treatments, PTT is a minimally
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invasive method characterized with simple procedures and mitigated side effects [4]. Selection of a suitable photo-absorbing agent (PTA) with prominent photothermal conversion efficiency is of great
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significance for the efficient tumor PTT [5]. Due to their splendid physical and chemical properties, inorganic materials such as two dimensional transition metal sulfides [6-8] and metal nanoparticles
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(NPs) [9] have been used as PTAs in vitro and in vivo. However, these inorganic PTAs are suffering
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from critical issues that limit their further application. For example, CuS NPs are not readily
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metabolized in organism, which raises a long-term biosafety issue [10]. Due to the “melting effect”,
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the morphology of Au based nanomaterials will be changed under laser irradiation, leading to a weak
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photothermal durability [11].
In contrast, organic compounds, typically polymeric materials such as PPy [12-16],
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polydopamine [17] and polyaniline [18, 19] have become innovative PTAs due to their good
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biocompatibility and excellent photothermal conversion performance [20-22]. Nevertheless, there remain many problems among organic PTAs. For example, although indocyanine green has been frequently studied, its applications in tumor PTT remain restricted owing to serious photobleaching
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and short circulation time in the bloodstream. Polyaniline nanoparticles have also been confronted with many obstacles including low photothermal conversion efficiency and complicated synthesis processes [23]. Thus, the design of novel biocompatibile and photothermal stable organic materials remains a challenge. 3
Nanomaterials have generated considerable research interest in biomedical fields in recent years [24-26]. Preparation of PPy-based nanomaterials has become a research hotspot in recent years attributing to their convenient synthesis approach and good biocompatibility [27-29]. PPy possesses an excellent optical adsorption in the NIR region, and moreover, it can convert the absorbed NIR
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light into heat energy. It has been reported that the application performance of PPy can be directly affected by its microscopic morphology [30]. Besides, bare PPy NPs have a poor colloidal stability.
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In order to promote their tumor PTT application, it is urgent to seek the optimized operational parameter to realize the morphology and properties-controllable preparation of PPy NPs. Pyrrole
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monomer has the same polymerization ability at α and β sites, as a consequence, to obtain PPy NPs
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with specific morphology, there must be a constraint to enable the guided polymerization in a certain
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dimension. Previous study proposed that certain dopants can guide the organized growth and
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formation of PPy with special microstructure (fibrous, tubular and microspheres, etc) [31-33]. In
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most cases, the dopants can be surfactants which are able to form a special molecular structure to confine the three-dimensional growth of PPy NPs. For example, using polyvinyl alcohol (PVA) as a
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stabilizer, researchers synthesized uniform PPy nanoparticles via a facile one-step aqueous
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dispersion polymerization [34, 35]. As a kind of nonionic surfactant with a long-standing and safe record in biomedicine, PVP has been frequently used as a template to confine the growth of nanomaterials [36-39]. Moreover, PVP can enhance the circulatory half-life of drug in plasma when
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used as a drug carrier [40]. In another research, Woo et al. found that the shape and size of PPy NPs can be controlled by changing the chain length of PVP [41]. However, the relationship between the chain length (molecular weight) and concentration of PVP and drug loading, tumor PTT and combined chemotherapy application potentials of PPy NPs has not been reported. 4
In this research, PPy NPs with synchronous surface PVP modification, high colloidal stability and good biocompatibility were synthesized via a one-step aqueous dispersion polymerization using iron (III) chloride hexahydrate (FeCl3·6H2O) as an oxidant and PVP as a template and surface modifier (Fig. 1a). Specifically, the relationship between the surface modified PVP and the
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physicochemical properties of PPy-PVP NPs were studied. Results showed that PVP can not only guide the growth of PPy NPs but also synchronously decorate the surface of PPy NPs. Moreover, the
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drug loading and tumor PTT efficiency can be tuned via reducing the chain length and concentration of PVP. We showed that PVP with a molecular weight of 360kDa and a concentration of 5 mg/mL is
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the experimental PVP chain length and concentration range.
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the optimized option to guarantee the highly efficient drug loading and tumor therapy application in
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2. Experimental section
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2.1 Materials
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Pyrrole was purchased from Adamas Chemical Reagent Co., Ltd. (Shanghai, China). FeCl3·6H2O was provided by Aladdin Reagent Co., Ltd (China). PVP (with molecular weights of 40 kDa, 360
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kDa and 1300 kDa) was obtained from Sigma-Aldrich (USA). Mice fibroblast cells (L929),
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pancreatic acinar cell line (266-6) and human colorectal carcinoma (HT29) cells were obtained from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin
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and phosphate buffer (PBS) were purchased from Gibco (Shanghai, China). The CCK-8 (cell counting kit-8) was purchased from Dojindo Laboratories (Japan). Experimental Balb/c nude mice and Kunming (KM) mice with body weight of ~20 g were bought from Shanghai Slac Laboratory Animal Center (Shanghai, China). Animals were raised according to protocols and policies of the 5
National Ministry of Health. Distilled water with a resistivity higher than 18.2 MΩ·cm used in this study was produced using the Pall Cascada laboratory water system. All chemicals were directly used as received. 2.2 Synthesis and Characterization of PPy-PVP NPs
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Three types (sample I-III) of PPy-PVP NPs were synthesized by a redox reaction using FeCl3·6H2O as an oxidant. Briefly, 1 g PVP with molecular weight of 360 kDa (sample I), or 1300
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kDa (sample II), or 0.2 g PVP with molecular weight of 360 kDa (sample III) was separately dissolved into 40 mL distilled water in a vial and magnetically stirred to obtain a homogenous
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solution at room temperature. Then, FeCl3·6H2O (2.48 g) was added in each vial and stirred to get a
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homogenous solution. After cooling the mixture solution to 4 °C using ice, pyrrole monomer (140 µL)
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was added in each vial and stirred for 4 h. The produced PPy-PVP NPs were centrifuged (13000 rpm,
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6 min) and rinsed with water for 3 times. The as-prepared PPy-PVP NPs can be re-dispersed in water
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and saline by an ultrasonic dispersing technology using the ultrasonic cell grinder (5 min, 400 w, Scientz, Ningbo, China). The size and morphology of PPy-PVP NPs were observed by the scanning
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electron microscopy (SEM, FEI Magellan 400). Before the observation, 10 µL aqueous solution
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(about 500 µg/mL) of each sample was dropped onto a mica sheet and air-dried. The hydrodynamic sizes of the PPy-PVP NPs were measured by using dynamic light scattering (DLS, Nano ZS 90, Malvern). FTIR (Nicolet Nexus 670) was used for the structural analysis of PPy-PVP NPs, the data
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were collected with 16 scans/min by transmission mode ranging from 4000 to 400 cm-1. Image J 1.40 G software were used to measure the diameter of PPy-PVP NPs. At least 50 individual NPs for each sample were randomly measured.
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2.3 Photothermal conversion performance The spectroscopic characteristic of PPy-PVP NPs in the wavelength range of 200-900 nm was measured by UV-Vis-NIR spectroscopy with a medium scanning speed (Lambda 25, PerkinElmer, USA). The as-synthesized PPy-PVP NPs (sample I-III) with concentration of 100 μg/mL or 50
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μg/mL were dispersed in holes of a 96-well plate and continuously irradiated with NIR laser (0.8 W/cm2, 808 nm, 5 min) using a high power multimode pump laser (Shanghai Connet Fiber Optics
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Company). Water was set as control. The temperature increase (△T) of PPy-PVP dispersion and water with irradiation time were measured by the FLIRTM E60 camera (FLIR, USA). To study the
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photothermal conversion efficiency, PPy-PVP NPs (100 μg/mL, sample I-III) were continuously
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irradiated with NIR laser (0.3 W/cm2, 808 nm, 5 min) and then naturally cooled to room temperature.
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The photothermal conversion efficiency was calculated according to literature [42]. To study the
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photo-stability, PPy-PVP NPs (sample III) with a concentration of 100 μg/mL were irradiated with
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NIR laser for 8 cycles (laser on: 5 min, 2 W/cm2; laser off: 5 min). The solution temperature was measured by the FLIRTM E60 camera (FLIR, USA).
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2.4 In vitro cyto-compatibility assay
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L929, HT29, and 266-6 cells were used as models to test the in vitro cyto-compatibility of PPy-PVP NPs by means of CCK-8 assay, cell morphological observation and cell staining according to previous studies [6, 8]. Briefly, cells were seeded in 96-well plates (density of 8000 cells/well) and
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cultured in a CO2 incubator for 24 h. After that, sample I-III dispersed in DMEM were added to the holes (100 μg/mL, 100 µL) and cultured for another 24 h. Cells treated with pure DMEM were set as control. The viability of treated cells was tested using CCK-8 kit according to the instruction. For cell morphological observation, cells incubated with sample I-III after 24 h were washed with PBS 7
and observed by phase contrast microscope (Leica DMIL LED). For the cell staining, cells after the morphological observation were stained with dead/live kit for 15 min. Then, cells were washed with PBS and intuitively observed by phase contrast microscope (Leica DMIL LED). 2.5 In vitro hemo-compatibility
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For in vitro hemo-compatibility assay, KM mice were anesthetized and their blood was collected by cardiac puncturing. Then, mice red blood cells (mRBCs) were separated by centrifugation and
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washed with PBS for 3 times. The mRBCs were dispersed into PBS for the hemo-compatibility assay. In this assay, 1.2 mL sample I - III (the final sample concentration: 100 μg/mL) were mixed with 0.3
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mL mRBCs and cultured at 37 oC for 2 h. 0.3 mL mRBCs treated with 1.2 mL distilled water and 1.2
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mL PBS was set as the positive and negative control, respectively. After incubation and
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centrifugation, the absorption at 570 nm of supernatant was measured by UV-Vis-NIR spectroscopy
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2.6 Drug loading and release
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(Lambda 25, Perkin Elmer, USA) to calculate the hemolysis percentage (HP).
For the in vitro drug loading evaluation, 1 mL of DOX aqueous solution was mixed with sample
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I-III (final DOX concentration: 100 μg/mL; final PPy NPs concentration: 500 μg/mL) and stirred for
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24 h. Then, the mixture solution was centrifuged to separate the drug-loaded PPy-PVP NPs. The unloaded DOX in the supernatants was measured using high performance liquid chromatography (HPLC, Ichrom 5100) to calculate the DOX loading capacity of sample I-III. The HPLC operation
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parameters were as follows: mobile phase A: acetonitrile solution (70%); mobile phase B: phosphoric acid solution of pH = 3.4 (30%); detection wavelength: 425 nm, flow rate: 0.8 mL/min. For the in vitro drug release, the drug loading PPy-PVP NPs (sample III, 3 mg) were packed with the dialysis bag (MWCO = 10000 kDa) and transferred into a sample vial that was filled with 4 mL 8
PBS (pH = 7.4) or sodium acetate-acetic buffer solution (pH = 5.4). Subsequently, the vials were incubated in a vapor-bathing vibrator at 37 oC or 50 oC. At predetermined time points, 1 mL buffer solution was sucked out from each vial using a pipettor and equal volume of fresh buffer solution was supplemented. Finally, the released DOX concentration was determined via measuring the
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solution absorbance at 480 nm using UV-Vis-NIR spectrophotometer (Lambda 25, PerkinElmer, USA).
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2.7 In vitro PTT and combined tumor therapy
HT29 cells were seeded and cultured at a density of 8000 cells/well in a 96-well plate and
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cultured in a CO2 incubator for 24 h. Then, sample III with different concentrations (25, 50, 75, 100
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and 200 μg/mL, in DMEM) were added to the cell culture holes (0.1 mL/hole). After incubated for 6
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h, cells were continuously irradiated with 808 nm NIR laser (1 W/cm 2) for 5 min and further
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incubated for 24 h. For the in vitro combined tumor therapy, sample III with a concentration of 200
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μg/mL, DOX loaded PPy-PVP NPs (PPy-PVP concentration: 200 μg/mL; DOX concentration: 10 μg/mL, according to the drug loading efficiency, pure DOX (10 μg/mL) were added to the cell
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culture holes. After 12 h incubation, cells treated with sample III and DOX loaded sample III were
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continuously irradiated with 808 nm NIR laser (1 W/cm2) for 5 min and further incubated for 24 h. Finally, CCK-8 and dead/live staining were used to evaluate the in vitro PTT effect. 2.8 In vivo biocompatibility
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In vivo biosafety of PPy-PVP NPs were evaluated from views of in vivo hemo and
histocompatibility. In vivo hemo-compatibility was studied via routine blood test and serum biochemistry assay using the Sysmex XS-800i automated hematology analyzer and DxC 800 automatic biochemical analyzer, respectively. The healthy KM mice that were intravenously (I. V.) 9
injected with sample III (200 μL, 1000 μg/mL) were anesthetized on 1, 7 and 14 days for cardiac puncturing to collect blood. KM mice treated with PBS were tested as control. Major organs including heart, liver, spleen, lung and kidney of the mice after the blood collecting were soaked in polyformaldehyde to perform hematoxylin and eosin (H&E) staining to evaluate the in vivo
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histocompatibility according to previous report [7]. 2.9 In vivo tumor PTT and combined chemotherapy
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For the construction of tumor model, ~107 HT29 cells in 0.1 mL serum-free DMEM were subcutaneously injected into the back of each Balb/c nude mouse. After 2 weeks feeding, a tumor
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nodule with diameter of 0.5 cm was formed. Then, tumor bearing Balb/c nude mice were randomly
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assigned to 6 groups including control group (I. V. injected with PBS) and experimental groups (5
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groups, group I-V). Then, mice in the first three experimental groups were separately injected (I. V.)
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with 200 μL of sample I, sample II, and sample III that were dispersed in PBS (material
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concentration: 1000 μg/mL). Mice in group IV and group V were I. V. and intratumorally (I. T.) injected with drug loaded PPy-PVP (sample III) (group IV: 200 μL, group V: 20 μL, PPy NPs
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concentration: 1000 μg/mL, respectively). Note that the I. V. or I. T. injected materials will be diluted
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by the mice blood (total volume: 1.5-2.0 mL) and the final concentration will not exceed 100 μg/mL. Finally, mice in the above 6 groups were continuously irradiated with NIR laser (1 W/cm2, 808 nm, 5 min). The temperature elevation and thermal images of mice were measured and recorded by the
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FLIRTM E60 camera (FLIR, USA). Mice after laser irradiation were continually fed to compare the in vivo tumor therapeutic efficiency. 2.10 Statistical analysis One way ANOVA statistical analysis was used to calculate the significant difference of data. 10
0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001.
3. Results and discussion
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3.1 Synthesis and characterization of PPy-PVP NPs Via selecting PVP as a template and modifier, and FeCl3·6H2O as an oxidant, PVP-modified PPy
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NPs were synthesized from the chemical oxidative polymerization of pyrrole at 4 oC. To search a process parameter that can endow PPy NPs with optimum photothermal performance, we
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investigated the influence of PVP molecular weight and concentration on the PPy morphology and
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application property. As show in Fig. 1b-c and Fig. 2, well-defined PPy NPs with spherical shape
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were obtained in all tests. The mean particle size of sample I, sample II and sample III was counted
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as 74.55 nm, 68.98 nm and 83.79 nm, respectively (Fig. 2a-d, 1b-c). Specifically, small-size
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PPy-PVP NPs were synthesized when PVP with a higher molecular weight and concentration was selected. As an ampholytic surfactant, PVP can form a polymer micelle to act as an emulsifying
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agent to increase the solubility of pyrrole monomer. When water-soluble oxidant (FeCl3•6H2O) were
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added into the aqueous solution, they diffused into the monomer containing micelle and initiate the polymerization of pyrrole monomer to form the PPy NPs. PVP with a higher molecular weight and concentration may form polymer micelles with small-size. Besides, PVP with higher molecular
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weight and concentration may act as a sticky template and exert a higher resistance to the NPs growth and thereby induce the formation of NPs with smaller size. In order to verify that PVP molecules were synchronously modified on the surface of PPy NPs during the formation, FTIR characterizations of PPy-PVP NPs (sample III was selected as the 11
representative) and pure PVP molecules (molecule weight: 360 kDa) were performed (Fig. S1). Peaks located at 1553 cm-1, 1473 cm-1, 1285 cm-1 and 1042 cm-1 corresponding to the characteristic C-C conjugation, C-N stretching vibration, C-H in-plane bend and C-H ring deformation of PPy respectively were detected from the spectrum of PPy-PVP NPs. Vibration signals located at 2950
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cm-1, 1646 cm-1, 1490-1450 cm-1 and 1291 cm-1 can be ascribed to the C-H stretching/bending vibrations, C=O stretching vibration, C-N stretching vibration and -CH2 deformation vibration of
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PVP respectively, clearly indicating that PVP has been successfully attached to the surface of PPy NPs. The peak located at 3370 cm-1 can be ascribed to characteristic peak of bounded water.
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Owing to the steric hindrance and hydrophilicity of these surface modified PVP molecules,
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PPy-PVP NPs showed a good colloidal stability. The DLS analysis revealed that all of the three kinds
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of PPy-PVP NPs were readily dispersed in water (Fig. S2a-c), PBS (Fig. 3a-c) and DMEM (Fig. 3d-f)
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and displayed the typical Tyndall effect (Fig. S2d and Fig. 3g). Moreover, the hydrodynamic size of
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three kinds of PPy-PVP NPs in different medium did not show obvious change over 48 h, laying a foundation for the evaluation of photothermal conversion and in vivo tumor killing capacity.
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3.2 Photothermal performance of PPy-PVP NPs
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Before moving to the tumor PTT assessment, the spectroscopic characteristics of three kinds of PPy-PVP NPs were studied using UV-Vis-NIR spectroscopy. As show in Fig. S3a, PPy-PVP NPs (sample I-III) showed an obvious absorption in the wavelength range of 200 - 900 nm. Notably,
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similar to the NPs size order, their light absorption level followed the order of sample II < sample I < sample III at the same PPy-NPs concentration. Providing that the surface modified PVP molecules has no contribution to the light absorption, the pure PPy contents of sample I-III were thus found to follow the same order with NPs size (sample II < sample I < sample III), while the surface modifed 12
PVP molecules follow the order of sample III < sample I < sample II, according to the Lambert-Beer's
Law.
Additionally,
the
light
absorption
of
the
PPy-PVP
NPs
was
concentration-dependent (Fig. S3b). The photothermal property of PPy-PVP NPs and their correlation with the surface modified PVP
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were investigated. At the concentration of 100 μg/mL and laser power density of 0.8 W/cm2, all kinds of PPy-PVP NPs induced a swift solution temperature increase (Fig. 4a,b). Due to the NIR laser
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inertness of PVP, the in vitro photothermal outcome of three kinds of PPy-PVP NPs was closely related to their PPy abundance. Over 5 min of laser irradiation, the solution temperature increased by
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38.1 and 32.4 oC for the solution of sample I or sample II, respectively; sample III generated the
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highest solution temperature elevation of 44.7 oC. Comparatively, the △T of distilled water was
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negligible. Besides, the photothermal performance of PPy-PVP NPs was concentration-dependent.
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When samples were diluted to 50 μg/mL, the △T of sample I, sample II and sample III was
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quantified as 28.3, 26.8, 22.9 and 41.2 oC respectively (Fig. 4c, d). The photothermal conversion efficiency of different PPy-PVP NPs was further compared to
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explore their correlation with the surface modified PVP. Similarly, the photothermal conversion
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efficiency of PPy-PVP NPs also increased with the PPy abundance and the η value of sample I, sample II and sample III was calculated to be 39.87%, 35.60% and 51.61%, respectively (Fig. 4e, f). The photothermal stability of PPy-PVP NPs was studied by selecting sample III as a representative,
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since it possesses the optimal photothermal conversion efficiency. It was found that the cycling △T of sample III kept almost similar during 8 consecutive laser on/off cycles, clearly suggesting the high tolerance of PPy-PVP NPs to NIR laser irradiation (Fig. 4g). The above results clearly suggest that the photothermal conversion ability of PPy-PVP NPs can be optimized by adjusting the molecular 13
weight and concentration of the PVP template. 3.3 Drug loading and release Beyond the spectroscopic characteristic and photothermal property, it was found that the surface modified PVP also affected the drug loading performance of PPy-PVP NPs. Similar with the pure
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PPy contents of sample I-III, the drug loading efficiency also follows the order (sample II < sample I < sample III). As show in Fig. 5a, when the concentration of DOX was 100 μg/mL, the drug loading
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efficiency of sample I, sample II and sample III was calculated as 13.5%, 11.9% and 26.4%, respectively. The difference in drug loading efficiency of sample I-III indicates that the surface
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modified PVP affected the physical adsorption of DOX: sample III has the lowest surface modified
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PPy contents and thereby the best DOX loading-efficiency. Typically, the encapsulation of
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chemotherapeutics within nanocarriers can alter their way into cancer cells from passive drug
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permeation to active endocytosis, therefore, the drug utilization can be enhanced while its
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side-effects to normal cells can be reduced [43].
The controlled DOX release was studied by choosing sample III as a representative. It was found
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that the cumulative release amount of DOX from sample III within 48 h was 20.3%, 23.7% and 25.0%
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at pH = 7.4/T = 37 oC, pH = 5.4/T = 37 oC and pH = 5.4/T = 50 oC, respectively, proving that the DOX release was increased with the rise of temperature and the decrease of pH value of surrounding solution (Fig. 5b). The increased DOX release rate is supposedly because of the enhanced DOX
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solubility in acidic environment and under a higher temperature. Such a pH and heat responsive drug release is beneficial for the enhancement of tumor therapeutic efficiency since the slightly acidic environment and the accumulated heat in tumor will locally trigger the DOX release.
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3.4 In vitro cytotoxicity and hemo-compatibility Ahead of the tumor photothermal study, the in vitro and in vivo biocompatibility of PPy-PVP NPs were analyzed. To assess the in vitro cytotoxicity, the standard CCK-8 assay, cell morphological observation and trypan blue staining were performed. It was found that the percentage of viable L929
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cells after incubated with different PPy-PVP NPs with a concentration of 100 μg/mL for 24 h were about 84.0 ± 1.3 % (sample I), 100 ± 4.1 % (sample II) and 99.8 ± 3.3 % (sample III, Fig. 6a).
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Similar with cells incubated with PBS (control), the L929 cell morphology and structural integrity were not destroyed (Fig. 6c). The cytocompatibility of PPy-PVP NPs was further studied using HT29
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and 266-6 cells as models. The percentage of viable HT29 and 266-6 cells after incubated with
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different PPy-PVP NPs with a concentration of 100 μg/mL for 24 h were about 99.14 % and 99.47%
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(sample I), 92.88 % and 95.55% (sample II), 97.09 % and 95.95% (sample III) (Fig. S4a,b).
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Dead/live staining which can stain the live and dead cells into green and red respectively indicated
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that almost all of the treated cells was green stained (Fig. 6c,d), evidences that PPy-PVP NPs are biocompatible in the experimental dosage.
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We next studied the in vitro hemo-compatibility by monitoring the HP of mRBCs that
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wereincubated with PPy-PVP NPs (Fig. 7a). Compare with saline, the HP of sample I, sample II and sample III was determined as 4.8%, 1.0% and 3.6% respectively, indicating that a small portion of mRBCs was destroyed by the PPy-PVP NPs. This is likely owing to the nonspecific interaction
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between mRBCs and PPy-PVP NPs. However, the HP of mRBCs treated with each kind of PPy-PVP NPs was lower than 5%, proving that the PPy-PVP NPs possess an outstanding in vitro hemo-compatibility in the experimental dosage [44]. Oppositely, mRBCs treated with water were completely damaged (insert of Fig. 7a). 15
3.5 In vitro tumor therapy Encouraged by the photothermal performance and in vitro biocompatibility of PPy-PVP NPs, we then explored the in vitro tumor PTT ability using sample III as a representative since it showed the highest photothermal conversion efficiency. The cell viability after PTT was evaluated using the
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standard CCK-8 assay and dead/live staining (Fig. S5a, b). After continuously irradiated with NIR laser, the HT29 cell viability gradually decreased with the concentration of PPy-PVP NPs and the
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viability decreased to less than 20% when the concentration of NPs was no less than 100 μg/mL (100 μg/mL: 16.7 %; 200 μg/mL: 12.7%; Fig. S5a). Consisted with the CCK-8 results, dead/live staining
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pictures displayed that the portion of red stained cells increased with materials concentration and
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almost all HT29 cells were red stained when the concentration was higher than 100 μg/mL (Fig. S5b),
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clearly suggesting the excellent in vitro cancer cell inhibition efficiency of PPy-PVP NPs.
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Since PPy-PVP NPs possess a tunable DOX loading and releasing ability, the in vitro combined
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tumor therapy of DOX-loaded PPy-PVP NPs was studied. As show in Fig. 6b, cells treated with saline kept healthy, while the viability of cells treated with pure DOX and DOX loaded PPy-PVP
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NPs (sample III) was significantly reduced (DOX: 79.39%, p < 0.05, versus control; sample III +
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DOX: 78.31%, p < 0.05, versus control) resulting from the cell killing effect of DOX. Meanwhile, proliferation of sample III treated cells was significantly suppressed after NIR irradiation (14.50%, p < 0.001, versus control), implying the excellent in vitro photothermal therapeutic efficiency of
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PPy-PVP NPs (sample III). Moreover, due to the NIR triggered photothermal ablation and DOX induced DNA damage, cancer cells treated with sample III + DOX + NIR presented nearly complete cell killing with a viability of 10.09% (p < 0.001, versus control), significantly lower than cells treated with sample III + DOX (p < 0.05), pure DOX (p < 0.01) and sample III + NIR (p < 0.01). In 16
accordance with CCK-8 results, dead/live staining pictures further demonstrated the synergistic photothermal and chemotherapy efficiency of DOX-loaded PPy-PVP NPs (Fig. 6d). 3.6 In vivo biocompatibility The long-term in vivo bio-safety of PPy-PVP NPs (sample III) was assessed via performing the
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routine blood test, serum biochemistry analysis and H&E staining. As show in Fig. S6 and Fig. 7b, the difference of all routine blood and serum biochemistry parameters between control and
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experimental group was meaningless, inferring that PPy-PVP NPs have an admirable in vivo hemo-compatibility. The in vivo biocompatible nature of PPy-PVP NPs was further explored by
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recording the body weight and the tissue lesion of major organs of KM mice that received the NPs
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injection. As illustrated in Fig. 7c, the weight fluctuation between the treatment group and the control
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group over the feeding period is insignificant. H&E staining images further confirmed that PPy-PVP
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NPs exerted no significant in vivo negative effect and pathological toxicity to the experimental
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animals (Fig. 7d). This fine biocompatibility together with the excellent photothermal conversion
tumor PTT.
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efficiency indicated the feasibility of employing the PPy-PVP NPs as an efficient platform for in vivo
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3.7 In vivo tumor therapy
The topic of the present study is the one-pot synthesis of PPy-PVP NPs with tunable photothermal conversion, drug loading capacity and antitumor efficiency. Therefore, the in vivo
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tumor PTT effectiveness of the PPy-PVP NPs was examined on tumor bearing Balb/c mice. Interestingly, the in vivo PTT performance of PPy-PVP NPs was related to their surface properties as well. The tumor temperature of mice that were injected with sample I and sample II increased by 9.3 o
C and 6.5 oC, respectively after 5 min of NIR irradiation (Fig. 8a and b). Followed the similar 17
tendency as the in vitro spectroscopic characteristic and photothermal property, sample III showed the most evident in vivo photothermal conversion performance with a △T of 14.2 oC. As a simple administration method, I. T. injection has been frequently used in laboratory investigations to directly demonstrate the in vivo therapeutic effects of nanomaterials [45]. As show in Fig. 8c, the in vivo
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PTT performance of mice I. T. injected with DOX-loaded sample III was most encouraging, with the temperature swiftly increased by 30 oC under laser irradiation for 5 min. Comparatively, the tumor
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temperature variation of mice with I. V. PBS injection was insignificant.
The in vivo tumor therapeutic performance was monitored by quantifying the tumor volume and
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recording the tumor appearance. After 14 days feeding, the tumor volume of mice in control group
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expanded to 6.8 times than initial. The tumor growth of the mice that were I. V. injected with sample
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I, sample II and sample III was partially inhibited with the final volume expanded about 5.3, 5.7 and
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4.5 times respectively than initial after 14 days feeding. Particularly, the PPy-PVP NPs can act as a
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promising synergistic anticancer platform after DOX loading. The tumor volume in mice injected with drug-loaded PPy-PVP NPs (sample III) was further inhibited than group I-III (Fig. 8c and d).
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More strikingly, ascribed to the multiple killing effects of PPy-PVP NPs and DOX, the tumor
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increase of mice that were I. T. injected with DOX-loaded PPy-PVP NPs was completely suppressed. It should be mentioned that the tumor cell killing effect was solely related to the PTT capacity of PPy NPs and chemotherapy effects of DOX, since pure PPy NPs have no influence on the viability of
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HT29 cells within the experimental concentration range (Fig. S4a, c). 4. Conclusions In general, we reported the structure and physicochemical property controlled synthesis of PPy-PVP NPs for highly efficient tumor PTT and chemotherapy. Different kinds (sample I-III) of 18
biocompatible PPy-PVP NPs were synthesized via a one-step aqueous dispersion polymerization using PVP as a template and surface modifier, and FeCl3·6H2O as an oxidant. The spectroscopic characteristic, photothermal property, drug loading performance, in vitro and in vivo antitumor therapy efficiency of the designed PPy-PVP NPs can be adjusted by changing the molecular weight
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and concentration of PVP. It was found that PVP with molecular weight of 360 kDa and concentration of 5 mg/mL is the most revealing option to facilitate the combined tumor photothermal
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and chemotherapy of PPy-PVP NPs. Findings in this study are anticipated to provide new ideas for the design of conjugated polymer NPs for tumor hyperthermia therapy and other clinical
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applications.
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Authors have no conflicts of interest to declare.
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Conflicts of interest
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Acknowledgements
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This work was supported by China National Natural Science Foundation of China (Grant No. 51702214), by Shanghai Sailing Program (17YF1412600) supported by the Shanghai Committee of
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Science and Technology.
References
[1] Q. Chen, J. Wen, H. Li, Y. Xu, F. Liu, S. Sun, Recent advances in different modal imaging-guided
A
photothermal therapy, Biomaterials. 106 (2016) 144-166. [2] B. Du, C. Ma, G. Ding, X. Han, D. Li, E. Wang, J. Wang, Cooperative strategies for enhancing performance of photothermal therapy (PTT) agent: optimizing its photothermal conversion and cell Internalization ability, Small. 13 (2017) 1603275. 19
[3] W. Wang, L. Wang, Y. Li, S. Liu, Z. Xie, X. Jing, Nanoscale polymermetal-organic framework hybrids for effective photothermal therapy of colon cancers, Adv. Mater. 28 (2016) 9320-9325. [4] J. Shen, X. Sheng, Z. Chang, Q. Wu, S. Wang, Z. Xuan, D. Li, Y. Wu, Y. Shang, X. Kong, L. Yu, L. Li, K. Ruan, H. Hu, Y. Huang, L. Hui, D. Xie, F. Wang, R. Hu, Iron Metabolism regulates p53
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signaling through direct heme-p53 interaction and modulation of p53 localization, stability, and function, Cell Rep. 7 (2014) 180-193.
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[5] X. Wang, Y. Ma, S. Xing, Y. Wang, H. Xu, Ultrathin polypyrrole nanosheets via space-confined synthesis for efficient photothermal therapy in the second near-infrared window, Nano Lett. 18 (2018)
U
2217-2225.
N
[6] H. Yang, J. Zhao, C. Wu, C. Ye, D. Zou, S. Wang, Chem. Facile synthesis of colloidal stable
A
MoS2 nanoparticles for combined tumor therapy, Chem. Eng. J. 351 (2018) 548-558.
M
[7] S. Wang, J. Zhao, H. Yang, C. Wu, F. Hu, H. Chang, G. Li, D. Ma, D. Zou, M. Huang, Bottom-up
ED
synthesis of WS2 nanosheets with synchronous surface modification for imaging guided tumor regression, Acta Biomater. 58 (2017) 442-454.
PT
[8] J. Zhao, P. Xie, C. Ye, C. Wu, W. Han, M. Huang, S. Wang, H. Chen, Outside-in synthesis of
CC E
mesoporous silica/molybdenum disulfide nanoparticles for antitumor application, Chem. Eng. J. 351 (2018) 157-168.
[9] H. Ke, J. Wang, Z. Dai, Y. Jin, E. Qu, Z. Xing, C. Guo, X. Yue, J. Liu, Ed Gold-nanoshelled
A
microcapsules: a theranostic agent for ultrasound contrast imaging and photothermal therapy, Angew. Chem., Int. , 123 (2011) 3017-3021. [10] J. Mou, P. Li, C. Liu, H. Xu, L. Song, J. Wang, K. Zhang, Y. Chen, J. Shi, H. Chen,
ltrasmall
Cu2-xS nanodots for highly efficient photoacoustic imaging-guided photothermal therapy, Small. 11 20
(2015) 2275. [11] B. Nikoobakht, J. Wang, M. A. El-Sayed, Surface-enhanced raman scattering of molecules adsorbed on gold nanorods: off-surface plasmon resonance condition, Chem. Phys. Lett. 366 (2002) 17-23.
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[12] J. Y. Hong, H. Yoon, J. Jang, Kinetic study of the formation of polypyrrole nanoparticles in water-soluble polymer/metal cation systems: a light-scattering analysis, Small. 6 (2010) 679-686.
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[13] X. Song, H. Gong, S. Yin, L. Cheng, C. Wang, Z. Li, Y. Li, X. Wang, G. Liu, Z. Liu, Ultra-small iron oxide doped polypyrrole nanoparticles for in vivo multimodal imaging guided photothermal
U
therapy, Adv. Funct. Mater. 24 (2014)1194-1201.
N
[14] Q. Wang, J. Wang, G. Lv, F. Wang, X. Zhou, J. Hu, Q. Wang, Facile synthesis of hydrophilic
A
polypyrrole nanoparticles for photothermal cancer therapy, J. Mater. Sci. 49 (2014) 3484-3490.
M
[15] Y. Tian, J. Zhang, S. Tang, L. Zhou, W. Yang, Polypyrrole composite nanoparticles with
ED
morphology-dependent photothermal effect and immunological responses, Small. 12 (2016) 721-726.
PT
[16] X. Song, C. Liang, L. Feng, K. Yang, Z. Liu, Iodine-131-labeled, transferrin-capped polypyrrole
CC E
nanoparticles for tumor-targeted synergistic photothermal-radioisotope therapy, Biomater. Sci. 5 (2017) 1828-1835.
[17] H.C. Zhang, X.D. Wang, P.Y. Wang, R. Liu, X.M. Hou, One-pot synthesis of biodegradable
A
polydopamine-doped mesoporous silica nanocomposites (PMSNs) as pH-sensitive targetingdrug nanocarriers for synergistic ch emo-photothermal therapy, RSC Adv. 8 (2018) 37433-37440. [18] J. Zhou, Z. Lu, X. Zhu, X. Wang, Y. Liao, Z. Ma, F. Li, NIR photothermal therapy using polyaniline nanoparticles, Biomaterials. 34 (2013) 9584-9592. 21
[19] B. P. Jiang, L. Zhang, Y. Zhu, X. C. Shen, S. C. Ji, X. Y. Tan, L. Cheng, H. Liang, Water-soluble hyaluronic acid–hybridized polyaniline nanoparticles for effectively targeted photothermal therapy, J. Mater. Chem. B. 3 (2015) 3767-3776. [20] Y. Shi, M. Liu, F. Deng, G. Zeng, Q. Wan, X. Zhang, Y. Wei, Recent progress and development
IP T
on polymeric nanomaterials for photothermal therapy: A brief overview, J. Mater. Chem. B. 5 (2016) 194-206.
SC R
[21] Y. Zhang, C. Y. Ang, Y. Zhao, Polymeric nanocarriers incorporating near-infrared absorbing agents for potent photothermal therapy of cancer, Polym. J. 48 (2015) 589-603.
U
[22] W. Zhou, L. Lu, D. Chen, Z. Wang, J. Zhai, R. Wang, G. Tan, J. Mao, P. Yu, C. Ning,
N
Construction of high surface potential polypyrrole nanorods with enhanced antibacterial properties, J.
A
Mater. Chem. B. 6 (2018) 3128-3135.
M
[23] J. F. Lovell, C. S. Jin, E. Huynh, H. Jin, C. Kim, J. L. Rubinstein, W. C. W. Chan, W. Cao, L. V.
ED
Wang, G. Zheng, Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents, Nat. Mater. 10 (2011) 324-332.
PT
[24] P. Ji, B. Zhou, Y. Zhan, Y. Wang, Y. Zhang, Y. Li, P. He, Multi-stimulative nanogels with
CC E
enhanced thermosensitivity for intracellular therapeutic delivery, ACS Appl. Mater. Interfaces. 5 (2017) 39143–39151.
[25] Y. Li, Y. Xiao, C. Liu, The Horizon of Materiobiology: A Perspective on Material-Guided Cell
A
Behaviors and Tissue Engineering, Chemical Reviews. 5 (2017) 4376−4421. [26] Y. Li, J. Rodrigues, H. Tomás, Injectable and Biodegradable Hydrogels: Gelation, Biodegradation and Biomedical Applications, Chem. Soc. Rev. 6 (2012) 2193-2221. [27] Z. Yang, W. He, H. Zheng, J. Wei, P. Liu, W. Zhu, L. Lin, L. Zhang, C. Yi, Z. Xu, One-pot 22
synthesis of albumin-gadolinium stabilized polypyrrole nanotheranostic agent for magnetic resonance imaging guided photothermal therapy, Biomaterials. 161 (2018) 1-10. [28] X. Luo, X. Liu, Y. Pei, Y. Ling, P. Wu, C. Cai, Leakage-free polypyrrole-Au nanostructures for combined raman detection and photothermal cancer therapy, J. Mater. Chem. B. 5 (2017) 7449-7962.
IP T
[29] T. T. V. Phan, N. Q. Bui and M. S. Moorthy, Synthesis and in vitro performance of polypyrrole-coated iron-platinum nanoparticles for photothermal therapy and photoacoustic imaging,
SC R
Nanoscale Res. Lett. , 12 (2017) 570.
[30] H. Liu, W. Li, Y. Cao, Y. Guo, Y. Kang, Theranostic nanoplatform based on polypyrrole
U
nanoparticles for photoacoustic imaging and photothermal therapy, J. Nanopart. Res. 20 (2018) 57.
using
carrageenan
as
a
polypyrrole/multi-walled
carbon
nanotube
M
nanocomposites, Polymers. 10 (2018) 632.
dopant:
A
polypyrrole
N
[31] M. Rahaman, A. Aldalbahi, M. Almoiqli, S. Alzahly, Chemical and electrochemical synthesis of
ED
[32] F. Khadem, M. Pishvaei, M. Salami-Kalajahi, F. Najafi, Morphology control of conducting polypyrrole nanostructures via operational conditions in the emulsion polymerization, J. Appl. Polym.
PT
Sci. 134 (2017) 44697.
CC E
[33] A. T. Mane, S. D. Sartale, V. B. Patil, Dodecyl benzene sulfonic acid (DBSA) doped polypyrrole (PPy) films: Synthesis, structural, morphological, gas sensing and impedance study, J. Mater. Sci.: Mater. Electron. 26 (2015) 1-10.
A
[34] K. Yang, H. Xu, L. Chng, C. Sun, J. Wang, Z. Liu, In vitro and in vivo near-infrared photothermal therapy of cancer using polypyrrole organic nanoparticles, Adv. Mater. 734 (2013) 5586-5592. [35] Z. Zha, X. Yue, Q. Ren, Z. Dai, Uniform polypyrrole nanoparticles with high photothermal 23
conversion efficiency for photothermal ablation of cancer cells, Adv. Mater. 5 (2013) 777-782. [36] W. Zhang, M. Tan, P. Zhang, L. Zhang, W. Dong, Q. Wang, J. Ma, E. Dong, S. Xu, G. Wang, One pot synthesis of Sb2S3 nanocrystalline films through a PVP-assisted hydrothermal process, Appl. Surf. Sci. 455 (2018) 1063-1069.
IP T
[37] S. M. Lam, M. W. Kee, J. C. Sin, Influence of PVP surfactant on the morphology and properties of ZnO micro/nanoflowers for dye mixtures and textile wastewater degradation, Mater. Chem. Phys.
SC R
212 (2018) 35-43.
[38] S. Bensaadi, O. Arous, H. Kerdjoudj and M. Amara, Evaluating molecular weight of PVP on
U
characteristics of CTA membrane dialysis, J. Environ. Chem. Eng. 4 (2016) 1545-1554.
N
[39] M. J. A. Oliveira, O. S. Estefânia, M. A. B. Lúcia, M. Regina, V. S. Amato, A. B. Lugão, D. F.
A
Parra, Influence of chitosan/clay in drug delivery of glucantime from PVP membranes, Radiat. Phys.
M
Chem. 94 (2014 )194-198.
ED
[40] D. Liu, B. Yu, G. Jiang, W. Yu, Y. Zhang, B. Xu, Fabrication of composite microneedles integrated with insulin-loaded CaCO3 microparticles and PVP for transdermal delivery in diabetic
PT
rats, Mater. Sci. Eng., C. 90 (2018) 180-188.
CC E
[41] H. Y. Woo, W. G. Jung, D. W. Ihm, J. Y. Kim, Synthesis and dispersion of polypyrrole nanoparticles in polyvinylpyrrolidone emulsion, Synth. Met. 160 (2010) 588-591. [42] S. Wang, Y. Wu, R. Guo, Y. Huang, S. Wen, M. Shen, J. Wang, X. Shi, Laponite nanodisks as an
A
efficient platform for doxorubicin delivery to cancer cells. Langmuir, 29 (2013) 5030-5036. [43] G. Yu, A. Liu, H. Jin, Y. Chen, D. Yin, R. Huo, S. Wang, J. Wang, Urchin-shaped Bi2S3/Cu2S/Cu3BiS3 composites with enhanced photothermal and CT imaging performance, J. Phys. Chem. C. 122 (2018) 3794-380. 24
[44] S. Wang, J. Zhao, F. Hu, X. Li, X, An, S. Zhou, Y. Chen, M. Huang, Phase-changeable and bubble-releasing implants for highly efficient HIFU-responsive tumor surgery and chemotherapy, J. Mater. Chem. B. 4 (2016) 7368-7378. [45] S. Wang, X. Li, Y. Chen, X. Cai, H. Yao, W. Gao, Y. Zheng, X. An, J. Shi, H. Chen, A Facile
A
CC E
PT
ED
M
A
N
U
SC R
Multi-Modality Tumor Imaging and Therapy, Adv. Mater. 27 (2015) 2775-2782.
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One-Pot Synthesis of a Two-Dimensional MoS2/Bi2S3 Composite Theranostic Nanosystem for
25
A
N
U
SC R
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Figures and Captions
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Fig. 1. (a) Schematic illustration of PVP guided synthesis of PPy-PVP NPs; (b, c) SEM micrograph
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and diameter distribution histogram of PPy-PVP NPs (sample III).
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Fig. 2. SEM micrographs and diameter distribution histograms of (a, b) sample I; (c, d) sample II.
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Fig. 3. (a-f) Time-dependent DLS profiles of sample I, sample II, sample III dispersed in PBS (a:
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sample I, b: sample II, c: sample III) and DMEM (d: sample I, e: sample II, f: sample III); (g)
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photographic images of Tyndall effect of sample I, sample II, sample III dispersed in saline (1: sample
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I, 3: sample II, 5: sample III) and DMEM (2: sample I, 4: sample II, 6: sample III). Left: after 24 h
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storage; right: after 48 h storage.
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Fig. 4. (a, c) Temperature curves of PPy-PVP NPs aqueous solutions (a: 100 μg/mL; c: 50 μg/mL)
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under laser irradiation of 808 nm laser at the power density of 0.8 W/cm2; (b, d) thermal images of
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PPy-PVP NPs aqueous solution corresponding to (a) and (c), respectively; (e) the η value of PPy-PVP NPs; (f) time constant for heat transfer of the sample III; (g) temperature curve of PPy-PVP
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NPs aqueous solution during 8 irradiation and cooling cycles.
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Fig. 5. (a) Drug loading efficiency (Mt/Mo × 100% (Mt and Mo represents the amount of loaded
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DOX and the total mass of DOX used for drug loading, respectively) of PPy-PVP NPs; (b) drug
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release of DOX loaded sample III. Data are presented as mean ± SD (n = 3).
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Fig. 6. (a) Viability of PPy-PVP NPs treated L929 cells; (b) in vitro combined tumor photothermal therapy of sample III; (c) morphology of PPy-PVP NPs treated L929 cells corresponding to (a); (d)
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morphology of dead/live staining of HT29 cells corresponding to (b). Data in panel (a) and panel (b)
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are presented as mean ± SD (n = 3).
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Fig. 7. (a) Hemolysis percentage (HP) of mRBCs co-incubated with PPy-PVP NPs solutions (100
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μg/mL, n = 3); (b) Body weight evolution of KM mice treated with PBS and sample III (1000
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μg/mL); (c) blood biochemistry parameters of KM mice that were I. V. injected with PBS or aqueous solution of sample III; (d) H&E staining images of heart, liver, spleen, lung, and kidney of KM mice.
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Data in pane (b) and panel (c) are presented as mean ± SD (n = 6).
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Fig. 8. (a) Temperature profiles of mice treated with PPy-PVP NPs and PBS (control) under NIR laser irradiation; (b) in vivo thermal images of mice under continuous irradiation for varied durations
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corresponding to (a); (c) time-dependent tumor growth profile of mice after various treatments as
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noted; (d) representative pictures of HT29 tumor bearing mice that received different treatments at
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day 1 and day 14. Data in panel (c) are presented as mean ± SD (n = 6).
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