Enhanced Photoacoustic and Photothermal Effect of Functionalized

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Enhanced photoacoustic and photothermal effect of functionalized polypyrrole nanoparticles for near-infrared theranostic treatment of tumor Wenchao Li, Xingyue Wang, Jingjing Wang, Yuan Guo, Shi-Yu Lu, Chang Ming Li, Yuejun Kang, Zhi-Gang Wang, Hai-Tao Ran, Yang Cao, and HUI LIU Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01453 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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Enhanced Photoacoustic and Photothermal Effect of Functionalized Polypyrrole Nanoparticles for NearInfrared Theranostic Treatment of Tumor Wenchao Li,a, ‡ Xingyue Wang,b’ ‡ Jingjing Wang,a Yuan Guo,b Shi-Yu Lu,a Chang Ming Li,a Yuejun Kang,a,c Zhi-Gang Wang,b Hai-Tao Ran,b Yang Cao,b,* and Hui Liua,c,* a

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University),

Ministry of Education, Faculty of Materials and Energy, Southwest University, Chongqing 400715, China b

Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging,

Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China; c

Chongqing Engineering Research Center for Micro-Nano Biomedical Materials and Devices,

Chongqing 400715, China.

ACS Paragon Plus Environment

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ABSTRACT

Functionalized nanomaterials with near-infrared (NIR) responsive capacity are quite promising for theranostic treatment of tumors, but formation of NIR responsive nanomaterials with enhanced theranostic ability and excellent biocompatibility is still very challenging. Herein, PEGylated indocyanine green (ICG)-loaded polypyrrole nanoparticles (PPI NPs) were designed and successfully formed through selecting polydopamine as the linkage between each component, demonstrating enhanced NIR responsive theranostic ability against tumor. Combining in vitro cell study with in vivo assay, the formed PPI NPs were proved being fantastic biocompatible while effectively internalizing in HeLa cells and retaining in HeLa tumor demonstrated by in vitro flow cytometry/confocal measurement and in vivo photoacoustic imaging assay. With the guidance of photoacoustic imaging, successful photothermal ablation of tumor was achieved when treating with PPI NPs plus laser, which was much more effective than the group treated with NPs free of ICG. The greatly combined enhanced photoacoustic and photothermal effect is mainly ascribed to the functionalized polypyrrole nanoparticles, which could accumulate in tumor site more effectively with a relative longer retention time taking advantage of the nanomaterial-induced endothelial leakiness phenomenon. All these results demonstrate the designed PPI NPs possess enhanced NIR responsive property are to hold a great promise for tumor NIR theranostic applications.


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1. INTRODUCTION The huge threaten from cancer to human health is rendering conventional treatment methods in predicament, which makes the development of desirable patient-friendly treatment techniques imperious. 1, 2 These kind of developed techniques always contain imaging and therapy functions in order to label the location of tumors clearly followed by effectively eliminating. The great progress of bio-nanotechnologies makes this possible.3, 4 Theranostic nanoplatforms containing both imaging and therapy functionalities are emerging as potential candidates against cancers.5-7 These developed nanoplatforms always contain single or multiple components that are able to response to inner (eg., pH, redox, enzyme) and/or external (eg., near-infrared (NIR) light, magnetic field, heat) stimuli to perform their imaging and therapy functions.8-12 Among all these kinds of developed candidates, NIR light responsive nanoplatforms are more attractive due to the “two-in-one” characteristic, which could act as agents for photoacoustic (PA) imaging and photothermal therapy (PTT) simultaneously.13-18 NIR light can be easily operated to focus on specific area with desirable tissue penetrate depth because its noninvasive property against skin and tissue.19, 20 Upon the irradiation of NIR light, these kinds of nanomaterials can generate PA signals for detection. Combining the high resolution of ultrasound detection and high sensitivity of optical imaging, PA imaging is of great potential in biomedical applications, especially in cancer diagnosis field.21-23 Meanwhile, the adsorbed NIR light can be transformed into heat by these nanomaterials to kill cancer cell by photothermal effect.24-27 Among various developed NIR light responsive nanomaterials (eg., metal-based, carbon-based, organic molecules-based),14, 28-31 polymer-based NIR theranostic nanomaterials are more attractive due to


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their effective NIR light responsive properties, desirable biocompatibility, and facile preparation process.32-35 Several kinds of polymers such as polyaniline, polypyrrole (PPy), and polydopamine (PDA) have been explored to be as NIR-responsive agents for cancer theranostic applications.36 In particular, PPy nanoparticles are currently attracting particular interest.37, 38 PPy NPs can be formed through a facile self-polymerization method with reported satisfactory stability and biocompatibility.39, 40 Their excellent NIR-responsive ability renders them to be promising tumor theranostic candidates. Following the pioneer work by Liu group and Zheng group,40, 41 PPy NPs have been designed as PA imaging agents or PTT agents against cancer, as well as theranostic PA imaging/PTT agents.42 However, few attempts have been made to improve their NIR responsive property. Indocyanine green (ICG) is a kind of cyanine dye owning prominent absorbance in the NIR region, which is biocompatible and approved by the Food and Drug Administration (FDA) in 1959 for clinical applications.43 Based on its excellent NIR responsive property, ICG is popular to be combined with other component for enhancing NIR responsive purpose.44-48 In this present study, we report a kind of theranostic PEGylated PPy-ICG (PPI) NPs for enhanced PA imaging guided PTT. The synthesis strategy was illustrated in Figure 1a. Each component was rational integrated using polydopamine coating as the linkage due to its excellent coating ability, active groups-providing ability, and small molecular adsorption ability. Upon the irradiation of NIR light, the obtained PPI NPs displayed enhanced photoacoustic and photothermal abilities with desirable photostability. The formed NPs could be effectively internalized by cancer cells showing effective killing effect. Strikingly, after intravenous injection, enhanced in vivo PA imaging and PTT were achieved, resulting in the effective


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elimination of tumor. Moreover, the biocompatibility of the formed PPI NPs was proved to be desirable. 2. EXPERIMENTAL SECTION 2.1. Materials Pyrrole (98%) and dopamine hydrochloride (98%) were purchased from J&K Scientific Ltd. Poly(vinyl alcohol) (PVA, MW 9K-10K) and Trizma were obtained from Sigma-Aldrich. Iron(III) chloride hexahydrate (FeCl36H2O, 99%) was obtained from Chongqing Aoshe Co., Ltd (Chongqing, China). Cardio-Green (98%) was purchased from Adamas (Titan, Shanghai, China). 2.2. Synthesis of PEGylated PPy (PP) NPs Typically, PVA (0.820 g) was dissolved in distilled water at 60 oC under vigorous stirring for 30 min. Then, the cooling-down solution was mixed with FeCl3 (1.384 g) to get a uniform yellow solution. After stirring for 1 h, pyrrole (154 μL) was introduced. The mixture was transferred to ice bath and kept for 4 h to perform the polymerization reaction. Finally, PPy NPs were separated and purified by centrifugation. Then, polydopamine coating of the formed PPy NPs was performed for facile polyethylene glycol (PEG) modification. Typically, PPy NPs (20 mg) was dissolved in Tris buffer (10 mM, pH 8.5) and mixed with dopamine aqueous solution (0.08 mL, 50 mg/mL). After 1 h stirring, the formed PDA coated PPy NPs were collected and purified. Then, mPEG-NH2 (MW 2000, 40 mg) was introduced and the reaction was performed for 1 day. The mixture was centrifuged to obtain PP NPs.


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2.3. Synthesis of PEGylated PPy-ICG (PPI) NPs The formed PP NPs were used to load ICG through the coated PDA shell. Briefly, PP NPs solution and freshly prepared ICG aqueous solution were mixed and stirred for 4 h. Then, the mixture was centrifuged to obtain PPI NPs. The feeding ICG/PP feeding weight ratios were set at 0.05/1, 0.10/1, 0.15/1, 0.20/1, 0.25/1, and 0.30/1. Unloaded free ICG was collected for analyzing the loading content and loading efficiency of ICG on the NPs.49 2.4. Characterization Techniques A field emission scanning electron microscopy (FESEM, JSM-7800F, Japan) and a transmission electron microscopy (TEM, JEM-2100, Japan) were employed to observe the morphology and size of the formed NPs. The PEG modification was studied by proton nuclear magnetic resonance (1H NMR) spectra (Bruker AV 300 NMR, Germany). Their hydrodynamic sizes and surface potentials were characterized by dynamic light scattering (DLS, Nano ZS90, Malvern Instruments, UK). Their UV-vis-NIR absorption spectra were recorded via a UV-1800 spectrophotometer (Shimadzu, Japan). The release profile of ICG from PP NPs was also analyzed to investigate the stability of the NPs. 2.5. NIR Performance Evaluation The photoacoustic ability of the formed NPs was firstly evaluated using a small animal photoacoustic imaging appliance (Vevo LAZR, Canada). The aqueous solutions of PP or PPI NPs ranging from 0.1 to 0.8 mg/mL were put into the pre-prepared agarose gel molds and scanned using a laser of 706 nm to acquire the PA signals.


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For photothermal performance evaluation, PP or PPI NP aqueous solutions of series concentrations were irradiated (808 nm, 1.0 W/cm2) for 10 min (LWIRL808, LASERWAVE). At certain time intervals during laser irradiation, the temperature changes were recorded. Through treating PPI NP solution (0.1 mg/mL) with 5 cycles of heating-cooling process, the corresponding temperature curve was recoded to evaluate their photothermal stability. For photothermal conversion efficiencies calculation, the formed NPs (PPy, PP, and PPI) were dispersed in water at a concentration of 100 ppm and irradiated with an 808 nm laser for 15 min to reach a temperature plateau. Then the laser was turn off and the solutions were cooled down. The temperatures of the solutions were recorded every 30 s. Their photothermal conversion efficiencies were calculated using the method developed by Roper,50, 51 demonstrating in detail in Supporting Information. 2.6. Cellular Uptake and Intracellular Localization Assay For cellular uptake analysis, flow cytometry assay was performed using HeLa cells as model. Briefly, 1.0 × 105 cells were seeded into each well and fed with PPI NPs with concentration range of 0.05-0.2 mg/mL. After 1 h or 3 h co-incubation, the cells were collected into special tubes followed by analyzing via flow cytometry (NovoCyte, ACEA). For intracellular localization analysis, HeLa cells (1.5 × 105 per well) were fed with PPI NPs with a concentration of 200 ppm for 12 h. Then, Hoechst 33342 solution was used to stain the cell nuclei. The fluorescence images was scanned and obtained using a confocal laser scanning microscopy (LSM 780, Carl Zeiss, Germany). 2.7. Cell Viability Assay and In Vitro PTT


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For cytocompatibility assay, 1.0 × 104 HeLa cells were seeded into each well and fed with different concentrations of PPI NPs ranging from 10 to 200 ppm. After co-incubation for 24 and 48 h, the viabilities of cells were evaluated using a standard CCK-8 assay 49. For each sample, the mean and standard deviation of measurements for triplicate wells were reported. Then, the in vitro PTT assay was performed against HeLa cells. PP or PPI NPs of different concentrations were fed into each well for 6 h following by laser irradiation for 10 min. The viabilities of cells were also evaluated using CCK-8 assay. 2.8. Animals and Tumor Models Nude mice (approximately 4 weeks old) were purchased from Beijing Huafukang Biological Technology Co. Ltd. All the procedures regarding animal maintenance and experiments are in strict accordance with the policy of the Institutional Animal Care and Use Committee of Chongqing Medical University. The mice bearing HeLa tumor models were constructed through subcutaneously injection of HeLa cell PBS suspension (0.2 mL, 1.0 × 106 cells). All the corresponding in vivo experiments were conducted till the tumor reached to the size around 150 mm3. 2.9. In Vivo Biochemical Assay and Hematology Analysis The in vivo biocompatibility of the formed PPI NPs was investigated. Briefly, nine KunMing mice with average weight of 25 mg were divided into three groups randomly (n = 3, saline group, PPI NP (1 day post-injection) group, PPI NP (7 days post-rejection) group). Each mouse was intravenously injected with 0.2 ml saline (control) and PPI NP solutions (5 mg/mL), respectively. The blood was collected after 1day and 7day post injection for analysis. For biochemical assay, three liver function markers and one renal function marker were tested, including aspartate


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transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and blood urea nitrogen (BUN). For hematology analysis, nine blood parameters were tested, including red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), white blood cells (WBC), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), red cell distribution width (RDW), platelet (PLT), and mean corpuscular hemoglobin concentration (MCHC). For hemolysis analysis, mice red blood cells were collected and purified according to the reference report.52 Then the cells were mixed with the PPI NPs at different concentrations. Water and PBS buffer at pH 7.4 were used as positive and negative controls, respectively. 2.10. In Vivo PA Imaging To demonstrate the PA imaging ability of PPI NPs for tumor model, the nude mice bearing HeLa tumor were injected intravenously with PP or PPI (0.2 mL, 5 mg/mL) NP solutions, respectively. PA images of tumor regions were collected at predetermined time points (0 (pre-injection), 1, 3, 6, 12, 24, and 48 h post-injection) using the above-mentioned PA imaging platform. By selecting regions of interests, PA signals of each time point were quantitatively analyzed. 2.11. In Vivo PTT The tumor-bearing mice with tumor volume approximately 150 mm3 were randomly divided into four groups (n = 5 per group). Then, 0.2 mL of saline (two groups), PP or PPI NP (5 mg/mL) saline solutions was intravenous injected into each group. Except for one saline group, the other three groups were laser irradiated (808 nm, 1.0 W/cm2) for 10 min. The thermal images and temperature changes of tumor regions for each group were monitored and recorded using an infrared thermal imaging system (Fluke T32, Fluke, USA). One day after the therapy, one mice of each group was randomly sacrificed. Main organs and tumors were collected for hematoxylin-


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eosin (H&E) staining. Meanwhile, the tumor sections were stained using terminal deoxynucleotidyl transferased dUTP nick end labeling (TUNEL). The tumor volume as well as body weight were recorded every other day for 21 days.

3. RESULTS and DISCUSSION 3.1. Preparation and Characterization of PPI NPs The design and formation process for PEGylated PPy-ICG NPs was illustrated in Figure 1a, as well as their performance as NIR theranostic agents. PPy NPs were firstly formed through the polymerization of pyrrole monomers in the presence of FeCl3 and PVA, which acted as oxidizing agent and stabilizer, respectively. The formed PPy NPs were special relatively uniform in size with a mean diameter of 65 nm (Figure S1a). The NP surface was then coated by a shell of polydopamine taking advantage of its facile surface-adherent property.53, 54 After coating, their size observed from FESEM kept similar (Figure S1b). The PDA shell was observed and estimated to be around 2 nm in the TEM image (Figure S1c-d). It should be mentioned that the thickness of PDA coating was designed to be quite thin to avoid the compromise of the NIR responsive ability of PPy nanoparticles.53 However their hydrodynamic size increased from 117.9 ± 0.7 nm to 250.2 ± 3.6 nm after coating (Figure S1e-f), which may be caused by the thicker hydration shell provided by PDA shell. In addition, the successful coating was also confirmed by the decrease of their surface potential from +30.0 ± 0.1 mV to -36.7 ± 1.2 mV (Figure S2). Based on the PDA coating, PEG chains were easily linked onto the NP surface via the Michael addition and/or Schiff base reaction between catechol/quinine groups of PDA and amine groups of PEG.49, 55 After linkage, a new signal appeared around 3.6 ppm in the 1H NMR


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spectrum (Figure S3), which was the chemical shift from -CH2- protons of the PEG chains. Meanwhile, the surface potential of the NPs increased to -27.1 ± 0.5 mV, indicating the neutralization of surface charges during PEG linkage. The formed PP NPs were uniform in shape and no obvious size or morphology change was observed (Figure 1b). Their hydrodynamic size was measured to be 280.6 ± 3.6 nm (Figure S4a) and this slightly increase may be caused by the introduced PEG shell. Then, the formed PP NPs were used to load ICG through the π−π/electrostatic interactions with the PDA coating.56 Data in Figure 1c demonstrated that the loading content of ICG increased continuously with the increase of ICG/PP NP feeding ratio and reached a plateau of around 9.3% at weight ratio of 0.25/1. Meanwhile, the measured loading efficiency decreased at the ICG/PP weight ratio of 0.30/1. Therefore, the weight ratio of 0.25/1 was chosen for further study. The finally obtained PPI NPs also owned uniform morphology with a mean diameter around 65 nm (Figure 1d). Their hydrodynamic size was measure to be 331.3 ± 3.7 nm with a surface charge of -37.0 ± 0.8 mV (Figure S4b and S2). After storage at 4 oC

for three months, the formed PPI NPs showed similar shape and dispersity to that of the as-

prepared NPs (Figure S5), indicating its desirable stability. The loaded ICG could enhance the absorbance around 808 nm of PP NPs notably (Figure 1e), which may be of potential to improve their NIR responsive property. The release profile of ICG from NP surface demonstrated the desirable stability of the final NPs (Figure S6), which could be attributed to the strong adsorption capacity of PDA shell.56


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Figure 1. (a) Schematic illustration of the formation and NIR theranostic applications of PPI NPs. FESEM images and size distributions (inset) of the formed PP NPs (b) and PPI NPs (d). Scale bar: 100 nm. (c) Loading efficiency and loading content of ICG on PP NPs. (e) UV-visNIR absorption spectra of the obtained NPs. 3.2. NIR Performance Evaluation


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Having successfully obtained the PPI NPs, we moved on to evaluate their NIR responsive property. Their PA imaging ability was firstly investigated using a 706 nm laser pulse. Displayed in Figure 2a, PP NPs yielded obvious PA signals, indicating the imaging ability of PPy NPs. Furthermore, the loaded ICG could enhance the PA signals of the NPs prominently. Quantitative data analysis shown in Figure 2b demonstrated that the signals for both PP and PPI NPs were enhanced with increasing concentration (Figure 2b). At certain concentration point, PPI NPs generated stronger signal than that of PP NPs. Notably, the PA signal of PPI NPs was two times than that of PP NPs at the concentration of 0.8 mg/mL.

Figure 2. PA images (a) and the corresponding signals (b) of PP and PPI NPs. (c) Temperature curves of the obtained PPI NPs with distinct concentrations and 100 ppm PPI NP solution under the designed heating-cooling process (808 nm, 1.0 W/cm2). The temperature change curve (e) of the formed PPI NP aqueous solution during the heating (808 nm, 1.0 W/cm2, 15 min) and cooling (laser shut off, 20 min) process. The linear time data versus –Ln(θ) obtained from the cooling period in (e)


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Next their photothermal performance was assessed under laser irradiation. At same concentrations, PPI NPs displayed a higher ability to raise temperature than PP NPs (Figure S7), also indicating the enhanced NIR responsive ability after ICG loading. To make this clear, the photothermal conversion efficiency (PCE) of each formed NPs was calculated. Firstly, the PCE of PPy NPs was calculated to be 39.9% (Figure S8a-b), which decreased to around 34.6% after PDA coating and PEG linkage (Figure S8c-d). The lower PCE of PP NPs could be attributed to the decreased percentage of photothermal agent component. After ICG loading, the calculated PCE increased to 38.5% (Figure 2c-d), indicating the synergistic photothermal effect between PPy NPs and ICG. With the increase of PPI NP concentration, the temperature evaluation was enhanced gradually (Figure 2e). During the designed heating-cooling process, the photothermal reproducibility of PPI NPs was assessed (Figure 2f). The constant temperature curve indicated that there was no photo-bleaching. Furthermore, the laser irradiated PPI NPs had similar morphology with the as-prepared NPs, as well as similar hydrodynamic size (338.7 ± 7.9 nm) and optical absorbance (Figure S9a-c). The color of their aqueous solution kept similar after laser irradiation with good dispersity (Figure S9d). In contrast, free ICG displayed decreased temperature evaluation during the five cycles of laser irradiation. Meanwhile, its solution color changed obviously after laser irradiation (Figure S10). In short, the obtained PPI NPs had desirable photothermal stability, which may facilitate their further applications. 3.3. In Vitro Cell Study Choosing HeLa cells as model, the interactions between cells and the formed PPI NPs were investigated. Flow cytometry analysis was proceeded to evaluate the cellular uptake behavior of PPI NPs. The number of fluorescence positive cell increased with NP concentration after 1 h coincubation (Figure 3a). When co-incubated with PPI NPs at 0.20 mg/mL, the percentage reached


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to 76.20%. Furthermore, the cellular uptake was enhanced by increasing the co-incubation time to 3 h (Figure 3b), reaching a percentage of 93.24% for the cells co-incubated with 0.20 mg/mL PPI NPs. Then, the intercellular localization of PPI NPs was observed using confocal laser scanning microscopy (Figure 3c). The obtained images reflected that most internalized PPI NPs were located in cytoplasma, surrounding the cell nuclei. CCK-8 assay was employed to assess whether the internalized NPs cause toxicity to cells. The cells showed viabilities similar to those of blank cell treated with PBS in the studied concentration range after 24 h or 48 h co-incubation (Figure 3d). This indicated the desirable cytocompatibility of the obtained PPI NPs. Subsequently, the PTT effect of PPI NPs in vitro was investigated using HeLa cells as model (Figure 3e). Under laser irradiation, both PP and PPI NPs could lead the decrease of cell viability, confirming their photothermal ability. The faster viability decrease of cells incubated with PPI NPs was observed, which could be attributed to the enhanced photothermal ability after ICG loading. It should be mentioned that 200 ppm PPI NPs plus laser resulted in the cell viability around 13.7%, showing an effectively cancer cell killing effect compared to PP NPs plus laser group (with a cell viability of 49.3% at 200 ppm NP concentration).


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Figure 3. Flow cytometry assay data after incubated with PPI NPs of different concentrations (0.05, 0.10, 0.15, 0.20 mg/mL) for 1 (a) and 3 h (b). (c) Confocal images of HeLa cells after 12 h co-incubation with PPI NPs. The fluorescence of Hoechst 33342 and PPI NPs were pseudolabelled with blue and red, respectively. (d) Cell viability data measured by CCK-8 assay. (e) In


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vitro PTT assay measured after co-incubated with the obtained NPs for 6 h followed by 10 min laser irradiation (808 nm, 1.0 W/cm2).

Figure 4. In vivo biochemical assay (a) and hematology analysis (b) of mice with distinct treatments (saline control, PPI NPs for 1 and 7 days, mean ± SD, n = 3).


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3.4. In Vivo Biochemical Assay and Hematology Analysis Prior to in vivo imaging and therapy assay, the biocompatibility of PPI NPs was evaluated using biochemical assay and hematology analysis. The level of three liver function markers and one renal function marker were tested, including ALP, ALT, AST, and BUN, which were found to be similar to that of control group (Figure 4a). Then, the hemocompatibility of the formed PPI NPs was evaluated. The obtained data revealed that PPI NP-treated mice for 1 d and 7 d show no prominent differences in these nine detected parameters when compared with saline control group (Figure 4b). Furthermore, no hemolysis phenomenon was observed in the studied condition (Figure S11). All assays demonstrated the excellent biological safety of the obtained PPI NPs. 3.5. In Vivo PA Imaging The desirable NIR responsive property and biocompatibility of the designed PPI NPs motivated us to investigate their PA imaging ability for tumor model in vivo. HeLa tumor bearing mice were injected with PP or PPI NP solutions intravenously, respectively. Then, the PA images of the tumor regions were collected at predetermined time points (0 (before injection), 1, 3, 6, 12, 24 and 48 h (Figure 5a). Bright PA signals could be observed for both groups at 3 h, 6 h, and 12 h post-injection. Furthermore, the PA signals at tumor regions were quantitative analyzed (Figure 5b). After injection PP NP solutions, PA signals increased till 3 h post injection, which was measured to decrease by 26.7% at 6 h post-injection. For PPI treated group, the PA signals kept increasing within 6 h post-injection. This implied that the formed PPI NPs could accumulate in tumor site effectively with relative longer retention time. This may be induced by the distinct surface charges of PP and PPI NPs when arrived tumor site owning a relative acid


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microenvironment (approximately pH 6.5).57 As shown in Figure S12, PPI NPs displayed a much lower surface charge (-24.1 ± 1.1 mV) than that of PP NPs (-8.7 ± 1.5 mV) when dispersed in pH 6.5 buffer. Negatively surface charge could cause nanomaterial-induced endothelial leakiness (NanoEL), which results in higher tumor accumulation of NPs.58, 59 The gradually decreased signals for both groups indicated that the metabolism of NPs by tumor. It can be seen that during the time range of 6-24 h post-injection, there existed significant difference between the PA signals of the two groups, demonstrating the superiority in PA imaging of the finally formed PPI NPs.

Figure 5. PA (top row) and merged PA/ultrasound (bottom row) images (a) and the corresponding quantitative analysis the PA signals (b) of HeLa tumor regions before and after


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intravenous injection of 0.2 mL of PP or PPI NPs (5 mg/mL) in HeLa tumor-bearing mice, respectively (* for p < 0.05, ** for p < 0.01, and *** for p < 0.001, respectively). 3.6. In Vivo PTT Based on the enhanced photothermal property in vitro and tumor region accumulation ability, the in vivo PTT assay of the formed PPI NPs was performed on HeLa tumor-bearing mice. At the highest accumulation time post-injection guided by PA imaging, the tumor region was laserirradiated (808 nm, 1.0 W/cm2) for 10 min. The infrared images of tumor regions were collected at predetermined time intervals during the laser irradiation process (Figure 6a). Compared with laser only group and PP plus laser group, the tumor temperature of PPI plus laser group evaluated prominently. Quantitative temperature monitoring revealed that the temperature of tumor regions increased approximately 5 oC and 10 oC for laser only group and PP plus laser group after 10 min laser irradiation, respectively, which was around 20 oC for PPI plus laser group (Figure 6b). The tumor temperature dramatically increased to 45.7 oC and 52.3 oC for PPI plus laser group after 4 min and 10 min laser irradiation respectively, which could cause cancer cell apoptosis irreversibly. In comparison, the corresponding temperature was 39.9 oC and 40.9 oC

for PP plus laser group. This remarkable increase of temperature for tumor region treated by

PPI plus laser may cause effective PTT effect in vivo. To demonstrate this, one day after treatment, relevant tumor tissues were collected for histological analysis. The absence of cell nucleus observed by H&E staining and abundant green dots in the TUNEL assay (Figure 6c) both revealed obvious cell damage and apoptotic caused by PPI plus laser treatment.


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Figure 6. Infrared thermal images (a) and the corresponding temperature changes (b) of HeLa tumors recorded at each time points during laser irradiation. (c) Histological H&E staining and TUNEL staining (The fluorescence of nucleus and apoptosis cells were pseudo-labelled with blue and green, respectively) of the tumor regions after different treatment. Scale bar: 100 m. To assess the therapy effect and recurrence of tumor, the tumor sizes were monitored every other day until 21 days after treatments (Figure 7a). The tumor sizes for blank group and laser only group increased in succession during 21 days after therapy, with no tend to regression. For PP plus laser group, only a slight degree of decrease was detected, which may be caused by their PTT effect. Remarkably, the tumor size of PPI plus laser group displayed dramatic regression within 21 days, indicating the strong PTT ability of PPI NPs against tumor. Quantitative analysis data further revealed that the tumor treated with PPI plus laser regressed prominently, while the tumors with the other three treatments kept growing within 21 days (Figure 7b). At 21 days after


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treatments, the tumors were collected to be photographed and weighted (Figure 7c-d). There was no observed recurrence of tumor for the mice treated with PPI plus laser. The existence of significant different for tumor size between PPI plus laser group with the other three groups demonstrated the effective PTT effect of PPI NPs against tumor in vivo.

Figure 7. (a) Representative photographs of tumor size changes of nude mice with different treatments during 21 days. (b) Tumor growth profile of mice after different treatments as noted (mean ± SD, n = 5). Digital photo (c) and weight (d, mean ± SD, n = 5) of tumor tissues collected from each treatment group 21 days after laser irradiation (* for p < 0.05, ** for p < 0.01, and *** for p < 0.001, respectively).


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Meanwhile, the biosafety of PPI NPs was further confirmed. The H&E staining sections of major organs displayed no apparent necrosis or physiological morphology changes when compared to blank group one day after treatment (Figure S13). Furthermore, the mice body weight of PPI plus laser group kept smooth and steady within 21 days after treatment, which was similar to that of control groups (Figure S14), indicating a negligible effect of this treatment on the body weight. Combined all these data, the biocompatibility of the designed PPI NPs was favorable. 4. CONCLUSIONS In summary, functionalized polypyrrole NPs with enhanced NIR responsive property were developed for cancer theranostic applications. The induced PDA shell could integrate PPy NP, PEG chain, and ICG as an organic nanoplatform. The absorbed ICG improved the PA imaging and PTT ability of PP NPs dramatically, reaching an enhanced NIR responsive property. Meanwhile, in vitro cellular assays and in vivo blood/histological assay demonstrated the elaborate biocompability of PPI NPs. After intravenous injection, PPI NPs could accumulate in tumor region effectively to act as PA imaging agent. Under PA imaging guidance, effective PTT of tumors could be realized. This designed multifunctional PPy-based NPs are promising for cancer NIR theranostic applications.

ASSOCIATED CONTENT Supporting Information


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Additional data of FESEM, TEM, DLS, zeta potential, NMR characterization of the formed NPs, photothermal conversion efficiency calculation and photothermal stability assessment, hemolytic assay, H&E staining images of tissues, and body weight of mice.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] E-mail: [email protected] ORCID Hui Liu: 0000-0002-7648-0915 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was funded by the National Natural Science Foundation of China (51703184, 31671037, 81630047), the Chongqing Research Program of Basic Research and Frontier Technology (cstc2017jcyjAX0066), the Fundamental Research Funds for the Central Universities from Southwest University (XDJK2018B007), a start-up grant from Southwest


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For Table of Contents Use Only

Enhanced Photoacoustic and Photothermal Effect of Functionalized Polypyrrole Nanoparticles for NearInfrared Theranostic Treatment of Tumor Wenchao Li,a, ‡ Xingyue Wang,b’ ‡ Jingjing Wang,a Yuan Guo,b Shi-Yu Lu,a Chang Ming Li,a Yuejun Kang,a,c Zhi-Gang Wang,b Hai-Tao Ran,b Yang Cao,b,* and Hui Liua,c,*


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Enhanced Photoacoustic and Photothermal Effect of Functionalized

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