Radiotherapy-Sensitized Tumor Photothermal Ablation Using Î³

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Radiotherapy-Sensitized Tumor Photothermal Ablation Using #-Polyglutamic Acid Nanogels Loaded with Polypyrrole Yiwei Zhou, Yong Hu, Wenjie Sun, Shiyi Lu, Chao Cai, Chen Peng, Jing Yu, Rachela Popovtzer, Mingwu Shen, and Xiangyang Shi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00184 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Biomacromolecules

Radiotherapy-Sensitized Tumor Photothermal Ablation Using γ-Polyglutamic Acid Nanogels Loaded with Polypyrrole

Yiwei Zhoua,b, Yong Hub, Wenjie Sunb, Shiyi Lub, Chao Caib, Chen Penga,d*, Jing Yud, Rachela Popovtzerc*, Mingwu Shenb, Xiangyang Shia,b*

a

Department of Radiology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine,

Shanghai 200072, P. R. China b

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China c

Faculty of Engineering and the Institute of Nanotechnology & Advanced Materials, Bar-Ilan

University, Ramat Gan, 5290002, Israel d

Ninghai First Hospital, Ningbo 315600, P. R. China

Keywords: γ-polyglutamic

acid; polypyrrole;

nanogels; tumors; photoacoustic

imaging;

photothermal therapy; radiotherapy

*

Authors

to

be

corresponded.

E-mail:

[email protected]

[email protected] (R. Ropovtzer) and [email protected] (X. Shi)

1

ACS Paragon Plus Environment

(C.

Peng),

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Abstract Development of versatile nanoscale platforms for cancer diagnosis and therapy is of great importance for applications in translational medicine. In this work, we present the use of γ-polyglutamic acid (γ-PGA) nanogels (NGs) to load polypyrrole (PPy) for thermal/photoacoustic (PA) imaging and radiotherapy (RT)-sensitized tumor photothermal therapy (PTT). First, a double emulsion approach was used to prepare the cystamine dihydrochloride (Cys)-crosslinked γ-PGA NGs. Next, the crosslinked NGs served as a reactor to be filled with pyrrole monomers that were subjected to in-situ oxidation polymerization in the existence of Fe(III) ions. The formed uniform PPy-loaded NGs having an average diameter of 38.9 ± 8.6 nm exhibited good water-dispersibility and colloid stability. The prominent near-infrared (NIR) absorbance feature due to the loaded PPy endowed the NGs with contrast enhancement in PA imaging. The hybrid NGs possessed excellent photothermal conversion efficiency (64.7%) and stability against laser irradiation, and could be adopted for PA imaging and PTT of cancerous cells and tumor xenografts. Importantly, we also explored the cooperative PTT and X-ray radiation-mediated radiotherapy (RT) for enhanced tumor therapy. We show that PTT of tumors can be more significantly sensitized by RT using the sequence of laser irradiation followed by X-ray radiation as compared to using the reverse sequence. Our study suggests a promising theranostic platform of hybrid NGs that may be potentially utilized for PA imaging and combination therapy of different types of tumors.

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1. Introduction Rapid development of nanofabrication enables facile design of multifunctional theranostic nanoplatforms that incorporate both molecular imaging and therapeutic elements inside one single nanoscale device.1-3 As one typical molecular imaging technology with powerful and non-invasive feature, photoacoustic (PA) imaging has recently attracted great attention due to the merits of deep tissue penetration capability, excellent spatial resolution, and fast, quantitative, and volumetric measurements.4, 5 For cancer therapy, one important therapeutic approach is photothermal therapy (PTT) that has been emerging due to its highly localized therapeutic efficacy and insignificant systemic toxicity.6-10 In general, PTT of cancer can be performed with assistance of nanomaterials that transform near-infrared (NIR) laser light into thermal energy.11 Inorganic nanomaterials including but not limited to carbon-based graphene and carbon nanotubes12-14 and gold nanoparticles (NPs) of various shapes (e.g., nanorods, nanocages, and nanoshells)15-17 have been used as PTT nanoagents owing to their superior photothermal conversion efficiency. Recently, different conductive polymers such as polypyrrole (PPy),18-22 polyaniline (PANI),23, 24 and polythiophene25 possessing good NIR absorption feature, biocompatibility, optical stability, and high thermal conversion efficiency26-28 have also been used as PTT agents for cancer therapy. Additionally, based on their prominent features, conducting polymers have also been exploited as coating materials for NPs. For instance, PPy was recently used for coating iron oxide (Fe3O4) and gold (Au) NPs, leading to improved molar extinction coefficient or photothermal conversion efficiency.29, 30 Nevertheless, in most cases, the PTT agents have to be functionalized with polymers or loaded within other nanocarriers to improve their stability, and to have targeting specificity and other functionalities.7, 31-34 For instance, Au nanostars can be modified with polydopamine to have enhanced photothermal converison properties for tumor PTT applications,35 or can be functionalized with dendrimers for specific CT imaging-guided synergistic PTT and gene therapy of tumor xenografts.11 PANI NPs can be hybridized with hyaluronic acid (HA) 3



by one-step aniline polymerization in the presence of HA that can be used as both a stabilizer and a targeting ligand for effective synergistic therapy applications.36 Interestingly, owing to the feature of NIR absorption, most phototherapeutic agents can be simultaneously employed for high performance PA imaging.32,

37

This feature of PTT agents in combination with appropriate polymer

functionalization or carrier loading makes it feasible to develop various unique nanoplatforms for theranostic applications. To improve the therapeutic outcomes, other therapeutic strategies have also been combined with PTT by judicious design of the nanoplatforms.7, 11, 34 Radiotherapy (RT) is a common therapeutic strategy with an advantage of high tissue penetration. In principle, RT employs X-ray or γ-ray to destroy cancerous cells through creation of oxygen radicals to attack important biomolecules (e.g., DNA).38 However, one of the main drawbacks of RT is that a significant anti-cancer efficacy cannot be achieved for cancer cells at S-phase, which are hypoxic cells that are the least radiation-responsive cells. Fortunately, all these radiation resistant cells are rather sensitive to hyperthermia.39,

40

Moreover, local hyperthermia treatment induced by PTT with NIR-absorbing

agents enhances the blood flow in the tumor tissue and increases the oxygen concentration in the microenvironment of tumors, and thus overcomes hypoxia-associated RT resistance.41-43 Therefore, the combination of RT and PTT is expected to offer high efficacy in cancer treatment, while decreasing the therapeutic dosage (radiation dose and photothermal agent dose), and hence reducing side effects. In our previous work, we constructed γ-polyglutamic acid (γ-PGA) nanogels (NGs) loaded with PANI for PTT of tumors under guidance of PA imaging.37 However, the prior study was restricted to the utilization of PANI as a photothermal agent, and only single-mode PTT of tumors was investigated. It is essential to develop hybrid NG-based platforms with other photothermal agents for combination PTT/RT of tumors under the guidance of imaging. In the present study, polymer NGs were adopted as a nanoreactor for PPy loading by in-situ 4


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oxidative polymerization for RT-sensitized PTT of tumors under the guidance of PA imaging. The cystamine dihydrochloride (Cys)-crosslinked γ-PGA (for short, CCPA) NGs were initially prepared using a double emulsion approach according to our previous work.37 Thereafter, CCPA NGs served as a nanoreactor to incorporate pyrrole monomers that were subjected to oxidation polymerization in the existence of Fe(III) ions (Scheme 1). The thus formed CCPA@PPy NGs were systematically characterized by different techniques. Their cytocompatibility, internalization by cancer cells, and potential use for PA imaging and RT-sensitized PTT of cancerous cells and tumor xenografts were thoroughly assessed. A thorough literature investigation leads us to claim that our work is the very first example dealing with the preparation of CCPA@PPy NGs for RT-sensitized PTT of tumors guided by PA imaging.

Scheme 1. Construction of CCPA@PPy NGs for RT-sensitized PTT of cancerous cells and tumor xenografts as guided by PA imaging.

2. Experimental Section 2.1. Synthesis of CCPA@PPy NGs In the first step, we synthesized CCPA NGs according to earlier work reported in our group.37, 44 5



Then, 15 mg of the CCPA NGs were dispersed in water (15 mL), and added with 70 µL of pyrrole (0.967 g/cm3) under stirring for 30 min in an ice bath. Afterwards, FeCl3·6H2O (350 mg, in 2 mL water) was dropwise placed into the above mixture for in-situ oxidative polymerization of pyrrole monomers. Three hours later, the raw product of CCPA@PPy NGs was subjected to dialysis against water (6 times, 4 L) using a regenerated cellulose dialysis membrane (molecular weight cut-off (MWCO) = 100 000) for 3 days. The purified CCPA@PPy NGs were concentrated through centrifugation (10 000 rpm, 10 min), dispersed in water or normal saline (NS), and kept at 4 oC. 2.2. In Vitro RT-Sensitized PTT of Cancer Cells Briefly, 4T1 murine breast cancer cells (for short, 4T1 cells) were plated into a 96-well plate with a density of 1 × 104 cells in each well and the cells were incubated at 37 °C under 5% CO2. After overnight culture, the medium in each well was substituted by CCPA@PPy NG-containing fresh medium. After additional 6 h, the adherent cells were washed with fresh medium for three times and allocated into five groups: Control groups (only X-ray radiation, laser irradiation, or the combination of X-ray radiation and laser irradiation in two opposite sequences in the absence of NGs), RT group (NGs + X-ray radiation), PTT group (NGs + laser irradiation), RT/PTT group (NGs + X-ray radiation + laser irradiation), and PPT/RT group (NGs + laser irradiation + X-ray radiation). The difference in treatment between RT/PTT group and PPT/RT group was the sequence of radiation and laser: in the RT/PTT group X-ray radiation was first and laser irradiation followed in sequence, while the treatment sequence in the PTT/RT group was reverse. Without specific clarification, an 808 nm laser with an output of 1.0 W/cm2 for 5 min was used for all cases of laser ablation. X-ray radiation treatment was carried out under 4 Gy of X-ray. After another 2 h incubation, cell counting kit-8 (CCK-8) assay was used to assess the cell viability according to procedures reported in the literature.45 2.3. In Vivo PA and Thermal Imaging of a Xenografted Tumor Model We carried out all animal experiments under the instructions of the animal care committee of 6


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Shanghai Tenth People’s Hospital and all animal experiments were approved by the ethical committee of the same hospital. Six-week-old female BALB/c nude mice (15-20 g) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China), and were utilized to build up xenografted tumors. Briefly, 2 × 106 4T1 cells in 0.2 mL of NS were inoculated into the right hind back of each mouse. Once the tumor volume reached approximately 90 mm3, 0.1 mL NS containing CCPA@PPy NGs (0.5 mg/mL) was intratumorally injected. As for PA imaging, the tumor regions were scanned before and at 30 min posttreatment using the CCPA@PPy NGs in a Vevo® LAZR photoacoustic imaging system equipped with an 808 nm laser (VisualSonics Inc., Toronto, Canada). For thermal imaging, the tumor-bearing mice were firstly anesthetized, and then intratumorally injected with 0.1 mL NS (control) or CCPA@PPy NGs (0.5 mg/mL, in 0.1 mL NS). Thereafter, the tumor site was irradiated by an 808 nm laser (1.0 W/cm2, 5 min). We collected the whole-body infrared thermal images and temperature change of tumor region by a FLIR A300 photothermal medical device (IRS Systems Inc., Shanghai, China) coupled with an infrared camera. 2.4. In Vivo RT-Sensitized PTT of 4T1 Tumors Mice bearing 4T1 tumors were assigned to 6 groups and subjected to different treatments (n = 4 for each group): Group 1 (injection of NS without any treatments, NS); Group 2 (injection of CCPA@PPy NGs alone, NGs); Group 3 (injection of CCPA@PPy NGs, followed by X-ray radiation, NGs + RT); Group 4 (injection of CCPA@PPy NGs, followed by 808 nm laser irradiation, NGs + PTT); Group 5 (injection of CCPA@PPy NGs, followed by X-ray radiation and laser irradiation in sequence, NGs + RT + PTT); and Group 6 (injection of CCPA@PPy NGs, followed by laser irradiation and X-ray radiation in sequence, NGs + PTT + RT). The concentration of CCPA@PPy NGs used in Groups 2-6 was kept similar (0.5 mg/mL, in 0.1 mL NS) and all materials for each group were intratumorally injected. Laser irradiation was performed by using an 808 nm laser with an output of 1.0 W/cm2 for 5 min, and 4 Gy of X-ray radiation was carried out. We measured the body weight, the tumor size, the relative tumor volume, and the survival rate of the mice in each 7



group according to the literature.32 See more experimental details in Supporting Information.

3. Results and Discussion 3.1. Characterization of the synthesized CCPA@PPy NGs Firstly, negatively charged CCPA NGs were prepared following our previous protocols,37 and were then adopted as a nanoreactor to incorporate positively charged pyrrole monomers via electrostatic interaction. After oxidative polymerization of pyrrole monomers inside NGs by Fe(III) ions, the PPy-loaded CCPA NGs were obtained (Scheme 1).

Figure 1. (a) UV-vis spectra of CCPA@PPy NGs before and after purification. (b) TEM image and (c) size distribution histogram of CCPA@PPy NGs. (d) PA images and linear fitting of PA values of the CCPA@PPy NGs at NG concentrations of 0, 0.125, 0.25, 0.5, and 1 mg/mL, respectively.

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After in-situ oxidative polymerization, the dark green color of the CCPA NG solution suggests the successful preparation of CCPA@PPy NGs. In order to verify the presence of PPy, we collected the UV-vis spectra of the PPy-loaded CCPA NGs before and after purification (Figure 1a). The results showed that CCPA@PPy NGs had very chaotic absorption peaks in the NIR region before purification, whereas purified CCPA@PPy NGs displayed prominent NIR absorption around 850 nm. Since CCPA NGs have no absorption peak in the NIR region as demonstrated in our prior work,37 we conclude that PPy was loaded successfully within the CCPA NGs. Thermal gravimetric analysis (TGA) was adopted to determine the percentage of PPy loaded in the CCPA NGs (Figure S1, Supporting Information). Clearly, CCPA NGs have a weight loss of 87.1% at 900 oC. After PPy loading, the weight loss of CCPA@PPy NGs was 74.5% at the same temperature. Hence, PPy loaded within NGs was calculated to be 12.6% by subtracting the weight loss of CCPA@PPy NGs from that of CCPA NGs. TEM (Figures 1b, c) and SEM (Figure S2, Supporting Information) were carried out to investigate the shape and size of the CCPA@PPy NGs. Apparently, the NGs displayed a spherical shape and the average sizes of the NGs were 38.9 nm 52.6 nm, respectively as measured by TEM and SEM. As revealed by dynamic light scattering (DLS) and zeta potential measurements, the CCPA@PPy NGs possessed a hydrodynamic size of 135 nm ± 6 nm with a quite narrow size distribution (Figure S3, Supporting Information) and a surface potential of 38.1 ± 0.6 mV (Table S1, Supporting Information). Notably, the measured TEM or SEM size of the CCPA@PPy NGs was much smaller than the hydrodynamic size of the NGs, presumably owing to the fact that the dried and hence shrunken NGs were measured by TEM or SEM, whereas DLS measures NGs at a swelled state in aqueous solution. In addition, the slightly larger size measured by SEM than that measured by TEM should be attributable to the SEM sample preparation process involving the sputter coating of 10 nm-thick gold film. Further, to monitor the colloidal stability of the CCPA@PPy NGs, we employed DLS to record their size change over 10 days (Figure S4, Supporting Information). 9



Obviously, the NGs in water, saline, PBS, and DMEM showed negligible changes in the hydrodynamic size, confirming their outstanding colloidal stability. The NGs with a relatively larger hydrodynamic size in DMEM could be caused by the slight protein adsorption onto their surface. 3.2. Photoacoustic (PA) Imaging Property and Photothermal Conversion Performance We next investigated the PA imaging property of the CCPA@PPy NGs (Figure 1d). Obviously, the PA signal intensity gradually increased with the increase of NG concentration under irradiation of an 808 nm laser (Figure 1d, inset). Furthermore, a linear relationship was found by fitting the PA values versus NG concentration. These data indicate that the CCPA@PPy NGs can be potentially utilized for PA imaging. The displayed NIR absorption characteristics of the CCPA@PPy NGs also endowed them with a property to convert light to heat. Figure 2a shows the temperature change of the NG suspension versus NG concentration after exposure to laser. The temperature of NG suspension increased by 21.8 °C and 31.7 °C at the NG concentration of 0.5 mg/mL and 2 mg/mL, respectively. As opposed, pure water just had a temperature increase of 4.4 oC after laser irradiation under the same conditions. At a given NG concentration, the temperature increase of the suspension was also dependent on the laser power density with a higher temperature increase trend at a higher laser power density (Figure 2b). The NG suspension had a temperature increase by 50.7 °C at 2 W/cm2, which was the highest laser output power density used. Next, we evaluated the photothermal stability of the CCPA@PPy NGs by five cycles of laser irradiation (laser on) and cooling down (laser off) of the NG suspension (Figure S5, Supporting Information). The results demonstrate that CCPA@PPy NGs exhibit robust photothermal stability. Moreover, we monitored the processes of laser irradiation for 65 s and cooling down of NG suspension (0.5 mg/mL) for 49 s to the initial temperature of 27.7 oC. By plotting of the cooling time vs -lnθ, the time constant τs for heat transfer (the slope of the plot) was measured to be 84 s based on the linear regression analysis. According to the previously reported formulas,32 the photothermal 10


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conversion efficiency (η) of the hybrid NGs was analyzed to be 64.7%, which is quite larger than those of other reported PTT agents in the literature.46-48

Figure 2. (a) Time-dependent temperature variation of the CCPA@PPy NG suspension at varying concentrations (0, 0.125, 0.25, 0.5, 1 and 2 mg/mL, respectively), under irradiation with an 808 nm laser at an output of 1.0 W/cm2. (b) Time-dependent temperature change of the CCPA@PPy NG suspension (0.5 mg/mL), under the 808 nm laser irradiation with different power densities (0.5, 1.0, 1.5 and 2.0 W/cm2, respectively). (c) Plot of the temperature versus time for the CCPA@PPy NGs (0.5 mg/mL) during laser irradiation (808 nm, 1.0 W/cm2) and cooling (laser off). (d) Plot of the cooling time versus -lnθ.

3.3. Cytotoxicity and Cellular Uptake Assays 11



As shown in Figure 3a, CCK-8 assay of cell viability was employed to evaluate the cytocompatibility of hybrid NGs. As compared with the NS-treated cells (control group), no significant change in the viability of 4T1 cells was observed after the cells were incubated with CCPA@PPy NGs in the studied range concentration (0.0625-1 mg/mL). At a concentration as high as 1 mg/mL, the cell viability was maintained at a level higher than 80%. However, CCPA@PPy NGs showed slight toxicity to 4T1 cells when the NG concentration reached 2 mg/mL. Due to the good cytocompatibility of NGs in the range of concentrations studied, the subsequent experiments were conducted in this NG concentration range.

Figure 3. (a) CCK-8 viability assay of 4T1 cells treated with the CCPA@PPy NGs at different concentrations for 24 h. (b) Absorbance at 850 nm for 4T1 cells treated with the CCPA@PPy NGs as a function of incubation time.

Our previous report has shown that contrast agent-loaded NGs could be taken up by cancer cells more significantly than the contrast agents before loading within the NGs attributable to the NG property of softness and fluidity.49, 50 To investigate the uptake of the CCPA@PPy NGs by 4T1 cells, the adherent cells were incubated with NGs for different time periods, followed by cell disruption. The absorbance was then gauged at 850 nm (Figure 3b). The absorbance of cell samples increased significantly with the increase of NG incubation time.

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Control

RT

PTT

RT/PTT

PTT/RT

100

Cell viability (%)

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80 60 40 20 0 0

0.0625 0.125 0.25

0.5

1

Concentration (mg/mL) Figure 4. CCK-8 viability assay of 4T1 cells treated with RT, PTT, RT/PTT, or PTT/RT at different concentrations of CCPA@PPy NGs.

3.4. In Vitro RT-Sensitized PTT of Cancer Cells Next, we used the CCPA@PPy NGs to treat cancer cells in vitro via RT-sensitized PTT (Figure 4). We found that cells treated with NGs alone or RT alone maintained high viability at different NG concentrations, likely due to the nourishing factor of the γ-PGA NGs for cell growth and the low dose of RT at 4 Gy that cannot immediately induce cell apoptosis. In sharp contrast, cells treated with PTT alone or the combination of RT and PTT show a marked concentration-dependent viability reduction at an order of control > RT > PTT > RT/PTT > PTT/RT under the same NG concentrations. The cell viabilities of these five groups were measured to be 95.35% (NGs alone), 90.28% (NGs + RT), 37.03% (NGs + PTT), 24.80% (NGs + RT + PTT), and 12.26% (NGs + PTT + RT) at an NG concentration of 0.5 mg/mL. Clearly, combined PTT and RT enhances cancer cell treatment when compared to a single mode of treatment. Moreover, the sequence of laser irradiation and X-ray radiation is important for effective treatment of cancer cells, as PTT/RT has better treatment outcome than RT/PTT at the same NG concentration. 13



3.5. In Vivo PA and Thermal Imaging of Tumors Next, the feasibility of CCPA@PPy NGs for tumor PA imaging in vivo was evaluated (Figure 5a). We can see that the tumor PA signal is greatly strengthened after intratumoral injection of the CCPA@PPy NGs. Moreover, the tumor PA signal value (0.820) was 10-fold higher than that before injection (0.081). These results indicate that the CCPA@PPy NGs are effective for PA imaging of tumors in vivo.

Figure 5. (a) PA images and (b) PA values of the tumor site before and at 30 min post intratumoral injection of the CCPA@PPy NGs (0.5 mg/mL, in 100 µL NS).

The whole-body temperature change of mice were also monitored (Figure 6a). As a control, NS was injected into the tumor site (Bx1) and a subtle temperature increase (1.4 oC and 1.6 oC at 2.5 min and 5 min post laser irradiation, respectively) was detected. To be opposed, the tumor site (Bx2) displayed a temperature increase by 12.1 oC and 13.6 oC after laser irradiation for 2.5 min and 5 min, respectively after the NGs were intratumorally injected. These data indicate that the designed 14


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CCPA@PPy NGs can be adopted for tumor thermal imaging and hold great potency for PPT of tumors as well.

Figure 6. (a) Thermal images of mice bearing 4 T1 tumors after intratumoral injection of 0.1 mL of NS (Bx1), or 0.1 mL of NS containing NGs (0.5 mg/mL) (Bx2) at 0.5 h postinjection under 808 nm laser irradiation with an output of 1.0 W/cm2 at 0 min, 2.5 min and 5 min, respectively. (b) The temperature elevation curves of tumor regions versus laser irradiation time.

3.6. In Vivo RT-Sensitized PTT of Tumors To investigate the RT-sensitized tumor PTT using the CCPA@PPy NGs, mice bearing 4T1 tumors were randomly assigned to 6 groups: Group 1 (NS), Group 2 (NGs), Group 3 (NGs + RT), Group 4 (NGs + PTT), Group 5 (NGs + RT + PTT), and Group 6 (NGs + PTT + RT). Tumor volume change after the different treatments was monitored every two days (Figure 7a). We found that tumors in Groups 1 and 2 unrestrictedly grew with time; in striking contrast, the tumors in Groups 3-6 were suppressed, to different degrees, suggesting that both RT and PTT contribute to the tumor therapy. Surprisingly, the relative tumor volume in Group 6 was the smallest, reaching 0.013 at 2 days posttreatment, while the relative tumor volumes for the other treatments were 1.02 in Group 3, 0.52 in Group 4, and 0.16 in Group 5, respectively. This suggests that a suitable level of hyperthermia could enhance the blood flow in tumor tissue and subsequently improve intratumoral oxygenation status, which may bring the cells to be more RT-sensitive,42, 51, 52 leading to the high efficiency of PTT/RT combination treatment. 15



Figure 7. The relative tumor volume variation (a), body weight change (b) and survival rate (c) of mice having 4T1 tumors versus time posttreatments: Group 1 (NS), Group 2 (NGs), Group 3 (NGs + RT), Group 4 (NGs + PTT), Group 5 (NGs + RT + PTT), and Group 6 (NGs + PTT + RT). (d) Quantitative analysis of the apoptotic rate of tumor cells in different groups of treatments.

Additionally, the in vivo toxicity of NGs was evaluated by uninterruptedly checking the body weight of mice bearing tumors (Figure 7b). The variation of body weight for the mice having tumors in each group was negligible, further demonstrating that all different treatments do not produce any significant toxicity in mice. To further validate the combination PTT/RT efficacy, the survival rate of mouse in all six groups was measured and the harvested tumor sections was histologically examined (Figure S6, Supporting Information). We found that Groups 2, 4, 5, and 6 maintained a 100% survival rate for over 13 days, whereas Groups 1 and 3 had a shorter average lifespan of 8.25 and 16


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11.75 days, respectively (Figure 7c). As can be seen in Figure S6a, H&E staining reveals that the NGs + PTT/RT-treated tumors display the most obvious necrotic cell morphology when compared with other groups. The degree of cell necrosis follows the order of control (Groups 1 and 2) < RT (Group 3) < PTT (Group 4) < RT/PTT (Group 5) < PTT/RT (Group 6). Meanwhile, TUNEL staining (Figure S6b) data show that only a quite rare amount of positively stained apoptotic cells were in the sections of Group 1 (1.2%), Group 2 (2.8%), and Group 3 (16.6%). On the contrary, a larger amount of apoptotic cells can be observed in the tumors of Group 4 (47.1%) after PTT treatment; Group 6 has the highest percentage of apoptotic cells (89.9%) after PPT/RT treatments, and 82.3% cells were apoptotic in Group 5 after RT/PPT treatments (Figure 7d). Taken together, our findings indicate that PTT is more effective than RT, not only for achieving high therapeutic efficacy, but also for prolonging the lifespan of tumor-bearing mice. Moreover, utilization of PTT/RT in sequence appears to be the best combination therapy strategy. The in vivo long-term toxicity of the CCPA@PPy NGs was also assessed. Thin sections of heart, liver, spleen, lung, and kidney of mice at 10 days post intravenous injection of NGs were H&E stained (Figure S7, Supporting Information). All main organs of the NG-treated mice displayed normal morphology, which are comparable with the NS-treated control group, suggesting that the designed CCPA@PPy NGs own a negligible toxic effect on mouse organs and have a good in vivo biocompatibility.

4. Conclusions We develop a simple approach to prepare highly water-dispersible and stable CCPA@PPy NGs for RT-sensitized tumor PTT under the guidance of PA imaging. The synthesized CCPA@PPy NGs having a size of 38.9 ± 8.6 nm show uniform distribution, and excellent cytocompatibility within the range of given concentrations. The designed CCPA@PPy NGs have excellent photothermal stability, prominent PA/thermal imaging properties, and high photothermal conversion efficiency (64.7%), and 17



can be utilized for PTT of tumors under the PA imaging guidance. In addition, the sequence of PTT/RT displays a better tumor therapeutic efficacy than either RT alone, PTT alone, or the sequence of RT/PTT. The CCPA@PPy NGs constructed in this research could be employed as a versatile nanoplatform for loading of other diagnosis/therapeutic elements, or for covalent conjugation of the functional biological molecules by virtue of their surface carboxyl groups for a variety of theranostic applications.

Acknowledgements This work has been supported financially by the Fundamental Research Funds for the Central Universities (for X. Shi, M. Shen and C. Peng), the National Natural Science Foundation of China (81761148028, 21773026 and 81470648), and the Science and Technology Commission of Shanghai Municipality (17540712000 and 15520711400).

Supporting Information: Additional details of materials and methods and data of zeta potential, hydrodynamic size, TGA, SEM images, DLS assessment of colloidal stability, photothermal stability assessment of NGs, H&E and TUNEL staining images of tumor sections, and H&E staining images of main organs of mice. This material is available free of charge via the Internet at http://pubs.acs.org.

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Radiotherapy-Sensitized Tumor Photothermal Ablation Using Î³

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