Cyanine-Curcumin Assembling Nanoparticles for Near-Infrared

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Cyanine-Curcumin Assembling Nanoparticles for Near-infrared Imaging and Photothermal Therapy Jianxu Zhang, Shi Liu, Xiuli Hu, Zhigang Xie, and Xiabin Jing ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00315 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 15, 2016

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ACS Biomaterials Science & Engineering

Cyanine-Curcumin Assembling Nanoparticles for Near-Infrared Imaging and Photothermal Therapy Jianxu Zhang, †,‡ Shi Liu, † Xiuli Hu,† Zhigang Xie, *,† Xiabin Jing†

†

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China ‡

University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, P. R.

China

Keywords: self-assembly, cyanine, NIR, photothermal therapy.

ABSTRACT: Near-infrared (NIR) imaging and photothermal therapy (PTT) based on the multifunctional cyanine dyes has shown great promise for cancer therapy. However, most of the PTT agents are often limited by low drug loading, short circulation time and low biocompatibility. Herein, we developed cyanine-curcumin assembling nanoparticles (CCNPs) via a single-step reprecipitation method. IR-780-C4 (Cyc4) was employed as a photothermal and NIR imaging agent. Self-assembly of Cyc4 and curcumin in aqueous solution could be performed in the absence of surfactants or adjuvants, which is a simple and efficient way to fabricate nanomedicine with high drug loading. Formed CCNPs showed monodispersity, good stability in physiological conditions, and lower cytotoxicity. Moreover, CCNPs possess the high

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loading (70%) of cyanine dyes and a higher photothermal conversion efficacy than free Cyc4, which contribute to decrease the application dosage of cyanine dyes in cancer therapy. Importantly, CCNPs exhibited excellent NIR imaging capacity and photothermal tumor ablation under laser irradiation in vitro and in vivo. This work highlights the potential of using selfassembling of drug molecules to develop functional nanoparticles for drug delivery and cancer therapy.

INTRODUCTION With the increasing of incidence and mortality, tumor has become the leading cause of death worldwide.1,2 Up to now, a lot of technologies for cancer treatments have been developed, such as gene therapy,3,4 chemotherapy,5-8 photodynamic therapy,9,10 photothermal therapy (PTT)

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and combination therapy.14-18 Among the various treatments, PTT has been paid great attention because of the noninvasiveness, low toxicity to normal tissues and highly efficient therapy.19-21 The photothermal agents with strong absorbance in the NIR region can efficiently convert light energy into thermal energy to induce hyperthermia (above 42oC) and kill the tumor cells.22,23 The ideal photothermal agents should have high photothermal conversion efficiency and robust photostability without rendering toxic side effects.21 Various materials with encouraging therapeutic efficacies have been widely explored as photothermal agents, including inorganic nanomaterials 24-35 and polymeric nanoparticles.36-41 Although most of photothermal agents have shown great promise in treatment of tumor, some properties such as non-biodegradable nature, poor photostability, insufficient photothermal conversion efficiency and short circulation time have prevented their further applications in cancer therapy.27


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Indocyanine green (ICG), a NIR cyanine dye, has been approved as a medical diagnostic agent by Food and Drug Administration.42-44 A lot of cyanine dyes have been constructed and studied in detail.45-49 They possess multifunctional properties including near-infrared fluorescence (NIRF), photoacoustic imaging and photothermal effect under light irradiation, which is ideal as an imaging and PTT agent.50,51 Nanoparticles made via polymer encapsulation or chemical conjugation can effectively incorporate cyanine dyes

with enhanced stability and

selectivity.17,22,44,52 Nevertheless, low drug loading capacity, complex compositions, and potential toxicity of carriers, are the major drawbacks for these nanoscale formulations.43,44 Recently, nanoparticles made directly from organic molecules without surfactants received more attentions.53-57 In our previous studies, several nanomaterials, including fluorescent nanoparticles for cellular imaging,53 nanomedicines54 and nanocapsule for drug delivery,43 have been made via self-assembly of small molecules. Very recently, Zhang et al. reported some hydrophobic molecules (like curcumin, camptothecine, and doxorubicin) could self-assemble into nanoparticles for cancer therapy.7,8,15,55 Considering the conjugated electron configuration, we hypothesized that versatile cyanine with similar structure could form nanoscale formulation through co-assembling with curcumin. IR-780-C4 (Cyc4) is a derivative of Cypate, which have shown great potential in NIRF and PTT.45, 46, 48 In this work, we used the hydrophobic curcumin as the matrix to co-assemble with Cyc4 to form the Cyc4-Curcumin Nanoparticles (CCNPs). The preparation of CCNPs and their application for NIR imaging and PTT were illustrated in Scheme 1. CCNPs with high stability showed strong NIRF and high photothermal conversion efficacy upon laser irradiation. Compared with free Cyc4, CCNPs exhibited higher anti-tumor efficacy in vitro and in vivo.


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Scheme 1. Schematic illustration on the preparation of the Cyc4-curcumin NPs (CCNPs) and their application for NIR imaging and PTT. EXPERIMENTAL SECTION Materials. All chemicals and reagents we used were obtained from commercial sources and used without further purification. The Milli-Q water we used here was collected from a Milli-Q system (Millipore, USA). Cyc4 was synthesized following the protocol has been reported.48 The instruments have been provided in our previous works.6, 43, 59 A Maestro 500FL in vivo optical imaging system (Cambridge Research & Instrumentation, Inc.) was used to record the NIR imaging on mice. Preparation of CCNPs. In a typical procedure, 3mM solutions of Cyc4 and 1.5 mM solutions of curcumin in ethanol were respectively prepared. Samples of 400 µL the Cyc4 and 800 µL of curcumin solutions were then mixed. Then the mixed solution was quickly dropwise dispersed


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into 5 mL of milli-Q water with vigorous stirring for 30 min at room temperature. Later, the water in CCNPs solution was volatilized to concentrate CCNPs solution. Photothermal generation detection. 0.2 mL CCNPs (containing Cyc4 at 20 µg/mL ) and free Cyc4 at 20 and 200 µg/mL in DMSO were irradiated using a laser (808 nm, 1.5 W/cm2) for 5 min. A temperature detector was used here to detect the temperature change with time. Cell experiments. The experimental procedure were given in our previous work.6, 43, 59 The experimental procedure in detail were showed in supporting information. Animals and tumor model. We complied with the NIH guidelines for laboratory animals to perform all animal experiments. CT26 cells were administered by subcutaneous injection into the armpit or the right flank region of the male BALB/c mice. Tumor volume was calculated as (tumor length) × (tumor width)2/2 and the tumor length and width were measured with calipers. In vivo NIRF imaging and organic distribution studies. We choose the mice bear the tumor in armpit to carry out this study. In order to detect the NIRF imaging capacity, we choose the depth of insertion at about 8 mm. So till the diameter of tumors above 10 mm, CCNPs (0.5 mg/kg Cyc4) were administrated into the mice via intratumor injection. Then, under anesthesia, the in vivo NIRF imaging was performed using an in vivo imaging system at 6, 24, 48, 72, 96, 120, 144 and 168 h post-injection, respectively. In addition, CCNPs (0.5 mg/kg Cyc4) were administrated into the mice via tail vein injection and whole body images of mice were acquired at 0.5, 6 and 24 h post-injection, respectively. After the in vivo NIRF imaging experiment, the mice were sacrificed to extract tumor and organs including heart, liver, spleen, lung and kidney. The fluorescence emission from the tissues were detected via an in vivo imaging system for measuring the ex vivo biodistribution of Cyc4 from CCNPs.


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In vivo photothermal treatments. The mice bearing the tumor in right flank region was used. In order to avoid the high temperature influence on normal tissue, we adjusted the dosage of Cyc4 and laser power to control the temperature below 44oC. When the tumors reached a size of ~200 mm3, the mice were divided into five groups: normal saline/irradiation, Cyc4, Cyc4/irradiation, CCNPs, CCNPs/irradiation. The mice were intratumor injected with normal saline (150 µL), free Cyc4 (0.5 mg/kg) and CCNPs (containing Cyc4 0.5 mg/kg), respectively. At 24 h post-injection, the tumors of the laser treatment groups suffered from 808 nm irradiation (5 min, 1.0 W/cm2). Then, the tumor volumes and body weight of each mouse were monitored every other day. After 15 days, the tumors were extracted from the mice and took photo to record.

RESULTS AND DISCUSSION Preparation and characterization of CCNPs. CCNPs were prepared through the reprecipitation method by adding an ethanol solution of curcumin and Cyc4 (molar ratio is 1:1) dropwise into water with vigorous stirring. The content of curcumin and Cyc4 in CCNPs were about 30 wt% and 70 wt% (see the Supporting Information, Table S1, Figure S1 and S2), which were determined by UV-vis standard curves. In this system, curcumin co-assemble with Cyc4 via strong intermolecular interactions such as π-π stacking, hydrophobic interactions.43 The transmission electron microscopy (TEM) image (Figure 1a) showed that the CCNPs were generally spherical in shape with a diameter of 90-110 nm. Dynamic light scattering (DLS) (Figure 1b) gave a hydrodynamic diameter of about 104 nm and a polydispersity index (PDI) of 0.2. The size of CCNPs was about 100 nm, which was larger than that of nanoparticles formed


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only by curcumin.7 The zeta potential of free curcumin, free Cyc4 and CCNPs were about -5, +76, and +43 mV, respectively (Figure 1c). These results confirm the formation of nanoparticles from Cyc4 and curcumin.

Figure 1. Physical properties of CCNPs. a) TEM image of CCNPs (scale bar = 200 nm). b) Size distribution of CCNPs (DLS and PDI measurements of CCNPs in aqueous medium). c) Zeta potential of free curcumin in ethanol, free Cyc4 in ethanol, and CCNPs in water.

The UV-vis absorption and photoluminescence (PL) spectra of curcumin, Cyc4 and CCNPs were shown in Figure 2. Compared with curcumin and Cyc4, the absorption peaks of CCNPs were red-shifted from 428 nm to 432 nm, and from 785 nm to 793 nm, which were the characteristic for curcumin and Cyc4, respectively (Figure 2a). These data suggested that both curcumin and Cyc4 maintained their optical properties in CCNPs. Curcumin and Cyc4 show a strong fluorescent intensity, while negligible fluorescence is seen for CCNPs (Figure 2b) due to the typical aggregation induced quenching effect. Photographs of Cyc4 and curcumin in ethanol and CCNPs in water (from left to right) under white light, UV light (365 nm) and NIR light (750 nm) were shown in Figure 2c. The original green fluorescence of curcumin and NIR fluorescence of Cyc4 were quenched after the formation of CCNPs. The self-assembling CCNPs


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consisted of two optically active components with independent fluorescence, and we could take this advantage to track the drugs delivery in vitro and in vivo for imaging and PTT.

Figure 2. Optical characterization of CCNPs. a) UV-vis absorption spectra and b) PL spectra of free curcumin, free Cyc4 in ethanol and CCNPs in water. c) photographs of Cyc4 solution, curcumin solution, and CCNPs under white light (upper photo), UV light (middle photo) at 365 nm and NIR light (lower photo) at 750 nm.

Photothermal effect. To demonstrate the photothermal conversion behavior of CCNPs, we monitored the temperature changes (△T) under laser irradiation. CCNPs had an obvious increase of temperature even at a low concentration of Cyc4 upon laser irradiation of 1.5 W/cm2 for 5 min (Figure 3a). CCNPs exhibited concentration-dependent temperature increasing, and could


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quickly trigger hyperthermia (above 42oC). We also compared the photothermal effect of free Cyc4 and CCNPs (Figure 3b). CCNPs had a bigger temperature changes than free Cyc4 at the same concentration. In order to reach the same photothermal result as CCNPs, 10 fold concentration of Cyc4 is needed. The fluorescence quantum yield and the photothermal conversion efficiency of the CCNPs were calculated. The quantum yield of CCNPs in water was about 0.5%, while free Cyc4 in ethanol was about 4%. The calculated η value of the CCNPs was 52% according to the published method,58 as shown in Figure S3, which was higher than that of Au nanorod (22%)58 and TPT-TT NPs (32%).59 The enhanced photothermal effect was ascribed to the aggregation of Cyc4 in CCNPs. Compared with the free Cyc4, the lower QY of Cyc4 in CCNPs result from the formation of nanoparticles, which further lead to the enhanced photothermal ability of CCNPs. Moreover, the higher condensed concentration may reduce heat dissipation after laser irradiation, and the excitation thermal radiation is also entrapped in the enclosure of NPs.

Figure 3. Photothermal effect of CCNPs. Photothermal conversion behavior of a) CCNPs containing different concentrations of Cyc4 and water was used as the control and b) CCNPs


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containing 20 µg/mL Cyc4 in water, free Cyc4 at 20 and 200 µg/mL in DMSO, and water under irradiation (808 nm, 1.5 W/cm2) for 5 min. Stability of CCNPs. Excellent stability is essential for keeping the shape and size of nanoparticles in blood circulation, which helps them maintain their intrinsic function before reaching the targeting sites.17 Here, we evaluated the stabilities of CCNPs in various conditions by DLS and TEM. As shown in Figure 4a, CCNPs maintain the initial hydrodynamic diameter and size distribution in water even in two weeks. The particles still possess isolate spherical shape after storage in water for four weeks (see the Supporting Information, Figure S4). CCNPs stored in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FBS) and 1% penicillin/streptomycin exhibited unchangeable sizes and size distributions (see the Supporting Information, Figure S5), suggesting a great stability of CCNPs in physiological condition. The diameter had a slight increase by about 6 nm in DMEM, which possibly was caused by the adsorption of the proteins on the surface of CCNPs. The stabilities of CCNPs were studied in solution with different pH values (from 5 to 9). DLS revealed no significant size changes when pH changed (see the Supporting Information, Figure S6), and UV-vis spectra proved the same optical character of CCNPs at different pH (Figure 4b), indicating that the CCNPs were able to maintain stable nanostructures in acid-base environment. In Figure S7 (see the Supporting Information), the surface potential ζ showed a slight increase from 23.5 to 35 ± 3 mV when pH values of media changed from 5 to 9. The reason is not clear now, but this change has no obvious effect on the stability of CCNPs. We tested the photothermal conversion behavior of CCNPs in solution with different pH values. As shown in Figure 4c, the temperature increasement is almost same under irradiation (808 nm, 1.5 W/cm2) for 5 min.


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The photostability of CCNPs was investigated by monitoring the absorbance changes upon continuous laser irradiation. As shown in Figure 4d, CCNPs exhibited slight decrease of absorbance under irradiation for 90 s, but the absorbance of free Cyc4 decreased about 35% of absorbance. Although CCNPs exhibited a modest reduction of absorbance during irradiation, it was slower than that of free Cyc4. We also detected the size and morphology of CCNPs after laser irradiation. As shown in Figure S8 (see the Supporting Information), no obvious dissociation occurred, and the particles still maintained sphere after irradiation for 10 min. There were no changes of the diameter of CCNPs under different time of irradiation as revealed in Figure S9 (see the Supporting Information), indicating CCNPs kept stable nanostructures under irradiation. In addition, we investigated the protein adsorption of CCNPs. The diameter of CCNPs had a little increase after being incubated with 1 mg/mL human serum albumin (HSA) for 6 h at 37oC (see the Supporting Information, Figure S10), which was in line with the results shown in Figure S4. While the surface potential changed from positive to negative due to the protein adsorption, which was beneficial for long circulation in blood and decreasing the toxicity induced by positive charge. All these results revealed that CCNPs possessed excellent stability in physiological condition.


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Figure 4. Stabilities of CCNPs in different conditions. a) The size stability test of CCNPs in water over 14 days. b) UV-vis spectra of CCNPs in solution with different pH value. c) photothermal conversion behavior of CCNPs containing 20 µg/mL Cyc4 in different pH solutions under irradiation (808 nm, 1.5 W/cm2) for 5 min. d) Normalized absorbance of Cyc4 in CCNPs and free Cyc4 under irradiation (808 nm, 1.5 W/cm2) for different time periods.

Cellular uptake. Cellular uptake is necessary for nanomaterials to exert their functions, and can provide a key index for tracking and predicting the in vivo behavior of imaging and PTT.51 Human cervical carcinoma HeLa cells and murine colon carcinoma CT26 cells were used to investigate the cellular uptake of CCNPs by confocal laser scanning microscopy (CLSM). After


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incubation with CCNPs for 6 h at 37oC, cellular nuclei were dyed by 4,6-diamidino-2Phenylindole (DAPI). As shown in Figure S11 (see the Supporting Information), the homogeneous green and red fluorescence locate in cytoplasm, suggesting that CCNPs distribute nonspecifically inside the cells. In the merge images, the yellow fluorescence revealed that curcumin and Cyc4 had a good colocalization in the two cancer cell lines. To study the imaging of CCNPs, we used CLSM to evaluate their time-dependent internalization. As presented in Figure 5a, the fluorescence intensity of curcumin and Cyc4 increased obviously from 1 h to 8 h, indicating that the CCNPs had a sustained cellular uptake and accumulation in HeLa cells. Similar results were obtained in CT26 cells (see the Supporting Information, Figure S12). The real-time monitoring of the fluorescence of Cyc4 can be used to decide when to start the laser irradiation. In addition, we detected the cellular uptake efficiency of CCNPs with UV-vis spectra. The adsorption of curcumin (Figure 5b) and Cyc4 (Figure 5c) extracted from the HeLa cells for different time increase from 0.5 h to 8 h. The results were in line with the observation in CLSM. These results suggested that CCNPs could be used as a fluorescence probe for imaging.


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Figure 5. Cellular uptake of CCNPs. a) confocal microscopy images showing changes in the signal of curcumin and Cyc4 in HeLa cells treated with CCNPs at 1, 4, and 8 h. Different imaging channels are displayed horizontally for each sample (from left to right): DAPI (405 nm excitation), Cur channel (488 nm excitation), Cyc4 (639 nm excitation), and merged images. Absorbance of b) curcumin and c) Cyc4 of the CCNPs internalized by HeLa cells after 0.5, 2, 4, and 8 h incubation.

Cytotoxicity of CCNPs. Firstly, we studied the potential cytotoxicity profiles of CCNPs toward cancer cells by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-Diphenyltetrazolium bromide) assay. Figure 6a and Figure S13 (see the Supporting Information) showed the high viabilities of cancer cells after being cultured with CCNPs (20 µg/mL of Cyc4) from 1 h to 8 h. With the increase of time, the cell viabilities slightly decreased, but still maintained above 90%. We further studied the photothermal therapeutic efficiency of CCNPs against HeLa cells and CT26 cells. As shown in Figure 6b and Figure S14, CCNPs exhibited concentration-dependent toxicity upon laser irradiation, but the control group only with laser irradiation had no significant cytotoxicity. Only 20% of cells survived upon laser irradiation with the concentration of Cyc4 in CCNPs at 80 µg/mL (Figure 6b), showing that CCNPs could serve as an effective photothermal agent. It was notable that CCNPs without irradiation also exhibited a concentration-dependent and time-dependent toxicity (Figure 6b, see the Supporting Information, Figure S14). The possible reason was the release curcumin and Cyc4 from CCNPs in living cells. To visually demonstrate the efficiency of CCNPs in photothermal ablation, the cells were stained with calcein-AM and Propidium Iodide to identify live (green) and dead/late apoptotic


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(red) cells, respectively. Figure 6c and Figure S15 (see the Supporting Information) exhibited that only laser irradiation did not affect cell viability, which agreed well with the MTT experiments. With the increase of concentration of CCNPs, the cells upon irradiation were killed more and more. These results suggested CCNPs had a great promise as a photothermal system for cancer therapy.

Figure 6. cytotoxic effects of CCNPs in HeLa cells. a) In vitro cytotoxicity of CCNPs incubated in HeLa cells for 1, 2, 4, 6, and 8 h, respectively. b) cytotoxic effects of only irradiation, CCNPs without irradiation, CCNPs with irradiation in HeLa cells with increasing NPs concentrations. c) Fluorescence microscope images of calcein AM (green, live cells) and Propidium Iodide (red, dead cells) co-cultured HeLa cells with only irradiation and different concentrations of CCNPs with irradiation. In vivo imaging. To demonstrate the in vivo NIRF imaging capacity, CT26 tumor-bearing BALB/c mice was administered by intratumor injection with CCNPs at the center of the tumor


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with the depth of insertion about 0.8 cm, and then imaged by in vivo optical imaging system. As shown in Figure 7a, fluorescence emission from the tumor was monitored for 7 days. CCNPs formed a highlight point at the injection site and exhibited significant NIRF signals at 6 h postinjection. As time goes on, the fluorescent area spread around to form a gradient intensity distribution at 24 h post-injection, and the intensity decreased gradually with time, but still maintained relative strong signals after 7 days. During the whole process, we can track the photothermal agent at the tumor site by virtue of the fluorescence. We also evaluated imaging capacity of CCNPs in tumor-bearing mice by tail intravenous injection. As displayed in Figure 7b, when the mice was imaged at 0.5 h post-injection, there was no fluorescence emission at tumor site, and the liver showed strong signals. At 6 h post-injection, CCNPs were observed in the tumor site, and the fluorescence in tumor sites increased continuously. However, the most of injected CCNPs were accumulated at liver, which would decrease the photothermal therapy efficacy. In order to get a high photothermal therapy efficacy, we used the intratumor injection for further anti-tumor study of CCNPs in vivo. Next, we studied the biodistribution of CCNPs by detecting the fluorescence in the tumor and major organs excised from the mice. As shown in Figure 7c, a strong fluorescence could be clearly observed in tumor while a weak fluorescence in liver. No fluorescence emission was seen from other organs at 168 h post-injection. These results indicated that CCNPs were retained around the tumor even after 7 days, in favor of maintaining the photothermal conversion efficacy at the tumor site. The distribution major organs and tumor by tail intravenous injection was shown in Figure 7d. CCNPs were distributed into tumor and normal organs at 24 h after injection, with the highest accumulations in the spleen and liver, followed by tumor and lung.


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The higher fluorescence in the liver and spleen suggested that CCNPs were mainly eliminated by macrophage cells of the liver and spleen. To sum up, the good NIRF imaging capacity of CCNPs in vivo for long-term period possibly results from the nanoscale formulation. Firstly, the nanoparticles enhance the accumulation and cellular uptake of CCNPs at tumors. Second, the assembling of Cyc4 with curcumin enhances the stability and reduces the degradation of Cyc4 in complex physiological conditions. Up to now, most of cyanine-based imaging-guided PTT agents exhibit limited imaging contrast owing to their low cyanine loading efficacy and quick elimination at tumors.22,45 This aggregate with high content of cyanine is promising as the imaging contrast for imaging-guided PTT.

Figure 7. In vivo imaging and biodistribution of CCNPs in BALB/c mice bearing CT26 tumors after injection. a) In vivo NIRF imaging of the mice intratumor injected with CCNPs at the dose of 0.5 mg/kg Cyc4 at 6, 24, 48, 72, 96, 120, 144, and 168 h, respectively. b) In vivo NIRF imaging of the mice intravenous injected with CCNPs at the dose of 0.5 mg/kg Cyc4 at


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0.5, 6, and 24 h, respectively. c) NIRF images of major organs and tumor after intratumor injection of CCNPs at 168 h. d) NIRF images of major organs and tumor after intravenous injection of CCNPs at 24 h.

Photothermal therapeutic effect in vivo. To evaluate the photothermal therapeutic effects in vivo, saline, free Cyc4 and CCNPs were intratumorally injected into mice and then suffered laser irradiation at 24 h post-injection. The tumor volume and body weight were measured every three days in 15 days, then the mice were sacrificed and the tumors were excised. As shown in Figure S16a, S16b and Figure S17 (see the Supporting Information), the tumor in the saline/irradiation group grew rapidly with a 5.6-fold increase of volume than its original, suggesting that irradiation had a negligible influence on tumor growth. Compared to the saline/irradiation group, the growth of tumor was slightly inhibited by free Cyc4, possibly induced by the inherent toxicity of Cyc4. CCNPs without irradiation showed a higher antitumor effect when compared with the Cyc4, which was ascribed to the curcumin in CCNPs. Cyc4/irradiation group inhibited tumor growth less effectively than CCNPs plus laser because of the lower PTT effect of Cyc4 (Figure 3b). Importantly, the tumor volume in CCNPs/irradiation was much smaller as compared to those treated with other formulations (Figure S17a and S17b). As shown in Figure S17, CCNPs/irradiation group indicated only 2.2 fold increase of tumor volume which was much smaller than that of saline/irradiation (5.6 fold), Cyc4 (4.8 fold), Cyc4/irradiation (3.2 fold) and CCNPs (3.8 fold) treated groups. These results suggested that CCNPs had excellent photothermal therapy efficacy with a single-dose treatment.


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The potential adverse effect of photothermal agents is evaluated by the changes of body weight of mice. As shown in Figure S16c, all groups showed very little body weight reduction (

Cyanine-Curcumin Assembling Nanoparticles for Near-Infrared

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