Dendrimer-Templated Ultrasmall and Multifunctional Photothermal

Dendrimer-Templated Ultrasmall and Multifunctional Photothermal Agents for Efficient Tumor Ablation Zhengjie Zhou,† Yitong Wang,† Yang Yan, Qiang Zhang,* and Yiyun Cheng* Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241, People’s Republic of China S Supporting Information *

ABSTRACT: Ultrasmall and multifunctional nanoparticles are highly desirable for photothermal cancer therapy, but the synthesis of these nanoparticles remains a huge challenge. Here, we used a dendrimer as a template to synthesize ultrasmall photothermal agents and further modified them with multifunctional groups. Dendrimer-encapsulated nanoparticles (DENPs) including copper sulfide, platinum, and palladium nanoparticles possessed a sub-5 nm size and exhibited an excellent photothermal effect. DENPs were further modified with TAT or RGD peptides to facilitate their cellular uptake and targeting delivery to tumors. They were also decorated with fluorescent probes for real-time imaging and tracking of the particles’ distribution. The in vivo study revealed RGD-modified DENPs efficiently reduced the tumor growth upon near-infrared irradiation. In all, our study provides a facile and flexible scaffold to prepare ultrasmall and multifunctional photothermal agents. KEYWORDS: photothermal therapy, ultrasmall, multifunctional, dendrimer, cancer targeting including diagnosis,26 imaging,27 targeting delivery,28 tracking the particle’s pharmacokinetics,29 and evaluating the therapeutic efficacy.30 Due to these advantages, multifunctional nanoparticles have emerged as an attractive preference to design optimized therapies for personalized therapy. However, highquality multiple functionalization is still a huge challenge. Multifunctionality means additional costs and multiple synthetic steps,31 and especially for the ultrasmall nanoparticles, it is very difficult to arrange diverse functional groups on their surface with controlled numbers and surface placements.32 As a result of this, the synthesized multifunctional nanoparticles might have an undesired pharmacokinetic behavior and cause severe side effects in vivo. Dendrimers have well-defined size and shape, hollow interior, and high density of surface functional groups,33−35 which are widely used as templates to synthesize ultrasmall nanoparticles (typically 40 nm at least in one dimension14−17 and are difficult to penetrate deeply in the tumor tissues and be cleared out of the body posttreatment,18,19 leading to decreased therapeutic outcomes and increased potential toxicity. However, this problem can be addressed by reducing the particle size. It is demonstrated that sub-10 nm nanoparticles can penetrate into the deep region of the tumors, be efficiently internalized by the tumor cells compared with the larger ones,19−21 and can also be rapidly cleared out of the body.22−24 Besides, the ultrasmall size also benefits nanoparticles by enabling them to escape capture from the reticuloendothelial system (e.g., liver and spleen) to enhance their accumulation in the tumors.25 Multiple functionalization endows PAs with additional capabilities, © XXXX American Chemical Society

Received: March 24, 2016 Accepted: April 7, 2016

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Figure 1. Characterization of three different DENPs. (a) Scheme depicts the preparation of multifunctional UPAs. TEM images of (b) DECuS, (c) DEPt, and (d) DEPd. The insets show the HRTEM images of single DENP. The scale bars in the inset images are 1 nm. (e) UV−vis spectra of three different DENPs. (f) Temperature changes of DENPs suspended in DI water (300 μM, 1 mL) while being irradiated by a NIR laser at a power density of 6.40 W·cm−2 for 10 min. DI water and dendrimer solution were used as the control. (g) Cytotoxicity of three DENPs on NIH3T3 cells.

with an average number of 100 acetyl groups on the surface to reduce its cationic toxicity (defined as G5-NH2-AC100 PAMAM dendrimer, Figure S1), which was further used as a model template to synthesize UPAs with diverse functional groups (Figure 1a). Dendrimer-encapsualted CuS nanoparticles (DECuS) were prepared by a coprecipitation method. The transmission electron microscopy (TEM) image shows that DECuS had an average ultrasmall size of 4.5 ± 3.0 nm (Figures 1b and S4a), and the high-resolution TEM (HRTEM) image reveals they possessed a lattice fringe of 0.33 nm (Figure 1b, inset), which is consistent with that of the (102) plane of hexagonal CuS.41 The powder X-ray diffraction (XRD) pattern of DECuS was consistent with that of the standard powder CuS with a hexagonal structure (JCPDE card no. 06-0464) (Figure S2), and the high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image and the elemental mapping images confirm that DECuS were composed of Cu and S (Figure S3). DEPt and dendrimerencapsulated Pd nanoparticles (DEPd) were prepared according to Crooks’s method.42 DEPt had an ultrasmall size of 1.4 ± 0.3 (Figures 1c and S4b), and DEPd had a size of 1.5 ± 0.5 nm

ultrasmall photothermal agents (UPAs), including copper sulfide (CuS), platinum (Pt), and palladium (Pd) nanoparticles, and then modified them with multifunctional groups. Dendrimer-encapsualted Pt nanoparticles (DEPt) were chosen as the optimal UPA due to their excellent photothermal effect and great biocompatibility. Given the high density of functional groups on the surface of the dendrimer, DEPt were further modified with cell-penetrating or tumor-targeting peptides (i.e., trans-activating transcriptional activator (TAT) and cyclic ArgGly-Asp (RGD) peptides, respectively) and fluorescent probes for different therapeutic purposes. The intracellular delivery of TAT-modified DEPt (TAT-DEPt) and RGD-modified DEPt (RGD-DEPt) and their in vitro cell-killing efficacies upon nearinfrared (NIR) irradiation were determined. Furthermore, RGD-DEPt were administered systematically, and their tumor-targeting efficiency and ability to ablate tumors were intensively investigated.

RESULTS AND DISCUSSION The amine-terminated generation 5 (G5-NH 2 ) poly(amidoamine) (PAMAM) dendrimer was partially modified B

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Figure 2. TAT peptide-enhanced cellular uptake of DEPt. (a) Schematic of TAT-mediated cellular uptake of DEPt. (b) TEM of TAT-DEPt. (c) ζ-Potential of G5-NH2 PAMAM dendrimer, DEPt, and TAT-DEPt. (d) Temperature changes of DEPt (250 μM) before and after the modification of the TAT peptide by NIR exposure. (e) Confocal images of PC-9 cells treated with dendrimer-RBITC (TAT−) and TATdendrimer-RBITC (TAT+) for 4 h. RBITC emits red fluorescence. The actin filaments were stained with phalloidin−FITC (green), and the nuclei were stained with DAPI (blue). (f) ICP-MS analysis of Pt content in PC-9 cells treated with DEPt and TAT-DEPt for 4 h; **p < 0.01 analyzed by student’s t test. (g) AO/EB double staining of PC-9 cells treated with DEPt (TAT−) and TAT-DEPt (TAT+) after NIR irradiation. The free DEPt and TAT-DEPt were removed before NIR irradiation. White circles indicate the irradiation regions.

(Figures 1d and S4c). HRTEM images reveal that the lattice fringes of DEPt and DEPd were 0.22 and 0.21 nm, respectively (Figure 1c,d, inset), which correspond to the (111) planes of Pt43 and Pd.44 The hydrodynamic sizes of DECuS, DEPt, and DEPd in deionized (DI) water were 6.5, 7.8, and 9.0 nm, respectively (Figure S5a−c), which suggests the three nanoparticles were highly monodisperse in solution. Furthermore, the time-elapsed size evolution of the three nanoparticles in 50% fetal bovine serum (FBS) containing phosphate buffer solution (PBS) was recorded. The result shows that there were no obvious size changes for these nanoparticles (Figure S5d−f), indicating that they were very stable in physiological conditions. The photothermal effect and biocompatibility of the three dendrimer-encapsulated nanoparticles (DENPs) was primarily evaluated. The ultraviolet−visible (UV−vis) spectra show that the three DENPs had considerable extinction in the NIR region (Figure 1e), indicating they might convert NIR light into heat. By being exposed to NIR light, the temperature of DECuS, DEPt, and DEPd suspensions quickly enhanced to a high value of 45.6, 47.0, and 43.8 °C, respectively, while that of DI water and dendrimer solution showed minimal enhancements (Figure 1f). This result suggests that DENPs could efficiently convert NIR light into heat. The cytotoxicity of DENPs was assessed on

a normal cell line of NIH3T3 and a cancer cell line of PC-9 using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The result shows that DEPt and DEPd had negligible toxicity on both cell lines in a broad concentration range of 0−400 μM (the concentrations of DENPs shown here and below were all metal ion concentrations except a special instruction; Figures 1g and S6), while DECuS were toxic at high concentration (Figures 1g and S6). The 1H nuclear magnetic resonance (NMR) spectra show that the G5-NH2-AC100 PAMAM dendrimer after being heated at 90 °C for 15 min and DECuS had the same NMR peaks as the fresh G5-NH2-AC100 PAMAM dendrimer, indicating that the dendrimer was not degraded during the synthesis of DECuS (Figure S7). The MTT assay shows that the G5-NH2-AC100 PAMAM dendrimer heated or not had a comparable toxicity on NIH3T3 cells (Figure S8). Therefore, the cytotoxicity of DECuS was not from G5-NH2-AC100 PAMAM but might be due to the release of trace amounts of copper ions. Based on the above evaluations, DEPt were chosen as the optimal UPA for the following studies. To evaluate the in vitro photothermal killing efficiency, PC-9 cells were incubated with 200 μM DEPt for 4 h, followed by NIR irradiation (6.4 W·cm−2, 10 min). After the treatments, the C

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Figure 3. RGD peptide-enhanced cellular uptake of DEPt. (a) Schematic of RGD-mediated cellular uptake of DEPt. (b) TEM of RGD-DEPt. (c) Temperature changes of DEPt before and after the modification of RGD by NIR exposure. (d) Confocal images and (e) flow cytometry analysis of MDA-MB231 cells and (f) flow cytometry analysis of NIH3T3 cells treated with dendrimer-RBITC (RGD−) and RGD-dendrimerRBITC (RGD+) for 4 h. (g) Competitive inhibition analysis. MDA-MB231 cells were pretreated with excess free RGD peptide following with the treatment of RGD-dendrimer-RBITC (RGD+ with free RGD). MDA-MB231 cells treated with dendrimer-RBITC (RGD−) was used as a negative control. (h) ICP-MS analysis of Pt content in MDA-MB231 cells treated with DEPt (RGD−) and RGD-DEPt (RGD+) for 4 h; *p < 0.05 analyzed by student’s t test. (i) AO/EB double staining of MDA-MB231 cells treated with DEPt (RGD−) and RGD-DEPt (RGD+) after NIR irradiation. The free DEPt and RGD-DEPt were removed before NIR irradiation. White circles indicate the irradiation regions.

positive ζ-potential of TAT-dendrimer did not lead to toxicity on NIH3T3 cells (Figure S12). PC-9 cells were further treated with TAT-DEPt and DEPt, and the intracellular Pt content was determined by inductively coupled plasma mass spectrometry (ICP-MS). The result reveals that Pt content in TAT-DEPttreated cells was much higher than that in DEPt-treated cells (Figure 2f). Subsequently, we performed the photothermal killing assay with the removal of free TAT-DEPt before NIR irradiation. PC-9 cells treated with TAT-DEPt were all killed in the irradiated region (Figure 2g, right, marked by a white dotted circle), while the cells treated with DEPt showed negligible cell death (Figure 2g, left). These studies confirm that the TAT peptide could enhance the cellular uptake of DEPt, leading to more efficient photothermal killing of cancer cells in vitro. RGD peptide can preferentially bind to cancer cells that overexpress αvβ3 integrin.46 In this case, an average number of 3.0 RGD peptides were modified on the surface of the G5NH2-AC100 PAMAM dendrimer (RGD-dendrimer, Figures 3a and S13). The as-prepared RGD-DEPt had a size and photothermal effect similar to that with DEPt (Figure 3b,c), and their ζ-potential was more positive than that of DEPt due to the existence of arginine in the RGD sequence (Figure S14). A significantly enhanced cellular uptake of dendrimer was observed in MDA-MB-231 cells (a mammary cancer line

cells were all killed, as revealed by an acridine orange/ethidium bromide (AO/EB) staining assay, while the cells incubated with DEPt or treated with NIR irradiation only were still alive (Figure S9a). Moreover, the cells could also be partially killed by adjusting the laser power density or irradiation time (Figure S9b). If the culture media were refreshed before NIR irradiation, the cells were no longer killed (data not shown), indicating DEPt were minimally internalized by cancer cells. The TAT peptide can penetrate cell membranes and facilitate the cellular uptake of diverse therapeutic agents.45 In our case, an average number of 2.5 TAT peptides were modified on the G5-NH2-AC100 PAMAM dendrimer (TATdendrimer, Figures S10 and 2a), and a fluorescent probe, rhodamine B isothiocyanate (RBITC), was further modified on the TAT-dendrimer for in situ monitoring (TAT-dendrimerRBITC, Figure 2a). Both NIH3T3 cells and PC-9 cells were treated with TAT-dendrimer-RBITC and dendrimer-RBITC, respectively. After 4 h incubation, only TAT-dendrimer-RBITC was more efficiently taken up by both cells (Figures S11 and 2e). TAT-dendrimer was further used to prepare TAT-DEPt, which had a size and photothermal effect similar to that with DEPt (Figure 2b,d). The significantly increased ζ-potential of TAT-DEPt compared with that of the G5-NH2 PAMAM dendrimer and DEPt was attributed to the abundant arginine and lysine in the TAT peptide (Figure 2c). However, the D

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Figure 4. Biodistribution of RGD-DEPt and DEPt. (a,b) IVIS images taken for mice (a) and excised organs and tumor tissues (b) harvested from mice 24 h after the intravenous injection of DEPt-Cy5.5 (RGD−) and RGD-DEPt-Cy5.5 (RGD+), respectively. (c,d) Biodistribution of DEPt (RGD−) and RGD-DEPt (RGD+) in tumors and different organs and tissues 12 (c) and 24 h (d) postinjection was determined by ICPMS analysis. (e) Tumor-site temperature changes while being irradiated by the NIR laser at a power density of 1.0 W·cm−2 for 5 min 24 h postinjection. (f) Corresponding thermographs of mice after NIR irradiation; **p < 0.01 and ***p < 0.001 analyzed by student’s t test.

overexpressing αvβ3 integrin) treated with RBITC-labeled RGD-dendrimer (RGD-dendrimer-RBITC), while few dendrimers were internalized in the cells treated with RBITClabeled dendrimer (dendrimer-RBITC) (Figure 3d). The flow cytometric analysis shows that there was a distinguished fluorescence enhancement for RGD-dendrimer-RBITC-treated MDA-MB-231 cells compared with control cells and the cells incubated with dendrimer-RBITC (Figure 3e), which indicates that RGD indeed increased the cellular uptake of the dendrimer. There were no distinguished fluorescence differences between NIH3T3 cells (without overexpression of αvβ3 integrin) treated with RGD-dendrimer-RBITC and dendrimerRBITC (Figure 3f). If MDA-MB-231 cells were pretreated with excess RGD peptides to inhibit the binding ability of αvβ3 integrin on the cell membrane, the cellular uptake of RGDdendrimer-RBITC was consequently decreased (Figure 3g). Moreover, MDA-MB-231 cells treated with RGD-DEPt showed an intracellular Pt concentration higher than those treated with DEPt, as revealed by ICP-MS analysis (Figure 3h). The photothermal killing assay was also conducted on MDA-MB231 cells with the removal of RGD-DEPt and DEPt before NIR irradiation. The cells treated with RGD-DEPt were completely

killed in the irradiated region, while the ones treated with DEPt showed minimal cell death (Figure 3i). All these data suggest that the RGD peptide enhanced the cellular uptake of DEPt in cancer cells overexpressing αvβ3. Finally, the stability of RGDDEPt in different media was analyzed by measuring their UV− vis spectra and photothermal conversion efficiencies at a series of time points, and the result suggests that they were highly stable in these media (Figure S15). The biodistribution and tumor-targeting efficiency of RGDDEPt were evaluated before the in vivo therapy. Tumor-bearing mice (MDA-MB-231 tumors stably expressing luciferase, MDAMB-231-Luc) were intravenously injected with cyanine 5.5 (Cy5.5)-labeled RGD-DEPt and DEPt (defined as RGD-DEPtCy5.5 and DEPt-Cy5.5, respectively) and were imaged by an in vivo imaging system (IVIS). A much higher fluorescent signal in the tumor was detected for a RGD-DEPt-Cy5.5-treated mouse (RGD+) compared with the one treated with DEPt-Cy5.5 (RGD−) 24 h postinjection (Figure 4a). IVIS images were also taken for tumors and main organs and tissues including heart, liver, spleen, lung, kidney, and brain (Figure 4b). The fluorescence intensities in liver and spleen were decreased in the RGD-DEPt-Cy5.5 group compared with that in DEPtE

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Figure 5. In vivo tumor ablation by RGD-DEPt-mediated PTT. (a) In vivo luminescence imaging of mice bearing MDA-MB231 tumors before and after PTT treatment. (b) Evolution of tumor volumes during the therapeutic period. (c) Weight and (d) photographs of the excised tumors after PTT treatment. (e) Body weight changes in the therapeutic period. (f) Apoptosis (red) of tumor cells after treatment analyzed by a TUNEL assay. The cell nuclei were stained by Hoechst 33342. The mice in the RGD+ group were intravenously injected with RGD-DEPt, and the ones in the RGD− group were injected with DEPt; ***p < 0.001 analyzed by student’s t test.

Cy5.5 group, while the tumor in RGD-DEPt-Cy5.5 group represented a much higher fluorescence intensity than the one in the DEPt-Cy5.5 group (Figure 4b). In order to quantitatively understand the biodistribution of RGD-DEPt and DEPt in the body, Pt contents in tumors and main organs and tissues including heart, liver, spleen, lung, kidney, and brain were determined using ICP-MS. As shown in Figure 4c,d, Pt contents in the RGD-DEPt group (RGD+) were significantly increased in tumor tissues but decreased in the liver and spleen compared with that in DEPt group (RGD−) at both time points of 12 and 24 h postinjection. Based on the above analyses, it is demonstrated that RGD could efficiently enhance the targeting delivery of DEPt to the tumors. Furthermore, the tumor-site temperature evolution in mice treated with RGD-DEPt and DEPt was recorded during NIR irradiation. The tumor-site temperature in the mice treated with RGD-DEPt increased faster than that in the mice treated with DEPt at the time points of 12 and 24 h postinjection (Figures S16a and 4e), and a final temperature of 45.4 °C in mice

treated with RGD-DEPt was detected at the time point of 24 h (Figure 4e), which is high enough to ablate the tumor in vivo.47 The thermographs of mice treated with RGD-DEPt also represent a temperature in the tumor much higher than that of mice treated with PBS and DEPt (Figures S16b and 4f). These results suggest that RGD-DEPt could efficiently accumulate in the tumors and conduct efficient PTT. The photothermal ablation of tumors using RGD-DEPt was assessed on mice bearing MDA-MB-231 tumors. Initially, NIR irradiation at a power density of 1.3 W·cm−2 for 10 min was applied to the mice, which decreased the tumor growth (Figure S17) but caused severe damage to the healthy tissues due to the high temperature in the tumor (58.1 °C). Therefore, NIR irradiation at a lower power density of 1.0 W·cm−2 and for a shorter time of 5 min was applied in the second round of experiments. The mice were intravenously injected with PBS, DEPt, and RGD-DEPt three times and were irradiated by the NIR laser twice after each injection. After the treatment, the luminescent intensities of tumors in the RGD-DEPt (RGD+) F

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being cooled at room temperature, the reaction solution was transferred into a dialysis bag (Biosharp, MWCO 3500) and intensively dialyzed against DI water. The final product was collected and stored at 4 °C. The concentration of metal was measured by ICPMS. DEPt were prepared according to a previous method.38 In a standard synthesis of DEPt, 0.553 mL of K2PtCl6 (20 mM) was added into 0.150 mL of G5-NH2-AC100 (0.573 mM) in aqueous solution under magnetic stirring. After 24 h, 0.553 mL of NaBH4 (200 mM) solution was added into the reaction solution. After another 2 h, the reaction solution was transferred to a dialysis bag (Biosharp, MWCO 3500) and intensively dialyzed against DI water. The final product was collected and stored at 4 °C. The concentration of metal ions was calibrated by ICP-MS. The same procedure was also used for the preparation of DEPd. Synthesis of TAT- or RGD-Modified Dendrimer. G5-NH2AC100 (37.00 mg, 1.117 μmol) was dispersed in 3.0 mL of PBS buffer containing 3 mM EDTA·2Na solution (pH 7.2). SMCC (2.24 mg, 7 μmol) dissolved in DMSO was added into the solution, and then the reaction solution was stirred at room temperature for 24 h. TAT (13.93 mg, 8 μmol) or RGD (4.85 mg, 8 μmol) was added into the reaction solution, and the mixture was stirred at room temperature for another 24 h. The solution was loaded into a dialysis bag (Biosharp USA, MWCO 3500) and dialyzed against DI water to remove the free peptides. 1H NMR spectrum of the final products shows that the double bonds of the maleimide group (δ = 6.70 ppm) disappeared, and the aromatic protons (δ = 7.30 ppm) on the phenylalanine of TAT or RGD appeared (Figures S10 and S13), which indicates TATand RGD-modified G5-NH2-AC100 dendrimers were successfully synthesized. According to the peak areas of the dendrimer and peptide signals, an average number of 2.5 TAT (Figure S10) and 3.0 RGD molecules (Figure S13) was conjugated on each G5-NH2-AC100. Preparation of TAT-Dendrimer-RBITC and RGD-DendrimerRBITC. TAT-dendrimer-RBITC was synthesized by adding RBITC (5.50 μM) into the TAT-dendrimer (1.10 μM) solution. The mixture was stirred in the dark at room temperature for 24 h. The unreacted RBITC was removed by intensive dialysis against DI water. RGDdendrimer-RBITC was prepared via the same procedure. Preparation of TAT-DEPt and RGD-DEPt. The preparation of TAT-DEPt and RGD-DEPt was similar to the synthesis of DEPt. Briefly, 0.553 mL of K2PtCl6 (20 mM) was added into 0.150 mL of TAT-dendrimer or RGD-dendrimer (0.573 mM). After 24 h, 0.553 mL of NaBH4 (200 mM) solution was added into the reaction solution and stirred for 2 h. Then the reaction solution was transferred to a dialysis bag (Biosharp USA, MWCO 3500) and intensively dialyzed against DI water. The metal ion concentration of the final product was calibrated by ICP-MS and stored at 4 °C. Preparation of RGD-DEPt-Cy5.5. RGD-DEPt-Cy5.5 was obtained by simply mixing 0.005 mL of Cy5.5 N-hydroxysuccinimide ester (0.7 mM) and 0.150 mL of RGD-DEPt (0.7 mM) in DI water for 24 h in the dark. To purify the product, the reaction solution was transferred into a dialysis bag (Biosharp USA, MWCO 3500) and intensively dialyzed against with DI water. The final product was stored at 4 °C. Characterization. TEM images of the samples were collected using a transmission electron microscope (HT7700, HITACHI, Japan) at an accelerating voltage of 100 kV. Digital images were obtained using the Gatan digital micrograph imaging system. X-ray diffraction was measured using a Siemens Kristalloflex 810 D-500 Xray diffractometer (Karlsruhe, Germany). HRTEM images, HAADFSTEM images, and elemental mapping images were obtained with a JEOL JEM-2100 electron microscope (Tokyo, Japan) with an accelerating voltage of 200 kV. The absorption spectra were recorded using an Agilent Technologies Cary 60 UV−vis spectrophotometer with a 1.0 cm optical path length quartz cuvette. ICP-MS was obtained with Neptune MC-ICP-MS (Thermo, Germany). NMR spectra were obtained with a 500.132 MHz NMR spectrometer (Bruker, Germany). Dynamic light scattering was recorded with Zetasizer Nano ZS90 (Malvern, UK).

group were notably reduced compared with that of the tumors in PBS and DEPt (RGD−) groups (Figures 5a and S18), which indicates that the tumor growth in the RGD-DEPt group was significantly suppressed. The photograph of tumor volume and weight of excised tumors confirms that the tumors in mice treated with RGD-DEPt were efficiently decreased (Figure 5b− d). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay suggests that the tumor cells in RGDDEPt groups were highly apoptotic after PTT, while the ones in PBS and DEPt groups were non-apoptotic and minimally apoptotic, respectively (Figure 5f). There were no obvious body weight changes during the therapeutic period (Figure 5e) and no observable pathological changes in the histological sections after PTT (Figure S19), indicating RGD-DEPt had minimal systematic toxicity.

CONCLUSION In summary, we developed a facile and versatile platform for the preparation of UPAs with multifunctionality. UPAs derived from DENPs including DECuS, DEPt, and DEPd possessed ultrasmall size, great monodispersity, and high stability and also represented excellent photothermal conversion efficiency. Benefitting from the high-density functional groups on the surface of the dendrimer, DENPs could be modified with diverse functional groups, such as cell-penetrating peptides, tumor-targeting peptides, and fluorescent probes, with controllable numbers and placements. Our investigation utilizes the dendrimers with their advantages of synthesizing ultrasmall nanoparticles and precise surface modifications and provides a facile route to prepare diversified UPAs for tailoring PTTs. EXPERIMENTAL SECTION Materials. G5-NH2 PAMAM dendrimers were purchased from Dendritech Inc. (Midland, MI), and their purity was checked by 13C NMR spectroscopy and the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy (Figure S20). Copper(II) chloride (CuCl2·2H2O), sodium hydrosulfide (NaHS), potassium hexachloroplatinate (K2PtCl6), sodium tetrachloropalladate (Na2PdCl4), sodium borohydride (NaBH4), 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), and RBITC were obtained from Sigma-Aldrich (St. Louis, MO). TAT peptide (NH2-CYGRKKRRQRRR-COOH) and RGD peptide with a sequence of Arg-Gly-Asp-(D-Phe)-Cys were purchased from GL Biochem Ltd. (Shanghai, China). All agents were used without further purification. Synthesis of the G5-NH2-AC100 PAMAM Dendrimer. G5-NH2 PAMAM dendrimers (200 mg, 0.007 mmol) were dissolved in 3 mL of absolute methanol, and then triethylamine (127 μL, 0.911 mmol) and acetic anhydride (69 μL, 0.728 mmol) were added. The mixture was stirred at room temperature for 48 h and then transferred into a dialysis bag (Biosharp USA, molecular weight cut off (MWCO) = 3500 Da, defined as MWCO 3500) and dialyzed against DI water 10 times. The average number of acetyl groups conjugated on the surface of the G5-NH2 PAMAM dendrimer was calculated according to the peak area ratio of dendrimer protons (Ha,a′) and acetyl protons (Hf) according to the 1H NMR (Figure S1). Synthesis of Dendrimer-Encapsulated Metal Nanoparticles. DECuS were prepared via a coprecipitation method.5 First, 0.354 mL of CuCl2 (100 mM) was added into 1.700 mL of G5-NH2-AC100 (0.163 mM) in aqueous solution, and then 0.884 mL of NaHS (80 mM) was dropwise added in the reaction solution under magnetic stirring at room temperature. The solution color changed from light blue to dark brown immediately upon the addition of NaHS. Thirty minutes after the addition of NaHS, the reaction solution was transferred into an oil bath and heated at 90 °C for 15 min until magnetic stirred. Finally, a dark green solution was obtained. After G

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ACS Nano Photothermal Efficiency of DENPs. A fiber-coupled NIR laser with continuous wave at 808 nm (MDL-III-808, Changchun New Industries Optoelectronics Technology Co., Ltd., China) was employed as the light source. The output power of the laser was calibrated using a hand-held model optical power meter (PM-8082000, Changchun New Industries Optoelectronics Technology Co., Ltd., China). The fiber output end was positioned 1.5 cm above the surface of the solution (solution volume was 1 mL; cross sectional dimension of vessel was 1 × 1 cm). DENPs were suspended in DI water, and dendrimer was also dissolved in DI water. An infrared thermal imaging camera was used to record the temperature evolution. Cell Culture. NIH3T3 cells (a mouse embryo fibroblast cell line, ATCC) were cultured in DMEM (Invitrogen, 10% FBS, 100 μg/mL streptomycin, and 100 μg/mL penicillin) at 37 °C under 5% CO2. PC9 cells (a human lung adenocarcinoma cell line, ATCC) were incubated in RPMI 1640 medium (Invitrogen) at 37 °C under 5% CO2. MDA-MB-231 cells (mammary cancer cells, ATCC) were cultured in MEM (GIBCO), containing 10% FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin at 37 °C under 5% CO2. Cytotoxicity. NIH3T3 and PC-9 cells were seeded in 96-well plates with a density of 10 000 cells per well 1 day before the experiment. Cells were washed with PBS once and then incubated with DENPs in a concentration range of 0−400 μM (the equivalent dendrimer concentration range is 0−3.906 μM) at 37 °C for 24 h. Cells without particle treatment were used as a control. A standard MTT assay was used to determine the cytotoxicity. Photothermal Killing of Cancer Cells Using DEPt. PC-9 cells were seeded in a 96-well plate with a density of 10 000 cells per well 1 day before the experiment. The cells were incubated with DEPt at a concentration of 200 μM (the equivalent dendrimer concentration is 1.953 μM, optical density at 808 nm (OD808) = 0.225) at 37 °C for 4 h and then were irradiated with an 808 nm NIR laser at a power density of 6.4 W·cm−2 for 10 min. Furthermore, the laser power densities and the irradiation times were varied to evaluate the cancer-killing efficiency. After NIR treatment, cells were cultured for another 2 h at 37 °C and then analyzed with the standard MTT assay. For the AO/EB double staining assay, cells were washed twice with cold PBS after NIR irradiation and then stained with AO (5 μg/mL) and EB (5 μg/mL) for 3 min. After that, the cells were again washed twice with cold PBS and observed by a fluorescent microscope (Olympus, Japan). In Vitro Cellular Uptake of TAT-Dendrimer-RBITC and RGDDendrimer-RBITC. The uptake of TAT-dendrimer-RBITC and RGD-dendrimer-RBITC by cancer cells was determined by confocal laser scanning microscopy (CLSM, TCS SP5, Leica, Germany) and flow cytometry. For CLSM observation, PC-9 cells (10 000 cells per well) were seeded in a 24-well plate with glass-bottom dishes (35 mm × 10 mm, Corning Inc., New York) for 24 h and then were treated with 0.3 mL of TAT-dendrimer-RBITC or dendrimer-RBITC at a dendrimer concentration of 1.953 μM (the equivalent metal ion concentration is 200 μM, OD808 = 0.001). After 4 h incubation, the culture media were removed and cells were washed twice with PBS. Then, 0.50 mL of paraformaldehyde (4%) was added to fix the cells for 15 min; phalloidin−FITC dissolved in DMSO was added to stain the cytoskeleton for 20 min, and 4,6-diamidino-2-phenylindole (DAPI) solution in methanol (10%) was added for another 15 min to stain the nuclei. After the treatment, cells were gently washed twice with PBS to remove excess dye. Finally, the cells were visualized under CLSM. The fluorescence images were taken under a 60× oil-immersion objective. Blue and green luminescent emissions from DAPI and FITC were excited at the wavelength from 425 to 475 nm for DAPI and 500 to 550 nm for FITC. For the αvβ3 integrin-blocking assay, MDA-MB-231 cells were incubated with excess free RGD (40 μg, 0.069 μmol) for 1 h followed by the treatment of 0.3 mL of RGD-dendrimer-RBITC (1.953 μM in dendrimer concentration, OD808 = 0.001). Two hours later, the cells were washed twice with PBS and analyzed by flow cytometry. In Vitro Cellular Uptake of TAT-DEPt and RGD-DEPt Analyzed by ICP-MS. PC-9 or MDA-MB-231 cells were seeded in 24-well plates with a density of 20 000 cells per well 1 day before the experiment. TAT-DEPt or RGD-DEPt dispersed in culture media

were added to the cells at a dendrimer concentration of 1.953 μM (the equivalent metal ion concentration is 200 μM, OD808 = 0.225). Four hours later, the culture media were removed, and the cells were washed three times with cold PBS. Then, the cells were treated with 0.20 mL of trypsin solution (containing 0.25% EDTA) and counted using flow cytometry. The washed cells were also digested with 0.50 mL of aqua regia to dissolve the cells and Pt nanoparticles. The digested samples were then diluted 100 times with 1% aqua regia. Pt contents were measured with ICP-MS (7500A, Thermo, USA). Quantification was carried out by an external five-point calibration with internal standard correction. The amounts of TAT-DEPt or RGD-DEPt were finally normalized to the cell number. Photothermal Killing of Cancer Cells Using TAT-DEPt or RGD-DEPt. PC-9 cells were seeded in a 96-well plate with a density of 10 000 cells per well 1 day before the experiment. Then, 0.1 mL of 200 μM TAT-DEPt or RGD-DEPt (the equivalent dendrimer concentration is 1.953 μM, OD808 = 0.225) dispersed in RPMI-1640 culture media were added into the cell culture. After 4 h incubation, the cell culture media were removed, and the cells were washed twice with cold PBS and added with fresh culture media. Cells without particle treatment were used as a control. Then, the cells were irradiated by NIR light at a power density of 25 W·cm−2 for 5 min. AO/EB double staining was processed to visualize the cell death as described above. In Vivo Photothermal Tumor Ablation. All the animals were housed under specific pathogen-free conditions within the animal care facility at East China Normal University. The animal experiments were performed in compliance with the Guidance Suggestions for the Care and Use of Laboratory Animals that was approved by the ethics committee of East China Normal University. Male BALB/c nude mice (4−6 weeks old) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). MDA-MB-231-Luc cells (1 × 106) were subcutaneously injected into the flank region of the mice. The nude mice grafted with subcutaneous MDA-MB-231-Luc tumors with an average volume of 130 mm3 were randomly divided into three groups (five mice per group). The three groups of mice were intravenously administrated with PBS, DEPt, and RGD-DEPt three times every 3 days, respectively, and were irradiated by the 808 nm laser (1.0 W· cm−2, 5 min) at a time point of 12 and 24 h after each injection. DEPt and RGD-DEPt in PBS at a metal mass concentration of 1.125 mg/mL were prepared. The dose for PBS was 1.6 mL/kg, and that for DEPt and RGD-DEPt was 1.5 mg Pt/kg; the injection dose for each mouse was adjusted according to individual body weight. The temperatures at the tumor site and thermographic images were obtained using an infrared thermal imaging camera (ThermoX, China). The tumor volume and body weight of each mouse were recorded every day. For ICP-MS analysis, the organs and tissues harvested from mice were weighted and milled. After that, the final samples were prepared according to the same protocol depicted in the section of in vitro cellular uptake of TAT-DEPt and RGD-DEPt analyzed by ICP-MS.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b02058. Additional figures and experimental details (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

Z.Z. and Y.W. contributed equally on this work.

Notes

The authors declare no competing financial interest. H

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