Photoacoustic Synergistic

In Vivo Imaging-Guided Photothermal/ Photoacoustic Synergistic Therapy with Bioorthogonal Metabolic GlycoengineeringActivated Tumor Targeting Nanoparticles Lihua Du, Huan Qin, Teng Ma, Tao Zhang,* and Da Xing* MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China S Supporting Information *

ABSTRACT: Developing multifunctional phototheranostics with nanoplatforms offers promising potential for effective eradication of malignant solid tumors. In this study, we develop a multifunctional phototheranostic by combining photothermal therapy (PTT) and photoacoustic therapy (PAT) based on a tumor-targeting nanoagent (DBCO-ZnPc-LP). The nanoagent DBCO-ZnPc-LP was facilely prepared by self-assembling of a single lipophilic near-infrared (NIR) dye zinc(II)-phthalocyanine (ZnPc) with a lipid-poly(ethylene glycol) (LP) and following modified further with dibenzyl cyclootyne (DBCO) for introducing the two-step chemical tumor-targeting strategy based on metabolic glycoengineering and click chemistry. The as-prepared DBCO-ZnPc-LP could not only convert NIR light into heat for effective thermal ablation but also induce a thermalenhanced ultrasound shockwave boost to trigger substantially localized mechanical damage, achieving synergistic antitumor effect both in vitro and in vivo. Moreover, DBCO-ZnPc-LP can be efficiently delivered into tumor cells and solid tumors after being injected intravenously via the two-step tumor-targeting strategy. By integrating the targeting strategy, photoacoustic imaging, and the synergistic interaction between PTT and PAT, a solid tumor could be accurately positioned and thoroughly eradicated in vivo. Therefore, this multifunctional phototheranostic is believed to play an important role in future oncotherapy by the enhanced synergistic effect of PTT and PAT under the guidance of photoacoustic imaging. KEYWORDS: nanoparticle, photothermal therapy, photoacoustic therapy, photoacoustic imaging, click chemistry, tumor-targeting

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To date, PTT has been combined with various therapeutics, such as chemotherapy,16−19 radiotherapy,20−23 and photodynamic therapy (PDT),24−26 another phototherapeutic killing cells by singlet oxygen generated with a light-activated photosensitizer. Considering the appealing features of phototherapeutics, in particular, the combined PTT and PDT has been frequently adopted by researchers.27−31 Despite satisfying synergistic PTT/PDT treatment results as presented by those reports, however, some imperfections still exist. For instance, efficient destruction of hypoxic solid tumors is actually still challenging due to the high dependence on oxygen of PDT.32,33 Furthermore, the therapeutic agents with two or more different components in one system usually require complicated synthetic routes, even though some single-component agents

hototherapy, benefiting from its strength of spatiotemporal selectivity, has increasingly attracted considerable attention in the personalized noninvasive treatment of cancers to minimize the side effects of conventional chemotherapy and radiotherapy.1−3 Among the existing phototherapies, photothermal therapy (PTT) is a well-established phototherapeutic modality that uses the heat generated from the absorbed optical energy by photothermal agents accumulated in the malignant tumor to ablate tumor cells.4−9 However, typically, the heat of PTT produces a killing effect to only a population of cancer cells, and residual cancer cells rapidly develop and acquire resistance to thermal stress, resulting in limited therapeutic efficacy so that PTT alone is capable of eradicating tumors totally.10,11 In view of this inadequacy, the combined therapy with synergistic effect is usually considered as a very promising strategy to improve the therapeutic efficacy.12−15 © 2017 American Chemical Society

Received: May 9, 2017 Accepted: September 11, 2017 Published: September 11, 2017 8930

DOI: 10.1021/acsnano.7b03226 ACS Nano 2017, 11, 8930−8943

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ACS Nano Scheme 1. Schematic Illustration of PA Imaging-Guided Synergistic PTT/PAT with the Bioorthogonal Metabolic Glycoengineering-Activated Tumor Targeting Nanoagent

promising potentials for enhanced phototherapeutic outcomes of cancers. In the present work, we develop the phototheranostic for PA imaging-guided synergistic PTT/PAT therapy, which has not yet been investigated to date, based on a tumor-targeting nanoagent containing only one single chromophore, a phthalocyanine compound, as the photoabsorbing agent (Scheme 1). Phthalocyanine complexes possess exceptional photophysical properties, such as large molar absorption coefficients and high photostabilities compared to cyanines, and have been explored for construction of PA imaging and PTT particles.47−49 In this design, a lipophilic zinc(II)phthalocyanine (ZnPc) was synthesized, assembled, and aggregated with an amino lipid-poly(ethylene glycol) (LP) DSPS-PEG2000-NH2 to afford a near-infrared (NIR) photoabsorbing nanomicelle with high photothermal conversion efficiency. In order to deliver the nanomicelle to the tumor site selectively and efficiently to conduct PTT and PAT, we then modified the nanomicelle further with dibenzyl cyclootyne (DBCO) to afford the aimed nanoagent (DBCO-ZnPc-LP) to introduce the two-step tumor-targeting strategy based on metabolic glycoengineering and click chemistry, a recently emerging tumor-targeting technique that has been demonstrated high tumor-specificity with remarkable advantage over biological receptors: the artificial chemical-receptor can be expressed on the cell surface in large quantities regardless of the types or subpopulations of tumor cells.50−54 First, tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), the precursor for the generation of the artificial “receptor-like” azide group by serving as a building block in cell metabolism to make sialic acids, 55 was herein loaded into a nanomicelle (Ac4ManNAz-LP). Then, Ac4ManNAz-LP was intravenously injected into tumor-bearing mice, which led to site-specific

for both PTT and PDT are developed, satisfying balance between the photothermal conversion efficiency, and the singlet oxygen quantum yield is generally challenging. Therefore, the development of a PTT-based multifunctional phototherapeutic with facile construction routes but effective therapeutic effect is still highly desirable for eradicating the target solid tumors. High-intensity focused ultrasound (HIFU) shockwave has been proved to induce damage to tissues and used for clinical cancer therapy.34,35 Thus, another phototherapeutic, photoacoustic therapy (PAT), has recently emerged by utilizing the enhanced photoacoustic (PA) shockwave generated from the pulsed-light excited agents accumulated selectively in the tumor site to destroy the tumor cells.36,37 Our previous works and others have demonstrated PAT as an effective modality for tumor treatments based on single-walled carbon nanotubes (SWCTs), gold nanorods (GNRs), and indocyanine green (ICG) loaded phospholipid-polyethylene glycol nanoparticles under the irradiation of a pulsed laser.38−40 This phototherapeutic technique provides promising potential advantages, such as mechanical damage to target cells without suffering from drug, radiation or thermal resistance, and no restriction of hypoxic tumor microenvironment. As PAT takes advantage of photothermally converted acoustic waves, PAT agents naturally consist in PTT agents.41−43 Thereby, PAT as well as PTT can be guided by PA imaging, a powerful optical imaging methodology providing higher spatial resolution and deeper tissue penetration over traditional optical imaging techniques (e.g., fluorescence imaging).44 Moreover, the PA intensity has been proved to increase linearly along with the rising of temperature.45,46 Thus, integrating PTT with the neoadjuvant PAT into one platform as a PA imaging-guided phototheranostic is expected to be facilely accomplished and has 8931


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Figure 1. Synthesis and characterization of the nanoagent DBCO-ZnPc-LP. (a) Schematic design of synthesis of DBCO-ZnPc-LP. (b) Size distribution and the morphology (TEM image) of DBCO-ZnPc-LP. (c,d) Absorption and fluorescence spectra of DBCO-ZnPc-LP in tetrahydrofuran (black) and water (red) at the same concentration.

to be 82% in the final DBCO-ZnPc-LP. The particle sizes of DBCO-ZnPc-LP were found to be ≈164.18 nm (Figure 1b) via dynamic light scattering (DLS) measurement. Transmission electron microscopy (TEM) also revealed that the DBCOZnPc-LP exhibited uniform morphology and sizes with diameters of 100−200 nm (Figure 1b inset and Figure S4). DBCO-ZnPc-LP was also characterized further by FT-IR, Raman, and zeta potential spectroscopy (Figures S5−S7). Typically, the appearance of the characteristic Raman scattering peak at ∼2159 cm−1 of the DBCO group due to CC stretching vibration57 of DBCO-ZnPc-LP together with the decease of surface charge density of the nanoparticles from positive (amine groups) to negative compared with ZnPc-LP after reaction with DBCO-NHS58 confirmed the successful synthesis of the final product DBCO-ZnPc-LP. The as-prepared water-soluble DBCO-ZnPc-LP was highly stable in various physiological solutions without showing significant release of ZnPc even after 15 days incubation with PBS. Then the optical properties of DBCO-ZnPc-LP were investigated by UV−vis absorption and emission spectra (Figure 1c,d). DBCO-ZnPc-LP exhibited a blunt, structureless absorption spectrum with a significant hypochromic change as compared to the ZnPc monomer, which showed a typical feature of the phthalocyanine chromophores with sharp absorption bands (a high-energy Soret transition at 340 nm and a low-energy Q bands at 680 nm). The broadened Q-band with red-shift wavelength at ∼750 nm is in close accordance to characteristic absorption changes of dimeric aggregation of phthalocyanines.59 Furthermore, the fluorescence of DBCOZnPc-LP was completely quenched as typically observed for the common organic dyes in the concentrated or aggregated state.60 This intraparticle aggregation can promote the internal conversion and vibrational relaxation of the excited state of dyes and thus efficiently blocks other competitive deactivation

generation of azide groups on tumor tissue by tumor-targeted delivery of Ac4ManNAz-LP due to the enhanced permeability and retention (EPR) effect, rapid uptake of the Ac4ManNAz-LP into tumor cells, and site-specific metabolic glycoengineering. Second, the nanoagent DBCO-ZnPc-LP was intravenously injected into the same mice. Copper-free click chemistry between DBCO on DBCO-ZnPc-LP and azide groups on tumor tissue then results in enhanced tumor-targeting of DBCO-ZnPc-LP. Therefore, benefiting from the high tumortargeting and photothermal conversion efficiency, DBCOZnPc-LP could not only produce significant amounts of cytotoxic heat but also simultaneously generate thermalenhanced ultrasound shockwave upon laser excitation, which would result in remarkable in vitro cell damage and in vivo tumor regression. Furthermore, DBCO-ZnPc-LP could serve as a natural contrast agent for PA imaging diagnosis of tumors to guide the target solid tumor killing. As a result, the multifunctional DBCO-ZnPc-LP offers a platform in exploring for simultaneous biomedical imaging and combined PTT and PAT of target tumors.

RESULTS AND DISCUSSION The multifunctional nanoagent DBCO-ZnPc-LP was constructed following our previous similar protocol (Figure 1a).56 First, in order to enhance the loading efficiency, a lipophilic phthalocyanine ZnPc with four hydrophobic alkyl chains was synthesized and characterized by MALDI-TOF mass and 1H NMR spectra (Figures S1−S3). Then, the lipophilic ZnPc was assembled and aggregated with DSPS-PEG2000-NH2 via van der Waals forces and π−π stacking interaction to afford a photoabsorbing and amine-bearing nanomicelle (ZnPc-LP) and following modified with N-hydroxysuccinimidyl functionalized DBCO (DBCO-NHS) afforded the final nanoagent DBCO-ZnPc-LP. The loading capacity of ZnPc was measured 8932


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Figure 2. Photothermal and photoacoustic properties of DBCO-ZnPc-LP. (a) The photothermal heating curves of pure water and DBCOZnPc-LP solutions with different concentrations (12.5, 25, 50, 100, and 200 μg/mL) under 808 nm continuous laser irradiation at the power density of 0.3 W/cm2. (b) Temperature change at 10 min in (a). (c) Temperature change of DBCO-ZnPc-LP under four irradiation/cooling cycles (0.3 W/cm2). (d) PA signals of DBCO-ZnPc-LP at various concentrations under 808 nm pulsed laser irradiation of 0.3 W/cm2. (e) Temperature-dependent PA signals measurement. (f) PA signals of DBCO-ZnPc-LP with irradiation for 50 min.

Figure 3. Azide group generation and click reaction activated cellular binding. (a) Fluorescence images of tumor cells pretreated with Ac4ManNAz or Ac4ManNAz-LP and then reacted with FITC-labeled DBCO (DBCO-FITC). (b) Flow cytometric analysis of (a). (c) Western blot analysis of cells treated Ac4ManNAz or Ac4ManNAz-LP. (d) Photoacoustic images of tumor cells pretreated with Ac4ManNAz or Ac4ManNAz-LP and then reacted with DBCO-ZnPc-LP. (e) Photoacoustic signals monitored in the cells of (d). (f) Photoacoustic signals of cells treated with different concentrations of DBCO-ZnPc-LP.

pathways, such as the fluorescence decay and intersystem crossing energy transfer, thereby, providing an optimal condition for high photothermal conversion efficiency. In view of the remarkable photoproperties of DBCO-ZnPcLP, we then evaluated its photoconverted thermal and acoustic effect in detail. As expected, the solution of DBCO-ZnPc-LP

could be effectively heated up with obvious concentrationdependent temperature increase under exposure to an 808 nm continuous NIR laser at 0.3 W/cm2 (Figure 2a,b). The temperature of DBCO-ZnPc-LP solution at a concentration of 25 μg/mL rose rapidly over 40 °C within 5 min. The photothermal conversion efficiency was then quantitatively 8933


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Figure 4. In vitro PTT, PAT, and PTT + PAT therapy. (a) Relative viabilities of A549 cells incubated with Ac4ManNAz-LP or DBCO-ZnPcLP. (b) Relative viabilities of cells treated by PTT with different 808 nm continuous laser power. (c) Relative viabilities of cells treated by PAT with 808 nm pulsed laser at the different power density. (d) Relative viabilities of cells for different treatment groups after being incubated with different concentrations of DBCO-ZnPc-LP, pretreated with Ac4ManNAz-LP. PTT was introduced by 808 nm continuous laser irradiation at 0.1 W/cm2 for 5 min, while PAT was conducted by 808 nm pulsed laser irradiation at 0.2 W/cm2 for 1 min. (e) Confocal fluorescence microscope images of calcein AM and PI co-stained A549 cells after various treatments indicated. Green and red colors represented live and dead cells, respectively. Data were shown as mean ± SD (n = 6). P-values were determined by Student’s t test. *P < 0.05, **P < 0.01.

2f).61 The high photostability of DBCO-ZnPc-LP was further confirmed by monitoring its absorption and emission spectra changes under the irradiation of 808 nm continuous and pulsed laser (Figure S10). The biostability of DBCO-ZnPc-LP was then evaluated by measuring its absorption and emission spectra in different pH (from 4 to 10) and presence of various biospecies (Ac4ManNAz, Zn2+, HCO3−, Cu2+, H2PO4−, and citrate) (Figure S11). The persistent absorption and emission of DBCO-ZnPc-LP in these conditions as found suggested its high stability in physiological environments. The remarkable photothermal and photoacoustic performance of DBCO-ZnPcLP makes it a promising multifunctional theranostic nanoagent for PA imaging-guided synergistic PTT/PAT anticancer treatment. As efficient delivery and specific targeting of therapeutic agents to the tumor region are utmost important in cancer treatments,62 before investigation of the synergistic phototherapeutic efficacy of DBCO-ZnPc-LP, its performance in the tumor targeting was thereby explored. To deliver Ac4ManNAz to the tumor site more efficiently, Ac4ManNAz was also encapsulated by DSPS-PEG2000-NH2 to obtain the nanomicelle Ac4ManNAz-LP (Figure S12). TEM images manifested that Ac4ManNAz-LP exhibited uniform morphology and sizes with

measured and calculated to be 44.39% (Figure S8). Evaluation of the photothermal performance of DBCO-ZnPc-LP indicated that our nanoagent could speedily and efficiently convert photoenergy into heat. The photothermal stability of DBCOZnPc-LP was also measured by irradiating the sample with the 808 nm continuous laser for 10 min and turning off the laser for four cycles (Figure 2c). We found that the temperature changes of DBCO-ZnPc-LP had no obvious reduction, indicating the high photothermal stability of such phthalocyanine-loaded DBCO-ZnPc-LP. On the other side, photothermally converted acoustic effect investigation revealed that the PA signals rose linearly with an increase in the concentration of DBCO-ZnPcLP upon exposure to 808 nm pulsed laser irradiation at 0.3 W/ cm2, which is beneficial to their future PA imaging and PAT treatment (Figure 2d). To verify the influence of temperature on the PA signal intensity, the PA signals of DBCO-ZnPc-LP as well as water were measured at different temperatures under the irradiation of laser. As manifested in Figure 2e, the PA signal of DBCO-ZnPc-LP rose in linearity with the temperature, while that of pure water exhibited negligible increase. Moreover, DBCO-ZnPc-LP also exhibited almost no thermal effect to the surroundings and good photoacoustic stability under irradiation of the 808 nm pulsed laser (Figures S9 and 8934


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Figure 5. In vivo photoacoustic and photothermal images. (a) Photoacoustic images of tumor in nude mice before and 6, 12, and 24 h intravenous postinjections of DBCO-ZnPc-LP. (b) Thermographic images of mice exposed to 808 nm continuous laser (0.1 W/cm2) taken at different times. (c) Average PA signal intensity of tumor tissues of (a). (d) Temperature change of tumors upon laser irradiation as a function of irradiation time.

diameters of ≈78.82 nm (Figure S13,S14). To verify the twostep chemical targeting efficiency for tumor cells, the metabolized generation of the artificial receptor azide groups on the cell surface and the subsequent click targeting were evaluated by cellular imaging on human lung adenocarcinoma A549 cells. After A549 cells treated with free Ac4ManNAz or Ac4ManNAz-LP for 3 days, DBCO-FITC was added to determine the azide groups.63 As illustrated in Figure 3a, clear fluorescence imaging on cell surface was captured in both free Ac4ManNAz and Ac4ManNAz-LP treatment groups. The optical signal observed inside the cells may be attributed to the intrinsic glycan internalization, followed by endocytosis of the probes.52,64 The fluorescence intensity of the cells pretreated Ac4ManNAz-LP was similar to those pretreated with free Ac4ManNAz and higher than the control cells with no pretreatment (Figure 3b). Western blot analysis revealed that abundant azide groups were generated via metabolic glycosylation of proteins on the cell surface for those treated with free Ac4ManNAz or Ac4ManNAz-LP (Figure 3c). Immunofluorescence assays of

the A549 cells treated with free Ac4ManNAz or Ac4ManNAzLP afforded further confirmation for the generation of the azide groups on the cell surface (Figure S15). Moreover, investigations of the expression level of glycoproteins on the cell surface by real-time PCR revealed that free Ac4ManNAz or Ac4ManNAz-LP had no obvious negative effects on the expression of the glycoproteins compared with the controls (Figure S16). Ac4ManNAz and Ac4ManNAz-LP also had a higher efficiency for the metabolic generation of azide groups in tumor cells than the normal cells (Figure S17). After that, we incubated DBCO-ZnPc-LP with the A549 cell treated with free Ac4ManNAz and Ac4ManNAz-LP. Photoacoustic microscopic imaging revealed that abundant DBCO-ZnPc-LP was bound to the cell membrane surface via copper-free click reaction at both conditions with no obvious difference (Figure 3d,e). In addition, the PA signal amplitude was enhanced with the increase of the concentration of Ac4ManNAz-LP (Figure 3f). Taken together, the results indicated that the strategy can artificially introduce large number of ‘receptor-like’ chemical groups for the efficient delivery of the nanoagent. 8935


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Figure 6. In vivo behaviors. (a) Accumulation of DBCO-ZnPc-LP in the tumors in the presence or absence of Ac4ManNAz-LP. (b) Blood circulation of DBCO-ZnPc-LP. (c) Biodistribution of DBCO-ZnPc-LP in A549-tumor-bearing mice. (d) Excretion profile of DBCO-ZnPc-LP. (e) Western blot analysis for the biodistribution and clearance of Ac4ManNAz-LP.

irradiated at different conditions (Figure 4d). It was found that the combination treatment of PTT and PAT was highly effective in destructing cancer cells at the current conditions, while the single treatment of either PTT or PAT alone could only induce partial cell death. Significantly, the cell viability in the combination therapy group was substantially decreased to 37% with the concentration of DBCO-ZnPc-LP at 25 μg/mL, which was 78% lower than that of PTT alone and was 80% lower than that of PAT alone (Figure 4d). The results confirmed the considerable PTT and PAT synergistic effect could be achieved with DBCO-ZnPc-LP and laser in lower concentration and power dose, respectively. The synergistic effect of PTT and PAT was also verified by calcein AM and propidium iodide (PI) double-staining live (green) and dead cells (red), respectively (Figure 4e). PA imaging is a promising diagnostic imaging technique to monitor the molecular distribution and tumor size/morphology for therapeutical guidance due to its high resolution and noninvasive visualization of tissue structures. The significant photothermal and photoacoustic effects of DBCO-ZnPc-LP motivated us to use PA imaging to investigate the accumulation behavior of DBCO-ZnPc-LP in vivo. After intravenous injection of DBCO-ZnPc-LP into the A549 tumor-bearing mice which were pretreated with either saline or Ac4ManNAz-LP for 3 days, PA images of tumor region were recorded at times of

Encouraged by the high photothermal and photoacoustic effect and the remarkable chemical tumor-targeting performance, we then evaluated the in vitro synergistic phototherapeutic efficacy of our nanoagent with different treatment protocols in detail. The standard cell counting kit 8 (CCK-8) assay was conducted to assess quantitatively the cell viability after the different treatments. First of all, the viability of A549 cells treated with DBCO-ZnPc-LP or Ac4ManNAz-LP was determined under dark conditions, and the results revealed no significant cytotoxicity was observed for both nanoparticles even at high concentrations, indicating their good cytocompatibility (Figure 4a). Next, as shown in Figure 4b,c, when exposure to 808 nm continuous laser or pulsed laser, light dosedependent cell killing effect of DBCO-ZnPc-LP after incubated for 1 h was recorded. Notably, the viability of those cells pretreated with Ac4ManNAz-LP and then incubated with DBCO-ZnPc-LP was inhibited dramatically to 37.2% with 0.2 W/cm2 continuous laser for 5 min and 35% with 0.3 W/cm2 pulsed laser for 1 min. The results demonstrated that DBCOZnPc-LP has great potential for use as both photothermal and photoacoustic therapy agent for treatment of the target tumors. The cellular synergistic effect of PTT and PAT caused by DBCO-ZnPc-LP was then investigated. After pretreatment with Ac4ManNAz-LP for 3 days, cells were incubated with various concentrations of DBCO-ZnPc-LP for 1 h in dark and then 8936


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Figure 7. In vivo PTT/PAT in tumor-bearing mice. (a) Photographs of mice in different groups before and after therapy: I, PBS + PTT/PAT; II, Ac4ManNAz-LP + DBCO-ZnPc-LP + PTT; III, Ac4ManNAz-LP + DBCO-ZnPc-LP + PAT; and IV, Ac4ManNAz-LP + DBCO-ZnPc-LP + PTT/PAT. (b) Measured tumor growth for 18 days after treatments. (c) Body weight of mice after various treatments.

tumor cells.65 Thereby, the DBCO-ZnPc-LP with the assistance of Ac4ManNAz-LP for the targeting of tumors indeed causes photoheat conversion efficiently in vivo, and it can be used as hyperthermia agents for cancer therapy. However, it is worth noting that persistent and exorbitant temperature can also harm some organ tissue, which decreases efficacy of PTT. Next, we investigated further the in vivo behaviors of DBCOZnPc-LP by measuring Zn2+ levels in the tumors, various organs, and blood samples using inductively coupled plasma mass spectrometry (ICP-MS). As shown in Figure 6a, the ICPMS data showed a 3.4 times higher accumulation of the nanoparticles in this Ac4ManNAz-LP pretreated group (6.30% of ID) than the untreated group (1.85% of ID) at 6 h and a longer retention than the untreated group at 24 h, which agreed well with the PA imaging results and confirmed further the high delivery efficiency of our nanoagent DBCO-ZnPc-LP using this two-step targeting strategy. Blood circulation investigation showed that the blood levels of DBCO-ZnPc-LP reduced gradually over time but were maintained at a relatively high level even at 24 h postinjection (Figure 6b). Biodistribution data of DBCO-ZnPc-LP at different time intervals postinjection revealed that DBCO-ZnPc-LP accumulated relatively low in heart and lung (Figure 6c). The accumulation of DBCO-ZnPcLP in liver and spleen was determined to be ∼8.5 and ∼6.6% ID at 24 h postinjection and further decreased to rather low levels later on after 7 days. Despite of the reticuloendothelial systems (RES), in contrast, high levels of Zinc element were observed in the tumor. The tumor uptake was measured to be about 15.3% ID at 24 h postinjection. This is likely due to the PEG coating on the surface of DBCO-ZnPc-LP, which can delay their macrophage clearance during blood circulation66−68 and favor accumulation in the tumor by the dual targeting effect of the EPR effect and subsequent binding reaction with the “receptor-like” azide groups on tumors. Moreover, the feces and urine were also collected to study the possible clearance

preinjection (0 h), 6, 12, and 24 h postinjection. It indicated that the PA signal in the tumor sites substantially increased in the group pretreated with Ac4ManNAz-LP, which could be attributed to the dual targeting effect of EPR and the subsequent binding reaction with the “receptor-like” azide on tumors along with the extension of time (Figure 5a,c). In contrast, only a slight accumulation of DBCO-ZnPc-LP driven by the passive EPR targeting effect alone was observed in the group without pretreated Ac4ManNAz-LP. Moreover, the PA signal strength in this Ac4ManNAz-LP pretreated group still maintained a gradual increasing trend after 24 h postinjection of DBCO-ZnPc-LP, providing outstanding PA imaging for definition of the tumor region and precise guidance for the subsequent laser irradiation. On the other side, in order to confirm the in vivo hyperthermic effect induced by laser irradiation with DBCOZnPc-LP, the photothermal conversion kinetics were assessed in mice bearing A549 tumors. Here, as controls, group 1 received a saline injection only, group 2 received a DBCOZnPc-LP injection but without pretreated with Ac4ManNAzLP, and group 3 received a DBCO-ZnPc-LP injection and pretreated with Ac4ManNAz-LP. Photothermal imaging of those groups under irradiation of 808 nm contiuous laser at a power density of 0.1 W/cm2 was recorded using an IR thermal camera. As manifested in Figure 5b, the local temperature of tumors with DBCO-ZnPc-LP and Ac4ManNAz-LP injected increased rapidly up to 45 °C within 4 min, whereas the temperature was raised up to 38 °C in groups injected with DBCO-ZnPc-LP, and no noticeable temperature rose in the control group injected with saline, which was consistent with PA imaging results and confirmed the higher tumor targeting efficiency of the two-step strategy than the EPR effect alone. In view of PTT, controlled localized temperature increments have shown signs of apoptosis on tumor cells within the 40−45 °C temperature range (Figure 5d), which is high enough to ablate 8937


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Figure 8. Histological examination of tumor after various treatments indicated. (a) H&E stained tumor sections from different therapy groups. (b) Histological staining of the heart, liver, spleen, lung, and kidney from different groups.

contrast, the combination therapy of PTT/PAT showed efficient tumor inhibition with tumor volume shrinked persistently (Figure 7a,b). Additionally, the side effect of DBCO-ZnPc-LP was also assessed by monitoring of the body weight (Figure 7c). Measurement of the body weight of those mice in test showed neither obvious body weight loss nor noticeable abnormality, indicating no significant organism toxicity of our theranostic nanoagent. Then, hematoxylin and eosin (H&E) staining of tumor sections was performed after the phototreatment on day 18, as shown in Figure 8. In either the PTT or PAT treated group, the histological section revealed most malignant tumor cells with highly pleomorphic nuclei and many mitoses, indicating limited destruction from the laser treatment that led to revival of tumor growth. In stark contrast, intensive necrosis area stained by eosin dominated tumor section in the combined PTT and PAT treated group and pyknosis can also be observed under this situation, resulting from irreversible condensation of chromation in the nuclei of tumor cells undergoing necrosis or apoptosis. To further assess the toxicity of our nanoagent DBCO-ZnPc-LP, the major organs including heart, liver, spleen, lung, and kidney from the mice at 18 day after treatment were collected, and H&E staining analysis indicated no obvious histopathological abnormalities or lesions were discovered in three test therapy group compared with the control group, suggesting a reasonable safety margin without noticeable toxicity for the phototherapy application. In brief, this combination phototreatment of PTT and PAT has achieved enhanced synergistic therapeutic efficacy over that of PTT or PAT alone and obtained a desirable phototherapy outcome in vivo.

pathway of DBCO-ZnPc-LP (Figure 6d). High levels of Zn were detected in both urine and feces, indicating that DBCOZnPc-LP could be excreted through both renal and fecal, leaving very little retention of remaining Zn-species in the mouse body after 9 days, which was in accordance with the relative high accumulation levels of DBCO-ZnPc-LP in liver, spleen, and kidney. Next the metabolic pathway of the adjuvant agent Ac4ManNAz-LP was evaluated by Western blot analysis (Figure 6e). The Ac4ManNAz-LP was found to accumulate dramatically in tumors due to the EPR effect, and the kidneys (renal excretion) and the liver (biliary excretion) can be predicted to be responsible for the clearance of Ac4ManNAzLP. Taken together, the results indicated that our nanoagent DBCO-ZnPc-LP and its adjuvant agent Ac4ManNAz-LP should not show any further long-term toxicity to mice at this dose and is suitable for further in vivo phototherapy investigations. As a proof-of-concept, to verify the synergistic therapeutic effect of PTT and PAT in vivo, mice bearing A549 tumors with initial volumes of 100−150 mm3 were chosen and randomly divided into four groups (n = 5 per group), where one group was injected with saline as a control and the other three groups received DBCO-ZnPc-LP injected and Ac4ManNAz-LP pretreated and then went through corresponding treatments. For the PTT treatment, mice were irradiated with 808 nm continuous laser at the power density of 0.1 W/cm2 for 5 min, while the mice for the PAT treated group were irradiated with 808 nm pulsed laser at 0.2 W/cm2 to for 1 min. As depicted in Figure 7, compared with the control group, either PTT or PAT alone only inhibited partially the tumor growth in the first 8 days or 6 days but failed to inhibit tumor growth sustainedly over time at such a power density. In significant 8938


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Synthesis of DBCO-ZnPc-LP. DBCO-ZnPc-LP was synthesized according to our previous work.56 First, ZnPc (10 mg) was dissolved in THF (1 mL) and added dropwise to the DSPE-PEG2000-NH2 (20 mg) in deionized distilled water/THF (v/v, 10:1, 10 mL) with 150 μL/min. After stirring for 30 min at room temperature and sonication, the organic solvent was evaporated under vacuum. Then, the mixture was filtered through a syringe filter membrane (cellulose, 0.45 μm) to remove unloaded ZnPc. Then, to the dark green filtrate, DBCO-NHS (10 mg) in dimethyl sulfoxide (DMSO) (2 mL) was added dropwise and stirred overnight at room temperature. After dialyzed using a cellulose membrane (MWCO 3500 Da) for 72 h, the mixture was freeze-dried for 9 h, and the product was obtained as a dark green power. Finally, it was dispersed in deionized distilled water to produce the aimed nanoagent DBCO-ZnPc-LP solution (1 mg/mL of ZnPc). The loading capacity of ZnPc was measured to be 82% in the final DBCO-ZnPc-LP. The absorption coefficient of DBCO-ZnPc-LP at 680 and 808 nm was measured to be 17.6 and 7.4 L/g·cm, respectively. Preparation of Ac4ManNAz-LP. Ac4ManNAz and Ac4ManNAzLP were prepared according to the previous literature.51,74 Ac4ManNAz (5 mg/150 μL) in THF was added dropwise to the DSPE-PEG2000-NH2 (20 mg) in deionized distilled water/THF (v/v, 10:1, 10 mL) with 150 μL/min. After stirring for 30 min at room temperature and sonication, the organic solvent was evaporated under vacuum and then filtered through a syringe filter membrane (cellulose, 0.45 μm) and dialyzed using a cellulose membrane (MWCO 3500 Da) for 72 h. The mixture was freeze-dried for 4 h, and the product was obtained as a white power. Finally, it was dispersed in deionized distilled water (pH 7.4) to produce the nanomicelle Ac4ManNAz-LP solution (1 mM of Ac4ManNAz). The loading capacity of Ac4ManNAz was measured to be 90.8% using high-performance liquid chromatography (HPLC) in Ac4ManNAz-LP. Characterizations. The optical characteristics of ZnPc and DBCO-ZnPc-LP were investigated by UV−vis absorption spectra (Lambda-35 UV−vis spectrophotometer, PerkinElmer, MA, USA). Fluorescence spectra of ZnPc and DBCO-ZnPc-LP were investigated by an LS-55 fluorescence spectrophotometer (PerkinElmer). Fourier transform infrared (FT-IR) spectra were recorded with KBr pellets on a Bio-Rad FTS 6000 spectrometer (Bio-Rad Company, Hercules, California, USA) at room temperature. Raman spectra of samples were measured using a Renishaw inVia microspectrometer using an excitation wavelength of 514 nm. NMR spectra were recorded on a Bruker Ultrashield 400 Plus NMR spectrometer. The TEM images were collected on a field emission high-resolution 2100F transmission electron microscope (JEOL, Japan) operating at an acceleration voltage of 200 kV. The sizes and zeta-potentials of micelle nanoparticles were measured using ZEN3690 zetasizer (Malvern, USA). Photothermal temperature was recorded by an IR thermal camera (E50, IRS Systems). Photoacoustic signal intensity of DBCOZnPc-LP at various concentrations was recorded by a 10 MHz, 10 mJ/ cm2, 384-element ring ultrasound array, and an optical parametric oscillator (OPO) (Surelite II-20, Continuum, Santa Clara, CA, USA) with 4−6 ns pulse duration and 20 Hz pulse repetition rate used as the light source. Cell Culture. Human lung adenocarcinoma cells (A549) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Cell density was determined using a hemocytometer before experimentation. In Vitro Fluorescence/PA Imaging. For fluorescence imaging, DBCO-FITC was prepared by mixing DBCO-NH2 with FITC in DMSO at room temperature in dark condition for 6 h. A549 cells or LO2 cells were incubated with Ac4ManNAz (50 μM) or Ac4ManNAzLP (50 μM Ac4ManNAz) for 3 days and then washed with PBS (pH 7.4) for twice, followed by incubation with DBCO-FITC (25 μM) for 1 h in dark condition. Cells were rinsed twice with PBS and fixed with 4% formaldehyde solution for 10 min at room temperature and washed twice, and images were obtained using a confocal laser scanning microscope (ZEISS LSM 510 META, Germany). DBCOFITC was excited at 488 nm and recorded at 500−530 nm.

CONCLUSION A multifunctional phototheranostic based on a tumor-targeting nanoagent has been fabricated as an unreported example of integrating nanomaterial-enhanced PA imaging and synergistic PTT/PAT treatment of tumors. Investigations show that the NIR light can be absorbed by the facile-prepared phthalocyanine-containing nanoagent DBCO-ZnPc-LP and transferred into considerable amounts of heat energy and thermalenhanced ultrasonic shockwave to achieve a remarkable synergistic PTT/PAT effect. Moreover, the nanoagent DBCO-ZnPc-LP is demonstrated to be delivered efficiently to the tumor cells and tissues by intravenous injection with the two-step tumor-targeting strategy via metabolic glycoengineering and click chemistry. With the significant tumor-targeting strategy, PA imaging, and the synergistic effect of PTT and PAT, the solid tumors are precisely positioned and completely eradicated with little side effect in vivo. We believe that this research offers the proof of concept for PA imaging-guided synergistic PTT/PAT on target tumors. In view of the broad application of PTT for the tumor ablation,69−72 in the future, this integration is expected to produce a powerful platform for the image-guided phototherapeutic alliance with significant therapeutic effect. EXPERIMENTAL SECTION Materials. 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamineamino carboxyl poly(ethylene glycol) (DSPE-PEG-NH2) was obtained from Avanti Polar Lipids Inc. (AL, USA); cell-counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan); 4nitrophthalonitrile fluorescein isothiocyanate (FITC), calcein-acetylmethoxylate (calcein-AM), and propidium iodide (PI) were obtained from Sigma-Aldrich Corporation (MO, USA). DBCO-NHS and DBCO-NH2 were purchased from Click Chemistry Tools (Scottsdale, AZ). Phosphine-PEG3-biotin, streptavidin-horseradish peroxidase (streptavidin-HRP), and streptavidin-FITC were purchased from Thermo Scientific (Rockford, IL, USA). All other chemicals used in this work were of analytical grade. All reagents were used without further purification. Synthesis of ZnPc. ZnPc was synthesized according to the previous literature.73 Briefly, 4-nitrophthalonitrile (1.73 g, 10 mmol) and nonyl alcohol (7.21 g, 50 mmol) were dissolved in anhydrous DMF (50 mL) under nitrogen atmosphere and stirred for 1 h at 60 °C. Then K2CO3 (5 g, 36 mmol) was added in three batches within 3 h. The mixture was stirred for 24 h under these conditions and monitored by thin-layer chromatography (TLC). When the reaction was completed, deionized water (150 mL) was added and stirred for further 30 min. Then chloroform (100 mL) was added for extraction four times, and the organic phase was collected and dried by anhydrous Na2SO4. After the solvent was removed under vacuum, the residue was collected and purified using a silica gel column eluted with dichloromethane and petroleum ether (v/v, 20:6) to afford the intermediate 4-nonoxylphthalonitrile 1.51 g (55.6%). Then, the intermidate (1.08 g, 4 mmol) was dissolved in anhydrous n-amyl alcohol (10 mL) under nitrogen atmosphere with stirring, and 1,8diazabicycloundeca-7-ene (DBU, 1.5 mL) was added. After the solution was stirred for 1 h at 140 °C, zinc(II) acetate dihydrate (0.438 g, 2 mmol) was added and stirred for 24 h at this temperature. After that, the reaction mixture was cooled down to room temperature and poured into a mixture of methanol/water (1:1, v/v, 50 mL). The formed precipitate was collected and washed with methanol for three times. The crude product was purified using a silica gel column eluted with petroleum ether and tetrahydrofuran (THF) (v/v, 20:8) to afford the final product as a dark green solid 0.33 g (28.9%). 1H NMR (CDCl3, 400 MHz): δ 7.47 (s, 2H), 6.13−7.05 (m, 10H), 3.47 (s, 8H), 1.62 (s, 6H), 1.39 (s, 46H), 1.25 (s, 4H), 0.99 (s, 12H). MALDI-TOFMS for [M]+ of C68H92N8O8O4Zn: calcd, m/z 1144.6220; found, m/z 1144.6224. UV−vis (THF, 25 °C): log ε (λmax) = 5.01 L/mol·cm. 8939


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PBS (pH 7.4) for twice, followed by incubating with DBCO-ZnPc-LP (25 μg/mL of ZnPc) for 1 h. After rinsing twice with PBS, cells were illuminated using a continuous laser (808 nm, 5 min) or pulsed laser (808 nm, 1 min) with different power density (0, 0.1, 0.2, 0.3, 0.4 W/ cm2). All group cells were incubated for another 24 h, the cell viability was detected by added of CCK-8 (10 μL), and the absorbance at a wavelength of 450 nm of each well was measured using a microplate reader (Infinite 200, TECAN, Switzerland). For the synergistic therapy, cells in 96-well plates at the density of 1 × 104 cells/well were preincubated with Ac4ManNAz-LP (50 μM of Ac4ManNAz) for 3 days, then cultured with different concentrations of DBCO-ZnPc-LP (0, 6.25, 12.5, 25, 50 μg/mL) for 1 h. Each group of cells were conducted with various treatments: control, PTT, PAT, PTT + PAT. PTT groups were irradiated by 808 nm continuous laser for 5 min at the power density of 0.1 W/cm2, while cells of the PAT groups were irradiated by 808 nm pulsed laser for 1 min at the power density of 0.2 W/cm2. Cells were further incubated for 24 h before the standard CCK-8 essay was conducted. For the co-staining of live and dead cells, cells were seeded in 35 mm plates at the density of 5 × 104 cells and incubated with Ac4ManNAz-LP (50 μM of Ac4ManNAz) for 3 days and then cultured with DBCO-ZnPc-LP (25 μg/mL of ZnPc) for 1 h. The cells were given four treatments mentioned previously: control, PTT, PAT, and PTT + PAT. After incubation for 12 h, each plate was incubated with 1 mL of dye solution (2 μM calcein AM and 4 μM PI), co-stained for 30 min at 37 °C, washed 3 times using PBS, and imaged by a confocal laser scanning microscope (calcein AM λex = 488 nm, λem = 515 nm; PI λex = 535 nm, λem = 617 nm). Animal Model. All animal experiments were performed in compliance with institutional guidelines of animal use and care and approved by institutional committees. 4-week-old female BALB/c nude mice were purchased from the Animal Experiment Center, Southern Medical University (20 g). A549 tumor cells (1.0 × 106 cells) suspended in PBS (100 μL) were subcutaneously injected into the flanks of each mice. When the tumors reached approximately about 100 mm3, animals were used in the experiments. The longest and shortest dimensions of tumors were monitored by digital calipers. In Vivo PA/PT Imaging. Ac4ManNAz-LP (40 mg/kg of Ac4ManNAz) was injected into the tail vein of A549 tumor-bearing mice once a day for 4 days as the testing group, and another group was injected with saline as the control. Then DBCO-ZnPc-LP (2.5 mg/kg of ZnPc) was injected into the tail vein of the above mouse. The mice were imaged and analyzed at 1, 6, 12, and 24 h after injection. Photoacoustic imaging was performed by a photoacoustic computed tomography system equipped with a 10 MHz, 10 mJ/cm2, 384element ring ultrasound array, and an optical parametric oscillator (OPO) (Surelite II-20, Continuum, Santa Clara, CA,USA) with 4−6 ns pulse duration and 20 Hz pulse repetition rate used as the light source. For photothermal imaging, the mice were treated similar to photoacoustic imaging treatment. The mice were divided into three groups, one group as the control group, one group with only intravenous injection of DBCO-ZnPc-LP (2.5 mg/kg of ZnPc), and another group with intravenous injection of DBCO-ZnPc-LP (2.5 mg/ kg of ZnPc) and pretreatment with Ac4ManNAz-LP (40 mg/kg of Ac4ManNAz). The A549 tumor bearing mice were irradiated by 808 nm continuous laser at 0.1 W/cm2 for 5 min 24 h after DBCO-ZnPcLP injection. Tumor surface temperature and thermal imaging with time were recorded by an IR thermal camera (E50, IRS Systems). In Vivo Accumulation Analysis. Saline or Ac4ManNAz-LP (40 mg/kg of Ac4ManNAz) was injected into the tail vein of A549 tumorbearing mice once a day for 4 days, as mentioned above. Then DBCOZnPc-LP (2.5 mg/kg of ZnPc) was injected into the tail vein of two groups (n = 3 per group). Then the mice were sacrificed at 6, 12, and 24 h postinjection. Tumors were dissected, digested in aqua regia, and measured for Zn content by ICP-MS to evaluate the delivery efficiency of DBCO-ZnPc-LP. The amount of Zn element was measured by Elan DRC II inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer, USA). In Vivo Kinetics, Biodistribution, and Clearance. A549 tumorbearing mice were intravenously injected with Ac4ManNAz-LP (40 mg/kg of Ac4ManNAz) once a day for 4 days, followed by DBCO-

For photoacoustic imaging, A549 cells were seeded onto 35 mm glass-bottom dishes at a density of 5 × 104 cells and incubated with Ac4ManNAz (50 μM) or Ac4ManNAz-LP (50 μM of Ac4ManNAz) for 3 days, then washed with PBS (pH 7.4) twice, followed by incubating with DBCO-ZnPc-LP (25 μg/mL of ZnPc) for 1 h. Fixation was done with the same conditions, and PA imaging was performed by photoacoustic computed tomography system. Western Blot Analysis of Cells. A549 cells were pretreated with Ac4ManNAz (50 μM) or Ac4ManNAz-LP (50 μM of Ac4ManNAz) for 3 days and washed with PBS (pH 7.4) twice, then harvested from the plates and centrifuged at 1800 rpm for 3 min. Cells were resuspended and incubated in 100 μL of lysis buffer (1% SDS, 100 mM Tris·HCl, pH 7.4) containing protease inhibitor cocktail (Complete, EDTA-free) at 4 °C for 30 min and centrifuged for 20 min at 11000 rpm to get supernatant solution. The solution lysate (5 mg/mL, 50 μL) were incubated with phosphine-PEG3-biotin (5 μL, 5 mM in DPBS) for 6 h at 37 °C. Then the samples were resolved by 12% SDS/PAGE and transferred to pure nitrocellulose blotting membranes (BioTrace NT; Pall Life Science, Pensacola, FL, USA). The membranes were blocked in 10 mM Tris/HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween-20 containing 5% nonfat milk for 1.5 h. Then, the membrane was incubated with streptavidin-HRP (diluted 1:2000 in TBST) overnight at 4 °C. The membrane was developed by using HPR-AEC Western Blotting Substrate. Flow Cytometry Analysis. A549 cells or LO2 cells were pretreated with Ac4ManNAz (50 μM) or Ac4ManNAz-LP and followed with DBCO-FITC, as mentioned above. Then, cells were lifted and washed with FACS buffer. 10,000 cells per sample were performed on a FACScanto II flow cytometer (Becton Dickinson, Mountain View, CA, USA) with excitation at 488 nm. Immunofluorescence Microscopy. A549 cells were incubation with Ac4 ManNAz (50 μM) or Ac4 ManNAz-LP (50 μM of Ac4ManNAz) for 3 days on 35 mm glass-bottom dishes, then fixed for 10 min in 4% paraformaldehyde, followed by permeabilization with 0.15% Triton X-100 in NaCl/Pi for 15 min. Cells were washed with PBS (pH 7.4) twice and incubated with phosphine-PEG3-biotin (50 μM) for 6 h. Finally, the cells were washed three times for 10 min each in blocking buffer and incubated with streptavidin-FITC (30 μM) for 2 h. Images were acquired with a confocal laser scanning microscope (ZEISS LSM 510 META, Germany) Real-Time PCR for mRNA Expression Analysis. A549 cells were incubated with Ac4ManNAz (50 μM) or Ac4ManNAz-LP (50 μM of Ac4ManNAz) for 3 days. Total mRNA was isolated from A549 cell lines using a commercial RNAiso Plus for RNA extracting kit (TakaRa, China). All operations followed the kit protocol, and the obtained small RNA was quantified by measuring the optical density at 260 nm/ 280 nm and then diluted with RNase-free water and stored at −80 °C. The reverse transcriptase (RT) reactions contained the total mRNA (800 ng, extracted from cell lines), 1 × RT buffer, 0.25 mM each of dNTPs, 10 U/μL RevertAid Premium reverse transcriptase, and 1 U/ μL RNase inhibitor, and the total volume is 20 μL. The RT reaction was carried out in the thermocycler at 37 °C for 60 min and 95 °C for 3 min. The quantitative real time PCR (qPCR) was performed using SYBR Green I fluorescence dye. The 20 μL real-time PCR reaction contained 1 μL of RT product, 1 × SYBR Green qPCR Mix, 0.8 μM forward primer. and 0.4 μM reverse primer. The reaction was carried out in Applied Biosystems real-time PCR at 95 °C for 3 min and 40 cycles of 95 °C for 5 s and 60 °C for 30 min. The sequences of primers used for qPCR analysis are as follows: NRCAM (5′-CGAGGCGTCTGAGCAGTAT TT-3′ and 5′-CATTCAAGGGCTTCCACGT-3′), MYO1C (5′-GCGGTGCCTGTTGTGAAATA-3′ and 5′-T GGCGTAATCAATCCTCTGCT-3′), and CNTN1 (5′-AACCTAAAGCTGCACCGAAAC-3′ and 5′-CTGCTGCTAT TGACAAGCCACT-3′). qPCR primers were commercially synthesized and purified by Sangon Biotechnology Co. (Shanghai, China). Cytotoxicity Evaluation in Vitro. Relative cell viabilities were determined by the standard thiazolyl tetrazolium (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay. A549 cells were seeded into 96-well plates at the density of 1 × 104 per well and incubated with Ac4ManNAz-LP (50 μM of Ac4ManNAz) for 3 days, then washed with 8940


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ACS Nano ZnPc-LP (2.5 mg/kg of ZnPc) injection. For blood circulation measurement, blood samples were collected at 0.5, 1, 1.5, 2, 2.5, 4, 6, 12, and 24 h postinjection, respectively. The Zn levels in blood were measured by ICP-MS and presented as the percentage of injected dose per gram of blood (% ID/g). For biodistribution measurement, the mice were sacrificed at various time points postinjection (each group has three mice as replicates), and then major organs of those mice were harvested at 1, 12, 24, 48, 72, and 168 h and solubilized by aqua regia for ICP-MS measurement to determine Zn contents. For excretion measurement, urine and feces of each mouse were collected at various time points and then solubilized by aqua regia for ICP-MS measurement. Western Blot Analysis of Tissues. A549 tumor bearing mice were intravenously injected with Ac4ManNAz-LP (40 mg/kg of Ac4ManNAz) once a day for 4 days. The mice were sacrificed 24, 48, 72, and 96 h postinjection, and the major organs and tumors were dissected, incubated with 500 μL lysis buffer (1% SDS, 100 mM Tris· HCl, pH 7.4) containing protease inhibitor cocktail at 4 °C for 30 min, and removed insoluble debris by centrifugation for 20 min at 11000 rpm. Then, Western blot analysis was performed with phosphinePEG3-biotin by the same method as the cell lysate analysis. In Vivo Combination Therapy. A tunable pulsed laser with a repetition rate of 20 Hz and a pulse width of 4 ns (Nd:YAG SurelightII-20 connected to Surelite OPO Plus, spectral tuning range 675−1000 nm, Continuum) was used as the light source for PAT with a laser fluence of ≈20 mJ cm−2. Typically, the spot size of the pulsed laser and continuous laser was adjusted as ∼0.8 cm2, and the output power was thereby set as 0.16 and 0.08 W for PAT and PTT, respectively. For in vivo therapy, mice bearing A549 tumors were randomly divided into four groups (n = 5 per group). Ac4ManNAz-LP (40 mg/ kg Ac4ManNAz) was injected into the tail vein of tumor bearing mice once a day for 4 days. Then, DBCO-ZnPc-LP (2.5 mg/kg of ZnPc) was injected into the tail vein of the above mouse. After 24 h, the mice treated with different therapy: Control (i), 808 nm continuous laser (0.1 W/cm2, 5 min) (ii), 808 nm pulsed laser (0.2 W/cm2, 1 min) (iii), 808 nm continuous laser (0.1 W/cm2, 5 min), and 808 nm pulsed laser (0.2 W/cm2, 1 min) (iv). The tumor sizes were measured using a digital caliper every other day, and tumor volume (V) calculated as V = (tumor length) × (tumor width)2 /2. Relative tumor volumes were counted as V/V0 (V0 was the initial tumor volume). Besides, the body weight of each mouse was weighted every other day using a digital balance and monitored the mice survival. H&E staining was performed according to a protocol provided by the vendor (BBC Biochemical). After the experiments were finished, the mice were sacrificed. The tumors and major organs (heart, liver, spleen, lung, and kidney) of the mice in four groups were retrieved and cut into 4 μm sections, then fixed in 4% formaldehyde solution for 8 h at room temperature, dehydrated with ethanol, and embedded with paraffin after slicing and staining with H&E, and then studied using an inverted fluorescence microscope (Nikon E200).

ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (21405052), the Program for Changjiang scholars and Innovative Research Team in University (IRT0829), the Key Program of the National Natural Science Foundation of China (613300033), and the Natural Science Foundation of Guangdong Province, China (2014A030310255). REFERENCES (1) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869−10939. (2) Jaque, D.; Maestro, L. M.; Del Rosal, B.; Gonzalez, P. H.; Benayas, A.; Plaza, J. L.; Rodríguez, E. M.; Solé, J. G. Nanoparticles for Photothermal Therapies. Nanoscale 2014, 6, 9494−9530. (3) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380−387. (4) Melamed, J. R.; Edelstein, R. S.; Day, E. S. Elucidating the Fundamental Mechanisms of Cell Death Triggered by Photothermal Therapy. ACS Nano 2015, 9, 6−11. (5) Chen, Q.; Xu, L.; Liang, C.; Wang, C.; Peng, R.; Liu, Z. Photothermal Therapy with Immune-Adjuvant Nanoparticles Together with Checkpoint Blockade for Effective Cancer Immunotherapy. Nat. Commun. 2016, 7, 13193. (6) Zhang, S.; Guo, W.; Wei, J.; Li, C.; Liang, X.; Yin, M. Terrylenediimide-Based Intrinsic Theranostic Nanomedicines with High Photothermal Conversion Efficiency for Photoacoustic ImagingGuided Cancer Therapy. ACS Nano 2017, 11, 3797−3805. (7) Zhang, C.; Bu, W.; Ni, D.; Zuo, C.; Cheng, C.; Li, Q.; Zhang, L.; Wang, Z.; Shi, J. A Polyoxometalate Cluster Paradigm with SelfAdaptive Electronic Structure for Acidity/Reducibility-Specific Photothermal Conversion. J. Am. Chem. Soc. 2016, 138, 8156−8164. (8) Zhang, S.; Sun, C.; Zeng, J.; Sun, Q.; Wang, G.; Wang, Y.; Wu, Y.; Dou, S.; Gao, M.; Li, Z. Ambient Aqueous Synthesis of Ultrasmall PEGylated Cu2‑xSe Nanoparticles as a Multifunctional Theranostic Agent for Multimodal Imaging Guided Photothermal Therapy of Cancer. Adv. Mater. 2016, 28, 8927−8936. (9) Zhang, J.; Yang, C.; Zhang, R.; Chen, R.; Zhang, Z.; Zhang, W.; Peng, S.; Chen, X.; Liu, G.; Hsu, C. S.; Lee, C. S. Biocompatible D-A Semiconducting Polymer Nanoparticle with Light-Harvesting Unit for Highly Effective Photoacoustic Imaging Guided Photothermal Therapy. Adv. Funct. Mater. 2017, 27, 1605094. (10) Feder, M. E.; Hofmann, G. E. Heat-Shock Proteins, Molecular Chaperones, and the Stress Response: Evolutionary and Ecological Physiology. Annu. Rev. Physiol. 1999, 61, 243−282. (11) Li, L.; Chen, C.; Liu, H.; Fu, C.; Tan, L.; Wang, S.; Fu, S.; Liu, X.; Meng, X.; Liu, H. Multifunctional Carbon-Silica Nanocapsules with Gold Core for Synergistic Photothermal and Chemo-Cancer Therapy under the Guidance of Bimodal Imaging. Adv. Funct. Mater. 2016, 26, 4252−4261. (12) Huang, S.; Duan, S.; Wang, J.; Bao, S.; Qiu, X.; Li, C.; Liu, Y.; Yan, L.; Zhang, Z.; Hu, Y. Folic-Acid-Mediated Functionalized Gold Nanocages for Targeted Delivery of Anti-miR-181b in Combination of Gene Therapy and Photothermal Therapy against Hepatocellular Carcinoma. Adv. Funct. Mater. 2016, 26, 2532−2544. (13) Lv, R.; Yang, P.; He, F.; Gai, S.; Li, C.; Dai, Y.; Yang, G.; Lin, J. A Yolk-like Multifunctional Platform for Multimodal Imaging and Synergistic Therapy Triggered by a Single Near-Infrared Light. ACS Nano 2015, 9, 1630−1647. (14) Yan, Y.; Björnmalm, M.; Caruso, F. Particle Carriers for Combating Multidrug-Resistant Cancer. ACS Nano 2013, 7, 9512− 9517. (15) Xing, R.; Liu, K.; Jiao, T.; Zhang, N.; Ma, K.; Zhang, R.; Zou, Q.; Ma, G.; Yan, X. An Injectable Self-Assembling Collagen-Gold Hybrid Hydrogel for Combinatorial Antitumor Photothermal/Photodynamic Therapy. Adv. Mater. 2016, 28, 3669−3676.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03226. Synthetic scheme, characteristics, additional graphs and images (Figures S1−S17) (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Da Xing: 0000-0002-5098-0487 Notes

The authors declare no competing financial interest. 8941


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