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Plasmonic Resonance Energy Transfer Enhanced Photodynamic Therapy with Au@SiO2@Cu2O/Perfluorohexane Nanocomposites Conghui Liu, HaiFeng Dong, Nianqiang Wu, Yu Cao, and Xueji Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00112 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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ACS Applied Materials & Interfaces

Plasmonic Resonance Energy Transfer Enhanced Photodynamic Therapy with Au@SiO2@Cu2O/Perfluorohexane Nanocomposites Conghui Liu, † Haifeng Dong, †,* Nianqiang Wu, ‡ Yu Cao, † Xueji Zhang†,* †

Research Center for Bioengineering & Sensing Technology, School of Chemistry &

Biological Engineering, University of Science & Technology Beijing, Beijing 100083, P. R. China. ‡

Department of Mechanical and Aerospace Engineering, West Virginia University,

Morgantown, West Virginia 26506-6106, USA.

KEYWORDS:

photodynamic therapy, plasmonic resonance energy transfer,

core-shell structure, self-enriched oxygen, perfluorocarbon

ABSTRACT: photosensitizers

Reactive and

oxygen

hypoxia

species

(ROS)

microenvironment

generation in

solid

efficiency tumor

of

hampers

photodynamic therapy (PDT) efficacy. Here, we introduce an efficient inorganic photosensitizer by incorporating plasmonic gold metal nanostructures into Cu2O semiconductors for PDT. By utilizing the plasmon-induced resonance energy transfer (PIRET) process from Au to Cu2O, the Au@SiO2@Cu2O (ASC) demonstrates a high singlet oxygen quantum yield of 0.71 under a 670 nm laser irradiation. The ASC is 1 ACS Paragon Plus Environment

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loaded into oxygen self-enriched perfluorohexane (PFH) droplets and coated with liposome

(Lip)

to

form

Lip(ASC/PFH)

nanocomposites.

The

achieved

(Lip(ASC/PFH) shows considerable anti-cancer efficacy for in vitro cancer cells and in vivo tumor growth. The proposed oxygen self-enriched PIRET-PDT concept has significant implication in PDT design.

Introduction Cancer burden is becoming one of the biggest challenges with aging and booming of the world population.1 Currently, chemotherapy and radiation therapy are two major clinic treatments. Both frequently induce high side effects and unexpected systemic toxicity.2,

3

Alternatively, photodynamic therapy (PDT) shows great potential in

meeting currently unmet medical needs.3-5Using wavelength-selected light-activated photosensitizers, PDT kills cancer cells by converting oxygen to high level singlet oxygen (1O2).6,

7

The later can quickly induce significant cytotoxicity, leading to

selective disruption of malignant tumors via apoptosis or necrosis.6, 8 PDT treatment of cancer requires the presence of photosensitizers with advanced 1O2 generation efficiency in the sufficient oxygen-containing environment.9

Organic photosensitizers are commonly used in PDT. However, they suffer from photo-induced and enzymatic degradation. Hence inorganic photosensitizers with better stability have been actively developed for PDT. Inorganic semiconductors such as TiO2, 10, 11 ZnO12 and CdSe13, 14 hold great potential as photosensitizers due to their tunable absorption spectra from ultraviolet (UV) to near-infrared (NIR) by changing 2 ACS Paragon Plus Environment

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particles size and chemical composition.13 Nevertheless, most of the reported inorganic photosensitizers are only activated by narrow ultraviolet-visible (UV-Vis) light, which has potential significant side effect in therapeutics and poor tissue penetration.15 The rapid development of surface plasmon resonance (SPR)16-19 has provided a new opportunity to design efficient inorganic photosensitizers with longer absorption wavelength by incorporating plasmonic metal nanostructures into semiconductors for PDT.

Recently

it

has

been

reported

that

plasmonic

metal/semiconductor

Au@SiO2@Cu2O (ASC) exhibit much higher photocatalytic activity than both pure Cu2O and [email protected] Incorporating plasmonic Au nanoparticles with Cu2O greatly extends the light absorption to the red–to-NIR region, of which the penetration depth into tissues is greatly improved than UV-Vis light.21,

22

Importantly, it efficiently

enhances the generation of electron−hole pairs in the Cu2O shell through a unique charge separation mechanism of plasmon-induced resonance energy transfer (PIRET), and consequently improves the photocatalytic activity.19,

20, 23

Meanwhile, Cu2O

exhibits effective ability to absorb O2 to consume the photogenerated electrons during water photocatalysis.24, 25 The generated electrons in Cu2O are readily donated to O2 to produce 1O2 since the conduction band (Ec) of Cu2O (1.92 eV) is higher than the redox potential (EH) of 1O2/ O2 (1.88 eV).26 In principle, the ASC nanostructure with the PIRET charge separation mechanism could serve as novel efficient photosensitizers for PDT, involving a similar process to photocatalysis.

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On the other side, hypoxia microenvironment in solid tumors is another challenge for PDT treatment because oxygen, an essential element, is insufficient.3, 27 Several strategies such as in situ generation of O2 from endogenous hydrogen peroxide using catalyst,28-31 enhancing intrablood flow for increasing the amount of available O2 by normalizing

tumor

vasculatures,32,

33

have

been

reported

to

oxygenate

microenvironment for improved therapeutics outcome. However, these approaches still suffer from either limited available amount of endogenous hydrogen peroxide or low O2 delivery capability of the red blood cell in the tumor vasculatures. A recent oxygen self-enriching photodynamic therapy strategy is promising to overcome these problems and to improve the PDT efficacy.34

In the present work, a unique Au@SiO2@Cu2O/ perfluorohexane nanocomposite, in which the plasmonic Au@SiO2@Cu2O photosensitizer is loaded into the perfluorohexane (PFH) nanodroplet coated with liposome (Lip(ASC/PFH), is designed to utilize the plasmonic effect combined with the oxygen self-enriching effect. (Scheme 1). By utilizing the PIRET process from the plasmonic Au to Cu2O,20, 23

the ASC core-shellnanostructure demonstrated much stronger 1O2 production

capacity than either Cu2O or Au@Cu2O nanoparticles under irradiation of the 670 nm laser (0.48 W cm-2) at an ultralow dose (20 µg/mL of ASC). The PFC maintain a higher oxygen content than the tumor matrix at a given oxygen partial pressure due to its high oxygen capacity,27, 34-36 which provided enriched oxygen for enhanced PDT. As a result, the Lip(ASC/PFH) displayed great PDT efficacy in treatment of cancer cells, multicellular tumor spheroids and solid tumors. 4 ACS Paragon Plus Environment

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Results and Discussion ASC NPs were synthesized according to previously methods with slight modification (See detailed in Experimental Section ).20, 37, 38 The transmission electron microscope (TEM) images of Figure 1a-c and S1 confirmed the morphology and size of the core-shell ASC NPs. The core-shell ASC NP was consisted of 15 nm diameter Au NPs coated with a ~3 nm thick SiO2 layer and a ~30 nm thick Cu2O shell in Figure 1a-c, which was consistent with the observation of dynamic light scattering in Figure S2a, S2b. The interfacial high resolution TEM (HRTEM) image in Figure 1d clearly confirmed the core-shell structure. Au core was single crystalline with clear exposed (111) plane with lattice fringe spacing of 0.23 nm, while the Cu2O was composed of small crystalline grains with a lattice fringe of 0.24 nm corresponding to the (111) plane. The different orientations of lattice fringes in Cu2O indicated that the Cu2O shell was polycrystalline. There were not distinct lattice fringes observed in the SiO2, indicating amorphous structure.20,

39

We also investigated the components of the

core-shell structure by X-ray photoelectron spectroscopy (XPS). As shown in Figure 1g, the XPS survey spectrum of ASC confirmed the chemical composition agreeing well with previous reports.20,

40

Figure 1h, 1i and S3 presented the detailed

information of elemental composition. In the high resolution XPS spectrum of Cu 2p (Figure 1h), a prominent Cu+ 2p3/2 peak (931.7 eV) and a strong Cu+ 2p1/2 (951.5 eV) can be observed, confirming the successful formation of Cu2O.41 While the weak shake-up satellite peak (about 945.4 eV) revealed the existence of small amount of



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Cu2+.42 No signal of Au 4f can be detected in Figure 1i, which may be shielded by the thick Cu2O shell.20

As control, Au@Cu2O core-shell nanostructure with a 15-nm diameter of Au NPs core directly coated with a ~30 nm thick Cu2O uniform shell was also achieved (Figure 1e). Figure 1f and S4a presented the UV-Vis absorbance spectra and optical images of different core-shell nanoparticles. The Au NPs displayed strong Localized SPR (LSPR) at 520 nm in Figure 1f, which had a negligible red-shift to 523 nm after coating with the SiO2 layer. The introduction of Cu2O caused large red-shift to 680 nm and 650 nm for Au@Cu2O and ASC, respectively, which was resulted from the increasing effective dielectric constant between the Au core and Cu2O shell.20,

43

Importantly, the spectral overlap of Au core with Cu2O shell extends light absorption to wider and longer red–to–NIR wavelengths,

20, 23

which are beneficial to biological

application. The 1O2 production capacity of ASC was first evaluated by the chemical probe of 1, 3-diphenylisobenzofuran (DPBF).44, 45 We compared the 1O2 generation of ASC to Cu2O, Au@SiO2 and Au@Cu2O. As shown in Figure 2b, the time-dependent absorption at 410 nm of pure Cu2O showed negligible change similar to control group. Compared to the pure Cu2O, the downtrend of Au@Cu2O at 410 nm absorption showed enhancement within 10 minutes, and that of ASC was much more steeply within 10 minutes under irradiation (670 nm, 0.48 W cm-2), indicating that ASC performed best in 1O2 production among these materials. Electron spin resonance 6 ACS Paragon Plus Environment

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(ESR) measurements were further employed to identify the 1O2 generation of ASC using 2, 2, 6, 6-tetramethylpiperidine (TEMP) as 1O2 trapping agent. It can be clearly observed that 1:1:1 triplet ESR signal of 1O2 for the ASC after irradiation with 670 nm laser, while no distinct signals can be detected for ASC without irradiation. These results effectively confirmed the 1O2 production ability of ASC (Figure 2c). To further quantify the 1O2 generation capability, a commercial photosensitizer of Chlorin e6 (Ce6) was used as the reference (1O2 quantum yield, ΦCe6 ≈ 0.65 in ethanol46). As shown in Figure 2d and Figure S5a, the absorbance intensity of DPBF gradually decreased with time after irradiation for both ASC and Ce6 solution due to the 1O2 generation. The rate constant for DPBF decomposition by ASC and Ce6 was 0.13 min−1 and 0.034 min−1 respectively. The corresponding 1O2 quantum yield was calculated to be 0.71 (See detailed calculation method and Figure S5a-c in Supporting Information ), which was slightly higher than that of Ce6, suggesting the good 1O2 generation efficacy of ASC. The mechanism of enhanced 1O2 generation of ASC core-shell structure was schematically described in Figure 2a. Generally, semiconductor metal-oxide NPs can only be photo-excited when their band gap (Eg) is less than the energy of incident light.26 Therefore, pure Cu2O NPs (Eg=2.2 eV) undoubtedly cannot be excited by the 670 nm laser (about 1.94 eV) for 1O2 generation. In the ASC and Au@Cu2O nanostructure, the energy of incident photons was converted into localized surface plasmon resonance oscillations by Au core, and then the generated plasmonic energy was transfered to the Cu2O semiconductor shell and directly induced the generation of 7 ACS Paragon Plus Environment


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electron-hole pairs in Cu2O via PIRET process, although the incident photon’s energy was below the band gap of Cu2O.

19, 20, 23

Then the photogenerated electrons in Cu2O

reacted with O2 to generate 1O2 for PDT.20 It is worth noting that the insulating layer of SiO2 between the Au NP core and Cu2O shell prevents the interfacial damping and the back charge transfer as well as the dephasing of the plasmon from hot-electron transfer. Hence the presence of the silica layer ensures the non-radiative transfer of plasmonic energy from the Au NP core to Cu2O, 19, 20, 23 lead to more efficient charge separation in Cu2O and enhanced 1O2 generation in PDT by ASC compared with that of [email protected], 23, 47 The PIRET mechanism in ASC nanostructure endowed it with high 1O2 generation capability for acting as PDT photosensitizers. To overcome the hypoxia microenvironment and ensure PDT efficacy, the resulted ASC was further loaded into PFH nanodroplets coated with liposome to fabricate an oxygen self-enriching Lip(ASC/PFH) nanostructure. The average diameter of resulting Lip(ASC/PFH) was ~193 nm (Figure S2d), which was appropriate for prolonged blood circulation and efficient accumulation in tumor for PDT due to the enhanced permeability and retention (EPR) effect.30,

48

In addition, the aqueous diameter of Lip(ASC/PFH)

showed negligible change in DMEM cell media and buffer solution with a pH ranging from 4 to 9 (Figure S6), which indicated good pH stability of Lip(ASC/PFH). The time-dependent degradation of DPBF at 410 nm caused by 1O2 generated by Lip(ASC/PFH) was also invesgated. Remarkably, the absorbance around 410 nm for Lip(ASC/PFH) solution dropped much more quickly than Lip(ASC) (Figure 3a, 3b). 8 ACS Paragon Plus Environment

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The rate constant for DPBF decomposition by Lip(ASC/PFH) was 0.17 min−1 (Figure S5d), and the corresponding 1O2 quantum yield was 1.01 referenced to Ce6. (See detailed calculation method and Figure S5d and S5e in Supporting Information). The higher 1O2 quantum yield of Lip(ASC/PFH) than that of ASC demonstrated the enhancement of PFH for 1O2 generation in Lip(ASC/PFH). Under irradiation with 670 nm laser, the absorbance of Lip(ASC/PFH) at 670 nm almost kept the same within 20 min, indicating good photostability of Lip(ASC/PFH) in the process of PDT (Figure S7). The cellular uptake of Lip(ASC/PFH) was then studied by incorporating a fluorescent and hydrophobic Ce6 to the hydrophobic liposomal lipid bilayer.27, 49 As shown in Figure S8, intracellular Ce6 fluorescence gradually increased in Ce6 modified- Lip(ASC/PFH)-treated MCF-7 cells along with the incubation time increasing. The cells exhibited strong fluorescence after 4 h, which efficient intracellular uptake of Lip(ASC/PFH). Singlet Oxygen Sensor Green (SOSG), a kind of fluorescent dye that can generate green fluorescence after oxidized by 1O2, was further used to investigate the effect of intracellular 1O2 generation efficacy of the proposed nanostructures. As shown in Figure 3c, the PBS-treated MCF-7 cells hardly showed any fluorescence related to 1O2 generation. The ASC (20 µg/mL) treated MCF-7 cells presented stronger fluorescence than the control group, while the Lip(ASC/PFH) (con-staining 20 µg/mL ASC) treated MCF-7 cells displayed strongest green fluorescence. These results suggested the powerful 1O2 production ability of ASC, the pivotal role of PFH in 1O2 generation and the potential of Lip(ASC/PFH) in vivo PDT treatment. 9 ACS Paragon Plus Environment


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Furthermore, in order to more directly confirm the strong oxygen carrying capacity of PFH, the oxygen concentration for Lip(ASC) and Lip(ASC/PFH) nanocomposites was evaluated. After addition of Lip(ASC) or Lip(ASC/PFH) into deoxygenated water under N2 atmosphere, the real-time oxygen concentration was measured by an oxygen electrode probe (Figure 3d), and the corresponding oxygen concentration when reached equilibrium was given in Figure 3e. The initial oxygen concentration of Lip(ASC) and Lip(ASC/PFH) was calculated to be 5.01 mg/L and 30.21 mg/L, respectively, and the oxygen loading capacity of PFH was calculated to be 168 mg/L (or 5.25 mmol/L) (see specific calculation method in Supporting Information), which suggested the good oxygen carrying capacity of PFH in line with previous reports.34, 50

We further performed an in vitro and in vivo hypoxia condition experiments to

demonstrate the oxygen self- enriched ability of Lip(ASC/PFH) for enhanced PDT performance. As shown in Figure S9, Lip(ASC/PFH) showed significant phototoxicity to MCF-7 cells in both normoxic and hypoxic conditions. On the contrary, the phototoxicity of Lip(ASC) to MCF-7 cells in hypoxic conditions was obviously reduced than that in the air condition because of inadequate supply of oxygen. Hence, these results suggested the oxygen self-enriched effect of Lip(ASC/PFH) nanocomposites could guarantee PDT efficacy in vitro. The remarkable 1O2 generation ability of Lip(ASC/PFH) inspired us to explore its PDT treatment effect of anti-cancer. The cytotoxicity of pure Cu2O, Au@Cu2O, ASC and Lip(ASC/PFH) at different concentrations with or without irradiation were first measured using a standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium 10 ACS Paragon Plus Environment

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bromide (MTT) viability assay (Figure S10a,b) and the corresponding half maximum inhibiting concentration (IC50 value) of each group was given in Figure S10c. All groups showed a dose-dependent cytotoxicity against MCF-7 cells, and the Lip(ASC/PFH) displayed a lowest IC50 value of 13.18 µg/mL under irradiation. We further compared the cytotoxicity and phototoxicity of Lip(ASC/PFH) with that of pure Cu2O, Au@Cu2O and ASC at the same concentration of 20 µg/mL. As shown in Figure 4a, the Cu2O itself presented significant cytotoxicity, in agreement with previous reports51-53 toward MCF-7 cells without irradiation, while Au@Cu2O, ASC displayed similar slight cytotoxicity to Lip(ASC/PFH) in the same condition. After irradiation with laser (670 nm, 0.48 W cm-2), the cells viability of PBS, Cu2O and Au@Cu2O-treated groups showed negligible changes compared to their counterparts without irradiation. Conversely, the ASC displayed obvious anti-cancer ability for MCF-7 cells, and Lip(ASC/PFH) showed strongest anti-cancer effect with the cell viability decreased to less than 20%. It is worthy to mention that the good performance was realized at the ultralow dosage of Lip(ASC/PFH).

The Calcein AM/ Prodium Iodide (PI) cell-survival assay distinguishing live (green) from dead (red) cells was further used to investigate the anti-cancer effect of Lip(ASC/PFH). The Lip(ASC/PFH) presented no effect on cells viability without irradiation, remarkably, most of the Lip(ASC/PFH)-treated cells were killed under irradiation due to the PDT effect (Figure 4c). It was also confirmed that the cytotoxicity of Lip(ASC/PFH) under irradiation was connected with apoptosis or necrosis54, 55 according to the fluorescence imaging of Annexin V-FITC/PI staining 11 ACS Paragon Plus Environment


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assays (Figure 4d). The caspase-3 protein activity, acted as a apoptosis initiator,56 was investigated by colorimetry using the substrate of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), which can be catalyzed by caspase-3 to generate yellow p-nitroanilide (pNA) with strong absorption at 405 nm. It was found that the laser irradiation induced the significant increase of caspase-3 protein activity in MCF-7 cells treated with Lip(ASC) or Lip(ASC/PFH) (Figure S11). Especially, the Lip(ASC/PFH)-treated MCF-7 cells irradiated with laser showed a 3.6-folds higher activity than that of without irradiation (Figure 4b). Furthermore, flow cytometry was used to quantitatively analyze the PDT-mediated cell death by Annexin V-FITC/PI dual fluorescence staining. As shown in Figure 4e, lower left, lower right, upper right and upper left in each panel represented living cells, early apoptotic cells, late apoptotic cells and necrotic cells respectively. Without laser irradiation, the proportion of dead and apoptosis of cells treated with PBS, Lip(ASC) and Lip(ASC/PFH) was 0.3%, 2.59% and 2.45% respectively, which indicated negligible cytotoxicity. After exposure to a laser, the abovementioned proportion was increased to 2.18%, 70.24% and 81.62%, respectively. The enhanced PDT performance of Lip(ASC/PFH) was a result of oxygen self-enriched effect of PFH. These results suggested Lip(ASC/PFH)-mediated PDT induced the significant apoptosis and death of cancer cells. What’s more, the photothermal effect on the death of cancer cells is negligible due to the slight temperature change of Lip(ASC/PFH) solution under irradiation (Figure S12). These results above prove that Lip(ASC/PFH) is an


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excellent photosensitizer for PDT anti-cancer treatment, which was attributed to the PIRET enhanced 1O2 generation efficacy and oxygen self-enriching PFH.

To identify the feasibility of Lip(ASC/PFH) for in vivo PDT therapeutics, we chose the multicellular tumor spheroids (MCTS) of MCF-7 cells as model. MCTs are spherical aggregation of tumor cells and generate by a liquid overlay cultivation technique, which adequately mimic and reflect the crucial properties of solid tumors and the microenvironment.30, 54, 57, 58 In our experiment, MCTS were incubated with PBS or Lip(ASC/PFH) for 24 h, then were co-stained by Calcein-AM and PI. As shown in Figure S13a, the PBS-treated MCTS showed strong green fluorescence from Calcein-AM in the whole tumor spheres that with or without irradiation. As for the Lip(ASC/PFH)-treated MCTS, although strong green fluorescence from Calcein-AM related to live cells can be observed before irradiation, while the whole tumor spheres exhibited were strong red fluorescence from PI (Figure S13b), suggesting that most of cancer cells were killed. These results reveal that Lip(ASC/PFH) is a promising photosensitizer for PDT in vivo anti-cancer treatment in term of its ultralow dosage and excellent PDT therapeutic effect.

The good in vitro anti-tumor efficiency of Lip(ASC/PFH) encouraged us to further investigated its in vivo performance using BALB/c nude mice (female, 4 weeks old) with MCF-7 tumor xenografts. The ability of Lip(ASC/PFH) to overcome hypoxia in vivo was first invesgated by conducting a hypoxia-probe (pimonidazole) immuno-histochemical assay. The nuclei and hypoxia areas were stained with DAPI 13 ACS Paragon Plus Environment


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(blue) and anti-pimonidazole antibody (green), respectively. As shown in Figure 5a and 5b, the MCF-7 tumors treated with PBS (pH=7.4, 10 mM) combined with or without irradiation, and Lip(ASC) without irradiation showed similar strong pimonidazole-stained (green) hypoxic signals, revealing hypoxic microenvironment in tumors. The laser irradiation induced the increase of green hypoxic signal for the Lip(ASC)-treated group, indicating the oxygen consumption in the PDT treatment. Notably, the hypoxic signals of tumors only treated with Lip(ASC/PFH) significantly reduced to 2.3%. After laser irradiation, the hypoxic signals were also lower than the group treated with Lip(ASC) and laser irradiation, powerfully proving that the strong oxygen carrying capacity and efficient PDT performance of Lip(ASC/PFH).

The blood circulation time and biodistribution in main organs of Lip(ASC/PFH) were then investigated. Blood samples were collected from the tail of mice received preinjection of Lip(ASC/PFH) at different time points, and the concentration of copper in the blood samples was measured by ICP-MS. As shown in Figure 6A, the concentrations of copper in the blood samples gradually decreased with the increase of the time, which was consistent with a two-compartment model. The measured first (t1/2) and second (t1/2) half-lives were 0.41 and 7.37, respectively, indicating relative long blood circulation time of Lip(ASC/PFH). It was beneficial to Lip(ASC/PFH) tumor accumulation for in vivo PDT. In order to quantitatively investigate the biodistribution of Lip(ASC/PFH), the mice received preinjection of Lip(ASC/PFH) for 24 h were sacrificed and the main organs were collected, and the concentration of copper was then analyzed by ICP-MS. Figure 6B revealed that 14 ACS Paragon Plus Environment

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Lip(ASC/PFH) nanocomposites were mainly distributed in reticuloendothelial systems such as the liver, spleen, and lung of mice. Notably, the copper content of tumor was also at a relatively high level, indicating that Lip(ASC/PFH) nanocomposites could be passively homed to tumor through the EPR effect.

We further studied the in vivo anti-tumor performance of Lip(ASC/PFH). As shown in Figure 6c and 6d, the laser and PBS showed negligible inhibition effect on tumor growth as expected. Without laser irradiation, neither Lip(ASC) nor Lip(ASC/PFH) showed obvious inhibition to tumor growth, similar to PBS control group. On the contrary, the tumor growth of mice injected with Lip(ASC) and treated with a 670 nm laser irradiation were significantly inhibited. The mice injected with Lip(ASC/PFH) and exposed to the laser exhibited the most effective tumor growth suppression among all six groups, indicating the advanced anti-tumor effect of the Lip(ASC/PFH)-mediated PDT. In addition, no obvious changes of the body weights of the mice were observed over a period of 14 days (Figure 6e).

Hematoxylin and eosin (H&E) staining was performed after various treatments (Figure 7). It was revealed that, significant antitumor effects and cell apoptosis in the Lip(ASC/PFH) and Lip(ASC)-treated and combined with the laser irradiation groups, especially the former, almost the cells were apoptotic/dead. On the contrary, no obvious antitumor growth behaviors were observed for the other groups. The H&E staining assay of the five major organs (heart, liver, spleen, lung, and kidney) after mice were scarified revealed no histopathological abnormalities, and the minimal 15 ACS Paragon Plus Environment


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systematic toxicity of Lip(ASC/PFH) indicated the good biocompatibility of Lip(ASC/PFH) with irradiation.

Conclusion In conclusion, a oxygen self-enriched plasmonic nanocomposite was developed by loading the Au@SiO2@Cu2O nanoparticles into PFH droplets and coating liposome to form Lip(ASC/PFH). Remarkable PDT therapeutic effect of Lip(ASC/PFH) nanocomposites was demonstrated on cancer cells and solid tumors, including high 1

O2 quantum yield of ASC, considerable oxygen carrying capacity of PFH,

photocytotoxicity of Lip(ASC/PFH) with ultralow dosage against cancer cells in vitro, and distinct anti-cancer effect on the tumor growth of MCF-7 tumor-bearing mouse. The enhanced PDT performance of Lip(ASC/PFH) was attributed to the PIRET-enhanced charge generation in the Cu2O shell and to a high oxygen content maintained in perfluorohexane. The attractive oxygen self-enriched PIRET-PDT can be exploited as a promising platform for cancer treatments, which provides new insight into the design of PDT systems.

Experimental Section

Materials and Reagents: HAlCl4·3H2O was obtained from Acros Oganics (Geel, Belgium).

Sodium

silicate

solution

(Na2SiO3),

perfluorohexane

(PFH),

N-(3-(dimethylamino)-propyl)-N-ethylcarbodiimide hydrochloride crystalline (EDC), Nhydroxysuccinimide (NHS) and thiazolyl blue tetrazolium blue (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA). Aminopropyltrimethoxysilane 16 ACS Paragon Plus Environment

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(APS) was obtained from J&K Scientific Co., Ltd. (Beijing, China). Copper chloride (CuCl2), sodium hydroxide (NaOH), hydroxylamine hydrochloride (NH2OH·HCl), sodium dodecyl sulfate (SDS), dichloromethane (CH2Cl2) and trisodium citrate dehydrate (Na3C6H5O7·2H2O) were purchased from Beijing Chemical Reagents Company (Beijing, China). Lecithin and cholesterol were supplied by Aladdin Industrial Corporation (Shanghai, P. R. China China). DSPE-PEG2000 was obtained from A.V.T. Pharm. Ltd (Shanghai, China). Singlet Oxygen Sensor Green was obtained from Molecular Probes, Inc. (Shanghai, China). 1,3-diphenylisobenzofuran (DPBF) was obtained from Alfa Aesar (Shanghai, China). Phosphate-buffered saline (PBS, pH=7.4, 10 mM), penicillin-streptomycin, standard fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Life Technologies Corporation (Los Angeles, CA, USA). Deionized (D.I.) water was generated using a Millipore Milli-Q system (Billerica, MA, USA). 4% Paraformaldehyde Fix Solution and Caspase 3 Activity Assay Kit) were purchased from Beyotime Biotechnology.

Characterizations: The morphology of nanoparticles was examined with an FEIF20 transmission electron microscope (TEM) (FEI, USA). The X-ray-photoelectron spectroscopy (XPS) analysis was recorded with an ESCALAB 250 spectrometer (Thermo-VG Scientific, USA). The UV−Visible (UV−Vis) absorption analysis was examined with an UV-1800 spectrophotometer (Shimadzu, Japan). Dynamic light scattering analysis (DLS) was performed on Nano ZS (Malvern, UK). All fluorescence measurements were performed on a confocal laser scanning fluorescence 17 ACS Paragon Plus Environment


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microscope (CLSM, FV1200, Olympus, Japan). 1O2 was quantified by an ESR spectrometer (JEOL FA-200). The oxygen concentrations in solutions were measured by a portable dissolved oxygen meter (METTLER TOLEDO, SevenGo pro, China). Flow cytometry (Beckman Coult, cytoflex) was used to analyze cell apoptosis and death. The concentration of Cu in blood samples and tissues was conducted by inductively coupled plasma source mass spectrometer (ICP-MS) (7700X, Agilent).

Synthesis of Au@SiO2 nanoparticles: Au@SiO2 nanoparticles were synthesized according to previously reported methods with some modification following by three main steps.37 Firstly, 6.6 mL of 1 wt% sodium citrate was added to 100 mL of 0.02 wt% HAuCl4 solution at boiling point to obtain the Au nanoparticles (~15 nm). Then, a freshly prepared methanol solution of APS (0.5 mL, 1 mM) was added to the resulting solution under vigorous magnetic stirring for 20 min followed by standing for 15 min. Finally, 4 mL of 0.54 wt % Na2SiO3 aqueous solution was added to 100 mL of the surface-modified Au solution under vigorous magnetic stirring for 10 min. The resulting dispersion was standed for two days before use.

Synthesis

of

Au@SiO2@Cu2O

(ASC)

nanoparticles:

The

ASC

core-shellnanoparticles were synthesized according to a previous report with some modification.20, 38 Briefly, 12.3 mL D.I. water, 0.2 mL of 0.1M CuCl2 solution, 5 mL of 0.06M SDS solution, 0.5 mL of Au@SiO2 solution and 0.5 mL of 1M NaOH solution were added into a conical flask in sequence. After dropping 1.5 mL of 0.2 M NH2OH·HCl with shaking, the solution changed from blue to light green. The color 18 ACS Paragon Plus Environment

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finally changed to yellow-green when the mixture was aged for 2 h. The aged solution was washed with D.I. water and centrifuged four times at 10000 rpm for 10 min to remove the surfactant. Finally, the precipitate was dispersed in 4 mL of ethanol. The synthesis procedure for the Au@Cu2O nanoparticles was similar to the ASC except that the Au@SiO2 solution was replaced with Au nanoparticles solution. Synthesis of Lip(ASC/PFH): Lip(ASC/PFH) was synthesized as follows.34 First, solution containing 24.65 mg lecithin, 4.28 mg cholesterol and 3.79 mg DSPE-PEG2000, the resulting mixture was sonicated to obtain milky mixture. 5 mL of Au@SiO2@Cu2O (400 µg/mL) solution was then added into the mixture under stirring for 10 min. Then, lipid film was formed by rotary evaporation and peeled off by sonication for 10 min with 8.5 mL pure water. Next, 1.5 mL PFH was gradually added and dispersed into the peeled lipid film solution with under high-speed dispersion (IKA, T25, German) at 25,000 r.p.m in an ice bath for 25 min to form 10 mL Lip(ASC/PFH) (15 v/v% PFH). Lip(ASC) was formed as the similar procedure of Lip(ASC/PFH) except that addition of pure water instead of PFH.

Singlet Oxygen Generation Efficiency Measurement: The DPBF was used as probe, and Chlorin e6 (Ce6, 1O2 quantum yield ΦCe6 ≈ 0.65 in ethanol) 46 was used as reference (ref) to measure the 1O2 generation efficiency of Lip(ASC/PFH). Before measurement, the UV-vis absorption maxima of ethanol solution containing sample was adjusted to ≈0.2 OD. Then, DPBF solution (20 µL, 10 mM) was added to the sample solution (2 mL) with irradiation (670 nm, 0.48 W cm-2) for 10 min. The 19 ACS Paragon Plus Environment


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decomposition rates of DPBF by sample was recorded every minute, and quantified by the absorbance intensity change of DPBF at 410 nm. The 1O2 quantum yield of ASC is calculated by the following equation:

ࢶ࡭ࡿ࡯ =

࢘࡭ࡿ࡯ /࡭࡭ࡿ࡯ ࢶ ࢘࡯ࢋ૟ /࡭࡯ࢋ૟ ࡯ࢋ૟

Where ΦASC and ΦCe6 are the 1O2 quantum yield of ASC and Ce6, respectively. rASC and rCe6 are the photodecomposition rates of DPBF caused by ASC and Ce6 under irradiation, respectively (Figure 2d, Figure S5a). AASC and ACe6 are the integration of the optical absorption bands of ASC and Ce6 in the wavelength range 450–700 nm, respectively (Figure S5b and S5c). Measurement of O2 release36: 1 mL Lip(ASC/PFH) (20 µg/mL) was added into 20 mL deoxygenated water under N2 atmosphere. The oxygen electrode probe inserted into deoxygenated water was used to measure real-time oxygen concentration of the solution under N2 atmosphere. Lip(ASC) (20 µg/mL) was also measured as control. The O2 concentration of Lip(ASC/PFH)=[C1*V(Lip(ASC/PFH)+Deoxygenated Deoxygenated water)]/V(Lip(ASC/PFH).

water)

– C3*V

The O2 oxygen loading capacity per 1 mL PFH =

(C1-C2)*V (Lip(ASC/PFH)+Deoxygenated water )/VPFH. Cytotoxicity assay: The MCF-7 cells were seeded in 96-well plates (104 cells per well) and cultured in DMEM culture medium containing 10% FBS and 1% penicillin/streptomycin at 37 oC under 5% CO2 for 24 h. Then, the cells were treated with pure Cu2O, Au@Cu2O, ASC and Lip(ASC/PFH) at different concentrations for 4 h and then irradiated by a 670 nm laser (0.48 W cm-2) for 10 min. After incubation for 20 ACS Paragon Plus Environment

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4 h, the media were replaced with fresh culture media and the cells were further incubated for 24 h. Then, 10 µL MTT solution (5 mg/mL MTT in PBS, pH 7.4) was added to each well and incubated for 4 h to measure the absorbance at 492 nm by microplate reader. The control was either the cells without addition of any nanocomposite or free of laser irradiation.

In vitro Hypoxic Experiment: MCF-7 cells seeded in 96-well plates were incubated in DMEM medium containing PBS, Lip(ASC) or Lip(ASC/PFH) (20 µg/mL). After 4 h, the 96-well plates of hypoxia groups were placed in a transparent box and ventilated with N2 for 20 min. Then the 96-well plates were irradiated under a 670 nm laser (0.48 W cm−2) for 10 min at N2 atmosphere condition. Finally, cells were transferred into fresh media and conducted with a standard MTT test to measure the relative cell viabilities after incubated for 12 h.

In vitro Cellular Uptake of Lip(ASC/PFH): To examine the cellular uptake of Lip(ASC/PFH) nanocomposites, Ce6-modified Lip(ASC/PFH) was prepared. Commercial Ce6 was conjugated with hexylamine in the presence of EDC and NHS to prepare hydrophobic Ce6 according to previous reports.27, 49 Other procedures were the same as preparation of Lip(ASC/PFH) except that the addition of 3 mg of hydrophobic Ce6 along with 24.65 mg lecithin, 4.28 mg cholesterol and 3.79 mg DSPE-PEG2000 to 5 mL CH2Cl2. MCF-7 cells seeded in culture dishes were incubated with Ce6-modified Lip(ASC/PFH) for different time periods (0 h, 1 h, 2.5 h, and 4 h), the resulting cells were washed thoroughly with PBS (10 mM, pH=7.4) and 21 ACS Paragon Plus Environment


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treated with 4% paraformaldehyde fix solution for 15 min. Afterwards, the cells were incubated with Hoechst 33342 for another 15 min, and the cells washed with PBS imaged using CLSM. SOSG Imaging of Intracellular 1O2: Intracellular 1O2 generation of the prepared nanocomposites irradiated by 670 nm laser (0.48 W cm-2) was detected using SOSG. MCF-7 cells (1 × 105 cells/mL) were seeded in culture dishes and incubated in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 oC under 5% CO2 for 24 h. The medium was replaced by OPTI-MEM containing PBS (pH=7.4, 10 mM) or Lip(ASC/PFH) (20 µg/mL) or ASC (20 µg/ml) and cultured for 4 h, and then the medium was replaced by fresh DMEM culture medium to incubate for 12 h. 2 µL of Hoechst 33342 and 2 µL of SOSG (1 mg/mL) were then added and incubated for 15 min respectively, the resulting cells were washed twice with PBS (pH=7.4, 10 mM) and irradiated with 670 nm laser (0.48 W cm-2) for 10 min. Afterwards, the medium was replaced with PBS (pH=7.4, 10 mM) and the cells were imaged using a CLSM. Intracellular PDT Performance: MCF-7 cells (2 × 105 cells/mL) were seeded on culture dishes and cultured for 24 h as aforementioned. The medium was replaced by OPTI-MEM containing PBS or Lip(ASC/PFH) (20 µg/mL) and cultured for 4 h. The medium was replaced with fresh DMEM culture medium to incubate for 12 h. Calcein-AM (2 µL) and PI (2 µL) was added and incubated for 15 min. Then the cells were washed twice with PBS (pH=7.4, 10 mM) and irradiated with a 670 nm laser 22 ACS Paragon Plus Environment

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(0.48 W cm-2) for 10 min. The medium was replaced with PBS (pH=7.4, 10 mM) and the cells were imaged using a CLSM.

Cell Apoptosis Analysis: The transfection of Lip(ASC/PFH) into MCF-7 cells were processed as aforementioned in vitro PDT performance. The transfected cells were stained by the Annexin V-FITC/ PI for 15 min and irradiated by a 670 nm laser (0.48 W cm-2) for 10 min. Then the fluorescence images of treated cells were acquired under a CLSM after incubating for 12 h. To quantitatively analyze the PDT-mediated cell apoptosis and death, flow cytometry analysis of MCF-7 cells with different treatments were further investigated. After seeding of MCF-7 cells in six-well plates (105 cells per well) for 24 h, and incubating with PBS (pH=7.4, 10 mM), Lip(ASC) or Lip(ASC/PFH) (20 µg/mL) for another 4 h, the cells of PDT groups were irradiated with a 670 nm laser (0.48 W cm-2) for 10 min and incubated for another 12 h. Afterwards, the cells were digested by trypsin and transferred into 1.5 mL plastic centrifuge tubes, and the Annexin V-FITC/ PI was added and incubated for another 15 min. Finally, the cells were analyzed by flow cytometry (Beckman Coult, cytoflex).

Caspase-3 Protein Activity Measurement: Caspase-3 Activity Assay Kit was purchased from Beyotime, and the measurement was performed according to the manufacturer’s instruction. Briefly, we measured the absorbance at 405 nm of pNA with different concentrations (0, 10, 20, 50, 100, 200 µM) by a microplate reader to obtain a standard curve. MCF-7 cells were seeded in six-well plates (106 cells per well) and incubated for 12 h, and the DMEM cell medium was replaced with 1 mL 23 ACS Paragon Plus Environment


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Opti-MEM solution containing PBS, Lip(ASC) or Lip(ASC/PFH) (20 µg/mL). After incubation for 4 h, cells were rinsed by PBS twice and treated with trypsin. Then, 1.5 mL of cold PBS was added to each well, and the solutions were centrifuged at 600 g for 5 min at 4 0C. The resulting cells were resuspended in 100 µL of lysate and kept on ice for 15 min. After centrifugation at 16,000 rpm for 10 min at 4 oC, the protein of the supernatant was determined by Bradford protein assay and adjusted the supernatant to enable the same concentration of protein. Afterwards, 90 µL of the protein solution mixed with 10 µL Ac-DEVE-pNA was added in each well and incubated for 2 hour at 37 oC. The real absorbance at 405 nm of pNA catalyzed by the caspased-3 in each sample was obtained by subtracting the OD value of the control blank group from the measured OD value.

Multicellular Tumor Spheroids (MCTSs) PDT Experiments: The MCTSs were generated according our previous report.54 The resulting MCTSs were incubated with PBS or Lip(ASC/PFH) (20 µg/mL) for 4 h. Then, MCTSs were washed with PBS and cultured for another 24 h. MCTSs were then stained with Calcein-AM and PI and irradiated a 670 nm laser (0.48 W cm-2) for 10 min for confocal imaging.

Tumor Models: MCF-7 cancer bearing female Balb/c mice (4 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and used under protocols approved by the Department of Laboratory Animal Science of Peking University Health Science Center. To build the MCF-7 tumor model, 2×106 MCF-7 cells in 100 µL PBS (pH=7.4, 10 mM) was subcutaneously injected to the 24 ACS Paragon Plus Environment

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right axilla of each mouse. When the tumor sizes reached about 50∼80 mm3, the mice were randomly divided into 6 groups and used for in vivo experiments (5 mice in each group). Then, the mice were intravenously injected with PBS, Lip(ASC) (200 µL, 20 µg/mL in terms of ASC) or Lip(ASC/PFH) (200 µL, 20 µg/mL in terms of ASC). After 24 h, the mice were irradiated with a 670 nm laser (0.48 W cm-2) for 10 min or without any treatment. The tumor sizes and body weight were recorded every other day for two weeks. The tumor sizes was calculated as followss: V = Width2×Length/2. The relative tumor volumes were normalized by the tumor volumes of the first day. After 14 days, the cancer tissues and the main organs were collected from sacrificed mice for histological analysis.

In Vivo Biodistribution: MCF-7 cancer bearing mice were intravenously injected with Lip(ASC/PFH) (200 µL, 20 µg/mL in terms of ASC). 50 µL of blood was extracted every time from the tail of each mouse at indicated time points (1, 2, 4, 8, 12, 24 h) after intravenous injection, the collected blood were weighted and then dissolved in digesting chloroazotic acid (HNO3:HCl = 3:1) to analyze the amount of copper using ICP-MS. After intravenous injection for 24 h, the mice were sacrificed and the liver, spleen, kidney, heart, lung and tumor of sacrificed mice were collected, weighted, and solubilized in chloroazotic acid to analyze the amount of copper using ICP-MS. As control, the copper levels in different organs of untreated mice were also measured.



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In Vivo Anti-Tumor Performance:To investigate the in vivo anti-tumor performance of Lip(ASC/PFH)-mediated PDT, MCF-7 tumor-bearing mice were randomly divided into 6 groups (5 mice in each group) when the tumor sizes reached 50∼80 mm3: group 1: PBS (mice injected with PBS only); group 2: PBS + laser (mice irradiated with a 670 nm laser after injection of PBS); group 3: Lip(ASC) (mice injected with Lip(ASC) only); group 4: Lip(ASC) + laser (mice irradiated with a 670 nm laser (0.48 W cm-2) after injection of Lip(ASC)); group 5: Lip(ASC/PFH) (mice injected with Lip(ASC/PFH) only); group 6: Lip(ASC/PFH) + laser (mice irradiated with a 670 nm laser after injection of Lip(ASC/PFH)). At 24 h postinjection, the group 2, 4 and 6 were irradiated with a 670 nm laser (0.48 W cm-2) for 10 min. The tumor sizes and the body weights of mice were recorded every other day without extra irradiation.

Immunohistochemistry: MCF-7 cancer bearing mice were intravenously injected with PBS, Lip(ASC) (200 µL, 20 µg/mL in terms of ASC) or Lip(ASC/PFH) (200 µL, 20 µg/mL in terms of ASC) and irradiated with a 670 nm laser (0.48 W cm-2) for 10 min after 24 h. These mice were then intraperitoneally injected with pimonidazole HCl solution (60 mg kg−1) (HypoxyprobeTM-1 plus kit, Hypoxyprobe Inc), which was reductively activated in hypoxic cells and formed stable adducts with thiol (sulfhydryl) groups in proteins, amino acids, and peptides. After 90 min, tumors were surgically excised and stained with a fluorescein-conjugated mouse IgG1 monoclonal antibody (MAb clone 4.3.11.3) as primary antibodies to label tumor hypoxia regions.


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Afterward, the slices were then stained with peroxidase conjugated anti-FITC secondary protocol and imaged using CLSM.

ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge. Additional characterization results (TEM, DLS, XPS and UV−Vis absorption) (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by Special Foundation for State Major Research Program of China (Grant Nos. 2016YFC0106602 and 2016YFC0106601); National Natural Science Foundation of China (Grant No. 21645005, 21475008); the Open Research Fund Program of Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University (PRRD-2016-YB2). 27 ACS Paragon Plus Environment


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Scheme 1. Schematic illustration of Lip(ASC/PFH) for singlet oxygen generation during PDT under laser irradiation.



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Figure 1. Characterization of the core-shell nanoparticles. (a-c) Transmission electron micrographs (scale bars: 10 nm) of uncoated Au nanoparticles, Au@SiO2, ASC, respectively. (d) The interfacial HRTEM image of ASC. (e) Transmission electron micrographs (scale bars: 10 nm) of Au@Cu2O. (f) UV-Vis absorbance spectra. (g) XPS survey spectrum and high resolution XPS of (h) Cu 2p and (i) Au 4f of ASC nanoparticles.

Figure 2. 1O2 generation measurement. (a) Schematic diagram of charge separation in ASC core-shell structure. (b) Time-dependent degradation of DPBF at 410 nm caused by 1O2 generated by Cu2O, Au@SiO2, Au@Cu2O and ASC under 670 nm laser irradiation. (c) ESR spectra of ASC in the presence of TEMP with or without irradiation for 10 min (670 nm, 0.48 W cm−2). (d) Time-dependent degradation of


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DPBF caused by 1O2 generated by ASC under irradiation (670 nm, 0.48 W cm−2, 10 min). The insets are rate constants for DPBF decomposition in the presence of ASC.

Figure 3. Comparing singlet oxygen production capacity in vitro. (a) Time-dependent degradation of DPBF caused by 1O2 generated by Lip(ASC/PFH) under irradiation (670 nm, 0.48 W cm−2, 10 min). (b) Time-dependent degradation of DPBF at 410 nm caused by 1O2 generated by ASC, Lip(ASC) and Lip(ASC/PFH) under 670 nm laser irradiation. (c) Detection of 1O2 in Hoechst 33342- and SOSG-costained MCF-7 cells respectively treated with PBS, ASC and Lip(ASC/PFH) under 670 nm laser irradiation for 10 min. Scale bars: 20 µm. (d) O2 concentration changes over time in 20 mL deoxygenated water after addition of 1 mL Lip(ASC) or Lip(ASC/PFH) (20 39 ACS Paragon Plus Environment


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µg/mL) under a N2 atmosphere. (e) The corresponding oxygen concentration of Lip(ASC/PFH), Lip(ASC) and deoxygenated water when the oxygen concentration reached the equilibrium of (d).


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Figure 4. Photodynamic therapy in vitro. (a) Cytotoxicity of different samples in the presence or absence of irradiation (670 nm, 0.48 W cm-2). (b) Relative activity multiple of caspase-3 protein in MCF-7 cells activated by Lip(ASC) and Lip(ASC/PFH) (20 µg/mL) with or without a 670 nm irradiation for 10 min. (c) 41 ACS Paragon Plus Environment


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Fluorescence images of Calcein AM- and PI-costained MCF-7 cells treated with Lip(ASC/PFH) to differentiate live (green fluorescence) and dead cells (red fluorescence) with or without 670 nm irradiation. Scale bars: 40 µm. (d) Fluorescence images of MCF-7 cells treated with Lip(ASC/PFH) and stained with Annexin V-FITC/ PI under 670 nm irradiation. Scale bars: 40 µm. (e) Flow cytometric analysis results of cell apoptosis of MCF-7 cells treated with PBS, Lip(ASC) and Lip(ASC/PFH) (20 µg/mL) with or without 670 nm irradiation for 10 min. Inserted numbers in the profiles indicate the percentage of the cells present in this area.

Figure 5. (a) Immunofluorescence images of tumor slices stained by the hypoxia-probe. The nuclei and hypoxia areas were stained with DAPI (blue), and anti-pimonidazole antibody (green), respectively. Scale: 200 µm. (b) Quantification of hypoxia area of tumor according to (a).


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Figure 6. Photodynamic therapy in vivo. (a) Blood circulation and (b) biodistribution of Lip(ASC/PFH) after intravenous injection into MCF-7 tumor-bearing mice by measuring Cu concentrations with ICP-MS. (c) Photographs of the mice taken before treatment (0 day) and at 14 days with different treatments, and tumors collected from different groups of mice at 14 days. (d) Tumor growth curves and (e) body weight curves of mice. Laser irradiation was conducted at 24 h after injection by a 670 nm laser (0.48 W cm−2) for 10 min.



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Figure 7. H&E stained slices of tumor tissues and other organs from different groups collected at 14 days. Scale bar: 100 µm.


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