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Bioinspired Hybrid Protein Oxygen Nanocarrier Amplified Photodynamic Therapy for Eliciting Anti-Tumor Immunity and Abscopal Effect Zhikuan Chen, Lanlan Liu, Ruijing Liang, Zhenyu Luo, Huamei He, Zhihao Wu, Hao Tian, Mingbin Zheng, Yifan Ma, and Lintao Cai ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04371 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018
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Key Lab for Health Informatics, Shenzhen Engineering Laboratory of Nanomedicine and Nanoformulations Cai, Lintao; Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences, Guangdong Key Laboratory of Nanomedicine, CAS Key Lab for Health Informatics, Shenzhen Engineering Laboratory of Nanomedicine and Nanoformulations
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Bioinspired Hybrid Protein Oxygen Nanocarrier Amplified Photodynamic Therapy for Eliciting Anti-Tumor Immunity and Abscopal Effect Zhikuan Chen1, 3, ‡, Lanlan Liu1, 3, ‡, Ruijing Liang1, ‡, Zhenyu Luo1, 3, Huamei He1, Zhihao Wu1, Hao Tian 2, Mingbin Zheng1, 2, *, Yifan Ma1 and Lintao Cai1, *
1
Guangdong Key Laboratory of Nanomedicine, CAS Key Lab for Health Informatics,
Shenzhen Engineering Laboratory of Nanomedicine and Nanoformulations, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences, Shenzhen 518055, P. R. China 2
Dongguan Key Laboratory of Drug Design and Formulation Technology, Key Laboratory for
Nanomedicine, Guangdong Medical University, Dongguan 523808, P. R. China 3
University of Chinese Academy of Sciences, Beijing 100049, P. R. China
‡
These authors contributed equally to this work.
*
Correspondence and requests for materials should be addressed to L.C. (
[email protected])
and M.Z. (
[email protected]).
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ABSTRACT An ideal cancer therapeutic strategy is expected to possess potent ability that not only can ablate primary tumor, but also can prevent the distance metastasis and relapse. In this study, human serum albumin was hybridized with hemoglobin by intermolecular disulfide bonds to develop a hybrid protein oxygen nanocarrier with chlorine e6 encapsulated (C@HPOC) for oxygen self-sufficient photodynamic therapy (PDT). C@HPOC realized the tumor-targeted co-delivery of photosensitizer and oxygen, which remarkably relieved tumor hypoxia. C@HPOC was favorable for more efficient PDT and enhanced infiltration of CD8+ T cells in tumors. Moreover, oxygen-boosted PDT of C@HPOC induced immunogenic cell death (ICD) with the release of danger-associated molecular patterns to activate dendritic cells, T lymphocytes, and natural killer cells in vivo. Notably, C@HPOC-mediated immunogenic PDT could destroy primary tumors and effectively suppress distant tumors and lung metastasis in a metastatic triple-negative breast cancer model by evoking systemic anti-tumor immunity. This study provides a paradigm of oxygen-augmented immunogenic PDT for metastatic cancer treatment.
KEYWORDS: oxygen nanocarrier, photodynamic therapy, tumor hypoxia, immunogenic cell death, metastasis
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Tumor metastasis becomes the leading challenge in the current clinical cancer treatment, accounting for over 90% of all cancer-caused deaths.1, 2 Particularly, metastatic triple-negative breast cancer (mTNBC) is nearly the most fatal breast cancer subtype due to its high recurrence rate, distant metastasis, and poor prognosis.3 Conventional cancer therapies, such as chemotherapy and surgery, have been employed to destroy the primary tumors, but fail to induce abscopal effect for metastasis inhibition.4 Thus, an effective cancer treatment strategy for mTNBC depends on eradicating the primary tumor and simultaneously eliminating the metastatic lesions. Photodynamic therapy (PDT) has emerged as a promising cancer therapeutic modality which kills cancer cells by employing photosensitizers and oxygen to generate reactive oxygen species (ROS), for instance singlet oxygen (1O2), under light irradiation.5 Compared to the conventional therapies, PDT has its merits of minimal invasiveness, high spatiotemporal selectivity and favorable safety and efficacy.6-11 In addition to inducing the local tumor regression, PDT has the potential to induce anti-tumor immunity to kill the residual or metastatic tumor cells.12, 13 To achieve strong anti-tumor effect for both primary tumors and distant metastasis, strategies which combine PDT with checkpoint inhibitors or immune adjuvants have attracted increasing attentions in recent years.14-16 However, these strategies are still limited in clinic applications due to their fairly high costs and serious adverse effects.17,
18
Besides, some studies have indicated that hypoxia in solid tumors not only
promotes tumor metastasis,19 but also severely limits the PDT efficiency.5 Hence, it is urgently needed to develop a more affordable, safe, and efficient strategy of immunogenic PDT against tumor growth and metastasis. ACS Paragon Plus Environment
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Recently, various nanomaterials, including oxygen carriers (e.g., perfluocarbon, hemoglobin) and catalyst-based oxygen generators (e.g., MnO2, CaO2), have been applied to relieve the hypoxic tumor microenvironment (TME).20-23 Meanwhile, the modulation of tumor hypoxia can improve therapeutic outcomes of PDT and reverse the immunosuppressive TME.24 Hemoglobin (Hb)-based oxygen carriers, well-known as blood substitutes in past decades,25 have recently been widely used in cancer treatments due to the inherent and reversible oxygen-transport capacity of Hb.21, 26, 27 However, free Hb is still far from being satisfactory as an oxygen carrier, owing to its short circulation time, poor stability, and potential side effect.25 Human serum albumin (HSA), a highly soluble and abundant plasma protein, has been developed as drug-delivery nanocarriers. Moreover, HSA shows native biocompatibility, excellent in vivo stabilities and active tumor-targeting capacity.28,
29
Therefore, it is of great interest to combine HSA and Hb to develop a safe and versatile oxygen nanocarrier for better therapeutic outcomes of PDT. Herein, we present a protein hybridization approach to develop a bioinspired hybrid protein oxygen nanocarrier with Ce6 loaded (C@HPOC) via intermolecular disulfide conjugations for oxygen-augmented immunogenic PDT (Figure 1). Benefited from the tumor-targeted co-delivery of oxygen and photosensitizer, C@HPOC overcame tumor hypoxia and generated massive
1
O2 to improve the PDT efficacy. Furthermore,
oxygen-boosted PDT with C@HPOC elicited immunogenic cell death (ICD) with enhanced release of danger-associated molecular patterns (DAMPs, including calreticulin (CRT), high-mobility group box 1 (HMGB1), adenosine triphosphate (ATP)). Afterwards, these DAMPs promoted the maturation of dendritic cells (DCs) and subsequently activated T lymphocytes and natural killer cells (NK). Consequently, C@HPOC-mediated PDT could ACS Paragon Plus Environment
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elicit effective anti-metastatic and abscopal effect in a 4T1 mTNBC murine model through stimulating strong and systemic anti-tumor immunity.
Figure 1. Schematic depiction of oxygen-augmented immunogenic PDT with C@HPOC for eliciting the anti-metastatic and abscopal effect. Human serum albumin (HSA) was hybridized with oxygen-carrying hemoglobin (Hb) via intermolecular disulfide bonds to form a hybrid protein oxygen nanocarrier with Ce6 loaded (C@HPOC). Under laser irradiation, oxygen self-supplied nanoparticles (C@HPOC) elevated the generation of cytotoxic 1O2 and moreover triggered immunogenic cell death (ICD). Then, enhanced release of damage-associated molecular patterns (DAMPs, e.g., CRT, HMGB1, and ATP) from dying tumor cells promoted the maturation of dendritic cells (DCs), and subsequently activated T lymphocytes and natural killer cells (NK). Thus, C@HPOC-mediated PDT not only destroyed the primary tumors, but also inhibited the distant tumors and lung metastasis by systemic anti-tumor immune responses.
RESULTS Fabrication and characterization of C@HPOC. Firstly, HSA was reduced by excessive glutathione (GSH) and the disulfide bonds from ACS Paragon Plus Environment
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HSA were cleaved to expose a mass of reactive sulfhydryl groups. Afterward, the reduced HSA and hemoglobin (Hb) were hybridized by intermolecular disulfide bonds to yield the hybrid protein oxygen carrier (HPOC) nanoparticles. During the rebuilding of disulfide bonds, photosensitizers (Ce6) were encapsulated into the HPOC nanoparticles via hydrophobic interaction,30 forming the C@HPOC (Figure S1). Ultimately, C@HPOC was oxygenated to serve as a bioinspired oxygen nanocarrier for oxygen-boosted PDT. The formation process of C@HPOC was verified by monitoring the changes of the amount of sulfhydryl groups with the previously reported method.31 Compared to the initial amount of free sulfhydryl groups (-SH) in HSA and Hb (about 1×10-5 mol/g), the reduced HSA with excessive GSH exhibited over 16 fold increase in the -SH amount, and the -SH level ultimately returned to about 1×10-5 mol/g after C@HPOC was formed, suggesting the rebuilding of the disulfide bonds (Figure 2a). The average diameter of C@HPOC measured by dynamic light scattering (DLS) was 35 ± 4 nm (Figure 2b). Transmission electron microscopy (TEM) image in the inset of Figure 2b revealed the obtained C@HPOC exhibited a well-defined spherical structure. The UV-vis absorption peaks of C@HPOC were located at 280 nm, 405 nm and 660 nm, indicating the characteristic absorption of HSA, Hb, and Ce6, respectively (Figure 2c). To accurately determine the Hb content in C@HPOC, Fe concentration of C@HPOC was measured by inductively coupled plasma optical emission spectrometer (ICP-OES), showing 17 wt% of Hb in C@HPOC. Figure 2d showed that the fluorescence emission peak of C@HPOC was similar to that of free Ce6. These results indicated the successful preparation of C@HPOC, and the encapsulated Ce6 remained its absorption and fluorescent properties. We further explored the photoacoustic responses of oxygenated C@HPOC at 850 nm. As shown in Figure 2e, the photoacoustic intensity of C@HPOC in aqueous solution was linearly ACS Paragon Plus Environment
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correlated with the oxygenated hemoglobin (HbO2) concentrations. The results indicated that C@HPOC could be monitored by photoacoustic imaging to evaluate the delivery and oxygenation process. Next, we investigated the oxygen release of the oxygenated-C@HOPC in hypoxic solution by a portable oxygen meter. The results in Figure 2f showed that both C@HPOC and free Hb realized the gradual release of oxygen in hypoxic solution, but the amount of oxygen released from C@HPOC was higher than that of free Hb. The reason may be that proteins hybridization could prevent the dissociation of Hb and improve its stability to keep a stable oxygen carrying capacity.25
Figure 2. Characterization of C@HPOC. (a) Differences of the amount of free sulfhydryl groups in HSA, reduced HSA, Hb or C@HPOC. The disulfide bonds of HSA were cleaved by a reducing agent (GSH) to obtain free sulfhydryl groups, and then the reduced HSA was cross-linked with Hb via the formation of disulfide bonds to form the C@HPOC. (b) Size distribution of C@HPOC measured by dynamic light scattering (DLS). Inset: TEM image of C@HPOC. (c) UV-vis absorption spectra of C@HPOC. (d) The fluorescence (FL) spectra of C@HPOC. (e) Quantitative curve of the photoacoustic (PA) intensity of oxygenated C@HPOC solution with different HbO2 concentrations at 850 nm. Inset: the corresponding PA images of oxygenated C@HPOC solution. (f) Oxygen release profiles of C@HPOC in hypoxic solution.
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Oxygen-boosted PDT performance of C@HPOC in vitro. ROS, particularly 1O2, which derives from oxygen molecules via the photosensitizers under the light activation, is responsible for killing cancer cells during PDT.7 Firstly, 1O2 generation in water solution was measured with singlet oxygen sensor green (SOSG). Figure S2 indicated that C@HPOC significantly enhanced the 1O2 generation under 660 nm laser irradiation (0.5 min, 0.1 W/cm2), compared to the groups of Ce6 and C@HSA. Afterward, the ROS production ability of C@HPOC in 4T1 tumor cells was further studied by confocal laser scanning microscopy (CLSM) and flow cytometry with a kind of classical ROS probe (DCFH-DA).32 As shown in Figure 3a, compared with the treatments of PBS + laser, Ce6 + laser, C@HSA + laser, and C@HPOC (without laser), stronger green fluorescence in cells was observed after treatment with C@HPOC + laser (2 min, 0.1 W/cm2), suggesting a greatly enhanced ROS generation. By comparing C@HSA + laser with C@HPOC + laser, it can be concluded that the principal contribution of ROS production was the hemoglobin-based oxygen supply other than nanoparticle-mediated uptake enhancement. Flow cytometry results further confirmed that C@HPOC + laser group exhibited greatly enhanced ROS generation in comparison with other groups (Figure 3b). To investigate the tumor cell killing effect of C@HPOC + laser in vitro, we determined the viability of 4T1 cells treated with free Ce6, C@HSA, and C@HPOC under 660 nm laser irradiation (2 min, 0.1 W/cm2). The results revealed that the cell viability of C@HPOC + laser group dramatically decreased to 11%, 7-fold lower than that of both Ce6 + laser group and C@HSA + laser group under Ce6 concentration of 1 µg/mL (Figure 3c), indicating that C@HPOC + laser exhibited fairly high phototoxicity against 4T1 cells compared with treatments of Ce6 + laser and C@HSA + laser. On the contrary, no obvious dark cytotoxicity was observed when 4T1 cells treated with ACS Paragon Plus Environment
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C@HPOC, suggesting good biocompatibility of our designed nanoparticles (Figure S3). We next assessed the PDT efficacy of C@HPOC in vitro by apoptosis assay. 4T1 cells after various treatments were stained with Annexin V and propidium iodide (PI) for examining the ratio of cell apoptosis using flow cytometry. Figure 3d,e demonstrated that C@HPOC + laser induced about 80% apoptosis ratio of 4T1 cells, over 13-fold higher than that of the groups of PBS + laser, Ce6 + laser, C@HSA + laser, and C@HPOC (without laser). All these data indicated that C@HPOC + laser exhibited oxygen-boosted PDT effect to kill tumor cells.
Figure 3. The oxygen-boosted PDT effect of C@HPOC for 4T1 tumor cells in vitro. (a) Confocal images of cellular uptake and ROS generation in 4T1 tumor cells. (b) Flow cytometry quantification of the ROS production in 4T1 tumor cells after various treatments. (c) Tumor cells viability after treatment with free Ce6, C@HSA, and C@HPOC plus laser irradiation (2 min, 0.1 W/cm2). (d) Flow cytometry analysis of 4T1 cell apoptosis. (e) Corresponding statistical data of percentage of apoptotic 4T1 cells. (+) refers to 660 nm laser irradiation. Data are expressed as means ± SD (n=3).
Immunogenic cell death induced by oxygen-boosted PDT in vitro. ACS Paragon Plus Environment
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Previous studies have reported that cancer therapies such as PDT, chemotherapy or radiotherapy might elicit ICD by inducing dying tumor cells to release immunogenic signals called DAMPs.33-35 Vital DAMPs, including CRT (translocated from endoplasmic reticulum to cell surface), HMGB1 (released from nuclei), and extracellular ATP, could stimulate the engulfment of dying tumor cells by immature DCs, leading to DC maturation (Figure 4a).36, 37 To investigate whether C@HPOC-mediated PDT can trigger ICD, 4T1 cells were treated with C@HPOC + laser, followed by the evaluation of CRT exposure, HMGB1 release, and ATP secretion. As shown in Figure 4b, 4T1 tumor cells treated with PBS + laser, Ce6 + laser, C@HSA + laser, and C@HPOC (without laser) showed few cell-surface CRT exposure (green). But significant cell-surface CRT exposure was observed in C@HPOC + laser treated group, which was attributed to the oxygen-boosted PDT efficacy of C@HPOC. The quantitative analysis of flow cytometry further confirmed that C@HPOC + laser dramatically induced more CRT exposure on 4T1 cells, compared to other treatments (Figure 4c). Next, HMGB1 release was analyzed by enzyme-linked immuno sorbent assay (ELISA), and ATP secretion was detected by a luciferin-based ATP assay. Compared with the groups of Ce6 + laser, C@HSA + laser, and C@HPOC (without laser), C@HPOC + laser group significantly enhanced the release of HMGB1 and the secretion of ATP from 4T1 cells (Figure 4d,e). Furthermore, after treatment with C@HPOC + laser, 4T1 tumor cells were incubated with immature DC, using MHC II and CD86 as markers for evaluating DC maturation. The results in Figure 4f,g revealed that the percentage of mature DCs (MHCII+CD86+) in C@HPOC + laser group was significantly higher than that in other treated groups. All the results taken together suggested that oxygen-boosted PDT of C@HPOC could significantly induce enhanced emission of DAMPs from 4T1 tumor cells and then promote DC maturation. ACS Paragon Plus Environment
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Figure 4. In vitro immunogenic cell death triggered by oxygen-boosted PDT. (a) Schematic illustration of mechanism of DC maturation facilitated by the major ICD-associated DAMPs from dying tumor cells. Tumor cells undergoing ICD released the DAMPs (including CRT, HMGB1 and ATP), which promoted the engulfment of tumor antigens by immature DC, followed by stimulating DC maturation. (b) Fluorescent imaging of CRT exposed on surface of 4T1 tumor cells after different treatments. (c) Flow cytometry quantification of CRT on 4T1 tumor cells. (d) Detection of HMGB1 extracellular release. (e) Detection of ATP extracellular secretion. (f,g) Flow cytometry analysis of DC maturation by two markers (MHC II and CD86) after the immature DCs incubated with C@HPOC + laser treated 4T1 tumor cells. Data are expressed as means ± SD (n=3).
Targeted C@HPOC for tumor oxygenation and oxygen-boosted PDT effect in vivo. ACS Paragon Plus Environment
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Before the therapeutic experiments, the in vivo biosafety of C@HPOC was first evaluated by hematoxylin and eosin (H&E) staining and blood biochemistry test. H&E staining showed no obvious tissue damage of C@HPOC treated group compared to PBS group (Figure S4). The serum alanine aminotransferase (ALT, liver functions index) and blood urea nitrogen (BUN, kidney function index) were further tested. The results demonstrated that there were no difference between C@HPOC treated group and PBS group in the serum level of ALT and BUN, suggesting the good biocompatibility of C@HPOC in vivo (Figure S5a,b). Next, to explore the tumor-targeted delivery efficiency of C@HPOC, the mice-bearing 4T1 tumors after intravenous injection of free Ce6 and C@HPOC were imaged by tracking Ce6 fluorescence with an in vivo imaging system. The fluorescence signals of free Ce6-injected mice vanished rapidly within 6 h due to its rapid clearance in circulation.30 But C@HPOC showed an obvious long-term circulation and more accumulation in tumor (Figure S6a,b). At 24 h post-injection, semiquantitative biodistribution validated the long-term retention of C@HPOC in liver and more importantly in tumor, compared to free Ce6 group (Figure 5a and S7). Owing to EPR effect and HSA-active tumor targeted capability, the C@HPOC facilitated the Ce6 delivery to tumor region.38, 39 Further, to testify whether oxygen could be effectively delivered for tumor oxygenation, the photoacoustic imaging was performed to measure the HbO2 signals with an 850 nm laser excitation.22 As shown in Figure 5b and S8, compared with the PBS group, the injection of C@HPOC exhibited a high level of the photoacoustic intensity of HbO2 in the surrounding of blood vessels at 2-6 h. Notably, the peak value of the photoacoustic intensity of HbO2 appeared at 4 h, with about 47% enhancement compared with that of pre-injection. The efficiency of tumor oxygenation with C@HPOC was further examined by hypoxyprobe immunofluorescence assay for mapping the ACS Paragon Plus Environment
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tumor hypoxia. In the groups of PBS and free Ce6, large areas of green signals indicated the extensively distributed hypoxic areas within the tumor sections. Dramatically, the hypoxia was barely detectable in the tumor section with administration of C@HPOC (Figure 5c). It revealed that the tumor hypoxia was drastically attenuated with the targeted oxygen supply of C@HPOC. The PDT effect in vivo was investigated by the fluorescent probe (SOSG) at 4 h post-injection of C@HPOC. The results in Figure 5d showed that the PDT effect was severely impaired in the Ce6 injected tumor due to the limited photosensitizer accumulation and lack of sustained oxygen supply. However, strong green fluorescence was detected in the C@HPOC group, indicating the sufficient 1O2 generation. The effective accumulation of oxygen and photosensitizer can be attributed to the advantages of C@HPOC: (i) The tumor-targeted C@HPOC with enhanced permeability and retention (EPR) effect allowed outstanding performance in tumor accumulation of oxygen and Ce6. (ii) The oxygen-carrying C@HPOC allowed oxygen preservation during circulation, and oxygen release in the hypoxic tumor, resulting in precise and sufficient tumor oxygenation for attenuating tumor hypoxia and enhanced PDT.
Figure 5. Targeted tumor oxygenation and oxygen-boosted PDT effect of C@HPOC in vivo. (a)
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Fluorescence images of major organs and tumors at 24 h after intravenous injection of free Ce6 or C@HPOC (n=3). (b) Tumor-oxygenated effect of C@HPOC was evaluated by photoacoustic images of oxygenated hemoglobin (HbO2, λ=850 nm) after intravenous administration of C@HPOC (n=3). (c) Ex
vivo immunofluorescence images of tumor sections of various groups after hypoxia staining assay. The hypoxic areas (green) were detected by hypoxyprobe (n=3). (d) In vivo singlet oxygen evaluation via SOSG probe (green) after treatment with PBS, free Ce6, and C@HPOC, followed by 660 nm laser irradiation (30 min, 0.1 W/cm2) (n=3).
Abscopal and anti-metastatic effect of oxygen-boosted PDT in 4T1 tumors. In the following study, we explored the abscopal and anti-metastatic effect of C@HPOC-mediated PDT. For evaluation of abscopal effect, a bilateral 4T1 mTNBC model was developed by subcutaneously injecting 4T1 cells into both the left and right flank regions of mice. The left tumor was set as the primary tumor (inoculated on day 0) and the right tumor was designated as the distant tumor (inoculated on day 6). On day 7, the primary tumors were treated with PBS + laser, Ce6 + laser, C@HPOC + laser, and C@HPOC (without laser), whereas the distant tumors were shielded from any treatments (Figure 6a). The volume of both primary and distant tumors were recorded from day 7. We found that C@HPOC + laser treatment could elicit a robust therapeutic efficacy in combating the primary tumors progression, compared with the groups of PBS + laser, Ce6 + laser, and C@HPOC (without laser) (Figure 6b). In addition, Figure S9 showed that C@HSA + laser displayed better tumor inhibition than Ce6 + laser, but was less efficient than C@HPOC + laser. And those results that reflected oxygen-boosted PDT with C@HPOC should mainly depend on the tumor oxygenation of hemoglobin. Notably, C@HPOC + laser treatment significantly inhibited the growth of distant tumors (without treatment) compared to the PBS + laser group. In contrast, both free Ce6 + laser and C@HPOC (without laser) treatments failed to delay distant tumors
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progression (Figure 6c). Additionally, the body weight of mice from the group of C@HPOC + laser was not significantly diminished, suggesting no severe systemic toxicity (Figure S10).
Figure 6. The abscopal and anti-metastatic effect of oxygen-boosted PDT in 4T1 tumors. (a) Schematic depicting the experimental approach for evaluation of the abscopal effect induced by C@HPOC-mediated PDT. (b) Growth curves of primary tumor on mice after various treatments. (c) Growth curves of distant tumor on mice in different treated groups. (d) Schematic illustrating the experimental approach for assessment of anti-metastatic effect caused by C@HPOC-mediated PDT. (e) Numbers of lung metastatic nodules from each group. (f) The inhibition rate of lung metastatic nodules of other treatments in comparison to the PBS plus laser group. (g) Photographs of metastatic nodules in lungs. The black circles denote the metastatic nodules. (h) Histological assessment of lung metastatic nodules via H&E staining. Data are expressed as means ± SD (n=5).
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Next, we sought to determine the anti-metastatic effect of C@HPOC-mediated PDT. Primary tumors were inoculated on day 0, and the lung metastatic tumors were developed by intravenous injection of 4T1 cells on day 6. Treatments of PBS + laser, Ce6 + laser, C@HPOC + laser, and C@HPOC (without laser) were respectively performed on day 7. The lungs of treated mice in each groups were harvested for assessment of anti-metastatic efficacy on day 25 (Figure 6d). As shown in Figure 6e, the average number of metastatic nodules per lung in C@HPOC + laser group (13 ± 2) was much fewer than those in the groups of PBS + laser (48 ± 7), Ce6 + laser (41 ± 6), and C@HPOC without laser (46 ± 3 ). Thus, the C@HPOC-mediated PDT treatment showed a 73% inhibition of lung metastases, which was 5- and 13-fold higher than that in the groups of free Ce6 + laser and C@HPOC (without laser) (Figure 6f). The anti-metastatic effect was further verified by the images of those harvested lungs and their sections. Lung metastatic nodules from C@HPOC + laser treated group were significantly less than that from other treated groups (Figure 6g). H&E staining assay further showed the significant decrease in the number and size of visible metastatic nodules from C@HPOC + laser treated group (Figure 6h). Overall, these results suggested that the potent efficacy of C@HPOC-mediated PDT on the primary tumors which could elicit not only abscopal effect on distant tumors, but also drastic suppression of metastatic tumors. Anti-tumor mechanism of oxygen-augmented immunogenic PDT. The recruitment of immune cells into TME is a key parameter directly correlated with the anti-tumor immune responses. Thus, we tested whether the observed anti-tumor effect of C@HPOC-mediated PDT was facilitated by the increased tumor infiltration of immune cells, using immunofluorescence staining assay and flow cytometry. As shown in Figure 7a, C@HPOC + laser treatment showed a mass of CD8+ T cells (red) infiltration in tumors, ACS Paragon Plus Environment
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compared to the PBS + laser group with negligible tumor-infiltrating CD8+ T cells. Besides, a small amount of tumor-infiltrating CD8+ T cells was also observed in the groups of Ce6 + laser and C@HPOC (without laser). Furthermore, flow cytometry assay showed that tumor-infiltrating CD8+ T cells of C@HPOC (without laser) group were significant higher than that of PBS + laser group, which indicated that tumor oxygenation by C@HPOC could moderate the immunosuppressive TME to promote the infiltration of CD8+ T cells in tumors.40 More importantly, C@HPOC + laser treatment not only elevated the percentage of tumor-infiltrating CD8+ T cells, which was about 2-fold higher than other groups, but also significantly increased the infiltration of CD4+ T and NK cells in tumors (Figure 7b). The notable results may be attributed to the TME modulation ability of C@HPOC and the subsequently oxygen-boosted PDT treatment which could induce the release of tumor antigens and endogenous adjuvants (e.g., DAMPs).41, 42
Figure 7. The immune cells infiltration in tumor tissues. (a) Immunofluorescence staining detection of CD8 T cells (red) in tumor tissues. (b) Flow cytometric analysis of CD8, CD4, and NK cells in tumors. Data are expressed as means ± SD (n=5).
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release and DC maturation in vitro, we explored whether C@HPOC-mediated PDT could induce effective ICD in vivo. As reported, CRT exposure, as a crucial indicator of ICD, could promote the DC maturation and subsequently trigger T lymphocyte-mediated anti-tumor immunity.43-45 Hence, the induction of CRT exposure by C@HPOC-mediated PDT was analyzed in vivo. The results in Figure 8a showed that C@HPOC + laser exhibited significantly enhanced CRT exposure in tumors compared to the groups of PBS + laser, Ce6 + laser and
C@HPOC
(without laser).
Next,
we
sought
to investigate
whether
C@HPOC-mediated PDT could lead to DC maturation in vivo. Tumor-draining lymph node (TDLN) is a crucial peripheral lymphoid organ where mature DCs present antigens to the T lymphocytes and then elicit systemic immune responses.13,
46
Thus, tumors and
tumor-draining lymph nodes (TDLNs) were harvested to analyze the expression of the activated DCs markers (CD86 and MHC II) in CD11c+ cells by flow cytometry. The results revealed that, with an enhanced CRT exposure of tumor cells, C@HPOC-mediated PDT promoted a much higher level of DC maturation both in tumors and TDLNs, in contrast with other groups (Figure 8b,c). Mature DCs play a vital role in initiating an effective adaptive immune response by presenting antigens to the T lymphocytes for further anti-cancer effect.47 We found that C@HPOC + laser treatment significantly increased the proportion of activated CD8+ T cells (CD8+CD69+), CD4+ T cells (CD4+CD69+), and NK cells (NK1.1+CD69+) both in tumors and TDLNs, when compared with the treatments of PBS + laser, Ce6 + laser, and C@HPOC (without laser) (Figure 8d,e). Collectively, our results suggested that C@HPOC-mediated PDT could induce potent anti-tumor immune responses via ICD with enhanced CRT exposure, DC maturation, and consequent activation of T lymphocytes and NK cells, which accordingly led to effective abscopal and anti-metastatic effect. ACS Paragon Plus Environment
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Figure 8. In vivo activation of immune responses elicited by oxygen-augmented immunogenic PDT. (a) Immunofluorescence imaging of CRT (the typical hallmark of ICD) exposure in 4T1 tumors treated with C@HPOC plus laser. (b) Flow cytometric analysis of activated dendritic cells (CD11c+CD86+ or CD11c+MHCII+) in tumors. (c) Flow cytometric analysis of activated dendritic cells (CD11c+CD86+ or CD11c+MHCII+) in TDLNs. (d) Flow cytometric analysis of effector cells (activated CD8+ T cells (CD8+CD69+), activated CD4+ T cells (CD4+CD69+) and activated NK cells (NK1.1+CD69+)) in tumors. (e) Flow cytometric analysis of effector cells (activated CD8+ T cells (CD8+CD69+), activated CD4+ T cells (CD4+CD69+) and activated NK cells (NK1.1+CD69+)) in TDLNs. Data are expressed as means ± SD (n=5).
DISCUSSION Cancer vaccine can offer many advantages including eliciting systemic anti-tumor responses to treat metastases and building a long-term anti-tumor immunological memory to prevent tumor recurrence.48-50 Existing protein- and peptide- based cancer vaccines have been widely studied for tumor therapy,51, 52 but their application in clinical treatments is still limited by the inefficient delivery of antigens and adjuvants or immune tolerance.53 Based on the bioinspired oxygen nanocarrier (C@HPOC), our study provided an oxygen-boosted immunogenic PDT strategy to generate vaccine-like functions in situ without ACS Paragon Plus Environment
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delivery of foreign antigens or adjuvants to tumors. Compared with other PDT-based anti-tumor studies,6,
54
we proposed that oxygen-augmented immunogenic PDT with
C@HPOC could induce highly efficient tumor regression of both primary tumors and distant tumors. (i) Oxygen played the main role in ameliorating tumor hypoxia and elevating the generation of singlet oxygen in tumors. (ii) Oxygen moderated the immunosuppressive TME leading to enhanced infiltration of CD8+ T cells in tumors, which favored the anti-tumor immunity.40 (iii) Oxygen helped to induce ICD by oxygen-boosted PDT, which activated DCs and effector cells (CD8+, CD4+ T and NK cells) in tumors and TDLNs. (iv) Oxygen amplified the efficacy of PDT, eliciting potent abscopal and anti-metastatic effect by systemic anti-tumor immune responses. Therefore, the local PDT treatment with C@HPOC could evoke robust and systemic anti-tumor immunity without adding immune-adjuvants or checkpoint inhibitors, thereby leading to effective abscopal effect and inhibition of metastasis. These findings highlight that oxygen-boosted immunogenic PDT of C@HPOC offers an alternative strategy for the treatment of metastatic cancers.
CONCLUSION In summary, we developed a bioinspired hybrid protein oxygen nanocarrier with Ce6 loaded (C@HPOC) for oxygen-augmented immunogenic PDT against the tumor growth and metastasis. With tumor-targeted co-delivery of oxygen and photosensitizer, C@HPOC was able to overcome the tumor hypoxia, and resulted in profound benefits for both improving the efficacy of PDT and increasing the tumor-infiltrating CD8+ T cells. More importantly, C@HPOC-mediated PDT could trigger potent ICD-based anti-tumor immune responses, as ACS Paragon Plus Environment
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evidenced by the enhanced release of DAMPs from tumor cells, elevated proportion of mature DCs, activated T lymphocytes and NK cells in tumors and TDLNs. The well-defined C@HPOC elicited oxygen-augmented immunogenic PDT, which not only destroyed the primary tumors, but effectively inhibited distant tumors and lung metastasis. This study might present an inspiration for improving the therapeutic outcomes and prognosis of metastatic cancers in clinic.
EXPERIMENTAL SECTION Materials. Human serum albumin (HSA), bovine hemoglobin (Hb), and glutathione (GSH) were purchased from Hefei Bomei Biotechnology (China). Chlorin e6 (Ce6) was purchased from J&K Scientific Ltd. (China). Singlet oxygen sensor green (SOSG) was obtained from Thermo Fisher Scientific (USA). 2’,7’-dichlorofluorescin diacetate (DCFH-DA) was bought from Sigma-Aldrich (USA). Cell Counting Kit-8 assay and hematoxylin and eosin (H&E) were purchased from Beyotime Institute of Biotechnology (China). Annexin V-FITC /Propidium Iodide (PI) Cell Apoptosis Kit was bought from BD Biosciences (USA). Rabbit anti-calreticulin antibody was bought from Cell Signaling Technology (USA). Alexa Fluor 488-conjugated secondary anti-rabbit antibody and Chemiluminescence ATP Determination Kit were purchased from Life technologies (USA). HMGB1 ELISA Kit was purchased from Aviva systems biology (USA). Roswell Park Memorial Institute (RPMI) 1640 medium,
fetal
bovine
serum
(FBS),
phosphate
buffered
PBS
(PBS)
and
penicillin-streptomycin were bought from Gibco Life Technologies (USA). Fabrication of C@HPOC. First, the reduced HSA was prepared as follows. 20 mg HSA was reduced with 6.7 mg GSH in 2 mL deionized water at room temperature for 1 h,
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following the solution was dialyzed (membrane cutoff Mw: 3500) for 12 h to remove excessive GSH. Then, 0.4 mg Ce6 and 3.6 mg Hb were added in the reduced HSA solution and mixed well at pH 8.0. Then, to precipitate the C@HPOC, 3 mL ethanol was added into the solution. The suspension was kept stirring adequately at room temperature for 30 min to rebuild the disulfide bonds. Finally, the suspension was dialyzed (membrane cutoff Mw: 100 kD) with deionized water for 24 h to remove ethanol, free Ce6, HSA, and Hb. Ce6-encapsulated HSA nanoparticles (C@HSA) were prepared following the similar process described above. Characterization of C@HPOC. The number of the free sulfhydryl of HSA, reduced HSA, Hb, and C@HPOC was measured with the previously reported method.31 Size distribution of C@HPOC was measured by a Zetasizer Nano ZS (Malvern, U.K.). The morphology of C@HPOC was characterized by TEM (FEI Tecnai G2 F20 S-Twin, USA). UV-vis absorption spectra of C@HPOC, Ce6, Hb, and HSA were measured by UV/vis spectrometry (Lambda25, Perkin-Elmer, USA) in PBS. The C@HPOC was diluted to make absorbance values of characteristic absorptions (280 nm for HSA, 405 nm for Hb and 660 nm for Ce6) at the range of 0.1-1.0. The concentrations of free Ce6, Hb, and HSA were 5 µg/mL, 100 µg/mL, and 500 µg/mL respectively, and their spectra were normalized to the corresponding characteristic peaks. To accurately determine the Hb content in C@HPOC, Fe concentration in C@HPOC or free Hb solution was measured by inductively coupled plasma optical emission spectrometer (ICP-OES, Perkin Elmer Optima 7000DV, USA). Before the ICP-OES measurement, samples were digested with 68% aqueous nitric acid solution and diluted with ultrapure water, and then filtered through 0.22 µm filters. The fluorescence spectra of free Ce6 and C@HPOC were obtained by fluorescence spectroscopy at 640 nm ACS Paragon Plus Environment
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excitation (F900, Edinburgh Instruments, Ltd., U.K.) in PBS. The photoacoustic (PA) imaging system (Endra Nexus 128, Ann Arbor, MI, USA) was applied to detect the PA intensity of C@HPOC with different HbO2 concentrations. The oxygen release profiles of C@HPOC and free Hb were measured using a dissolved oxygen detector (Mettler Toledo, Switzerland). Tumor Cell Culture. 4T1 murine breast tumor cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under 5% CO2. In Vitro Singlet Oxygen Detection. In vitro singlet oxygen generation from free Ce6, C@HSA, and C@HPOC (1 µg/mL of Ce6) in water solution (660 nm laser irradiation for 0.5 min, 0.1 W/cm2) was detected with SOSG (1 µM) according to the manufacturer’s protocol. Intracellular ROS detection was analyzed by CLSM and flow cytometry using DCFH-DA as the ROS sensor. For CLSM observation, 4T1 cells were seeded into 8-well chambered slides (Thermo Scientific, USA) at a density of 8×103 cells per well and cultured for 12 h. After removing the medium, cells were treated with PBS, free Ce6, C@HSA, and C@HPOC for another 2 h at an equivalent Ce6 concentration (5 µg/mL, Ce6 was employed to detect the Ce6 fluorescence by confocal imaging) and then irradiated with a 660 nm laser for 2 min (0.1 W/cm2). The cells were further incubated with DCFH-DA (20 µM) for 30 min. Subsequently, the cells were stained with 1 µM Hoechst 33342 (Invitrogen, USA), and then observed via a TCS SP5 laser confocal microscope (TCS SP5II, Leica, Ernst-Leitz-Strasse, Germany). For flow cytometry, 4T1 cells were seeded into 24-well plates (5×104 cells/well) and cultured for 12 h. After treatment with PBS, free Ce6 , C@HSA, and C@HPOC for another 2 h at an equivalent Ce6 concentration (1 µg/mL), 4T1 cells were subjected to a 660 nm laser ACS Paragon Plus Environment
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irradiation for 2 min (0.1 W/cm2), followed by incubation with DCFH-DA (20 µM) for 30 min. C@HPOC without laser irradiation served as the dark control. Afterward, the cells were harvested and the fluorescence signals of ROS generation were analyzed by flow cytometer (Becton Dickinson, San Jose, CA, USA). Cytotoxicity and Apoptosis Assay. For cell viability, 4T1 cells were seeded into 96-well plates at a density of 8×103 cells per well and cultured for 12 h. The cells were treated with free Ce6, C@HSA, and C@HPOC at various concentrations of Ce6. After incubation for 2 h, the cells were irradiated with a 660 nm laser for 2 min (0.1 W/cm2) or kept in dark, respectively. The cells were further incubated for 24 h and the cell viability was detected via CCK-8 assay (Beyotime, China), according to the manufacturer’s protocol. Apoptosis of tumor cells was assessed by Annexin V-FITC/PI Cell Apoptosis Kit. Cells were seeded into 24-well plates (5×104 cells/well) and cultured for 12 h. Cells were treated with PBS, free Ce6, C@HSA, and C@HPOC at an equivalent Ce6 concentration (1 µg/mL). After 2 h of incubation, the cells were irradiated with a 660 nm laser for 2 min (0.1 W/cm2), followed by a further 24 h incubation. C@HPOC without laser irradiation served as the dark control. The cell apoptosis was detected by Annexin V-FITC/PI Cell Apoptosis Kit using flow cytometry, according to the manufacturer’s protocol. Detection of Crucial ICD Biomarkers. The surface-exposure of CRT was assessed by immunofluorescence and flow cytometry. For immunofluorescence analysis, 4T1 cells were seeded into 8-well chambered slides (8×103 cells/well) and cultured for 12 h. The cells were treated with PBS, free Ce6, C@HSA, and C@HPOC (1 µg/mL of Ce6). After 2 h incubation, cells were irradiated with a 660 nm laser for 2 min (0.1 W/cm2). Cells treated with C@HPOC without laser irradiation served as dark control. After further 24 h incubation, the ACS Paragon Plus Environment
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cells were washed twice with PBS and then incubated with anti-calreticulin antibody for 2 h at 4 °C. Subsequently, the cells were further washed twice with PBS and incubated with Alexa Fluor 488-conjugated secondary antibody (Life technologies, USA) for 1 h. After staining with DAPI, the cells were observed under CLSM. For flow cytometry analysis, 4T1 cells were seeded into 24-well plates (5×104 cells/well) and cultured for 12 h. The cells were treated with PBS, free Ce6, C@HSA, and C@HPOC (1 µg/mL of Ce6). After 2 h incubation, cells were irradiated with a 660 nm laser for 2 min (0.1 W/cm2). Cells treated with C@HPOC without laser irradiation served as dark control. After further 24 h incubation, the cells were collected, followed by incubation with anti-calreticulin antibody for 2 h. Subsequently, the cells were washed, incubated with Alexa Fluor 488-conjugated secondary antibody for 1 h. Finally, the samples were analyzed via flow cytrometry. The extracellular released HMGB1 and ATP was examined using the HMGB1 ELISA Kit and Chemiluminescence ATP Determination Kit, respectively. Briefly, 4T1 cells were seeded into 24-well plates (5×104 cells/well) and cultured for 12 h. The cells were treated with PBS, free Ce6, C@HSA, and C@HPOC (1 µg/mL of Ce6). After 2 h incubation, cells were irradiated with a 660 nm laser for 2 min (0.1 W/cm2). Cells treated with C@HPOC without laser irradiation served as dark control. Following incubation for an additional 4 h, the cell supernatant was collected. The release of HMGB1 and ATP in the cell supernatant was detected by the HMGB1 ELISA Kit and Chemiluminescence ATP Determination Kit, according to the manufacturer’s protocols, respectively. In Vitro Induction of DC Maturation. DCs were generated from the 6-week old BALB/c female mice using a previously reported method.55 Briefly, mouse bone marrow cells were obtained via flushing the tibia and femur with PBS containing 2% FBS. ACS Paragon Plus Environment
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Subsequently, the cells were collected and cultured in X-vivo 15 medium (Lonza, Switzerland) containing GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) for 5 days to acquire the immature DCs. On day 6, immature DCs were co-incubated with 4T1 cells treated with PBS + laser, Ce6 + laser, C@HSA + laser, C@HPOC + laser, and C@HPOC (without laser) at an equivalent Ce6 concentration (1 µg/mL), respectively. After 24 h, DCs were stained with anti-mouse MHC II-PE and anti-mouse CD86-Percp antibodies (eBioscience, USA), and then analyzed by flow cytometry. Animal and Tumor Models. Female BALB/c mice (4-6 weeks) were purchased from Vital River Animal Technology Co. Ltd and used under the protocols approved by the Animal Care and Use Committee (Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences). For the unilateral subcutaneous tumor model, 4T1 cells (1×106) were subcutaneously injected into the left flank of female BALB/c mice. For bilateral subcutaneous tumor model, the primary tumor (first) was established via subcutaneous injection of 4T1 cells (1×106) into the left flank of a female BALB/c mouse, and the distant tumor (second) was conducted via subcutaneously injecting 4T1 cells (2×105) into the right flank of the same mouse on day 6. For pulmonary metastasis model, mice were subcutaneously injected with 4T1 cells (1×106) into the left flanks, and intravenously injected with 2×105 4T1 cells via the tail vein on day 6. The tumor volume was calculated in accordance with the following formula: width2 × length × 0.5. In Vivo PA/FL Imaging and Biodistribution Analysis. When the tumor volumes reached about 0.2 cm3, BALB/c mice were randomly divided into two groups. Mice were intravenously administrated with free Ce6 (2.5 mg/kg of Ce6) or C@HPOC (2.5 mg/kg of Ce6). After injection, the FL signals of Ce6 were recorded on IVIS spectrum imaging system ACS Paragon Plus Environment
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(Xenogen, USA) (ex: 640 nm; filter: 680 nm) at 2, 4, 6, 8, and 12 h. The FL images of mice before injection with samples served as blank control. For the biodistribution study, mice were sacrificed after 24 h post injection, and the tumors and normal tissues were harvested and imaged. Similarly, for PA imaging analysis, tumor-bearing mice were injected with PBS or C@HPOC via the tail vein. After injection, PA images of the tumor sites were recorded on photoacoustic computerized tomography scanner (850 nm) (Endra Nexus 128, Ann Arbor, MI, USA) at 2, 4, 6, and 24 h. The PA images of mice before injection with samples served as blank control. In Vivo Singlet Oxygen Detection. Tumor-bearing mice were intravenously administrated with 200 µL of PBS, free Ce6 (5 mg/kg Ce6), and C@HPOC (5 mg/kg Ce6), respectively. After 3.5 h, the mice of various groups were intraperitoneal injected with singlet oxygen sensor green (SOSG, 6.25 µg/mice, Invitrogen), followed by irradiation with a 660 nm laser for 30 min (0.1 W/cm2). Subsequently, tumors from various groups were harvested and cryosectioned onto slides. The fluorescence signals of the oxidized SOSG (ex/em=488/525 nm) were observed by CLSM. In Vivo Anti-Tumor Study. To study the abscopal effect of C@HPOC, seven days after the primary tumor inoculation, the bilateral 4T1 tumor bearing mice (n = 5) were intravenously injected with PBS, free Ce6 (5 mg/kg Ce6), C@HSA (5 mg/kg Ce6), and C@HPOC (5 mg/kg Ce6), respectively. 4 h later, the primary tumors were irradiated with a 660 nm laser for 30 min (0.1 W/cm2). The mice treated with C@HPOC without laser irradiation served as dark control. The primary and distant tumor size and body weight were monitored every 3 day. For anti-metastatic activity of C@HOPC, one day after intravenous injection with 4T1 ACS Paragon Plus Environment
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cells, the mice (n = 5) were i.v. injected with PBS, free Ce6, and C@HPOC at a Ce6 dose of 5 mg/kg. At 4 h post-injection, the mice were irradiated with a 660 nm laser for 30 min (0.1 W/cm2). At 18 days after the PDT-treatment for the primary tumors, the mice were sacrificed. The lungs of the mice were harvested and observed for the metastasis nodules, then sectioned and subjected to H&E staining. Ex Vivo Immunofluorescence Staining. To evaluate the tumor oxygenation ability of C@HPOC, tumor-bearing mice were i.v. injected with PBS, free Ce6, and C@HPOC at a Ce6 dose of 5 mg/kg, respectively. At 3 h post-injection, mice were intraperitoneal injected of pimonidazole hydrochloride (60 mg/kg) (Hypoxyprobe-1 plus kit, Hypoxyprobe Inc.). One hour later, the tumors of various groups were harvested. Subsequently, the tumors were sectioned and incubated with mouse anti-pimonidazole primary antibody and then Alex 488-conjugated goat anti-mouse secondary antibody (KPL, USA) according to the manufacturer’s protocol. After staining with DAPI, the obtained slices were observed by CLSM. For detection of CRT exposure in vivo, 2 days after various treatments, the mice were sacrificed and the tumors were harvested and sectioned into slices. After incubation with anti-calreticulin antibody, the slices were further incubated with Alexa Fluor 488-conjugated secondary antibody. The obtained slices were stained with DAPI, following observing by CLSM. Similarly, to study the tumor-infiltrating CD8+ T cells induced by C@HPOC, the slices were incubated with anti-mouse CD8a-PE antibodies (eBiosciences, CA, USA). After staining with DAPI, the obtained slices were observed under CLSM. Flow Cytometry Analysis for Anti-Tumor Immune Responses. Tumor-bearing mice were i.v. injected with PBS, free Ce6, and C@HPOC at a Ce6 dose of 5 mg/kg, followed by ACS Paragon Plus Environment
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irradiation with a 660 nm laser for 30 min (0.1 W/cm2). For analysis of the DCs, NK Cells, and T Cells, the tumors and tumor-draining lymph nodes were harvested from the treated mice on day 2. The TDLNs were grinded with glass slides or the tumor tissues were cut into small pieces and digested with collagenase (Sigma-Aldrich, USA). The tissue suspension was filtered with nylon mesh filters to acquire the single-cell suspension. To analyze the activated DCs, the cells were stained with anti-mouse CD11c-FITC, anti-mouse MHC II-PE, and anti-mouse CD86-Percp-Cy5.5 antibodies. For analysis of the active T cells and NK cells, the cells were stained with anti-mouse CD4-FITC, anti-mouse CD8a-APC, anti-mouse NK1.1-Percp-Cy5.5, and anti-mouse CD69-PE antibodies (all from eBiosciences, USA). The immune cell populations above were determined via the flow cytometry. In Vivo Biosafety of C@HPOC. Female BALB/c mice were i.v. injected with 200 µL PBS or C@HPOC (5 mg/kg Ce6). At 48 h postinjection, the liver or kidney function was assessed by determining the serum level of alanine aminotransferase (ALT) or urea nitrogen (BUN) using the ALT or BUN activity Assay Kit (JianCheng Biotech, China) according to the manufacturer’s protocol. Major organs were harvested and sectioned for H&E staining and then observed via an Olympus microscope (Olympus BX53, Japan). Statistical Analysis. Data are expressed as means ± SD. Differences among groups were analyzed using one-way ANOVA analysis followed by Tukey’s post-test. Asterisks: differences between PBS plus laser and other treatments statistically significant. *p < 0.05, **p < 0.01. #: differences between two different treatment are statistically significant, #p < 0.05, ##p
