Enhanced Immunotherapy Based on Photodynamic Therapy for Both

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Enhanced Immunotherapy Based on Photodynamic Therapy for Both Primary and Lung Metastasis Tumor Eradication Wen Song, Jing Kuang, Chu-Xin Li, Mingkang Zhang, Diwei Zheng, Xuan Zeng, Chuanjun Liu, and Xian-Zheng Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b09112 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 9, 2018

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Enhanced Immunotherapy Based on Photodynamic Therapy for Both Primary and Lung Metastasis Tumor Eradication Wen Song,† Jing Kuang, ‡,# Chu-Xin Li, † Mingkang Zhang, † Diwei Zheng, † Xuan Zeng, † Chuanjun Liu,†,* and Xian-Zheng Zhang†,§,*

†

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

‡

Department of Pathophysiology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430072, P. R. China

#

Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei 430079, P. R. China

§

The Institute for Advanced Studies (IAS), Wuhan University, Wuhan 430072, P. R. China

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ACS Paragon Plus Environment

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ABSTRACT: Metastasis and recurrence are two unavoidable and intractable problems in cancer therapy, despite of various robust therapeutic approaches. Currently, it seems that immunotherapy is an effective approach to solve the problems, but the high heterogeneity of tumor tissue, inefficient presentation of tumor antigen and deficient targeting ability of therapy usually blunt the efficacy of immunotherapy and hinder its clinical application. Herein, an approach based on combining photodynamic and immunological therapy was designed and developed. We synthesized a chimeric peptide PpIX-1MT, which integrated photosensitizer PpIX with immune checkpoints inhibitor 1MT via a caspase responsive peptide sequence Asp-Glu-Val-Asp (DEVD), to realize cascaded synergistic effect. The PpIX-1MT peptide could form nanoparticles in PBS and accumulate in tumor areas via enhanced penetration retention (EPR) effect. Upon 630nm light irradiation, the PpIX-1MT nanoparticles produced ROS, induced apoptosis of cancer cells, and thus facilitated the expression of caspase-3 and the production of tumor antigens, which could trigger the intense immune response. The subsequently released 1MT upon caspase-3 cleavage could further strengthen the immune system and help to activate CD8+ T cells effectively. This cascaded synergistic effect could inhibit both primary and lung metastasis tumor effectively, which may provide the solution for solving tumor recurrence and metastasis clinically.

Keywords: chimeric peptide, photodynamic therapy, immunotherapy, tumor, anti-metastasis.

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The therapeutic strategy dealing with cancer clinically at present includes surgical resection, chemotherapy, radiotherapy, all of which could indeed inhibit the tumor growth intensely in a short term. However, what usually leads to therapeutic failure or even death is the recurrence and metastasis of cancer in a few years.1 Particularly, the patients with triple negative breast cancer (TNBC), a highly metastatic cancer type, usually have poor prognosis after surgical resection and low 5-year survival rate clinically.2-4 Many strategies based on photodynamic therapy (PDT) for cancer metastasis inhibition currently have been reported.5,6 The cancer model in these reports was usually in an early stage and the mechanism of metastasis inhibition was based on the inhibition of primary tumor.7 However, the cancer has already been in mid and late stage in most cases when metastasis and recurrence appear.8 It is incapable to eliminate metastasis using PDT because the metastasis often occurs in deep location that the light could not penetrate through. Till now there is no effective management to the existing cancer metastasis clinically.1 Thus, developing the strategy to solve this problem remains an unmet need in the field of cancer therapy. Immunotherapy has become a promising strategy for cancer therapy in recent years, and it shows effective efficacy to mid and late stage cancer patients especially.9-11 That is mainly due to the fact that the immunosuppressive immune system could re-recognize and eradicate tumor cells, and produce durable response to tumor antigen for a long time.12 The blockade of immune checkpoints is one of the 3


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most effective modality among immunotherapy.13,

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14

Indoleamine 2,3-dioxygenase

(IDO), an immunosuppressive enzyme highly expressed in tumors, is such an immune checkpoint that causes the anergy and apoptosis of CD8+ T cells (also known as cytotoxic T lymphocytes (CTLs)) and enhances the immunosuppression modulated by CD4+ T cells (also named as regulatory T cells (Treg)).15,16 The small molecule 1-methyl-tryptophan (1MT),17,18 an IDO inhibitor, could effectively block IDO downstream, which could be used to strengthen durable immune response and avoid tumor recurrence for a long time.19 However, modest anticancer immunity was shown when using 1MT as monotherapy due to the ineffective antigen presentation and intratumoral immunosuppressive microenvironment.20 PDT could repair the deficiencies of immunotherapy mentioned above, since the exogenous light is controllable to target tumor tissues for precise therapy,21-24 and the in-situ explosive release of tumor antigens due to PDT cytotoxicity could significantly initiate immune response.20, 25-29 1MT mediated immune checkpoint blockade together with PDT could effectively complement with each other in anti-tumor process. Since PDT could prime an intense immune response within the primary tumor while IDO inhibitor help assure the effectiveness and durability and eliminate both primary and metastatic cancer cells. We conceived that a caspase responsive peptide Asp-Glu-Val-Asp (DEVD) could be used as a bridge to combine the photosensitizer PpIX with IDO inhibitor 1MT,30-32 and thus constructed a cascaded reaction for rational synergistic photodynamic and immunological therapy. Herein, we reported a chimeric peptide C16-K(PpIX)-PEG8-KDEVD-1MT 4


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(designed as PpIX-1MT). As Scheme 1A shows, the chimeric peptide PpIX-1MT consists of three parts. Palmitic acid and PpIX were used as a whole hydrophobic part to form the hydrophobic core of the nanoparticle in self-assembly. The PEG segment works for one hand as a linker to stabilize the molecule’s structure, and for another as a hydrophilic shell of the nanoparticles. To link the caspase-3 sensitive DEVD sequence with 1MT by an amide bond, PpIX-1MT nanoparticles could continuously release 1MT when caspase-3 is activated. In this work, the pulmonary tumorous metastasis mice model was established via intravenously injecting of murine colon carcinoma cells. As shown in Scheme 1B, it was envisioned that PpIX-1MT nanoparticles through intravenous injection could accumulate in primary tumor under the enhanced permeability and retention (EPR) effect. Then PpIX-1MT nanoparticles could produce ROS which induces tumor cells apoptosis by light irradiation. Afterwards, the caspase-3 given off from the tumor cells could promote the release of 1MT from PpIX-1MT nanoparticles to activate CD8+ T cells via inhibiting the IDO pathway. This could inhibit and eradicate effectively both the primary tumor and the lung metastasis.

RESULTS AND DISCUSSION Synthesis and Characterization of PpIX-1MT Nanoparticles. First of all, the compound Fmoc-Trp (Me)-OH was obtained through a four-step synthetic reaction (Scheme S1, Supporting Information) which is described in the Experimental Section. Then the chimeric peptide was obtained via standard solid-phase peptide synthesis 5


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(SPPS) method.33-35 The detailed structure of Fmoc-Trp (Me)-OH and PpIX-1MT was confirmed by 1H NMR (Figure S1-S3, Supporting Information), 13C NMR (Figure S4, Supporting Information) and electrospray ionization mass spectrometry (ESI-MS) (Figure S5, Supporting Information) respectively. The DEVD-1MT sequence in chimeric peptide was supposed to undergo a cleavage of the DEVD/1MT (/ represented the cleavage site) in the presence of caspase-3, which could release 1MT and therefore strengthen the effectiveness of tumor immunotherapy. As shown in transmission electron microscope (TEM) image

(Figure

1A),

chimeric peptide PpIX-1MT could form stable and well-dispersed spherical nanoparticles that the diameter is approximate 50 nm in phosphate buffer solution (PBS). Similar to the results of TEM, dynamic light scattering (DLS) shows the positive dispersity and the hydrodynamic size of PpIX-1MT nanoparticles is 128.5±1.2 nm. The minor discrepancy between the results from TEM and DLS is because of the shrinkage in vacuum condition during the TEM sample preparation stage.36 We also monitor the diameter of PpIX-1MT nanoparticles in H2O, DMEM and PBS with 10% fetal bovine serum. The diameter was almost unchanged within 48 hours (Figure S6, Supporting Information). This results confirmed the good stability of PpIX-1MT nanoparticles in physiological conditions. The ROS generating capability of PpIX-1MT nanoparticles under the light irradiation was identified from the change of fluorescence of ROS sensitive probe, 2’,7’-dichlorofluorescin diacetate (DCFH-DA). The nonfluorescent DCFH could be rapidly oxidized into fluorescent molecular dichlorofluorescein (DCF) under the 6


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existence of ROS.37 With the same condition of light irradiation, the PBS group showed little change in fluorescence intensity, and the same went to PpIX group due to the aggregation self-quenching effect.38 The PpIX-1MT group, however, demonstrates noticeable increase in fluorescence intensity, which indicating that PpIX-1MT nanoparticles could effectively avoid self-quenching of PpIX (Figure 1B). This experimental results confirmed PpIX-1MT nanoparticles could firmly promote the production of ROS and enhance the efficacy of PDT. In addition, singlet oxygen sensor green (SOSG) was employed to detect the generation of singlet oxygen, as shown in Figure 1C. The results were similar to measurement using DCFH. The amount of singlet oxygen in PpIX-1MT group was extremely greater than the one by PpIX as well as PBS. Furthermore, the intracellular ROS generation was studied in murine colon adenocarcinoma cell CT26 via confocal laser scanning microscopy (CLSM). The DCFH-DA was employed to identify the generation of ROS as well. As shown in Figure 1D, under the same condition of light irradiation, the stronger intensity of green fluorescence in PpIX-1MT (+) group was observed while there is nearly no green fluorescence in PBS, PpIX group. Without light irradiation, the negligible green fluorescence was detected in PpIX-1MT (-) and 1MT (-) group. These experimental results indicated that the apparent green fluorescence was due to the generation of ROS by PpIX-1MT nanoparticles under the condition of irradiation, which is consistent with the results in Figure 1B and Figure 1C, and confirmed that the PpIX-1MT nanoparticles also possess the capability to generate ROS in the 7


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intracellular environment during the light irradiation. After proving that PpIX-1MT could generate ROS, the release behavior of 1MT from PpIX-1MT nanoparticles was investigated in PBS in the presence of caspase-3 by using high-performance liquid chromatography (HPLC). The amount of 1MT released from PpIX-1MT nanoparticles incubated with caspase-3 at the temperature of 37 oC was increased with the extension of time (Figure 1E). The accumulative release amount of 1MT reaches approximate 83% after about 50 hours. This results fully testified that the IDO inhibitor 1MT could be firmly released from PpIX-1MT nanoparticles in the presence of caspase-3 in vitro, and the strategy that combined PDT and immune checkpoints inhibitor through a caspase responsive sequence peptide was feasible. Phototoxicity of PpIX-1MT Nanoparticles and Immune Response in Vitro. After confirming the results that PpIX-1MT nanoparticles could both generate ROS in vitro and intracellularly, the phototoxicity of PpIX-1MT nanoparticles was then evaluated via 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphe-nyltetrazolium-bromide (MTT) assay. CT26 cells were incubated with different concentrations of PpIX-1MT nanoparticles. As Figure 2A displays, the cell viability was about less than 50% while the concentration was 2.5µM within 2 minutes light irradiation, which further confirmed the eminent capability of ROS generation by PpIX-1MT nanoparticles. On the contrary, without the light irradiation, negligible change in cell viability was observed. The PpIX-1MT nanoparticles that possess the capability of high phototoxicity and low dark cytotoxicity is sufficiently suitable for clinical usage, since 8


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both the outstanding PDT efficacy and prominent biocompatibility are the firm foundation for in vivo application. Whether the combination of the PDT and 1MT could facilitate the immune response was then investigated by coculture of CT26 tumor cells and lymphocytes. Briefly, the splenocytes was isolated from Balb/c mice and cocultured with CT26 cells for 48 hours. PBS, 1MT, PpIX and PpIX-1MT were then added respectively and the cocultured cells of PBS (+), PpIX (+) and PpIX-1MT (+) group received 2 minutes light irradiation while there are two groups of PpIX-1MT (-) and 1MT (-) not receiving light irradiation as control. As shown in Figure 2B, the percentage of CD3+CD4+ T cells considerably decreased after being treated with PpIX-1MT nanoparticles for 2 minutes light irradiation comparing to other groups. The percentage of CD3+CD8+ T cells after being treated shows an opposite result against above. Whereafter, the ratio of CD3+CD8+ T cells to CD3+CD4+ T cells was observed and the PpIX-1MT (+) group showed large value than other groups (Figure 2E). However, small changes of the percentage of CD3+CD4+ T cells and the ratio of CD3+CD4+ T cells to CD3+CD8+ T cells appeared in 1MT (-) group (Figure 2D), which could further confirm the fact that 1MT shows a slight efficiency when it is used as a single agent. All the data suggested that, PpIX-1MT nanoparticles not only possess the excellent phototoxicity to induce apoptosis of tumor cells but could, effectively recruit CD8+ T cells via blocking IDO pathways after a series cascaded reaction to release 1MT. It was reported that PDT could induce immunogenic cell death (ICD) via 9


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apoptosis and necrosis.39,

40

We also investigated the cell-surface exposure of

calreticulin (CRT), an ICD associated proteins which serves as an “eat me” signal to mediate antitumor immune response,41, 42 by flow cytometry and immunofluorescence respectively after PDT treatment. As shown in Figure 2F, the cells only treated with PpIX-1MT and irradiation have exposed the calreticulin on the cell surface. It was further confirmed by the results of flow cytometry as Figure 2G displayed. Negative exposure of CRT was observed in PBS (+), PBS (-) and PpIX-1MT (-) groups. However, the cells showed positive exposure of CRT only in PpIX-1MT (+) group. From both the results from flow cytometry and immunofluorescence, the capability that PpIX-1MT nanoparticles could initiate the immune response during the PDT process was further verified. Tumor Imaging and Biodistribution of PpIX-1MT Nanoparticles in Vivo. The anticancer feasibility of PpIX-1MT nanoparticles in vivo was afterwards investigated. Firstly, we explored the PpIX-1MT nanoparticles in vivo biodistribution and tumor imaging by using CT26 tumor-bearing mice as animal models. As shown in Figure 3A, the distinctly fluorescent signals were observed in tumor areas after a two-hour intravenous injection of PpIX-1MT nanoparticles. The PpIX-1MT nanoparticles are equipped with good stability, enabled with excellent long blood circulation and achieved EPR effect effectively. The fluorescent intensity reaches its maximum value at the time of 12 hours. It is clearly shown that there is still strong fluorescence at the time of 24 hours, which could declare that the PpIX-1MT nanoparticles could retain for a long time after accumulation in tumor areas. 10


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After 24 hours intravenous injection, the mouse was peeled off and the biodistribution of PpIX-1MT nanoparticles in tumor and other tissues was studied. The main fluorescence was located in tumor. Meanwhile, some fluorescence exists in liver and lung. On the contrary, there is little fluorescence in heart, spleen and kidneys (Figure 3B). The mean fluorescent signal of PpIX-1MT nanoparticles in tumor tissue and other organs is demonstrated in Figure 3C, and the MFI value of tumor tissue is much higher than that of other organs. In a word, PpIX-1MT nanoparticles were able to efficiently accumulate and retain in tumor areas after intravenous injection, whose outcome is beneficial for anticancer application in vivo. Anticancer Efficacy by PpIX-1MT Nanoparticles via PDT in Primary Tumor. After confirming the capability of accumulation and retention in tumor areas of PpIX-1MT nanoparticles, the anticancer efficacy of PpIX-1MT nanoparticles was then explored after intravenous injection. The experiment lasted 21 days and the treatment was conducted at 1st, 5th and 10th days respectively, as shown in Figure 4A. Irradiation time point was chosen at 12 hours after the intravenous injection of PpIX-1MT nanoparticles since it has the most amount of accumulation in tumor area, which is referred to the results of Figure 3A. The change of absolute tumor size with the time is shown in Figure 4C. The mice treated by PpIX-1MT could hardly inhibit tumor growth without light irradiation as well as the mice treated with PBS. Comparing to the PBS (+) group, the other two groups of 1MT (-) and PpIX (+) only show slight inhibition effect. Nevertheless, what sharply contrasts with these four groups is that tumor growth witnessed significant inhibition in PpIX-1MT (+) group 11


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within 10 minutes light irradiation in every treatment. The results fully authenticate the strategy of the combination of PDT phototoxicity and immune activation by 1MT could inhibit tumor growth to the maximum extent. Tumor tissues of each group were then peeled off, as shown in Figure 4B. Similar to the results of Figure 4C, the image of tumor tissues shows the excellent therapeutic efficacy of PpIX-1MT nanoparticles more directly under the condition of light irradiation. The relative body weight shows unobvious changes until the end of the treatment in PpIX-1MT (+) group (Figure 4D). Besides, the blood biochemistry and blood routine test are normal after the 21-day treatment (Figure 4G), which could entirely declare that there is rare systematic toxicity via intravenous injection of PpIX-1MT nanoparticles. The mTOR pathway is known to be sensitive to depletion of tryptophan. 43 As the results of tryptophan decreased, the phosphorylation of certain proteins are interferential and the P-S6 kinase (P-S6K) is activated resulting in the immunosuppression. 44, 45 The expression of phosphorylated p70S6K (P-S6) after PDT treatment was then investigated by western blot. The expression of P-p70S6K was significantly decreased in PpIX-1MT (+) group compare to PBS (+) and PpIX-1MT (-) groups (Figure 4E). Furthermore, the level of cleaved caspase-3 was also analyzed with western blot. As Figure 4F shows, PpIX-1MT (+) group shows positive expression of cleaved caspase-3 which could cleave the IDO inhibitor 1MT from PpIX-1MT nanoparticles. We further confirmed the therapeutic efficacy via hematoxylin and eosin (H&E) staining. More necrosis regions were observed in PpIX-1MT (+) group than the other 12


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groups. Meanwhile, the in situ terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was applied to investigate the mechanism of therapy (Figure 4H). Cells that were treated with PpIX-1MT nanoparticles under light irradiation send out bright green fluorescent signals, similar to the results of H&E staining, which means the maximum amount of cell apoptosis appeared in PpIX-1MT (+) group comparing to other groups. Efficacy of Metastasis Inhibition by PpIX-1MT Nanoparticles. As well known, the main reason that leads to therapy failure and the death of patients is the cancer metastasis. Therefore, whether PpIX-1MT nanoparticles could inhibit and eradicate cancer metastasis by activating immune system or not was central issue, and investigated as following. The pulmonary tumorous metastasis mice model was established by intravenous injection of CT26 luciferase cells at the day before therapy began. By irradiating to the primary tumor, the situation of pulmonary tumorous node was examined with bioluminescence imaging after intraperitoneal injection of D-luciferin potassium solution at the last day of treatment. Comparing to the other groups, observed in Figure 5A, PpIX-1MT (+) group didn’t show any luminescence, which means PpIX-1MT nanoparticles could both effectively inhibit and eradicate the primary tumor and pulmonary tumorous metastasis simultaneously during the light irradiation (Figure S7, Supporting Information). On the contrary, the other groups show strong luminescence in lung tissues. In the next step, we peeled off the lung tissues of mice that had been treated by Indian ink. There is almost no pulmonary tumorous node in PpIX-1MT (+) group comparing to the large amount of pulmonary 13


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tumorous node in the other groups (Figure 5B, red arrow shows the pulmonary tumorous node). The appearance of white tumorous node particularly in PpIX (+) and 1MT (-) groups suggested that whether use PDT or 1MT alone is incapable of eradicating the lung metastasis. H&E staining of lung tissues was further to be confirmed (Figure 5C, blue arrow shows the pulmonary tumorous metastasis). Comparing to the positive results of PpIX-1MT (+) group, other four groups show different degrees of cancerization. It is suggested that 1MT applied in this nanoplatform could tackle the problems which PDT couldn’t achieve as using alone. All these data could be firmly confirmed that the activation of immune system during PDT process via blocking IDO downstream by 1MT resulting from cell apoptosis could inhibit not only primary tumor but lung metastasis. Immune Response in Vivo. After verifying the efficacy of PpIX-1MT nanoparticles to eliminate both primary and lung metastasis tumor, whether the in vivo immune response can be elicited by PpIX-1MT nanoparticles was then investigated. Briefly, the fresh blood and spleen were obtained from each group mice after the treatment had been finished, and the relative immune cells were measured via flow cytometry. As shown in Figure 6A (also in Figure S8, Supporting Information), the ratio of CD3+CD8+ T cells to CD3+CD4+ T cells was significantly increased in PpIX-1MT (+) group comparing to other groups. Similar tendency was also displayed in Figure 6B (also in Figure S8, Supporting Information), the percentage of CD3+CD4+ T cells was conspicuously declined in PpIX-1MT (+) group, at the peak of 27.8%, while the other groups were showed unobvious changes comparing to PBS 14


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treated group. The same results were observed on the cells extracted from spleen. High percentage of CD3+CD8+ T cells and high ratio of CD3+CD8+ T cells to CD3+CD4+ T cells were observed in Figure 6C and D. All the experimental results proved that PpIX-1MT nanoparticles could elicit systematic immune response in vivo by blocking the IDO pathways. The effector cytotoxic T lymphocytes (CD8+ T cells) were observably activated by IMT and ultimately could enhance antitumor efficacy completely. Mechanism of Anticancer Efficacy in Vivo. Then, the mechanism of anticancer efficacy

by

PpIX-1MT

nanoparticles

was

further

investigated

via

immunofluorescence assay. As is well-known that the generation of antitumor immune response compose of a series of stepwise events.46 Briefly, dendritic cells (DCs) captured tumor antigens from dead tumor cells and presented intracellularly to activate immature T cells to effector T cells (cytotoxic T lymphocytes (CTLs)). Effector T cells then recognize and kill targeted tumor cells by inducing apoptotic pathways.47 Thus, caspase-3, CD86, CD8 and granzyme-B antibodies were used to label the dead cells, DC cells, CD8+ T cells and granzyme-B respectively, which stand for the extent of immune response elicited by each group. Comparing to PBS (+) group, the PpIX-1MT (+) group in primary tumor or lung showed more coexisting red and green fluorescence (Figure 7A), which indicated that the caspase-3, DC cells, CD8+ T cells and granzyme-B were effectively activated both in primary tumor and lung. However, by observing the fluorescence among PpIX-1MT (+) groups of primary tumor and lung respectively, more green fluorescence was observed in lung 15


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than in primary tumor, which could demonstrate that PpIX-1MT nanoparticles could significantly inhibit primary tumor growth mainly due to PDT process and eradicate lung metastasis because of the activated immune system. Whereafter, the immune-associated factors were analyzed via western blot. The immune-facilitate indexs include tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-17 (IL-17), CD-8, CD-86, granzyme-B were showing an upward tendency from PBS (+) group to PpIX-1MT (+) group regardless it is in lung or primary tumor (Figure 7B). While the IL-10, a mark standing for immunosuppression, showed an opposite tendency. By comparing the data above, we concludedthat the group after PpIX-1MT treatment with light irradiation was able to efficiently recruit much more DCs and CD8+ T cells, and thus enhanced antitumor efficacy. From this, it could be completely verified that PpIX-1MT nanoparticles possess the capability to activate immune system during PDT process. The strategy that the immunotherapy was induced by tumor cell apoptosis was successfully applied within both primary and lung metastasis tumor inhibition. CONCLUSIONS In summary, chimeric peptide PpIX-1MT combining PDT and an immune checkpoints inhibitor was designed and developed. PpIX-1MT nanoparticles could be formed via self-assembly and exhibited eminent dispersity and stability, effective accumulation and retention at tumor site. Then ROS could be generated upon 630 nm light irradiation and induce apoptosis of tumor cells. Subsequently, the promoted production of caspase-3 could cleave 1MT, an immune checkpoints inhibitor, from 16


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PpIX-1MT nanoparticles, thus strengthen the therapeutic efficacy for inhibition of primary tumor and lung metastasis. The strategy combined photosensitizer PpIX and immune checkpoints inhibitor 1MT was complementing each other with advantages. Such as the precise therapy of PDT could induce tumor apoptosis accurately and expression of tumor antigens effectively which could improve the precision and efficacy of immunotherapy. While the 1MT possessed capability to activate immune response that could efficiently recruit CD8+ T cells both in vitro and in vivo, which could solve the limitation of light. This synergistic system exhibited outstanding results for existing metastasis inhibition. Significantly, the simple preparation of chimeric PpIX-1MT and the cascaded reaction for cancer therapy could provide a solution to address cancer metastasis and recurrence clinically.

MATERIALS AND METHODS Materials.

N-fluorenyl-9-methoxycarbonyl

(Fmoc)

protected

L-amino

acid

Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, 2-cholrotriyl

chloride

1-hydroxybenzotriazole

resin

(100-200 (HOBt),

mesh,

loading:

and

0.914

mmol

g-1),

o-benzotriazole-N,N,N’,N’-

tetramethyluroniumhexafluorophosphate (HBTU) were purchased from GL Biochem. Ltd. (Shanghai, China). N,N’-dimethylformamid (DMF), Diisopropylethylamine (DIEA), protoporphyrin (PpIX), trifluoroacetic acid (TFA), piperdine, iodomethane, chlorotrimethylsilane

(TMS-Cl)

and

9H-fluoren-9-ylmethyl

carbonochloridate

(Fmoc-Cl) were provided from Shanghai Chemical Co. (China). Caspase-3 (human 17


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recombinant) was purchased from Biovision Corp. DCFH-DA was purchased from Beyotime Biotechnology Co., Ltd. (China). Roswell Park Memorial Institute (RPMI) 1640 medium, trypsin, fetal bovine serum (FBS), and Penicillin–streptomycin were provided by Biological Industries (USA). MTT was purchased from Genview Scientific Inc. (USA). All other reagents were used without further purification. The antibodies of CD3, CD4 and CD8 were purchased from 4A Biotech Co., Ltd (Beijing, China). Synthesis

of

Methyl

N-(tert-butoxycarbonyl)-1-Methyl-Tryptophanate.

N-Boc-Tryptophan (3.04 g, 10 mmol) and NaOH (0.8 g, 20 mmol) were dissolved in 20 mL dried DMSO. The mixture was stirred at the temperature of 40 ℃ for 2 hours. Then the iodomethane (4.26 g, 30 mmol) was added and the mixture was stirred for another 4 hours. 120 mL distilled water was added to stop the reaction and the mixture was extracted by EtOAc for 3 times. Then the organic phase was collected, dried by anhydrous Na2SO4 overnight, and the crude compound (1.8 g) was obtained by evaporating the solvent. The crude compound was purified by column chromatography on silica gel. 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 7.9 Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.24 – 7.18 (m, 1H), 7.11 (dd, J = 11.0, 3.9 Hz, 1H), 6.85 (s, 1H), 5.08 (d, J = 7.8 Hz, 1H), 3.77 – 3.71 (m, 3H), 3.68 (s, 3H), 3.27 (d, J = 5.1 Hz, 2H), 1.53 – 1.29 (m, 9H). Synthesis of 1-Methyl-Tryptophan. methyl N-(tert-butoxycarbonyl)-1-methyltryptophanate (2 g 6 mmol) was dissolved in the mixture of 1M NaOH (9 mL, 1.5 equiv) and 20 mL DMSO, and stirred for 5 hours at room temperature. Distilled water 18


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was then added and the aqueous was washed with EtOAc for 3 times. The pH was adjusted to 2.4 and the aqueous was extracted by EtOAc for 3 times. The collecting organic phase were concentrated under reduced pressure and got the crude product. The crude product was dissolved in CH2Cl2-CF3COOH (4:1 20 mL) and stirred for 2 hours. The solvent was evaporated and the crude product was dissolved in distilled water (10 mL), then filtered after adjusting pH to 7.0 to give pure 1-methyl-tryptophan as white solid. 1H NMR (400 MHz, DMSO) δ 7.57 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.18 – 7.11 (m, 2H), 7.02 (dd, J = 11.0, 3.9 Hz, 1H), 3.72 (s, 3H), 3.26 (d, J = 4.0 Hz, 2H), 2.93 (dd, J = 15.1, 8.9 Hz, 1H). Synthesis of Fmoc-Trp(Me)-OH. 1-methyl-tryptophan (0.82 g 3.75 mmol) was dissolved in 8.75 mL CH2Cl2. Then 1 mL (7.5 mmol) TMS-Cl was added. The mixture was cooled down in an ice bath after 1 hour refluxing. Diisopropylethylamine (1.2 mL 6.5 mmol) and Fmoc-Cl (0.65 g 2.5 mmol) were added. The solution was stirred in ice bath for 1 hour and warm to room temperature. The mixture was concentrated and then distributed in 20 mL ether and 25 mL 2.5% NaHCO3. The phases were separated. Aqueous layer was acidified to pH 2 and extracted by EtOAc. The yellow powder Fmoc-Trp(Me)-OH was obtained by evaporating the solvent. 1H NMR (400 MHz, DMSO) δ 12.73 (s, 1H), 7.90 (s, 1H), 7.88 (s, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.67 (t, J = 8.3 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.40 (dt, J = 11.7, 5.6 Hz, 3H), 7.29 (ddd, J = 15.1, 7.4, 3.7 Hz, 2H), 7.16 – 7.11 (m, 2H), 7.02 (t, J = 7.4 Hz, 1H), 4.28 – 4.13 (m, 4H), 3.71 (s, 3H), 3.18 (dd, J = 14.5, 4.5 Hz, 1H), 3.02 (dd, J = 14.6, 9.7 Hz, 1H). 19


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Synthesis of Chimeric Peptide PpIX-1MT. chimeric peptide PpIX-1MT was synthesized using standard Solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin. Briefly, the resin was soaked in anhydrous DMF for 30 minutes, and then 2 equiv. (relative to the loading content of resins) of Fmoc-Trp(Me)-OH and 6 equiv. of DIEA was added and reacted in DMF for 3 hours. Unreacted solution was removed by using anhydrous DMF three times. Unreacted reactive sites of resins were capped by 30 minutes reaction with a mixture of CHOH, DIEA and DMF (v/v/v=1.5:2:6.5). The resins were treated with 20% piperidine in DMF to remove the Fmoc protecting group. The next Fmoc-protected amino acid Asp(OtBu) was added to react with resins with 4.8 equiv. HOBt, 4.8 equiv. HBTU and 8 equiv. DIEA for 2 hours. Then Val, Glu(OtBu), Asp(OtBu) Lys(Boc), PEG8, Lys(Dde) and PA (C16) were conjugated to the resins by the same method. PpIX was conjugated to the resins after removing the Dde protected group by 2% hydrazine hydrate in DMF. Peptide was cleaved from resins by the solution of TFA, H2O and TIS with the ratio of 0.95:0.25:0.25 for 2 hours. Finally, the filtrate was concentrated and the pure peptide was precipitated in cold ether. In Vitro 1MT Release Behavior of PpIX-1MT Nanoparticles Incubated with Caspase-3. PpIX-1MT nanoparticles (5×10-6M) and caspase-3 (human recombinant 300×10-12M) were incubated in PBS and the mixture solution was dialyzed at 37 ℃. Dialysate which is from the mixture solution at different time point was analyzed by HPLC. The amount of 1MT released from PpIX-1MT nanoparticles was calculated by referencing to the standard curve of 1MT, and the percentage of 1MT release = the 20


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amount of released 1MT / the amount of 1MT in PpIX-1MT nanoparticles × 100%. Cell Culture. CT26 (colon adenocarcinoma cell line) cells were incubated in RPMI 1640 medium containing 10% FBS and 1% antibodies (penicillin–streptomycin, 10000 U mL−1), and cultured in humidified atmosphere that the temperature was 37 ℃ with 5% CO2. In Vitro Singlet Oxygen Generation Detection. By using the

1

O2 probe

DCFH-DA, the 1O2 generation detection capability of chimeric PpIX-1MT was studied. Briefly, the solution of DCFH-DA was firstly treated with NaOH (0.01M) for 30 minutes. Then the samples (PBS, PpIX-1MT, 0.5×10-6M, PpIX, 0.5×10-6M with 0.5% DMSO) in PBS, pH 7.4) were added into the treated solution of DCFH-DA. The solutions were irradiated with the red light (630 nm, 30mW cm-2) for different time respectively, and the fluorescence change was recorded with the excitation wavelength at 488 nm and the emission wavelength at 530 nm. By using the singlet oxygen sensor green (SOSG) to detect the generation of singlet oxygen by PpIX-1MT under light irradiation, 100 µg of SOSG was diluted in 330 µL DMSO to achieve 500 µM SOSG stock for preparation. Adding 10 µL SOSG stock into 1mL sample with certain concentration. The samples were then irradiated by red light (630 nm, 30mW cm-2) for different time respectively, and the fluorescence change was recorded with the excitation wavelength at 504 nm and the emission wavelength at 550 nm. For Visualization of Intracellular

1

O2 Generation by Chimeric Peptide

PpIX-1MT. CT26 cells were incubated with different drugs (PBS, PpIX-1MT 21


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1×10-6M, 1MT 1×10-6M, PpIX 1×10-6M with 0.5% DMSO) for 4 hours. Then replaced the medium and the cells were washed the three times. DCFH-DA containing the RPMI 1640 medium was added and further incubated for 0.5 h. In the next, light irradiation (630 nm, 30mW cm-2, 1 minute) was employed to the solutions (PpIX-1MT with no light as negative control). The cells were observed by CLSM as soon as possible (excitation wavelength: 488 nm, emission band pass: 500–550 nm). Cytotoxicity. CT26 cells were seeded into a 96-well plate (5000 cells per well) and cultured in RPMI 1640 medium containing 10% FBS (100 µL) incubated for 24 hours (37 ℃, 5% CO2). Then RPMI 1640 (100 µL) containing different concentrations of the PpIX-1MT nanoparticles were added in each well. For phototoxicity, after 12 hours incubation, the cells were irradiated by a diode laser (630 nm, 30mW cm-2) for 1 minute and further incubated for 24 hours at 37 ℃. For dark toxicity, the cells were further

incubated

for

24

hours

at

37

℃.

Then

MTT

(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) solution (20 µL, 5 mg mL−1) was added to each well and further incubated for 4 hours. Subsequently, the MTT media were removed and DMSO (150 µL) was added to each well. The optical density (OD) was measured at 570 nm with a microplate reader (Thermo Scientific Multiskan Go). The relatively cell viability was calculated as follows: viability= (OD sample/OD control) × 100%. The OD sample was obtained from the drug treated cells, and OD control was obtained from the cells without any treatments. CRT Exposure. The CRT exposure on cell surface was analyzed by flow cytometry. CT26 cells were incubated with PpIX-1MT for 12 hours and then received 22


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irradiation for 1 minute (630 nm, 30mW cm-2) and incubated in 37 ℃ for 4 hours. The cells were collected and washed with PBS twice before incubated with CRT antibody for 45 minutes. After that cells were washed with PBS twice and incubated with secondary antibody for 30min. For flow cytometry detection, cells were analyzed on FACS Aria III flow cytometer (BD Co., America) and data were analyzed with FlowJo (Tree Star). For observation by immunofluorescence via confocal laser scanning microscopy (CLSM). The cells were fixed in paraformaldehyde for 5 minutes after being treated with PpIX-1MT under 1 minute irradiation. The fixed cells were then stained by CRT antibody for 45 minutes and then stained with secondary antibody for 30 minutes after washes with PBS. The nuclei were stained with DAPI before observation. In Vitro Immune Response Analysis. the influence of immune related cells was assessed for measures of absolute lymphocyte count (ALC). Splenocytes were isolated from bal/bc mice and cocultured with CT26 cells (10:1 ratio of splenocytes to cancer cells) for 48 hours in the presence of PBS, 1MT, PpIX, and PpIX-1MT. Then the cells with PpIX-1MT were divided into two groups with or without the light irradiation (630 nm, 30mW cm-2) for 3 minutes respectively, and the cells with PBS and PpIX were under the same condition of light irradiation. Cells were collected and stained with indicated antibodies include CD3 (17A2, 1:500), CD4 (GK1.5, 1:500) and CD8 (53-6.7, 1:500). Cells were analyzed on a BD Fortessa (BD Bioscience) and data were analyzed with FlowJo (Tree Star). In vivo Immune Response Analysis. To measure the ALC in vivo, the mice were 23


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sacrificed at last day of treatment. Mice were acquired from Animal Experiment Center of Wuhan University (Wuhan, China), and fed with standard chow. All study protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal Experiment Center of Wuhan University (Wuhan, China). All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China. The blood and spleens were collected and the grinding solution of spleen or serum by each group were stained with indicated antibodies include CD3 (17A2, 1:500), CD4 (GK1.5, 1:500) and CD8 (53-6.7, 1:500). Cells were analyzed on a BD Fortessa (BD Bioscience) and data were analyzed with FlowJo (Tree Star). In vivo Tumor Imaging and Biodistribution. CT26 tumor-bearing mice were intravenously injected with PpIX-1MT (3mg kg-1 of PpIX in each mouse) nanoparticles when the tumor volume was reached about 200 mm3. The mice were imaged by the IVIS Spectrum (PerkinElmer) at the time point of 0, 2nd, 4th, 8th, 12th, 24th hour postinjection respectively (excitation wavelength: 630nm, fluorescence emission signal wavelength: 680nm). After 24 hours injection, the mice were sacrificed and the heart, liver, spleen, lung, kidney and tumor were peeled off. Then the organs were imaged by IVIS Spectrum (PerkinElmer) at the same condition. In

Vivo

Antitumor

Efficacy

and

Immunofluorescence

Assay.

CT26

tumor-bearing mice were randomly divided into five groups (PBS/hv, 1MT/dark, PpIX/hv, PpIX-1MT/hv and PpIX-1MT/dark groups) when the tumor volume was 24


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reached about 100mm3. Mice were then intravenously injected with PBS, 1MT (1mg kg-1 of 1MT), PpIX (3mg kg-1 of PpIX with 5% DMSO) and PpIX-1MT (3mg kg-1 of PpIX) solution respectively at the 1st, 5th, 10th days and the PBS/hv, PpIX/hv, PpIX-1MT/hv groups were received light irradiation (630nm wavelength, 340mW cm-2) for 10 minutes at 12 hours postinjection. The tumor volume and mice weight of each group were measured in every other day. The tumor volume was calculated by following formula: V=(tumor length)×(tumor width)2/2. Relative tumor volume was calculated as V/V0, V0 was the tumor volume on the first day before treatment. The mice were sacrificed at the 21th day. The solid tumors and organs were peeled off for histological observation by standard H&E staining, immunofluorescence staining and Western blot. The relative methods and manufacturer’s protocol were based on our previous study.7 The blood serums were collecting on the 21th day and the relative index were analyzed by biochemical auto analyzer (MNCHIP, Tianjin, China). The lung metastasis was imaged by bioluminescence analyzed via IVIS Spectrum (PerkinElmer) after intraperitoneal injection of D-luciferin solution at 21th day. To evaluate the anti-lung metastasis efficacy, the mice were injected with India ink (15%) through the trachea and the lungs were then peeled off and photographed at the last day of treatment. Statistical Analysis. Statistical analysis was performed using a Student's t test. The differences were considered to be statistically significant for a p value

Enhanced Immunotherapy Based on Photodynamic Therapy for Both

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