Efficient Synthesis of Chlorin e6 and its Potential Photodynamic Immunotherapy in Mouse Melanoma by the Abscopal Effect

DOI: https://doi.org/10.21203/rs.3.rs-2153107/v1

Abstract

Background

Photodynamic therapy (PDT) can eradicate not only cancer cells but also stimulate an anti-tumor immune response. Herein, we describe two efficient synthetic methodologies for the preparation of the second generation of photosensitizer Chlorin e6 (Ce6) from Spirulina platensis in higher yield and purity, and we address the phototoxic effect of Ce6 in vitro along with anti-tumor activity due to photodynamic therapy in vivo.

Methods

The use of different solvents, the duration of extraction/reaction, and the yield were analyzed and compared between the two methods during the synthesis of Ce6. The synthesized Ce6 was evaluated by TLC, HPLC, LC/MS, NMR, and studied for the anti-tumor activity of melanoma in vitro and in vivo. Melanoma B16F10 cells were seeded and phototoxicity was monitored by the MTT assay. C57BL/6 mice were transplanted with B16F10 cells for the tumor allograft model. The mice were subcutaneously inoculated on the left and right flank with 0.1 mL of B16F10 cells (1×106 cells/mL). The treated mice were intravenously injected with Ce6 of 2.5 mg/kg and then exposed to red light (660 nm) on the left flank tumors at 3 h after the injection.

Results

Our results revealed that the tumor was suppressed not only in the left flank but also in the right flank, where no PDT was given. The immune response was also studied by analyzing Interferon-gamma (IFN-γ), Tumor necrosis factor-alpha (TNF-α), and Interleukin-2 (IL-2) of the right flank tumors through qPCR. The upregulated expression of IFN-γ, TNF-α, and IL-2 revealed the local anti-tumor immunity due to Ce6-PDT.

Conclusion

The finding of this study suggest an efficient methodology of Ce6 preparation and the efficacy of Ce6-PDT as a promising anti-tumor immune response.

Background

Melanoma is an extremely aggressive malignant tumor that originates from melanocytes, and its advancement is difficult to forecast as it can also develop in other tissues like the eyes, nasal cavity, anal canal, digestive tract, and genitourinary tract [1, 2]. Despite the current guideline-based therapies for patients with melanoma including surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapy [3], photodynamic therapy (PDT) has been successfully used to treat patients having non-melanoma skin cancer [4]. In recent years, PDT has evolved as a promising alternative to the existing therapeutic modality for the treatment of various cancers due to its less invasiveness and minimal systemic toxicity [5]. PDT comprises the use of photosensitizers (PSs), appropriate light (600–800 nm) along with molecular oxygen to generate reactive oxygen species (ROS) or singlet oxygen (1O2) which are responsible for the cancerous cell death by apoptosis, necrosis, or destruction of blood vessels [6]. PDT has the ability to trigger a localized immune response that fights against cancers, but it also has a systemic abscopal effect that promotes the regression of metastatic and distant contralateral malignancies [7, 8]. An increase in antigen presentation following a higher ratio of immunologic cell death within the tumor microenvironment can enhance anti-tumor immunity via release of primary cytokines like IFN-γ, TNF-α, and IL-2 followed by activation of dendritic cells, triggering CDLs to stimulate CD4 + and CD8 + T cells and further CTLs (Cytotoxic T lymphocytes) and natural killer (NK) cell proliferation [9, 10].

Since the photosensitizers (PSs) have played a vital role in PDT activities, various photosensitizers have been developed, mostly belonging to the tetrapyrrole structure, whose absorbance wavelength is in between 600–800 nm and exhibit no dark toxicity as well as relatively rapid clearance from normal tissues after PDT, thereby minimizing phototoxic side-effects [11]. However, the development of PSs with a highly selective affinity towards tumor-killing activities is always demanded. In this regard, Ce6 has been developed as a promising second-generation photosensitizer due to not only its higher phototoxic potential but also displaying a strong absorption in the red region of the visible spectrum, leading to penetrating deeper cancer tissue layers [12]. Thus, Ce6-PDT demonstrates significant clinical achievement in the treatment of various cancers, including pancreatic [13], melanoma [14], bladder [15], and breast cancers [16].

Considering the therapeutic efficiency, researchers have explored various methods to synthesize Ce6. Notably, Ce6 is prepared from chlorophyll a. The extraction of chlorophyll a can be achieved from different algae or plants, including Erythrina variegate [17], Bamboo leaves [18], Erythrina oricntalis leaves, Mulberry leaves, Pennisetum purpureum, and Chlorella ellipsoidea [19] etc. In this regard, Zhang’s group extracted crude chlorophyll from crude silkworm excrement and prepared Ce6 through reverse Claisen condensation, hydrolysis, and acidification [20]. In addition, the accumulated findings suggest that Ce6 was prepared by either methyl pheophorbide a (Scheme 1) [2123] or pheophytin a [24, 25], which was converted from chlorophyll a. However, the method of preparing pheophytin is much easier and simpler to handle the reaction.

In the course of developing the efficient method to prepare Ce6, we followed two methods via pheophytin a from chlorophyll a, which is extracted from Spirulina platensis. The methods were modified in various ways by altering the quantity of solvents, extraction or reaction duration, and chemical reagents. Therefore, our first aim was to find the best methodological strategy to prepare Ce6 and analyze its PDT in vitro by using melanoma B16F10 cells. We also studied the immune response in vivo by irradiating light into the left flank of mice, inducing the regression of untreated right flank tumors. In addition, the primary cytokines such as IFN-γ, TNF-α, and IL-2 were also examined through qPCR to support the antitumor immunity by Ce6-PDT.

Materials And Methods

Synthesis of Ce6

We developed two new methods, i.e., method 1 and method 2, which included three steps: a) extraction of chlorophyll a from spirulina powder, b) demetallation of chlorophyll a to convert it into pheophytin a, and c) conversion to Ce6 from pheophytin a. These methods were accomplished by altering the reaction procedure as well as different conditions with each other (Scheme 2).

Method 1. Chlorophyll a extraction and Synthesis of Ce 6

a) Extraction of chlorophyll a

Spirulina (200 g) was dispersed in EtOH (2L) and stirred overnight (12 h) in a continuous nitrogen environment. The spirulina was filtered and the filtered ethanol solution was evaporated through a vacuum evaporator until the volume of ethanol remained to 400 mL. Then, water (200 mL) and hexane (500 mL) were added to the chlorophyll a containing EtOH solution, stirred for 10 min and kept in the refrigerator overnight. Later, chlorophyll a was extracted in hexane by liquid-liquid layer separation three times.

b) Conversion to pheophytin a

To the hexane solution of chlorophyll a, 1N HCl (7 mL) was added dropwise and stirred for 4 h at room temperature. 1M NaOH (10 mL) was added dropwise after the completion of the reaction analyzed by TLC. Then, 70% EtOH (400 mL) was added to the reaction mixture, which was again kept in the refrigerator for 12 h. The hexane layer was separated by liquid-liquid separation with 70% EtOH three times, vacuum evaporated, and then dried to obtain pheophytin a.

c) Conversion and purification of Ce6

The pheophytin a (4.92 g) was dissolved in acetone (400 mL) and nitrogen bubbled into the solution of pheophytin a for 30 min. 1M NaOH (20 mL) was added dropwise and refluxed overnight under inert conditions. After completion of the reaction monitored by TLC, the reaction mixture (Ce6) was filtered and washed with acetone. The black solid of Ce6 was collected and dried for 4 h. The dried Ce6 (3.92 g) was dissolved in 25 mL of water and stirred for 2 h at room temperature under inert conditions. 1N HCl (7.5 mL) was added to the solution to maintain pH 7.0 and stirred for 2 h. The reaction mixture was centrifuged at 10,000 rpm for 1.5 h to remove the junk like carbohydrates, peptides, and others. The supernatant was filtered, and then the filtrate was treated with 1N HCl to maintain pH ~ 3.5 and the Ce6 was collected through filtration. It was vacuum dried at 35 oC for 4 h to get 1.75 g yield (1% of spirulina powder). The Ce6 was analyzed through NMR and LC/MS. 1H-NMR (600 MHz, DMSO-d6); δ 9.75 (s, 1H), 9.65 (s, 1H), 9.10 (s, 1H), 8.23 (dd, 1H, J = 18, 18 Hz), 6.41 (dd, 1H, J = 18, 18 Hz), 6.13 (dd, 1H, J = 18, 18 Hz), 5.37–5.41 (q, 2H, J = 18 Hz), 4.61–4.65 (q, 1H, J = 6 Hz), 4.45–4.47 (q, 1H, J = 6 Hz), 3.71–3.75 (q, 2H, J = 6, 12 Hz), 3.59 (s, 3H), 3.49 (s, 3H), 3.25 (s, 3H), 2.63–2.68 (m, 1H), 2.29–2.34 (m, 1H), 2.14–2.19 (m, 1H), 1.68 (d, 6H, J = 6 Hz), 1.65 (t, 1H, J = 6, 12 Hz) Mass spectra m/z C34H36N4O6 (M + H)+: 597. Yield: 1.75 g, < 1% of spirulina powder

Method 2. Chlorophyll a extraction and Synthesis of Ce 6

a) Extraction of chlorophyll a

Spirulina (100 g) was dispersed in hexane (200 mL) for 30 min, 96% EtOH (800 mL) was added, and stirred overnight (12 h) in a continuous flow of nitrogen. Then, spirulina was filtered, water (200 mL) was added to the filtrate, and the hexane layer was separated through the liquid-liquid separation method to obtain chlorophyll a.

b) Conversion to pheophytin a

To the hexane solution of chlorophyll a, 5 mL of 2N HCl was added dropwise and stirred for 2 h. After completion of the reaction checked by TLC, the reaction mixture was washed through liquid-liquid separation by using 80% EtOH (200 mL) twice. The pheophytin a in the hexane layer was collected, vacuum evaporated, and dried for an hour.

c) Conversion and purification of Ce6

The pheophytin a (2.5 g) was dissolved in acetone (60 mL) and nitrogen bubbled into the solution of pheophytin a for 30 min. Then, 13% KOH in ethanol (378 mL) was added dropwise and refluxed for 10 min. After the completion of the reaction checked by TLC, the reaction mixture was cooled down to 17 oC and kept on an ice-bath. 11% v/v HCl was added to the reaction mixture to maintain pH 0.2 and stirred for an hour for acidification. Then 30% NaOH solution was added to the solution to maintain pH 0.6 and stirred for 30 min for Ce6 cleaning while maintaining a temperature of 17–19 oC. The reaction solution was filtered where almost all the junk like carbohydrates, peptides, carotenoids, and others was removed. The filtered solution was again treated with the addition of 30% NaOH solution to maintain the pH ~ 3.0 and stirred for an hour. Thus, the formed precipitate of Ce6 was filtered and dried for 24 h at 25 oC. The synthesized Ce6 was analyzed through NMR and LC/MS. 1H-NMR (600 MHz, DMSO-d6); δ 9.73 (s, 1H), 9.61 (s, 1H), 9.10 (s, 1H), 8.21 (dd, 1H, J = 18 Hz, 18 Hz), 6.39 (dd, 1H, J = 18), 6.12 (dd, 1H, J = 18, 18 Hz), 5.37–5.39 (q, 2H, J = 7.2 Hz), 4.61–4.65 (q, 1H, J = 6 Hz), 4.45–4.47 (q, 1H, J = 12 Hz), 3.71–3.74 (q, 2H, J = 6 Hz), 3.59 (s, 3H), 3.48 (s, 3H), 3.22 (s, 3H), 2.63–2.69 (m, 1H), 2.30–2.34 (m, 1H), 2.14–2.19 (m, 1H), 1.68 (d, 6H, J = 6 Hz), 1.63–1.66 (t, 1H, J = 6, 12 Hz) Mass spectra m/z C34H36N4O6 (M + H)+: 597. Yield: 0.8 g, < 1% of spirulina powder.

NMR, HPLC, LC/MS, and UV analysis

1H-NMR spectra were taken at a Bruker 600 MHz in deuterated DMSO using the solvent chemical shift of 2.5 ppm and water peak at 3.35 ppm. Thin-layer chromatography was performed on silica gel 60 F254 (Merck). Spirulina platensis was purchased from SHAAN XI FREESUN TRADING CO. LTD, China. Ethanol, acetone, and hexane were purchased from Daejung Chemicals, South Korea. HPLC grade water, methanol and acetonitrile were purchased from Burdick and Jackson, USA. Compounds were analyzed using Waters Alliance separation module e2695 (Waters, Milford, MA), coupled with 2998 PDA detector (Empower® 3 software). Capcell pak UG120 C18 (4.6x150 mm, 5 µm) using a linear gradient of 45–100% B (acetonitrile) in 0.1% TFA water (A) over 20 min at a flow rate of 1 mL/min (Ce6). The detection wavelength was set to 407 nm for Ce6 and 430 nm for chlorophyll a and pheophytin a. For chlorophyll a and pheophytin a, isocratic elution of acetonitrile, methanol, and ethyl acetate mixture (3:1:1) was used. The column temperature was set at 23 ± 2°C. Mass analysis (m/z 200–800) was performed by online HPLC–MS using a Waters ZQ2000 mass detector (Waters, Milford, MA) with ESI positive ionization mode. For the ultraviolet-visible (UV) spectrophotometry (Thermo-scientific, Skanlt software 5.0), the samples were prepared in 95% ethanol solvent (Duksan, HPLC grade pure) at a concentration of 10 µM. The data was corrected for solvent background by the instrument’s calibration using the 95% ethanol as a blank. The absorption spectra of the sample in solution was obtained in the range of 300–800 nm at a 1 nm interval in three determinations using three trial samples.

Cell viability assay

B16F10 cells were purchased from Korean Cell Line Bank (KCLB, Seoul, Republic of Korea). They were grown in DMEM supplemented with 10% fetal bovine serum (life technologies corporation, USA) and 1% Penicillin & Streptomycin (life technologies corporation, USA). These cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. B16F10 cells were plated at a density of 5 x 103 cells per well in 96-well plates and then incubated for 3 h. After treatment of the cells with various concentrations of Ce6, the cells were or were not exposed to irradiation of a 660 nm laser with power densities of 1 J/cm2 and were further incubated for 72 h. An MTT assay was carried out to determine the cell viability at 590 nm by using a microplate reader.

Animal model

6 week old male C57BL/6 mice (n = 10) weighing 21 g were purchased from Orient Bio (Seongnam, South Korea) and the mice were housed in a standard environment (20 ± 2˚C; 50 ± 5% humidity; 12, 12 h light/dark cycle; diet; and filtered water ad libitum) in the animal house facility of Dongsung Cancer Center, Daegu for 7 days. Each experimental group consisted of randomly grouped mice of similar weight. All the mouse experiments were reviewed and carried out with the approval of the Institutional Animal Care and Use Committee of the Dongsung Cancer under protocol IACUC #ds002106117-2.

Allograft mouse model using B16F10 melanoma cell line

10 mice were chosen at random for the study after a week of acclimation according to the previous literature [26]. Mice received a subcutaneous injection of 0.1 mL of B16F10 cells (1 × 106 cells/mL) into both the left and right flank. When the tumor mass reached the range of 50–70 mm3 after inoculation, a total of 10 tumor-bearing mice were randomly divided into control (n = 5) and Ce6-induced PDT (Ce6-PDT) groups (n = 5).

PDT in the animal model

The mice were administered saline or 2.5 mg/kg Ce6 intravenously through mouse tail veins in the vehicle control and Ce6-PDT groups after applying anesthesia. After staying in the dark room for 3 h, the mice were irradiated by a red light at a rate of 100 J/cm2 for 8 min 20 s. To evaluate the therapeutic efficacy of Ce6-PDT, the tumor volumes of the mice were recorded on days 8, 11, 15, and 18 respectively. The tumor volumes (V) were measured by a vernier caliper and were calculated by using the following formula: V = L x W2/2, (L: length and W: width). When the tumor reached a size of over 2000 mm3, the mice were euthanized by cervical dislocation. Since 2 mice each from the control and Ce6-PDT groups were found to be dead during the experiment, they were not included in the analysis. Only 3 mice from each group were included for the analysis.

Cytokine mRNA expression assay

The mRNA expression of IFN-γ, TNF-α, and IL-12 was determined by qPCR. Total RNA was isolated from cells according to the manufacturer’s recommendations using TRIzol reagent and the Absolutely RNA kit from Takara Bio. Total RNA (2 µg) was reverse transcribed in a total volume of 20 µL using 200 U of Superscript II reverse transcriptase (Invitrogen), 100 pmol oligo-dT, 0.5 mM dNTP, and 40 U RNasin (Promega, Madison, WI, USA). The resulting complementary DNA was diluted 1:10 with nuclease-free water. Five microliters of diluted cDNA was used in subsequent PCR reactions. The PCR amplification protocol was 500C for 2 min and 950C for 10 min followed by the 40 cycles of 95 oC for 30 s, 600C for 30 s and 720C for 30 s. The qPCR analysis was performed on a C1000™ Thermal Cycler (CFX 96™ Real-Time System, BioRad, Munich, Germany). All qRT-PCR reactions were carried out in triplicate. Relative quantification with the data obtained was performed according to the user's manual. The results are presented as transcript levels relative to the levels in untreated control cells, with average mRNA levels of the internal control gene, glyceraldehyde-3-phosphate dehydrogenase, used as the normalization control. To verify changes in gene expression, qPCR was carried out on selected genes. All primers were designed based on nucleotide sequences retrieved from Genbank using the Primer Express software (Applied Biosystems, Foster City, CA, USA). The fold-change (ratio) in gene expression was calculated by using the threshold cycle (CT). The primer pairs used in this study are listed in Table 1.

Table 1

The primer sequences used for qPCR.

Oligo name

Oligo sequences

TNF-α FP

AGC CCA TGT AGC AAA CC

TNF-α RP

GGA AGA CCC CTC CCA GAT AG

IFN-γ FP

TTC AGC TCT GCA TCG TTT TG

IFN-γ RP

CAT GTA TTG CTT TGC GTT GG

IL-2 FP

TGC AAC TCC TGT CTT GCA GC

IL-2 RP

ATG GTT GCT GTC TCA TCA GC

Results

Synthesis of Ce6

The Ce6 was prepared through two different methodological strategies. Method 1 and method 2 were analyzed, compared, and summarized in Table 2. Method 1 involved the extraction of chlorophyll a for 12 h by using 99.9% EtOH, whereas in method 2, 96% EtOH and hexane were used for 12 h. Although the same solvent, hexane, was used in both methods for synthesis of pheophytin a from chlorophyll a, the addition of double strength of HCl (2N) in method 2 reduced the reaction time from 4 h to 2 h. When 13% ethanolic solution of KOH was used in acetone reflux of pheophytin a in method 2, the reaction was completed in 10 min, but in method 1, it took 12 h for the completion of the reaction by using 1M NaOH. After getting the Ce6, the purification process was also performed in two different ways. Method 1 included a series of steps, i.e., pH change of Ce6 solution to 7, centrifugation of solution followed by filtration of the supernatant, pH change of the Ce6 solution to ~ 3.5, and filtration. Similarly, method 2 included pH alteration of Ce6 solution to 0.6, filtration, pH change of filtered Ce6 solution to ~ 3, and again filtration. The consumption of 11% v/v HCl and 30% NaOH was greater during the purification process in method 2 than in method 1. Among the two methods, the preparation time duration of Ce6 was reduced in method 2 than in method 1. The final yield was similar but the purity of Ce6 in method 1 was slightly improved over method 2.

Table 2

Comparison of method 1 and method 2

Parameters

Method 1

Method 2

The solvent used in the extraction

99.9% EtOH

96% EtOH, hexane

Extraction time duration of chlorophyll a

12 h

12 h

Solvent/acid strength/time for demetallation (Preparation of pheophytin a)

Hexane, 1N HCl, 4 h

Hexane, 2N HCl, 2 h

Base used for Ce6 synthesis

1M NaOH

13% ethanolic solution of KOH

Time duration for Ce6 synthesis

12 h

10 min

Ce6 purification

pH change, centrifugation, and filtration

pH change, filtration

Total time

5 days

3 days

Yield % and Purity %

< 1% of Spirulina mass, and < 97%

< 1% of Spirulina mass, and < 94%

NMR, HPLC, and LC/MS analysis

The synthesized Ce6 from two different methods were analyzed and compared through NMR, HPLC, and LC/MS. During the HPLC analysis of Ce6, it came at RT 4.9 at 407 nm (Figs. 1A and 1B). Similarly, the two compounds were also confirmed by LC/MS H+ as 597 (Figs. 1C and 1D). In addition, during the reaction, the chlorophyll a and pheophytin a were analyzed and compared through HPLC. They were detected at 5.1 and 9.53 RT in 430 nm (S1, S2, and S4, S5 Figs) respectively. From the 1H NMR analysis of two Ce6, both revealed similar peak values at the same chemical shift including –OH peaks of 3 carboxylic acids at δ 9.75 (s), 9.65 (s), and 9.10 (s) of method 1- Ce6 and δ 9.73 (s), 9.61 (s), and 9.10 (s) of method 2- Ce6 (S3 and S6 Figs). The purity of methods 1 and 2 were found as 97% and 94%.

Figure 1 HPLC chromatogram and mass spectrogram of Ce6 by method 1 and method 2. Separation condition is linear gradient of 45–100% (acetonitrile) in 0.1% TFA water in a reverse-phase HPLC column. A. HPLC chromatogram of Ce6 by method 1. B. HPLC chromatogram of Ce6 by method 2. C. LC/MS of Ce6 by method 1. D. LC/MS of Ce6 by method 2.

UV absorbance of Ce6 at different pH

Dihydroporphyrin (chlorin) compounds exhibit absorption maxima of the two most intense bands, which are located in the visible range near 402 ± 4 nm (Soret band) and 660 ± 5 nm (Q-band) [27]. Therefore, the synthesized Ce6 was further characterized by intense absorption bands in the visible and UV regions of the spectrum at different pH, ranging from pH 1 to pH 9 (Fig. 2).

Figure 2 Absorption spectra of Ce6 at different pH (pH 1- pH 9)

The Ce6 absorption spectrum was characterized by a maximum at 402 nm (Soret bands) and a shoulder at 662 nm (Q bands). On increasing the acidification of the solution from pH 4 to pH 1, this resulted in the gradual bathochromic shift of the Soret band to 405 nm and 406 nm and the hypsochromic shift of Q bands to 642 nm. On increasing pH from pH 5 to pH 9, the soret band was noticed at 402 nm and the Q1 band at 662 nm along with the Q2 band at 502 nm.

In vitro cytotoxicity of Ce6-PDT in melanoma cancer

To investigate the cytotoxic effect of Ce6 under laser irradiation against melanoma cancer cells (B16F10), the MTT assay was performed (Fig. 3A-3C). Ce6 in the dark showed the highest cytotoxicity at 768 µM concentrations (IC50 = 519.6 µΜ) (Fig. 3A), whereas at the concentrations of 25 µM, the combination of Ce6 and 660 nm laser irradiation at 50 mW for 200 s (1 J/cm2), induced phototoxicity in B16F10 cells (IC50 = 20.98 µΜ) (Fig. 3B). As a result, the finding suggests that Ce6 in the dark has minimal toxicity, which is good for the ideal photosensitizer. On the other hand, Ce6-PDT demonstrated high cytotoxicity.

Figure 3 Ce6-mediated cytotoxicity on melanoma cancer cells by the MTT assay. A. Cytotoxic activity of Ce6 against B16F10 cells in the dark. B. Cytotoxic activity of Ce6-PDT against B16F10 cells. C. Morphological changes of B16F10 cells at concentration of the Ce6 (192 µM, 384 µM, and 768 µM) in the dark and (6 µM, 12.5 µM, and 25 µM) in Ce6-PDT.

In vivo effect of Ce6-PDT in allograft mouse model

Since the Ce6-PDT demonstrated the phototoxicity in vitro, we used an allograft mouse model using B16F10 melanoma cells to investigate Ce6 efficacy as a PDT agent. In this regard, C57BL/6 mice were transplanted with B16F10 cells for the tumor allograft model. Tumor-bearing mice in the vehicle control and Ce6-PDT groups were injected intravenously with vehicle (normal saline) and 2.5 mg/kg Ce6 solution via the tail vein, respectively. As shown in Figs. 4A and 4B, the tumor volume increased after the subcutaneous transplantation in the control group. As shown in Fig. 4C, after Ce6-PDT treatment on the left flank, the tumor volume was considerably decreased. On the other hand, the growth of the right tumor was also suppressed without Ce6-PDT treatment on the right flank (Fig. 4D) compared to the control group on day 11, but it then resumed growing till the 18th day. Suppressed tumor growth of the right flank was due to the immune responses generated by Ce6-PDT treatment on the left flank.

Figure 4 Tumor volumes of control and Ce6-PDT groups with tumors in the left and right flanks. The mice were subcutaneously injected at the left and right flank with 0.1 mL of B16F10 cells (1×106 cells/mL). The vehicle control was injected with normal saline. A. Changes in volume of the left tumor in the control group. B. Changes in volume of the right tumor in the control group. C. Changes in volume of irradiated left tumor in Ce6-PDT group. D. Volume changes of the unirradiated right tumor in the Ce6-PDT group.

Cytokines response after Ce6-PDT

We aimed to identify the immune response involved in PDT's anti-tumor effect since we discovered that the unirradiated right tumor also showed reduced growth along with the irradiated left tumor. Real-time PCR analysis was performed on the tumor tissues of unirradiated tumors in the right flank. When compared to the control in the right unirradiated tumor, IFN-γ production was upregulated on the fourth and seventh days after PDT treatment of the left tumor (Fig. 5A). TNF-α and IL-2 expression were found to be elevated, as was IFN-γ (Figs. 5B and 5C). However, IL-2 levels on the fourth day of Ce6-PDT were less than those of control. These results suggest an antitumor immune response of Ce6-PDT. Moreover, to further evaluate the systemic effects of Ce6-PDT, blood serum (10 µL) was also used for determining the cytokine mRNA expressions. TNF-α and IFN-γ expression levels were unchanged and were comparable to control levels on the fourth and seventh days, but IL-2 levels increased on both days following Ce6-PDT therapy as in tumor tissues (Fig. 5D). According to previous and current findings, cytokines including TNF-α, IFN-γ, and IL-2 are powerful inducers of antitumor immunity [28]. Therefore, the results here indicate that due to the elevated expression of TNF-α, IFN-γ, and IL-2, the growth of the unirridiated right flank tumors was decelerated.

Figure 5 Real-time PCR expression of IFN-γ, TNF-α, and IL-2 after Ce6-PDT. A. IFN-γ level on the 4th and 7th day after Ce6-PDT from the unirradiated right flank tumors. B. TNF-α level on the 4th and 7th day after Ce6-PDT from the unirradiated right flank tumors. C. IL-2 level on the 4th and 7th day after Ce6-PDT from the unirradiated right flank tumors. D. Relative expression levels of Th1 cytokines (IFN-γ, TNF-α, and IL-2) in the serum on the 4th and 7th day after Ce6-PDT

Discussion

Ce6 is the second-generation photosensitizer that is predominantly utilized for the treatment of various cancers, including melanoma [29]. Melanoma is a destructive form of skin cancer that has a very low rate of patient survival due to resistance to most therapeutic strategies [2]. Therefore, in order to assess the anticancer effectiveness of Ce6- PDT both in vitro and in vivo utilizing a melanoma mouse model, this study concentrated on the efficient synthesis of Ce6 with high yield and purity. For the development of Ce6, we have studied two methodological strategies for the synthesis of Ce6, analyzed each step, and compared them with each other. When comparing the two methods of preparing Ce6, method 1 was introduced as the superior strategical protocol as revealed by its 97% purity and simplicity in handling the reaction. However, method 1 took longer than method 2 in overall steps. Although Ce6 absorption in the red light region has been significantly demonstrated [30], the current study tested Ce6 absorbance in various pH ranges (1–9) and found that Ce6 has good stability with an absorbance of 640–665 nm.

Ideal PSs shows minimal toxicity in the absence of light and is only cytotoxic in the presence of light at a defined wavelength [31]. To achieve an optimal cytotoxic response of Ce6 and its PDT in melanoma cancer, B16F10 cells were studied. For the PDT, the melanoma cells were exposed to Ce6 (0-768 µM) in the dark and Ce6 (0-100 µM) with irradiation at 660 nm (50 mW, 1 J/cm2) for 200 s. Notably, Ce6 caused a significant PDT response at the concentration of 25 µM with an IC50 of 20.98 µΜ. The result also demonstrated the lower toxicity of Ce6 in the absence of light with an IC50 of 519.6 µΜ, which is a beneficial factor for PDT.

Inspired by the encouraging response of Ce6-PDT in vitro, its anti-tumors effectiveness and abscopal effect were also examined by successfully establishing tumors derived from B16F10 cells in both flanks of syngeneic mice. Only the primary tumors on each mouse's left side received Ce6-PDT treatment. The tumors on the right side were examined for a potential abscopal impact because they were regarded as distant tumors. Tumors and blood serum were collected on the fourth and seventh days of Ce6-PDT treatment to evaluate the cytokine response. Rapid tumor growth was observed in the control group, but the tumors on the left flank, where the light was irradiated, were significantly diminished, whereas those on the right flank grew slowly. Our findings showed that Ce6-PDT treatment on the left flank of mice led to a restriction in the growth of tumors on the right flank, signifying the antitumor immune responses of Ce6-PDT treatment. Oh and his team demonstrated similar antitumor immune responses by using Ce6-PDT coupled with an anti-CD25 monoclonal antibody [32]. Taken together, Ce6-PDT had an abscopal effect in addition to having a direct effect on melanoma tumor growth.

PDT stimulates the immune system through a variety of mechanisms, including the release of previously concealed tumor-associated antigens (TAAs) and immune-stimulatory molecules from tumors, which could activate and prompt an anticancer immune response. In addition, PDT also encourages chemokine and cytokine release to produce systemic influence and further activate the immune system’s anti-tumour activities [33]. An earlier study discovered that combining TNF-α and IFN-γ causes inflammatory cancer cell death via PANoptosis, as well as an adaptive immune response on the levels of their increment [34, 35]. Moreover, the production of IL-2 contributes to immune stimulation by leading to rapid lymphocyte proliferation and amplification of antigen-specific responses [36]. Therefore, for additional investigation of the antitumor immune response of Ce6-PDT, we analyzed the gene expression of Th1 cytokines such as TNF-α, IFN-γ, and IL-2 in unirradiated tumors of Ce6-PDT groups. On the fourth and seventh days of Ce6-PDT treatment compared to the vehicle control, mRNA expression was significantly increased. In addition, the expression of IL-2 was marginally downregulated on the 4th day, but on the 7th day, it was upregulated fourfold compared to that of the control. These findings imply that the increased levels of TNF-α, IFN-γ, and IL-2 in the unirradiated tumor indicated Ce6-PDT's antitumor immune response. Also we found that the integrated density values of TNF-α, IFN-γ, and IL-2 were similar to the control on the fourth and seventh days after Ce6-PDT. Our findings were also consistent with previous reports that pyrolipid nanoparticle-PDT contributed to the enhancement of antitumor immunity by elevation of TNF-α, IFN-γ, and IL-2 on the first day after PDT treatment, but cytokine levels were found to decrease on the second day [37]. According to previous studies, Ce6 associated with hybrid protein oxygen nanocarrier [38] and incorporated dissolving microneedles-mediated PDT [39] have shown a significant abscopal effect elicited by activating immunogenic cell death (ICD), which in turn promoted dendritic cells (DCs) maturation and the subsequent antigen presentation, thereby facilitating the T-cell-mediated immune response. However, for the first time, we have reported the abscopal effect of Ce6-PDT without capsuling any nanoparticles or in combination with other therapies.

Conclusion

Ce6 was successfully synthesized by two new methods and compared the methodological strategy, yield and purity. Among them, method 1 could be better protocol of preparation of Ce6 in terms of solvents, and laboratory handling, however, the purity and yields of both methods are comparable. Taken together, our results suggest that Ce6-PDT has local antitumor activity and its immune response that the production of pro-inflammatory (Th1) cytokines like TNF-α, IFN-γ, and IL-2 might restrict tumors growth on the unirradiated right flank of the mice due to the abscopal effect. Therefore, these findings might serve as an experimental support for future studies into the anticancer and abscopal effects of Ce6-PDT.

Abbreviations

Chlorin e6                   Ce6.

Photodynamic therapy                        PDT.

Chlorin e6-induced photodynamic therapy                Ce6-PDT.

Interferon-gamma                   IFN-γ.

Tumor necrosis factor-alpha               TNF-α.

Interleukin-2               IL-2.

Reactive oxygen species                     ROS.

Cytotoxic T lymphocytes                    CTLs.

Declarations

Ethics approval and consent to participate

The study protocol and animal procedures were approved by the Animal Care and Use Committee of Dongsung Cancer Center under protocol IACUC #ds002106117-2. For the reporting of animal experiments, all methods are reported in accordance with ARRIVE guidelines.

Consent for publication

Not applicable

Availability of data and materials

All datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests 

The authors declare that they have no competing interests.

Funding 

This work was supported by grants from the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (NTIS Number: 1711135018, RS-2020-KD000106).

Author’s contribution

YWK was responsible for the conception of this present work. SKM, JL and RS conducted the chemistry and biological experiments, analyzed the data, drafted the manuscript, and created the image. PG and TBTM reviewed and made significant contributions to the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

References

  1. Larribere L, Utikal J. Stem cell-derived models of neural crest are essential to understand melanoma progression and therapy resistance. Front Mol Neurosci. 2019;12:111.
  2. Tripp MK, Watson M, Balk SJ, Swetter SM, Gershenwald JE. State of the Science on Prevention and Screening to Reduce Melanoma Incidence and Mortality: The Time Is Now. CA Cancer J Clin. 2016;66:460–80.
  3. Bibbins-Domingo K, Grossman DC, Curry SJ, Davidson KW, Ebell M, Epling JW Jr, et al. Screening for Skin Cancer: US Preventive Services Task Force Recommendation Statement. JAMA. 2016;26;316(4):429-35.
  4. Lu Y-G, Wang Y-Y, Yang Y-D, Zhang X-C, Gao Y, Yang Y, et al. Efficacy of topical ALA-PDT combined with excision in the treatment of skin malignant tumor. Photodiagnosis Photodyn Ther. 2014;11:122–6.
  5. Santos AF, Almeida DRQ, Terra LF, Baptista MS, Labriola L. Photodynamic therapy in cancer treatment - an update review. J Cancer Metastasis Treat. 2019;5:25.
  6. Gunaydin G, Gedik ME, Ayan S. Photodynamic Therapy for the Treatment and Diagnosis of Cancer – A Review of the Current Clinical Studies. Front Chem. 2021;2:9:686303.
  7. Reginato E, Wolf P, Hamblin MR. Immune response after photodynamic therapy increases anticancer and antibacterial effects. World J Immunol. 2014;27:4(1):1-11.
  8. Lou J, Aragaki M, Bernards N, Kinoshita T, Mo J, Motooka Y, et al. Repeated porphyrin lipoprotein-based photodynamic therapy controls distant disease in mouse mesothelioma via the abscopal effect. Nanophotonics. 2021;10(12):3279-3294.
  9. Anzengruber F, Avci P, Freitas LF, Hamblin MR. T-cell mediated anti-tumor immunity after photodynamic therapy: Why does it not always work and how can we improve it? Photochem Photobiol Sci. 2015;14(8):1492-1509.
  10. Jin F, Liu D, Xu X, Ji J, Du JY. Nanomaterial-Based Photodynamic Therapy with Combined Treatment Improves Antitumor Efficacy Through Boosting Immunogenic Cell Death. Int J Nanomed. 2021;16:4693-4712.
  11. Kumar A, Moralès O, Mordon S, Delhem N, Boleslawski E. Could Photodynamic Therapy Be a Promising Therapeutic Modality in Hepatocellular Carcinoma Patients? A Critical Review of Experimental and Clinical Studies. Cancers. 2021;13(20):5176.
  12. Ryu A-R, Kim Y-W, Lee M-Y. Chlorin e6-mediated photodynamic therapy modulates adipocyte differentiation and lipogenesis in 2T3-L1 cells. Photodiagnosis Photodyn Ther. 2020;31:101917.
  13. Wang Y, Wang H, Zhou L, Lu J, Jiang B, Liu C, et al. Photodynamic therapy of pancreatic cancer: Where have we come from and where are we going? Photodiagnosis Photodyn Ther. 2020;31:101876.
  14. Naidoo C, Kruger CA, Abrahamse H. Photodynamic Therapy for Metastatic Melanoma Treatment: A Review. Technol Cancer Res Treat. 2018;17:1533033818791795.
  15. Yavari N, Andersson-Engels S, Segersten U, Malmstrom, PU. An overview on preclinical and clinical experiences with photodynamic therapy for bladder cancer. Can J Urol. 2011;18(4):5778-86.
  16. Ostanska E, Aebisher D, Bartusik-Aebisher D. The potential of photodynamic therapy in current breast cancer treatment methodologies. Biomed Pharmacother. 2021;137:111302.
  17. Javed SB, Anis M. Cobalt induced augmentation of in vitro morphogenic potential in Erythrina variegata L.: a multipurpose tree legume. Plant Cell Tiss Organ Cult. 2015;120:463-474.
  18. Wu J-H, Wang S-Y, Chang S-T. Extraction and determination of chlorophylls from moso bamboo (Phyllostachys pubescens) culm. J Bamboo and Rattan. 2002;1(2):171-180.
  19. Doan DT, Le TG, Nguyen QT, Nguyen VT, Montforts F-P. An Improved preparation of Methylpheophorbide A and Chlorin-E6 Trimethylester from Spirulina and Silkworm Waste. Chem Res J. 2018;3(6):82 – 87.
  20. Zhang G, Yang J, Hu C, Zhang X, Li X, Shan A, et al. Green synthesis of Chlorin e6 and tests of photosensitive bactericidal activities. J For Res. 2019;30:2349–2356.
  21. Bui HTH, Pham TT, Nguyen HTT, Do TM, Nga VT, Bac ND, et al. Transformation Chlorophyll a of Spirulina platensis to Chlorin e6 Derivatives and Several Applications. J Med Sci. 2019;7(24):4372-4377.
  22. Dias LD, Mfouo-Tynga IS. Learning from Nature: Bioinspired Chlorin-Based Photosenitizers Immobilized on Carbon Materials for Combined Photodynamic and Photothermal Therapy. Biomimetics. 2020;5:53.
  23. Guo X, Wang L, Wang S, Li Y, Zhang F, Song B, et al. Syntheses of new chlorin derivatives containing maleimide functional group and their photodynamic activity evaluation. Bioorg Med Chem Lett. 2015;25:4078-4081.
  24. Pumilia G, Cichon MJ, Cooperstone JL, Giuffrida D, Dugo G, Schwartz SJ. Changes in chlophylls, chlorophyll degradation products and lutein in pistachio kernels (Pistacia vera L.) during roasting. Food Res Int. 2014;65:193-198.
  25. Jenisova Z, Braniša J. Scientific experiment focused at pigment degradation by polyvinyl chloride combustion in science education. J Technol Sci Educ. 2019;9(3):458-466.
  26. Charan J, Kantharia ND. How to calculate sample size in animal studies? J Pharmacol Pharmacother. 2013;4(4):303-6. doi: 10.4103/0976-500X.119726.
  27. Cunderlikova B, Gangeskar L, Moan J. Acid-base properties of chlorin e6: relation to cellular uptake. J Photochem Photobiol B: Biol. 1999;53:81-90.
  28. Havunen R, Santos JM, Sorsa S, Rantapero T, Lumen D, Siurala M, et al. Abscopal effect in non-injected tumors achieved with cytokine-armed oncolytic adenovirus. Mol Ther Oncolytics. 2018;6:11:109-121.
  29. Kulbacka J, Chodaczek G, Rossowska J, Szewczyk A, Saczko J, Bazylinska J. Investigating the photodynamic efficacy of chlorin e6 by millisecond pulses in metastatic melanoma cells. Bioelectrochemistry. 2021;138:107728.
  30. Mocanu MN, Yan F. Ultrasound-assisted interaction between chlorin-e6 and human serum albumin: pH dependence, singlet oxygen production, and formulation effect. Spectrochim Acta A Mol Biomol Spectrosc. 2018;5:190:208-214.
  31. Yoon II, Li JZ, Shim YK. Advance in Photosensitizers and Light Delivery for Photodynamic Therapy. Clin Endosc. 2013;46:7–23.
  32. Oh DS, Kim H, Oh JE, Jung HE, Lee YS, Park JH, et al. Intratumoral depletion of regulatory T cells using CD25-targeted photodynamic therapy in a mouse melanoma model induces antitumoral immune responses. Oncotarget. 2017;8(29):47440.
  33. Nkune NW, Simelane NWN, Montaseri H, Abrahamse H. Photodynamic Therapy-Mediated Immune Responses in Three-Dimensional Tumor-Models. Int J Mol Sci. 2021;22:12618.
  34. Malireddi RKS, Karki R, Sundaram B, Kancharana B, Lee SJ, Samir P, et al. Inflammatory cell death, PANoptosis, mediated by cytokines in diverse cancer lineages inhibits tumor growth. Immunohorizons. 2022;5(7):568-580.
  35. Mroz P, Szokalska A, Wu MX, Hamblin MR. Photodynamic Therapy of Tumors Can Lead to Development of Systemic Antigen-Specific Immune Response. PLoS ONE. 2010;5(12): e15194.
  36. Briukhovetska D, Dorr J, Endres S, Libby P, Dinarello CA, Kobold S. Interleukins in cancer: from biology to therapy. Nat Rev Cancer. 2021;21(8): 481-499.
  37. He C, Duan X, Guo N, Chan C, Pooon C, Weichselbaum RR, et al. Core-shell nanoscale coordination polymers combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy. Nat Commun. 2016;7:12499.
  38. Chen Z, Liu L, Liang R, Luo Z, He H, Wu Z, et al. Bioinspired Hybrid Protein Oxygen Nanocarrier Amplified Photodynamic Therapy for Eliciting Anti-tumor Immunity and Abscopal Effect. ACS Nano. 2018;12:8633-8645.
  39. Bian Q, Huang L, Xu Y, Wang R, Gu Y, Yuan A, et al. A Facile Low-Dose Photosensitizer-Incorporated Dissolving Microneedles-Based Composite System for Eliciting Antitumor Immunity and the Abscopal Effect. ACS Nano. 2021;28:15(12):19468-19479.

Schemes

Schemes 1 and 2 are available in the Supplementary Files section.