Study on the Photodynamic Efficiency of Sodium Chlorophyllin from Spirulina

Jianjiao CHEN Zhiqiang CHEN Jun WEN Jian WEN Jingfei FENG Jing YI Dengfeng ZOU

Abstract [Objectives]This study was conducted to investigate the photodynamic technology （PDT） of water-soluble sodium chlorophyllin extract from Spirulina and its photodynamic sterilization efficiency on Gram bacteria and photodynamic antitumor effect on rat glioma C6 cells. [Methods]The absorption spectrum and fluorescence spectrum of sodium chlorophyllin were measured by an ultraviolet spectrophotometer and a fluorescence spectrophotometer; the plate count method was used to investigate the photodynamic sterilization efficiency of sodium chlorophyllin on Escherichia coli and Staphylococcus aureus; and the MTT method was used to determine the photodynamic antitumor effect of sodium chlorophyllin on rat glioma cell C6. [Results]The sterilization rates of sodium chlorophyllin with the 100 J/cm2 photodynamic treatment were 98.96% 1.284 and 100% 0 on S. aureus and E. coli， respectively. The half maximal inhibitory concentration （IC50） of the sodium chlorophyllin photodynamic therapy with 5， 10 and 20 J/cm2 on C6 were 69.9， 48.21 and 47.56 μg/ml respectively， and the dark toxicity was extremely low at 0 J/cm2. [Conclusions]The photodynamic treatment mediated by the alcohol-extracted sodium chlorophyllin from Spirulina showed excellent inhibitory effects on bacteria and tumor cells. This study initially reveals its excellent photodynamic performance and provides a reference for its in-depth application in the field of photodynamic therapy.

Key words Photodynamic technology; Spirulina; Sodium chlorophyllin; Staphylococcus aureus， Escherichia coli; Glioma

Photodynamic technology （PDT） is a non-invasive treatment technology that activates a photosensitizer in the presence of molecular oxygen to produce toxic reactive-oxygen species （ROS） including singlet oxygen （1O2）， which selectively kill specific cell tissues and microbial-enriched infection sites[1-3].

Compared with terrestrial plants， Spirulina， as an aquatic plant， receives less light energy for photosynthesis， so the chlorophyll content in its body is higher to maintain its normal growth[4]. In nature， the chlorophyll content is about 0.3%-0.5% in the leaves of terrestrial plants， about 0.8%-1.0% in silkworm excrement， and 1%-2% in Spirulina[5]. In addition， the components in algae are relatively simple and the extraction process is easier to control， so Spirulina is an ideal source of chlorophyll components.

Among pathogenic microorganisms， bacteria are often more common than fungi. Bacteria can be divided into Gram-negative bacteria and Gram-positive bacteria according to the Gram staining method. Escherichia coli， as a representative strain of Gram-negative bacteria， often causes diseases by producing endotoxins. Staphylococcus aureus is a representative strain of Gram-positive bacteria， which often causes purulent lesions by producing exotoxin. In the treatment of bacterial infections， antibiotics are usually the first choice， and the premise of antibiotic treatment is to strictly distinguish the negative or positive types of pathogenic bacteria， in order to carry out targeted treatment[6]. However， the inevitable drug resistance caused by the abuse of antibiotics and the emergence of "super bacteria" have made new antibacterial methods and antibacterial drugs imminent. Antibacterial photodynamic therapy （APDT） is a new type of antibacterial treatment. During the treatment， because bacteria cannot resist the effects of drugs by stopping the intake of small molecules of photosensitizers， increasing the metabolic detoxification rate， or accelerating the efflux of small molecules of photosensitizers， antibacterial photodynamic therapy is less likely to make the body resistant to drugs than traditional antibiotic therapy[7]， and there are no reports of photodynamic resistant strains.

Glioma is the most common primary intracranial tumor， accounting for about 70% of primary intracranial malignant tumors. Its pathogenesis is not clear， and the incidence rate in adults is 8 per 100 000 people per year[8]. Glioma has the characteristics of high degree of malignancy， rapid proliferation， poor prognosis， and high recurrence rate. Surgical removal of the tumor is the most common method for treating glioma and prolonging the life of patients. However， in the central nervous system， gliomas grow diffusely， and there is no obvious boundary with normal brain tissue， so it cannot be completely removed by surgery. Currently， radiotherapy and chemotherapy are often supplemented after surgery. However， glioma cells are very insensitive to radiotherapy and chemotherapy， resulting in a very poor prognosis and a median survival time of only 12 to 18 months[9]. It is reported that photodynamic therapy can greatly improve this situation， and can improve and prolong the survival period of patients[10]. Therefore， we explored the antibacterial and antitumor efficiency of sodium chlorophyllin from Spirulina in photodynamic therapy.

Materials and Methods

Materials

Food-grade Spirulina powder was purchased from Lanyuan Spirulina Co.， Ltd.， Wuchuan City， Guangdong Province; E. coli and S. aureus were purchased from Guangdong Huankai Microbial Technology Co.， Ltd.; tryptone， yeast extract， agar powder， and NaCl powder were purchased from Beijing Soleibao; rat-derived glioma cells C6 were purchased from Procell Life Science&Technology Co.， Ltd.; sodium chlorophyllin was independently synthesized by our laboratory; and the reagents used in the experiment were purchased from Xilong Science Co.， Ltd. and were free of any pretreatment.

Instruments

Ultraviolet spectrophotometer （Shimadzu UV-2700， Japan）; fluorescence spectrophotometer （Hitachi F-7000， Japan）; vertical laminar flow clean bench （Suzhou Purification Equipment Co.， Ltd.）; 37 ℃， 5% CO2 incubator （Thermo， USA ）; fluorescence microplate reader （Thermo， USA）; laser photodynamic therapy device （Guilin Xingda Medical Equipment Co.， Ltd.）.

Methods

Determination of ultraviolet absorption spectrum of sodium chlorophyllin

Sodium chlorophyllin was prepared to a working solution with a concentration of 100 μg/ml， and the ultraviolet absorption spectrum and fluorescence spectrum were obtained by scanning with an ultraviolet spectrophotometer and a fluorescence spectrophotometer， respectively.

Determination of photodynamic sterilization efficiency of sodium chlorophyllin

The experiment was divided into the PBS dark light group， the PBS illumination group， the sodium chlorophyllin dark light group and the odium chlorophyllin illumination group. The sodium chlorophyllin concentration was 15 mg/ml， and the light dose was 100 J/cm2. Into the sodium chlorophyllin solution and PBS solution， 0.1 ml of the Escherichia coli and Staphylococcus aureus bacterial suspensions with a concentration of about 106 cfu/ml were inoculated， respectively. After the photodynamic treatment， 50 μl was applied to LB agar medium， and the bacteria were then cultured in an inverted state in a constant temperature incubator at 37 ℃ for 24 h， and then taken out for colony count. The sterilization rate was calculated according to the following equation： Sterilization rate = （The number of colonies in the dark light group-The number of colonies in the illumination group） / The number of colonies in the dark light group.

Detection of the photodynamic antitumor activity of sodium chlorophyllin by thiazolyl blue （MTT）

The C6 cells in the logarithmic growth phase were inoculated into a 96-well cell culture plate at a density of 6×105 cells/ml. After 12 h， the culture solution was removed and discarded， and the cultures solution containing different concentrations of sodium chlorophyllin （15， 30， 45， 60， 75， 90， 105， 120 μg/ml） were added， followed by incubating in the dark for 24 h and the photodynamic therapy （5， 10， 20 J/cm2）. At the end of the treatment， the drug-containing culture media were discarded and replaced with fresh medium， and culture was continued for 12 h with illumination， while the dark light group was only subjected to medium replacement， but not illuminated. The culture solution was removed， and 200 μl of serum-containing medium containing 20% MTT was added to each well to continue the culture in the cell incubator for 4 h in the dark. Next， the supernatant was carefully sucked off， and 150 μl of DMSO solution was added to each well， followed by shaking for 10 min in the dark to fully dissolve formazan produced in each well. The OD value of each well of the culture plate was measured at 570 nm using a microplate reader.

Statistical methods

All data was analyzed with SPSS 22.0 statistical software， and the comparison between groups was performed by t test， with P<0.05 indicating that the difference is statistically significant.

Results and Analysis

UV absorption spectrum and fluorescence spectrum

The ultraviolet absorption spectrum and fluorescence spectrum of sodium chlorophyllin were shown in Fig. 1. The ultraviolet absorption spectrum showed ultraviolet absorption at 401， 501 and 655.5 nm. The fluorescence excitation wavelength of sodium chlorophyllin was 337.2 nm， and the emission wavelength was 680.4 nm.

Evaluation of photodynamic antibacterial activity of sodium chlorophyllin

The photodynamic treatment with sodium chlorophyllin at 100 J/cm2 had obvious bactericidal effect on S. aureus and E. coli. The sterilization rates are shown in Fig. 2. Compared with corresponding dark light group， the sodium chlorophyllin illumination group had an inhibition rate of 98.87%±1.284 （n=3） against S. aureus， and an inhibition rate of 100%±0 （n=3） against E. coli. In the pure solution control groups， the number of bacteria in the illumination group after photodynamic treatment showed a certain increase compared with the dark light group. It was guessed that the extra heat generated by the small temperature rise caused by laser irradiation accelerated the reproduction speed of bacteria.

MTT Determination of photodynamic antitumor activity of sodium chlorophyllin

Fig. 3 shows the survival curves of C6 cells mediated by sodium chlorophyllin. Specifically， sodium chlorophyllin in the dark light group showed very low toxicity. After light stimulation， the toxicity of the illumination group increased with the increases of light dose and drug concentration. The photodynamic IC50 of sodium chlorophyllin to C6 under the light doses of 5， 10， and 20 J/cm2 were， respectively， 69.9， 48.21， and 47.56 μg/ml， of which the IC50 with 20 and 10 J/cm2 were very close， suggesting that the optimal light dose of sodium chlorophyllin-mediated photodynamic therapy against rat-derived glioma C6 cells was 10 J/cm2.

Conclusions and Discussion

The experimental data showed that the sodium chlorophyllin-mediated photodynamic technology had good bactericidal effects on such two representative gram bacteria as E. coli and S. aureus， and the effect on E. coli was better. In the antitumor activity assay， sodium chlorophyllin showed excellent photosensitizer properties with low dark toxicity and high phototoxicity to rat brain glioma cells C6. Therefore， as a photosensitizer with a wide range of sources， sodium chlorophyllin has good photodynamic effects， and simultaneously shows bactericidal and anti-tumor activity， which lays a solid theoretical basis or further exploration of the in-depth research and application of sodium chlorophyllin in the field of photodynamic therapy.

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