Get 20M+ Full-Text Papers For Less Than $1.50/day. Subscribe now for You or Your Team.

Learn More →

Cyanobacteria-Based Bio-Oxygen Pump Promoting Hypoxia-Resistant Photodynamic Therapy

Cyanobacteria-Based Bio-Oxygen Pump Promoting Hypoxia-Resistant Photodynamic Therapy fbioe-08-00237 March 21, 2020 Time: 15:12 # 1 ORIGINAL RESEARCH published: 24 March 2020 doi: 10.3389/fbioe.2020.00237 Cyanobacteria-Based Bio-Oxygen Pump Promoting Hypoxia-Resistant Photodynamic Therapy 1,2† 3† 3 3 3 3 Tao Sun , Yingying Zhang , Chaonan Zhang , Hanjie Wang , Huizhuo Pan , Jing Liu , 2,4,5 2,4,5 3 1,2,4,5 Zhixiang Li , Lei Chen , Jin Chang and Weiwen Zhang * * 1 2 Center for Biosafety Research and Strategy, Tianjin University, Tianjin, China, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin, China, School of Life Sciences, Tianjin University, Tianjin, China, Laboratory of Synthetic Microbiology, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, China Hypoxia not only alters tumor microenvironment but leads to the tumor progression and metastasis as well as drug resistance. As a promising strategy, photodynamic Edited by: therapy (PDT) can inhibit tumor by catalyzing O to cytotoxic reactive oxygen species. Chun Li, However, its effects were limited by hypoxia and in turn deteriorate hypoxia due Beijing Institute of Technology, China to O consumption. Hereon, aiming to alleviate hypoxia and promote PDT, a bio- Reviewed by: Jianping Yu, oxygen pump was created based on cyanobacteria, which are the only prokaryotic National Renewable Energy organisms performing oxygenic photosynthesis. Detailly, controlled-release PDT via Laboratory (DOE), United States loading indocyanine green into mesoporous silica nanoparticles was established. Then Konstantinos Vavitsas, National and Kapodistrian University bio-oxygen pump based on a fast-growing cyanobacterium Synechococcus elongatus of Athens, Greece UTEX 2973 was tested and further packaged together with PDT to create an injectable *Correspondence: hydrogel. The packaged hydrogel showed stable oxygen production and synergetic Jin Chang therapy effect especially toward hypoxia 4T1 cells in vitro. More importantly, strong jinchang@tju.edu.cn Weiwen Zhang in vivo therapeutic effect reaching almost 100% inhibition on tumor tissues was realized wwzhang8@tju.edu.cn using PDT equipped with oxygen pump, with only negligible in vivo side effect on healthy These authors have contributed mice from S. elongatus UTEX 2973. The new photo-oxygen-dynamic therapy presented equally to this work here provided a promising strategy against hypoxia-resistant tumor and may worth Specialty section: further modifications for therapeutic application. This article was submitted to Synthetic Biology, Keywords: oxygenic cyanobacteria, hypoxia, photodynamic therapy, indocyanine green, nanoparticles, injectable a section of the journal hydrogels Frontiers in Bioengineering and Biotechnology Received: 28 January 2020 INTRODUCTION Accepted: 06 March 2020 Published: 24 March 2020 Cancer is a leading cause of death worldwide, accounting for an estimated 9.6 million deaths in Citation: 2018 (Bray et al., 2018). For several decades, various strategies targeting oncotherapy have been Sun T, Zhang Y, Zhang C, developed and evaluated (Dolmans et al., 2003; Gorrini et al., 2013; Desterro et al., 2019). Among Wang H, Pan H, Liu J, Li Z, Chen L, them, photodynamic therapy (PDT), which contains two individually non-toxic components, Chang J and Zhang W (2020) i.e., photosensitizer and light illumination with specific wavelength to transfers energy from Cyanobacteria-Based Bio-Oxygen light to molecular O to generate cytotoxic reactive oxygen species (ROS), has been used for Pump Promoting Hypoxia-Resistant more than 100 years (Dolmans et al., 2003). Nevertheless, a common feature of most tumors is Photodynamic Therapy. the low oxygen level called hypoxia, which leads to the progression, metastasis, radiation and Front. Bioeng. Biotechnol. 8:237. doi: 10.3389/fbioe.2020.00237 drug resistance of cancer cells, as well as the altering tumor microenvironment (Harris, 2002; Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 2 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT Muz et al., 2015). Meanwhile, effect of PDT has been found sodium alginate were purchased from Aladdin Bio-Chem restricted by hypoxia due to the limited oxygen available and its Technology Co., Ltd. (Shanghai, China). Propidium iodide (PI), conversion of O to ROS may in turn deteriorate the hypoxia calcein-AM, Hoechst 33528 were obtained from Sigma-Aldrich degree. Hence the improvement of oxygen availability in tumor (MO, United States). IL-2, IL-6, IFN-b and IFN-g detection kits tissues could not only alter the tumor microenvironment but also were bought from H-Y Biological Co., Ltd. (Wuhan, China). enhance the PDT therapeutic effect. CCK-8 cell proliferation and cytotoxicity assay kit and Annexin Cyanobacteria are the only prokaryote microorganism capable V-FITC apoptosis detection kit was both obtained from Solarbio of oxygenic photosynthesis via respectively taking sunlight Technology Co., Ltd. (Beijing, China). and CO as the sole energy and carbon source (Stanier and Cyanobacterium S. 2973 was cultivated with BG11 liquid Bazine, 1977). Besides their roles as primary producers on earth medium (pH = 7.5) under a light intensity of 100 mmol 2 1 (Giordano et al., 2005), cyanobacteria have been considered photons m s in an illuminating shaking incubator (HNYC- as model organisms for photosynthesis research and even 202T, Honour, Tianjin, China) at 130 rpm and 37 C (Li et al., “photosynthetic cell factories” to produce renewable chemicals 2018). 4T1 cells were cultured in RPMI-1640 medium (Gibco) (Lea-Smith et al., 2015; Gao et al., 2016). More interestingly and supplement with 10% fetal bovine serum (FBS) and maintained excitingly, biomedical applications based on cyanobacteria have at 37 C, 5% CO in a humidified atmosphere. Female BALB/c been evaluated in recent years (Raja et al., 2016). For example, mice (about 20 g) were bought from SPF Biotechnology Co., Cohen et al. (2017) presented a novel system that rescued Ltd (Beijing, China) and maintained in common environment. the myocardium from acute ischemia using photosynthesis All animal experiments were operated following the Guidance through intramyocardial delivery of the model cyanobacterium Suggestions for the Care and Use of Laboratory Animals and Synechococcus elongatus PCC 7942 (Cohen et al., 2017). In principle of 3Rs. All animals were subjected to an anesthesia addition, Yin et al. (2019) reported the role of S. elongatus experiment with a small animal anesthesia machine (Ruiwode PCC 7942 in accelerating cutaneous wound healing by secreting Life Technology Co., Ltd., Shenzhen, China). All animals were extracellular vesicles to promote angiogenesis (Yin et al., 2019). sacrificed by spine dislocation, and the bodies were chilled at All these studies demonstrated the promising therapeutic effects 20 C and uniformly incinerated by the animal center. of S. elongatus PCC 7942. In 2015, Synechococcus elongatus UTEX 2973 (hereafter S. 2973), whose genome only contained 55 single Cell Density, Absorption Spectrum and nucleotide polymorphisms and insertion-deletions compared to Dissolved O Measurement that of S. elongatus PCC 7942 (Yu et al., 2015), was isolated Cell density of S. 2973 was measured at OD using a 750 nm and demonstrated preferable properties like fast growing and UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). Besides, high light tolerance. More importantly, as optimized growing the images of S. 2973, ALG-S2973 and ALG-MI-S2973 with or temperature of S. 2973 was similar to that of human body, it may without red laser irradiation were took to study the effect of represent a more robust and promising chassis for biomedical 640 nm red light on the activity of S. 2973. applications than S. elongatus PCC 7942. Absorption spectrum of S. 2973 was investigated to evaluate In this study, aiming to simultaneously alleviate the tumor the viability of S. 2973 in different conditions using the hypoxia and the effect of PDT, a photo-oxygen-dynamic spectrophotometer. To identify the optimal power intensity of therapy (PODT) strategy combined of a light-driven bio-oxygen 640 nm laser, S. 2973 were exposed to the irradiation dose of 0, pump based on the fast-growing cyanobacterium S. 2973 and 0.1, 0.25, and 0.5 W/cm for 5 min. After that, the irradiation PDT was developed and investigated (Figure 1). Firstly, the time for 1, 5, and 10 min under the same irradiation dose of controlled-release PDT via loading indocyanine green (ICG) as 0.25 W/cm was also evaluated, respectively. photosensitizer into mesoporous silica nanoparticles (MSN) was To evaluate the elimination of S. 2973 by ICG, strains were established and investigated in vitro. Then, bio-oxygen pump incubated with ICG of different concentrations (0, 5, 10, and using S. 2973 was developed and further packaged together with 20 mg/mL). First of all, S. 2973 were irradiated with 0.25 W/cm PDT to create an injectable hydrogel. The in vivo biosafety of S. of 640 nm laser for 5 min in order to generate oxygen. Then 2973 was further measured and the PODT effect was evaluated the cells were exposed to 808 nm laser with the power intensity using a 4T1 tumor model. Excitingly, the packaged hydrogel of 2.0 W/cm for 5 min. At last, the absorptions spectrum showed strong in vivo therapeutic effect on tumor tissues. The and images of S. 2973 with different treatments were recorded. photo-oxygen-dynamic oncotherapy presented here provided a Dissolved O was detected using a Dissolved Oxygen Meter promising strategy against hypoxia-resistant tumor cells and (SMART SENSOR, Guangdong, China). worth further optimization for therapeutic application as mature genetic tools have been developed for S. 2973. AM/PI Staining AM/PI staining was utilized to evaluate the cytotoxic effect of MATERIALS AND METHODS S. 2973, ICG and MI against 4T1 cells. The effect of S. 2973 on cellular activity was determined under the irradiation dose Materials of 0.25 W/cm . 4T1 cells were seeded in 48-well plate and Indocyanine green (ICG), cetyltrimethylammonium bromide cultivated in normoxic or hypoxic condition. Cells were treated (CTAB), tetraethylorthosilicate (TEOS), diethanolamine and with ICG and MI (equivalence to 20 mg/mL of ICG) with or Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 2 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 3 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 1 | Schematic illustration of the photo-oxygen-dynamic therapy. (A) S. 2973 and MSN loaded with photosensitizer ICG were mixed with sodium alginate to make injectable hydrogels (ALG-MI-S2973). (B) The hydrogels ALG-MI-S2973 was intratumorally injected and then formed gels at the tumor site. The tumor tissue was exposed to 640 nm laser to support survival of S. 2973 to generate O , which alleviated the hypoxia microenvironment and further enhanced PDT effects. After releasing, ICG excited by 808 nm laser could exert efficient PDT effects to inhibit tumor growth. MSN, mesoporous silica nanoparticles; ICG, indocyanine green; ALG, hydrogels based on sodium alginate; MI, ICG-loaded MSN; ALG-MI-S2973, hydrogels packaging MSN-ICG and S. 2973. without 808 nm irradiation for 24 h. After removing the medium, A total of 4 mg of MSN-ICG and ALG-MI-S2973 was dispersed 1 mM Calcein-AM and PI were added and then observed by in 1 mL ddH O and then put into a dialysis bag. At certain times, inverted fluorescence microscope. The live/dead staining assay 200 mL of the samples was collected to measure the absorbance at was also conducted for 4T1 cells treated with ALG-MI-S2973 and 780 nm. The cumulative release of ICG was calculated according respectively irradiated with only 640 nm laser for 5 min, only to ICG calibration curve. 808 nm laser for 5 min or both of them. Detection of Reactive Oxygen Species Preparation and Characterization of (ROS) and Hypoxia MSN and Hydrogels 4T1 cells were grown in normoxic condition and incubated with ICG and MSN-ICG containing 20 mg/mL ICG for 24 h. For preparation of MSN-ICG, MSN were synthesized first according to our previous studies (Zheng et al., 2016). In order Then the cells were exposed to 808 nm irradiation for 5 min. Finally, cells were stained with hypoxia/ROS kits and observed to load ICG, 40 mg of MSN were re-dispersed with ddH O and stirred with 5 mg of ICG for 12 h in dark. After the reaction, using a confocal laser scanning microscope. Similar analysis the product was obtained by centrifugation and washing. For was also performed for 4T1 cells treated with ALG-MI-S2973 preparation of sodium alginate gel co-loaded with MSN-ICG and respectively irradiated with 640 nm laser, 808 nm laser and cyanobacteria (ALG-MI-S2973), ALG aqueous solution was or both of them. mixed with Glucono delta-lactone (GDL) using a vortex mixer. And then MSN-ICG and S. 2973 were added and stirred to mix CCK-8 Proliferation Assay 2C evenly. PBS containing 1.8 mM Ca was injected to form gel. The biosafety of S. 2973 on 4T1 cells were determined by CCK-8 For characterization of nanoparticles, transmission electron assay. Briefly, 4T1 cells were seeded in 96-well plate and cultured microscope (TEM) images were acquired by JEOL JEM100CXII in normoxic condition. Cells were co-cultured with S. 2973 at operating voltage of 100 kV. The particle size distribution and irradiated with 640 nm laser. After that cells were treated was measured using dynamic light scattering (DLS) on a with CCK-8 reagent (10 mL/well) at 37 C for 2 h. At last, the Zetasizer Analyzer (Malvern). UV-Vis spectroscopic absorbance absorbance at 450 nm was measured by Thermo Synergy HT was captured on a UV-2450 spectrophotometer. Microplate Reader. The in vitro Release of ICG Cell Apoptosis Analysis The dialysis bag diffusion technique was used to measure the For analysis of apoptosis, 4T1 cells were seeded in 6-well plate and in vitro release of ICG from MSN-ICG and ALG-MI-S2973. cultured in normoxic or hypoxic condition. Cells were treated Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 4 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 2 | The preparation and characterization of MSN-ICG. (A) The preparation process of MSN-ICG. (B) TEM images of MSN before and after ICG loading. (C,D) Live/dead staining and cell apoptosis analysis of 4T1 cells treated with control, ICG or MSN-ICG with or without 808 nm laser irradiation under normoxic or hypoxic condition. (E) Measurements of hypoxia and ROS for 4T1 cells treated with control, ICG or MSN-ICG with 808 nm laser irradiation under normoxia. *p < 0.05, **p < 0.01 compared with control group by Student’s t-test. MSN-ICG, indocyanine green-loaded mesoporous silica nanoparticles. with ICG and MSN-ICG (equivalence to 20 mg/mL of ICG) with with ELISA kits (H-Y biological) according to the instructions. or without 808 nm irradiation for 24 h. Then cells were stained Besides, blood was collected on the 1st, 7th, and 14th day of with 5 mL of Annexin V-FITC and PI for 15 min in the dark treatment to conduct blood routine examination. In the end, mice and then subjected to flow cytometry. Cells treated with ALG- were sacrificed and main organs (heart, liver, spleen, lung, and MI-S2973 and respectively irradiated with 640 nm laser, 808 nm kidney) were collected for pathological analysis. laser or both of them were also tested by flow cytometry to discuss the cell apoptosis induced by the PDT effect of ALG-MI-S2973. In vivo PDT Effects Each female BALB/c mouse was subcutaneously injected 2 10 The Biosafety Investigation of S. 2973 4T1 cells into the axilla. When the tumor volume reached 50– Female BALB/c mice were randomly divided into four groups 100 mm , mice were randomly divided into four groups. ALG- and subcutaneously injected with S. 2973 and ALG-S2973. After MI and ALG-MI-S2973 (equivalence to 20 mg/mL ICG) were 1, 4-, and 7-days’ injection, blood was collected and analyzed intratumorally injected at 0 day. Tumors were irradiated with Red Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 4 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 5 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 3 | Construction and characterization of the bio-oxygen pump in vitro. (A) Scheme of oxygen generation system based on S. 2973 under 640 nm red laser irradiation. (B) Absorption spectrum and OD of S. 2973 irradiated using 640 nm laser with different intensities. (C) Absorption spectrum and OD of S. 750 nm 750 nm 2973 irradiated using 640 nm laser with 0.25 W/cm for different times. (D) Oxygen evolution of S. 2973 under 640 nm red laser irradiation. (E) Cell viability and AM/PI staining of 4T1 cells with or without treatments (i.e., 640 nm laser irradiation, co-cultivation with S. 2973 or both of them) for 24 h. (F) Phenotypes of S. 2973 treated with different concentrations of ICG exposed to 2 W/cm of 808 nm laser for 5 min. decrease the injection frequency and prolong the functional time laser (0.25 W/cm , 5 min) for 3 days. 808 nm laser was used on of ICG when used in vivo, we established a controlled-release the 4th day. The body weight and tumor volume were measured system MSN-ICG by loading ICG into MSN (Table 1). TEM every 3 days. After 21 days’ treatment, mice of all groups were images of MSN before and after ICG loading was shown in sacrificed and then tumors and main organs were collected to Figure 2B and Supplementary Figure S1, suggesting the good study the pathological morphology. Besides, TUNEL staining and uniformity of the nanoparticle. In addition, continuous release of HIF-1a analysis were carried out to assess the apoptosis and ICG from MSN as long as 24 h could be detected (Supplementary hypoxia induced by PDT effect of ALG-MI-S2973. Figure S2). To evaluate the PDT effect, 4T1 cells were treated with control, ICG or MSN-ICG with or without 808 nm laser Histopathology TUNEL and (NIR) irradiation under both normoxia and hypoxia conditions. Immunohistochemical Analysis As illustrated in Figures 2C,D, the controlled-release MSN-ICG Main organs of mice were fixed and embedded in paraffin blocks. system reached similar PDT effects as ICG alone after irradiation, The sections were stained with hematoxylin and eosin (H&E) and resulting in37% apoptosis of 4T1 cells under normoxia, which examined by a microscope. TUNEL analysis was used to evaluate indicated the feasibility of PDT as well as the controlled-release the cell apoptosis in tumor tissues. And then the tumor sections system. In addition, the PDT effect was also demonstrated were immune-stained with a rat anti-mouse HIF-1a protein and by the ROS measurement (Figure 2E). On the contrary, the observed by a microscope. apoptosis rate was dramatically decreased to below 12%14% under hypoxia (Figure 2D), suggesting the low oxygen content seriously restricted the PDT. Meanwhile, for 4T1 cells under RESULTS normoxia, the hypoxia measurement revealed that PDT in turn aggravated hypoxia due to the consumption of O (Figure 2E). Establishment and Investigation of the Controlled-Release PDT System Evaluation of the Bio-Oxygen Pump To construct the PDT system (Figure 2A), we utilized a near- infrared fluorescent dye ICG that has been approved by the Based on Cyanobacteria Food and Drug Administration of United States for various Aiming at alleviating hypoxia and enhancing the PDT effect, clinical applications (Bradley and Barr, 1968; Sauda et al., 1986). we tested the bio-oxygen pump in vitro first (Figure 3A). To Meanwhile, we used near-infrared light to ensure relatively strong ensure the viability of S. 2973 and sustainable O generation penetrability toward tissues and to avoid killing S. 2973 when when used in vivo, a 640 nm red laser (Red) was chosen given used visible light to support the in vivo survival of S. 2973. To the utilized spectrum of cyanobacteria and the penetrability of Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 6 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 4 | Construction and characterization of injectable hydrogels packing MSN-ICG and/or S. 2973. (A) The preparation of injectable hydrogels packing MSN-ICG and S. 2973 (i.e., ALG-MI-S2973). (B) Growth investigation of S. 2973 directly in BG11 or packaged in ALG-MI-S2973 cultivated under normal or 640 nm irradiation. (C) The ROS generation in 4T1 cells after treatment of ALG-MI-S2973 with different amounts of S. 2973. (D,E) AM/PI staining and cell apoptosis analysis of 4T1 cells treated with or without ALG-MI-S2973 under control, 808 nm irradiation or both 640 and 808 nm irradiation. (F) Measurements of hypoxia and ROS of normoxia 4T1 cells treated with or without ALG-MI-S2973 under control, 808 nm irradiation or both 640 and 808 nm irradiation. *p < 0.05, **p < 0.01 compared with untreated group by Student’s t-test. MSN-ICG, indocyanine green-loaded mesoporous silica nanoparticles; ALG-MI-S2973, hydrogels packaging MSN-ICG and S. 2973. light toward tissue. Then red lasers with different intensity of 0, was demonstrated via detecting the dissolved O irradiated with 2 2 2 0.1, 0.25, and 0.5 W/cm were tested. Density of 0.25 W/cm the red laser of 0.25 W/cm (Figure 3D). Although no literature was found feasible as growth of S. 2973 was accelerated with evidence of Synechococcus-derived cytotoxins or cytotoxicity was the increasing laser density but was inhibited when it reached found (Klemenci ˇ c ˇ et al., 2017), we evaluated the cytotoxicity of up to 0.5 W/cm (Figure 3B). In addition, correlated with the S. 2973 to ensure its biosafety. As expected, cell viability assay condition under normal light, longer irradiation time increased and AM/PI staining assay didn’t find significant cytotoxicity from the growth of S. 2973 (Figure 3C). Meanwhile, O generation S. 2973 or red laser irradiation (Supplementary Figure S3 and Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 7 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT Figure 3E), indicating the biosafety is in general assured. To Long-Term Biosafety Investigation of S. further guarantee the biosafety when used in vivo, the eliminating 2973 on Healthy Mice effect caused by PDT was also tested. As illustrated in Figure 3F, The biosafety of ICG has been demonstrated previously by complete inactivation of S. 2973 could be observed when treated several independent studies (Bradley and Barr, 1968; Sauda with 20 mg/mL ICG after 5 min irradiation with 808 nm laser, et al., 1986). Therefore, before testing the PODT in vivo, long- indicating PDT could synchronously inhibit the tumor and term potential toxicity from S. 2973 on healthy mice was first eliminate S. 2973. investigated (Figure 5A). Detailly, female BALB/c mice were randomly divided into four groups (each group containing three Creation and Evaluation of the mice) and two of them were subcutaneously injected with S. Controlled-Release Injectable Hydrogels 2973 or ALG-S2973 (Table 1). All groups except control were irradiated with 640 nm laser on the 1st, 4th, and 7th day, the Packaging With S. 2973 and MSN-ICG investigation of injection site suggesting the well survival of To provide an oxygen pump for PDT, we created a combined S. 2973 (Figure 5B). After 1, 4-, and 7-days’ injection, blood system packing both S. 2973 and MSN-ICG into injectable was collected to analyze the immune factors including IL-2, IL- hydrogels named ALG-MI-S2973 (Figure 4A and Table 1). 6, IFN-g and IFN-b (Figure 5C). Besides, blood was collected As shown in Figure 4B, though slightly slower than the S. on the 1st, 7th, and 14th day after injection to conduct blood 2973 cultivated under normal condition with white light or routine examination (Figure 5D). Finally, mice were sacrificed red light (640 nm), S. 2973 can grow well in the hydrogel and main organs including heart, liver, spleen, lung and kidney system. In addition, continuous release of ICG from MSN as were collected for pathological analysis (Supplementary Figure long as 72 h could be detected due to the package of MSN and S4). Although certain properties like immune factor IL-6 showed ALG (Supplementary Figure S2). Further, we treated 4T1 cells slight change in mice group injected with S. 2973 or ALG-S2973 respectively with control and ALG-MI-S2973 to determine its compared to the control group, we believe that it may be due effect in vitro. Mixture of 4T1 cells and ALG-MI-S2973 led to to the limiting number of mice used in the biosafety evaluation strong ROS signal orderly after 640 and 808 nm laser irradiation, experiments, which may lead to wide error bars. Although further suggesting the increasing O evolution generated from ALG- biosafety experiments are certainly necessary in the future, our MI-S2973 with the increasing amount of S. 2973 (Figure 4C). study found that consistent with the previous studies (Cohen Besides, as illustrated in Figures 4D,E, compared with PDT et al., 2017), neither injection of S. 2973 nor ALG-S2973 caused based on MSN-ICG, apoptosis rate of 4T1 cells based on ALG- any effect on immune factors, hematological index or main MI-S2973 was decreased from 37 to 22% with only 808 nm organs of mice, suggesting of in general good biosafety of both irradiation under normoxia, indicating the slower release of ICG the S. 2973 and ALG-S2973 in vivo. from ALG-MI-S2973 than that from MSN-ICG. Nevertheless, after cascaded irradiation with 640 nm for 5 min then following 808 nm excitation to induce the oxygen production and PDT Evaluation of the PODT in vivo Using 4T1 sequentially, apoptosis rate of 4T1 cells based on ALG-MI-S2973 Tumor Model was dramatically increased up to 56%. More importantly, the Finally, we investigated in vivo anti-tumor effects of ALG-MI- new PODT system could significantly increase the effect under S2973 using the 4T1 tumor model (Figure 6A and Table 1). hypoxia (22% apoptosis rate) (Figures 4D,E), suggesting the Detailly, female BALB/c mice were randomly divided into four feasibility of the photo-oxygen-dynamic oncotherapy in vitro. groups and each group contained five mice. Besides the control Consistently, hypoxia and ROS measurements for 4T1 cells group (Group I), one group were subcutaneously injected only under normoxia showed that the addition of red laser could with MSN-ICG (Group II) while the other two groups were significantly alleviate the hypoxia and promote the PDT due to subcutaneously injected with ALG-MI-S2973 (Group III and IV). the O evolution, compared with ALG-MI-S2973 without 640 nm After injection, 640 nm laser irradiation was conducted during irradiation (Figure 4F). the next 3 days to induce the oxygen generation then the 808 nm laser was used to induce the PDT on the 4th day for Group I and IV. While only 808 nm laser was used for Group II and TABLE 1 | Detailed characteristics of the biomaterials used in this study. III. Consistently, fluorescence imaging of S. 2973 in tumor tissue showed that they were accumulated in the first 3 days due to Name Description the radiation of red laser and were killed in the 5th day due to ICG Indocyanine green directly for PDT the PDT (Figure 6B). Body weight of 4T1 tumor-bearing mice S. 2973 Wild type S. 2973 for oxygen generation with different treatments was recorded, suggesting no significant MSN-ICG Nanoparticles via loading indocyanine green into fluctuation during the process (Figure 6C and Supplementary mesoporous silica Figure S5). In addition, the volume and weight of tumor tissue ALG-S2973 Hydrogels via packaging S. 2973 with sodium was recorded and pictured then the inhibition rate was calculated alginate (Figures 6D–G). Though all treatments showed therapy effect ALG-MI Hydrogels via packaging MSN-ICG with sodium of varying degrees, traditional PDT for group II could only alginate achieve an average tumor inhibition rate at 50%. Excitingly, ALG-MI-S2973 Hydrogels via packaging MSN-ICG and S. 2973 with sodium alginate the addition of oxygen pump remarkably enhanced the PDT, Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 7 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 8 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 5 | The in vivo long-toxicity evaluation of S. 2973 and ALG-S2973. Each group contained three mice. (A) The schedule of animal experiments for toxicity evaluation. (B) Photos of the injection site of BALB/c mice. (C) Detection of immune factors of mice with different treatments at the 1st, 4th, and 7th day. (D) Hematological index of the mice with different treatments at the 1st, 7th, and 21st day. *p < 0.05, **p < 0.01 compared with untreated group by Student’s t-test. ALG-S2973, hydrogels packaging S. 2973. showing amazing effect as the tumor tissue disappeared just from bio-oxygenic cyanobacterium S. 2973 with traditional PDT based the 4th day for Group IV. The inhibition rate on 4T1 tumor were on ICG allowed us to avoid the endogenous and the PDT-induced almost 100% for 4 of 5 mice in Group IV using photo-oxygen- hypoxia, achieving an inhibition rate up to almost 100% on 4T1 dynamic therapy, reaching an average inhibition rate at 86%. tumor cells. More importantly, the in vivo toxicity evaluation Tumor tissue for 1 of 5 mice in Group III was also disappeared demonstrated the biosafety of the strategy, thus probably at last, which may be due to the original O production from S. providing a promising oncotherapy strategy. Most recently, 2973 just after injection. Finally, H&E and TUNEL demonstrated Zhou et al. (2019) reported a light triggered oxygen-affording the apoptosis and elimination of tumor tissue in Group IV engine for hypoxia-resistant oncotherapy via combining PDT (Figure 6H). Furthermore, immunohistochemical staining of with Chlorella pyrenoidosa (Zhou et al., 2019). Compared with HIF-1a protein in tumor tissues showed that hypoxia induced the reported work, this study innovatively divided the growth factor HIF-1a was significantly decreased in Group IV due to the of S. 2973 and the trigger of PDT via two lasers with different oxygen pump (Figure 6H). wavelengths, avoiding the killing of S. 2973 by PDT-induced ROS in the early stage thus ensuring the maximum oxygen generation. Meantime, the induction of PDT via 808 nm laser simultaneously cleaned the S. 2973, further guaranteeing the biosafety of DISCUSSION the strategy. In addition, the construction of control released All types of solid tumors, especially malignant solid tumors are injectable hydrogels prolonged the functional time of the therapy, decreased the dose used and may improve the therapy effects subject to hypoxia, whose O levels are remarkably lower than their original tissue. Hypoxia affects tumor microenvironment further. Finally, compared with other oxygenic microorganism and increases blood vessel formation, aggressiveness, metastasis, like Chlorella, mature genetic toolboxes have been developed for and resistance to treatment, which further restricts the model cyanobacteria like S. 2973 (Sun et al., 2018), allowing for oncotherapy effect like PDT. In this study, combining the the further optimization of the cyanobacteria-based bio-oxygenic Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 9 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 6 | In vivo investigation of anti-tumor effects based on ALG-MI-S2973. Each group contained five mice. (A) Schematic diagram of animal experiments. (B) Viability measurement of S. 2973 in tumor sections using fluorescence imaging excited by 561 nm. (C,D) Body weight changes and tumor growth curves of control and 4T1 tumor-bearing mice with different treatments. **p < 0.01 compared with untreated group by Student’s t-test. (E,F) Volumes and weight of tumors from 4T1 tumor-bearing mice with different treatments; (G) Tumor inhibition rates of different treatments. (H) H&E, TUNEL and immunohistochemical staining of HIF-1a protein in tumor tissues from 4T1 tumor-bearing mice with different treatments. ALG-MI, hydrogels packaging MSN-ICG; ALG-MI-S2973, hydrogels packaging MSN-ICG and S. 2973. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 9 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 10 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT systems. Most Recently, Huo et al. (2019) first demonstrated the ETHICS STATEMENT role of cyanobacterium S. elongatus PCC 7942 in promoting PDT effect. As a complementary study, our research also demonstrated The animal study was reviewed and approved by the the similar results using a more promising cyanobacterium Experimental Animal Ethics Committee of Institute of Radiation S. 2973. Although the study here only evaluated the PODT Medicine, Chinese Academy of Medical Sciences. using subcutaneous tumor model, the positive results suggested further efforts by increasing the oxygen generation of S. 2973, deleting its endotoxin for tail vein injection or even enabling AUTHOR CONTRIBUTIONS its ability for synthesizing anti-tumor chemicals via synthetic TS, YZ, and CZ performed the experiments and wrote the biology may enhance the efficiency of the photo-oxygen-dynamic manuscript. TS, YZ, CZ, HW, HP, JL, and ZL analyzed oncotherapy in the future. the data. LC, JC, and WZ designed the study and revised the manuscript. CONCLUSION In this study, an injectable hydrogel packaging both an oxygen FUNDING pump based on S. 2973 and ICG-loaded mesoporous silica This research was supported by grants from the National nanoparticles of controlled-release was created and evaluated Key R&D Program of China (Nos. 2018YFA0903600, in vivo. The packaged hydrogel showed strong therapeutic effect 2018YFA0903000, and 2019YFA0904600) and the National on tumor tissues with only negligible side effect on healthy Natural Science Foundation of China (Nos. 31901017, 31770100, mice. The PODT presented here provided a promising therapy 31972931, 91751102, 21621004, 31370115, and 31470217). strategy against hypoxia-resistant tumor and may worth further modifications for therapeutic application. SUPPLEMENTARY MATERIAL DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found All datasets generated for this study are included in the online at: https://www.frontiersin.org/articles/10.3389/fbioe. article/Supplementary Material. 2020.00237/full#supplementary-material Harris, A. L. (2002). Hypoxia–a key regulatory factor in tumour growth. Nat. Rev. REFERENCES Cancer 2, 38–47. doi: 10.1038/nrc704 Bradley, E. C., and Barr, J. W. (1968). Determination of blood volume using Huo, M., Wang, L., Zhang, L., Wei, C., Chen, Y., and Shi, J. (2019). Photosynthetic indocyanine green (cardio-green) dye. Life Sci. 7, 1001–1007. doi: 10.1016/ tumor oxygenation by photosensitizer-containing cyanobacteria for enhanced 0024- 3205(68)90108- 90102 photodynamic therapy. Angew Chem. Int. Ed. Engl. 206:108854. doi: 10.1002/ Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., and Jemal, A. anie.201912824 (2018). Global cancer statistics 2018: globocan estimates of incidence and Klemenci ˇ c ˇ, M., Nielsen, A. Z., Sakuragi, Y., Frigaard, N. U., Celešnik, H., Jensen, mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, P. E., et al. (2017). Synthetic biology of cyanobacteria for production of biofuels 394–424. doi: 10.3322/caac.21492 and high-value products. Microalgae Based Biofuels Bioprod. 2017, 305–325. Cohen, J. E., Goldstone, A. B., Paulsen, M. J., Shudo, Y., Steele, A. N., Edwards, Lea-Smith, D. J., Biller, S. J., Davey, M. P., Cotton, C. A. R., Perez Sepulveda, B. M., B. B., et al. (2017). An innovative biologic system for photon-powered Turchyn, A. V., et al. (2015). Contribution of cyanobacterial alkane production myocardium in the ischemic heart. Sci. Adv. 3:e1603078. doi: 10.1126/sciadv. to the ocean hydrocarbon cycle. Proc. Natl. Acad. Sci. U.S.A. 112, 13591–13596. 1603078 doi: 10.1073/pnas.1507274112 Desterro, J., Bak-Gordon, P., and Carmo-Fonseca, M. (2019). Targeting mRNA Li, S., Sun, T., Xu, C., Chen, L., and Zhang, W. (2018). Development processing as an anticancer strategy. Nat. Rev. Drug Discov. 19, 112–129. doi: and optimization of genetic toolboxes for a fast-growing cyanobacterium 10.1038/s41573- 019- 0042- 43 Synechococcus elongatus UTEX 2973. Metab. Eng. 48, 163–174. doi: 10.1016/j. Dolmans, D. E., Fukumura, D., and Jain, R. K. (2003). Photodynamic ymben.2018.06.002 therapy for cancer. Nat. Rev. Cancer 3, 380–387. doi: 10.1038/nrc Muz, B., de la Puente, P., Azab, F., and Azab, A. K. (2015). The role of hypoxia in 1071 cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia Gao, X., Sun, T., Pei, G., Chen, L., and Zhang, W. (2016). Cyanobacterial 3, 83–92. doi: 10.2147/HP.S93413 chassis engineering for enhancing production of biofuels and chemicals. Raja, R., Hemaiswarya, S., Ganesan, V., and Carvalho, I. S. (2016). Recent Appl. Microbiol. Biotechnol. 100, 3401–3413. doi: 10.1007/s00253-016-7374- developments in therapeutic applications of Cyanobacteria. Crit. Rev. Microbiol. 7372 42, 394–405. doi: 10.3109/1040841X.2014.957640 Giordano, M., Beardall, J., and Raven, J. A. (2005). CO2 concentrating Sauda, K., Imasaka, T., and Ishibashi, N. (1986). Determination of protein mechanisms in algae: mechanisms, environmental modulation, and evolution. in human serum by high-performance liquid chromatography with Annu. Rev. Plant Biol. 56, 99–131. doi: 10.1146/annurev.arplant.56.032604.14 semiconductor laser fluorometric detection. Anal. Chem. 58, 2649–2653. 4052 doi: 10.1021/ac00126a016 Gorrini, C., Harris, I. S., and Mak, T. W. (2013). Modulation of oxidative stress Stanier, R. Y., and Bazine, G. C. (1977). Phototrophic prokaryotes: the as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947. doi: 10.1038/ Cyanobacteria. Ann. Rev. Microbiol. 31, 225–274. doi: 10.1146/annurev.mi.31. nrd4002 100177.001301 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 10 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 11 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT Sun, T., Li, S., Song, X., Diao, J., Chen, L., and Zhang, W. (2018). Toolboxes Zhou, T. J., Xing, L., Fan, Y. T., Cui, P. F., and Jiang, H. L. (2019). for cyanobacteria: recent advances and future direction. Biotechnol. Adv. 36, Light triggered oxygen-affording engines for repeated hypoxia-resistant 1293–1307. doi: 10.1016/j.biotechadv.2018.04.007 photodynamic therapy. J. Control Rel. 307, 44–54. doi: 10.1016/j.jconrel.2019. Yin, H., Chen, C. Y., Liu, Y. W., Tan, Y. J., Deng, Z. L., Yang, F., et al. (2019). 06.016 Synechococcus elongatus PCC7942 secretes extracellular vesicles to accelerate cutaneous wound healing by promoting angiogenesis. Theranostics 9, 2678– Conflict of Interest: The authors declare that the research was conducted in the 2693. doi: 10.7150/thno.31884 absence of any commercial or financial relationships that could be construed as a Yu, J., Liberton, M., Cliften, P. F., Head, R. D., Jacobs, J. M., Smith, R. D., et al. potential conflict of interest. (2015). Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO(2). Sci. Rep. 5:8132. doi: 10.1038/ Copyright © 2020 Sun, Zhang, Zhang, Wang, Pan, Liu, Li, Chen, Chang and Zhang. srep08132 This is an open-access article distributed under the terms of the Creative Commons Zheng, B., Chen, H. B., Zhao, P. Q., Pan, H. Z., Wu, X. L., Gong, Attribution License (CC BY). The use, distribution or reproduction in other forums X. Q., et al. (2016). Persistent luminescent nanocarrier as an accurate is permitted, provided the original author(s) and the copyright owner(s) are credited tracker in vivo for near infrared-remote selectively triggered photothermal and that the original publication in this journal is cited, in accordance with accepted therapy. ACS Appl. Mater. Interf. 8, 21603–21611. doi: 10.1021/acsami.6b0 academic practice. No use, distribution or reproduction is permitted which does not 7642 comply with these terms. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 11 March 2020 | Volume 8 | Article 237 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Bioengineering and Biotechnology Pubmed Central

Cyanobacteria-Based Bio-Oxygen Pump Promoting Hypoxia-Resistant Photodynamic Therapy

Frontiers in Bioengineering and Biotechnology , Volume 8 – Mar 24, 2020

Loading next page...
 
/lp/pubmed-central/cyanobacteria-based-bio-oxygen-pump-promoting-hypoxia-resistant-EdHZPLEvRi

References (52)

Publisher
Pubmed Central
Copyright
Copyright © 2020 Sun, Zhang, Zhang, Wang, Pan, Liu, Li, Chen, Chang and Zhang.
ISSN
2296-4185
eISSN
2296-4185
DOI
10.3389/fbioe.2020.00237
Publisher site
See Article on Publisher Site

Abstract

fbioe-08-00237 March 21, 2020 Time: 15:12 # 1 ORIGINAL RESEARCH published: 24 March 2020 doi: 10.3389/fbioe.2020.00237 Cyanobacteria-Based Bio-Oxygen Pump Promoting Hypoxia-Resistant Photodynamic Therapy 1,2† 3† 3 3 3 3 Tao Sun , Yingying Zhang , Chaonan Zhang , Hanjie Wang , Huizhuo Pan , Jing Liu , 2,4,5 2,4,5 3 1,2,4,5 Zhixiang Li , Lei Chen , Jin Chang and Weiwen Zhang * * 1 2 Center for Biosafety Research and Strategy, Tianjin University, Tianjin, China, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin, China, School of Life Sciences, Tianjin University, Tianjin, China, Laboratory of Synthetic Microbiology, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, China Hypoxia not only alters tumor microenvironment but leads to the tumor progression and metastasis as well as drug resistance. As a promising strategy, photodynamic Edited by: therapy (PDT) can inhibit tumor by catalyzing O to cytotoxic reactive oxygen species. Chun Li, However, its effects were limited by hypoxia and in turn deteriorate hypoxia due Beijing Institute of Technology, China to O consumption. Hereon, aiming to alleviate hypoxia and promote PDT, a bio- Reviewed by: Jianping Yu, oxygen pump was created based on cyanobacteria, which are the only prokaryotic National Renewable Energy organisms performing oxygenic photosynthesis. Detailly, controlled-release PDT via Laboratory (DOE), United States loading indocyanine green into mesoporous silica nanoparticles was established. Then Konstantinos Vavitsas, National and Kapodistrian University bio-oxygen pump based on a fast-growing cyanobacterium Synechococcus elongatus of Athens, Greece UTEX 2973 was tested and further packaged together with PDT to create an injectable *Correspondence: hydrogel. The packaged hydrogel showed stable oxygen production and synergetic Jin Chang therapy effect especially toward hypoxia 4T1 cells in vitro. More importantly, strong jinchang@tju.edu.cn Weiwen Zhang in vivo therapeutic effect reaching almost 100% inhibition on tumor tissues was realized wwzhang8@tju.edu.cn using PDT equipped with oxygen pump, with only negligible in vivo side effect on healthy These authors have contributed mice from S. elongatus UTEX 2973. The new photo-oxygen-dynamic therapy presented equally to this work here provided a promising strategy against hypoxia-resistant tumor and may worth Specialty section: further modifications for therapeutic application. This article was submitted to Synthetic Biology, Keywords: oxygenic cyanobacteria, hypoxia, photodynamic therapy, indocyanine green, nanoparticles, injectable a section of the journal hydrogels Frontiers in Bioengineering and Biotechnology Received: 28 January 2020 INTRODUCTION Accepted: 06 March 2020 Published: 24 March 2020 Cancer is a leading cause of death worldwide, accounting for an estimated 9.6 million deaths in Citation: 2018 (Bray et al., 2018). For several decades, various strategies targeting oncotherapy have been Sun T, Zhang Y, Zhang C, developed and evaluated (Dolmans et al., 2003; Gorrini et al., 2013; Desterro et al., 2019). Among Wang H, Pan H, Liu J, Li Z, Chen L, them, photodynamic therapy (PDT), which contains two individually non-toxic components, Chang J and Zhang W (2020) i.e., photosensitizer and light illumination with specific wavelength to transfers energy from Cyanobacteria-Based Bio-Oxygen light to molecular O to generate cytotoxic reactive oxygen species (ROS), has been used for Pump Promoting Hypoxia-Resistant more than 100 years (Dolmans et al., 2003). Nevertheless, a common feature of most tumors is Photodynamic Therapy. the low oxygen level called hypoxia, which leads to the progression, metastasis, radiation and Front. Bioeng. Biotechnol. 8:237. doi: 10.3389/fbioe.2020.00237 drug resistance of cancer cells, as well as the altering tumor microenvironment (Harris, 2002; Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 2 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT Muz et al., 2015). Meanwhile, effect of PDT has been found sodium alginate were purchased from Aladdin Bio-Chem restricted by hypoxia due to the limited oxygen available and its Technology Co., Ltd. (Shanghai, China). Propidium iodide (PI), conversion of O to ROS may in turn deteriorate the hypoxia calcein-AM, Hoechst 33528 were obtained from Sigma-Aldrich degree. Hence the improvement of oxygen availability in tumor (MO, United States). IL-2, IL-6, IFN-b and IFN-g detection kits tissues could not only alter the tumor microenvironment but also were bought from H-Y Biological Co., Ltd. (Wuhan, China). enhance the PDT therapeutic effect. CCK-8 cell proliferation and cytotoxicity assay kit and Annexin Cyanobacteria are the only prokaryote microorganism capable V-FITC apoptosis detection kit was both obtained from Solarbio of oxygenic photosynthesis via respectively taking sunlight Technology Co., Ltd. (Beijing, China). and CO as the sole energy and carbon source (Stanier and Cyanobacterium S. 2973 was cultivated with BG11 liquid Bazine, 1977). Besides their roles as primary producers on earth medium (pH = 7.5) under a light intensity of 100 mmol 2 1 (Giordano et al., 2005), cyanobacteria have been considered photons m s in an illuminating shaking incubator (HNYC- as model organisms for photosynthesis research and even 202T, Honour, Tianjin, China) at 130 rpm and 37 C (Li et al., “photosynthetic cell factories” to produce renewable chemicals 2018). 4T1 cells were cultured in RPMI-1640 medium (Gibco) (Lea-Smith et al., 2015; Gao et al., 2016). More interestingly and supplement with 10% fetal bovine serum (FBS) and maintained excitingly, biomedical applications based on cyanobacteria have at 37 C, 5% CO in a humidified atmosphere. Female BALB/c been evaluated in recent years (Raja et al., 2016). For example, mice (about 20 g) were bought from SPF Biotechnology Co., Cohen et al. (2017) presented a novel system that rescued Ltd (Beijing, China) and maintained in common environment. the myocardium from acute ischemia using photosynthesis All animal experiments were operated following the Guidance through intramyocardial delivery of the model cyanobacterium Suggestions for the Care and Use of Laboratory Animals and Synechococcus elongatus PCC 7942 (Cohen et al., 2017). In principle of 3Rs. All animals were subjected to an anesthesia addition, Yin et al. (2019) reported the role of S. elongatus experiment with a small animal anesthesia machine (Ruiwode PCC 7942 in accelerating cutaneous wound healing by secreting Life Technology Co., Ltd., Shenzhen, China). All animals were extracellular vesicles to promote angiogenesis (Yin et al., 2019). sacrificed by spine dislocation, and the bodies were chilled at All these studies demonstrated the promising therapeutic effects 20 C and uniformly incinerated by the animal center. of S. elongatus PCC 7942. In 2015, Synechococcus elongatus UTEX 2973 (hereafter S. 2973), whose genome only contained 55 single Cell Density, Absorption Spectrum and nucleotide polymorphisms and insertion-deletions compared to Dissolved O Measurement that of S. elongatus PCC 7942 (Yu et al., 2015), was isolated Cell density of S. 2973 was measured at OD using a 750 nm and demonstrated preferable properties like fast growing and UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). Besides, high light tolerance. More importantly, as optimized growing the images of S. 2973, ALG-S2973 and ALG-MI-S2973 with or temperature of S. 2973 was similar to that of human body, it may without red laser irradiation were took to study the effect of represent a more robust and promising chassis for biomedical 640 nm red light on the activity of S. 2973. applications than S. elongatus PCC 7942. Absorption spectrum of S. 2973 was investigated to evaluate In this study, aiming to simultaneously alleviate the tumor the viability of S. 2973 in different conditions using the hypoxia and the effect of PDT, a photo-oxygen-dynamic spectrophotometer. To identify the optimal power intensity of therapy (PODT) strategy combined of a light-driven bio-oxygen 640 nm laser, S. 2973 were exposed to the irradiation dose of 0, pump based on the fast-growing cyanobacterium S. 2973 and 0.1, 0.25, and 0.5 W/cm for 5 min. After that, the irradiation PDT was developed and investigated (Figure 1). Firstly, the time for 1, 5, and 10 min under the same irradiation dose of controlled-release PDT via loading indocyanine green (ICG) as 0.25 W/cm was also evaluated, respectively. photosensitizer into mesoporous silica nanoparticles (MSN) was To evaluate the elimination of S. 2973 by ICG, strains were established and investigated in vitro. Then, bio-oxygen pump incubated with ICG of different concentrations (0, 5, 10, and using S. 2973 was developed and further packaged together with 20 mg/mL). First of all, S. 2973 were irradiated with 0.25 W/cm PDT to create an injectable hydrogel. The in vivo biosafety of S. of 640 nm laser for 5 min in order to generate oxygen. Then 2973 was further measured and the PODT effect was evaluated the cells were exposed to 808 nm laser with the power intensity using a 4T1 tumor model. Excitingly, the packaged hydrogel of 2.0 W/cm for 5 min. At last, the absorptions spectrum showed strong in vivo therapeutic effect on tumor tissues. The and images of S. 2973 with different treatments were recorded. photo-oxygen-dynamic oncotherapy presented here provided a Dissolved O was detected using a Dissolved Oxygen Meter promising strategy against hypoxia-resistant tumor cells and (SMART SENSOR, Guangdong, China). worth further optimization for therapeutic application as mature genetic tools have been developed for S. 2973. AM/PI Staining AM/PI staining was utilized to evaluate the cytotoxic effect of MATERIALS AND METHODS S. 2973, ICG and MI against 4T1 cells. The effect of S. 2973 on cellular activity was determined under the irradiation dose Materials of 0.25 W/cm . 4T1 cells were seeded in 48-well plate and Indocyanine green (ICG), cetyltrimethylammonium bromide cultivated in normoxic or hypoxic condition. Cells were treated (CTAB), tetraethylorthosilicate (TEOS), diethanolamine and with ICG and MI (equivalence to 20 mg/mL of ICG) with or Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 2 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 3 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 1 | Schematic illustration of the photo-oxygen-dynamic therapy. (A) S. 2973 and MSN loaded with photosensitizer ICG were mixed with sodium alginate to make injectable hydrogels (ALG-MI-S2973). (B) The hydrogels ALG-MI-S2973 was intratumorally injected and then formed gels at the tumor site. The tumor tissue was exposed to 640 nm laser to support survival of S. 2973 to generate O , which alleviated the hypoxia microenvironment and further enhanced PDT effects. After releasing, ICG excited by 808 nm laser could exert efficient PDT effects to inhibit tumor growth. MSN, mesoporous silica nanoparticles; ICG, indocyanine green; ALG, hydrogels based on sodium alginate; MI, ICG-loaded MSN; ALG-MI-S2973, hydrogels packaging MSN-ICG and S. 2973. without 808 nm irradiation for 24 h. After removing the medium, A total of 4 mg of MSN-ICG and ALG-MI-S2973 was dispersed 1 mM Calcein-AM and PI were added and then observed by in 1 mL ddH O and then put into a dialysis bag. At certain times, inverted fluorescence microscope. The live/dead staining assay 200 mL of the samples was collected to measure the absorbance at was also conducted for 4T1 cells treated with ALG-MI-S2973 and 780 nm. The cumulative release of ICG was calculated according respectively irradiated with only 640 nm laser for 5 min, only to ICG calibration curve. 808 nm laser for 5 min or both of them. Detection of Reactive Oxygen Species Preparation and Characterization of (ROS) and Hypoxia MSN and Hydrogels 4T1 cells were grown in normoxic condition and incubated with ICG and MSN-ICG containing 20 mg/mL ICG for 24 h. For preparation of MSN-ICG, MSN were synthesized first according to our previous studies (Zheng et al., 2016). In order Then the cells were exposed to 808 nm irradiation for 5 min. Finally, cells were stained with hypoxia/ROS kits and observed to load ICG, 40 mg of MSN were re-dispersed with ddH O and stirred with 5 mg of ICG for 12 h in dark. After the reaction, using a confocal laser scanning microscope. Similar analysis the product was obtained by centrifugation and washing. For was also performed for 4T1 cells treated with ALG-MI-S2973 preparation of sodium alginate gel co-loaded with MSN-ICG and respectively irradiated with 640 nm laser, 808 nm laser and cyanobacteria (ALG-MI-S2973), ALG aqueous solution was or both of them. mixed with Glucono delta-lactone (GDL) using a vortex mixer. And then MSN-ICG and S. 2973 were added and stirred to mix CCK-8 Proliferation Assay 2C evenly. PBS containing 1.8 mM Ca was injected to form gel. The biosafety of S. 2973 on 4T1 cells were determined by CCK-8 For characterization of nanoparticles, transmission electron assay. Briefly, 4T1 cells were seeded in 96-well plate and cultured microscope (TEM) images were acquired by JEOL JEM100CXII in normoxic condition. Cells were co-cultured with S. 2973 at operating voltage of 100 kV. The particle size distribution and irradiated with 640 nm laser. After that cells were treated was measured using dynamic light scattering (DLS) on a with CCK-8 reagent (10 mL/well) at 37 C for 2 h. At last, the Zetasizer Analyzer (Malvern). UV-Vis spectroscopic absorbance absorbance at 450 nm was measured by Thermo Synergy HT was captured on a UV-2450 spectrophotometer. Microplate Reader. The in vitro Release of ICG Cell Apoptosis Analysis The dialysis bag diffusion technique was used to measure the For analysis of apoptosis, 4T1 cells were seeded in 6-well plate and in vitro release of ICG from MSN-ICG and ALG-MI-S2973. cultured in normoxic or hypoxic condition. Cells were treated Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 4 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 2 | The preparation and characterization of MSN-ICG. (A) The preparation process of MSN-ICG. (B) TEM images of MSN before and after ICG loading. (C,D) Live/dead staining and cell apoptosis analysis of 4T1 cells treated with control, ICG or MSN-ICG with or without 808 nm laser irradiation under normoxic or hypoxic condition. (E) Measurements of hypoxia and ROS for 4T1 cells treated with control, ICG or MSN-ICG with 808 nm laser irradiation under normoxia. *p < 0.05, **p < 0.01 compared with control group by Student’s t-test. MSN-ICG, indocyanine green-loaded mesoporous silica nanoparticles. with ICG and MSN-ICG (equivalence to 20 mg/mL of ICG) with with ELISA kits (H-Y biological) according to the instructions. or without 808 nm irradiation for 24 h. Then cells were stained Besides, blood was collected on the 1st, 7th, and 14th day of with 5 mL of Annexin V-FITC and PI for 15 min in the dark treatment to conduct blood routine examination. In the end, mice and then subjected to flow cytometry. Cells treated with ALG- were sacrificed and main organs (heart, liver, spleen, lung, and MI-S2973 and respectively irradiated with 640 nm laser, 808 nm kidney) were collected for pathological analysis. laser or both of them were also tested by flow cytometry to discuss the cell apoptosis induced by the PDT effect of ALG-MI-S2973. In vivo PDT Effects Each female BALB/c mouse was subcutaneously injected 2 10 The Biosafety Investigation of S. 2973 4T1 cells into the axilla. When the tumor volume reached 50– Female BALB/c mice were randomly divided into four groups 100 mm , mice were randomly divided into four groups. ALG- and subcutaneously injected with S. 2973 and ALG-S2973. After MI and ALG-MI-S2973 (equivalence to 20 mg/mL ICG) were 1, 4-, and 7-days’ injection, blood was collected and analyzed intratumorally injected at 0 day. Tumors were irradiated with Red Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 4 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 5 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 3 | Construction and characterization of the bio-oxygen pump in vitro. (A) Scheme of oxygen generation system based on S. 2973 under 640 nm red laser irradiation. (B) Absorption spectrum and OD of S. 2973 irradiated using 640 nm laser with different intensities. (C) Absorption spectrum and OD of S. 750 nm 750 nm 2973 irradiated using 640 nm laser with 0.25 W/cm for different times. (D) Oxygen evolution of S. 2973 under 640 nm red laser irradiation. (E) Cell viability and AM/PI staining of 4T1 cells with or without treatments (i.e., 640 nm laser irradiation, co-cultivation with S. 2973 or both of them) for 24 h. (F) Phenotypes of S. 2973 treated with different concentrations of ICG exposed to 2 W/cm of 808 nm laser for 5 min. decrease the injection frequency and prolong the functional time laser (0.25 W/cm , 5 min) for 3 days. 808 nm laser was used on of ICG when used in vivo, we established a controlled-release the 4th day. The body weight and tumor volume were measured system MSN-ICG by loading ICG into MSN (Table 1). TEM every 3 days. After 21 days’ treatment, mice of all groups were images of MSN before and after ICG loading was shown in sacrificed and then tumors and main organs were collected to Figure 2B and Supplementary Figure S1, suggesting the good study the pathological morphology. Besides, TUNEL staining and uniformity of the nanoparticle. In addition, continuous release of HIF-1a analysis were carried out to assess the apoptosis and ICG from MSN as long as 24 h could be detected (Supplementary hypoxia induced by PDT effect of ALG-MI-S2973. Figure S2). To evaluate the PDT effect, 4T1 cells were treated with control, ICG or MSN-ICG with or without 808 nm laser Histopathology TUNEL and (NIR) irradiation under both normoxia and hypoxia conditions. Immunohistochemical Analysis As illustrated in Figures 2C,D, the controlled-release MSN-ICG Main organs of mice were fixed and embedded in paraffin blocks. system reached similar PDT effects as ICG alone after irradiation, The sections were stained with hematoxylin and eosin (H&E) and resulting in37% apoptosis of 4T1 cells under normoxia, which examined by a microscope. TUNEL analysis was used to evaluate indicated the feasibility of PDT as well as the controlled-release the cell apoptosis in tumor tissues. And then the tumor sections system. In addition, the PDT effect was also demonstrated were immune-stained with a rat anti-mouse HIF-1a protein and by the ROS measurement (Figure 2E). On the contrary, the observed by a microscope. apoptosis rate was dramatically decreased to below 12%14% under hypoxia (Figure 2D), suggesting the low oxygen content seriously restricted the PDT. Meanwhile, for 4T1 cells under RESULTS normoxia, the hypoxia measurement revealed that PDT in turn aggravated hypoxia due to the consumption of O (Figure 2E). Establishment and Investigation of the Controlled-Release PDT System Evaluation of the Bio-Oxygen Pump To construct the PDT system (Figure 2A), we utilized a near- infrared fluorescent dye ICG that has been approved by the Based on Cyanobacteria Food and Drug Administration of United States for various Aiming at alleviating hypoxia and enhancing the PDT effect, clinical applications (Bradley and Barr, 1968; Sauda et al., 1986). we tested the bio-oxygen pump in vitro first (Figure 3A). To Meanwhile, we used near-infrared light to ensure relatively strong ensure the viability of S. 2973 and sustainable O generation penetrability toward tissues and to avoid killing S. 2973 when when used in vivo, a 640 nm red laser (Red) was chosen given used visible light to support the in vivo survival of S. 2973. To the utilized spectrum of cyanobacteria and the penetrability of Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 6 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 4 | Construction and characterization of injectable hydrogels packing MSN-ICG and/or S. 2973. (A) The preparation of injectable hydrogels packing MSN-ICG and S. 2973 (i.e., ALG-MI-S2973). (B) Growth investigation of S. 2973 directly in BG11 or packaged in ALG-MI-S2973 cultivated under normal or 640 nm irradiation. (C) The ROS generation in 4T1 cells after treatment of ALG-MI-S2973 with different amounts of S. 2973. (D,E) AM/PI staining and cell apoptosis analysis of 4T1 cells treated with or without ALG-MI-S2973 under control, 808 nm irradiation or both 640 and 808 nm irradiation. (F) Measurements of hypoxia and ROS of normoxia 4T1 cells treated with or without ALG-MI-S2973 under control, 808 nm irradiation or both 640 and 808 nm irradiation. *p < 0.05, **p < 0.01 compared with untreated group by Student’s t-test. MSN-ICG, indocyanine green-loaded mesoporous silica nanoparticles; ALG-MI-S2973, hydrogels packaging MSN-ICG and S. 2973. light toward tissue. Then red lasers with different intensity of 0, was demonstrated via detecting the dissolved O irradiated with 2 2 2 0.1, 0.25, and 0.5 W/cm were tested. Density of 0.25 W/cm the red laser of 0.25 W/cm (Figure 3D). Although no literature was found feasible as growth of S. 2973 was accelerated with evidence of Synechococcus-derived cytotoxins or cytotoxicity was the increasing laser density but was inhibited when it reached found (Klemenci ˇ c ˇ et al., 2017), we evaluated the cytotoxicity of up to 0.5 W/cm (Figure 3B). In addition, correlated with the S. 2973 to ensure its biosafety. As expected, cell viability assay condition under normal light, longer irradiation time increased and AM/PI staining assay didn’t find significant cytotoxicity from the growth of S. 2973 (Figure 3C). Meanwhile, O generation S. 2973 or red laser irradiation (Supplementary Figure S3 and Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 7 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT Figure 3E), indicating the biosafety is in general assured. To Long-Term Biosafety Investigation of S. further guarantee the biosafety when used in vivo, the eliminating 2973 on Healthy Mice effect caused by PDT was also tested. As illustrated in Figure 3F, The biosafety of ICG has been demonstrated previously by complete inactivation of S. 2973 could be observed when treated several independent studies (Bradley and Barr, 1968; Sauda with 20 mg/mL ICG after 5 min irradiation with 808 nm laser, et al., 1986). Therefore, before testing the PODT in vivo, long- indicating PDT could synchronously inhibit the tumor and term potential toxicity from S. 2973 on healthy mice was first eliminate S. 2973. investigated (Figure 5A). Detailly, female BALB/c mice were randomly divided into four groups (each group containing three Creation and Evaluation of the mice) and two of them were subcutaneously injected with S. Controlled-Release Injectable Hydrogels 2973 or ALG-S2973 (Table 1). All groups except control were irradiated with 640 nm laser on the 1st, 4th, and 7th day, the Packaging With S. 2973 and MSN-ICG investigation of injection site suggesting the well survival of To provide an oxygen pump for PDT, we created a combined S. 2973 (Figure 5B). After 1, 4-, and 7-days’ injection, blood system packing both S. 2973 and MSN-ICG into injectable was collected to analyze the immune factors including IL-2, IL- hydrogels named ALG-MI-S2973 (Figure 4A and Table 1). 6, IFN-g and IFN-b (Figure 5C). Besides, blood was collected As shown in Figure 4B, though slightly slower than the S. on the 1st, 7th, and 14th day after injection to conduct blood 2973 cultivated under normal condition with white light or routine examination (Figure 5D). Finally, mice were sacrificed red light (640 nm), S. 2973 can grow well in the hydrogel and main organs including heart, liver, spleen, lung and kidney system. In addition, continuous release of ICG from MSN as were collected for pathological analysis (Supplementary Figure long as 72 h could be detected due to the package of MSN and S4). Although certain properties like immune factor IL-6 showed ALG (Supplementary Figure S2). Further, we treated 4T1 cells slight change in mice group injected with S. 2973 or ALG-S2973 respectively with control and ALG-MI-S2973 to determine its compared to the control group, we believe that it may be due effect in vitro. Mixture of 4T1 cells and ALG-MI-S2973 led to to the limiting number of mice used in the biosafety evaluation strong ROS signal orderly after 640 and 808 nm laser irradiation, experiments, which may lead to wide error bars. Although further suggesting the increasing O evolution generated from ALG- biosafety experiments are certainly necessary in the future, our MI-S2973 with the increasing amount of S. 2973 (Figure 4C). study found that consistent with the previous studies (Cohen Besides, as illustrated in Figures 4D,E, compared with PDT et al., 2017), neither injection of S. 2973 nor ALG-S2973 caused based on MSN-ICG, apoptosis rate of 4T1 cells based on ALG- any effect on immune factors, hematological index or main MI-S2973 was decreased from 37 to 22% with only 808 nm organs of mice, suggesting of in general good biosafety of both irradiation under normoxia, indicating the slower release of ICG the S. 2973 and ALG-S2973 in vivo. from ALG-MI-S2973 than that from MSN-ICG. Nevertheless, after cascaded irradiation with 640 nm for 5 min then following 808 nm excitation to induce the oxygen production and PDT Evaluation of the PODT in vivo Using 4T1 sequentially, apoptosis rate of 4T1 cells based on ALG-MI-S2973 Tumor Model was dramatically increased up to 56%. More importantly, the Finally, we investigated in vivo anti-tumor effects of ALG-MI- new PODT system could significantly increase the effect under S2973 using the 4T1 tumor model (Figure 6A and Table 1). hypoxia (22% apoptosis rate) (Figures 4D,E), suggesting the Detailly, female BALB/c mice were randomly divided into four feasibility of the photo-oxygen-dynamic oncotherapy in vitro. groups and each group contained five mice. Besides the control Consistently, hypoxia and ROS measurements for 4T1 cells group (Group I), one group were subcutaneously injected only under normoxia showed that the addition of red laser could with MSN-ICG (Group II) while the other two groups were significantly alleviate the hypoxia and promote the PDT due to subcutaneously injected with ALG-MI-S2973 (Group III and IV). the O evolution, compared with ALG-MI-S2973 without 640 nm After injection, 640 nm laser irradiation was conducted during irradiation (Figure 4F). the next 3 days to induce the oxygen generation then the 808 nm laser was used to induce the PDT on the 4th day for Group I and IV. While only 808 nm laser was used for Group II and TABLE 1 | Detailed characteristics of the biomaterials used in this study. III. Consistently, fluorescence imaging of S. 2973 in tumor tissue showed that they were accumulated in the first 3 days due to Name Description the radiation of red laser and were killed in the 5th day due to ICG Indocyanine green directly for PDT the PDT (Figure 6B). Body weight of 4T1 tumor-bearing mice S. 2973 Wild type S. 2973 for oxygen generation with different treatments was recorded, suggesting no significant MSN-ICG Nanoparticles via loading indocyanine green into fluctuation during the process (Figure 6C and Supplementary mesoporous silica Figure S5). In addition, the volume and weight of tumor tissue ALG-S2973 Hydrogels via packaging S. 2973 with sodium was recorded and pictured then the inhibition rate was calculated alginate (Figures 6D–G). Though all treatments showed therapy effect ALG-MI Hydrogels via packaging MSN-ICG with sodium of varying degrees, traditional PDT for group II could only alginate achieve an average tumor inhibition rate at 50%. Excitingly, ALG-MI-S2973 Hydrogels via packaging MSN-ICG and S. 2973 with sodium alginate the addition of oxygen pump remarkably enhanced the PDT, Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 7 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 8 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 5 | The in vivo long-toxicity evaluation of S. 2973 and ALG-S2973. Each group contained three mice. (A) The schedule of animal experiments for toxicity evaluation. (B) Photos of the injection site of BALB/c mice. (C) Detection of immune factors of mice with different treatments at the 1st, 4th, and 7th day. (D) Hematological index of the mice with different treatments at the 1st, 7th, and 21st day. *p < 0.05, **p < 0.01 compared with untreated group by Student’s t-test. ALG-S2973, hydrogels packaging S. 2973. showing amazing effect as the tumor tissue disappeared just from bio-oxygenic cyanobacterium S. 2973 with traditional PDT based the 4th day for Group IV. The inhibition rate on 4T1 tumor were on ICG allowed us to avoid the endogenous and the PDT-induced almost 100% for 4 of 5 mice in Group IV using photo-oxygen- hypoxia, achieving an inhibition rate up to almost 100% on 4T1 dynamic therapy, reaching an average inhibition rate at 86%. tumor cells. More importantly, the in vivo toxicity evaluation Tumor tissue for 1 of 5 mice in Group III was also disappeared demonstrated the biosafety of the strategy, thus probably at last, which may be due to the original O production from S. providing a promising oncotherapy strategy. Most recently, 2973 just after injection. Finally, H&E and TUNEL demonstrated Zhou et al. (2019) reported a light triggered oxygen-affording the apoptosis and elimination of tumor tissue in Group IV engine for hypoxia-resistant oncotherapy via combining PDT (Figure 6H). Furthermore, immunohistochemical staining of with Chlorella pyrenoidosa (Zhou et al., 2019). Compared with HIF-1a protein in tumor tissues showed that hypoxia induced the reported work, this study innovatively divided the growth factor HIF-1a was significantly decreased in Group IV due to the of S. 2973 and the trigger of PDT via two lasers with different oxygen pump (Figure 6H). wavelengths, avoiding the killing of S. 2973 by PDT-induced ROS in the early stage thus ensuring the maximum oxygen generation. Meantime, the induction of PDT via 808 nm laser simultaneously cleaned the S. 2973, further guaranteeing the biosafety of DISCUSSION the strategy. In addition, the construction of control released All types of solid tumors, especially malignant solid tumors are injectable hydrogels prolonged the functional time of the therapy, decreased the dose used and may improve the therapy effects subject to hypoxia, whose O levels are remarkably lower than their original tissue. Hypoxia affects tumor microenvironment further. Finally, compared with other oxygenic microorganism and increases blood vessel formation, aggressiveness, metastasis, like Chlorella, mature genetic toolboxes have been developed for and resistance to treatment, which further restricts the model cyanobacteria like S. 2973 (Sun et al., 2018), allowing for oncotherapy effect like PDT. In this study, combining the the further optimization of the cyanobacteria-based bio-oxygenic Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 9 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT FIGURE 6 | In vivo investigation of anti-tumor effects based on ALG-MI-S2973. Each group contained five mice. (A) Schematic diagram of animal experiments. (B) Viability measurement of S. 2973 in tumor sections using fluorescence imaging excited by 561 nm. (C,D) Body weight changes and tumor growth curves of control and 4T1 tumor-bearing mice with different treatments. **p < 0.01 compared with untreated group by Student’s t-test. (E,F) Volumes and weight of tumors from 4T1 tumor-bearing mice with different treatments; (G) Tumor inhibition rates of different treatments. (H) H&E, TUNEL and immunohistochemical staining of HIF-1a protein in tumor tissues from 4T1 tumor-bearing mice with different treatments. ALG-MI, hydrogels packaging MSN-ICG; ALG-MI-S2973, hydrogels packaging MSN-ICG and S. 2973. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 9 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 10 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT systems. Most Recently, Huo et al. (2019) first demonstrated the ETHICS STATEMENT role of cyanobacterium S. elongatus PCC 7942 in promoting PDT effect. As a complementary study, our research also demonstrated The animal study was reviewed and approved by the the similar results using a more promising cyanobacterium Experimental Animal Ethics Committee of Institute of Radiation S. 2973. Although the study here only evaluated the PODT Medicine, Chinese Academy of Medical Sciences. using subcutaneous tumor model, the positive results suggested further efforts by increasing the oxygen generation of S. 2973, deleting its endotoxin for tail vein injection or even enabling AUTHOR CONTRIBUTIONS its ability for synthesizing anti-tumor chemicals via synthetic TS, YZ, and CZ performed the experiments and wrote the biology may enhance the efficiency of the photo-oxygen-dynamic manuscript. TS, YZ, CZ, HW, HP, JL, and ZL analyzed oncotherapy in the future. the data. LC, JC, and WZ designed the study and revised the manuscript. CONCLUSION In this study, an injectable hydrogel packaging both an oxygen FUNDING pump based on S. 2973 and ICG-loaded mesoporous silica This research was supported by grants from the National nanoparticles of controlled-release was created and evaluated Key R&D Program of China (Nos. 2018YFA0903600, in vivo. The packaged hydrogel showed strong therapeutic effect 2018YFA0903000, and 2019YFA0904600) and the National on tumor tissues with only negligible side effect on healthy Natural Science Foundation of China (Nos. 31901017, 31770100, mice. The PODT presented here provided a promising therapy 31972931, 91751102, 21621004, 31370115, and 31470217). strategy against hypoxia-resistant tumor and may worth further modifications for therapeutic application. SUPPLEMENTARY MATERIAL DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found All datasets generated for this study are included in the online at: https://www.frontiersin.org/articles/10.3389/fbioe. article/Supplementary Material. 2020.00237/full#supplementary-material Harris, A. L. (2002). Hypoxia–a key regulatory factor in tumour growth. Nat. Rev. REFERENCES Cancer 2, 38–47. doi: 10.1038/nrc704 Bradley, E. C., and Barr, J. W. (1968). Determination of blood volume using Huo, M., Wang, L., Zhang, L., Wei, C., Chen, Y., and Shi, J. (2019). Photosynthetic indocyanine green (cardio-green) dye. Life Sci. 7, 1001–1007. doi: 10.1016/ tumor oxygenation by photosensitizer-containing cyanobacteria for enhanced 0024- 3205(68)90108- 90102 photodynamic therapy. Angew Chem. Int. Ed. Engl. 206:108854. doi: 10.1002/ Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., and Jemal, A. anie.201912824 (2018). Global cancer statistics 2018: globocan estimates of incidence and Klemenci ˇ c ˇ, M., Nielsen, A. Z., Sakuragi, Y., Frigaard, N. U., Celešnik, H., Jensen, mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, P. E., et al. (2017). Synthetic biology of cyanobacteria for production of biofuels 394–424. doi: 10.3322/caac.21492 and high-value products. Microalgae Based Biofuels Bioprod. 2017, 305–325. Cohen, J. E., Goldstone, A. B., Paulsen, M. J., Shudo, Y., Steele, A. N., Edwards, Lea-Smith, D. J., Biller, S. J., Davey, M. P., Cotton, C. A. R., Perez Sepulveda, B. M., B. B., et al. (2017). An innovative biologic system for photon-powered Turchyn, A. V., et al. (2015). Contribution of cyanobacterial alkane production myocardium in the ischemic heart. Sci. Adv. 3:e1603078. doi: 10.1126/sciadv. to the ocean hydrocarbon cycle. Proc. Natl. Acad. Sci. U.S.A. 112, 13591–13596. 1603078 doi: 10.1073/pnas.1507274112 Desterro, J., Bak-Gordon, P., and Carmo-Fonseca, M. (2019). Targeting mRNA Li, S., Sun, T., Xu, C., Chen, L., and Zhang, W. (2018). Development processing as an anticancer strategy. Nat. Rev. Drug Discov. 19, 112–129. doi: and optimization of genetic toolboxes for a fast-growing cyanobacterium 10.1038/s41573- 019- 0042- 43 Synechococcus elongatus UTEX 2973. Metab. Eng. 48, 163–174. doi: 10.1016/j. Dolmans, D. E., Fukumura, D., and Jain, R. K. (2003). Photodynamic ymben.2018.06.002 therapy for cancer. Nat. Rev. Cancer 3, 380–387. doi: 10.1038/nrc Muz, B., de la Puente, P., Azab, F., and Azab, A. K. (2015). The role of hypoxia in 1071 cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia Gao, X., Sun, T., Pei, G., Chen, L., and Zhang, W. (2016). Cyanobacterial 3, 83–92. doi: 10.2147/HP.S93413 chassis engineering for enhancing production of biofuels and chemicals. Raja, R., Hemaiswarya, S., Ganesan, V., and Carvalho, I. S. (2016). Recent Appl. Microbiol. Biotechnol. 100, 3401–3413. doi: 10.1007/s00253-016-7374- developments in therapeutic applications of Cyanobacteria. Crit. Rev. Microbiol. 7372 42, 394–405. doi: 10.3109/1040841X.2014.957640 Giordano, M., Beardall, J., and Raven, J. A. (2005). CO2 concentrating Sauda, K., Imasaka, T., and Ishibashi, N. (1986). Determination of protein mechanisms in algae: mechanisms, environmental modulation, and evolution. in human serum by high-performance liquid chromatography with Annu. Rev. Plant Biol. 56, 99–131. doi: 10.1146/annurev.arplant.56.032604.14 semiconductor laser fluorometric detection. Anal. Chem. 58, 2649–2653. 4052 doi: 10.1021/ac00126a016 Gorrini, C., Harris, I. S., and Mak, T. W. (2013). Modulation of oxidative stress Stanier, R. Y., and Bazine, G. C. (1977). Phototrophic prokaryotes: the as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947. doi: 10.1038/ Cyanobacteria. Ann. Rev. Microbiol. 31, 225–274. doi: 10.1146/annurev.mi.31. nrd4002 100177.001301 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 10 March 2020 | Volume 8 | Article 237 fbioe-08-00237 March 21, 2020 Time: 15:12 # 11 Sun et al. Cyanobacterial Bio-Oxygen Pump Promoting PDT Sun, T., Li, S., Song, X., Diao, J., Chen, L., and Zhang, W. (2018). Toolboxes Zhou, T. J., Xing, L., Fan, Y. T., Cui, P. F., and Jiang, H. L. (2019). for cyanobacteria: recent advances and future direction. Biotechnol. Adv. 36, Light triggered oxygen-affording engines for repeated hypoxia-resistant 1293–1307. doi: 10.1016/j.biotechadv.2018.04.007 photodynamic therapy. J. Control Rel. 307, 44–54. doi: 10.1016/j.jconrel.2019. Yin, H., Chen, C. Y., Liu, Y. W., Tan, Y. J., Deng, Z. L., Yang, F., et al. (2019). 06.016 Synechococcus elongatus PCC7942 secretes extracellular vesicles to accelerate cutaneous wound healing by promoting angiogenesis. Theranostics 9, 2678– Conflict of Interest: The authors declare that the research was conducted in the 2693. doi: 10.7150/thno.31884 absence of any commercial or financial relationships that could be construed as a Yu, J., Liberton, M., Cliften, P. F., Head, R. D., Jacobs, J. M., Smith, R. D., et al. potential conflict of interest. (2015). Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO(2). Sci. Rep. 5:8132. doi: 10.1038/ Copyright © 2020 Sun, Zhang, Zhang, Wang, Pan, Liu, Li, Chen, Chang and Zhang. srep08132 This is an open-access article distributed under the terms of the Creative Commons Zheng, B., Chen, H. B., Zhao, P. Q., Pan, H. Z., Wu, X. L., Gong, Attribution License (CC BY). The use, distribution or reproduction in other forums X. Q., et al. (2016). Persistent luminescent nanocarrier as an accurate is permitted, provided the original author(s) and the copyright owner(s) are credited tracker in vivo for near infrared-remote selectively triggered photothermal and that the original publication in this journal is cited, in accordance with accepted therapy. ACS Appl. Mater. Interf. 8, 21603–21611. doi: 10.1021/acsami.6b0 academic practice. No use, distribution or reproduction is permitted which does not 7642 comply with these terms. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 11 March 2020 | Volume 8 | Article 237

Journal

Frontiers in Bioengineering and BiotechnologyPubmed Central

Published: Mar 24, 2020

There are no references for this article.