Chlorella Hydrogel With Photosynthetic Oxygen Production For Enhanced Local Photodynamic Therapy

The hypoxic microenvironment in solid tumors will limit the efficacy of photodynamic therapy（PDT）.Therefore,increasing the oxygenation level in tumor will take advantage on PDT efficiency.To increase the oxygenation level at the tumor site,many strategies by delivering oxygen sources have been proposed to reverse tumor hypoxia.The results show that the reversed tumor hypoxia can enhance the therapeutic effect of PDT.However,studies have shown that the process of PDT will consume numerous oxygen,and simultaneously will damage blood vessels.This indicates that supply oxygen alone can still cause further hypoxia within tumor,which can not fully exert the efficacy of PDT.More importantly,the hypoxia after PDT also poses difficulties on tumor killing during the subsequent treatment.In this article,we design Chlorella hydrogels（C-Gel）that can produce oxygen through photosynthesis as an oxygen source for continuous oxygen supply under light control.The C-Gel can not only improve the photodynamic efficacy,but also improve tumor hypoxia and prognosis after PDT.The content of this article is mainly divided into four parts:Part One:C-Gel preparation and characterization.We make use of the property of sodium alginate interacting with Ca²⁺to form the C-Gel to prepare a gel containing Chlorella.The experiments we conducted prove that the C-Gel can be formed when the concentration of sodium alginate concentration reached 20mg/ml.The C-Gel with strong mechanical force can be formed by measuring the elastic modulus.The cell viability was determined by the SYTO-9 staining kit.Even after 13 days after the formation of C-Gel in vitro or after 24 hours after the formation of C-Gel in vivo,the survival rate remained the same as the day 0（surviving rate:100%）,indicating that the C-Gel can protect Chlorella from outer environment.And through the cell viability experiment,it was found that after incubating w/or w/o C-Gel with tumor cells,the activity of the tumor cells was not different from that of the group w/o C-Gel.The cell viability was all around 100%.Chlorella had no direct toxicity on the tumor cells.The above results indicate that C-Gel can protect the activity of Chlorella in good manner.Part two:Characterization of oxygen production capacity of C-Gel.To determine the oxygen production capacity of C-Gel,the oxygen production capacity of C-Gel in vivo and in vitro was measured using an oxygen electrode.When C-Gel was irradiated with light irradiation in vitro,the oxygen production of C-Gel and free Chlorella can reach more than 300μM within 30min,and oxygen production of C-Gel is much higher than free Chlorella.When using 660nm light,the C-Gel can produce 150μM of oxygen within 10 minutes,and oxygen production can be controlled by controlling the light on and off.By staining and quantifying the protein HIF-1αin the tumor cells,it was found that the oxygen produced by the photosynthesis of C-Gel after light irradiation can significantly reduce the amount of HIF-1αin hypoxic cells or hypoxic tissues by about30%.And the oxygen produced by C-Gel can improve the hypoxia at the tumor site.The above results indicate that the C-Gel can achieve cyclically oxygen production under the light control in vivo and in vitro,and improve the tumor hypoxia,and has the potential to improve the therapeutic effect of PDT in tumor treatment.Part three:Preparation and characterization of photosensitize nanoparticles.Todelivering the photosensitizer to the tumor cell,the photosensitizer Ce6 and perfluorocarbon are co-loaded into albumin to form Ce6 nanoparticles（Ce6-NPs）by the folding-unfolding method.The morphology and size of Ce6-NPs were investigated using dynamic light scattering（DLS）instrument and transmission electron microscope（TEM）.The results showed that the prepared Ce6 nanoparticles have an average particle size of 115nm.The results of evaluating the in vivo distribution of Ce6-NPs showed that after 8-12 hours of intravenous injection,the nanoparticles mainly accumulated in the tumor site.The above results show that the prepared Ce6-NPs have uniform particle size and have strong tumor targeting ability.Part four:Oxygen production by C-Gel increases photodynamic therapy efficiency.To verify the synergistic effect of C-Gel and Ce6-NPs on tumor cells during the PDT process,the use of singlet oxygen detection proved that oxygen production of C-Gel under light can increase the singlet oxygen production of Ce6-NPs in vivo and in vivo by three times,which then poses more than two times the photodynamic killing ability on tumor cells.The cell viability was verified in the CT26 cell line.The results showed that oxygen production of C-Gel can indeed enhance the inhibition rate of CT26 tumors,and 30%of the tumors were completely cured.This combined therapy was proved to have high biological safety.The above results showed that the photosynthetic oxygen production of C-Gel can increase the efficiency of PDT to kill tumors.This article aims to solve the problem of tumor hypoxia on restricting the efficacy of PDT,and solve the further local hypoxia caused after PDT process which even affects the follow-up therapeutic effect.C-Gel,which have the ability to generate oxygen under light control,can be taken as an oxygen factory to relieve tumor hypoxia and enhance the efficacy of PDT.The results showed that C-Gel achieved controlled and efficient oxygen supply for long time,thus relieving tumor hypoxia and enhancing PDT.Therefore,the C-Gel with the ability of light-controlled oxygen production provides a new aspect for improving tumor hypoxia and enhancing therapeutic effect during PDT process.For the good therapeutic effect and biological safety,C-Gel can indeed enhance photodynamic therapy and have great potential transforming into clinic.