Photosensitizer-loaded Photosynthetic Macrophage-mediated Targeted Oxygen Self-supply Photodynamic Therapy

As two major diseases menacing human health,malignant tumor and bacterial infection have brought enormous pressure to global medical systems.Trapped by the complexity of tumor treatment and bacterial drug resistance,existing treatment methods face more difficulties in meeting treatment needs.Therefore,it is of great practical significance to develop novel and effective treatment schemes.Photodynamic therapy,as a clinically recognized treatment procedure of minimal invasion,represents a valuable treatment equipped with virtues such as low side effects,remarkable effect and no drug resistance.However,hypoxic environment of tumor and bacterial infection impede its further application.Although the rapidly developing micro-nano biomedicine provides a new solution for hypoxia remission,the relevant high potential toxicity,external microenvironment dependent oxygen generation and instability of biomaterials still loom large.Recently,Chlorella（Chl）,has won more and more interest of researchers as a kind of photosynthetic single-celled microorganism by virtue of its stable particle size,excellent biosafety and robust oxygen generation capacity.However,simple Chl registers low passive-targeting efficiency due to its liability to be recognized and cleared by the immune system.Improved schemes such as magnetic materials armed Chl for magnetic targeting,erythrocyte membrane coating to evade recognition by immune system have been propounded one after another,which eventually failed to achieve thorough amelioration in Chl delivery.Living cell-mediated drug delivery especially via macrophage（MΦ）,is able to actively deliver drugs to inflammatory locations and hypoxic areas of tumor with vasculature deficiency by means of MΦ’s natural tropism to these sites.So far,MΦ-mediated photothermal therapy,chemotherapy and immunotherapy have been explored in existing research home and abroad,but there are few reports on its photodynamic therapy.Enlightened by the theory of endosymbiosis origin of chloroplasts,namely chloroplasts come from the combination of phagocytes and cyanobacteria and MΦ’s strong ability of microorganisms phagocytosis,this study,for the first time,has proposed a kind of endosymbiosis-like system:Chl endocytosed by MΦ,a type of photosynthetic MΦ（Chl/MΦ）.Further,photosensitizer Chlorin e6（Ce6）was loaded into Chl/MΦwith SiO₂ as the carrier,so the as-prepared Chl/MΦloaded with SiO₂-Ce6（（Chl,SiO₂-Ce6）/MΦ）was constructed to mediate the tumor and bacterial infection targeted oxygen self-supply photodynamic therapy.Specially,（1）The endocytosis efficiency of MΦand MΦactivity after endocytosis were set as reference standards for the following exploration of the optimal concentration of Chl and time for co-culture.Chl/MΦgenerating from Chl and MΦcultured at an optimal ratio 3:1 for 2 h maintained excellent cell viability.At this time endocytosed Chl located in cytoplasm of MΦand endocytosis efficiency reached 96.7%.The oxygen production capacity of Chl/MΦwas equivalent to that of pure Chl and compared with MΦ,Chl/MΦshowed stronger ability of nanoparticles phagocytosis.（2）Three drug-loaded nanoparticles were structured:SiO₂ loaded with Ce6（SiO₂-Ce6）,mesoporous SiO₂ with Ce6 and graphene quantum dots with Ce6.The endocytosis efficiency of MΦ,MΦactivity after endocytosis and the ability of Ce6/MΦto produce reactive oxygen species were set as reference standards for the following exploration of the type of Ce6 carriers and time for co-culture.SiO₂ was selected as the appropriate carrier and 3 h was selected as the appropriate time,so as-prepared（Chl,SiO₂-Ce6）/MΦmaintained favorable cell viability and endocytosed SiO₂-Ce6 located in cytoplasm of MΦ.（3）Under hypoxic environment with light in vitro,（Chl,SiO₂-Ce6）/MΦmaintain killing effects on laryngeal cancer cells by reversing the hypoxia with oxygen production to maintain reactive oxygen species generation ability of Ce6.When exposed to normal oxygen with light in vitro,（Chl,SiO₂-Ce6）/MΦenhanced killing activity by generating oxygen.（4）A tumor-bearing mice model was established to prove that（Chl,SiO₂-Ce6）/MΦassumed effective tumor-targeting power by in vivo imaging and presented effective hypoxia remission of solid tumor under laser by immunofluorescence staining.The datas including morphology and growth curve of tumor,morphology,quality,histopathological characteristic of isolated tumor showed that（Chl,SiO₂-Ce6）/MΦoffered tumor-targeting oxygen self-supply photodynamic therapy and exhibited noticeable tumor growth inhibition.（5）Similarly,the datas including morphology,growth curve and histopathological characteristic of abcess comfirmed that（Chl,SiO₂-Ce6）/MΦcould effectively inhibit abscess caused by bacterial infection development with laser irradiation（6）The biosafety of（Chl,SiO₂-Ce6）/MΦwas systematically investigated in terms of in vitro hemolysis test,body temperature,body weight,morphology of main tissues and organs,organ coefficients,pathological characteristics and physiological and biochemical blood indexes of mice after injection.The results meant favorable biocompatibility of（Chl,SiO₂-Ce6）/MΦ.In conclusion,for the first time,this study prepared photosynthetic MΦ（Chl/MΦ）and selected SiO₂ as the very carrier of Ce6 from GQDs、m SiO₂and SiO₂ followed by Ce6 loading into Chl/MΦto obtain Chl/MΦloaded with photosensitizer.The multifunctional（Chl,SiO₂-Ce6）/MΦpossesses the capability of photosynthesis of Chl,photodynamic effect of Ce6 and the active targeting character of MΦto tumor,inflammatory and hypoxic locations.Hence the（Chl,SiO₂-Ce6）/MΦ-mediated targeted oxygen self-supply photodynamic therapy can not only remarkably inhibit the growth of solid tumors,but also cure abscesses caused by bacterial infection.This study offers more options for photodynamic therapies,displays a paradigm for the combination of MΦ-mediated drug delivery and photodynamic therapy.Probably,the prepared Chl/MΦwill further serve as a platform for loading chemical drugs,radiosensitizers and sonosensitizers,thus rendering novel yet effective choices for hypoxia-associated therapies.