Study On Targeted Photodynamic Therapy Against Glioma By Biomembrane-Coated Lipid Nano-Drug Delivery System

Objective:Glioblastoma(GBM)is a highly lethal primary brain cancer,which is characterized by high invasiveness,high invasiveness and high recurrence rate.At present,the clinical treatment methods for glioma mainly include surgical resection,drug chemotherapy,radiotherapy,etc.However,surgical resection is difficult and prone to recurrence.Drug chemotherapy has poor selectivity,high toxic side effects,and easy to produce drug resistance.Radiotherapy has great side effects and individual variation,and the commonly used treatment methods can not meet the needs of glioma treatment.Therefore,finding efficient and safe treatments for glioma is a key scientific problem that needs to be solved urgently.Methods:In this study,ICG-loaded solid lipid nanoparticles(GLP@ICG)were prepared by the emulsified solvent diffusion method,and the biofilm-coated lipid nanodelivery system(GLP@ICG@M)was prepared by the liposome extrusion method.The retention of membrane proteins of GLP@ICG@M was detected by SDS-PAGE and WB analysis.The particle size and surface potential of GLP@ICG@M were measured by particle size and surface potential analyzer,the particle size and morphology of GLP@ICG@M were observed by transmission electron microscope,and the drug release behavior of GLP@ICG@M in vitro was investigated by dialysis bag method.The rat glioma cell line C6 was used as a model cell.Laser confocal microscopy and flow cytometry were used to qualitatively and quantitatively study the tumor cell targeting ability of GLP@ICG@M.MTT assay and scratch test were used to determine the cytotoxicity and inhibition of tumor migration ability of GLP@ICG@M before and after near-infrared laser treatment.Singlet oxygen sensor green(SOSG)probe was used to determine the ability of GLP@ICG@M to induce C6 cells to produce Reactive oxygen species(ROS)by near infrared laser.C6 ectopic tumor Balb/c nude mice were used as model animals.The distribution and tumor fluorescence imaging of GLP@ICG@M in the model animals were observed by small animal in vivo fluorescence imaging system.The antitumor effect of GLP@ICG@M was further investigated under the action of near-infrared laser.H&E staining was used for histopathological analysis,and immunohistochemistry was used to study the molecular mechanism of GLP@ICG@M inducing tumor cell apoptosis.Results:1.The particle sizes of GLP,GLP@ICG and GLP@ICG@M were 69.2 ± 1.0 nm,60.1 ± 0.7 nm and 118.0 ± 1.1 nm,respectively.The surface potentials were-3.3 ± 0.7m V,0.004 ± 0.1 m V and-36.3 ± 1.8 m V.Tem showed that GLP,GLP@ICG,and GLP@ICG@M nanoparticles were spherical in shape and had uniform particle size distribution,among which GLP@ICG@M had an obvious core-shell structure.2.Similar protein bands were observed in C6 cell lysates,C6 cell membrane(M)and GLP@ICG@M by SDS-PAGE,indicating that the cell membrane proteins were well retained on GLP@ICG@M.WB results showed that M and GLP@ICG@M retained the membrane surface specific protein Na+/K+-ATPase.Compared with C6 cells,M and GLP@ICG@M hardly expressed GAPDH(cytoplasmic protein),Cytochrome C(mitochondrial protein)and Histone H3(nuclear protein).These results indicate that GLP@ICG@M retains C6 cell membrane protein,which can be used as a homologous target for subsequent tumor therapy.3.The results of in vitro release study showed that GLP@ICG@M could rapidly release the drug in response to the slightly acidic tumor environment.Under the condition of p H 6.8,the release rate of ICG from GLP@ICG@M increased significantly after near-infrared laser irradiation,and the cumulative release rate of ICG reached 92.5% within 48 hours.In contrast,without laser irradiation,the cumulative drug release from GLP@ICG@M over 48 hours was only 62.5%.The possible reason is that near-infrared laser irradiation may induce photothermal effect of ICG,and the increase of temperature will cause destruction of GLP@ICG@M nanometer,which in turn will promote the release of ICG.4.At the cellular level,the uptake of GLP@ICG@M in C6 cells was time-dependent,and the biofilm modification could significantly enhance the targeted uptake of GLP@ICG@M in C6 cells.Given near-infrared laser irradiation,GLP@ICG@M can induce cells to produce a large amount of ROS,and show obvious cytotoxicity and inhibition of tumor migration.5.The results of in vivo fluorescence imaging of small animals showed that the biofilm modification could significantly enhance the targeted distribution of GLP@ICG@M in tumor tissues.Given near-infrared laser irradiation,GLP@ICG@M could significantly inhibit tumor growth,and the tumor inhibition rate was 96.8%.H&E staining,immunohistochemistry and immunofluorescence results further showed that the apoptosis of tumor tissues in the GLP@ICG@M group was obvious under the action of near-infrared excitation light,the expression of Bax,C-Caspase-3,and Caspase-9 was significantly increased,and the expression of Bcl-2was significantly decreased.These results indicate that GLP@ICG@M can induce the activation of mitochondria-mediated apoptotic pathway and inhibit tumor growth under the action of specific wavelength near-infrared laser.In addition,there were no significant differences and lesions in the main organs of each experimental group,indicating that GLP@ICG@M had good biocompatibility.Conclusion:In this study,a biofilm-coated lipid nanodrug delivery system(GLP@ICG@M)was constructed for targeted photodynamic therapy of glioma.The results of in vitro and in vivo experiments show that the lipid nanodrug delivery system(GLP@ICG@M)coated with biofilms has good biocompatibility,low systemic toxicity,and good homologous targeting ability.Under near-infrared laser,GLP@ICG@M can induce obvious phototoxicity and tumor growth inhibition in tumor cells.The experiments further verified that GLP@ICG@M combined with near-infrared laser irradiation could inhibit tumor growth by inducing and activating the mitochondrial apoptosis pathway in tumor cells.In summary,this study provides a novel strategy for targeted photodynamic therapy of GBM.