Nose-to-brain Delivery Of Temozolomide-loaded Nanoparticles Functionalized With Anti-EPHA3 For Glioblastoma Targeting

Most of glioblastomas multiforme?GBM?are primary central nervous system tumors.Many treatments are combined with resection and drug chemotherapy.However,due to the malignant invasion of GBM,the quality of life of the patients is poor,and the chemotherapy drugs can not achieve satisfactory results,which are limited by many factors,such as poor penetrability and poor targeting.Therefore,it is necessary to develop an effective treatment method suitable for clinical application.The non-invasive nasal brain drug delivery system can avoid the obstruction of the blood-brain barrier?BBB?,improve the brain rate of the drug and reduce the side effects of drugs,and is a more effective way to treat brain tumors.Temozolomide?TMZ?is currently one of the most effective drugs for the treatment of GBM,although TMZ could be reported through BBB and had high bioavailability,due to the large dose and systemic administration,the side effects are more serious.Therefore,this study designed and constructed a delivery system of of tyrosine protein kinase receptor A3 antibody?anti-EPHA3?modified,trimethyl chitosan?TMC?coated poly?lactic-co-glycolic acid??PLGA?nanoparticles?NPs?to treat GBM by intranasal administration,aiming at increasing drug delivery efficiency in the brain,improve the targeting of lesion sites,reduce the distribution of drug peripheral tissues,and achieve safe and effective treatment of brain tumors.Due to the poor liposolubility of TMZ,it has been prepared as temozolomide butyl ester?TBE?,which is easier to use in the preparation of new formulations while increasing lipid solubility.The research content mainly includes the preparation and characterization of nanoparticles,in vivo and in vitro targeting evaluation and pharmacodynamics,which lays a foundation for the feasibility of temozolomide through the nose-to-brain targeted delivery system for the treatment of GBM.The research topics mainly include the following contents:1.Expression detection of EPHA3 receptorIn this study,the expression of EPHA3 receptor was determined in rat glioma cell C6 and its formed glioma tissues,and human bronchial epithelial cells 16HBE was used as a control.The results showed that the EPHA3 receptor was present in glioma cells and tissues and was not expressed in normal cells,demonstrating that this receptor can be used as a specific functional target for the treatment of GBM.2.Synthesis of temozolomide and preparation of coating materialTMZ was hydrolyzed and then esterified with n-butanol to obtain TBE.The structure of TMZ was verified by ¹H-NMR.The cytotoxicity was examined using glioma cell C6 as a model,and the results indicate that TBE has a cell growth inhibitory activity comparable to that of TMZ.In order to increase the adhesion of NPs in nasal cavity,TMC was synthesized by reducing methylation of chitosan and was verified by ¹H-NMR,and then applied to the surface of the NPs to increase the nasal retention of the NPs.3.Preparation and characterization of temozolomide butyl ester nanoparticlesThe TBE NPs were prepared by emulsion solvent evaporation method and the optimal prescription was determined by single factor investigation.The optimal prescription obtained from the experimental results is:carrier material PLGA is 10 mg,drug TBE is 1 mg,oil-water ratio is 1:3,PVA concentration is 1%,and ultrasonic power is 300 w.The PLGA nanoparticles?P-NPs?were prepared by this optimal formulation and process,T/P-NPs were prepared by TMC coating,anti-EPHA3 was thiolated and conjugated with the maleimide group?Mal?on the surface of T/P-NPs to prepare anti-EPHA3-T/P-NPs.The all prepared NPs were uniform light blue solution,and the characteristic parameters were as follows:particle size?145.9±8.7?nm,potential?+23.08±2.5?mV,encapsulation efficiency?34.83±1.53?%,drug loading?3.02±0.68?%,the anti-EPHA3 attachment rate was?8.1±1.5?%.Under the transmission electron microscope,the particles were smooth and spherical,and there was no adhesion state.Anti-EPHA3-T/P-NPs can be slowly released to 48 h in PBS?pH=7.4?release medium.4.In vitro evaluation of nanoparticlesIn this study,the cytotoxicity of blank NPs and drug-loaded NPs was investigated,and the qualitative and quantitative uptake of NPs by using glioma cell C6 as a model cell was observed.The results showed that the concentration of PLGA was in the range of 0.23².35 mg·mL^-1,and the blank NPs were not cytotoxic to C6,indicating that the carrier material was safe.The results showed that the concentration of TBE was in the range of 10¹00?g·mL^-1,and the anti-EPHA3-T/P-NPs had the highest cytotoxicity.Qualitative and quantitative studies of cellular uptake showed that the cells in the anti-EPHA3-T/P-NPs group had stronger fluorescence and showed significant cell targeting compared with the P-NPs and T/P-NPs groups?p<0.01?.5.In vivo evaluation of nanoparticlesIn this study,the fluorescence distribution of glioma model rats was investigated by using DiR-loaded NPs.The results of in vivo imaging showed that after nasal administration,the fluorescence distribution was significantly concentrated in the brain,and other organs had no obvious fluorescence signal,indicating that the NPs can increase the amount of drug into the brain and reduce the peripheral distribution after nasal administration.Among them,the anti-EPHA3-T/P-NPs group had the strongest fluorescence in the brain.The fluorescence distribution in the brain of glioma model rats was investigated by using coumarin-6-loaded NPs.The results of brain fluorescence section showed that anti-EPHA3-T/P-NPs had strong fluorescence signal in the brain glioma site.It showed that anti-EPHA3 could significantly enhance the targeting of GBM.The pharmacodynamic study of NPs was studied by investigating the survival and tumor cell apoptosis of glioma rats.After nasal administration in rats,the brain section showed that TBE-loaded anti-EPHA3-T/P-NPs could cause more apoptosis of glioma cells and prolong the median survival time of rats to 26 days,which was 1.53 and 1.44times higher than that of the control group and the TBE solution group,respectively,indicating that TBE-loaded anti-EPHA3-T/P-NPs had better better therapeutic effect on GBM.6.ConclusionThe anti-EPHA3-T/P-TBE-NPs constructed in this study have good physical and chemical properties,and can be released in vitro for 48 h,showing a certain sustained release effect.In vitro cell uptake studies showed that anti-EPHA3-T/P-NPs have strong targeting to glioma cell C6,resulting in strong fluorescence aggregation and strong inhibition growth of C6 cells in cytotoxicity studies.In vivo studies have shown that nasal delivery of anti-EPHA3-T/P-NPs can significantly increase the amount of drug into the brain,reduce systemic distribution,and have high targeting of glioma lesions,showing unique advantages in the treatment of GBM.