Self-assembled Angiopep-2 Modified Lipid-poly(hypoxic Radiosensitized Polyprodrug)nanoparticles Delivery Temozolomide For Glioma Synergistic Temozolomide And Radiation Therapy

Objective:The hypoxic radiosensitizer encapsulates temozolomide(TMZ)hydrophobic anticancer drug,which could enhance the radiosensitizing effect and cellular uptake the TMZ to achieve the synergistic treatment of chemoradiotherapy for glioma.Methods:1.1H NMR spectra assay was used to confirm if P(MIs)25 was synthesized correctly.2.Malvern Zetasizer Nano ZS and TEM were used to test whether Angiopep-2(A2)-P(MIs)25 and A2-PLGA nanoparticle were constructed successfully.3.The loading efficiency of TMZ was tested by UV-vis spectrophotometer.4.The toxicity of A2-P(MIs)25 and A2-PLGA were measured by MTT assay.5.To research intracellular DOX release from P(MIs)25/DOX and A2-P(MIs)25/DOX,C6 cells were incubated.The cellular release of DOX from P(MIs)25/DOX and A2-P(MIs)25/DOX was tested by fluorescence microscopy and flow cytometry.6.We tested the effects of radiotherapy on A2-P(MIs)25 by colony-forming and Immunofluorescence test.7.Tumor growth of C6 bearing mice was monitored by live bioluminescence imaging.8.To further evaluate the antiglioma effects of these NPs on tumor growth,TUNEL was used to show cell apoptosis in xenografts.Throughout the study,mice were weighted regularly.The survival time was calculated from the day of glioma cell inoculation(0 day)to the death day.9.Histological and microscopic examinations were used to study whether A2-P(MIs)25 A2-P(MIs)25/TMZ was fine biocompatibility.Results:1.Preparation and characterization of lipid-polymer(1)1H NMR spectra assay suggested that P-(MIs)25 was synthesized correctly.(2)The average diameters of A2-P(MIs)25 and A2-PLGA were approximately.The TEM images suggested that the A2-P(MIs)25 and A2-PLGA were spherical NPs with a unimodal size distribution.(3)The results of enzyme labeling showed that A2-P(MIs)25 could effectively load TMZ.2.In Vitro analysis:(1)The MTT assay results showed that when the concentration of A2-P(MIs)25 reached 1000 ug/m L,the survival rate of C6 cell line was still higher than 90%,which proved that the toxicity of A2-P(MIs)25 lipid-polymer nanoparticles was very low.(2)The experimental results of fluorescent microscope and flow cytometry both indicated that the fluorescence intensity of A2-P(MIs)25/DOX group was stronger than that of P(MIs)25/DOX group(P < 0.01),indicating that A2 has the ability to target glioma.(3)The results of colony formation assay showed that,compared with PBS group and A2-PLGA group,the clone formation rate of C6 cells in A2-P(MIs)25 group was significantly decreased under hypoxia(P < 0.01),and the expression of ?-H2 AX was significantly enhanced under hypoxia(P < 0.01)by A2-P(MIs)25 combined with radiotherapy.It shows that A2-P(MIs)25 can enhance the therapeutic effect of X-ray ionizing radiation under hypoxic environment and is an effective radiosensitizer.3.In Vivo analysis:(1)The orthotopic transplantation tumor model was successfully established with C6/Luci/GFP cells.(2)According to the statistical results of fluorescence intensity,survival time and weight change of C6 cells in mice brain glioma model,the fluorescence intensity of DOX was the strongest in A2-P(MIs)25/DOX group,and the glioma proliferation rate was the slowest in A2-P(MIs)25/TMZ + radiation therapy(RT)group,the weight loss was the slowest and the median survival time was the longest(P <0.01).These results indicate that A2-P(MIs)25 can more effectively carry chemotherapeutic drugs to glioma tissues,P(MIs)25 is an effective radiosensitizer,and A2-P(MIs)25 + RT can effectively enhance the therapeutic effect of X-ray ionizing radiation.TUNEL results showed that compared with PBS group,PBS+RT group,A2-PLGA+RT group,free TMZ+RT group,A2-P(MIs)25+RT group and A2-PLGA/TMZ+RT group,A2-P(MIs)25/TMZ+RT group had the most significant inhibitory effect on the proliferation of glioma cells and the promotion of glioma cell apoptosis(P<0.01).(3)No obvious damage of A2-P(MIs)25/TMZ lipid-polymer nanoparticles to heart,liver,spleen,lung and kidney tissues was observed by HE staining,which proved that A2-P(MIs)25/TMZ had good biocompatibility and low toxicity and side effects.Conclusions:A-2 modified lipid-poly(hypoxic radiosensitized polyprodrug)nanoparticles were developed to deliver TMZ(A2-P(MIs)25/TMZ)for glioma synergetic TMZ and RT therapy.The in vitro and in vivo results demonstrate that these A2-P(MIs)25/TMZ can efficiently target glioma.The therapeutic studies show that A2-P(MIs)25/TMZ can effectively inhibit the growth of glioma and significantly improve mice survival time without causing obvious adverse effects.Therefore,A2-P(MIs)25/TMZ can provide a powerful new strategy for enhancing the RT sensitivity of glioma and achieving the synergistic combination of radiation and TMZ for glioma.