| Background:PAE nanoparticles with good biocompatibility have a responsive degradation function in an acidic environment,and can be used to deliver anti-tumor drugs.The modification of PEG allows PAE-PEG to evade immune detection and to increase the capability of drug loading.The modification of transferrin(Tf)can improve the targeting and permeability of PAE-PEG,thereby increasing the uptake rate of tumor tissues.Temozolomide(TMZ)is the main chemotherapeutic drug for the clinical treatment of glioma,but TMZ has weak targeting ability and cannot maintain an effective drug concentration in tumor tissues,which is prone to drug resistance.Epidermal growth factor receptor(EGFR)is highly expressed in glioblastoma.The downstream signaling pathway mediated by EGFR plays an important role in promoting tumor cell proliferation,migration and invasion.Meanwhile,EGFR also leads to temozolomide resistance.And all these are closely related to the treatment and prognosis of glioma.Based on the importance of NPs,EGFR and TMZ in the treatment of glioma,we constructed a co-delivery system of siEGFR and temozolomide based on PAEPEG.In cell experiments,we aim to study the effect of siEGFR,temozolomide and nanoparticle co-delivery system on the proliferation and apoptosis of glioma cells.In animal experiments,we aim to study the therapeutic effect of siEGFR,temozolomide and nanoparticle co-delivery system on glioma orthotopic model.Purpose:This study introduces a pH-responsive nanopolymer carrier that simultaneously delivers temozolomide and the sensitizing gene siEGFR to treat glioma.The study aims to observe its ability in inhibiting tumor cell proliferation and the underlying mechanism.Methods:(1)Preparation and detection of PAE-PEG-Tf polymer:①.NMR was utilized to detect the successful synthesis of molecules containing poly(β-aminoester)group(PAE-PEG-Tf);②.Malvern particle size analyzer was utilized to detect the changes of particle size of poly(β-amino ester)nanocomposite(PAE-PEG-Tf)and the poly(β-amino ester)nanocomposite(PLGA-PEG-Tf)in non-acidic and acidic environments.The morphological change of the nanocomposite was detected by transmission electron microscope;③.Enzyme-linked immunoassay was to study the encapsulation efficiency of PAE-PEGTf/TMZ@siEGFR and its release in acidic environment.(2)Cell study:①.The toxicity of PAE-PEG-Tf was detected by MTT experiment.②.The amount of TMZ released by PAEPEG-Tf/TMZ@siEGFR and PAE-PEG/TMZ@siEGFR with or without transferrin targeting peptide modification to U87 cells was observed by flow cytometry and fluorescence microscope.③.The inhibition effect of PAE-PEG-Tf/TMZ@siEGFR on the proliferation of U87 cells was observed by colony cloning experiment.④.qRT-PCR and Western blot techniques were utilized to detect the down-regulation effect of PAE-PEGTf/TMZ@siEGFR on the target gene expression;⑤.The effect of PAE-PEGTf/TMZ@siEGFR on promoting cell apoptosis was detected by flow cytometry and live and dead cell staining technology.(3)Animal study:①.Orthotopic nude mouse model of glioma was successfully constructed;②.The targeting ability and the organ distribution of PAE-PEG-Tf/TMZ@siEGFR nanocarrier were detected with small animal in vivo imager;③.The changes in the fluorescence intensity of in situ nude mouse glioma was observed by in vivo imaging technology,with the weight and survival information recorded and drawn.④.Pathological tissue sections were to detect damages of the PAE-PEG-Tf/TMZ@siEGFR nanocarrier on major organs,and tumor tissue apoptosis in each experimental group was observed by TUNEL detection.Results:(1)Preparation and properties of PAE-PEG-Tf polymer:①.The experimental group containing poly(β-aminoester)molecules(PAE-PEG-Tf)was successfully synthesized;②.Construction of nanocarrier PAE-PEG-Tf in the experimental group and PLGA-PEG-Tf in the control group were successful and both morphologies are uniform spherical structures.The diameter of PAE-PEG-Tf was 95.34±3.25 nm,and the Tf-PLGA-PEG-Tf was 108.44±3.48 nm.Compared with the normal pH environment,the particle size of PAE-PEGTf under the acidic environment became larger,and the morphology under the transmission electron microscope also changed,with the polymer structure obviously damaged.However,PLGA-PEG-Tf had no change in the particle size and morphology in acidic environment;③.The respective encapsulation efficiency of PAE-PEG-Tf/TMZ@siEGFR to TMZ and siEGFR was 88.7%and 77.9%.Through in vitro drug release,PAE-PEG-Tf/TMZ@siEGFR responsively cleaved and released TMZ and siEGFR in an acidic environment.(2)Cell sduty:①.MTT results showed that the survival rate of U87 glioma cells was still higher than 83%when the concentration of PAE-PEG-Tf reaches 1.0 μg mL-1,indicating the low toxicity of PAE-PEG-Tf vector.②.The results of flow cytometry and fluorescence microscopy showed that PAE-PEG-Tf/TMZ@siEGFR released more TMZ into U87 cells responsively than PAE-PEG/TMZ@siEGFR.③.The colony formation experiment results showed that compared with the PBS,TMZ,TMZ@siEGFR and PLGA-PEG-Tf/TMZ@siEGFR groups,the clonal formation rate of PAE-PEG-Tf/TMZ@siEGFR group decreased significantly.④.The results of qRT-PCR and Western blot showed that PAE-PEG-Tf/TMZ@siEGFR had the effect of down-regulating the expression of the target gene EGFR;⑤.The results of flow cytometry and live and dead cell staining confirmed that PAE-PEG-Tf/TMZ@siEGFR promoted cell apoptosis.(3)Animal study:①.The orthotopic nude mouse model of U87 glioma cells stably transduced with luciferase was successfully constructed.②.Brain targeting test results showed that,compared with TMZ and siEGFR,PAE-PEGTf/TMZ@siEGFR was more effectively targeted to the inside of brain tumor tissue;TMZ,siEGFR and PAE-PEG-Tf/TMZ@siEGFR were mostly distributed in liver and kidney tissues.③.Statistical results of fluorescence intensity of orthotopic tumor model showed that compared to PBS group,TMZ group,TMZ@siEGFR group,PAE-PEG/TMZ@siEGFR group and PLGA-PEG-Tf/TMZ@siEGFR group,the growth rate of glioma in PAE-PEGTf/TMZ@siEGFR group was the slowest with the survival time of tumor-bearing nude mice the longest.And the difference was statistically significant(P<0.01).④.The PAE-PEG-Tf/TMZ@siEGFR group nanocarriers were not observed to cause damage to the main organs in HE tissue sections,indicating that the PAE-PEG-Tf/TMZ@siEGFR group had low toxicity and good biocompatibility.⑤.TUNEL staining of tumor tissue sections in each experimental group showed that the PAE-PEG-Tf/TMZ@siEGFR group had the highest cell apoptosis rate.Conclusion:We successfully designed and prepared a novel nanocarrier PAE-PEG-Tf/TMZ@siEGFR with pH response characteristics that can simultaneously down-regulate the EGFR gene and target-deliver temozolomide,which can effectively adsorb the siEGFR gene,and encapsulate TMZ to achieve synergy between genes and chemotherapy inhibition of the proliferation of glioma. |
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