ApoE-targeted And Redox-sensitive Polymersomal Doxorubicin For The Treatment Of Glioma

Glioma is the most common malignancies of the central nervous system.The current principle for glioma treatment is on the basis of surgical treatment,combined with radiotherapy and chemotherapy.The biggest challenge for glioma chemotherapy is how to get the drug cross the blood-brain barrier(BBB)to reach the tumor site.PEG-P(TMC-DTC)has good biocompatibility and reduction responsiveness and is expected to become an efficient drug delivery vehicle.In our study,we found that Apo E,a protein polypeptide,can be better targeted to the LRP1 receptor co-expressed in BBB and glioma cells.So Apo E was selected as a targeting molecule.The current clinical application of gliomas is TMZ.Because GBM tumors resist or do not respond to TMZ,most GBM patients(60-75%)cannot benefit from TMZ therapy,and there was no other drug selection for glioma patients with TMZ-resistant.Based on this clinical problem,our group has successfully constructed a novel vesicle nanoparticle drug delivery system targeting glioma,which has been validated in both in vivo and in vitro experiments.Through the use of a PEG-P(TMC-DTC)diblock polymer as a nanocarrier to encapsulate the chemotherapeutic drug doxorubicin to prepare vesicle nanomedicine,letting DOX crosses the BBB to reach the tumor site and released to achieve the purpose of treating glioma.Part I: Preparation and Characterization of Biodegradable and Redox-sensitive Vesicle NanoparticlesObjective: To use protein-polypeptide Apo E as a targeting molecule and PEG-P(TMC-DTC)as a nanocarrier to encapsulate doxorubicin to prepare stable vesicle nanoparticles with reduced response.Method: First,the Apo E molecule was modified to PEG-P(TMC-DTC)polymer by an addition reaction.After dissolving the Apo E-PEG-P(TMC-DTC)and PEG-P(TMC-DTC)polymers in DMF at a concentration of 10 mg/m L,the two polymers were dropped into the citric acid buffer(p H 4,10 m M)in appropriate proportions.Then placed at 37°C for 1-2 hours.After the vesicle solution had a uniform blue light,the p H of the vesicle solution was adjusted to 7.8 with a saturated Na2HPO4 solution.After approximately 3 seconds,DOX solution was dropped into the the solution and stirred for 5-10 minutes.After standing overnight,self-crosslinked was accomplished and finally dialyzed in PB(p H 7.4,10 m M)for 6-8 hours at room temperature.After preparation of vesicle nanoparticles,the particle size and PDI of the nanoparticles were measured with a DLS instrument,and the entrapment efficiency and drug loading of DOX in the nanoparticles were measured with an ultraviolet spectrophotometer.The DLS instrument was used to determine the stability of the nanoparticles under different conditions(diluted 100-fold,10% FBS,placed for a week).The DLS was also used to detect the reduction response of nanoparticles at 10 m M GSH.Results: The size of the vesicle nanoparticles was about 80 nm,and the different Apo E targeting ratio had no obvious effect on the size and size distribution of the nanoparticles.Dox-loaded has little effect on the particle size and particle size distribution of different Apo E-targeting nanoparticles(Apo E: 0,10,20,30%).The particle size and PDI of Apo E20-PS did not change much under the conditions of 10% FBS,100-fold dilution,200-fold dilution and stay for 1 week,suggesting that our nanoparticles are relatively stable.In the absence of GSH,the particle size and PDI of the nano-particles did not change significantly after shaken in the shaker for 24 h.But after 12 h under the condition of 10 m M GSH,the nanoparticles began to swell and size of nanoparticles increased.The fully swelled particle size significantly increased to more than 1000 nanometers after 24 h,suggesting that the nanoparticles have been responsive.From the DOX release experiments we observed that within 24 hours,70% and 83% drug were released from PS-DOX and Apo E-PS-DOX under reducing conditions,respectively;while only 16% and 21% were released under physiological conditions.Conclusion: A novel brain-targeted vesicle nano-drug Apo E20-PS-DOX was successfully prepared in this study.The nano-drug has good stability and reductive response and DOX can be released efficiently under the reducing environment of GSH.Part II: In Vitro Bioactivity Detection of Biodegradable and Redox-sensitive Vesicle NanoparticlesObjective: To evaluate the in vitro biological activity of the novel polymersomal drug Apo E20-PS-DOX constructed in this study.Evaluation methods include biocompatibility,cytotoxicity,competitive inhibition assay,intracellular uptake quantitative assay,apoptosis assay,and transwell assay.Method: The toxicity of PS and Apo E-PS on U87 and normal astrocytes cells was detected by MTT assay at a polymer concentration of 0.1-0.5 mg/m L to evaluate their biosafety.The effect of Apo E20-PS-DOX,Lipo-DOX and PS-DOX on the apoptosis of U87 cells was detected by Annexin V-FITC/7AAD method,and the related molecular mechanism was explored by WB assay.Transwell assay was used to detect the effect of polymersomal Apo E20-PS-DOX,Lipo-DOX and PS-DOX on invasion of U87 cells.The endocytotic effect of Lipo-DOX,PS-DOX and Apo E-PS-DOX with different Apo E targeted ratios was detected by FACS.The endocytosis and intracellular release of vesicle nanoparticles were detected by CLSM.Results: 1.In U87 cells,the IC50 s of the nanovesicular drugs Apo E10-PS-DOX,Apo E20-PS-DOX,Apo E30-PS-DOX,PS-DOX,and Lipo-DOX was: 1.7,1.0,1.8,3.6,and 10.2 μg/m L,respectively.Apo E20-PS-DOX can cause the most significant cytotoxicity.2.In the nanovesicular drug,the Apo E20-PS-DOX group had the best targeting effect on U87 cells,and its targeting effect was 5 times than that of Lipo-DOX.3.PS-DOX caused about 40% cell apoptosis,Apo E-PS-DOX caused about 80% cell apoptosis,and the pro-apoptotic effect of Apo E20-PS-DOX is most obvious;4.Transwell experiments showed that Apo E20-PS-DOX group had the most significant effect on inhibiting U87 cell invasion,which was stronger than PS-DOX group and Lipo-DOX group.Conclusion: The novel brain-targeted vesicle nanoparticle drug Apo E-PS-DOX has a certain degree of glioma targeting,and when Apo E ratio is 20%,the targeting effect of U87 cells is the best;the vesicle nano-drug can significantly promote the apoptosis of U87 cells and inhibit its invasion.Part III: In Vivo Bioactivity Detection of Biodegradable and Redox-sensitive Vesicle NanoparticlesObjective: To evaluate the in vivo biological activity of the novel polymersomal drug Apo E20-PS-DOX constructed in this project.To detect the biodistribution,blood circulation time and therapeutic effect on glioma model of this vesicle nanoparticle.Methods: 1.Blood circulation experiment: detecting the circulation time of different drug-loaded nanoparticles injected into tumor-bearing nude mice;2.Biodistribution experiment: different drug-loaded nanoparticles injected into tumor-bearing nude mice,distribution of DOX·HCl in organs and tumors were detected;3、Tolerability of drugs: To explore the tolerance of DOX in nude mice;4.To detect changes of intracranial tumors during the course of treatment in living imaging experiments;5、Hematoxylin and eosin staining was used to evaluate the effect of tumor treatment and cardiotoxicity;6.Tunel assay was used to detect the apoptosis in tumor cells.Results: 1.From the blood circulation experiment: we observed that the half-life of Lipo-DOX is about 8.2 hours,and the Apo E20-PS-DOX had a long circulation time with an elimination half life of 3.9 hours,which is longer than that of DOX(0.2 hours);2.From the biodistribution experiment : we observed Apo E20-PS-DOX had a remarkably high DOX·HCl brain tumor accumulation,which was 4.8 times that of the PS-DOX,demonstrating that Apo E has a very good glioma targeting effect;3.On the 12 th day of the treatment,from the in vivo imaging experiments,the size of intracranial tumors,were ranked as follows: PBS group,Lipo-DOX group,PS-DOX group,Apo E20-PS-DOX group,TMZ group;4.The treatment effect of Apo E-PS-DOX group was comparable to TMZ group.The median survival time of nude mice was: TMZ group(51 days),Apo E-PS-DOX group(44 days),no targeted PS-DOX group(35 days),and Lipo-DOX group(28 days),PBS control group(23 days).Conclusion:The new brain-targeted vesicle nanocarriers developed in this project can improve the tolerance of mice to DOX,and the vesicle nanoparticles have a longer blood circulation time;biodistribution experiments show that compared with the PS-DOX group and Lipo group,Apo E-PS-DOX group had a remarkably high DOX·HCl brain tumor accumulation;the new brain-targeted vesicle nanoparticle Apo E20-PS-DOX developed in this study not only has a better therapeutic effect on orthotopic gliomas,but also can reduce the toxic side effects of DOX on the heart.