Preparation Of Vincristine Sulfate Apoferritin Nanoparticles And Treatment On Brain Gliomas

Objective In order to provide a theoretical basis for nano-administration preparations on treating brain gliomas,our study was aimed to prepare and characterize vincristine sulfate apoferritin nanoparticles,and investigated the ability of the drug-loaded apoferritin nanoparticles to cross the blood-brain barrier in vitro and in vivo.Furthermore,we researched its targeting and anti-tumor effects on brain glioma in vitro and in vivo.Methods First of all,we prepared vincristine sulfate apoferritin nanoparticles by pH gradient method.HPLC was used to measure encapsulation efficiency of vincristine sulfate apoferritin nanoparticles.Secondly,L9(3~3)Orthogonal test was used to optimize the best preparation process on the basis of single-factor investigation,with the encapsulation efficiency as an evaluation index.And then,we observed morphology of vincristine sulfate apoferritin nanoparticles by transmission electron microscopy.The particle size and zeta potential of apoferritin nanoparticles were measured by laser particle size analyzer.Dialysis method was used to investigate the release of nanoparticles in pH7.4 and pH5.0 in vitro,and the dissolution curve fitting equation was calculated.The cellular uptake in vitro and tumor-penetrating experiments of apoferritin nanoparticles were observed by laser confocal microscopy later,using C6 cells as an model in vitro.Four kinds of endocytosis inhibitors were used to treat C6cells and the mechanism of internalization of C6 cells was investigated by laser confocal microscope and flow cytometry.The blood-brain barrier cell model in vitro was established by bEnd.3/C6 co-culture in order to investigate cross-BBB status of apoferritin nanoparticles in vitro.At the same time,mammalian zebrafish was used as a cross-body blood-brain barrier model,and we observed the movement of apoferritin nanoparticles in real time by laser confocal microscopy.MTT assay and microplate reader were used to examine the anti-proliferation ability of apoferritin nanoparticles on C6 cells in vitro.In addition,C6 cells were used to establish the orthotopic mice brain glioma model,regarding cy5.5 as a fluorescent probe to label apoferritin nanoparticles,and then we observed the distribution of cy5.5-APO by a small animal imager in vivo.After administration,we investigated the change of mice weight,inhibition rate and survival curve.Finally,the size of brain tumors in tumor-bearing mice measured by NMR and the pathological sections were used to detect the therapeutic effect of apoferritin nanoparticles on brain gliomas.Results Vincristine sulfate apoferritin nanoparticles were prepared by pH gradient method,the best preparation process as follows:the stirring speed was 350 rpm,the stirring time was 60min and the pH value during polymerization was 7.4.Three batches samples prepared according to the best preparation process had an encapsulation efficiency of(39.49±0.36)%and a drug loading of(1.45±0.28)%.The appearance of vincristine sulfate apoferritin nanoparticles was milky white.The morphology of apoferritin nanoparticles was spherical.The particle size of apoferritin nanoparticles was(26.41±0.42)nm and their zeta potential was(-20.30±0.81)mV.The stability of apoferritin nanoparticles was good within 7 days.And then,the cumulative release amount of apoferritin nanoparticles was(16.50±1.18)%and(68.90±1.76)%at 36 h under pH7.4 and 5.0 conditions in vitro,which was in line with the Weibull model’s release pattern(R=0.9660,R=0.9759).Cell experiments in vitro demonstrated that apoferritin nanoparticles can be taken up by C6 cells and bEnd.3 cells,and penetrate C6tumor spheres to the center of the sphere.The uptake mechanism of C6 cells with four endocytosis inhibitors was via caveolin-mediated endocytosis,accompanied by giant pinocytosis,and the entire process was energy-dependent.Both the blood-brain barrier model in vitro and the zebrafish experiment confirmed that apoferritin nanoparticles can effectively cross the blood-brain barrier.Vinblastine sulfate apoferritin nanoparticles had significant proliferation inhibitory effects on C6 tumor cells.The brain glioma situ model of mice was successfully established with C6 cells.After intravenous injection of cy5.5-APO,a strong fluorescence signal was detected in the brain at 2 h,and signal intensity was still present at 4 h.Compared with the free cy5.5 group,cy5.5-APO was found in the brain with a strong accumulation capacity.Anti-tumor in vitro results showed that vinblastine sulfate apoferritin nanoparticles can significantly prolong the survival of mice,whose median survival time was31 days.Compared with the control group,the weight in the nanoparticles group was significantly lower,and the tumor inhibition rate to(89.72±3.69)%.Nuclear magnetic and pathological sections also showed that apoferritin nanoparticles can reduce the volume of brain gliomas in mice and observe less tumor cells.Conclusion The diameter of fervidin sulfate apoferritin nanoparticles prepared by pH gradient method is according with design requirements.The morphology of apoferritin nanoparticles is round.The encapsulation efficiency and drug loading of apoferritin nanoparticles are favorable.The stability of apoferritin nanoparticles is good,and it is beneficial to the release of drugs in an acidic environment.The apoferritin nanoparticles can be taken in by C6 cells and bEnd.3 cells and penetrate C6 cell tumor spheres.At the same time,they can successfully cross the blood-brain barrier and has a brain targeting ability in vivo.In conclusion,fervidin sulfate apoferritin nanoparticles has a significant therapeutic effect on mice brain glioma,which provides a theoretical basis for treating brain glioma.