MRI-visible Harmine Loaded Hyaluronic Acid Polymer Micelles And Its Antitumor Activity In Vitro

Objective:To prepare pH-sensitive amphiphilic polymer micelles imagingprobe loaded with harmine and to investigate its properties and tumor activities in vitro.Method:Enzyme-sensitive amphiphilic polymer micelles were prepared.Firstly,the carboxyl group of hyaluronic acid and the amino group of 3,5 bis?trifluoromethyl?benzylamine were conjugated to obtain a polymer of hyaluronic acid and 3,5 bis?trifluoromethyl?benzylamine.The characteristics of this polymer were proved by infrared spectroscopy,¹H-NMR and ¹⁹F-NMR.The NMR method was used to characterize the HA-F structure and its substitution rate.And then blank micelles and harmine-loaded micelles were prepared by dialysis method,and the particle sizes,zeta potential,morphology,release rate and enzyme responsiveness of micelles were characterized.Two cell models of human breast cancer MADA-MB231 and MCF-7 were established to investigate the cytotoxicity,cell uptake,apoptosis of micelles In vivo imaging of animals were investigated using balc/c mice.Results:The results of NMR and IR spectroscopy showed that HA-F was successfully synthesised.The average particle sizes measured by dynamic light scattering particle size analyzer were?344.4?25.46?nm,the zeta potential was?-23.5?1.47?mv,PDI was?0.283±0.15?for blank micelle.The optimal ratio of HA?:F was 10?10,the best ratio of HM?HA-F was 10?15,the drug loading efficiency was?2.78±0.81?%,and the average particle sizes of drug loaded micelles were?255.1±27.8?nm?zeta potential was?-23.84±0.56?mv?PDI was?0.271±0.06?.The cumulative release amount of the drug in the drug-loaded micelles in the enzyme-treated pH 5.0 PBS solution was significantly higher than that of micelles enzyme added pH 5.0 PBS solution,which indicated that the micelles had enzyme responsiveness.Cell uptake experiments showed that compared with human breast cancer cell MCF-7,Harminemicelles were uptakenmore by human breast cancer MDA-MB231 which overexpressed CD44 receptor,and Lysosome colocalization;After treatment with blank micelles plus hyaluronidase for 3 h,the F signal became strong and the signal at the control group was almost zero,which verified the enzyme responsiveness;Blank micelles in each concentration were almost non-toxic at 24 h?48 h and 72 h to MCF-7 and MDA-MB231 cells.MCF-7 were incubated with 20?g·mL^-11 drug-loaded micelles for48 h,the cell viability was about 50%.After 72 h,the cell viability decreased to 25%,which showed the sustained release of the drug-loaded micelles.IC50 of MCF-7 and MDA-MB231 was 20?g·mL ¹ and 10?g·mL^-1at 24 h respectively.Obviously,HM inhibited MDA-MB-231 more strongly.Before injection of HA-F polymer micelles,there was no obvious signal at the tumor site;at 4 h after injection of HA-F polymer micelles,the imaging signal at the tumor site was the strongest,and at 8 h,the nanoparticles were metabolized and showed dispersed-signal image.Conclusion:HA-F polymer can self-assemble into micelles in water and entrap harmine successfully.The formed micelles are pH and enzyme sensitive,HA-F has certain encapsulation efficiency and drug loading efficiency.In vivo small animal imaging results show that HA-F polymer micelles can target to tumor sites and can act as medical probe.Themicells can be used as a novel carrier for entrapping hydrophobic drugs to achieve slow release of drug,and played a role in the integration of treatment and diagnosis.