Pullulan-based Nano-drug Carrier For Ultrasound Controlled Drug Release

The conventional nano-drug delivery systems were difficult to control the rapid drug release in the lesions area,resulting in the unsatisfactory therapeutic effect and limiting their clinical application as a potential drug delivery system.Therefore,the nano-drug delivery system that can precisely control drug release in the spatiotemporal level by various stimulus response has been a hot research topic in recent years.Amongst various stimuli,the ultrasound-sensitive nano-drug delivery system can respond to the external ultrasonic stimulus and undergo structural change,leading to the rapid drug release in the tumor site with a spatiotemporal manner,thereby improving the anti-tumor effect.In this paper,a new type of delivery carrier for ultrasound controlled drug release was synthesized with pullulan as the backbone,and the properties of the drug-loaded carrier were studied.1.In this study,we for the first time grafted hydrophobic stearic acid onto pullulan backbone using O-alkylhydroxylamine,an ultrasound-sensitive bond,as a linker to form an amphiphilic molecule named P-OC.The chemical structure of P-OC was successfully confirmed by ¹H NMR、HRMS and FTIR.It was observed that P-OC copolymer can self-assemble into nanomicelles easily in the distilled water by dialysis.TEM and DLS demonstrated that P-OC nanomicelles was uniformly distributed in spherical shape with a mean particle size of 202.43±3.37 nm and zeta potential of-17.00±0.14 mV.The P-OC molecules was fractured to produce 1-hydroxyl-2,2,6,6-tetramethylpiperidine stearate after ultrasound irradiation.The hemolysis analysis and MTT assay demonstrated that the blank P-OC nanomicelles showed excellent blood compatibility and cell compatibility respectively.2.Doxorubicin（DOX）,as a hydrophobic anticancer drug,was successfully loaded into P-OC nanomicelles by dialysis method.The optimal encapsulation efficiency（65.65±0.02%）and drug loading content（7.46±0.02%）were achieved at 1:10 drug/carrier ratio（w/w）.The cumulative release of DOX up to 73.66±1.31%from P-OC/DOX nanomicelles was significantly accelerated with 30 min ultrasound irradiation（1.0 MHz,9.9 W）,while the DOX release was only 37.71±1.78%under without ultrasound irradiation condition.The excellent ultrasound controlling DOX release behavior of P-OC/DOX in vitro was conducive to achieving accurate spatial and temporal DOX release at the tumor site and improving the effectiveness of cancer therapy.Further,the results of CLSM and FCM indicated that the P-OC/DOX nanomicelles could be effectively internalized by MCF-7 cells and the release of DOX was quickly triggered by the external ultrasound stimulus.MTT assay demonstrated that the P-OC/DOX nanomicelles showed higher cytotoxicity against both MCF-7 and HepG2cells under external ultrasound irradiation than that of P-OC/DOX nanomicelles without ultrasound irradiation,and the half inhibitory concentration IC₅₀ were 0.98μg/mL and 1.73μg/m L,respectively,which were less than that of free DOX.3.The antitumor efficacy of 4T1 xenograft BALB/c mice displayed that the P-OC/DOX nanomicelles exhibited high therapeutic efficacy with ultrasound irradiation,the mean tumor weight inhibition rate was 80.98%significantly higher than that of the P-OC/DOX nanomicelles（43.28%）without ultrasound.To some extent,P-OC/DOX nanomicelles can reduce the side effects of DOX on body and gain idealistic therapy effects.The above results demonstrated that P-OC/DOX nanomicelles exhibits excellent ultrasound controlling DOX release behavior both in vitro and in vivo and is potential for widely clinical tumor treatment application as a smart drug delivery system.