Ultrasound-responsive Polymer Micelles Targeted Delivery Of Curcumin To Inhibit Breast Cancer Proliferation

Targeting drug delivery remains a challenge in various disease treatment including cancer.Based on the target enrichment of lesions,achieving space-time controlled release of drug to achieve precise treatment has important innovative significance and application value.Here we develop pluronic P123/F127 polymeric micelles(M)encapsulating curcumin(Cur)that are permeabilized directly by focused ultrasound,in which ultrasound triggers drug release.Tumor preferential accumulation and site-specific sonochemotherapy were then evaluated.Cur-loaded P123/F127 mixed micelles(Cur-M)exhibited longer circulating time and increased cellular uptake compared to free Cur.With the assistance of focused ultrasound treatment,Cur-M showed tumor-targeting deposition in a time-dependent manner following systemic administration.This was due to enhanced permeabilization of tumor regions and increased penetration of Cur-M in irradiated tumor cells by ultrasound sonoporation.Furthermore,Cur-M self-assembly could be regulated by ultrasound irradiation.In vitro Cur release from mixed micelles was greatly dependent on ultrasound intensity but not on duration,suggesting the cavitational threshold was necessary to initiate subsequent sonochemotherapy.In vivo site-specific drug release was demonstrated in dual-tumor models,which showed spatial-temporal release of entrapped drugs following intratumoral injection.The sonoporation-assisted site specific chemotherapy significantly inhibited tumor growth.In conclusion,the established ultrasound-guided nanomedicine targeting deposit and local release may represent a new strategy to improve chemotherapy efficiency.In this parper,we mainly study from the design and synthesis of mixed micelles,ultrasound-controlled drug release,in vitro cytotoxicity,sonoporation-guided drug accumulation and release in tumor sites,in vivo antitumor effects and so on.From the following aspects:Part 1 Preparation and Characterization of Cur-MThe pluronic mixed micelles was chosen as a drug loading platform for the antitumor drug Cur,which was encapsulated into the hydrophobic core.Cur-M was prepared by thin-film hydration methods.The particle size and surface charge of Cur-M in an aqueous medium were measured by DLS,transmission electron microscop was used to observe the micelles morphology,the encapsulation efficiency of Cur within Cur-micelles was determined through a spectrophotometric method.Meanwhile,in order to evaluate the stability of Cur-M in blood circulation,we used saline containing fetal bovine serum(FBS)to simulate the blood environment.The results showed that the preparation procedure of Cur-M by thin-film hydration methods was simple and the results were stable and reliable.The morphology of Cur-M was characterized by TEM confirming their nearly spherical shape with nice monodispersity,DLS indicates the average particle size of Cur-M was approximately 23 ± 1.06 nm,which are favorable for passive targeting in tumor via the enhanced permeability and retention effect(EPR).In this system,the loading efficiency of Cur was 4.56%,and the encapsulation efficiency was 86.67%.No significant change in the particle size and zeta potential could be observed within 48 h,suggesting Cur-M stability.Furthermore,the result also showed that the micelles displayed excellent colloidal stability after 24 h storage in PBS buffer containing 10%,20%,or 40%FBS at 37 ? in vitro,indirectly supporting the good stability of Cur-M in blood circulation.All of the results showed than Cur-M could effectively overcome the Cur clinical application of low bioavaiability and other issues.Part 2 Ultrasound Triggered Drug Release from MicellesFirst,we estimated the ultrasonic cavitation using TA(terephthalic acid)dosimetry.Subsequently,most fluorescent molecules loaded in micelles lead to high local concentrations with fluorescence quenching,then we examined the fluorescence changes under different conditions.Drug release studies:the same amount of Cur-M were divided into two groups(ultrasound group and the control group),the control group is not with irradiation,and the ultrasound frequency of 1.90 MHz,the load power(LP)indicated as 1(Usl),2(Us2),and 3 W(Us3)of ultrasound group.The drug release profiles of Cur-M upon dialysis against PBS were determined to evaluate the influence of Us on Cur-M.Results showed:The fluorescence intensity of Cur in Cur-M was much lower than free Cur,indicating the aggregation state after being encapsulated in micelles;However,the fluorescence intensity of Cur in Cur-M was significantly increased after Us3 exposure,suggesting that micelles were disrupted by Us irradiation and the cargos could be freely released.The measurement of hydroxyl radicals using TA method indirectly demonstrates cavitational events,which indeed occurred under the applied Us intensities.A gradual increase in hydroxyl radicals by Us1,Us2,and Us3 treatment was found.Therefore,the pronounced Cur leakage in the present study was mainly attributed to Us-induced cavitation.Ultrasound triggered drug release results suggested that the Us intensity was a determinant factor in initiating cargo release form P123/F123-mixed micelles,which may be related to Us-induced cavitation.Part 3 In Vitro Cellular Uptake and CytotoxicityWe chose human breast cancer cell line MDA-MB-231as the model.In order to evaluate its cellular uptake performance,intracellular Cur content was determined in MDA-MB-231 cells by flow cytometry and confocal laser scanning microscope,SEM was used to observe the cells damage after 24 h treatment.Cell viability was measured by regular MTT assay at 24 h and cell apoptosis was determined through Annexin V-FITC/PI double staining with flow cytometry.Results showed:(1)Intracellular Cur content was determined in MDA-MB-231 cells by flow cytometry.Cur-M displayed more intracellular uptake than free Cur,and Us treatment(0.4 W/cm2)further enhancedcellular internalization(p<0.01),Confocal images show much brighter fluorescent signal in Cur-M plus Us treated MDA-MB-231 cells.(2)Scanning microscopy demonstrated that well-adherent cells in control group displayed typical fusiform morphology.Notably,the cell morphology in Cur-M+Us group were seriously damaged,showing cytoskeletal collapse.(3)To determine whether the increased Cur uptake caused serious cytotoxicity,MTT assay and apoptotic analysis-were performed.Cell cytotoxicity revealed does-dependent tendencies against both free Cur and Cur-M.Free Cur caused no obvious decrease in MDA-MB-231 cells viability at the selected dose range.The cell viability loss caused by Cur-M was approximately 10.60%at the concentration of 10 ?M and the presence of Us(0.4 W/cm2)increased the cell viability loss to 59.57%(p<0.01),FITC/PI double staining with flow cytometry,and the result demonstrated that Cur-M plus Us irradiation obviously induced apoptosis of MDA-MB-231 cells.Part 4 Sonoporation-Guided Drug Accumulation and Release in Tumor SiteFirstly,to assess whether the micelles have a long circulation lifetime was carried out using a 4T1 xenograft mice.Cur and Cur-M were intravenously injected,at differert time after injection,the mice were euthanized,and tissues were excised,followed by homogenization and extraction with methanol.Subsequently,build dual-xenograft model as autologous control experiment.To investigate the biodistribution of micelles in vivo and whether Us could promote drug accumulation in tumor tissue,a dye,DiR was used to replace Cur due to its strong red fluorescence in the NIR region.Finally,build dual-xenograft model as autologous control experiment,the mice with dual tumors were intratumorally injected with DiR-micelles to investigate whether Us could triggered drug release of micelles in vivo.Results showed:(1)Cur-M exhibited significantly prolonged blood retention time over a span of 48 h compared to free Cur.More than 18.42%of Cur-M was found in blood vessels after 48 h injection,whereas the free Cur was almost eliminated from the blood 12 h after injection,meanwhile,Cur-M exhibited significant retention time in the major organs compared to free Cur.(2)Drug accumulation results show,after 5 min post injection,the right tumor was treated with exposure system(Us2,LP = 2 W,1.90 MHz,5 min).The NIR fluorescence signal in left tumor site of xenograft mice after injection of DiR-loaded micelles increased gradually and reached a maximum at 24 h.By comparison,the DiR signal in right tumor site reached a maximum at 12 h after intravenous injection followed by immediate Us2 treatment.Furthermore,the fluorescent signals were far higher than the self-left contrast.(3)drug release and.penetration results show:The mice with dual tumors were intratumorally injected with DiR-micelles,then the left tumor was treated with ultrasonic exposure system(Us3,LP = 3 W,1.90 MHz,3 min),the fluorescent signals were much stronger than without Us(p<0.01),which indirectly suggests that Us could trigger drug release in the tumor mass.Then,tumor tissues were collected and continuously frozen sectioned and Cur fluorescence was observed by a Stereo Fluorescence Microscope.Interestingly,green fluorescence of Cur diffused throughout the tumor section after Us treatment,while fluorescence only gathered brightly in a small region in the untreated group,suggesting that Us can promote intratumoral diffusion and penetration of Cur.Part 5 In Vivo Antitumor Effects4T1 tumor-burdened was established to evaluate following research.Respectively from the tumor volume,weight in mice,main viscera observation,chip morphology observation and immunohistochemical aspects discusses the Us on 4T1 transplantation tumor growth inhibition effect.The experimental results showed that:The Cur-M synergistic with twice Us significantly inhibited tumor growth in tumor-bearing mice(The first Us:Us2(LP = 2 W)guided tumor targeting accumulation and Us3(LP =3 W)further triggered efficient Cur release from Cur-M).Different treatment had no obvious effect on body weight in mice and main organs,which prompted the processing pattern is relatively safe and reliable.ConclusionsNovel Cur-loaded polymeric micelles with Us-responsive properties were successfully developed to conduct chemotherapy in cancer.The present study confirmed that the mixed micelles possess superior anticancer activity through sonoporation-assisted chemotherapy.Sonoporation induced by Us could significantly increase intracellular Cur accumulation.Both in vivo and in vitro studies confirmed that Us-triggered drug release from Cur-M was intensity-dependent.Furthermore,Us could guide the targeting accumulation and deep penetration of mixed micelles in situ.Localized Us irradiation following systemic injection of Cur-M effectively suppressed tumor with no adverse effect in xenograft mice.Therefore,the potential therapeutic effects of this platform will facilitate its future clinical applications.