Thermosensitive And Passive Targeted Nanomicelles Loading Shikonin: Synthesis And Anti-Tumor Effect On Breast Cancer

ObjectiveIncidence of breast cancer has become highest in the women with cancer.Breast cancer alone is expected to account for 29% of all new cancers in women.Breast cancer is the most common cause of cancer death in women aged 20-59.Thus,breast cancer has seriously affect women’s health.Shikonin is the main active ingredient of boraginaceae,which is one of traditional Chinese medicine.In recent years,the anti-tumor effect and mechanism of Shikonin has been confirmed by a large number of studies.The experiments in vitro have confirmed that shikonin has strong anti-cancer effect in a time and dose dependent manner.However,shikonin is difficult to dissolve in water.It has been reported that mice after intravenous injection of 3H-labeled shikonin,widely distributed content in this order: bile,liver > lung,spleen,kidney,heart,skin> testis,muscle,brain.This suggested that shikonin may also have some side effects on normal tissue.To alleviate the toxicity of shikonin on normal tissue and enhance its breast tissue targeting,one temperature responsive block copolymers micelles(BCMs)have been synthesized without changing the structure.The kernels of BCMs have been adjusted,thus enhancing shikonin drug loading by interacting with the kernel.Meanwhile,the tumor microenvironment with higher the temperature,the internal thermosensitive BCMs enhance endocytosis achieve thermosensitive passive targeting,which is different from the condition of normal tissue.Finally,the biocompatibility and anti-tumor effect of shikonin thermosensitive nanomicelles on breast cancer in vivo has been evaluated.MethodsFirstly,thermosensitive BCMs(PID118-b-PLA39)was synthesized a temperature.Its volume phase transition temperature(VPTT)was controlled at about 40 ℃,which is beneficial for thermosensitive passive targeting function.Dialyzed against water by self-assembling,adjusting morphology PID118-b-PLA39,preparation thermosensitive nanomicelles.The use of PLA micelle hydrophobic core shikonin soluble form can load shikonin thermosensitive targeting nanomicelles.In addition,the dynamic/static light scattering instrument(DLS)and transmission electron microscopy(TEM)on the properties and structure of the thermosensitive nanomicelles prepared was measured.In the experiments in vitro,the distribution of particle size measurement by combining the first thermosensitive nanomicelles and BSA after the measured serum stability.Use inverted fluorescence microscope and laser scanning confocal microscopy degree thermosensitive nanomicelles swallow and enrichment within the MCF cells.The CCK-8 experimental evaluation of thermosensitive nanomicelles toxicity,and cell killing effect after loading shikonin.The flow cytometry wrapped shikonin thermosensitive nanomicelles apoptosis in breast cancer MCF-7 cells.Confocal microscopy thermosensitive nanomicelles cause autophagic cell MCF varied by laser.In vivo,the nude mice model bearing breast cancer was constructed and observed by a small animal in vivo imaging instrument imaging system to evaluate the in vivo distribution of thermosensitive nanomicelles in tumor-bearing mice(BALB/C mice).Construction of further breast tumor-bearing mice were observed after the thermosensitive nanomicelles,nude body weight,different size and survival time of intravenous injection of tumor.Groups are blow: Control group(PBS),free shikonin group,thermosensitive nanomicelles group,and thermosensitive nanomicelles group(heating in vitro).ResultsWithout changing the structure of the thermosensitive nanomicelles,we have successfully synthesized thermosensitive nanomicelles.In addition,the stability tests in vitro also show that the thermosensitive nanomicelles have good stability in serum.Inverted fluorescence microscope and confocal microscopy results show that in the case of confocal passive temperature control of targeted guidance,in the case of thermosensitive nanomicelles 40 ℃,the annexation of breast cancer cells MCF-7 enrichment significantly stronger than the 37 ℃ groups and free shikonin group.The results of CCK-8 experiments show that the thermosensitive nanomicelles toxicity is very low.However,thermosensitive nanomicelles loading shikonin is able to kill breast cancer MCF-7 cells effectively.Thermosensitive nanomicelles loading shikonin show more efficient in 40 ℃ group than 37 ℃ group.Flow cytometry results show that the thermosensitive parcels shikonin nanomicelles in the case of 40 ℃,can induce apoptosis in breast cancer MCF cells effectively.Studies also show that autophagy,wrapped shikonin thermosensitive Nanomicelles in the case of 40 ℃ of autophagy significantly more than the control group.Distribution of the results found in the body,thermosensitive nanomicelles in nude mice intravenously into the 24 h,can be observed in solid tumors in mice to a very significant nanomicelles aggregation.Antitumor experiments show that the thermosensitive nanomicelles treatment,breast cancer growth of solid tumors has been significantly reduced(PBS = 3859 mm3,FS = 2372 mm3,STN(37 ℃)= 2278 mm3,STN(40 ℃)= 1235 mm3;STN(40 ℃)vs PBS: p < 0.001,STN(40 ℃)vs FS: p < 0.001,STN(40 ℃)vs STN(37 ℃): p < 0.05).ConclusionThe higher thermosensitive and passive targeted nanomicelles loading shikonin can penetrate the vessel wall and enriched in breast cancer tissue specifically.It can kill the breast cancer cells significantly by inhibiting cell proliferation and inducing apoptosis and autophagy.