Gold Nanorods Modified Celastrol And Sodium Tanshinone ⅡA Sulfonate Co-transfer Temperature Sensitive Lipid Complex For Synergistic Anti-breast Cancer Treatment

Celastrol is a natural component of five-ring triterpenoids with various biological activities.Althrough its strong and wide anti-tumor activities against different types of cancers,the pratical application remains limition by severe toxicities.Liposomes as drug carriers have many advantages,such as reducing drug toxicity,low immunogenicity,and good biocompatibility;however,it seems to be trapped in a bottleneck of anticancer efficacy.The main cause of such issue is associated with the complexity of the tumor microenvironment.In order to solve this problem,the thermo-sensitive liposomal system was employed as the vehicle and the gold nanorod was used as a thermal trigger in this study.Such drug delivery system encapsulates“sodium tanshinone ⅡA sulfonate”as an improver for tumor microenvironmen and“celastrol”acting as an anti-tumor component,that were located in liposome’s hydrophilic cavity and lipid bilayer,respectively.The photothermal transformation effect of gold nanorods was induced by the 808 nm external laser,leading to the increase in local temperature which influences the bilayer stability of thermo-sensitive liposomes.According to our design,such liposomal system is capable of releasing hydrophilic sodium tanshinone ⅡA sulfonate and hydrophobic celastrol squentially under radiation of 808nm laser in a manner of optimized collaborative treatment,and thereby of significant improving the anti-tumor effect.The main research contents of this study are as follows:1.Preparation,characterization and cytotoxicity evaluation of GNRGNR were reportedly synthiesized by a seed growth method.We optimized the preparation by adjusting the concentration of Ag NO₃.When the concentration of Ag NO₃ is 0.023 mol/L,the length and width of prepared GNR are are 37.74±0.06 nm and 1.59±0.07 nm,respectively with the zeta potential of 30.02±4.21 m V and the LSPR at 911 nm.After irradiated by 808 nm near-infrared laser,the temperature of GNR rised remarkably and was proportional to its concentration and the irradiation time.MTT assay was used to investigate the toxicity of GNR against MCF-7 cells.The results indicate that GNR can significantly inhibit the proliferation of MCF-7 cells.The cell inhibition was enhanced after 808 nm near-infrared laser irradiation,and the IC₅₀values were 14.73±0.56μg/m L and 7.93±0.30μg/m L,respectively.2.Preparation and characterization of gold nanorods modified celastrol and sodium tanshinone ⅡA sulfonate temperature sensitive codelivery lipid complex systemThe best method to prepare the gold nanoroads modified temperature sensitive liposomes was selected by stirring,hydration and film forming.Finally,the film forming method was used.G-G-TL and G-C/T-TL were successful prepared by film dispersion method,with the particle size of 146.91±1.14 nm and 111.52±1.19 nm,narrow PDI and the zeta potential of 23.31±0.52 m V、-33.53±0.42 m V,respectively.In vitro drug release of G-C-TL and G-C/T-TL showed that tanshinone ⅡA sodium sulfonate release rate is significantly faster than celastrol.The 12-hour cumulative release amount reaches more than 95%without thermo-specific difference.But the release rate of celastrol at 42℃was significantly higher than the rate at 37℃.The cumulative release amount of celastrol in G-C-TL and G-C/T-TL was 82.75±1.71%and 78.91±5.25%at 42℃,and was 51.52±7.97%and 51.62±3.59%at 37℃.3.Cellular studies of celastrol and sodium tanshinone ⅡA sulfonate temperature sensitive codelivery lipid complex systemThe localization of C-TL,C/T-TL,G-C-TL and G-C/T-TL in the cells was examined by laser confocal microscopy.It was found that apart from lysosomes,mitochondria also can internalize temperature sensitive liposomes.The toxicity of TL and GNR-TL on MCF-7 cells was investigated via MTT assay.The IC₅₀ value of TL and GNR-TL on MCF-7 cells were 13802.00±0.32μg/m L and 84.79±0.11μg/m L,and the IC₅₀value of GNR-TL is 51.26±0.12μg/m L after irradiated by 808 nm NIR laser.In addition,the effect of Cel,C+T,C-L,C-TL,C/T-TL,G-C-TL and G-C/T-TL on the proliferation of MCF-7 cells was also examined by MTT assay.The results showed that each celastrol preparation group can inhibit the proliferation of MCF-7 cells and it was positively correlated with the concentration of celastrol.G-C-TL and G-C/T-TL were more potent in inhibiting cell proliferation after irradiated by 808 nm near-infrared laser,significantly stronger than the free drug-treated group.The IC₅₀ value of Cel,G-C-TL and G-C/T-TL were 3.97±0.03μM,5.11±0.08μM and 4.03±0.18μM.After irradiated by 808 nm near-infrared laser,the IC₅₀ value of G-C-TL and G-C/T-TL were2.37±0.11μM and 1.53±0.09μM.To investigate the uptake of MCF-7 cells in each celastrol preparation group,we measured the protein content of the cells by BCA kit and the content of intracellular drugs by HPLC.The results showed that intracellular cel was only 0.14±0.01μg/mg,which was even a little lower than C+T.However,t the uptake of nano preparation on MCF-7 cells was significantly increased.Compared with Cel group,the intake of C-L,C-T,C/T-TL,G-C-TL and G-C/T-TL increased by2.0times,2.5 times,3 times,0.6 times and 0.7 times,respectively.The apoptosis of MCF-7 cells at different time was induced by Cel,C+T,C-L,C-T,C/T-TL,G-C-TL and G-C/T-TL with the concentration of celastrol at 5μM.The results showed that the apoptosis rate increased with the prolongation of incubation time.4.Antitumor effect and mechanism evaluationThe biodistribution of codelivery lipid complex system was investigated with the help of in vivo near-infraded imaging system using Di D as a fluoresecence probe.The results showed that Di D/TL and GNR/Di D/TL significantly accumulated in the tumor site compared with free Di D.After 24 h of injection,the tumor site of GNR/Di D/TL-treated mice exhibited the overwhelming fluorescence among all the groups.The results of antitumor efficacy showed that treatments with various celastrol-related preparations could inhibit tumor growth,especially G-C/T-TL group.The tumor inhibition rate of G-C/T-TL increased by 1.35 times in comparision with that of G+C+T.Notably,all tumors of mice were eradicated under radiation with 808 nm NIR laser.The immunohistochemistry and immunofluorescence staining showed that G-C/T-TL can promote tumor cell apoptosis,reduce collagen content in tumor cells,decrease vascular density and the number of tumor-associated fibroblasts.Besides,compared with the saline-treated group,the treatment of G-C/T-TLcould decrease the levels of CCL2,IL-6,IL-10 and TGF-βand elevate theconcentration of IL-12.5.Evaluation of systemic safetyThe blood of the nude mice was measured by a fully automatic blood analyzer,and the liver function indexes such as aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,renal function indicators such as uric acid（UA）,blood urea nitrogen（BUN）and creatinine（CREA）were measured by blood biochemical analyzer.The results showed that none of the treatments obviously changed the concentration of ALT,AST,UA,BUN,CREA compared with the saline group.The blood routine indexes of each treatment group were normally.The results of HE staining showed that the heart,liver,spleen,lung and kidney of each preparation group had no obvious abnormal.In a word,the preparation was safely.