Study On Antitumor Activity Of Ruthenium Complex-Loaded Liposomes And A Composited Drug Delivery System Combining Scaffolds With Liposomes

In recent years,metal complexes represented by cisplatin have played a prominent role in clinical medication,but their adverse effects have also drew wide concern.Therefore,ruthenium complexes have gradually exhibited great potential in cancer treatment due to its high efficiency as well as low toxicity and have also been made major breakthroughs in the research.In our study,a novel ruthenium complex[Ru?ttbpy?₂?PIP?]?ClO₄?₂?Ru?was designed and synthesized.The chemical structure of the complex was confirmed by mass spectrometry,¹H-NMR and ¹³C-NMR.The UV-visible absorption spectrum and luminescence emission properties of Ru were characterize by a UV-Vis spectrophotometer and a fluorescence spectrophotometer,respectively.There was two absorption peak of the ruthenium complex at 286 nm and 461 nm respectively and a property of emitting red fluorescence.Additonally,its poor solubility in water was proved by the solubility test.In addition,the long-circulating liposomes were developed to cope with the poor solubility,improve the anti-tumor effect and increase the commercial value of the ruthenium complex.Formulations and preparation technologies of the liposomes were investigated by single factor test.Ru-loaded long-circulating liposomes?Ru-Lip?were prepared by thin-film method.The optimal liposomes were obtained while the mass ratio of phospholipid to Ru was 30:1,the ratio of phospholipid to cholesterol was 3:1 and the dosage of phospholipid was 30 mg.The encapsulation efficiency was?86.3±1.0?%,the particle size was 234.9±10.1 nm,the PDI was 0.269±0.046,and the zeta potential was-14.67±0.46 mV.In the anti-tumor activity study,it was found that the half inhibitory concentration(IC₅₀)of Ru-Lip on HeLa cervical cancer cells was significantly lower than that of Ru,which was 5.3?M and 10.8?M,respectively.In addition,apoptosis assays showed that Ru-Lip could induce apoptosis in HeLa cells.Further investigation of its apoptotic mechanism revealed that Ru-Lip could significantly raise the level of reactive oxygen species?ROS?in HeLa cells from 5.5 to 12.3,increase the cellular uptake of Ru.Further,the Ru acted on mitochondria,and then reduced mitochondrial membrane potential?the intensity ratio of green light to red light rose from 1.6 to 3.3?.Intracellular Ca²⁺assay showed that Ru-Lip could destroy the balance of Ca²⁺in HeLa cells in a concentration-dependent manner.When the Ru-Lip concentration increased to 10.8?M,the fluorescence intensity of Fluo-3 soared from13.2 to 134.5.The results of cytochrome C test were similar to those of Ca²⁺test.As the concentration of Ru-Lip increased from 5.4?M to 10.8?M,intracellular cytochrome C increased markedly?fluorescence intensity increased from 1.6 to 4.8 and 55.5,respectively?.Western blotting showed that Ru-Lip induced the expression of Bax and Caspase-3 protein with an increase of 20%and 11%respectively in grayscale.In conclusion,the Ru-Lip constructed in this study can induce apoptosis of HeLa cells through mitochondrial pathway,remarkably enhancing in vitro anti-cervical cancer effect of the Ru.In view of the strong metastasis and recurrence of osteosarcoma,a composite Ru-Lip-scaffolds drug delivery system using scaffolds to load Ru-Lip was also designed in this study.A relatively uniform and interconnected pore structure of the blank scaffolds and Ru-Lip-scaffolds was ovserved by scanning electron microscopy.As shown in the compression test,the mechanical properties of the Ru-Lip-scaffolds were better than those of blank scaffolds.The Young's modulus of scaffold-1,Ru-Lip-scaffold-1,scaffold-2 and Ru-Lip-scaffold-2 were 59.6,83.0,61.5 and 81.1 KPa,respectively,and all of them were able to withstand the stress during the recovery period of implantation by surgery.A desirable sustained release effect of the composite Ru-Lip-scaffolds drug delivery system was shown in the in vitro release test.Inhibition of MG-63 osteosarcoma cells treated with the Ru-Lip-scaffolds for 24 h was also demonstrated.The IC₅₀ of the two Ru-Lip-scaffolds were 32.04±0.69 and 27.70±0.39 respectively.In addition,AO/EB double staining test also revealed that it had a certain apoptotic effect on MG-63 cells.The detection of reactive oxygen species,mitochondrial membrane potential and cytochrome C suggested that the composite Ru-Lip-scaffolds drug delivery system could increase intracellular reactive oxygen species,decrease mitochondrial membrane potential and induce the leakage of cytochrome C.That preliminarily revealed the composite Ru-Lip-scaffolds drug delivery system also causes apoptosis by inducing mitochondrial dysfunction.