Study Of Metabolism And Tumor-suppression Effects Of Drug-loadable Mesoporous Bioactive Glass

Objectives:The development of nano-drug carriers with biosafety and the function of tumor suppression is one of the most notable core problems in the nano biomedical field nowadays.However,although the existing gold nanoparticles and carbon nanomaterials can inhibit tumor growth by photothermal effects of themselves,they still have the deficiencies of not easy to degrade,hard to excrete from the body and toxic reaction.To avoid these problems and develop nano-drug carriers with biosafety and the function of tumor suppression,this study focused on mesoporous bioactive glass（MBG）nanosphere and regarded radioisotope labeling tracer technique as a cutting point.The study also revealed the metabolic rules of MBG nanosphere and system evaluated the effects on body physiological function and main organs.The effects and mechanisms of MBG nanosphere on tumor suppressionanti were illustrated at the overall,cell and molecular biology levels.The drug delivery system of drug-loadable MBG nanosphere wasdeveloped to estimate the pharmacokinetics and the treatment effects of the tumor in vivo.The present study would eventually provide important and scientific basis on the possibility of MBG nanosphere for clinical use.Materials and Methods:MBG nanosphere with the main component of calcium and silicon was fabricated by sol-gel method combined with hydrothermal reaction.It was characterized by SEM、TEM、EDS and nitrogen adsorption stripping method.Radionuclide ⁴⁵Ca was introduced to establish the tracing method on MBG nanosphere.After MBG nanosphere got into the mice for 30 days,liquid scintillation technique was used to study the distribution,accumulation and excretion pathway of MBG nanosphere in each major organization organs.It was emphasized on the location of MBG nanosphere in the liver cells（the nucleus,cytoplasm and mitochondrial）and its effects on the function of mitochondrial in liver cells.The biosecurity of MBG nanosphere was system evaluated by combining clinical biochemical analysis and histopathology examination.Surface-active agent and alkaline environmen of the synthetic reaction were further adjusted to optimize the radial hole structure of r MBG nanosphere.The mice tumor transplanted model was established and used to reveal the anti-tumor effects by TUNEL fluorescence and mmunohistochemical.The cell model in vitro was also used to illustrate the mechanism of apoptosis pathway mediated by calcium ion through the method of flow cytometry and western blot.Besides,r MBG nanosphere was loaded with doxorubicin hydrochloride,and its pharmacokinetics was analyzed by high performance liquid chroma-mass spectrography（HPLC-MS）.The treatment effects of the tumor and the body toxic effects were also observed.Results:（1）⁴⁵Ca-MBG nanosphere with worm hole like mesoporous structure was successfully labeled and fabricated.The particle size range of the ⁴⁵Ca-MBG nanosphere was from 50 to 100 nm and the purity of the labeled nanosphere was>95%.（2）⁴⁵Ca-MBG nanosphere got into the mice and then distributed to organs such as heart,lung,liver,spleen,kidney and intestine though blood circulation.It was mainly accumulated in liver（maximum can attain 13.37±2.50 ID/g）.A small number of ⁴⁵Ca-MBG nanosphere was uptake by liver cells in the form of exogenous phagosome and located in cytoplasm（about 96%）.Traces of ⁴⁵Ca-MBG nanosphere involved mitochondria,but do not induce oxidative stress response in liver cells.（3）The accumulated amount of ⁴⁵Ca-MBG nanosphere in each organ at30 day significantly decreased to 0.3～2.3%ID/g.And the ⁴⁵Ca-MBG nanosphere distributed in vivo can excrete out of the body follow with faeces（main）and urine.（4）The tail vein of mice was injected with ⁴⁵Ca-MBG nanosphere for continuous 7 days,which did not induce the abnormal of clinical biochemical indicators and the alteration of pathological in main organizations.（5）r MBG nanosphere was successfully fabricated with radial hole structure.The specific surface area is 593.766 m²/g and the mesoporous range is from 2.656 to 10.986 nm.The r MBG nanosphere can release2-4-fold calcium ion in the simulated acidic conditions of the tumor over that of the normal condition.（6）r MBG nanosphere can promote the expressions of Caspase-3 and TUNEL positive cells in tumors in vivo and inhibit the tumor growth.The rate of the inhabitation reaches 63.66±18.75%.（7）r MBG nanosphere with a certain concentration did not induce the toxic and apoptosis response in normal cells.But it can up-regulate the level of calcium in tumor cells via p H response of itself to release calcium ions.It further activates Calcineurin A and inhibits the activity of Bcl-2,eventually induces the high expression of Caspase-3.（8）The drug loading ratio of r MBG nanosphere reaches 43.00±0.11%.At each time point in 72 h,the max of drug concentration of r MBG nanosphere in blood was 9-fold over than that of free drugs,which increased the duration time of drugs in vivo for 2 fold.（9）r MBG nanosphere can effectively cooperated with drugs to exert the tumor suppression effects with the tumor inhibition rate of 76.53±10.70%.It can significantly relive the systemic effect induced by free drugs.Conclusion:（1）The labeling technique of ⁴⁵Ca-MBG nanosphere was successfully developed,and the radioactive isotope tracer method was used to objectively reveal its biological metabolism pathway in vitro and in vivo.（2）The distribution and accumulation of ⁴⁵Ca-MBG nanosphere in each major organization was revealed and it was found that the faeces is the main excretion pathway.It proved that ⁴⁵Ca-MBG nanosphere did not induce pathological change and dysfunction in involved tissue,which showed good biosecurity.（3）It was the first to develop r MBG nanosphere with high specific surface area,radial hole structure and the capability of p H response to release calcium.The r MBG nanosphere was proved to have higher loading efficiency.（4）It was found for the first time that r MBG nanosphere had specificity tumor suppression effect.The mechanism might be that nanosphere interfered the calcium homeostasis in tumor cells and activate the calcium signaling downstream apoptotic pathway,which lead to the apoptotic of the tumor cell.（5）It was found for the first time that r MBG nanosphere have three characters such as can increase drug bioavailability,cooperate with durg to treat tumor and relieve the toxic and side effect of chemotherapeutics.It has application advantages in nano-drug carriers.This study provides important scientific evidence for the clinical use of MBG nanosphere.