Nanolization Of Borneol And Its Apoptosis-inducing Ability And Enhancement Effects On Sorafenib’s Anticancer Activity

As a result of the activation of proto-oncogenes in normal cells,normal cells mutate into tumor cellswhich rapidly proliferate,migrate and invade various tissues and organs of human body and thus cause complications.Human health and normal life was seriouslythreatened.At present,a variety of clinical treatments for cancer have been established,such as chemotherapy and radiotherapy,but traditional treatment all have some limitations: Incompletely remove cells and susceptible to damage normal cells.As a result,scientists have developed new treatments for medical,for example,targeted drug therapy and immunotherapy.New anti-cancer therapies are more effective,but costly and MDR.Therefore,we need to find more convenient and faster anti-tumor methods with fewer side effects.As a commonly used adjuvant traditional Chinese medicine,borneol itself has the advantages of antibacterial and anti-inflammation,analgesia,protecting central nervous system,promoting drug passage through barrier,enhancing drug sensitivity and so on.The preparation of drugs into nano-drug loading system can improve the solubility or bioavailability of insoluble drugs,improve the stability of drugs,reduce adverse drug reactions,delay drug release and improve drug targeting.Therefore,in this research,we will focus on nanopaticle of borneol induced tumor apoptosis and anti-tumor activity of sorafenib sensitized by borneol.The main results of studywere as follow:1.Two different type nanopartcle of borneol were synthesized by different methods,respectively:NBNPs-1 was synthesized from RNB,ethanol,PLGA,dichloromethane,Tween-80(TW80)and ultrapure water by emulsifying solvent volatilization.NBNPs-2 was synthesized from RNB,ethanol,olive oil,TW80 and ultrapure water by lipid emulsions.We made a comparation between these two type NBNPs by MTT assay,particle size test and TEM morphology observation forselecting a moresuitable NBNPs for further experiments.The results showed that NBNPs-2 had fairly stable physical properties and good anti-tumor properties which meant NBNPs-2could be used for further research.2.We further compared the intracellular absorption between NBNPs-2 and RNB,and the results showed that the uptake of NBNPs-2 by cells was stronger than that of RNB at the equivalent concentration(for example,7.5 μg/m L NBNPS-2≈40 μg/m L RNB),possibly because NBNPs-2 could more easily change the mobility and permeability of cell membrane,making it easier for NB to penetrate cells.We also analyzed the causes of cell apoptosis through intracellular ROS(reactive oxygen species)content detection,mitochondrial injury experiment and flow cytometry.The final scratch migration assay and transwell invasion assay were performed to determine the ability of NBNPs to block the migration and invasion of tumor cells.Through the mitochondrial fragmentation assay,we found that NBNPS-2 could cause the previously linear fusion mitochondria fragmentation more quickly and thoroughly than NB,leading to the reduction or even destruction of the synergistic effect between mitochondria and the inability to produce enough ATP to maintain the activity of tumor cells,which is one of the reasons for accelerated cell apoptosis.In addition,under the effect of 20μg/m L NBNPS-2,the ROS content of cells increased by 20% on average,and the excessive rise of ROS content induced cell apoptosis.On the other hand,the ROS content of cells treated with NB was no significant change.Flow cytometry showed that the cell content of sub-G1 phase was increased to 12.62% under the action of 30 μg/m L NBNPS-2,and the cell content of sub-G1 phase was further increased to 80% when the concentration of NBNPS-2 was increased to 60μg/m L,which confirmed that NBNPS-2 had stronger apoptosis-inducing ability than NB.The scratch migration experiment also showed that the cell mobility decreased to 59% at 28 μg/m L,which was basically consistent with the effect of 160 μg/m L LNB.When the concentration of NBNPS-2 was increased to 56 μg/m L,the cell migration distance was significantly reduced and the mobility was reduced to only 25%,which verified that NBNPS-2 had a strong anti-migration effect.Finally,transwell invasion experiment proved that the invasion rate of cells almost dropped to zero under the action of n BNPS-2 with a concentration of 56 μg/m L,which also showed that NBNPS-2 had a strong anti-invasion effect.The above experimental results show that NBNPS-2 is a good anti-tumor nanodrug carrier.3.We compared the anti-tumor effects between L-Natural Borneol(LNB)+ Sorafenib(Sora)and D-Natural Borneol(RNB)+ sorafenib(Sora)—MTT assay,cellular uptake between LNB and RNB,measurement of ROS generation,mitochondrial fragmentation analysis,flow cytometric analysis,and Western Blot for analysisingthe regulation effect of LNB+Sora and RNB+Sora on related pathways.The results showed that compared with Sora alone,the cytotoxicity of LNB+Sora was stronger when treated with 2 and 4 μM.But when Sora concentration was 6 and 8 μM,The cytotoxicity of RNB+Sora was stronger.In other words,NB+Sora’s anti-tumor activity mainly depended on the concentration of Sora.Moreover,the increase of NB concentration was avail to improve Sora’s anti-tumor activity.Then,we analysedthe cellular uptakeof LNB and RNB.When the cells were treated with 40 μg/m L NB,the intracellular uptake of RNB was higher than that of cells treated by LNB for 1 h,2 h,4 h and 8 h,respectively.When the cells were treated with 80 μg/m L NB,there’s no significant change.The cellular uptake of LNB by HepG-2 cells exceeded thecellular uptake of RNB after the treatment of 160 μg/m L NB.We could realize that as the concentration of NB increased,the permeability and fluidity of cell membranes would be strengthened.Besides,the strengthening of the permeability and fluidity of cell membranes between same concentration of LNB and RNB treatment was different.In other words,the optical rotation direction of NB would also influence the fluidity and permeability of cell membrane,and ultimately affect the cell uptake of NB.Moreover,measurement of ROS generation and mitochondrial fragmentation analysis also confirmed that NB +Sora could induce cell apoptosis by producing excessive ROS and mitochondrial fragmentation.Flow cytometry analysis showed that Sora could mainlyinduce G0/G1 phase cell cycle arrest,thencell apoptosis.The result of Western Blot also showed that NB+Sora could effectively inhibit the expression of VEGFR2 and Raf kinase and regulate the expression of p-ATM and p-MDM2,thereby activating the p53 pathway to induce cell apoptosis and thereby inhibit cell growth.In a word,natural borneol can not only be prepared as a nanometer drug to make it a good anti-tumor drug,but also can be used in combination with other drugs to realize anti-tumor synergistic sensitization,which reveals the application prospect of natural borneol in anti-tumor application.It provides a new research direction and basis for tumor treatment in the future.