Synthesis Of The PH-sensitive Nanoformulated Triptolide And The Treatment Research Of Hepatocellular Carcinoma

PurposeAs one of the most common malignancies,the global mortality rate of liver cancer is next only to lung cancer,ranking second in the world.With the increasing incidence of liver cancer year by year,it is reported that there are approximately 782,000 new cases of liver cancer patients in the world each year.China has a large number of chronic hepatitis B patients and hepatitis B virus carriers.More than 50%of liver cancer patients are in China,and the incidence of liver cancer ranks first in the world.At present,the therapeutic model of liver cancer has developed into a multidisciplinary comprehensive treatment of tumors.The establishment of the multidisciplinary comprehensive treatment team contributes to achieving optimal individualized comprehensive treatment of liver cancer patients.Nano-medicine technology,an emerging technology,provides new ideas for the treatment of diseases.Due to its unique advantages,nanocarriers have become a hotspot in nanomedicine research.Triptolide(TP)is a diterpenoid triepoxide with a broad-spectrum anti-tumor effect,which has a strong lethality on lung cancer,breast cancer,colon cancer,and gastric cancer.However,severe systemic organ toxicities limit its clinical application.This topic combines nanotechnology with triptolide drugs to construct a stable nano-drug carrier,targeted at hepatoma tissue.Evaluate the biosecurity of the newly mixed nano-carrier drugs and the validity of the treatment of liver cancer via simplification and optimization of synthesis methods,researches of sensitivity experiments on cells and targeted therapy on animal liver cancer models,providing a new alternative for individualized comprehensive treatment of liver cancer in the future.MethodsThis paper could mainly divide into three sections.At the first section,the method,utilizing Octadecylamine,β-benzyl-L-aspartate(BLA)and 1-(3-aminopropyl)imidazole(API)to form Octadecylamine-p(API-ASP)10,a high molecular polymer,via a series of chemical reactions,is characterized.Taking use of the ionization of API in different PH conditions,the Octadecylamine-p(API-ASP)10 acquires the capacity of PH sensitivity.Finally,the micelle structured nanogel drug which is highly loaded with triptolide is synthetized by Pluronic F127,Triptolide and Octadecylamine-p(API-ASP)10 via the film dispersive hydration method and its stability as well as the PH sensitivity is characterized.At the second section,a series experiments are conducted to verify the high efficiency of liver cancer cells lethality of nanoformulated triptolide(Nf-Trip).Firstly,the capacity of several different chemotherapeutic drugs in killing liver cancer cells is evaluated and triptolide is found to be a highly effective chemotherapeutic agent compared to other chemotherapeutic drugs.To further explore the mechanism of killing cancer cells of Nf-trip,the free triptolide is taken into comparison.And a series of experiments such as CCK-8,flow cytometry,cell cycle,caspase-3 and Western-Blot are investigated.Finally,the PH sensitivity of Nf-trip is verified.At the third section,a Hepatocellular Carcinoma Orthotopic model is established to take the nanoformulated triptolide(Nf-Trip)and conventional chemotherapeutics(Oxaliplatin)into comparison.Taking advantage of in vivo bioluminescence imaging system,evaluate the therapeutical effect of nanoformulated triptolide(Nf-Trip)on liver cancer in Orthotopic model nude mice by comparing the fluorescence intensity of the hepatocarcinoma in nude mice.Besides,investigate the temporal distribution of nanoformulated triptolide(Nf-Trip)in nude mice via fluorescence-based Nf-Trip nanocarrier drug and clarify the biosecurity and targeted lethality of Nf-Trip by means of observing weight variation,liver and kidney function indicators and survival status of nude mice in different groups.Finally,a multiple transfer model in vivo of nude mice is conducted to further verify the conclusion.Results1.Successfully synthetize Octadecylamine-p(API-ASP)10,a pH sensitive high molecular polymer which is verified by using 1H-NMR.2.Successfully synthetize the Nf-Trip,a micelle structured nanogel which is composed of hydrophilic segment at the outside and hydrophobic segment inside packing the triptolide.In the SEM observation,the Nf-Trip is at the spherical state of uniform size in the neutral environment when the pH is 7.4.The hydrodynamic radius of the Nf-Trip is measured to be(113.7 ± 15.3)nm and its zeta potential is(-11.45±2.14)mV.The loading efficiency of Nf-trip is measured to be(9.25 ± 0.21)%with a good dispersivity,the concentration of triptolide is about 560ug/ml.When it changes to the acid environment,the pH-sensitiv Nf-trip is observed to be ionize and disperse with the dramatically smaller size.3.The release of Nf-Trip is tested in different pH environments via in vitro dynamic membrane dialysis.It is observed that the release of Nf-Trip is measured to be lower than 10%after 30 days put in the 4℃ refrigerator when the pH is about to be 7.4 which suggests that the Nf-Trip is stable in the neutral environment.And the Nf-trip is observed to release nearly 80%after 36 hours,put in the acid environment when the pH equals 6.0 which suggests the Nf-Trip is disperse in the acid environment4.Comparing to conventional chemotherapeutic drugs,triptolide which has the very low IC50(about 0.005ug/ml)is observed to contain a dramatically stronger ability to inhibit the cancer cell viability and facilitate the apoptosis of cancer cells.Besides,Nf-Trip has limited effect on cancer cells viability when pH is about to be 7.4 while it obviously inhibits the cell cycle and activates the apoptosis via the mitochondria pathway when the pH is about to be 6.0.In addition,the Nf-Trip is measured to be more efficient than the free triptolide to facilitates the apoptosis of the cancer cells at the same concentration.5.Compared with Oxaliplatin,Nf-Trip nano-carriers drug inhibits the growth of liver cancer in Orthotopic model nude mice.The Nf-Trip is observed to accumulate in the liver cancer tissue 6h after the tail vein injection and the H&E stain suggests the obviously necrosis of liver tissue while it shows limited toxicity on the heart,kidney,spleen and testis of nude mice.In addition,the survival rate of the HCC orthotopic nude mice is highly increased by Nf-Trip.ConclusionsA pH sensitive micelle structured nanogel Nf-Trip is synthesized by a relatively simple method,which performs well in preparation technology and reproductivity,efficiently promoting the solubility and bioavailability of triptolide,realizing the positioning release of tumor cells in acid environment,enhancing the targeting of triptolide,meanwhile,reducing the toxicity of this drug.Besides,the ability of Nf-Trip to induces apoptosis in hepatoma cells via mitochondrial pathway is proven in this paper,increasing the clinical application value of triptolide in antitumor ability,providing a new idea for individualized comprehensive treatment of liver cancer patient in thefuture.