Study On Glycyrrhetic Acid Modified Ph-sensitive Curcumin Mixed Micelle For Anti-hepatoma Cell

Object:Primary liver cancer was a malignant tumor that seriously threatened the health of our people.Current treatment strategies included surgery,chemotherapy,radiation therapy,and interventional therapy.Among them,chemotherapy had certain effects on early,middle and advanced liver cancer,and it was often used with other treatment methods,which was a common strategy for treating liver cancer.However,due to the problems of non-specific drug delivery,toxicity and side effects,chemotherapy did not obtain desireable effects.Therefore,seeking a safe and effective treatment for liver cancer was a key issue which needed to be solved urgently.According to the results of ancient literature researchs and modern pharmacological researchs,the curcumin in rhizoma curcumae longae,a blood-activating and blood-stasis medicine,was considered to be a safe and effective broad-spectrum antitumor drug.Curcumin had shown good antitumor efficacy in both in vivo and in vitro experiments.The pharmacological effects of CUR on tumor cells include inhibiting proliferation,inducing apoptosis,inhibiting metastasis and invasion,and reversing multidrug resistance.In addition,CUR could protect liver cells,and significantly reduce the levels of ALT and AST in the serum of mice,and reduce the degree of acute liver cell necrosis in mice.However,the clinical application of curcumin was limited by its own physical and chemical properties,such as poor water solubility,low bioavailability,and poor light stability.With the development of nanotechnology,these problems had been improved in a certain extent.Curcumin-loaded micelles had certain advantages in improving antitumor efficacy and passively targeting tumor tissues by the EPR effect.However,most of the inchoate micelles were not functional,so there were some inadequacies such as active targeting,intelligent drug release,and drug loading rates.Therefore,a Pluronic F6 8-based glycyrrhetic acid modified and pH sensitive mixed micelles was constructed and evaluated by synthesizing the polymer materials,confirming the structure of polymer materials,researching the thermodynamic properties of polymer materials,preparing the mixed micelles,formulation evaluation,and in vitro efficacy evaluation.In order to reducing the critical micelle concentration of F68,increasing its drug loading and stability,achieving the active targeted delivery of curcumin to hepatocellular carcinoma cells and rapid release of drugs in response to the weakly acidic environment within the cells.So as to further reduce the toxicity of curcumin to normal liver cells and enhances its antitumor effects.And try to use the second law of thermodynamics to explain the effect of hydrophobic group modification and formulation temperature on the micellization.Method:1 FAP,FBP,FAC,FBC,and FGA materials were synthesized by esterification reaction and electrophilic addition reaction,and their chemical structures were verified by 1H-NMR and Fourier infrared spectroscopy(FTIR)Determin if the materials were successful synthesized.2 First,the feasibility of the CUR content detection method was verified with the 2015 Chinese Pharmacopoeia Part Ⅳ 9101.Second,the method was used to measure the saturation concentration of CUR in water and n-octanol,then calculate the logP value of its ratio,which was the oil-water partition coefficient of CUR.Third,the 1H-NMR spectra of a series of surfactants with different hydrophilic-lipophilic balance(HLB)values were detected and processed to obtain a linear function of the HLB value and the hydrophilic group coefficient(R)of different surfactants.And the R value of F68 and its derivatives were used to substitute into the linear function to calculate the HLB value of F68 and its derivatives.Last,the encapsulation efficiency(EE)and drug loading(DL)of CUR drug-loaded micelles prepared with different materials were measured by HPLC,and the relationship between HLB value with EE and DL was analyzed3 The single-factor experiment was used to investigate the optimal preparation conditions for mixed micelles.Dynamic light scattering(DLS)was employed to measure the particle size,potential,and multi-distribution coefficients of the micelles HPLC was used to detect the EE and DL of the micelles.These data were analyzed and contrasted to obtain the best process conditions4 Isothermal titration calorimetry(ITC)was used to measure the heat change during the micellization of different materials,and compared the changes in entropy,enthalpy,and Gibbs free energy during the micellization to evaluate the difficult degree of micellization.It was analyzed that the effects of PCL and CLR modification on the thermodynamic function of F68,and the most suitable polymer materials were screened for the micelles preparation experiments.At the same time,comparing the micellization entropy,enthalpy,and Gibbs free energy at different temperatures to estimate the effect of temperature on micellization.And analyze the influence of three variables △H,△S and T on micellization in the formula of △G=△H-T·△S.5 The performance of mixed micelles was evaluated through a series of experiments.Dynamic light scattering(DLS)was used to investigate the particle size,potential,pH sensitivity,and stability of micelles;Transmission electron microscopy(TEM)was used to observe the appearance of micelles;dialysis experiments were used to investigate accumulative drug release curve;The hemolysis of the micelles solution were detected by microplate reader;the critical micelle concentration(CMC)of the polymer was measured by pyrene fluorescence probe method;Tyndall phenomenon was employed to evalutate the anti-dilution ability of the micelles.6 In vitro pharmacodynamic experiments were used to evaluate the antitumor efficacy of CUR drug-loaded micelles at the cellular level.To investigate the cytotoxicity of FAP/CUR,FBP/CUR,and MIX/CUR micelles on hepatocellular carcinoma cells and normal liver cells,MTT experiments had been employed;flow cytometry was used to compare pro-apoptotic status of free curcumin,FAP/CUR,FBP/CUR,and MIX/CUR micelles on liver cancer cells;The high-content cell imaging system was used to observe the nucleus morphology which treated with different micelles,and compare the cellular uptake capacity of liver cancer cells on different micelles;the fluorescence was used to detect the levels of ROS in liver cancer cells which was treatment with different micelles,the above experiments were used to evaluate the anti-cancer effects of mixed micelles in vitro.Result:1 Confirmed by H-NMR and FTIR,FAP,FBP,FAC,FBC,and FGA were successfully synthesized.2 It was good that the results of methodological experiment of the CUR content detection method,and the CUR content in the preparation could be accurately detected.The log P of CUR was 3.77,and it was belong to a lipophilic drug.The linear function of HLB value and hydrophilic group coefficient(R)of different surfactants was HLB=18.947*R+2.210,and r=0.9987 shown a good linear relationship.The HLB value of F68 and its derivatives decreased as the molecular weight of the hydrophobic segment increased.The experimental results show that under the same preparation conditions,the change of EE and DL were closely knitted with the HLB value of the materials.Within the six kinds of materials involved in the experiment,as the HLB value decreased,the EE and DL of the micelles gradually increased.Among them,FAP and FBP micelles had the highest EE and DL.3 The best conditions for preparing FAP/CUR micelles which obtained by single factor experimental were as follows:FAP 10mg,CUR 1mg,put them into a 250ml eggplant-shaped bottles,add 5ml acetone to dissolve them by ultrasound.evaporate the acetone at 60℃,-0.1Mpa,100rpm,then add 10ml PBS(pH=7.4)at 60℃,spin and hydrate at 60℃,atmospheric pressure,100rpm for 30min,cool the micelles solution to room temperature and made up to 10ml.After passing through a 0.22μm filter,store it at 4℃ before test.After experimental verification,this condition could be used for preparing FBP/CUR micelles solution with good reproducibility.The optimal ratio of FAP/FGA for MIX/CUR micelles preparation was 1:1,and other parameters were the same as the conditions of FAP/CUR micelles.4 ITC results showed that as the molecular weight of the hydrophobic segment increases,the enthalpy and entropy of polymer materials to form micelles were increased,and the Gibbs free energy decreases.According to the second law of thermodynamics,the increasing of molecular weight of hydrophobic segments made it easier that the material to self-assemble into micelles,and might have better thermodynamic stability.At the same time,the ITC results of MIX micelles at different temperatures showed that as the temperature increases,the negative value of the micellization free energy was lower,indicating that the mixture of FAP and FGA was more likely to self-assemble at higher temperatures to form micelles.In the experimental temperature range,△H was a positive value.This phenomenon proved that the micellization in water by a mixture of FAP and FGA was an endothermic process.Because △H and △S of MIX micelles increased together before 30℃ and decreased together after 30℃.For the three variables △H,△S,and T in the △G formula,the partial derivatives were obtained.The negative change of △G was mainly affected by △S and T,while the negative change of △H had no positive effect.Therefore,the increase of entropy played an important role in driving MIX micellization.5 Preparing a mixed micellar solution by a thin film hydration method,the particle size measured by DLS was 91.06±1.37nm and the potential was-9.79±0.47mV.The CMC of mixed micelles which prepared by FAP and FGA was 1.702mg/L.It was far less than F68.The EE and DL of drug-loaded micelles were 75.13±1.36%and 6.31±0.09%,respectively,which were determined by HPLC.The mixed micelles were stored for 15 days under storage conditions and placed in a medium containing 10%FBS for 24h,then the particle size of the micelles did not change significantly,indicating that the stability of the mixed micelles was good.In addition,the mixed micelles showed significant pH sensitivity.After incubation 6h in a medium with pH=5.0,the diameter distribution diagram changed from a narrow single peak to a widened double peaks or multiple peaks,which indicated that micelles was broke,aggregated,and the particle size was changed.In vitro accumulative drug release experiments showed that compared to a medium with pH=7.4,a medium with pH=5.0 enables more drugs to be released.The results of hemolysis experiments indicated that the polymer materials which constituted mixed micelles did not have remarkable hemolytic and have good biological safety.6 MTT experiments showed that compared with FAP/CUR micelles,FBP/CUR micelles,and free CUR,MIX/CUR micelles had more toxic to liver cancer cells,but less toxic to normal liver cells than free CUR.At the same time,under the same conditions,blank micelles have no significant cytotoxic effect on liver cancer cells and normal liver cells.Flow cytometry results showed that after 24 hours of treatment,there were significant differences in the pro-apoptotic effects of free CUR,FAP/CUR,FBP/CUR,and MIX/CUR micelles on hepatocellular carcinoma cells.The apoptosis rate from low to high as following:free CUR<FBP/CUR<FAP/CUR<MIX/CUR,indicating that pH-sensitive and GA-mediated active targeting could improve the anti-tumor effect of CUR.By comparing the high-content cell imager results of CUR-loaded micelles and free CUR,it can be seen that in hepatocellular carcinoma cells,the number of nuclear deformation and shrinkage of the CUR drug-loaded micelle group was higher,indicated that micelles could improve the pro-apoptotic effect of CUR.In the ROS level test,the order of fluorescence intensity in the cell as following:free CUR<FBP/CUR<FAP/CUR<MIX/CUR.The results of flow cytometry cell uptake experiments showed that the fluorescence intensity of the NR drug-loaded micelle group in hepatoma cells was higher than that of the free NR group.The experimental results which observed by high-content cell imager also showed that the intracellular red fluorescence intensity of MIX/NR with active targeting function was the highest.Conclusion:Through confirming the chemical structure of polymer materials,evaluating and characterizing the micellar formulations,and evaluating the in vitro effect of the micellar formulations,F68 based glycyrrhetic acid modified and pH-sensitive mixed micelles were successfully established.It was used to improve the water solubility of curcumin,specifically delivered curcumin to liver cancer cells,and responsed the acidic environment for rapid drug release.It could enhance the curative effect of curcumin on liver cancer cells and reduces the toxic and side effects on normal liver cells.And all so provide experimental basis for development and application of CUR nano preparation.In addition,the research around the thermodynamic properties and functions of F68 and its homologues,such as HLB value,CMC,△H,△S,and △G,which preliminarily explained that increasing entropy plays an key role in driving MIX micellization.