| ObjectiveLiver cancer is a type of malignant tumor that threatens human health.At present,several potential chemotherapeutic drugs that can treat tumour have toxic side effects and low specificity,which is the direct reason for limiting their use or dosage.Damage to human cells,such as blood cells and lymphoid tissue cells is the main cause of collapse of the human immune defense system.Therefore,it is a long-term goal and arduous task to achieve early diagnosis,early treatment,effective prevention,and precise treatment of liver cancer.Moreover,it is a long-term research focus for pharmacists to identify an antitumor drug with low toxicity and high efficacy.Tanshinone ?A(Tan ?A),a quinone compound,is a representative of the fat-soluble components of Salvia miltiorrhiza Bge(molecular formula:C19H18O3),which has a potent effect on inducing apoptosis and broad-spectrum anticancer activity.Although tanshinones ?A have little toxic side effects,the insolubility in water and the short half-life of it significantly limits its clinical use.How to extend the action time and improve the targeting of tanshinone ?A with proper methods is the key for its application in anti-hepatoma therapy.Therefore,to improve its bioavailability and physicochemical properties,enhance its anticancer activity,and make its clinical application prospect more promising,it is considered to encapsulate tanshinone ?A by niosomes.In order to encapsulate tanshinone ?A by niosomes to increase the antihepatoma effect,we hypothesize that the tanshinone ?A can be concentrated in the liver and then be fully released in tumor cells,this requires our vesicles to be "smarter",so they can identify the liver site and have a unique ability to "sense" tumor cells.Successful targeted preparations should possess characteristics,including localized concentration,controlled release,low toxicity,and biodegradability.Niosomes have key attributes,such as low toxicity and biodegradability and in order to enhance their ability to localize and control drug release,niosomes are modified with galactose to make them livertargeting.Moreover,pH-sensitive excipients are added to the niosomes to control drug release at the tumor site.MethodsIn this study,targeted niosomes loaded with tanshinone ?A were prepared by ethanol injection and pharmacodynamic studies in vitro and in vivo were carried out.Then the pharmacokinetic characteristics of the preparations were analyzed.Finally,the targeting effect of the preparation was further verified by tissue distribution studies.1.Targeted niosomes loaded with tanshinone ?A were prepared by an ethanol injection method.With the encapsulation efficiency as an index,prescription factors,such as the amount of nonionic surfactant and cholesterol,as well as the technology factors,such as the stirring speed and hydration temperature were investigated.Finally,the optimal formulation and technology were determined by orthogonal experiments.The quality of the prepared vesicles was evaluated,including morphological examination,particle size,and potential examination,encapsulation efficiency,leakage examination,etc.In addition,an in vitro release analysis method was established to verify the pH sensitivity of the vesicles.2.The uptake of targeted niosomes by hepatoma cells was evaluated by inverted fluorescence microscopy and flow cytometry.The in vitro targeting ability of the vesicles was analyzed,and the dosage of galactosylated stearate was determined based on the results of the targeting study.The CCK8 assay was used to determine the survival rate of hepatoma cells,and flow cytometry was performed to detect the apoptosis and cell cycle phase of cells,and the intracellular level of reactive oxygen species(ROS)and mitochondrial membrane potential were determined to further verify the effect of tanshinone?A and its preparation in inducing cell apoptosis.Through the above-mentioned experiments,the in vitro efficacy of targeted niosomes loaded with tanshinone?A was fully evaluated.3.An Akt/c-Met-induced HCC mouse model was established by means of hydrodynamic transfection to evaluate the in vivo anti liver cancer effect of the preparation,and additional studies were performed to investigate its underlying mechanisms of anti-tumor activity.The drug was administered via tail vein injection,and blood samples were collected from the rat orbital veins,followed by determination and analysis of pharmacokinetic parameters.Furthermore,the distribution of drugs in rat tissues was evaluated,and the targeting effect of drugs in vivo was analyzed.Results1.Targeted niosomes loaded with tanshinone ?A were prepared by the ethanol injection method,according to the optimal formulation and technology.The appearance of niosomes is round and smooth,the distribution is uniform,the average particle size is 53.72 nm,the zeta potential is-28.31 m V,the encapsulation efficiency is 84.70%,and the leakage after 60 days is only 3.43%.The results of the in vitro release test showed that drug-loaded vesicles exhibited significant sustained release characteristics relative to free drugs in alkaline medium.In weakly acidic media,pH-sensitive vesicles released slightly faster compared to ordinary vesicles.Furthermore,in acidic medium,the release rate of pH-sensitive vesicles was much faster compared to that in alkaline medium.In addition,the release process was closer to that of free drugs,and the release rate was significantly greater than that of ordinary vesicles.2.The analysis results showed that A2780 cells and HCT8 cells treated with 10%Gal-pH-NR-NSVs showed fluorescence characteristics that were not very intense.In contrast,hepatoma cells treated the same,showed intense fluorescence characteristics.Fluorescence intensity of cells was enhanced with the increase of the dosage of galactosylated stearate and was higher compared to that of cells treated with free Nile red.Taking the IC50(?g/m L)as an indicator of anti-tumor activity,the anti-tumor activity of different tanshinone ?A preparations in Hep G2 and Huh7 cell was as follows: Gal-pH-Tan?ANSVs(1.481,1.539)> Gal-Tan?A-NSVs(2.185,2.252)? Free Tan?A(2.256,2.505)> pH-Tan?A-NSVs(3.042,3.627)> Tan?A-NSVs(4.419,5.108).The results of intracellular ROS levels and the mitochondrial membrane potential,as well as the flow cytometry results,further proved that galactose-modified pH-sensitive niosomes loaded with tanshinone ?A showed the strongest effect in inducing apoptosis.The results of the in vivo pharmacodynamics study showed that galactose-modified pH-sensitive niosomes loaded with tanshinone ?A inhibited the growth of liver tumors to the greatest extent.The most obvious improvement was observed in the tumor lesion area,which depended on the inhibition of the phosphorylation of pathway-related proteins as well as the expression of FASN,thereby blocking the signaling of related pathways and inhibiting tumor development.3.Pharmacokinetic test results showed that tanshinone ?A encapsulated by niosomes had a larger area under the curve(AUC,Free Tan?A:8.81,Tan?ANSVs:28.23,Gal-pH-Tan?A-NSVs:25.03,?g/m L·h),a longer mean residence time(MRT,Free Tan?A:3.53,Tan?A-NSVs:18.56,Gal-pH-Tan?ANSVs:14.64,h)and half-life(T1/2,Free Tan?A:3.09,Tan?A-NSVs:11.66,GalpH-Tan?A-NSVs:8.43,h),and theoretically a better systemic drug effect compared with free drugs.Although the Gal-pH-Tan ?A-NSVs group had a longer MRT and lower clearance rate(CL)than the free Tan ?A group,the volume of distribution(Vd)was smaller.Determination of the tanshinone ?A content per unit mass in the,heart,liver,spleen,lung and kidney showed that the drug elimination of free tanshinone ?A was faster in vivo,and tanshinone ?A encapsulated by niosomes accumulated significantly more in the liver compared to free tanshinone ?A.Moreover,compared with common vesicles,galactose-modified targeted vesicles showed a significant tendency to accumulate in the liver.ConclusionIn this study,galactose-modified pH-sensitive niosomes loaded with tanshinone ?A were successfully prepared.These vesicles have an encapsulation efficiency around 85% and a low leakage rate,and the particle size is around 50 nm.Compared with free drugs and plain vesicles,which are characterized by liver targeting and rapid drug release at the tumor site,they exhibited excellent anti-hepatoma activity in vitro and in vivo.In vivo,and compared with the free drug,this preparation exhibited a larger area under the curve,and longer mean residence time and half-life,which effectively prolonged the retention of tanshinone ?A in the liver and significantly increased the distribution of tanshinone ?A in liver tissue. |
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