Preparation Of Kaempferol Liposome And Its Anti-Tumor Research

Kaempferol,as a common natural active ingredient in nature,has a wide range of pharmacological effects and has the value of preparation development and clinical application,and has certain prospects in the treatment of various diseases.In the treatment of tumors,anti-cancer mechanisms such as kaempferol induce apoptosis and block cell cycle have been widely reported.However,due to the poor water solubility of kaempferol and low bioavailability in vivo,it has seriously affected the development and application of kaempferol.In the treatment of cancer,kaempferol can induce apoptosis,block the cycle and other anti-cancer mechanisms have been widely reported.The use of new nano-drug carriers for targeted delivery of kaempferol to cancer sites can improve the anti-cancer efficacy of kaempferol.As a nano-medicine carrier with good biocompatibility,liposomes can be used to load kaempferol and improve its water solubility and stability to solve the problem of solubility.In addition,the amphiphilic targeting polymer DSPE-PEG₂₀₀₀-cRGD is used to modify the kaempferol liposomes,which is achieved by the specific binding between the targeting ligand cRGD and the integrins overexpressed on the surface of cancer cells Targeting cancer cells can improve the anti-tumor cancer efficacy of kaempferol and kaempferol.So as to solve the problem of low bioavailability in the body.Therefore,in this project,the kaempferol kaempferol-loaded tumor cRGD targeting liposome was prepared,and the morphology,particle size,drug loading,encapsulation rate,and drug release performance of the liposome were investigated.Corresponding characterization of stability and safety,and a preliminary discussion on its anti-ovarian cancer activity and mechanism.The main contents are as follows:1.Establish a method for the detection and analysis of kaempferol and kaempferol.It was also investigated that the solubility of kaempferol in PBS solution could not reach the detection standard,and it was a poorly soluble substance.However,the solubility of kaempferol and kaempferol in PBS with2%Tween-80 increased significantly to 121.72μg/m L.Drug release media for subsequent in vitro drug release.In addition,the targeted polymer DSPE-PEG₂₀₀₀-cRGD was prepared by addition reaction,and its structure can be verified and characterized by proton nuclear magnetic resonance spectroscopy and infrared spectroscopy.Finally,it was verified by fluorescence microscopy and flow cytometry.Finally,the ratio of the targeted polymer was screened by fluorescence microscopy and flow cytometry,and it was found that the best ratio of DSPE-PEG2000-cRGD to phospholipid was 1∶31.Taking particle size,dispersion coefficient and encapsulation efficiency as indicators,through single factor investigation,it is obtained that the molar ratio of phospholipid,cholesterol,and drug is 120∶7.5∶6 as the optimal prescription ratio.2.Kaempferol kaempferol liposomes（LP@KAE）and targeted kaempferol kaempferol liposomes（cRGD-LP@KAE）were prepared by thin film hydration method combined with high pressure homogenization technology.Carry out macroscopic and microscopic morphology observations.When observed with the naked eye,it can be seen that the two liposome solutions are translucent light greenish blue opalescent solutions.When the solution is irradiated with a laser pointer,the Tyndall effect can be observed in the direction perpendicular to the light.Observed by electron microscope,both of the two liposomes have a spherical structure with uniform particle size distribution.And the particle diameters of LP@KAE and cRGD-LP@KAE liposomes in aqueous solution are 163.73±3.12 nm and 148.60±0.20 nm,respectively,and their PDI are0.058±0.016 and 0.096±0.011,respectively,and their zeta potentials are respectively-38.07±0.38 m V,-21.97±0.15 m V,drug loading was 1.32%,1.12%,and encapsulation efficiency was 98.88±0.15%,98.97±0.27%.The in vitro drug release behaviors of both free KAE and two liposomal preparations and free KAE were investigated respectively,and it was found that LP@KAE and cRGD-LP@KAE have obvious sustained-release behaviors,and the release curves are different.After fitting the mathematical model,it was found that both the free KAE and the two liposome preparations conformed to the Weibull equation,and their drug release behaviors were significantly different.Using particle size and encapsulation efficiency as indicators to investigate the stability of liposomes,it was found that cRGD-LP@KAE exhibited good stability after 15 days storage at 4 ^oC,while the encapsulation efficiency of LP@KAE decreased..In order to improve the stability of liposomes during the freeze-drying process and after rehydration,we used the particle size,PDI and encapsulation efficiency of liposomes after reconstitution as indicators,and examined sucrose,trehalose and mannitol.The effect of dry protectant.The results showed that compared with trehalose and mannitol,the lyoprotectant is an important factor to ensure the reconstitution effect of liposomal drugs during long-term storage.The particle size,PDI,and PDI of the lyophilized product of cRGD-LP@KAE are used for reconstitution.The encapsulation rate is used as an indicator,and it is found that sucrose has a better freeze-drying protection effect than trehalose and mannitol,and the effect is best when its dosage is 4times the mass of phospholipids.Finally,the hemolysis rate was used as a criterion to evaluate the biological safety of nano-drug blank liposomes and drug-loaded liposomes,and it was found that the hemolysis of LP@KAE and cRGD-LP@KAE blank ordinary liposomes and targeted liposomes All have a hemolysis rate lower than 2%,The hemolysis rate of the corresponding blank drug-loaded liposomes LP@KAE and cRGD-LP@KAE without KAE are both lower than 2%.It also has a low hemolysis rate,which proves that the four liposomes in this article have good blood compatibility and are safe nano-medicine carriers or preparations.3.Using ovarian cancer A2780 cells as an experimental model,the in vitro anti-tumor efficacy of free KAE,LP@KAE and cRGD-LP@KAE was investigated by MTT method.Detected by MTT method,the median inhibitory concentration（IC50）of KAE,LP@KAE and cRGD-LP@KAE on A2780 cells were 24.55μg/m L,18.93μg/m L and 11.95μg/m L,respectively,which proved that the kaempferol lipid can have better anti-tumor efficacy of improving kaempferol,and cRGD-LP@KAE with targeted liposome preparation polymer has the best efficacy.Using coumarin 6 as a fluorescent indicator,fluorescence microscopy and flow cytometry were used to detect the encapsulation in liposomes,and the effects of ovarian cancer A2780 cells on common liposomes LP@C6 and targeted liposomes within 2 h were investigated.The uptake of cRGD-LP@C6.The results show that cRGD-LP@C6 enters the A2780 cell cRGD-LP@C6 group with higher fluorescence intensity,indicating that its uptake is much stronger.In order to verify that the uptake behavior is indeed caused by cRGD,the A2780 cells were pretreated with cRGD solution to saturate first,and then cRGD-LP@C6 was added.It was found that the cell uptake was greatly reduced,which proved that cRGD and the over-expressed integrin on the cell membrane surface.The specific recognition between A2780cells is the main reason for increasing the uptake of cRGD-LP@C6 by A2780cells.cRGD can significantly improve the targeting of liposomes.Finally,flow cytometry was used to investigate the effects of free KAE,LP@KAE and cRGD-LP@KAE on cell apoptosis and cell cycle.The apoptosis and cell cycle of ovarian cancer A2780 cells were detected,and it was found through flow cytometry that after treatment with free KAE drugs,LP@KAE and cRGD-LP@KAE,the total apoptotic rate of the cells was 10.41%,31.39%,and 61.17%respectively,indicating that the targeted preparation has the strongest effect on inducing cell apoptosis.In cell cycle testing,it was found that kaempferol can significantly induce G2/M phase arrest.The G2/M phase ratios of the three types of cells were 26.44%,30.44%,and 40.46%,respectively.The result table proves that cRGD-LP@KAE is in Apoptosis induction and cell cycle arrest have the best significant effect.The cRGD-LP@KAE liposome prepared in this paper can significantly improve the water solubility of kaempferol and kaempferol,and has good biological safety,stability and biological safety.Compared with free kaempferol and LP@KAE liposomes,the liposomes can increase the uptake of kaempferol by ovarian cancer cells,and significantly improve the kaempferol and have significant anti-tumor efficacy.It is a kaempferol mountain.The development of new naphthol preparations provides experimental basis and new ideas.