Study On The Preparation Of DMDD Targeting Liposomes And Its Mechanism In The Treatment Of Breast Cancer

Objective2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione(DMDD)is one of the main active ingredients isolated from Averrhoa carambola L.Previous studies have confirmed that DMDD has played a significant anti-tumor role.However,due to the disadvantages of insolubility in water,low bioavailability and poor tumor targeting ability,its clinical development and application were limited.In this study,DMDD tumor targeting liposomes(HA/TN-DLP)modified by hyaluronic acid(HA)and TAT-NBD peptide(TN)were prepared,and its characterization and targeting in vivo and in vitro were investigated.The effects and underlying mechanisms of HA/TN-DLP against breast cancer were investigated by transcriptomics,metabolomics,apoptosis and signaling pathways in cell and animal experiments.Method1.Formulation and quality evaluation of DMDD common liposomes(DLP)HPLC was used to establish the method for the determination of DMDD content,and the methodology of precision,repeatability and stability were investigated;low-speed centrifugation was used to detect the Encapsulation Efficiency(EE)of DMDD liposome;the EE,particle size,PDI and Zeta potential as the evaluation index to investigate the preparation method of DLP;the single factor test was used to screen the influencing factors of DLP;Box-Behnken response surface design was used to optimize the formulation of DLP and verify the formulation;the particle size,PDI and Zeta potential of DLP were detected by Malvern Zetasizer Nano ZS90;the morphology of DLP was observed;the storage stability,the dilution stability and in vitro medium stability of DLP were investigated.2.Formulation and quality evaluation of DMDD targeting liposomes(HA/TN-DLP)The EE,particle size,PDI and Zeta potential as the evaluation index,the single factor test was used to screen the influencing factors of HA/TN-DLP and optimize the formulation;the particle size,PDI and Zeta potential of HA/TN-DLP were detected by Malvern Zetasizer Nano ZS90;the morphology of HA/TN-DLP was observed;the storage stability,the dilution stability and in vitro medium stability hemolytic of HA/TN-DLP were investigated.3.Formulation and quality evaluation of HA/TN-DLP freeze-dried liposomesHA/TN-DLP freeze-dried liposomes were prepared by freeze-drying method;the appearance,redispersibility,particle size,PDI and EE were used as the evaluation indexes to select the optimal freeze-drying process,type and dosage of freeze-dried protective agent,and the quality of the freeze-drying liposomes was evaluated.4.In vitro and in vivo targeted study of DMDD targeting liposomesIn vitro targeted study,the uptake effect of different targeting liposomes in4T1 cells for qualitative study was detected by a laser scanning confocal microscope;the uptake effect of different targeting liposomes in 4T1 cells for quantitative study was detected by fluorescent spectrophotometer;the expression of CD44 protein on the surface of 4T1 cells was detected by Western blot;in vivo targeted study,the sustained-release effect and targeting effect of DMDD liposomes were detected by UPLC-q Tof-MS.5.In vivo pharmacodynamics and mechanism of DMDD targeting liposomes(HA/TN-DLP)In vivo pharmacodynamic evaluation,4T1 cells were used to establish a mouse breast cancer subcutaneous xenograft model,and the same dosage and different DMDD liposomes were given for treatment to investigate its inhibitory effect on mouse breast cancer;moreover,the potential targets of the liposomes on breast cancer were predicted by transcriptomic analysis and then verified by multiple examinations in breast cancer in mice.6.In vitro pharmacodynamics and mechanism of DMDD targeting liposomes(HA/TN-DLP)In vitro pharmacodynamic evaluation,the toxicity of different DMDD liposomes to 4T1 cells was observed by MTT method,LDH viability assay and cell colony formation assay;the effects of migration and invasion of 4T1 cells were observed by scratch experiments and Transwell experiments;AO-EB fluorescence staining,caspase 3/caspase 9 activity assay and flow cytometry were used to detect the effects of the same dosage and different DMDD liposomes on apoptosis,mitochondrial membrane potential and cell cycle;the effects of the same dosage and different DMDD liposomes on PI3K/Akt and NF-κB signaling pathways were detected by Western blot and RT-PCR.Result1.Formulation and quality evaluation of DMDD common liposomes(DLP)HPLC method for the determination of DMDD was established,the precision,repeatability and stability of the method were accord with testing requirements;15min low-speed centrifugation(3000 rpm)method was finally determined to be the method for the determination of the EE of DMDD liposomes;the ethanol injection way was finally determined to be the method for the preparation of DLP;the single factor test method was used to determine that the factors affecting the shaping process of DLP were ratio of egg yolk lecithin to cholesterol,ratio of egg yolk lecithin to drug and reaction temperature;through the box-Behnken response surface investigation,the optimum preparation process of DLP was determined as follows: ratio of egg yolk lecithin to cholesterol was 7.1:1,ratio of egg yolk lecithin to drug was 7.1:1,and the reaction temperature was 52.7℃;the particle size distribution of DLP was uniform,and PDI,EE and DL were accord with liposome requirements;the results of stability investigation showed that DLP had good storage stability,dilution stability and in vitro medium stability.2.Formulation and quality evaluation of DMDD targeting liposomes(HA/TN-DLP)HA/TN-DLP were prepared via electrostatic attraction;the single factor test method of HA/TN-DLP was used to determine the optimal preparation process;the particle size distribution of HA/TN-DLP was uniform,and PDI,EE and DL were accord with liposome requirements;the results of stability investigation showed that HA/TN-DLP had good storage stability,dilution stability and in vitro medium stability;the hemolysis test showed that HA/TN-DLP had no hemolysis phenomenon and good biocompatibility.3.Formulation and quality evaluation of HA/TN-DLP freeze-dried liposomesHA/TN-DLP freeze-dried liposomes were prepared with 5% mannitol as freeze-dried protective agents,and the particle size,PDI,Zeta potential and EE did not change significantly before and after lyophilization;the stability results showed that HA/TN-DLP was better after freeze-dried,and it could be stored at4℃ for more than 3 months.4.In vitro and in vivo targeted study of DMDD targeting liposomesThe results of targeted study in vitro showed that the uptake intensity of targeted group was higher than that of un-targeted group(P<0.05 or P<0.01).The results of targeted study in vivo showed that HA/TN-DLP had sustained-release and tumor targeting effects.5.In vivo pharmacodynamics and mechanism of DMDD targeting liposomes(HA/TN-DLP)In vivo pharmacodynamic evaluation showed that HA/TN-DLP could significantly inhibit breast cancer and induced cell apoptosis and necrosis(P<0.05 or P<0.01),its effects were more significant than free DMDD and non-targeted DMDD liposomes at the same administered dose;HA/TN-DLP affected the glycerophospholipid metabolism pathway by reducing the biosynthesis of phosphatidylcholine and 1-Acyl-sn-glycero-3-phosphocholine through regulating the expressions of CEPT1 and LYPLA1;HA/TN-DLP significantly decreased the levels of PI3 K,Akt,m TOR,P70S6 K,FAK,TLR4,My D88,NF-κB p65,IκBαand IKKα/β(P<0.05 or P<0.01),thereby regulating the expression of PI3K/Akt and NF-κB signaling pathway to inhibit tumor cell growth.6.In vitro pharmacodynamics and mechanism of DMDD targeting liposomes(HA/TN-DLP)In vitro pharmacodynamic evaluation showed that HA/TN-DLP significantly inhibited the proliferation(P<0.05 or P<0.01),colony formation,migration and invasion of 4T1 cells,and its effects were more significant than free DMDD and non-targeted DMDD liposomes at the same administered dose;HA/TN-DLP induced apoptosis of 4T1 cells,decreased mitochondrial membrane potential,and blocked cells in G0/G1 phase;HA/TN-DLP significantly decreased the levels of PI3 K,Akt,m TOR,P70S6 K,FAK,TLR4,My D88,NF-κB p65,IκBαand IKKα/β(P<0.05 or P<0.01),thereby regulating the expression of PI3K/Akt and NF-κB signaling pathway to inhibit the proliferation of 4T1 cells.ConclusionHA/TN-DLP is successfully prepared by ethanol injection and electrostatic attraction,and the freeze-dried liposomes are prepared.The particle size distribution,EE,DL,stability and safety in vitro are up to the mustard of liposomes.Compared with the DMDD and DLP,HA/TN-DLP has better targeting properties to 4T1 cells and tumor tissues in vitro and in vivo.In addition,HA/TN-DLP significantly inhibits the growth of breast cancer in vitro and in vivo,which may be related to the regulation of glycerophospholipid metabolism pathway,PI3K/Akt and NF-κB signaling pathways.