Dequalinium chloride-polyethylene glycol-distearoyl phosphatidyl ethanolamine conjugated compound and resveratrol liposome modified thereby

Abstract

The invention discloses a DQA-PEG 2000-DSPE (Dequalinium Chloride-Polyethylene Glycol-Distearoyl Phosphatidyl Ethanolamine) conjugated compound and resveratrol liposome modified thereby. The structural formula of the compound is shown by a formula I. In terms of composition, the resveratrol liposome comprises resveratrol and a fat material, wherein the mass ratio of the resveratrol to the fat material is (1:20)-(1:40); and the fat material consists of egg yolk lecithin, cholesterol and the compound shown by the formula I in the molar ratio of (63-67):(18-22):(2-4.35) in sequence. Pharmacodynamic tests prove that mitochondrial targeted resveratrol liposome has an extremely strong cell toxic effect in in-vitro cell experiments of human lung adenocarcinoma A549 cells and drug-resistant A549 / cDDP cells thereof, a tumor sphere model and an in-vivo transplantation tumor model and can penetrate through the core of the tumor sphere. The anti-tumor effect of vinorelbine liposome on the drug-resistant A549 / cDDP cells can be obviously improved by combining the resveratrol liposome with the vinorelbine liposome for use (formula I).

Description

technical field

[0001] The invention belongs to the field of medicine, in particular to a dequalinium chloride-polyethylene glycol-distearoylphosphatidylethanolamine conjugate compound and a preparation method thereof, and resveratrol liposomes modified with the conjugate compound Background technique

[0002] Tumor multidrug resistance is the main reason for the failure of chemotherapy, which can lead to drug resistance, recurrence and metastasis of tumor cells, especially endogenous multidrug resistance. Tumor cells can cause genetic and epigenetic changes by changing the functions of proteins encoded by pre-apoptotic and apoptotic genes (such as Bcl-2 family proteins, Caspase proteins in the apoptosis signaling pathway, etc.), resulting in endogenous of multidrug resistance.

[0003] Cancer chemotherapy mainly adopts two basic methods to eliminate tumor cells: one is to kill tumor cells by directly acting on chemotherapy drugs, and the other is to activate the apoptosis ...

Examples

Embodiment 1

[0071] Embodiment 1, dequalinium chloride-polyethylene glycol-distearoylphosphatidylethanolamine (DQA-PEG 2000 Synthesis and Characterization of -DSPE) Conjugated Compounds

[0072] DQA-PEG 2000 -DSPE consists of amino groups and COOH-PEG on DQA 2000 -Carboxyl groups on DSPE were condensed by acylation, using DCC and HOBt as reactive condensing agents. 10 μmol DQA, 49.2 μmol DMAP, 20 μmol DCC, 20 μmol HOBt and 2 μmol COOH-PEG 2000 -DSPE was dissolved in 2ml of 50% (v / v) acetonitrile-water solution, the reaction solution mixture was stirred gently with a magnetic stirrer at room temperature for 12h to obtain the crude product, then the crude product was transferred to the regenerated cellulose dialysis bag (retention molecular weight 2000), dialyzed in deionized water for 48h to remove unreacted DQA, DMAP, DCC, HOBt and acetonitrile. Next, the reaction solution was lyophilized to obtain a white powder of the dry product mixture, and the H NMR spectrum ( 1 H NMR), laser des...

Embodiment 2

[0077] Embodiment 2, preparation and characterization of liposome

[0078] 1) Preparation of liposomes:

[0079] Mitochondria-targeted resveratrol liposomes were prepared by film dispersion method and extrusion through the membrane. In addition, unloaded mitochondria-targeted liposomes, resveratrol liposomes, vinorelbine liposomes , coumarin liposomes, and mitochondria-targeted coumarin liposomes were used as controls, cytotoxic drugs, and fluorescent probes, respectively.

[0080] With egg yolk lecithin (EPC), cholesterol and DQA-PEG 2000 -DSPE is used as the resin material, and the molar ratio of the three is 65:20:4.35. Lipid material and resveratrol (lipid material: drug = 20: 1, mass / mass) are put in an eggplant-shaped bottle and dissolved with methanol, and the methanol is removed by rotary evaporation. Beijing Co., Ltd., product code is 63008816) solution hydration, water bath ultrasonic 10min. Then, further ultrasonication was performed in the cell disruptor, and t...

Embodiment 3

[0098] Embodiment 3, the efficacy experiment of mitochondria-targeted resveratrol liposome

[0099] (1) Cell culture

[0100] Human lung adenocarcinoma A549 cells (purchased from Cancer Institute, Cancer Hospital, Chinese Academy of Medical Sciences) were cultured in F-12 medium containing 10% fetal bovine serum and 1% double antibody (100 U / ml penicillin, 100 μg / ml streptomycin) ; Multidrug-resistant lung cancer A549 / cDDP cells (purchased from Cancer Institute, Cancer Hospital, Chinese Academy of Medical Sciences) were treated with RPMI- 1640 culture medium, in order to maintain the drug resistance of the cells, cisplatin with a final concentration of 4 μM was added to the culture medium, and cultured with the above-mentioned medium without cisplatin one week before the experiment. Both cells were cultured under 5% CO 2 , 37°C.

[0101] (2) Cytotoxicity

[0102] A549 cells or A549 / cDDP cells in 5.4 × 10 3 The concentration of cells / well was inoculated in 96-well cell cul...

technical field

[0001] The invention belongs to the field of medicine, in particular to a dequalinium chloride-polyethylene glycol-distearoylphosphatidylethanolamine conjugate compound and a preparation method thereof, and resveratrol liposomes modified with the conjugate compound Background technique

[0002] Tumor multidrug resistance is the main reason for the failure of chemotherapy, which can lead to drug resistance, recurrence and metastasis of tumor cells, especially endogenous multidrug resistance. Tumor cells can cause genetic and epigenetic changes by changing the functions of proteins encoded by pre-apoptotic and apoptotic genes (such as Bcl-2 family proteins, Caspase proteins in the apoptosis signaling pathway, etc.), resulting in endogenous of multidrug resistance.

[0003] Cancer chemotherapy mainly adopts two basic methods to eliminate tumor cells: one is to kill tumor cells by directly acting on chemotherapy drugs, and the other is to activate the apoptosis ...

Examples

Embodiment 1

[0071] Embodiment 1, dequalinium chloride-polyethylene glycol-distearoylphosphatidylethanolamine (DQA-PEG 2000 Synthesis and Characterization of -DSPE) Conjugated Compounds

[0072] DQA-PEG 2000 -DSPE consists of amino groups and COOH-PEG on DQA 2000 -Carboxyl groups on DSPE were condensed by acylation, using DCC and HOBt as reactive condensing agents. 10 μmol DQA, 49.2 μmol DMAP, 20 μmol DCC, 20 μmol HOBt and 2 μmol COOH-PEG 2000 -DSPE was dissolved in 2ml of 50% (v / v) acetonitrile-water solution, the reaction solution mixture was stirred gently with a magnetic stirrer at room temperature for 12h to obtain the crude product, then the crude product was transferred to the regenerated cellulose dialysis bag (retention molecular weight 2000), dialyzed in deionized water for 48h to remove unreacted DQA, DMAP, DCC, HOBt and acetonitrile. Next, the reaction solution was lyophilized to obtain a white powder of the dry product mixture, and the H NMR spectrum ( 1 H NMR), laser des...

Embodiment 2

[0077] Embodiment 2, preparation and characterization of liposome

[0078] 1) Preparation of liposomes:

[0079] Mitochondria-targeted resveratrol liposomes were prepared by film dispersion method and extrusion through the membrane. In addition, unloaded mitochondria-targeted liposomes, resveratrol liposomes, vinorelbine liposomes , coumarin liposomes, and mitochondria-targeted coumarin liposomes were used as controls, cytotoxic drugs, and fluorescent probes, respectively.

[0080] With egg yolk lecithin (EPC), cholesterol and DQA-PEG 2000 -DSPE is used as the resin material, and the molar ratio of the three is 65:20:4.35. Lipid material and resveratrol (lipid material: drug = 20: 1, mass / mass) are put in an eggplant-shaped bottle and dissolved with methanol, and the methanol is removed by rotary evaporation. Beijing Co., Ltd., product code is 63008816) solution hydration, water bath ultrasonic 10min. Then, further ultrasonication was performed in the cell disruptor, and t...

Embodiment 3

[0098] Embodiment 3, the efficacy experiment of mitochondria-targeted resveratrol liposome

[0099] (1) Cell culture

[0100] Human lung adenocarcinoma A549 cells (purchased from Cancer Institute, Cancer Hospital, Chinese Academy of Medical Sciences) were cultured in F-12 medium containing 10% fetal bovine serum and 1% double antibody (100 U / ml penicillin, 100 μg / ml streptomycin) ; Multidrug-resistant lung cancer A549 / cDDP cells (purchased from Cancer Institute, Cancer Hospital, Chinese Academy of Medical Sciences) were treated with RPMI- 1640 culture medium, in order to maintain the drug resistance of the cells, cisplatin with a final concentration of 4 μM was added to the culture medium, and cultured with the above-mentioned medium without cisplatin one week before the experiment. Both cells were cultured under 5% CO 2 , 37°C.

[0101] (2) Cytotoxicity

[0102] A549 cells or A549 / cDDP cells in 5.4 × 10 3 The concentration of cells / well was inoculated in 96-well cell cul...

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