CN113288880B - Tea saponin/chitosan coated dihydromyricetin liposome and preparation method and application thereof - Google Patents

Tea saponin/chitosan coated dihydromyricetin liposome and preparation method and application thereof Download PDF

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CN113288880B
CN113288880B CN202110622492.8A CN202110622492A CN113288880B CN 113288880 B CN113288880 B CN 113288880B CN 202110622492 A CN202110622492 A CN 202110622492A CN 113288880 B CN113288880 B CN 113288880B
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dihydromyricetin
chitosan
tea saponin
solution
dmy
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CN113288880A (en
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舒绪刚
吴紫倩
陈任翔
罗帆
任艳丽
曾丹丹
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Zhongkai University of Agriculture and Engineering
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Abstract

The invention provides a tea saponin/chitosan coated dihydromyricetin liposome and a preparation method and application thereof, belonging to the technical field of pharmaceutical chemistry. The tea saponin/chitosan coated dihydromyricetin liposome provided by the invention comprises an active component, a first coating layer and a second coating layer, wherein the first coating layer and the second coating layer are sequentially coated on the surface of the active component; the active component is dihydromyricetin, the first coating layer is a phospholipid compound modified with polyethylene glycol, and the second coating layer is chitosan grafted with tea saponin. The phospholipid compound modified with polyethylene glycol is used as the first coating layer, so that the problems of low DMY hydrolysis degree, short biological half-life period and poor membrane permeability can be solved, meanwhile, the chitosan grafted with tea saponin is used as the second coating layer, so that the slow-release tea saponin/chitosan-coated dihydromyricetin liposome with strong permeability to bacteria is obtained, the slow-release effect is good, and the drug resistance of the bacteria can be avoided.

Description

Tea saponin/chitosan coated dihydromyricetin liposome and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to a tea saponin/chitosan coated dihydromyricetin liposome and a preparation method and application thereof.
Background
Dihydromyricetin (DMY) is used as the most abundant (about 30wt% maximum) nutritional supplement in Ampelopsis grossedentata, and has pharmacological activities of preventing alcohol hangover, resisting cancer, resisting inflammation, resisting bacteria, etc. However, the low solubility, low permeability and low bioavailability properties of DMY severely limit its application in clinical medicine. Liu et al (Liu D, maoY, ding L, zeng XA.Dihydromycin: A review on identification and qualification methods, biological activities, chemical stability, metabolism and aprroaches to environments biological activities. Trends Food Sci technology.2019; 91. In the prior art, DMY is coated with a single soybean phospholipid as a coating to prepare a liposome, but the bioavailability of DMY still needs to be improved.
Disclosure of Invention
The tea saponin/chitosan-coated dihydromyricetin liposome provided by the invention takes phospholipid compounds modified with polyethylene glycol as a first coating layer and takes chitosan grafted with tea saponin as a second coating layer, so that the bioavailability of dihydromyricetin can be effectively improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a tea saponin/chitosan-coated dihydromyricetin liposome, which comprises an active component, a first coating layer and a second coating layer, wherein the first coating layer and the second coating layer are sequentially coated on the surface of the active component; the active component is dihydromyricetin, the first coating layer is a phospholipid compound modified with polyethylene glycol, and the second coating layer is chitosan grafted with tea saponin.
Preferably, the content of the dihydromyricetin in the tea saponin/chitosan-coated dihydromyricetin liposome is 30-45 wt%.
Preferably, the polyethylene glycol is polyethylene glycol 4000, polyethylene glycol 2000 or polyethylene glycol 4500.
Preferably, the phospholipid compound is egg yolk lecithin or soybean lecithin.
Preferably, the molar ratio of the tea saponin to the chitosan is 1: (0.5-2.0).
The invention provides a preparation method of tea saponin/chitosan coated dihydromyricetin liposome in the technical scheme, which comprises the following steps:
mixing a first emulsifier, polyethylene glycol and a phosphoric acid buffer solution to obtain an aqueous phase solution; mixing phospholipid compound, cholesterol, dihydromyricetin and organic solvent to obtain dihydromyricetin phase solution; dropwise adding the dihydromyricetin phase solution into the water phase solution to obtain an emulsion; removing the organic solvent in the emulsion to obtain dihydromyricetin liposome solution;
mixing tea saponin, chitosan, acetic acid, a second emulsifier and water to obtain a tea saponin-chitosan mixed solution, and then carrying out a grafting reaction to obtain a tea saponin/chitosan solution;
and (3) dropwise adding the dihydromyricetin liposome solution into the tea saponin/chitosan solution, and carrying out encapsulation treatment to obtain the tea saponin/chitosan-coated dihydromyricetin liposome.
Preferably, the volume fraction of the first emulsifier in the aqueous phase solution is 0.05-0.25%, and the concentration of the polyethylene glycol is 0.0005-0.015 g/mL; the concentration of the phospholipid compound in the dihydromyricetin phase solution is 0.01-0.2 g/mL, the concentration of cholesterol is 0.001-0.02 g/mL, and the concentration of dihydromyricetin is 0.001-0.005 g/mL; the volume ratio of the dihydromyricetin phase solution to the water phase solution is 1: (2-10).
Preferably, the concentration of the tea saponin in the tea saponin-chitosan mixed solution is 0.004-0.006 g/mL, the concentration of the chitosan is 0.004-0.006 g/mL, the volume fraction of the acetic acid is 0.8-1.2%, and the volume fraction of the second emulsifier is 0.4-0.6%.
Preferably, the volume ratio of the dihydromyricetin liposome solution to the tea saponin/chitosan solution is 1: (0.5-2.0).
The invention provides application of the tea saponin/chitosan-coated dihydromyricetin liposome in the technical scheme or the tea saponin/chitosan-coated dihydromyricetin liposome prepared by the preparation method in the technical scheme in preparation of an anti-pathogen preparation.
The invention provides a tea saponin/chitosan-coated dihydromyricetin liposome, which comprises an active component, a first coating layer and a second coating layer, wherein the first coating layer and the second coating layer are sequentially coated on the surface of the active component; the active component is dihydromyricetin, the first coating layer is a phospholipid compound modified with polyethylene glycol, and the second coating layer is chitosan grafted with tea saponin. The phospholipid compound modified with polyethylene glycol is used as a first coating layer, so that the problems of low hydrolysis degree of DMY, short biological half-life period and poor membrane permeability can be solved, and meanwhile, the Chitosan (CTS) grafted with Tea Saponin (TS) is used as a second coating layer to obtain the slow-release tea saponin/chitosan coated dihydromyricetin liposome (TS/CTS) with strong permeability to bacteria (such as escherichia coli (E.coli) and staphylococcus aureus (S.ausreus)), wherein TS/CTS @ DMY-lips and E.coli outer-layer lipopolysaccharide or S.ausreus outer-layer teichoic acid have an electrostatic effect, so that a smooth road is laid for a nano-carrier to permeate the outer layer of bacterial peptidoglycan, and the characteristic of slowly and continuously releasing the medicament provides a basis for DMY to exert the maximum medicament effect and avoid the medicament resistance of bacteria.
Drawings
FIG. 1 is a schematic representation of the preparation of TS/CTS based on a grafting reaction in accordance with the present invention;
FIG. 2 is a schematic representation of the action of TS/CTS @ DMY-lips on bacteria according to the present invention;
FIG. 3 is a graph of the topography and drug loading results of DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips;
FIG. 4 is a comprehensive structural representation diagram of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips;
FIG. 5 is a graph of the release rate of drugs from DMYLips, CTS @ DMY-lips, and TS/CTS @ DMY-lips at pH =3.0, pH =5.0, pH =6.0, and pH =7.0;
fig. 6 is a graph of the bacteriostatic activity results of free DMY on e.coli and s.aureus;
FIG. 7 is a graph showing the results of the bacteriostatic activity of DMY lips, blank CTS-lips, blank TS/CTS-lips, CTS @ DMY lips and TS/CTS @ DMY-lips on E.coli and S.aureus;
FIG. 8 is a graph showing the growth of E.coli and S.aureus colonies cultured 24h after treatment with DMYLips, CTS @ DMYLips and TS/CTS @ DMY-lips;
FIG. 9 is OD of E.coli and S.aureus after treatment with TS/CTS @ DMY-lips of different concentrations 600 Growth curves and colony growth situation graphs;
FIG. 10 is an analysis chart of the bacteriostatic mechanism of TS/CTS @ DMY-lips.
Detailed Description
The invention provides a tea saponin/chitosan-coated dihydromyricetin liposome, which comprises an active component, a first coating layer and a second coating layer, wherein the first coating layer and the second coating layer are sequentially coated on the surface of the active component; the active component is dihydromyricetin, the first coating layer is a phospholipid compound modified with polyethylene glycol, and the second coating layer is chitosan grafted with tea saponin.
In the invention, the active component in the tea saponin/chitosan-coated dihydromyricetin liposome is dihydromyricetin, and the content of the dihydromyricetin in the tea saponin/chitosan-coated dihydromyricetin liposome is preferably 30-45 wt%. According to the invention, the phospholipid compound modified with polyethylene glycol is used as a first coating layer, the chitosan grafted with tea saponin is used as a second coating layer, and the second coating layer is used as a drug loading system, so that the bioavailability of dihydromyricetin can be effectively improved.
In the invention, the component of the first coating layer in the tea saponin/chitosan coated dihydromyricetin liposome is a phospholipid compound modified with polyethylene glycol, and the polyethylene glycol is preferably polyethylene glycol 4000, polyethylene glycol 2000 or polyethylene glycol 4500; the phospholipid compound is preferably egg yolk lecithin or soybean lecithin. The invention adopts polyethylene glycol to modify phospholipid compounds, can stabilize the mechanical skeleton of the whole liposome, is beneficial to the loading of medicaments, can prevent the phagocytosis of the liposome by an immune system, and promotes the whole medicament-carrying system to completely transport the medicaments to the infection parts of pathogens (such as bacteria) in vivo.
In the invention, the component of the second coating layer in the tea saponin/chitosan-coated dihydromyricetin liposome is chitosan grafted with tea saponin, and the mol ratio of the tea saponin to the chitosan is preferably 1: (0.5-2.0). According to the invention, the tea saponin is grafted on the chitosan, so that the stability of the chitosan under neutral physiological conditions of a human body is favorably improved, the mechanical strength of the whole drug-loading system is favorably improved, the tea saponin/chitosan-coated dihydromyricetin liposome is ensured to have a better dihydromyricetin slow-release effect within a wider pH value range (pH = 3-7), and the permeability of the drug-loading system to bacteria is effectively improved.
In the invention, the entrapment rate of the tea saponin/chitosan-coated dihydromyricetin liposome is preferably 30-45%, and the particle size of the tea saponin/chitosan-coated dihydromyricetin liposome is preferably 50-300 nm.
The invention provides a preparation method of tea saponin/chitosan coated dihydromyricetin liposome in the technical scheme, which comprises the following steps:
mixing a first emulsifier, polyethylene glycol and a phosphoric acid buffer solution to obtain an aqueous phase solution; mixing phospholipid compound, cholesterol, dihydromyricetin and organic solvent to obtain dihydromyricetin phase solution; dropwise adding the dihydromyricetin phase solution into the water phase solution to obtain an emulsion; removing the organic solvent in the emulsion to obtain dihydromyricetin liposome solution;
mixing tea saponin, chitosan, acetic acid, a second emulsifier and water to obtain a tea saponin-chitosan mixed solution, and then carrying out a grafting reaction to obtain a tea saponin/chitosan solution;
and (3) dropwise adding the dihydromyricetin liposome solution into the tea saponin/chitosan solution, and carrying out encapsulation treatment to obtain the tea saponin/chitosan-coated dihydromyricetin liposome.
In the present invention, the starting materials are all commercially available products well known to those skilled in the art unless otherwise specified.
According to the invention, a first emulsifier, polyethylene glycol and a phosphoric acid buffer solution are mixed to obtain an aqueous phase solution. In the present invention, the first emulsifier is preferably tween 80 or span 60, more preferably tween 80; the first emulsifier is adopted in the invention, which is beneficial to improving the drug loading rate of the liposome. In the present invention, the volume fraction of the first emulsifier in the aqueous phase solution is preferably 0.05 to 0.25%, more preferably 0.1 to 0.2%; the concentration of polyethylene glycol is preferably 0.0005 to 0.015g/mL, more preferably 0.0005 to 0.005g/mL. In the present invention, the pH of the phosphoric acid buffer solution is preferably 6.5.
The mixing mode of the first emulsifier, the polyethylene glycol and the phosphoric acid buffer solution is not particularly limited, and all the components can be uniformly mixed.
The phospholipid compound, cholesterol, dihydromyricetin and an organic solvent are mixed to obtain a dihydromyricetin phase solution. In the present invention, the concentration of the phospholipid compound in the dihydromyricetin phase solution is preferably 0.01 to 0.2g/mL, more preferably 0.02 to 0.1g/mL; the concentration of cholesterol is preferably 0.001 to 0.02g/mL, more preferably 0.003 to 0.01g/mL; the concentration of dihydromyricetin is preferably 0.001 to 0.005g/mL, more preferably 0.002 to 0.004g/mL. In the present invention, the organic solvent is preferably absolute ethanol or absolute methanol. In the present invention, the cholesterol can improve the mechanical strength of the liposome and stabilize the liposome so that it is not easily decomposed.
The method for mixing the phospholipid compound, the cholesterol and the dihydromyricetin with the organic solvent is not particularly limited, and the components can be uniformly mixed.
After obtaining the aqueous phase solution and the dihydromyricetin phase solution, the invention adds the dihydromyricetin phase solution into the aqueous phase solution dropwise to obtain the emulsion. In the present invention, the volume ratio of the dihydromyricetin phase solution to the aqueous phase solution is preferably 1: (2 to 10), more preferably 1: (2.5-5). In the present invention, the dihydromyricetin phase solution is preferably added dropwise to the aqueous phase solution, the dropwise addition is preferably performed under stirring conditions, and the rotation speed of the stirring is preferably 200 to 400rpm, and more preferably 250 to 300rpm. In the invention, in the dropping process of the dihydromyricetin phase solution, the water phase solution gradually turns yellow and is slightly turbid; and (3) after the dihydromyricetin phase solution is dropwise added, obtaining emulsion.
After the emulsion is obtained, the invention removes the organic solvent in the emulsion to obtain dihydromyricetin liposome (DMY-lips) solution. In the invention, the emulsion is preferably heated to remove the organic solvent, the heating is preferably carried out under the condition of stirring, and the rotating speed of the stirring is preferably 80-120 rpm, more preferably 100rpm; the heating temperature is preferably 40-45 ℃, and more preferably 42-43 ℃; the heating time is based on the complete removal of the organic solvent, and in the embodiment of the invention, the heating time can be specifically 1-5 h; the invention promotes the volatilization of the organic solvent under the conditions of low-temperature heating and stirring, the water is not easy to volatilize under the conditions, the organic solvent is considered to be completely removed when the volume of the system is kept constant, and the heating is stopped, so that the dihydromyricetin liposome solution is obtained.
The method comprises the steps of mixing tea saponin, chitosan, acetic acid, a second emulsifier and water to obtain a tea saponin-chitosan mixed solution, and then carrying out grafting reaction to obtain a tea saponin/chitosan (TS/CTS) solution. In the invention, in the tea saponin-chitosan mixed solution, the concentration of the tea saponin is preferably 0.004-0.006 g/mL, more preferably 0.005g/mL; the concentration of the chitosan is preferably 0.004-0.006 g/mL, and more preferably 0.005g/mL; the volume fraction of acetic acid is 0.8-1.2%, more preferably 1%; the volume fraction of the second emulsifier is 0.4 to 0.6%, more preferably 0.5%. In the present invention, the second emulsifier is preferably tween 80 or span 60, more preferably tween 80; the second emulsifier adopted by the invention is beneficial to improving the drug loading rate of the liposome. The invention adopts a small amount of acetic acid to ensure that the chitosan is dissolved on the basis of not damaging the chitosan structure.
According to the invention, acetic acid, a second emulsifier and water are preferably mixed to obtain an acetic acid-emulsifier mixed solution; and then mixing the acetic acid-emulsifier mixed solution with tea saponin and chitosan to obtain a tea saponin-chitosan mixed solution.
In the present invention, the temperature of the grafting reaction is preferably 80 to 85 ℃, more preferably 82 to 83 ℃; the time of the grafting reaction is preferably 1.5 to 2.5 hours, and more preferably 2 hours; the grafting reaction is preferably carried out under stirring conditions, the rotation speed of the stirring preferably being 150 to 250rpm, more preferably 200rpm.
After the grafting reaction, the obtained product system is preferably cooled to room temperature, and then dialysis is carried out to remove residual tea saponin in the system, so as to obtain a tea saponin/chitosan solution. In the examples of the present invention, the room temperature is specifically 25 ℃. In the invention, the molecular weight cut-off of a dialysis bag used for dialysis is preferably 5000Da, deionized water is preferably used as dialysate for dialysis, and the dialysis time is preferably 4h.
In the present invention, the molecular structural formula of the TS is as follows:
Figure BDA0003100437390000061
FIG. 1 is a schematic diagram of the preparation of TS/CTS based on the grafting reaction in the present invention, and it can be seen from FIG. 1 that carboxyl group in TS reacts with hydroxyl group and amino group in CTS respectively, thereby obtaining TS/CTS.
After obtaining a dihydromyricetin liposome solution and a tea saponin/chitosan solution, the invention adds the dihydromyricetin liposome solution into the tea saponin/chitosan solution dropwise for encapsulation treatment to obtain the tea saponin/chitosan coated dihydromyricetin liposome. In the invention, the volume ratio of the dihydromyricetin liposome solution to the tea saponin/chitosan solution is preferably 1: (0.5 to 2.0), more preferably 1:1. in the present invention, the dihydromyricetin liposome solution is preferably added dropwise to the tea saponin/chitosan solution. In the present invention, the encapsulation treatment is preferably performed at room temperature, and the time of the encapsulation treatment is preferably 3.5 to 4.5 hours, and more preferably 4 hours; the encapsulation treatment time is counted by the completion of the dropping of the dihydromyricetin liposome solution. In the present invention, the encapsulation treatment is preferably carried out under stirring conditions, and the stirring rate is preferably 150 to 250rpm, more preferably 200rpm. In the invention, after the encapsulation treatment is finished, a solution containing tea saponin/chitosan coated dihydromyricetin liposome is obtained, and the tea saponin/chitosan coated dihydromyricetin liposome is obtained after drying; the drying is preferably freeze drying.
The invention provides application of the tea saponin/chitosan-coated dihydromyricetin liposome in the technical scheme or the tea saponin/chitosan-coated dihydromyricetin liposome prepared by the preparation method in the technical scheme in preparation of an anti-pathogen preparation, wherein the anti-pathogen preparation is preferably an antibacterial agent.
The phospholipid compound modified with polyethylene glycol is used as the first coating layer, so that the problems of low DMY hydrolysis degree, short biological half-life period and poor membrane permeability can be solved, and meanwhile, the Chitosan (CTS) grafted with Tea Saponin (TS) is used as the second coating layer to obtain the slow-release tea saponin/chitosan coated dihydromyricetin liposome (TS/CTS @ DMY-lips) with strong permeability to bacteria (such as escherichia coli (E.coli) and staphylococcus aureus). FIG. 2 is a schematic diagram of the action of TS/CTS @ DMY-lips on bacteria (yolk lecithin and polyethylene glycol 4000 are taken as examples), and electrostatic interaction exists between TS/CTS @ DMY-lips and E.coli outer layer lipopolysaccharide or S.ausreus outer layer teichoic acid, so that a road is paved for a nano carrier to permeate the outer layer of bacterial peptidoglycan, and the characteristic of slowly and continuously releasing the drug provides a basis for DMY to exert the maximum drug effect and avoids drug resistance of bacteria.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental equipment and reagents used in the following examples and test examples are shown in tables 1 and 2, respectively.
TABLE 1 Experimental apparatus
Name of instrument Instrument type Manufacturer of the product
Vacuum freeze drier Alpha1-2LDPlus Christ, germany
Ultraviolet spectrophotometer S22PC SHANGHAI LENGGUANG TECHNOLOGY Co.,Ltd.
Fourier infrared spectrometer Spectrum100 PerkinElmer Inc. of USA
Raman spectrometer DXR2 ThermoFisher company, USA
Powder X-ray diffraction D8Advance Bruker, germany
Synchronous thermal analyzer TGA2 Mettler-Toledo, USA
Nanometer granularity tester 90PlusPALS Brookhacen instruments USA
Perspective electron microscope TecnaiG2F20 FeI USA Inc
Dissolution instrument RC12AD Tiandatianfa Technology Co.,Ltd.
Conductivity meter CT-3030 Shenzhen Koedida electronics Co Ltd
TABLE 2 test reagents
Name of reagent Purity of Manufacturer of the product
Polyethylene glycol-4000 (PEG-4000) Analytical purity TIANJIN DAMAO CHEMICAL REAGENT FACTORY
Cholesterol Analytical purity SHANGHAI MACKLIN BIOCHEMICAL Co.,Ltd.
Tea Saponin (TS) Analytical purity SHANGHAI MACKLIN BIOCHEMICAL Co.,Ltd.
Chitosan (CTS) Analytical purity SHANGHAI MACKLIN BIOCHEMICAL Co.,Ltd.
Tween 80 Cell culture grade ALADDIN REAGENT (SHANGHAI) Co.,Ltd.
Egg yolk lecithin Biological pharmaceutical grade Evertop (Shanghai) Pharmaceutical Technology Co.,Ltd.
LB nutrient agar Cell culture grade BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.
LB Broth Cell culture grade BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.
In addition, the ultramicro Na +/K + -ATP enzyme assay kit and the total protein assay kit (Coomassie brilliant blue method) are purchased from Nanjing institute of bioengineering; coli and s.aureus were taken from sec happy agro-engineering institute biochemical laboratories.
Dihydromyricetin (DMY) is extracted from Ampelopsis grossedentata by hydrothermal extraction method, and comprises the following steps:
adding 20g of stem and leaf of camellia oleifera abel cake and 400mL of deionized water into a round-bottom flask, uniformly stirring, adjusting the pH value of a system to 7 by using NaOH, placing the round-bottom flask into a constant-temperature heating magnetic stirrer, and extracting in a water bath at 100 ℃ for 0.5h; filtering while hot, removing filter residues, transferring the extracting solution into a rotary flask for concentration until the volume of the obtained concentrated solution is 1/4 of that of the extracting solution, placing the concentrated solution in a refrigerator at 4 ℃ for cooling for 12h, filtering, and removing filtrate to obtain a DMY crude extract;
dissolving the DMY crude extract in 8mL of absolute ethyl alcohol, uniformly stirring, filtering, removing filter residues, slowly pouring the filtrate into 400mL of deionized water, standing and storing at room temperature (25 ℃) until DMY crystals are separated out, and obtaining a suspension containing DMY crystals; and (3) carrying out suction filtration on the suspension by using a reduced pressure suction filter, discarding the filtrate, and placing the filter residue in an oven for forced air drying to obtain DMY (the purity is 94.3%).
Example 1
The preparation method of the tea saponin/chitosan coated dihydromyricetin liposome comprises the following steps:
(1) The DMY-lip solution is prepared by adopting an oil-in-water method, which comprises the following steps:
adding 30 μ L of tween 80 and 0.01g of PEG-4000 to 20mL of phosphate buffer solution (pH = 6.5), and stirring uniformly to obtain an aqueous phase solution; adding 0.2g of egg yolk lecithin, 0.025g of cholesterol and 0.02g of DMY into 8mL of absolute ethyl alcohol, and stirring to fully dissolve the mixture to obtain a DMY phase solution; under the condition of magnetic stirring at 300rpm, dropwise adding the DMY phase solution into the water phase solution, and obtaining an emulsion after dropwise adding; the emulsion was stirred at 43 ℃ for 3h at 100rpm until the absolute ethanol was removed from the emulsion and a constant volume of DMY-lips solution was obtained (DMY content in DMY-lips was 26.49 wt%).
(2) The TS/CTS solution was prepared as follows:
adding 100 mu L of acetic acid and 50 mu L of Tween 80 into water to obtain 10mL of acetic acid-Tween 80 mixed solution; accurately weighing 0.05g of Chitosan (CTS) and 0.05g of Tea Saponin (TS) to dissolve in 10mL of the acetic acid-Tween 80 mixed solution, and placing the obtained system in a water bath kettle at 83 ℃ for reaction for 2 hours under the condition of magnetic stirring at 200 rpm; after the reaction was completed, the reaction mixture was cooled to room temperature (25 ℃ C.), and the resulting product system was placed in a 5000Da dialysis bag and dialyzed in deionized water for 4 hours to remove excess TS, to obtain a TS/CTS solution (TS/CTS content: 1 wt%).
(3) The preparation method comprises the following steps of preparing TS/CTS @ DMYLips:
and (3) dropwise adding 10mL of the DMY-lips solution in the step (1) into 10mL of the TS/CTS solution in the step (2) under the magnetic stirring condition of 200rpm at room temperature, and continuously stirring for 4 hours to obtain a TS/CTS @ DMYLips solution.
Comparative example 1
The procedure of example 1 was followed, except that TS was omitted, to finally prepare a chitosan-supported dihydromyricetin liposome (CTS @ DMYLips) solution;
the procedure of example 1 was followed, except that TS and DMY were omitted, to finally prepare a solution of non-loaded chitosan liposomes (Blank CTS-lipids);
the procedure of example 1 was followed except that DMY was omitted and a solution of tea saponin/chitosan liposome (BlankTS/CTS-lips) without drug loading was finally prepared.
The data of the following test examples are statistically analyzed by using SPSS 25.0 statistical software, all results are expressed by numerical values +/-standard error, and P <0.05 is used for judging that the numerical values have statistical significance.
Test example 1
(1) Drug loading assay
Accurately weighing 5mL of drug-loaded solution (DMY lips solution, CTS @ DMY-lips solution or TS/CTS @ DMY-lips solution) and placing in a 10mL centrifuge tube, centrifuging for 20min at 6000r/min, sucking 1mL of supernatant and diluting with phosphoric acid buffer solution to constant volume to 25mL; the absorbance of the unencapsulated DMY was measured using an ultraviolet spectrophotometer at λ =293nm against the background of phosphate buffer solution. The linear equation of the standard concentration (C) and the absorbance (A) of DMY is A =48.3085C-0.00829; r 2 =0.9988. The solution drug loading (EE) was calculated according to formula 1:
Figure BDA0003100437390000091
in formula 1, m total Represents the total amount of DMY in the system, mg; m is free Represents the amount of unencapsulated DMY in the system, mg; EE stands for drug loading,%.
(2) Structural characterization and microscopic morphology observation
The particle size and Zeta potential of the liposome in the DMY lips solution, the CTS @ DMY-lips solution and the TS/CTS @ DMY-lips solution are measured by adopting a nano-particle size tester, and the microscopic morphology and the particle size distribution of the DMY lips, the CTS @ DMY-lips and the TS/CTS @ DMY-lips are observed by adopting a transmission electron microscope.
And (3) freeze-drying the DMY lips solution, the CTS @ DMY-lips solution and the TS/CTS @ DMY-lips solution by using a vacuum freeze dryer to obtain corresponding solid samples. At 4000-400 cm -1 In the range, performing infrared analysis on DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips by using a Fourier infrared spectrometer according to a KBr tablet method; carrying out Raman analysis on DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips by adopting a micro Raman spectrometer; taking a Cu target K alpha as a radiation source, and carrying out XRD analysis on DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips by using an X-ray powder diffractometer, wherein the wavelength (lambda) is 0.154056nm, the tube voltage is 40kV, and the tube current is 15mA; in N 2 Under protection, a synchronous thermal analyzer is used to examine the thermal stability of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips at the temperature of 40-900 ℃ at the temperature rise rate of 10 ℃/min.
(3) Analysis of results
FIG. 3 is a result graph of the morphology structures and drug loading rates of DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips. This is now explained in more detail with reference to fig. 3.
FIG. 3 (a) is a schematic diagram of the synthesis of TS/CTS @ DMY-lips, and the coating of TS/CTS on the surface of liposome in the present invention can improve the stability and penetration of liposome.
FIG. 3 (b) is a macroscopic view of the DMYLips (i) solution, the CTS @ DMY-lips (ii) solution and the TS/CTS @ DMY-lips (iii) solution, and the results show that the DMYLips solution and the CTS @ DMY-lips solution are pale yellow transparent solutions, while the TS/CTS @ DMY-lips solution is yellow transparent solution, indicating that the substance microstructure is changed.
FIG. 3 (c) is a graph showing the particle size distribution of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips, showing that the particle size of DMY lips is 71.25nm and the particle size is uniform; in contrast, the sizes of CTS @ DMY-lips and TS/CTS @ DMY-lips particles increased to 144.90nm and 266.49nm, respectively, the increase in particle size was attributed to the formation of CTS or TS/CTS coatings on the liposome surface. Driven by the positively charged amino group and the negatively charged phosphate group of the egg yolk lecithin, the TS/CTS can automatically cover the surface of the liposome and can form a hydrogen bond with PEG-4000 on the surface of the liposome to stably exist on the surface of the carrier.
FIG. 3 (d) is a Zeta potential map of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips, showing that the Zeta potentials of DMY lips and CTS @ DMY-lips are-14.28 mV and 31.33mV, respectively, and surface charge reversal confirms successful coverage of CTS on the liposome surface. Furthermore, the Zeta potential of TS/CTS @ DMY-lips is 16.05mV, which is likely that the formation of the TS/CTS complex consumes the amino functionality on the CTS.
It is worth noting that the construction of the cationic drug delivery system not only facilitates the penetration of bacteria, but also promotes the drug loading. FIG. 3 (e) is a graph showing the drug loading results of DMYlips, CTS @ DMY-lips and TS/CTS @ DMY-lips, showing that the drug loading of DMY lips is 26.49%, and the drug loading of CTS @ DMY-lips and TS/CTS @ DMY-lips are increased to 45.29% and 41.93%, respectively. The increase of the drug loading rate is caused by the saturated absorption effect of CTS on the surface of the liposome, and the positive CTS and the negative DMY have ionic interaction, so that the absorption of the liposome to the DMY is promoted.
TEM images (scales are all 200 nm) of (f), (g) and (h) in FIG. 3 are respectively DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips, and the results show that DMYLips are nano-scale spherical structures, contain a plurality of vesicles, and have a bilayer structure; CTS @ DMY-lips presents an elliptical structure, the particle size is slightly larger than DMYLlips, and the particle size is consistent with the particle size test result; whereas TS/CTS @ DMY-lips present a dendritic cell-type structure, with the liposome surface completely covered. The drug delivery systems are connected by extended "tentacles" and maintain relative spatial separation. This behavior is mainly caused by two reasons, firstly, the high dispersion phase generated by the high molecular weight CTS causes the carrier surface to have rheological properties (high viscoelasticity and good interfacial properties), and secondly, the abundant hydroxyl groups on the sugar group in TS and CTS form hydrogen bonds to promote their cross-linking.
FIG. 4 is a comprehensive structural characterization diagram of DMY lips, CTS @ DMY-lips, and TS/CTS @ DMY-lips, wherein (i) DMYlips; (ii) CTS @ DMY-lips; (iii) TS/CTS @ DMY-lips. This is now explained in more detail with reference to fig. 4.
FIG. 4 (a) is an infrared analysis chart of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips, showing that DMYLips is located at 3431cm -1 2919cm of stretching vibration peak with wide peak of-OH -1 And 2873cm -1 The absorption peak is generated by the stretching vibration of C-H on the shell PEG-4000; in addition, 1735cm -1 And 1104cm -1 The absorption peak can be attributed to the tensile vibration of C = O and C-O-C. After CTS encapsulation, the-OH stretching vibration peak on the liposome is shifted to 3416cm -1 This is probably due to the-OH of PEG-4000 and the-OH and-NH of CTS 2 The mutual interference between them. CTS @ DMY-lips at 1670cm -1 Where (tensile vibration of C = O in amide I) and 1645cm -1 The characteristic absorption band of the amide bond (bending vibration of N-H in amide II) appears, and the presence of CTS is well confirmed. In the infrared curve of TS/CTS @ DMY-lips, -OH absorption peak shifts to 3410cm -1 And the absorption band becomes wider, indicating that intermolecular hydrogen bonds increase; the CTS amido bond absorption peak shifts to 1672cm -1 Point sum 1641cm -1 And the C-H absorbance continued to decrease, indicating that TS was successfully attached to CTS.
FIG. 4 (b) is a Raman analysis chart of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips, showing that the Raman curve of DMYLips and the Raman curve of CTS @ DMY-lips are 1092cm -1 The absorption peak can be attributed to the stretching vibration of C-O-C; in contrast, newly formed absorption peaks of ester groups or amido groups (1374 cm) in the Raman curve of TS/CTS @ DMY-lips -1 ) The successful synthesis of TS/CTS @ DMY-lips was directly demonstrated.
FIG. 4 (c) is an XRD pattern of DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips, showing that the XRD curves of DMY lips exhibit a broad and overlapping pattern, indicating that they are amorphous structures; the XRD curve of CTS @ DMY-lips shows partial Bragg peak, which indicates that the surface structure of the substance is changed; in contrast, the Bragg peak of the XRD curve of TS/CTS @ DMY-lip disappears again and presents amorphous phase structural characteristics, which are probably related to the non-directional recombination of TS and CTS, and the drug with small particle size and low crystallinity is easier to dissolve and absorb, which indicates that the amorphous phase TS/CTS @ DMY-lip prepared by the invention is possibly favorable for permeating and entering the interior of bacteria.
FIG. 4 shows thermogravimetric analysis curves for (d) DMYLips, CTS @ DMY-lips, and TS/CTS @ DMY-lips, and derivative thermogravimetric curves for (e) DMYLips, CTS @ DMY-lips, and TS/CTS @ DMY-lips. The results show that the weight loss of DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips in the range of 40-150 ℃ is caused by evaporation of free water and crystal water on the surface of the substance, and the weight loss rates are respectively 8.32%, 6.56% and 6.63%. Continuously raising the temperature, wherein the second-stage weight loss (12.49%) of CTS @ DMY-lips occurs at 220-320 ℃, and the corresponding DTA peak is 274.86 ℃, which is mainly related to the fracture of the C-O-C bond of the main chain of CTS. In contrast, the DTA peak at this stage for TS/CTS @ DMY-lips increased to 284.79 deg.C, probably due to the establishment of ester or amide linkages that improved the overall thermal stability of CTS. When the temperature is in the range of 320-450 ℃, obvious weight loss occurs in DMY lips, CTS @ DMY-lips and TS/CTS @ DMY-lips, which is caused by thermal disintegration of the liposome skeleton. The DTA peaks of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips at the stage respectively appear at 400.98 ℃, 389.56 ℃ and 405.95 ℃, which shows that TS/CTS @ DMY-lips have higher thermal stability, and the basis is provided for TS successful grafting of CTS.
Test example 2 sustained Release Performance test
The release rates of DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips in phosphate buffered solutions at pH =3, 5, 6 and 7 were investigated with free DMY as a control group. Specifically, 500mL of phosphoric acid buffer solution is accurately prepared and poured into a dissolution bottle, and the mechanical paddle is adjusted to the most appropriate height according to the operation instruction of the dissolution instrument. 2mL of the sample solution was put into a 5000Da dialysis bag, and the dialysis bag was settled on the bottom of the dissolution flask and dissolved by mechanical stirring (100 rpm) at a constant temperature of 37 ℃. Every other one10mL of the eluate was collected over time and the machine automatically compensated for pure phosphoric acid buffer. Measuring DMY absorbance (A) of the collected sample solution by using an ultraviolet spectrophotometer, wherein a standard working curve A =48.3085C-0.00829 of the DMY absorbance (A) is obtained, and calculating the cumulative release rate (R) of the DMY according to formula 2 i )。
Figure BDA0003100437390000131
In formula 2,. Rho i Represents the mass concentration of DMY in the sample, mg/mL; m is a unit of DMY Is the total mass of DMY in 2mL of sample, mg.
Fig. 5 is a graph of the release rate of the drug in the environment of pH =3.0, pH =5.0, pH =6.0 and pH =7.0 for DMYlips, cts @ dmy-lips and TS/cts @ dmy-lips, fig. 5 (a) being pH =3.0, (b) being pH =5.0, (c) being pH =6.0, (d) being pH =7.0; wherein, (i) free DMY, (ii) DMYLips, (iii) CTS @ DMY-lips, (iv) TS/CTS @ DMY-lips. As can be seen from fig. 5, free DMY rapidly diffuses within 1h without the protection of the drug delivery system and the release rate increases with time, indicating that the diffusion of DMY is not affected by the dialysis bag; the cumulative release rates of free DMY in the four pH environments were 82.39%, 79.04%, 84.77%, and 72.31%, respectively. In contrast, the three drug delivery systems DMYLips, CTS @ DMY-lips and TS/CTS @ DMY-lips can effectively relieve the release of DMY, wherein the TS/CTS @ DMY-lips has the best effect, and the property of the drug delivery system has a larger influence on the drug release. The CTS is coated or embedded in a phospholipid bilayer on the surface of the liposome, so that the liposome can be prevented from disintegrating to cause the leakage of the wrapped medicament. And TS and CTS are combined to form amide bond, so that CTS (pKa = 6.3-6.5) can be further inhibited from being hydrolyzed under neutral condition, and the whole delivery system is further consolidated. After the DMY lips are dissolved out for 29h in four pH environments, the total release rates are 60.35%, 64.08%, 63.83% and 56.50%, respectively. After TS/CTS encapsulation, the drug release rate of TS/CTS @ DMY-lips in four pH environments is respectively reduced to 28.10%, 34.36%, 34.82% and 22.65%, which means that most of DMY is reserved for subsequent bacterial absorption. As most of bacterial infection sites exist in weak acid or neutral environment, TS/CTS @ DMY-lips with broad-spectrum pH slow release performance has great potential in the field of biological medicine.
Test example 3 bacteriostatic Property test
(1) Study of antibacterial Activity
0.02g of DMY is accurately weighed and dissolved in 20mL of phosphoric acid buffer solution (pH = 6.5), the obtained suspension is heated at 60 ℃ until the DMY is completely dissolved, and the mixture is kept stand and cooled to room temperature to obtain 1.00mg/mL of DMY solution, and the DMY solution is ready to use.
The antibacterial ability of the DMY lips solution, the CTS @ DMY-lips solution and the TS/CTS @ DMY-lips solution to E.coli and S.aureus is measured by adopting an agar diffusion method. Specifically, 25mL of sterilized agar medium was poured into a sterilized glass petri dish on a sterile super clean bench, and naturally cooled to solidify, followed by aspiration of 100. Mu.L of viable bacterial suspension (E.coli: 1.0X 10) 8 CFU/mL；S.aureus：1.0×10 6 CFU/mL) and evenly inoculated on the surface of an agar plate, three round holes with the diameter of 6mm are dug on a solid medium by using a puncher, and 80 mu L of an antibacterial sample is added into each hole; packaging the culture dish, placing the culture dish in an incubator, and incubating at 37 ℃; after 24h, the petri dish was removed, and the diameter of the zone of inhibition was measured by the crossover method after several experiments (see fig. 7 for specific results, which will be described in detail later).
To further evaluate the antibacterial ability of DMY tips, CTS @ DMY-tips and TS/CTS @ DMY-tips, the OD of the two bacterial suspensions after 24h drug treatment was determined using a microplate reader with sterile phosphate buffer (pH = 6.5) as a blank control 600 Growth curves. Adding 20 mu L of bacterial suspension and 80 mu L of sterilized LB broth liquid culture medium into a hole of a sterile 96-hole plate in a sterile ultra-clean bench, slightly shaking and shaking uniformly, and adding 100 mu L of liquid medicine sample; packaging 96-well plate, culturing at 37 deg.C for 24 hr in shaking table, and measuring OD of bacteria liquid with microplate reader at intervals during culturing 600 The value is obtained. Each treatment was performed in 5 parallel experiments (see fig. 8 for specific results, which will be described in detail later).
(2) Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays
Determination of M of TS/CTS @ DMY-lips solution on E.coli and S.aureus by standard microdilution methodIC and MBC. Adding 20 μ L of bacterial suspension and 80 μ L of sterilized LB broth into a hole of a 96-well plate, and then respectively adding 100 μ L of TS/CTS @ DMY-lips solution with different concentrations to ensure that the final concentrations of the medicaments are respectively 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.10mg/mL, 0.2mg/mL, 0.3mg/mL and 0.5mg/mL; packaging 96-well plate, culturing at 37 deg.C for 24 hr in shaking table, and measuring OD of bacteria liquid with microplate reader at intervals during culturing 600 The value is obtained. After 24h incubation, 100. Mu.L of drug-containing bacterial solution was collected and spread evenly on the surface of 25mL agar solid medium, incubated at 37 ℃ for 24h, photographed and recorded for colony formation on agar. Where MIC is interpreted as the lowest sample concentration that inhibits bacterial growth and MBC is interpreted as the lowest drug concentration that kills 99.9% of the bacteria in the bacterial suspension. Each treatment was performed in 5 replicates.
(3) Biological perspective electron microscope observation
Under the aseptic condition, sucking 1mL of bacterial suspension and 1mL of 2MIC TS/CTS @ DMY-lips solution into a 2mL sterile centrifuge tube, packaging the centrifuge tube, placing the centrifuge tube in a shaking table, culturing for 6h at 37 ℃ and centrifuging for 10min at 3000r/min, removing the solution, filling 2.5% glutaraldehyde solution into the centrifuge tube to fix the bacteria precipitated at the bottom of the tube, pouring out the fixing solution after storing for 24h at 4 ℃, washing the resuspended bacteria for 3 times by using 0.1mol/L phosphoric acid buffer solution (pH = 7.0), re-fixing the sample for 2h by using 1% osmium acid solution, and further washing the resuspended bacteria for 3 times by using the phosphoric acid buffer solution; and (3) gradient eluting a bacteria sample by adopting an ethanol solution, embedding bacteria by using an embedding medium, slicing, dyeing, airing, and observing the microscopic morphological change of the bacteria by adopting a biological transmission electron microscope.
(4) Determination of conductivity of bacterial liquid
Accurately pipette 4mL of the bacterial suspension into a 15mL sterile centrifuge tube, centrifuge at 3000r/min for 10min, pour off the solution, wash the bacteria 3 times with phosphate buffered saline (pH = 7.0), and redisperse the clean bacterial pellet in 15mL phosphate buffered saline for use. 2mL of clean bacterial suspension is sucked into a 5mL sterile centrifuge tube, then 2mL2MIC TS/CTS @ DMY-lips solution or isometric sterile water is added, the centrifuge tube is placed in a shaking table after being fully shaken up, and the culture is carried out at the temperature of 37 ℃; and then placing the drug-containing bacterial liquid in an ultracentrifuge, centrifuging for 10min under the condition of 3000r/min, collecting 1mL of supernatant, diluting to 25mL by using ultrapure water, and measuring the conductivity of the bacterial liquid after the drug treatment for 0h, 1h, 2h, 4h and 6h by using a conductivity meter. Each treatment was performed in parallel for 3 experiments.
(5) Determination of intracellular nucleic acid dissolution
Obtaining the bacterial liquid treated by TS/CTS @ DMY-lips according to the method in the step (4), and measuring the absorbance of the bacterial liquid at 260nm by using an ultramicro ultraviolet spectrophotometer to judge the dissolution condition of the intracellular nucleic acid. Each treatment was done in parallel for 3 experiments.
(6) Determination of intracellular adenosine triphosphate (Na +/K + -ATP)
Washing and resuspending 4mL of bacterial suspension by using normal saline, fixing the volume to 15mL, sucking 2mL of clean bacterial suspension into a 5mL sterilized centrifugal tube, adding 2mL2MIC TS/CTS @ DMY-lips solution or isometric sterile water, fully shaking up, placing the centrifugal tube in a shaking table, and culturing at 37 ℃; and then placing the drug-containing bacterial liquid in an ultracentrifuge, centrifuging for 10min under the condition of 3000r/min, discarding the filtrate, retaining the bottom bacterial precipitate, adding physiological saline to resuspend until the volume of the solution is 0.2mL, and then crushing the bacteria by using an ultrasonic crusher. And (3) preparing an indicator and a display agent according to the operation instruction of the purchased Na +/K + -ATP kit, and measuring the absorbance of the solution at 636nm by using an ultramicro ultraviolet spectrophotometer after incubation. Each treatment was done in parallel for 3 experiments.
(7) Analysis of results
Fig. 6 is a graph showing the results of bacteriostatic activity of free DMY on e.coli and s.aureus, and (a) in fig. 6 is e.coli treated with free DMY and (b) is s.aureus treated with free DMY. FIG. 7 is a result chart of the bacteriostatic activity of DMYLips, blank CTS-lips, blank TS/CTS-lips, CTS @ DMY lips and TS/CTS @ DMY-lips on E.coli and S.aureus in FIG. 7, wherein (a) is E.coli treated with different drugs, (b) is S.aureus treated with different drugs, and (c) is OD of E.coli treated with different drugs 600 Growth curve, (d) OD of s.aureus after treatment with different drugs 600 Growth curves. FIG. 8 is a graph showing the growth of E.coli and S.aureus colonies cultured 24 hours after treatment with DMY lips, CTS @ DMY lips and TS/CTS @ DMY-lips, and FIG. 8 (a) is a graph showingGrowth pattern of coli colonies, (b) growth pattern of s.
As can be seen from fig. 6, free DMY had no significant bacteriostatic effect on e. As can be seen from (a) and (b) in fig. 7, the inhibition zone of DMYlips on e.coli is 10.82 ± 2.34mm, mainly because the coating of liposome improves the solubility of DMY and slows down the drug release. However, DMYlips did not find a zone of inhibition in s. In addition, the Blank CTS-lips and the Blank TS/CTS-lips drug holes are generated in E.coli and S.aureus culture media, and CTS and TS/CTS coats in a surface drug loading system have no obvious bacteriostatic action on bacteria. In contrast, the zone of inhibition for e.coli and s.aureus by CTS @ DMY lips was 14.35 ± 0.26mm and 21.67 ± 1.69mm, respectively, which is likely due to the CTS outer garment increasing the membrane penetration of DMY lips to e.coli and s.aureus. CTS is capable of electrostatic interaction with lipopolysaccharides of gram negative bacteria (e.coli) and teichoic acids of gram positive bacteria (s.aureus), resulting in osmosis. The inhibition zones of TS/CTS @ DMYLips on E.coli and S.aureus are 15.44 +/-0.49 mm and 20.29 +/-2.01 mm respectively, which indicates that the interaction of TS and CTS does not reduce the antibacterial activity of the whole drug delivery system.
As shown in fig. 7 (c) and (d), the e.coli and s.aureus bacterial liquid ODs after DMYlips treatment were obtained 600 The values are improved along with the time extension, and after 24 hours, the OD of E.coli and S.aureus bacterial liquid is obtained 600 The values were 0.81 and 0.63, respectively, indicating that the bacteriostatic effect of DMYlips was not significant. In contrast, the growth curves of the two bacteria treated by the CTS @ DMYLips and the TS/CTS @ DMY-lips show no growth or even negative growth trends, which are mainly caused by killing or completely inhibiting all bacteria in the bacterial liquid. As can be seen from FIG. 8, the bacterial liquid of E.coli co-cultured with CTS @ DMYLips for 24 hours still had the colony count after being cultured in the solid medium, indicating that CTS @ DMYLips could not completely kill E.coli cells. In contrast, no colony count was produced in E.coli and S.aureus cultures co-cultured with TS/CTS @ DMY lips for 24h, indicating that TS/CTS @ DMYlips had 100% killing capacity against E.coli and S.aureus. Therefore, based on the antibacterial analysis, the successful preparation of TS/CTS @ DMY-lips is proved to greatly improve the antibacterial activity of the DMY to E.coli and S.aureus.
FIG. 9 shows the use of different concentrationsOD of E.coli and S.aureus after treatment with TS/CTS @ DMY-lips 600 FIG. 9 (a) is a graph showing the OD of E.coli treated with TS/CTS @ DMY-lips at different concentrations 600 Growth curve, (b) OD of S.aureus after treatment with different concentrations of TS/CTS @ DMY-lips 600 Growth curves, (c) are plots of growth of E.coli and S.aureus colonies grown after 24h treatment with TS/CTS @ DMY-lips at different concentrations. Wherein, the OD is high 600 Values are interpreted as high bacterial concentration and poor bacteriostatic activity. As is clear from FIGS. 9 (a) and (b), the E.coli and S.aureus bacterial strains OD co-cultured with TS/CTS @ DMY-lips were compared with the control group (0 mg/mL) 600 The values drop significantly and decrease over time. When the treatment concentration of TS/CTS @ DMY-lips is more than or equal to 0.05mg/mL, the bacterial liquid of E.coli and S.aureus shows no growth or even negative growth trend, which indicates that the thalli are killed or completely inhibited. To further confirm whether the bacterial suspension after TS/CTS @ DMY-lips treatment has bacterial activity, the bacterial suspension co-cultured with the drug for 24 hours was spread and cultured in a solid medium to observe the number of colonies, as shown in (c) of FIG. 9, the number of colonies of E.coli bacterial suspension treated with 0.025mg/mLTS/CTS @ DMY-lips was significantly reduced compared to the control group, and no E.coli colonies were produced in the medium after the concentration of TS/CTS @ DMY-lips treatment was higher than 0.20mg/mL, indicating that MIC and MBC of TS/CTS @ DMY-lips to E.coli were 0.025mg/mL and 0.20mg/mL, respectively. Similarly, the MIC and MBC of TS/CTS @ DMY-lips to S.aureus were 0.025mg/mL and 0.05mg/mL, respectively.
FIG. 10 is an analysis chart of the bacteriostatic mechanism of TS/CTS @ DMY-lips, and FIG. 10 (a) is a scanning electron micrograph (scale: 1 μm) of E.coli after treatment with TS/CTS @ DMY-lips; (b) Is a graph of the effect of TS/CTS @ DMY-lips on the extracellular conductivity of E.coli; (c) Is a graph of the influence of TS/CTS @ DMY-lips on the release amount of intracellular nucleic acid in E.coli; (d) Scanning electron microscope image (0.5 μm scale) of S.aureus after TS/CTS @ DMY-lips treatment; (e) The influence of TS/CTS @ DMY-lips on the extracellular conductivity of S.aureus is shown in the figure; (f) Is a graph of the influence of TS/CTS @ DMY-lips on the release amount of intracellular nucleic acid in S.aureus; (g) Pictures of E.coli and S.aureus bacterial liquid after 6h treatment of different medicaments are shown, wherein (i) the E.coli bacterial liquid is treated by sterile water, (ii) the E.coli bacterial liquid is treated by TS/CTS @ DMY-lips, (iii) the S.aureus bacterial liquid is treated by sterile water, and (iv) the S.aureus bacterial liquid is treated by TS/CTS @ DMY-lips; (h) The influence graph of TS/CTS @ DMY-lips on the ATP content in E.coli cells is shown; (i) Is a graph of the influence of TS/CTS @ DMY-lips on the intracellular ATP content of S.aureus. The values between the asterisked histograms in the plots were statistically different (p < 0.05).
As can be seen from (a) and (d) in fig. 10, e.coli and s.aureus suffered from severe disruption by TS/cts @ DMY-lips, both cells exhibited morphological atrophy or distortion, cell membrane damage and leakage of plasma within the cells, and these morphological changes further demonstrate that TS/cts @ DMY-lips were more effective in disrupting bacterial biofilms than the untreated DMY sample. The killing activity of TS/CTS @ DMY-lips to E.coli and S.aureus is assisted by CTS on the outer layer of the carrier. CTS can interact electrostatically with lipopolysaccharides of gram negative bacteria (e.coli) or with teichoic acids of gram positive bacteria (s.aureus), thus promoting penetration of the entire drug delivery system. As shown in FIGS. 10 (b) and (e), the conductivities of the two bacterial liquids co-cultured with TS/CTS @ DMY-lips increased with time as compared with the control (0 mg/mL); after treatment for 6 hours, the conductivity of E.coli bacterial liquid is increased from 1025.33 mu s/cm to 1690.33 mu s/cm, and the conductivity of S.aureus bacterial liquid is increased from 1035.33 mu s/cm to 1820.67 mu s/cm. As is clear from (c) and (f) in FIG. 10, the intracellular nucleic acid release amount of E.coli and S.aureus cells co-cultured with TS/CTS @ DMY-lips rapidly increased, and the OD of E.coli cell suspension was 6 hours after the treatment 260 The OD of S.aureus bacterial liquid is increased to 0.57 from 0.02 rapidly 260 Rapidly increased from 0.02 to 0.73. This revealed a high efficiency of damaging effect of DMY on e.coli and s.aureus biofilms. As shown in FIG. 10 (g), the bacterial suspension co-cultured with TS/CTS @ DMY-lips was stained with an ATP revealing agent and then reduced in color, indicating that the ATP content in the cells was significantly reduced. According to the instruction of the commercial ATP kit, the absorbance of the bacterial liquid at 636nm is measured by an ultramicro ultraviolet spectrophotometer, and the results are shown in (h) and (i) in FIG. 10, compared with the control group (0 mg/mL), the ATP content of the E.coli bacterial liquid co-cultured with TS/CTS @ DMY-lips for 6h is reduced from 66.15gprot/L to 22.79gprot/L, and the ATP content of the S.aureus bacterial liquid co-cultured with TS/CTS @ DMY-lips for 6h is reduced from 78.26gprot/L to 47.12gprot/L. This indicates that TS/CTS @ DMY-lips causes the bacteria not to normally decompose glucose to obtain energy, and further causes ATP synthesis to be hindered.
As can be seen from the above examples and test examples, the present invention prepares DMY lips with phospholipid compounds as carriers and polyethylene glycol as modifiers, and then functionalizes the whole nano-carrier with CTS and TS as encapsulating agents to finally obtain TS/CTS @ DMY-lips. The construction of the whole drug-loading system not only effectively improves the hydrophilicity and the hydrolysis degree of the DMY, but also promotes the DMY to permeate the bacterial membrane. Electrostatic interaction exists between TS/CTS @ DMY-lips with positive charges and E.coli and S.aureus with negative charges, and paves the way for nano-carriers to stay and enter bacteria. And the broad-spectrum pH slow release performance provides a stage for the long-acting release of DMY in bacteria, so that TS/CTS @ DMY-lips has 100% killing activity on E.coli and S.aureus. TS/CTS @ DMY-lips irreversibly damages the cell membrane, leading to bacterial protoplast dissolution and death. Meanwhile, TS/CTS @ DMY-lips effectively inhibit bacterial oxidative respiration and energy metabolism, and kill the possibility of bacterial division and diffusion. Different from the traditional bacteriostatic agent, the main wall materials and the drugs which form the TS/CTS @ DMY-lips are natural products, thereby improving the compliance of patients on antibacterial treatment. Therefore, the long-acting slow-release drug-carrying system has unlimited prospect in future biological medical research.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (8)

1. A tea saponin/chitosan coated dihydromyricetin liposome comprises an active component, a first coating layer and a second coating layer, wherein the first coating layer and the second coating layer are sequentially coated on the surface of the active component; the active component is dihydromyricetin, the first coating layer is a phospholipid compound modified with polyethylene glycol, and the second coating layer is chitosan grafted with tea saponin;
the preparation method of the tea saponin/chitosan coated dihydromyricetin liposome comprises the following steps:
mixing a first emulsifier, polyethylene glycol and a phosphoric acid buffer solution to obtain an aqueous phase solution; mixing phospholipid compound, cholesterol, dihydromyricetin and organic solvent to obtain dihydromyricetin phase solution; dropwise adding the dihydromyricetin phase solution into the water phase solution to obtain an emulsion; removing the organic solvent in the emulsion to obtain dihydromyricetin liposome solution; the volume fraction of the first emulsifier in the aqueous phase solution is 0.05 to 0.25 percent, and the concentration of the polyethylene glycol is 0.0005 to 0.015g/mL; the concentration of the phospholipid compound in the dihydromyricetin phase solution is 0.01-0.2g/mL, the concentration of cholesterol is 0.001-0.02g/mL, and the concentration of dihydromyricetin is 0.001-0.005g/mL; the volume ratio of the dihydromyricetin phase solution to the water phase solution is 1: (2 to 10); the first emulsifier is tween 80 or span 60;
mixing tea saponin, chitosan, acetic acid, a second emulsifier and water to obtain a tea saponin-chitosan mixed solution, and then carrying out a grafting reaction to obtain a tea saponin/chitosan solution; the concentration of the tea saponin in the tea saponin-chitosan mixed solution is 0.004-0.006g/mL, the concentration of the chitosan is 0.004-0.006g/mL, the volume fraction of acetic acid is 0.8-1.2%, and the volume fraction of the second emulsifier is 0.4-0.6%; the second emulsifier is tween 80 or span 60;
and (3) dropwise adding the dihydromyricetin liposome solution into the tea saponin/chitosan solution, and carrying out encapsulation treatment to obtain the tea saponin/chitosan-coated dihydromyricetin liposome.
2. The tea saponin/chitosan-coated dihydromyricetin liposome of claim 1, wherein the content of dihydromyricetin in the tea saponin/chitosan-coated dihydromyricetin liposome is 30-45wt%.
3. The tea saponin/chitosan-coated dihydromyricetin liposome of claim 1, wherein the polyethylene glycol is polyethylene glycol 4000, polyethylene glycol 2000 or polyethylene glycol 4500.
4. The tea saponin/chitosan-coated dihydromyricetin liposome of claim 1, wherein the phospholipid compound is egg yolk lecithin or soybean lecithin.
5. The tea saponin/chitosan-coated dihydromyricetin liposome of claim 1, wherein the molar ratio of the tea saponin to the chitosan is 1: (0.5 to 2.0).
6. The method for preparing the theasaponin/chitosan coated dihydromyricetin liposome of any one of claims 1~5, comprising the steps of:
mixing a first emulsifier, polyethylene glycol and a phosphoric acid buffer solution to obtain an aqueous phase solution; mixing phospholipid compound, cholesterol, dihydromyricetin and organic solvent to obtain dihydromyricetin phase solution; dropwise adding the dihydromyricetin phase solution into the water phase solution to obtain an emulsion; removing the organic solvent in the emulsion to obtain dihydromyricetin liposome solution; the volume fraction of the first emulsifier in the aqueous phase solution is 0.05 to 0.25 percent, and the concentration of the polyethylene glycol is 0.0005 to 0.015g/mL; the concentration of the phospholipid compound in the dihydromyricetin phase solution is 0.01-0.2g/mL, the concentration of cholesterol is 0.001-0.02g/mL, and the concentration of dihydromyricetin is 0.001-0.005g/mL; the volume ratio of the dihydromyricetin phase solution to the water phase solution is 1: (2 to 10); the first emulsifier is tween 80 or span 60;
mixing tea saponin, chitosan, acetic acid, a second emulsifier and water to obtain a tea saponin-chitosan mixed solution, and then carrying out a grafting reaction to obtain a tea saponin/chitosan solution; the concentration of the tea saponin in the tea saponin-chitosan mixed solution is 0.004-0.006g/mL, the concentration of the chitosan is 0.004-0.006g/mL, the volume fraction of acetic acid is 0.8-1.2%, and the volume fraction of the second emulsifier is 0.4-0.6%; the first emulsifier is tween 80 or span 60;
and (3) dropwise adding the dihydromyricetin liposome solution into the tea saponin/chitosan solution, and carrying out encapsulation treatment to obtain the tea saponin/chitosan-coated dihydromyricetin liposome.
7. The method of claim 6, wherein the volume ratio of dihydromyricetin liposome solution to tea saponin/chitosan solution is 1: (0.5 to 2.0).
8. Use of the tea saponin/chitosan coated dihydromyricetin liposome of any one of claims 1~5 or the tea saponin/chitosan coated dihydromyricetin liposome prepared by the preparation method of any one of claims 6~7 in preparing an anti-pathogenic agent preparation.
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