CN114588127A - A modified zein nano-drug-loading system encapsulated by bacterial outer membrane vesicles and its preparation method and application - Google Patents

Abstract

The invention discloses a modified zein nanometer drug-carrying system wrapped by bacterial outer membrane vesicles, and a preparation method and application thereof. Dissolving the drug in absolute ethanol, adding deionized water after ultrasonic dissolving, adding zein, and ultrasonicating; in the obtained mixed solution, rapidly adding sodium caseinate solution under stirring, and stirring to obtain modified zein. Protein drug-loaded nanoparticle solution; mix the bacterial outer membrane capsule and the modified zein drug-loaded nanoparticle solution evenly, use high-pressure nitrogen as the extrusion pressure source, and pass the mixed solution through a polycarbonate membrane of 200 nm for 2-8 times Extrusion, the obtained product is centrifuged, and the precipitate is collected, which is a modified zein nano-drug-carrying system encapsulated by bacterial outer membrane vesicles. The nano-drug loading system provided by the invention has a narrow particle size distribution range and good stability, and can load hydrophobic drugs soluble in ethanol, so as to enhance its solubility in water and improve the stability and bioavailability of the drugs.

Description

A modified zein nano-drug-carrying system encapsulated by bacterial outer membrane vesicles and its preparation method and application

technical field

The invention relates to a biomimetic natural biopolymer nano-carrier, in particular to a modified zein nano-drug-carrying system wrapped by bacterial outer membrane vesicles, and a preparation method and application thereof.

Background technique

In recent years, interest in protein biopolymers for drug delivery systems has greatly increased. Proteins have amphiphilic properties and can interact well with drugs and solvents. They are one of the ideal materials for the preparation of nano-drug delivery systems, and have great application prospects in the field of biomedicine. The key features that make native proteins potentially efficient nanocarriers are their availability of natural resources, inherent biocompatibility, biodegradability, non-toxicity, and their biological functions. Zein was listed by the FDA as one of the safest biomaterial excipients in 1985. Due to its good safety and biocompatibility, it is currently widely used in drug controlled release and biomedical drug delivery systems. Zein drug-loaded nanoparticles can effectively improve drug absorption after oral administration. The hydrophobic nature of zein makes it a good candidate for encapsulating hydrophobic drugs, which can be encapsulated in nanoparticles through hydrophobic, electrostatic, and hydrogen bonding interactions. However, the zein drug-loaded nanoparticles have poor stability, are easy to aggregate, and have the problem of poor re-solubility after drying.

Synthetic nanoparticles are quite different from endogenous substances in the human body. Usually, the interaction between cells is less effective, and they cannot exert their due advantages. They may even be regarded as exogenous substances and cause an immune response. Three major types of biomimetic drug delivery systems for endogenous proteins, pathogens and cells have emerged, among which cell membrane biomimetic nanoparticles have been widely used in the study of drug delivery vehicles. When the free drug binds directly to the cell membrane, the toxicity of the drug may affect the cell membrane, resulting in uncontrolled drug release. Therefore, cell membranes are often coated on the surface of synthetic nanoparticles. This not only solves the problem of drug toxicity, but also has the physicochemical properties of synthetic nanoparticles and the complex biological functions of cell membranes, which can effectively reduce the immunogenicity of nanoparticles, improve the targeting of drugs, and increase the residence time of drugs in vivo. .

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a modified zein nano-drug-carrying system wrapped in bacterial outer membrane vesicles, which has a narrow particle size distribution range and good stability, and can be used for embedding hydrophobic ethanol-soluble vesicles. Drugs to enhance their solubility in water and improve the stability and bioavailability of embedded components.

The second purpose of the present invention is to provide a preparation method of a modified zein nano-drug loading system wrapped by bacterial outer membrane vesicles.

The technical scheme adopted in the present invention is: a preparation method of a modified zein nanometer drug-carrying system wrapped by bacterial outer membrane vesicles, comprising the following steps:

1) Dissolve the drug in absolute ethanol, ultrasonically dissolve it completely, add deionized water, then add zein, and ultrasonicate for 5-15min; in the obtained mixed solution, quickly add sodium caseinate under stirring solution, stirring for 0.5-1.5h to obtain a modified zein drug-loaded nanoparticle solution;

2) Mix the bacterial outer membrane sac with the modified zein drug-loaded nanoparticle solution obtained in step 1) uniformly, and the obtained mixed solution uses high-pressure nitrogen gas as the extrusion pressure source, and the polycarbonate membrane of 200 nm is passed through 2-8 times. After extrusion, the obtained product was centrifuged at 6000 r/min for 20 min at 4°C, and the precipitate was collected, which was a modified zein nano-drug delivery system wrapped by bacterial outer membrane vesicles.

Further, in the above-mentioned preparation method, in step 1), by volume ratio, absolute ethanol: deionized water=4:1.

Further, in the above preparation method, in step 1), the sodium caseinate solution is rapidly added at a stirring speed of 10-20 r/min.

Further, in the above preparation method, in step 1), the ultrasonic frequency is 80 Hz.

Further, in the above preparation method, in step 2), the bacterial outer membrane sac is Escherichia coli DH5a outer membrane sac.

Further, in the above preparation method, in step 2), by volume ratio, bacterial outer membrane sac: modified zein drug-loaded nanoparticle solution=4:1.

The application of the modified zein nanometer drug loading system wrapped by bacterial outer membrane vesicles provided by the invention as a drug carrier.

Further, the drug is a hydrophobic drug.

Further, the hydrophobic drug is an alcohol-soluble hydrophobic drug.

Further, the hydrophobic drug is paclitaxel.

The present invention has the following beneficial results:

1. The modified zein nano-drug loading system wrapped by bacterial outer membrane vesicles prepared by the present invention has good stability, and the particle size is about 175 nm.

2. The modified zein nano-drug-carrying system wrapped by bacterial outer membrane vesicles prepared by the present invention has a smooth spherical structure, the particle size is highly concentrated, and the particle size distribution range is narrow.

3. The modified zein nanometer drug-carrying system wrapped by bacterial outer membrane vesicles prepared by the present invention can resist the damage of part of gastric acid, so that the drug will not leak prematurely.

4. The modified zein nano-drug-carrying system wrapped by bacterial outer membrane vesicles prepared by the present invention has no burst-release phenomenon in artificial intestinal fluid, and has good sustained-release characteristics.

5. In the modified zein nano-drug-carrying system prepared by the invention, which is wrapped by bacterial outer membrane vesicles, sodium caseinate is the sodium salt of casein, which is odorless, tasteless, and safe. It is a soluble mixture of various proteins, containing various hydrophobic and hydrophilic groups, and is a natural stabilizer and emulsifier. Sodium caseinate enhanced the stability and dispersibility of zein drug-loaded nanoparticles in water through surface adsorption. In the process of preparing zein drug-loaded nanoparticles, an appropriate amount of sodium caseinate is added as a stabilizer, so that the zein drug-loaded nanoparticles are encapsulated by sodium caseinate, which provides steric hindrance and static electricity. It can effectively improve the stability and resolubility of zein drug-loaded nanoparticles.

Description of drawings

Figure 1 is a scanning electron microscope image of the modified zein nano-drug delivery system encapsulated by bacterial outer membrane vesicles.

Figure 2 is a graph showing the change in particle size and PDI of the modified zein nano-drug delivery system encapsulated by bacterial outer membrane vesicles in PBS buffer for 48 hours.

Figure 3 is a graph showing the release rate of drugs encapsulated in the modified zein nano-drug delivery system encapsulated by bacterial outer membrane vesicles in artificial gastric juice.

Figure 4 is a graph showing the release rate of drugs encapsulated in the modified zein nano-drug delivery system encapsulated by bacterial outer membrane vesicles in artificial intestinal fluid.

Detailed ways

Example 1 A modified zein nano-drug-loading system wrapped by bacterial outer membrane vesicles

(1) The modified zein nano-drug loading system wrapped by bacterial outer membrane vesicles, the preparation method includes the following steps:

1. Preparation of modified zein drug-loaded nanoparticle solution

Accurately weigh 12.5 mg of sodium caseinate with an analytical balance, dissolve it in 7 mL of deionized water, and ultrasonically dissolve it completely to obtain a sodium caseinate solution.

Precisely weigh 3 mg of paclitaxel with an analytical balance and dissolve it in 1.6 mL of absolute ethanol, ultrasonically dissolve it completely, add 0.4 mL of deionized water to make the ethanol concentration 80% (volume), and then add 10 mg of zein, at 80 Hz Frequency ultrasonic for 7min to obtain a mixed solution. Quickly pour 7 mL of sodium caseinate solution into the mixed solution with a rotating speed of 15 r/min, stir for 1 h, and after the ethanol is volatilized, the modified paclitaxel-loaded zein nanoparticles modified with sodium caseinate can be obtained solution, labeled Zine-CAS-PTX.

2. Preparation of modified zein nanoparticle-loaded paclitaxel system encapsulated by bacterial outer membrane vesicles

The outer membrane vesicles of Escherichia coli DH5a and the modified zein-loaded paclitaxel nanoparticle solution, by volume ratio, bacterial outer membrane vesicle: modified zein-loaded paclitaxel nanoparticle solution = 4:1, mixed evenly , and then using high-pressure nitrogen as the extrusion pressure source, the mixed solution was passed through a polycarbonate membrane of 200 nm, and extruded 6 times. The modified zein nano-paclitaxel-loaded system encapsulated by membrane vesicles, labeled as OMVs-NPs.

(3) Detection

1. Characterization of OMVs-NPs Morphology

The morphology of OMVs-NPs nanoparticles was observed by transmission electron microscopy. Pipette 10 μL of the OMVs-NPs nanoparticle solution with a pipette, drop it on the carbon support membrane, and after it is dry, stain with 1% uranyl acetate solution, place the sample in the air, dry it naturally, and use Transmission electron microscope observation.

As shown in Figure 1, it can be seen that the nanoparticles wrapped by OMVs have a spherical structure. OMV-NPs have obvious shell-core structure, which proves that OMVs are successfully wrapped on the surface of Zein-CAS-PTX nanoparticles.

2. Investigation of the stability of OMVs-NPs

The OMVs-NPs were placed in a refrigerator at 4°C to stand, and the changes in particle size and PDI were measured within 48 h.

As shown in Figure 2, the particle size and PDI of OMVs-NPs did not change significantly in PBS buffer, indicating that OMVs-NPs had better stability.

3. Investigation of the release of OMVs-NPs in vitro

Prepare artificial gastric juice and artificial intestinal juice, and then take 30 mL of each and place them in a 50 mL centrifuge tube respectively, and add 1% sodium lauryl sulfate to make it reach the sink condition. Pipette 2 mL of Zein-CAS-PTX nanoparticle solution and OMV-NPs solution into the treated dialysis bag (molecular weight 3500 Da), respectively, and then place the dialysis bag into a centrifuge tube. The release rate was determined by the shaking table method. The experimental conditions were 37°C and 120 rpm. The artificial gastric juice group absorbed the release solution at 0.5, 1, 1.5, and 2 h; the artificial intestinal juice group was at 1, 2, 3, 6, 8, 10, 12, 24, 48h suction release solution. At the same time, after each release solution was taken, the same volume of release medium was supplemented. The ultraviolet absorption value of the release solution was measured, and the PTX concentration at different sampling time points was calculated by substituting it into the standard curve. Then, according to the cumulative drug release formula, the cumulative drug release percentage was calculated, and the in vitro release curve was drawn.

As shown in Figure 3, the cumulative drug release amount of Zein-CAS-PTX nanoparticles in artificial gastric juice for 2 h was 12.58%, and the cumulative drug release amount was low, indicating that Zein-CAS-PTX nanoparticles were weakly eroded by gastric acid environment. This is because Zein is a hydrophobic protein that protects the drug from an acidic environment, preventing drug release in the stomach. The cumulative release of OMVs-NPs in artificial gastric juice for 2 h was 8.89%, which was lower than that of Zein-CAS-PTX nanoparticles, indicating that OMVs played a protective role and could resist the damage of some gastric acid, so that the drug would not leak prematurely.

As shown in Figure 4, the cumulative drug release amount of Zein-CAS-PTX nanoparticles in artificial intestinal fluid for 2 hours was 15.87%, and the cumulative drug release amount was higher than that in artificial gastric fluid, indicating that the stability of Zein in artificial gastric fluid was higher than that in artificial gastric fluid. Intestinal fluid. Zein-CAS-PTX nanoparticles were released in artificial intestinal fluid after 48 hours, and the cumulative release amount could reach 68.97%, and there was no burst release phenomenon, indicating that Zein-CAS-PTX nanoparticles had good sustained-release properties. The cumulative release of OMVs-NPs in the first 2 hours in artificial intestinal juice was 3.14%. Compared with artificial gastric juice, OMVs-NPs had better stability. For the Zein-CAS-PTX nanoparticle group, it shows that the package of OMVs can increase the sustained release effect of the drug.

Claims (10)

1. the preparation method of the modified zein nanometer drug-carrying system that a bacterial outer membrane vesicle is wrapped, it is characterised in that the preparation method comprises the steps: 1) Dissolve the drug in absolute ethanol, ultrasonically dissolve it completely, add deionized water, then add zein, and ultrasonicate for 5-15min; in the obtained mixed solution, quickly add sodium caseinate under stirring solution, stirring for 0.5-1.5h to obtain a modified zein drug-loaded nanoparticle solution; 2) Mix the bacterial outer membrane sac with the modified zein drug-loaded nanoparticle solution obtained in step 1) uniformly, and the obtained mixed solution uses high-pressure nitrogen gas as the extrusion pressure source, and the polycarbonate membrane of 200 nm is passed through 2-8 times. After extrusion, the obtained product was centrifuged at 6000 r/min for 20 min at 4°C, and the precipitate was collected, which was a modified zein nano-drug delivery system wrapped by bacterial outer membrane vesicles. 2. preparation method according to claim 1 is characterized in that, in step 1), by volume ratio, dehydrated alcohol: deionized water=4:1. 3. The preparation method according to claim 1 is characterized in that, in step 1), the sodium caseinate solution is rapidly added at a stirring speed of 10-20r/min. 4. The preparation method according to claim 1, wherein in step 1), the ultrasonic frequency is 80Hz. 5. The preparation method according to claim 1, wherein in step 2), the bacterial outer membrane sac is Escherichia coli DH5a outer membrane sac. 6. preparation method according to claim 1 is characterized in that, in step 2), by volume ratio, bacterial outer membrane sac: modified zein drug-loaded nanoparticle solution=4:1. 7. Application of the modified zein nano-drug loading system prepared by the method according to any one of claims 1-6, which is wrapped by bacterial outer membrane vesicles, as a drug carrier. 8. The use according to claim 7, wherein the drug is a hydrophobic drug. 9 . The application according to claim 8 , wherein the hydrophobic drug is an ethanol-soluble hydrophobic drug. 10 . 10. The use according to claim 9, wherein the hydrophobic drug is paclitaxel.

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