CN109717186B

CN109717186B - Accurate controlled release method of environment-friendly biomembrane inhibitory molecules

Info

Publication number: CN109717186B
Application number: CN201910154661.2A
Authority: CN
Inventors: 郭晓燕; 范守港; 李铖昊
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2021-06-18
Anticipated expiration: 2039-02-28
Also published as: CN109717186A

Abstract

The invention discloses an accurate controlled release method of an environment-friendly biological membrane inhibitory molecule D-amino acid. According to the method, D-amino acid is loaded on acid-modified nanoclay halloysite, then a composite fiber material with a core-shell structure is constructed by the halloysite nanotube loaded with the D-amino acid and an hydrophilic/hydrophobic polymer through a coaxial electrospinning method, and the shell structure of polymer fiber is regulated through electrospinning, so that accurate controlled release of the D-amino acid for inhibiting molecules of a biological membrane is realized, and a new strategy is provided for efficiently controlling microbial membrane pollution.

Description

Accurate controlled release method of environment-friendly biomembrane inhibitory molecules

Technical Field

The invention belongs to the field of pollution control, and particularly relates to a novel method for regulating and controlling biological membrane pollution, namely, an electrostatic spinning technology is adopted to realize accurate controlled release of biological membrane inhibitory molecules, so that the biological membrane pollution is efficiently controlled.

Background

Biofilm contamination is usually caused by nonspecific adsorption of proteins, polysaccharides, microorganisms and the like on the surface of a substrate and continuous growth and reproduction of microorganisms, and is widely present in the fields of biomedicine, human health, food safety, environmental pollution control and the like. Controlling biofilm contamination has been a very problematic issue for researchers. At present, the control methods aiming at the pollution of the biological membrane comprise physical removal, development of new antibacterial materials, use of bactericides and the like. The above methods all achieve certain control effects, but have the problems of low efficiency, high cost, environmental toxic and side effects and the like. Therefore, there is a need to develop a new method for controlling biofilm pollution, which is efficient and environmentally friendly.

The D-amino acid is a novel environment-friendly biological membrane inhibiting molecule discovered in recent years, can inhibit the formation of biological membranes, promotes the disintegration of the formed biological membranes, can effectively inhibit gram-positive bacteria and gram-negative bacteria from forming biological membranes at a concentration of 5nM, and shows great application potential in the aspect of biological membrane pollution control. However, the problems of low utilization rate and high cost of the existing use mode of the D-amino acid become bottlenecks for limiting the application of the D-amino acid in the control of the pollution of the biological membrane. Therefore, the development of efficient utilization of D-amino acids is an urgent problem to be solved to advance the application of D-amino acids in biofilm pollution control.

The concept of drug sustained release provides a new idea for controlling the pollution of the biological membrane by efficiently utilizing D-amino acid. The utilization rate of the D-amino acid can be obviously improved by selecting a proper carrier to slowly release the D-amino acid. Nanoclay materials are often used as slow release carriers due to their large specific surface area and inexpensive characteristics. Among them, the halloysite nanotube has received wide attention in the aspect of sustained release of drugs due to its unique loading lumen structure and good biocompatibility. However, the independent use of halloysite for the sustained release of D-amino acid is liable to cause a burst release phenomenon, which is not favorable for the efficient use of D-amino acid. Recent research shows that the composite carrier constructed by combining the nano material and the polymer can effectively inhibit the burst release of the drug and improve the release stability. The electrostatic spinning technology has more advantages in the aspect of constructing the nano material/polymer composite carrier, and the release rate of the drug can be further regulated and controlled by changing the electrospinning parameters to regulate and control the structure of the nano material/polymer composite fiber, so that the precise controlled release of the drug is realized.

Based on the analysis, the invention adopts the nanoclay halloysite to load D-amino acid, then constructs the D-amino acid loaded halloysite nanotube and the hydrophilic/hydrophobic polymer into the composite fiber material with the core-shell structure by a coaxial electrospinning method, and realizes the accurate controlled release of the molecular D-amino acid inhibited by the biological membrane by regulating and controlling the structure of the polymer fiber, thereby providing a new strategy for efficiently controlling the microbial membrane pollution.

Disclosure of Invention

The invention aims to provide a precise controlled release method for an environment-friendly biological membrane inhibitory molecule D-amino acid, so that biological membrane pollution is efficiently controlled.

The invention provides a preparation method of a halloysite-based core-shell structure composite fiber carrier, which comprises the following steps:

(1) dissolving a hydrophobic polymer in an organic solvent to obtain a shell electrospinning solution;

(2) blending a hydrophilic polymer and D-amino acid loaded halloysite to obtain inner core electrospinning liquid;

(3) and (3) carrying out coaxial electrostatic spinning on the shell layer spinning solution and the core layer spinning solution obtained in the steps (1) and (2) to obtain the prepared composite fiber membrane.

Further, the concentration of the hydrophobic polymer in the step (1) is 10-15%.

Further, the concentration of the hydrophilic polymer in the step (2) is 10-15%.

Further, the organic solvent used for the polymer in the step (1) and the step (2) is one or more of N, N-dimethylformamide, N-dimethylacetamide, dichloromethane and tetrahydrofuran.

Further, the D-amino acid loaded by the halloysite in the step (2) is one or more of D-tyrosine, D-methionine, D-leucine and D-lysine.

Further, the D-amino acid-containing halloysite added in step (2) is 2 to 10 wt% of the hydrophilic polymer powder by mass.

Further, in the step (3), the feeding speed of the inner core electrospinning liquid in the electrospinning is 0.5-0.7mL/h, and the feeding speed of the outer layer electrospinning liquid is 0.5-1.5 mL/h.

Furthermore, the voltage of the coaxial electrostatic spinning is 15-20KV, the temperature is 20-25 ℃, and the humidity is 40-55%. The rotating speed of the coaxial electrostatic spinning rotary drum receiving device is 300-500 rpm.

The precise controlled release method of the biological membrane inhibitory molecules provided by the invention has the following advantages:

(1) the halloysite-based core-shell structure composite fiber carrier material constructed by the invention can protect the activity of loaded D-amino acid;

(2) the core-shell structure composite fiber carrier material prepared by the invention shows good D-amino acid controlled release effect, and improves the utilization rate of D-amino acid;

(3) the invention realizes the accurate controlled release of the biological membrane inhibitory molecule D-amino acid, thereby efficiently controlling the biological membrane pollution.

Drawings

FIG. 1 is a Scanning Electron Microscope (SEM) image of the PAN/S-HNTs/DAA-PVDF core-shell electrospun fiber obtained in example 1;

FIG. 2 is a diameter distribution diagram of the PAN/S-HNTs/DAA-PVDF core-shell electrospun fiber obtained in example 1;

FIG. 3 is a Transmission Electron Microscope (TEM) image of the PAN/S-HNTs/DAA-PVDF core-shell electrospun fiber obtained in example 1;

FIG. 4 shows the cumulative release rate of controlled-release D-amino acids from the PAN/S-HNTs/DAA-PVDF core-shell electrospun fiber obtained in example 1;

Detailed Description

The present invention is further described in the following specific examples, which are provided only for the purpose of illustration and are not intended to limit the scope of the present invention.

Example 1

The method comprises the following specific steps:

(1) acid modified halloysite (S-HNTs)

3g of HNTs were weighed out and 300mL of 1M H was added₂SO₄The solution was magnetically stirred at 200rpm for 5 hours at 50 ℃ and the mixture was centrifuged at 4000 rpm. The centrifuged product was then rinsed with deionized water and the pH was adjusted with 1M NaOH solution until the pH of the aqueous wash was neutral, and then the centrifuged product was dried in a drying oven at 105 ℃ for 24 hours to give acid-modified halloysite (S-HNTs).

(2) Acid-modified HNTs loaded with D-tyrosine (DAA)

0.4g of acid modified HNTs powder is weighed and added into 300mL of D-tyrosine solution with the concentration of 0.4g/L, 40KHz ultrasonic dispersion is carried out for 20min, and then the mixed solution is magnetically stirred for 24h at the rotating speed of 200rpm under the condition of 25 ℃. Standing the mixed solution for 2h, performing suction filtration on the mixed solution by using a 0.45-micron filter membrane under the negative pressure condition of 0.05Mpa, and drying the product at 30 ℃ to obtain S-HNTs/DAA. The D-tyrosine loading was determined at 570nm using ninhydrin colorimetry.

(3) Preparation of core electrospinning blending liquid

Weighing 45g N, adding N-Dimethylformamide (DMF) into a three-neck flask, then adding 0.25g S-HNTs/DAA powder (5 wt% of the mass of polyacrylonitrile powder), ultrasonically dispersing at 25 ℃ for 20min, then adding 5g of Polyacrylonitrile (PAN) powder, then mechanically stirring at 50 ℃ and 200rpm for 12h, then putting the blended polymer solution into a vacuum drying oven, and degassing at room temperature at-3 MPa for 1h for later use.

(4) Preparation of shell electrospinning liquid

Weighing 36g N, adding N-Dimethylformamide (DMF) solvent and 9g tetrahydrofuran into a three-neck flask, slowly adding 5g polyvinylidene fluoride (PVDF) powder, mechanically stirring at 50 ℃ and 200rpm for 12h, then placing the PVDF polymer solution into a vacuum drying oven, degassing at 25 ℃ and-3 MPa for 1h, and preparing into 10 wt% PVDF electrospinning solution for later use.

(5) Preparation of core-shell structure composite fiber based on halloysite

The vacuum defoamed inner core electrospinning blend liquid is added into a 5mL syringe and is marked as a syringe 1. The housing electrospinning solution was added to another syringe of the same specification, which was designated syringe 2. Electrospinning parameters: the voltage is 20KV, the distance from the needle to the receiving rotary drum is 20cm, the rotating speed of the receiving rotary drum is 500rpm, the electrospinning temperature is 25 ℃, and the electrospinning humidity is 55 degrees. The advancing speed of the electrospinning liquid in the syringe 1 is 0.9mL/h, and the advancing speed of the electrospinning liquid in the syringe 2 is 1.1 mL/h. The electrospinning time was 4 h. And after spinning is finished, taking down the constructed nanofiber membrane, and drying the nanofiber membrane in a vacuum drying oven at the temperature of 25 ℃ for 12 hours.

(6) DAA sustained Release test

Weighing 20mg of pure HNTs loaded with DAA and 20mg of acid modified HNTs loaded with DAA respectively, shearing 0.05g of halloysite-based core-shell structure composite fiber membrane, respectively adding the core-shell structure composite fiber membrane into a 10mL centrifuge tube, then adding 5mL of pure water, fixing the centrifuge tube on a shaking table, oscillating at the speed of 150rpm at the temperature of 25 ℃, centrifuging the centrifuge tube at the speed of 4000rpm for 10min at regular intervals, then taking 1mL of supernatant, measuring the absorbance of the solution at the position of 570nm by using a ninhydrin colorimetric method, and calculating the cumulative release amount of the DAA medicament. At the end of each centrifugation, the centrifugate was aspirated and then 5mL of purified water was added again for the release experiment.

The core-shell structure composite fiber based on halloysite prepared in the method is endowed with a composite fiber surface pore structure (shown in figure 1) by regulating and controlling the proportion of N, N-Dimethylformamide (DMF) and tetrahydrofuran in an organic solvent in a shell layer electrospinning solution, and the diameter of the composite fiber is changed by regulating and controlling the core-shell feeding speed. Finally, compared with the halloysite nanotube sustained release D-amino acid, the composite fiber material realizes the effective controlled release of the D-amino acid (see figure 4).

The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to be as broad in scope as possible. It is the intention of the applicant that the appended claims are not to be limited by the choice of examples of features of the invention.

Claims

1. A controlled release method of an environmental-friendly biomembrane inhibiting molecule D-amino acid is characterized in that,

selecting a natural halloysite nano material, modifying and expanding a halloysite inner cavity by sulfuric acid to load D-amino acid, and then constructing a composite fiber material with a core-shell structure by using a coaxial electrospinning method for the halloysite nanotube loaded with the D-amino acid and a polymer;

the specific preparation method of the composite fiber material with the core-shell structure comprises the following steps: (1) dissolving a hydrophobic polymer in an organic solvent to obtain a shell electrospinning solution; (2) blending a hydrophilic polymer and a D-amino acid loaded halloysite nanotube to obtain a kernel electrospinning solution; (3) performing coaxial electrostatic spinning on the shell electrospinning liquid and the core electrospinning liquid obtained in the steps (1) and (2); the hydrophobic polymer is polyvinylidene fluoride powder, and the hydrophilic polymer is polyacrylonitrile powder; the addition amount of the D-amino acid loaded halloysite is 2-10 wt% of the mass of the hydrophilic polymer powder;

the feeding speed of the core electrospinning liquid for coaxial electrospinning is 0.5-0.7mL/h or 0.9mL/h, and the feeding speed of the shell electrospinning liquid is 0.5-1.5 mL/h; electrospinning parameters: the voltage is 15-20KV, the temperature is 20-25 deg.C, and the humidity is 40-55%.

2. The method for controlled release of an environmentally friendly biofilm inhibiting molecule, D-amino acid, according to claim 1, wherein said D-amino acid is one or more of D-tyrosine, D-methionine, D-leucine and D-lysine.