CN111450309B

CN111450309B - Anti-infection silicon dioxide biological tissue adhesive and application thereof

Info

Publication number: CN111450309B
Application number: CN201910045956.6A
Authority: CN
Inventors: 高亦鲲; 王思玲; 林伟康; 刘迎春
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2019-01-18
Filing date: 2019-01-18
Publication date: 2022-06-07
Anticipated expiration: 2039-01-18
Also published as: CN111450309A

Abstract

The invention relates to a preparation method of a drug-loaded antibacterial nano mesoporous silica biological tissue adhesive and application of the adhesive in tissue adhesion. The preparation method of the drug-loaded mesoporous silica comprises the following steps: preparing oil-in-water emulsion by using n-octane, hexadecyl trimethyl ammonium bromide and water, polymerizing a styrene monomer and an ethyl orthosilicate monomer in oil phase droplets, and removing styrene through demulsification, centrifugation and high-temperature calcination to prepare mesoporous silica nanoparticles; adding the prepared silicon dioxide into lysozyme solution to load lysozyme, and centrifugally drying to prepare the anti-infection silicon dioxide biological tissue adhesive. The invention aims to solve the defects of secondary injury caused by surgical suture in wound treatment, the limitation that certain organs and tissues are not suitable for suture, poor effect of the traditional organic biological adhesive and the like. Has the advantages of rapid action, no secondary wound and the like, and has wide application prospect in the field of wound treatment.

Description

Anti-infection silicon dioxide biological tissue adhesive and application thereof

Technical Field

The invention belongs to the field of biomedicine, relates to a preparation method and application of a novel biological adhesive, and particularly relates to preparation and application of lysozyme-loaded anti-infection nano mesoporous silica.

Background

Chronic intractable wounds and injuries such as visceral surgery incisions place a heavy burden on the patient and the health care system. At present, surgical suture and polymer adhesive are generally used as treatment means for wounds caused by liver, lung, heart and other viscera operations. However, for soft tissues such as liver and lung, the surgical suture can cause certain damage to organs and leave incision gaps; polymeric binders have also limited their range of application due to their limited utility in a wet in vivo environment and the complexity of the conditions necessary to control the polymerization or crosslinking reaction in vivo. Research shows that under the condition of room temperature, one drop of silicon oxide nano particle solution can generate rapid and strong adhesion between two independent hydrogels through a 'nano bridging' effect, and the effect can still be verified on skin wound and visceral surgery wound models.

In addition, in most cases, wound and infection always occur simultaneously, and due to the influence of factors such as wound environment, the incidence rate of infection after wound is extremely high, and new secondary infection and cross infection can be caused in subsequent treatment. At present, the method commonly adopted for controlling bacterial infection is to apply antibiotics, but the antibacterial effect of the antibiotics on local wound surfaces is often limited, and meanwhile, the problems of drug resistance, toxic and side effects, abuse of the antibiotics and the like exist. Therefore, the development of a novel biological adhesive with antibacterial activity has important significance for clinical application. Lysozyme, also known as muramidase, is an alkaline enzyme that hydrolyzes mucopolysaccharides in pathogenic bacteria. The bacterial lysis is achieved by breaking the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, breaking down the insoluble mucopolysaccharide of the cell wall into soluble glycopeptides, which in turn leads to the escape of the contents of the broken cell wall. Lysozyme can also be directly combined with virus protein with negative charge, and forms double salt with DNA, RNA and apoprotein to inactivate virus. Therefore, the enzyme has the functions of antibiosis, antiphlogosis, antivirus, etc. The lysozyme is protein with relatively stable species and has relatively strong heat resistance; the lysozyme has low production cost and wide antibacterial spectrum, is not limited to gram-negative bacteria, and has an inhibiting effect on partial gram-positive bacteria.

In conclusion, the research establishes a novel biological adhesion treatment system based on the mesoporous silicon dioxide nano material, and the double functions of the mesoporous silicon nano particles on the nano bridging effect of the wound tissues and the antibacterial activity of lysozyme loaded on the wound tissues are utilized to promote incision and tissue adhesion and anti-infection repair.

Disclosure of Invention

The invention aims to provide an anti-infection silicon dioxide biological tissue adhesive, which is lysozyme-loaded mesoporous silicon dioxide nanoparticles with antibacterial activity, and adopts the following preparation technical scheme:

(1) cetyl Trimethyl Ammonium Bromide (CTAB) is stirred and dissolved in water at the temperature of 20-60 ℃, then n-octane is dropped in, and the stirring is continued to prepare an oil-in-water heterogeneous system.

(2) Tetraethoxysilane (TEOS) was dropped into the above system, and lysine was added.

(3) And (3) dripping a styrene monomer into the system in the step (2), and adding azodiisobutyramidine hydrochloride (AIBA) under the protection of nitrogen.

(4) After stirring the reaction mixture, the reaction mixture was cooled to room temperature and allowed to stand overnight.

(5) The reaction solution was mixed with absolute ethanol and centrifuged.

(6) And calcining the precipitate in a muffle furnace and grinding to obtain the mesoporous silica nanoparticles.

(7) The prepared mesoporous silica nanoparticles are dissolved in phosphate buffer solution of lysozyme with pH of 6.8 to obtain lysozyme-loaded mesoporous silica nanoparticles.

Wherein the content of the first and second substances,

n-octane in step (1): the mass ratio of the hexadecyl trimethyl ammonium bromide aqueous solution is 0.2-0.4;

the temperature in the step (1) is 20-60 ℃;

the concentration of the hexadecyl trimethyl ammonium bromide aqueous solution in the step (1) is as follows: 1-5 mg/mL.

The weight-volume ratio of lysine to tetraethoxysilane in the step (2) is (g/mL): 1:25 to 1:80, preferably 1:25-1: 50.

cetyl trimethyl ammonium bromide in step (3): the mass ratio of styrene monomer is 1: 15.15-40.45.

The final concentration of the azodiisobutyramidine hydrochloride added in the step (3) is as follows: 0.4-1.5 mg/mL.

The reaction time in the step (4) is 3-6 hours;

the volume ratio of the reaction liquid to the absolute ethyl alcohol in the step (5) is 1: 1; the centrifugal rotating speed is as follows: 5000-.

And (6) putting the precipitate into a muffle furnace, and calcining at 500-600 ℃ for 5-6 hours.

The concentration of lysozyme in the step (7) is as follows: 1-25mg/mL, the mesoporous silica nanoparticle concentration is: 0.5-15 mg/mL.

Specifically, the method comprises the following steps:

(1) cetyl trimethylammonium bromide (CTAB), a surfactant, is added to distilled water, stirred at 20-60 ℃ (preferably 60 ℃) for 2 hours, and then a certain amount of n-octane, n-octane: the mass ratio of the hexadecyl trimethyl ammonium bromide aqueous solution is 0.2-0.4 (preferably 0.4), the stirring is continued for 2 hours at the temperature, a water-CTAB-n-octane oil-in-water heterogeneous system is formed, and the concentration of the hexadecyl trimethyl ammonium bromide aqueous solution is as follows: 2.13X 10^-3g/mL。

(2) Tetraethoxysilane (TEOS) was dropped into the above system at a rate of 2 drops/sec, and lysine was added in a weight volume ratio (g/mL) of lysine to tetraethoxysilane: 1:48.5.

(3) And (3) dropping a styrene monomer into the system at the speed of 2 drops/second, wherein the concentration of the styrene monomer in the system is 0.219-0.585 mg/mL (preferably 0.585mg/mL), and adding the azo-bis-isobutyramidine hydrochloride AIBA under the condition of nitrogen protection. Ethyl orthosilicate: lysine: cetyl trimethylammonium bromide: the mass ratio of styrene monomer is 10: 0.22: 1: 15.15-40.45, preferably: 10: 0.22: 1: 40.45 of; the weight-volume ratio of the added azodiisobutyramidine hydrochloride in the volume is as follows: 0.84mg/mL

(4) Stirring for reaction for 3-6 hours (preferably 3 hours), cooling the reaction solution to room temperature, and standing overnight.

(5) The reaction solution of step (5) was mixed with absolute ethanol 1:1, centrifuged once at 12000rpm for 15 minutes, and the precipitate was mixed with 50mL of absolute ethanol, ultrasonically dispersed for 20 minutes, and centrifuged once again at 12000rpm for 15 minutes. The precipitate was air dried at room temperature.

(6) And placing the precipitate dried at room temperature into a muffle furnace, calcining at 500-600 ℃ for 5-6 hours, and grinding to obtain the mesoporous silica nanoparticles.

(7) The mesoporous silica nano-particle is dissolved in 5mg/mL lysozyme phosphate buffer solution with pH of 6.8, and the concentration of the mesoporous silica nano-particle is 2.5 mg/mL. Stirring at 200rpm for 3 hours at 4 ℃, and finally centrifuging at 8000rpm for 3 minutes to obtain the lysozyme-loaded mesoporous silica nanoparticles.

The mesoporous silica nanoparticles prepared by the method are monodisperse, the particle size is 97-550nm, the pore diameter is 5-11nm, and the specific surface area is 397-1059 m²Per g, preferably with a prescribed pore size of 10.49nm and a specific surface area of 610.69m²/g。

The drug loading rate of the mesoporous silica nanoparticle loaded with lysozyme protein prepared by the invention is 15-30%, the drug release rate reaches 20-50% within 5 hours, and meanwhile, the released drug still maintains higher activity. The maximum average anti-pulling force of the self-made PDMA bionic gel and the Wistar rat liver after adhesion respectively reaches 0.065 and 0.049N/cm²Mg, about 11 times and 8.5 times the average tensile resistance after adhesion of the commercially available non-porous silica nanoparticles TM-50. The bonding effect is obviously improved.

Drawings

FIG. 1 is a TEM micrograph of sample 3.

FIG. 2 is an SEM micrograph of sample 3.

Fig. 3 shows the results of

samples

2, 3, and 4 when the concentrations of lysozyme to the samples are 1:2, the drug loading temperature is 4 ℃, and the calculated loading curve is obtained within 3 hours under the condition of the rotation speed of 200 rpm.

FIG. 4 is a graph showing the percentage of drug released in 25mL of pH6.8 phosphate buffer in water bath at 37 ℃ for 5 hours for

samples

2, 3 and 4 after loading.

Fig. 5 is a schematic diagram of an apparatus for testing the tensile resistance using a tensile tester after bonding two PDMA hydrogels using sample 3.

Figure 6 is the average anti-pull force after bonding PDMA hydrogel or Wistar rat liver using sample 2, sample 3, sample 4, respectively.

FIG. 7 is a schematic diagram of an apparatus for testing anti-pull force using a tensile testing machine after adhering two Wistar rat livers using sample 3.

FIG. 8 shows the activity of lysozyme released within five hours from three drug-loaded samples in 25mL of pH6.8 phosphate buffer in a water bath at 37 ℃.

Detailed Description

EXAMPLE 1 preparation of mesoporous silica

Sample No. 1

Weighing 0.3g of CTAB into a 500mL three-necked bottle, adding 96mL of distilled water, protecting the interior of the reaction device with nitrogen, magnetically stirring at 70 ℃ for 1 hour to fully dissolve the CTAB, weighing 45mL of n-octane, and dripping into the CTAB solution system at the speed of 2 drops/second. Stirring was continued for 20 minutes after dropping to form a homogeneous emulsion. 3.2mL of tetraethylorthosilicate TEOS was measured and dropped into the above solution at a rate of 2 drops/sec. After dropping, 0.066g of lysine was weighed out and added to the above solution. Then, 8.9mL of methyl methacrylate monomer was measured and dropped into the above solution at a rate of 2 drops/sec. After dropping, 113.4mg of azobisisobutyramidine hydrochloride AIBA was weighed into the reaction solution. After the addition, the reaction was continued for 4 hours with magnetic stirring in a water bath at 70 ℃. And (3) stopping heating after the reaction is finished, transferring the reaction liquid to a beaker after the reaction liquid is cooled to room temperature, sealing the beaker by using a preservative film, and standing overnight. The next day, centrifugation at 15000rpm for 15 minutes resulted in no precipitate fraction and failed preparation.

Sample 2

0.3g CTAB was weighed into a 500mL three-necked flask, 93mL of distilled water was added, the mixture was magnetically stirred at 20 ℃ for 2 hours to be sufficiently dissolved, 42mL of n-octane was measured, and the CTAB solution system was dropped at a rate of 2 drops/sec. Stirring was continued for 2 hours after dropping to form a homogeneous emulsion. 3.2mL of tetraethylorthosilicate TEOS was measured and dropped into the above solution at a rate of 2 drops/sec. After dropping, 0.066g of lysine was weighed out and added to the above solution. Then, 8.9mL of styrene monomer was measured and dropped into the above solution at a rate of 2 drops/sec. After dropping, the inside of the reaction apparatus was protected with nitrogen, and 113.4mg of azobisisobutyramidine hydrochloride AIBA was weighed and added to the reaction solution. After the addition, the reaction was continued for 6 hours with magnetic stirring in a water bath at 60 ℃. And (3) stopping heating after the reaction is finished, transferring the reaction liquid to a beaker after the reaction liquid is cooled to room temperature, sealing the beaker by using a preservative film, and standing overnight. The next day, the ratio of 1: anhydrous ethanol was added at a volume ratio of 1, and after stirring well, centrifugation was carried out at 12000rpm for 15 minutes, and the resulting precipitate was sonicated using 50mL of anhydrous ethanol for 20 minutes, followed by further centrifugation at 12000rpm for 15 minutes. The supernatant was discarded and the precipitate was left to air dry at room temperature. After air drying, the precipitate was transferred to, for example, a ceramic crucible and placed in a muffle furnace, and calcined at 500 ℃ for 5 hours. And (3) fully grinding the solid matter after calcining and sintering to obtain the mesoporous silica nanoparticles.

The specific surface area of the sample is 1058.73m measured by a specific surface area and pore size distribution tester²The pore diameter per gram was 7.03 nm. The particle size of the sample was determined to be 550.3nm using a dynamic laser light scattering apparatus.

Sample 3

0.3g of cetyltrimethylammonium bromide CTAB is weighed into a 500mL three-necked flask, 93mL of distilled water is added, the mixture is magnetically stirred for 2 hours in a water bath at 60 ℃ to be fully dissolved, 42mL of n-octane is weighed, and the CTAB solution system is dripped into the mixture at the speed of 2 drops/second. Stirring was continued for 2 hours after dropping to form a homogeneous emulsion. 3.2mL of tetraethylorthosilicate TEOS was measured and dropped into the above solution at a rate of 2 drops/sec. After dropping, 0.066g of lysine was weighed out and added to the above solution. 13.35mL of styrene monomer was then metered in and dropped into the above solution at a rate of 2 drops/sec. After dropping, the inside of the reaction apparatus was protected with nitrogen, and 113.4mg of azobisisobutyramidine hydrochloride AIBA was weighed and added to the reaction solution. After the addition, the reaction was continued for 3 hours with magnetic stirring in a water bath at 60 ℃. And (3) stopping heating after the reaction is finished, transferring the reaction liquid to a beaker after the reaction liquid is cooled to room temperature, sealing the beaker by using a preservative film, and standing overnight. The next day, the ratio of 1: anhydrous ethanol was added at a volume ratio of 1, and after stirring well, centrifugation was carried out at 12000rpm for 15 minutes, and the resulting precipitate was sonicated using 50mL of anhydrous ethanol for 20 minutes, followed by further centrifugation at 12000rpm for 15 minutes. The supernatant was discarded and the precipitate was left to air dry at room temperature. After air drying, the precipitate was transferred to, for example, a ceramic crucible and placed in a muffle furnace, and calcined at 500 ℃ for 5 hours. And (3) fully grinding the solid matter after calcining and sintering to obtain the mesoporous silica nanoparticles.

The specific surface area of the sample is 610.69m by adopting a specific surface area and pore size distribution tester²Per g, pore size 10.49 nm. The particle size of the sample was determined to be 170.4nm using a dynamic laser light scattering apparatus. The clear mesoporous channel structure can be seen by observing the sample by adopting a transmission electron microscope and a scanning electron microscope. (see attached FIG. 1 and FIG. 2)

Sample No. 4

0.3g CTAB was weighed into a 500mL three-necked flask, 93mL of distilled water was added, the mixture was magnetically stirred at 20 ℃ for 2 hours to be sufficiently dissolved, 42mL of n-octane was measured, and the CTAB solution system was dropped at a rate of 2 drops/sec. Stirring was continued for 2 hours after dropping to form a homogeneous emulsion. 3.2mL of tetraethylorthosilicate TEOS was measured and dropped into the above solution at a rate of 2 drops/sec. After dropping, 0.066g of lysine was weighed out and added to the above solution. Then, 5.0mL of styrene monomer was measured and dropped into the above solution at a rate of 2 drops/sec. After dropping, the inside of the reaction apparatus was protected with nitrogen, and 113.4mg of azobisisobutyramidine hydrochloride AIBA was weighed and added to the reaction solution. After the addition, the reaction was continued for 6 hours with magnetic stirring in a water bath at 60 ℃. And (3) stopping heating after the reaction is finished, transferring the reaction liquid to a beaker after the reaction liquid is cooled to room temperature, sealing the beaker by using a preservative film, and standing overnight. The following day, the rate of 1: anhydrous ethanol was added at a volume ratio of 1, and after stirring well, centrifugation was carried out at 12000rpm for 15 minutes, and the resulting precipitate was sonicated using 50mL of anhydrous ethanol for 20 minutes, followed by further centrifugation at 12000rpm for 15 minutes. The supernatant was discarded and the precipitate was left to air dry at room temperature. After air drying, the precipitate was transferred to, for example, a ceramic crucible and placed in a muffle furnace, and calcined at 500 ℃ for 5 hours. And after calcining, fully grinding the solid matter to obtain the mesoporous silica nanoparticles.

The specific surface area of the sample is 397.53m measured by a specific surface area and pore size distribution tester²The pore diameter per gram was 5.25 nm. The particle size of the sample was measured to be 97.4nm using a dynamic laser scattering apparatus.

The differences in specific surface area, pore size and particle size of the different samples are mainly determined by the amount of styrene monomer and the reaction time after addition of styrene and AIBA when the ratio of hexaalkyltrimethylammonium bromide: the mass ratio of styrene monomer is 1: 15.15-40.45, when the reaction time is 3-6h, the aperture of the prepared sample can reach 5-11nm, the specific surface area can reach 397-1059nm, and the particle size can reach 97-550 nm.

EXAMPLE 2 preparation of lysozyme-loaded mesoporous silica nanoparticles

Weighing 40mg lysozyme in 10mL penicillin bottle, and adding 8mL pH6.8 PBS solution, at 4 degrees C under 200rpm magnetic stirring for 1 hours to fully dissolve the lysozyme. Then, 20mg of the mesoporous silica nanoparticles prepared in example 1 was weighed and placed in the lysozyme solution described above, and magnetic stirring was continued at 200rpm for 3 hours at 4 ℃. And after stirring, centrifuging at 8000rpm for 3 minutes, and removing supernatant to obtain the lysozyme protein loaded mesoporous silica nanoparticles.

In the preparation process of the mesoporous silica nanoparticle loaded with lysozyme, samples are taken at 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours and 3 hours respectively. The sampling mode is that 500 mul of stirring liquid is sucked and centrifuged for 3 minutes at 8000rpm, and then 200 mul of supernatant is taken and stored at 4 ℃ to be tested. Preparing a Coomassie brilliant blue G-250 staining solution, weighing 100mg of Coomassie brilliant blue G-250, placing into a 1000mL brown measuring flask, adding 50mL of 95% ethanol, ultrasonically dissolving, adding 100mL of 85% phosphoric acid, diluting to 1000mL with distilled water, mixing uniformly, and storing at 4 ℃ for later use.

Precisely absorbing 7 mu L of supernatant obtained at each time point, diluting the supernatant to 500 mu L by using phosphate buffer PBS (pH6.8), mixing the supernatant with 3mL of the Coomassie brilliant blue staining solution, shaking the mixture uniformly, dyeing the mixture for 3 minutes in a dark place, testing ultraviolet-visible absorbance at 595nm (500 mu L of phosphate buffer with pH6.8, which is dyed by an ultraviolet-visible absorbance photometer in the same way, correcting a zero point), substituting the obtained absorbance-lysozyme concentration standard curve drawn in advance, calculating the actual protein concentration and the mass of lysozyme not loaded in a solution system, and reversely deducing the mass of the loaded lysozyme.

After 3 hours of drug loading, the loading ratios of sample 2 and sample 3 to lysozyme reached 27.0% and 28.0%, respectively (the drug loading curves are shown in fig. 3).

Example 3 lysozyme-loaded mesoporous silica nanoparticle drug release assay

After loading lysozyme in example 2 for 3 hours, the remaining solution was centrifuged at 8000rpm for 3 minutes, the solid precipitate was transferred to a 50mL flat-bottomed flask, and 25mL phosphate buffer pH6.8 was added and the drug release was continued for 24 hours with magnetic stirring in a water bath at 37 ℃. Samples were taken at 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, and 5 hours, respectively. The sampling mode is that 1000 mul of stirring liquid is sucked and centrifuged for 3 minutes at 8000rpm, then 500 mul of supernatant is taken to be stored at 4 ℃ for testing, 500 mul of PBS with pH6.8 is added to the residual liquid and solid, then the mixture is blown evenly and added back to the drug release system again.

mu.L of the supernatant obtained at each time point was mixed with the Coomassie brilliant blue staining solution described in example 2, shaken well, and after 3 minutes of dark staining, the UV-visible absorbance was measured at 595nm (the UV-visible absorptiometry had used 500. mu.L of phosphate buffer pH6.8, which had been stained in the same way, to correct the zero point), and the absorbance-lysozyme concentration standard curve was plotted in advance to calculate the actual protein concentration and the amount of protein released in the solution system. After 5 hours of drug release, the release ratios of sample 2 and sample 3 to the loaded lysozyme reached 48.4% and 51.1%, respectively (the drug release curves are shown in figure 4).

EXAMPLE 4 mesoporous silica nanoparticle bonded biomimetic hydrogel PDMA anti-pulling test

The poly-N, N ' -dimethylacrylamide hydrogel PDMA was prepared by mixing 42.5mL of distilled water with 6.8mL of N, N ' -dimethylacrylamide monomer, adding 9.2mg of N, N ' -methylenebisacrylamide MBA and 164mg of potassium persulfate KPS under magnetic stirring at 25 ℃ and nitrogen protection, stirring thoroughly for 5 minutes, adding 90. mu.L of tetramethylethylenediamine TEMED to the reaction solution system, stirring rapidly for 20 seconds, transferring the reaction solution to a 10cm X10 cm plastic petri dish, and standing horizontally overnight under nitrogen protection.

The following day, the PDMA hydrogel was diced in pieces (length × width × thickness) 5cm × 1.5cm × 0.5cm and weighed. Sample 2 and sample 3 carrying lysozyme were measured at 2mg/cm²Uniformly spreading on the surface of the PDMA cut block, and covering a rectangular surface (length. times. width) of 0.7 cm. times.1.5 cm. Another piece of PDMA was overlaid and bonded on the above covered rectangular surface, and pressed for 30 seconds. The PDMA bonded by the above method was vertically fixed in a tensile tester (see fig. 5), and the maximum anti-pull force until slipping was tested, and the actual anti-pull force was obtained by summing the maximum anti-pull force and the gravity of a single PDMA hydrogel.

The maximum tensile resistance after PDMA bonding of sample 2 and sample 3 reaches 0.050 and 0.065N/cm²Mg. (FIG. 6)

Example 5 mesoporous silica nanoparticle-bonded liver anti-drag test

Male Wistar rats weighing 300g were fasted for 12 hours, and right liver lobes were picked after dislocation of cervical vertebrae, and after hemostasis with gauze, width and thickness of liver lobes were recorded, and the cross-sectional area was estimated as half the width x thickness. The liver was bisected by a longitudinal cut at the length of the liver 1/2.

Samples

2 and 3 were measured at 2mg/cm²Spreading on the cut surface of the liver. The two sections were superimposed and pressed for 1 minute. The adhered liver lobes were mounted vertically in a tensile tester (see fig. 7) and tested for maximum tensile resistance until breakage. And after the test is finished, weighing and recording the mass of the half liver lobe positioned below the liver lobe during the test, wherein the actual anti-pulling force is obtained by adding the maximum anti-pulling force and the gravity of the half liver lobe.

The maximum anti-pulling force of sample 2 and sample 3 carrying lysozyme after being adhered to the right side liver lobe of male Wistar rat reaches 0.026 and 0.049N/cm respectively²Mg. (FIG. 6)

EXAMPLE 6 measurement of antimicrobial Activity of lysozyme released from mesoporous silica nanoparticles

10.4g of sodium dihydrogen phosphate, 7.86g of disodium hydrogen phosphate and 0.37g of ethylenediaminetetraacetic acid were weighed and dissolved in 1000mL of distilled water to obtain phosphate buffered saline PBS having a pH of 6.2.

Taking the supernatant with the drug release time of 1 hour, 2 hours, 3 hours, 4 hours and 5 hours respectively, and diluting until the protein concentration is 50 mug/mL to be detected.

2.5mg of lysozyme Lys is precisely weighed and placed in a 50mL measuring flask, phosphate buffer solution is added for dissolution, and the volume is fixed. 200mg of Micrococcus muralis is weighed, about 3mL of phosphate buffer (pH 6.2) is added, grinding is carried out for about 3min, phosphate buffer is added to the total volume of 200mL, equilibrium heat preservation is carried out in a water bath at 25 ℃, 3mL is precisely measured in a 1cm colorimetric cell, and absorbance is measured at a wavelength of 450nm as a reading a of 0 second. Accurately weighing 0.15mL of test solution which is subjected to balance heat preservation in a water bath at 25 ℃, adding the test solution into the colorimetric pool, quickly mixing the test solution uniformly, timing by using a stopwatch, and measuring the absorbance A when the time is 60 seconds. Meanwhile, 0.15mL of phosphate buffer (pH 6.2) was measured precisely, and the same procedure was followed to obtain a blank, and a reading a 'at 0s and a reading A' at 60s were measured, and the titer was calculated by dividing the 60-second drop by the sample volume and corrected by a blank experiment. The calculation formula of the lysozyme activity is as follows:

the lysozyme released by the two drug-loaded mesoporous silica nanoparticles still keeps higher bacteriostatic activity (figure 7).

Claims

1. An anti-infective mesoporous silica biological tissue adhesive, characterized in that: the anti-infection mesoporous silica biological tissue adhesive is prepared by the following method:

weighing 0.3g of Cetyl Trimethyl Ammonium Bromide (CTAB) into a 500mL three-necked bottle, adding 93mL of distilled water, magnetically stirring for 2 hours in a water bath at 60 ℃ to fully dissolve the CTAB, then measuring 42mL of n-octane, dripping into the CTAB solution system at the speed of 2 drops/second, and continuously stirring for 2 hours after dripping to form uniform emulsion; weighing 3.2mL of tetraethoxysilane TE0S, dripping into the solution at the speed of 2 drops/second, weighing 0.066g of lysine into the solution after dripping is finished, then weighing 13.35mL of styrene monomer, dripping into the solution at the speed of 2 drops/second, carrying out nitrogen protection in a reaction device after dripping is finished, and then weighing 113.4mg of azodiisobutyl amidine hydrochloride AIBA into the reaction solution; after the addition, the reaction is continued for 3 hours under the water bath of 60 ℃ by magnetic stirring; stopping heating after the reaction is finished, transferring the reaction liquid to a beaker after the reaction liquid is cooled to room temperature, sealing the beaker by using a preservative film, and standing overnight; the next day, the ratio of 1: adding absolute ethyl alcohol into the mixture in a volume ratio of 1, fully stirring the mixture, centrifuging the mixture for 15 minutes at 12000rpm, performing ultrasonic treatment on the precipitate obtained by centrifuging the mixture for 20 minutes by using 50mL of absolute ethyl alcohol, and continuing to centrifuge the precipitate for 15 minutes at 12000 pm; discarding the supernatant, placing the precipitate at room temperature for airing, transferring the precipitate into a ceramic crucible after airing, placing the ceramic crucible into a muffle furnace, calcining for 5 hours at 500 ℃, and fully grinding the solid matter after calcining to obtain mesoporous silica nanoparticles; the prepared mesoporous silica nanoparticles are dissolved in a phosphate buffer solution of lysozyme with the pH value of 6.8 to obtain the lysozyme-loaded mesoporous silica nanoparticles.

2. The anti-infective mesoporous silica biological tissue adhesive of claim 1, wherein: the concentration of lysozyme is: 1-25mg/mL, the mesoporous silica nanoparticle concentration is: 0.5-15 mg/mL.

3. Use of the anti-infective mesoporous silica biological tissue adhesive of claim 1 or 2 for the preparation of a biological adhesive for promoting incision, tissue adhesion and anti-infective repair.