CN106693072B - Preparation method of infection response type guided tissue regeneration membrane - Google Patents

Abstract

The invention discloses a preparation method of an infection-responsive guided tissue regeneration membrane. The matrix material of the guiding tissue regeneration membrane is a degradable polyester material and a natural degradable polymer material, and an ester bond is used to chemically combine the antibacterial drug with a hydroxyl group to the surface of the guiding tissue regeneration membrane. The specific method is: by dopamine coating, a large number of hydroxyl groups are introduced on the surface of the membrane; the silicon-oxygen bond on the silane coupling agent reacts with the hydroxyl groups on the surface of the membrane, so as to introduce amino groups on the surface of the membrane; then the antibacterial drugs with hydroxyl groups are reacted with acryloyl chloride , introducing an ester bond and a carbon-carbon double bond on the drug; finally, the carbon-carbon double bond and the amino group on the surface of the membrane are subjected to Michael addition reaction, so that the drug can be grafted to the surface of the fiber membrane through the ester bond. The infection-responsive guided tissue regeneration membrane of the present invention has excellent biocompatibility and the ability to controllably release antibacterial drugs according to the degree of infection, thereby achieving the purpose of effectively inhibiting infection.

Description

A kind of preparation method of infection-responsive guided tissue regeneration membrane

technical field

The invention belongs to the field of material surface modification, and in particular relates to a preparation method of an infection-responsive guided tissue regeneration membrane that releases antibacterial drugs.

Background technique

Guide Tissue Regeneration (GTR) technology is a new technology developed in the late 1980s and early 1990s. The principle is to use the physical barrier function of the membrane to isolate the damaged part from the surrounding tissue, creating a relatively closed tissue environment, so that osteoblasts can migrate and grow preferentially. The application of GTR provides a new and effective way for the treatment of periodontal disease, the restoration of insufficient bone mass in the dental implant area and other bone defects, and the healing of fractures.

Although GTR technology can achieve long-term and stable curative effect, the infection caused by anaerobic bacteria and aerobic bacteria introduced into the injury site will affect the acquisition of new tissue during and after surgery and during the secondary removal process. At present, systemic drugs are mainly used for postoperative anti-infection, but the drug utilization rate is low, and it is easy to cause gastrointestinal side effects. Local drug delivery can enhance the therapeutic effect and reduce adverse reactions.

The drug blending method is often accompanied by the phenomenon of drug burst release, which cannot achieve the purpose of sustained drug release. The continuous release of the drug can be achieved by the covalent bond loading method, and the relevant chemical bonds can be selectively cut off by pH or enzymes, and the local target The drug release can enhance the therapeutic effect, thereby reducing adverse reactions.

When the infection occurs, a large number of macrophages will aggregate, and the aggregated macrophages will secrete a large amount of cholesterol esterase (CE). And ester bonds have selective enzymatic hydrolysis. Grafting of drugs to the surface of the guide membrane through ester bonds is responsive to enzymes produced during infection. When the infection occurs, the ester bond is broken under the action of the enzyme, and the drug is released; the higher the infection degree, the more macrophages gathered around, the higher the content of the released enzyme, and the more the amount of the drug released. Using the selective enzymatic hydrolysis of ester bonds by CE, and immobilizing drug molecules on the surface of the guiding tissue regeneration membrane through ester bonds, it is possible to construct a guiding tissue regeneration membrane that can release drugs in response to infection. So as to overcome the clinical differences of different patients' onset time and severity, and to effectively prevent and suppress infection.

Dopamine is widely used in material surface modification. For hydrophobic materials, through dopamine modification, a large number of functional groups: amino groups (-NH ₂ ) and hydroxyl groups (-OH) can be introduced on the surface of hydrophobic materials, which greatly improves the hydrophilicity of the hydrophobic surface.

SUMMARY OF THE INVENTION

1. A method for surface modification of an infection-responsive guided tissue regeneration membrane, characterized in that: using degradable aliphatic polyester and degradable natural polymer guided tissue regeneration membrane as matrix materials, and by a series of chemical modification methods, the antibacterial The drug is grafted on the membrane surface by ester bond, and the modified membrane surface has the effect of responsive drug release to the esterase secreted by the body under the physiological environment of infection.

The specific method is as follows: by coating with dopamine, a hydroxyl group is introduced on the surface of the film; the silicon-oxygen bond on the silane coupling agent reacts with the hydroxyl group on the surface of the film, thereby introducing an amino group on the surface of the film; An ester bond and a carbon-carbon double bond are introduced into the drug; finally, the carbon-carbon double bond and the amino group on the surface of the membrane are subjected to Michael addition reaction, so that the drug can be grafted to the surface of the fiber membrane through the ester bond.

2. a series of described chemical modification methods are characterized in that, its concrete operation steps are:

(1) Soak the guiding tissue regeneration membrane in 0.05g/L-10g/L dopamine solution (dopamine hydrochloride: tris(hydroxymethylaminomethane)=5:3 (molar ratio), pH=5-10), After stirring and reacting for 6-48 hours, the membrane was taken out, soaked and washed to remove the unreacted dopamine solution.

(2) Immerse the guided tissue regeneration membrane obtained in step (1) in a silane coupling agent solution with a concentration of 1.25g/L-5.0g/L, and stir under a magnetic stirrer at a temperature of 25°C-50°C After the reaction was carried out for 3h-72h, the membrane was taken out, and the unreacted silane coupling agent was removed by dipping.

(3) The antibacterial drug is reacted with acryloyl chloride to introduce an ester bond on the metronidazole molecule. The reaction process is as follows: ① Weigh the medicine metronidazole: acryloyl chloride: triethylamine=1:1:1 (molar ratio) and chloroform. ② Dilute acryloyl chloride with chloroform and transfer it to a constant pressure dropping funnel; transfer the weighed metronidazole, triethylamine and chloroform into a container. ③ Pass N ₂ to make the whole system in the atmosphere of N ₂ . ④Put the container in an ice bath, and dropwise add the mixed solution of acryloyl chloride and chloroform with a volume ratio of 1:15-1:20; after dropping, place it in the ice bath for 20min-30min, remove the ice bath, and react at room temperature 12h. ⑤ Add 2ml-5ml of deionized water dropwise to the container to terminate the reaction. ⑥ Transfer the mixed liquid in the container into a separating funnel and extract with dichloromethane. ⑦Add enough anhydrous Na ₂ SO ₄ to the solution obtained from the extraction until the liquid is clear, and let it stand for 2-3 hours; the obtained solution is rotary-evaporated at 30°C-40°C to obtain the desired product.

(4) Soak the guided tissue regeneration membrane obtained in step (2) in a system of methanol:water (volume ratio)=4:1, weigh the product obtained in step (3) and dissolve it, at 30°C- Under the condition of 50 ℃, stirring continuously, the reaction was carried out for 6h-72h. The membrane is taken out, and the unreacted product obtained in step (3) is removed by dipping.

3. The guided tissue regeneration membrane has a thickness of 100-500 μm, a non-porous or porous structure, and a pore size of 1-10 μm. The preparation methods include but are not limited to: electrospinning, melt casting and vacuum molding.

4. The prepared material is degradable aliphatic polyester, including: polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-caprolactone copolymer, polylactic acid-glycolic acid-caprolactone copolymer The degradable natural macromolecular materials include: type I collagen, gelatin, chitosan, starch, cellulose, elastin, or a mixture of two or more.

5. described antibacterial drug is characterized in that, contains hydroxyl (-OH) in structure, is easily soluble in organic solvent, and has bacteriostatic or bactericidal properties, including but not limited to: metronidazole, erythromycin, piperazine cillin, chloramphenicol, etc.

6. The related enzyme, characterized in that the enzymes that are sensitive to ester groups secreted by the body under the physiological reaction of infection include but are not limited to: cholesterol esterase and phospholipase A2 (PLA2).

7. The silane coupling agent, characterized in that the structure contains amino groups (-NH ₂ ) and alkoxy groups, including but not limited to: γ-aminopropyltriethoxysilane (KH550), 3-amino Propyltrimethoxysilane (A-1110), N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane, etc. (A-1120).

Description of drawings

Figure 1 is a schematic diagram of the reaction steps of the electrospinning polycaprolactone fiber membrane surface grafting metronidazole.

Figure 2 is the SEM images of the surface morphology of the membrane before and after modification; the left image is the guided tissue regeneration membrane before modification, and the right image is the modified guided tissue regeneration membrane.

Figure 3 is the infrared spectrum of the membrane obtained by each step of the reaction. Wherein: a refers to the polycaprolactone electrospinning film; b refers to the polycaprolactone electrospinning film coated with dopamine; c refers to the polycaprolactone obtained by modification of the silane coupling agent KH550 on the basis of b Ester electrospinning film; d represents the film obtained by grafting the drug metronidazole (MNA) onto polycaprolactone electrospinning film through Michael addition reaction on the basis of c.

Comparing the FTIR curves of a and b, b has an indole structure peak in polydopamine at the position of 1620cm ^-1 , and the absorption peak of hydroxyl (-OH) clearly appears at the wavenumber of 3000-3500cm ^-1 , indicating that the dopamine After soaking in the solution, the fibers were coated with a polydopamine shell.

Comparing b and c, the absorption peak of hydroxyl (-OH) disappears at 3000-3500 cm ^-1 wavenumber. Because the silane coupling agent KH550 is grafted onto the polycaprolactone nanofibers through the reaction between the ethoxy group on KH550 and the hydroxyl group (-OH) on the polydopamine shell layer, the absorption peak of the hydroxyl group (-OH) disappears , indicating that b is grafted with silane coupling agent KH550. At the same time, the absorption peak of Si-O-Si bond at the wavenumber of 1000-1100cm ^-1 indicates that KH550 has been grafted onto the polycaprolactone nanofibers.

According to the data, the antisymmetric stretching vibration peak of the nitro group of aliphatic nitro compounds is 1565-1530cm ^-1 (s); the symmetric stretching vibration peak: 1380-1340cm ^-1 (s), while comparing c and d, there are obvious The difference shows that the drug metronidazole is grafted on.

Figure 4 is the drug release curve at different CE enzyme concentrations. With the increase of enzyme concentration, the drug release rate increased significantly.

Detailed ways

The present invention is further described below through specific examples, but is not limited to the following examples. After reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, but all within the spirit and principle of the present invention, the changes, modifications, etc. of these equivalent forms should be included in the present invention within the scope of protection.

Example 1

(1) Cut the PCL nanofibers prepared by electrospinning into discs with a diameter of 2 cm, and soak them in 1.0 g/L dopamine solution (with dopamine hydrochloride: tris(hydroxymethylaminomethane)=5:3 (molar ratio) ), the pH value of the solution is 8.5), after 12 hours of reaction under stirring, the spinning membrane is taken out, and the unreacted dopamine solution is removed by dipping.

(2) The polycaprolactone nanofibers prepared by electrospinning obtained in step (1) were soaked in a 5.0 g/L silane coupling agent KH550 solution, reacted at 40° C. for 24 h respectively, and the spinning The silk membrane was taken out, and the unreacted KH550 was removed by dipping.

(3) introduce ester bond on drug molecule: metronidazole reacts with acryloyl chloride, thereby introducing ester bond into drug metronidazole; reaction process is as follows: 1. take by weighing drug metronidazole: acryloyl chloride: triethylamine=1 : 1:1 (molar ratio), chloroform. ② Dilute acryloyl chloride with chloroform and transfer it to a constant pressure dropping funnel; transfer the weighed metronidazole, triethylamine and chloroform into an eggplant-shaped bottle and set up the device. ③ Pass N ₂ to make the whole system in the atmosphere of N ₂ . ④Put the eggplant-shaped bottle in the ice bath, add a magnet and keep stirring, and dropwise add the mixed solution of acryloyl chloride and chloroform; after the drop, take the ice bath for 30 minutes, remove the ice bath, and react at room temperature for 12 hours. ⑤ Add deionized water dropwise to the eggplant-shaped bottle to terminate the reaction. ⑥ Transfer the mixed liquid in the eggplant-shaped bottle into a separating funnel and extract with dichloromethane. ⑦Add anhydrous Na ₂ SO ₄ to the solution obtained by extraction until the liquid is clear, and let it stand for 3 hours; the obtained solution is rotary-evaporated at 30°C-40°C to obtain the desired product.

(4) soak the fiber membrane obtained in step (2) in a system of methanol and water with a volume ratio of 4:1, take by weighing the product obtained in step (3), dissolve and add, under the condition of 40 ° C, keep stirring, Reaction for 24h. The spinning membrane is taken out, and the unreacted product obtained in step (3) is removed by dipping.

Example 2

The polycaprolactone nanofibers in Example 1 were replaced with cellulose films, and other experimental conditions were the same as those in Example 1. By comparing the SEM images of the fibrous membrane a before dopamine coating and the fibrous membrane b after dopamine coating, it can be found that the surface of b is coated with a layer, and with the prolongation of immersion in dopamine, the coating amount increases, indicating that dopamine is successful Grafted to the surface of the fiber membrane; the fiber membrane after grafting modification by KH550 is denoted as c. Comparing the infrared spectra of b and c, the absorption peak of hydroxyl (-OH) disappears at the wave number of 3000-3500cm ^-1 , indicating that KH550 The ethoxy group on KH550 reacts with the hydroxyl group (-OH) on the polydopamine shell and is grafted to the fiber membrane, so that the absorption peak of the hydroxyl group (-OH) disappears; ^Si- The absorption peak of the O-Si bond indicates that KH550 has been grafted to the fiber membrane; the fiber membrane d is obtained after drug grafting and modification, and the drug release curve is measured. The modified membrane d releases the drug under the condition of enzyme, The drug was not released without the enzyme, indicating that the infection-responsive drug release effect was achieved.

Example 3

The γ-aminopropyltriethoxysilane (KH550) in Example 1 was replaced with 3-aminopropyltrimethoxysilane (A-1110), and other experimental conditions were the same as those in Example 1. Comparing the infrared spectra of the polycaprolactone nanofiber film obtained by dopamine coating and the polycaprolactone nanofiber film obtained by further grafting modification with A-1110, hydroxyl groups (-OH) at 3000-3500cm ^-1 wavenumber The absorption peak disappeared, indicating that 3-aminopropyltrimethoxysilane (A-1110) was grafted to the polycaprolactone nanoparticle through the reaction between the methoxy group on A-1110 and the hydroxyl group (-OH) on the polydopamine shell layer. Therefore, the absorption peak of hydroxyl (-OH) disappears; the absorption peak of Si-O-Si bond is at the wavenumber of 1000-1100 cm ^-1 , indicating that A-1110 has been grafted onto polycaprolactone nanofibers.

Example 4

Infection-responsive surface modification of the polycaprolactone-guided tissue regeneration film obtained by vacuum molding: the polycaprolactone electrospinning film in Example 1 was replaced with a polycaprolactone-guided tissue regeneration film prepared by vacuum molding, Other experimental conditions were the same as in Example 1. Note: a refers to the polycaprolactone-guided tissue regeneration membrane obtained by vacuum molding; b refers to the dopamine-coated polycaprolactone-guided tissue regeneration membrane; c refers to b, modified by silane coupling agent KH550 The obtained polycaprolactone guided tissue regeneration membrane; d represents the membrane obtained by grafting the drug metronidazole (MNA) onto the polycaprolactone guided tissue regeneration membrane through Michael addition reaction on the basis of c. By comparing the SEM images of the polycaprolactone-guided tissue regeneration membrane before and after dopamine coating, it can be found that the surface of the polycaprolactone-guided tissue regeneration membrane is coated with a layer, and with the prolongation of immersion in dopamine, the coating amount increases , indicating that dopamine was successfully grafted to the surface of the polycaprolactone-guided tissue regeneration membrane; compared with the KH550 grafted modified polycaprolactone-guided tissue regeneration membrane, compared with the infrared spectra of b and c, at 3000-3500cm ^-1 wavenumber The absorption peak of hydroxyl (-OH) disappears, indicating that KH550 is grafted to the polycaprolactone-guided tissue regeneration membrane through the reaction between the ethoxy group on KH550 and the hydroxyl group (-OH) on the polydopamine shell, thus making the hydroxyl group The absorption peak of (-OH) disappears; the wavenumber of 1000-1100 cm ^-1 is the absorption peak of Si-O-Si bond, indicating that KH550 has been grafted onto the polycaprolactone-guided tissue regeneration membrane; after drug grafting modification, it is obtained d, Determination of drug release curve, the modified film released the drug under the condition of enzyme, but not without enzyme, indicating that the effect of infection-responsive drug release was achieved.

Example 5

The antibacterial drug metronidazole in Example 1 was replaced with erythromycin, and other experimental conditions were the same as those in Example 1. The mass spectrum and NMR spectrum of the reaction product of erythromycin and acryloyl chloride were measured, and it was found that a substance with a molecular weight of 787 appeared in the mass spectrum, and by comparing the chemical shift of the corresponding H in the NMR spectrum, it was confirmed that the desired product was obtained. The modified membrane was tested for drug release, and the modified membrane released the drug erythromycin under the condition of enzyme, but did not release the drug under the condition of no enzyme.

Example 6

The antibacterial drug metronidazole in the embodiment 1 is replaced with chloramphenicol, and an ester bond is introduced on the drug molecule: chloramphenicol reacts with acryloyl chloride, thereby the ester bond is introduced into the drug chloramphenicol; The reaction process is as follows: 1. say Take medicine chloramphenicol: acryloyl chloride: triethylamine=1:1:1 (molar ratio) and acetone. ② Dilute acryloyl chloride with acetone and transfer it to a constant pressure dropping funnel; transfer the weighed chloramphenicol, triethylamine and acetone into an eggplant-shaped bottle, and set up the device. ③ Pass N ₂ to make the whole system in the atmosphere of N ₂ . ④ As for the eggplant-shaped bottle in the ice bath, add a magnet and keep stirring, and drop the mixed solution of acryloyl chloride and acetone; ⑤ Add deionized water dropwise to the eggplant-shaped bottle to terminate the reaction. ⑥ Add the mixed liquid in the eggplant-shaped bottle dropwise to deionized water whose volume is 10-15 times the volume of the mixed solution to precipitate, filter the precipitate, and wash it with deionized water for several times to remove residual acryloyl chloride and triethylamine. The product was obtained by drying at 50°C. Other experimental conditions are the same as in Example 1. The mass spectrum and NMR of the reaction product of chloramphenicol and acryloyl chloride were measured, and it was found that a substance with a molecular weight of 377.12 appeared in the mass spectrum, and by comparing the chemical shift of the corresponding H in the NMR image, it was confirmed that the desired product was obtained. The modified membrane was tested for drug release, and the modified membrane released the drug chloramphenicol under the condition of enzyme, but did not release the drug under the condition of no enzyme.

Claims (6)

1. An infection response type guided tissue regeneration membrane surface modification method is characterized in that: the degradable aliphatic polyester or degradable natural polymer guided tissue regeneration membrane is taken as a matrix material, and hydroxyl is introduced on the surface of the membrane through dopamine coating; leading amino on the surface of the film through the reaction of a silicon-oxygen bond on the silane coupling agent and hydroxyl on the surface of the film; then, the antibacterial drug with hydroxyl reacts with acryloyl chloride, and ester bonds and carbon-carbon double bonds are introduced into the drug; and finally, carrying out Michael addition reaction on the carbon-carbon double bond and the amino on the surface of the membrane, thereby grafting the medicine to the surface of the fiber membrane through an ester bond.

2. The method according to claim 1, characterized in that it comprises the following specific operating steps:

(1) soaking the guided tissue regeneration membrane in 0.05-10 g/L dopamine solution, stirring and reacting for 6-48h, taking out the membrane, soaking and cleaning to remove unreacted dopamine solution; dopamine hydrochloride in dopamine solution: tris-hydroxymethyl aminomethane in a 5: 3, pH is 5-10;

(2) soaking the guided tissue regeneration membrane obtained in the step (1) in a silane coupling agent solution with the concentration of 1.25-5.0 g/L, stirring and reacting for 3-72 h at the temperature of 25-50 ℃ under a magnetic stirrer, taking out the membrane, and washing to remove the unreacted silane coupling agent;

(3) the antibacterial drug and acryloyl chloride are reacted to introduce ester bonds on metronidazole molecules, the reaction process is that ① weighs the medicines metronidazole, acryloyl chloride and triethylamine are 1:1:1, the molar ratio is the ratio, ② dilutes acryloyl chloride with trichloromethane, transfers the acryloyl chloride to a constant pressure dropping funnel, the weighed metronidazole, triethylamine and trichloromethane are transferred to a container, ③ leads N to₂So that the whole system is N₂④ placing the container in ice bath, dropping the mixed solution of acryloyl chloride and trichloromethane in the volume ratio of 1:15-1:20, placing the container in ice bath for 20min-30min after dropping, removing ice bath, reacting at normal temperature for 12h, dropping deionized water into the container to terminate the reaction ⑤, ⑥ transferring the mixed solution in the container into a separating funnel, extracting with dichloromethane, ⑦ adding anhydrous Na into the solution obtained by extraction₂SO₄Standing for 2-3h after the liquid is clear; the obtained solution is steamed in a rotary manner at the temperature of 30-40 ℃ to obtain a required product;

(4) soaking the guided tissue regeneration membrane obtained in the step (2) in methanol: weighing the product obtained in the step (3) and dissolving the product in a system with the water ratio of 4:1 and the volume ratio, continuously stirring the mixture at the temperature of between 30 and 50 ℃, and reacting for 6 to 72 hours; and (4) taking out the membrane, and washing to remove the unreacted product obtained in the step (3).

3. The method as claimed in claim 1, wherein the guided tissue regeneration membrane has a thickness of 100-500 μm, a non-porous or porous structure, and a pore size of 1-10 μm, and is prepared by electrospinning, melt casting, or vacuum molding.

4. The method of claim 1, wherein the degradable aliphatic polyester comprises: one or a mixture of more than two of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-caprolactone copolymer and polylactic acid-glycolic acid-caprolactone copolymer; the degradable natural polymer material comprises: one or a mixture of more than two of type I collagen, gelatin, chitosan, starch, cellulose and elastin.

5. The method of claim 1, wherein the antimicrobial agent comprises metronidazole, erythromycin, piperacillin, chloramphenicol.

6. The method of claim 1, wherein the silane coupling agent comprises: gamma-aminopropyltriethoxysilane (KH550), 3-aminopropyltrimethoxysilane (A-1110) or N-beta- (aminoethyl) -gamma-aminopropyltrimethoxysilane (A-1120).

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