CN112316158B - Method for closing antibacterial agent activity in collagen solution by using supermolecule encapsulating agent - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 22
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- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims abstract description 31
- 229960003405 ciprofloxacin Drugs 0.000 claims abstract description 16
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 claims abstract description 12
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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Abstract
本发明公开了一种利用超分子包合剂关闭胶原溶液中抗菌剂活性的方法。该方法将金刚烷胺偶联于环丙沙星哌嗪环上的仲胺基团，利用葫芦[7]脲及其衍生物与金刚烷胺间特异性超分子包合作用，阻止环丙沙星通过革兰氏阴性菌外膜孔蛋白OmpF通道跨外膜转运，实现胶原溶液所含抗菌剂活性按需关闭，以协调胶原溶液的储存稳定性和使用安全性，解决天然胶原抗菌溶液防腐必要性与维持生物相容性完整间由来已久的矛盾，为突破医用胶原溶液的应用限制奠定基础。
The invention discloses a method for shutting down the activity of antibacterial agent in collagen solution by using supramolecular inclusion agent. In this method, amantadine is coupled to the secondary amine group on the piperazine ring of ciprofloxacin, and the specific supramolecular inclusion between cucurbit[7] urea and its derivatives and amantadine is used to prevent ciprofloxacin Stars are transported across the outer membrane through the outer membrane porin OmpF channel of Gram-negative bacteria, and the antibacterial agent activity contained in the collagen solution can be closed on demand, so as to coordinate the storage stability and safety of the collagen solution, and solve the need for antiseptic solutions of natural collagen antibacterial solutions. The long-standing conflict between sex and maintaining the integrity of biocompatibility lays the foundation for breaking through the limitations of medical collagen solutions.
Description
Technical Field
The invention relates to a method for closing the activity of an antibacterial agent in a collagen solution by using a supramolecular encapsulating agent, belonging to the field of biomass materials.
Background
Collagen is a renewable resource with unique structure and multiple functions, has excellent biocompatibility compared with other industrial materials, and is widely applied in clinical scenes such as injection wrinkle removal, ophthalmic treatment and the like after being prepared into solution. However, natural collagen is rich in nutrition, is a good carrier for the growth of microorganisms, and has the defect of easy mildew growth. Therefore, the antibacterial and antiseptic properties are necessary prerequisites for high-value utilization of collagen solution in the biomedical field.
In order to prevent the collagen solution from mildewing and growing, the most common method is to add an antibacterial mildew preventive externally. However, the traditional antibacterial mildew preventive has low biological selectivity, can kill harmful microorganisms such as bacteria and the like, has toxicity to eukaryotic cells, can cause adverse reactions such as allergy, inflammation and even nerve injury, and can damage the inherent biocompatibility of collagen. Therefore, the contradiction exists between the necessity of the antibiosis and mildew prevention of the collagen solution and the maintenance of the biocompatibility integrity for a long time; how to close the activity of the contained antibacterial agent before clinical application so as to avoid toxic and side effects of the antibacterial agent is a key and difficulty for fully utilizing the inherent biocompatibility of the medical collagen solution.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides a method for closing the activity of an antibacterial agent in a collagen solution by using a supramolecular encapsulating agent, which is characterized in that the method comprises the following synthesis steps and conditions, and the following materials are used in parts by weight:
(1) dispersing 1-5 parts of ciprofloxacin in 30-80 parts of solvent, adding 40-80 parts of distilled water dissolved with 1-8 parts of catalyst, and uniformly stirring to obtain a component 1; meanwhile, 4-25 parts of connecting agent is dissolved in 10-50 parts of solvent, slowly added into the component 1 within 1-2 hours at 0-5 ℃, continuously stirred for 1-4 hours, heated to normal temperature and stirred for 12-14 hours; finally, washing the product with 5-10% acid water solution and distilled water in turn, taking an organic layer, and drying the organic layer in vacuum to constant weight to obtain a first-step synthesized product;
(2) dissolving 1-3 parts of the first-step synthesized product in 10-40 parts of solvent, adding 2-5 parts of amantadine into the solution, continuously stirring for 12-14 hours at 40-60 ℃, and drying the obtained product in vacuum to constant weight to obtain a target product;
(3) adding 0.1-0.5 part of the target product into 100 parts of collagen solution, and continuously stirring for 30-60 minutes at 4-25 ℃ to prepare antibacterial collagen solution which can prevent the collagen from mildewing and growing bacteria in the storage process;
(4) before the antibacterial collagen solution is used as a biomedical material, 1-5 parts of a supermolecule coating agent is added into 100 parts of the antibacterial collagen solution, and the mixture is continuously stirred for 30-60 minutes at 4-25 ℃, so that the antibacterial activity of the antibacterial agent in the collagen solution can be closed;
in the method, the catalyst in the step (1) is one or more of potassium carbonate, sodium carbonate and calcium bicarbonate;
in the method, the connecting agent in the step (1) is one or more of bromoacetyl bromide and bromoacetyl chloride;
in the method, the solvent in the steps (1) and (2) is one or more of dichloromethane, toluene, n-butanol and chloroform;
in the method, the acid in the step (1) is one or more of hydrochloric acid, sulfuric acid and nitric acid;
in the method, the supermolecule encapsulating agent in the step (4) is one or more of cucurbit [7] urea, monohydroxylated cucurbit [7] urea and perhydroxylated cucurbit [7] urea.
Compared with the prior art, the invention has the following positive effects:
(1) ciprofloxacin is a third-generation quinolone antibacterial agent, the parent nucleus domain of ciprofloxacin is a pyridonic acid A ring, the synergistic group is 1-site cyclopropyl and 6-site fluorine atoms, and the antibacterial mechanism is inhibition of DNA gyrase and topoisomerase IV. To contact intracellular targets, ciprofloxacin is typically transported across the outer membrane using the gram-negative outer membrane porin, OmpF channel, which has an upper exclusion limit of about 700 Da. Therefore, after the secondary amine group on the ciprofloxacin piperazine ring is chemically modified and coupled with the amantadine, the mother nucleus structure domain and each synergistic group are not damaged, the molecular weight is 522Da and is not more than 700Da, the transfer of ciprofloxacin across an outer membrane porin OmpF channel and the contact with an intracellular target are not influenced, the antibacterial activity is not greatly reduced, the ciprofloxacin can be used as an active antibacterial agent to be added into a collagen solution, and the collagen is prevented from being corroded by microorganisms in the storage process.
(2) Gourds [7]Urea is a rigid macrocyclic compound formed by bridging seven glycoside urea units through methylene, has a hydrophobic cavity and two electron-rich holes surrounded by carbonyl groups, can selectively include amantadine by means of hydrophobic interaction and ion-dipole interaction, and has a binding constant as high as 1012M-1. When calabash [7]]After carbamide and derivatives thereof perform inclusion on amantadine supramolecules coupled on the cyclopropane sargassum, the molecular weight reaches 1684Da at the lowest, the amantadine supramolecules cannot smoothly pass through an outer membrane porin OmpF channel, and the activity is closed along with the passage.
(3) Before the collagen solution is used for injection wrinkle removal and ophthalmic treatment, the cucurbit [7] urea and the supermolecule thereof are used for including the adamantylamine group covalently coupled on the ciprofloxacin molecule, so that the activity of the ciprofloxacin antibacterial agent in the collagen solution can be closed, the toxic and side effects of the ciprofloxacin antibacterial agent can be effectively avoided, and the long-term contradiction between the necessity of antibacterial and antiseptic of the collagen solution and the maintenance of biocompatibility integrity can be coordinated. The method is simple and easy to operate, and the used supermolecule encapsulating agent is non-toxic and does not influence the three-strand helical structure and the biological function of the procollagen.
Drawings
FIG. 1 is a schematic view showing the steps of synthesizing an antibacterial agent for a collagen solution according to the present invention.
FIG. 2 is a schematic diagram showing the mechanism of activity shutdown of the antibacterial agent for collagen solution according to the present invention.
Detailed Description
The invention is described in detail below with reference to examples, which are intended to be illustrative only and not to be construed as limiting the scope of the invention, and many insubstantial modifications and variations of the invention can be made by an engineer skilled in the art based on the teachings of the invention.
Example 1:
(1) dispersing 2 parts of ciprofloxacin in 40 parts of dichloromethane, adding 50 parts of distilled water dissolved with 3 parts of potassium carbonate, and uniformly stirring to obtain a component 1; meanwhile, 8 parts of bromoacetyl bromide is dissolved in 20 parts of dichloromethane, slowly added into the component 1 within 1.5 hours at the temperature of 0 ℃, continuously stirred for 2 hours, and then heated to normal temperature and stirred for 13 hours; finally, washing the product with 5% hydrochloric acid aqueous solution and distilled water in sequence respectively, taking an organic layer, and drying the organic layer in vacuum until the weight is constant to obtain a first-step synthesized product;
(2) dissolving 2 parts of the first-step synthesized product in 30 parts of dichloromethane, adding 3 parts of amantadine into the solution, continuously stirring for 13 hours at 50 ℃, and drying the obtained product in vacuum to constant weight to obtain a target product;
(3) adding 0.2 part of the target product into 100 parts of collagen solution, and continuously stirring for 40 minutes at 10 ℃ to prepare antibacterial collagen solution which can prevent the collagen from mildewing and growing bacteria in the storage process;
(4) before the antibacterial collagen solution is used as a biomedical material, 2 parts of cucurbit [7] uril are added into 100 parts of the antibacterial collagen solution, and the mixture is continuously stirred for 40 minutes at 10 ℃, so that the antibacterial activity of an antibacterial agent in the collagen solution can be closed;
the antibacterial effect of the antibacterial collagen solution prepared in the embodiment 1 is measured by adopting a colony total number measuring method specified in GB4789.2-94 standard, the antibacterial rate of the antibacterial collagen solution to escherichia coli is more than or equal to 98%, and the collagen solution can be effectively prevented from mildewing and growing during storage; meanwhile, the in vitro cytotoxicity of the collagen is detected by adopting ISO10993-5 international standard, and the solution is found to be 2-grade toxicity to 3T3 fibroblasts; after the addition of the supramolecular encapsulating agent cucurbit [7] urea, the collagen solution was 0-grade toxic to 3T3 fibroblasts, and the activity of the contained antibacterial agent was turned off.
Example 2:
(1) dispersing 1 part of ciprofloxacin in 30 parts of toluene, adding 40 parts of distilled water dissolved with 2 parts of sodium carbonate, and uniformly stirring to obtain a component 1; meanwhile, 5 parts of bromoacetyl bromide is dissolved in 10 parts of toluene, slowly added into the component 1 within 1 hour at the temperature of 2 ℃, continuously stirred for 1 hour, and then heated to normal temperature and stirred for 12 hours; finally, sequentially washing the product with 5% sulfuric acid aqueous solution and distilled water respectively, taking an organic layer, and drying the organic layer in vacuum to constant weight to obtain a first-step synthesized product;
(2) dissolving 1 part of the first-step synthesized product in 10 parts of toluene, adding 2 parts of amantadine into the solution, continuously stirring for 12 hours at 40 ℃, and drying the obtained product in vacuum to constant weight to obtain a target product;
(3) adding 0.1 part of the target product into 100 parts of collagen solution, and continuously stirring for 30 minutes at 8 ℃ to prepare antibacterial collagen solution which can prevent the collagen from mildewing and growing bacteria in the storage process;
(4) before the antibacterial collagen solution is used as a biomedical material, 1 part of monohydroxy cucurbit [7] urea is added into 100 parts of the antibacterial collagen solution, and the mixture is continuously stirred for 30 minutes at the temperature of 8 ℃, so that the antibacterial activity of the antibacterial agent in the collagen solution can be closed;
the antibacterial effect of the antibacterial collagen solution prepared in the example 2 is measured by adopting a colony total number measuring method specified in GB4789.2-94 standard, the inhibition rate of the antibacterial collagen solution to escherichia coli is more than or equal to 96%, and the collagen solution can be effectively prevented from mildewing and growing during storage; meanwhile, the in vitro cytotoxicity of the collagen is detected by adopting ISO10993-5 international standard, and the solution is found to be 2-grade toxicity to 3T3 fibroblasts; after the addition of supramolecular mixture monohydroxylated cucurbit [7] urea, the collagen solution was 0-grade toxic to 3T3 fibroblasts and the activity of the contained antibacterial agent was switched off.
Example 3:
(1) dispersing 4 parts of ciprofloxacin in 60 parts of n-butanol, adding 70 parts of distilled water dissolved with 6 parts of calcium bicarbonate, and uniformly stirring to obtain a component 1; simultaneously, 13 parts of bromoacetyl chloride is dissolved in 40 parts of n-butanol, slowly added into the component 1 within 2 hours at 4 ℃, continuously stirred for 4 hours, and then heated to normal temperature and stirred for 14 hours; finally, sequentially washing the product with 10% nitric acid aqueous solution and distilled water respectively, taking an organic layer, and drying the organic layer in vacuum until the weight is constant to obtain a first-step synthesized product;
(2) dissolving 3 parts of the first-step synthesized product in 40 parts of n-butanol, adding 4 parts of amantadine into the solution, continuously stirring for 14 hours at 60 ℃, and drying the obtained product in vacuum to constant weight to obtain a target product;
(3) adding 0.4 part of the target product into 100 parts of collagen solution, and continuously stirring for 50 minutes at 16 ℃ to prepare antibacterial collagen solution which can prevent the collagen from mildewing and growing bacteria in the storage process;
(4) before the antibacterial collagen solution is used as a biomedical material, 4 parts of full-hydroxylated cucurbit [7] urea is added into 100 parts of the antibacterial collagen solution, and the mixture is continuously stirred for 50 minutes at the temperature of 16 ℃, so that the antibacterial activity of the antibacterial agent in the collagen solution can be closed;
the antibacterial effect of the antibacterial collagen solution prepared in the embodiment 3 is measured by adopting a colony total number measuring method specified in GB4789.2-94 standard, the inhibition rate of the antibacterial collagen solution to escherichia coli is more than or equal to 99%, and the collagen solution can be effectively prevented from mildewing and growing during storage; meanwhile, the in vitro cytotoxicity of the collagen is detected by adopting ISO10993-5 international standard, and the solution is found to be 2-grade toxicity to 3T3 fibroblasts; after the addition of the supermolecule encapsulating agent of the total hydroxylated cucurbit [7] urea, the collagen solution has grade 0 toxicity to 3T3 fibroblasts, and the activity of the contained antibacterial agent is closed.
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