CN107304233B - Non-cytotoxic high-molecular antibacterial agent and preparation method thereof - Google Patents
Non-cytotoxic high-molecular antibacterial agent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a non-cytotoxic macromolecular antibacterial agent and a preparation method thereof. The molecular structural formula of the product is as follows:
wherein x, y and n are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is more than or equal to 1 and less than or equal to 30. The preparation method comprises the following steps: reacting carboxymethyl chitosan with (oligo) arginine under a catalytic condition to obtain the (oligo) arginine modified carboxymethyl chitosan derivative. Then, the final product is obtained after separation and purification. The invention improves the antibacterial property of carboxymethyl chitosan by grafting (oligo) arginine on the main chain, further improves the biocompatibility of carboxymethyl chitosan and ensures that the product has no cytotoxicity.
Description
Technical Field
The invention relates to the field of carboxymethyl chitosan chemical modification. More particularly, relates to a (oligo) arginine-based modified carboxymethyl chitosan, non-cytotoxic green polymer antibacterial agent and a preparation method thereof.
Background
In recent years, epidemic caused by pathogenic bacteria is frequently generated in the global range, which greatly improves the health consciousness of people. Therefore, the antibacterial material and product industry is rapidly developing in China. The core part of the antibacterial material is an antibacterial agent which can effectively inhibit the growth and reproduction of microorganisms or kill bacteria. Currently, the antibacterial agent is mainly an inorganic system and an organic system in China. Inorganic antibacterial agents such as nano silver, zinc oxide, titanium oxide, etc., and organic antibacterial agents are mainly small molecular compounds such as quaternary ammonium salts, quaternary phosphonium salts, guanidine groups, etc. With the increasing concern over bacterial resistance and environmental pollution caused by overuse of antimicrobial agents, the problem of poor biological safety of inorganic and organic small molecule antimicrobial agents has been increasingly revealed. Thus, the demand for the performance of antibacterial agents is also increasing. The antibacterial agent not only has good antibacterial performance, but also has good biological safety, does not generate drug resistance and is environment-friendly.
The chitosan has certain antibacterial property, good biocompatibility and biodegradability. However, chitosan is insoluble in water, so that the application of chitosan in the antibacterial field is greatly limited. Chitosan can be carboxymethylated under alkaline conditions to give carboxymethyl chitosan (j.adv.drug.deliv.rev.2001,50,591.). The introduction of carboxymethyl group endows chitosan with good water solubility, however, the change of chemical structure influences the antibacterial performance of chitosan to a certain extent.
Arginine is alkaline positively charged water-soluble amino acid, has good anticoagulation property, adsorptivity and excellent biocompatibility, and can effectively improve the cell membrane penetrating efficiency of the material. Therefore, in the prior art, arginine is generally introduced into chitosan to improve the anticoagulation performance of chitosan (CN1226054C), the adsorption capacity of heavy metals (201510203082.4), and the transfection efficiency of chitosan gene vectors (CN 103539866A). However, since chitosan is difficult to be dissolved in water and insoluble in organic solvent, the reaction of chitosan grafting arginine is usually carried out under heterogeneous phase (organic solvent and water mixed system) or lower pH value, which not only complicates the reaction process, but also results in undesirable yield.
If the carboxymethyl chitosan can be subjected to arginine modification reaction in an aqueous solution to generate a high-molecular antibacterial agent which is easily soluble in water and has no cytotoxicity, the carboxymethyl chitosan has great potential application value and prospect.
Disclosure of Invention
An object of the present invention is to provide a polymer antibacterial agent free from cytotoxicity. The antibacterial agent is a brand new (oligo) arginine modified carboxymethyl chitosan derivative. The carboxymethyl chitosan has good water solubility, and the modification of (oligo) arginine on the main chain can simultaneously improve the antibacterial performance and the biological safety.
Another object of the present invention is to provide a method for preparing arginine or oligoarginine-modified carboxymethyl chitosan derivative. The preparation method has mild reaction conditions, does not need high temperature and high pressure, does not use organic solvent in the preparation process, saves energy and protects the environment in the whole production process, and can realize large-scale industrial production.
In order to achieve the first purpose, the invention adopts the following technical scheme:
a non-cytotoxic polymeric antimicrobial agent, the molecular structural formula of the antimicrobial agent is shown as formula (1):
wherein x, y and n are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is more than or equal to 1 and less than or equal to 30.
As is common general knowledge in the art, carboxymethyl chitosan is the carboxymethylation product of chitosan. Because of the different degree of substitution of the carboxymethyl groups, carboxymethyl chitosans contain varying proportions of non-carboxymethylated units, e.g.
Because of the different degree of deacetylation of the chitosan starting material, carboxymethyl chitosan will also contain varying proportions of non-deacetylated units, e.g.
As is common knowledge in the art, the general formula
To represent carboxymethyl chitosan. However, it will be appreciated by those skilled in the art that the carboxymethyl chitosan material will contain some non-carboxymethylated units in the molecular chain, as well as some non-deacetylated units.
In the present invention, in the molecular structural formula (1), the arrangement order of the two repeating units in the polymer chain is not completely in the order indicated in the structural formula, but is arranged and combined in the polymer chain in a random arrangement manner. That is, the (oligo) arginine substituents in the molecular chain of the (oligo) arginine-modified carboxymethyl chitosan are randomly arranged.
The invention successfully prepares the (oligo) arginine modified carboxymethyl chitosan derivative for the first time, and improves the antibacterial property and the biological safety of the carboxymethyl chitosan on the premise of keeping the good water solubility of the carboxymethyl chitosan. The product is a high molecular green antibacterial agent without cytotoxicity.
The invention introduces arginine or arginine oligomer on the main chain of the carboxymethyl chitosan, not only can keep the water solubility of the carboxymethyl chitosan, but also can improve the antibacterial performance and the biological safety.
In order to achieve the second object, the preparation method of the invention is to react carboxymethyl chitosan with (oligo) arginine under a catalytic condition to obtain the modified carboxymethyl chitosan derivative. Wherein carboxymethyl chitosan can be selected from various commercially available products, and has a molecular weight of 103～3×105Da. Preferably, carboxymethyl chitosan with carboxymethylation degree of 50% or more, more preferably deacetylation degree of 95% or more, and molecular weight of 10 is used4～2×105Carboxymethyl chitosan between Da. And (oligo) arginine modified carboxymethyl chitosan derivative obtained by the reaction is separated and purified to obtain the final product.
Further, the preparation method comprises the following steps:
(1) dissolving carboxymethyl chitosan in water to prepare carboxymethyl chitosan solution;
(2) activating (oligo) arginine under the condition of aqueous solution to obtain (oligo) arginine activated solution;
(3) adding the (oligo) arginine activating solution into the carboxymethyl chitosan solution for reaction;
(4) adding a terminator to end the reaction;
(5) and (3) separating and purifying the reaction liquid to obtain a product with the molecular weight of more than 5000Da, namely the (oligomeric) arginine modified carboxymethyl chitosan derivative.
The preparation method has simple process and mild reaction condition, does not need to add organic solvent or strong acid and strong alkali, and has relatively simple treatment process of the production wastewater. Through comparison experiments, the antibacterial performance of carboxymethyl chitosan or arginine is not ideal, but the antibacterial performance of the carboxymethyl chitosan derivative modified by arginine is greatly improved, and the carboxymethyl chitosan derivative has good biocompatibility.
Preferably, in step (1), the carboxymethyl chitosan has a number average molecular weight of 103～106The degree of substitution of carboxymethyl is 50-100% and the degree of deacetylation is 80-100% between Da. More preferably, the carboxymethyl chitosan has a number average molecular weight of 104～106The degree of substitution of carboxymethyl is 80-100%, and the degree of deacetylation is 95-100%. The preferable molecular weight range can ensure the high molecular property of the carboxymethyl chitosan, and the antibacterial performance is weakened along with the reduction of the water solubility when the molecular weight is too large.
Preferably, in the step (1), the concentration of the carboxymethyl chitosan in the carboxymethyl chitosan solution is 0.1-30% by mass/volume percentage. More preferably, the concentration of the carboxymethyl chitosan in the carboxymethyl chitosan dilute acid solution is 1 to 20 percent by mass volume percentage. This preferred carboxymethyl chitosan concentration range ensures high production efficiency of the reaction and high grafting yield of the product. Too high concentration of carboxymethyl chitosan increases the viscosity of the reaction system, which reduces the reaction conversion rate and has poor reaction uniformity.
Preferably, in the step (2), the molar ratio of the N-hydroxysuccinimide (hereinafter abbreviated as NHS) to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (hereinafter abbreviated as EDC) is 1: 0.1 to 10. More preferably, the molar ratio of NHS to EDC is 1: 1 to 1.0. This preferred molar ratio range of NHS to EDC may ensure optimal activation efficiency. Too low a molar ratio results in low grafting; too high a molar ratio does not further increase the reaction conversion rate, but instead results in waste of raw materials.
Preferably, in the step (2), the pH value of the activated (oligo) arginine is 5.5-7.4. More preferably, the pH value is 6.0-6.5. This preferred pH range ensures optimum catalytic efficiency. Both too low and too high a pH value can affect the activation efficiency and thus the reaction conversion.
Preferably, in step (2), the arginine oligomer has a number average molecular weight <5000 Da. More preferably, the arginine oligomer has a number average molecular weight <3000 Da. The optimized molecular weight range can ensure that the arginine oligomer has better antibacterial performance. Too large a molecular weight will affect the conversion of the reaction and the antimicrobial properties of the product will be reduced.
Preferably, in step (2), the molar ratio of (oligo) arginine to EDC is 1: 0.5 to 10. More preferably, the molar ratio of (oligo) arginine to EDC is 1: 0.5 to 5. This preferred range of molar ratios ensures optimum conversion of the reaction. Too low or too high a molar ratio can affect the activation efficiency and thus the reaction conversion.
Preferably, in the step (3), the molar ratio of the carboxymethyl chitosan to the (oligo) arginine is 1: 0.02 to 1. More preferably, 1: 0.02 to 0.5. This preferred range of molar ratios ensures the best antimicrobial properties of the final product. The low molar ratio can reduce the grafting amount of (oligomeric) arginine and has poor antibacterial performance; the grafting rate cannot be obviously improved when the molar ratio is higher than the optimized range, and the raw material waste is caused.
The chemical reaction formula involved in the invention is as follows:
in the prior art, arginine is generally directly introduced into chitosan to improve the anticoagulation performance of the chitosan, the adsorption capacity of heavy metals and the transfection efficiency of chitosan gene vectors. However, since chitosan is difficult to be dissolved in water and insoluble in organic solvent, the reaction of chitosan grafting arginine is usually carried out under heterogeneous phase (organic solvent and water mixed system) or lower pH value, which not only complicates the reaction process, but also results in undesirable yield.
The technical scheme of the invention is different from the prior art and is improved in that:
because the carboxymethyl chitosan has good water solubility, the preparation process of the (oligo) arginine modified carboxymethyl chitosan is completely carried out in an aqueous solution environment, strong acid or organic solvent is not needed, the reaction condition is mild, and the preparation process can be continuously carried out in the same reaction kettle. This not only greatly simplifies the reaction process, but also minimizes the environmental pollution. Meanwhile, the reaction is carried out under the weak acidic condition, which is beneficial to the activation of (oligo) arginine, greatly improves the grafting efficiency, and further can reduce the dosage of the catalyst and the (oligo) arginine, reduce the raw material cost, shorten the reaction time and reduce the energy consumption.
Arginine and carboxymethyl chitosan have better biocompatibility. Compared with the (oligo) arginine modified chitosan, under the condition of similar (oligo) arginine grafting rate, the (oligo) arginine modified carboxymethyl chitosan has no cytotoxicity and more excellent biocompatibility, is a more ideal high-molecular green antibacterial agent, and has great market development prospect.
The invention improves the antibacterial property of carboxymethyl chitosan by grafting arginine or oligoarginine on the main chain, further improves the biocompatibility of carboxymethyl chitosan, and leads the product to have no toxicity to cells. The product is suitable for the application field with higher requirements on the biological safety of the antibacterial material.
It should be noted that: in all molecular structural formulas appearing in the specification of the invention, the arrangement sequence of the respective repeating units is not completely in the order noted in the structural formulas, but is arranged and combined in a random arrangement manner in a polymer chain.
It is further noted that any range recited herein includes the endpoints and any values therebetween and any subranges subsumed therein or any values therebetween unless otherwise specified.
The invention has the following beneficial effects:
1. the molecular chain of the carboxymethyl chitosan derivative is modified with (oligo) arginine with antibacterial property and no cytotoxicity, and the antibacterial property and the biological safety are improved;
2. the carboxymethyl chitosan has good water solubility, biodegradability, low toxicity and less pollution in the production process;
3. compared with unmodified carboxymethyl chitosan, the antibacterial performance of the product can be improved from the promotion of the growth of escherichia coli to 99% of the bacterial reduction rate under the same concentration, and the survival rate of mouse fibroblast L929 is still higher than 90% under the condition of 10mg/mL concentration;
drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the preparation of carboxymethyl chitosan as a raw material in comparative example 31H NMR spectrum.
FIG. 2 shows arginine-modified carboxymethyl chitosan derivative prepared in example 1 of the present invention1H NMR spectrum.
FIG. 3 is a photograph showing the results of measuring the antibacterial activity of Escherichia coli using a plate-coating method using the arginine-modified carboxymethyl chitosan derivative prepared in example 1 of the present invention.
FIG. 4 shows the results of the test of L929 cells, mouse fibroblasts, using the arginine-modified carboxymethyl chitosan derivative prepared in example 1 of the present invention.
FIG. 5 is a photograph showing the results of the antibacterial property test of the comparative example product against Escherichia coli by the plate coating method.
FIG. 6 shows the results of the test of comparative product on mouse fibroblast cells L929.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
An arginine modified carboxymethyl chitosan derivative has a molecular structural formula as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, and y is more than or equal to 1 and less than or equal to 10000.
The specific preparation method of the arginine modified carboxymethyl chitosan derivative comprises the following steps:
0.35g carboxymethyl chitosan (number average molecular weight 2X 10) was weighed out5Da, the degree of substitution of carboxymethyl is 100%, and the degree of deacetylation is 95%) is added into 35mL of water to obtain a solution with the mass-volume percentage concentration of 1%; arginine was activated in 15mL of pH 5.5 solution (NHS to EDC molar ratio 1: 1); pouring the arginine activating solution into a carboxymethyl chitosan solution, and reacting for 24 hours; adding hydroxylamine hydrochloride with the same molar ratio with arginine into the reaction solution to terminate the reaction; separating and purifying the reaction product to obtain the molecular weight>5000Da final product, namely the arginine modified carboxymethyl chitosan derivative. In the reaction, the molar ratio of carboxymethyl chitosan, arginine and EDC is 1: 0.05: 0.25.
comparative example 1
An arginine-modified chitosan derivative, the molecular structural formula of which is as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, and y is more than or equal to 1 and less than or equal to 10000.
The preparation method of the arginine modified chitosan derivative comprises the following steps:
the specific preparation process comprises the following steps:
0.35g of chitosan (number average molecular weight 10)5Da, degree of deacetylation 95%) was dispersed in 35mL of isopropanol/water (volume ratio 1: 1) mixing the solution; arginine was activated in 15mL of pH 5.5 solution (NHS to EDC molar ratio 1: 1); pouring the arginine activating solution into the chitosan solution, and reacting for 48 hours; will react with arginineAdding hydroxylamine hydrochloride with equal molar ratio into the reaction solution to terminate the reaction; separating and purifying the reaction product to obtain the molecular weight>5000Da final product, namely the arginine modified carboxymethyl chitosan derivative. In the reaction, the molar ratio of chitosan, arginine and EDC is 1: 0.05: 0.25.
comparative example 2
A chitosan has a molecular structural formula as follows:
wherein x is a natural number, and x is more than or equal to 1 and less than or equal to 15000. The chitosan was purchased commercially.
Comparative example 3
The carboxymethyl chitosan has the following molecular structural formula:
wherein x is a natural number, and x is more than or equal to 1 and less than or equal to 15000. The carboxymethyl chitosan was purchased commercially and used as a starting material for the reaction in example 1.
FIG. 1 shows the preparation of carboxymethyl chitosan as a raw material in comparative example 31H NMR spectrum. FIG. 2 shows arginine-modified carboxymethyl chitosan derivative prepared in example 1 of the present invention1H NMR spectrum. By comparing the starting materials and the products1H NMR spectrum can show that arginine modified carboxymethyl chitosan derivative1The H NMR spectrum shows a characteristic peak of arginine at 1.1ppm, which indicates that arginine is successfully grafted to the molecular chain of chitosan through an amino group.
FIG. 3 is a photograph showing the results of the antibacterial activity test of Escherichia coli using the arginine-modified carboxymethyl chitosan derivative prepared in example 1 of the present invention. From left to right in the figure are a blank control sample and a sample using 5mg/mL of arginine-modified carboxymethyl chitosan, respectively.
The test method comprises the following steps: the arginine-modified carboxymethyl chitosan derivative aqueous solution prepared in this example (the blank was deionized water with the same volume) was mixed with the bacterial solution, cultured on a constant temperature and humidity shaker at 37 ℃ for 12 hours, and then plated. The plates were incubated at 37 ℃ for 24 hours in a constant temperature and humidity cabinet and the viable colonies were counted. The results show that: the arginine-modified carboxymethyl chitosan derivative prepared in this example has good killing performance on escherichia coli.
The statistical results of the plate-coating method for the reduction rate of the bacteria in Escherichia coli for the product prepared in this example 1 are shown in the following table:
TABLE 1 bacteria reduction rate test results for arginine-modified carboxymethyl chitosan
FIG. 4 shows the results of toxicity test of arginine-modified carboxymethyl chitosan derivative prepared in example 1 of the present invention against mouse fibroblast L929 cells.
The test method comprises the following steps: mouse fibroblast cells L929 were seeded into 96-well plates at approximately 5000 cells/well for 3-5 replicate spots. After the cells adhere to the wall, arginine modified carboxymethyl chitosan solutions with different concentrations are added. Cells were in 5% CO2And cultured at 37 ℃ for 24 hours. The relative proliferation rate of the cells is detected by using a CCK-8 kit. The test data shows that: under the condition that the concentration of the arginine modified carboxymethyl chitosan derivative is less than or equal to 10mg/mL, the L929 cell survival rate>90％。
The above data results illustrate that: the arginine-modified carboxymethyl chitosan derivative not only has good antibacterial performance, but also can ensure that cells can normally grow under effective antibacterial concentration, and has good biological safety.
The products in comparative examples 1-3 were tested for antibacterial activity against E.coli and toxicity against mouse fibroblast L929 cells according to the methods described above, and the results are shown in FIGS. 5 and 6, respectively.
FIG. 5 is a photograph showing the results of the antibacterial property test of the comparative example product against Escherichia coli by the plate coating method. The test effect of the product of comparative example 1, the product of comparative example 2 and the product of comparative example 3 are shown in the figure from left to right, and the concentration is 5 mg/mL.
FIG. 6 shows the results of the test of comparative product on mouse fibroblast cells L929. The test effect data of the product of the comparative example 1, the product of the comparative example 2 and the product of the comparative example 3 are shown in the figure from left to right, and the concentration of the test effect data is 1 mg/mL.
The results of the above tests are compared with those of example 1 and are tabulated below:
comparison of test results shows that: the bacterial reduction rate of the arginine modified carboxymethyl chitosan on escherichia coli reaches 99% when the concentration of the arginine modified carboxymethyl chitosan is 5 mg/mL. Under the same concentration, the bacteria reduction rate of the arginine modified chitosan and the chitosan raw material is only 72.9 percent and 7.8 percent; the carboxymethyl chitosan material has no antibacterial property and promotes the growth of bacteria. Similarly, under the condition of 1mg/mL, the arginine modified carboxymethyl chitosan and the carboxymethyl chitosan raw material have better biocompatibility, the relative proliferation rate of mouse fibroblast L929 is more than 100%, and the relative proliferation rate of the arginine modified chitosan and the chitosan raw material L929 is only about 55% and 10%. Therefore, the antibacterial property and the safety of the arginine modified carboxymethyl chitosan are superior to those of the arginine modified chitosan and raw material products.
Example 2
An oligoarginine-modified carboxymethyl chitosan derivative, the molecular structural formula of which is shown as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is 10.
The specific preparation method of the oligoarginine-modified carboxymethyl chitosan derivative comprises the following steps:
7g of carboxymethyl chitosan (number average molecular weight 10) was weighed4Da, the substitution degree of carboxymethyl is 80%, and the deacetylation degree is 100%), adding 35mL of water, and obtaining a solution with the mass volume percentage concentration of 20%; activating oligoarginine in 15mL of pH 6.5 solution (NHS and EDC molar ratio 1: 10); pouring the arginine activating solution into a carboxymethyl chitosan solution, and reacting for 48 hours; after the reaction is finished, the molecular weight is obtained through separation and purification>5000Da final product, namely the oligoarginine modified carboxymethyl chitosan derivative. In the reaction, the molar ratio of carboxymethyl chitosan, arginine and EDC is 1: 1: 0.5. the oligoarginine-modified carboxymethyl chitosan derivative prepared in this example was tested for antibacterial properties and safety properties with reference to example 1, and the results were similar to example 1.
Example 3
An oligoarginine-modified carboxymethyl chitosan derivative, the molecular structural formula of which is shown as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is equal to 30.
The specific preparation method of the oligoarginine-modified carboxymethyl chitosan derivative comprises the following steps:
10.5 g of carboxymethyl chitosan (number average molecular weight 10) was weighed out3Da, the substitution degree of carboxymethyl is 80%, and the deacetylation degree is 100%), adding 35mL of water, and obtaining a solution with the mass volume percentage concentration of 30%; activating oligoarginine in 15mL pH6.0 solution (NHS and EDC molar ratio is 1: 10); pouring the oligoarginine activating solution into a carboxymethyl chitosan solution, and reacting for 48 hours; adding hydroxylamine hydrochloride with the same molar ratio with the oligoarginine into the reaction solution to terminate the reaction; separating and purifying the reaction product to obtain the molecular weight>5000Da final product, namely the oligoarginine modified carboxymethyl chitosan derivative. In the reaction, the molar ratio of carboxymethyl chitosan, arginine and EDC is 1: 0.05: 0.5. the oligoarginine-modified carboxymethyl chitosan derivative prepared in this example was subjected to the following procedure in reference to example 1The antibacterial performance and safety performance test were carried out, and the results were similar to those of example 1.
Example 4
An oligoarginine-modified carboxymethyl chitosan derivative, the molecular structural formula of which is shown as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is 15.
The specific preparation method of the oligoarginine-modified carboxymethyl chitosan derivative comprises the following steps:
0.35g of carboxymethyl chitosan (number average molecular weight 10) was weighed out3Da, the degree of substitution of carboxymethyl is 50%, and the degree of deacetylation is 95%) is added into 35mL of water to obtain a solution with the mass-volume percentage concentration of 1%; activating oligoarginine in 15mL of pH 4.5 solution (NHS and EDC molar ratio 1: 0.1); pouring the oligoarginine activating solution into a carboxymethyl chitosan solution, and reacting for 6 hours; adding hydroxylamine hydrochloride with the same molar ratio with the oligoarginine into the reaction solution to terminate the reaction; separating and purifying the reaction product to obtain the molecular weight>5000Da final product, namely the oligoarginine modified carboxymethyl chitosan derivative. In the reaction, the molar ratio of carboxymethyl chitosan, arginine and EDC is 1: 0.02: 0.2. the oligoarginine-modified carboxymethyl chitosan derivative prepared in this example was tested for antibacterial properties and safety properties with reference to example 1, and the results were similar to example 1.
Example 5
An oligoarginine-modified carboxymethyl chitosan derivative, the molecular structural formula of which is shown as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is 20.
The specific preparation method of the oligoarginine-modified carboxymethyl chitosan derivative comprises the following steps:
0.035 g of carboxymethyl chitosan (number average molecular weight 3X 10) was weighed out5Da, the degree of substitution of carboxymethyl is 100%, and the degree of deacetylation is 50%), adding 35mL of water to obtain a solution with a mass-volume percentage concentration of 0.1%; activating oligoarginine in 15ml of pH 7.4 solution (NHS and EDC molar ratio 1: 1); pouring the oligoarginine activating solution into a carboxymethyl chitosan solution, and reacting for 12 hours; adding hydroxylamine hydrochloride with the same molar ratio with the oligoarginine into the reaction solution to terminate the reaction; separating and purifying the reaction product to obtain the molecular weight>5000Da final product, namely the oligoarginine modified carboxymethyl chitosan derivative. In the reaction, the molar ratio of the carboxymethyl chitosan to the oligoarginine to the EDC is 1: 0.5: 5. the oligoarginine-modified carboxymethyl chitosan derivative prepared in this example was tested for antibacterial properties and safety properties with reference to example 1, and the results were similar to example 1.
Example 6
An oligoarginine-modified carboxymethyl chitosan derivative, the molecular structural formula of which is shown as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is 10.
The specific preparation method of the oligoarginine-modified carboxymethyl chitosan derivative comprises the following steps:
0.035 g of carboxymethyl chitosan (number average molecular weight 10) was weighed out6Da, the degree of substitution of carboxymethyl is 100%, and the degree of deacetylation is 80%), adding 35mL of water to obtain a solution with a mass-volume percentage concentration of 0.1%; activating oligoarginine in 15mL pH6.0 solution (NHS and EDC molar ratio is 1: 1); pouring the oligoarginine activating solution into a carboxymethyl chitosan solution, and reacting for 8 hours; adding hydroxylamine hydrochloride with the same molar ratio with the oligoarginine into the reaction solution to terminate the reaction; separating and purifying the reaction product to obtain the molecular weight>5000Da final product, namely the oligoarginine modified carboxymethyl chitosan derivative. In the reaction, carboxymethyl chitosan and oligoarginineMolar ratio to EDC 1: 1: 5. the oligoarginine-modified carboxymethyl chitosan derivative prepared in this example was tested for antibacterial properties and safety properties with reference to example 1, and the results were similar to example 1.
Example 7
An oligoarginine-modified carboxymethyl chitosan derivative, the molecular structural formula of which is shown as follows:
wherein x and y are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is 10.
The specific preparation method of the oligoarginine-modified carboxymethyl chitosan derivative comprises the following steps:
0.35g carboxymethyl chitosan (number average molecular weight 3X 10) was weighed out5Da, the substitution degree of carboxymethyl is 20%, and the deacetylation degree is 95%) is added into 35mL of water, and a solution with the mass volume percentage concentration of 1% is obtained; activating oligoarginine in 15mL pH6.0 solution (NHS and EDC molar ratio is 1: 0.5); pouring the oligoarginine activating solution into a carboxymethyl chitosan solution, and reacting for 8 hours; adding hydroxylamine hydrochloride with the same molar ratio with oligoarginine into the reaction solution; separating and purifying the reaction product to obtain the molecular weight>3000Da, namely the final product is the oligoarginine modified carboxymethyl chitosan derivative. In the reaction, the molar ratio of the carboxymethyl chitosan to the oligoarginine to the EDC is 1: 1: 5. the oligoarginine-modified carboxymethyl chitosan derivative prepared in this example was tested for antibacterial properties and safety properties with reference to example 1, and the results were similar to example 1.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (9)
1. A non-cytotoxic polymeric antimicrobial agent characterized by: the molecular structural formula of the antibacterial agent is shown as a formula (1):
wherein x, y and n are natural numbers, x is more than or equal to 1 and less than or equal to 10000, y is more than or equal to 1 and less than or equal to 10000, and n is more than or equal to 1 and less than or equal to 30;
the preparation method of the non-cytotoxic polymer antibacterial agent comprises the following steps:
(1) dissolving carboxymethyl chitosan in water to prepare carboxymethyl chitosan solution; the weight average molecular weight of the carboxymethyl chitosan is 103～106Da, the substitution degree of carboxymethyl is 20-100%, and the deacetylation degree is 50-100%; the concentration of the carboxymethyl chitosan solution is 0.1-30% by mass percent;
(2) activating oligoarginine or arginine under the condition of aqueous solution to obtain an activated solution of oligoarginine or arginine; the number average molecular weight of the oligoarginine is less than 5000 Da;
(3) adding the activated solution of oligoarginine or arginine into a carboxymethyl chitosan solution for reaction, wherein the molar ratio of carboxymethyl chitosan to oligoarginine or arginine is 1: 0.02 to 1;
(4) adding a terminator to end the reaction;
(5) and (3) separating and purifying the reaction liquid to obtain a product with the molecular weight of more than 5000Da, namely the oligoarginine or arginine modified carboxymethyl chitosan derivative.
2. The method of claim 1, wherein the method comprises the steps of:
(1) dissolving carboxymethyl chitosan in water to prepare carboxymethyl chitosan solution;
(2) activating oligoarginine or arginine under the condition of aqueous solution to obtain an activated solution of oligoarginine or arginine;
(3) adding the oligoarginine or the activated solution of arginine into the carboxymethyl chitosan solution for reaction;
(4) adding a terminator to end the reaction;
(5) and (3) separating and purifying the reaction liquid to obtain a product with the molecular weight of more than 5000Da, namely the oligoarginine or arginine modified carboxymethyl chitosan derivative.
3. The method for preparing a non-cytotoxic polymeric antibacterial agent according to claim 2, wherein: in the step (2), the pH value of the oligoarginine or arginine during activation is 4.5-7.4.
4. The method for preparing a non-cytotoxic polymeric antibacterial agent according to claim 2, wherein: in the step (2), the activation comprises the following steps: dissolving N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in water to obtain a catalytic solution; adding oligoarginine or arginine into the catalytic solution to obtain an activating solution of oligoarginine or arginine.
5. The method for preparing a non-cytotoxic polymeric antibacterial agent according to claim 4, wherein: the molar ratio of the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: 0.1 to 10; the molar ratio of the oligoarginine or arginine to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: 0.5 to 10.
6. The method for preparing a non-cytotoxic polymeric antibacterial agent according to claim 2, wherein: in the step (3), the reaction time is 6-48 hours.
7. The method for preparing a non-cytotoxic polymeric antibacterial agent according to claim 2, wherein: in the step (4), the terminating agent is hydroxylamine hydrochloride; the mol ratio of the terminator to the oligoarginine or the arginine is 1: 1.
8. the method for preparing the non-cytotoxic polymeric antibacterial agent according to any one of claims 2 to 7, wherein the method comprises the following steps:
(1) dissolving carboxymethyl chitosan in water to prepare carboxymethyl chitosan solution; the weight average molecular weight of the carboxymethyl chitosan is 104～3×105Between Da, the substitution degree of carboxymethyl is 50-100%, and the deacetylation degree is 80-100%; the concentration of the carboxymethyl chitosan solution is 1-20% by mass percent;
(2) activating oligoarginine or arginine under the condition of aqueous solution to obtain an activated solution of oligoarginine or arginine; the pH value of the oligoarginine or arginine during activation is 5.5-7.4; the molar ratio of the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: 1-10; the molar ratio of the oligoarginine or arginine to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: 0.5 to 5;
(3) adding the oligoarginine or the activated solution of arginine into the carboxymethyl chitosan solution for reaction; the molar ratio of the carboxymethyl chitosan to the oligoarginine or arginine is 1: 0.02 to 0.5; the reaction time is 6-48 hours;
(4) adding a terminator to end the reaction; the terminator is hydroxylamine hydrochloride; the mol ratio of the terminator to the oligoarginine or the arginine is 1: 1;
(5) and (3) separating and purifying the reaction liquid to obtain a product with the molecular weight of more than 5000Da, namely the oligoarginine or arginine modified carboxymethyl chitosan derivative.
9. Use of the non-cytotoxic polymeric antibacterial agent of claim 1 in the preparation of an antibacterial material.
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| CN101624427A (en) * | 2009-08-12 | 2010-01-13 | 华中科技大学 | Arginine-chitosan with high degree of substitution, preparation method and application thereof |
| CN105218699A (en) * | 2014-07-04 | 2016-01-06 | 中国医学科学院药物研究所 | The chitosan of oligomerization arginine covalent modification, preparation method, screening and application |
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| CN101624427A (en) * | 2009-08-12 | 2010-01-13 | 华中科技大学 | Arginine-chitosan with high degree of substitution, preparation method and application thereof |
| CN105218699A (en) * | 2014-07-04 | 2016-01-06 | 中国医学科学院药物研究所 | The chitosan of oligomerization arginine covalent modification, preparation method, screening and application |
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| "Preparation and characterization of antimicrobial chitosan-N-arginine with different degrees of substitution";Bo Xiao等;《Carbohydrate Polymers》;20100724;第83卷(第1期);第144-150页 * |
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