A kind of intelligent response chitosan oligosaccharide-based nanogel and preparation method thereof

Abstract

The present invention discloses a kind of intelligent response chitosan oligosaccharide-based nanogel and its preparation method, and belongs to the technical field of carrier preparation. The preparation method of a kind of intelligent response chitosan oligosaccharide-based nanogel described in the present invention specifically comprises the following steps: firstly, gelatin nanoparticles are prepared by a two-step desolvation method, and then gelatin-chitosan oligosaccharide-pectin composite nanogel is prepared by a layer-by-layer self-assembly method, and then it is heated in a water bath at 40°C, and the free gelatin dissolved by heating is removed by centrifugation to obtain a hollow nanogel carrier, and after loading the active substance, it is finally sealed with chitosan oligosaccharide to obtain the final chitosan oligosaccharide-based nanogel. The chitosan oligosaccharide-based nanogel provided by the present invention has a wide range of pH responsiveness, and can also realize accurate quantitative loading of active substances by regulating the size of gelatin nanoparticles, and has the advantages of being green, safe and efficient.

Description

Technical Field

[0001] The invention relates to the technical field of carrier preparation, and in particular to a chitosan oligosaccharide-based nanogel with intelligent response and a preparation method thereof. Background Art

[0002] Smart nanogels are gel particles with a particle size of 1 to 1000 nm that can respond to external stimuli. They have good biocompatibility, unique cross-linked network structure and functionality. External stimuli usually include slight changes in ambient temperature, pH, ionic strength, etc., as well as light, magnetic field, specific chemical substances or biological substances. Some smart nanohydrogels can also respond to two or more of these stimuli. This type of smart nanohydrogel is also called multi-stimulus responsive nanohydrogel. The form in which smart nanogels respond to external stimuli is usually manifested as changes in physical and chemical properties such as volume, water content, refractive index, colloidal stability, hardness, permeability of the internal gel network, and hydrophilicity-hydrophobicity. In addition to the above stimulus responsiveness, they also have the characteristics of small particle size, large specific surface area, surface functional groups that can be coupled to other components with special functions, and good biocompatibility. Therefore, smart nanogels have good application prospects in many fields such as medicine, food, and materials. Since the first report of temperature stimulus responsive nanogels in the 1980s, the research on smart nanogels has begun to receive widespread attention at home and abroad. Especially in the past 10 years, with the invention and research of various stimulus-responsive nanogels, the literature reports on the research of smart nanogels have almost shown an exponential growth trend.

[0003] Currently, pH-responsive nanogels have attracted much attention in the pharmaceutical industry, and have been widely studied as drug carriers for targeted delivery. However, most of the prepared pH-responsive nanogel carriers have limitations such as single functionality, the inability to flexibly adjust the pH response range, and the inability to accurately control the loading amount. Summary of the invention

[0004] Technical problem to be solved: The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a smart-responsive chitosan oligosaccharide-based nanogel and a preparation method thereof, by adjusting the pH value of gelatin nanoparticles during preparation, controlling their size, and then regulating the internal cavity of the hollow nanogel and the overall size, to achieve precise quantitative loading of active substances; in addition, by changing the volume ratio of the nanogel loaded with active substances and the chitosan oligosaccharide solution to control the pH response range of the final chitosan oligosaccharide-based nanogel, different pH response requirements of the nanogel are achieved, giving the nanogel a variety of functions.

[0005] Technical solution: A method for preparing a chitosan oligosaccharide-based nanogel with intelligent response, comprising the following steps:

[0006] S1: dissolving gelatin powder in deionized water and stirring evenly to obtain a gelatin solution of a certain mass concentration, adding acetone to the gelatin solution, stirring evenly, centrifuging at 5000 rpm for 10 min, removing the supernatant, dissolving the obtained precipitate in deionized water, adjusting the pH value, and then adding glutaraldehyde to cross-link the gelatin to form particles, performing refrigerated centrifugation at 10000 rpm for 30 min, and volatilizing and removing the acetone to obtain gelatin nanoparticles;

[0007] S2: dissolving chitosan oligosaccharide in deionized water, fully dissolving and stirring to obtain a chitosan oligosaccharide solution, re-dispersing gelatin nanoparticles in deionized water to obtain a gelatin nanoparticle suspension, and adding the suspension to the chitosan oligosaccharide solution to obtain a gelatin-chitosan oligosaccharide composite nanogel;

[0008] S3: adding gelatin-chitosan oligosaccharide composite nanogel into the pectin solution and stirring well to obtain gelatin-chitosan oligosaccharide-pectin composite nanogel;

[0009] S4: heating the gelatin-chitosan oligosaccharide-pectin composite nanogel in a water bath at 40°C, centrifuging at 5000 rpm for 10 min, removing the heated and dissolved free gelatin, and obtaining a hollow nanogel;

[0010] S5: adding active substances to the hollow nanogel, stirring thoroughly to obtain nanogel loaded with active substances, and then adding the nanogel to the chitosan oligosaccharide solution, stirring thoroughly to obtain chitosan oligosaccharide-based nanogel.

[0011] Preferably, the gelatin in step S1 is type B gelatin.

[0012] Preferably, the mass concentration of the gelatin solution in step S1 is 1% to 10%, the volume ratio of the gelatin solution to acetone is 1:1, and the pH value is adjusted to 6 to 12.

[0013] Preferably, all operations except the centrifugation operation in step S1 are performed in a 40° C. water bath.

[0014] Preferably, the mass concentration of the chitosan oligosaccharide solution in step S2 is 1% to 5%, and the volume ratio of the gelatin nanoparticle suspension to the chitosan oligosaccharide solution is 1:1.

[0015] Preferably, the mass concentration of the pectin solution in step S3 is 0.5% to 5%, and the volume ratio of the gelatin-chitosan oligosaccharide composite nanogel to the pectin solution is 5:1.

[0016] Preferably, the water bath heating time in step S4 is 30 to 120 minutes.

[0017] Preferably, the active substances in step S5 include ketoprofen, paclitaxel, thymol, melatonin and tea polyphenols.

[0018] Preferably, in step S5, the mass concentration of the active substance in the hollow nanogel is 1% to 30%, and the volume ratio of the nanogel loaded with the active substance to the chitosan oligosaccharide solution is (1 to 5):(1 to 5).

[0019] The present invention also provides a nanogel, which is prepared by using the method for preparing the intelligently responsive chitosan oligosaccharide-based nanogel as described above.

[0020] Beneficial effects:

[0021] 1. The chitosan oligosaccharide-based nanogel provided by the present invention has a flexibly adjustable pH response range. The pH response characteristics of the final chitosan oligosaccharide-based nanogel can be adjusted by changing the volume ratio of the nanogel loaded with active substances and the chitosan oligosaccharide solution, thereby achieving different pH response requirements of the nanogel and giving the nanogel a variety of functions.

[0022] 2. The present invention weakens the hydration of gelatin molecules by using acetone to make them curled, and then adjusts the pH value and adds a cross-linking agent, glutaraldehyde, to produce cross-linking reaction to prepare gelatin nanoparticles, which have strong hydrophilicity and thermal solubility, and the size of the gelatin nanoparticles can be controlled by adjusting different pH values.

[0023] 3. The present invention uses gelatin nanoparticles as templates and utilizes the temperature sensitivity of gelatin nanoparticles to prepare hollow nanogels with uniform particle sizes. The size of the gelatin nanoparticles can be adjusted to achieve quantitative loading of active substances.

[0024] 4. The chitosan oligosaccharide-based nanogel provided by the present invention is composed of active substances and natural biomacromolecule chitosan oligosaccharides and pectin, and has the advantages of being green, safe and efficient. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments, which are explanations of the present invention and are not limited to the following embodiments:

[0026] Example 1

[0027] S1: Dissolve gelatin powder in deionized water, stir evenly at 40°C to obtain a gelatin solution with a mass concentration of 3%, add acetone to the gelatin solution at a volume ratio of 1:1, stir evenly at 40°C, centrifuge at 5000rpm for 10min, remove the supernatant, and dissolve the obtained precipitate in deionized water again, adjust the pH value to 6, then add glutaraldehyde to cross-link the gelatin to form particles, perform refrigerated centrifugation at 10000rpm for 30min, and volatilize and remove the acetone to obtain gelatin nanoparticles;

[0028] S2: Dissolve chitosan oligosaccharide in deionized water, fully dissolve and stir to obtain a chitosan oligosaccharide solution with a mass concentration of 3%, redisperse gelatin nanoparticles in deionized water to obtain a gelatin nanoparticle suspension, and add the suspension to the chitosan oligosaccharide solution at a volume ratio of 1:1 to obtain a gelatin-chitosan oligosaccharide composite nanogel;

[0029] S3: adding gelatin-chitosan oligosaccharide composite nanogel to a pectin solution with a mass concentration of 2% at a volume ratio of 1:1, and stirring the mixture thoroughly to obtain gelatin-chitosan oligosaccharide-pectin composite nanogel;

[0030] S4: heating the gelatin-chitosan oligosaccharide-pectin composite nanogel in a water bath at 40°C for 60 min, and centrifuging at 5000 rpm for 10 min to remove the free gelatin dissolved by heating to obtain a hollow nanogel;

[0031] S5: adding ketoprofen to the hollow nanogel, stirring thoroughly to obtain a ketoprofen-loaded nanogel, making the mass concentration of ketoprofen in the nanogel to be 5%, and then adding it to the chitosan oligosaccharide solution, making the volume ratio of the ketoprofen-loaded nanogel to the chitosan oligosaccharide solution to be 5:1, stirring thoroughly to obtain a chitosan oligosaccharide-based nanogel.

[0032] Example 2

[0033] S1: Dissolve gelatin powder in deionized water, stir evenly at 40°C to obtain a gelatin solution with a mass concentration of 3%, add acetone to the gelatin solution at a volume ratio of 1:1, stir evenly at 40°C, centrifuge at 5000rpm for 10min, remove the supernatant, and dissolve the obtained precipitate in deionized water again, adjust the pH value to 8, then add glutaraldehyde to cross-link the gelatin to form particles, perform refrigerated centrifugation at 10000rpm for 30min, and volatilize and remove the acetone to obtain gelatin nanoparticles;

[0034] S2: Dissolve chitosan oligosaccharide in deionized water, fully dissolve and stir to obtain a chitosan oligosaccharide solution with a mass concentration of 3%, redisperse gelatin nanoparticles in deionized water to obtain a gelatin nanoparticle suspension, and add the suspension to the chitosan oligosaccharide solution at a volume ratio of 1:1 to obtain a gelatin-chitosan oligosaccharide composite nanogel;

[0035] S3: adding gelatin-chitosan oligosaccharide composite nanogel to a pectin solution with a mass concentration of 2% at a volume ratio of 1:1, and stirring the mixture thoroughly to obtain gelatin-chitosan oligosaccharide-pectin composite nanogel;

[0036] S4: heating the gelatin-chitosan oligosaccharide-pectin composite nanogel in a water bath at 40°C for 60 min, and centrifuging at 5000 rpm for 10 min to remove the free gelatin dissolved by heating to obtain a hollow nanogel;

[0037] S5: Add paclitaxel to the hollow nanogel, stir thoroughly to obtain a nanogel loaded with paclitaxel, so that the mass concentration of paclitaxel in the nanogel is 5%, and then add it to the chitosan oligosaccharide solution so that the volume ratio of the paclitaxel-loaded nanogel and the chitosan oligosaccharide solution is 3:1, stir thoroughly to obtain a chitosan oligosaccharide-based nanogel.

[0038] Example 3

[0039] S1: Dissolve gelatin powder in deionized water, stir evenly at 40°C to obtain a gelatin solution with a mass concentration of 3%, add acetone to the gelatin solution at a volume ratio of 1:1, stir evenly at 40°C, centrifuge at 5000rpm for 10min, remove the supernatant, and dissolve the obtained precipitate in deionized water again, adjust the pH value to 9, then add glutaraldehyde to cross-link the gelatin to form particles, centrifuge at 10000rpm for 30min, evaporate and remove the acetone, and obtain gelatin nanoparticles;

[0040] S2: Dissolve chitosan oligosaccharide in deionized water, fully dissolve and stir to obtain a chitosan oligosaccharide solution with a mass concentration of 3%, redisperse gelatin nanoparticles in deionized water to obtain a gelatin nanoparticle suspension, and add the suspension to the chitosan oligosaccharide solution at a volume ratio of 1:1 to obtain a gelatin-chitosan oligosaccharide composite nanogel;

[0041] S3: adding gelatin-chitosan oligosaccharide composite nanogel to a pectin solution with a mass concentration of 2% at a volume ratio of 1:1, and stirring the mixture thoroughly to obtain gelatin-chitosan oligosaccharide-pectin composite nanogel;

[0042] S4: heating the gelatin-chitosan oligosaccharide-pectin composite nanogel in a water bath at 40°C for 60 min, and centrifuging at 5000 rpm for 10 min to remove the free gelatin dissolved by heating to obtain a hollow nanogel;

[0043] S5: adding thymol to the hollow nanogel and stirring thoroughly to obtain a nanogel loaded with thymol, so that the mass concentration of thymol in the nanogel is 5%, and then adding it to the chitosan oligosaccharide solution so that the volume ratio of the nanogel loaded with thymol to the chitosan oligosaccharide solution is 1:1, stirring thoroughly to obtain a chitosan oligosaccharide-based nanogel.

[0044] Example 4

[0045] S1: Dissolve gelatin powder in deionized water, stir evenly at 40°C to obtain a gelatin solution with a mass concentration of 3%, add acetone to the gelatin solution at a volume ratio of 1:1, stir evenly at 40°C, centrifuge at 5000rpm for 10min, remove the supernatant, and dissolve the obtained precipitate in deionized water again, adjust the pH value to 10, then add glutaraldehyde to cross-link the gelatin to form particles, perform refrigerated centrifugation at 10000rpm for 30min, and volatilize and remove the acetone to obtain gelatin nanoparticles;

[0046] S2: Dissolve chitosan oligosaccharide in deionized water, fully dissolve and stir to obtain a chitosan oligosaccharide solution with a mass concentration of 3%, redisperse gelatin nanoparticles in deionized water to obtain a gelatin nanoparticle suspension, and add the suspension to the chitosan oligosaccharide solution at a volume ratio of 1:1 to obtain a gelatin-chitosan oligosaccharide composite nanogel;

[0047] S3: adding gelatin-chitosan oligosaccharide composite nanogel to a pectin solution with a mass concentration of 2% at a volume ratio of 1:1, and stirring the mixture thoroughly to obtain gelatin-chitosan oligosaccharide-pectin composite nanogel;

[0048] S4: heating the gelatin-chitosan oligosaccharide-pectin composite nanogel in a water bath at 40°C for 60 min, and centrifuging at 5000 rpm for 10 min to remove the free gelatin dissolved by heating to obtain a hollow nanogel;

[0049] S5: adding melatonin to the hollow nanogel, stirring thoroughly to obtain a nanogel loaded with melatonin, so that the mass concentration of melatonin in the nanogel is 5%, and then adding it to the chitosan oligosaccharide solution, so that the volume ratio of the nanogel loaded with melatonin and the chitosan oligosaccharide solution is 1:3, stirring thoroughly to obtain a chitosan oligosaccharide-based nanogel.

[0050] Example 5

[0051] S1: Dissolve gelatin powder in deionized water, stir evenly at 40°C to obtain a gelatin solution with a mass concentration of 3%, add acetone to the gelatin solution at a volume ratio of 1:1, stir evenly at 40°C, centrifuge at 5000rpm for 10min, remove the supernatant, and dissolve the obtained precipitate in deionized water again, adjust the pH value to 12, then add glutaraldehyde to cross-link the gelatin to form particles, perform refrigerated centrifugation at 10000rpm for 30min, and volatilize and remove the acetone to obtain gelatin nanoparticles;

[0052] S2: Dissolve chitosan oligosaccharide in deionized water, fully dissolve and stir to obtain a chitosan oligosaccharide solution with a mass concentration of 3%, redisperse gelatin nanoparticles in deionized water to obtain a gelatin nanoparticle suspension, and add the suspension to the chitosan oligosaccharide solution at a volume ratio of 1:1 to obtain a gelatin-chitosan oligosaccharide composite nanogel;

[0053] S3: adding gelatin-chitosan oligosaccharide composite nanogel to a pectin solution with a mass concentration of 2% at a volume ratio of 1:1, and stirring the mixture thoroughly to obtain gelatin-chitosan oligosaccharide-pectin composite nanogel;

[0054] S4: heating the gelatin-chitosan oligosaccharide-pectin composite nanogel in a water bath at 40°C for 60 min, and centrifuging at 5000 rpm for 10 min to remove the free gelatin dissolved by heating to obtain a hollow nanogel;

[0055] S5: Add tea polyphenols to the hollow nanogel and stir thoroughly to obtain nanogel loaded with tea polyphenols, so that the mass concentration of tea polyphenols in the nanogel is 5%, and then add it to the chitosan oligosaccharide solution so that the volume ratio of the nanogel loaded with tea polyphenols and the chitosan oligosaccharide solution is 1:5, stir thoroughly to obtain chitosan oligosaccharide-based nanogel.

[0056] Performance test 1: pH responsiveness determination of chitosan oligosaccharide-based nanogels

[0057] After the chitosan oligosaccharide-based nanogels in Examples 1-5 were adjusted to pH 1-15, their average particle sizes were measured. The results are shown in Table 1.

[0058] Table 1 Average particle size of chitosan oligosaccharide-based nanogels at different pH values

[0059]

[0060]

[0061] As can be seen from Table 1, the volume ratios of the nanogel loaded with active substances and the chitosan oligosaccharide solution in the preparation of the chitosan oligosaccharide-based nanogels in Examples 1-5 are different, so that the prepared chitosan oligosaccharide-based nanogels show different average particle sizes and different change trends and ranges at different pH values. This shows that by changing the volume ratio of the nanogel loaded with active substances and the chitosan oligosaccharide solution, the pH response characteristics of the final chitosan oligosaccharide-based nanogel can be regulated, different pH response requirements of the nanogel can be achieved, and the functional diversity of the nanogel can be given.

[0062] Performance test 2: Determination of the average particle size of gelatin nanoparticles and hollow nanogels and the maximum loading amount of active substances

[0063] The average particle size of the gelatin nanoparticles and hollow nanogels in Examples 1-5 and the maximum loading amount of the active substance were measured. The results are shown in Table 2.

[0064] Table 2 Average particle size of gelatin nanoparticles and hollow nanogels and maximum loading of active substances

[0065]

[0066] As can be seen from Table 2, the average particle size of the gelatin nanoparticles in Examples 1-5 shows a gradually increasing trend with the increase of pH during preparation, so that the average particle size of the prepared hollow nanogel also shows a gradually increasing trend, and its maximum loading capacity for active substances also shows a gradually increasing trend. This shows that when preparing gelatin nanoparticles, the size of the gelatin nanoparticles can be controlled by adjusting different pH preparation conditions, thereby regulating the internal cavity and the overall size of the hollow nanogel to achieve accurate quantitative loading of active substances.

[0067] In addition, the PDI of the hollow nanogels in Examples 1-5 were all less than 0.15, indicating that hollow nanogels with uniform particle size can be prepared by using gelatin nanoparticles as templates and utilizing the temperature sensitivity of gelatin nanoparticles.

[0068] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing a chitosan oligosaccharide-based nanogel with intelligent response, characterized in that: The following steps are involved: S1: dissolving gelatin powder in deionized water, stirring evenly to obtain a gelatin solution of a certain mass concentration, adding acetone to the gelatin solution, stirring evenly, centrifuging at 5000rpm for 10min, removing the supernatant, dissolving the obtained precipitate in deionized water, adjusting the pH value, then adding glutaraldehyde to cross-link the gelatin to form particles, performing refrigerated centrifugation at 10000rpm for 30min, and volatilizing and removing the acetone to obtain gelatin nanoparticles; the gelatin is type B gelatin; S2: dissolving chitosan oligosaccharide in deionized water, fully dissolving and stirring to obtain a chitosan oligosaccharide solution, re-dispersing gelatin nanoparticles in deionized water to obtain a gelatin nanoparticle suspension, and adding the suspension to the chitosan oligosaccharide solution to obtain a gelatin-chitosan oligosaccharide composite nanogel; S3: adding gelatin-chitosan oligosaccharide composite nanogel into the pectin solution and stirring well to obtain gelatin-chitosan oligosaccharide-pectin composite nanogel; S4: heating the gelatin-chitosan oligosaccharide-pectin composite nanogel in a water bath at 40°C, centrifuging at 5000 rpm for 10 min, removing the heated and dissolved free gelatin, and obtaining a hollow nanogel; S5: adding active substances to the hollow nanogel, stirring thoroughly to obtain nanogel loaded with active substances, and then adding the nanogel to the chitosan oligosaccharide solution, stirring thoroughly to obtain chitosan oligosaccharide-based nanogel.

2. The method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to claim 1, characterized in that: The mass concentration of the gelatin solution in step S1 is 1% to 10%, the volume ratio of the gelatin solution to acetone is 1:1, and the pH value is adjusted to 6 to 12.

3. The method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to claim 1, characterized in that: All operations except the centrifugation operation in step S1 are performed in a 40° C. water bath.

4. The method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to claim 1, characterized in that: The mass concentration of the chitosan oligosaccharide solution in step S2 is 1% to 5%, and the volume ratio of the gelatin nanoparticle suspension to the chitosan oligosaccharide solution is 1:

1.

5. The method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to claim 1, characterized in that: The mass concentration of the pectin solution in step S3 is 0.5% to 5%, and the volume ratio of the gelatin-chitosan oligosaccharide composite nanogel to the pectin solution is 1:

1.

6. The method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to claim 1, characterized in that: The water bath heating time in step S4 is 30 to 120 minutes.

7. The method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to claim 1, characterized in that: The active substances in step S5 include ketoprofen, paclitaxel, thymol, melatonin and tea polyphenols.

8. The method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to claim 1, characterized in that: In step S5, the mass concentration of the active substance in the hollow nanogel is 1% to 30%, and the volume ratio of the nanogel loaded with the active substance to the chitosan oligosaccharide solution is (1 to 5): (1 to 5).

9. The nanogel prepared by the method for preparing a chitosan oligosaccharide-based nanogel with intelligent response according to any one of claims 1 to 8.

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