Sodium alginate / chitosan microsphere for loading cells and preparation method thereof

Abstract

The invention discloses a sodium alginate / chitosan microsphere for loading cells and a preparation method, the preparation method comprises the following steps: adding a pore-foaming agent into a sodium alginate solution, and stirring overnight to obtain a sodium alginate solution containing the pore-foaming agent; the preparation method comprises the following steps: dissolving chitosan in an acetic acid solution, adding calcium chloride, and stirring until the calcium chloride is completely dissolved to obtain a chitosan mixed solution; dropwise adding a sodium alginate solution containing a pore-foaming agent into the chitosan mixed solution, standing, and filtering to obtain a microsphere precipitate; soaking the microsphere precipitate in a hydrochloric acid solution until no bubble overflows, filtering, cleaning and drying; according to the sodium alginate / chitosan microspheres for loading the cells, natural polymer materials, namely the sodium alginate and the chitosan, are adopted, so that excellent biocompatibility is ensured, and the rejection reaction risk is reduced; through a calcium carbonate pore-forming technology, the microspheres have an accurate pore structure, so that loading of cells and stable release of drugs are facilitated, the release rate is controlled, and the treatment effect is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to sodium alginate / chitosan microspheres for loading cells and a preparation method thereof. Background Art

[0002] In the biomedical field, existing drug and cell carrier materials face challenges such as low encapsulation efficiency, poor biocompatibility, difficulty in structural control, and complex preparation processes. With the rise of precision medicine and cell therapy, there is an urgent need for efficient, safe, and controllable carrier systems.

[0003] In recent years, chitosan-based cell microcarriers have been reported, including solid microcarriers and porous microcarriers. Their preparation techniques often use cross-linking agents such as glutaraldehyde and epichlorohydrin or pore-forming agents such as polyethylene glycol, which increases the biological toxicity and preparation cost of the microcarriers.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a sodium alginate / chitosan microsphere for loading cells and a preparation method thereof. The sodium alginate / chitosan microsphere for loading cells of the present invention adopts natural polymer materials sodium alginate and chitosan as a matrix, and constructs a microsphere system with a controllable pore structure by introducing calcium carbonate as a porogen. This method not only improves the cell or drug loading capacity of the microsphere, but also realizes the precise delivery and controllable release of drugs at the lesion site, thereby significantly improving the therapeutic effect.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The first aspect of the present invention provides a method for preparing sodium alginate / chitosan microspheres for loading cells, and the method for preparing sodium alginate / chitosan microspheres for loading cells comprises the following steps:

[0008] (a) adding a porogen to a sodium alginate solution and stirring overnight to obtain a sodium alginate solution containing a porogen;

[0009] (b) dissolving chitosan in an acetic acid solution, adding calcium chloride and stirring until completely dissolved to obtain a chitosan mixed solution;

[0010] (c) under stirring conditions, adding a sodium alginate solution containing a porogen dropwise into the chitosan mixture, and then allowing the mixture to stand and filter to obtain a microsphere precipitate;

[0011] (d) soaking the microsphere precipitate in a hydrochloric acid solution until no bubbles overflow, filtering, washing and drying to obtain the sodium alginate / chitosan microspheres for loading cells.

[0012] Preferably, in step (a), the concentration of the sodium alginate solution is 1.5% to 2.5%.

[0013] Preferably, in the step (a), the mass volume concentration of the porogen in the sodium alginate solution containing the porogen is 10% to 20%.

[0014] Preferably, in step (a), the porogen is selected from at least one of calcium carbonate, egg shell powder and shell powder.

[0015] Preferably, in step (b), the chitosan concentration in the chitosan mixed solution is 4-8 mg / mL, and the calcium chloride concentration is 15-25 mg / mL.

[0016] Preferably, in step (b), the concentration of the acetic acid solution is 0.8% to 1.2%.

[0017] Preferably, in step (c), the mass ratio of sodium alginate to chitosan is 1:(0.2-0.8);

[0018] The stirring speed is 450-600 rpm.

[0019] Preferably, in step (d), the concentration of the hydrochloric acid solution is 1 to 3 mol / L.

[0020] The second aspect of the present invention provides a sodium alginate / chitosan microsphere for loading cells obtained by the above preparation method.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least:

[0022] The sodium alginate / chitosan microspheres for loading cells of the present invention adopt natural polymer materials sodium alginate and chitosan, thereby ensuring excellent biocompatibility and reducing the risk of rejection reaction; through the calcium carbonate pore-forming technology, the microspheres have a precise pore structure, which is beneficial to the loading of cells and the stable release of drugs, and controls the release rate to improve the therapeutic effect; in addition, the degradability of the sodium alginate / chitosan microspheres for loading cells of the present invention avoids secondary surgery and reduces the burden on patients, and the structure is stable, the preparation process is simple, and it is easy to realize industrial production and reduce the cost; in addition, the sodium alginate / chitosan microspheres for loading cells of the present invention have brought revolutionary progress to the fields of clinical precision medicine and cell therapy by virtue of their comprehensive advantages in biocompatibility, loading efficiency, degradability, structural stability and preparation efficiency, and show broad application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0024] Figure 1 This is an electron microscopic image of sodium alginate / chitosan microspheres for loading cells prepared by the method of Example 1 of the present invention;

[0025] Figure 2 This is an electron microscopic image of sodium alginate / chitosan microspheres for loading cells prepared by the method of Example 2 of the present invention;

[0026] Figure 3 This is an electron microscopic image of sodium alginate / chitosan microspheres for loading cells prepared by the method of Example 3 of the present invention;

[0027] Figure 4 This is an electron microscopic image of sodium alginate / chitosan microspheres for loading cells prepared by the method of Example 4 of the present invention. DETAILED DESCRIPTION

[0028] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0029] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0030] The embodiment of the present invention provides a method for preparing sodium alginate / chitosan microspheres for loading cells. The method for preparing sodium alginate / chitosan microspheres for loading cells comprises the following steps:

[0031] (a) adding a porogen to a sodium alginate solution and stirring overnight to obtain a sodium alginate solution containing a porogen;

[0032] (b) dissolving chitosan in an acetic acid solution, adding calcium chloride and stirring until completely dissolved to obtain a chitosan mixed solution;

[0033] (c) under stirring conditions, adding a sodium alginate solution containing a porogen dropwise into the chitosan mixture, and then allowing the mixture to stand and filter to obtain a microsphere precipitate;

[0034] (d) soaking the microsphere precipitate in a hydrochloric acid solution until no bubbles overflow, filtering, washing and drying to obtain the sodium alginate / chitosan microspheres for loading cells.

[0035] The sodium alginate / chitosan microspheres for loading cells of the present invention adopt natural polymer materials sodium alginate and chitosan, thereby ensuring excellent biocompatibility and reducing the risk of rejection reaction; through the calcium carbonate pore-forming technology, the microspheres have a precise pore structure, which is beneficial to the loading of cells and the stable release of drugs, and controls the release rate to improve the therapeutic effect; in addition, the degradability of the sodium alginate / chitosan microspheres for loading cells of the present invention avoids secondary surgery and reduces the burden on patients, and the structure is stable, the preparation process is simple, and it is easy to realize industrial production and reduce the cost; in addition, the sodium alginate / chitosan microspheres for loading cells of the present invention have brought revolutionary progress to the fields of clinical precision medicine and cell therapy by virtue of their comprehensive advantages in biocompatibility, loading efficiency, degradability, structural stability and preparation efficiency, and show broad application prospects and market value.

[0036] In some embodiments, in step (a), the concentration of the sodium alginate solution is 1.5% to 2.5%.

[0037] In some embodiments, in step (a), the mass volume concentration of the porogen in the sodium alginate solution containing the porogen is 10% to 20%.

[0038] In some embodiments, in step (a), the porogen is selected from at least one of calcium carbonate, egg shell powder and shell powder.

[0039] In some embodiments, in step (b), the chitosan concentration in the chitosan mixed solution is 4-8 mg / mL, and the calcium chloride concentration is 15-25 mg / mL.

[0040] In some embodiments, in step (b), the concentration of the acetic acid solution is 0.8% to 1.2%.

[0041] In some embodiments, in step (c), the mass ratio of sodium alginate to chitosan is 1:(0.2-0.8);

[0042] The stirring speed is 450-600 rpm.

[0043] In some embodiments, in step (d), the concentration of the hydrochloric acid solution is 1 to 3 mol / L.

[0044] Another embodiment of the present invention provides a sodium alginate / chitosan microsphere for loading cells prepared by the above preparation method.

[0045] The technical solution of the present invention is further described in detail below through specific embodiments.

[0046] Example 1

[0047] This embodiment is a method for preparing sodium alginate / chitosan microspheres for loading cells, and the method for preparing sodium alginate / chitosan microspheres for loading cells comprises the following steps:

[0048] (a) dissolving 2 g of sodium alginate in 100 mL of deionized water, stirring to completely dissolve the sodium alginate, to obtain a sodium alginate solution with a mass fraction of 2.0%;

[0049] Adding 10% calcium carbonate to the sodium alginate solution and stirring overnight to obtain a sodium alginate solution containing a porogen;

[0050] (b) dissolving 1.2 g of chitosan in 200 mL of 1% acetic acid solution, adding 4 g of calcium chloride and stirring until completely dissolved to obtain a chitosan mixed solution;

[0051] (c) under stirring at 500 rpm, adding the sodium alginate solution containing the porogen to the chitosan mixture, and then standing for 2 h and filtering to obtain microsphere precipitation;

[0052] (d) Soaking the microsphere precipitate in a 1 mol / L hydrochloric acid solution until no bubbles overflow, filtering, and then transferring to deionized water for soaking and washing, each soaking time is 20 min, washing by vacuum filtration until the washing solution is neutral, and freeze-drying to obtain the sodium alginate / chitosan microspheres for loading cells.

[0053] Example 2

[0054] This embodiment is a method for preparing sodium alginate / chitosan microspheres for loading cells, and the method for preparing sodium alginate / chitosan microspheres for loading cells comprises the following steps:

[0055] (a) dissolving 2 g of sodium alginate in 100 mL of deionized water, stirring to completely dissolve the sodium alginate, to obtain a sodium alginate solution with a mass fraction of 2.0%;

[0056] Add 20% calcium carbonate to the sodium alginate solution and stir overnight to obtain a sodium alginate solution containing a porogen;

[0057] (b) dissolving 1.2 g of chitosan in 200 mL of 1% acetic acid solution, adding 4 g of calcium chloride and stirring until completely dissolved to obtain a chitosan mixed solution;

[0058] (c) under stirring at 500 rpm, adding the sodium alginate solution containing the porogen to the chitosan mixture, and then standing for 2 h and filtering to obtain microsphere precipitation;

[0059] (d) Soaking the microsphere precipitate in a 1 mol / L hydrochloric acid solution until no bubbles overflow, filtering, and then transferring to deionized water for soaking and washing, each soaking time is 20 min, washing by vacuum filtration until the washing solution is neutral, and freeze-drying to obtain the sodium alginate / chitosan microspheres for loading cells.

[0060] Example 3

[0061] This embodiment is a method for preparing sodium alginate / chitosan microspheres for loading cells, and the method for preparing sodium alginate / chitosan microspheres for loading cells comprises the following steps:

[0062] (a) dissolving 2 g of sodium alginate in 100 mL of deionized water, stirring to completely dissolve the sodium alginate, to obtain a sodium alginate solution with a mass fraction of 2.0%;

[0063] Add 20% calcium carbonate to the sodium alginate solution and stir overnight to obtain a sodium alginate solution containing a porogen;

[0064] (b) dissolving 1.2 g of chitosan in 200 mL of 1% acetic acid solution, adding 4 g of calcium chloride and stirring until completely dissolved to obtain a chitosan mixed solution;

[0065] (c) under stirring at 500 rpm, adding the sodium alginate solution containing the porogen to the chitosan mixture, and then standing for 2 h and filtering to obtain microsphere precipitation;

[0066] (d) Soaking the microsphere precipitate in a 2 mol / L hydrochloric acid solution until no bubbles overflow, filtering, and then transferring to deionized water for soaking and washing, each soaking time is 20 min, washing by vacuum filtration until the washing solution is neutral, and freeze-drying to obtain the sodium alginate / chitosan microspheres for loading cells.

[0067] Example 4

[0068] This embodiment is a method for preparing sodium alginate / chitosan microspheres for loading cells, and the method for preparing sodium alginate / chitosan microspheres for loading cells comprises the following steps:

[0069] (a) dissolving 2 g of sodium alginate in 100 mL of deionized water, stirring to completely dissolve the sodium alginate, to obtain a sodium alginate solution with a mass fraction of 2.0%;

[0070] Add 20% calcium carbonate to the sodium alginate solution and stir overnight to obtain a sodium alginate solution containing a porogen;

[0071] (b) dissolving 1.2 g of chitosan in 200 mL of 1% acetic acid solution, adding 4 g of calcium chloride and stirring until completely dissolved to obtain a chitosan mixed solution;

[0072] (c) under stirring at 500 rpm, adding the sodium alginate solution containing the porogen to the chitosan mixture, and then standing for 2 h and filtering to obtain microsphere precipitation;

[0073] (d) Soaking the microsphere precipitate in a 3 mol / L hydrochloric acid solution until no bubbles overflow, filtering, and then transferring to deionized water for soaking and washing, each soaking time is 20 min, washing by vacuum filtration until the washing solution is neutral, and freeze-drying to obtain the sodium alginate / chitosan microspheres for loading cells.

[0074] Experimental example

[0075] Obtain the sodium alginate / chitosan microspheres for loading cells prepared in Examples 1 to 4 respectively;

[0076] The above-mentioned different sodium alginate / chitosan microspheres used for loading cells were scanned by electron microscope, and the scanning results are as follows: Figures 1 to 4 As shown;

[0077] The cell adsorption rates of the above-mentioned different sodium alginate / chitosan microspheres used to load cells were tested at different time periods. The testing method is as follows:

[0078] Step 1, preparing microspheres: taking 0.2 g of each of the microspheres prepared in Examples 1-4, sterilizing them with ultraviolet light and pouring them into sterile cell culture bottles, preparing three groups each;

[0079] Step 2: Prepare cells: Prepare a suspension of human umbilical cord mesenchymal stem cells (3 × 10 6 Resuspend each cell in 30 mL of complete medium for later use. Prepare twelve copies.

[0080] Step 3, inoculating cells: the above cell suspensions are mixed into cell culture bottles respectively, and mixed with microspheres;

[0081] Step 4, cell adsorption: Place the cell culture flask on a low-speed stirrer and place it in a 37°C, 5% carbon dioxide incubator for stirring at 50 rpm to allow the cells to adhere to the microspheres by rotation;

[0082] Step 5: Count cells and calculate adsorption rate: After stirring for 4 hours, 8 hours, and 12 hours, the three groups of microcarriers mixed with the cell suspension were collected through a 100 μm cell sieve, and the total number of cells in the cell suspension was detected.

[0083] The calculation formula of the adsorption rate is: adsorption rate = [1-(number of unadsorbed cells / total number of cells)] × 100%. The test results are shown in Table 1.

[0084] Table 1 Cell adsorption rate of microspheres in different embodiments at different time periods

[0085]

[0086] From Table 1, we can see that:

[0087] The sodium alginate / chitosan microspheres for loading cells prepared in the embodiments of the present invention have excellent adsorption performance for cells. It can be determined by comparing the various embodiments that Example 2 can better improve the adsorption rate of the prepared microspheres for cells by limiting the amount of porogen and the concentration of the hydrochloric acid solution.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for preparing sodium alginate / chitosan microspheres for loading cells, characterized in that: The method for preparing sodium alginate / chitosan microspheres for loading cells comprises the following steps: (a) adding a porogen to a sodium alginate solution and stirring overnight to obtain a sodium alginate solution containing a porogen; (b) dissolving chitosan in an acetic acid solution, adding calcium chloride and stirring until completely dissolved to obtain a chitosan mixed solution; (c) under stirring conditions, adding a sodium alginate solution containing a porogen dropwise into the chitosan mixture, and then allowing the mixture to stand and filter to obtain a microsphere precipitate; (d) soaking the microsphere precipitate in a hydrochloric acid solution until no bubbles overflow, filtering, washing and drying to obtain the sodium alginate / chitosan microspheres for loading cells.

2. The method for preparing sodium alginate / chitosan microspheres for loading cells according to claim 1, characterized in that: In the step (a), the concentration of the sodium alginate solution is 1.5% to 2.5%.

3. The method for preparing sodium alginate / chitosan microspheres for loading cells according to claim 1, characterized in that: In the step (a), the mass volume concentration of the porogen in the sodium alginate solution containing the porogen is 10% to 20%.

4. The method for preparing sodium alginate / chitosan microspheres for loading cells according to claim 1, characterized in that: In the step (a), the porogen is selected from at least one of calcium carbonate, egg shell powder and shell powder.

5. The method for preparing sodium alginate / chitosan microspheres for loading cells according to claim 1, characterized in that: In the step (b), the chitosan concentration in the chitosan mixed solution is 4-8 mg / mL, and the calcium chloride concentration is 15-25 mg / mL.

6. The method for preparing sodium alginate / chitosan microspheres for loading cells according to claim 1, characterized in that: In the step (b), the concentration of the acetic acid solution is 0.8% to 1.2%.

7. The method for preparing sodium alginate / chitosan microspheres for loading cells according to claim 1, characterized in that: In the step (c), the mass ratio of sodium alginate to chitosan is 1:(0.2-0.8); The stirring speed is 450-600 rpm.

8. The method for preparing sodium alginate / chitosan microspheres for loading cells according to claim 1, characterized in that: In the step (d), the concentration of the hydrochloric acid solution is 1 to 3 mol / L.

9. Sodium alginate / chitosan microspheres for loading cells obtained by the preparation method according to any one of claims 1 to 8.

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