Co-grafted dendronized chitosan, temperature-sensitive hydrogel and preparation method and application of co-grafted dendronized chitosan and temperature-sensitive hydrogel

Abstract

The invention relates to co-grafted dendronized chitosan, temperature-sensitive hydrogel as well as a preparation method and application of the co-grafted dendronized chitosan and the temperature-sensitive hydrogel. The co-grafted dendronized chitosan has the following structural general formula: # imgabs0 #, the temperature-sensitive hydrogel is prepared by dissolving and heating the co-grafted dendronized chitosan, and the solid content of the co-grafted dendronized chitosan in the temperature-sensitive hydrogel is 0.2-15 wt%. Compared with the prior art, the co-grafted dendritic chitosan prepared by the method has good solubility in a physiological buffer solution and a culture medium, can form transparent hydrogel after being heated, has excellent temperature response characteristics, and can be used for supporting in-situ entrapment and three-dimensional culture of cells.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a co-grafted dendritic chitosan, a thermosensitive hydrogel, and a preparation method and application thereof. Background Art

[0002] Cell culture systems are crucial in biological research, effectively reducing the use of experimental animals and providing a powerful foundation for the discovery and screening of new drugs and further exploration of life sciences. However, cells cultured on a 2D surface cannot mimic the true tissue structure and natural microenvironment of cells in a living organism. With the increasing demand for disease model construction, drug toxicity analysis, and drug development, 3D cell culture is gaining increasing attention.

[0003] Hydrogels are three-dimensional networks composed of hydrophilic polymers that provide a water environment similar to the natural extracellular matrix, making them the most widely used 3D cell culture matrix. However, existing hydrogel-based 3D cell in vitro models generally face the following key challenges: (1) The cell encapsulation process is highly damaging to cells, and the use of free radicals or toxic cross-linkers can destroy the normal cell structure. (2) Cell release relies on destructive conditions such as UV or chemical degradation. (3) Hydrogels lack a biomimetic extracellular matrix structure. These challenges have hindered the application of the above-mentioned hydrogels in cell 3D culture. In recent years, temperature-responsive hydrogels have become an important 3D cell culture matrix. Sol-gel transformation can be easily achieved by adjusting the temperature, thereby achieving lossless release and collection of cells.

[0004] Chitosan, a natural cationic polysaccharide, is highly favored in the fields of biomedicine and tissue engineering due to its excellent biocompatibility, degradability and antibacterial activity. Chitosan can be given temperature sensitivity by adding sodium β-glycerophosphate to the solution or modifying it with hydroxypropyl or hydroxybutyl groups. However, the introduction of this temperature-sensitive property is usually only applicable to aqueous solutions. In physiological solutions, the above-mentioned substances are usually difficult to dissolve and usually precipitate after heating, making it difficult to form a transparent hydrogel. This is because the physical cross-linking process of the above-mentioned added substance chitosan forms a heterogeneous network structure or a salting-out effect, resulting in low transparency of the gel, which limits the real-time observation of cells under an optical microscope and is therefore not suitable for three-dimensional cell culture.

[0005] CN108484797A discloses an alkoxy ether dendronized chitosan, its hydrogel material, and its preparation method. This method grafts temperature-sensitive alkoxy ether dendrons onto chitosan side groups, and produces chitosan derivatives with varying grafting ratios by varying the chitosan-alkoxy ether ratio. High-grafting polymers can form gels by direct heating, while low-grafting polymers can form hydrogels by adding a crosslinker. However, the alkoxy ether dendronized chitosan is a single-grafted chitosan, suitable only for aqueous solutions and unable to dissolve in physiological solutions or cell culture media. This limits the application of these temperature-sensitive dendronized chitosan hydrogels in three-dimensional cell culture and observation.

[0006] Therefore, a new type of temperature-sensitive chitosan hydrogel remains to be developed, which can maintain low viscosity at low temperatures to encapsulate cells and maintain high transparency after gelation to observe cells in real time. Summary of the Invention

[0007] The purpose of the present invention is to provide a co-grafted dendritic chitosan, a thermosensitive hydrogel, a preparation method and an application thereof in order to overcome the defects of the prior art.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The present invention first provides a co-grafted dendritic chitosan having the following general structural formula:

[0010]

[0011] Wherein, q = 10-6500, z = 10-6500, r = 10-6500;

[0012] R1 is or—COO - H3N + -;

[0013] R2 is H or

[0014] R3 and R4 are

[0015] wherein n=3-4, m=0-2, X and Y are independently selected from hydroxyl or C 1-6 of alkoxy.

[0016] In the structural formula of the above-mentioned co-grafted dendronized chitosan, R1 is derived from the amide bond or electrostatic interaction formed between the chitosan amino group and the carboxyl group of the dendron unit during the preparation process; R2 is derived from the structure of the chitosan molecule itself and depends on the degree of deacetylation of the chitosan itself; R3 and R4 are derived from the introduction of different dendronized alkoxy ether units, respectively.

[0017] Furthermore, the molecular weight range of the co-grafted dendritic chitosan is 1×10 4 -1×10 6 .

[0018] The present invention also provides a method for preparing co-grafted dendritic chitosan, which is selected from method A.

[0019] or any of Method B;

[0020] The method A comprises the following steps:

[0021] S1.1: Mix chitosan, reaction aid and solvent until chitosan is completely dissolved;

[0022] S1.2: Adding the dendrimer alkoxy ether moieties I and II corresponding to R3 and R4, respectively, and an activator, followed by a condensation reaction;

[0023] S1.3: After the reaction is completed, the product is dialyzed and freeze-dried to obtain the co-grafted dendritic chitosan;

[0024] The method B comprises the following steps:

[0025] S2.1: Mix chitosan, reaction auxiliary agent and solvent until chitosan is completely dissolved;

[0026] S2.2: Adding the dendrimer alkoxy ether moiety I corresponding to R3 and an activator, followed by a condensation reaction;

[0027] S2.3: After the reaction is completed, the product is dialyzed and freeze-dried to obtain single-grafted dendritic chitosan;

[0028] S2.4: After redissolving the mono-grafted dendritic chitosan, the dendritic alkoxy ether unit II corresponding to R4 and an activator are added, followed by a secondary condensation reaction;

[0029] S2.5: After the reaction is completed, the product is dialyzed and freeze-dried to obtain the co-grafted dendritic chitosan;

[0030] The structural formulas of the branched alkoxy ether units I and II are:

[0031]

[0032] Furthermore, the reaction auxiliary agent is any one or more of 1-hydroxybenzotriazole, 1-oxybenzotriazole, 1,1-dimethyl-3-oxy-1,2,3-triazabenzotriazole, O-benzotriazole-N,N,N',N"-tetramethyluronium hexafluorophosphate, O-benzotriazole-N-tetramethyluronium tetrafluoroborate, benzotriazole-1-acyl-1-methylpyridinium hexafluorophosphate, benzotriazole-1-acyloxytris(dimethylamino)phosphonium hexafluorophosphate, 3-[(trisdimethylamino)sulfonyl]-2-oxy-4-benzotriazole, O-benzotriazole-NN,N'-trimethyluronium trifluoroacetate, and 3-[(diphenylphosphoryl)oxy]-4-benzotriazole.

[0033] Furthermore, the activator is any one or more of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide, cyanodimethylethylcarbodiimide, n-propylphosphonic anhydride, ethylmethylphosphinic anhydride, diphenylphosphoryl chloride, di(2-oxo-3-oxazolidinyl)phosphoryl chloride, N,N-carbonyldiimidazole, and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-7-yl-2,2-dimethylpropyl ester.

[0034] Furthermore, in method A and method B, the molar ratio of the repeating unit of chitosan to the reaction auxiliary agent is 1:(1-2).

[0035] Furthermore, in method A, the molar ratio of the chitosan repeating unit, the dendronized alkoxy ether unit I, the dendronized alkoxy ether unit II and the activator is 1:(0.1-2):(0.1-2):(1-1.5).

[0036] Furthermore, in method B, the molar ratio of the chitosan repeating unit, the dendronized alkoxy ether unit I and the activator is 1:(0.1-2):(1-1.5).

[0037] Furthermore, in method B, the molar ratio of the repeating unit of the mono-grafted dendronized chitosan, the dendronized alkoxy ether unit II and the activator is 1:(0.1-2):(1-1.5).

[0038] Furthermore, in method A and method B, the condensation reaction time is 4-96 hours, and the reaction temperature is 15-100°C.

[0039] Furthermore, in method A and method B, the dialysis time is 5-7 days, and the dialysis molecular weight cut-off is 3.5 kDa.

[0040] The present invention also provides a thermosensitive hydrogel, which is prepared by dissolving the co-grafted dendritic chitosan and then heating it.

[0041] Furthermore, the solid content of the co-grafted dendritic chitosan in the thermosensitive hydrogel is 0.2-15 wt %.

[0042] Furthermore, the thermosensitive hydrogel is prepared from any of the following components:

[0043] 0.1-10 wt% of co-grafted dendritic chitosan, 0.1-10 wt% of buffer, and the balance being water;

[0044] Alternatively, 0.1-10 wt % of dendritic chitosan, 0.1-99.9 wt % of cell culture medium, and the balance being water are co-grafted;

[0045] Alternatively, 0.1-10 wt% of dendritic chitosan is co-grafted, and the balance is water.

[0046] Furthermore, the buffer includes any one or more of the following salts or organic solvents: disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium β-glycerophosphate, sodium acetate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium chloride, potassium nitrate, sodium sulfate, sodium sulfite, glycerol, propylene glycol, and dimethyl sulfoxide.

[0047] Furthermore, the cell culture medium is any one or more of DMEM culture medium, MEN culture medium, RPMI 1640 culture medium, DMEM / F12 culture medium, IMDM culture medium, HamF10 culture medium, M199 culture medium, McCoy5A culture medium, and L15 cell culture medium.

[0048] The present invention also provides an application of a thermosensitive hydrogel in three-dimensional cell culture, wherein the application specifically comprises the following steps:

[0049] S3.1: Dissolve the co-grafted dendritic chitosan and mix it evenly with the cell suspension to be cultured, and then incubate until a gel is formed;

[0050] S3.2: Place the gel in culture medium and perform three-dimensional cell culture at a suitable temperature;

[0051] S3.3: After the culture is completed, the cells in the hydrogel are released by cooling, and the cultured cells are collected by centrifugation.

[0052] Furthermore, the cells may be co-cultured with one or more cells.

[0053] Furthermore, the cells can be cultured in the gel or on the surface of the gel.

[0054] Furthermore, the cell culture medium may contain active substances such as growth factors, proteins, nucleic acids, and drugs.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The co-grafted dendritic chitosan hydrogel of the present invention is a thermosensitive co-grafted dendritic chitosan hydrogel prepared by introducing two different dendritic alkoxy ether units. The co-grafted dendritic chitosan hydrogel has good solubility in physiological buffer solution and culture medium at room temperature, and can form a transparent hydrogel when the temperature is raised to near body temperature. It has excellent temperature response characteristics and can be used to support in situ encapsulation and three-dimensional culture of cells.

[0057] (2) The co-grafted dendritic chitosan hydrogel of the present invention introduces two different dendritic alkoxy ether units, which makes chitosan have adjustable radial amphiphilicity and prevents chitosan from forming intermolecular hydrogen bonds. When the temperature rises, it can assemble into fibers and physically cross-link to form a transparent hydrogel.

[0058] (3) The present invention can achieve controllable regulation of the solution-gel transition temperature, mechanical properties and solubility of the co-grafted dendron chitosan hydrogel by adjusting the co-grafting ratio of the dendrons and the structure of the dendrons.

[0059] (4) The co-grafted dendritic chitosan hydrogel synthesized in the present invention exhibits excellent biocompatibility, enabling the mixing and dispersion of cells at room temperature. It forms a gel when heated above the gelation temperature, enabling 3D cell culture, and releases the cells when the temperature is lowered back to room temperature. Furthermore, the co-grafted dendritic chitosan hydrogel of the present invention maintains high transparency after gelation, overcoming the opacity of other temperature-sensitive hydrogels and supporting real-time observation of cells during culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the infrared spectrum of the co-grafted dendritic chitosan of Example 1 of the present invention.

[0061] Figure 2 Schematic diagram of the sol-gel formation of the co-grafted dendritic chitosan in Example 1 of the present invention in a phosphate buffer solution and a cell culture medium.

[0062] Figure 3 Schematic diagram of the dissolution effect of chitosan in comparative examples 1 and 2 in phosphate buffer solution.

[0063] Figure 4 3 is a curve showing the change of storage modulus and loss modulus of the co-grafted dendritic chitosan in Example 1 of the present invention in phosphate buffer (pH 7.4) as a function of temperature.

[0064] Figure 5 Schematic diagram of the co-grafted dendritic chitosan used in the present invention for three-dimensional cell culture.

[0065] Figure 6 This is an optical microscope photo of cells cultured in three dimensions using the co-grafted dendritic chitosan hydrogel of the present invention on the 7th day.

[0066] Figure 7 3D stacked fluorescence images of cells cultured on day 7 using co-grafted dendritic chitosan hydrogels in this invention.

[0067] Figure 8 This is a photo of cells released from the co-grafted dendritic chitosan by lowering the temperature after the three-dimensional cell culture of the present invention is completed. DETAILED DESCRIPTION

[0068] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0069] The first aspect of the present invention provides a co-grafted dendritic chitosan having the following general structural formula:

[0070]

[0071] Wherein, q = 10-6500, z = 10-6500, r = 10-6500;

[0072] R1 is or—COO - H3N + -;

[0073] R2 is H or

[0074] R3 and R4 are

[0075] wherein n=3-4, m=0-2, X and Y are independently selected from hydroxyl or C 1-6 Alkoxy groups such as methoxy, ethoxy, propoxy, butoxy, etc.

[0076] In the structural formula of the above-mentioned co-grafted dendronized chitosan, R1 is derived from the amide bond or electrostatic interaction formed between the chitosan amino group and the carboxyl group of the dendron unit during the preparation process; R2 is derived from the structure of the chitosan molecule itself and depends on the degree of deacetylation of the chitosan itself; R3 and R4 are derived from the introduction of different dendronized alkoxy ether units, respectively.

[0077] In some specific embodiments, the molecular weight of the co-grafted dendritic chitosan is in the range of 1×10 4 -1×10 6 .

[0078] The second aspect of the present invention provides a method for preparing co-grafted dendritic chitosan. The preparation method is selected from either method A or method B;

[0079] The method A comprises the following steps:

[0080] S1.1: Mix chitosan, reaction aid and solvent until chitosan is completely dissolved;

[0081] S1.2: Adding the dendrimer alkoxy ether moieties I and II corresponding to R3 and R4, respectively, and an activator, followed by a condensation reaction;

[0082] S1.3: After the reaction is completed, the product is dialyzed and freeze-dried to obtain the co-grafted dendritic chitosan;

[0083] The method B comprises the following steps:

[0084] S2.1: Mix chitosan, reaction auxiliary agent and solvent until chitosan is completely dissolved;

[0085] S2.2: Adding the dendrimer alkoxy ether moiety I corresponding to R3 and an activator, followed by a condensation reaction;

[0086] S2.3: After the reaction is completed, the product is dialyzed and freeze-dried to obtain single-grafted dendritic chitosan;

[0087] S2.4: After redissolving the mono-grafted dendritic chitosan, the dendritic alkoxy ether unit II corresponding to R4 and an activator are added, followed by a secondary condensation reaction;

[0088] S2.5: After the reaction is completed, the product is dialyzed and freeze-dried to obtain the co-grafted dendritic chitosan;

[0089] The structural formulas of the branched alkoxy ether units I and II are:

[0090]

[0091] In some specific embodiments, the reaction auxiliary agent is any one or more of 1-hydroxybenzotriazole, 1-oxybenzotriazole, 1,1-dimethyl-3-oxy-1,2,3-triazabenzotriazole, O-benzotriazole-N,N,N',N"-tetramethyluronium hexafluorophosphate, O-benzotriazole-N-tetramethyluronium tetrafluoroborate, benzotriazole-1-acyl-1-methylpyridinium hexafluorophosphate, benzotriazole-1-acyloxytris(dimethylamino)phosphonium hexafluorophosphate, 3-[(trisdimethylamino)sulfonyl]-2-oxy-4-benzotriazole, O-benzotriazole-NN,N'-trimethyluronium trifluoroacetate, and 3-[(diphenylphosphoryl)oxy]-4-benzotriazole, preferably 1-hydroxybenzotriazole.

[0092] In some specific embodiments, the activator is any one or more of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide, cyanodimethylethylcarbodiimide, n-propylphosphonic anhydride, ethylmethylphosphinic anhydride, diphenylphosphoryl chloride, di(2-oxo-3-oxazolidinyl)phosphoryl chloride, N,N-carbonyldiimidazole, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-7-yl-2,2-dimethylpropyl ester, preferably 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride.

[0093] In some specific embodiments, in method A and method B, the molar ratio of the repeating unit of chitosan to the reaction auxiliary agent is 1:(1-2), such as 1:1, 1:1.5, and 1:2.

[0094] In some specific embodiments, in method A, the molar ratio of the chitosan repeating unit, the dendronized alkoxy ether unit I, the dendronized alkoxy ether unit II, and the activator is 1:(0.1-2):(0.1-2):(1-1.5). The solubility and gelation temperature can be adjusted by adjusting the co-grafting ratio of the dendrons, the dendron structure, etc.

[0095] In some specific embodiments, in method B, the molar ratio of the chitosan repeating unit, the dendronized alkoxy ether unit I, and the activator is 1:(0.1-2):(1-1.5).

[0096] In some specific embodiments, in method B, the molar ratio of the repeating unit of the mono-grafted dendronized chitosan, the dendronized alkoxy ether unit II, and the activator is 1:(0.1-2):(1-1.5).

[0097] In some specific embodiments, in method A and method B, the condensation reaction time is 4-96 hours, and the reaction temperature is 15-100°C.

[0098] In some specific embodiments, in method A and method B, the dialysis time is 5-7 days, and the dialysis molecular weight cut-off is 3.5 kDa.

[0099] A third aspect of the present invention provides a thermosensitive hydrogel, which is prepared by dissolving co-grafted dendritic chitosan and then heating it.

[0100] In some specific embodiments, the solid content of the co-grafted dendritic chitosan in the thermosensitive hydrogel is 0.2-15 wt %.

[0101] In some specific embodiments, the pH value of the co-grafted dendritic chitosan in the thermosensitive hydrogel is 5-11.

[0102] In some specific embodiments, the thermosensitive hydrogel is prepared from any of the following components:

[0103] 0.1-10 wt% of co-grafted dendritic chitosan, 0.1-10 wt% of buffer, and the balance being water;

[0104] Alternatively, 0.1-10 wt % of dendritic chitosan, 0.1-99.9 wt % of cell culture medium, and the balance being water are co-grafted;

[0105] Alternatively, 0.1-10 wt% of dendritic chitosan is co-grafted, and the balance is water.

[0106] In some specific embodiments, the buffer includes any one or more of the following salts or organic solvents: disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium β-glycerophosphate, sodium acetate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium chloride, potassium nitrate, sodium sulfate, sodium sulfite, glycerol, propylene glycol, and dimethyl sulfoxide. For example, a phosphate buffer solution with a pH of 7.4, is isotonic with human blood, and its main components are sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and potassium chloride.

[0107] In some more specific embodiments, the cell culture medium is any one or more of DMEM medium, MEN medium, RPMI1640 medium, DMEM / F12 medium, IMDM medium, HamF10 medium, M199 medium, McCoy5A medium, and L15 cell culture medium.

[0108] A fourth aspect of the present invention provides an application of a thermosensitive hydrogel in three-dimensional cell culture, wherein the application specifically comprises the following steps:

[0109] S3.1: Dissolve the co-grafted dendritic chitosan and mix it evenly with the cell suspension to be cultured, and then incubate until a gel is formed;

[0110] S3.2: Place the gel in culture medium and perform three-dimensional cell culture at a suitable temperature;

[0111] S3.3: After the culture is completed, the cells in the hydrogel are released by cooling, and the cultured cells are collected by centrifugation.

[0112] The above embodiments can be implemented individually or in any combination of two or more. The above embodiments will be described in more detail below with reference to specific examples.

[0113] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0114] In the preparation methods of the following examples and comparative examples, the structural formulas of the dendronized alkoxy ether units I and II are:

[0115]

[0116] The specific preparation methods of the dendronized alkoxy ether units I and II are described in Li, W; Zhang, A; Schlüter, AD Chem. Commun. 2008, 5523.

[0117] Example 1:

[0118] This embodiment provides a co-grafted dendritic chitosan, the preparation method of which is as follows:

[0119] (1) At room temperature (20°C), 100 mg of chitosan (molecular weight 1.5×10 5 , deacetylation degree> 95%) and 84 mg of 1-hydroxybenzotriazole were dissolved in 30 mL of deionized water and stirred until clear.

[0120] (2) 250 mg each of dendronized alkoxy ether moieties I (X is ethoxy, n is 3) and II (Y is ethoxy, n is 2) were added to the chitosan solution and stirred for 30 min. 130 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was then added dropwise to the chitosan solution and allowed to react at room temperature for 48 h.

[0121] (3) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and then freeze-dried to obtain the co-grafted dendritic chitosan of this embodiment.

[0122] The prepared co-grafted dendritic chitosan sample was dissolved in dimethyl sulfoxide, and the solution was dropped onto a potassium chloride tablet. The structure of the co-grafted dendritic chitosan was tested using a Fourier transform infrared spectrometer.

[0123] like Figure 1 As shown, at 2981, 2926, 2872cm -1 The absorption peak at 1108 cm is attributed to the stretching vibration of methyl and methylene CH in the alkoxy ether structure. -1 Stretching vibration peak at 1649cm -1 The C=O stretching vibration peak of the amide bond at 400 nm was enhanced, indicating that an amide bond was formed between the dendronized alkoxy ether and chitosan, indicating that the dendrons I and II were successfully bound to chitosan.

[0124] Example 2:

[0125] This embodiment provides a co-grafted dendritic chitosan, the preparation method of which is as follows:

[0126] (1) At room temperature (20°C), 100 mg of chitosan (molecular weight 1.5×10 5 , deacetylation degree> 95%) and 42 mg of 1-hydroxybenzotriazole were dissolved in 30 mL of deionized water and stirred until clear.

[0127] (2) 125 mg of dendrimer alkoxy ether moiety I (X is ethoxy, n is 3) was added to the chitosan solution and stirred for 30 min. 65 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was then added dropwise to the chitosan solution and allowed to react at room temperature for 48 h.

[0128] (3) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and then freeze-dried to obtain the single-grafted dendritic chitosan of this embodiment.

[0129] (4) After the mono-grafted dendronized chitosan was dissolved again, 125 mg of dendronized alkoxy ether unit II (Y is ethoxy, n is 2) and 42 mg of 1-hydroxybenzotriazole were added, and then 65 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was added dropwise to the chitosan solution, followed by reaction at room temperature for 48 h.

[0130] (5) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and freeze-dried after dialysis to obtain the co-grafted dendritic chitosan of this embodiment.

[0131] Characterization by infrared spectroscopy shows that this embodiment can also achieve successful combination of dendritic alkoxy ether units I and II with chitosan.

[0132] It can be seen from this that the two different dendrons I and II of the present invention can be added at once for simultaneous grafting, or added separately in sequence, and both can successfully achieve efficient synthesis of co-grafted dendronized chitosan.

[0133] Example 3:

[0134] This embodiment provides a co-grafted dendritic chitosan, the preparation method of which is as follows:

[0135] (1) At room temperature (20°C), 100 mg of chitosan (molecular weight 1.5×10 5 , deacetylation degree> 95%) and 84 mg of 1-hydroxybenzotriazole were dissolved in 30 mL of deionized water and stirred until clear.

[0136] (2) 250 mg each of dendronized alkoxy ether moieties I (X is methoxy, n is 3) and II (Y is methoxy, n is 2) were added to the chitosan solution and stirred for 30 min. 130 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was then added dropwise to the chitosan solution and allowed to react at room temperature for 48 h.

[0137] (3) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and then freeze-dried to obtain the co-grafted dendritic chitosan of this embodiment.

[0138] Example 4:

[0139] This embodiment provides a co-grafted dendritic chitosan, the preparation method of which is as follows:

[0140] (1) At room temperature (20°C), dissolve 100 mg of chitosan (molecular weight 1.5×105, degree of deacetylation >95%) and 84 mg of 1-hydroxybenzotriazole in 30 mL of deionized water and stir until clear.

[0141] (2) 250 mg each of dendronized alkoxy ether moieties I (X is methoxy, n is 4) and II (Y is propoxy, n is 2) were added to the chitosan solution and stirred for 30 min. 130 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was then added dropwise to the chitosan solution and allowed to react at room temperature for 48 h.

[0142] (3) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and then freeze-dried to obtain the co-grafted dendritic chitosan of this embodiment.

[0143] Example 4:

[0144] This embodiment provides a co-grafted dendritic chitosan, the preparation method of which is as follows:

[0145] (1) At room temperature (20°C), 100 mg of chitosan (molecular weight 1.5×10 5 , deacetylation degree> 95%) and 84 mg of 1-hydroxybenzotriazole were dissolved in 30 mL of deionized water and stirred until clear.

[0146] (2) 250 mg each of dendronized alkoxy ether moieties I (X is methoxy, n is 4) and II (Y is propoxy, n is 2) were added to the chitosan solution and stirred for 30 min. 130 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was then added dropwise to the chitosan solution and allowed to react at room temperature for 48 h.

[0147] (3) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and then freeze-dried to obtain the co-grafted dendritic chitosan of this embodiment.

[0148] Example 5:

[0149] This embodiment provides a co-grafted dendritic chitosan, the preparation method of which is as follows:

[0150] (1) At room temperature (20°C), 100 mg of chitosan (molecular weight 1.5×10 5 , deacetylation degree> 95%) and 84 mg of 1-hydroxybenzotriazole were dissolved in 30 mL of deionized water and stirred until clear.

[0151] (2) 250 mg each of dendronized alkoxy ether moieties I (X is hydroxyl, n is 4) and II (Y is butoxy, n is 2) were added to the chitosan solution and stirred for 30 min. 130 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was then added dropwise to the chitosan solution and allowed to react at room temperature for 48 h.

[0152] (3) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and then freeze-dried to obtain the co-grafted dendritic chitosan of this embodiment.

[0153] Comparative Example 1:

[0154] This comparative example provides a pure chitosan without any dendron grafting, with a molecular weight of 1.5×10 5 , deacetylation degree>95%.

[0155] Comparative Example 2:

[0156] This comparative example provides a single-grafted dendritic chitosan, the preparation method of which is as follows:

[0157] (1) At room temperature (20°C), 100 mg of chitosan (molecular weight 1.5×10 5 , deacetylation degree> 95%) and 84 mg of 1-hydroxybenzotriazole were dissolved in 30 mL of deionized water and stirred until clear.

[0158] (2) 250 mg of dendrimer alkoxy ether moiety I (X is ethoxy, n is 2) was added to the chitosan solution and stirred for 30 min. 130 mg of the activator 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride was then added dropwise to the chitosan solution and allowed to react at room temperature for 48 h.

[0159] (3) The reaction solution was dialyzed for 7 days (molecular weight cut-off: 3.5 kDa), and then freeze-dried to obtain the single-grafted dendritic chitosan of this embodiment.

[0160] The present invention conducted the following tests on the dendritic chitosan or chitosan prepared in Examples 1 to 5 and Comparative Examples 1 to 2:

[0161] (1) Temperature sensitivity test:

[0162] The prepared chitosan sample was dissolved in phosphate buffer (pH=7.4, isotonic with human blood, main components of which are sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and potassium chloride) at room temperature, and then the temperature was raised to 37° C. and incubated for 3 minutes.

[0163] The effect comparison of the co-grafted dendritic chitosan of Example 1 before and after gelation is as follows: Figure 2 As shown in the figure, the co-grafted dendritic chitosan of Example 1 is soluble in phosphate buffer at room temperature and forms a transparent hydrogel near body temperature without any loss of transparency. The co-grafted dendritic chitosan prepared in Examples 2 to 5 can all be dissolved in phosphate buffer and form transparent hydrogels at 37°C, showing good temperature-sensitive properties. Because the present invention introduces two different dendritic alkoxy ether moieties, the chitosan has adjustable radial amphiphilicity and prevents the chitosan from forming intermolecular hydrogen bonds. When the temperature rises, the chitosan can be assembled into fibers and physically cross-linked to form a transparent hydrogel.

[0164] In addition, if Figure 2 As shown in FIG. 1 , the co-grafted dendritic chitosan of Example 1 can also be dissolved in DMEM culture medium and form a transparent gel.

[0165] like Figure 3As shown, the pure chitosan in Comparative Example 1 could not be dissolved and had no temperature responsiveness. The chitosan grafted with a single dendritic alkoxy ether in Comparative Example 2 swelled to form a gel at room temperature and had no temperature responsiveness either.

[0166] (2) Gel temperature test

[0167] The gel point of the thermosensitive hydrogel prepared in Example 1 was tested using a temperature sweep mode of a rotational rheometer, wherein the intersection of the storage modulus (G′) and the loss modulus (G″) is the gel temperature.

[0168] like Figure 4 As shown in the figure, G′ gradually increases with increasing temperature and eventually exceeds G″, indicating that the co-grafted dendritic chitosan transforms from solution to gel, and the gelation temperature is about 30°C.

[0169] application:

[0170] Given that the co-grafted dendritic chitosan thermosensitive hydrogel prepared in the present invention is soluble in buffer solutions or cell culture media and forms a transparent hydrogel at approximately body temperature, the co-grafted dendritic chitosan thermosensitive hydrogel of the present invention has excellent application prospects in three-dimensional cell culture. The co-grafted dendritic chitosan thermosensitive hydrogel prepared in Example 1 was used as an example to test its specific application in three-dimensional cell culture.

[0171] The method of three-dimensional cell culture in the present invention is as follows Figure 5 Specifically, the co-grafted dendritic chitosan was dissolved in phosphate buffer to prepare a chitosan solution with a solid content of 2.5 wt%. The co-grafted dendritic chitosan solution was mixed with an A549 cell suspension (DMEM medium, cell concentration 1×10 7 The mixture was then transferred to a 37°C incubator to form a gel to encapsulate the cells. DMEM medium was then added to submerge the gel and continue cell culture.

[0172] like Figure 6 As shown in the optical microscope photos, on the 7th day of culture, the number of A549 cells increased and cell clusters were formed.

[0173] like Figure 7 As shown in the 3D stacked fluorescence images, on day 7 of culture, A549 cells were evenly distributed in 3D space, with no sedimentation observed. Subsequent staining using AO / EB staining, with live cells indicated in green and dead cells indicated in red, revealed that after seven days of culture, the cells still maintained a high viability of over 95%, demonstrating that the co-grafted dendritic chitosan thermosensitive hydrogel prepared in Example 1 successfully enabled three-dimensional cell culture.

[0174] When the cultured cells need to be collected after the culture is completed, the co-grafted dendritic chitosan thermosensitive hydrogel is placed in a refrigerator at 4° C. for 30 minutes, and phosphate buffer is added to dilute the gel after it is liquefied.

[0175] like Figure 8 As shown, the cells were then separated by centrifugation (3000 rpm, 5 min), and the cells still maintained the morphology of cell pellets.

[0176] In summary, the co-grafted dendritic chitosan hydrogel produced by the present invention exhibits reversible sol-gel transition properties controlled by temperature and can be dissolved in a variety of buffers and culture media. The co-grafted dendritic chitosan hydrogel of the present invention exhibits excellent temperature responsiveness and is suitable for 3D cell culture. It also has potential applications in a variety of fields, including cell therapy, drug encapsulation, drug protection, tissue engineering, and wound dressing.

[0177] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A co-grafted dendritic chitosan, characterized in that: It has the following general structural formula: Wherein, q = 10-6500, z = 10-6500, r = 10-6500; R1 is or -COO - H3N + -; R2 is H or R3 and R4 are wherein n=3-4, m=0-2, X and Y are independently selected from hydroxyl or C 1-6 of alkoxy.

2. A method for preparing the co-grafted dendritic chitosan according to claim 1, characterized in that: The preparation method is selected from either method A or method B; The method A comprises the following steps: S1.1: Mix chitosan, reaction aid and solvent until chitosan is completely dissolved; S1.2: Adding the dendrimer alkoxy ether moieties I and II corresponding to R3 and R4, respectively, and an activator, followed by a condensation reaction; S1.3: After the reaction is completed, the product is dialyzed and freeze-dried to obtain the co-grafted dendritic chitosan; The method B comprises the following steps: S2.1: Mix chitosan, reaction auxiliary agent and solvent until chitosan is completely dissolved; S2.2: Adding the dendrimer alkoxy ether moiety I corresponding to R3 and an activator, followed by a condensation reaction; S2.3: After the reaction is completed, the product is dialyzed and freeze-dried to obtain single-grafted dendritic chitosan; S2.4: After redissolving the mono-grafted dendritic chitosan, the dendritic alkoxy ether unit II corresponding to R4 and an activator are added, followed by a secondary condensation reaction; S2.5: After the reaction is completed, the product is dialyzed and freeze-dried to obtain the co-grafted dendritic chitosan; The structural formulas of the branched alkoxy ether units I and II are:

3. The method for preparing co-grafted dendritic chitosan according to claim 2, wherein: The reaction auxiliary agent is any one or more of 1-hydroxybenzotriazole, 1-oxybenzotriazole, 1,1-dimethyl-3-oxy-1,2,3-triazabenzotriazole, O-benzotriazole-N,N,N',N"-tetramethyluronium hexafluorophosphate, O-benzotriazole-N-tetramethyluronium tetrafluoroborate, benzotriazole-1-acyl-1-methylpyridinium hexafluorophosphate, benzotriazole-1-acyloxytris(dimethylamino)phosphonium hexafluorophosphate, 3-[(trisdimethylamino)sulfonyl]-2-oxy-4-benzotriazole, O-benzotriazole-NN,N'-trimethyluronium trifluoroacetate, and 3-[(diphenylphosphoryl)oxy]-4-benzotriazole.

4. The method for preparing co-grafted dendritic chitosan according to claim 2, wherein: The activator is any one or more of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide, cyanodimethylethylcarbodiimide, n-propylphosphonic anhydride, ethylmethylphosphinic anhydride, diphenylphosphoryl chloride, di(2-oxo-3-oxazolidinyl)phosphoryl chloride, N,N-carbonyldiimidazole, and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-7-yl-2,2-dimethylpropyl ester.

5. The method for preparing co-grafted dendritic chitosan according to claim 2, characterized in that: In both method A and method B, the molar ratio of the chitosan repeating unit to the reaction auxiliary agent is 1:(1-2); In method A, the molar ratio of the chitosan repeating unit, the dendronized alkoxy ether unit I, the dendronized alkoxy ether unit II and the activator is 1:(0.1-2):(0.1-2):(1-1.5); In method B, the molar ratio of the repeating unit of chitosan, the dendronized alkoxy ether unit I and the activator is 1:(0.1-2):(1-1.5), and the molar ratio of the repeating unit of the mono-grafted dendronized chitosan, the dendronized alkoxy ether unit II and the activator is 1:(0.1-2):(1-1.5).

6. The method for preparing co-grafted dendritic chitosan according to claim 2, characterized in that: In method A and method B, the condensation reaction time is 4-96h, and the reaction temperature is 15-100°C; The dialysis time is 5-7 days, and the dialysis molecular weight cut-off is 3.5 kDa.

7. A thermosensitive hydrogel, characterized in that The co-grafted dendritic chitosan according to claim 1 is dissolved and then heated; The solid content of the co-grafted dendritic chitosan in the thermosensitive hydrogel is 0.2-15 wt %.

8. The thermosensitive hydrogel according to claim 7, characterized in that The thermosensitive hydrogel is prepared from any of the following components: 0.1-10 wt% of co-grafted dendritic chitosan, 0.1-10 wt% of buffer, and the balance being water; Alternatively, 0.1-10 wt % of dendritic chitosan, 0.1-99.9 wt % of cell culture medium, and the balance being water are co-grafted; Alternatively, 0.1-10 wt% of dendritic chitosan is co-grafted, and the balance is water.

9. The thermosensitive hydrogel according to claim 8, characterized in that The buffer comprises any one or more of the following salts or organic solvents: disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium β-glycerophosphate, sodium acetate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium chloride, potassium nitrate, sodium sulfate, sodium sulfite, glycerol, propylene glycol, dimethyl sulfoxide; The cell culture medium is any one or more of DMEM culture medium, MEN culture medium, RPMI 1640 culture medium, DMEM / F12 culture medium, IMDM culture medium, HamF10 culture medium, M199 culture medium, McCoy5A culture medium, and L15 cell culture medium.

10. Use of the thermosensitive hydrogel according to claim 7 in three-dimensional cell culture, characterized in that: The application specifically includes the following steps: S3.1: Dissolve the co-grafted dendritic chitosan and mix it evenly with the cell suspension to be cultured, and then incubate until a gel is formed; S3.2: Place the gel in culture medium and perform three-dimensional cell culture at a suitable temperature; S3.3: After the culture is completed, the cells in the hydrogel are released by cooling, and the cultured cells are collected by centrifugation.

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