A glucose system chitosan hydrogel and its preparation method and application

Abstract

The present application relates to a kind of glucose system chitosan hydrogel, the hydrogel is with glucose solution as solvent, by chitosan chloride, hydroxyethyl cellulose, namely chitosan chloride / hydroxyethyl cellulose glucose hydrogel.The hydrogel uses the preparation method of physical crosslinking, reduces the use of chemical crosslinking agent, reduces cytotoxicity.The glucose system chitosan hydrogel is injectable hydrogel, uses glucose to do solvent, higher safety, while its porosity is big, aperture is big, can load drug, cell etc., with excellent biocompatibility, can be safely injected to target site, can be used as biosensor, drug delivery carrier, tissue engineering cell carrier etc. In the medical field.

Description

Technical Field

[0001] This invention relates to the field of hydrogels and their preparation and application, and in particular to a glucose-based chitosan hydrogel, its preparation method and application. Background Technology

[0002] Chitosan is a natural cationic polymer and the only basic polysaccharide among natural polysaccharides. It is obtained from chitin through partial deacetylation using chemical or enzymatic methods. In vivo, it can be degraded by enzymes into non-toxic chitosan oligosaccharides, glucosamine, and other products, all of which are biocompatible and readily absorbed by the human body. The positive charge on the free amino groups of chitosan endows it with excellent adhesion and gel-forming abilities, allowing it to electrostatically adsorb onto most negatively charged surface matrices of living tissues. It is widely used as an injectable scaffold material in tissue engineering and other fields. Acidification of chitosan yields chitosan hydrochloride (CsCl, i.e., chlorinated chitosan). Chlorinated chitosan effectively overcomes the limitation of chitosan's solubility only under acidic conditions, but the properties of the two are almost identical. Therefore, chlorinated chitosan is widely used in the preparation and research of biomedical materials, such as medical dressings, cell scaffolds, sponges, and hydrogels with controlled drug delivery.

[0003] Cellulose is a large polysaccharide composed of glucose and is the most widely distributed polysaccharide in nature. Hydroxyethyl cellulose (HEC) is one of the best examples of cellulose, a non-ionic cellulose ether obtained by alkalizing and etherifying cellulose. It possesses excellent hydrophilicity, biocompatibility, and biodegradability. HEC is produced by reacting the hydroxyethyl groups on the cellulose backbone with acryloyl chloride (or vinyl chloride). Its long chains contain numerous hydroxyl groups, which can cross-link with other biomolecules through hydrogen bonds to form hydrogel networks.

[0004] Injectable hydrogels possess a highly functional three-dimensional structure, and by altering the material composition, concentration, and cross-linking method, specific requirements under different conditions can be met. Hydrogels can absorb large amounts of water and swell, making them excellent as antibacterial dressings or sponges for absorbing wound exudate. Not only are hydrogels soft, similar to human soft tissue and extracellular matrix, exhibiting good biocompatibility and biodegradability, but their porous structure also allows for the loading of drugs, growth factors, nucleic acids, cells, and other substances. Therefore, injectable hydrogels have become potential candidates for biosensors, drug delivery carriers, and tissue-engineered cell carriers.

[0005] Glucose is non-toxic and non-irritating, an essential component of the body, an energy source for living cells, and a metabolic intermediate. It is a necessary substance in the cell repair process and is a commonly used excipient in clinical practice, offering higher safety. A search revealed no reports of using glucose as a solvent to prepare injectable hydrogels. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a glucose-based chitosan hydrogel. This invention provides a novel injectable hydrogel, namely, a chlorinated chitosan / hydroxyethyl cellulose glucose hydrogel. This hydrogel uses chlorinated chitosan instead of regular chitosan, improving the solubility of chitosan. Simultaneously, it has high porosity and large pore size, allowing for the loading of drugs, cells, etc. Using glucose as a solvent enhances safety. This hydrogel exhibits excellent biocompatibility and can be safely injected into targeted sites, showing great promise for future research.

[0007] The hydrogel uses glucose solution as a solvent and is composed of chlorinated chitosan and hydroxyethyl cellulose.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned glucose system chitosan hydrogel.

[0009] Step 1, Preparation of chlorinated chitosan (CsCl) solution:

[0010] Accurately weigh CsCl powder and dissolve it in D-Glu solution. After stirring to dissolve, add NaHCO3 powder to make the final concentration of NaHCO3 in the solution 0.2% to 0.4%. Stir to obtain a clear solution.

[0011] Step 2: Preparation of hydroxyethyl cellulose (HEC) solution:

[0012] Accurately weigh HEC powder and dissolve it in an aqueous solution containing D-Glu and 0.4%–0.7% NaHCO3. Stir to obtain a clear solution and store at low temperature for later use.

[0013] Step 3: Preparation of chlorinated chitosan / hydroxyethyl cellulose (CsCl / HEC) hydrogel:

[0014] Mix the CsCl solution and HEC solution thoroughly and allow them to gel at 36–40°C to obtain the CsCl / HEC hydrogel.

[0015] Preferably, in step one, the concentration of the chlorinated chitosan (CsCl) solution is 1% to 5%, and more preferably, the concentration of the chlorinated chitosan (CsCl) solution is 2%.

[0016] Preferably, in step two, the concentration of the hydroxyethyl cellulose (HEC) solution is 1% to 5%, and more preferably, the concentration of the hydroxyethyl cellulose (HEC) solution is 2.25%.

[0017] Preferably, in step three, the mass ratio of chlorinated chitosan to hydroxyethyl cellulose is 1:0.1 to 0.25, and more preferably, the mass ratio of chlorinated chitosan to hydroxyethyl cellulose is 1:0.175.

[0018] Preferably, the isotonic concentration of the glucose solution is 4-6%, and more preferably, the isotonic concentration of the glucose solution is 4%.

[0019] Preferably, in step one, the concentration of NaHCO3 in the solution is 0.32%.

[0020] Preferably, in step two, the concentration of the NaHCO3 solution is 0.6%.

[0021] The third objective of this invention is to apply the glucose system chitosan hydrogel as a biosensor, drug delivery carrier, tissue-engineered cell carrier, etc., in the medical field.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. This invention provides an injectable hydrogel based on a glucose system, namely, a chlorinated chitosan / hydroxyethyl cellulose glucose hydrogel. This hydrogel utilizes chitosan hydrochloride to increase its solubility, and then adjusts the pH by adding NaHCO3 to achieve the pH required for cell culture, thereby reducing cytotoxicity.

[0024] 2. Both chlorinated chitosan and hydroxyethyl cellulose have abundant hydroxyl groups on their main chains, which can be easily cross-linked through hydrogen bonds to form hydrogels and meet the basic requirements of injectable hydrogels. The use of physical cross-linking methods to prepare hydrogels reduces the use of chemical cross-linking agents and reduces cytotoxicity.

[0025] 3. This injectable hydrogel uses glucose as a solvent, which enhances its safety. Furthermore, its high porosity and large pore size allow it to load drugs, cells, etc., exhibiting excellent biocompatibility and enabling safe injection into target sites. It holds immense promise for future research. Attached Figure Description

[0026] Figure 1 Infrared spectra of different raw materials and hydrogels.

[0027] Figure 2 The gelation times of the three hydrogels in Examples 1-3, where a, b, and c represent Examples 1, 2, and 3, respectively.

[0028] Figure 3 Examples 1-3 show the sequential injection of three types of hydrogels, from left to right: Examples 1, 2, and 3.

[0029] Figure 4 Examples 1-3 illustrate the self-healing process of three hydrogels, from left to right: Examples 1, 2, and 3.

[0030] Figure 5Pore ​​structure diagrams of the three hydrogels in Examples 1-3, where a, b, and c represent Examples 1, 2, and 3, respectively.

[0031] Figure 6 Examples 1-3 show hemolysis images of three hydrogels, with the left image showing the hemolysis effect and the right image showing the hemolysis rate. 1#, 2#, and 3# represent Examples 1, 2, and 3, respectively. - represents the negative control and + represents the positive control.

[0032] Figure 7 Example 2: Cell viability of hydrogel. Detailed Implementation

[0033] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the technical solutions of this invention, should all be covered within the protection scope of this invention.

[0034] Example 1

[0035] Take 1g of CsCl powder, accurately weigh it, and dissolve it in 50mL of 4% D-Glu solution. After stirring and dissolving, add 0.16g of NaHCO3 powder to make the final concentration of NaHCO3 in the solution 0.32%. Stir to obtain a clear 2% CsCl solution.

[0036] Accurately weigh 0.225 g of HEC powder and dissolve it in 10 mL of an aqueous solution containing 4% D-Glu and 0.6% NaHCO3. Stir to obtain a clear 2.25% HEC solution. Store at 4°C for later use.

[0037] Take 4g of 2% CsCl solution, add 0.5g of 2.25% HEC solution, mix well, place in a 37℃ water bath, and wait for it to gel to form a CsCl / HEC hydrogel.

[0038] Example 2

[0039] Take 1g of CsCl powder, accurately weigh it, and dissolve it in 50mL of 4% D-Glu solution. After stirring and dissolving, add 0.16g of NaHCO3 powder to make the final concentration of NaHCO3 in the solution 0.32%. Stir to obtain a clear 2% CsCl solution.

[0040] Accurately weigh 0.225 g of HEC powder and dissolve it in 10 mL of an aqueous solution containing 4% D-Glu and 0.6% NaHCO3. Stir to obtain a clear 2.25% HEC solution. Store at 4°C for later use.

[0041] Take 4g of 2% CsCl solution, add 0.7g of 2.25% HEC solution, mix well, place in a 37℃ water bath, and wait for it to gel to form a CsCl / HEC hydrogel.

[0042] Example 3

[0043] Take 1g of CsCl powder, accurately weigh it, and dissolve it in 50mL of 4% D-Glu solution. After stirring and dissolving, add 0.16g of NaHCO3 powder to make the final concentration of NaHCO3 in the solution 0.32%. Stir to obtain a clear 2% CsCl solution.

[0044] Accurately weigh 0.225 g of HEC powder and dissolve it in 10 mL of an aqueous solution containing 4% D-Glu and 0.6% NaHCO3. Stir to obtain a clear 2.25% HEC solution. Store at 4°C for later use.

[0045] Take 4g of 2% CsCl solution, add 0.9g of 2.25% HEC solution, mix well, place in a 37℃ water bath, and wait for it to gel to form a CsCl / HEC hydrogel.

[0046] Example 4

[0047] Accurately weigh 0.5g of CsCl powder and dissolve it in 50mL of 5% D-Glu solution. After stirring and dissolving, add 0.1g of NaHCO3 powder to make the final concentration of NaHCO3 in the solution 0.2%. Stir to obtain a clear 1% CsCl solution.

[0048] Accurately weigh 0.1 g of HEC powder and dissolve it in 10 mL of an aqueous solution containing 5% D-Glu and 0.4% NaHCO3. Stir to obtain a clear 1% HEC solution. Store at 4°C for later use.

[0049] Take 4g of 1% CsCl solution, add 0.4g of 1% HEC solution, mix well, place in a 36℃ water bath, and wait for it to gel to form a CsCl / HEC hydrogel.

[0050] Example 5

[0051] Accurately weigh 2.5g of CsCl powder and dissolve it in 50mL of 6% D-Glu solution. After stirring and dissolving, add 0.2g of NaHCO3 powder to make the final concentration of NaHCO3 in the solution 0.4%. Stir to obtain a clear 5% CsCl solution.

[0052] Accurately weigh 0.5 g of HEC powder and dissolve it in 10 mL of an aqueous solution containing 6% D-Glu and 0.7% NaHCO3. Stir to obtain a clear 5% HEC solution. Store at 4°C for later use.

[0053] Take 4g of 5% CsCl solution, add 1g of 5% HEC solution, mix well, place in a 40℃ water bath, and wait for it to gel to form a CsCl / HEC hydrogel.

[0054] Example 1: Fourier Transform Infrared Spectroscopy Characterization (FT-IR)

[0055] CsCl powder, D-Glu powder, CsCl-D-Glu lyophilized product, HEC powder, a physical mixture containing D-Glu, CsCl and HEC, and CsCl / HEC hydrogel lyophilized product were each mixed with an appropriate amount of KBr, compressed into tablets, dried, and then analyzed using a Fourier transform infrared spectroscopy at 400 cm⁻¹. -1 -4000cm -1 Perform spectral scanning within the range.

[0056] Appendix Figure 1 Infrared spectra of the various raw materials, Glu, HEC, and CsCl, and the CsCl / HEC hydrogel. The characteristic absorption peak of CsCl is at 3500 cm⁻¹. -1 The peaks of the -OH and -NH2 stretching vibrations overlapped on both sides; at 1635, 1527, and 1378 cm⁻¹. -1 The absorption bands at these locations represent the C=O stretching vibration of amide I peak, the N–H bending vibration of amide II peak, and the CN stretching vibration of amide III peak in the CsCl structure.

[0057] D-Glu 3441cm -1 The peak at 2941 cm⁻¹ is a broad and strong peak representing the stretching vibration of the hydroxyl group. -1 Several weak peaks are due to CH stretching vibration, 1108 cm⁻¹ -1 The peaks around the left and right of the point represent CO stretching vibrations, which split into several peaks.

[0058] HEC at 3452cm -1 There is an absorption peak at 1067 cm⁻¹ due to the stretching vibration of -OH. -1 and 1049cm -1 The absorption peak is due to the stretching vibration of CO.

[0059] Comparison of the FT-IR spectra of CsCl and Gel shows that the -OH peak changes from 3500 cm⁻¹. -1 Shift down to 3420, 3423

[0060] cm -1 Furthermore, the peak broadening and absorption intensity enhancement indicate the formation of hydrogen bonds between CsCl and HEC.

[0061] Example 2: CsCl / HEC hydrogel gelation time

[0062] Using the inverted test tube method, the hydrogels of Examples 1-3 were added to vials, placed in a 37°C water bath, and timed. Observations were taken every 5 minutes. The point at which the vials showed no flowability after inverting was defined as "gel formation." (See attached image) Figure 2 The gelation time is for hydrogels with different proportions in Examples 1-3.

[0063] Example 3: Injectability and self-healing properties of CsCl / HEC hydrogel

[0064] Add 5 drops of methylene blue to each of the three hydrogel precursor solutions in Examples 1-3, transfer them to a 5mL syringe, write English letters on the culture dish, and test whether they can be injected continuously.

[0065] Three types of transparent or methylene blue hydrogel were placed in a petri dish. After they solidified, each was torn in half with a syringe. The transparent half and the blue half were then placed together in equal proportions. The healing process was observed and timed.

[0066] Appendix Figure 3 The results show that the hydrogel can be continuously extruded and injected using conventional injection techniques, indicating that the experiment successfully prepared an injectable hydrogel that can be easily injected into the target site using a syringe.

[0067] Appendix Figure 4 The results show that the hydrogels prepared in Examples 1-3 have the potential for self-healing. By placing the hydrogels in the same proportion together, all three hydrogels successfully healed within 2 minutes and could be easily picked up with a syringe.

[0068] Example 4: Hydrogel porosity

[0069] Porosity was determined using the liquid displacement method. The disc-shaped freeze-dried hydrogels from Examples 1-3 were weighed and recorded as m0. They were then immersed in anhydrous ethanol for 24 hours, and their weight was recorded as m. S Calculate the porosity P according to formulas (1) and (2). Make three samples for each group and take the average value.

[0070]

[0071] V0=πr 2 h (2)

[0072] Where ρ is the density of anhydrous ethanol at 26℃ (0.79 g·cm³). -3 V0 is the volume of the disc-shaped hydrogel.

[0073] Porosity is of great significance for the loading of drugs and cells onto hydrogels; the greater the porosity, the stronger the loading capacity. Figure 5The results show that the porosities of the hydrogels in Examples 1-3 are 52%, 37%, and 25%, respectively. As the amount of HEC added increases, the crosslinking density of the hydrogel increases, and the porosity decreases.

[0074] Example 5: Hemolysis assay

[0075] Whole blood was collected from the inner canthus of rats and transferred to an EP tube containing 10 μL of heparin. The tube was gently shaken and centrifuged at 5000 rpm for 10 min (centrifugation conditions, same below). The supernatant plasma was discarded. The lower red blood cell layer was washed once with physiological saline and several times with PBS solution, centrifuged after each wash, and the supernatant was discarded, until a clear supernatant was observed. The red blood cell sediment was diluted to 5% (v / v) with PBS solution for later use. 0.3 mL of each of the three hydrogel precursor solutions was added to 1.5 mL of EP. After gelation, 0.7 mL of 5% red blood cell suspension was added. Red blood cell suspensions incubated with 0.1% Triton and PBS were used as positive and negative controls, respectively. After incubation at 37°C for 1 h, the tube was centrifuged. The supernatant was transferred to a new EP tube and photographed. The color intensity was compared with the positive and negative controls. The supernatant was then added to a 96-well plate, and the absorbance was measured at 540 nm using a microplate reader. Three samples were prepared for each group, and the average value was taken. The hemolysis rate was calculated using the following formula.

[0076]

[0077] As verified by experiments (see attached). Figure 6 As shown, the supernatants of the three hydrogels with different proportions were as clear and transparent as the negative control, while the 0.1% Triton caused hemolysis of red blood cells, and the solution turned a bright red, indicating that the hydrogels prepared in this experiment have good blood compatibility.

[0078] Example 6: Cytotoxicity Assessment

[0079] Under aseptic conditions, add 94.5 mL of DMEM, 0.5 mL of penicillin-dextrose antibody, and 5 mL of Australian fetal bovine serum (FBS) to a centrifuge tube to prepare complete culture medium for later use. Incubate the culture flask containing the complete culture medium and AC16 cells at 37°C (containing 5% CO2), changing the complete culture medium every two days. After passage, wash the cells with PBS buffer, add trypsin for digestion, observe the digestion under a microscope, transfer to a centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells in the complete culture medium, mix well, add 10 μL of cell suspension to a cell counter, and count the cell number.

[0080] Sterilize centrifuge tubes and CsCl powder under UV light. Prepare 2% CsCl and 2.25% HEC in DMEM under aseptic conditions. Add 100 μL of hydrogel to each well of a 24-well plate and allow it to gel. Add 1000 μL of cell suspension (containing 5000 cells) to each well, and set up a control group. Incubate at 37°C for 1, 3, and 7 days, changing the culture medium every two days.

[0081] At the predetermined time point, remove the incubated 24-well plates and add 100 μL of CCK-8 to each well for cell viability assay. Incubate at 37°C and 5% CO2 for 1-2 hours. Transfer 100 μL of supernatant from each well to a new 96-well plate and measure the absorbance at 450 nm using a microplate reader. An absorbance of 0.8-1.5 is ideal. Calculate cell viability using the following formula:

[0082]

[0083] In a light-protected environment, 15 μL of LAM dye and 45 μL of LPI dye were added to 15 mL of 10× Buffer and mixed thoroughly to obtain Live & Dead staining solution. At the predetermined time point, after the supernatant was aspirated, 500 μL of staining solution was added to each well, and the cells were incubated at 37°C for 15 min. The staining status of the cells was observed under an inverted fluorescence microscope (green light Ex = 488 nm Em = 530 nm, red light Ex = 535 nm Em = 617 nm).

[0084] The biocompatibility of the hydrogel with AC16 cells was investigated using the CCK-8 assay kit and the Calcein-AM / PI live / dead cell double staining kit. (See attached...) Figure 7 As shown, when cells were co-cultured with the hydrogel, the viability of AC16 cells increased over time, consistently exceeding 80%. In live / dead staining, good cell growth was observed under an inverted fluorescence microscope, consistent with the CCK-8 results, indicating that the hydrogel prepared in this experiment has excellent cell compatibility.

Claims

1. A method for preparing a glucose-based chitosan hydrogel with injectable self-healing properties, wherein... The hydrogel is characterized by using D-Glu solution as a solvent and being composed of chlorinated chitosan and hydroxyethyl cellulose. Its preparation method includes the following steps: Step 1, Preparation of chlorinated chitosan solution: Accurately weigh CsCl powder and dissolve it in D-Glu solution. After stirring to dissolve, add NaHCO3 powder to make the final concentration of NaHCO3 in the solution 0.2%~0.4%. Stir to obtain a clear solution. Step 2: Preparation of hydroxyethyl cellulose solution: Accurately weigh HEC powder and dissolve it in an aqueous solution containing D-Glu and 0.4%~0.7% NaHCO3. Stir to obtain a clear solution and store it at low temperature for later use. Step 3: Preparation of chlorinated chitosan / hydroxyethyl cellulose hydrogel: Mix CsCl solution and HEC solution, and allow them to gel at 36~40℃ to obtain CsCl / HEC hydrogel. In step one, the concentration of the chlorinated chitosan solution is 1% to 5%; in step two, the concentration of the hydroxyethyl cellulose solution is 1% to 5%; and in step three, the mass ratio of the chlorinated chitosan solution to the hydroxyethyl cellulose solution is 1:0.1 to 0.

25.

2. The method for preparing the glucose-based chitosan hydrogel with injectable self-healing properties according to claim 1, characterized in that, The isotonic concentration of D-Glu solution is 4-6%.

3. The method for preparing the glucose-based chitosan hydrogel with injectable self-healing properties according to claim 1, characterized in that, In step one, the final concentration of NaHCO3 in the solution is 0.32%.

4. The method for preparing the glucose-based chitosan hydrogel with injectable self-healing properties according to claim 1, characterized in that, In step two, the concentration of the NaHCO3 solution is 0.6%.

5. The application of the injectable self-healing glucose-based chitosan hydrogel prepared according to any one of claims 1-4 as a cell transplantation scaffold material in myocardial tissue engineering.

6. The application of the injectable self-healing glucose-based chitosan hydrogel according to claim 5 in cell transplantation scaffold materials in myocardial tissue engineering, characterized in that: The glucose-based chitosan hydrogel with injectable self-healing properties can be used as a biosensor, drug delivery carrier, or cell carrier for various tissue engineering processes.

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