Use of hyaluronic acid in improving performance of polysaccharide-polypeptide composite hydrogel

Abstract

The present invention pertains to the technical field of novel biomaterials. Disclosed is use of hyaluronic acid in improving the performance of a polysaccharide-polypeptide composite hydrogel. The present invention, on the basis of the self-assembly characteristics of polypeptides, prepares polysaccharide-polypeptide composite hydrogels with hyaluronic acid (HA) at different concentrations. It has been verified that compared to a single polypeptide hydrogel free of HA, HA reduces the mechanical properties of the composite gels, but can still slow the degradation of the composite hydrogels and improve the stability thereof. HA can also improve the ability of the composite hydrogels to load quercetin and hesperidin, playing a role in slowing the in vitro release of the two, which is conducive to the sustained release of active substances. Moreover, the addition of HA does not affect the secondary structures and micro-nano structures of the composite hydrogels, and can also improve the strength of non-covalent interactions between molecules, improving the stability of the composite hydrogels. The present invention explores the effect of HA on the performance of the composite hydrogels and provides a theoretical basis for the research of hydrophobic drug delivery carriers.

Description

Application of hyaluronic acid in improving performance of polysaccharide-polypeptide composite hydrogel TECHNICAL FIELD

[0001] The application belongs to the technical field of novel biomaterials, and relates to application of hyaluronic acid in improving performance of polysaccharide-polypeptide composite hydrogel. BACKGROUND

[0002] Self-assembly of polypeptides refers to formation of nanoscale fiber structures by using various non-covalent binding forces between molecules under specific conditions. Self-assembled polypeptide hydrogel is a semi-solid transparent gel formed by polypeptide molecules as gel factors and self-assembly of polypeptides.

[0003] Polysaccharide-polypeptide composite hydrogel is a composite hydrogel taking polysaccharides and polypeptides as core functional components. As a novel biomaterial, the composite hydrogel has good biocompatibility, good hydrophilicity and degradability, and has good application potential in fields such as drug or active biological macromolecule delivery, tissue repair and artificial organs. Hyaluronic acid (HA) is a natural mucopolysaccharide and a main component of natural cell matrix, and is widely present in related tissues such as skin, vitreous humor of the eye and synovial fluid. HA has excellent biocompatibility, water absorption, high viscoelasticity, non-antigenicity and non-toxicity to degradation process, and is widely used in fields such as food, medicine and health products.

[0004] At present, there are still many problems in the application of polypeptide composite hydrogel, for example, when polypeptide composite hydrogel is used as a drug delivery carrier, the hydrogel has unstable properties, poor drug loading capacity, low drug encapsulation rate, and the drug components cannot be continuously and stably released. Therefore, how to improve the stability of polypeptide composite hydrogel, improve its drug loading capacity, and slow down the release rate of drugs, especially hydrophobic drug components, is a problem to be solved at present. SUMMARY

[0005] In order to solve the problems that polypeptide hydrogel has unstable properties, poor drug loading capacity and drug components cannot be continuously and stably released when the polypeptide hydrogel is used as a drug delivery carrier, the application provides application of hyaluronic acid in improving performance of polypeptide hydrogel.

[0006] The application studies the influence of different concentrations of HA on the structure and properties of the polypeptide composite hydrogel of different concentrations of polysaccharides (i.e. HA / Fmoc-FRGDF) by formulating the polypeptide composite hydrogel of different concentrations of polysaccharides. First, the influence of HA on the rheological properties of HA / Fmoc-FRGDF is analyzed by the change of storage modulus (G') and loss modulus (G''), and then the influence of HA on the secondary structure of HA / Fmoc-FRGDF is determined by Fourier infrared spectroscopy (FTIR) and circular dichroism spectrometer (CD), and the internal structure of the gel is observed by transmission electron microscopy (TEM). Secondly, the in vitro degradation test of HA / Fmoc-FRGDF is carried out to determine the influence of HA on the in vitro degradation rate of HA / Fmoc-FRGDF. Finally, Que and hesperetin (Hes) are used as hydrophobic drug models to test the loading capacity and controlled release effect of HA / Fmoc-FRGDF on Que and Hes. Based on the above technical ideas, the application provides the following technical solutions.

[0007] The application provides the application of hyaluronic acid in improving the performance of polysaccharide-polypeptide composite hydrogel, and specifically includes: HA is used for reducing the mechanical performance of polysaccharide-polypeptide composite hydrogel, or for delaying the degradation rate of polysaccharide-polypeptide composite hydrogel, or for improving the loading capacity of polysaccharide-polypeptide composite hydrogel for hydrophobic drugs, or for delaying the release rate of hydrophobic drugs in polysaccharide-polypeptide composite hydrogel.

[0008] Further, in the application, in the concentration range of 0.1-1.5 mg / mL, HA can reduce the storage modulus (G') and loss modulus (G'') of the polysaccharide-polypeptide composite hydrogel.

[0009] Further, in the application, in the concentration range of 0.1-1.5 mg / mL, HA can delay the degradation rate of the polysaccharide-polypeptide composite hydrogel.

[0010] Further, in the application, in the concentration range of 0.1-1.5 mg / mL, HA can improve the loading capacity of the polysaccharide-polypeptide composite hydrogel for hydrophobic drugs.

[0011] Further, in the application, in the concentration range of 0.1-1.5 mg / mL, HA can delay the release rate of hydrophobic drugs in the polysaccharide-polypeptide composite hydrogel.

[0012] Further, in the application, the polypeptide is Fmoc-FRGDF, and the hydrophobic drug is a hydrophobic substance that is soluble in an alkaline solution, such as Que or hesperetin.

[0013] Further, in the application, the preparation method of the polysaccharide-polypeptide composite hydrogel comprises: dissolving hyaluronic acid and polypeptide in water, and adding NaOH solution, HCl solution, PBS buffer, oscillating and dissolving and defoaming.

[0014] Further, in the application, the preparation raw material is prepared in a ratio of 70 μL of NaOH solution, 65 μL of HCl solution, 465 μL of PBS buffer and 10 mg of polypeptide per 400 μL of water.

[0015] Further, in the application, the concentration of the NaOH solution is 0.5 M, the concentration of the HCl solution is 0.1 M, the concentration of the PBS buffer is 0.1 M, and the pH is 7.4.

[0016] Further, in the application, the content of hyaluronic acid in the polysaccharide-polypeptide composite hydrogel is ≥0.1 mg / mL.

[0017] Compared with the prior art, the application of hyaluronic acid in improving the performance of polysaccharide-polypeptide composite hydrogel has the following beneficial effects:

[0018] HA has the effect of reducing the mechanical properties of HA / Fmoc-FRGDF under the premise of delaying the degradation rate of HA / Fmoc-FRGDF. The design and construction of hydrogels with tissue-specific mechanical properties play an important role in the stability of cell microenvironment and the exertion of biological functions. Compared with polysaccharide-polypeptide composite hydrogels with high mechanical strength, composite hydrogels with lower mechanical properties are beneficial to be applied to parts of the extracellular matrix with lower mechanical strength, such as the brain. The elasticity of different parts of the human body is very different, such as the brain elasticity modulus is 100~1000Pa, the muscle elasticity modulus is 8000~17000Pa, and the cross-linked collagen protein elasticity modulus is 25000~40000Pa. Polysaccharide-polypeptide composite hydrogels with low mechanical strength have better biocompatibility and higher plasticity, and are easier to be processed into various shapes and sizes to meet different application requirements. The HA / Fmoc-FRGDF hydrogel provided by the application reduces the mechanical properties under the premise of delaying the degradation rate of HA / Fmoc-FRGDF, and can be used for extracellular matrix with lower mechanical strength.

[0019] HA can delay the degradation rate of HA / Fmoc-FRGDF. The in vitro degradation test of HA / Fmoc-FRGDF shows that the A0 remaining mass percentage decreases fastest with the extension of time, and only remains 4.34% at 192h, indicating that its degradation rate is the fastest. A2 remains 10.46% at 192h, indicating that the addition of HA can delay the degradation rate of HA / Fmoc-FRGDF and improve the stability of the hydrogel, which is of great significance for the sustained release of active substances.

[0020] HA can improve the loading capacity of HA / Fmoc-FRGDF for Que and Hes. The encapsulation efficiency of Que in the HA / Fmoc-FRGDF hydrogel increases with the increase of the concentration, and the maximum is 90.29%, which is 17.00% higher than that of the polypeptide hydrogel without adding HA (i.e. A0). The encapsulation efficiency of Hes in the HA / Fmoc-FRGDF hydrogel increases with the increase of the concentration, and the maximum is 97.60%, which is 50.79% higher than that of the polypeptide hydrogel without adding HA (i.e. A0). It is shown that HA in HA / Fmoc-FRGDF can improve its loading capacity for Que and Hes, and accommodate more Que and Hes.

[0021] HA can delay the release rate of Que in HA / Fmoc-FRGDF. In the polypeptide hydrogel without adding HA (i.e. A0), the release rate of Que is the fastest, followed by A2, A1, A4 and A3 with HA added, which shows that HA can significantly reduce the release rate of Que and alleviate the problem that hydrophobic drugs cannot be released stably and continuously in the composite hydrogel.

[0022] In addition, through Fourier infrared spectroscopy (FTIR) and circular dichroism spectrometer (CD) analysis, the secondary structure of HA / Fmoc-FRGDF is β-pleated arrangement, and the addition of HA does not affect the secondary structure of HA / Fmoc-FRGDF. Moreover, the addition of HA promotes the improvement of the interaction strength between molecules and the stability of HA / Fmoc-FRGDF, and the greater the concentration of HA, the stronger the absorption peak. In addition, in the circular dichroism spectrum, the 230~270nm region indicates that the polypeptide nanofiber forms a supramolecular arrangement, and the addition of Que greatly enhances the peak intensity in the 230~270nm region, indicating that Que enhances the ordering of the supramolecular structure.

[0023] Transmission electron microscopy (TEM) results show that the hydrogel with HA added forms nanofibers, and HA does not change the micro-nano structure of the polysaccharide-polypeptide composite hydrogel, and the nanometer structure of the composite hydrogel loaded with Que is also not changed. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the change of the storage modulus (G') of HA / Fmoc-FRGDF under different HA concentrations.

[0025] Figure 2 is the change of the loss modulus (G") of HA / Fmoc-FRGDF under different HA concentrations.

[0026] Figure 3 is the Fourier infrared spectrum (FTIR) of HA / Fmoc-FRGDF.

[0027] Figure 4 is a FTIR zoom-in of HA / Fmoc-FRGDF in the 1550~1750 cm -1 frequency range.

[0028] Figure 5 is a circular dichroism (CD) analysis of single Fmoc-FRGDF hydrogel, HA / Fmoc-FRGDF (A1~A4) and Que-loaded HA / Fmoc-FRGDF hydrogel (B2) in the 190~300 nm wavelength range.

[0029] Figure 6 is a plot of the remaining mass percentage of HA / Fmoc-FRGDF in PBS buffer over time.

[0030] Figure 7 is a plot of the release amount of Que-loaded HA / Fmoc-FRGDF hydrogel over time.

[0031] Figure 8 is a transmission electron microscope (TEM) image of Que-loaded HA / Fmoc-FRGDF hydrogel.

[0032] BRIEF DESCRIPTION OF DRAWINGS A0 represents single Fmoc-FRGDF hydrogel without HA; A1 represents HA / Fmoc-FRGDF with a HA concentration of 0.1 mg / mL; A2 represents HA / Fmoc-FRGDF with a HA concentration of 0.5 mg / mL; A3 represents HA / Fmoc-FRGDF with a HA concentration of 1.0 mg / mL; and A4 represents HA / Fmoc-FRGDF with a HA concentration of 1.5 mg / mL. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present application.

[0034] In the embodiments, the polysaccharide acid has a molecular weight of 40~100 kDa and a purity of 97%; the polypeptide is Fmoc-FRGDF with a purity of >95%; the quercetin and dihydroflavonol have a purity of 95%; and the potassium phosphate buffer (PBS) has a concentration of 0.1 M and a pH of 7.4. Embodiment 1

[0035] This embodiment provides preparation of polysaccharide-polypeptide composite hydrogel (i.e. HA / Fmoc-FRGDF).

[0036] Accurately weigh 0, 0.1, 0.5, 1.0, 1.5 mg of hyaluronic acid into a small glass bottle, and the corresponding numbers are A0 (HA, 0 mg / mL), A1 (HA, 0.1 mg / mL), A2 (HA, 0.5 mg / mL), A3 (HA, 1.0 mg / mL), A4 (HA, 1.5 mg / mL).

[0037] Add 10 mg of polypeptide (Fmoc-FRGDF), 400 μL of ultrapure water, 70 μL of 0.5 M NaOH solution, 65 μL of 0.1 M HCl solution, and 465 μL of PBS buffer to each small glass bottle, and the listed reagents are added while shaking.

[0038] Vortex until the HA and Fmoc-FRGDF are completely dissolved. After ultrasonic defoaming, stand at room temperature for 24 h to obtain HA / Fmoc-FRGDF. Example 2

[0039] This example provides a determination of the rheological properties of HA / Fmoc-FRGDF.

[0040] Prepare HA / Fmoc-FRGDF using the method in Example 1. Prepare the HA / Fmoc-FRGDF samples (A0~A4) and determine the storage modulus (G') and loss modulus (G") in the linear viscoelastic range using a HAAKE MARS modular advanced rheometer, with a shear stress of 1%, a frequency of 0.1~100 Hz, and a temperature of 25°C.

[0041] Figure 1 shows the change in storage modulus (G') of HA / Fmoc-FRGDF at different HA concentrations. Figure 2 shows the change in loss modulus (G") of HA / Fmoc-FRGDF at different HA concentrations. As shown in Figures 1~2, the HA / Fmoc-FRGDF prepared at different concentrations of HA all show viscoelastic gel behavior, and the storage modulus (G') is higher than the loss modulus (G") within the frequency range shown in the figures. The storage modulus (G') of the single Fmoc-FRGDF hydrogel (A0) is the highest, and the storage modulus (G') of the composite hydrogel after adding HA is lower than that of the single Fmoc-FRGDF hydrogel at different frequencies, indicating that HA reduces the mechanical properties of the hydrogel. This change in storage modulus (G') is due to the good water absorption of hyaluronic acid, which increases the water content of the gel and reduces the elasticity. Example 3

[0042] This example provides a secondary structure analysis of HA / Fmoc-FRGDF.

[0043] 1. After freeze-drying of the HA / Fmoc-FRGDF sample, a small amount of the dried sample was mixed with dried potassium bromide and ground, pressed into a tablet, and analyzed using a Tensor 27 Fourier Transform Infrared Spectrometer (FTIR) with a pure potassium bromide tablet as a blank control. The analysis conditions were: a wave number range of 400-4000 cm -1 , a resolution of 4 cm -1 , and 32 scans.

[0044] Figure 3 is an FTIR spectrum of HA / Fmoc-FRGDF. The Fmoc-FRGDF has N-H and O-H stretching vibration peaks of the amide A band at 3294 cm -1 , characteristic absorption peaks of a β-sheet at 1636 cm -1 and 1690 cm -1 , and a C-H C-N (amine) stretching peak at 1079 cm -1 .

[0045] Figure 4 is an FTIR zoom-in view of HA / Fmoc-FRGDF in the 1550-1750 cm -1 frequency range. As can be seen from Figure 4, the characteristic absorption peaks of the Fmoc-FRGDF and the HA / Fmoc-FRGDF of different concentrations are similar, and all have a β-sheet absorption peak at 1636 cm -1 . The vibrations in the 1600-1700 cm -1 range are usually used to determine the secondary structure of a peptide chain, and thus it can be seen that no new chemical bonds are formed in the HA / Fmoc-FRGDF, indicating that the addition of HA does not affect the secondary structure of the polypeptide in the gel. In addition, the addition of HA promotes the improvement of the intermolecular interaction force strength and the stability of the HA / Fmoc-FRGDF, and the greater the concentration of HA, the stronger the absorption peak.

[0046] 2. The HA / Fmoc-FRGDF sample was diluted to 0.01 wt%, and a circular dichroism spectrometer was used to record the spectrum of the sample, with a wavelength of 190-300 nm and a resolution of 1 nm at room temperature. All tests were repeated 3 times, and the average value was taken.

[0047] Figure 5 is a circular dichroism (CD) analysis diagram of single Fmoc-FRGDF hydrogel, HA / Fmoc-FRGDF (A1~A4) and Que-loaded HA / Fmoc-FRGDF hydrogel (B2) in the wavelength range of 190~300 nm. As can be seen from Figure 5, the negative peak in the region of 190~220 nm indicates the presence of β-sheet, and the region of 230~270 nm indicates that the polypeptide nanofiber forms a supramolecular arrangement structure, and macroscopically forms a gel. The addition of Que greatly enhances the peak intensity in the region of 230~270 nm, indicating that the addition of Que enhances the strength of the supramolecular arrangement structure. Example 4

[0048] This example provides in vitro degradation test of HA / Fmoc-FRGDF.

[0049] According to the method in Example 1, HA / Fmoc-FRGDF samples with concentrations of A0, A1, A2, A3, A4 were prepared. Take 1 mL of HA / Fmoc-FRGDF sample, add 2 mL of PBS buffer respectively, keep in a closed container, place in a 37℃, 100r / min shaking incubator, remove 2.0 mL of supernatant at 0, 24, 48, 72, 96, 120, 144, 168, 192h, wipe with paper towel to dehydrate, and weigh the sample mass, finally supplement with equal volume of fresh PBS buffer, set 3 repeats for each group. Record the weight loss of the sample during the whole process and draw the degradation curve. The remaining weight ratio of the hydrogel is calculated as follows:

[0050] The remaining weight ratio of the hydrogel (%) = (W t / W0) x 100%;

[0051] In the formula, W t and W0 represent the remaining weight of the hydrogel at the set time point, and W0 represents the initial weight of the hydrogel. The weight value is the average of 3 measurement results.

[0052] Figure 6 is the change of the remaining mass percentage of HA / Fmoc-FRGDF in PBS buffer with time. As can be seen from Figure 6, with the extension of time, the remaining mass percentage of A0 decreases the fastest, only remaining 4.34% at 192h, indicating that its degradation rate is the fastest. A2 remains 10.46% at 192h, indicating that the addition of HA can delay the degradation rate of HA / Fmoc-FRGDF and improve the stability of the hydrogel, which is of great significance for the sustained release of active substances. Example 5

[0053] This example provides the loading capacity and controlled release capacity test of HA / Fmoc-FRGDF for quebrachitol (Que).

[0054] 1. EE of Que-loaded HA / Fmoc-FRGDF hydrogel

[0055] Que powder 30.76 mg, Fmoc-FRGDF powder 10 mg and 0, 0.1, 0.5, 1.0, 1.5 mg of HA were weighed into centrifuge tubes, corresponding to A0 (HA, 0 mg / mL), A1 (HA, 0.1 mg / mL), A2 (HA, 0.5 mg / mL), A3 (HA, 1.0 mg / mL), A4 (HA, 1.5 mg / mL).

[0056] Each centrifuge tube was added with: 400 μL ultrapure water, 70 μL 0.5 M NaOH solution, 65 μL 0.1 M HCl solution, 465 μL PBS buffer, and all reagents were added while shaking.

[0057] Vortex shaking was used until HA and Fmoc-FRGDF were completely dissolved. After ultrasonic defoaming, the room temperature was placed for 24 h, and the Que-loaded HA / Fmoc-FRGDF hydrogel was obtained.

[0058] The Que-loaded HA / Fmoc-FRGDF hydrogel was vortexed at 1500 r / min for 2 min and centrifuged at 10000 g for 10 min, and the supernatant was collected. The spectrophotometer (TU-1901 double-beam ultraviolet visible spectrophotometer) was used to determine the absorbance at 374 nm. The concentration was calculated by using the Que standard curve. Under the same conditions, Y=1.2914X-0.0032 (R 2 =1.0000) was obtained. The absorbance of samples of different concentrations was determined and the EE value of Que was calculated to characterize the loading capacity (see Table 1).

[0059] EE (%)=(weight of Que in the gel / total weight of Que)×100%.

[0060] Table 1. EE value of Que-loaded HA / Fmoc-FRGDF hydrogel

[0061] Sample name EE (%) A0 (HA, 0 mg / mL) 73.29±0.03 a A1 (HA, 0.1 mg / mL) 85.82±0.01 b A2 (HA, 0.5 mg / mL) 86.48±0.04 c A3 (HA, 1.0 mg / mL) 87.68±0.01 d A4 (HA, 1.5 mg / mL) 90.29±0.00 e

[0062] Note: The EE in Table 1 is the average value ± standard deviation (n=3).

[0063] Table 1 is the EE value of the HA / Fmoc-FRGDF hydrogel loaded with Que for Que. As can be seen from Table 1, with the increase of the concentration of HA, the EE value also increases, and the maximum is 90.29%, which is 17% higher than that of the polypeptide hydrogel without HA (A0). It shows that in the HA / Fmoc-FRGDF hydrogel loaded with Que, HA can improve its loading capacity for Que and accommodate more Que.

[0064] 2. Test of controlled release ability of HA / Fmoc-FRGDF hydrogel loaded with Que

[0065] 1.0 mL of HA / Fmoc-FRGDF hydrogel loaded with Que was placed in a dialysis bag (100-500 Da), and then the dialysis bag was immersed in a beaker containing 50 mL of PBS solution (HA / Fmoc-FRGDF hydrogel loaded with Que contained the same concentration of HCl and NaOH solution), and sealed. The beaker was placed in a 37℃, 100r / min shaker, and 2mL of PBS buffer in the beaker was taken at 0, 6, 12, 18, 24, 30, 36, 42, 48h, respectively, and the absorbance at 286nm was measured (Y=1.2914X-0.0032; R 2 =1.0000; 2.5-100.0µg / mL), and an equal amount of PBS buffer was added in the beaker.

[0066] Figure 7 is the change of the release amount of HA / Fmoc-FRGDF hydrogel loaded with Que at different times. As can be seen from Figure 7, the release speed of Que in A0 group without adding HA is the fastest, followed by A2, A1, A4 and A3. It shows that HA has obvious delaying effect on the in vitro release of Que.

[0067] Example 6

[0068] This example provides the loading capacity of HA / Fmoc-FRGDF for dihydrogen flavonoid (Hes).

[0069] 2mg of Hes powder, 10mg of Fmoc-FRGDF powder and 0, 0.1, 0.5, 1.0, 1.5mg of HA were weighed in a centrifuge tube, corresponding to A0 (HA, 0mg / mL), A1 (HA, 0.1mg / mL), A2 (HA, 0.5mg / mL), A3 (HA, 1.0mg / mL), A4 (HA, 1.5mg / mL).

[0070] To each centrifuge tube, respectively, add: 400 μL ultrapure water, 70 μL of 0.5 M NaOH solution, 65 μL of 0.1 M HCl solution, 465 μL of PBS buffer, and all the listed reagents are added while shaking.

[0071] Shake with vortex shaker until HA and Fmoc-FRGDF are completely dissolved. After ultrasonic defoaming, stand at room temperature for 24 h, and the Hes-loaded HA / Fmoc-FRGDF hydrogel is obtained.

[0072] Centrifuge the Hes-loaded HA / Fmoc-FRGDF hydrogel at 1500 r / min for 2 min, and centrifuge at 10000 g for 10 min, and collect the supernatant. Use a spectrophotometer to measure the absorbance at 286 nm. Calculate the concentration of Hes using the Hes standard curve. Under the same conditions, Y=0.011X-0.0242 (R 2 =0.9988). Measure the absorbance of samples of different concentrations and calculate the EE value of Hes to characterize the loading capacity (see Table 2 for results). The EE is calculated as follows:

[0073] EE (%)=(weight of Hes in the gel / total weight of Hes) x 100%.

[0074] Table 2. EE value of Hes-loaded HA / Fmoc-FRGDF hydrogel for Hes

[0075] Sample name EE (%) A0 (HA, 0 mg / mL) 46.81 ± 0.01 e A1 (HA, 0.1 mg / mL) 88.61 ± 0.01 c A2 (HA, 0.5 mg / mL) 90.56 ± 0.00 b A3 (HA, 1.0 mg / mL) 97.60 ± 0.01 a A4 (HA, 1.5 mg / mL) 85.65 ± 0.00 d

[0076] Table 2 is the statistical result of EE value of Hes-loaded HA / Fmoc-FRGDF hydrogel for Que. As can be seen from Table 2, with the increase of the concentration of HA, the EE value also increases, and the maximum is 97.60%, which is 50.79% higher than that of the polypeptide hydrogel without adding HA (A0). It shows that in the Hes-loaded HA / Fmoc-FRGDF hydrogel, HA can improve its loading capacity for Hes and accommodate more Hes. Example 7

[0077] This example provides a TEM image of the HA / Fmoc-FRGDF hydrogel loaded with Que.

[0078] HA / Fmoc-FRGDF samples with concentrations of A0, A1, A2, A3, A4 respectively were prepared according to the method in Example 1. The HA / Fmoc-FRGDF hydrogel sample B2 loaded with Que (HA concentration of 0.5 mg / mL) was prepared according to the method in Example 5.

[0079] TEM images were measured using a JEOL-2100 LaB6 transmission electron microscope (JEOL Ltd., Japan) with a working voltage of 100 Kv. A 300-mesh lacey carbon film-coated copper grid was used as a sample holder. The hydrogel sample was diluted 5 times (20 μL hydrogel + 80 μL distilled water), and 12 μL of the diluted hydrogel sample was taken with a pipette and placed on the copper grid, and allowed to adsorb for 30 s, and then the excess liquid was absorbed with a separate Whatman filter paper (No. 1). One drop of negative NanoVan stain (Bio-Scientific Pty) was dropped on parafilm M sealing film, and then the copper grid with the adsorbed hydrogel sample was placed on the NanoVan stain on the parafilm M sealing film, with the carbon side facing down, and stained for 5 min, and then dried in air for 2 min with the carbon side facing up, and finally the copper grid was placed in a copper grid box and dried overnight for TEM imaging.

[0080] Figure 8 is a TEM image of the HA / Fmoc-FRGDF hydrogel loaded with Que. As can be seen from Figure 8, all the gel systems form nanofibers, indicating that the addition of HA and Que does not destroy the nanofiber structure of the polypeptide.

[0081] In summary, the present application studies the effects of different concentrations of HA on the structure and properties of polysaccharide-polypeptide composite hydrogel HA / Fmoc-FRGDF by preparing different concentrations of polysaccharide-polypeptide composite hydrogel HA / Fmoc-FRGDF. The results show that HA has a direct impact on the network structure and drug release kinetics of HA / Fmoc-FRGDF.

[0082] Firstly, HA reduces the storage modulus and energy dissipation modulus of HA / Fmoc-FRGDF. The HA / Fmoc-FRGDF hydrogel provided by the present application reduces the mechanical properties of HA / Fmoc-FRGDF under the premise of delaying the degradation rate of HA / Fmoc-FRGDF, and can be used for extracellular matrix with lower mechanical strength. The secondary structure of HA / Fmoc-FRGDF is β-sheet arrangement, and the addition of HA does not affect its secondary structure. The addition of HA promotes the improvement of the strength of intermolecular forces and the stability of HA / Fmoc-FRGDF. The greater the concentration of HA, the stronger the absorption peak.

[0083] In addition, in vitro degradation test of HA / Fmoc-FRGDF shows that the addition of HA can delay the degradation rate of HA / Fmoc-FRGDF and improve the stability of the hydrogel, which is of great significance for the sustained release of active substances.

[0084] In addition, in vitro degradation test of HA / Fmoc-FRGDF shows that the addition of HA can delay the degradation rate of HA / Fmoc-FRGDF and improve the stability of the hydrogel, which is of great significance for the sustained release of active substances.

[0085] Finally, the transmission electron microscopy (TEM) results show that the Que-loaded HA / Fmoc-FRGDF hydrogel does not destroy the nanofiber structure of the polypeptide.

[0086] The above-described embodiments are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. All other embodiments obtained by relevant deduction and replacement made by those skilled in the art under the condition of the concept of the present application, without making creative efforts, belong to the scope of protection of the present application.

Claims

1. Use of hyaluronic acid for improving the properties of polysaccharide- polypeptide composite hydrogels, characterized in that, Hyaluronic acid is used for reducing the mechanical property of polysaccharide-polypeptide composite hydrogel, or for delaying the degradation rate of polysaccharide-polypeptide composite hydrogel, or for improving the loading capacity of polysaccharide-polypeptide composite hydrogel for hydrophobic drugs, or for delaying the release rate of hydrophobic drugs in polysaccharide-polypeptide composite hydrogel.

2. Use according to claim 1, characterized in that, In the concentration range of 0.1-1.5 mg / mL, hyaluronic acid can reduce the storage modulus and energy dissipation modulus of polysaccharide-polypeptide composite hydrogel.

3. Use according to claim 1, characterized in that, In the concentration range of 0.1-1.5 mg / mL, hyaluronic acid can delay the degradation rate of polysaccharide-polypeptide composite hydrogel.

4. Use according to claim 1, characterized in that, In the concentration range of 0.1-1.5 mg / mL, hyaluronic acid can improve the loading capacity of polysaccharide-polypeptide composite hydrogel for hydrophobic drugs.

5. The use according to claim 1, characterized in that, In the concentration range of 0.1-1.5 mg / mL, hyaluronic acid can delay the release rate of hydrophobic drugs in polysaccharide-polypeptide composite hydrogel.

6. Use according to claim 1, characterized in that, The polypeptide is Fmoc-FRGDF, and the hydrophobic drug is quercetin or dihydroflavonoid.

7. The use according to claim 1, characterized in that, The preparation method of the polysaccharide-polypeptide composite hydrogel comprises: dissolving hyaluronic acid and polypeptide in water, adding NaOH solution, HCl solution and PBS buffer, oscillating to dissolve and defoaming.

8. Use according to claim 7, characterized in that, The ratio of the preparation raw materials is: 70 μL of NaOH solution, 65 μL of HCl solution, 465 μL of PBS buffer and 10 mg of polypeptide are used for every 400 μL of water.

9. Use according to claim 8, characterized in that, The concentration of NaOH solution is 0.5 M, the concentration of HCl solution is 0.1 M, and the concentration of PBS buffer is 0.1 M, pH=7.

4.

10. Use according to claim 9, characterized in that, The content of hyaluronic acid in the polysaccharide-polypeptide composite hydrogel is ≥0.1 mg / mL.

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