Histamine sustained-release dopamine-hyaluronic acid injectable hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses histamine sustained-release dopamine-hyaluronic acid injectable hydrogel, a preparation method and application. According to the histamine slow-release dopamine-hyaluronic acid injectable hydrogel, after histamine is added into an HA-DA conjugate, H2O2 / HRP is used as an initiator system, and a hydrogel network is formed through oxidative coupling of catechol groups. According to the histamine sustained-release dopamine-hyaluronic acid hydrogel, the solubility of histamine can be improved, the half-life period of histamine acting is prolonged, and stable and continuous release of histamine is achieved. Hyaluronic acid is a component of an extracellular matrix and is beneficial to repair of damaged tissues. The injection can be directly injected into an area adjacent to ischemic injury muscle tissue, and is simple and quick.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to a histamine sustained-release dopamine-hyaluronic acid injectable hydrogel, a preparation method and an application thereof. Background Art

[0002] Histamine is a small molecule biogenic amine that can exert a variety of physiological or pathological effects by binding to histamine receptors on the cell membrane surface. Previous studies have shown that supplementing a certain concentration of histamine in an appropriate time window after muscle tissue injury has been proven to be an effective strategy for intervening in ischemic skeletal muscle injury and myocardial injury in mice. Angiogenesis plays a key role in tissue repair after ischemic injury to muscle tissue. Studies have found that histamine deficiency inhibits the regeneration and repair of ischemic skeletal muscle tissue in mice, which is related to histamine regulation of angiogenesis. Intraperitoneal or subcutaneous injection of a certain amount of histamine into mice can alleviate the inflammatory response disorder, angiogenesis and delayed tissue repair caused by histamine deficiency. However, histamine has toxic effects when the concentration is too high. Systemic application of histamine may cause side effects such as diarrhea, headache, hypotension, arrhythmia, skin urticaria and itching when the concentration is too high. The half-life of histamine in different tissue environments is different, it is easy to degrade, and it is difficult to control the concentration. In previous studies, histamine was injected intraperitoneally or subcutaneously once a day, which was not easy to maintain the stability and effectiveness of histamine concentration in local tissues.

[0003] Therefore, how to maintain the stability and effectiveness of histamine concentration in local tissues while ensuring the use of histamine is one of the urgent problems to be solved. Summary of the invention

[0004] In order to maintain the stability and effectiveness of histamine concentration in local tissues while ensuring the use of histamine, the present application provides an injectable histamine sustained-release hydrogel, which can improve inflammatory disorders and delayed angiogenesis caused by histamine deficiency in the injured area, and promote tissue repair after injury.

[0005] The present invention cross-links dopamine and hyaluronic acid through EDC / NHS conjugation chemistry, adds histamine phosphate into the dopamine-hyaluronic acid mixture, uses H2O2 / HRP as an initiator system, and forms a hydrogel network through oxidative coupling of catechol groups.

[0006] Hyaluronic acid is a component of the extracellular matrix, which provides a good microenvironment for cell growth and promotes tissue regeneration and repair. The hydrogel is hydrophilic, and histamine is a water-soluble small molecule. The hydrogel with hyaluronic acid as the main component will increase its solubility. The hyaluronic acid-dopamine hydrogel has a long half-life and a low degradation rate, and can be retained in the tissue for a long time. Histamine is loaded into the hyaluronic acid-dopamine hydrogel and injected into the muscle tissue adjacent to the damaged tissue by intramuscular injection. As the hydrogel degrades, the encapsulated histamine is gradually released, which plays a role in stabilizing and continuously replenishing histamine.

[0007] Specifically, the scheme of the present invention is as follows:

[0008] In a first aspect, the present application provides an injectable histamine sustained-release hydrogel, which adopts the following technical solution:

[0009] An injectable histamine sustained-release hydrogel, wherein the sustained-release hydrogel is a histamine sustained-release dopamine-hyaluronic acid injectable hydrogel, which is prepared by adding histamine to a HA-DA conjugate, using H2O2 / HRP as an initiator system, and forming a hydrogel network through oxidative coupling of catechol groups.

[0010] In a second aspect, the present application provides a method for preparing the injectable histamine sustained-release hydrogel in the first aspect, using the following technical solution:

[0011] The preparation method of the injectable histamine sustained-release hydrogel comprises the following steps: dissolving hyaluronic acid in deionized water, adding EDC-HCl and NHS and mixing, adding dopamine, and then adding histamine solution, adjusting the pH after mixing, reacting, and finally using H2O2 / HRP as an initiator system to form a hydrogel network through oxidative coupling of catechol groups.

[0012] Optionally, the usage ratio of hyaluronic acid, EDC-HCl and NHS is 1:1.48:0.9 (by weight).

[0013] Optionally, the usage ratio of hyaluronic acid to dopamine is 1:1.48 (by weight).

[0014] Optionally, the ratio of hyaluronic acid to histamine phosphate is 1:1 (by weight).

[0015] Optionally, after adding histamine phosphate, the pH is adjusted to 5.0-5.5 and the reaction is carried out for 8 hours.

[0016] Further optionally, after the reaction is completed, the final solution obtained by the reaction is treated with a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyzed in pure water for 48 hours to completely remove unreacted reagents and salts.

[0017] In a third aspect, the present application provides use of the injectable histamine sustained-release hydrogel in the first aspect in the preparation of a drug.

[0018] Optionally, the drug refers to a drug that improves inflammatory disorders and delayed angiogenesis caused by histamine deficiency at the injured site, and promotes tissue repair after injury.

[0019] In summary, this application has the following beneficial effects:

[0020] Histamine sustained release dopamine-hyaluronic acid hydrogel can improve the solubility of histamine, increase the half-life of histamine, and achieve stable and sustained release of histamine. Hyaluronic acid is a component of the extracellular matrix and is beneficial to the repair of damaged tissues. It can be directly injected into the adjacent area of ​​ischemic damaged muscle tissue, which is simple and quick. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Schematic diagram of the preparation and functional effects of histamine sustained-release dopamine-hyaluronic acid injectable hydrogel;

[0022] Figure 2 :Physicochemical characteristics and sustained-release properties of histamine-dopamine-hyaluronic acid injectable hydrogel;

[0023] A: Electron microscopy results of histamine sustained-release hydrogel: 50 μm on the left, 20 μm on the right; B: Degradation rate of histamine sustained-release hydrogel in PBS at 37°C; C: Histamine release results of histamine sustained-release hydrogel; D: In vivo imaging results and statistical analysis of mouse lower limbs after local injection of histamine sustained-release hydrogel; E: Immunofluorescence confocal results and statistical analysis of mouse lower limbs after local injection of histamine sustained-release hydrogel; F: Elisa results of mouse serum after local injection of histamine sustained-release hydrogel; G: Elisa results of mouse lower limb muscle tissue after local injection of histamine sustained-release hydrogel (P<0.05; n=6);

[0024] Figure 3 : Functional effects of histamine-slow-release dopamine-hyaluronic acid injectable hydrogel in mice;

[0025] A: Doppler results and statistical analysis of lower limb ischemia injury in WT mice, HDCKO mice, intraperitoneal injection of histamine, and injection of histamine sustained-release gel; B: Lower limb condition scores after LI in WT and HDCKO mice, intraperitoneal injection of histamine, and injection of histamine sustained-release gel; C: Masson staining results and statistical analysis of muscle tissue of lower limb ischemia injury in WT mice, HDCKO mice, intraperitoneal injection of histamine, and injection of histamine sustained-release gel; D: HE staining results and statistical analysis of muscle tissue of lower limb ischemia injury in WT mice, HDCKO mice, intraperitoneal injection of histamine, and injection of histamine sustained-release gel; E: CD31 immunofluorescence staining results and statistical analysis of muscle tissue of lower limb ischemia injury in WT mice, HDCKO mice, intraperitoneal injection of histamine, and injection of histamine sustained-release gel; F: alpha-SMA immunofluorescence staining results and statistical analysis of muscle tissue of lower limb ischemia injury in WT mice, HDCKO mice, intraperitoneal injection of histamine, and injection of histamine sustained-release gel (P<0.05; n=6). DETAILED DESCRIPTION

[0026] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0027] Example 1: Preparation of histamine sustained-release dopamine-hyaluronic acid injectable hydrogel

[0028] 1.0g hyaluronic acid (HA) was completely dissolved in 100mL deionized water, and the resulting HA solution was then stored in a nitrogen environment. Subsequently, 1.48g EDC-HCl and 0.9g NHS were slowly added to the HA solution and stirred vigorously for 20min. Subsequently, 1480mg dopamine (DA) and 1000mg histamine phosphate were added to the solution and vortexed for 1min. The pH of the solution was adjusted to 5.0-5.5 and reacted for 8h. The final solution obtained from the reaction was then divided into dialysis bags with a molecular weight cutoff of 8000-14000Da. Dialysis was performed in pure water for 48h to completely remove unreacted reagents and salts. Finally, 0.5mM H2O2 and 0.5U / mL horseradish peroxidase (HRP) were added to crosslink the hydrogel.

[0029] Example 2: Physicochemical characteristics and sustained release properties of histamine sustained-release dopamine-hyaluronic acid injectable hydrogel

[0030] (1) The morphology of freeze-dried hydrogel samples was observed using a projection electron microscope. Figure 2 -A.

[0031] (2) The degradation rate and histamine release results of the histamine sustained-release dopamine-hyaluronic acid injectable hydrogel in PBS were tested at 37°C. The initial weight W0 of the hydrogel and the weight Wt at different time points (0 day, 1 day, 3 days, 7 days, 14 days, 21 days, 28 days) after immersion in 37°C PBS were weighed respectively, and the formula was used to calculate: Degradation rate (%) = (W0-Wt) / W0×100%; the sample should be freeze-dried at -20°C before weighing. Histamine was fluorescently labeled with Cy5.5, and the fluorescence intensity of the solution at different times (0 day, 1 day, 3 days, 7 days, 14 days, 21 days, 28 days) after the hydrogel was immersed in 37°C PBS was measured using an enzyme-labeled instrument. The results are as follows: Figure 2 -As shown in B and C.

[0032] (3) Cy5.5 fluorescently labeled histamine hydrogel and Cy5.5 fluorescently labeled histamine aqueous solution were injected into the lower limbs of mice, and the fluorescence intensity was measured using a small animal in vivo imager at different time points (0 day, 1 day, 3 days, 7 days, 14 days, and 21 days); the results are shown in Figure 2. Figure 2 -D as shown.

[0033] (4) Cy5.5 fluorescently labeled histamine hydrogel and Cy5.5 fluorescently labeled histamine aqueous solution were injected into the lower limbs of mice, respectively. Seven days later, the gastrocnemius muscle tissue of the mice was obtained, and the frozen sections were immunofluorescently stained. The binding of Cy5.5 fluorescently labeled histamine to blood vessels was observed under a confocal microscope. The results are as follows: Figure 2 -E as shown.

[0034] (5) Histamine sustained-release hydrogel was injected into the lower limbs of HDCKO mice. After 7 days, the serum and gastrocnemius muscle tissue of the mice were collected. The histamine content in the serum and gastrocnemius muscle tissue of the mice was measured by Elisa, and compared with that of WT mice, HDCKO control group and intraperitoneal injection of histamine solution group. The results are shown in Figure 2 -F, G as shown.

[0035] Example 3: Functional effects of histamine sustained-release dopamine-hyaluronic acid injectable hydrogel in mice

[0036] (1) The hind limb ischemia model was established in WT mice and HDCKO mice by femoral artery ligation, and histamine solution was injected intraperitoneally or histamine sustained-release hydrogel was injected intramuscularly. The laser Doppler perfusion imager (MoorLDI2-HIR) was used to non-invasively evaluate the reperfusion of the hind limbs at different time points (1, 7, 14, and 21 days). The images were evaluated and statistically analyzed using MLDIMainV53 software. The perfusion rate was calculated by comparing the ratio of the ischemic limb (IL) to the non-ischemic limb (NIL). The results are shown in Table 1. Figure 3 -A.

[0037] (2) The hind limb ischemia model was established in WT mice and HDCKO mice by femoral artery ligation, and histamine solution was injected intraperitoneally or histamine sustained-release hydrogel was injected intramuscularly. The hind limb condition of the mice was scored 21 days after ligation: limb function score (0 means the same as the contralateral hind limb, 1 means mild discoloration, 2 means moderate discoloration, 3 means severe discoloration; 4 means amputation); walking disorder score (0 means toe flexion; 1 means plantar flexion; the foot does not drag; 2 means dragging; 3 means dragging). The higher the score, the more severe the damage to limb function. The results are shown in Table 2. Figure 3 -B.

[0038] (3) The limb ischemia model was established in WT mice and HDCKO mice by femoral artery ligation, and histamine solution was injected intraperitoneally or histamine sustained-release hydrogel was injected intramuscularly. The gastrocnemius muscle tissue of mice was obtained 21 days after ligation, and paraffin sections were stained with Masson and HE, and the collagen area and muscle fiber area were statistically analyzed. The results are as follows: Figure 3 -As shown in C and D.

[0039] (4) Femoral artery ligation was performed to establish a hind limb ischemia model in WT mice and HDCKO mice, and histamine solution was injected intraperitoneally or histamine sustained-release hydrogel was injected intramuscularly. Gastrocnemius muscle tissue was obtained from mice 3 days after ligation, and frozen sections were immunofluorescently stained, and CD31 was expressed. + Cells and alpha-SMA + The cell area was statistically analyzed. Figure 3 -E,F as shown.

[0040] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. An injectable histamine sustained-release hydrogel, characterized in that: The sustained-release hydrogel is a histamine sustained-release dopamine-hyaluronic acid injectable hydrogel, which is prepared by adding histamine to a HA-DA conjugate and using H2O2 / HRP as an initiator system to form a hydrogel network through oxidative coupling of catechol groups.

2. The method for preparing the injectable histamine sustained-release hydrogel according to claim 1, characterized in that: Hyaluronic acid was dissolved in deionized water, and EDC-HCl and NHS were added and mixed, dopamine was added, and then histamine solution was added. After mixing, the pH was adjusted and reacted. Finally, H2O2 / HRP was used as the initiator system to form a hydrogel network through oxidative coupling of catechol groups.

3. The method according to claim 2, characterized in that The usage ratio of hyaluronic acid, EDC-HCl and NHS is 1:1.48:0.9 (by weight).

4. The method according to claim 2, characterized in that The dosage ratio of hyaluronic acid to dopamine is 1:1.48 (by weight).

5. The method according to claim 2, characterized in that: The dosage ratio of hyaluronic acid to histamine phosphate is 1:1 (by weight).

6. The method according to claim 2, characterized in that After adding the histamine solution, the pH was adjusted to 5.0-5.5 and the reaction was allowed to proceed for 8 hours.

7. The method according to claim 6, characterized in that After the reaction, the final solution obtained by the reaction was treated with a dialysis bag with a molecular weight cutoff of 8000-14000Da and dialyzed in pure water for 48h to completely remove unreacted reagents and salts.

8. Use of the injectable histamine sustained-release hydrogel according to claim 1 in the preparation of medicines.

9. The use according to claim 8, characterized in that: The drug refers to a drug that improves inflammatory disorders and delayed angiogenesis caused by histamine deficiency at the injured site and promotes tissue repair after injury.

Images