阅读说明：本技术 一种用于治疗慢性创伤的组合制剂及其制备方法 (Combined preparation for treating chronic wounds and preparation method thereof ) 是由 侯豹 戚梦婷 朱雪锐 于晓依 班雅婷 胡江涛 康心勉 蔡维维 邱丽颖 于 2021-08-16 设计创作，主要内容包括：本发明公开了一种用于治疗慢性创伤的组合制剂及其制备方法,属于生物工程技术领域。本发明的组合制剂分为A和B两个制剂,A为洗剂；B为喷剂,其中,所述洗剂A为壳聚糖纳米水凝胶负载0.5％-2％的王不留行黄酮苷和0.5％-2％的王不留行刺桐碱,所述喷剂B为壳聚糖纳米水凝胶负载3％-5％的王不留行黄酮苷和3％-5％的王不留行刺桐碱溶液。本发明的组合制剂对慢性创伤部位具有显著的抗炎作用,对创伤部位血管再生具有显著的促进作用。(The invention discloses a combined preparation for treating chronic wounds and a preparation method thereof, belonging to the technical field of biological engineering. The combined preparation of the invention is divided into two preparations A and B, wherein A is lotion; and B is a spray, wherein the lotion A is formed by loading 0.5-2% of cowherb seed flavonoid glycoside and 0.5-2% of cowherb seed erythrina alkali on chitosan nano hydrogel, and the spray B is formed by loading 3-5% of cowherb seed flavonoid glycoside and 3-5% of cowherb seed erythrina alkali solution on chitosan nano hydrogel. The combined preparation of the invention has obvious anti-inflammatory effect on chronic wound parts and obvious promotion effect on revascularization of wound parts.)

1. A combined preparation for treating chronic wounds is characterized in that the combined preparation is divided into two preparations A and B, wherein A is lotion; and B is a spray, wherein the lotion A is formed by loading 0.5-2% of cowherb seed flavonoid glycoside and 0.5-2% of cowherb seed erythrine on chitosan nano hydrogel, and the spray B is formed by loading 3-5% of cowherb seed flavonoid glycoside and 3-5% of cowherb seed erythrine on chitosan nano hydrogel.

2. The combination preparation for treating chronic wounds according to claim 1, wherein lotion A is chitosan nano hydrogel loaded with 2% of cowherb flavonoid glycoside and 2% of cowherb erythrine, and spray B is chitosan nano hydrogel loaded with 5% of cowherb flavonoid glycoside and 5% of cowherb erythrine.

3. The combination for treating chronic wounds according to claim 1, wherein the viscosity of lotion A is 10^-4～10^-1Pa · s.

4. A method of preparing a combined preparation for the treatment of chronic wounds according to any one of claims 1 to 3, comprising:

(a) firstly, adding span 80 into liquid paraffin, and then mixing in a water bath at 50-70 ℃ to serve as an oil phase for later use;

(b) dissolving cowherb seed flavonoid glycoside and cowherb seed erythrine in a chitosan solution, adding the mixed chitosan solution into the oil phase obtained in the step (a), and stirring for 2-6 h;

(c) adding sodium tripolyphosphate into the stirring liquid obtained in the step (b), and continuously stirring for 0.5-2 hours to obtain a mixed liquid;

(d) washing the mixed solution obtained in the step (c) for several times by using petroleum ether with the volume 2-5 times, centrifuging, and drying in vacuum to obtain drug-loaded nanoparticles;

(e) dissolving the chitosan nanoparticles loaded with the drugs obtained in the step (d) in an aqueous solution, and ultrasonically stirring to obtain the nano hydrogel loaded with the cowherb seed flavonoid glycoside and the cowherb seed erythrine, wherein the lotion A and the spray B can be obtained according to different proportions of the flavonoid glycoside and the erythrine, and the spray B is uniformly sprayed for use through a spray bottle.

5. The preparation method of claim 4, wherein the chitosan hydrogel comprises span 80, liquid paraffin, a chitosan solution and a sodium tripolyphosphate solution, and the volume ratio of the span 80 to the liquid paraffin to the chitosan solution is 4-6: 36-40: 20-25: 5-10.

6. The method according to claim 4, wherein the concentration of the chitosan solution is 4-6 mg/mL.

7. The method according to any one of claims 4 to 6, wherein the concentration of the sodium tripolyphosphate solution is 1 to 3 mg/mL.

8. The method according to any one of claims 4 to 7, wherein the nanoparticle size of the combined preparations A and B is (64.97 ± 4.39) nm, and the zeta potential is (-1.83 ± 0.32) mV.

9. Use of a combined preparation according to any one of claims 1 to 3 or a method according to any one of claims 4 to 8 in the manufacture of a medicament for the treatment of chronic wounds.

10. A medicament comprising a combination preparation for the treatment of chronic wounds as claimed in any one of claims 1 to 3 or a combination preparation prepared by the process as claimed in any one of claims 4 to 8.

Technical Field

The invention relates to a combined preparation for treating chronic wounds and a preparation method thereof, belonging to the technical field of biological engineering.

Background

Chronic trauma not only causes the quality of life of the patient to be reduced, but also brings great economic burden to the family members of the patient. From a demographic perspective, the number of patients with chronic wounds and impaired healing is reaching epidemic proportions and will become more burdened with human health and economy. Therefore, an increasing number of researchers are concerned with the study of chronic wounds. The process of wound healing can be summarized as the formation of blood clots, inflammation, tissue regeneration and tissue remodeling. During the inflammatory phase, the early inflammatory response mobilizes local inflammatory factors and systemic defense responses to the wound site. The inflammatory phase of chronic wounds lasts for a long time. Researchers believe that these wounds may be in a chronic inflammatory state and not transition to the tissue regeneration phase. The chronic wound healing inflammatory phase block is closely related to activation of NLRP3 inflammasome and activation of TLR 4/NF-kB signaling pathway, and not only inhibits activation of NLRP3 inflammasome but also blocks activation of TLR 4/NF-kB signaling pathway in the treatment process of chronic wounds. The anti-endothelial cell inflammation process of erythrina is realized only by inhibiting the expression of inflammatory factors such as IL-1 beta and TNF-alpha.

Research shows that inflammatory cells composed of neutrophils and macrophages infiltrate the surface of a wound, so that IL-1 beta and TNF-alpha are increased, metalloproteinase is further increased, local ECM is excessively degraded, cell migration is damaged, and host tissues are seriously damaged. In the tissue regeneration phase, angiogenesis is difficult and nutrients and various growth factors cannot be transported to the wound site, resulting in impaired wound healing.

Currently, there are a number of growth factors, gene delivery therapies, and cell therapy strategies to treat wound healing, but few effective drugs are available. The treatment of chronic wounds has been a problem for researchers, and despite the large number of topical therapeutic/antimicrobial drugs and dressings available to clinicians, there is little prospective data support. Thus, clinicians tend to use their habits or to determine effective treatments based on personal experience.

Disclosure of Invention

In order to solve the problem of difficult treatment of the chronic wounds, the invention takes the nano hydrogel loaded medicine as the basis, loads the medicine flavonoid glycoside for promoting the angiogenesis and the anti-inflammatory medicine erythrina alkali, prepares the lotion A and the spray B, and has the medicine effect and the functions of nano hydrogel of antibiosis, moisture retention and anti-inflammation. Lotion A has debridement effect on chronic wound, and mainly plays a role of chitosan. The spray B is sprayed to the wound surface to form a layer of protective film, so that the effect of effective medicine for promoting wound healing is exerted, the gaps between the protective films can be subjected to gas exchange, and the reproduction of anaerobic bacteria at the wound part is inhibited.

In order to achieve the above objects, the present invention firstly provides a combined preparation for treating chronic wounds, which is divided into two preparations, a and B, wherein a is a lotion; and B is a spray, wherein the lotion A is formed by loading 0.5-2% of cowherb seed flavonoid glycoside and 0.5-2% of cowherb seed erythrine on chitosan nano hydrogel, and the spray B is formed by loading 3-5% of cowherb seed flavonoid glycoside and 3-5% of cowherb seed erythrine on chitosan nano hydrogel.

In one embodiment of the invention, preferably, the lotion a is chitosan nano hydrogel loaded with 2% of cowherb flavonoid glycoside and 2% of cowherb erythrina alkali, and the spray B is chitosan nano hydrogel loaded with 5% of cowherb flavonoid glycoside and 5% of cowherb erythrina alkali.

In one embodiment of the invention, the viscosity of lotion A is 10^-4～10^-1Pa · s.

The invention also provides a preparation method of the combined preparation, which comprises the following steps:

(a) firstly, adding span 80 into liquid paraffin, and then mixing in a water bath at 50-70 ℃ to serve as an oil phase for later use;

(b) dissolving cowherb seed flavonoid glycoside and cowherb seed erythrine in a chitosan solution, adding the mixed chitosan solution into the oil phase obtained in the step (a), and stirring for 2-6 h;

(c) adding sodium tripolyphosphate into the stirring liquid obtained in the step (b), and continuously stirring for 0.5-2 hours to obtain a mixed liquid;

(d) washing the mixed solution obtained in the step (c) for several times by using petroleum ether with the volume 2-5 times, centrifuging, and drying in vacuum to obtain drug-loaded nanoparticles;

(e) dissolving the drug-loaded nanoparticles obtained in the step (d) in an aqueous solution, and ultrasonically stirring to obtain the chitosan nano hydrogel loaded with the cowherb flavonoid glycoside and the erythrine from cowherb, wherein the lotion A and the spray B can be obtained according to different proportions of the flavonoid glycoside and the erythrine, and the spray B is uniformly sprayed for use through a spray bottle.

In one embodiment of the invention, the chitosan hydrogel is composed of span 80, liquid paraffin, a chitosan solution and a sodium tripolyphosphate solution, wherein the volume ratio of the span 80 to the liquid paraffin to the chitosan solution is 4-6: 36-40: 20-25: 5-10.

In one embodiment of the invention, the concentration of the chitosan solution is 4-6 mg/mL.

In one embodiment of the invention, the concentration of the sodium tripolyphosphate solution is 1-3 mg/mL.

In one embodiment of the invention, the nanoparticle size of combination preparations A and B is (64.97. + -. 4.39) nm and the zeta potential is (-1.83. + -. 0.32) mV.

The invention also provides application of the combined preparation or the preparation method in preparing a medicament for treating chronic wounds.

In one embodiment of the invention, in the treatment of chronic wounds, the wounds of the chronic wounds are first cleaned with the agent A, the wounds are cleaned and then sprayed with the agent B, and the wounds are treated once a day with the agent A and the agent B.

The invention also provides a medicament containing the combined preparation or the combined preparation prepared by the preparation method.

The invention has the following beneficial effects:

1. the combined preparation provided by the invention has a remarkable effect on the treatment of chronic wounds.

2. The combined preparation provided by the invention has a remarkable anti-inflammatory effect on a chronic wound part and a remarkable promoting effect on blood vessels of the wound part.

Drawings

Figure 1 is a photograph of a type a lotion and a type B spray of the present invention.

FIG. 2.a is the transmission electron microscope image of the B type spray, and B and c are the viscosity distribution diagrams of the hydrogel of the A type lotion under different shearing rates at 25 ℃ and 37 ℃.

FIG. 3 MTT results statistical plots for NPS-VAC + HYP, NPS and HYP + VAC groups, where CON is the control group; NPS is chitosan nanometer hydrogel group; HYP + VAC is (5% of Royal jelly erythrina alkali + 5% of Royal jelly flavonoid glycoside normal saline solution group); NPS-VAC + HYP is chitosan nanometer hydrogel loaded erythrina alkali and flavonoid glycoside group (A and B dosage form combined medicine).

Fig. 4a is a statistical graph of the accelerated chronic wound healing of a type a lotion and a type B spray, and B is a graph of the healing of chronic wounds at different time points. CON is a control group; NPS is chitosan nanometer hydrogel group; HYP + VAC is 5% of semen Vaccariae erythrinae alkali + 5% of semen Vaccariae flavonoid glycoside group normal saline solution group; NPS-VAC + HYP is chitosan nanometer hydrogel loaded erythrina alkali and flavonoid glycoside group (A and B dosage form combined medicine); HT as positive drug Huatuo trauma ointment group.

FIG. 5 is a graph of NPS-VAC + HYP accelerated chronic wound re-epithelialization (A) representing images of day 6 wound HE staining. (B) Graph represents image of day 9 wound HE staining (C) represents that of day 12 wound HE staining; the magnification was 40 times and 200 times, respectively. In the figure, I represents inflammatory cells, B represents blood vessels, and F represents fibroblasts.

FIG. 6NPS-VAC + HYP down-regulates the expression levels of the pro-inflammatory cytokines IL-1 β and TNF- α. (A) Represents the IL-1 beta immunohistochemical staining image of the wound surface on the 6 th, 9 th and 12 th days after the wound (400-fold amplification). Black arrows indicate IL-1 β staining (B) the image represents immunoblot detection of protein expression of TNF- α and IL-1 β. Values are mean ± sd. P <0.05, P < 0.01. Compared with the control group.

Detailed Description

The present invention is further described below with reference to examples, but the embodiments of the present invention are not limited thereto.

EXAMPLE 1 preparation of Combined preparations

(a) Firstly, adding 4mL of span 80 into 36mL of liquid paraffin, and then mixing in a water bath at 55 ℃ to serve as an oil phase for later use;

(b) dissolving 5mg of flavonoid glycoside and 5mg of erythrina indica alkali in 20mL of chitosan solution (5mg/mL), adding the mixed chitosan solution into the above oil phase, and stirring at 680 rpm for 3 h;

(c) slowly adding 5mL of sodium tripolyphosphate (2mg/mL) into the stirred solution, and continuously stirring for 0.5h to obtain a uniform mixed solution;

(d) washing the obtained mixed solution with petroleum ether with 3 times of volume for 3 times, centrifuging, and drying in vacuum to obtain drug-loaded nanoparticles;

(e) dissolving nanoparticles in an aqueous solution, performing ultrasonic stirring to obtain a nano hydrogel loaded with flavonoid glycoside and erythrina alkali, and obtaining a lotion A and a spray B according to different proportions of the flavonoid glycoside and the erythrina alkali, wherein the lotion A is prepared by loading 2% of cowherb flavonoid glycoside and 2% of erythrina alkali on chitosan nano hydrogel, the spray B is prepared by loading 5% of cowherb flavonoid glycoside and 5% of erythrina alkali solution on the chitosan nano hydrogel, the lotion A is placed in an EP tube for use, and the spray B is uniformly sprayed for use through a spray bottle. The product of the type A lotion and type B spray is shown in figure 1.

Particle size and zeta potential measurements

The nano hydrogel solution obtained by the above preparation method was measured by a Zetapals analyzer to have an average nanoparticle size of (64.97 + -4.39) nm and a zeta potential of (-1.83 + -0.32) mV. The transmission electron microscope image of B-type spray obtained by transmission electron microscope is shown in FIG. 2a, and the viscosity of lotion A is 10 as shown in FIG. 2B and FIG. 2c after the viscosity test result^-4～10^-1Pa · s.

Second, in vitro safety test

Cytotoxicity was detected with raw264.7 macrophages: mix raw246.7 (1X 10)⁴Cells/well) were seeded in 96-well plates and cultured at 37 ℃. NPS-VAC + HYP (combination of dosage forms a and B), NPS (chitosan nano-hydrogel group, prepared as in example 1 of the present invention, but without erythrine and flavonoid glycoside) and VAC + HYP (5% erythrine vaccaria + 5% flavonoid glycoside group saline solution group) were added to the wells at different concentrations (5, 10, 25 μ g/mL), respectively. After 24 hours of cell culture, 10. mu.L of MTT solution at a concentration of 5mg/mL was added to each well, and the cells were incubated for 4 hours, followed by addition of 150. mu.L of dimethyl sulfoxide (DMSO). The Optical Density (OD) value was measured at 570nm, and the cell viability was calculated.

The results show that: the cytotoxicity of HYP-NPS on RAW264.7 cells was measured by MTT method, and the results are shown in FIG. 3. Within the concentration range (5-25 μ g/mL), neither VAC + HYP, NPS nor NPS-VAC + HYP are cytotoxic. These results indicate that HYP-NPS has good biocompatibility and safety and can be used as a wound healing material. In vitro cytotoxicity assays are important methods for assessing the biocompatibility of biomaterials.

Third, animal experiment

48 SPF male SD rats (6 weeks old, 200-220 g) were purchased from Shanghai Slek laboratory animals, Inc. and were fed adaptively for 7 days. Intraperitoneal injection of Streptozotocin (STZ) induced a diabetic rat model. STZ was dissolved in citrate buffer (10mM, pH 4.5) and injected intraperitoneally at a dose of 70mg/kg body weight. After 2 weeks of STZ treatment, rats were considered diabetic when blood glucose was above 300mg/dL, and an open wound model was made.

The application method of the nano hydrogel applied to the open wound model comprises the steps of firstly coating the lotion A on a wound part, cleaning necrotic tissues of the wound part, and then spraying the spray B on the wound part to form a protective film capable of performing gas exchange.

The results are shown in fig. 4, where CON is the control group (no treatment); NPS is a chitosan group (chitosan nanoparticle solution smeared); HYP + VAC is the combination of the Royal seed Royal tree alkali and the Royal seed flavone glycoside (smearing); NPS-VAC + HYP is a chitosan loaded erythrina and flavonoid glycoside group (combination of dosage forms a and B, example 1); HT as positive drug Huatuo trauma ointment group.

As can be seen from fig. 4a, the combination of dosage forms a and B promoted healing of chronic wounds significantly faster than the other groups on days 6 and 9 after the wound; from fig. 4B, it can be seen that the combination of dosage forms a and B has a significant effect of promoting the healing of chronic wounds.

Fourth, histological evaluation

The skin tissue at the wound site was prepared into 5 μm tissue sections. Hematoxylin-eosin staining was used for pathology analysis. Histopathological changes in wound healing were observed under a microscope as shown in figure 5.

The results show that the NPS-VAC + HYP group promotes re-epithelialization of wound tissue by inhibiting chronic wound inflammatory reaction, and the revascularization speed is obviously higher than that of other groups.

Fifth, proteomics evaluation

Extracting protein tissue of the wound part, and detecting the expression levels of IL-1 beta and TNF-alpha of the wound part by an immunoblotting method. The results indicate that NPS-VAC + HYP can down-regulate the expression of proinflammatory cytokines IL-1 beta and TNF-alpha during the wound healing process, as shown in FIG. 6.

In general, the combined preparation provided by the invention has no cytotoxicity, has a remarkable anti-inflammatory effect on a chronic wound part, and has a remarkable promoting effect on vascular proliferation of the wound part.

In addition, when the lotion A is prepared by loading 0.5-2% of cowherb seed flavonoid glycoside and 0.5-2% of cowherb seed erythrine on the chitosan hydrogel and the spray B is prepared by loading 3-5% of cowherb seed flavonoid glycoside and 3-5% of cowherb seed erythrine alkali solution on the chitosan hydrogel, the effects of obvious anti-inflammatory effect on a chronic wound part and obvious promotion effect on the vascular proliferation of the wound part can be realized.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.