CN112076343A

CN112076343A - Alginate-carboxymethyl cellulose gel sponge and preparation method and application thereof

Info

Publication number: CN112076343A
Application number: CN202010820957.6A
Authority: CN
Inventors: 李炳林; 章莹; 陆遥; 李展; 王万舜; 夏琼; 卓荣锋
Original assignee: Southern Theater Command General Hospital of PLA
Current assignee: Southern Theater Command General Hospital of PLA
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-12-15

Abstract

The invention discloses alginate-carboxymethyl cellulose gel sponge and a preparation method and application thereof. The alginate-carboxymethyl cellulose gel sponge comprises the following components in parts by weight: 30-50 parts of sodium alginate, 30-50 parts of carboxymethyl cellulose, 10-20 parts of calcium salt and 0.1-0.3 part of nano copper powder. The alginate-carboxymethyl cellulose gel sponge prepared by mutually matching sodium alginate, carboxymethyl cellulose, calcium salt and nano copper powder has a porous structure, higher strength, a lasting antibacterial effect and higher biological safety, can prevent wound adhesion, and has the functions of stopping bleeding and promoting healing.

Description

Alginate-carboxymethyl cellulose gel sponge and preparation method and application thereof

Technical Field

The invention relates to the field of medical materials, in particular to alginate-carboxymethyl cellulose gel sponge and a preparation method and application thereof.

Background

Wound bleeding caused by trauma is easy to cause wound infection in later period, and clinically common treatment methods are debridement and antibiotic application. However, the infective wound surface usually needs multiple surgical treatments and long-term antibiotic use to effectively control infection, and the excessive use of antibiotic easily causes the generation of drug-resistant bacteria. The growth of bacteria is inhibited in the early stage, and the severity of infection in the later stage can be effectively controlled. The traditional gauze has no antibacterial ability and poor biocompatibility, and is easy to adhere to tissues to cause unnecessary consequences. Alginate and cellulose are natural polysaccharides which are common in the natural world, and have good application prospect in hemostatic materials. Previous researches prove that alginate and cellulose have good biological safety, can degrade and promote platelet activation and other functions, and can better improve the conditions of the traditional gauze for treating the wound surface.

Copper is a metal element widely existing in the nature and is also a trace element required by the human body. A large number of researches show that copper has a strong antibacterial effect and does not cause the generation of drug-resistant bacteria, and open wounds are not applicable to dressings containing antibiotics, so that copper has a good application prospect as an antibacterial material. In daily life, 2-3 parts of nano copper can be added in the alloy preparation process to achieve an effective antibacterial effect, but in the field of medical materials, the amount of copper contained in the material applied to wounds cannot be too high, otherwise toxic reaction can be caused. Chinese patent application CN107519541A discloses a hydrogel for preventing postoperative adhesion of abdominal cavity, which is prepared by dissolving sodium alginate and carboxymethyl cellulose in deionized water, and introducing multivalent cations for crosslinking, wherein the multivalent cations include copper ions. The hydrogel can effectively prevent postoperative wound fibrous adhesion. However, the loss of copper is easy to occur in the preparation process of the copper ion antibacterial material, so that the copper concentration of the product is variable, and the copper ion solution is directly added into an anionic polymer solution (such as an alginate solution and a carboxymethyl cellulose solution) to cause instantaneous crosslinking, so that the copper is unevenly distributed. When the ion-form antibacterial dressing is applied to wet wound surfaces, the ion release of the dressing is fast, and the situations that the concentration of local metal ions is too high in the early stage, cytotoxicity is caused, the concentration of local metal ions is low in the later stage, and the antibacterial performance is reduced easily occur.

Therefore, it is required to develop an anti-adhesion gel material having stable antibacterial effect and good biosafety.

Disclosure of Invention

In order to overcome the defect of unstable antibacterial effect in the prior art, the invention provides the alginate-carboxymethyl cellulose gel sponge which has a lasting antibacterial effect and higher biological safety, and can prevent wound adhesion and promote wound healing.

Another object of the present invention is to provide a method for preparing the alginate-carboxymethylcellulose gel sponge.

The invention also aims to provide the application of the alginate-carboxymethyl cellulose gel sponge in the wound repair material for infected bleeding.

In order to solve the technical problems, the invention adopts the technical scheme that:

an alginate-carboxymethyl cellulose gel sponge comprises the following components in parts by weight: 30-50 parts of sodium alginate, 30-50 parts of carboxymethyl cellulose, 10-20 parts of calcium salt and 0.1-0.3 part of nano copper powder.

Preferably, the average particle size of the nano copper powder is 10-30 nm.

The nanometer copper powder has good dispersibility, so that the copper in the prepared alginate-carboxymethyl cellulose gel sponge is uniformly distributed. The nanometer copper powder belongs to elemental metal copper, has stable chemical property and stable and lasting copper ion release rate, so that the alginate-carboxymethyl cellulose gel sponge can keep lasting and good antibacterial effect, can slowly release copper ions after being contacted with wound blood seepage, effectively avoids sudden release of the copper ions, reduces the toxic effect of heavy metal ions, and avoids the condition of cytotoxicity caused by overhigh concentration of local copper ions. The alginate and the carboxymethyl cellulose can induce the activation of blood platelets and accelerate the blood coagulation process; the copper has the effect of promoting the erythrocyte aggregation, and the addition of the nano-copper can synergistically enhance the hemostatic effect of the alginate-carboxymethyl cellulose gel sponge.

Therefore, the alginate-carboxymethyl cellulose gel sponge contains nano copper, so that the antibacterial capability of the gel sponge can be enhanced, the bacterial growth in the wound surface and blood clots can be inhibited, the wound surface infection can be prevented, the skin healing can be effectively promoted, and the exposure time of the wound surface can be shortened.

Preferably, the calcium salt is one or more of calcium chloride, calcium bicarbonate or calcium nitrate.

Preferably, the weight ratio of the sodium alginate to the carboxymethyl cellulose is (0.7-1.4): 1.

More preferably, the weight ratio of the sodium alginate to the carboxymethyl cellulose is 1: 1.

The inventor finds that the gel sponge prepared from alginate alone is weak in strength, and the gel sponge prepared from carboxymethyl cellulose alone is loose in structure and easy to collapse. When the two are applied in combination, a synergistic effect appears, and the defects of single application can be well compensated. The gel sponge prepared by the synergism of sodium alginate and carboxymethyl cellulose has increased porosity, enhanced strength and soft texture.

Meanwhile, the inventor also finds that when the addition amounts of the sodium alginate and the carboxymethyl cellulose are lower or the proportion is unequal, the strength of the gel sponge is reduced; when the addition amount of the sodium alginate and the carboxymethyl cellulose is too large, the dissolution time of the sodium alginate and the carboxymethyl cellulose is remarkably prolonged, more lumps are generated, and the nano copper powder is easy to be unevenly distributed.

Preferably, the alginate-carboxymethyl cellulose gel sponge comprises the following components in parts by weight: 40-45 parts of sodium alginate, 40-45 parts of carboxymethyl cellulose, 10-15 parts of calcium salt and 0.1-0.15 part of nano copper powder.

More preferably, the alginate-carboxymethyl cellulose gel sponge comprises the following components in parts by weight: 42.5 parts of sodium alginate, 42.5 parts of carboxymethyl cellulose, 15 parts of calcium salt and 0.125 part of nano copper powder.

The invention also provides a preparation method of the alginate-carboxymethyl cellulose gel sponge, which comprises the following steps:

s1, dispersing sodium alginate, carboxymethyl cellulose and nano copper powder in water, and uniformly stirring to obtain a mixed solution;

s2, performing freeze casting on the mixed solution, and performing freeze drying to obtain initial sponge;

s3, adding the calcium salt water solution into the initial sponge for crosslinking, and cleaning to obtain crosslinked sponge;

s4, freeze-drying the cross-linked sponge to obtain the alginate-carboxymethyl cellulose gel sponge.

The alginate-carboxymethyl cellulose gel sponge prepared by the freeze drying technology has a porous structure, and the structure is favorable for keeping the sponge to have higher water absorption and can quickly absorb the oozing blood of a wound surface.

Preferably, the crosslinking temperature in the step S3 of the preparation method is 20-25 ℃, and the crosslinking time is 30-60 min.

The invention also protects the application of the alginate-carboxymethyl cellulose gel sponge in the wound repair material for infected bleeding.

The alginate-carboxymethyl cellulose gel sponge has high water absorption and biological safety, good hemostatic performance, antibacterial performance and healing promotion performance, can quickly absorb blood seepage, promote hemostasis and prevent wound infection when being applied to bleeding wounds, and has the effect of promoting wound healing.

Compared with the prior art, the invention has the beneficial effects that:

the invention creatively prepares the alginate-carboxymethyl cellulose gel sponge by mutually matching sodium alginate, carboxymethyl cellulose, calcium chloride and nano copper powder. The nano copper powder is used as elemental copper and can be uniformly mixed in a gel sponge system; after contacting with wound bleeding, copper ions can be slowly released, thereby effectively avoiding copper ion burst release and reducing the toxic effect of heavy metal ions. The alginate-carboxymethyl cellulose gel sponge has a porous structure, is high in strength, has a lasting antibacterial effect and biological safety, can prevent wound adhesion, and has the functions of stopping bleeding and promoting healing.

Drawings

FIG. 1 is the appearance of the alginate-carboxymethylcellulose gel sponge prepared in example 1.

FIG. 2 is the appearance of the alginate-carboxymethylcellulose gel sponge prepared in example 1 in a gel solution.

FIG. 3 is a scanning electron micrograph of the alginate-carboxymethylcellulose gel sponge prepared in example 1.

Fig. 4 is an appearance of the alginate-carboxymethylcellulose gel sponge prepared in comparative example 4.

FIG. 5 is the appearance of the alginate-carboxymethylcellulose gel sponge prepared in comparative example 4 in a gel solution.

Fig. 6 is a graph showing the results of mechanical tests on alginate-carboxymethylcellulose gel sponges prepared in example 1 and comparative example 3.

FIG. 7 is a scanning electron microscope image of the effect of alginate-carboxymethylcellulose gel sponges prepared in example 1 and comparative example 2 on the erythrocyte interface.

Fig. 8 is a graph of wound healing for treatment of SD rat skin wounds using alginate-carboxymethylcellulose gel sponges prepared in example 1 and comparative example 2, respectively.

Detailed Description

The present invention will be further described with reference to the following embodiments.

The starting materials in the examples are all commercially available, and are specified below:

sodium alginate Mecline, S817372, CP grade, viscosity 200 + -20 mpa.s;

carboxymethyl cellulose michelin, C804622, m.w.250000(DS ═ 0.7), 1500-;

anhydrous calcium chloride mcrine, C805225, AR grade, 96%;

the nano copper powder Michael, C804490, has an average particle size of 10-30 nm.

Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Examples 1 to 9

Examples 1 to 9 each provide an alginate-carboxymethylcellulose gel sponge, and the amounts of the components of the alginate-carboxymethylcellulose gel sponge added are shown in table 1.

TABLE 1 addition amount (in parts by weight) of each component in examples 1 to 9

Examples 1-9 methods for preparing Zhonghai alginate-carboxymethyl cellulose gel sponges are all as follows:

s1, weighing sodium alginate, carboxymethyl cellulose and nano copper powder, adding the sodium alginate, carboxymethyl cellulose and nano copper powder into 40mL of water, and fully stirring and uniformly mixing to obtain a mixed solution A;

s2, weighing calcium chloride, adding the calcium chloride into 40mL of water, and fully and uniformly stirring to obtain a mixed solution B;

s3, pouring the mixed solution A obtained in the step S1 into a mold, enabling the thickness of the solution to be 0.6mm, ensuring that the mold is in a horizontal position, freezing the mold at-80 ℃ for 8 hours, and then carrying out vacuum freeze drying for 48 hours to obtain initial sponge;

s4, adding the homogeneous liquid B in the step S2 into the initial sponge, crosslinking for 60min at 25 ℃, and cleaning with deionized water to obtain crosslinked sponge;

s5, freezing the cross-linked sponge obtained in the step S4 at-80 ℃ for 8h, and then carrying out vacuum freeze drying for 24h to obtain the alginate-carboxymethyl cellulose gel sponge.

Comparative example 1

Comparative example 1 provides an alginate-carboxymethylcellulose gel sponge, which is different from example 1 in that the alginate-carboxymethylcellulose gel sponge copper nanoparticles of comparative example 1 are added in an amount of 0.35 parts.

The addition amounts of the components and the preparation method of the gel sponge except for the nano copper powder were the same as those of example 1.

Comparative example 2

Comparative example 2 provides an alginate-carboxymethylcellulose gel sponge, which is different from example 1 in that no copper nanoparticles are added to the alginate-carboxymethylcellulose gel sponge of comparative example 2.

Comparative example 3

Comparative example 3 provides an alginate-carboxymethylcellulose gel sponge, which is different from example 1 in that sodium alginate and carboxymethylcellulose are added in an amount of 25 parts each in the alginate-carboxymethylcellulose gel sponge of comparative example 3.

The addition amounts of the components and the preparation method of the gel sponge are the same as those in example 1 except for sodium alginate and carboxymethyl cellulose.

Comparative example 4

Comparative example 4 provides an alginate-carboxymethylcellulose gel sponge, which is different from example 1 in that the alginate-carboxymethylcellulose gel sponge of comparative example 4 uses copper sulfate or the like in place of the nano-copper powder by weight.

Application testing

The alginate-carboxymethylcellulose gel sponges prepared in the examples and comparative examples were tested for use according to the following test methods:

cell proliferation activity: mixing alginate-carboxymethyl cellulose gel sponge with 10⁴Co-culturing individual umbilical vein endothelial cells (HUVEC), sucking out culture medium in 1, 4 and 7 days respectively, adding 9.09% CCK-8 solution into the sponge group and the sponge-free group (blank group), incubating for 1h in dark, placing the reaction solution into an enzyme labeling instrument, and measuring the absorbance at 450 nm;

the hemolysis rate: respectively co-culturing alginate-carboxymethyl cellulose gel sponge, phosphate buffer solution (negative control group) and pure water (positive control group) with diluted normal adult blood for 1h, centrifuging at high speed, taking out supernatant, placing the supernatant into an enzyme-labeling instrument, and measuring absorbance at 545 nm;

the antibacterial rate is as follows: mixing alginate-carboxymethyl cellulose gel sponge with 10⁶Coculture of CFU with Staphylococcus aureus for 24h, and spongeless 10⁶Culturing Staphylococcus aureus (blank group) of CFU for 24h, diluting by 100 times, plating, and counting colonies of sponge group and sponge-free group after bacteria culture for 24 h;

alginate-carboxymethylcellulose gel sponge structure scanning electron microscope: firstly, adhering a conductive adhesive to a sample table, then respectively placing gel sponges on the conductive adhesive and adhering the gel sponges to the conductive adhesive, then adhering the conductive adhesive to a part of the upper surface of a material, adhering the conductive adhesive to a bottom sample table along the side wall of a sample, then carrying out vacuum gold spraying treatment on the surface of the sample, and observing the sample by using a scanning electron microscope; the scanning electron microscope equipment is Nova NanoSEM 430;

alginate-carboxymethylcellulose gel sponge acts on erythrocyte interface: placing alginate-carboxymethyl cellulose gel sponge in PBS buffer solution for 1h, adding normal adult blood into PBS buffer solution, incubating for 5min, washing sponge with PBS buffer solution, removing non-adhered red blood cells, fixing, dehydrating, drying, and observing interface action of red blood cells and gel sponge with scanning electron microscope.

Gel sponge strength: the sponge to be tested is cut into proper size according to the requirements of testing electronic universal testing machine (nonmetal direction, model: CMT6103) equipment, placed on a testing platform, compressed at the speed of 1mm/min, and tested for the compressive stress when the sponge is damaged.

Test results (I) alginate-carboxymethylcellulose gel sponge appearance characterization

The alginate-carboxymethylcellulose gel sponge prepared in example 1 is shown in fig. 1 in appearance, the gel solution without lyophilization is shown in fig. 2 in appearance, and the scanning electron microscope picture is shown in fig. 3 in image. It can be seen that the gel solution can be kept in a liquid state all the time when the gel sponge is not freeze-dried, the nano copper powder is uniformly distributed in the solution, and instantaneous crosslinking does not occur; the scanning electron microscope image shows that the alginate-carboxymethyl cellulose gel sponge prepared in example 1 has a porous structure, and the distribution of the porous structure is uniform.

The alginate-carboxymethylcellulose gel sponge containing copper ions prepared in comparative example 4 is shown in fig. 4, the gel solution without lyophilization is shown in fig. 5, and copper ions are represented as dark colors in the gel sponge. It can be seen that, in the preparation process, the copper ions are instantaneously crosslinked with the sodium alginate and the carboxymethyl cellulose, so that the copper ions cannot be uniformly distributed, and the gel sponge is not uniformly formed. Due to the fact that the concentration of copper ions in each part of the gel sponge is uneven, local too high metal ion concentration is easy to occur, and cytotoxicity is caused.

(di) alginate-carboxymethylcellulose gel sponge strength test

The results of mechanical property tests on the alginate-carboxymethylcellulose gel sponges prepared in example 1 and comparative example 3 are shown in fig. 6.

In fig. 6, the ordinate represents the compressive stress and the abscissa represents the deformation, and the gel sponge prepared in example 1 requires a larger stress under the same deformation. When a deformation of 60% was reached, the gel sponge prepared in example 1 required a stress of 0.195MPa, while the gel sponge in comparative example 3 required only 0.048 MPa. This indicates that the gel sponge prepared in comparative example 3 has poor strength.

Effect of (tri) alginate-carboxymethylcellulose gel sponge on erythrocytes

FIG. 7 is a scanning electron microscope image of the effect of alginate-carboxymethyl cellulose antibacterial hemostatic gel sponge prepared in example 1 and comparative example 2 of the present invention on erythrocyte interface, and the arrows cause the aggregation and overlapping of erythrocytes. It can be seen that the gel sponge prepared in example 1 significantly promoted the aggregation of red blood cells, compared to the gel sponge of comparative example 2 containing no copper.

(IV) alginate-carboxymethyl cellulose gel sponge for healing wound

A rat skin defect model was made, and the mouse skin wounds were treated with the alginate-carboxymethyl cellulose antibacterial hemostatic gel sponges prepared in example 1 and comparative example 2, respectively, and wound healing was observed on days 1, 7, and 14. The test results are shown in fig. 8.

By comparison, it can be seen that the wound surface healed faster with the gel sponge of example 1 compared to a gel sponge without copper. The nano copper powder added into the gel sponge can effectively promote wound healing.

(V) detection of cytotoxicity, hemolysis and antibacterial property of alginate-carboxymethylcellulose gel sponge

The alginate-carboxymethyl cellulose gel sponges prepared in examples 1 to 9 and comparative examples 1 to 3 were subjected to cytotoxicity, hemolytic rate and antibacterial rate detection, and the cytotoxicity is represented by cell proliferation activity, and the detection results are shown in table 2.

TABLE 2 Performance test results for examples 1-9 and comparative examples 1-3

As can be seen from table 2, each of the gel sponges in examples 1 to 9 has good cell proliferation activity, which indicates that the gel sponges prepared in the examples of the present application have no significant cytotoxicity; the hemolysis rate is less than or equal to 2.0 percent, which indicates that the gel sponge prepared by the embodiments of the application has no obvious hemolysis; the antibacterial rate is not less than 97.58, and the antibacterial agent has excellent antibacterial performance. The gel sponges prepared in examples 1-9 had good stability of the drug effect within 7 days.

In examples 1 to 9, the gel sponge prepared in example 1 has excellent antibacterial property, and the antibacterial rate is 99.64%; low hemolytic property, the hemolytic rate is 1.56%; meanwhile, the cell proliferation activity D1-D7 is at a higher level, and the cell proliferation activity reaches 98.34% in the seventh day, which indicates that the biological safety is very good.

The cell proliferation activities of the gel sponge of comparative example 1 on days 1, 4 and 7 were 67.43%, 55.91% and 45.59%, respectively, and the cell proliferation activities were decreased with the lapse of time, indicating that the gel sponge was cytotoxic and could not be used for wound healing in medical treatment. The antibacterial rate of the gel sponge of comparative example 2 was only 0.18%, and it had almost no antibacterial property. The gel sponge of comparative example 3 has good cell proliferation activity, hemolysis rate and antibacterial rate, but the gel sponge has poor strength, loose structure and easy collapse as shown by the strength test of the gel sponge.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The alginate-carboxymethyl cellulose gel sponge is characterized by comprising the following components in parts by weight: 30-50 parts of sodium alginate, 30-50 parts of carboxymethyl cellulose, 10-20 parts of calcium salt and 0.1-0.3 part of nano copper powder.

2. The alginate-carboxymethylcellulose gel sponge of claim 1, wherein the average particle size of the copper nanopowder is 10-30 nm.

3. The alginate-carboxymethylcellulose gel sponge of claim 1, wherein the calcium salt is one or more of calcium chloride, calcium bicarbonate or calcium nitrate.

4. The alginate-carboxymethylcellulose gel sponge of claim 1, wherein the weight ratio of sodium alginate to carboxymethylcellulose is (0.7-1.4): 1.

5. The alginate-carboxymethylcellulose gel sponge of claim 4, wherein the weight ratio of sodium alginate to carboxymethylcellulose is 1: 1.

6. The alginate-carboxymethylcellulose gel sponge of claim 1, comprising the following components in parts by weight: 40-45 parts of sodium alginate, 40-45 parts of carboxymethyl cellulose, 10-15 parts of calcium salt and 0.1-0.15 part of nano copper powder.

7. The alginate-carboxymethylcellulose gel sponge of claim 1, comprising the following components in parts by weight: 42.5 parts of sodium alginate, 42.5 parts of carboxymethyl cellulose, 15 parts of calcium salt and 0.125 part of nano copper powder.

8. The method of preparing the alginate-carboxymethylcellulose gel sponge of any one of claims 1-7, comprising the steps of:

9. The method according to claim 8, wherein the crosslinking temperature in S3 is 20-25 ℃ and the crosslinking time is 30-60 min.

10. Use of the alginate-carboxymethylcellulose gel sponge of any one of claims 1-7 in wound repair materials for infected bleeding.