CN114681656B

CN114681656B - Antibacterial repair absorbable composite polysaccharide biological material and preparation method and application thereof

Info

Publication number: CN114681656B
Application number: CN202011633452.5A
Authority: CN
Inventors: 宋德利; 郭梓炀; 梁阿辉; 郑雯; 曲洪媛; 赵芳; 吴昌琳
Original assignee: SUZHOU BOCHUANG TONGKANG BIOLOGICAL ENGINEERING CO LTD
Current assignee: SUZHOU BOCHUANG TONGKANG BIOLOGICAL ENGINEERING CO LTD
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2023-07-07
Anticipated expiration: 2040-12-31
Also published as: CN114681656A

Abstract

The invention provides an antibacterial repair absorbable composite polysaccharide biological material, a preparation method and application thereof. The absorbable composite polysaccharide biological material comprises denatured polysaccharide and an efficacy component; the efficacy component comprises polylysine and/or collagen. According to the invention, the modified polysaccharide, the polylysine and the collagen are compounded in a physical way to form polyelectrolyte, the obtained biological material has a porous network structure, has good water absorbability, and obviously improves liquid absorbability and liquid absorption rate, and the modified polysaccharide, the polylysine and the collagen are matched in a synergistic way and promoted mutually along with the addition of the polylysine and the collagen, so that the biological material also has good biocompatibility, antibacterial capability, repair capability, tissue filling capability, tissue supporting capability and wound healing promotion capability.

Description

Antibacterial repair absorbable composite polysaccharide biological material and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a medical biological material, and particularly relates to an antibacterial repair absorbable composite polysaccharide biological material, and a preparation method and application thereof.

Background

Arterial bleeding, organ damage (e.g., bleeding of liver, spleen, etc.) during surgery and accidental trauma can seriously endanger the life of the patient if not handled in time, and therefore, good biological means are critical to the success of the surgery. Development of degradable and absorbable biological materials which are safe, effective, good in biocompatibility, capable of promoting wound healing, resistant to infection, convenient to use and low in cost is extremely important for improving the success rate of operations.

The biological materials currently in use and reported mainly include: (1) Biological gums, such as fibrin glue and synthetic glue; (2) Biological sponges such as gelatin sponge, collagen sponge, chitosan biological sponge carboxymethyl cellulose biological sponge, thrombin-and fibrin-containing biological sponge, etc.; (3) Polysaccharide biological powder such as chitosan biological powder, starch type polysaccharide biological powder, algae biological powder, etc.; (4) Biological gauze or biological membranes, such as oxidized cellulose and oxidized regenerated cellulose biological membranes, oxidized cellulose biological gauze containing carboxymethyl cellulose, and the like. Different materials have different biological mechanisms and corresponding advantages and limitations, and in clinical application, various factors including bleeding sites, wound shapes, wound packing requirements, compatibility of the materials with the body and the like are considered.

Starch is a kind of glucan, generally insoluble in water, non-water-absorbing or difficult to absorb at normal temperature, and natural starch swells in hot water at 60 ℃ or higher to become a semitransparent colloidal solution with viscosity. The raw starch is processed to have isomerism, and new chemical and physical characteristics are obtained, so that the modified starch is obtained. Starch is classified into potato starch, corn starch and the like according to sources, contains amylose and amylopectin, has a particle size of 1-100 μm and an average diameter of 15-30 μm. The starch is widely used as a source, can be extracted from corn, potato, cassava, sweet potato, kudzuvine root and other plants, has low price and good biocompatibility and degradability. In recent years, starch is used as a raw material, so that adverse reactions caused by animal-derived material hybrid proteins can be avoided, glucose is easily catalyzed and hydrolyzed by human body enzymes, and is absorbed and metabolized by human bodies, and the method is an ideal raw material for preparing absorbable biological materials.

For example, CN106822984a discloses a degradable absorbing antibacterial biological composition with strong biocompatibility, tissue adhesion prevention and wound healing promotion and application thereof, which is prepared by taking amylopectin, sodium carboxymethyl starch, epsilon-polylysine and chitosan as main raw materials, optimizing a certain proportion, mixing for a period of time, filtering, drying and sterilizing. The invention has the defect that chitosan is added, but the enzyme for rapidly and effectively degrading chitosan is lacking in a human body, and the chitosan cannot be used in a surgical operation. In addition, the application of chitosan and other animal-derived materials to human bodies can increase the risks of virus transmission, immunogenicity and the like, and has more strict requirements on the safety evaluation of animal-derived medical devices.

CN110115776a discloses an absorbable starch microsphere biological powder with antibacterial activity and application thereof, wherein soluble starch and sodium trimetaphosphate are used as raw materials, and starch and sodium trimetaphosphate are reversely emulsified and crosslinked to form a three-dimensional network structure, so that the soluble starch forms a microporous structure, the water absorption of the whole starch microsphere biological powder is enhanced, and the biological effect of using single soluble starch is improved. However, the gel formed by the soluble starch which is not subjected to ionic crosslinking is reversible in the solution, so that the biological microspheres formed by the emulsifier are poor in integrity and uneven in dispersion, adhesion is easy to occur among particles, the particle formation of the biological powder microspheres is affected, and the addition of sodium trimetaphosphate is accompanied with the residue of an organic solvent, so that the safety of the biological powder is affected to a certain extent.

CN105770969a discloses an absorbable starch biological powder, which is prepared by mixing plant raw starch with carboxymethyl starch after microwave treatment, absolute ethyl alcohol washing and drying, emulsifying and dispersing span 80, tween 80 and vegetable oil, crosslinking sodium trimetaphosphate, extracting and separating liquid with ethyl acetate, and the like. The biological powder is added with carboxymethyl starch, and can quickly form gel after absorbing plasma in blood, so that damaged tissues and blood vessels can be effectively plugged, the blood concentration is improved, the aggregation of blood platelets is accelerated, and the living beings are promoted. However, due to the addition of the crosslinking agent and the modifier, the biological safety is inferior to that of pure starch-based raw materials by dissolution of the organic solvent, and the biological powder has no antibacterial property.

The natural raw starch is in powder form, and is small in particles and light in particles, so that the natural raw starch is sprayed on the surface of bleeding blood surface and often floats on the surface of bleeding blood, and when the natural raw starch is subjected to blood, denatured starch is often agglomerated, and the natural raw starch is not easy to permeate into bleeding parts, particularly for active bleeding, the biological purpose is difficult to achieve. Thus, researchers have modified starches to prepare polysaccharide biopowders from modified starches, including concentrated specific modified starches such as cationic starches, epichlorohydrin crosslinked starches, carboxymethyl starches, hydroxyethyl starches, pregelatinized modified starches, pregelatinized hydroxypropyl distarch phosphate, acrylic acid-carboxymethyl starch graft copolymers, and the like. When the cationic starch is used as a biological material, the cationic starch is used for attracting negatively charged red blood cells due to positive charges on the surface of the cationic starch and is interacted with the negatively charged red blood cells, so that the coagulation process is accelerated; on the other hand, the positively charged modified starch can be tightly adhered to tissues after contacting with blood, and the wound is closed, so that the hemostasis is rapid.

US6060461 discloses a technique called Arista ^TM The active ingredient of the absorbable biological material is crosslinked and denatured starch-based microporous polysaccharide, including glucan. The microporous polysaccharide is prepared by extracting purified natural plant starch, is powder-like and biocompatible, and does not contain any animal or human components, so that allergy risk can be avoided. Arista (Arista) ^TM The absorbable biological material is prepared by crosslinking soluble starch and epichlorohydrine, and the epichlorohydrine with hydroxyl reacts with starch molecules to generate ethyl glycerol, so that the starch molecules can be crosslinked into a three-dimensional network structure. Arista (Arista) ^TM Biological powders, while having good biological effects, still present some problems themselves. Epichlorohydrin is added in the preparation process, is colorless oily liquid, has pungent smell like ether and chloroform, has toxicity and anesthetic property, has safety risk and has higher cost; in addition, the material has the advantages of insufficient water absorption, low water absorption multiple, slower water absorption rate and influence on biological effect.

In addition, du Baotang and the like carry out gelatinization, enzymolysis, emulsification crosslinking and other treatments on raw potato starch to prepare the MMPH porous microsphere, the process is complex, the prepared starch microsphere has slower water absorption rate and poor adhesiveness, and the biological effect on large-area blood permeation is poor (see medical and health equipment, 2014, 35 (3): 23-25 for details).

The existing biological products have the defects of low liquid absorption rate, incapability of achieving good biological effects and the like in clinical application, and wound infection cannot be avoided; in addition, the product is added with a cross-linking agent, and has potential toxicity. In surgical operation, most of the wounds and infections always occur simultaneously, and the incidence of infection after the wounds is extremely high due to the influence of factors such as wound environment, and the subsequent treatment not only greatly increases the medical cost of patients, but also causes operation failure and death for serious patients.

Therefore, a biological material which has antibacterial property, can be fast biological, has strong adhesiveness, has positive effect on infection, good biocompatibility and degradability and absorbability, and has important significance for clinical application is developed.

Disclosure of Invention

In view of the problems existing in the prior art, the invention aims to provide an antibacterial and repairable absorbable composite polysaccharide biological material, and a preparation method and application thereof. The absorbable composite polysaccharide biological material has excellent biological effect, good biocompatibility, antibacterial effect and capability of promoting tissue growth by mixing the modified polysaccharide with the polylysine solution or collagen or the polylysine and the collagen solution and cooperatively matching the modified polysaccharide with the polylysine solution or the collagen solution or the polylysine and the collagen solution.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an antimicrobial repair absorbable composite polysaccharide biomaterial comprising a denatured polysaccharide and an efficacy component; the efficacy component comprises polylysine and/or collagen.

The absorbable composite polysaccharide biological material obtained by mixing polylysine and/or collagen with denatured polysaccharide has rich loose structure and good water absorbability, and can quickly form gel after absorbing plasma in blood, thereby effectively blocking damaged tissues and blood vessels, improving blood concentration, accelerating the aggregation of blood platelets and promoting biology. Therefore, the absorbable composite polysaccharide biomaterial has good liquid absorption, liquid absorption rate, adhesiveness and healing promotion capability.

When only polylysine is mixed with modified polysaccharide, the obtained material has slightly bad repairability but good antibacterial effect, and when only collagen is mixed with modified polysaccharide, the obtained material has good repairability but bad antibacterial property, so that the polylysine, the modified polysaccharide and the collagen are reasonably proportioned and mutually cooperated to obtain the biological material with good antibacterial repairability.

Preferably, the absorbable composite polysaccharide biomaterial comprises the following components in percentage by mass:

20 to 95 percent of modified polysaccharide

Polylysine 2-70%

Collagen 0.8-10%.

In the invention, the absorbable composite polysaccharide biological material comprises 20-95% of modified polysaccharide by mass percent, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and the like; comprises 2 to 70% of epsilon-polylysine, such as 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, etc.; comprises 0.8-10% collagen, for example, 0.8%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% collagen, etc.

Preferably, the polylysine comprises epsilon-polylysine.

Preferably, the absorbable composite polysaccharide biomaterial comprises denatured polysaccharide, epsilon-polylysine and collagen.

According to the invention, epsilon-polylysine, modified polysaccharide and collagen are mixed to prepare the biological material, so that the hydrophilicity, pasting viscosity stability, coagulation property and freeze thawing stability of the modified polysaccharide are improved, and the antibacterial performance of epsilon-polylysine is utilized, so that the biological purpose is achieved, and meanwhile, the antibacterial effect is improved; in addition, collagen is added to enable the absorbable composite polysaccharide biological material to interact with coagulation factors in the initial stage of inflammation, cause platelet aggregation, play a biological role, promote wound healing after biology, induce the activity of fibroblasts, activate and regulate the functions of different blood cells, promote the regeneration and arrangement of collagen in vivo and enhance the absorption of exudates in the middle stage of inflammation, namely the growth period of granulation tissue, form a bracket of epithelial cells in the later stage of inflammation, induce the generation and arrangement of fibroblasts and differential collagen fibers, and promote the generation of granulation tissue; promoting the formation of blood vessels and new scar tissues and completing the wound repair and healing.

As a preferred technical scheme of the invention, the modified polysaccharide comprises any one or a combination of at least two of modified starch, glycogen, cellulose, chitin, polyfructose, gelatin, hyaluronic acid or sodium alginate.

Preferably, the modified starch comprises any one or a combination of at least two of etherified modified starch, oxidized starch, esterified starch, acid modified starch or cross-linked starch.

Preferably, the etherified modified starch comprises any one or a combination of at least two of cationic starch, carboxymethyl starch or hydroxyethyl starch, preferably carboxymethyl starch.

The modified starch is added, wherein carboxymethyl starch is taken as an example, carboxymethyl starch is taken as a linear polymer, and the modified starch is etherified modified starch, and the structural formula is shown as formula I:

the carboxymethyl starch is clinically used as a blood plasma substitute, has good biocompatibility, high safety, strong water absorbability and expansibility, stable aqueous solution and excellent performance, has the functions of bonding, thickening, water retention, emulsification, suspension, dispersion and the like, and is a purely natural thickening agent and emulsifying agent.

Preferably, the cellulose comprises any one or a combination of at least two of oxidized cellulose, carboxymethyl cellulose or oxidized regenerated cellulose.

Preferably, the collagen comprises any one or a combination of at least two of type I collagen, type II collagen, or type III collagen.

Preferably, the collagen comprises recombinant human collagen.

Or, the collagen is prepared from any one of fish skin, fish gelatin or skin bone of mammal.

In the invention, the collagen comprises collagen prepared from skin bones of mammals such as cow bones, cow skins, pig skins and the like, fish skins, fish gelatin and the like as raw materials.

Preferably, the molecular weight of the collagen is 2 to 300kDa, for example, 2kDa, 20kDa, 50kDa, 80kDa, 100kDa, 120kDa, 140kDa, 150kDa, 180kDa, 200kDa, 220kDa, 250kDa or 300kDa, etc., preferably 200 to 300kDa.

As a preferable technical scheme of the invention, the absorbable composite polysaccharide biological material comprises the following components in percentage by mass:

20 to 75 percent of modified polysaccharide

Polylysine 20-70%

Collagen 0.8-10%.

30 to 70 percent of modified polysaccharide

Polylysine 25-50%

Collagen 0.8-5%.

Preferably, the mass ratio of polylysine to denatured polysaccharide is 1 (0.5-1), which may be 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7, 1:0.75, 1:0.8, 1:0.85, 1:0.9, 1:0.95 or 1:1, for example.

In the present invention, if the ratio of polylysine to denatured polysaccharide is outside this range, a higher polylysine ratio may result in a decrease in gel transparency and eventually a collapse of the network structure.

Preferably, the mass ratio of polylysine to collagen is 1 (0.02-0.06), which can be 1:0.02, 1:0.025, 1:0.03, 1:0.035, 1:0.04, 1:0.045, 1:0.05, 1:0.055 or 1:0.06, for example.

In the invention, epsilon-polylysine with molecular weight of 3600-4300 has antibacterial activity, and degradation product is one of eight amino acids necessary for human body, and the safety of epsilon-PL has been proved in mouse experiments, and has no toxicity to nerves, reproduction, embryo and immune organs, and the growth of fetuses, the growth of fetuses offspring and the development of embryo of two generations. Studies based on absorption, distribution, metabolism and excretion of epsilon-PL by rats have shown that uptake of epsilon-PL is safe.

Therefore, the epsilon-PL can be added into the absorbable composite polysaccharide biological material to have excellent anti-infection property, and can be matched with collagen to play roles in promoting coagulation, accelerating biology and promoting tissue growth.

Preferably, the molecular weight of the absorbable composite polysaccharide biomaterial is 1×10 ⁴ ～5×10 ⁶ g/mol, for example, may be 1X 10 ⁴ g/mol、2×10 ⁴ g/mol、5×10 ⁴ g/mol、1×10 ⁵ g/mol、2×10 ⁵ g/mol、5×10 ⁵ g/mol、8×10 ⁵ g/mol、1×10 ⁶ g/mol、2×10 ⁶ g/mol、5×10 ⁶ g/mol or 5X 10 ⁶ g/mol, etc.

Preferably, the particle size of the absorbable composite polysaccharide biomaterial is 10 to 800 μm, for example, 10 μm, 20 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, or the like.

In a second aspect, the present invention provides a method for preparing an absorbable composite polysaccharide biomaterial as described in the first aspect, the method comprising:

mixing polylysine water solution with modified polysaccharide, adding collagen solution, stirring, settling, pre-freezing, freeze drying, pulverizing, and sterilizing to obtain the absorbable composite polysaccharide biological material.

In the invention, the absorbable composite polysaccharide biological material is realized by epsilon-polylysine, modified polysaccharide and collagen in a physical composite mode, does not add any cross-linking agent or auxiliary agent, can directly act on a wound surface with blood, comprises organisms of tissues and organs on the body surface, in vivo and in a body cavity, can be rapidly absorbed by a human body, and has the function of viscous blocking. Meanwhile, the composite biological material is subjected to freeze-drying treatment, so that the surface of the starch particles is roughened, the specific surface area is increased, the particle structure is fluffy, and the degradation of enzymes on the starch particles in vivo is accelerated.

In a preferred embodiment of the present invention, the mixing time is 1 to 16 hours, and may be, for example, 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, or the like.

Preferably, the rotation speed during the mixing is 150 to 1000rpm, and may be, for example, 150rpm, 200rpm, 250rpm, 300rpm, 350rpm, 400rpm, 500rpm, 600rpm, 700rpm, 800rpm, 900rpm, 1000rpm, or the like.

Preferably, the collagen solution is an acetic acid solution containing collagen.

Preferably, the mass fraction of collagen in the collagen solution is 0.4% -10%, for example, 0.4%, 0.8%, 1%, 1.5%, 2%, 3%, 5%, 6%, 7%, 8% or 10% or the like.

Preferably, the stirring time is 1 to 10 hours, and may be, for example, 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or the like.

Preferably, the rotation speed during stirring is 300 to 750rpm, and may be, for example, 300rpm, 350rpm, 400rpm, 450rpm, 500rpm, 550rpm, 600rpm, 650rpm, 700rpm, 750rpm, or the like.

As a preferable technical scheme of the invention, the prefreezing temperature is-80 to-20 ℃, and can be-80 ℃, -70 ℃, -60 ℃, -50 ℃, -40 ℃, -30 ℃, -25 ℃ or-20 ℃ and the like.

Preferably, the pre-freezing time is 2-48 h, and can be 2h, 4h, 5h, 8h, 10h, 12h, 16h, 20h, 25h, 30h, 35h, 40h, 42h, 45h or 48h, for example.

Preferably, the freeze-drying step is followed by a step of shaping the resulting material, i.e. the material can be made into a corresponding form after freeze-drying.

Preferably, the method of sterilization comprises irradiation sterilization.

Preferably, the irradiation dose of the irradiation sterilization is 10 to 25kGy, and may be, for example, 10kGy, 12kGy, 15kGy, 18kGy, 20kGy, 22kGy, 24kGy, 25kGy, or the like.

As a preferable technical scheme of the invention, the preparation method comprises the following steps:

(1) Dissolving epsilon-polylysine in water, and stirring for 1-16 h to obtain epsilon-polylysine water solution;

(2) Mixing the modified polysaccharide with the epsilon-polylysine water solution, and stirring for 1-10 h to obtain a mixed solution;

(3) Dissolving collagen in acetic acid solution with the volume fraction of 0.1-5%, and stirring until the collagen is dissolved to obtain collagen solution with the mass fraction of 0.4-10%;

(4) Mixing the mixed solution obtained in the step (2) with the collagen solution, and stirring;

(5) And (3) performing precipitation, pre-freezing at-80 to-20 ℃ for 2-48 hours, freeze drying, forming and packaging on the solution obtained in the step (4), and performing radiation sterilization at the radiation dose of 10-25 kGy to obtain the absorbable composite polysaccharide biological material.

In a third aspect, the present invention also provides the use of an absorbable composite polysaccharide biomaterial as described in the first aspect in the preparation of a medical biomaterial;

preferably, the medical biomaterial comprises any one of a particle, gel, sponge, patch or film; preferably, the medical biomaterial comprises any one of hemostatic powder, hemostatic gel, hemostatic sponge, hemostatic membrane, hemostatic patch, repair membrane or repair patch.

The medical biological material can be gel, and the preparation of the medical biological material can be obtained by diluting, swelling and dissolving powder in other liquid which is not limited by water according to a certain proportion;

the medical biological material can also be hemostatic sponge, hemostatic membrane, hemostatic patch, repair membrane and repair patch, wherein the sponge, patch and membrane can be made into membrane or layer, or can be columnar, sheet, block or flocculent.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The antibacterial repair absorbable composite polysaccharide biological material is realized by epsilon-polylysine, modified polysaccharide and collagen in a physical composite mode, and no cross-linking agent or auxiliary agent is added; the inside of the porous material has rich loose structure and good water absorbability, and after the material absorbs plasma in blood, gel can be quickly formed, damaged tissues and blood vessels can be effectively plugged, meanwhile, the blood concentration is improved, the aggregation of blood platelets is accelerated, and the living things are promoted; meanwhile, the absorbable composite polysaccharide biological material also has antibacterial effect and wound healing promoting capability;

(2) In the preparation method provided by the invention, the composite biological material is subjected to freeze-drying treatment, so that the surface of starch particles becomes rough, the specific surface area is increased, the particle structure becomes fluffy, and the degradation of enzymes on the starch particles in vivo is accelerated; the preparation method is high in operability, simple to manufacture and suitable for mass production;

(3) The absorbable composite polysaccharide biological material provided by the invention has antibacterial property and tissue healing promoting capability, can be used for general bleeding, deep bleeding and difficult-to-reach parts of various surgical operations, and is convenient to use; and after the organisms are finished, the biological agent can be degraded and absorbed by human bodies or can be directly cleaned by normal saline or pure water, has no residue and no damage to wound surfaces, and has good market prospect.

Drawings

FIG. 1 is a scanning electron microscope (scale 100 μm) of the absorbable composite polysaccharide biomaterial prepared in example 1.

FIG. 2 is a scanning electron microscope (scale 2 μm) of the absorbable composite polysaccharide biomaterial prepared in example 1.

Detailed Description

The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present invention and do not represent or limit the scope of the invention, which is defined by the claims.

In the following examples, epsilon-polylysine was used as available from Zhejiang New silver image bioengineering Co., ltd, with CAS number 25104-18-1;

the carboxymethyl starch is purchased from Anhui mountain river pharmaceutic adjuvant Co., ltd, and the CAS number is 9063-38-1;

the sodium carboxymethyl cellulose is purchased from Anhui mountain river pharmaceutic adjuvant Co., ltd, and the CAS number is 9004-32-4;

the collagen used is purchased from Tianjin Xeronine bioengineering technology Co., ltd, and has CAS number of 9064-67-9;

the remaining materials and reagents, without specific description, are available from manufacturers of reagents conventional in the art.

Example 1

The embodiment provides an antibacterial repair absorbable composite polysaccharide biological material, which is prepared from the following raw materials:

component (A)	Quality (g)	Mass fraction (%)
			Epsilon-polylysine	2.5	33
Carboxymethyl starch	5	66
			Collagen	0.05	1

The preparation method comprises the following steps:

(1) Adding epsilon-polylysine into 100mL of distilled water, and uniformly stirring to prepare solution A;

(2) Adding carboxymethyl starch into the solution A, and stirring to prepare solution B;

(3) Adding collagen into acetic acid solution with the volume percentage of 3% to prepare collagen solution with the mass percentage of 0.5mg/mL, and stirring until the collagen is dissolved to obtain solution C:

(4) Adding the solution C into the solution B, stirring to obtain solution D, precipitating, placing into a freeze dryer, and performing ultralow temperature prefreezing and vacuum freeze drying to obtain degradable starch biological material;

(5) Crushing, sieving and packaging: drying the freeze-dried product for preparing the biological powder, sieving by a 500-mesh sieve, and packaging;

(6) And (3) sterilization: and (3) sterilizing the packaged material by adopting radiation, wherein the radiation dose is 25kGy, and obtaining the antibacterial absorbable composite polysaccharide biological material.

The form of the absorbable composite polysaccharide biological material prepared in the embodiment is detected and analyzed by a scanning electron microscope, the obtained results are shown in fig. 1 and 2, wherein fig. 1 is a view field picture under 100 μm, and fig. 2 is a view field picture under 2 μm.

Example 2

component (A)	Quality (g)	Mass fraction (%)
			Epsilon-polylysine	5	49.5
Carboxymethyl starch	5	49.5
			Collagen	0.1	1

The preparation method comprises the following steps:

(2) Weighing carboxymethyl starch, adding into the solution A, and stirring to prepare solution B;

(3) Adding collagen into acetic acid solution with the volume percentage of 3% to prepare collagen solution with the mass percentage of 0.1mg/mL, and stirring until the collagen is dissolved to obtain solution C:

(4) Adding the solution C into the solution B, stirring for a period of time to obtain solution D, precipitating, placing into a freeze dryer, pre-freezing, and sublimating and drying to obtain the degradable starch biological material.

(5) Crushing, sieving and packaging: drying the freeze-dried product for preparing the biological powder, crushing, screening and packaging;

Example 3

component (A)	Quality (g)	Mass fraction (%)
			Epsilon-polylysine	5	41.3
Carboxymethyl starch	7	57.9
			Collagen	0.1	0.8

The preparation method comprises the following steps:

(2) 7g of carboxymethyl starch is weighed and added into the solution A, and the solution B is prepared by stirring;

(3) Adding collagen into acetic acid solution with the volume percentage of 3 percent to prepare collagen solution with the mass percentage of 0.5mg/mL, and stirring until the collagen is dissolved to obtain solution C:

(6) And (3) sterilization: and (3) sterilizing the packaged material by adopting radiation with the radiation dose of 15kGy to obtain the antibacterial absorbable composite polysaccharide biological material.

Example 4

the preparation method comprises the following steps:

(6) And (3) sterilization: and (3) sterilizing the packaged material by adopting irradiation with the irradiation dose of 20kGy to obtain the antibacterial absorbable composite polysaccharide biological material.

Example 5

The present example provides an antibacterial restorable absorbable composite polysaccharide biomaterial, which is different from example 1 in that the carboxymethyl starch is replaced with carboxymethyl cellulose (manufactured by Anhui mountain river pharmaceutical excipients Co., ltd.); the content of each component and the preparation method were the same as in example 1.

Example 6

The present example provides an antibacterial restorable absorbable composite polysaccharide biomaterial, differing from example 1 in that the mass of epsilon-polylysine is 5g, and the mass of carboxymethyl starch is 2.5g;

the remaining component contents and preparation method were the same as in example 1.

Example 7

The present example provides an antibacterial restorable absorbable composite polysaccharide biomaterial, differing from example 1 in that the mass of epsilon-polylysine is 5.5g, and the mass of carboxymethyl starch is 2g;

Example 8

The present example provides an antibacterial restorable absorbable composite polysaccharide biomaterial, differing from example 1 in that the epsilon-polylysine has a mass of 2g and collagen has a mass of 0.55g;

Example 9

The present example provides an antibacterial repair absorbable composite polysaccharide biomaterial, which is different from example 1 in that the preparation method of the absorbable composite polysaccharide biomaterial is that the step (4) does not adopt prefreezing and vacuum freeze drying methods, but adopts a vacuum drying method.

Comparative example 1

This comparative example provides an absorbable complex polysaccharide biomaterial, i.e., commercially available Arista ^TM Biological powder.

Comparative example 2

This comparative example provides an absorbable composite polysaccharide biomaterial, differing from example 1 in that epsilon-polylysine is not contained therein, and the carboxymethyl starch content is increased to 7.5g; the remaining components were identical to the preparation method as in example 1.

Comparative example 3

This comparative example provides an absorbable composite polysaccharide biomaterial, differing from example 1 in that it does not contain collagen and increases the carboxymethyl starch content to 5.1g; the remaining components were identical to the preparation method as in example 1.

Comparative example 4

This comparative example provides an absorbable composite polysaccharide biomaterial, differing from example 1 in that carboxymethyl starch was replaced with potato starch (Anhui mountain river pharmaceutical excipients Co., ltd.); the remaining components were identical to the preparation method as in example 1.

Comparative example 5

This comparative example provides an absorbable composite polysaccharide biomaterial, differing from example 1 in that epsilon-polylysine was replaced with chitosan (Anhui Shanhe pharmaceutical excipients Co., ltd.); the remaining components were identical to the preparation method as in example 1.

Performance test 1 cytotoxicity test

According to GB/T16886.5-2016 medical device biological evaluation part 5: in vitro cytotoxicity test MTT colorimetric method cytotoxicity detection was carried out on the biological materials prepared in examples 1 to 9 and comparative examples 1 to 5.

The method comprises the following specific steps:

(1) Inoculating the prepared L929 fibroblast suspension into a culture plate, culturing for 24 hours, and removing the supernatant;

(2) The positive control group was added with the biomaterials provided in examples 1 to 9; adding the biological materials provided in comparative examples 1 to 5 into a negative control group; adding the same amount of fresh cell culture solution into a blank control group;

(3) Culturing was continued for 72 hours, and the relative proliferation rate of cells was calculated by observing the cell morphology. The results obtained are shown in Table 1:

TABLE 1

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As shown in the table, the absorbable composite polysaccharide biological material biological powder provided by the invention has small cytotoxicity, and the relative cell proliferation rate is 68.9-110.5%, so that the absorbable composite polysaccharide biological material biological powder meets the clinical use requirement.

Performance test 2 Water absorption Rate comparative test

The absorbable composite polysaccharide biomaterials prepared in examples 1 to 9 and comparative examples 1 to 5 were used for comparison test of water absorption rate and water absorption rate, and were measured by a natural filtration method.

The results obtained are shown in table 2 below:

TABLE 2

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As can be seen from the table, the absorbable composite polysaccharide biological material provided by the invention has a better water absorption rate, the water absorption rate (10 min) is 2212-4929, the water absorption rate (24 h) can reach 3654-4929 in a preferred range (examples 1-5), the water absorption rate (24 h) is 2816-5425, the water absorption rate (30 s) can reach 3756-5428 in a preferred range (examples 1-5), and the water absorption rate (30 s) is 115-212, and the water absorption rate (30 s) can reach 186-212 in a preferred range (examples 1-5).

As can be seen from comparison of example 1 and example 5, the carboxymethyl starch is replaced by carboxymethyl cellulose, and the obtained product still has better water absorption performance, so that the material has more various choices for modified polysaccharide in the invention;

as can be seen from the comparison of examples 1 and examples 6 to 8, although the absorbable composite polysaccharide biomaterials provided in examples 6 to 8 also have better water absorption capacity, they are still worse than example 1, and analyzed that the reasons are that the epsilon-polylysine, carboxymethyl starch and collagen are not in optimal proportion, so that the coordination effect between the components is affected;

as is clear from comparison of example 1 with example 9, when vacuum spray drying is employed, the lyophilization efficiency is high and the time is short, but the materials are easily adhered to the container, resulting in a decrease in the yield;

compared with the commercialized same material, the absorbable composite polysaccharide biological material provided by the invention has better water absorption rate and water absorption rate as shown in comparison of the example 1 and the comparative example 1;

as can be seen from comparison of example 1 and comparative examples 2 to 5, if epsilon-polylysine, collagen or modified polysaccharide is not added to the absorbable composite polysaccharide biomaterial or other substances are used for substitution, the water absorption effect of the absorbable composite polysaccharide biomaterial cannot be achieved, which indicates that the epsilon-polylysine, collagen and modified polysaccharide have a synergistic effect, and the absorption effect of the absorbable composite polysaccharide biomaterial on liquid is promoted.

Performance test 3 bacteriostasis test

Antibacterial tests were performed using the biological materials prepared in examples 1 to 9 and comparative examples 1 to 5, and the antibacterial properties of the samples were qualitatively evaluated according to the degree of bacterial reproduction at the contact points of the culture medium and the samples.

The results obtained are shown in Table 3 below:

TABLE 3 Table 3

As can be seen from the table, the absorbable composite polysaccharide biological material provided by the invention has antibacterial capability, the range of the antibacterial zone of the escherichia coli in the preferred range (examples 1-5) is 0.74-1.25 mm, and the range of the antibacterial zone of the staphylococcus aureus is 1.89-3.75 mm;

as can be seen from the comparison of examples 1 and examples 6 to 8, although the absorbable composite polysaccharide biomaterials provided in examples 6 to 8 also have antibacterial ability, they are still inferior to example 1, and analyzed that the ratio of epsilon-polylysine, carboxymethyl starch and collagen is not optimal, thereby affecting the cooperation between the components;

as can be seen from comparison of example 1 and comparative example 1, the absorbable composite polysaccharide biomaterial provided by the invention has antibacterial property compared with the commercialized homogeneous materials;

as is clear from comparison of example 1 and comparative examples 2 to 5, if epsilon-polylysine, collagen or modified polysaccharide is not added to the absorbable composite polysaccharide biomaterial or other substances are used for substitution, the water absorption effect described in the invention cannot be achieved, which indicates that the epsilon-polylysine, collagen and modified polysaccharide have a synergistic effect, and the antibacterial property of the absorbable composite polysaccharide biomaterial is improved.

In summary, the antibacterial absorbable composite polysaccharide biological material provided by the invention mixes the polylysine solution, the modified polysaccharide and the collagen, and the modified polysaccharide, the polylysine and the collagen are compounded to form the polyelectrolyte to form the porous network structure, and the three are cooperated with each other, so that the defect of poor mechanical property of the pure modified polysaccharide is overcome, and the defect of poor liquid absorption of starch is overcome.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims

1. The antibacterial repair absorbable composite polysaccharide biological material is characterized by comprising the following components in percentage by mass:

modified polysaccharide 33%

Epsilon-polylysine 66%

Collagen 1%;

wherein the modified polysaccharide is carboxymethyl starch, and the absorbable composite polysaccharide biological material does not comprise hyaluronic acid or polypeptide.

2. The absorbable composite polysaccharide biomaterial of claim 1, wherein the collagen comprises any one or a combination of at least two of type I collagen, type II collagen, or type III collagen.

3. The absorbable composite polysaccharide biomaterial of claim 1, wherein the collagen comprises recombinant human collagen.

4. The absorbable composite polysaccharide biomaterial of claim 1, wherein the collagen is prepared from any one of fish skin, fish gelatin, or mammalian skin bone.

5. The absorbable composite polysaccharide biomaterial of claim 1, wherein the collagen has a molecular weight of 2-300 kDa.

6. The absorbable composite polysaccharide biomaterial of claim 5, wherein the collagen has a molecular weight of 200-300 kDa.

7. The absorbable composite polysaccharide biomaterial of claim 1, wherein the molecular weight of the absorbable composite polysaccharide biomaterial is 10000-5000000 g/mol.

8. The absorbable composite polysaccharide biomaterial of claim 1, wherein the absorbable composite polysaccharide biomaterial has a particle size of 10-800 μm.

9. A method for preparing the absorbable composite polysaccharide biomaterial of any one of claims 1-8, comprising:

mixing polylysine water solution with modified polysaccharide, adding collagen solution, stirring, settling, pre-freezing, freeze drying and sterilizing to obtain the absorbable composite polysaccharide biological material.

10. The method of claim 9, wherein the mixing time is 1 to 16 hours.

11. The method according to claim 9, wherein the rotational speed during mixing is 150 to 1000 rpm.

12. The method of claim 9, wherein the collagen solution is an acetic acid solution containing collagen.

13. The preparation method according to claim 9, wherein the mass fraction of collagen in the collagen solution is 0.4% -10%.

14. The method according to claim 9, wherein the stirring time is 1 to 10 hours.

15. The method according to claim 9, wherein the rotation speed during stirring is 300 to 750 rpm.

16. The method according to claim 9, wherein the pre-frozen temperature is-80 to-20 ℃.

17. The method of claim 9, wherein the pre-freezing time is 2-48 hours.

18. The method of claim 9, further comprising the step of forming the resulting material after freeze-drying.

19. The method of claim 9, wherein the method of sterilization comprises irradiation sterilization.

20. The method of claim 19, wherein the irradiation dose for irradiation sterilization is 10-25 kGy.

21. The preparation method according to claim 9, characterized in that the preparation method comprises the steps of:

(1) Dissolving epsilon-polylysine in water, and stirring for 1-16 h to obtain epsilon-polylysine aqueous solution;

(2) Mixing the modified polysaccharide with the epsilon-polylysine water solution, and stirring for 1-10 hours to obtain a mixed solution;

(3) Dissolving collagen in an acetic acid solution with the volume fraction of 0.1% -5%, and stirring until the collagen is dissolved to obtain a collagen solution with the mass fraction of 0.4% -10%;

22. The use of an absorbable composite polysaccharide biomaterial as claimed in any one of claims 1 to 8 in the preparation of a medical biomaterial.

23. The use of claim 22, wherein the medical biomaterial comprises any one of a particle, gel, sponge, patch or film.

24. The use of claim 23, wherein the medical biomaterial comprises any one of hemostatic powder, hemostatic gel, hemostatic sponge, hemostatic film, or hemostatic patch.

25. The use of claim 23, wherein the medical biomaterial comprises any one of a repair film or a repair patch.