CN113577400A

CN113577400A - Preparation method of marine plant polysaccharide anti-adhesion material

Info

Publication number: CN113577400A
Application number: CN202110938200.1A
Authority: CN
Inventors: 王宝群; 刘桂亭; 邹圣灿; 林莎莎
Original assignee: Qingdao Chenland Marine Biotechnology Co ltd
Current assignee: Qingdao Chenland Marine Biotechnology Co ltd
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-11-02
Anticipated expiration: 2041-08-16
Also published as: CN113577400B

Abstract

The invention discloses a preparation method of a marine plant polysaccharide anti-adhesion material, belonging to the technical field of biomedical materials. The invention discloses a preparation method of marine plant polysaccharide anti-adhesion material, which comprises the steps of taking pharmaceutical adjuvant grade alginate as a material, adsorbing by active carbon, centrifuging, crosslinking supernatant by calcium ions to form gel microspheres, soaking the gel microspheres in acidic solution and alkaline solution, performing spray drying to obtain powder, filling, and sterilizing to obtain the marine plant polysaccharide anti-adhesion material. The invention has simple and safe components, simple and easy preparation process, short preparation time and good biocompatibility, and can form gel hemostasis adhesion after imbibition; the degradation time of the adhesive can be consistent with the key period of adhesion formation, the adhesion formation is prevented in the whole process, and the integral adhesion prevention effect is achieved.

Description

Preparation method of marine plant polysaccharide anti-adhesion material

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a preparation method of a marine plant polysaccharide anti-adhesion material.

Background

Tissue adhesions are prone to occur after surgery, a common clinical phenomenon in the surgical field, and also a process that patients must undergo during the healing process. Adhesions are abnormal structures formed by the connective tissue fiber band joining together adjacent tissues or organs. The size of the adhesions can range from a thin piece of film to a dense vascular scar. Postoperative tissue adhesions occur in almost any body part that is surgically treated, especially after abdominal and pelvic surgery, tendon and spinal surgery, and the like. It has been reported that abdominal and pelvic surgeries, in 50% -100%, can result in varying degrees of adhesion. If the adhesion phenomenon appears in operations of abdominal cavity, pelvic cavity, bone and the like, serious complications can be caused, such as adhesive intestinal obstruction in the abdominal cavity, the pelvic cavity and the like, recurrent laryngeal nerve injury after thyroid gland operation and female infertility caused by pelvic cavity tissue adhesion. The purpose of preventing adhesions is to eliminate or reduce the incidence and extent of adhesions while ensuring normal wound healing and avoiding infection.

At present, anti-adhesion products on the market mainly comprise sodium hyaluronate, chitosan anti-adhesion films, polylactic acid and the like, the sodium hyaluronate is easy to cause fever, and the degradation time in vivo is too short to effectively prevent the adhesion of tissues; after the chitosan is used for preventing adhesion, a patient feels uncomfortable, can feel that foreign bodies exist at an operation position, and the chitosan belongs to animal-derived materials and has the risks of immunogenicity and virus transmissibility; the polylactic acid has long degradation time, is easy to cause inflammation after being stored in a human body for a long time, and lactic acid which is one of degradation products can stimulate the organism; in addition, the above products are all used after complete hemostasis.

Therefore, the problem to be solved by the technical personnel in the field is how to provide a preparation method of an anti-adhesion material which has simple and safe components, simple and easy preparation process, short preparation time, good biocompatibility and can realize the integral anti-adhesion effect.

Disclosure of Invention

In view of the above, the invention provides a preparation method of marine plant polysaccharide anti-adhesion material, which has simple and safe components, simple and easy preparation process, short preparation time and good biocompatibility, and can form gel hemostasis adhesion after imbibition; the degradation time of the adhesive can be consistent with the key period of adhesion formation, the adhesion formation is prevented in the whole process, and the integral adhesion prevention effect is achieved.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of marine plant polysaccharide anti-adhesion material comprises the following steps:

(1) dissolving alginate in 0.3-1.0% w/w soluble sodium salt solution to make alginate solution with concentration of 2% w/w; adding activated carbon, wherein the mass ratio of the activated carbon to the alginate is 3:1, stirring for 0.5-4h at 30-50 ℃, centrifuging the mixed solution, and taking the supernatant to obtain alginate solution;

(2) adding 0.1-10% w/w calcium ions into the alginate solution obtained in the step (1) through a micro injection needle with the specification of 20G-30G under the stirring of 500-2000rpm at the temperature of 30-50 ℃ to form gel microspheres (reverse phase droplet technology), continuing stirring for 0.5-2h, centrifuging, taking out precipitates, and fully washing the gel microspheres by using distilled water;

(3) stirring and soaking the gel microspheres cleaned in the step (2) in an acid solution with the pH value of 2-4 for 1-10h, centrifuging, and fully cleaning precipitates with distilled water;

(4) placing the gel microspheres treated in the step (3) in an alkaline solution with the pH value of 9-10, soaking for 2-5h, centrifuging, and taking out a precipitate to be fully cleaned by distilled water;

(5) and (4) dispersing the gel microspheres treated in the step (4) in distilled water, drying the gel microspheres by using a spray drying method, and sterilizing to obtain the anti-adhesion material.

Further, the preparation method of the marine plant polysaccharide anti-adhesion material further comprises the step (6) of filling the dried powder obtained in the step (5) into a powder spray bottle and sterilizing.

Further, the alginate in the step (1) is one or two of sodium alginate, potassium alginate and derivatives of alginate, and the relative molecular weight of the alginate is 5000-100000.

Further, the soluble sodium salt in the step (1) is sodium chloride, sodium nitrate, sodium acetate or sodium lactate.

Further, the calcium ions in the step (2) are from calcium gluconate, calcium nitrate or calcium chloride.

Further, the centrifugation in the steps (1) to (4) is performed at 9000-15000rpm for 10-30 min.

According to the technical scheme, compared with the prior art, the preparation method of the marine plant polysaccharide anti-adhesion material disclosed by the invention has the advantages that the raw materials are simple and safe, the preparation process is simple, the operation is easy, the materials are easy to obtain, and the anti-adhesion material can be prepared in a short time. The anti-adhesion material is used as injectable dry powder, and alginate has the function of resisting cell adhesion, so that after operation, the material can be injected to a wound part to quickly absorb liquid to form gel, so that the material can play a role in three-dimensional network separation in a surgical operation, prevent postoperative tissue adhesion, stop bleeding and reduce operation in the operation. The anti-adhesion material can be gradually and completely degraded in vivo for 14-30 days, the degradation time of the anti-adhesion material is consistent with the key period of adhesion formation, the adhesion formation is prevented in the whole process, the integral anti-adhesion effect is realized, and the anti-adhesion material is a novel anti-adhesion material. The preparation of the material combines the reversed-phase liquid drop technology and the spray drying technology, and the prepared microspheres have smaller and more uniform particle size, so that the microspheres are of a microporous structure, and the final powder can quickly absorb liquid to stop bleeding to form gel to prevent adhesion; meanwhile, the combination of the spray drying technology is more beneficial to realizing industrialization and improving the productivity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the pathological analysis of HE staining according to the present invention;

wherein, A: 7 d; b: 14 d; c: 30 d.

FIG. 2 is a graph showing the effect of adhesion prevention of the adhesion-preventing material prepared in example 1 of the present invention after 14 days of operation in an adhesion model of injured abdominal wall-cecum of SD rat;

wherein, A: adhesion occurred between the injured cecum and abdominal wall of SD rats in the control group using physiological saline; 1: adhesion; b: the SD rats using the anti-adhesion powder material prepared in example 1 did not develop adhesion between the injured cecum and abdominal wall; 2: a healed abdominal wall; 3: the healed cecum.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) dissolving commercial pharmaceutic adjuvant grade sodium alginate with the molecular weight of 1 ten thousand in 0.4% w/w sodium chloride aqueous solution to prepare solution with the concentration of 2% w/w; adding activated carbon, wherein the mass ratio of the activated carbon to the sodium alginate is 3:1, stirring for 4h at 30 ℃, centrifuging the mixed solution for 30min at 9000rpm, and taking supernatant to obtain a sodium alginate solution;

(2) adding 1% w/w calcium chloride into the sodium alginate solution obtained in the step (1) through a 26G injection needle under the stirring of 500rpm at 50 ℃ to form gel microspheres, continuing stirring for 2h, centrifuging at 9000rpm for 30min, and taking out precipitate to fully wash the gel microspheres with distilled water;

(3) stirring and soaking the gel microspheres cleaned in the step (2) in a lactic acid solution with the pH value of 2 for 3h, centrifuging at 9000rpm for 15min, and fully cleaning precipitates with distilled water;

(4) soaking the gel microspheres processed in the step (3) in a sodium phosphate solution with the pH value of 10 for 2h, centrifuging at 9000rpm for 15min, and fully cleaning precipitates with distilled water;

(5) dispersing the gel microspheres treated in the step (4) in distilled water, and finally drying the gel microspheres by using a spray drying method;

(6) and (4) filling the powder dried in the step (5) into a powder spray bottle, and sterilizing to obtain the anti-adhesion sterile product.

Example 2

(1) dissolving medicinal adjuvant grade potassium alginate with the molecular weight of 3 ten thousand sold in a 0.6% w/w sodium nitrate water solution to prepare a solution with the concentration of 2% w/w; adding activated carbon, wherein the mass ratio of the activated carbon to the potassium alginate is 3:1, stirring for 3h at 40 ℃, centrifuging the mixed solution for 20min at 10000rpm, and taking supernatant to obtain a potassium alginate solution;

(2) adding 5% w/w of calcium gluconate into the potassium alginate solution obtained in the step (1) through a 20G injection needle under the stirring of 1000rpm at 50 ℃ to form gel microspheres, continuing stirring for 1.5h, centrifuging at 10000rpm for 20min, and taking out precipitates to fully wash the gel microspheres with distilled water;

(3) stirring and soaking the gel microspheres cleaned in the step (2) in a lactic acid solution with the pH value of 3 for 4 hours, centrifuging at 10000rpm for 20min, and fully cleaning precipitates with distilled water;

(4) soaking the gel microspheres processed in the step (3) in a sodium carbonate solution with the pH value of 9 for 4h, centrifuging at 10000rpm for 20min, and fully cleaning precipitates with distilled water;

Example 3

(1) dissolving sodium alginate oxide with molecular weight of 10 ten thousand in 0.9% w/w sodium lactate water solution to obtain 2% w/w solution; adding activated carbon, wherein the mass ratio of the activated carbon to the oxidized sodium alginate is 3:1, stirring at 50 ℃ for 0.5h, centrifuging the mixed solution at 12000rpm for 15min, and taking supernatant to obtain an oxidized sodium alginate solution;

(2) adding 10% w/w calcium nitrate into the oxidized sodium alginate solution obtained in the step (1) through a 24G injection needle under the stirring of 1500rpm at the temperature of 30 ℃ to form gel microspheres, continuing stirring for 1h, centrifuging at 12000rpm for 15min, and taking out precipitate to fully wash the gel microspheres with distilled water;

(3) stirring and soaking the gel microspheres cleaned in the step (2) in a lactic acid solution with the pH value of 4 for 10 hours, centrifuging at 12000rpm for 15min, and fully cleaning precipitates with distilled water;

(4) soaking the gel microspheres treated in the step (3) in a sodium monohydrogen phosphate solution with the pH value of 9 for 5h, centrifuging at 12000rpm for 15min, and fully cleaning precipitates with distilled water;

Example 4

(1) dissolving commercial pharmaceutical adjuvant grade sodium alginate with molecular weight of 6000 in 0.5% w/w sodium acetate water solution to obtain 2% w/w solution; adding activated carbon, wherein the mass ratio of the activated carbon to the sodium alginate is 3:1, stirring for 4h at 30 ℃, centrifuging the mixed solution at 15000rpm for 10min, and taking supernatant to obtain a sodium alginate solution;

(2) adding 10% w/w calcium gluconate into the sodium alginate solution obtained in step (1) through a 26G injection needle under the stirring of 1800rpm at 50 ℃ to form gel microspheres, continuing stirring for 0.5h, centrifuging at 15000rpm for 10min, and taking out the precipitate to sufficiently clean the gel microspheres with distilled water;

(3) stirring and soaking the gel microspheres cleaned in the step (2) in a lactic acid solution with the pH value of 2 for 3h, centrifuging at 15000rpm for 10min, and fully cleaning precipitates with distilled water;

(4) soaking the gel microspheres processed in the step (3) in a sodium phosphate solution with the pH value of 10 for 2h, centrifuging at 15000rpm for 10min, and fully cleaning precipitates with distilled water;

Comparative example 1

A preparation method of an anti-adhesion material comprises the following specific steps:

(1) dissolving commercially available pharmaceutical adjuvant grade potassium alginate with molecular weight of 4 ten thousand in 0.6% w/w sodium nitrate water solution to prepare solution with concentration of 2% w/w; adding activated carbon, wherein the mass ratio of the activated carbon to the potassium alginate is 3:1, stirring at 50 ℃ for 0.5h, centrifuging the mixed solution at 9000rpm for 15min, and taking supernatant to obtain potassium alginate solution;

(2) adding 10% w/w calcium nitrate into the potassium alginate solution obtained in the step (1) through a 24G injection needle under the stirring of 1000rpm at the temperature of 30 ℃ to form gel microspheres, continuing to stir for 30min, centrifuging at 9000rpm for 15min, and taking out precipitate to fully wash the gel microspheres with distilled water;

(3) stirring and soaking the gel microspheres cleaned in the step (2) in a lactic acid solution with the pH value of 4 for 10 hours, centrifuging at 9000rpm for 15min, and fully cleaning precipitates with distilled water;

(4) soaking the gel microspheres treated in the step (3) in a sodium monohydrogen phosphate solution with the pH value of 9 for 5h, centrifuging at 9000rpm for 15min, and fully cleaning precipitates with distilled water;

(5) drying the precipitate cleaned in the step (4) by adopting a vacuum drying method;

Comparative example 2

(1) dissolving commercially available pharmaceutic adjuvant grade sodium alginate with the molecular weight of 1 ten thousand in water to prepare a solution with the concentration of 2% w/w, adding activated carbon, stirring for 3 hours at 40 ℃ with the mass ratio of the activated carbon to the sodium alginate being 3:1, centrifuging the mixed solution at 9000rpm for 15min, and taking supernatant to obtain a sodium alginate solution;

(2) adding the sodium alginate solution obtained in the step (1) into 2% w/w calcium gluconate through a 20G injection needle under the stirring of 1000rpm at 50 ℃ to form gel microspheres, continuing stirring for 30min, centrifuging at 9000rpm for 15min, and taking out precipitates to fully wash the gel microspheres with distilled water;

(3) stirring and soaking the gel microspheres cleaned in the step (2) in a lactic acid solution with the pH value of 3 for 4 hours, centrifuging at 9000rpm for 15min, and fully cleaning precipitates with distilled water;

(4) soaking the gel microspheres treated in the step (3) in a sodium carbonate solution with the pH value of 9 for 4h, centrifuging at 9000rpm for 15min, and fully cleaning precipitates with distilled water;

(5) dispersing the gel microspheres treated in the step (4) in distilled water, and finally drying the gel microspheres by using a spray drying method.

As a result, the gel microspheres prepared by the method have high hardness, obvious agglomeration phenomenon and no influence on the spray drying, and the sample is always blackened, so that the next filling and sterilizing work is not carried out.

Comparative example 3

(1) dissolving commercially available pharmaceutic adjuvant grade sodium alginate with the molecular weight of 1 ten thousand in water to prepare a solution with the concentration of 2% w/w; adding activated carbon, wherein the mass ratio of the activated carbon to the sodium alginate is 3:1, stirring for 5h at 30 ℃, centrifuging the mixed solution at 9000rpm for 15min, and taking supernatant to obtain a sodium alginate solution;

(2) adding 1% w/w calcium chloride into the sodium alginate solution obtained in the step (1) through a 26G injection needle under the stirring of 1000rpm at 50 ℃ to form gel microspheres, continuing stirring for 30min, centrifuging at 9000rpm for 15min, and taking out precipitate to sufficiently clean the gel microspheres with distilled water;

Test examples

1) The main properties of the anti-blocking materials obtained in examples 1 to 4 and comparative examples 1 to 3, such as gel formation time, pH (0631 in the pharmacopoeia of the people's republic of China (2020 version)), heavy metals (2321 in the pharmacopoeia of the people's republic of China (2020 version), bacterial endotoxins (1143 in the pharmacopoeia of the people's republic of China (2020 version)) and sterility (1101 in the pharmacopoeia of the people's republic of China (2020 version)) were examined within 10 days, and the results are shown in Table 1.

TABLE 1 Main Properties (as physicochemical indices for 0 month) of anti-blocking materials obtained in examples 1 to 4 and comparative examples 1 to 3

Note: comparative examples 1 and 3 were not tested for other items due to the failure in the properties and gel formation time; in contrast, comparative example 2 was not tested for various items because filling and sterilization were not performed.

The results of comparative example 1 show that the powder meeting the requirements can be obtained only by combining the reversed phase droplet technology with the spray drying technology, and if the reversed phase droplet technology is combined with the vacuum drying technology, the gel forming time of the formed powder is obviously prolonged, and the properties are not qualified.

The results of comparative example 3 show that the direct dissolution of sodium alginate in water affects the gel formation time of the anti-blocking material, presumably due to the presence of salt which may affect its surface structure.

2) Product stability test of anti-adhesion material

The anti-adhesion materials prepared in examples 1 to 4 were stored at 40 ℃ under the accelerated stability condition of RH 60%, and the properties, pH, heavy metals, bacterial endotoxin and sterility of the preparations were examined under the accelerated conditions for significant changes, with the stability data shown in Table 2.

TABLE 2 stability test results

The results in Table 2 show that the anti-adhesion materials prepared in examples 1 to 4 have good product stability, and have no significant changes in the storage properties, pH, heavy metals, bacterial endotoxin and sterility under the accelerated stability conditions of 40 ℃ and RH 60%.

3) In vivo degradation and local reaction assay

The samples from example 1 were taken and tested by subcutaneous implantation as specified in GB/T16886.6-2015, using rabbits as the animal. No abnormality is found in the tissue structure of the implanted part of the muscle by visual observation of the test sample and the negative control. Histopathological examination showed that the samples and negative control 7d were slightly irritant, and 14d and 30d were both non-irritant. The muscle implantation 7d sample was gel-like, 14d was gel-like, the volume decreased from the previous one, and the test sample was not observed for 30d (fig. 1). The material is predicted to be gradually and completely degraded in vivo for 14-30 days, the degradation time of the material is consistent with the key period of adhesion formation, the adhesion formation is prevented in the whole process, the integral anti-adhesion effect is realized, and the material is a novel anti-adhesion material.

4) Evaluation of anti-blocking effectiveness

Anesthetizing and sterilizing SD rat as experimental animal, opening abdomen at the position of leucorrhea, and peeling 1x2 cm away from abdominal wall with ophthalmic scissors²The superficial muscle of the size, forming a bleeding surface; then rubbing the surface of the cecum corresponding to the wound surface of the abdominal wall by using an operation brush until the serosal layer of the cecum is damaged and obvious punctate bleeding occurs; then, the mesentery of the cecum is sutured and fixed at the right upper corner of the wound surface of the abdominal wall by using a 3-0 suture line so as to ensure the abdominal wall and the wound surface of the abdominal wallSufficient contact between the wounds of the cecum is enabled. Before closing the abdomen, 1.0g of the anti-adhesion powder prepared in example 1 was sprinkled on the wound surface of the abdominal wall and the cecum.

The abdomen was opened for 14 days, and the results are shown in fig. 2; as a result, it was found that SD rats using the anti-adhesion powder prepared in example 1 did not develop any form of adhesion between the injured cecum and abdominal wall, and the injured cecum and abdominal wall had completely healed; in contrast, in the control group using physiological saline, extensive and dense adhesion occurred between the injured cecum and abdominal wall of the SD rat.

Meanwhile, the anti-adhesion powder materials prepared according to the present invention as in examples 2 to 4 and others not described in detail also showed similar anti-adhesion effects in the same kind of experiments using rats, mice, new zealand white rabbits, etc. as experimental animals.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of marine plant polysaccharide anti-adhesion material is characterized by comprising the following steps:

(2) adding 0.1-10% w/w of calcium ions into the alginate solution obtained in the step (1) through a micro injection needle with specification of 20G-30G under the stirring of 500-2000rpm at 30-50 ℃ to form gel microspheres, continuing stirring for 0.5-2h, centrifuging, taking out the precipitate, and fully washing the gel microspheres with distilled water;

2. The method for preparing marine plant polysaccharide anti-adhesion material according to claim 1, further comprising the step (6) of filling the dried powder of step (5) into a powder spray bottle and sterilizing.

3. The method for preparing marine plant polysaccharide anti-adhesion material as claimed in claim 1 or 2, wherein the alginate in step (1) is one or two of sodium alginate, potassium alginate and derivatives of alginate, and the relative molecular weight of the alginate is 5000-100000.

4. The method for preparing marine plant polysaccharide anti-adhesion material according to claim 1 or 2, wherein the soluble sodium salt in step (1) is sodium chloride, sodium nitrate, sodium acetate or sodium lactate.

5. The method for preparing marine plant polysaccharide anti-adhesion material according to claim 1 or 2, wherein the calcium ions in step (2) are derived from calcium gluconate, calcium nitrate or calcium chloride.

6. The method for preparing marine plant polysaccharide anti-adhesion material as claimed in claim 1 or 2, wherein the centrifugation in steps (1) to (4) is performed at 9000-15000rpm for 10-30 min.