CN112972752A - Method for stopping bleeding by mixing frustules with different structural characteristics - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/0005—Ingredients of undetermined constitution or reaction products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0015—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
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Abstract
The invention discloses a method for improving the hemostatic performance of diatom shells by mixing diatom shells with different structural characteristics, which comprises the following steps: (1) preparing algae mud; (2) treating the algae mud by an acid-thermal method; (3) washing and drying to obtain diatom shells; (4) the diatom shells with different structural characteristics are fully mixed according to a certain proportion, and the hemostatic effect of a single diatom shell is improved. The method of the invention adopts a method of mixing various diatom shells to improve the hemostatic performance, does not need special chemical group modification, has simple and easy operation process, does not have harmful substance residue, and is energy-saving and environment-friendly. The diatom shells with different structural characteristics are mixed, the structural advantages of the diatom shells can be fully utilized, the maximum hemostatic potential of the diatom shells is exerted, and certain reference is provided for people to search for the best hemostatic material.
Description
Technical Field
The invention relates to the field of hemostasis of mixed frustules, in particular to a method for improving hemostasis effect by mixing frustules with different structural characteristics.
Background
Diatoms, a unicellular eukaryote present in fresh and sea water, was observed as early as the 18 th century. The diatom is characterized by the nanometer siliceous cell wall, which is connected by the upper and lower shell surfaces and a hoop siliceous structure called as a ring belt structure. Diatoms are the first model organism to study silica mineralization, with well-defined siliceous walls comprising amorphous silica (amophorus silica) and specific biomolecules. The shell structure has good mechanical strength and various types, has multistage micro-nano pores, huge specific area and various optical characteristics, and is a new material in the field of biological manufacturing. With the discovery of the nanoscale porous shell surface structure of diatoms, more and more potential applications of frustules have been developed, such as: bionic material synthesis, water quality monitoring, adsorption, photocatalysis, genetic engineering, drug slow release and the like.
Recent research shows that the diatom shells have important application value in the development field of hemostatic materials. Research of the Von super et al (DOI:10.1021/acsami.6b12317) of China ocean university in 2016 shows that the frustules of the round sieve algae have excellent liquid adsorption performance, and have a remarkable effect in promoting blood coagulation because the frustules carry a large amount of negative charges due to the presence of surface silanol groups.
At present, the research of using the frustules as the hemostatic material at home and abroad is very little, and in order to overcome the defects of the self structure of the frustules and further improve the hemostatic performance, the chemical group modification is carried out on the frustules of the rotundifolia by the Von super et al (DOI: 10.1021/acsami.6b12317). For example, chitosan is used for modifying diatom shells of the round sifting algae, so that partial positive charges are carried on the surfaces of the diatom shells, and the in-vivo and in-vitro hemostatic effects of the diatom shells are improved by utilizing the electrostatic effect; in addition, due to Ca2+Can be used as a blood coagulation factor, has important effect in blood coagulation cascade, and is selected from Lysium shimeji (DOI:10.1039/c8tb00667a) for Ca treatment of frustules of round screen algae2+Modification of Ca2+Coupled with the frustules, the hemostatic performance of the frustules of the round sifting algae is further improved; the research results of the royal ya Nan and the like (DOI:10.1016/j. carbpol.2018.07.065) in 2018 show that the composite microspheres formed by crosslinking the spirulina shells of the round sieve algae with dopamine and chitosan can obviously shorten the external hemostasis time and improve the biocompatibility of the spirulina shells; lijing et al (10.1002/adhm.202000951) prepared chitosan/frustules from dopamine as a cross-linking agent by simple alkaline precipitation and tert-butanol displacementThe gel particles change the porosity of the gel particles by controlling the concentration of the tert-butyl alcohol, so that the hemostatic performance of the frustules is improved. The defects of the modification mode are as follows:
1) time and labor are wasted, the modification process is complicated, and the shell structure can be broken.
2) The surface of the diatom shell is provided with a micro-nano structure with distinct levels, and after chemical modification, the blockage of a porous structure with levels can be caused, so that the liquid absorption performance of the diatom shell is influenced, and the hemostatic effect is influenced to a certain extent.
3) The process of modifying the shell relates to the use of chemicals, is not environment-friendly, and also considers the problem of no harmful reagent residue after modification, and has low efficiency.
Recently, the pearson linear correlation analysis results on the hemostatic effect and physicochemical characteristics of the frustules show that the specific surface area, particle size, pore size, liquid adsorption performance and shape index of the frustules are the main factors influencing the hemostatic performance (table 1). For example: the coagulation starting time of the frustules is in obvious negative correlation with the specific surface area and the liquid adsorption performance of the frustules and is in obvious positive correlation with the pore diameter; the blood coagulation rate is in a significant negative correlation with the particle size, pore size and shape index. If the structure advantages of the frustules are fully utilized on the premise of not involving the use of chemicals and keeping the original appearance and microstructure of the frustules, the maximum hemostatic potential of the frustules can be exerted, and certain reference can be provided for people to improve the hemostatic performance of the frustules and search for an optimal hemostatic material.
Disclosure of Invention
The invention is based on the defects of the prior art and provides a method for mixing the frustules with different structural characteristics (complementary advantages) to further improve the hemostatic effect. The method mixes two kinds of diatom shells with proper specific surface area, particle size, pore size, liquid adsorption performance and shape index, fully utilizes the advantages of the diatom shells and further improves the hemostasis effect of the single diatom shell. The method is simple to operate, does not damage the shell structure, does not need to use chemicals, is energy-saving and environment-friendly, and has high efficiency.
The invention firstly improves the extraction method of the diatom shells to obtain complete and pure diatom shells, then researches the hemostasis effect of the diatom shells with different structural characteristics, mixes the diatom shells with different structural characteristics, and further improves the hemostasis effect of single diatom shells.
In order to solve the technical problems, the invention adopts the technical scheme that:
a method for mixing frustules having different structural characteristics to further enhance hemostasis, comprising the steps of:
(1) preparing algae mud;
(2) treating the algae mud by an acid-thermal method;
(3) washing and drying to obtain the diatom shell.
(4) Mixing the frustules with different structural characteristics according to a certain proportion.
The specific method for preparing the algae mud in the step (1) is to precipitate algae liquid of marine unicellular diatoms in a centrifugal or filtering mode to obtain the algae mud.
The specific method for treating the algae mud by the acid thermal method in the step (2) comprises the following steps: adding mixed solution of concentrated sulfuric acid and concentrated nitric acid into the algae mud for acid-heat treatment.
The mass fractions of the concentrated sulfuric acid and the concentrated nitric acid are respectively 98% and 65%.
The mixed solution of concentrated sulfuric acid and concentrated nitric acid is prepared from the following components in percentage by volume: sulfuric acid: nitric acid 1:1 (V/V).
The acid-thermal method comprises the steps of adding concentrated sulfuric acid into the marine unicellular diatom, fully and uniformly mixing, heating in a water bath at 60 ℃ for 15-60min, and standing at room temperature for 24-48h by utilizing most organic matters on the surfaces of the carborundum frustules due to strong oxidizing property, dehydration property and corrosiveness of the concentrated sulfuric acid; adding equal volume of concentrated nitric acid, heating to 60 deg.C for clarification, oxidizing organic matter and pigment on the surface of frustules by strong oxidizing property of concentrated nitric acid, and standing at room temperature for 12-24 hr.
The specific method for washing and drying in the step (3) is as follows: in order to avoid the damage of centrifugal force to the diatom shells, the diatom shells subjected to acid treatment are naturally filtered through bolting silk, and are washed for 3-10 times by distilled water, so that the residual acid in the diatom shells is removed; washing with anhydrous ethanol for 3-10 times, removing residual water and organic matter, and drying at 60 deg.C to obtain pure frustules.
The marine unicellular diatom is a marine unicellular organism containing a siliceous shell, and specifically comprises the following components: navicula (Navicula avium), Navicula (Navicula sp.), oval algae (coconeiopsis orthoneoides), and prodenia litura (pleurosporigma indicum).
The frustules with different structural characteristics have particle size (10-120 μm), pore diameter (7-11 nm) and pore volume (0.2cc g)-1-0.5cc g-1) Specific surface area (43 m)2 g-1-190m2 g-1) The liquid adsorption performance (2600-3500%), the potential (-24 mV-33 mV), and the shape index (14-26).
The diatom shells with different structures are fully mixed according to the proportion of A: B ═ 1-4:1-4(M: M).
Further preferred are:
collecting 1L of algae solution by centrifuging or filtering, adding 25mL concentrated sulfuric acid into diatom ooze, mixing well, heating at 60 deg.C for 45min, and standing at room temperature for 24 h. Continuously adding the concentrated nitric acid with the same volume, heating to be clear at 60 ℃, and standing for 24 hours at room temperature. Collecting the treated algae cells by filtering, washing with distilled water for 5 times, washing with absolute ethyl alcohol for 5 times, and drying at 60 ℃ to obtain pure diatom shells. Adhering the diatom ooze to a double-sided adhesive tape for spraying gold, and observing the structural integrity of the diatom ooze under a scanning electron microscope without organic matter residue. Analyzing the main components of the frustules of the diatom as Si and O by using an energy chromatography X-ray spectrometer, wherein the main components can be regarded as pure SiO2A material.
The specific surface area is 120m2 g-1A particle diameter of 10 μm, an average pore diameter of 7.25 μm, a shape index of 15, a liquid adsorption rate of 3010% and a pore volume of 0.2202cc g-1Navicula hull with a potential of-24.6 mV and a specific surface area of 190m2 g-1A particle diameter of 22 μm, an average pore diameter of 7.27 μm, a shape index of 14, a liquid adsorption rate of 3500%, and a pore volume of 0.3475cc g-1Mixing the ootheca body with-32.7 mV potential with the mixture at a ratio of 1:2(M: M), and coagulating in vitro and in vivo respectivelyBlood tests show that compared with single diatom shell, the mixed frustules can obviously shorten the in vitro and in vivo blood coagulation time.
The invention has the beneficial effects that:
1) the operation process is simple. The material used in the invention is the processed diatom shells, the operation process is simple and easy to implement, and no special processing step is needed.
2) No chemical use is involved. The invention can improve the hemostatic performance of the diatom ootheca by only mixing a plurality of diatom oothecas, does not need to add special reagents in the whole process, does not need to consider harmful substance residue, and is energy-saving and environment-friendly.
3) The efficiency is high. The diatom shells with different physical and chemical characteristics are mixed, the hemostasis advantages of single diatom shells can be fully exerted, the advantage complementation is realized, and the hemostasis effect can be obviously improved by the mixed diatom shells.
4) Lays a foundation for further exploring the dominant algae species with excellent hemostatic effect.
Drawings
FIG. 1 shows the complete shell structure of the oval algae (a-b) and navicula (c-d).
FIG. 2 is an energy spectrum of the hull of the oval algae (a) and navicula (b).
FIG. 3 is a statistical plot of the in vitro clotting times of 4 diatoms and mixed frustules.
FIG. 4 is a statistical chart of the in vivo clotting (rat tail broken) hemostasis time (a) and bleeding volume (b) of 4 kinds of Lucilia and mixed diatom shells.
FIG. 5 is a statistical graph of the total (a) and repeat (b) amounts of blood clotting (femoral artery damage) in vivo for 4 types of Botrytis pinnata and mixed frustules.
Detailed Description
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
4 Marine diatoms used in the experiment are inoculated into f/2 liquid Culture medium (Guillard RL (1975) Culture of cultivation for Feeding Marine Animals, in Culture of Marine Animals, eds Smith WL, Chanley MH (Plenum, New York), pp 29-60.) according to a certain proportion, and are cultured by passing air at 23 ℃ (ventilation volume is 150 ml/min), illumination intensity is controlledIs 40 mu mol m-2s-1The light-dark period is 12h:12 h.
Example 1
Cultured 1L of Navicula sp and Cocconeis orthoneoides were respectively put into 100mL centrifuge tubes and centrifuged at 2000g for 5 minutes. After the centrifugation is finished, the supernatant is poured off, the diatom oozes in each centrifuge tube are combined, and the diatom oozes are washed for 3 times by distilled water to remove inorganic salts and impurities. Adding 25mL of concentrated sulfuric acid as treating solution to mix algae cell and concentrated sulfuric acid, and heating in 60 deg.C water bath for 45 min. The heated mixture was left at room temperature for 24 h. Adding the concentrated nitric acid with the same volume into the mixed solution again, heating to be clear at 60 ℃, and standing for 24 hours at room temperature. Collecting the treated algae cells by a filtering method, washing with distilled water for 5 times, washing with absolute ethyl alcohol for 5 times, and drying in a 60 ℃ drying oven to obtain white powder, namely the frustules. Adhering the shells of the two kinds of algae to a double-sided adhesive tape, spraying gold on the double-sided adhesive tape, and observing under a scanning electron microscope.
FIG. 1 shows the results of scanning electron micrographs of two diatoms. As can be seen from fig. 1, the diatom shell obtained by the method provided by this embodiment has a complete structure, clear pores, and substantially no organic matter remains.
FIG. 2 shows the analysis results of the elemental compositions of the hulls of Navicula and Odoomenium by energy dispersive X-ray spectroscopy (EDXS), from which it can be seen that the hulls of diatom purified by this method have Si and O as the main components and can be regarded as pure SiO2。
The specific surface area is 120m2 g-1A particle diameter of 10 μm, an average pore diameter of 7.25 μm, a shape index of 15, a liquid adsorption rate of 3010% and a pore volume of 0.2202cc g-1Navicula hull with a potential of-24.6 mV and a specific surface area of 190m2 g-1A particle diameter of 22 μm, an average pore diameter of 7.27 μm, a shape index of 14, a liquid adsorption rate of 3500%, and a pore volume of 0.3475cc g-1The ootheca of-32.7 mV potential was mixed well with the navicula, ootheca 1-4:1-4(M: M), and the external blood coagulation effect of the mixed frustules was evaluated.
The specific evaluation method comprises the following steps:
accurately weighing 2.5mg mixed frustules in 2mL ep tube, adding 500. mu.L blood, and rapidly adding 50. mu.L CaCl2The ep tube was placed in a 37 ℃ water bath, turned 180 degrees every 10 seconds until blood coagulated, and the coagulation time was recorded, the results are shown in fig. 3. The results in FIG. 3 show that the mixed frustules can significantly shorten the clotting time of a single frustule, and the in vitro clotting time can be shortened by 40-60s, compared to a single frustule.
Table 1 shows the pearson correlation analysis of physicochemical characteristics of frustules with hemostasis time (. beta. represents p < 0.05) and the specific results are as follows:
TABLE 1 Pearson correlation analysis of physicochemical characteristics of Diatom shells with hemostasis time (. sup. < 0.05)
Example 2
The cultured Navicula (Navicula sp.) and oval algae (coconeeiopsis orthoidea) were dispensed into 100mL centrifuge tubes and centrifuged at 2000g for 5 minutes. After the centrifugation was completed, the supernatant was poured off, and the algal slurry in each tube was combined and washed 3 times with distilled water. The frustules were obtained and characterized in the same manner as in example 1.
The specific surface area is 120m2 g-1A particle diameter of 10 μm, an average pore diameter of 7.25 μm, a shape index of 15, a liquid adsorption rate of 3010% and a pore volume of 0.2202cc g-1Navicula hull with a potential of-24.6 mV and a specific surface area of 190m2 g-1A particle diameter of 22 μm, an average pore diameter of 7.27 μm, a shape index of 14, a liquid adsorption rate of 3500%, and a pore volume of 0.3475cc g-1The ootheca body having a potential of-32.7 mV was mixed well with the mixture at a ratio of 1:2(M: M) to evaluate the hemostatic effect of the mixed frustules in the tailgating model.
The specific evaluation method comprises the following steps:
250-g anesthetized male SD rats with 300g are fixed in an anatomical frame, the tail of the rat with half the length is cut off by surgical scissors, the tail end of the severed tail is placed in the air for 10s to ensure normal bleeding, then the severed tail is placed in 100mg mixed diatom shell (Navicula: oval algae: 1:2), and the hemostasis time and the blood loss are recorded.
FIG. 4 shows the time to hemostasis and the amount of blood lost for the mixed frustules. The results show that mixing the frustules significantly reduces the time to hemostasis and reduces the amount of bleeding compared to the frustules alone.
Example 3
The cultured Navicula (Navicula sp.) and oval algae (coconeeiopsis orthoneoides) were dispensed into 100mL centrifuge tubes and centrifuged at 2000g for 5 minutes. After the centrifugation was completed, the supernatant was poured off, and the algal slurry in each tube was combined and washed 3 times with distilled water. The frustules were obtained and characterized in the same manner as in example 1. The mixed frustules were mixed in the same manner as in example 1 to evaluate the hemostatic effect in the mixed frustules femoral artery model.
The specific evaluation method comprises the following steps:
placing 250-300g of anesthetized male SD rat on an anatomical table for fixation, and cutting the skin of the right lower limb by using surgical scissors to expose femoral artery; the femoral artery was dissected with a scalpel, 50mg of mixed frustules (navicula: oval 1:2) were quickly applied, the bleeding was aspirated with pre-weighed filter paper, the filter paper was changed every 15s, the weight of the wet filter paper was weighed and recorded in time, and the entire procedure lasted 15 min. The bleeding volume and the bleeding time were calculated from the weight of the wet filter paper and the results are shown in Table 2.
TABLE 2 hemostasis time and bleeding volume for single and mixed frustules following femoral artery injury
As can be seen from Table 2, the mixed frustules had the shortest hemostasis time (180s) and the lowest total bleeding in the femoral artery injury model. FIG. 5 is a graph of bleeding from mixed frustules following femoral artery injury. The results in FIG. 5 show that the total blood loss of the mixed frustules was minimal and that the secondary bleeding was minimal.
The method for improving the hemostatic effect by mixing the diatom shells with different structures is also suitable for other marine unicellular organisms containing siliceous shells, such as: navicula (Navicula avium), Navicula (Navicula sp.), oval algae (coconeiopsis orthoneoides), and prodenia litura (pleurosporigma indicum).
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