NL2032483B1

NL2032483B1 - Preparation method for swim bladder-based myocardial patch

Info

Publication number: NL2032483B1
Application number: NL2032483A
Authority: NL
Inventors: Wang Wei; Dai Jian
Original assignee: Zju Hangzhou Global Scientific And Tech Innovation Center
Priority date: 2022-01-27
Filing date: 2022-07-14
Publication date: 2023-08-07
Also published as: CN114569791B; CN114569791A

Abstract

The present invention discloses a preparation method, for swim bladder—based myocardial patch: (1) depositing metal nanoparticles on a swim bladder in situ to obtain a deified conductive swim bladder substrate; (2) encapsulating one side of the conductive 5 swiH1 bladder substrate wittl a hydrogel prepared, front fish glue collagen, and coating and encapsulating the edge contact position of the conductive swim bladder substrate with the hydrogel with a fish glue—based biological glue, so as to obtain the swim bladder— based, myocardial patch. The SWiHI bladder—based, myocardial patch 10 provided by the present invention, with relaxation and contraction of heart, generates piezoelectric polarization inside the swim bladder to trigger a piezoelectric effect, so that the electrically conducting swim bladder substrate can perform electrical stimulation therapy on the attached damaged part of the 15 myocardium in heartbeats, so as to promote repair of the damaged part of the myocardium. (+ Fig.)

Description

PREPARATION METHOD FOR SWIM BLADDER-BASED MYOCARDIAL PATCH

TECHNICAL FIELD

The present invention relates to the technical field of myo- cardial repair, and in particular to a preparation method for swim bladder-based myocardial patch.

BACKGROUND ART

At present, all treatments for heart diseases are designed to alleviate damage to the heart, and there is no way to repair the damage done because once the muscle on the heart dies, it will not regrow. But cardiac patches can repair the lost heart muscle. Car- diac patches may provide mechanical support and restore electrome- chanical coupling at the site of myocardial infarction, reducing myocardial reconfiguration and maintaining normal cardiac func- tion.

In order to perform cardiac repair, many existing methods re- quire open surgery, which further requires secondary or even mul- tiple treatments, and is likely to cause secondary damage to the patient. Although it has been reported that the myocardial patch can exert repair effect on the damaged myocardial area, it is still necessary to perform another open surgery to remove the myo- cardial patch after the completion of myocardial repair due to the fact that it is in the site of heart, thus causing secondary sur- gical trauma to the patient. Therefore, the prior art has yet to be further improved.

The swim bladder is a biomass material. Some existing studies have shown that the swim bladder has good biocompatibility, bioab- sorbability and biodegradability. For example, Chinese patent with

Publication Number CN108578781A relates to a swim bladder-derived biovalve material and preparation method and application thereof, where the preparation method includes the following steps: cutting and decellularizing a swim bladder to obtain the decellularized swim bladder; fixing and cross-linking the decellularized swim bladder, after which rinsing is performed to obtain the swim blad-

der-derived biovalve material. For example, Chinese patent with

Publication Number CN112961374A discloses a preparation and appli- cation of conductive hydrogel derived from natural swim bladder. A preparation method for the conductive hydrogel includes the fol- lowing steps: (1) mixing a swim bladder with H,0; for 15 min-16 h, and then mixing the swim bladder with an alkali solution for 4-8 h to obtain a hydrogel; (2) mixing the hydrogel with monomeric pyr- role; and then mixing the hydrogel with an iron ion solution for reaction.

Therefore, it is a research hotspot on how to apply swim bladder to myocardial patch in the field.

SUMMARY

In view of the above deficiencies of the prior art, it is an object of the present invention to provide a preparation method for swim bladder-based myocardial patch for solving the problem that the existing myocardial patch causes secondary surgical trau- ma to a patient. Since swim bladder is a biomass material, and some existing studies have shown that swim bladder has good bio- compatibility, bioabsorbability and bicdegradability, the swim bladder-based myocardial patch of the present invention can be gradually absorbed and degraded in the body while treating the damaged myocardium. It will not bring about immune rejection and other damages to human body, and meanwhile, it is not necessary to perform a secondary surgery to remove the myocardial patch. There- fore, the myocardial patch is beneficial to cardiac repair of pa- tients with damaged myocardium, can reduce the pain of patients and reduce the risk of secondary damage.

The present invention provides a preparation method for swim bladder-based myocardial patch which includes: (1) depositing metal nanoparticles on a swim bladder in situ to obtain a modified conductive swim bladder substrate; (2) encapsulating one side of the conductive swim bladder substrate with a hydrogel prepared from fish glue collagen, and coating and encapsulating the edge contact position of the conduc- tive swim bladder substrate with the hydrogel with a fish glue- based biological glue, so as to obtain the swim bladder-based myo-

cardial patch.

Further, the metal nanoparticles are metals such as Ag and Mg which are harmless to human body and have good conductivity.

Further, the source of the swim bladder for the swim bladder- based myocardial patch is not limited to freshwater fish such as silver carp and grass carp, and the swim bladder of deep-sea fish and the like can also be used.

Further, in the preparation method for the conductive swim bladder substrate, the swim bladder is modified: soaking the swim bladder in a silver nitrate solution, and adding dropwise a vita- min C solution to the swim bladder to deposit silver nanoparticles in situ. The swim bladder surface is thereby rendered conductive.

Specifically, the swim bladder substrate soaked with silver ni- trate is placed on a filter paper, and then it is connected to a suction filtration device for suction filtration; after adding dropwise the vitamin C solution to the swim bladder substrate, the water in the mixed solution can be quickly suction filtered, leav- ing the Ag particles deposited in situ on the swim bladder, so as to obtain the modified conductive swim bladder substrate.

Concentrations of the silver nitrate solution and the vitamin

C solution are at a chemical molar ratio of 2:1.

The concentration of the silver nitrate solution is 0.2 mol/L to 4 mol/L and the concentration of the vitamin C solution is 0.1 mol/L to 2 mol/L.

In the step (2), the hydrogel has an area larger than that of the conductive swim bladder substrate.

Further, the specific preparation method for the swim blad- der-based myocardial patch is as follows: cleaning the fresh silver carp swim bladder with deionized water and then cutting it into circular pieces with a diameter of 2 cm to obtain the swim bladder substrate; preparing separately a silver nitrate solution with a concen- tration of 2 mol/L and a vitamin C solution with a concentration of 1 mol/L for later use; soaking the swim bladder substrate in the silver nitrate solution for 24 h to completely infiltrate it, then sonicating it for 10 minutes, and then soaking it again for 12 h so that the internal fibers of the swim bladder substrate are all in contact with the silver nitrate solution ions; placing the swim bladder substrate soaked with silver nitrate on a filter paper, and then connecting it to a suction filtration device for suction filtration; after adding dropwise the vitamin C solution to the swim bladder substrate, the water in the mixed so- lution can be quickly suction filtered, leaving the Ag particles deposited in situ on the swim bladder; after obtaining the modified conductive swim bladder sub- strate, encapsulating one side of the conductive swim bladder sub- strate with a hydrogel prepared from fish glue collagen, and cov- ering the modified swim bladder substrate with the hydrogel in a plane area ratio of 1:1.21, and coating a ring of a width of 0.2 cm on the edge contact position of the swim bladder substrate with the hydrogel with a fish glue-based biological glue, thereby en- capsulating the conductive swim bladder substrate with the fish glue-based biological glue.

Further, in the step (2), the preparation method for the swim bladder collagen hydrogel uses swim bladder as a source of the collagen, and dissolves swim bladder collagen protein in an acidic solution and then prepares the swim bladder collagen hydrogel, which specifically includes: dissolving a prescribed amount of swim bladder collagen pro- tein in ag. HCl (pH 2.0) to prepare a collagen protein solution; allowing the mixture to stand at room temperature for 2 days without external interference to obtain a homogeneous solution; placing 1 mL of the collagen protein solution on a glass plate which is controlled with two silicone gaskets, covering it with a glass plate with a spacing of 0.5 mm, and contacting the disc-shaped collagen protein solution with the glass plate with an initial diameter of about 2.2 cm; introducing 0.1 M sodium-phosphate buffer (pH 7.2) into the reaction cell from the peripheral edge portion of the collagen protein solution to perform collagen protein gelation; advancing the gelation process from the periphery to the cen- ter of the collagen protein solution by the diffusion of the buff- er; forming a disc-shaped collagen hydrogel with a diameter of about 2.2 cm and a thickness of 0.5 mm after 1 hour.

Further, in the step (2), the preparation method for the fish glue-based biological glue is as follows: digesting, beating and squeezing the swim bladder collagen to prepare the fish glue-based 5 biological glue.

The step of preparing the biological glue based on swim blad- der collagen specifically includes: cutting the cleaned swim bladder into thin strips and trans- ferring them into a mortar, and then manually beating and grinding them with a pestle; steaming the grinded fish glue pieces in a steamer for more than half an hour, and then continuing to grind them to obtain a swim bladder paste; putting the swim bladder paste into a fine mesh gauze, wrap- ping it well, putting it into a beaker with water bath at 80°C, and squeezing the gauze as hard as possible to make the glue ooze out and into the beaker; pouring the liquid glue into a glass bottle, and the glue can be used as a biological glue, and the biological glue can be rap- idly solidified at room temperature.

The present invention provides a swim bladder-based myocardi- al patch which uses swim bladder, a biological material, as a sub- strate that is then modified on the surface thereof so as to be rendered conductive. The modification method used is in-situ depo- sition of metal nanoparticles, and the fish glue-based biological glue of the present invention is used to glue the patch to the surface of the heart, thereby expanding the myocardial patch dur- ing relaxation and contraction of heart; the swim bladder induces a piezoelectric effect due to an external force, and a piezoelec- tric signal inside the material is exported, so as to perform electrical stimulation treatment on the damaged part of the myo- cardium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of a preparation method for swim blad- der-based myocardial patch according to an embodiment of the pre- sent invention.

FIG. 2 is a schematic diagram of structural composition of a swim bladder-based myocardial patch according to an embodiment of the present invention.

FIG. 3 is a graph showing an experiment testing a conductivi- ty of a swim bladder-based material before and after modification according to an embodiment of the present invention.

FIG. 4 is a test on piezoelectric power generation perfor- mance of a swim bladder-based material according to an embodiment of the present invention.

FIG. 5 is a graph recording voltage and current generated by piezoelectric power generation of a swim bladder-based material according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides a preparation method for swim bladder-based myocardial patch, and in order to make the objects, technical solutions and effects of the present invention clearer, the present invention is described in further detail below. It should be understood that the specific embodiments described here- in are illustrative only and are not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the specification of the invention herein are for the purpose of describing specific implementations only and are not intended to limit the invention.

As shown in FIG. 1, in one implementation of the present in- vention, a preparation method for swim bladder-based myocardial patch specifically includes:

S100: after cleaning the fresh swim bladder, modifying it with a silver nitrate solution and a vitamin C solution to obtain a conductive swim bladder;

S200: dissolving swim bladder collagen protein in an acidic solution and then preparing a swim bladder collagen hydrogel;

S300: digesting, beating and squeezing the swim bladder col- lagen to prepare a swim bladder-based biological glue (fish glue- based biological glue);

S400: encapsulating the conductive swim bladder substrate, swim bladder collagen hydrogel and swim bladder-based biological glue to obtain the swim bladder-based myocardial patch.

In the present experimental example, the swim bladder used includes, but is not limited to, swim bladders of freshwater fish such as silver carp and grass carp, as well as swim bladders of deep-sea fish such as cod.

The preparation method of the piezoelectric catalyst of the present invention will be further explained by the following spe- cific preparation embodiments.

Cleaning the fresh silver carp swim bladder with deionized water and then cutting it into circular pieces with a diameter of 2 cm to obtain the swim bladder substrate; soaking the swim blad- der substrate in a silver nitrate solution with a concentration of 2 mol/L for 24 h to completely infiltrate it, then sonicating it for 10 minutes, and then soaking it again for 12 h so that the in- ternal fibers of the swim bladder substrate are all in contact with the silver nitrate solution ions; placing the swim bladder substrate soaked with silver nitrate on a filter paper, and then connecting it to a suction filtration device for suction filtra- tion; adding dropwise a vitamin C solution with a concentration of l mol/L to the swim bladder substrate; the water in the mixed so- lution can be quickly suction filtered, leaving the Ag particles deposited in situ on the swim bladder, so as to obtained a modi- fied conductive swim bladder substrate.

Weighing and dissolving 3 g of swim bladder collagen protein in ag. HCl (pH 2.0) to prepare a collagen protein solution; allow- ing the mixture to stand at room temperature for 2 days to obtain a homogeneous solution; placing 1 mL of the collagen protein solu- tion on a glass plate which is controlled with two silicone gas- kets, covering it with a glass plate with a spacing of 0.5 mm, and contacting the disc-shaped collagen protein solution with the glass plate with an initial diameter of about 2.2 cm; introducing 0.1 M sodium-phosphate buffer (pH 7.2) into the reaction cell from the peripheral edge portion of the collagen protein solution to perform collagen protein gelation; advancing the gelation process from the periphery to the center of the collagen protein solution by the diffusion of the buffer; forming a disc-shaped collagen hy- drogel with a diameter of about 2.2 cm and a thickness of 0.5 mm after 1 hour.

Cutting the cleaned swim bladder into thin strips and trans- ferring them into a mortar, and then manually beating and grinding them with a pestle; steaming the grinded fish glue pieces in a steamer for more than half an hour, and then continuing to grind them to obtain a swim bladder paste; putting the swim bladder paste into a fine mesh gauze, wrapping it well, putting it into a beaker with water bath at 80°C, and squeezing the gauze as hard as possible to make the glue ooze out and into the beaker; pouring the liquid glue into a glass bottle, and the glue can be used as a biological glue.

After obtaining the modified conductive swim bladder sub- strate, encapsulating one side of the conductive swim bladder sub- strate with a hydrogel prepared from fish glue collagen, and cov- ering the modified swim bladder substrate with the hydrogel in a plane area ratio of 1:1.21, and coating a ring of a width of 0.2 cm on the edge contact position of the swim bladder substrate with the swim bladder collagen hydrogel with a fish glue-based biologi- cal glue, thereby encapsulating the swim bladder substrate with the fish glue hydrogel.

FIG. 2 is a schematic diagram of structural composition of a swim bladder-based myocardial patch of the present invention, and it specifically includes: front view A, left view B, fish glue hy- drogel (hydrogel) 1, coating ring 2 formed by swim bladder-based biological glue, and conductive swim bladder substrate 3 with sil- ver deposited on the surface. The bulge seen from the left view B is a hollow area left after the fish glue hydrogel 1 and the con- ductive swim bladder substrate 3 are bonded by the fish glue-based biological glue 2, so that a certain air layer is reserved for the conductive swim bladder substrate during the relaxation and con- traction process with the heart, and the spatial change of the air layer can enhance the piezoelectric effect of the swim bladder substrate.

In summary, the swim bladder-based myocardial patch prepared by the preparation method of the present invention is entirely prepared based on swim bladder in terms of the swim bladder sub- strate, the encapsulating material, and the adhesive. Therefore, the swim bladder-based myocardial patch of the present invention has good biocompatibility and biodegradability, and does not cause secondary damage to human body.

As shown in FIG. 3 and FIG. 4, the conductivity test and the piezoelectric power generation performance test on the conductive swim bladder substrate are performed in the swim bladder-based em- bodiment, and (a), (b), {c) and (d) in FIG. 3 are respectively a cleaned swim bladder substrate, a swim bladder substrate with sil- ver deposition (conductive swim bladder substrate), a conductivity test on the swim bladder substrate, and a conductivity test on the swim bladder with silver deposition. It can be seen from ({c) and (d}) in FIG. 3 that the unmodified swim bladder substrate cannot conduct the circuit and light up the small bulb, while the swim bladder with silver deposition has an improved conductivity and can conduct the circuit and light up the small bulb. (a), (b), (Cc) and (d) in FIG. 4 are respectively a swim bladder substrate and a swim bladder substrate with silver deposition, and it can be seen that under finger pressure for the piezoelectric test, the swim bladder substrate can light up small LED bulb, and under finger pressure for the piezoelectric test, the swim bladder substrate with silver deposition can light up small LED bulb, and the bulb can always keep bright with finger pressure or being hold with hands, i.e., under external forces.

As shown in FIG. 5, a swim bladder substrate with silver dep- osition can generate a micro-current of about 14 nA and a micro- voltage of about 55 mV under hand pressure, which provides direct data support for swim bladder substrate being used as a myocardial patch to provide electrical stimulation treatment.

It is worth noting that the application of the present inven- tion is not limited to the examples described above, and that im- provements and variations may be suggested to one of ordinary skill in the art in light of the above description, all of which are intended to be included within the protection scope of the ap- pended claims of the present invention.

Claims

CONCLUSIESCONCLUSIONS

1. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch, met het kenmerk, dat de werkwijze voor het be- reiden omvat: (1) het in situ afzetten van metalen nanodeeltjes op een zwemblaas om een gemodificeerd geleidend zwemblaassubstraat te verkrijgen; (2) het inkapselen van één zijde van het geleidende zwemblaassub- straat met een hydrogel bereid uit vislijmcollageen, en het coaten en inkapselen van de randcontactpositie van het geleidende zwem- blaassubstraat met de hydrogel met een op vislijm gebaseerde bio- logische lijm, om de op de zwemblaas gebaseerde myocardiale patch te verkrijgen.A method of preparing a swim bladder-based myocardial patch, characterized in that the method of preparation comprises: (1) depositing metal nanoparticles in situ on a swim bladder to obtain a modified conductive swim bladder substrate; (2) encapsulating one side of the conductive swim bladder substrate with a hydrogel prepared from isinglass collagen, and coating and encapsulating the edge contact position of the conductive swim bladder substrate with the hydrogel with a isinglass based biological glue, to swim bladder-based myocardial patch.

2. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 1, met het kenmerk, dat in stap (1) een bereidingswerkwijze voor het geleidende zwemblaassub- straat als volgt is: het doordrenken van de zwemblaas in een zil- vernitraatoplossing, en het druppelsgewijs toevoegen van een vita- mine C-oplossing aan de zwemblaas om zilveren nanodeeltjes in situ af te zetten.A method of preparing a swim bladder-based myocardial patch according to claim 1, characterized in that in step (1), a preparation method for the conductive swim bladder substrate is as follows: soaking the swim bladder in a silver nitrate solution , and adding a vitamin C solution dropwise to the swim bladder to deposit silver nanoparticles in situ.

3. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 2, met het kenmerk, dat de concentraties van de zilvernitraatoplossing en de vitamine C- oplossing een chemische molaire verhouding van 2:1 hebben.A method for preparing a swim bladder based myocardial patch according to claim 2, characterized in that the concentrations of the silver nitrate solution and the vitamin C solution have a chemical molar ratio of 2:1.

4. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 2, met het kenmerk dat de concentratie van de zilvernitraatoplossing 0,2 mol/L tot 4 mol/L is en de concentratie van de vitamine C-oplossing 0,1 mol/L tot 2 mol/L is.The method for preparing a swim bladder based myocardial patch according to claim 2, characterized in that the concentration of the silver nitrate solution is 0.2 mol/L to 4 mol/L and the concentration of the vitamin C solution is 0. 1 mol/L to 2 mol/L.

5. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 2, met het kenmerk, dat het met zilvernitraat doordrenkte zwemblaassubstraat op een filtreer-A method of preparing a swim bladder-based myocardial patch according to claim 2, characterized in that the silver nitrate-soaked swim bladder substrate is placed on a filtering

papier wordt geplaatst en de vitamine C-oplossing druppelsgewijs aan het zwemblaassubstraat wordt toegevoegd, gevolgd door zuigfil- tratie om de zilveren nanodeeltjes in situ op de zwemblaas af te zetten.paper is placed and the vitamin C solution is added dropwise to the swim bladder substrate, followed by suction filtration to deposit the silver nanoparticles in situ on the swim bladder.

6. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 1, met het kenmerk, dat in stap (1) de zwemblaas afkomstig is van zoetwatervissen of diepzee- vissen.A method of preparing a swim bladder based myocardial patch according to claim 1, characterized in that in step (1) the swim bladder is derived from freshwater fish or deep sea fish.

7. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 2, met het kenmerk, dat in stap (2) de hydrogel een groter oppervlak heeft dan dat van het geleidende zwemblaassubstraat.A method of preparing a swim bladder based myocardial patch according to claim 2, characterized in that in step (2) the hydrogel has a surface area greater than that of the conductive swim bladder substrate.

8. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 1, met het kenmerk, dat in stap (2) een bereidingswerkwijze voor de hydrogel als volgt is: het gebruik van de zwemblaas als een bron van het collageen, het oplossen van het zwemblaas collageeneiwit in een zure oplossing en het vervolgens bereiden van de hydrogel.A method of preparing a swim bladder-based myocardial patch according to claim 1, characterized in that in step (2), a preparation method for the hydrogel is as follows: using the swim bladder as a source of the collagen, dissolving the swim bladder collagen protein in an acidic solution and then preparing the hydrogel.

9. Werkwijze voor het bereiden van een op een zwemblaas gebaseerde myocardiale patch volgens conclusie 1, met het kenmerk, dat in stap (2) een bereidingswerkwijze voor de op vislijm gebaseerde bi- ologische lijm als volgt is: het verteren, kloppen en knijpen van het zwemblaascollageen om de op vislijm gebaseerde biologische lijm te bereiden.A method for preparing a swim bladder-based myocardial patch according to claim 1, characterized in that in step (2), a preparation method for the isinglass-based biological glue is as follows: digesting, beating and squeezing the swim bladder collagen to prepare the isinglass based biological glue.

10. Myocardiale patch gebaseerd op een zwemblaas verkregen door de werkwijze voor het bereiden volgens een van de conclusies 1 tot 9.A myocardial patch based on a swim bladder obtained by the method of preparation according to any one of claims 1 to 9.