WO2016159500A1 - Filter for portable peritoneal dialysate regeneration system using enzyme-included silk fibroin and method for manufacturing same - Google Patents

Abstract

The present invention relates to a method for manufacturing a filter for a peritoneal dialysate regeneration system, using silk fibroin or silk protein and, more specifically, to a filter comprising silk fibroin or silk protein including an enzyme such as urease therein and to a method for manufacturing a filter for a portable peritoneal dialysate regeneration system. Silk fibroin, unlike the other polymers, does not induce an immune response in vivo, and was approved to be safe by FDA, and thus silk fibroin has been used for other medical materials including a suture thread. The filter according to the present invention can regenerate a peritoneal dialysate very effectively by effectively removing low-molecular materials. In addition, the filter of the present invention can be used simultaneously with an existing activated carbon filter, thereby allowing more effective regeneration of dialysate. The silk fibroin filter according to the present invention has advantages of mass productivity, regulatory degradability, and enzyme immobility, and thus has an excellent dialysate regeneration effect as a filter for a portable peritoneal dialysate regeneration system, in which an enzyme such as urease is immobilized.

Description

Filter for portable peritoneal dialysis solution regeneration system using silk fibroin entrapped with enzyme and method for producing same

The present invention relates to a filter for a portable peritoneal dialysis solution regeneration system, and more particularly, to a filter for a portable peritoneal dialysis solution regeneration system in which an enzyme is encapsulated in silk fibroin or silk protein.

Due to the spread of westernized eating habits and lifestyles, lifestyle-type metabolic diseases such as diabetes and hypertension have steadily increased and the number of patients with kidney disease has increased rapidly. In particular, 30% of the 4 million Korean diabetic patients have a potential increase in renal disease.

The kidney is a biological biological organ that performs the functions necessary to maintain life, and the function of filtering the metabolites in the body and excreted in the urine as well as regulating acid and base equilibrium. Dysfunction of these kidneys builds up waste products in the blood, causing uremia and life threatening. End stage renal failure is permanently reduced in the glomerular filtration function of the kidney is impossible to recover the kidney function is inevitable kidney replacement therapy. Therefore, patients with end-stage renal failure may receive urinary tract disease through transplantation of another person, dialysis of blood through artificial kidneys, or peritoneal dialysis with a glucose solution containing fluid-like components in the peritoneum and exchanged at regular intervals. Remove your back.

However, the current financial burden of insurance due to hemodialysis treatment is increasing significantly, and according to the data of the Korean National Criminal Plain, the total medical expenses paid by patients with end-stage renal failure are similar to those of cancer patients. It is 3 times the highest in Korea.

Kidney transplantation can remove the medium-molecular substances that cannot be removed by dialysis, and can produce hormones such as hematopoietic hormones and can eat and live normally, but the number of organ donors is significantly lower than the number of patients waiting for organ transplantation. to be. In addition, there is a limitation that it is difficult to consider renal transplantation in cases of old age, untreated malignant tumors, and uncontrolled infections, rejection after transplantation, and drug-related drugs, including the need for life-long immunosuppressants or cancer. The disadvantage is that side effects can occur. Dialysis, on the other hand, is known as a safe and effective way to immediately improve symptoms such as volume overload due to kidney failure, hyperkalemia and intoxication by nitrogen waste.

Activated carbon is widely used for water purification, and it uses carbonaceous materials such as coconut shell, various woods, lignite, anthracite coal and bituminous coal as raw materials to develop fine pores of molecular size through the activation process. It is characterized by having an internal surface area of more than 1000m ² per g. Such activated carbon can adsorb low molecular materials and high molecular materials, but it has the selectivity of adsorbing the low molecular materials and then desorbing the small molecules that are adsorbed first and adsorbing the larger molecules when larger molecules come into contact. In order to increase the selectivity, activated carbon may improve the properties for adsorption through acid treatment and the like.

The peritoneal dialysis solution of the patient is urea (Calcium), creatinine (Creatinine), glucose (Glucose), phosphorus (Phosphorus), uric acid (Uric acid), albumin (Albumin) and the like. Each component is divided into small molecules, medium molecules, and high molecular materials. Urea (60 Dalton), calcium (40 Dalton), and phosphorus (30 Dalton) belong to low molecular materials, creatinine (113 Dalton), glucose (180 Dalton), and uric acid. (168 Dalton) can be classified as a medium molecule, and albumin can be classified as a polymer at 65,000 Dalton. However, in case of filtering peritoneal dialysis solution using activated carbon, creatinine and uric acid, which are medium molecules, can be adsorbed and removed by activated carbon.However, in the case of urea, which is a low molecular material, even if initially adsorbed due to adsorption selectivity of activated carbon, Desorption has a disadvantage that does not exhibit a removal effect.

Silk is a natural polymer that can be obtained from silkworms. It is a structure in which two strands of fibroin protein are surrounded by sericin protein, and sericin is removed through a process called refining to obtain fibroin protein. Silk fibroin, unlike other polymers, does not induce an immune response in the living body and is used for other medical materials such as sutures because it is recognized for safety by the FDA. Due to these advantages, various materials using silk fibroin have been developed one after another.

Silk fibroin is a protein extracted from silkworm cocoon, which can be mass-produced and low in cost, and is a polymer that can control the degree of decomposition depending on the production form and crystallization method. In addition, there is an advantage that can effectively fix enzymes such as urease during the crystallization process. Taking advantage of such mass productivity, controllable degradability, enzyme fixation, and the like of the silk fibroin, the present inventors have completed the present invention by preparing a filter for a portable peritoneal dialysis solution regeneration system to which an enzyme such as urease is immobilized.

The present invention is to improve the quality of life of patients by effectively regenerating the dialysis solution already used by using the enzyme-immobilized silk fibroin and activated carbon filter when patients with end-stage renal failure periodically undergo peritoneal dialysis. have.

The present invention provides a filter for regenerating a peritoneal dialysis solution, wherein the enzyme comprises immobilized silk fibroin nanofibers. Preferably, a polymer is added to the silk fibroin nanofibers, and the polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyvinyliden fluoride, and the like. More preferably. The enzyme may be urease. The silk fibroin nanofibers may further include activated carbon.

The present invention also provides a portable peritoneal dialysis solution regeneration system comprising an enzyme-immobilized silk fibroin nanofiber filter layer and an activated carbon filter layer. Preferably, the two filter layers are included in multiple layers.

The present invention also provides a method for preparing silk fibroin-polymer-enzyme mixed solution by mixing silk fibroin aqueous solution, polymer and enzyme; Preparing a filter by selecting the mixed aqueous solution from a crowd consisting of electrospinning, electrospraying, and lyophilization; And it provides a method for producing a portable peritoneal dialysis solution regeneration filter comprising the step of insolubilizing the prepared filter. The polymer is preferably at least one selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyvinyliden fluoride, and the like. The insolubilization step may recrystallize the filter by selecting one or more of the group consisting of ethanol, methanol, propanol. The insolubilization step may also be treated with water vapor to recrystallize the filter. In the insolubilization step, the step of immobilizing the enzyme to the filter can be performed simultaneously. In the step of making the silk fibroin-polymer-enzyme mixed aqueous solution, activated carbon may be further added to form a mixed aqueous solution.

The filter according to the present invention can effectively remove the low molecular weight material and very effectively regenerate the peritoneal dialysis solution. In addition, since it can be used simultaneously with the existing activated carbon filter, more effective dialysis fluid regeneration is possible.

The silk fibroin filter according to the present invention has advantages such as mass productivity, controllable degradability, enzyme fixation, and the like, and has an excellent dialysis solution regeneration effect as a filter for a portable peritoneal dialysis solution regeneration system to which an enzyme such as urease is immobilized.

1 is a graph showing the removal rate of the peritoneal dialysis solution according to the change in the stirring time of the peritoneal dialysis solution and activated carbon.

Figure 2 is a graph of the removal rate of the peritoneal dialysis solution according to the change in the amount of activated carbon when stirring the peritoneal dialysis solution and activated carbon.

3 is a result of observing the change of the peritoneal dialysis solution by filling the activated carbon filter in the column and continuously flowing the peritoneal dialysis solution.

Figure 4 is a photograph observing the structure of the silk fibroin filter produced by electrospinning through SEM.

5 is a graph showing the porosity of the silk fibroin filter produced by the electrospinning.

Figure 6 illustrates the structure of a portable peritoneal dialysis solution regeneration system.

The present invention relates to a filter for removing an element that is not removed from an activated carbon filter layer in a peritoneal dialysis solution of a patient, maintaining a proper pore and support structure, and effectively removing the element and a method of manufacturing the same. In order to achieve the above object, the present invention relates to a filter for a regeneration system of a dialysate using silk fibroin, and a method for manufacturing the same, for example, urease, an urea removal enzyme for removing urea contained in the dialysate (urease) can be mixed to fix urease to the silk fibroin nanofibrous membrane. Other enzymes can be immobilized on the filter to remove materials other than urea. Silk fibroin filter according to the present invention can control the decomposition rate through the crystallization process, and can effectively fix enzymes such as urease during the crystallization process. The silk fibroin aqueous solution is preferably in a concentration of 1 to 25 w / v%.

The present invention also provides a dialysis solution regeneration filter, characterized in that the urease, urease, which is directly mixed with an aqueous solution of silk fibroin or coated on an electrospun nanofiber membrane, for example, in order to promote urea decomposition. It relates to a manufacturing method.

In order to maximize the effect of the filter for the peritoneal dialysis solution regeneration system, the urea degrading enzyme may be immobilized in the process of producing nanofibers using silk fibroin, which is a natural polymer. For example, urease may be mixed together in a silk fibroin solution and then electrospun, so that the urease is evenly encapsulated in the silk fibroin nanofibers, and through crystallization, a membrane for urease-fixed filter may be manufactured. In addition, the shape of the filter may be prepared in the form of nanofibers as well as nanofibers-beads, simple lyophilized sponges, size-controlled particles as well as nanofiber membranes. The filter for the peritoneal dialysis solution regeneration system according to the present invention prepared as described above may be usefully used as a filter for effectively removing the elements in the dialysate. In addition, the silk fibroin filter and the activated carbon filter layer may be used in combination to be used as a portable peritoneal dialysis solution regeneration filter.

The present invention also preferably further comprises insolubilizing the filter provided above.

For example, the filter according to the present invention preferably has a structure in which a filter in which urease is immobilized and an activated carbon filter are separated (FIG. 6). In order to effectively regenerate the peritoneal dialysis solution of the patient, for example, as shown in Figure 9 divided into urease-immobilized filter layer and activated charcoal filter layer, by removing the urea, creatinine, uric acid, etc. by allowing the peritoneal dialysis solution to go through each step can do.

The present invention will be described by taking an embodiment as an example.

Preparation of Nanofiber Stock

Silk fibroin protein is made by removing sericin from the cocoon. In order to remove sericin protein and impurities from the cocoon (Bombyx mori), the cocoon is heated in a basic aqueous solution such as sodium carbonate, heated and washed with distilled water. Silk fibroin obtained through the above process is dissolved in a salt solution such as lithium bromide or calcium chloride to prepare a silk fibroin aqueous solution. It is then dialyzed with distilled water to remove ionic components from the solution. Dialysis time is preferably 36 to 72 hours. If the dialysis time is less than 36 hours, the removal of ionic components may be insufficient, and exceeding 72 hours is an unnecessary process. The silk fibroin aqueous solution is preferably in a concentration of 1 to 25 w / v%. When the concentration of the aqueous solution of silk fibroin is less than 1 w / v%, the structure of the support is not firm, and when it exceeds 25 w / v%, the silk fibroin solution may become unstable and gel.

The silk fibroin aqueous solution is used to prepare a filter for a peritoneal dialysis solution regeneration system. Polymer may be added to improve the properties of silk fibroin. For example, an aqueous solution of silk fibroin / polyethylene glycol is prepared by adding polyethylene glycol to increase the viscosity and radioactivity of the silk fibroin solution. The volume ratio of the polymer and silk fibroin added at this time is preferably 1: 4 to 1: 5. When the silk fibroin is less than 4, it may be difficult to form a nanofiber structure because the viscosity is low, and when the silk fibroin is greater than 5, the viscosity may be too high to reduce radioactivity. As the polymer, for changing physical properties, one or more selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyvinyliden fluoride, and the like may be selected and added. For example, silk fibroin / polyethylene glycol / urease aqueous solution is prepared by mixing urease as urease. In addition, when preparing a filter further comprising activated carbon, activated carbon may be further mixed with the silk fibroin / polyethylene glycol / urease aqueous solution.

Process for producing peritoneal dialysis fluid regeneration filter

Peritoneal dialysis solution regeneration filter comprises a nanofiber type using an electrospinning method; A type in which nanofibers and beads are mixed using an electrospray method; Sponge type through lyophilization process; It can be produced in various forms, such as a particle type with adjustable size.

For example, in the manufacturing method of the filter using the electrospinning method, the aqueous solution is placed in a syringe and discharged using a delivery pump. At this time, connect the electrode to the syringe needle and apply a constant voltage to the DC power supply. The formed silk nanofibers are collected on a rotating drum or stationary plate surface.

For example, in the filter manufacturing method using the electric injection method, the aqueous solution is placed in an electric injector and discharged using a pressure. At this time, connect the electrode to the discharge port and apply a constant voltage to the DC power supply. The formed silk nanofibers and beads are collected on the stationary plate surface.

For example, in the filter manufacturing method using the freeze-drying method, the aqueous solution is placed in a petri dish and frozen at an ultra low temperature, and then freeze-dried for 12 to 72 hours using a freeze dryer.

For example, the particle type is prepared by slowly dropping the solution into droplet liquid nitrogen and quenching it, followed by a lyophilization process.

Silk fibroin insolubilization and enzyme immobilization process

Silk fibroin filters have a low strength and are very susceptible to moisture and are preferably subjected to insolubilization. The silk fibroin filter produced for this purpose is recrystallized. In this insolubilization process, the degree of degradation of the silk fibroin filter can be controlled and the enzyme can be immobilized on the silk fibroin. It can also control the dissolution rate of enzymes. The resolution and elution rate can be controlled according to the silk fibroin crystallization time and method. The crystallization time is preferably 3 to 24 hours. If it is less than 3 hours, crystallization may not occur, and if it exceeds 24 hours, maximum crystallinity is reached and unnecessary time is required. If the crystallization time of the silk fibroin is shortened, the release rate and degradation rate of the enzyme are increased. If the crystallization time is extended, the release rate and resolution of the silk fibroin are decreased.

In the present invention, the crystallization process for insolubilization of the manufactured silk fibroin filter may be crystallized by immersing in alcohol or treating with alcohol vapor. The alcohol is preferably selected from one or more of the group consisting of methanol, ethanol, propanol and the like. In addition, the crystallization for insolubilization may be carried out by treatment with water vapor. When crystallized by exposure to water vapor, the crystallinity is the lowest since the crystallization degree is the lowest compared to the crystallization with ethanol, methanol, propanol, etc., so that the release rate and degradation rate of the enzyme proceed rapidly. And crystallization is the highest when immersed in ethanol, methanol, propanol and the like crystallization.

Example 1

1) Preparation of Nanofiber Stock Solution

Silk fibroin protein was prepared by removing sericin from the cocoon. To remove sericin protein and impurities from the cocoon (Bombyx mori), the cocoon was added to a basic aqueous solution such as sodium carbonate, heated, and washed with distilled water. Silk fibroin obtained through the above process was dissolved in CaCl ₂ solution to prepare a silk fibroin aqueous solution of 10 w / v%. It was then dialyzed for 40 hours with distilled water to remove ionic components from the solution.

To 20 mL of the silk fibroin aqueous solution, 4 mL of a 30% polyethylene glycol aqueous solution having a molecular weight of 200,000 was added to prepare a silk fibroin / polyethylene glycol aqueous solution.

The aqueous solution of silk fibroin / polyethylene glycol / urease was prepared by mixing urease which is a urease enzyme with the aqueous solution.

2) Process for manufacturing peritoneal dialysis solution regeneration filter

As a method for producing a filter using an electrospinning method, the aqueous solution was placed in a syringe and discharged using a delivery pump. At this time, the electrode was connected to the syringe needle and a constant voltage was applied to the DC power supply. The formed silk nanofibers were collected in a rotating drum.

3) Silk fibroin insolubilization and enzyme immobilization process

Crystallization for insolubilization of the silk fibroin filter prepared above was crystallized by steam treatment of ethanol.

4) Adsorption experiment of activated carbon using peritoneal dialysis solution

Batch and continuous experiments were performed to observe the peritoneal dialysis solution regeneration effect of activated carbon filters. Each experiment was carried out as follows.

① Removal Characteristics of Peritoneal Dialysis Solution According to the Change of Stirring Time (Batch Test I)

Activated carbon was added to 200 mL of the peritoneal dialysis solution, and the stirring time was adjusted to 0, 5, 10, 15, 20, 30, 50, 70, 100 and 120 minutes to observe the change of the peritoneal dialysis solution. Over time, the removal rate of uric acid, calcium and creatinine increased. Among them, creatinine, which is used as an indicator for renal dysfunction, showed the highest elimination rate of about 67%. Uric acid also showed an excellent removal rate of about 64% (FIG. 1).

② Removal Characteristics of Peritoneal Dialysis Solution According to the Change of Activated Carbon Intake (Batch Experiment II)

Activated charcoal in 200mL peritoneal dialysis solution was adjusted to 0.1, 0.2, 0.5, 1.0, 2.0, 3.0g and stirred for 2 hours, and then the change of components of the peritoneal dialysis solution was observed. As activated charcoal input increased, the removal rate of each component increased. Among them, creatinine and uric acid were confirmed to be removed more than 90% (Fig. 2).

③ continuous experiment

In the continuous experiment, 1 g of activated carbon was charged to the column, and the peritoneal dialysis solution was flowed at a rate of 2 mL / min by a pump. Samples were taken at 0, 10, 20, 30, 60, 90, 120, 150, 180, 210, and 240 minutes to observe changes in the components of the peritoneal dialysis solution. When peritoneal dialysis solution was passed through the activated carbon filter, the removal rate of creatinine and uric acid was the highest at 10 minutes, and the removal rate gradually decreased with time, indicating that the adsorption capacity of activated carbon decreased. In the case of phosphorus, the adsorption removal rate was the highest at 60 minutes, and the removal rate was maintained over time (FIG. 3).

5) Structure of silk fibroin / urease filter

The structure of the membrane filter electrospun by mixing silk fibroin and urease was observed through FE-SEM. (FIG. 4) In FIG. 4, the upper photograph is a cross section of the membrane filter and the lower photograph is enlarged. It shows the shape. In the control bottle, the bottle top filter (Poyethersulfone) is in the form of microfibers with voids, and the silk fibroin filter (A), silk fibroin + activated carbon filter (B), silk fibroin + activated carbon + Urease filter (C) are all voids and nano A structure in which fibers and beads are mixed is formed. The pores allow the peritoneal dialysis solution to pass through, and the beads exhibit a structure containing urease or activated carbon.

6) Porosity of Silk Fibroin Filter

In order to control the voids of the silk fibroin filter, electrospinning was performed by dividing the electrospinning collector into a roller type and a plate type, and the porosity of each electrospinning filter was confirmed. Porosity was measured by the following equation. (n = 3)

The porosity was less than 20% for the filter that was electrospun on the roller, and the porosity was over 50% for the plate type. The porosity represents the extent to which voids are formed per unit area. The higher the porosity, the better the permeability of the peritoneal dialysis solution.

7) Peritoneal Dialysis Solution Regeneration Effect of Silk Fibroin Filter

In order to observe the peritoneal dialysis solution regeneration effect of the filter produced by electrospinning the silk fibroin, the silk fibroin electrospun membrane was cut into a diameter of 25 mm and fixed to the filter holder, and then the peritoneal dialysis solution was removed by using a micro pump at a rate of 7 mL / min. Passed through and filtered. After 10 minutes, the filtered peritoneal dialysis solution was taken for blood biochemical measurements. Table 1 The concentrations of urea and creatinine were decreased and the removal rates were 3.0% and 1.83%, respectively.

8) Peritoneal Dialysis Solution Regeneration Effect of Filters Mixed with Silk Fibroin and Activated Carbon

In order to observe the peritoneal dialysis solution regeneration effect of the filter produced by electrospinning the silk fibroin and activated carbon by the 3-way method, the silk fibroin electrospun membrane was cut to a diameter of 25 mm and fixed to the filter holder, and then the peritoneal dialysis solution was 7 mL / min. Filtered by speed. After 10 minutes, the filtered peritoneal dialysis solution was taken for blood biochemical measurements (Table 1). The concentrations of urea and creatinine were decreased, and the removal rates were 3.46% and 3.47%, respectively.

9) urease ( Urease ) Is fixed silk  Peritoneal Dialysis Solution Regeneration Effect of Fibroin and Activated Carbon Filter

In order to observe the peritoneal dialysis solution regeneration effect of the filter produced by electrospinning the solution of silk fibroin and urease and activated carbon by 3way method, the silk fibroin electrospun membrane was cut into 25mm diameter and fixed to the filter holder. After the peritoneal dialysis solution was filtered at a rate of 7 mL / min. After 10 minutes, the filtered peritoneal dialysis solution was taken for blood biochemistry measurement. (Table 1) The concentrations of urea and creatinine were decreased, and the removal rate was 5.46% and 3.62%, respectively.

	Urea concentration			Creatinine Concentration
	Sample	After 10 minutes filter	Removal rate	Sample	After 10 minutes filter	Removal rate
Silk Fibroin Filter	16.31	15.82	3.00%	2.79	2.74	1.83%
Silk Fibroin and Activated Carbon Blend Filter	17.93	17.31	3.46%	2.77	2.67	3.47%
Urease Fixed Silk Fibroin and Activated Carbon Filter	16.31	15.42	5.46%	2.79	2.69	3.62%

 The above examples are only for facilitating the understanding of the present invention, and the present invention should not be considered as being limited thereto.

Claims (12)

1. Portable peritoneal dialysis fluid regeneration filter, characterized in that it comprises an enzyme-immobilized silk fibroin nanofibers.
2. The filter of claim 1, wherein a polymer is added to the silk fibroin nanofibers.
3. The portable peritoneal dialysis solution according to claim 2, wherein the polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyvinyliden fluoride, and the like. Filter for playback.
4. The portable peritoneal dialysis fluid regeneration filter according to claim 1, wherein the enzyme is urease.
5. The portable peritoneal dialysis fluid regeneration filter according to claim 1, wherein the silk fibroin nanofiber further comprises activated carbon.
6. A portable peritoneal dialysis fluid regeneration system, comprising an enzyme-immobilized silk fibroin nanofiber filter layer and an activated carbon filter layer.
7. The portable peritoneal dialysis solution regeneration system according to claim 6, wherein the silk fibroin nanofiber filter layer and the activated carbon filter layers are included in a multilayer.
8. Mixing a silk fibroin aqueous solution, a polymer, and an enzyme to form a silk fibroin-polymer-enzyme mixed aqueous solution;

   Preparing a filter by selecting the mixed aqueous solution from a crowd consisting of electrospinning, electrospraying, and lyophilization; And

   Method for producing a portable peritoneal dialysis solution regeneration filter comprising the step of insolubilizing the prepared filter.
9. The portable peritoneal dialysis solution according to claim 8, wherein the polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyvinyliden fluoride, and the like. Method for producing filter for regeneration.
10. The method of claim 8, wherein the insolubilization step comprises recrystallizing the filter by selecting one or more selected from the group consisting of ethanol, methanol, and propanol.
11. 9. The method according to claim 8, wherein the insolubilization step comprises recrystallizing the filter by treating with water vapor.
12. The method of claim 8, wherein in the insolubilization step, the step of immobilizing the enzyme to the filter is performed simultaneously.

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