KR101535751B1 - Self-assembled microfiber and preparing thereof - Google Patents

Abstract

The present invention relates to nanoparticles using the self-associating property of tropoelastin extracted from human fat, and microfibers produced by binding PEG to the tropoelastin and then self-assembling to induce PEGylation, And microfibers, which can be used as drug delivery vehicles, tissue engineering agents, and biosensors.

Description

≪ Desc / Clms Page number 1 > Self-assembled microfibers and preparing them

The present invention relates to nanoparticles formed by self-association of water-soluble elastin and tropoelastin extracted from adipose tissue of a mammal, and microfibers formed by self-association using tropoelastin and PEG (polyethylene glycol).

Conventional water soluble elastin and tropoelastin extraction methods require five or more steps (high temperature sterilization, removal of water-soluble proteins using NaCl solution, lipid removal using organic solvents, protein removal through toxic reagents, etc.) to remove proteins other than tropoelastin in the recovered tissue Degradation, protein degradation through enzymes, etc.), and then hydrolyzing through oxalic acid at a high temperature of 100 ° C or higher to obtain tropoelastin. Because of the various procedures required, it took more than two weeks and the risk of toxic reagents could not be ruled out. The extraction process of tropoelastin has a great difference in characteristics and components of the finally obtained by-products depending on the conditions of solvent selection, treatment process and time. In the prior art, the tissue used for the extraction of tropoelastin, which is a basic unit of elastin fiber, is mainly an animal ligament or aorta, and it is difficult to mass-process the raw material because it is difficult to supply raw materials. To solve this problem, synthetic elastin Elastin-like Polypeptide (ELP) has been used for recombination, but there has been a problem in that a complicated procedure and synthesis yield based on recombinant methods are low.

Methods for manufacturing nano / microfibers include nanofiber fabrication using electrospinning, and nanofiber growth using chemical vapor deposition (CVD). The method of utilizing electrospinning is to obtain a fiber by collecting the raw material to be electrospun on one side by applying a voltage through a nozzle and collecting the fiber on the opposite side, but it is mainly unsuitable for using individual nanofibers Method. The chemical vapor deposition method is a method of growing nanofibers such as stalactite in a limestone cave by attaching molecules moving on the substrate using a catalyst to form a single component (in particular, metal) nanofiber such as carbon nanofiber Therefore, there have been limitations in application to biotechnology fields such as electronic materials and sensors.

Accordingly, the present inventors have fabricated microfibers capable of forming a fiber while self-assembling a PEG polymer, which is a hydrophilic polymer, to a tropoelastatin having hydrophilic and hydrophobic moieties as if they were block polymers, and self-assembling at a specific temperature. Can be utilized in drug delivery systems, biosensors, and tissue engineering preparations.

It is an object of the present invention to provide a method for producing nanoparticles.

It is still another object of the present invention to provide a method of manufacturing a microfiber.

It is still another object of the present invention to provide a drug delivery system in which a protein or a drug is supported on nanoparticles produced by the nanoparticle production method.

Still another object of the present invention is to provide a method for manufacturing the drug delivery system.

Still another object of the present invention is to provide a biosensor including a microfiber fabricated by the microfiber manufacturing method according to the present invention.

Yet another object of the present invention is to provide a tissue engineering preparation comprising microfibers produced by the microfiber manufacturing method according to the present invention.

In order to achieve the above object, the present invention provides a method for producing nanoparticles.

In addition, the present invention provides a microfiber manufacturing method.

In addition, the present invention provides a drug delivery system in which proteins or drugs are supported on nanoparticles prepared by the method of the present invention.

The present invention also provides a method for manufacturing the drug delivery system.

In addition, the present invention provides a biosensor including a microfiber fabricated by the microfiber manufacturing method according to the present invention.

In addition, the present invention provides a tissue engineering preparation comprising microfibers produced by the microfiber manufacturing method according to the present invention.

The nanoparticles and the microfibers using the self-assembly of the present invention can be produced in a simple manner on a receiving medium as compared with the prior art, and since the produced nanoparticles and microfibers are proteins contained in large amounts in the tissues of the human body, And can also be used as the main material of damaged tissues or tissues that are artificially manufactured for regeneration. In addition, by controlling the reaction ratio of tropoelastin and PEG, the length of the microfibers can be adjusted, so that the artificial tissue can be made as large as the actual damaged tissue size applicable to the clinic. In addition, PEG-elastin has potential for application in biosensors and catalyst fields besides drug delivery and tissue engineering, and will be utilized as useful biomaterials.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing a step of extracting water-soluble elastin and tropoelastin from human adipose tissue.
Fig. 2 is a view showing a step of extracting water-soluble elastin and tropoelastin from fat adipose tissue.
Figure 3 compares the structure of the tropoelastin of the present invention with a conventional tropoelastin product;
Solid line: tropoelastin of the present invention;
Dotted line: Conventional tropoelastin product;
(a): C = O stretching amide;
(b): CN stretch amide; And
(c): NH amide in plane deformation.
FIG. 4 is a photograph showing a particle formation according to the temperature of the tropoelastin of the present invention through a digital camera (disital camera), optical microscopy and scanning electron microscopy (SEM);
A and C: tropoelastin at 4 DEG C;
B, D and E: Tropoelastin at 37 ° C.
5 is a graph showing the particle size according to the concentration and temperature of the tropoelastin of the present invention;
A: A graph showing the particle formation temperature according to the concentration of tropoelastin of the present invention; And
B: Graph showing the size of particles formed according to the concentration and temperature of tropoelastin of the present invention.
FIG. 6 is a photograph showing the size of a formed particle according to the concentration of tropoelastin of the present invention by DLS equipment; FIG.
A: Particle size according to 5 mg / ml concentration of tropoelastin;
B: particle size according to concentration and temperature of 7.5 mg / ml of tropoelastin; And
C: Particle size according to concentration and temperature of 10 mg / ml of tropoelastin.
7 is a photograph showing that the tropoelastin of the present invention is loaded with insulin;
A: Amount of insulin loaded at a concentration of 7.5 mg / ml of tropoelastin; And
B: Insulin carried in tropoelastin particles identified by confocal microscopy.
8 is a photograph showing the microparticles produced by the tropoelastin of the present invention;
A and B: SEM photographs of microparticles formed at a concentration of 7.5 mg / ml of tropoelastin; And
C and D: SEM photographs of microparticles formed by combining 7.5 mg / ml of tropoelastin with PEG (one functional group) in a molar concentration ratio of 1000: 1.
9 is a SEM image of a sphere and an ellipse among the microparticles produced by the tropoelastin of the present invention;
A: SEM photograph of microparticles formed at a concentration of 7.5 mg / ml of tropoelastin;
B: SEM photograph of microparticles formed by binding PEG (one functional group) at a molar concentration ratio of 1000: 1 at a concentration of 7.5 mg / ml of tropoelastin.
10 is a photograph showing a fiber produced according to the reaction ratio of tropoelastin and PEG of the present invention according to SEM magnification.
11 is a photograph showing the length of microfibers produced according to the reaction ratio of tropoelastin and PEG of the present invention.

Hereinafter, the present invention will be described in detail.

The tropoelastin of the present invention can be extracted from adipose tissue of a mammal by the following method.

1) physically grinding mammalian adipose tissue to obtain an extracellular matrix (ECM);

2) adding an aqueous NaCl solution to the extracellular matrix of step 1) and stirring to obtain a residue from which the water-soluble protein has been removed;

3) autoclaving the residue of step 2) to denature the components other than elastin;

4) treating the residue of step 3) with a solution of oxalic acid, hydrolyzing and centrifuging to obtain a supernatant;

5) dialysis of the supernatant of step 4) to replace the acid solvent with an aqueous solvent;

6) lyophilizing the product after dialysis in step 5) to obtain water soluble elastin;

7) adding sodium acetate to the aqueous elastin of step 6) to obtain a precipitate;

8) recovering the precipitate of step 7) with distilled water and removing impurities by a recrystallization method;

9) dialyzing the supernatant of step 8) to remove salts; And

10) A method for extracting tropoelastin comprising lyophilization.

The mammal includes cows, pigs, mice, rabbits, dogs, cats, monkeys, humans and the like, more preferably a human or a pig, and most preferably human.

The present invention provides a method for producing nanoparticles comprising the step of inducing self association by maintaining water-soluble elastin or tropoelastin extracted from adipose tissue of a mammal at a temperature within 40 ° C as described above.

The water-soluble elastin is a mixture of elastin components including tropoelastin, and tropoelastin is a purified protein of the same molecular weight range in water-soluble elastin, both of which have self-associating properties.

It is preferable to adjust the size of the nanoparticles by adjusting the concentration of the water soluble elastin or tropoelastin, but the present invention is not limited thereto.

As the concentration of the water soluble elastin or tropoelastin increases, the size of the nanoparticles increases.

The water-soluble elastin or tropoelastin has an amphipathic structure and a high proportion of specific amino acids (Ala, Gly, Pro, Val) constituting the hydrophobic part may cause self-association at a specific temperature.

Self-assemble phenomena or sol-particle phase transition behavior refers to the phenomenon that a single tropoelastin aggregates several molecules at a lower critical solution temperature (LCST), which is a specific transition temperature. The self-associated molecules have an amphipathic structure like the extract and each part has the following characteristics. Hydrophilic moieties form intermolecular interactions between water molecules on the water solvent and hydrogen bonds. Even if the kinetic energy of the water molecules increases due to the increase of temperature, interaction of some water molecules around the hydrophilic moieties exist. On the other hand, the hydrophobic part, unlike the hydrophilic part, has a strong interaction part strong enough to form a hydrogen bond with the water molecule, so that the interaction with the water molecule is broken due to the temperature rise. As a result, So that the portions cohere. The phenomenon that occurs as a result of agglomeration of hydrophobic parts on water solvent is called self-association or sol-particle phase transition phenomenon.

Fundamentally, tropoelastin refers to the basic unit of elastin fibers. It is a unit that aggregates into aggregated particles by self-association in the body and attaches to the surface of the microfibrils that form the skeleton to form an elastin fiber strand do.

In addition,

1) dissolving tropoelastin and PEG polymer in DW;

2) calculating and mixing the molar concentration ratio between the dissolved tropoelastin and PEG;

3) stirring the mixed solution to bind the tropoelastin and PEG;

4) inducing a self-association at a temperature within 40 ° C of the PEG-tropoelastin polymer synthesized in step 3) to form a microfiber in the longitudinal direction.

The molar ratio of the tropoelastin to the PEG in the step 2) depends on the number of functional groups of the PEG polymer. In the case of one functional group, the molar ratio is preferably 10: 1, and in the case of two functional groups, 1 to 1: 2, but is not limited thereto.

The binding between tropoelastin and PEG in step 3) is preferably a covalent bond, but is not limited thereto.

The method may further include lyophilizing the microfibers formed after step 4), but the present invention is not limited thereto.

The microfibers formed after step 4) may be cross-linked to maintain the shape for mixing with other materials, but the present invention is not limited thereto.

The PEG preferably has a molecular weight (MW) of 3,000 to 30,000, but is not limited thereto.

The self-association induction temperature of step 4) is most preferably 37 ° C, but is not limited thereto.

In the present invention, microfibers refer to fibers having a diameter of 1 占 퐉 or less and a length of more than 1 占 퐉.

The present invention also relates to a method for producing a nanoparticle or microfiber of the present invention, which comprises adding a protein, a polymer (insulin, growth factor, etc.), DNA, RNA, siRNA or a drug Etc.) are supported on the surface of the drug carrier.

Since nanoparticles formed by self-assembly have a porous or hollow structure, it is possible to encapsulate various substances according to needs, such as drugs, proteins and growth hormones, so that they can be utilized in a drug delivery system.

The present invention also provides a method for preparing a drug delivery vehicle, which comprises mixing a protein or a drug with tropoelastin extracted from a fat of a mammal of the present invention, and then maintaining the temperature at 35 to 40 ° C to induce self association do.

In addition, the present invention provides a biosensor including a microfiber fabricated by a microfiber manufacturing method.

According to the method of the present invention, since the surface area from the spherical particle to the fiber shape can be controlled, the sensing ability of the sensor can be controlled and the microfiber of the present invention can be used as a fiber type having a large aspect ratio (width to length ratio, aspect ratio) And can be used for a biosensor of a specific pattern according to purposes.

In addition, the present invention provides a tissue engineering preparation comprising microfibers produced by the microfiber manufacturing method according to the present invention.

Since the main component of the fiber bundle existing in the muscle tissue of the human body is elastin, the nanofiber using the human tropoelastin of the present invention is easy to form the texture of the fiber bundle, so that it can be used as a support for tissue formation damaged or regenerated .

In a specific embodiment of the present invention, the inventors extracted tropoelastin from the adipose tissue of a discarded mammal (see FIGS. 1 and 2) and compared it with conventional tropoelastin to confirm the possibility of self association (FIGS. 3 - 5). As a result of confirming the self association (sol-particle phase transition behavior) of the tropoelastin at various temperatures, it was confirmed that nanoparticles were formed at 37 ° C (see FIG. 4). As the concentration of tropoelastin increased, (See FIGS. 5 and 6), and it was confirmed that proteins or drugs can be carried in nanoparticles formed by tropoelastin (see FIG. 7). Nanoparticles composed of the self-associating tropoelastin of the present invention can be used as drug delivery vehicles.

The tropoelastin of the present invention and the hydrophilic polymer PEG were combined at various molar ratios, and the microfibrils were formed by self-association at 37 ° C (see FIGS. 8 and 9) And the ratio and length were controlled by the reaction ratio of tropoelastin and PEG polymer (see FIG. 9). Therefore, the microfibers using the tropoelastin and PEG of the present invention can be used as a biosensor or tissue engineering preparation.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> from adipose tissue Tropoelastin  extraction

&Lt; 1-1 > Tropoelastin  extraction

The present inventors have extracted tropoelastin from human fatty tissue which is discarded.

Specifically, the extracellular matrix (ECM) was obtained by physically pulverizing the abandoned human adipose tissue (disused adipose tissue recovered according to the patient's consent after plastic surgery). To the obtained extracellular matrix was added 1 M NaCl aqueous solution, and the mixture was stirred at 4 ° C for 2 days and then centrifuged at 4 ° C for 5 minutes to obtain a residue that did not dissolve. The obtained residue was washed with DW (deionized water), and the washed extracellular matrix residue was autoclaved at a temperature of 100 ° C or more for 45 minutes under high pressure for 5 times to remove components other than the elastin component (collagen, polysaccharide, etc. ) Was removed. Thereafter, the remaining extracellular matrix was treated with 0.25M oxalic acid solution at 90 ° C for 1 hour for 10 times to hydrolyze the crosslinked elastin component to recover the water-soluble elastin component. The supernatant obtained by hydrolysis was dialyzed at 4 ° C for 2 days to replace the acid solvent with water. After dialysis, the product was freeze-dried to extract a water-soluble elastin component in the form of a yellow powder at a yield of 10 mg per 100 mg of ECM. The extracted water-soluble elastin was mixed with 0.01 M sodium acetate solution at a ratio of 1: 1 (v / v), incubated at 37 캜 for 10 minutes, and centrifuged at the same temperature for 20 minutes. The resulting yellow viscous precipitate was recovered by DW and the precipitate recovered was recrystallized to remove impurities. The supernatant, from which impurities were removed, was collected and dialyzed at 4 ° C for 2 days to remove salt components. After dialysis, the product was lyophilized to obtain a tropoelastin component in the form of a yellow powder at a yield of 10 mg per 100 mg of water soluble elastin (FIG. 1).

&Lt; 1-2 > Tropoelastin  extraction

The present inventors extracted tropoelastin from swine fat tissue.

Specifically, the pig fat tissue to be discarded (fat tissue recovered by the slaughterhouse in the wholesale market) was minced, 1M NaCl aqueous solution was added, and the mixture was stirred at 4 ° C for 2 days and then centrifuged at 4 ° C for 5 minutes To give a residue that did not dissolve. The obtained residue was washed with DW (deionized water), and the washed extracellular matrix residue was autoclaved at a temperature of 100 ° C or more for 45 minutes under high pressure for 5 times to remove components other than the elastin component (collagen, polysaccharide, etc. ) Was removed. Thereafter, the remaining extracellular matrix was treated with 0.25M oxalic acid solution at 90 ° C for 1 hour for 10 times to hydrolyze the crosslinked elastin component to recover the water-soluble elastin component. The supernatant obtained by hydrolysis was dialyzed at 4 ° C for 2 days to replace the acid solvent with water. After dialysis, the product was freeze-dried to extract a water-soluble elastin component in the form of a yellow powder at a yield of 100 mg per 100 g of tissue. The extracted water-soluble elastin was mixed with 0.01 M sodium acetate solution at a ratio of 1: 1 (v / v), incubated at 37 캜 for 10 minutes, and centrifuged at the same temperature for 20 minutes. The resulting yellow viscous precipitate was recovered by DW and the precipitate recovered was recrystallized to remove impurities. The supernatant, from which impurities were removed, was collected and dialyzed at 4 ° C for 2 days to remove salt components. After dialysis, the product was lyophilized to obtain a tropoelastin component in the form of a yellow powder at a yield of 20 mg per 100 mg of water soluble elastin (FIG. 2).

< Example  2> derived from human fat Tropoelastin  analysis

<2-1> Analysis through amino acid analysis Tropoelastin  Component analysis

In order to confirm whether human tropoelastin (hELN) extracted from <Example 1> can have a self-association at a specific temperature, the inventors of the present invention used a conventional commercially available animal ligament tissue (Elastin, soluble from bovine neck ligament / Sigma Aldrich / Cat. NO E6527) were analyzed and compared with commercial talloelastin (Comm. Elastin).

As a result, the extracted tropoelastin (hELN) of the present invention was also found to be a major component causing the sol-particle phase transition behavior (self-association phenomenon), Ala, Gly , Pro, and Val.

Therefore, it is judged that the human adipose-derived tropoelastin of the present invention can also be self-assembled.

^a Products that are extracted and produced from animal ligament tissue.

<2-2> IR  Through analysis Tropoelastin  Structure analysis

The present inventors performed IR spectroscopy to predict the structure of the tropoelastin extracted in Example 1 above.

Specifically, the structure of the two tropoelastins was compared and analyzed by IR analysis of the tropoelastin extracted from the commercialized animal tissue and the tropoelastin extracted in Example 1 above.

As a result, a peak was found in the same section, which means that the two tropoelastins have the same functional group, thereby confirming the possibility of having a similar structure (FIG. 3).

<2-3> Tropoelastin  Self-Association Evaluation

The inventors of the present invention have confirmed the self-assembly properties of the specific amino acids (Ala, Gly, Pro, Val) constituting the hydrophobic part of the tropoelastin extracted in Example 1 above.

Specifically, in order to confirm the sol-particle phase transition behavior of tropoelastin extracted by the method of the present invention, the temperature was raised at 37 ° C (B, D and E in FIG. 4) at 4 ° C The formation of tropoelastin particles was confirmed.

As a result, it was confirmed by SEM analysis that the tropoelastin particles were mainly formed into spheres at 37 ° C (FIG. 4).

<2-4> Tropoelastin  Determine particle size by concentration and temperature

The present inventors confirmed the particle size of spheres formed according to the concentration of tropoelastin in the self-association of the tropoelastin extracted in Example 1 above.

Specifically, the sol-particle phase transition behavior of tropoelastin extracted from UV-Vis analysis equipment and DLS analysis equipment at 37 ° C was confirmed in order to conduct self-association according to the concentration and temperature of tropoelastin.

As a result, it was confirmed that the self-association temperature was lowered as the concentration of tropoelastin was increased through the UV-Vis analysis equipment (Fig. 5A). As the concentration of tropoelastin was increased through DLS analysis equipment, It was confirmed that the average particle size of the elastin particles was increased (Fig. 5B and Fig. 6).

<2-5> Tropoelastin  Identification of protein or drug loading in particles

The present inventors confirmed the carrying of proteins or drugs into the particles formed by the self-association of the tropoelastin of the present invention.

Specifically, 0.03 mg / ml of insulin was dissolved in 7.5 mg / ml of the tropoelastin solution extracted in Example 1, and self-association was performed at 37 ° C. The insulin-impregnated particles in the tropoelastin were observed under a confocal fluorescence microscope Lt; / RTI &gt;

As a result, it was confirmed that insulin was successfully carried by the self-association of the tropoelastin of the present invention, and the loading amount was 0.03 mg / ml most efficiently (Fig. 7).

< Example  3> Tropoelastin and PEG Micro Fiber  synthesis

<3-1> Tropoelastin  And PEG of Mole ratio  Confirmation of formation of nano / microparticles or fibers

The present inventors confirmed the self association by reacting tropoelastine with PEG, which is a hydrophilic polymer, at a calculated molar ratio.

Specifically, after dissolving the tropoelastin (molecular weight: 75,000) and the PEG polymer (the molecular weight of the PEG having one functional group: 5,000 / 10,000 / 20,000 / 30,000 and the molecular weight of the PEG polymer having two functional groups: 3400) The two polymers dissolved at a reaction ratio according to the molar ratio of the molar ratio shown in Table 2 were mixed. The mixed solution was stirred at 4 캜 with a vortex equipment (equipment name: Vortex Genie 2, manufactured by Scientific Industries) to bind the tropoelastin and PEG. After the coupling reaction, the unreacted material after the coupling was removed through a centrifugal separation filter system having a filtering molecular weight of 50,000. The DWs that were lost after the initial reaction volume were corrected by adding a new DW. The synthesized PEG-elastin polymer was placed in a water bath at 37 ° C for 10 minutes and then lyophilized by rapid freezing in liquid nitrogen. Powdered samples were examined by Scanning Electron Microscopy (SEM) equipment to determine whether nano / microparticles or fibers were formed.

As a result, spherical nanoparticles were formed at a molar ratio of tropoelastin and PEG (one functional group) of 1,000: 1 (FIG. 8), and spherical nanoparticles other than spherical nanoparticles were formed (FIG. 9 ). Microfibers were formed at reaction ratios other than 1,000: 1 (Fig. 10).

PEG  Molecular Weight
(Weight average, g / mol ) Reaction ratio
( ELN : PEG ) Reaction time
(time) Number of functional groups
( succinimidyl succinate , amount) 3,400 2: 1 48 2 1: 2 24 5,000 1,000: 1
10: 1 24 One 10,000 20,000 30,000

(Tropoelastin (ELN): 7.5 mg / ml)

<3-2> Tropoelastin  And PEG  Depending on the reaction rate Micro Fiber  length adjusting

The present inventors confirmed the length of the microfibers formed according to the reaction ratio of tropoelastin and PEG.

Specifically, the PEG polymer (molecular weight: 3,400, two functional groups) was bound to 7.5 mg / ml of the extracted tropoelastin by the method of Example <3-1> (reaction ratio, tropoelastin: PEG = 2: 2) Microfibers produced by self-association. The microfibers formed by the SEM image were confirmed to have a microfiber length of 1: 2 (FIG. 11B) shorter than that of the tropoelastin: PEG polymer at a reaction ratio of 2: 1 (Fig. 11).

Thus, it can be seen that the length of the generated fiber can be controlled by controlling the reaction ratio of the tropoelastin: PEG polymer.

Claims (7)

1) physically grinding mammalian adipose tissue to obtain an extracellular matrix (ECM);
2) adding an aqueous NaCl solution to the extracellular matrix of step 1) and stirring to obtain a residue;
3) autoclaving the residue of step 2);
4) treating the residue of step 3) with oxalic acid solution, hydrolyzing and centrifuging to obtain a supernatant;
5) dialysis of the supernatant of step 4) to replace the acid solvent with an aqueous solvent;
6) lyophilizing the product after dialysis in step 5) to obtain water soluble elastin;
7) adding sodium acetate to the aqueous elastin of step 6) to obtain a precipitate;
8) recovering the precipitate of step 7) with distilled water and removing impurities by a recrystallization method to obtain a supernatant;
9) dialyzing the supernatant of step 8) to remove salts;
10) lyophilizing the product after dialysis in step 9) to obtain tropoelastin;
11) dissolving the tropoelastin and the polyethylene glycol (PEG) polymer of step 10) in the DW;
12) calculating and mixing a molar concentration ratio between the dissolved tropoelastin and polyethylene glycol (PEG);
13) stirring the mixed solution to bind the tropoelastin and the polyethylene glycol (PEG);
14) The polyethylene glycol (PEG) -tropoelastin polymer synthesized in the step 13) is put into a water tank at a temperature of 35 to 40 ° C to induce self association, and polyethylene glycol (PEG) bonded to the tropoelastin is pegylated, ; And
15) lyophilizing the self-assembled polyethylene glycol (PEG) -tropoelastin polymer in step 14).
The method according to claim 1,
Wherein the molecular weight of the polyethylene glycol (PEG) in the step 11) is 3,000 to 30,000 Mw.
The method according to claim 1,
Wherein the functional group of the polyethylene glycol (PEG) in step (11) is one or two.
The method according to claim 1,
Wherein the molar concentration ratio of tropoelastin and polyethylene glycol (PEG) in step 12) is in the range of 10: 1 to 1:10.
The method according to claim 1,
Wherein the microfiber length is adjustable by adjusting the molar ratio of the reaction mole of the tropoelastin and the polyethylene glycol (PEG) in step 12).
A biosensor comprising a microfiber fabricated by the microfiber manufacturing method of claim 1.
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