KR102033331B1 - Cell-instructed delivery system using tuning the toughness of an alginate microgel - Google Patents

Abstract

The present invention relates to a cell-driven delivery system through the control of the mechanical properties of the alginate microgel, according to the present invention, a microgel cross-linked with calcium ions by introducing methacrylate to the alginate molecule and mixed with the pure alginate molecule, and polyethylene The addition of glycol diacrylate and the addition of chemical crosslinking together with the ionic crosslinking can produce microgels having various strengths and toughness through grafting of methacrylate and control of chemical crosslinking. Prepared by ion crosslinking, the microgel system has a toughness (W _T ) of 213 J / m ² and a compressive strength (E) of 7.5 kPa to ensure that the neurons break up and release the gel after proliferation and differentiation. In addition, the cell-driven delivery system of the present invention systematically regulates the mechanical properties of the gel according to the cell towing and can control the release according to the degree of differentiation of the gel. Can be utilized.

Description

Cell-instructed delivery system using tuning the toughness of an alginate microgel}

The present invention relates to a cell-driven delivery system through the control of mechanical properties of alginate microgels.

Conquering diseases using cells is an important task in the life sciences, and has attracted attention in most medical fields, including the cardiovascular system, nervous system, and blood. In particular, the application of stem cell therapy to degenerative diseases considered incurable has led to many positive results. From these results, cell utilization is being evaluated as a state-of-the-art medical technology that can bring about dramatic changes in drug and surgical-oriented clinical treatment.

However, the procedure that is performed by using only a single cell has been pointed out various problems by clinical results, the biggest problem is the target and efficiency. In detail, in the case of a conventional injectable therapeutic agent, there is a possibility that the injected cells may be scattered with the problem that it may not be correctly delivered to the target site, and there is a report that it is difficult to settle and differentiate. There is a bar. Therefore, as a study for solving the problem of efficiency of cell delivery, researches on how to accurately deliver in a stable form to the site to be treated is progressing through cooperation in various fields of research.

In other words, cell therapeutics include the use of autologous, allogenic, or xenogenic cells, such as immune cells and stem cells, for the purpose of treating, diagnosing, and preventing disease. In order to maximize the effectiveness of therapeutic agents, research on cell delivery systems that deliver and transplant cells safely to target sites in the human body is emerging. In general, cell delivery systems use cell-derived extracellular matrix (ECM) -derived biomaterials to attach or encapsulate cells to protect them from external stimuli, proliferation and differentiation. It is delivered to the human body. To this end, research on customized cell carriers suitable for various cells such as immune cells, stem cells, nerve growth cells and retinal cells is underway. Recently, research has been reported to control cell delivery by an external magnetic field by attaching cells to a porous magnetic hydrogel. As such, unlike general drugs, cells grow, proliferate, and differentiate, and thus, a technique for controlling the cells to be released at an optimal time from the carrier to a target site in the human body is required.

Republic of Korea Patent No. 10-1239782 (Registered Feb. 27, 2013)

An object of the present invention is to provide a cell-driven delivery system through the control of mechanical properties of alginate microgels.

In order to achieve the above object, the present invention comprises a first step of preparing an alginate and methacryl alginate mixture; Dropping the mixture into a calcium chloride solution to prepare a microgel solution through an ion crosslinking reaction; And a third step of enclosing the nerve cells in the microgel by reacting the microgel solution with the nerve cells.

The present invention also provides a microgel for neuron delivery prepared by the above method.

According to the present invention, a microgel cross-linked with calcium ions by introducing methacrylate into an alginate molecule and mixed with a pure alginate molecule, and a microgel added with crosslinking chemicals by adding polyethylene glycol diacrylate to Microgels with varying strength and toughness can be prepared by controlling the degree of grafting and chemical crosslinking of acrylates. Prepared by ion crosslinking, the microgel system has a toughness (W _T ) of 213 J / m ² and a compressive strength (E) of 7.5 kPa to ensure that the neurons break up and release the gel after proliferation and differentiation. In addition, the cell-driven delivery system of the present invention systematically regulates the mechanical properties of the gel according to the cell towing and can control the release according to the degree of differentiation of the gel. Can be utilized.

Figure 1 shows a cell-driven delivery system through the attraction of the alginate microgels.
Figure 2 shows (a) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1-ethyl-3- (3) between the carboxylate group of alginate and the primary amine group of 2-aminoethyl methacrylate. -dimethylaminopropyl) carbodiimide (EDC) chemistry through the synthesis of methacryl alginate, (b) alginate and (c) ¹ H NMR (D ₂ O) spectra of methacryl alginate (MA) -5.
Figure 3 (a) using a universal testing machine (universal testing machine, UTM) to confirm the force and strain displacement applied to the alginate microgel and (b) compressive strength (E).
Figure 4 shows (a) the force and displacement curves applied to the alginate microgels as stress-strain conversion curves, and (b) the area under the stress-strain curves as fracture (W _T ) values.
5 is a microscopic image of PC12 cells encapsulated in (a) a microgel prepared by mixing methacryl alginate and alginate, (b) a field emission scanning electron microscope (FE) of lyophilized MA-G. -SEM image, (c) fluorescence microscopy image of PC12 cells grown and proliferated in MA-G and (d) X-MA-G (microgel cross-linked with UV after preparation of MA-G by addition of PEGG and photoinitiator) ) Shows microscopic images and MTT images of cells encapsulated.
6 shows (a) microscopic images of PC12 cells in MA-G broken at day 14, (b) FE-SEM images of broken MA-G, (c) fluorescence microscopy images of PC12 cells in MA-G, ( d) Phase change image of cells after neuronal growth factor (Nerve Growth Factor-β, NGF) treatment and (e) neurite growth identified by the length of neurites in the cells.

The inventors of the present invention designed a cell-driven delivery system capable of controlling the release according to the degree of differentiation of cells by controlling the mechanical properties of the cell delivery system. Alginate microgels with varying strengths and toughnesses were prepared by controlling the degree of methacrylate grafting and chemical crosslinking, and compared with alginate microgels, the alginate microgels had a similar level of strength but had higher toughness and lower toughness than other toughness gel systems. Gel system was prepared and applied as a neuronal growth carrier. In a microgel system having a W _T of 213 J / m ² and an E of 7.5 kPa, the cell-derived delivery system of the present invention can be used as a cell therapeutic agent by confirming that the cells that have differentiated into neurons are released from the gel. Confirming the present invention was completed.

The present invention comprises a first step of preparing an alginate and methacryl alginate mixture; Dropping the mixture into a calcium chloride solution to prepare a microgel solution through an ion crosslinking reaction; And a third step of enclosing the nerve cells in the microgel by reacting the microgel solution with the nerve cells.

The method for preparing methacryl alginate of the first step comprises the steps of: i) dissolving alginate in 2- (N-morpholino) ethanesulfonic acid to prepare a melt; ii) adding and stirring 1-hydroxybenzotriazole, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 2-aminoethyl methacrylate to the lysate to prepare a reaction; And iii) dialysis of the reactant with distilled water and freeze-drying to prepare methacryl alginate.

The methacryl alginate of the first step may be, but is not limited to, the grafting degree of the methacrylate molecules introduced into the alginate molecule may be 3 to 7 mol%. If the grafting degree of the methacrylate molecule is out of the above range, the mechanical properties of the microgel may be undesirable to encapsulate the cells.

The mixture of the first step specifies that alginate and methacryl alginate may be included in a weight ratio of 1: 1 to 2: 1, but is not limited thereto.

The neuron of the third step may be reacted at a cell concentration of 1 × 10 ⁴ to 1 × 10 ⁶ per mL of the microgel solution, but is not limited thereto.

The present invention also provides a microgel for neuron delivery prepared by the above method.

The microgels have a work to fracture (W _T ) of 150 to 250 J / m ² , and may have an elastic modulus (E) of 6.5 to 8.5 kPa, but are not limited thereto. . When the microgel has the toughness and compressive strength of the above range, the structural stability of the gel is maintained to allow the growth and proliferation of neurons encapsulated inside the microgel to proceed stably. Increasing traction forces nerve cells to break up the microgel and be released to the target site.

The average particle diameter of the microgel may be 100 to 5000 μm, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

EXAMPLE  1: Synthesis of Methacrylic Alginate (MA)

Alginate (Sigma) was added to 1.0% (w / v) in 0.1 M 2- (N-morpholino) ethanesulfonic acid (MES, Sigma) buffer at pH 6.4. It melt | dissolved fully at 30-40 degreeC, adding and stirring. When completely dissolved, 1-hydroxybenzotriazole (HOBt, Sigma), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1-ethyl-3- (3) -dimethylaminopropyl) carbodiimide, EDC, Sigma) and 2-aminoethyl methacrylate (2-aminoethyl methacrylate, AEMA, Sigma) were added slowly and reacted with stirring for 18 hours. After the reaction, the reaction solution was dialyzed with distilled water for 3 days and then lyophilized to obtain methacrylate-grafted alginate molecules (Methacrylic Alginate, MA).

Synthesized MA was dissolved in D ₂ O and analyzed by ¹ H NMR (Avance II, Bruker Biospin) at 400 MHz. The degree of substitution (DS) of MA was calculated by measuring free carboxylate groups that were not reacted by titration with sodium hydroxide (NaOH, Sigma).

Example 2: Alginate Microgel Preparation

A pre-polymer solution was prepared at 1.5% (w / v) in aqueous solution by mixing pure alginate molecules alone or with MA. Alginate microgels were prepared by dropping the gel solution into 0.5 M calcium chloride (calcium chloride, CaCl ₂ , trielectrochemical) solution using a 26 gauge needle into droplets.

In order to introduce IPN through chemical crosslinking inside the microgel, 2% (w / v) polyethylene glycol diacrylate (PEDA) (M _n : 575 g / mol, Sigma) and a photoinitiator Irgacure 2959 (Sigma) 0.1% (w / v) was added to prepare a microgel in the same manner, and then irradiated with UV (365 nm) for 10 minutes.

Example 3: Structure and Characterization of Alginate Microgels

Mechanical properties of alginate microgels were performed using a universal testing machine (UTM, DrTech). The set value of the universal testing machine was applied equally to the test speed of 0.5 mm / min and the load range of 1.0 kg _f . Compressive strength (Young's elastic modulus, E) was measured using the following formula 1 (Hertz contact theory) corresponding to the spherical elastic body.

[Calculation 1]

F is the force applied to the alginate microgel by height, R is the radius of the alginate microgel, h is the displacement, and ν is the Poisson's ratio of the microgel, and is based on rubbery theory. Is assumed to be 0.5.

The toughness of the alginate microgels was measured by compressing in a uniaxial direction until the microgels fractured. The applied force (F) and displacement (ΔD) curves were converted into ‘pseudo’ stress (σ) and strain (ε) curves (Equations 2 and 3). This is due to the assumption that given a high strain on the microgel, it can be regarded as a cylinder form.

[Calculation 2]

[Calculation 3]

A ₀ is the central cross-sectional area (area) of the microgel before compression, and D ₀ is the diameter of the microgel before compression. Toughness was measured by calculating the W _T (work to fracture, J / m ² ) value from the stress and the area of the strain curve until the microgel fractured (ΔD _T , ultimate displacement).

The microstructure of the microgel was observed using a scanning microscope (FEscanning electron microscope, JEOL-7001F).

Example 4 Observation of Neuronal Cell Inclusion and Cell Differentiation

Nerve cell lines (PC12, ATCC) were treated with 10% horse serum (HS, Biowest), 5% fetal bovine serum (FBS, Biowest), 1% penicillin in a Roswell Park Memorial Institute medium (RPMI-1640, Biowest) in a 75 cm ² flask. -Streptomycin (P / S, Biowest) was added to the culture medium at 37 ℃, 5% CO ₂ environment using.

Cultured nerve growth cells were encapsulated in each alginate microgel at a concentration of 2 × 10 ⁵ cells in 1.0 mL alginate gel solution. At this time, 0.3 mL of pre-treated collagen (collagen, 6.0 mg / mL, Advanced Bio-Matrix) solution was added to the gel solution to provide a growth environment for the enclosed cells. Microgels encapsulated with cells were washed twice with phosphate buffered saline (PBS, Biowest), grown and grown in culture.

After 5 days of culture, 1.0 μM of dexamethasone (Dexamethasone, BioReagent) and 50 ng / mL of neuronal growth factor (Nerve Growth Factor-β, NGF, Sigma) were added to promote differentiation into neurons. At this time, NGF was dissolved in PBS containing 0.1% bovine serum albumin (BSA, Sigma). The culture was replaced every two days, and the growth and neurite growth of the cells were observed by optical microscopy (Nikon Eclipse TS100).

The cells were encapsulated and the microgels cultured for 14 days were washed twice with PBS and fixed with 4% (v / v) formaldehyde solution at room temperature for 10 minutes. Treatment with 0.2% (v / v) Triton-X 100 (Sigma) solution increased cell permeability. Cells of the microgels were blocked with 1% (w / v) BSA solution for 30 minutes and blocked and washed again with PBS. Thereafter, FITC tagged phalloidin (Sigma) and DAPI (4,6-diamidino-2-phenylindole, Sigma) were treated for 12 hours and stained. Stained cells were observed using a fluorescent microscope (Nikon Eclipse Ti) and images were analyzed by ImageJ.

Example 5: Mesenchymal Stem Cell Inclusion and Cell Differentiation

Mesenchymal stem cells (D1, MSC, ATCC) were cultured in a 75 cm ² flask at 37 ° C using Dulbecco's Modified Eagle's Medium (DMEM, ATCC) added with 10% HS, 5% FBS, 1% P / S. Incubated in a 5% CO ₂ environment.

Cultured mesenchymal stem cells were encapsulated in each alginate microgel at a concentration of 2 × 10 ⁵ cells in 1.0 mL alginate gel solution, and the same experiment as that of the neuron of Example 4 was performed. The mesenchymal stem cells were encapsulated inside the gel and grown, and then the gels were broken and released according to differentiation (cartilage, fat and bone cells).

Experimental Example 1 Synthesis of Methacrylic Alginate (MA)

In the present invention, by controlling the mechanical properties of the cell transporter, a cell-instructed delivery system designed to control the release according to the degree of differentiation of the cell (Fig. 1). Alginate microgels (microgel, alginate-calcium chelate complex) cross-linked with Ca ²⁺ ions were used to encapsulate the cells and to allow proliferation and differentiation. The physical properties of alginate microgels can be controlled by various methods such as addition of polymer or fiber reinforcement, external coating of gel with polymer film, use of heavy ion crosslinking agents such as Zn ²⁺ , Sr ²⁺ , Ba ²⁺ , and formation of IPN (interpenetrating network) inside gel The study was conducted.

Methacrylic alginate (MA) was successfully synthesized by introducing AEMA into pure alginate molecules (FIG. 2 (a)). As shown in the ¹ H NMR analysis results of FIGS. 2 (b) and 2 (c), it can be confirmed that acrylates have been successfully incorporated into the alginate backbone. The degree of graft can be determined by using the characteristic peak (A: -CH-) represented by the repeating unit of the alginate molecule and the characteristic peak (E: -CH _2- ) analysis value of AEMA. The degree of substitution (DS) is defined as the number of molecules substituted with acrylate molecules per 100 repeating unit molecules of alginate. In Formula 4, n means the number of unit molecules substituted with acrylate molecules, m means the number of unit molecules not substituted.

[Calculation 4]

In the present invention, two kinds of MA-5 and MA-30 were synthesized such that the grafting degree of acrylates was 5 mol% and 30 mol%, respectively. The synthesized MA was titrated with NaOH and the number of unreacted COOH was measured to confirm that the grafting degree of the acrylates introduced into the alginate was 4.5 mol% (MA-5) and 32.0 mol% (MA-30), respectively.

Experimental Example 2: Preparation of alginate microgels and physical property analysis

Alginate microgels were prepared by dropping a gel solution into CaCl ₂ solution in droplet form by mixing pure alginate molecules alone or with MA.

As shown in Table 1, the microgels made of alginate molecules alone were named alginate-G and the microgels made of MA, respectively. Furthermore, after preparing MA-G by adding photoinitiator and PEGDA, the microgel cross-linked with UV inside the gel was named X-MA-G (cross-linked MAG).

In this case, the MA molecule may use MA-5 having a acrylates grafting degree of 5 mol and MA-30 having a 30 mol%, but in the present invention, the microgel is limited to MA-5 (MA-G, X-MA-G). ) Was prepared. When MA-G was prepared with MA-30, the mechanical properties of the microgel showed insufficient results for encapsulating the cells. This is because the number of free COOH that can be ion-crosslinked decreases by increasing the number of unit molecules of MA substituted with acrylates. However, in the preparation of X-MA-G, the use of MA-30 molecules with a large number of acrylates can increase the mechanical properties of the gel due to UV crosslinking.

Sample Diameter
(mm) Modulus
(kPa) Ultimate
strength (kPa) Ultimate
strain (%) Work to fracture (J / m ² ) Alginate-G 3.0 ± 0.3 7.7 ± 0.4 490.1 ± 34.9 94.1 ± 2.2 491.5 ± 6.4 MA-G 3.0 ± 0.1 7.5 ± 1.1 242.5 ± 9.7 87.1 ± 3.1 213.3 ± 15.7 X-MA-G 2.7 ± 0.2 10.9 ± 2.5 186.1 ± 51.9 78.8 ± 2.3 159.5 ± 68.5

Alginate-G and MA-G prepared as described above did not show a significant difference in size on the average of 3.0 mm, but X-MA-G decreased slightly in size of 2.7 mm (Table 1). This is because the inside of X-MA-G formed IPN between MA and PEGDA by UV crosslinking.

Figure 3 (a) is a result showing the force and strain (displacement) applied to the spherical microgel with UTM. Based on this, the compressive strength (E) of each microgel was calculated by Formula 1 (Hertz contact theory).

3 (b) shows that the E-value of MA-G is 7.5 ± 1.1 kPa, which is not significantly different from alginate-G showing 7.7 ± 0.4 kPa. When the MA-5 and alginate were mixed in the same amount using MA-5 having 5 mol% acrylates, the number of ion-crosslinkable free COOH was 97.5% compared to alginate-G. Because. However, in the case of X-MA-G, it can be seen that the E value is significantly increased to 10.9 ± 2.5 kPa, because not only physical crosslinking due to Ca ²⁺ but also chemical crosslinking due to MA and PEGDA occurred inside the gel. In the present invention, 5 mol% of MA-5 and 2% (w / v) of PEGDA were used. By controlling the grafting of MA and the amount of PEGDA, microgels having various E values could be prepared. .

4 (a) is a curve obtained by converting the force and displacement curves applied to the microgel into 'pseudo' stress (σ) and strain (ε). Given a significant deformation to the spherical microgel can be regarded as a cylindrical shape as shown in Fig. 4 (a-1), it was converted using the formula (2, 3). The stress and strain at break of the microgel are named as ultimate strength and ultimate strain, respectively. In addition, the toughness of the gel was measured by calculating the W _T (work to fracture, J / m ² ) value from the stress and area of the strain curve until the microgel was broken.

4 (b), it can be seen that the W _T value (159.5 ± 68.5 J / m ² ) of X-MA-G is the lowest. The compressive strength (E) is increased by crosslinking with IPN inside the gel, but as the chemical crosslinking increases, the plastic zone is relatively reduced, which can be interpreted as a lower W _T value. In general, according to the Maxwell model, gels formed by ion crosslinking exhibit spring-damper behavior, while gels formed by covalent crosslinking exhibit simple spring behavior. Therefore, alginate-G has relatively high toughness due to easy energy dissipation through decross-linking and re-cross-linking process due to deformation. Therefore, X-MA-G is a gel with high compressive strength and low toughness compared to alginate-G. Further, interestingly, it was confirmed that significantly decreased than the value W _T of MA-G (213.3 ± 15.7 J / m 2) W T value of the alginate-G (491.5 ± 6.4 J / m 2). Compared to the E-value of alginate-G which did not show a significant difference from the E-value of alginate-G because it was made using MA-5 (Fig. 4 (b)), the W _T value was about 43% of alginate-G. It was confirmed that the decrease. This means that it is possible to manufacture microgels with low toughness while maintaining similar strengths. This is because 2.5% of the molecular units substituted with acrylates act as defects. In other words, compared to alginate-G, it can be interpreted as the deformation acts on MA-G, which undergoes de-cross-linking and accelerates deformation when it encounters defects.

In conclusion, in the present invention, alginate-G, MA-G, and X-MA-G were prepared for application in cell-driven delivery systems, and (1) gel systems having similar strength but different toughness and (2) strength. Was used as a model for the gel system with high but low toughness.

Experimental Example 3: Preparation of Cell Driven Delivery System

Neurons were enclosed in each alginate microgel at a concentration of 2 × 10 ⁵ in 1.0 mL alginate gel solution. The neural growth cells were encapsulated inside the gel and then grown to design a cell-driven delivery system that could break and release the gel when a certain level of differentiation progressed.

Alginate-G with E of about 7.7 kPa and W _T of 491.5 J / m ² maintains the structural stability of the gel for about two weeks as the cells undergo proliferation and differentiation within the gel, preventing cells from being released out of the gel. Could not. This is because alginate-G has sufficient toughness compared to cell towing. In addition, X-MA-G, which has a low W _T value of 159.5 J / m ² but has a relatively increased E (10.9 kPa), also degrades the growth and proliferation of the cells itself, so that the encapsulated cells are gelled. It could not be broken and released (Fig. 5 (d)).

On the other hand, in the case of MA-G, a gel system having a strength similar to that of alginate-G but lowering the W _T value to 43%, cell growth and proliferation within the gel proceeded stably (Fig. 6 (a)). Then, when a certain level of cell differentiation progressed, it was confirmed that the microgels were broken and the cells were released.

First, as shown in FIG. 5 (a), about 2 × 10 ³ neuronal growth cells were packed per MA-G gel and cultured in 37 ° C. and 5% CO ₂ . As shown in the SEM image of Figure 5 (b), since the pores (pore) well developed in the form of a three-dimensional network inside the gel, the proliferation of the cells could be actively progressed. Nerve growth cells were stably grown and proliferated to form a cell cluster of about 100 μm, as shown in the staining image of FIG. 5 (c). At 5 days of culture, dexamethasone and NGF were added to the culture to promote differentiation into neurons. As a result, as cell differentiation progressed, it was confirmed that the cell towing increased and the gel began to break (FIG. 6 (a)). When the broken gel is confirmed by the SEM image, it can be observed that the fracture progressed from the inside of the gel (FIG. 6 (b)). This is because, as shown in FIG. 6 (c), neurites grew from the cell clusters and the cell towing force was increased (FIG. 6 (e)). On the other hand, gels without cells were retained structural stability in the culture. That is, when the total traction force through the proliferation and differentiation of cells is greater than the W _T value of MA-G, it can be interpreted that the hatching of the gel proceeds. The transfer of differentiated neurons can be utilized for neuromuscular junction (NMJ) research by grafting muscle cells at the target site.

On the other hand, in the case of alginate-G and MA-G encapsulated with mesenchymal stem cells rather than neurons, the gel was broken before reaching differentiation. This is because cell towing due to growth and spreading in mesenchymal stem cells is much larger than that of neurons. Therefore, MA-G can be used as a customized cell carrier according to the degree of differentiation of neurons, but it can be seen that mesenchymal stem cells require a gel system having stronger toughness than Alginate-G and MA-G.

Therefore, the cell-driven delivery system of the present invention can be applied to various cells such as stem cells, immune cells, and can be utilized as an effective cell delivery system by measuring and controlling the cell towing and the mechanical properties of the gel according to the released cell state. In addition, it can be developed as an integrated cell transporter by breaking the gel and conducting research on the behavior and movement of the released cells to the target site.

As described above in detail certain parts of the present invention, it is apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

The scope of the present invention is represented by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.

Claims (9)

A first step of preparing an alginate and methacryl alginate mixture;
Dropping the mixture into a calcium chloride solution to prepare a microgel solution through an ion crosslinking reaction; And
A third step of enclosing the neurons inside the microgel by reacting the microgel solution with the neurons;
Wherein, the methacryl alginate has a grafting degree of 3 to 7 mol% of the methacrylate molecule introduced into the alginate molecule, and neurons encapsulated inside the microgel proliferate and differentiate to break the gel and release to the target site. Method for producing a microgel for neuronal cell delivery, characterized in that. The method of claim 1, wherein the first step of preparing methacryl alginate is
i) dissolving alginate in 2- (N-morpholino) ethanesulfonic acid to prepare a lysate;
ii) adding and stirring 1-hydroxybenzotriazole, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 2-aminoethyl methacrylate to the lysate to prepare a reaction; And
iii) dialysis of the reactant with distilled water and lyophilization to prepare methacryl alginate. delete The method of claim 1, wherein the mixture of the first step comprises alginate and methacryl alginate in a weight ratio of 1: 1 to 2: 1. The method of claim 1, wherein the neurons of the third step are reacted at a cell concentration of 1 × 10 ⁴ to 1 × 10 ⁶ per 1 mL of the microgel solution. A microgel for neuronal cell delivery prepared by the method of any one of claims 1, 2, 4 and 5. The nerve according to claim 6, wherein the microgel has a work to fracture (W _T ) of 150 to 250 J / m ² and an elastic modulus (E) of 6.5 to 8.5 kPa. Microgels for Cell Delivery. The method of claim 6, wherein the average particle diameter of the microgel is a microgel for neuronal cell delivery, characterized in that 100 to 5000 μm. delete

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