KR102180045B1 - dual-mode drug release hydrogel and method of fabricating the same - Google Patents

Abstract

It relates to a dual-mode drug-releasing hydrogel and a method of manufacturing the same, wherein the drug release is sequentially controlled by a swelling-controlled and decomposition-controlled mechanism due to the adjustable mechanical and biodegradable properties of the hydrogel. As it shows kinetics, it can be usefully used in the field of drug delivery technology.

Description

Dual-mode drug release hydrogel and method of fabricating the same}

It relates to a dual mode drug release hydrogel and a method for preparing the same.

Hydrogels have high biocompatibility due to their high moisture content, porous structure, and similarity with extracellular matrix. Due to these characteristics, it has been extensively studied in biomedical fields such as drug delivery systems and tissue engineering.

As a drug delivery system, hydrogel can maximize the therapeutic effect with minimal administration by continuously maintaining a specific drug concentration in surrounding tissues for a long time. Therefore, in order to slowly release the drug for a desired period, the strength and porosity of the hydrogel, the degree of decomposition, and the like are adjusted.

Hydrogels used for drug delivery are generally formed outside the body and mixed with the drug prior to injection into the body. However, due to the nature of the gel, it is difficult to inject through injection, and there is a problem that the gel must be formed outside and implanted into the body. And it is also difficult to induce complete biodegradation of the implanted hydrogel. In order to solve this problem, many studies are being conducted on the development of a formulation that is in the form of a solution outside the body and is easy to inject, and a gel is formed after injection into the body to sustainably release the drug (Korean Patent Registration No. 10-1815780). , The situation is still insufficient.

One aspect is poly (ethylene glycol) diacrylate (poly(ethylene glycol) diacrylate: PEGDA) at a concentration of 5% to 30% (w/v); And it is to provide a composition for drug delivery comprising a hydrogel in which polyethyleneimine (polyehtleneimine: PEI) of 2.8% to 10% (w/v) concentration is crosslinked with each other.

Another aspect is (a) preparing a hydrogel precursor solution by mixing a PEGDA solution at a concentration of 5% to 30% (w/v), a PEI solution at a concentration of 2.8% to 10% (w/v), and a solvent ; And (b) comprising the step of dissolving the drug in the prepared hydrogel precursor solution, to provide a method for producing a hydrogel for drug delivery.

One aspect is poly (ethylene glycol) diacrylate (poly(ethylene glycol) diacrylate: PEGDA); And polyethyleneimine (polyehtleneimine: PEI) provides a composition for drug delivery comprising a hydrogel crosslinked with each other (Crosslinking).

As used herein, the term "poly(ethylene glycol) diacrylate (PEGDA)" refers to the hydroxyl end group and acryloyl chloride (poly(ethylene glycol): PEG). acryloyl chloride) may be generated by a nucleophilic substitution reaction between, and has a structure as shown in Formula 1 below.

[Formula 1]

In Formula 1, n may be an integer of 5 to 400. For example, 5 to 300, 5 to 200, 5 to 180, 5 to 160, 5 to 140, 5 to 120, 5 to 100, 5 to 80, 6 to 400, 6 to 300, 6 to 200, 6 to 180, 6 to 160, 6 to 140, 6 to 120, 6 to 100, 6 to 80, 7 to 400, 7 to 300, 7 to 200, 7 to 180, 7 to 160, 7 to 140, 7 to 120, 7 to 100, 7 to 80, 8 to 400, 8 to 300, 8 to 200, 8 to 180, 8 to 160, 8 to 140, 8 to 120, 8 to 100, 8 to 80, 9 to 400, 9 to 300, 9 to 200, 9 to 180, 9 to 160, 9 to 140, 9 to 120, 9 to 100, 9 to 80, 10 to 400, 10 to 300, 10 to 200, 10 to 180, 10 to 160, It may be 10 to 140, 10 to 120, 10 to 100, 10 to 80.

As used herein, the term "polyethyleneimine (PEI)" has an amine as a functional group as shown in Formula 2 below, and may be a branched polyethyleneimine containing primary, secondary and tertiary amine groups.

[Formula 2]

As used herein, the term "crosslinking" may refer to a physical crosslinking method and a chemical crosslinking method. Even if the same polymer is used, a hydrogel with completely different properties can be obtained depending on the crosslinking method. The physical crosslinking method refers to the reversible crosslinking method by ionic bonding, hydrogen bonding, Van der Waals force, hydrophobic interaction, and crystal of a polymer. The chemical crosslinking method refers to an irreversible crosslinking method by covalent bonds, and forms a more stable crosslinking structure than the physical crosslinking method. According to an embodiment, the crosslinking may be a chemical bond (covalent bond).

In one embodiment, the crosslinking may occur by forming a covalent bond between carbon-nitrogen between the acryl group of PEGDA and the amine group of PEI, through which a hydrogel may be formed.

As used herein, the term "hydrogel", also referred to as hydrogel or hydrogel, is a hydrophilic polymer network that forms three-dimensional crosslinks, and has a high moisture content and thus exhibits almost similar elasticity to natural tissues. In addition, since it is not dissolved in an aqueous environment and can be made from various polymers, it has various chemical compositions and properties. In addition, it is easy to process, so it can be transformed into various shapes depending on the application. Hydrogels have high biocompatibility due to their high water content and physicochemical similarity with the extracellular matrix.

As used herein, the term "drug delivery" means delivering a pharmaceutically effective amount of a pharmacologically active ingredient into a living body for the purpose of generating a desired pharmacological effect.

In one embodiment, the concentration of the PEGDA may be 5% to 30% (w/v), for example, 5% to 25%, 10% to 20%, or 8% to 11%. .

In one embodiment, the concentration of PEI may be 2.8% to 10% (w/v). For example, it may be 2.8% to 3.2%, 2.8% to 6%, or 4% to 9%. Here, when the concentration of PEI is less than 2.8%, amine groups involved in hydrogel bonding are insufficient, and when the concentration of PEI is more than 10%, the viscosity in the solution increases, thereby delaying the gel formation time. .

In one embodiment, the PEGDA may have a molecular weight of 400 to 6000 g / mol, for example, 600 to 2000 (2K), 600 to 3000 (3K), 600 to 4000 (4K), 600 to 6000 ( 6K), 700 to 2000 (2K), 700 to 3000 (3K), 700 to 4000 (4K), 800 to 2000 (2K), 800 to 3000 (3K), or 800 to 4000 (4K) g/mol have. Here, when the PEGDA molecular weight is 400 g/mol or less, the hydrophilic ethylene oxide chain is shorter than that of a high molecular weight, so that the level of hydrophobicity of the entire molecule is relatively increased, and accordingly, crosslinking with polyethyleneimine having hydrophilicity is weakened. Can be. In addition, when the molecular weight of the PEGDA is 3000 (3K) or more, the number of acryl groups that can be bonded to the amine group of polyethyleneimine is relatively insufficient, and thus, gel formation may be significantly inhibited.

As used herein, the term "dual mode sustained release" is a method for increasing the drug exposure period in vivo, and the "dual mode" is composed of two separate release modes that occur sequentially. I can. The "sustained release" refers to maintaining the effective concentration of the drug in the body for a long time, for example, 48 hours or more in one administration. The "release" may induce initial drug release in response to temperature, PH, light, ultrasound, glucose, etc. at the site of action. Thus, the drug can be slowly released in a bimodal mode in response to a specific stimulus at the site of action by the initial swelling-control mechanism and the subsequent decomposition-control mechanism.

In one embodiment, the swelling-control mechanism indicates that the initial drug release is influenced by the swelling action of the hydrogel. Swelling refers to the process of increasing the moisture content of the hydrogel through the penetration of the solution due to the network structure of the hydrogel and the osmotic pressure with the surrounding solution, and the relaxation of the chains. The swelling-control mechanism is the process that is included in the hydrogel during this process. It may mean that the drug is released to the outside through diffusion. In the swelling-control mechanism, the rate of drug release can be achieved through control of the diffusion rate through cross-link density control.

In one embodiment, the mechanism of degradation-control indicates that late drug release is dominated by the action of degradation. This may mean that the rate of drug release is rapidly increased as the biodegradable hydrogel is decomposed. When the biodegradation rate is similar to or slower than the diffusion rate, drug release occurs in a way that the sieve rate maintains the existing swelling-control mechanism, and when the biodegradation rate is high, it is rapidly unrelated to the swelling-control mechanism. Can be promoted.

According to an embodiment, the drug release behavior in the hydrogel decreases the crosslinking density as the molecular weight of PEGDA increases, thereby increasing the drug release rate. Therefore, by controlling the molecular weight of the PEGDA, it is possible to control the drug release behavior in detail.

As used herein, the term "elastic moduli (E)" refers to the ratio of stress/strain obtained through tension or compression of a hydrogel, which is the ratio of PEGDA-PEI hydrogel. It means mechanical stiffness. In addition, this indicates the crosslinking density in the crosslinked hydrogel through sufficient time for the completion of the crosslinking reaction, and since it is measured before biodegradation of the hydrogel occurs, it may mean the initial strength of the hydrogel. Since hydrogels contain a large amount of water in their structure, they have low mechanical strength. Therefore, in order to use the hydrogel in various fields such as tissue engineering, regenerative medicine, drug delivery, and immunotherapy, it is necessary to improve mechanical properties. In particular, when a hydrogel is used as a drug delivery vehicle, it may be difficult to maintain its structure for a long time due to swelling in an environment supplied with water or body fluid. Therefore, in order to apply to a case where drug delivery is required for a long period of time or to a field requiring high mechanical strength, the concentration and molecular weight of PEGDA and the concentration of PEI according to an embodiment can be adjusted to improve mechanical properties. The hydrogel may have an elastic modulus of 0.4 to 600 KPa. For example, 0.4 to 500, 0.4 to 450, 0.4 to 400, 0.4 to 350, 0.4 to 300, 0.4 to 250, 0.6 to 600, 0.6 to 500, 0.6 to 450, 0.6 to 400, 0.6 to 350, 0.6 to 300, 0.6 to 250, 0.8 to 600, 0.8 to 500, 0.8 to 450, 0.8 to 400, 0.8 to 350, 0.8 to 300, 0.8 to 250, 1 to 600, 1 to 500, 1 to 450, 1 to 400, 1 to 350, 1 to 300, 1 to 250, 10 to 600, 10 to 500, 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 50 to 600, 50 to 500, 50 to It may be 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 100 to 600, 100 to 500, 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250 KPa.

As used herein, the terms "storage moduli (G')" and loss moduli (G ") are measured through rheological repeated shear measurements, and the elastic component and viscosity of the hydrogel. Each component represents the crosslinking reaction rate (the point at which G'and G'meet) and the crosslinking ratio (the ratio of the portion crosslinked with a gel in the total polymer solution (G' vs. G')) in the gel formation process. In addition, the larger the storage coefficient, the more elastic and difficult to break under repeated shear conditions, The storage coefficient of the hydrogel may be 2 to 80 KPa, for example, 2 to 75, 2 to 70, 2 To 65, 2 to 60, 2 to 55, 2 to 50, 2 to 40, 2 to 30, 4 to 80, 4 to 75, 4 to 70, 4 to 65, 4 to 60, 4 to 55, 4 to 50 , 4 to 40, 4 to 30, 6 to 80, 6 to 75, 6 to 70, 6 to 65, 6 to 60, 6 to 55, 6 to 50, 6 to 40, 6 to 30, 10 to 80, 10 To 75, 10 to 70, 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 40 KPa.

As used herein, the term "drug" may be any one or more selected from the group consisting of nucleic acids, proteins, peptides, compounds, and cells, and for drug delivery purposes in the pharmaceutical field or artificial tissue transplantation in the tissue engineering field. It may be used for tissue regeneration purposes. In addition, it is not limited to hydrophilic drugs, and a hydrophobic drug can be loaded by introducing a molecular structure having a hydrophobic internal cavity.

In one embodiment, the protein may be insulin, growth factor, or antibody. The compounds are anticancer agents, antibiotics, anti-inflammatory agents, analgesics, epithelial regeneration accelerators, anesthetic agents, hemostatic agents, antibacterial agents, antibacterial agents, antiviral agents, antifungal agents, anti-inflammatory agents, antioxidants, preservatives, antihistamines, antitussive agents, antipyretics, immunostimulants, and dermatology It may be a formulation. The cells may be stem cells.

As used herein, the term "initiator" may mean light and/or heat, a crosslinking agent or a catalyst. In general, the crosslinking reaction to form a hydrogel involves the use of an initiator. For example, radical polymerization using vinyl-based functional groups requires radical initiators such as persulfates and phenones. However, these reagents often exhibit cytotoxicity depending on the concentration, so the dosage must be carefully selected. According to one embodiment, PEGDA-PEI hydrogel is harmless to the human body because it can form crosslinks by Michael addition reaction without the use of a separate crosslinking agent or catalyst having toxicity, and thus it is said that it is highly useful as a drug delivery system. I can.

As used herein, the term "in situ forming hydrogel" may generally mean a bio-injectable hydrogel. Injection-type hydrogels have the characteristics of conventional hydrogels, have fluidity like ordinary fluids outside the body, and are gelled by chemical or physical stimulation after injection, and are used as injection-type hydrogels in the body. According to an embodiment, PEGDA-PEI hydrogel may be suitable as an in situ hydrogel because it can be formed under mild conditions without a separate crosslinking agent or catalyst by forming crosslinks by Michael addition reaction.

Another aspect is (a) preparing a hydrogel precursor solution by mixing a PEGDA solution, a PEI solution, and a solvent; And (b) dissolving a drug in the prepared hydrogel precursor solution, providing a method of preparing a hydrogel for drug delivery.

In the above method, the concentration and molecular weight of the PEGDA, PEI, PEGDA solution, and the concentration of the PEI solution, drugs, drug delivery, hydrogel, and the like are as described above.

The term "solvent" as used herein refers to a material used to dissolve a polymer in order to prepare a hydrogel precursor solution, and various solvents commonly used in the art may be used, for example For example, it may be an equilibrium saline solution such as phosphate buffered saline (PBS).

The term "hydrogel precursor solution" as used herein may mean a liquid state before incubation to form a gel or before being injected into the body.

In one embodiment, after step (b), it may further include forming a hydrogel under conditions of pH 7 to 8, and 35 to 38 °C, for example, the condition is a physiological condition , Or may be in vivo.

The hydrogel according to an aspect exhibits dual-mode drug release kinetics in which drug release is sequentially controlled by a swelling-controlled and decomposition-controlled mechanism due to the adjustable mechanical and biodegradable properties of the hydrogel. , It can be usefully used in the field of drug delivery technology.

1 is a representative 1H-NMR spectrum of PEGDA. The table shows the peak integral ratio between the acrylic group peak and the (a, b, c) hydroxyethyl peak (d).
Figure 2 is a schematic diagram of a crosslinkable PEGDA-PEI hydrogel prepared under physiological conditions.
3 is a result of confirming the viscosity of PEI at various concentrations measured at room temperature or 37°C.
Figure 4 shows (a) A: 10% PEGDA-5% PEI, B: 20% PEGDA-5% PEI, C: 10% PEGDA-3% PEI, D: 20% PEGDA-3% PEI PEGDA-PEI Hydro This is a fluorescence microscope image of the gel. (b) After incubation in PBS, (E) PEGDA-PEI hydrogel image conjugated with FITC, or (F) PEGDA-PEI hydrogel image not conjugated with FITC.
5 is a rheological property of a reaction mixture of 10% PEGDA-5% PEI evaluated by changes in storage (G') and loss (G") coefficients measured over time, measured by concentration of PEGDA (( a) PEGDA400, (b) PEGDA700, (c) PEGDA1K, (d) PEGDA2K), and the arrow indicates the gel formation point (e) is the gelation time of 10% PEGDA or 20% PEGDA when 5% PEI ( tgel), and (f) is the gelation time (tgel) of 20% PEGDA700 according to various concentrations of PEI.
6 shows the rheological properties of the reaction mixture of 10% PEGDA-5% PEI evaluated by the change of the storage (G') and loss (G") coefficients measured over time, which is by concentration of PEGDA. It was measured ((a) PEGDA3K, (b) PEGDA6K).
7 shows the rheological properties of the reaction mixture of 20% PEGDA-5% PEI evaluated by the change of the storage (G') and loss (G") coefficients measured over time, which is by concentration of PEGDA. It was measured ((a) PEGDA400, (b) PEGDA700, (c) PEGDA1K, (d) PEGDA2K, (e) PEGDA3K, (f) PEGDA6K), and the arrows indicate the gel formation points.
Figure 8 is a 20% PEGDA700 and (a) 3% PEI, (b) 5% PEI, (c) 10% evaluated by changes in the storage (G') and loss (G") coefficients measured over time. Confirming the rheological properties of the reaction mixture of PEI, the arrow indicates the gel formation point.
9 shows the elastic modulus of (a) 10% or 20% PEGDA-5% PEI hydrogel according to the PEGDA molecular weight (E), (b) 10% PEGDA or (c) 20% PEGDA and various concentrations of PEI hydrogel. The modulus of elasticity according to the molecular weight of PEGDA (E), (d) The ratio of the number density (ФAc / Am) of the acryl group of PEGDA and the amine group of PEI according to the molecular weight of PEGDA is shown.
Figure 10 is the storage coefficient (G') of the PEGDA-PEI hydrogel taken from the highest G'value after gelation in the rheological experiment, (a) 10% or 20% PEGDA and 5% PEI hydrogel according to the molecular weight of PEGDA It shows the storage coefficient (G') of (b) 20% PEGDA700 and the storage coefficient (G') of PEI hydrogel of various concentrations.
Figure 11 shows (a) the decomposition mechanism of the PEGDA-PEI hydrogel, (b) the change over time of the normalized elastic modulus (E / E0) of 10% PEGDA-5% PEI hydrogel for various PEGDA molecular weights, (c ) Decomposition rate constant (kd) obtained by substituting FIGS. 11(b) and 12 into Equation (1), (d) 10% PEGDA, (e) 20% PEGDA combined with various PEI concentrations ( kd).
Figure 12 shows the change over time of the normalized elastic modulus (E / E0) of 20% PEGDA-5% PEI hydrogel for the molecular weight of various PEGDA.
13 is a drug release profile of (a) PEGDA-PEI hydrogel (10% PEGDA-5% PEI), showing two separate release modes that occur sequentially (initial swelling-control mechanism, late decomposition-control mechanism). This is the result of confirmation. (b) Initial drug release profile, (c) Reaction rate constant obtained by substituting the values of Figs. 13(b) and 14(b) into equation (2) (k1), (d) late drug release profile, (e) The reaction rate constant (k2) obtained by substituting the values of Figs. 13(d) and 14(c) into Equation (3) is shown.
14 is a drug release profile of (a) PEGDA-PEI hydrogel (20% PEGDA, 5% PEI), showing two separate modes of release that occur sequentially (initial swelling-control mechanism, then decomposition-control mechanism). This is the result of confirmation. (b) Early drug release profile, (c) Late drug release profile.
Figure 15 shows the k1 and k2 values obtained from the drug release profile of the PEG1-PEI hydrogel, showing the results at various concentrations of PEI and (a), (b) 10% PEGDA and (c), (d) 20% PEGDA. Is shown.
Figure 16 is a drug release profile of PEGDA-PEI hydrogel, (a) 3% PEI-10% PEGDA, (b) 10% PEI-10% PEGDA, (c) 3% PEI-20% PEGDA, (d) Results at 10% PEI-20% PEGDA are shown.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Reference Example 1. Experimental Materials

1-1. Synthesis of poly(ethylene glycol) diacrylate (PEGDA)

The acrylate was conjugated to PEG by a nucleophilic substitution reaction between the hydroxyl end group of the PEG and acryloyl chloride. Briefly, poly(ethylene glycol) (PEG, Sigma Aldrich) (MW 1000, 2000, 3000 and 6000 g mol-1) was first dissolved in dichloromethane at 30%. 2 molar equivalents of acryloyl chloride and triethylamine (Sigma Aldrich) were added to the mixture with respect to the hydroxyl group of PEG and stirred at room temperature with dry N2 for 24 hours. After the reaction, the insoluble salt formed was filtered, and the mixture was concentrated via rotary evaporation. The product was precipitated by addition of excess diethyl ether. The product was washed 3 times with diethyl ether and dried under vacuum. As shown in Fig. 1, the acrylate conjugate was confirmed by 1H-NMR (400MHz, Avance III HD, Bruker).

Reference Example 2. Experimental Procedure

2-1. PEGDA-PEI hydrogel preparation

A precursor solution was prepared by mixing a solution of PEGDA and PEI in phosphate buffered saline (PBS, pH 7.4) in various ratios. The precursor solution was immediately put into a mold with a 1 mm spacer and incubated at 37° C. until a hydrogel was formed. Hydrogel discs (8 mm in diameter) were punctured and immersed in PBS until further characterization was achieved.

2-2. Rheological characterization

Rheological characterization was performed to analyze the gelation kinetics of PEGDA-PEI hydrogel formation. Mix the PEGDA and PEI solutions and place them on a sample holder of a rotating disk rheometer (Kinexus, Malvern), and change the storage coefficient (G') and loss coefficient (G ") to a small amplitude vibration of 37 °C. Measurements were made during the crosslinking reaction in shear mode. During the experiment, the frequency and strain of the rotating disk were 1 Hz and 10%, respectively. The gelation time ( t _gel ) was intersected by the curves of G 'and G "indicating the gelation time. Expressed as a point.

2-3. Compression experiment

The rigidity of various PEGDA-PEI hydrogels was measured by calculating the modulus of elasticity obtained from compression experiments. Briefly, a hydrogel disk was uniaxially compressed at a rate of 1 mm min-1, and a strain versus stress curve was obtained (model 3343, Instron). The modulus of elasticity was determined as the slope of the straight region of the curve (first 10%).

2-4. Measures the change in modulus of elasticity over time

Biodegradability was analyzed by measuring the change in elastic modulus over time. PEGDA-PEI hydrogels were incubated with PBS at 37° C., and their elastic modulus was measured at the designated time points. The decomposition rate constant ( k _d ) is obtained by applying the following exponential decay model to the coefficient versus time curve,

[Equation (1)]

Where E _t is the elastic modulus measured at time t, and E ₀ is the initial elastic modulus.

2-5. Fluorescence microscopy imaging

The homogeneity of PEGDA and PEI after formation of the hydrogel was evaluated by observing the distribution in the hydrogel after conjugating a phosphor. Each PEGDA-PEI hydrogel disk was added to a solution of fluorescein isothiocyanate (FITC, Sigma Aldrich) in PBS (0.1 mg mL-1) and incubated for 3 hours at room temperature in the dark. The reaction was induced. Conjugation was confirmed by the presence of a fluorescent color remaining in the hydrogel after washing. The FITC-conjugated hydrogel was visualized with a fluorescence microscope (XDS-3FL, Optika) to evaluate the distribution of fluorescent substances in the hydrogel. In addition, the degradation of the FITC-conjugated hydrogel was observed under the same conditions as mentioned above and compared with the unmodified hydrogel.

2-6. BCATM Protein Assay

Drug release from various PEGDA-PEI hydrogels was measured to evaluate the effects of mechanical properties and biodegradability. The model protein drug bovine serum albumin (3 mg mL-1) was dissolved in a precursor solution before hydrogel preparation and encapsulated in a PEGDA-PEI hydrogel. The hydrogel was incubated in PBS at 37 °C, and the amount released was measured at various time points (BCATM Protein Assay, Thermo Fisher). The following two models were applied to the accumulated drug release profile.

[Equation (2)]

[Equation (3)]

Where M _t is the amount released at time t, M _∞ is the total amount in the hydrogel, k ₁ and k ₂ are reaction rate constants, and n is the exponent related to the release mechanism. Equation (2) applies the fractional time-dependent power-law release model ('Ritger-Peppas model') to represent the initial part of the drug release, while the exponential release model, Equation (3), It was applied to indicate accelerated release.

Example 1. Preparation of PEGDA-PEI hydrogel with different concentration and molecular weight

In this example, in order to control the mechanical properties of the hydrogel, a solution of PEGDA and PEI was mixed in various ratios to prepare a PEGDA-PEI hydrogel capable of crosslinking in physiological conditions and in situ conditions. Each hydrogel was prepared by the same procedure as described in "2-1. PEGDA-PEI Hydrogel Preparation" of "Reference Example 2. Experimental Procedure", except that the concentration and molecular weight were different.

The molecular weight of PEGDA was changed from 400 (PEGDA400) to 6000 (PEGDA6K) g/mol while adjusting the concentration of PEGDA to 10% or 20%.

The PEI concentration was also adjusted from 3% to 10%. When the concentration of PEI is less than 3%, the amine group involved in the hydrogel bonding is insufficient, thereby inhibiting gel formation, and when it is more than 10%, the mechanical properties of the hydrogel begin to decrease, and as shown in FIG. , As the viscosity in the solution increased, the gel formation time was delayed.

Example 2. Confirmation of homogeneity of PEGDA-PEI hydrogel by fluorescence microscopic imaging

In this example, in order to confirm the homogeneity of the PEGDA-PEI hydrogel, the unreacted amine group of PEI in the hydrogel and a fluorescent probe (eg FITC) were chemically conjugated, and then the fluorescence distributed in the hydrogel. The material was analyzed through microscopic imaging.

As a result, as shown in Fig. 4a, it was confirmed that a consistent fluorescence signal appeared throughout the hydrogel structure. This indicates that PEGDA and PEI are homogeneously mixed and distributed within the network.

Example 3. Confirmation of the influence of the molecular weight and concentration of PEGDA and the concentration of PEI on the crosslinking reaction rate between PEGDA and PEI

In this example, rheological characterization was performed to confirm the influence of the molecular weight and concentration of PEGDA and the concentration of PEI on the crosslinking reaction rate between PEGDA and PEI. It was evaluated by measuring the change in the viscoelastic properties, storage modulus (G', elastic component) and loss modulus (G'', viscous component) of the mixture over time.

3-1. Effect of molecular weight and concentration of PEGDA on gel formation

The concentrations of PEGDA and PEI were maintained at 10% and 5%, respectively, and the gelation point was observed up to PEGDA6K while increasing the molecular weight of PEGDA.

As a result, as shown in Figure 5a-d, until PEGDA 2K, a gelation point where G'crosses G'' appeared, but as shown in FIG. 6, it was confirmed that the gelation point did not appear in PEGDA3K and PEGDA6K. I did.

This indicates that when the molecular weight increases above 3K of PEGDA, the number of acrylic groups of PEGDA is relatively insufficient compared to the amine group of PEI, and thus a gel is not formed.

On the other hand, as shown in FIG. 7, when the PEGDA concentration was increased to 20% while maintaining 5% PEI, it was confirmed that gelation points appeared at the molecular weights of all PEGDAs. This indicates that as the PEGDA concentration increased to 20%, the total number of acrylic groups increased even in the high molecular weight PEGDA molecule of PEGDA3K or higher to form a gel.

3-2. Effect of molecular weight and concentration of PEGDA on gelation time (crosslinking reaction rate)

As shown in FIG. 5e, it was confirmed that the gelation time ( t _gel ) gradually decreased as the molecular weight of PEGDA increased in both 10% and 20% PEGDA. In addition, the reduction rate of t _gel relative to the molecular weight of PEGDA was observed to be higher when 20% of PEGDA was used.

This is contrary to the expectation that the reaction rate will be accelerated due to the relatively large number of acrylic groups in the low molecular weight PEGDA. In the low molecular weight PEGDA, there is a large amount of hydrophobic acrylic groups compared to the hydrophilic ethylene oxide chain, so that the level of hydrophobicity of the PEGDA molecule is relatively increased, and thus, the stable and homogeneous mixing between PEGDA and the hydrophilic PEI may have played a role. Is expected.

As a result, it can be seen that high molecular weight PEGDA having greater hydrophilicity can have an advantageous interaction with PEI and can induce an initial crosslinking process more stably.

3-3. Effect of the concentration of PEI on the gelation time (crosslinking reaction rate)

As shown in FIGS. 5F and 8, in order to confirm the effect of the PEI concentration on the gelation time ( t _gel ), the PEI concentration reacted with 20% PEGDA700 was changed from 3% to 10%.

As a result, it was confirmed that the t _gel value decreased as the PEI concentration increased, because the number of amine groups usable for crosslinking increased with the PEI concentration. However, as mentioned in Example 1, when the concentration of PEI exceeds 10%, the viscosity in the solution increases, and it is predicted that the gel formation time will be delayed.

Example 4. Confirm the influence of the molecular weight and concentration of PEGDA and the concentration of PEI on the strength of the hydrogel

In this example, in order to confirm the strength of the PEGDA-PEI hydrogel, the elastic modulus was measured by performing a uniaxial compression experiment of the fully-crosslinked hydrogel through a sufficient time for the completion of the crosslinking reaction.

4-1. Influence of molecular weight of PEGDA on the strength of hydrogel

In order to confirm the effect of the PEGDA molecular weight on the rigidity of the PEGDA-PEI hydrogel, the modulus of elasticity was measured while increasing the molecular weight of PEGDA in a 10% or 20% PEGDA solution while maintaining the concentration of PEI at 5%. .

As a result, as shown in FIG. 9A, it was confirmed that the modulus of elasticity decreased as the molecular weight of PEGDA increased in PEGDA700 or higher. For example, in the case of 10% PEGDA, the modulus of elasticity decreased from PEGDA700 to PEGDA2K, and in the case of 20% PEGDA from PEGDA700 to PEGDA6K. However, it was confirmed that the elastic modulus of the PEGDA400-PEI hydrogel was significantly lower than that of the PEGDA700-PEI hydrogel.

This indicates that PEGDA400 has more acrylic groups than PEGDA700, so that the hydrophobicity increases, preventing homogeneous mixing with hydrophilic PEI, and thus, the degree of crosslinking reaction is reduced.

In addition, the overall tendency of the modulus of elasticity according to the molecular weight of PEGDA was similar to that shown in FIG. 9A at all PEI concentrations regardless of the PEGDA concentration, as shown in FIGS. 9b-c.

4-2. Influence of the ratio of the number density of acrylic groups and amine groups on the strength of the hydrogel

In order to evaluate the influence of the stoichiometry of acryl and amine groups on the mechanical properties of PEGDA-PEI hydrogel, the theoretical number density ratio (ФAc/Am) of acryl groups to amine groups was calculated.

As a result, as shown in FIG. 9D, it was confirmed that ФAc/Am decreased as the molecular weight of PEGDA increased. In addition, in the 20% PEGDA400 with the highest number of acrylic groups at the same concentration, the фAc/Am value appears closer to 1 (0.86) compared to 3% PEI (0.43) and 10% PEI (1.4) in the case of 5% PEI. Confirmed.

This is because the crosslinking reaction occurs through a stoichiometric reaction between an acrylic group and an amine group, so it can be seen that the crosslinking density increases as the ratio is closer to 1. Likewise, it can be seen that the crosslinking density decreases as the φAc/Am value deviates from 1, and as a result, the hydrogel strength decreases. Therefore, it was found that the highest strength was observed for 5% PEI in 20% PEGDA400, which has the highest number of acrylic groups at the same concentration.

4-3. Effect of the concentration of PEGDA and PEI on the strength of the hydrogel

In order to confirm the effect of the PEGDA and PEI concentrations on the strength of the PEGDA-PEI hydrogel, the PEI concentration was changed from 3% to 10% for 10% or 20% PEGDA.

As a result, as shown in Fig. 9b-c, it was confirmed that the hydrogel having 20% PEGDA generally has a greater elastic modulus than that having 10% PEGDA in PEGDA of all molecular weights. In addition, it was confirmed that at a given PEI concentration, the molecular weight range of PEGDA in which a hydrogel can be formed is also larger in 20% PEGDA. For example, for 3% PEI, the maximum molecular weight of PEGDA to form a gel was PEGDA2K in 10% PEGDA and PEGDA3K in 20% PEGDA. Similarly, for 10% PEI, the maximum molecular weight of PEGDA for gel formation was PEGDA1K in 10% PEGDA and PEGDA2K in 20% PEGDA.

On the other hand, as shown in Fig.9b-c, the elastic modulus of the hydrogel with 3% PEI is much lower than that with 5% PEI, whereas when the PEI concentration increases to 10%, the elastic modulus for PEGDA of all molecular weights It was confirmed that was significantly reduced.

This indicates that in the case of 3% PEI, the amine content is lower than that of 5% PEI, so that the modulus of elasticity is lower. In addition, the number of acrylic groups in PEGDA is all consumed by the amine groups of 5% PEI, indicating that further increase in PEI concentration results in a significant stoichiometric excess of PEI molecules resulting in a limiting effect on the crosslinking reaction. That is, the presence of an excess of PEI reactants means that it acts as a blocker of crosslinking. This also means that although the modulus of elasticity of hydrogels with 10% PEI is generally greater than those with 3% PEI, the PEGDA of the highest molecular weight for gelation is at 3% PEI (PEGDA3K) than 10% PEI (PEGDA2K). It helps to explain the fact that it is higher. That is, a very large excess of PEI in 10% PEI hinders hydrogel formation by limiting the crosslinking reaction in the fewest number of acrylic groups in 10% PEI.

4-4. Hydrogel strength evaluation

The strength of the hydrogel was also evaluated as G'value under repeated shear conditions after complete gelation as shown in FIGS. 5-8.

As a result, as shown in FIG. 10A, the trend of G'in the hydrogel of 10% PEGDA-5% PEI was similar to that of the elastic modulus. However, when the PEGDA concentration increased to 20%, it was confirmed that the G'value continuously increased up to PEGDA2K as the molecular weight of PEGDA increased.

This is because the hydrogel cross-linked with high molecular weight PEGDA was more resilient even though the strength was low, so structural integrity could be maintained under shear conditions. On the other hand, hydrogels crosslinked with low molecular weight PEGDA have a much higher modulus of elasticity, but are also much more brittle. Therefore, it was found that the G'value decreased due to the occurrence of extensive breakage of the hydrogel during repeated shear measurement.

As shown in FIG. 10B, similar results were also observed for the hydrogel in which the PEI concentration was changed while maintaining the concentration and molecular weight of PEGDA (20% PEGDA700) (G' value continuously increased as the concentration of PEI increased). . At 10% PEGDA700, the trend of G'was similar to that of the elastic modulus (of PEGDA 700 shown in FIG. 9B) shown in FIG. 9B. However, at 20% PEGDA700, even though 5% PEI had the highest modulus of elasticity, G'was lower than other conditions (than 10% PEI), which is because the hydrogel with high strength also possesses considerable brittleness. It is due to the failure of the structure in the given repeated shear test conditions.

Example 5. Confirmation of decomposition behavior of PEGDA-PEI hydrogel

5-1. Decomposition mechanism of PEGDA-PEI hydrogel

Since it is unlikely that all amines in the PEI chain will react with the acryl groups of PEGDA, a significant number of unreacted amine groups may remain in the PEGDA-PEI hydrogel. This is because the reactivity decreases as the conformational and translational mobility of the polymer chain decreases as the crosslinking reaction proceeds. Therefore, as shown in FIG. 11A, the ester bond of the PEGDA-PEI hydrogel is shown to be hydrolyzed due to the unreacted amine group directly nucleophilic attack or the protonated amine group and increased hydroxide ions, accordingly, Hydrogel degradation is accelerated.

5-2. Effect of Molecular Weight of PEGDA on the Degradation Behavior of PEGDA-PEI Hydrogel

In this example, in order to confirm the decomposition behavior of the PEGDA-PEI hydrogel, the hydrogel was immersed in a physiological buffer (ie, PBS) and the change in elastic modulus was evaluated over time. The change in the fraction of the elastic modulus (E/E0) versus time (t) profile was substituted into the exponential reduction model (Equation (1)) to obtain the decomposition rate constant ( k _d ). Under all conditions, PEGDA-PEI hydrogel showed complete structural degradation at various rates.

11b-c and 12, the concentration of PEI was maintained at 5% while changing the molecular weight and concentration of PEGDA.

As a result, as shown in Fig. 11c, in the case of 10% and 20% PEGDA, the decomposition rate was greatly affected by the molecular weight of PEGDA, and it was confirmed that the k _d value generally increases as the molecular weight of PEGDA increases. . This is because the hydrogel with low crosslinking degree showed faster decomposition, because there were fewer ester bonds that had to be broken for decomposition in the hydrogel, and there was a greater osmotic pressure in the hydrogel with a higher degree of swelling in the high molecular weight PEGDA. Was expected.

In addition, as shown in Fig. 11d-e, decomposition was also observed for the PEGDA-PEI hydrogel in which the PEI concentration was changed. In general, it was confirmed that the k _d value increased with the molecular weight of PEGDA in 10% and 20% PEGDA regardless of the PEI concentration. For PEGDAs of the highest molecular weight (i.e., 10% PEGDA2K and 20% PEGDA3K), the k _d values at 5% PEI were significantly higher than at 3% PEI, although the modulus of elasticity was much higher at 5% PEI. In addition, as shown in FIG. 11d, the change in k _d according to the molecular weight of 10% PEGDA with 3% PEI was significantly lower than that of 5% PEI. Similarly, as shown in Fig. 11e, the increase of k _d to the molecular weight of PEGDA at 20% PEGDA was more pronounced with the concentration of PEI. It was confirmed that, regardless of the difference in the initial strength of the hydrogel, there is a possibility that a larger amount of unreacted amine groups may accelerate the decomposition process in a high concentration of PEI.

5-3. Influence of unreacted amine groups on the decomposition behavior of PEGDA-PEI hydrogel

On the other hand, as shown in Figure 4b, the effect of the unreacted amine group on the decomposition through hydrolysis was further confirmed by removing the amine group from the hydrogel by a chemical reaction. In the PEGDA-PEI hydrogel, unreacted amine groups were conjugated with FITC as mentioned above.

As a result, as shown in FIG. 4B, it was confirmed that the FITC-linked hydrogel (E) remained stable in PBS, but the unmodified hydrogel (F) was completely decomposed. This showed that the FITC-linked hydrogel was not decomposed and remained stable because all of the free amine groups were consumed, and as a result, it was shown that the unreacted amine groups of the PEGDA-PEI hydrogel caused hydrogel decomposition.

Example 6. Confirmation of bimodal drug release profile of PEGDA-PEI hydrogel

Combined with the tunable mechanical properties and degradation behavior of the in situ crosslinkable PEGDA-PEI hydrogel, it is ideal as a dosage form for injection. Therefore, in this example, as shown in FIGS. 13 and 14, the release of bovine serum albumin, a model drug, from the PEGDA-PEI hydrogel was measured over time, and the cumulative drug release versus time (t) Profiles were obtained and analyzed.

As a result, as shown in FIGS. 13A and 14A, it was confirmed that the overall release profile of the PEGDA-PEI hydrogel at all molecular weights of PEGDA consisted of two different phases occurring in sequence. That is, (1) an initial power law curve with fractional time dependence (~tn, 0 <n <1), and (2) an exponential curve indicating a rapid rise in the release rate before reaching the end point. This means that, as shown in FIG. 13A, the initial drug release is influenced by the swelling action of the hydrogel due to the combination of Fickian diffusion and chain relaxation of the network by polymerization, and subsequent drug release is promoted by hydrogel degradation. will be.

6-1. Effect of molecular weight of PEGDA on initial drug release rate

13b-c and 14b, the drug release profile for the initial 12 hours was obtained by applying the Ritger-Peppas model (Equation (2)) to obtain a reaction rate constant ( k ₁ ).

As a result, as shown in Fig. 13c, it was confirmed that the reaction rate constant ( k ₁ ) value increased as the molecular weight of PEGDA increased. For example, the reaction rate constant increased as the molecular weight increased from PEGDA700 to PEGDA3K for 20% PEGDA and from PEGDA700 to PEGDA2K for 10% PEGDA. This indicates that this is generally due to the rapid release from hydrogels with a small crosslinking density. On the other hand, it was confirmed that the release rate from the PEGDA400-PEI hydrogel was much higher than any other conditions despite having the largest crosslinking density. This suggests that the highly increased hydrophobicity in the lowest molecular weight PEGDA facilitated the extraction of hydrophilic molecules from the hydrogel than in other conditions.

6-2. Effect of molecular weight of PEGDA on late drug release rate

As shown in FIGS. 13D-E and 14C, the reaction rate constant ( k ₂ ) was obtained by applying the exponential drug release model (Equation (3)) to the subsequent drug release profile showing a rapid increase in the release rate. , Through this, it was possible to confirm the trend of drug release dominated by hydrogel degradation.

As a result, as shown in Figure 13e, the reaction rate constant ( k ₂ ) value is similar to the trend of the decomposition rate constant ( k _d ) in Figure 11, as the molecular weight of PEGDA increases in 10% and 20% PEGDA It was confirmed that it increased. This indicates that the release kinetics after the initial swelling-control mechanism is largely influenced by hydrogel degradation.

6-3. Identification of drug release profiles at various PEI concentrations

As shown in FIGS. 15 and 16, this sequential drug release profile could be confirmed even in a hydrogel crosslinked by 10% and 20% PEGDA and various PEI concentrations.

As shown in FIGS. 15A and 15C, the trend of k ₁ values for both 10% and 20% PEGDA indicates that initial drug release is mostly influenced by hydrogel diffusion, and the mechanical strength and general characteristics of the hydrogel shown in FIG. 9 It was confirmed that there is an inverse relationship.

As shown in FIGS. 15B and 15D, it was confirmed that the increase in the k ₂ value according to the increase in the molecular weight of PEGDA was more pronounced at the higher PEI concentration. This means that at accelerated drug release, higher degradation rates are shown at higher PEI concentrations.

In particular, as shown in Figure 15b, it was confirmed that the k ₂ value for 10% PEGDA1K-10% PEI was significantly higher than other conditions. This may have resulted from the combination of weak mechanical strength identified by the low modulus of elasticity shown in FIG. 9 and a high content of unreacted amine groups that promote hydrogel degradation. On the other hand, the tendency of the k ₂ value according to the molecular weight of PEGDA of 10% at low PEI concentration is closer to the tendency of mechanical strength, suggesting that the effect of diffusion is greater than that of degradation in drug release.

As shown in FIG. 15D, it was confirmed that the dependence of the k ₂ value on hydrogel degradation in 20% PEGDA closely follows the trend of the k ₂ value in FIG. 13. This suggests that hydrogels of higher crosslinking density with higher mechanical strength and lower diffusion have the effect of degradation on drug release far more important than that of diffusion.

Claims (19)

Poly(ethylene glycol) diacrylate (PEGDA) at a concentration of 8% to 11% (w/v); And as a composition for drug delivery comprising a hydrogel in which polyethyleneimine (polyehtleneimine: PEI) at a concentration of 2.8% to 10% (w/v) is crosslinked with each other,
The molecular weight of the PEGDA is 600 to 6000 g / mol, the drug is a drug delivery composition that is sustained release in a dual mode (dual mode).
delete The composition for drug delivery according to claim 1, wherein the concentration of PEI is 2.8% to 3.2% (w/v).
delete delete The composition for drug delivery according to claim 1, wherein the dual mode is sequentially controlled by swelling-control and decomposition-control mechanisms.
The composition for drug delivery according to claim 1, wherein the drug release behavior is controlled by the molecular weight of PEGDA.
The composition for drug delivery according to claim 1, wherein the hydrogel has an elastic modulus of 0.4 to 600 KPa.
The composition for drug delivery according to claim 1, wherein the hydrogel has a storage coefficient of 2 to 80 KPa.
The composition for drug delivery of claim 1, wherein the hydrogel further comprises a drug.
The composition for drug delivery according to claim 10, wherein the drug is any one or more selected from the group consisting of nucleic acids, proteins, peptides, compounds, and cells.
The composition for drug delivery according to claim 1, wherein the crosslinking is crosslinked in the absence of an initiator.
The composition for drug delivery according to claim 1, wherein the crosslinking is crosslinked by Michael addition reaction.
The composition for drug delivery according to claim 1, wherein the hydrogel is formed in situ.
(a) preparing a hydrogel precursor solution by mixing a PEGDA solution having a concentration of 8% to 11% (w/v), a PEI solution having a concentration of 2.8% to 10% (w/v), and a solvent; And
(b) As a method for producing a hydrogel for drug delivery comprising the step of dissolving a drug in the prepared hydrogel precursor solution,
The molecular weight of the PEGDA is 600 to 6000 g / mol, the drug is a method of producing a hydrogel for drug delivery that is sustained release in a dual mode (dual mode).
delete The method of claim 15, wherein the concentration of the PEI solution is 2.8% to 3.2% (w/v).
delete The method of claim 15, further comprising the step of forming a hydrogel under conditions of pH 7 to 8, and 35 to 38° C. after step (b).

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