KR102479259B1 - Injectable Hydrogels into injured tissue sites and uses thereof - Google Patents

Abstract

It relates to a hydrogel that can be injected directly into the damaged area and its use, since the hydrogel has appropriate properties and can be directly injected into the damaged tissue area together with physiologically active substances, it can recover the damaged tissue and treat various diseases including spinal cord injury effective in the prevention or treatment of

Description

Injectable Hydrogels into injured tissue sites and uses thereof {Injectable Hydrogels into injured tissue sites and uses thereof}

It relates to a hydrogel that can be directly injected into a damaged tissue site and its use.

Spinal cord injuries (SCI) are a type of mechanical trauma (primary injury). Primary damage that causes secondary damage, such as an increase in neuroinflammatory factors, apoptosis of oligodendrocytes, and loss of myelin, can damage neighboring tissues and lesions that are not damaged, especially in the neuroinflammatory process. make it worse On the other hand, methylprednisolone (methylprednisolone, MP) is the only approved anti-neuritis treatment for use in treatment, and by administering the MP, secondary damage caused by the neuroinflammatory process can be suppressed. However, MP has a short half-life during blood circulation and is rapidly eliminated by the liver or kidney, and MP treatment has problems with various harmful effects including infection, acute corticosteroid myopathy, and pneumonia.

Hydrogels are water content matrices and can be used to encapsulate drugs or cells. Therefore, when hydrogels are injected into the lesion for treatment, the directly injected hydrogels can create and preserve a hydrated structure that provides an environment suitable for recovery. However, until now, there has been little research on how to treat neuroinflammation after SCI by directly injecting the hydrogel, and direct injection of the hydrogel has a problem in that it entails risks such as infection due to residual substances. For example, there is a problem that additional damage may occur when the hydrogel injected into the spinal cord swells.

Therefore, there is a need for research on a hydrogel that can be directly injected into a damaged area where the above problems are improved.

Korean registered patent KR 10-1368736 B1

One aspect is to provide a hydrogel comprising glycol chitosan and oxidized hyaluroninate.

Another aspect is to provide a composition for preventing or treating spinal cord injury comprising the hydrogel.

Another aspect is mixing a sodium periodate solution with a hyaluronic acid solution to prepare an oxidized hyaluronic acid solution; and mixing the oxidized hyaluronate solution and the glycol chitosan solution.

One aspect provides a hydrogel comprising glycol chitosan and oxidized hualuroninate.

In one embodiment, the hydrogel may further include metal nanoparticles. The metal may be a metal oxide. In addition, the metal is an iron group metal element (Fe, Ni, Co), a rare earth element (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu), Monetary metal elements (Cu, Ag, Au), zinc group elements (Zn, Cd, Hg), aluminum group elements (Al, Ga, In, Tl), alkaline earth metal elements (Ca, Sr, Ba, Ra) and platinum group elements It may be made of one or more metals selected from (Pt, Pd, etc.) or alloys thereof.

In one embodiment, the hydrogel may further include a physiologically active material. The bioactive material may be bound to the surface of the metal nanoparticle. Examples of the physiologically active substance are anti-inflammatory agents, anti-cancer agents, contrast agents, hormones, anti-hormonal agents, vitamins, calcium agents, mineral agents, saccharides, organic acid agents, protein amino acid agents, detoxifying agents, enzyme agents, metabolic agents, diabetes concomitant agents, tissues Resurrection medicines, chlorophyll preparations, pigment preparations, tumor medicines, tumor treatment agents, radiopharmaceuticals, tissue cell diagnosis agents, tissue cell therapy agents, antibiotics preparations, antiviral agents, combined antibiotics preparations, chemotherapeutic agents, vaccines, toxins, toxoids, antitoxins . In a specific embodiment, the physiologically active substance may be a spinal cord injury treatment agent, for example, an anti-inflammatory agent, more specifically ursodeoxycholic acid.

In one embodiment, the glycol chitosan and oxidized hyaluroninate may form an imine bond by a Schiff base reaction between the amino group of glycol chitosan and the aldehyde group of oxidized hyaluroninate. The glycol chitosan and the oxidized hyaluroninate may be mixed in a weight ratio of 1 to 10:1. For example, the glycol chitosan and oxidized hyaluroninate are 1 to 10:1, 1 to 9:1, 1 to 8:8, 1 to 7:1, 2 to 10:1, 2 to 8:1, 3 to 10:1, 3 to 8:1, 3 to 8:1, 4 to 10:1, 4 to 8:1, 5 to 10:1, 5 to 9:1, 6 to 10:1, or 6 to It may be mixed in a weight ratio of 8:1. At this time, when the mixing ratio of glycol chitosan and oxidized hyaluronate is less than or exceeds the above range, the glycol chitosan and oxidized hyaluronate are not sufficiently cross-linked to form a liquid state fluid with low viscosity, thus forming a solid or semi-solid There is a problem that a gel in the state is not formed. Therefore, when the hydrogel is injected into the damaged area, the rate of absorption into the living body is very fast or it is easily dissolved in the living body, so the efficacy as a therapeutic agent may not be exhibited.

In one embodiment, the hydrogel has a storage modulus (G ' ) may have.

In one embodiment, the hydrogel may have a loss modulus (G ") of 10 to 150 Pa, 10 to 100 Pa, 10 to 40 Pa, 15 to 40 Pa, 15 to 30 Pa or 20 to 30 Pa. there is.

The modulus of elasticity may be measured with a viscometer (eg, a rotating rheometer) at a compressibility of 2 mm/min until the strain % of 8.1 is reached.

Another aspect is the hydrogel; And it provides a composition for preventing or treating spinal cord injury, including a therapeutic agent for spinal cord injury.

Another aspect is the hydrogel; And it provides a method for preventing or treating spinal cord injury comprising administering a spinal cord injury treatment agent to a subject. In one embodiment, the method may further include irradiating near infrared rays after administering the hydrogel to the subject. Details of the hydrogel are as described above.

In this specification, "spinal cord injury (SCI)" may be damage caused by compression or dislocation of the spinal cord. Spinal cord injuries include, for example, traumatic spinal cord injury, spinal degenerative diseases (spondylosis, etc.), spinal inflammatory diseases (spondylitis, chronic rheumatoid arthritis, etc.), tumors (spinal cord tumors, spinal tumors, etc.), vascular diseases (spinal hemorrhage, stroke, bone marrow) spinal cord paralysis due to external vascular disorders, etc.), myelitis (arachnoid, viral myelitis, bacterial myelitis, etc.), multiple sclerosis, amyotrophic lateral sclerosis, etc. In one embodiment, the composition may have a therapeutic effect on chronic spinal cord injury as well as acute spinal cord injury. In addition, the composition may inhibit secondary spinal cord injury caused by spinal cord injury treatment.

A composition according to one embodiment may be for photothermal therapy.

In this specification, "photothermal therapy" is to inject a drug with a photoreactive substance that generates heat by absorbing light into the damaged area, accumulates it in a location where hyperthermia therapy is needed, and irradiates a laser to induce heat generation of the photoreactive substance to treat the surrounding area. It is a treatment method that kills malignant cells.

In the present specification, “near-infrared” is absorbed by skin and tissues, has the ability to penetrate deep tissues in a non-penetrating manner, and is used for treating diseases with excellent heat transfer. In particular, it can be converted into heat by gold nanomaterials for efficient heat removal of diseased tissues. According to one embodiment, it was confirmed that the recovery of motor function, inhibition of neuroinflammation, or recovery of damaged tissue by near-infrared irradiation after the composition was injected into the damaged area was increased. In addition, the composition may inhibit phosphorylation of ERK, JNK or p38 in the MAPKs pathway or inhibit inflammatory cytokines such as TNF-α and IL-1β. Therefore, since the hydrogel according to one embodiment includes gold nanoparticles, heat generation is induced by the gold nanoparticles when irradiated with near-infrared rays, it is possible to suppress nerve inflammation caused by activated macrophages after spinal cord injury.

The dosage of the composition according to one embodiment is 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 100 mg. It may be 1 mg. However, the dosage may be variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art If so, the dosage can be appropriately adjusted by considering these factors. The number of administrations can be once or twice or more within the range of clinically acceptable side effects, and administration can be performed at one or two or more sites. For animals other than humans, the same dosage per kg as for humans is used, or the above dosage is converted by, for example, the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human. A single dose can be administered. Possible routes of administration include oral, sublingual, parenteral (eg, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or implantation. It may involve the implantation of devices or substances. Examples of animals to be treated according to one embodiment include humans and other target mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc. included

A pharmaceutical composition according to one embodiment may include a pharmaceutically acceptable carrier and/or additives. For example, sterile water, physiological saline, common buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, tonicity agents, or preservatives, etc. can do. For topical administration, it may also include combining organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. can

The pharmaceutical composition according to one embodiment, if necessary according to the administration method or dosage form, suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, Buffers, reducing agents, antioxidants and the like may be appropriately included. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The pharmaceutical composition according to one embodiment is formulated in unit dosage form by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. It can be prepared as, or it can be prepared by inserting into a multi-dose container. The formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of a powder, granule, tablet or capsule.

Another aspect includes preparing an oxidized hyaluroninate solution by mixing a solution containing sodium periodate with a solution containing hyaluroninate; and mixing a solution containing the oxidized hyaluronicate with a solution containing glycol chitosan.

The method according to one embodiment may include mixing a solution containing the glycol chitosan and metal nanoparticles. In one embodiment, the metal nanoparticle may combine with mercapto betacyclodextrin and polyethylene glycol-SH to form PEG-s-GNP-s-CD.

In one embodiment, the solution containing glycol chitosan and the oxidized hyaluroninate solution may be mixed in a volume ratio of 1:10 to 10:1. For example, the solution containing glycol chitosan and the solution containing oxidized hyaluroninate are 1:10 to 10:1, 1:9 to 9:1, 2:8 to 8:2, 3:7 to 7 :3, 4:6 to 6:4 or 5:5 may be mixed in a volume ratio. At this time, when the mixing ratio of the solution containing glycol chitosan and the solution containing oxidized hyaluronate is less than or exceeds the above range, the glycol chitosan and oxidized hyaluronate are not sufficiently cross-linked, resulting in a low viscosity liquid Since it forms a fluid phase, there is a problem that a solid or semi-solid gel is not formed. Therefore, when the hydrogel is injected into the damaged area, the rate of absorption into the living body is very fast or it is easily dissolved in the living body, so the efficacy as a therapeutic agent may not be exhibited.

According to one embodiment, an imine bond may be formed between the aldehyde group of the oxidized hyaluroninate and the amino group of the glycol chitosan. The imine bond may be formed by a Schiff base reaction between an aldehyde group and an amino group.

The hydrogel prepared by the above method can be directly injected into the damaged area for treatment by patients with spinal injuries, and no additional damage due to infection or swelling of the hydrogel due to residual substances occurs after spinal cord injury. effective in the treatment of neuroinflammation.

The hydrogel according to one aspect has appropriate properties and has an effect that can be directly injected with a physiologically active substance into a damaged tissue site.

1a is a schematic diagram of a method for preparing glycol chitosan containing ursodeoxycholic acid.
Figure 1b is a schematic diagram of a method for producing a hydrogel according to one aspect.
Figure 1c is a schematic diagram of the process of irradiating near-infrared rays after injecting a hydrogel according to one aspect to a rat inducing spinal cord injury.
Figure 1c is a schematic diagram of the photothermal treatment effect by near-infrared ray irradiation of the hydrogel according to one aspect.
2 is a photograph confirming the hydrogel according to one aspect by SEM.
3 is a graph confirming the rheological properties of a hydrogel according to one aspect.
Figure 4 is a graph confirming the volume change of the hydrogel according to one aspect.
Figure 5a is a photograph of the hydrogel incorporated into cells observed as a fluorescence image for 24 hours or 48 hours.
Figure 5b is a graph measuring the cell viability of the hydrogel according to one aspect.
Figure 6a is a photograph taken by an infrared camera of the temperature of the lesion site after injecting the hydrogel according to one aspect to a spinal cord injury (SCI) rat and irradiating NIR.
Figure 6b is a graph showing the temperature change of the lesion site after injecting the hydrogel according to one aspect to spinal cord injury (SCI) rats and irradiating NIR.
Figure 7 is a graph showing the results of evaluating motor function recovery on the Basso-Beattie-Bresnahan (BBB) scale after injecting a hydrogel according to one aspect to spinal cord injury (SCI) rats.
8a is a result of confirming the phosphorylation of JNK, ERK, and p38 after injecting the hydrogel according to one aspect into the injured spinal cord.
Figure 8b is a graph showing the expression levels of TNK-α and IL-1β after the hydrogel according to one aspect was injected into the injured spinal cord.
9 is a photograph showing the results of H&E staining after injecting the hydrogel according to one aspect into the injured spinal cord.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Production Example]

Preparation Example 1. Preparation of oxidized hyaluronate and glycol chitosan

Hyaluronate (HA) (molecular weight (MW) = 1,000 kDa) (Humedix (Korea) was oxidized with sodium periodate (Sigma-Aldrich, USA) to obtain oxidized hyaluronate (oHA) ) was synthesized. Specifically, 3.8 g of HA was dissolved in 360 ml of ultrapure deionized water (DW, 18.2 MΩ) 1.068 g of sodium periodate was dissolved in another 40 ml of DW. Dissolved periodic acid Sodium acid was slowly added to HA in the dark and mixed, then the mixture was stirred for 24 hours, and 1 ml of ethylene glycol (Sigma-Aldrich, USA) was added to the mixture to obtain HA and unreacted HA. Sodium iodate was neutralized. Then, the solution was dialyzed using a dialysis membrane (Spectrum Spectra, molecular weight cut off (MWCO): 12-14K) for 7 days, and freeze-dried. Then, ¹ H -NMR spectrometer (AVANCE-400 MHz, Bruker, Germany) was used to confirm the synthesis of oHA from HA. As a result, three peaks were observed at 5.0 ppm in the ¹ H-NMR spectrum, indicating that the aldehyde group of oHA In addition, the amount of aldehyde groups in oHA was quantified using 2,4,6-trinitrobenzene sulfonate (TNBS), and oHA was about 30% (29.3±2.3%). Therefore, it can be seen that when HA with a molecular weight of 1,000 kDa is oxidized, oHA with a molecular weight of 300 kDa is formed.

0.2 g of glycol chitosan (gC) (Sigma-Aldrich, USA) was dissolved in 10 ml of DW and purified in the same manner as above to prepare a gC 2% (w/v) solution. On the other hand, since the degree of polymerization of the purchased gC is 2,000, the molecular weight of the gC polymer is the degree of polymerization (=2,000) x the molecular weight of a single unit of gC (=173), so it is 346,000. Since it was used at 2% (w/v), the molecular weight of the gC polymer used is 6,920 Da.

Therefore, assuming that 1000 mL of gel is made, if 2% (w/v) gC and 3% (w/v) oHA are mixed in a ratio of 9:1, the molecular weight of gC is 0.02 x 346,000 x 900 = 6,228,000 Da, and the oHA molecular weight is 0.03 x 100 x 300,000 = 900,000 Da.

Preparation Example 2. Preparation of Gold Nanoparticles Containing Ursodeoxycholic Acid

136 mg of Chloroauric acid (HAuCl ₄ ) powder (Sigma-Aldrich, USA) was dissolved in 800 mL of DW, and 300 mg of trisodium citrate powder (Sigma-Aldrich, USA) was dissolved in 15 mL of DW. dissolved. Thereafter, the HAuCl ₄ solution was refluxed at 110 °C, and a trisodium citrate solution was quickly added to prepare a gold nanoparticle (GNP) solution. Then, 9.2 mg of Mono mercapto beta cyclodextrin (SH-β-CD) (Zhiyuan Biotechnology, China) was added to 500 mL of the gold nanoparticle solution and vigorously stirred for 24 hours to induce GNP-S bonding (GNP -s-CD) was formed. 9.5 mg of polyethylene glycol (PEG)-SH (MW: 2000 g/mol) (SunBio, Korea) was added to the GNP-s-CD solution and vigorously stirred for 24 hours to obtain PEG-s-GNP- s-CD was formed. Then, the PEG-s-GNP-s-CD solution was centrifuged and the precipitate was washed with DW three times. Subsequently, unconjugated SH-β-CD and PEG-SH were removed by dialysis against a cellulose membrane for 7 days. Then, 40 mg of ursodeoxycholic acid (UDCA) (MW: 392.58 g/mol) (Tokyo Chemical Industry Co., Japan) was dissolved in 50.9 mL of DMSO to form a 2 mM UDCA solution. After adding 100 μL of UDCA solution to 900 μL of PEG-s-GNP-s-CD solution, it was sonicated for 5 minutes and centrifuged at 13,000 rpm for 15 minutes. The precipitate was washed three times with fresh DW and GNP (1.45 nM) containing UDCA precipitate was stored at 4°C.

[Example]

Example 1. Preparation of Hydrogel Containing Gold Nanoparticles

After dissolving gC 2% (w / v) and oHA 3% (w / v) prepared in Preparation Example 1 in DPBS (Gibco, USA) and centrifuging at 13,000 rpm for 30 seconds to remove air bubbles, the hydrogel ( CHA gel) was prepared and stored at -20 °C. The formation of cross-links in the hydrogel was confirmed using Fourier transform infrared spectroscopy. As a result, an absorption peak was observed at 1456 cm ^-1 , indicating that the aldehyde group of oHA and the amino group of gC formed an amine bond. Thereafter, the GNP prepared in Preparation Example 2 was mixed with gC 2% (w/v) and then cross-linked with oHA 3% (w/v) to form a hydrogel containing gold nanoparticles (CHA-GNP gel) was formed.

Example 2. Confirmation of viscosity, rheological and biodegradation properties

2-1. Viscosity properties

The volume ratio of gC 2% (w/v) and oHA 3% (w/v) with or without GNP for optimizing the viscosity of the hydrogel according to one aspect was investigated. Specifically, the gC 2% (w / v) and the oHA 3% (w / v) are mixed at a volume ratio of 7: 3, 8: 2 or 9: 1 so that the total volume is 300 μL hydrogel (CHA gel and CHA-GNP gels) were prepared. Thereafter, the prepared hydrogel was passed through a 28 gauge (G) needle and gently detached in a Petri dish. The hydrogel was frozen at -80 °C for 24 hours and lyophilized for 2 days under reduced pressure, and SEM images were acquired at 10K magnification.

2 is a photograph confirming by SEM whether or not a hydrogel suitable for injection is formed. As shown in Figure 2, it was confirmed that the hydrogels of all volume ratios were formed from the solution within 1 minute after mixing. However, in the case of the CHA gel in which gC and oHA containing no GNP were mixed at a ratio of 7:3 or 8:2, respectively, the pore size was relatively irregular, and the viscosity exceeded 1,500 Pa, so it could not pass through a 28G needle. On the other hand, in the case of the CHA-GNP gel in which gC and oHA containing no GNP were mixed at a ratio of 9:1, respectively, it was confirmed that the gel passed through a 28G needle.

2-2. Rheological properties

The rheological properties of the hydrogel according to one aspect were confirmed. Specifically, 300 μL of a cylinder-shaped hydrogel (CHA gel and CHA-GNP gel) having a diameter of 12 mm and a height of 55 mm was prepared at the same volume ratio of gC and oHA as in Example 2-1. Then, the strain-dependent storage modulus (G') and loss modulus (G") were measured using a rotating rheometer (AR-G2, TA Instruments, USA) at a compression rate of 2 mm/min until a strain of 8.1% was reached. did

3 is a graph confirming the rheological properties of a hydrogel according to one aspect.

As a result, as shown in FIG. 3, in the case of CHA gel in which gC and oHA were mixed in volume ratios of 7:3, 8:2, and 9:1, respectively, G' was 2841.6±105.3 Pa, 1570.7±45.0 Pa, and 493.9 It decreased in the order of ±28.1 Pa. On the other hand, in the case of the CHA-GNP gel in which gC and oHA containing GNP were mixed at a volume ratio of 9:1, it was confirmed that G' increased compared to the CHA gel by showing 576.5±5.4 Pa. In addition, in the case of CHA gel in which gC and oHA were mixed in volume ratios of 7:3, 8:2, and 9:1, respectively, G˝ decreased in the order of 116.4±11.7 Pa, 56.6±3.3 Pa, and 25.4 Pa. In addition, the CHA-GNP gel in which gC and oHA containing GNP were mixed at a volume ratio of 9:1 showed 20.4±1.7 Pa.

2-3. biodegradation properties

Biodegradability was confirmed by measuring the hydrolysis rate of the hydrogel. Specifically, 400 μL of hydrogel (CHA gel and CHA-GNP gel) was prepared with gC and oHA containing or not containing GNP at a volume ratio of 9:1, respectively, and then each hydrogel was mixed with DPBS (pH 7.4 or pH 6.4). ) in 3 mL (n = 3). Thereafter, while culturing the hydrogel in a 100 RPM incubator for 14 days at 37° C., DPBS was replaced every other day. At predetermined time points (1h, 1d, 3d, 7d or 14d), each hydrogel was weighed and the weight of the remaining hydrogel was calculated at each time point using the equation below.

[mathematical expression]

% of remaining gel ₌ (Wp/ _Wo ) × 100,

Here, W _p is a weight at a predetermined point in time and W _o is an initial weight.

Figure 4 is a graph confirming the volume change of the hydrogel according to one aspect.

As a result, as shown in FIG. 4, it was confirmed that the CHA and CHA-GNP gels hardly swelled compared to the initial mass. In the case of CHA gel, it slightly increased to 103.6 ± 0.8% (pH 7.4) and 102.7 ± 0.4% (pH 6.4), respectively, for 1 hour, and in the case of CHA-GNP gel, 103.1 ± 0.6% at pH 7.4 and pH 6.4, respectively, A slight increase to 102.5±0.7% was confirmed. After that, it was confirmed that the mass% of each hydrogel continuously decreased for 14 days.

That is, as the hydrogel according to one aspect is hydrolyzed in vivo, it can be usefully used as a delivery system for treating damaged areas.

Example 3. Cell viability

The cytotoxicity of the hydrogel according to one aspect was evaluated. First, NIH-3T3 cells (ATCC, USA) were cultured in growth medium (GM) consisting of DMEM (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin-streptomycin. Thereafter, NIH-3T3 cells (2 x 10 ⁵ ) were seeded in 90 μL of a 2% (w/v) solution of gC with or without GNP and mixed with 10 μL of 3% (w/v) oHA to form a hydrogel (CHA and CHA-GNP gel) was formed (n = 5). Thereafter, the hydrogel containing the cells was dispensed into a 48-well culture plate, and incubated with a growth medium for 1-2 days. The hydrogel (n = 4) was tested for cytotoxicity using a cell viability assay kit (EZ-Cytox, Daeil Labservice, Korea), and the hydrogel was stained with Alexa Fluor 488 phalloidin and examined under a confocal laser scanning microscope (LSM). 880, Germany).

Figure 5a is a photograph of a hydrogel incorporated into cells observed as a fluorescence image for 24 to 48 hours, and Figure 5b is a graph measuring cell viability in a hydrogel according to one aspect.

As a result, as shown in Figure 5a, the number of cells in the CHA gel and CHA-GNP gel increased at 48 hours compared to 24 hours. In addition, as shown in Figure 5b, the cell viability in the CHA gel group calculated at 24 hours and 48 hours was 100 ± 9.8% and 146.7 ± 12.8%, respectively, and the cell viability in the CHA-GNP gel group was 113.3 ± 12.8%, respectively. 3.9% and 161.7±9.3% were confirmed.

That is, it can be seen that the hydrogel according to one aspect has excellent biocompatibility because it does not exhibit cytotoxicity.

Example 4. In vivo Experiment

4-1. Preparation of laboratory animals

Eighty-one adult female Sprague-Dawley (SD) rats (210-240 g) (Orient Bio Inc., Korea) were housed at a humidity of 55-65% and a temperature of 24±3° C. with a light/dark cycle of 12 hours. Rats were given free access to water/food. Thereafter, the rat was anesthetized by intraperitoneal administration of Rompun® mg/kg, Bayer, Korea)/Zoletil® (50 mg/kg, Virbac Laboratories, France) solution, and the ninth thoracic (T9) spinal cord segment of the anesthetized rat was exposed by minotomy. The vertebrae were stabilized by supporting them with Allis clamps at the T7 and T11 spinous processes by a known method. A 50 g metal impounder was then gently applied onto the T9 dura for 5 minutes to induce moderate weight compression and damage.

All surgical interventions and postoperative animal care procedures are in accordance with the Guidelines and Policies for Rodent Survival Surgery provided by the Institutional Animal Care and Use Committee (IACUC) at CHA University (IACUC180146) and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health (NIH), USA).

4-2. Check the temperature of the lesion after near-infrared irradiation

Thermal therapy using near-infrared (NIR) irradiation has been widely used mainly to treat tumors. Therefore, the treatment effect by NIR of the hydrogel according to one aspect was confirmed. Specifically, within 1 minute after injury, a 28G needle was inserted to a depth of 2.5 mm in the dura at the center of the lesion of the injured spinal cord in the 9 rats whose spinal cord was injured in Example 4-1 and left there for 10 seconds. Then, the needle was retracted from the dura to 0.5 mm, and DPBS (n = 3), CHA gel ( n=3) or 8 μl of CHA-GNP gel (n=3) was injected for 120 seconds each, then the needle was left in place for 20 seconds to prevent leakage. After that, the surgical site was sutured, irradiated with a NIR laser (powder density: 2 W/cm2, model: FC-W-808 nm, Changchun New Industries Optoelectronics Technology, China) for 5 minutes, and performed once a day for 5 days. did At 1, 3, and 5 days after injury (DPI), thermal images were acquired with an infrared camera (FLIR ONE PRO, FLIR Systems, Inc., OR, USA), and the temperature of the lesion was measured.

Figure 6a is a photograph of the temperature of the lesion site taken with an infrared camera after injecting a hydrogel into a spinal cord injury (SCI) rat and irradiating NIR, and Figure 6b is a photograph of a hydrogel injected into a spinal cord injury (SCI) rat and NIR After irradiation, it is a graph showing the temperature change of the lesion site.

As a result, as shown in Figure 6b, the temperature of the group injected with DPBS was 37.3 ± 1.5 on the 5th day, and the temperature of the group injected with CHA gel was 37.2 ± 1.3 ℃ (1 DPI), 39.8 ± 1.6 ℃ (3 DPI), 39.1 ± 1.4 °C (5 DPI), and the temperatures of the group injected with CHA-GNP gel were 44.5 ± 1.7 °C (1 DPI), 48.7 ± 1.3 °C (3 DPI) and 47.5 ± 1.8 °C (5 DPI). On the other hand, the temperature of the lesion before infrared irradiation was 30.4 ± 0.3 ° C, and it was confirmed that the temperature of the lesion increased the most in the group injected with CHA-GNP gel among the three groups.

4-3. Assessment of recovery of motor function

It was confirmed whether the hydrogel according to one aspect recovers the motor function of the damaged spinal cord. Specifically, DPBS (n = 18), Example 2-1 and DPBS (n = 18) in the same manner as Example 4-2, except that 72 rats with spinal cord injuries were used in Example 4-1. 8 μl of CHA gel (n = 18) or CHA-GNP gel (n = 36) mixed with gC and oHA with or without GNP in a volume ratio of 9:1 were respectively injected. Thereafter, NIR laser was irradiated on 18 rats injected with CHA-GNP gel in the same manner as in Example 4-2, and then 3 rats per group were randomly selected to treat 1, 2, 4, 7, and 10 rats after injury. And on day 14, recovery of hind limb motor function was evaluated. Specifically, behavioral scores were assessed on the Basso-Battie-Bresnahan (BBB) scale by two experienced investigators who were unaware of the experimental conditions.

7 is a graph showing the results of evaluation of motor function recovery after hydrogel injection in spinal cord injury (SCI) rats using the Basso-Beattie-Bresnahan (BBB) scale.

As a result, as shown in FIG. 7, the average scores in the four groups showed an increasing pattern for 14 days. Specifically, the group injected with CHA-GNP gel and then irradiated with NIR showed 9.6±1.4, 14.2±1.2, and 16.2±0.8 at 7, 10, and 14 days, respectively, and the group without NIR irradiation showed 6.7±1.5, respectively. , 9.2±1.9 and 10.8±1.7. That is, it can be seen that the motor function recovery of the CHA-GNP gel is improved by NIR irradiation.

4-4. Neuroinflammation inhibitory effect

It was confirmed whether the hydrogel according to one aspect could inhibit neuroinflammation in the injured spinal cord. Specifically, with respect to the rats of Example 4-3, 3 rats per group were sacrificed on days 1, 4, and 14 after injury. Then, spinal cord segments (10 mm) including the lesion epicenter were collected, washed with DPBS, and stored at 4°C. Then thoroughly homogenized in 1x RIPA lysis buffer (Sigma). It was centrifuged at 4 °C and 13,000 rpm for 15 minutes and the supernatant was removed. This washing process was repeated 4 times. Protein concentrations in tissue lysates were calibrated by the BCA Protein Assay Kit (Thermo Scientific, IL). Thereafter, phosphorylation of JNK, ERK, and p38 was confirmed by Western blot, and expression levels of TNF-α and IL-1β were measured by ELISA.

8a shows the result of confirming the phosphorylation of JNK, ERK, and p38 after hydrogel injection into the injured spinal cord.

As a result, as shown in FIG. 8a, it was confirmed that the phosphorylation of JNK, ERK, and p38 was inhibited in the spinal cord injured by the injection of the CHA gel or the CHA-GNP gel.

Figure 8b is a graph showing the expression levels of TNF-α and IL-1β after irradiation of the hydrogel on the injured spinal cord.

As a result, as shown in Figure 8b, the expression levels of TNF-α and IL-1β were significantly reduced in the group irradiated with NIR after injecting the CHA-GNP gel compared to the group injected with DPBS. .

Therefore, the hydrogel according to one company inhibits phosphorylation of JNK, ERK, and p38 in the injured spinal cord and reduces the level of inflammatory cytokines, such as TNF-α and IL-1β, thereby reducing neuroinflammation in the injured spinal cord. can be suppressed

4-5. Restoration effect of damaged tissue

It was confirmed whether the hydrogel according to one aspect promotes the recovery of damaged tissue. Specifically, 3 rats per group were sacrificed in the same manner as in Example 4-4. Thereafter, rats were perfused with 4% PFA followed by saline, and segments (10 mm) were collected from the snout to the tail, including the lesion epicenter, post-fixed overnight in 4% PFA, and embedded in paraffin. After that, they were dewaxed for histological analysis. Serial longitudinal sections (5 μm thick) through the dorsal axis of the spinal cord were collected at 150 μm (day 30) and 155 μm (day 31) lengths per rat. The 30th section for each rat was stained with H&E solution (BBC Biochemical, USA) and analyzed. The most severely damaged spinal cord region in each section was designated as a region of interest (ROI, 1500×1000 μm ² ). Morphological changes of the H&E-stained tissues were observed at ×4 (scale bar: 500 μm) and ×10 (scale bar: 200 μm) magnifications using an optical microscope (Olympus IX71, Japan). The rectangular area designated as the ROI represents 100%. Cavity tissue volume was calculated and quantified using ImageJ software (NIH).

9 is a photograph showing the results of H&E staining of the injured spinal cord.

As a result, as shown in FIG. 9, it was confirmed that the size of the cavity gradually decreased in the four groups. In particular, it was confirmed that cavity damage was significantly inhibited in the group irradiated with NIR after injecting the CHA-GNP gel. In addition, significant recovery was shown in the group irradiated with NIR after injection of CHA-GNP gel on the 4th day after injury.

Therefore, it can be seen that the hydrogel according to one aspect promotes the recovery of damaged spinal cord tissue.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (14)

glycol chitosan, oxidized hyaluronate; and a metal nanoparticle or a metal nanoparticle and a physiologically active substance bound to a surface of the metal nanoparticle, wherein the glycol chitosan and oxidized hyaluronate are mixed in a weight ratio of 1 to 10:1. The hydrogel of claim 1, wherein the weight ratio is 3 to 10: 1. delete The hydrogel of claim 1, wherein the solution containing the glycol chitosan and the solution containing the oxidized hyaluronate are mixed in a volume ratio of 1:10 to 10:1. The hydrogel of claim 1, wherein the hydrogel has a storage modulus (G′) of 300 to 800 Pa or a loss modulus (G″) of 10 to 40 Pa. The hydrogel of claim 1, wherein the glycol chitosan and oxidized hyaluronate form an imine bond. The hydrogel of claim 1, wherein the hydrogel is directly injectable into damaged tissue. A hydrogel comprising glycol chitosan and oxidized hyaluronate, wherein the glycol chitosan and oxidized hyaluronate are mixed in a weight ratio of 1 to 10:1; and
A drug carrier for drug delivery to a damaged tissue site containing a physiologically active substance bound to the surface of metal nanoparticles. delete Containing glycol chitosan, oxidized hyaluronate, metal nanoparticles and a treatment for spinal cord injury as active ingredients, wherein the glycol chitosan and oxidized hyaluronate are mixed in a weight ratio of 1 to 10:1 Phosphorus, a pharmaceutical composition for preventing or treating spinal cord injury. The method according to claim 10, wherein the spinal cord injury is traumatic spinal cord injury, spinal degenerative disease, spinal inflammatory disease, spinal cord tumor, spinal tumor, spinal cord hemorrhage, stroke, spinal cord paralysis due to extramedullary vascular disorder, myelitis, multiple sclerosis, amyotrophic lateral sclerosis A pharmaceutical composition for preventing or treating spinal cord injury that is selected from. The pharmaceutical composition for preventing or treating spinal cord injury according to claim 10, which is for photothermal therapy. The pharmaceutical composition for preventing or treating spinal cord injury according to claim 10, wherein the spinal cord injury treatment agent is bound to the surface of the metal nanoparticle. The pharmaceutical composition for preventing or treating spinal cord injury according to claim 10, wherein the metal nanoparticles are gold nanoparticles, and the spinal cord injury treatment agent is ursodeoxycholic acid.

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