KR102283681B1 - Lens shape drug delivery system and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a lens-shaped biodegradable drug delivery system and a method for manufacturing the same, and more particularly, hydrogel 1 to 70 w/v%, conjunctival cells 5×10 ⁶ to 50×10 ⁶ /ml, and treatment of eye diseases By using a bio-ink composition containing a drug for a drug, it is possible to manufacture a drug delivery system that exhibits excellent biocompatibility, can continuously release the drug, and has less damage to conjunctival cells.

Description

Lens-shaped drug delivery system and manufacturing method thereof {LENS SHAPE DRUG DELIVERY SYSTEM AND MANUFACTURING METHOD THEREOF}

The present invention relates to a lens-shaped drug delivery system and a method for manufacturing the same.

As the incidence rate of various eye diseases increases due to a rapid increase in the aging population and worsening air pollution, the market for eye disease treatment and related R&D is growing.

When a drug for treating eye diseases is administered as an eye drop, a low corneal penetration rate of the drug is shown, and the residence time of the drug on the corneal surface is only within 10 minutes, so there is a problem of the inconvenience of continuously instilling the drug. In addition, as the drug is administered in a significant amount, serious side effects such as vascular disease or heart disease may appear, and in many cases, the drug stays above the toxic concentration on the corneal surface immediately after the administration of the eye drops. As such, treatment of eye diseases through eye drops has problems such as cumbersome and side effects due to high dosing frequency, drug waste, and low corneal permeability. Therefore, there is a need to develop a new drug delivery method capable of maintaining the intraocular drug concentration and improving drug absorption.

On the other hand, 3D bioprinting technology, also called cell printing or organ printing, enables the production of computer-designed 3D structures using various types of cells, biomaterials, and biomolecules. Research to regenerate necessary functional tissues through the production of cell structures similar to tissues constituting the human body using 3D bioprinting technology is being actively conducted. In the 3D bioprinting process, bio-ink is the most important key factor for precise patterning of bio-cells and guarantees viability, and the characteristics of the ink are directly linked to the process.

'Bio-ink' is a generic term for a material that contains living cells or biomolecules and can be applied to bioprinting technology to fabricate necessary structures. Therefore, the bio-ink must provide physical properties for 3D processing and a biological environment for the cells to perform their intended functions. The bio-ink must first have good cell affinity. In the case of artificial tissue and organ regeneration, a biological environment favorable for proliferation and differentiation of printed cells must be provided from the bio-ink. When the printing process is long, the supply of nutrients and oxygen necessary for the survival of cells in the cartridge must be properly performed. In addition, it should be able to protect the cells from the physical stress that occurs during the printing process. In addition, bio-ink must have physical properties required in the printing process, such as repeatability of 3D patterning, productivity, and no clogging of nozzles. As described above, the characteristics that inks require for 3D bioprinting technology using living cells are very diverse and are intricately intertwined with each other. In other words, it can be said that the development of excellent bio-ink is the key to the development of bio-printing technology.

Korean Patent Publication No. 2018-0107218

An object of the present invention is to provide a drug delivery system for biodegradable ophthalmic diseases.

1. Hydrogel 1 to 70 w/v%, conjunctival cells 5×10 ⁶ to 50×10 ⁶ /ml, and a lens-shaped base matrix consisting of a bio-ink composition containing a drug for treating eye diseases for treatment of eye diseases A lens-shaped biodegradable drug delivery system loaded with drugs.

2. The method of 1 above, wherein the hydrogel comprises 1 to 30 w/v% of gelatin, 10 to 50 w/v% of collagen, and 1 to 30 w/v% of polyethylene glycol, a lens-shaped biodegradable drug delivery system .

3. The biodegradable drug delivery system of the above 1, wherein the bio-ink composition further comprises 1 to 20 w/v% of fibrinogen and 1 to 20 w/v% of glycerol.

4. The lens-shaped biodegradable drug delivery system according to the above 1, wherein the drug is acetylcysteine or tobramycin.

5. The lens-shaped biodegradable drug delivery system according to the above 1, wherein the drug is acetylcysteine in an amount of 0.01 to 10 mM.

6. The lens-shaped biodegradable drug delivery system according to the above 1, wherein the drug is 0.01-5 mM tobramycin.

7. Hydrogel 1 to 70 w/v %, conjunctival cells 5×10 ⁶ to 50×10 ⁶ /ml, and a bio-ink composition containing a drug for treating eye disease is injected into a 3D printer, so that the drug for treating eye disease is A method of manufacturing a lens-shaped biodegradable drug delivery system comprising; preparing a mounted lens-shaped base matrix.

8. The biodegradable drug delivery system in the form of a lens according to the above 7, wherein the hydrogel comprises 1 to 30 w/v% of gelatin, 10 to 50 w/v% of collagen, and 1 to 30 w/v% of polyethylene glycol manufacturing method.

9. The method of manufacturing a lens-shaped biodegradable drug delivery system according to the above 7, wherein the bio-ink composition further comprises 1 to 20 w/v% of fibrinogen and 1 to 20 w/v% of glycerol.

10. The method of manufacturing a lens-shaped biodegradable drug delivery system according to the above 7, wherein the drug is 0.01 to 10 mM acetylcysteine.

11. The method of manufacturing a lens-shaped biodegradable drug delivery system according to the above 7, wherein the drug is 0.01 to 5 mM tobramycin.

The bio-ink composition of the present invention has excellent printability and can protect cells from physical stress generated during the printing process.

The lens-shaped drug delivery system prepared from the bio-ink composition of the present invention has excellent biocompatibility and bioabsorption by using a human-derived material.

The lens-shaped drug delivery system prepared from the bio-ink composition of the present invention can continuously release a certain amount of drug within the eye, and thus has excellent therapeutic effect.

The manufacturing method of the lens-shaped drug delivery system of the present invention can mass-produce lenses with less damage to conjunctival cells by using a 3D bioprinter.

1 shows a lens manufacturing process using a 3D printer (Bio X 3D Print).
Figure 2 is a lens-shaped base matrix (Figure 2A) that is not loaded with a drug printed by a bioprinter, a drug delivery system loaded with acetylcysteine on the base matrix (Figure 2B), and a drug delivery system with tobramycin loaded on the base matrix (Figure 2B). 2C) shows the image.
3 shows the ATP concentration values of conjunctival cells according to the concentration of acetylcysteine.
4 is a result of measuring the LDH value of conjunctival cells according to the concentration of acetylcysteine.
5 shows the cell survival and death degree of conjunctival cells according to the concentration of acetylcysteine.
6 is a result of measuring the LDH value of conjunctival cells according to the concentration of tobramycin.
7 shows the cell survival and death degree of conjunctival cells according to the concentration of tobramycin.

Hereinafter, the present invention will be specifically described.

The present invention provides a drug delivery system in the form of a biodegradable lens.

The term 'biodegradability' refers to a property that can be decomposed by body fluids, enzymes or microorganisms in a living body.

The term 'lens-shaped drug delivery system' refers to a lens-shaped structure in which a drug is loaded, and refers to a material that can be worn on the eye.

The term 'lens' means a structure that can be placed on or in the eye of a wearer and includes lenses that can correct, improve, or change the user's field of vision. The term ‘lens’ may be used interchangeably with ‘contact lens’. The lens may be a hard lens or a soft lens.

The lens-shaped drug delivery system may be manufactured using a bio-ink composition.

The lens-shaped drug delivery system may be composed of components included in the bio-ink composition.

The term 'bio-ink composition' is a biomaterial that transports living cells during bioprinting, and refers to a material that contains living cells or biomolecules and can be applied to bioprinting technology to fabricate necessary structures.

The bio-ink composition requires excellent biocompatibility to be used in bio-printing, and must have physical properties that can be printed in a desired pattern by smoothly passing through a micro-diameter dispensing nozzle, and provide a cell-specific signal after printing It should be able to maintain the role of mechanical support while doing so.

The bio-ink composition should be able to provide physical properties for three-dimensional processing and a biological environment in which cells can perform a desired function, and should have excellent cell affinity.

The bio-ink composition may include a hydrogel and conjunctival cells.

The term 'hydrogel' is a hydrophilic polymer crosslinked by cohesive force such as covalent bond, hydrogen bond, van der Waals bond or physical bond, and has a three-dimensional polymer network structure that can absorb a large amount of water and swell in an aqueous solution. is a substance Hydrogels may have various shapes and properties depending on components and manufacturing methods, and may have intermediate properties between liquids and solids because they generally contain a large amount of moisture. In addition, the hydrogel may be a material that exhibits biodegradability and biocompatibility properties.

The term 'biocompatibility' refers to the property of being non-toxic to the human body and chemically inert.

The hydrogel may be natural or synthetic. The hydrogel can be used by purchasing commercially available products, or by directly synthesizing it.

The hydrogel is made of hyaluronic acid, gelatin, collagen, alginate, cellulose, chitosan, chitin, synthetic peptide, polyethylene glycol, polyethylene glycol diacrylate (PEGDA), polycaprolactone and glycerol. It may include at least one selected from the group consisting of.

The term 'hyaluronic acid' is used interchangeably with 'hyaluronan', 'hyaluronate' or 'HA', for example sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, hyaluronic acid potassium and combinations thereof.

The hyaluronic acid has a molecular weight of 1,000 g/mol to 100,000 g/mol, 2,000 g/mol to 90,000 g/mol, 3,000 g/mol to 80,000 g/mol, 4,000 g/mol to 70,000 g/mol, 5,000 g/mol to It may be 60,000 g/mol or 6,000 g/mol to 50,000 g/mol.

The term 'gelatin' may refer to a protein obtained by partial hydrolysis of collagen, which is a major protein component of connective tissues such as bones, cartilage, and leather of animals. Gelatin may include a gelatin derivative in addition to pure gelatin.

The gelatin derivative may be at least one selected from the group consisting of phthalated gelatin, esterified gelatin, amidated gelatin, and formylated gelatin.

The gelatin derivative may be gelatin methacrylate.

The gelatin may be derived from mammalian, fish, bovine bone, bovine skin, porcine bone or porcine skin.

The content of gelatin can be changed to control solubility.

Collagen may be included in an amount of 10 mg/ml to 25 mg/ml, 25 mg/ml to 35 mg/ml, or 35 mg/ml to 45 mg/ml per total volume of the bio-ink composition.

PEGDA may be included in the bio-ink composition together with alginate, and PEGDA may be included in 3 wt% with respect to alginate.

PEG or PEGDA may be included in the bio-ink composition having a viscosity of 0.5 dL/g to 1.5 dL/g.

The cellulose may be nano-sized nanocellulose.

Cellulose may be included in an amount of 3 to 5 wt% based on the total mass of the bio-ink composition.

Chitosan may have a molecular weight of 140 kDa to 300 kDa.

‘Polycaprolactone’ is a biocompatible and semi-crystalline fatty polyester.

Polycaprolactone has a molecular weight of 4,000 g/mol to 150,000 g/mol, 5,000 g/mol to 140,000 g/mol, 6,000 g/mol to 130,000 g/mol, 7,000 g/mol to 120,000 g/mol, 8,000 g/mol to 110,000 g/mol, 9,000 g/mol to 100,000 g/mol, 10,000 g/mol to 90,000 g/mol, 20,000 g/mol to 80,000 g/mol, or 30,000 g/mol to 70,000 g/mol.

Glycerol is one of the aliphatic trihydric alcohols, and may exhibit an anti-clogging effect during bioprinting.

Glycerol may be included in an amount of 1 to 40w/v%, 5 to 35w/v%, 10 to 30w/v%, or 15w/v% to 25w/v% per total volume of the bio-ink composition.

In one embodiment, the hydrogel may include 1 to 30 w/v% of gelatin, 10 to 50 w/v% of collagen, and 1 to 30 w/v% of polyethylene glycol, and to adjust solubility and/or transparency It is possible to change the content for

The term 'conjunctiva' is a thin, transparent mucous membrane that covers the inside of the eyelids and the white part of the eyeball, and is distributed to the edge of the cornea.

The conjunctival cell may be a conjunctival epithelial cell.

The conjunctival cells may be isolated from animals, for example, isolated from humans.

Conjunctival cells may be isolated by any method known in the art.

The conjunctival cells may be cultured in any manner known in the art.

For example, the conjunctival cells may be cultured under aseptic conditions from a living individual.

The present invention can mass-produce a drug delivery system more easily and quickly using conjunctival cells instead of corneal cells.

The bio-ink composition of the present invention may contain 1 to 70 w/v% of hydrogel relative to the total volume of the composition, and 5×10 ⁶ to 50×10 ⁶ /ml of conjunctival cells.

The bio-ink composition of the present invention may further include at least one selected from the group consisting of a cell culture medium, a physiologically active material, a decellularized extracellular matrix (dECM), an additive, and a drug for treating eye diseases.

The cell culture medium includes any medium suitable for the desired cells, and may be, for example, MEM medium, RPMI1640 medium, DMEM medium, IMDM medium, and the like. The cell culture medium may further contain vitamins, growth factors, cytokines, antibiotics, and the like.

The physiologically active substance is for proliferating cells or promoting the secretion of extracellular matrix, for example, transforming growth factor-beta (TGF-β), fibroblast growth factor (FGF), bone morphogenic protein (BMP), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), placental growth factor (PIGF), granulocyte colonies It may be at least one selected from the group consisting of a cell culture additive such as granulocyte colony stimulating factor (G-CSF) and ascorbate 2-phosphate.

The term 'extracellular matrix' (ECM) is a matrix that surrounds the outside of the cell, occupies between cells, and has a network structure mainly composed of proteins and polysaccharides.

The term 'decellularization' is a process of separating the extracellular matrix components contained in living tissue through chemical treatment. It can be decellularized by immersing it in a cell to remove the nucleus and leaving only the extracellular matrix.

The decellularized extracellular matrix may be obtained by decellularizing tissues extracted from mammals such as humans, pigs, cattle, rabbits, dogs, goats, sheep, chickens and horses.

The additive may be at least one of fibrinogen, thrombin, tissue adhesive, and polyethylene oxide-polypropylene oxide copolymer (Pluronic F127).

Fibrinogen is a protein belonging to globulin, and can create an environment suitable for cell adhesion and differentiation.

Fibrinogen or thrombin may have a molecular weight of 340 kDa or less.

The bio-ink composition of the present invention may further include 1 to 20 w/v% of fibrinogen and 1 to 20 w/v% of glycerol.

The term 'tissue adhesive' is an adjuvant for surgical wound closure, which can help in reconstructing a wound during surgery inside and outside the living body, and can be used not only in the field of surgery but also for drug delivery.

The tissue adhesive may be selected from the group consisting of cyanoacrylate, fibrin, and biodendrimer.

Pluronic F127 can be used to control the thickness of the printed structure.

Pluronic F127 per total volume of bio-ink composition 5mg/ml to 95mg/ml, 10mg/ml to 90mg/ml, 15mg/ml to 85mg/ml, 20mg/ml to 80mg/ml, 25mg/ml to 75mg/ml, 30 mg/ml to 70 mg/ml, 35 mg/ml to 65 mg/ml, 40 mg/ml to 60 mg/ml, or 45 mg/ml to 55 mg/ml may be included.

The term 'eye disease' means a disease, condition or condition that affects or is implicated in the ocular region. Generally speaking, the ocular includes the eyeball and the tissues and fluids that make up the eyeball, the periocular muscles and the portion of the optic nerve in or adjacent to the eyeball. “Eye region” means any area of the eyeball, including the anterior and posterior portions of the eye.

Eye diseases include dry eye syndrome, cataract, thyroid ophthalmopathy, cataract, retinitis, herpetic keratitis, bacterial keratitis, primary open-angle glaucoma, angle-closure glaucoma, neovascular glaucoma, proliferative vitreous retinopathy, macular degeneration, retinopathy pigmentosa, diabetes mellitus. Retinopathy, choroidal neovascularization, ischemic optic nerve disorder, retinopathy of prematurity, retinopathy of prematurity, epidemic keratoconjunctivitis, neovascular iris disease, retrocapsular fibroplasia, atopic keratitis, superior limbal keratitis, phase psoriatic keratitis, plictenoid keratitis, scleritis, diabetic macular edema, and the like.

The drug for treating eye disease may be DNA, RNA, cell, protein, polypeptide, or compound, but any known drug for preventing or treating eye disease may be applied without limitation.

Drugs for the treatment of eye diseases include, for example, acetylcysteine, antibiotics (tobramycin, bigamox, etc.), anti-inflammatory drugs (loteprednoletabonide, fluorometholone, etc.), Anti VEGF (Avastin, Lucentis eilia, etc.), dexametha Zone (Tamsetone, Markade, Ozerdex, etc.), intraocular injection, and a combination thereof may be at least one.

The bio-ink composition of the present invention is 0.01 to 10mM, 0.01 to 9mM, 0.01 to 8mM, 0.01 to 7mM, 0.01 to 6mM, 0.01 to 5mM, 0.01 to 4mM, 0.01 to 3mM, 0.01 to 2mM, 0.01 to 1mM, 0.01 to 0.5mM , 0.01 to 0.1 mM or 0.01 to 0.05 mM of the drug.

In one embodiment, the bio-ink composition of the present invention may contain 0.01 to 10 mM acetylcysteine. For example, the bio-ink composition of the present invention is 0.01 to 10mM, 0.01 to 9mM, 0.01 to 8mM, 0.01 to 7mM, 0.01 to 6mM, 0.01 to 5mM, 0.01 to 4mM, 0.01 to 3mM, 0.01 to 2mM or 0.01 to 1mM of acetylcysteine.

In one embodiment, the bio-ink composition of the present invention may contain 0.01 to 5 mM of tobramycin. For example, the bio-ink composition of the present invention is 0.01 to 5mM, 0.01 to 4mM, 0.01 to 3mM, 0.01 to 2mM, 0.01 to 1mM, 0.01 to 0.5mM, 0.01 to 0.4mM, 0.01 to 0.3mM, 0.01 to 0.2mM, 0.01 to 0.1 mM tobramycin may be included.

Cell viability can be maintained in the bioprinted construct by including the drug at the above-mentioned concentration in the bio-ink composition.

The lens-shaped drug delivery system of the present invention may be manufactured by bio-printing the above-described bio-ink composition with a 3D printer.

The term ‘3D bioprinting’ refers to a technology that uses 3D printing technology to produce biological structures such as cornea, liver, skin, and blood vessels. 3D printing may be using methods and devices generally known to those skilled in the art.

The lens-shaped drug delivery system manufactured using 3D printing technology has excellent adhesion to the eye, excellent biocompatibility and bioabsorption, and may exhibit biodegradable properties. In addition, by using 3D printing technology, it is possible to quickly and easily manufacture a lens-shaped drug delivery system in a large quantity.

The lens-shaped drug delivery system of the present invention may include a lens-shaped base matrix and a drug for treating eye diseases.

The term 'base matrix' refers to a base material on which a drug for preventing or treating eye disease can be mounted, and the base matrix of the present invention includes components other than the drug for treating eye disease among the components of the bio-ink composition described above. can

The substrate matrix may be in the form of commonly used soft lenses or hard lenses.

The drug delivery system of the present invention may be in a form in which a drug for treating eye diseases is mounted on a base matrix, for example, the drug may be located inside and/or on the surface of the base matrix.

The loading of the drug onto the substrate matrix can be performed through methods known in the art.

For example, the drug can be mounted on the substrate matrix by printing bio-ink containing the drug with a 3D printer.

For example, a drug-free bio-ink may be printed with a 3D printer to prepare a base matrix, and then the drug may be loaded onto the base matrix by immersing the base matrix in a drug solution.

A lens-shaped drug delivery system in which a drug is mounted on a base matrix may be decomposed in vivo, and the loaded drug may be slowly released.

In addition, the present invention provides a method for producing a lens-shaped biodegradable drug delivery system comprising: preparing a lens-shaped base matrix by printing a bio-ink composition comprising a hydrogel and conjunctival cells into a 3D printer to provide.

The bio-ink composition may contain 1 to 70 w/v % of hydrogel and 5×10 ⁶ to 50×10 ⁶ /ml of conjunctival cells relative to the total volume of the composition.

According to one embodiment, by using a bio-ink composition further comprising a drug for treating eye disease in addition to the hydrogel and conjunctival cells, a lens-shaped base matrix loaded with a drug for treating eye disease can be prepared.

Hydrogel, conjunctival cells, bio-ink composition, lens-shaped base matrix, ophthalmic diseases, and drugs have been described above, and detailed descriptions thereof will be omitted.

The manufacturing method of the present invention may further include loading a drug for treating eye diseases on a lens-shaped base matrix.

The loading of the drug may include immersing the base matrix onto a drug solution for treating eye diseases.

The step of loading the drug is 0.01 to 10mM, 0.01 to 9mM, 0.01 to 8mM, 0.01 to 7mM, 0.01 to 6mM, 0.01 to 5mM, 0.01 to 4mM, 0.01 to 3mM, 0.01 to 2mM, 0.01 to 1mM, 0.01 to 0.5mM , 0.01 to 0.1 mM or 0.01 to 0.05 mM of drug may be loaded.

In one embodiment, the step of loading the drug may include 0.01 to 10 mM acetylcysteine. For example, the bio-ink composition of the present invention is 0.01 to 10mM, 0.01 to 9mM, 0.01 to 8mM, 0.01 to 7mM, 0.01 to 6mM, 0.01 to 5mM, 0.01 to 4mM, 0.01 to 3mM, 0.01 to 2mM or 0.01 to 1mM. It may be loaded with acetylcysteine.

In one embodiment, the step of loading the drug may include 0.01 to 5 mM of tobramycin. For example, the bio-ink composition of the present invention is 0.01 to 5mM, 0.01 to 4mM, 0.01 to 3mM, 0.01 to 2mM, 0.01 to 1mM, 0.01 to 0.5mM, 0.01 to 0.4mM, 0.01 to 0.3mM, 0.01 to 0.2mM, 0.01 to 0.1 mM tobramycin may be loaded.

Hereinafter, examples will be given to describe the present invention in detail.

Example

1. Preparation of bio-ink composition

A concentrated solution of the hydrogel precursor was prepared as follows. 10 w/v% gelatin type A (Sigma-Aldrich), 10 w/v% gelatin methacrylate, 10 w/v% fibrinogen (Sigma-Aldrich), 20 w/v% in phosphate-buffered saline solution (PBS) 4-PEG amine (10,000 g/mol, Laysan Bio) was concentrated at 37 °C. After neutralizing a 10-50 w/v% collagen solution at 4°C in 1M NaOH and room temperature PBS, it was mixed with warm gelatin. After 1 h, the gelatin was diluted to 2% and dialyzed at 40 °C for 1 week. The solution was then lyophilized and stored dry at -20 °C. A bio-ink composition was prepared by mixing various amounts of a hydrogel precursor, a PEG crosslinking agent, PBS, extracellular matrix (ECM), conjunctival cells, and drugs for treating eye diseases (acetylcysteine and tobramycin).

Table 1 shows the contents of the components constituting the prepared bio-ink composition.

division Gelatin Type A gelatin methacrylate 4-PEG amine fibrinogen conjunctival epithelial cells drug Acetylcysteine tobramycin Example 1 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml 0.5 mM - Example 2 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml 1.0 mM - Example 3 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml 5.0 mM - Example 4 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml 10 mM - Example 5 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml - 0.1 mM Example 6 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml - 0.5 mM Example 7 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml - 1.0 mM Example 8 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml - 5 mM comparative example 10w/v% 10w/v% 20w/v% 10w/v% 5×10 ⁶ to 50×10 ⁶ /ml - -

2. Manufacturing of drug delivery system (drug delivery system) using bioprinter

Each of the bio-ink compositions (Example, Comparative Example) described in Table 1 was printed with a bio-printer to prepare a lens-shaped drug delivery system. The specific method is as follows.

After filling the bio-ink composition in a 3D bio-printer (Bio X 3D Print), the temperature was maintained at 37°C. Within 1 to 2 hours, a printing nozzle having a diameter of 200 to 300 μm was fixed to the cartridge, and the bio-ink composition was printed at a printing speed of 5 mm/s under a pressure of 1 to 1.25 kPa. 1 shows a process of printing a bio-ink composition with a 3D bio-printer. Figure 2 is a lens-shaped base matrix (control group) without a drug printed by a bioprinter (Figure 2A), a drug delivery system with acetylcysteine loaded on the base matrix (Figure 2B), and tobramycin loaded on the base matrix An image of the drug delivery system (FIG. 2C) is shown.

Experimental example

The following analysis was performed using Examples and Comparative Examples.

1. ATP analysis

The ATP concentration of conjunctival cells was measured using the EZ-ATP assay kit (DoGenBio DG-ATP100, KOREA).

Cells of Examples 1 and 2 of Examples 1-4 in which acetylcysteine was used as a drug ATP levels were significantly higher than Examples 3 and 4 (see FIG. 3 ). Through this, it was found that the survival effect of conjunctival cells was excellent when 1 mM or less of acetylcysteine was used as a drug.

2. LDH Assay

The amount of lactate dehydrogenase (LDH) released from apoptotic or damaged cells due to apoptosis or necrosis was measured to measure the degree of cytotoxicity/cytolysis. The amount of LDH was measured using an EZ-LDH assay kit (DoGenBio DG-LDH1000, KOREA).

In Examples 1 and 2 of Examples 1 to 4 in which acetylcysteine was used as a drug, the amount of LDH was significantly lower than in Examples 3 and 4 (see FIG. 4 ). Through this, it was found that the survival effect of conjunctival cells was excellent when 1 mM or less of acetylcysteine was used as a drug.

Among Examples 5 to 8 in which tobramycin was used as a drug, the amount of LDH in Example 5 was significantly lower than in Examples 6 to 8 (see FIG. 6 ). Through this, it was found that when 0.1 mM or less of tobramycin was used as a drug, the conjunctival cell survival effect was excellent.

3. Confirmation of cell viability and apoptosis

The degree of cell viability and apoptosis in lenses loaded with acetylcysteine and tobramycin in a construct containing cells produced through bioprinting using LIVE/DEAD™ Reduced Biohazard Cell Viability Kit #1 (Thermofisher L7013, USA) was evaluated.

5 shows the evaluation results of Examples 1 to 4 in which acetylcysteine was used as a drug. Here, green fluorescence means living cells and red fluorescence means dead cells. In Examples 1 and 2, the cell viability was significantly higher.

7 shows the evaluation results of Examples 5 to 8 in which tobramycin was used as a drug. Here, green fluorescence means living cells and red fluorescence means dead cells. In Example 5, the cell viability was significantly higher.

As described above, it was confirmed that the bio-ink composition of the present invention exhibits excellent printability. In addition, it was confirmed that, by using the bio-ink composition of the present invention, stress according to the bio-printing temperature condition or pressure condition can be minimized, and the survival rate of conjunctival cells is high even after the printing process. The drug delivery system prepared by using the bio-ink composition of the present invention is expected to be usefully used for the treatment of eye diseases.

Claims (11)

On a lens-shaped base matrix consisting of a bio-ink composition comprising 1 to 70 w/v% of hydrogel, 5×10 ⁶ to 50×10 ⁶ /ml of conjunctival cells, and a drug for treating eye diseases, a drug for treating eye diseases is A lens-shaped biodegradable drug delivery system mounted on the lens, wherein the hydrogel comprises 1 to 30 w/v% of gelatin, 10 to 50 w/v% of collagen, and 1 to 30 w/v% of polyethylene glycol Shaped biodegradable drug delivery system.
delete The biodegradable drug delivery system of claim 1, wherein the bio-ink composition further comprises 1 to 20 w/v% of fibrinogen and 1 to 20 w/v% of glycerol.
The lens-shaped biodegradable drug delivery system according to claim 1, wherein the drug is acetylcysteine or tobramycin.
The biodegradable drug delivery system of claim 1, wherein the drug is acetylcysteine in an amount of 0.01 to 10 mM.
The lens-shaped biodegradable drug delivery system according to claim 1, wherein the drug is 0.01-5 mM tobramycin.
Hydrogel 1 to 70 w/v %, conjunctival cells 5×10 ⁶ to 50×10 ⁶ /ml, and a bio-ink composition containing a drug for treating eye disease is injected into a 3D printer, and the drug for treating eye disease is loaded Including; preparing a lens-shaped substrate matrix;
The hydrogel is 1 to 30 w/v% of gelatin, 10 to 50 w/v% of collagen, and 1 to 30 w/v% of polyethylene glycol. The method of manufacturing a lens-shaped biodegradable drug delivery system.
delete The method of claim 7, wherein the bio-ink composition further comprises 1 to 20 w/v% of fibrinogen and 1 to 20 w/v% of glycerol.
The method according to claim 7, wherein the drug is 0.01 to 10 mM acetylcysteine.
The method of claim 7, wherein the drug is 0.01-5 mM tobramycin.

Images