KR101807938B1 - The method for preparation of hydrogel-based drug delivery system - Google Patents
The method for preparation of hydrogel-based drug delivery system Download PDFInfo
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- KR101807938B1 KR101807938B1 KR1020160014501A KR20160014501A KR101807938B1 KR 101807938 B1 KR101807938 B1 KR 101807938B1 KR 1020160014501 A KR1020160014501 A KR 1020160014501A KR 20160014501 A KR20160014501 A KR 20160014501A KR 101807938 B1 KR101807938 B1 KR 101807938B1
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
Abstract
According to an aspect of the present invention, there can be provided a method of manufacturing a hydrogel drug delivery system, which comprises the steps of preparing a hydrogel containing a drug and forming a multilayered thin film on a surface of the hydrogel by layered self-assembly.
Description
The present invention relates to a method of manufacturing a hydrogel-based drug delivery system, and more particularly, to a method of manufacturing a hydrogel-based drug delivery system coated with a multi-layered thin film capable of preventing leakage of a drug contained in a hydrogel .
Hydrogel means a material having a three-dimensional hydrophilic polymer network structure in which the polymer network forms a three-dimensional structure and contains a large amount of water. Since the hydrogel has various forms, physical properties and chemical properties depending on its constituent components, degree of crosslinking, degree of swelling, etc., hydrogel has been developed in various fields such as tissue engineering, drug delivery, sensor and protein separation Has been applied.
Natural polymers used as materials for hydrogels are polymers derived from natural materials, animals, and human bodies and have excellent biocompatibility. Therefore, hydrogels made of natural polymers can provide excellent biocompatibility and biodegradability as well as a less inflammatory reaction after transplantation into a living body. Hydrogels prepared from natural polymers, proteins, polysaccharides and the like are low in biocompatibility and toxicity, and are used variously in biomedical fields such as tissue engineering and drug delivery. When used in such fields, functional materials such as various drugs and proteins are included in the hydrogel.
Korean Patent No. 10-1368590 (hereinafter referred to as Patent Document 1) proposes a hydrogel and a drug delivery vehicle using the same. This patent discloses that hydrogels having biocompatibility, biodegradability and non-toxicity can be prepared by including various polymers. However, the hydrogel disclosed in
Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for preparing a hydrogel-based drug delivery system having a multi-layered thin film laminated on a hydrogel, which has excellent structural stability and has an increased release time of a drug contained in the hydrogel.
According to an aspect of the present invention,
Preparing a hydrogel comprising the drug; And
Forming a multilayer thin film on the surface of the hydrogel by layer self-assembly,
The forming of the multilayer thin film may include forming a first layer by spraying a first solution containing a biocompatible material on the surface of the hydrogel, And
Spraying a second solution comprising the biocompatible material onto the first layer and then drying to form a second layer;
Forming the first layer and the second layer by repeating one or more times to form a first double layer in which the first layer and the second layer are alternately repeated one or more times,
A method of manufacturing a hydrogel based drug delivery system is provided.
According to one embodiment of the present invention, it is possible to stably produce a hydrogel-based drug delivery system in which a multi-layered thin film laminated layer having an extended drug release time and excellent structural stability is laminated.
1 is a flowchart schematically illustrating a method of manufacturing a drug delivery system for hydrogel according to an embodiment of the present invention.
FIG. 2 is a schematic view illustrating a step of forming a first double layer on a surface of a hydrogel according to an embodiment of the present invention.
FIG. 3 is a schematic view showing an example in which a drug delivery system manufactured by a method according to an embodiment of the present invention is applied to cancer tissue. FIG.
4 is an optical image of the collagen hydrogel drug delivery system prepared in Example 1 of the present invention and the drug delivery system prepared in Example 2. Fig.
FIG. 5A is an SEM image of the collagen hydrogel prepared in Example 1 of the present invention, FIG. 5B is an SEM image of the drug delivery system prepared in Example 1, FIG. 5C is a SEM image of the drug prepared in Comparative Example 1 of the present invention SEM image of the delivery system.
FIG. 6A is an SEM image of the drug delivery system prepared in Example 2 of the present invention, and FIG. 6B is an SEM image of the drug delivery system prepared in Comparative Example 2 of the present invention.
7 is a SEM image of the (TA / BPEI) 20 multilayer thin film deposited on the hydrogel and (TA / BPEI) 20.5 / (PDAC / lignin) 20 multilayer thin film after 7 days of 1x PBS treatment.
8 is a graph showing the number of double layers stacked in the spraying method and the thicknesses according to the number of double layers stacked by the dipping method.
9 is a graph quantitatively showing a (BPEI / TA) n multilayer thin film and a (PDAC / lignin) m multilayer thin film adsorbed on a gold electrode by an injection method.
10 is a graph showing the cytotoxicity test results of (BPEI / TA) 20 multilayered membrane and (BPEI / TA) 20 / (PDAC / lignin) 20 multilayered membrane using HeLa cells.
11 is a graph showing the anticancer effect of collagen hydrogel obtained by stacking multilayer thin films using untreated collagen hydrogel containing DOX and spray-LbL after 48 hours.
Of DOX Figure 12a is that the untreated collagen hydrogel, (TA / BPEI) 20 multi-layered thin film is laminated collagen hydrogel, and (TA / BPEI) 19.5 / ( PDAC / lignin) released from the collagen hydrogel 5 multilayers are laminated FIG. 12B is a graph showing an enlargement of the accumulation amount of DOX for 6 hours from the beginning of FIG. 12A. FIG.
13 is an image showing the antimicrobial effect of (BPEI / TA) 25 multilayered membrane and (BPEI / TA) 25 / (PDAC / lignin) 25 multilayered membrane on Gram positive bacteria and gram negative bacteria.
14 is a graph showing load compression displacement curves of a multilayer thin film collagen hydrogel laminated using a layered self-assembly method with untreated collagen hydrogel.
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.
The terms used in this specification will be briefly described and the present invention will be described in detail.
While the present invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.
When an element is referred to as "including" an element throughout the specification, it is to be understood that the element may include other elements, without departing from the spirit or scope of the present invention.
According to an aspect of the present invention, a drug delivery system
Preparing a hydrogel comprising the drug; And
Forming a multilayer thin film on the surface of the hydrogel by layer self-assembly,
The forming of the multilayer thin film may include forming a first layer by spraying a first solution containing a biocompatible material on the surface of the hydrogel, And
Spraying a second solution comprising the biocompatible material onto the first layer and then drying to form a second layer;
Forming the first layer and the second layer is repeated one or more times to form a first double layer in which the first layer and the second layer are alternately repeated one or more times .
According to an aspect of the present invention, a multi-layered thin film is formed on the surface of a hydrogel to delay the time for releasing a drug contained in the hydrogel, thereby effectively manufacturing a structurally stable drug delivery system.
As the hydrogel, collagen hydrogel can be used. Collagen is the most abundant component of the extracellular matrix (ECM) protein, and it can form long-term structures of the body as structural proteins and skeletal proteins, and collagen hydrogels can possess high biocompatibility. However, the kind of the hydrogel that can be used in the present invention is not limited.
Hydrogels containing drugs can be prepared by conventional methods. For example, pig skin is put into an acidic solution to extract collagen, the drug is added to the obtained collagen extract, and the mixture is neutralized with an alkaline solution, .
In the case of a hydrogel containing a drug, the cured hydrogel is treated with a drug-containing solution (0.1 mg / mL) for about 12 hours to allow the drug to enter the hydrogel by diffusion.
According to one embodiment of the present invention, the drug contained in the hydrogel is doxorubicin hydrochloride, dexamethasone, transforming
The drug contained in the hydrogel can be used as long as it has a medicinal effect without any particular limitation. Drugs include, for example, doxorubicin hydrochloride, dexamethasone, Transforming
Forming a first layer by spraying a first solution containing a biocompatible material onto the surface of the hydrogel, spraying a second solution containing the biocompatible material onto the first layer, and drying to form a second layer And the first and second layers are repeated one or more times to form a first double layer in which the first layer and the second layer are alternately repeated one or more times.
The multilayer thin film including the first and second layers forming the first bilayer may be formed by electrostatic interaction, hydrogen bonding, covalent bonding, hydrophobic interaction, or the like. Can be combined and laminated. For example, a hydrogen bond and a hydrophobic interaction may be formed between the surface of the hydrogel and tannic acid (TA), which is an example of a biocompatible material contained in the first solution, and the biocompatible material contained in the first solution Electrostatic attraction and hydrogen bonding can be formed between branched tannin acid and branched polyethylene imine (BPEI), which is an example of a biocompatible material contained in the second solution.
The hydrogel and the first layer of the first double layer and the first layer and the second layer have excellent bonding strength and are structurally very stable and can form a multilayer thin film on the surface of the hydrogel regardless of the size and shape of the hydrogel have.
Also, the first layer and the second layer may be sequentially deposited on the hydrogel surface to include different biocompatible materials.
The first solution and the second solution may be sprayed in an amount such that the thickness of the first and second layers after drying is 1 to 1.5 nm, respectively.
The first solution and the second solution may include, without limitation, a solvent capable of dissolving the biocompatible material. For example, water may be included.
According to an embodiment of the present invention, there is provided a hydrogel drug delivery system comprising: spraying a third solution containing a biocompatible material on a first bilayer and then drying to form a third layer; Spraying a fourth solution comprising a biocompatible material onto the third layer, and then drying to form a fourth layer; Forming the third layer and the fourth layer is repeated one or more times to form a second double layer in which the third layer and the fourth layer are alternately repeated one or more times, .
In the case of the first solution, there may be mentioned tannic acid, ellagitannin, flavonoid, lignin, isoflavone, curcumin, N-feruloyl serotonin, resveratrol, piceatannol, pterostilbene, pinosylvin, alginic acid sodium salt, polyacrylic acid, dextran sulfate, a biocompatible polyphenolic material or an anionic biocompatible polymer selected from dextran sulfate, hyaluronic acid, and heparin sodium salt.
The second solution may be a branched polyethylenimine, poly allyamine hydrochloride, chitosan, poly diallyldimethyl ammonium chloride, poly L-lysine, ), Poly beta amino acid, and the like.
And the third solution forming the second bilayer is selected from the group consisting of poly diallyldimethyl ammoniumchloride, chitosan, poly L-lysine, poly allylamine hydrochloride, And one type of cationic biocompatible material selected from poly beta amino acids.
The fourth solution is selected from the group consisting of lignin, graphene oxide, tannic acid, aromatic polyamide, polyphthalamide, alginic acid sodium salt, polyacrylic acid an aromatic biocompatible material or an anionic biocompatible polymer selected from polyacrylic acid, dextran sulfate, hyaluronic acid, heparin sodium salt, and the like. .
The multilayer thin film including the third and fourth layers forming the second double layer may be formed by electrostatic interaction, hydrogen bonding, covalent bonding, hydrophobic interaction, pi (?) - cation interaction or the like. For example, TA, which is an example of a biocompatible material contained in the first layer included in the first bilayer, and polydialyldimethyl ammonium chloride (PDAC), which is an example of a biocompatible material included in the third layer, (Pi) - cation (cation) between the PDAC, which is one example of the biocompatible material contained in the third layer, and the lignin, which is an example of the biocompatible material contained in the fourth layer, ) Interaction can be formed and combined.
For example, tannic acid and branched polyethylenimine are repeatedly laminated in
The third solution and the fourth solution may be sprayed in an amount such that the thickness of the third layer and the fourth layer after drying is 1 to 1.5 nm, respectively.
The third solution and the fourth solution may include, without limitation, a solvent capable of dissolving the biocompatible material. For example, water may be included.
By using a substance having a predetermined effect as a biocompatible material contained in the first double layer and the second double layer, a multifunction drug delivery system can be provided. For example, a tinnitus that causes a hemostatic effect can be used to have a hemostatic effect. In addition, for example, lignin, which is a structurally stable substance, can be used to provide a drug delivery system having excellent structural stability.
1 is a flowchart schematically illustrating a method of manufacturing a drug delivery system for hydrogel according to an embodiment of the present invention.
1, a drug, such as doxorubicin (DOX), is added to a collagen hydrogel (Col-H) to obtain a drug-containing hydrogel, for example, a tannic acid (TA) and a branched polyethyleneimine BPEI) to form a first double layer containing TA / BPEI by layer-by-layer self-assembly, and a solution containing, for example, polydialyldimethyl ammonium chloride (PDAC) and lignin, respectively, A second bilayer with PDAC / lignin is formed by self-assembly.
FIG. 2 is a schematic view illustrating a step of forming a first double layer on a surface of a hydrogel according to an embodiment of the present invention.
According to one embodiment of the present invention, a first solution comprising tannic acid and a second solution comprising BPEI may be sequentially sprayed onto the hydrogel surface containing the drug and then dried to form a first bilayer. By forming the multilayer thin film by the spraying method, the thin film can be laminated on the surface of the hydrogel quickly, and the swelling of the hydrogel does not occur unlike the conventional dipping method, so that the first layer and the second layer are alternately repeated one or more times .
According to one embodiment of the present invention, the hydrogel may be conical. The hydrogel can be produced, for example, in a conical shape or a disk shape. However, the shape of the hydrogel described above is only an illustrative example and does not limit the shape of the hydrogel.
The position of the first solution and the second solution may be varied depending on the shape of the hydrogel. For example, in the case of a disk-shaped hydrogel, a solution may be injected in a direction perpendicular to the upper surface of the disk and in a direction perpendicular to the lower surface of the disk, and in the case of the conical hydrogel, , The solution may be injected in a direction perpendicular to the conical bottom surface.
According to one embodiment of the present invention, the multilayer thin film may have a total thickness of 10 to 500 nm. For example, the multilayer thin film on the surface of the hydrogel may be laminated with about 10 to about 200 layers, about 10 to about 140 layers, so that the total thickness of the thin film is about 10 nm to about 500 nm, . However, the total thickness of the above-mentioned thin film is only an illustrative example and does not limit the total thickness of the thin film.
According to one embodiment of the present invention, a hydrogel drug delivery system can be applied to internal, external surfaces or tissues of a living body. For example, the outer surface of a living body such as skin, or the surface of an internal organ exposed during a surgical procedure. The present invention also relates to a method of treating or preventing a disease or disorder in a tissue, such as skin, bone, oral cavity, muscle, fascia, brain, nerve, axon, cartilage, blood vessel, cornea, prostate, lung, spleen, small intestine, Lymph node, bone marrow, or kidney.
In addition, the hydrogel-based drug delivery system manufactured by the method according to an embodiment of the present invention can be applied to cancer cells and periodontal disease sites by changing the components of the drug included in the hydrogel, the molar ratio of the drug, and the like.
FIG. 3 is a schematic view showing an example in which a drug delivery system manufactured by a method according to an embodiment of the present invention is applied to cancer tissue. FIG.
As shown in FIG. 3, a hydrogel-based drug delivery system having an anticancer effect, an antibacterial effect and a structurally excellent stability can be obtained, for example, by incorporating DOX having an anticancer effect into a hydrogel, , The multilayered thin film includes TA which is excellent in antimicrobial effect against bacteria and lignin which is a structurally stable substance, the strength of the hydrogel can be increased, and the antibacterial effect against bacteria can be excellent.
Accordingly, the hydrogel-based drug delivery system manufactured by the method according to an embodiment of the present invention suppresses the initial burst release phenomenon of the drug contained in the hydrogel by including the multi-layered film formed on the surface of the hydrogel It is possible to provide a drug delivery system which can induce sustained release for a certain period of time and has excellent antimicrobial effect and structural stability depending on the properties of the biocompatible material contained in the multilayered film.
Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are merely illustrative and do not limit the technical scope of the present invention.
Preparation of polymer solution
The polymer is composed of tannic acid (TA, Mw: 1701.20), branched polyethylenimine (BPEI, Mw: ~ 25,000), poly diallyldimethyl ammonium chloride (PDAC, Mw: BPEI and PDAC and lignin polymers were independently dissolved in distilled water to prepare a polymer solution. Using a solution of NaOH and HCl, the BPEI solution and TA aqueous solution were adjusted to pH 7.5, the PDAC solution was adjusted to a pH of 10, and the lignin solution was adjusted to a pH of 7.
Example 1
Drug-containing collagen Hydrogel Produce
Collagen was extracted from pig skin using hydrochloric acid (HCl) as an acid solution, and 1N NaOH was added to the extracted collagen to prepare a collagen
The multi- Laminated Collagen Hydrogel Manufacture of a Containing Drug Delivery System
The collagen hydrogel containing the drug can be bound to the tannic acid (TA) by hydrogen bonding and hydrophobic interaction, so that TA is laminated to the first layer.
The multilayer thin films were laminated in the following order.
(1) A 1 mg / mL TA solution, a BPEI solution, and a distilled water adjusted pH were independently charged into a spray container, and collagen hydrogel was added to a stainless steel sieve.
(2) The TA solution was sprayed at a distance of 10 cm from the collagen hydrogel, and after having a waiting time of 30 seconds, washing with water having a pH-adjusted distilled water and a waiting time of 15 seconds was repeated twice.
(3) The BPEI solution was repeatedly subjected to the above process (2).
(4) The lamination process of the double layer formed in the process of (2) and (3) was repeated 20 times.
Comparative Example 1
(1) The collagen hydrogel containing the drug prepared in Example 1 was placed in a stainless steel sieve, immersed in 1 mg / mL of TA solution for 30 seconds, immersed in distilled water adjusted for 15 seconds for 15 seconds, And repeated.
(2) The above process (1) was repeatedly performed on the BPEI solution.
(3) The lamination process of the double layers formed in the process of (1) and (2) was repeated 20 times in total.
Example 2
The multilayer thin films were laminated in the following order.
(1) Tanning acid solution of 1 mg / mL, BPEI solution, PDAC solution and lignin solution and distilled water adjusted in pH were independently filled into a spray container, and collagen hydrogel was put into a stainless steel sieve.
(2) The TA solution was sprayed at a distance of 10 cm from the collagen hydrogel, and after having a waiting time of 30 seconds, washing with water having a pH-adjusted distilled water and a waiting time of 15 seconds was repeated twice.
(3) The BPEI solution was repeatedly subjected to the above process (2).
(4) The lamination process of the double layer formed in the process of (2) and (3) was repeated 20 times in total.
(5) The PDAC solution was sprayed at a distance of 10 cm from the collagen hydrogel, and after having a waiting time of 30 seconds, the pH-adjusted distilled water was sprayed and the washing process with a waiting time of 15 seconds was repeated twice.
(6) The lignin solution was repeatedly subjected to the above process (5).
(7) The lamination process of the bilayer formed in the processes of (5) and (6) was repeated 20 times.
Comparative Example 2
(1) The collagen hydrogel containing the drug prepared in Example 1 was placed in a stainless steel sieve, immersed in a 1 mg / mL TA solution for 30 seconds, and immersed in distilled water adjusted for 15 seconds. And repeated twice.
(2) The above process (1) was repeatedly performed on the BPEI solution.
(3) The lamination process of the double layers formed in the process of (1) and (2) was repeated 20 times in total.
(4) Subsequently, the plate was immersed in a 1 mg / ml PDAC solution and immersed in distilled water adjusted for 15 seconds.
(5) The lignin solution was repeatedly subjected to the above process (4).
(6) The lamination process of the double layer formed in the processes of (4) and (5) was repeated 20 times.
The multi- Laminated Collagen Hydrogel Analysis method
FE-SEM (SIGMA, Carl Zeiss) and AFM (NX-10, Park Systems) were used to analyze the morphology of multilayer thin films stacked using Dip-LbL or Spray-LbL. The thickness of the multilayer thin film was confirmed using a shape measuring device (Dektak 150, Veeco). QCM (
Hydrogel system Morphology analysis of drug delivery system
Fig. 4 is an optical image of the collagen hydrogel prepared in Example 1, the drug delivery system, and the drug delivery system prepared in Example 2. Fig.
FIG. 5A is an SEM image of the dried collagen hydrogel prepared in Example 1, FIG. 5B is an SEM image of the dried drug delivery system prepared in Example 1, FIG. 5C is a SEM image of the dry drug prepared in Comparative Example 1 SEM image of the delivery system.
FIG. 6A is an SEM image of the dried drug delivery system prepared in Example 2, and FIG. 6B is an SEM image of the dried drug delivery system prepared in Comparative Example 2. FIG.
As shown in FIG. 4, it was found that the collagen hydrogel having a multi-layered thin film gradually changed to light brown as the number of bilayer layers stacked on the collagen hydrogel increased. TA has a light blue color and lignin has a brown color, so the more multilayer thin films are laminated, the more TA and lignin colors appear.
As shown in FIG. 5B, the phenomenon of interdiffusion of the polymer in the multilayer thin film was small in the course of the multilayer thin film being laminated by the injection method, and it was confirmed that the smooth surface morphology was maintained compared with the collagen hydrogel alone as compared with FIG. 5A.
On the other hand, as shown in FIG. 5C, when the multilayer thin film is laminated by the conventional dipping method, it is confirmed that the surface is very rough due to interdiffusion of the polymer in the multilayer thin film. When the multilayer thin film is stacked according to the first embodiment of the present invention, the multilayer thin film is formed along the fibrils to fill the holes of the collagen hydrogel surface, and the collagen fibers (PDAC / lignin) 20 multilayer thin films are stacked And it was confirmed that it was completely covered.
Similar results were obtained when a multi-layered film of (TA / BPEI) 20 and (PDAC / lignin) 20 was laminated on the hydrogel as in the case of Figs. 6A and 6B.
7A and 7B show the results of a
As shown in Figure 7a, (TA / BPEI) in 20 multi-layered thin film is laminated collagen hydrogel PBS treated 7 days (TA / BPEI) 20 thin film may partially remain, the collagen hydrogel was exposed. As shown in FIG. 7B, the collagen hydrogel (TA / BPEI) 20.5 / (PDAC / lignin) 20 multilayer thin film laminated with 20.5 / (PDAC / lignin) 20 was left on the collagen hydrogel And only a small amount of polymer aggregate was generated due to PBS solute.
This (PDAC / lignin) 20 The in vivo (TA / BPEI) 20. It can be seen that the 5 prevent the decomposition.
Multi-layer Thickness Analysis and Quantitative Analysis
8 is a graph comparing the number of double layers laminated using a layered self-assembly method (Spray-LbL) by a spraying method and the thicknesses according to the number of double layers laminated using a layered self-assembly method (Dip-LbL) Fig.
In order to measure the thickness of the multilayer thin film according to the number of bilayer layers, the multilayer thin film was laminated on the Si wafer by layer self-assembling by the dipping method and the injection method. The lamination method was carried out in the same manner as in Example 1, Example 2, Comparative Example 1, and Comparative Example 2, except that a Si wafer having a negative charge by an oxygen plasma treatment was used instead of the collagen hydrogel.
As shown in FIG. 8, the thickness of the multilayer thin film was faster than that in the case of using the injection method in the case of the dipping method. This is probably due to the interdiffusion phenomenon of the polymer chains in the multilayer thin film in the case of the dipping method.
For the quantitative analysis of the multilayer thin film, the multilayer thin film was formed by the same method as in Example 1 and Example 2, except that the gold electrode was laminated instead of the collagen hydrogel, and then the multilayer thin film was measured using QCM.
9 is a graph quantitatively showing a (BPEI / TA) n multilayer thin film and a (PDAC / lignin) m multilayer thin film adsorbed on a gold electrode by an injection method.
As can be seen from FIG. 9, as the number of bilayers increases, the frequency of the multilayer thin film decreases. As a result, it is confirmed that each layer is linearly adsorbed, and that the thickness increase pattern of the multilayer thin film is linear.
Cytotoxic and Anticancer Effects of Multilayer Thin Films
To analyze the cytotoxicity and anticancer effect, HeLa cells were cultured on a 100 mm dish in an incubator at 37 ° C, 5% CO 2 for 2 to 3 days. Cell culture growth media consisted of Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic and 1% penicillin-streptomycin-glutamine. All of the above products were purchased from Gibco Life Technologies.
(BPEI / TA) 20 and (BPEI / TA) 20 / (PDACs) on a Si wafer negatively charged by oxygen plasma treatment to test the cytotoxicity of the laminated multilayer thin films using the layered self- / lignin) 20 were laminated in a spraying manner as in Example 1 and Example 2. [ The multi-layered wafer was immersed in 5 ml of 1x PBS solution and stored for 7 days in a 37 ° C incubator. After 7 days, the samples were collected and stored in a refrigerator.
HeLa cells were dispensed in a 24 well plate at a concentration of 5 x 10 4 cells / well. After 1 day, the growth medium was removed and replaced with a sample / growth medium mixture containing 20% of the collected sample. Cells were cultured for one day and cell viability was measured by MTT assay (absorbance: 540 nm).
In addition, the anticancer effect analysis was carried out in the same manner as the cytotoxicity analysis described above. More specifically, HeLa cells were dispensed into 96-well plates at a concentration of 1 × 10 4 cells / well, collagen hydrogel containing about 0.6 μg / mL of dexamethasone was impregnated in 1 × PBS for about one week, HeLa cells were cultured in PBS containing the drug, and then cultured for one day. Cell viability was measured by MTT assay (absorbance: 540 nm).
Cell viability of (BPEI / TA) 20 and (BPEI / TA) 20 / (PDAC / lignin) 20 multilayer thin films were analyzed using cervical cancer HeLa cells to confirm the applicability to the biomedical field.
10 is a graph showing the cytotoxicity test results of (BPEI / TA) 20 multilayered membrane and (BPEI / TA) 20 / (PDAC / lignin) 20 multilayered membrane using HeLa cells. As shown in FIG. 10, the (BPEI / TA) 20 multilayer thin film has 83.8% to 94.2% cell viability and the (BPEI / TA) 20 / (PDAC / lignin) 20 multilayer thin film has 77.49% to 109.91% Cell viability, and showed almost no cytotoxicity similar to the PBS treatment group. In particular, the (BPEI / TA) 20 multilayer film was biocompatible despite being partially degraded in PBS.
A drug delivery system (BPEI / TA) 19.5 / (PDAC / lignin) 5 multilayer thin film comprising a collagen hydrogel in which a multilayer film of (BPEI / TA) 20 was deposited in the same manner as in Example 1 and Example 2, The drug delivery system containing the laminated collagen hydrogel and the untreated collagen hydrogel of Example 1 were immersed in 2 mL of 1x PBS and stored in the incubator. Six days later, the DOX-released samples were collected. HeLa cells were dispensed into 96-well plates at a concentration of 1 x 10 cells / well. After 1 day, the growth medium was removed and replaced with a sample / growth medium mixture in which the collected samples were contained at 50%. Cells were cultured for 2 days with medium exchanged, and cell viability was measured by MTT assay (absorbance: 540 nm).
11 is a graph showing the anticancer effect of the untreated collagen hydrogel and the drug delivery system of the present invention after 48 hours.
As shown in Figure 11, through the MTT assay, cell viability of the untreated collagen hydrogel containing DOX drug was measured to 46.37% to 48.17%, (TA / BPEI) collagen hydrogel with 20 multilayers are laminated cell viability in drug delivery system comprising were measured by 75.13% to 76.31%, (TA / BPEI) 19.5 / (PDAC / lignin) cell viability in drug delivery systems containing a 5 multi-layer thin film is laminated collagen hydrogel Was measured from 75.13% to 76.31%.
Cell viability of DOX at a concentration of 0.6 μg / mL was measured as 51.9% to 55.46%, similar to the untreated collagen hydrogel containing DOX. When compared to untreated collagen hydrogel containing DOX and DOX, (TA / BPEI) 20 The multi-layered thin film is laminated collagen hydrogel and (TA / BPEI) 19.5 / ( PDAC / lignin) 5 multi-layered thin film is laminated collagen hydro The anticancer effect of the gel decreased slightly, but the amount of DOX released was found to be almost the same. This is due to the fact that the (TA / BPEI) 20 multilayer and (TA / BPEI) 19.5 / (PDAC / lignin) 5 multilayer thin films deposited on the collagen hydrogel are slightly degraded and the negatively charged BPEI and PDAC are positively charged DOX, and thus the anticancer effect is slightly reduced.
Hydrogel system Drug Release Analysis of Drug Delivery Systems
Of DOX Figure 12a is that the untreated collagen hydrogel, (TA / BPEI) 20 multi-layered thin film is laminated collagen hydrogel, and (TA / BPEI) 19.5 / ( PDAC / lignin) released from the collagen hydrogel 5 multilayers are laminated FIG. 12B is a graph showing an enlargement of the accumulation amount of DOX for 6 hours from the beginning of FIG. 12A. FIG.
In order to determine the release rate of DOX contained in the collagen hydrogel, collagen hydrogel was immersed in a DOX aqueous solution having a concentration of 0.1 mg / ml for one day so that the DOX could be homogenously diffused into collagen .
DOX release rate analysis was performed in the following manner. The collagen hydrogel layered with the multilayer thin film and the collagen hydrogel (untreated hydrogel) without the multilayer thin film laminated were layered by layered self-assembly method and cultured at 37 DEG C in 2 mL of 1x PBS aqueous solution. 0.5 mL of sample was collected at predetermined time intervals, and 0.5 mL of fresh 1x PBS was added. The collected samples were stored in a refrigerator. To determine the DOX emission profile, the amount of DOX was measured using a plate reader (λ ex = 490 nm, λ em = 590 nm, Synergy H1, Biotek).
Specifically, the non-treated DOX saturated hydrogel, (TA / BPEI) 20 multi-layered thin film is laminated with the hydrogel, and (TA / BPEI) 19.5 / ( PDAC / lignin) 5 multilayers DOX released from the stacked hydrogel The speed was measured. In the PBS environment, the DOX emission profile in a hydrogel layered with a (TA / BPEI) 19.5 / (PDAC / lignin) 5 multilayer thin film with untreated collagen hydrogel, (TA / BPEI) 20 multi- 12A and 12B.
As shown in Figs. 12A and 12B, the untreated collagen hydrogel immediately released the DOX contained in the collagen hydrogel after the PBS treatment, and the release curve reached its maximum point within 6 hours. On the contrary, the hydrogel in which the (TA / BPEI) 20 multilayer thin film laminated hydrogel and the (TA / BPEI) 19.5 / (PDAC / lignin) 5 multilayer thin film were stacked had a continuous DOX emission profile, The results are shown.
(TA / BPEI) 20 multilayer thin film-laminated hydrogel and (TA / BPEI) 19.5 / (PDAC / lignin) were measured by comparing the times when the DOX contained in the collagen hydrogel was 85% 5 multilayer thin film was increased by about 10 to 12 times longer than that of untreated collagen hydrogel.
(TA / BPEI) 20 multilayer thin film laminated hydrogel and (TA / BPEI) 19.5 / (PDAC / lignin) 5 multilayer thin film were prevented from releasing the DOX to PBS for 6 days. Therefore, the number of multi-layered films stacked on the collagen hydrogel can be controlled to adjust the time for which the DOX is released.
Analysis of antibacterial effect of multilayer thin film
Staphylococcus aureus (KTCT1621, ATCC25923) and gram negative bacteria (Pseudomonas aeruginosa, KTCT2513, ATCC9027) were cultured in order to analyze the antibacterial effect of the laminated multilayer thin films using Spray-LbL. Gram bacteria were purchased from the Microbiological Resource Center (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). The bacteria were cultured in 2.5 mL of Trypticase ™ soy broth media (BBL ™). The cultured bacteria were subcultured in a 6-well plate (Cell density: OD 600nm = 0.6-0.7) and cultured for 12 hours. A silicon wafer treated with oxygen plasma, a wafer laminated with a (BPEI / TA) 25 multilayered film and a wafer laminated with a (BPEI / TA) 25 / (PDAC / lignin) 25 multilayered film were treated with a plate cultured with bacteria, And cultured for 24 hours. After the incubation, the multi-layered thin film was collected from the bacterial culture medium, and the biofilm formed by attaching the bacteria was fixed with 99% ethanol and washed with water. To stain the multilayer thin film biofilm using layered self-assembly, samples were treated with 0.1% of crystal violet (Sigma Aldrich) for 30 minutes and rinsed with distilled water.
13 is an image showing the antimicrobial effect of (BPEI / TA) 25 multilayered membrane and (BPEI / TA) 25 / (PDAC / lignin) 25 multilayered membrane on Gram positive bacteria and gram negative bacteria.
As shown in FIG. 13, in the control group, the (BPEI / TA) 25 multilayer thin film and (BPEI / TA) 25 / (PDAC / lignin) 25 multilayer thin film had a distinctive color due to the thickness of the multilayer thin film itself. In the group treated with Gram bacteria, (BPEI / TA) 25 multilayer film and (BPEI / TA) 25 / (PDAC / lignin) 25 multilayer film were strongly stained with crystal violet compared with Si wafer. This indicates that the multilayer thin film including TA and lignin has an antibacterial effect.
(BPEI / TA) 25 / (PDAC / lignin) 25 multi-layer thin films, which contain more TA and lignin, than the (BPEI / TA) 25 multilayer thin films.
Hydrogel system Structural stability analysis of drug delivery system
Loaded compressive displacement test of the drug-containing untreated collagen hydrogel prepared in Example 1 and the drug delivery system prepared in Example 1 and Example 2 were performed.
FIG. 14 is a graph showing load compression displacement curves of the drug-containing untreated collagen hydrogel prepared in Example 1 and the drug delivery system prepared in Examples 1 and 2. FIG.
As shown in Fig. 14, the drug delivery system of Example 2 contained a structurally stable material, lignin, which increased the strength by 1.5 to 2 times. Referring to Fig. 14, the load required to compress the drug delivery system of Example 1 and the drug delivery system of Example 2 was found to be large, as compared with the drug-containing untreated collagen hydrogel. This indicates that the multilayered film included in the drug delivery system enhances structural stability. Also, the structural stability of the drug delivery system was found to increase as the number of laminated multilayer films increased.
It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.
The scope of the present invention is defined by the appended claims rather than the detailed description and all changes or modifications derived from the meaning and scope of the claims and their equivalents are to be construed as being included within the scope of the present invention do.
Claims (12)
Forming a multilayer thin film on the surface of the hydrogel by layer self-assembly,
The forming of the multilayered thin film may include forming a first layer by spraying a first solution containing tannic acid as a biocompatible material on the surface of the hydrogel, And
Spraying a second solution containing branched polyethylenimine as a biocompatible material on the first layer and drying to form a second layer;
Forming the first layer and the second layer by repeating one or more times to form a first double layer in which the first layer and the second layer are alternately repeated one or more times,
A method for manufacturing a hydrogel based drug delivery system.
Wherein the first solution and the second solution are sprayed in an amount such that the thickness of the first layer and the second layer becomes 1 to 1.5 nm after drying.
The forming of the multilayer thin film may include forming a third layer by spraying a third solution containing polydiaryldimethyl ammoniumchloride as a biocompatible material on the first bilayer and then drying the third layer; And
Spraying a fourth solution comprising lignin as a biocompatible material on the third layer and drying to form a fourth layer;
Further comprising repeating the forming of the third layer and the fourth layer one or more times to form a second double layer wherein the third layer and the fourth layer are alternately repeated one or more times,
A method for manufacturing a hydrogel based drug delivery system.
Wherein the third solution and the fourth solution are sprayed in an amount such that the thickness of the third layer and the fourth layer becomes 1 to 1.5 nm after drying.
These drugs include doxorubicin hydrochloride, dexamethasone, transforming growth factor beta 1, basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor ), Insulin, leukocyte proliferative factor (G-SCF), human growth hormone (hGH), EPO, Symlin, Exendin, Somatokine, Paclitaxal, One selected from the group consisting of Chlorambucil, Interferon, Monoclonal antibodies, Vaccines, Antimicrobials, Steroids, Antiinflammatory agents, Sex hormones, Immunosuppressive agents, Antiviral agents, Anesthetics, Antagonists and Antihistamines Or more of the drug delivery system.
Wherein the hydrogel is conical in shape.
Wherein the multi-layered thin film has a total thickness of 10 to 500 nm.
Wherein the hydrogel is a collagen hydrogel.
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