KR102088809B1 - A nitric oxide carrier and a method for manufacturing the same - Google Patents

Abstract

One aspect of the present invention includes a unit portion in which a first layer made of a cationic polymer having an amine group in a side chain and a second layer made of an anionic polymer having a carboxyl group in the side chain are stacked, and at least a part of the amine group is diazenium Provided is a nitrogen oxide transporter substituted with a dioleate group and a method for manufacturing the same.

Description

Nitric oxide carrier and its manufacturing method {A NITRIC OXIDE CARRIER AND A METHOD FOR MANUFACTURING THE SAME}

The present invention relates to a nitrogen oxide transporter and a method for manufacturing the same, and more specifically, in a multi-layer film in which a cationic polymer and an anionic polymer are cross-layered, the ionization degree of the cation and anionic polymer is controlled to control the amount and release of nitrogen oxide according to structural differences. The present invention relates to a nitrogen oxide carrier capable of easily adjusting the speed and a method for manufacturing the same.

Nitrogen monoxide (NO) is a highly reactive radical molecule produced by nitrogen monoxide generating enzymes in cells, and is an important cellular signaling molecule responsible for various physiological or pathological processes in the body. Nitrogen monoxide began to attract attention in the fields of neuroscience, physiology, and immunology as it was selected as the "molecule of the year" by the American journal Science in 1992. A study that revealed the cardiovascular signaling function of nitrogen monoxide won the Nobel Prize in Physiology in 1998. Nitrogen monoxide acts as a powerful vasodilator in blood vessels, and in addition, it has various functions such as immune response, nerve transmission, erectile regulation, antibacterial action, and wound healing.

Since nitrogen monoxide performs and regulates various roles in the body, studies have been conducted to find a method for artificially delivering nitrogen monoxide from the outside. In particular, nitrogen monoxide exhibits various effects in the gas state, and since the half-life is very short within 6 seconds, studies on nitrogen monoxide release in the form of compounds have been conducted.

The most studied compounds are diazeniumdiolate (NONOate) and S-nitrosothiol (RSNO). In the case of S- ^nitrosothiol , the release is promoted by temperature, light and copper ions (Cu ²⁺ ), and has the advantage of high nitrogen monoxide emission efficiency. On the other hand, there is a disadvantage that it is difficult to obtain nitrogen monoxide in a pure state because it is not released in vivo or the material is not stable. Diazenium dioleate can be stably stored in a solid state, has high solubility in water, and can easily produce nitrogen monoxide at biological temperature and pH conditions. However, the decomposition products can exhibit toxicity, and have the disadvantage that nitrogen monoxide is released simultaneously with contact with water.

Representative groups conducting research on nitrogen monoxide transporters include Mark E. Meyerhoff of the University of Michigan, USA and Mark H. Schoenfisch of the University of North Carolina, Chapel Hill. The Meyerhoff group developed silica nanoparticles that release hundreds of nanometers of nitrogen oxides based on diazenium diolate using a sol-gel method using aminoalkylsilane. Subsequently, in the Schoenfisch group, silica nanoparticles were first prepared, and then the secondary amine group on the surface was converted to a diazenium diol group to convert the particles showing the release of nitrogen monoxide on the surface and the aminoalkoxysilane molecule to the diazenium diol group. After the conversion, two types of silica nanoparticles were developed to release nitrogen monoxide from the inside of the particles. After preparing the particles using various aminoalkoxysilanes, the size and the nitrogen monoxide emission efficiency were summarized and reported. When there are aminoalkoxysilane groups in the particles, the nitrogen monoxide emission efficiency is maximized, resulting in high efficiency nitrogen oxide nano Particles can be prepared, and there is a difference in efficiency depending on the molecular structure of aminoalkoxysilane. However, the aminoalkoxysilane-based nitric oxide nanoparticles are manufactured according to the sol-gel principle, so the temperature and moisture must be thoroughly controlled during the experiment, so the process is difficult, costly, and likely to fail.

In Korea, POSTECH's Professor Kim Won-jong's team is currently conducting research to control the release of nitrogen monoxide by diazenium diolate-based nitrogen oxide nanoparticles. A porous silica nanoparticle made of aminoalkoxysilane was changed into a structural form by UV, and a 2-nitro-benzaldehyde molecule emitting hydrogen ions was added, followed by preparation of a capping nanoparticle with calcium phosphate. Nitrogen oxide nanoparticles were prepared by reacting for 3 days under a high pressure nitrogen monoxide gas. The nanoparticles thus prepared are not spontaneously released of nitrogen monoxide because the penetration of external hydrogen ions is inhibited. When UV is irradiated, 2-nitro-benzaldehyde inside the nanoparticles releases hydrogen ions to lower the pH. Accordingly, as the capping layer decomposes, nitrogen monoxide is released. However, even in this study, the process of using aminoalkoxysilane is complicated and difficult.

That is, the synthesis of nitrogen oxide nanoparticles based on the sol-gel reaction, which has been studied a lot, uses materials sensitive to temperature and moisture, so there are many conditions to control in the experimental process, the process is difficult, costly, and unsuccessful. The probability is very high.

The present invention is to solve the problems of the prior art described above, the object of the present invention is to provide a nitrogen oxide delivery system that can easily control the amount and rate of release of nitrogen oxides.

Another object of the present invention is to use a biocompatible ionic polymer instead of a material that is sensitive to the external environment, and apply a layer-by-layer (LbL) lamination method to easily and economically nitrogen oxides with a simple principle. It is to provide a method for producing a carrier.

One aspect of the present invention includes a unit portion in which a first layer made of a cationic polymer having an amine group in a side chain and a second layer made of an anionic polymer having a carboxyl group in the side chain are stacked, and at least a part of the amine group is diazenium Provided is a nitrogen oxide transporter substituted with a dioleate group.

In one embodiment, 30% or more of the amine group may be substituted with a diazenium dioleate group.

In one embodiment, the cationic polymer is a branched polyethyleneimine (bpei), chitosan, gelatin, collagen, collagen, fibrinogen, silk fibroin, casein ( casein, elastin, laminin, fibronectin, polydopamine, polyethyleneimine, poly-l-lysine, poly (vinylamine) hydrochloride (poly (vinylamine) hydrochloride), poly (amino acid), desaminotyrosylctylester, and a combination of two or more of them.

In one embodiment, at least a portion of the carboxyl group is deprotonated (deprotonated), and the anionic polymer may have a two-dimensional structure by repulsion between the deprotonated carboxyl groups.

In one embodiment, all of the carboxyl groups can be deprotonated.

In one embodiment, the anionic polymer is hyaluronic acid (hyaluronic acid), poly-l-lactic acid (poly-l-lactic acid), alginic acid (alginic acid), dextran sulfate (dextran sulfate), lignin (lignin) , Tannic acid, chondroitin sulfate, cellulose polymers, fucoidan, heparin, and combinations of two or more of them.

In one embodiment, the unit unit is two or more, and the first and second layers may be cross-layered.

Another aspect of the present invention, (a) adjusting the pH of a solution containing a cationic polymer having an amine group in a side chain to a predetermined range to prepare a first solution; (b) preparing a second solution by adjusting the pH of a solution containing an anionic polymer having a carboxyl group in a side chain to a predetermined range; (c) alternately coating the first and second solutions on a substrate; And (d) reacting with nitrogen monoxide; provides a method for producing a nitrogen oxide carrier comprising a.

In one embodiment, the pH of the first solution may be 7-10.

In one embodiment, the cationic polymer is a branched polyethyleneimine (bpei), chitosan, gelatin, collagen, collagen, fibrinogen, silk fibroin, casein ( casein, elastin, laminin, fibronectin, polydopamine, polyethyleneimine, poly-l-lysine, poly (vinylamine) hydrochloride (poly (vinylamine) hydrochloride), poly (amino acid), desaminotyrosylctylester, and a combination of two or more of them.

In one embodiment, the pH of the second solution may be 4-9.

In one embodiment, the anionic polymer is hyaluronic acid (hyaluronic acid), poly-l-lactic acid (poly-l-lactic acid), alginic acid (alginic acid), dextran sulfate (dextran sulfate), lignin (lignin) , Tannic acid, chondroitin sulfate, cellulose polymers, fucoidan, heparin, and combinations of two or more of them.

In one embodiment, the substrate may have a three-dimensional shape, a two-dimensional shape, or a combination of these.

In one embodiment, in step (c), the coating is electrostatic attraction between the cation and anion polymers, van der Waals forces, hydrogen bonding, covalent bonding, ionic bonding, hydrophobic bonding, and charge-transfer. One selected from the group consisting of binding, π-interaction, coordination chemical coupling, stereocomplexation, host-guest, biological binding, and combinations of two or more of these can be used.

In one embodiment, the reaction in the step (d) may be made at 5 ~ 300 ℃ and 1 ~ 30atm.

The nitrogen oxide transporter according to an aspect of the present invention can easily control the amount and rate of release of nitrogen oxides according to structural differences by controlling the degree of ionization of the cation and anion polymers in a multi-layer film in which a cationic polymer and an anionic polymer are cross-stacked. have.

In addition, in the method of manufacturing a nitrogen oxide delivery system according to another aspect of the present invention, the ionization degree of a biocompatible ionic polymer is adjusted to a predetermined range, and a layer-by-layer (LbL) lamination method is applied. A simple principle makes it easy and economical to produce a nitrogen oxide carrier.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a schematic diagram of a nitrogen monoxide delivery system and a method for manufacturing the same according to an embodiment of the present invention;
2 is a result of evaluating structural properties according to the number of double layers of a nitrogen monoxide delivery system according to an embodiment of the present invention;
3 is an SEM image of a surface shape of a nitrogen monoxide delivery system according to an embodiment of the present invention;
4 to 5 are the results of chemical analysis of the nitrogen monoxide delivery system according to an embodiment of the present invention;
6 is a result of evaluating the nitrogen monoxide emission characteristics of the nitrogen monoxide delivery system according to an embodiment of the present invention.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and thus is not limited to the embodiments described herein. In addition, in order to clearly describe the present invention in the drawings, parts irrelevant to the description are omitted, and like reference numerals are assigned to similar parts throughout the specification.

Throughout the specification, when a part is "connected" to another part, this includes not only "directly connected" but also "indirectly connected" with another member in between. . Also, when a part is said to “include” a certain component, this means that other components may be further provided instead of excluding the other component unless otherwise stated.

Nitric oxide carrier

One aspect of the present invention includes a unit portion in which a first layer made of a cationic polymer having an amine group in a side chain and a second layer made of an anionic polymer having a carboxyl group in the side chain are stacked, and at least a part of the amine group is diazenium Provided is a nitrogen oxide transporter substituted with a diol group (RR'NN (O) = NOR).

In the present invention, "nitrogen oxide transporter" means a material capable of delivering and releasing nitrogen monoxide, and may also be referred to as a "nitrogen oxide complex". The nitrogen oxide transporter may be applied to and coated on the surface of the substrate to deliver and release nitrogen monoxide. As the substrate, one or more selected from the group consisting of inorganic components that are stable in vivo, biodegradable polymers that are biodegradable, micelles, and bio-derived materials may be used.

The polymer containing the diazenium dioleate group can be stably stored and stored as a solid form, has high solubility in water, and a release rate and method depending on the structure of the rest of the diazenium dioleate group. The release form of can be controlled. In addition, the polymer containing the dianium diol group not only decomposes at biological temperature and pH conditions, but also has various types of release depending on the pH and releases 2 molecules of nitrogen monoxide per unit of dianium diol group. When included in the carrier, it can deliver and release relatively high concentrations of nitrogen monoxide.

The cationic polymer may be attached to the surface of a (-) charged substrate, and some of the amine groups located in the side chain of the cationic polymer, preferably, 30% or more, more preferably, 50% or more, and more preferably Preferably, 65% or more may be substituted with a diazenium dioleate group. Specifically, the cationic polymer is a solution, preferably, coated on the surface of the substrate in the form of an aqueous solution to form a first layer, and the pH of the solution is adjusted to a predetermined range to determine the degree of ionization of the cationic polymer, that is, protons. You can adjust the degree of fire. When the pH of the solution is adjusted to a range of 7 to 10, preferably 8.5 to 9.5, 10 to 40%, preferably 20 to 30% of the amine group may be protonated with -NH ₃ ⁺ , In addition, 60 to 90%, preferably 70 to 80%, is present as an unprotonated free amine group (free amine group, -NH ₂ ). In the present invention, “ionization degree” refers to a ratio of protonation and / or deprotonation among functional groups located in a side chain of a polymer.

Since the first layer has a (+) charge, the anionic polymer can be attached to the surface. The anionic polymer is also a solution, preferably, coated on the surface of the first layer in the form of an aqueous solution to form a second layer, and the pH of the solution is adjusted to a predetermined range to ionize the anionic polymer, that is, deproton. You can adjust the degree of fire. In addition, since the sequential and cross-linking of the first and second layers can be performed without a separate drying process, the degree of ionization of the anionic polymer may also affect the degree of ionization of the cationic polymer forming the first layer previously formed. have. When the pH of the solution is adjusted to a range of 4 to 9, preferably 7 to 8, 50% or more of the carboxyl group, preferably 90% or more, more preferably 95% or more, advantageously, 100 % Can be deprotonated. Deprotonation of the carboxyl group (-COO ^-) is the cation are combined by a protonated amine group (-NH ₃ ⁺⁾ and the electrostatic attraction of the polymer, and some residual non-deprotonated to screen a carboxyl group (-COOH) is It can be bonded by hydrogen bonding with the free amine group. However, since such a hydrogen bond may lower the possibility of substitution of the free amine group with the diazenium dioleate group, it is preferable to minimize the deprotonated carboxyl group (-COOH) so that it does not substantially exist.

In addition, when the second layer is formed of an anionic polymer in which all of the carboxyl groups are deprotonated, the degree of ionization of the cationic polymer contained in the first layer is 70% or less, preferably 50% or less, more preferably May be partially up-regulated to 35% or less, and conversely, a free amine group in which an amine group of 30% or more, more preferably, 50% or more, and more preferably 65% or more is not protonated. -NH ₂ ).

The first and second layers may be combined with each other to form one unit, and at least a portion, preferably, all of the free amine groups included in the first layer may be substituted with the diazenium diol group. have. In addition, the anionic polymer may have a substantially linear two-dimensional structure due to repulsion between the deprotonated carboxyl groups, and may be optional such as folding or coiling of molecules by electrostatic attraction in the molecule. Since it does not cause phosphorus deformation, it is possible to effectively obtain a high-density multi-layer film having a nanometer thickness.

In addition, two or more unit units may be provided, and the first and second layers may be cross-layered. Such a cross-layer structure may be implemented by a layer-by-layer (LbL) layering method, which will be described later.

The cationic polymer is branched polyethyleneimine (bpei), chitosan, gelatin, collagen, collagen, fibrinogen, silk fibroin, casein, elastin ), Laminin, fibronectin, polydopamine, polyethyleneimine, poly-l-lysine, poly (vinylamine) hydrochloride ), Poly (amino acid), desaminotyrosylctylester, and a combination of two or more of them, preferably, branched polyethyleneimine, , But is not limited thereto.

The anionic polymer is hyaluronic acid, poly-l-lactic acid, alginic acid, dextran sulfate, lignin, tannic acid , Chondroitin sulfate, cellulose polymers, fucoidan, heparin, and combinations of two or more of them, preferably, alginic acid. It is not limited.

The nitric oxide transporter can easily control the release rate and release rate of nitrogen monoxide according to the difference in the microstructure of the multi-layer film by controlling the ionization degree of the polymer, thereby expanding blood vessels, healing wounds, preventing aging, activating neurons, antibacterial, anticancer, etc. It can be used for medical purposes. In addition, the biocompatible polymer and the surface charge using the same can be modified to a surface having high biocompatibility, and thus can be widely applied to various fields.

Method of manufacturing a nitrogen oxide carrier

Another aspect of the present invention, (a) adjusting the pH of a solution containing a cationic polymer having an amine group in a side chain to a predetermined range to prepare a first solution; (b) preparing a second solution by adjusting the pH of a solution containing an anionic polymer having a carboxyl group in a side chain to a predetermined range; (c) alternately coating the first and second solutions on a substrate; And (d) reacting with nitrogen monoxide; provides a method for producing a nitrogen oxide carrier comprising a.

In the steps (a) and (b), first and second solutions for forming the first and second layers, respectively, constituting the unit part of the nitrogen oxide transporter are prepared, and the pH of each solution can be adjusted to a predetermined range. have. As the solvent in the first and second solutions, water, ethanol, sodium acetate buffer, phosphate buffered saline (PBS) or culture media may be used.

The cationic polymer may be attached to the surface of a (-) charged substrate, and some of the amine groups located in the side chain of the cationic polymer, preferably, 30% or more, more preferably, 50% or more, and more preferably Preferably, 65% or more may be substituted with a diazenium dioleate group. Specifically, the cationic polymer is coated on the surface of the substrate through a first solution to form a first layer, and the pH of the first solution is adjusted to a predetermined range to ionize the cationic polymer, that is, protonated You can adjust the degree. When the pH of the solution is adjusted to a range of 7 to 10, preferably 8.5 to 9.5, 10 to 40%, preferably 20 to 30% of the amine group may be protonated with -NH ₃ ⁺ , In addition, 60 to 90%, preferably 70 to 80%, is present as an unprotonated free amine group (free amine group, -NH ₂ ).

Since the first layer has a (+) charge, the anionic polymer can be attached to the surface. The anionic polymer is coated on the surface of the first layer through a second solution to form a second layer, and the pH of the second solution is adjusted to a predetermined range to ionize the anionic polymer, that is, deprotonated. You can adjust the degree. In addition, since the sequential and cross-linking of the first and second layers can be performed without a separate drying process, the degree of ionization of the anionic polymer may also affect the degree of ionization of the cationic polymer forming the first layer previously formed. have. When the pH of the solution is adjusted to a range of 4 to 9, preferably 7 to 8, 50% or more of the carboxyl group, preferably 90% or more, more preferably 95% or more, advantageously, 100 % Can be deprotonated. Deprotonation of the carboxyl group (-COO ^-) is the cation are combined by a protonated amine group (-NH ₃ ⁺⁾ and the electrostatic attraction of the polymer, and some residual non-deprotonated to screen a carboxyl group (-COOH) is Hydrogen bonds may be formed with the free amine groups. However, since such a hydrogen bond may lower the possibility of substitution of the free amine group with the diazenium dioleate group, it is preferable to minimize the deprotonated carboxyl group (-COOH) so that it does not substantially exist.

In addition, when the second layer is formed of an anionic polymer in which all of the carboxyl groups are deprotonated, the degree of ionization of the cationic polymer contained in the first layer is 70% or less, preferably 50% or less, more preferably May be partially up-regulated to 35% or less, and conversely, a free amine group in which an amine group of 30% or more, more preferably, 50% or more, and more preferably 65% or more is not protonated. -NH ₂ ).

Each of the layers formed by the first and second solutions may be combined with each other to form one unit, and at least a part, preferably, all of the free amine groups included in the first layer may be followed by (d ) May be substituted with a diazenium dioleate group. In addition, the anionic polymer may have a substantially linear two-dimensional structure due to repulsion between the deprotonated carboxyl groups, and may be optional such as folding or coiling of molecules by electrostatic attraction in the molecule. Since it does not cause phosphorus deformation, it is possible to effectively obtain a high-density multi-layer film having a nanometer thickness.

In the step (c), the first and second solutions are alternately coated on the substrate, that is, the first and second layers are mutually layer-by-layer (layer-by-layer, LbL). Cross-stacked structures can be implemented. The layer and layer lamination method is a method that can be freely controlled at the molecular level and does not limit the properties of various materials, and can easily produce a multi-layer nanofilm by molecular mutual attraction without the need for heat or strong stimulation. . The attraction of each other is that they are physical bonds, not chemical bonds, so they do not denature the properties of materials.

Since the layer and layer lamination method is a method of using a substrate rather than mixing solutions together, a film in which phase separation does not occur can be prepared. In addition, the layer and layer lamination method is not limited to the size or shape of not only nanoparticles, but also particles or flat substrates of all sizes, and modifies the surface to be coated through surface treatments such as oxygen plasma, RCA and piranha. There are almost no limits.

The layer-to-layer stacking method, for example, electrostatic attraction between the cation and anionic polymers, van der Waals forces, hydrogen bonding, covalent bonding, ionic bonding, hydrophobic bonding, charge-transfer bonding, π -Interactive (π-interaction), coordination chemical bonds, stereocomplexation (stereocomplexation), host-guest (host-guest), biological bonds, and any one selected from the group consisting of two or more of these can be used, but free amine groups It is preferable to exclude the binding of the type that inhibits the substitution with the dianium diolate group.

In addition, the coating is a dip coating (dip coating), spray coating (spray coating), roll coating (roll coating), bar coating (bar coating), brush coating (brush coating), deposition coating, sputtering, electrospinning and among them One method selected from the group consisting of two or more combinations, preferably, may be formed by dip coating, but is not limited thereto.

The types of the cation and anion polymers and their effects are as described above.

The substrate may have a three-dimensional shape, a two-dimensional shape, or a combination of these. The 3D shape may be, for example, particles such as nanoparticles or microparticles, and the particles may further include a shape of an irregular star shape, a trapezoid, an octahedron, or the like. In addition, the shape of the particles is not limited to those exemplified as above, and other two or more of rods, spheres, squares, and flakes are mutually combined, or one of them is partially modified, for example, a hemisphere, It may include elliptic spheres, hollow bodies, rectangles, trapezoids, rhombuses, parallelograms, and the like. The two-dimensional shape may include, for example, a sheet, a film, and a plate.

On the other hand, when the substrate does not have a (-) charge, a surface treatment step for imparting a (-) charge to the substrate may be additionally performed. The surface treatment is a high-frequency spark discharge treatment such as corona treatment or plasma treatment, heat treatment, flame treatment, coupling agent treatment, primer treatment or chemical activation using a vapor phase Lewis acid (ex. BF ₃ ), sulfuric acid or high temperature sodium hydroxide, etc. Treatment, and the like.

In the step (d), at least a part of the free amine group included in the first layer is replaced with a diazenium diol group by reacting a multilayer film including one or more unit parts including the first and second layers with nitrogen monoxide. To obtain a nitrogen oxide transporter. The reaction with the nitrogen monoxide can be made at 5 ~ 300 ℃ and 1 ~ 30atm. The reaction time may be 1-5 days, preferably 2-4 days or 3 days.

In the step (d), a film-coated substrate and a catalyst are put in a solvent and then introduced into a reactor; Evacuating the reactor with an inert gas; And introducing nitrogen monoxide into the reactor to react. Water, ethanol, methanol, THF or DMF may be used as the solvent, and a base catalyst such as sodium methoxide may be used as the catalyst. The exhaust to the inert gas may be performed to remove the gas contained in the reactor and the solution, and argon (Ar) may be used as the inert gas. The exhaust may be performed by repeatedly injecting and discharging an inert gas two or more times, and the product after the reaction may be dried and packaged and stored.

Hereinafter, embodiments of the present invention will be described in detail.

Example 1

Branched polyethyleneimine (BPEI, M _W 25,000) and alginic acid (ALG, M _W 75,000) were dissolved in purified water at a concentration of 1 mg / mL, respectively. The pH of the BPEI solution was adjusted to 9 with 1M HCl, and the pH of the ALG solution was adjusted to 8 with 0.1M HCl or NaOH. In the same way, the pH of the purified water was adjusted to 4, 8 and 9.

In order to clean the substrate, a 1 cm * 4 cm non-oxidized silicon wafer was sonicated in the presence of ethanol. The quartz glass and QCM (quartz crystal microbalance) electrode were washed with a Pirana solution (H ₂ SO ₄ : H ₂ O ₂ , 3: 1 vol / vol%) for 5 minutes.

The substrate was immersed in a (+) charged BPEI solution for 10 minutes, and immersed in purified water of pH 9 having the same pH as the BPEI solution three times for 2 minutes, 1 minute and 1 minute, respectively. Subsequently, the substrate was immersed in an ALG solution having a pH of 8 and immersed in purified water of pH 8 in the same manner. Through this process, BPEI and ALG were cross-stacked on the substrate, and when the cross-stacking was completed, it was dried with compressed air to form a multi-layer film (BPEI9 / ALG8) _n (n: number of repetitions of the process).

Example 2

Branched polyethyleneimine (BPEI, M _W 25,000) and alginic acid (ALG, M _W 75,000) were dissolved in purified water at a concentration of 1 mg / mL, respectively. The pH of the BPEI solution was adjusted to 9 using 1M HCl, and the pH of the ALG solution was adjusted to 4 using 0.1M HCl or NaOH. In the same way, the pH of the purified water was adjusted to 4, 8 and 9.

In order to clean the substrate, a 1 cm * 4 cm non-oxidized silicon wafer was sonicated in the presence of ethanol. The quartz glass and QCM (quartz crystal microbalance) electrode were washed with a Pirana solution (H ₂ SO ₄ : H ₂ O ₂ , 3: 1 vol / vol%) for 5 minutes.

The substrate was immersed in a (+) charged BPEI solution for 10 minutes, and immersed in purified water of pH 9 having the same pH as the BPEI solution three times for 2 minutes, 1 minute, and 1 minute, respectively. Subsequently, the substrate was immersed in an ALG solution having a pH of 4 and immersed in purified water of pH 4 in the same manner. Since there is no separate drying in the above process, the cleaning solution having a different pH can induce irregular assembly of the polymer on the base by changing the pH of the polymer solution. It was immersed once. Through this process, BPEI and ALG were cross-stacked on the substrate, and when the cross-stacking was completed, it was dried with compressed air to form a multi-layer film (BPEI9 / ALG4) _n (n: number of repetitions of the process).

Experimental Example 1: Structural analysis of multilayer film

Since branched polyethyleneimine (BPEI) and alginic acid (ALG) are weak polyelectrolytes, two different film structures can be prepared by controlling the degree of ionization. Referring to FIG. 1 (a), the multi-layer film can be assembled by mostly electrostatic interaction and additional hydrogen bonding between the amine of BPEI and the carboxylic acid of alginic acid. The weight average molecular weight of BPEI is 25,000, and the molecular weight of alginic acid is estimated to be about 3 times larger than that of BPEI. The structures of the multilayer films (BPEI9 / ALG8) _n and (BPEI9 / ALG4) _n were compared. (BPEI9 / ALG4) _n was expected to be more heterogeneous and thicker due to stronger interaction between BPEI and ALG than (BPEI9 / ALG8) _n .

Figure 1 (b) shows the film structure according to the degree of ionization of the polyelectrolyte between the growth of the film and the adhesion of the polyelectrolyte. Referring to Figure 1 (b), in order to explain the manufacturing process of the film, BPEI was about 21.5% ionized at pH 9 and formed a coiled structure. BPEI can be adsorbed by electrostatic interaction on a negatively charged substrate. In addition, in the ALG solution at pH 8, the adsorbed BPEI is ionized to about 31.9%, and can be electrostatically bound to fully charged (ionized) ALG in a linear structure by intermolecular repulsion between ionized carboxyl groups (Film 1 , (BPEI9 / ALG8) _n ). Since the linear ALG polymer is bound to (+) charged BPEI on the substrate, the film structure can be uniform. Then, in the BPEI solution, the absorbed ALG is fully charged (ionized) at pH 9, and the 21.5% charged (ionized) BPEI has specificity to the surface of the ALG. To maintain charge balance, the coiled BPEI polyelectrolyte can be adsorbed and its growth induced.

In the case of Film 2 ((BPEI9 / ALG4) _n ) of FIG. 1 (b), the first layer made of BPEI shows the same adsorption properties on the substrate at pH 9, but in the ALG solution at pH 4, the surface amine group is highly protonated Protonated, half-ionized ALG exhibits greater adsorption at the surface by charge compensation. Consequently, in a BPEI solution at pH 9, 22% of ionized BPEI is attached to the fully ionized carboxyl surface until charge balance is achieved. In this way, the (BPEI9 / ALG4) _n film exhibits a high growth pattern and a rough surface structure due to strong adsorption behavior of BPEI and ALG in a continuous charge compensation process.

The growth patterns of the two multilayer films were analyzed using a surface shape measuring device (profilometer, Dektak 150, Veeco) and QCM (QCM 200, 5 MHz, Stanford Research System). In FIG. 2 (a), since the multilayer film can generate a nitrogen monoxide donor in the amine group of BPEI, the thickness of the film was measured at a thickness of 2.5 double layers. Therefore, the outermost surface needs to be BPEI in order to create a donor with high efficiency, and the film has a different thickness, ie, the content of BPEI, at a double layer spacing of 2.5. Using the properties of the film, the nitrogen monoxide release behavior was controlled. As a result, the thickness of the (BPEI9 / ALG4) film is at least about 5 times that of the (BPEI9 / ALG8) film, because a large amount of polymer was continuously adsorbed for charge compensation. In FIG. 2 (b), the growth process of the film was confirmed through the QCM frequency shift of each layer constituting the film, and it can be seen that it has the same pattern as the thickness growth pattern. In FIG. 2 (c), the mass change of each layer was compared, and the mass of the ALG layer in both films was significantly changed compared to the BPEI layer. From this, it can be seen that the molecular weight of ALG is higher than that of BPEI.

The surface shape of the two multilayer films was observed using SEM (SIGMA, Carl Zeiss), and the results are shown in FIG. 3. (BPEI9 / ALG8) Although there is no characteristic shape in the film, referring to FIGS. 3 (i) and 3 (j), a circular microstructure having a diameter of several μm in a (BPEI9 / ALG4) film having a double layer of 8.5 or more Was observed. The microstructure grew with the number of double layers. This is consistent with (BPEI9 / ALG4) film, in which a polyelectrolyte having a low ionization degree is attached to the surface in a large amount and induces high roughness on a micrometer scale.

Preparation Example 1

Nitrogen monoxide release characteristics were imparted to the multilayer film (BPEI9 / ALG8) _n prepared in Example 1 through a high pressure reaction. Multilayer film (BPEI9 / ALG8) _n in a vial with 0.232 mmol of sodium methoxide (equal to the molar number of secondary amines contained in BPEI 10 mg) in a mixture of absolute ethanol and methanol (4: 1 vol / vol%) Immersed. Subsequently, the vial was placed in a high-pressure reactor, and the chamber was treated with 10 atm of argon gas to remove reactive gas molecules such as oxygen. Thereafter, the chamber was filled with 10 atm of nitrogen monoxide and left at room temperature for 3 days. The chamber was again treated with 10 atm argon gas to remove unreacted nitrogen monoxide gas and the film was taken out of the chamber. In order to remove sodium methoxide remaining in the obtained film, it was washed with anhydrous ethanol, dried quickly under vacuum conditions, and then stored in vacuum at -20 ° C.

Preparation Example 2

Nitrogen monoxide release characteristics were imparted to the multilayer film (BPEI9 / ALG4) _n prepared in Example 2 through a high pressure reaction. In a vial, a multi-layer film (BPEI9 / ALG4) _n in a mixture of anhydrous ethanol and methanol (4: 1 vol / vol%) with 0.232 mmol of sodium methoxide (equal to the molar number of secondary amines contained in BPEI 10mg) Immersed. Subsequently, the vial was placed in a high-pressure reactor, and the chamber was treated with 10 atm of argon gas to remove reactive gas molecules such as oxygen. Thereafter, the chamber was filled with 10 atm of nitrogen monoxide and left at room temperature for 3 days. The chamber was again treated with 10 atm argon gas to remove unreacted nitrogen monoxide gas and the film was taken out of the chamber. In order to remove sodium methoxide remaining in the obtained film, it was washed with anhydrous ethanol, dried quickly under vacuum conditions, and then stored in vacuum at -20 ° C.

Experimental Example 2: Evaluation of nitrogen monoxide release behavior

The multilayer film prepared as described above was applied as a two-dimensional platform for nitrogen monoxide delivery. In order to characterize the relationship between the film structure and the nitrogen monoxide release behavior, a diazonium diolate group was generated at the amine positions of the (BPEI9 / ALG8) and (BPEI9 / ALG4) films. The multi-layer film processed through the preparation example has an N-diazenium dioleate group at the amine position.

In Figure 4, comparing the film structure before and after the high pressure reaction, it can be confirmed whether the N-diazenium diolate functional group is generated. Referring to FIG. 4 (a), the (BPEI9 / ALG8) film has a free amine group that does not have any molecular interaction from a physicochemical point of view, and thus has more N-diazenium not only on its surface but also on the inside. It was assumed to have dioleate. 4 (b) and 4 (c) are FT-IR spectra (FT / IR 4700, Jasco) for films before and after the high pressure reaction. FIG so 4 (c) from, ONNO, NO, NN peaks each observed in a wave number of ^{1360cm -1, 1220cm -1, 945cm -1} , it can be seen that N- diamond jenyum diol acrylate groups generated. In addition, Figure 4 (d) and 4 (e) is a UV-vis spectrum (Evolution 300, Thermo Fisher Scientific) for the film before and after the high pressure reaction, N- diazenium diolate from the absorption band at 252 nm after the high pressure reaction It can be seen that the group was formed. UV absorbance differs depending on the amount of N-diazenium diolate group produced in the film, which increases in proportion to the thickness of the film.

On the other hand, (BPEI9 / ALG4) N-diazenium diolate group was produced on the film and analyzed in the same manner. Referring to FIG. 5 (a), it was assumed that the (BPEI9 / ALG4) film had a smaller amount of N-diazenium diol group than the (BPEI9 / ALG8) film even though the thickness was thicker. As seen through FIG. 1, the (BPEI9 / ALG4) film is prepared by strong electrostatic interaction between two types of polymers, and the polymer has a coiled structure because it lacks repulsion between molecules or molecules. Even if the final film has a thicker thickness and a rougher surface, the film structure is difficult to properly produce an N-dianium diol group because of lack of free amine groups and physical space. 5 (b) and 5 (c), weak peaks corresponding to the N-diazenium diol group appear in the FT-IR spectrum. 5 (d) to 5 (f), the UV absorbance is proportional to the thickness of the film, and after the reaction, the absorbance of the N-diazenium diolate group increased with the number of double layers. However, the difference in UV absorbance before and after the reaction did not show a tendency according to the thickness or number of the double layers, and the difference was significantly smaller than the (BPEI9 / ALG8) film.

From this, it can be seen that the (BPEI9 / ALG8) film has advantageous properties to realize the controlled release behavior of nitrogen monoxide compared to the (BPEI9 / ALG4) film.

Phosphate-buffered saline at 37 ° C (phosphate-buffered saline, PBS, 0.01M), the physiological conditions of the nitrogen monoxide release behavior of the multilayer film using a nitrogen monoxide analyzer (Sievers NOA 280i, GE Analytical Instruments) according to the chemiluminescence principle pH 7.4). The analyzer was calibrated with a zero filter (0 ppm of nitrogen monoxide) and standard nitrogen monoxide gas (AIRKOREA Inc.) before each sample measurement. The nitrogen monoxide released from the multilayer film in the sealed flask was transferred to the analyzer with an argon gas flow (70 mL min ^-1 ) moving along the reaction cell, and was measured until nitrogen monoxide emission dropped below 5 ppb.

6 (a) and 6 (c) are real-time measurement results of nitrogen monoxide gas, and FIGS. 6 (b) and 6 (d) are total nitrogen monoxide emission measurement results obtained by summing real-time measurement results. The (BPEI9 / ALG8) film shows a tendency to increase in proportion to the number of bilayers, while the (BPEI9 / ALG4) film shows unclear nitrogen monoxide release behavior. In order to compare the nitrogen monoxide release result with the yield of the nitrogen monoxide donor, in FIG. 6 (e), UV-vis absorbance of the N-diazenium dioleate of the (BPEI9 / ALG8) and (BPEI9 / ALG4) films are collectively shown. In Fig. 6 (f), the total nitrogen monoxide emission amount is shown. When comparing the UV absorbance of each film and the total nitrogen monoxide emission, all graphs have a consistent tendency, from which the UV-vis absorbance reflects the quantification of the nitrogen monoxide donor, as expected, of the (BPEI9 / ALG8) film It can be seen that the nitrogen monoxide emission can be controlled by increasing the thickness.

The specified values for the nitrogen monoxide release behavior are shown in Table 1 below.

division 2nd floor
(n) t [NO]
(μmol cm ^-2 ) t _1/2
(hr) [NO] _m
(ppb cm ^-2 ) t _m
(min) t _d
(hr) Example 1 2.5 0.04 ± 0.01 3.9 ± 0.4 145.3 ± 36.6

±3.19.3

± 3.1 11.4

± 2.2 Example 1 4.5 0.12 ± 0.00 5.4 ± 0.5 227.6 ± 0.6 23.8 ± 20.4 22.6 ± 1.5 Example 1 6.5 0.11 ± 0.04 4.3 ± 1.0 341.3 ± 78.9 14.5 ± 11.1 19.0 ± 0.7 Example 1 8.5 0.26 ± 0.01 4.8 ± 0.5 1044.9 ± 111.7 15.6 ± 2.0 27.6 ± 6.0 Example 1 10.5 0.30 ± 0.02 3.6 ± 0.4 1236.8 ± 41.6 13.1 ± 1.8 30.1 ± 4.6 Example 2 2.5 0.04 ± 0.01 4.5 ± 0.3 111.8 ± 26.7 7.0 ± 2.7 12.6 ± 0.2 Example 2 4.5 0.09 ± 0.01 5.9 ± 0.5 216.9 ± 10.7 10.5 ± 5.0 19.5 ± 2.1 Example 2 6.5 0.07 ± 0.04 3.2 ± 0.1 273.4 ± 33.9 11.2 ± 3.8 14.1 ± 1.4 Example 2 8.5 0.07 ± 0.00 5.2 ± 1.3 159.1 ± 63.2 11.1 ± 7.1 18.5 ± 3.0 Example 2 10.5 0.08 ± 0.01 5.8 ± 0.6 130.1 ± 2.8 18.2 ± 0.3 19.1 ± 2.1

-t [NO]: Total number of moles of nitrogen monoxide produced per unit area of film -t _1/2 : Half-life of nitrogen monoxide release

-[NO] _m : Maximum nitrogen monoxide concentration in real-time emission behavior

-t _m : maximum nitrogen monoxide concentration time

-t _d : Nitrogen monoxide release duration

The total amount of nitrogen monoxide released from the (BPEI9 / ALG8) film is 0.04 to 0.3 μmol / cm ² , and the half-life is 3.6 to 5.4 hours. In general, N-Diazenium Dioleate is a thermodynamically unstable molecule, so it quickly releases nitrogen monoxide gas within a half-life of several minutes, and the trigger is a proton. However, regardless of the structure of the multi-layer film, the half-life was significantly extended to several hours, and this sustained release behavior was analyzed because N-diazenium diolate was stabilized by chemically bonding to adjacent amine groups.

As such, the multi-layer film can increase the nitrogen monoxide emission amount by generating a nitrogen monoxide donor inside the film structure, so that the conventional small surface area can solve the disadvantages of the low nitrogen monoxide emission film, and the small surface area film Even in this case, the half-life can be extended to achieve continuous nitrogen monoxide emission behavior.

The above description of the present invention is for illustration only, and those of ordinary skill in the art to which the present invention pertains 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, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (15)

delete delete delete delete delete delete delete (A) preparing a first solution by adjusting the pH of the solution containing a cationic polymer having an amine group in the side chain in the range of 7 to 10;
(b) preparing a second solution by adjusting the pH of a solution containing an anionic polymer having a carboxyl group on the side chain to a range of 7 to 8;
(c) alternately coating the first and second solutions on a substrate; And
(d) reacting with nitrogen monoxide; a method for producing a nitrogen oxide carrier comprising a. delete The method of claim 8,
The cationic polymer is branched polyethyleneimine (bpei), chitosan, gelatin, collagen, collagen, fibrinogen, silk fibroin, casein, elastin ), Laminin, fibronectin, polydopamine, polyethyleneimine, poly-l-lysine, poly (vinylamine) hydrochloride ), Poly (amino acid), desaminotyrosylctylester, and a method for producing a nitrogen oxide transporter, one selected from the group consisting of two or more of these. delete The method of claim 8,
The anionic polymer is hyaluronic acid, poly-l-lactic acid, alginic acid, dextran sulfate, lignin, tannic acid , Chondroitin sulfate, cellulose polymers, fucoidan, heparin, and a method for producing a nitrogen oxide transporter selected from the group consisting of two or more of these. The method of claim 8,
The substrate is a method of manufacturing a nitrogen oxide carrier having a three-dimensional shape, a two-dimensional shape, or a combination of these. The method of claim 8,
In the step (c), the coating is electrostatic attraction between the cation and anion polymers, van der Waals forces, hydrogen bonding, covalent bonding, ionic bonding, hydrophobic bonding, charge-transfer bonding, π-interaction (π-interaction), coordination chemical bonding, stereocomplexation (stereocomplexation), host-guest (host-guest), biological binding, and a method for producing a nitrogen oxide carrier using one selected from the group consisting of two or more of them. The method of claim 8,
In the step (d), the reaction is a method for producing a nitrogen oxide carrier made at 5 to 300 ° C and 1 to 30 atm.

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