WO2018080155A2 - Method for producing polymer coating-based nitrogen oxide delivery composite, and use of same - Google Patents

Abstract

The present invention relates to a nitrogen oxide delivery composite in which the surface of a base material such as nano-particles or the like is modified by a polymer having a secondary amine group. A secondary amine forms a diazeniumdiolate functional group through a high pressure reaction with nitrogen monoxide, and the functional group can function as a nitrogen oxide provider. According to the present invention, the surface of a base material can be modified by a polymer to be single layer or multilayer through a layer-by-layer (LbL) coating method. The production technique involves simple processes, is economical, and can be applied in a wide range of fields. In addition, the emission speed and emission amount of nitrogen monoxide can be easily adjusted, and the distribution of a base material such as nano-particles or the like can be increased. The nitrogen oxide delivery composite produced according to the present invention is not cytotoxic, and thus is expected to be usable for medical purposes, such as for enlarging blood vessels, treating wounds, or killing bacteria and the like, in vivo, through adjustment of the emission amount and emission speed of nitrogen monoxide.

Description

Polymeric coating-based composite manufacturing method for nitric oxide delivery and its application

The present invention relates to a complex for delivery of nitric oxide and a method for producing the same.

Nitric monoxide (NO) is a highly reactive radical molecule produced by nitric oxide-producing 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 neuroscience, physiology, and immunology when it was named “Molecule of the Year” by the American journal Science in 1992. The study, which revealed the cardiovascular signaling function of nitric oxide, won the Nobel Prize in Physiology in 1998. Nitric oxide acts as a powerful vasodilator in the blood vessels, and in addition, it functions in various functions such as immune response, neurotransmission, erectile control, antibacterial action and wound healing.

Necessity of Nitric Oxide Delivery Through Nanoparticles

Since nitrogen monoxide plays a variety of roles in the body, people have conducted studies to find ways to artificially deliver nitrogen monoxide externally. In particular, nitrogen monoxide has various effects in the gas state, and since the half-life is very short within 6 seconds, studies on the release of nitrogen monoxide in the form of compounds have been conducted.

The most studied compounds include diaeniumdiolate (NONOate) and S-nitrosothiol (RSNO). In the case of S-nitrosothiol, the emission is promoted by temperature, light, and copper ions (Cu 2+), and the nitrogen monoxide emission efficiency is high. On the other hand, it is difficult to obtain nitrogen monoxide in a pure state because nitrogen monoxide is not released into the living body or the material is not stable. Diagenium dioleate can be stored stably in a solid state, has high solubility in water, and can easily produce nitrogen monoxide at a living temperature and pH conditions. However, degradation products may be toxic and have the disadvantage of releasing nitrogen monoxide upon contact with water.

Nitrogen Monoxide Delivery Through Nanoparticles

Representative foreign groups conducting research on nitrogen monoxide carriers include Mark E. Meyerhoff Group of the University of Michigan and Mark H. Schoenfisch Group of Chapel Hill, North Carolina State University. Meyerhoff Group has developed silica nanoparticles that release hundreds of nanometers of nitrogen oxides based on diazenium dioleate in a sol-gel manner using aminoalkylsilane (Non-Patent Document 1). Subsequently, the Schoenfisch group first manufactured silica nanoparticles, and then converted the secondary amine group on the surface into a diagenium diolate group, thereby converting the particles showing the release of nitrogen monoxide from the surface, and the aminoalkoxysilane molecules into the diagenium diolate group Next, silica nanoparticles were fabricated and developed two forms in which nitrogen monoxide was released from inside the particles. After preparing the particles using various aminoalkoxysilanes, the size and nitrogen monoxide emission efficiency were reported. When there is an aminoalkoxysilane group inside the particle, the efficiency of nitrogen monoxide emission is maximized, thereby making it possible to manufacture high-efficiency nitric oxide nanoparticles. The efficiency varies depending on the molecular structure of aminoalkoxysilane. However, since the aminoalkoxysilane-based nitric oxide nanoparticles are manufactured according to the sol-gel principle, the process must be thoroughly controlled for temperature and moisture, which makes the process difficult, costly, and likely to fail (Non-Patent Document 2).

In Korea, Professor Kim Won-jong of POSTECH is conducting research to control the nitrogen monoxide release of diazenium dioleate-based nitric oxide nanoparticles. The porous silica nanoparticles made of aminoalkoxysilane changed the structural form by UV, 2-nitro-benzaldehyde, to release hydrogen ions, and then capped the nanoparticles with calcium phosphate. The reaction was carried out for 3 days under to prepare nitric oxide nanoparticles. The nanoparticles produced as described above are inhibited from penetrating external hydrogen ions by capping, so the release of nitrogen monoxide does not occur spontaneously. In addition, when irradiated with UV, 2-nitro-benzaldehyde in the nanoparticles releases hydrogen ions, resulting in the release of nitrogen monoxide as the capping layer decomposes as the pH is lowered (Non-Patent Document 3). However, there is still a disadvantage in this study that the process of using aminoalkoxysilane is complicated and difficult.

In other words, the synthesis of nitric oxide nanoparticles based on the sol-gel reaction, which has been studied a lot, uses materials that are sensitive to temperature and moisture. The probability of failure is very high.

[Non-Patent Documents]

1. J. Am. Chem. Soc. 2003, 125, 5015-5024

2. Chem. Mater. 2008, 20, 239-249

ACS Nano 2016, 10, 4199-4208

In the present invention, in order to solve the above problems, using a biocompatible polymer instead of a material that reacts sensitively to the external environment, by applying a layer-by-layer (LbL) lamination method, it is easy and inexpensive with a simple principle. An object of the present invention is to provide a method for producing a composite for nitric oxide delivery.

In the present invention; And

It is formed on the substrate, and comprises a polymer thin film containing a diazenium dioleate group,

The polymer thin film provides a composite for transferring nitric oxide formed in at least one layer.

In addition, the present invention (A) forming at least one polymer thin film containing a secondary amine group on the substrate; And

(B) at a temperature of 25 to 300 ℃ and a pressure of 1 to 30 atm, it provides a method for producing a nitric oxide delivery complex comprising the step of reacting with nitrogen monoxide.

The nitric oxide delivery composite produced by the present invention has the same or higher nitrogen monoxide emission efficiency as conventional sol-gel based nitric oxide particles. In addition, the manufacturing process is simple and not limited, so the probability of manufacturing success is high and mass production is possible.

Polymer coating of the layer and layer (LbL) lamination method can be applied to any size or shape, such as microparticles and flat substrates as well as nanoparticles, it is possible to manufacture a variety of nitric oxide delivery complex.

In addition, the LbL method can coat not only desired biocompatible polymers but also graphene, proteins, drugs and growth factors based on various intermolecular attraction. Therefore, by applying this, it is possible to give a multi-functionality in which the release of the drug or growth factor simultaneously with the release of nitrogen oxide gas.

Furthermore, the surface coating can freely control the surface charges and functional groups of the particles or specific surfaces to increase the distribution ability and biocompatibility of the particles. The rate and amount of nitrogen monoxide release can also be controlled.

Figure 1 is a schematic diagram showing the manufacturing process of the composite for the delivery of nitrogen oxide of various forms, such as particles, thin film and hydrogel.

Figure 2 is a schematic diagram showing the chemical structure of the diazenium dioleate functional groups produced when a polymer such as BPEI reacted under high pressure nitrogen monoxide at room temperature for 3 days.

3 is an SEM image of the BPEI coated nanoparticles prepared in the Examples.

4 is a SEM image of the nitric oxide nanoparticles prepared in the example.

5 is a graph of surface charge analysis of silica nanoparticles, BPEI coated nanoparticles, and nitric oxide nanoparticles.

6 is a graph analyzing the amount of nitrogen monoxide released from the nitric oxide nanoparticles in real time with a nitrogen monoxide analyzer.

7 is a graph showing cumulative amounts of nitrogen monoxide released from nitric oxide nanoparticles.

8 is a table summarizing the total amount of nitrogen monoxide released from the nitric oxide nanoparticles, half-life, the maximum release of nitrogen monoxide and the total release time.

9 is a graph evaluating toxicity for myoblasts for 24 hours and 48 hours according to the concentrations of nitric oxide nanoparticles (B-NO) and BPEI coated nanoparticles (B-Si).

FIG. 10 is a graph evaluating toxicity for myoblasts with higher concentrations of silica nanoparticles (Si), nitric oxide nanoparticles (B-NO) and BPEI coated nanoparticles (B-Si) for 24 hours.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention; And a polymer thin film formed on the substrate and including a diazenium dioleate group.

The polymer thin film relates to a composite for transferring nitric oxide formed in at least one layer.

In the present invention, the "nitric oxide delivery complex" means a material capable of delivering nitrogen monoxide, and may be referred to as a "nitrogen oxide complex" or a "nitrogen oxide transporter."

In the present invention, the substrate is not particularly limited as long as it is a material to be coated on the surface to control and release nitrogen monoxide, and may be a stable inorganic component in vivo, a biodegradable polymer degradable in vivo, micelles and bio-derived materials. One or more selected from the group consisting of:

At this time, the inorganic component may be silica, hydroxyapatite or gold, and the like, and the biodegradable polymer may be poly-β-hydroxy butyrate (PHB), polylactic acid (PLA), Poly-DL-lactide-co-glycolide (PLGA), aliphatic polyesters, polycaprolactone (PCL), polymethyl methacrylate (polymethyl methacrylate, PMMA), poly-ethyleneglycol (PEG), poly-vinylalcohol (PVA), poly-alkyl-cyano-carylates (PAC) ), Chitosan or gelatin. In addition, micelles are Phopholipid-based liposomes, emulsion-type particles formed on water / oil including surfactants, PS-b-PAA, PS-b-P4VP, PEO-b-PCL, and the like. The block copolymer may be a block copolymer and the like, and the bio-derived material may be DNA / RNA, phospholipid or liposome.

In one embodiment, silica may be used as the substrate.

The shape of the substrate is not particularly limited, and may have a nanoparticle, microparticle, or film shape. When having the shape of the nanoparticles, the composite for delivering nitric oxide may be referred to as nitric oxide nanoparticles, and may be referred to as nitric oxide microparticles when having the shape of microparticles, and as a nitric oxide film when it has a film shape. .

In one embodiment, the substrate may be made of a material that can be inserted into the body (hereinafter, referred to as a body insert). The body insert means any article that can be placed in a position where the physiological effect can be seen by the release of nitric oxide in the body of an animal including a human or a mammal, and can have a carrier for controlled release of nitric oxide on its surface. Such inserts do not necessarily have to be present in the body as long as a part is inserted into the body. For example, connections such as pipes and wires may be pushed out of the body. These implants include stents, catheters, subcutaneous implants, chemical sensors, leads, heart pacemakers, vascular grafts, wound dressings, Penile implants, implantable pulse generators, implantable cardiac defibrillators, nerve stimulators, and the like.

In the present invention, a polymer thin film including a diazenium dioleate group (RR′N-N (O) = NOR) capable of storing and releasing nitrogen monoxide is formed on the substrate. The polymer containing the diazenium dioleate group can be stably stored as a solid form, has high solubility in water, and release forms such as release rate and method according to the structure of the remaining portion to which the diazenium dioleate group is connected. Can be adjusted. In addition, the polymer containing the diazenium dioleate group not only decomposes at the temperature and pH conditions of the body, but also has various forms of release depending on the pH. In addition, since two molecules of nitrogen monoxide are released per unit of diazenium dioleate group, relatively high concentrations of nitrogen monoxide can be generated when included in the complex.

The polymer thin film including the diazenium dioleate group may be formed in one or more layers, and specifically, may be formed in a multilayer of 1 to 500 layers or 2 to 200 layers.

When the thin film is formed of two or more layers, one thin film may be formed by a neighboring thin film and a layer-by-layer (LbL) lamination method.

Layer-by-layer lamination is a technique that can be freely controlled at the molecular level, does not limit the properties of various materials, and can produce multilayer nano thin films by simple molecular mutual attraction without applying heat or strong stimulus. . Since the attraction between each other is not a chemical bond but a physical bond, it does not denature the intrinsic properties of the materials. Since the layer and the layer lamination method use a substrate rather than mixing the solutions with each other, a thin film (polymer layer) without phase separation may be produced.

In addition, the layer and layer stacking method is not limited to the size and shape, such as not only nanoparticles, but also particles of any size or planar substrate, and by modifying the surface to be coated by surface treatment such as oxygen plasma, RCA and piranha There is almost no limit.

 The layer-to-layer stacking method includes electrostatic attraction, van der Waals forces, hydrogen bonds, covalent bonds, ionic bonds, hydrophobic bonds, charge-transfer bonds, π-interaction, and coordination. Chemical bonds, stereocomplexation, host-guest or biological bonds can be used.

In the host-guest, the host may be cyclodextrins, cucurbiturils, calixarenes, pillarararenes, crown ethers, or porphyrins. The guest may be ferrocene, adamantine or azobenzene. In addition, the biological binding may be avidin-Biotin binding, antigen-antibody binding, lectin-carbohydrate binding or DNA complementary binding.

In one embodiment, the layer and layer stacking method may use an electrostatic attraction or hydrogen bonding.

In the present invention, the total thickness of the thin film may be 1 nm to 1000 μm, or 1 nm to 10 μm. It is stable at this thickness and does not exhibit cytotoxicity, and may have excellent nitrogen monoxide release effect.

In the present invention, the polymer thin film including the diazenium dioleate group may further include one or more components selected from the group consisting of proteins, growth factors, graphene, and drugs in the thin film. These components can impart new functions in addition to nitrogen monoxide delivery.

In addition, the nitric oxide complex in the present invention may further include a thin film composed of one or more components selected from the group consisting of a second polymer, a protein, a growth factor, graphene, and a drug.

The protein, growth factor, graphene, and drugs can be used without limitation materials used in the art.

In one embodiment graphene has a gas barrier effect, it is possible to control the rate and amount of nitrogen monoxide released.

In one embodiment, when the multilayer polymer thin film is formed by electrostatic attraction, the nitric oxide complex may further include a thin film including a second polymer, wherein the second polymer may be a polymer having a negative charge. have.

The second polymer is poly (ethylene glycol), hyaluronic acid, poly-L-lactic acid, alginic acid, dextran Dextran sulfate, lignin, tannic acid, chondroitin sulfate, cellulose polymers, fucoidan, poly (acrylic acid), poly (Sodium-4-styrenesulfonate), poly (vinylidene fluoride), poly (methacrylic acid), poly (glycol Acid) (poly (glycolic acid)), poly (lactic acid), poly (caprolactone), poly (ortho ester) II, poly [(carboxyphenoxy) Propane sebacic acid], poly (alkyl cyanoacrylate), polyphosphoesters, polyester Imide may be used one or more selected from the group consisting of (polyester amides) and polyurethanes (polyurethane).

The present invention also relates to a method for preparing the nitric oxide complex described above.

The nitric oxide composite according to the present invention comprises the steps of: (A) forming at least one polymer thin film containing a secondary amine group on a substrate; And

(B) at a temperature of 25 to 300 ° C. and a pressure of 1 to 30 atm, it may be prepared through a step of reacting with nitrogen monoxide.

In the present invention, as described above, the substrate may use one or more selected from the group consisting of an inorganic component stable in vivo, a biodegradable polymer degradable in vivo, micelles, and a bio-derived material. It may be used, the shape may be nanoparticles, micro particles or film shape. When having the shape of the nanoparticles, the composite for delivering nitric oxide may be referred to as nitric oxide nanoparticles, and may be referred to as nitric oxide microparticles when having the shape of microparticles, and as a nitric oxide film when it has a film shape. .

Step (A) in the present invention can be carried out by immersing the substrate in a polymer solution containing a secondary amine group. The solvent in the polymer solution containing the secondary amine group may be water, ethanol, sodium acetate buffer (PBS), phosphate buffered saline (PBS) or culture media, and the like. It is positively charged.

In one embodiment, when silica is used as the substrate, the silica is negatively charged by the OH- group, and thus, the silica may be bonded to the secondary amine group through electrostatic attraction.

In one embodiment, when the substrate is not negatively charged, a surface treatment step for forming a negatively charged surface on the substrate may be further performed. The surface treatment may include a high frequency spark discharge treatment such as corona treatment or plasma treatment; Heat treatment; Flame treatment; Coupling agent treatment; Primer activation or chemical activation treatment using gas phase Lewis acid (ex. BF3), sulfuric acid or hot sodium hydroxide and the like.

In the present invention, the polymer containing the secondary amine group is branched polyethyleneimine (BPEI), chitosan (chitosan), gelatin (gelatin), collagen (collagen), fibrinogen, silk fibroin (silk fibroin), casein (casein), elastin, laminin, fibronectin, poly dopamine, poly ethyleneimine, poly-L-lysine, poly (vinyl) Amine) hydrochloride (poly (vinylamine) hydrochloride), poly (amino acids) and desaminotyrosyl octyl ester (desaminotyrosyl octyl ester) may be at least one selected from the group, specifically BPEI Can be.

In addition, the polymer solution including the secondary amine group may further include one or more selected from the group consisting of proteins, growth factors, graphene, and drugs, in addition to the polymer including the secondary amine group.

In the present invention, when the thin film is formed in multiple layers, one thin film may be formed by a neighboring thin film and a layer-by-layer (LbL) lamination method. The layer-to-layer stacking method includes electrostatic attraction, van der Waals forces, hydrogen bonds, covalent bonds, ionic bonds, hydrophobic bonds, charge-transfer bonds, π-interaction, and coordination. Chemical bonds, stereocomplexation, host-guest or biological bonds may be used, specifically electrostatic attraction or hydrogen bonds.

In one embodiment, when the thin film is formed of two or more multilayers through hydrogen bonding, the thin film may be formed by stacking a polymer solution containing a secondary amine group two or more times.

In addition, in one embodiment, when the thin film is formed into two or more multilayers through electrostatic attraction, the thin film may be formed by repeatedly stacking a polymer solution and a negatively charged polymer solution including a secondary amine group.

The negatively charged polymer in the negatively charged polymer solution refers to a polymer that can dissolve in a solvent and take a negative charge. The kind of the negatively charged polymer is not particularly limited, and polymers used in the art may be used without limitation. For example, negatively charged polymers include hyaluronic acid, poly-L-lactic acid, alginic acid, dextran sulfate, lignin, tannic acid natural polymer and poly (acrylic acid, PAA) including tannic acid, chondroitin sulfate, cellulose polymer, fucoidan, heparin, and HEP , Poly (sodium4-styrenesulfonate), poly (vinylidene fluoride), poly (methacrylic acid), poly (Glycolic acid), poly (lactic acid), poly (caprolactone), poly (ortho ester) II, poly [(carboxyphenoxy) Poly ((carboxyphenoxy) propane sebacic acid], poly (alkyl cyanoacrylate), polyphosphoester s), synthetic polymers such as polyester amides and polyurethanes, or mixtures thereof.

In the present invention, after forming one thin film, washing may be further performed.

In addition, the present invention may further comprise the step of forming a thin film comprising at least one selected from the group consisting of a second polymer, a protein, a growth factor, graphene and a drug. As the second polymer, the above-described polymer may be used.

In the present invention, step (B) is a step of reacting the substrate coated with the polymer thin film containing the secondary amine group prepared in step (A) with nitrogen monoxide. In the present invention, the nanoparticles or microparticles formed with the polymer thin film may be encapsulated in a hydrogel and then reacted with nitrogen monoxide.

In the present invention, the reaction with nitrogen monoxide may be carried out at a temperature of 25 to 300 ℃ and a pressure of 1 to 30 atm. The reaction time can be 1 to 5 days, 2 to 4 days or 3 days. In addition, the amount of the product prepared in (A) may vary depending on the size of the reaction vessel. For example, when the size of the reaction vessel is 1 ml to 10 L, the amount of the product may be 1 mg to 100 g. By this reaction, the secondary amine group can be converted to the diazenium diolate group (FIG. 2).

In one embodiment step (b) is a step of putting a thin film coated substrate and a catalyst in a solvent to the reactor;

Evacuating the reactor with an inert gas; And

Nitrogen monoxide in the reactor may be carried out through the step of reacting.

As the solvent, water, ethanol, methanol, THF, or DMF may be used, and as the catalyst, a base catalyst such as sodium methoxide may be used.

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 releasing an inert gas a plurality of times.

The reaction with nitrogen monoxide may be carried out for the above temperature, pressure and time.

After the reaction is completed, the product can be stored after packaging dry.

In the present invention, Figure 1 is a schematic diagram showing the manufacturing process of the nitric oxide composite of various forms such as particles, films and hydrogels.

As shown in FIG. 1, particles or films in which a polymer thin film is formed may be manufactured as a nitrogen monoxide composite through a layer-to-layer lamination method. In addition, the nitric oxide complex can also be produced in an applied form such as a hydrogel containing particles having a thin film formed thereon.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. These embodiments are provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art, and the invention will be defined by the scope of the claims. It is only.

Example

Example  1. Preparation of Nitric Oxide Nanoparticles

(1) Silica Nanoparticle Synthesis

Silica nanoparticles with a diameter of 100 nm were fabricated with reference to a study reported by K. Nozawa et al. Based on Stober principle (Langmuir 2005, 21, 1516).

Tetraethyl orthosilicate (TEOS), ammonia and ethanol were used in a volume ratio of 1: 1: 10. First, TEOS was added rapidly while ammonia was added to ethanol and the solution was stirred at 400 rpm. After stirring for 12 hours at the same rate, the supernatant was removed by centrifugation to identify the sinking silica nanoparticles. Silica nanoparticles were finally prepared by further washing and centrifuging the nanoparticles by adding ethanol and washing.

(2) Surface Modification of Silica Nanoparticles Using Amine-based Polymers) BPEI  Manufacturing coated nanoparticles)

The surface of the silica nanoparticles was modified using BPEI, a polymer having secondary amine functionality.

Aqueous solution of BPEI at a concentration of 10 mg / mL was prepared using water as a solvent. 1 L of BPEI aqueous solution was added to 500 mg of silica nanoparticles, and then dispersed for 5 minutes using a vortexer and a sonicator. The silica nanoparticles were then precipitated for 5 minutes using a small centrifuge. After removing the BPEI aqueous solution of the upper layer, 1 L of distilled water of the same pH was added, and then dispersed for 30 minutes to proceed with the washing process. In the same manner, the supernatant was removed by precipitating silica nanoparticles. This process was repeated five times to wash the silica nanoparticles to form a layer of BPEI on the surface of the nanoparticles.

In this way, by repeating the process using a material having a BPEI and attractive force it was possible to form a multilayer on the surface of the nanoparticles.

(3) Nitric Oxide Nanoparticle Synthesis

Nitric oxide nanoparticles were synthesized by reacting BPEI-coated nanoparticles under high pressure monoxide gas.

BPEI-coated nanoparticles were placed in a vial containing 30 mL of methanol, and sonication was performed for 30 minutes to disperse well. Sodium methoxide was added at the same ratio as the amount of BPEI. The vial was placed in a high-pressure reactor, sealed, and then rapidly injected and discharged with argon gas at 3 atm and repeated for 30 minutes to remove gases contained in the reactor and the solution.

Thereafter, nitrogen monoxide gas was injected into the reactor at 3 atmospheres, and the reaction was performed at room temperature for 5 days. After 5 days, the nitrogen monoxide gas was discharged, and then, the argon gas was rapidly injected and discharged at 3 atmospheres five times to remove the unreacted nitrogen monoxide gas from the solution. After removing the supernatant by centrifugation at 6,000 rpm for 30 minutes, the nanoparticles were dispersed by dissolving 100 ml of a solvent that could not be dissolved and then centrifuged twice in the same manner to wash the nitric oxide nanoparticles. . Through the above-described process, as shown in FIG. 2, the secondary amine group of the BPEI polymer is converted to a diaeniumdiolate group and may release nitric oxide.

Experimental Example  1.particle size and Surface charge  Measure

The surface charges of the silica nanoparticles, BPEI coated nanoparticles, and nitric oxide nanoparticles prepared in Examples were measured.

The nanoparticles were dried on a silicon wafer and analyzed for morphology and size by SEM, and the surface charge was measured by zeta potential.

3 is an SEM image of BPEI coated nanoparticles, in which silica nanoparticles having a diameter of about 100 nm were observed.

Figure 4 is a SEM image of the nitric oxide nanoparticles, it can be seen that there is no difference in the shape and size of the silica particles according to the high-pressure reaction of nitrogen monoxide.

In addition, Figure 5 is a graph measuring the charge charge of the nanoparticles. Since SiO ⁻ on the surface of the prepared silica nanoparticles is negatively charged, it is possible to coat positively charged BPEI on the nanoparticles using electrostatic attraction. This can be confirmed that the surface charge of the BPEI coated nanoparticles are positively charged. Subsequently, when the surface charge of the nitric oxide nanoparticles is measured, it is confirmed that the secondary amine is converted to a diazeniumdiolate in a large part and has a negative charge. The diazenium dioleate group is in the zwitter form in the pH 7 environment, but the negative charge is more activated in the pH 8 environment where zeta potential is measured.

Experimental Example  2. Measurement of Nitric Oxide Emissions from Nitric Oxide Nanoparticles

1 mg of the nitric oxide nanoparticles prepared in Example was quantified and dispersed in 0.1 ml of distilled water at pH 8. 0.01 M PBS (0.05 M NaCl) at pH 7.4 was added to the round flask and soaked in a constant temperature water bath to create a living environment at 37 ° C. A bubbler was installed inside the round flask to release argon gas, which serves to deliver nitrogen monoxide, onto the PBS. The other connection of the round flask was connected to a nitrogen monoxide analyzer. When ready in the analyzer, the analysis was started by software, and then nitric oxide nanoparticles dispersed in distilled water were added to the PBS inside the flask using a pipette.

In FIG. 6, the concentration (ppb) of nitrogen monoxide released in real time is measured and graphed at 1 second intervals. In FIG. In addition, in Figure 8 based on the graph of Figures 6 and 7, the total amount of nitrogen monoxide released, half-life, the maximum release and total release time of nitrogen monoxide are shown in a table.

 The total nitrogen monoxide emissions of the nitric oxide delivery nanoparticles were 3.5 umol / mg, which was high compared to previous studies. In addition, it can be said that the efficiency is very high considering the release of nitrogen monoxide only on the surface of the particles. In addition, the half-life of 151 minutes was 3 to 4 times slower than the existing diazenium diolate particles. Since BPEI contains many amine groups, it is expected that primary amines or unreacted secondary amines form hydrogen bonds with diazenium diolate groups, thereby stabilizing the release rate of nitrogen monoxide through stabilization.

Experimental Example  3. Evaluation of Biocompatibility of Nitric Oxide Nanoparticles

Rat myoblast cells were used to evaluate the biocompatibility of the BPEI coated nanoparticles and nitric oxide nanoparticles prepared in the examples.

First, myoblasts were seeded (2000) per well in four 24 well plates and cultured in an incubator for 12 hours. After removing the cell culture medium from each well, 640 μl of fresh culture medium was added, and then various concentrations (0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 5 μg / ml and 10 μg / ml) were added to PBS at pH 7.4. 160 μl of BPEI-coated nanoparticles dispersed in) were treated in the wells. Each concentration was treated with six wells. Nitric oxide nanoparticles were also dispersed in various concentrations in PBS at pH 8 and treated in wells in the same manner. After the well plate was incubated for 24 hours, the culture medium was removed, and 2.5 mg / ml of MTT solution and the culture solution were prepared in a 1: 9 ratio, and 800 μl was added to each well. After incubation for 2 hours, the culture solution was removed, DMSO was added to dissolve the dye, and 100 μl of the solution was transferred to a 96 well plate. The absorbance of the solutions at 540 nm was measured with a plate reader. The higher the absorbance, the higher the cell survival rate. The results obtained by treating the media and nanoparticles were calculated as percentages and compared. After 48 hours, cell viability was analyzed through the same procedure.

The analysis results are shown graphically in FIG. 9.

As shown in FIG. 9, when the nitric oxide nanoparticles were compared with the negative control group and the BPEI-coated nanoparticles at 24 hours and 48 hours, biocompatibility was confirmed because they did not show toxicity at the highest concentration. One unusual feature is that cell viability was slightly lower than the other concentrations when 5 μg / ml nitric oxide particles were treated in both graphs. Since nitric oxide gas is often involved in cell signaling and variously at low concentrations, it is likely that it is a specific nitric oxide concentration that causes other changes besides proliferation in myoblasts, including the possibility of differentiation, rather than causing cell death.

 The same myoblasts were used to assess the biocompatibility of nitric oxide nanoparticles in higher concentration ranges. Silica nanoparticles and BPEI-coated nanoparticles were used as a comparative test group, and the concentrations of the particles were 25, 50, and 100 μg / ml. MTT assay was performed after 24 hours in the same manner and the results are shown graphically in FIG. 10.

As shown in Figure 10, as the concentration of the nitric oxide nanoparticles increases it can be seen that the cell viability is rapidly reduced to 68%, 55%, 38%. This is because high concentrations of nitric oxide can be released rapidly and can be toxic to cells. Therefore, this concentration range is high enough to bring positive results to the cells, and can be applied to anticancer or antibacterial activity.

Since the nitric oxide delivery complex prepared according to the present invention has no cytotoxicity, it is expected to be used for medical purposes such as vasodilation, wound healing, and antibacterial through the regulation of the release amount and release rate of nitric oxide.

Claims (14)

1. materials; And

   It is formed on the substrate, and comprises a polymer thin film containing a diazenium dioleate group,

   The polymer thin film is a composite for delivering nitric oxide formed more than one layer.
2. The method of claim 1,

   The substrate is silica, hydroxyapatite, gold, poly-β-hydroxy butyrate (PHB), polylactic acid (PLA), poly-DL-lactide-co-glyco Lead (poly-DL-lactide-co-glycolide (PLGA)), aliphatic polyesters, polycaprolactone (PCL), polymethyl methacrylate (PMMA), polyethylene glycol ( poly-ethyleneglycol (PEG), poly-vinylalcohol (PVA), poly-alkyl-cyano-carylates (PAC), chitosan, gelatin, Complex for delivery of nitric oxide, which is formed based on one or more selected from the group consisting of micelles, DNA / RNA, phospholipids and liposomes.
3. The method of claim 1,

   The substrate is a composite for nitric oxide delivery having a nanoparticle, microparticle or film shape.
4. The method of claim 1,

   A polymer thin film comprising a diazenium dioleate group is formed of 1 to 500 layers.
5. The method of claim 1,

   A polymer thin film comprising a diazenium dioleate group is formed in a multi-layer by a layer-by-layer (LbL) lamination method.
6. The method of claim 5, wherein

   Layer-by-layer (LbL) lamination allows for electrostatic attraction, van der Waals forces, hydrogen bonds, covalent bonds, ionic bonds, hydrophobic bonds, charge-transfer bonds, and π-interactions. Complex for nitric oxide delivery using (π-interaction), coordination chemical bonds, stereocomplexation, host-guest or biological bonds.
7. The method of claim 1,

   The polymer thin film comprising a diazenium dioleate group further comprises at least one selected from the group consisting of proteins, growth factors, graphene and drugs in the thin film.
8. The method of claim 1,

   Nitric oxide delivery complex further comprises a thin film comprising at least one selected from the group consisting of a second polymer, a protein, a growth factor, graphene and a drug.
9. (A) forming at least one polymer thin film containing a secondary amine group on the substrate; And

   (B) a method for producing a composite for delivering nitric oxide, comprising the step of reacting with nitrogen monoxide at a temperature of 25 to 300 ° C. and a pressure of 1 to 30 atm.
10. The method of claim 9,

    The substrate is a method for producing a composite for delivering nitric oxide having a nanoparticle, microparticle or film shape.
11. The method of claim 9,

    Polymers containing secondary amine groups include branched polyethyleneimine (BPEI), chitosan, gelatin, collagen, fibrinogen, silk fibroin, casein , Elastin, laminin, fibronectin, poly dopamine, poly ethyleneimine, poly-L-lysine, poly (vinylamine) hydro A method for preparing a complex for delivery of nitric oxide, which is at least one selected from the group consisting of chloride (poly (vinylamine) hydrochloride), poly (amino acids) and desaminotyrosyl octyl ester.
12. The method of claim 9,

    If the thin film in step (A) is formed into two or more multilayers through hydrogen bonding,

    The thin film is a method for producing a nitric oxide delivery composite formed by stacking a polymer solution containing a secondary amine group two or more times.
13. The method of claim 9,

    If in step (A) the thin film is formed into two or more multilayers via electrostatic attraction,

    The thin film is a method for producing a nitric oxide delivery composite formed by repeatedly stacking a polymer solution and a negatively charged polymer solution containing a secondary amine group.
14. The method of claim 10,

    After performing step (A), the method for producing a composite for nitric oxide delivery further comprising the step of encapsulating nanoparticles or microparticles formed with a polymer thin film in a hydrogel.

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