1. A polymer comprising a biodegradable polymer having at least one group from a hydroxyl group and a carboxyl group as a main chain, wherein the biodegradable polymer has a functional group combined with a nitric oxide releasing compound including an NO donor and another functional group substituted with a photopolymerizable functional group.

2. The polymer of claim 1, wherein the biodegradable polymer is a natural polymer comprising at least one selected from the group consisting of: hyaluronic acid, gelatin, starch, chitin, cellulose, alginate, collagen, heparin, and chitosan; or a synthetic polymer comprising at least one selected from the group consisting of: such as polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), Polycaprolactone (PCL), Polydioxanone (PDO), polytrimethylene carbonate (PTMC), and Polyhydroxyalkanoate (PHA).

3. The polymer of claim 1, wherein the NO donor comprises at least one selected from the group consisting of: organic nitrites, organic nitrates, nitrosothiols, C-nitroso compounds, N-hydroxynitrosamines, diazacyclobutene dioxides, oxatriazole-5-imines, N-nitrosamines, sydnonimines, oximes, hydroxylamines, N-hydroxyguanidines, hydroxyureas, nitrosamines, N-hydroxynitrosamines, NO-metal complexes and N-diazeniumdiolates (NONONAtes).

4. The polymer of claim 1, wherein the nitric oxide releasing compound comprises a NO donor modified from an amine based compound comprising at least one selected from the group consisting of: n-methylethylenediamine (N-MEDN), N-ethylethylenediamine (N-EEDN), N-isopropylethylenediamine (N-IPED), N-isopropyl-1, 3-propanediamine (N-IPPDN), and N-benzylethylenediamine (N-BEDN).

5. The polymer of claim 1, wherein the nitric oxide-releasing compound comprises at least one selected from the group consisting of: (MEDN) -NONONOate modified by N-MEDN, (EEDN) -NOate modified by N-EEDN, (IPED) -NOate modified by N-IPED, (IPPDN) -NOate modified by N-IPPDN and (BEDN) -NOate modified by N-BEDN.

6. The polymer of claim 1, wherein the photopolymerizable functional groups comprise at least one selected from the group consisting of: methacrylic, ethacrylic, crotonic, cinnamic, vinyl ether, vinyl ester, vinyl arylene, dicyclopentadienyl, norbornenyl, isopentenyl, isopropenyl, allyl, or butenyl; a vinyl arylene ether group, a dicyclopentadienyl ether group, a norbornene ether group, an isopentenyl ether group, an isopropenyl ether group, an allyl ether group, or a butenyl ether group; and a vinyl arylene ester group, a dicyclopentadienyl ester group, a norbornenyl ester group, an isopentenyl ester group, an isopropenyl ester group, an allyl ester group, a butenyl ester group, or a glycidyl methacrylate ester group.

7. The polymer of claim 1, wherein the biodegradable polymer has both hydroxyl and carboxyl groups, wherein the carboxyl groups are bound to the nitric oxide releasing compound and the hydroxyl groups are substituted with the photopolymerizable functional groups.

8. The polymer of claim 1, wherein the biodegradable polymer bears only hydroxyl groups, wherein a portion of the hydroxyl groups are substituted with carboxyl groups and then combined with the nitric oxide releasing compound, and another portion of the hydroxyl groups are substituted with the photopolymerizable functional groups.

9. The polymer according to claim 1, wherein the biodegradable polymer has only carboxyl groups, wherein a part of the carboxyl groups are bound to the nitric oxide releasing compound and another part of the carboxyl groups are substituted with hydroxyl groups and then substituted with the photopolymerizable functional groups.

10. A nanofiber for storing and transferring nitric oxide, wherein the nanofiber is modified by a biodegradable compound.

11. The nanofiber of claim 10, wherein the biodegradable polymer has at least one group from a hydroxyl group and a carboxyl group, wherein the biodegradable polymer has a functional group combined with a nitric oxide releasing compound including an NO donor and another functional group substituted with a photopolymerizable functional group.

12. The nanofiber of claim 10, wherein the biodegradable polymer is a natural polymer comprising at least one selected from the group consisting of: hyaluronic acid, gelatin, starch, chitin, cellulose, alginate, collagen, heparin, and chitosan; or a synthetic polymer comprising at least one selected from the group consisting of: such as polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), Polycaprolactone (PCL), Polydioxanone (PDO), polytrimethylene carbonate (PTMC), and Polyhydroxyalkanoate (PHA).

13. A process for preparing a polymer, the process comprising the steps of:

reacting a biodegradable polymer with a polymer having photopolymerizable functional groups to synthesize an intermediate, wherein at least one functional group of the biodegradable polymer is substituted with the photopolymerizable functional group; and

the intermediate is mixed with a solvent and a nitric oxide releasing compound.

14. The method of claim 13, wherein the method further comprises a step of preparing an intermediate salt after the step of synthesizing the intermediate, the intermediate salt designed to solubilize the intermediate in an organic solvent.

15. The method of claim 13, wherein at least one carboxyl group of the biodegradable polymer is bound to the nitric oxide-releasing compound in the mixing step.

16. A method of making a nanofiber comprising the steps of:

preparing a polymer having a functional group bound to a nitric oxide releasing compound comprising an NO donor and another functional group substituted with a photopolymerizable functional group;

preparing a polymer precursor comprising the polymer, an additive and a photoinitiator; and

electrospinning the polymer precursor.

17. The method of claim 16, wherein the method further comprises the step of photopolymerizing the polymer precursor after the electrospinning step.

18. The method of claim 16, wherein the polymer precursor further comprises a base comprising at least one selected from the group consisting of: ammonium hydroxide (NH)₄OH), sodium methoxide (NaOMe), sodium ethoxide (NaOEt) and sodium propoxide (NaOPr).

Background

Disclosure of Invention

Various embodiments of the present disclosure provide a polymer having a novel structure for nanofibers that are highly biodegradable and capable of controlling the storage amount and release amount of nitric oxide, a method of preparing the same, nanofibers fabricated therefrom, and a method for manufacturing the nanofibers.

The polymer with novel structure according to the present disclosure is based on a biodegradable polymer.

The polymer having a novel structure according to the present disclosure may be a biodegradable polymer having at least one of a hydroxyl group and a carboxyl group, in which one functional group is combined with a nitric oxide releasing compound and the other functional group is substituted with a photopolymerizable functional group.

Nanofibers according to various embodiments of the present disclosure may be nanofibers modified with biodegradable polymers.

In particular, nanofibers according to various embodiments of the present disclosure may be fabricated by electrospinning the polymer having a novel structure.

According to various embodiments of the present disclosure, a nanofiber that is highly biocompatible and capable of controlling the amount of nitric oxide stored and released is provided. In particular, nanofibers according to various embodiments of the present disclosure are capable of controlling the payload of nitric oxide to from 5nmol mg^-1To 5,000 nmol. mg^-1Within a wide range of (a). Furthermore, the nanofibers according to various embodiments of the present disclosure are not cytotoxic, thereby enabling a reduction in potential toxicity when applied in vivo.

Since the nanofibers according to various embodiments can be applied to various sites in the body due to high biodegradability at various concentrations of hyaluronidase (HAse) measured in an actual biological system. In addition, nanofibers according to various embodiments of the present disclosure promote cell migration to promote wound healing, finding applications in various regenerative medicine fields, including burn treatment, kidney transplantation, and the like.

Drawings

The above and other aspects, features and advantages of the present disclosure will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

Fig. 1 shows a schematic diagram of a method of making a polymer, according to various embodiments of the present disclosure (a), along with its proton NMR data (B);

FIG. 2 shows FT-IR spectrum (A) and UV-Vis spectrum (B) of MA-HA- (MEDN) -NONONOATE;

fig. 3 and 4 show schematic diagrams of methods of making nanofibers according to various embodiments of the present disclosure;

fig. 5 shows SEM images of nanofibers in the dry state and confocal microscopy images of nanofibers in the swollen state (a) and a plot of nanofiber diameter (B);

figure 6 illustrates a plot of nitric oxide flux and total amount of nitric oxide as a function of time, according to various embodiments of the present disclosure;

fig. 7 shows the results of testing the biodegradability of a nanofiber for storing or releasing nitric oxide according to an embodiment;

fig. 8 shows a microscopic image (a) for analyzing cytotoxicity of nanofibers for storing and releasing nitric oxide according to an embodiment, and a cell proliferation test result (B) thereof;

fig. 9 shows an optical image (a) of the wound healing effect of the nanofibers for storing and releasing nitric oxide, and the quantitative analysis result (B) of the same;

FIG. 10 shows nanofibers according to NH₄Storage and release profiles of OH molarity;

FIG. 11 shows the storage and release curves of nanofibers according to the molar concentration of NaOMe;

FIG. 12 shows the storage and release curves of nanofibers as a function of molar concentration of NaOEt; and

fig. 13 shows the storage and release curves of nanofibers according to the molar concentration of NaOPr.

Detailed Description

Hereinafter, various embodiments herein will be described. The embodiments and terms used herein are not intended to limit the technology described in this disclosure to particular embodiments, and it should be understood that the embodiments and terms include modifications, equivalents, and/or alternatives to the respective embodiments described herein.

Polymer having novel structure and method for producing same

A polymer according to various embodiments of the present disclosure is based on a biodegradable polymer having at least one of a hydroxyl group and a carboxyl group.

The biodegradable polymer may be a natural polymer such as hyaluronic acid, gelatin, starch, chitin, cellulose, alginate, collagen, heparin or chitosan, or a synthetic polymer such as polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), Polycaprolactone (PCL), Polydioxanone (PDO), polytrimethylene carbonate (PTMC) or Polyhydroxyalkanoate (PHA).

Specifically, the polymers according to various embodiments of the present disclosure carry at least one of hydroxyl and carboxyl groups, wherein one functional group is combined with a nitric oxide releasing compound and the other functional group is substituted with a photopolymerizable functional group.

For example, when the biodegradable polymer carries both a hydroxyl group and a carboxyl group, the carboxyl group may be combined with a nitric oxide releasing compound, and the hydroxyl group may be substituted with a photopolymerizable functional group. The biodegradable polymer having both hydroxyl and carboxyl groups may be, for example, hyaluronic acid. Hyaluronic acid has the following chemical formula.

Hyaluronic acid is biocompatible, hydrophobic and biodegradable and is involved in the cellular processes of proliferation, inflammation and wound healing. In addition, hyaluronic acid is rich in hydroxyl groups and carboxyl groups, and thus can be easily modified with various functional groups.

In an alternative, when the biodegradable polymer carries only hydroxyl groups, a part of the hydroxyl groups may be substituted with carboxyl groups and then combined with a nitric oxide releasing compound, and another part of the hydroxyl groups may be substituted with photopolymerizable functional groups. Examples of biodegradable polymers with only hydroxyl groups include starch, chitin and chitosan.

Starch has the following chemical formula.

Chitin has the following chemical formula.

Chitosan has the following chemical formula.

In another alternative, when the biodegradable polymer carries only carboxyl groups, a part of the carboxyl groups may be bound to the nitric oxide releasing compound, and another part of the carboxyl groups may be substituted with hydroxyl groups and then with photopolymerizable functional groups. Examples of biodegradable polymers with only carboxyl groups include gelatin, alginate, heparin, polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), polytrimethylene carbonate (PTMC), Polydioxanone (PDO), and Polyhydroxyalkanoate (PHA).

Gelatin has the following chemical formula.

Alginates have the following chemical formula.

Heparin has the following chemical formula.

PLA has the following chemical formula.

PGA has the following chemical formula.

PLGA has the following chemical formula.

PTMC has the following chemical formula.

PDO has the following chemical formula.

PHA has the following chemical formula.

Meanwhile, the photopolymerization functional group may include at least one selected from the group consisting of: methacrylic, ethacrylic, crotonic, cinnamic, vinyl ether, vinyl ester, vinyl arylene, dicyclopentadienyl, norbornenyl, isopentenyl, isopropenyl, allyl, or butenyl; a vinyl arylene ether group, a dicyclopentadienyl ether group, a norbornene ether group, an isopentenyl ether group, an isopropenyl ether group, an allyl ether group, or a butenyl ether group; and a vinyl arylene ester group, a dicyclopentadienyl ester group, a norbornenyl ester group, an isopentenyl ester group, an isopropenyl ester group, an allyl ester group, a butenyl ester group, or a glycidyl methacrylate ester group. In other words, the photopolymerizable functional group contains an unsaturated double bond.

The nitric oxide releasing compound may be a substance capable of storing and releasing nitric oxide under specific conditions. For example, the nitric oxide releasing compound may be a compound comprising a NO donor.

In this regard, the NO donor may include at least one item selected from: organic nitrites, organic nitrates, nitrosothiols, C-nitroso compounds, N-hydroxynitrosamines, diazacyclobutene dioxides, oxatriazole-5-imines, N-nitrosamines, sydnonimines, oximes, hydroxylamines, N-hydroxyguanidines, hydroxyureas, nitrosamines, N-hydroxynitrosamines, NO-metal complexes and N-diazeniumdiolates (NONONAtes).

By way of example, a representative NO donor may have the following formula.

When the NO donor NONOate is included, the nitric oxide releasing compound is capable of releasing nitric oxide by the following process. I.e. it decomposes under aqueous conditions to release nitric oxide.

Meanwhile, the amine-based compound may include at least one selected from the group consisting of: n-methylethylenediamine (N-MEDN), N-ethylethylenediamine (N-EEDN), N-isopropylethylenediamine (N-IPED), N-isopropyl-1, 3-propanediamine (N-IPPDN), and N-benzylethylenediamine (N-BEDN).

According to embodiments of the present disclosure, the nitric oxide releasing compound may be modified from amine based compounds and include N-diazeniumdiolate (NONOate) in the NO donor. For example, the nitric oxide releasing compound may comprise at least one selected from the group consisting of: (MEDN) -NONONOate modified by N-MEDN, (EEDN) -NOate modified by N-EEDN, (IPED) -NOate modified by N-IPED, (IPPDN) -NOate modified by N-IPPDN and (BEDN) -NOate modified by N-BEDN.

Preferably, the nitric oxide releasing compound may be (MEDN) -NONOate modified with N-methyl ethylenediamine (N-MEDN). In this regard, (MEDN) -NONOate may have the following chemical formula.

The polymer according to various embodiments of the present disclosure, which is a polymer having hyaluronic acid as a main chain, may have the following chemical formula:

wherein n is 1 or greater.

Referring to the chemical formula, hydroxyl groups in hyaluronic acid are substituted with methacrylate groups, and carboxyl groups are bound to (MEDN) -NONOate.

Hereinafter, the preparation method of the polymer according to various embodiments will be described. The preparation method of the polymer can comprise the following steps: synthesizing an intermediate; generating intermediate salt; and mixing the intermediate with a solvent and a nitric oxide releasing compound.

In the step of synthesizing the intermediate, the biodegradable polymer is reacted with the polymer having the photopolymerization functional group to substitute the photopolymerization functional group for at least one of the functional groups of the biodegradable polymer.

For example, when the biodegradable polymer carries both a hydroxyl group and a carboxyl group, the hydroxyl group may be substituted with a photopolymerizable functional group.

In an alternative, when the biodegradable polymer has only hydroxyl groups, a part of the hydroxyl groups may be substituted with photopolymerizable functional groups.

In another alternative, when the biodegradable polymer has only carboxyl groups, a part of the carboxyl groups may be substituted with photopolymerizable functional groups.

According to one embodiment, hyaluronic acid, which is a biodegradable polymer, is reacted with a polymer having a photopolymerizable functional group such that the photopolymerizable functional group replaces at least one hydroxyl group of hyaluronic acid. For example, referring to fig. 1(a), hyaluronic acid is reacted with methacrylic anhydride to replace hydroxyl groups of hyaluronic acid with photopolymerizable methacrylic acid groups to synthesize methacrylic acid hyaluronic acid (MA-HA). That is, hydroxyl groups of hyaluronic acid are substituted with methacrylic groups by transesterification to provide hyaluronic acid methacrylate (MA-HA). The reaction may be carried out while maintaining the pH at 8 to 11. Further, after completion of the reaction, purification was performed by precipitation, centrifugation and dialysis.

After the step of synthesizing the intermediate, a step of producing an intermediate salt may be performed. This is a pre-treatment step to solubilize the intermediate in an organic solvent. For example, referring to FIG. 1(A), the intermediate MA-HA can be converted to a Tetrabutylammonium (TBA) salt (MA-HA-TBA) using an ion exchange resin. This pre-treatment step is necessary because the final polymer synthesis should be carried out in an organic solvent due to the high compatibility of the nitric oxide releasing compound to aqueous solutions.

In the mixing step, the intermediate salt is mixed with a solvent and a nitric oxide releasing compound.

In this connection, when the biodegradable polymer carries both hydroxyl and carboxyl groups, the carboxyl groups may be combined with the nitric oxide releasing compound.

Alternatively, when the biodegradable polymer carries only hydroxyl groups, the hydroxyl groups, which have not been substituted in the previous synthetic steps, can be converted beforehand into carboxyl groups, which are then combined with the nitric oxide releasing compound.

Further alternatively, when the biodegradable polymer carries only carboxyl groups, carboxyl groups which have not been substituted in previous synthetic steps may be combined with the nitric oxide releasing compound.

In a particular embodiment, the nitric oxide-releasing compound may be MEDN-NONONOate. The molar ratio of intermediate salt (MA-HA-TBA) and nitric oxide releasing compound may be 1:0.5 to 1: 30. In particular, the molar ratio of intermediate salt (MA-HA-TBA) and nitric oxide releasing compound may be 1:2, 1:5 or 1: 7. When made from polymers in such molar ratios, the nanofibers used for storing and releasing nitric oxide are capable of storing and releasing nitric oxide in various concentration ranges, optimizing the release time.

Referring to fig. 1(a), when the biodegradable polymer is hyaluronic acid, the carboxyl group of hyaluronic acid may be combined with the nitric oxide releasing compound through a mixing step.

The molar ratio of the biodegradable polymer and the nitric oxide releasing compound may be variously adjusted depending on the application of the polymer of the present disclosure. For example, the degradation rate of the nanofibers can be controlled by adjusting the content of the biodegradable polymer. On the other hand, the amount of nitric oxide released can be controlled by adjusting the content of the nitric oxide releasing compound. The amount of nitric oxide released may vary depending on the content of the nitric oxide releasing compound. Because of the ease of controlling these molar ratios, the polymers of the present disclosure can find a variety of applications depending on their use.

Nano-fiber for storing and transferring nitric oxide and manufacturing method thereof

Nanofibers according to various embodiments of the present disclosure may be nanofibers modified with biodegradable polymers. Specifically, the polymer having the novel structure as described above may be electrospun into nanofibers.

Hereinafter, methods for making nanofibers according to various embodiments of the present disclosure are described.

A method for making nanofibers may include the steps of: preparing the polymer with the novel structure; preparing a polymer precursor comprising the polymer, an additive and a photoinitiator; and electrospinning the polymer precursor.

The polymer precursor can be a mixture of a polymer of the present disclosure, poly (ethylene oxide) (PEO), 4-arm poly (ethylene glycol) thiol (4-arm PEG-SH), a photoinitiator, and a base. In this polymer precursor mixture, the polymer may be contained at a concentration of 1 to 50% (w/v). When made of such a concentration of polymer, the nanofibres for storing and releasing nitric oxide are able to cover the storing and releasing of nitric oxide of various concentrations and for this reason the release time is optimized.

PEO can be included in an amount of 0.1% (w/v) to 10% (w/v), based on the total weight of the polymer precursor mixture. The molecular weight of the PEO can be from 1,000 to 1,000,000 g/mol.

The 4-arm PEG-SH may be included in an amount of 0.1% (w/v) to 10% (w/v) based on the total weight of the polymer precursor mixture. The molecular weight of the 4-arm PEG-SH may be in the range of 1,000 to 1,000,000 g/mol. One or both of linear PEG-SH and 6-arm PEG-SH may be used in place of 4-arm PEG-SH.

The photoinitiator may be Irgacure 2959. Irgacure 2959 can be used at a concentration of 0.01% (w/v) to 10% (w/v).

The base may comprise at least one item selected from: ammonium hydroxide (NH)₄OH), sodium methoxide (NaOMe), sodium ethoxide (NaOEt), sodium propoxide (NaOPr). The nanofibers can alter the nitric oxide storage and release profile depending on the type and molarity of the base added. In other words, the kind and molar concentration of the base added to the polymer precursor may be adjusted in order to store and release nitric oxide in a desired concentration range.

Referring to fig. 3 and 4, the polymer precursor may be electrospun. Electrospinning is a complex process that can be influenced by various parameters including polymer concentration, surface tension, conductivity, solvent, applied voltage, flow rate, needle gauge, etc.

The process may add a photo-polymerization step before or after the electrospinning step. The photopolymerization step may stabilize the structure of the nanofibers.

Hereinafter, the present disclosure will be described in detail with reference to examples and experimental examples. However, the following examples and experimental examples are illustrative and should not be construed as limiting the present disclosure.

Examples of the invention

Step 1: synthesis of hyaluronic acid methacrylate (MA-HA)

500mg of Hyaluronic Acid (HA) (40kDa) was dissolved in 50mL of filtered deionized water (DIW) to make a 1% (w/v) HA solution. A5-fold molar excess (0.931mL) of methacrylic anhydride was added to the solution and reacted in the dark at 4 ℃ for 12 hours while maintaining the pH between 8 and 11 using 5N or 1N NaOH. The final product (HA-MA) was precipitated in a 10-fold excess of cold ethanol (EtOH). After centrifugation at 5,000rpm for 5 minutes at 4 ℃, the supernatant was removed and the pellet was redissolved in 50mL DIW. To remove unreacted reagents, MA-HA was purified by dialysis of DIW for 3 days using a dialysis membrane (cut-off concentration of 3.5kDa Mw).

Step 2: synthesis of MA-HA-TBA

15g (75mmol) of Dowex 50WX-8-400 ion exchange resin was suspended with 250mL of DIW in a 500mL round flask. Then, 29.335mL of TBA-OH (112.5mmol, 1.5 molar excess) was added to Dowex resin in a round flask, followed by reaction for 30 minutes. Dowex-TBA resin was filtered using filter paper and vacuum pump to remove impurities. For pH normalization, the resin was washed with sufficient DIW. Subsequently, 500mg of purified MA-HA in 100mL DIW was transferred to a 250mL round flask and the prepared Dowex-TBA resin (6.25g, 5 molar excess) was poured into the MA-HA solution. After 3 hours of mixing, the resulting product (MA-HA-TBA) was first filtered through filter paper and then filtered through a 0.45 μm filter to remove Dowex resin. Thereafter, the product was lyophilized for 3 days and stored at-20 ℃ until further use.

And step 3: synthesis of (MEDN) -NONONOATE

0.4593mL (5.0mmol) of N-MEDN and 0.9259mL (5.0mmol) of NaOMe were dissolved in 3.6148mL of EtOH to make the total volume 5 mL. The resulting solution was placed in a closed chamber and exposed to 10atm of NO gas for 3 days. After purging the chamber with Ar, the solution was drawn out of the chamber, vacuum sealed, and then stored in a refrigerator.

And 4, step 4: synthesis of MA-HA- (MEDN) -NONONONAte

0.2g (0.5mmol dimer, 1 equiv.) of MA-HA-TBA was dissolved in 20mL DMSO. To this solution, for example, 0.221mL of EDC (MW 155.24g · mol) is added in sequence^-11.25mmol, 2.5 equivalents), NHS 0.1438g (MW 115.09g · mol)^-11.25mmol, 2.5 equivalents) and (MEDN) -NONONAte 3.5mL (3.5mmol, 7 equivalents) and mixed at 25 ℃ for 3 hours. The amount of each reactant added to MA-HA-TBA may be varied to effect modification of the carboxyl groups of hyaluronic acid to NONOate groups. Finally, the reaction is carried outThe product was precipitated in excess diethyl ether and washed thoroughly with diethyl ether. MA-HA- (MEDN) -NONONONAte was dried under cold vacuum for 3 hours to evaporate the organic residue, which was then stored in a sealed container at-20 ℃ until use.

And 5: synthesis of nanofibers

To synthesize nanofibers, a polymer precursor for electrospinning was prepared as follows. 2% (w/v) of PEO as a polymer blend and 2% (w/v) of 4-arm PEG-SH as a crosslinking agent are dissolved in DIW/NH in sequence₄OH (1: 1 volume ratio). To the polymer solution obtained here, 0.1% (w/v) of Irgacure 2959 was added as a photoinitiator, the pH of the polymer solution was adjusted to 11 using 1N HCl, and 4, 7 or 10% (w/v) of MA-HA- (MEDN) -NONOate was added to the polymer precursor solution. Then, the mixed solution prepared above was loaded into a plastic syringe and flowed through a 25-gauge needle, and nanofibers were fabricated using a high voltage power supply (ESR200PR2D, nanc co., seoul, korea) under the following conditions: the volume flow rate is 20 mu L min^-1The applied voltage was 17.5kV and the tip-to-collector distance was 15 cm. After electrospinning, the electrospun nanofibers were photo-crosslinked for 5 minutes by using a UV LED lamp, vacuum sealed, and then stored in a refrigerator at-20 ℃.

Experimental example 1: characterization of MA-HA- (MEDN) -NONONONAte

Referring to fig. 1(a), hydroxyl groups of hyaluronic acid are substituted with methacrylic groups through step 1 to provide hyaluronic acid methacrylate (MA-HA). Referring to FIG. 1(B), methacrylate proton NMR peaks at 5.6 and 6.1ppm were detected. In addition, a methyl proton NMR peak of N-acetyl group in HA was detected at 1.9 ppm.

In step 2, MA-HA is converted into tetrabutylammonium salt (MA-HA-TBA) using ion exchange resin to be dissolved in an organic solvent (e.g., DMSO). Since the NONOate group is easily decomposed in aqueous solution, the synthesis of MA-HA- (MEDN) -NONOate should be performed under organic solvent conditions to minimize the decomposition of NONOate. Referring to fig. 1(a) and 1(B), the TBA binding process did not affect the modification of the methacrylate groups, as indicated by the methacrylate proton peaks before and after the reaction, indicating a reliable synthesis of MA-HA-TBA. In addition, TBA proton NMR peaks appear at 3.0ppm and 1.5 to 0.7 ppm.

As shown in FIG. 1(B), MA-HA-MEDN-NONONOATE was synthesized by coupling MA-HA-TBA with preformed MEDN-NONONONAte.

In FIG. 2(A), a characteristic peak of NONONOate was observed in the FR-IR spectrum. As shown in fig. 2(B), the formation of NONOate group was confirmed by UV-Vis spectroscopy. MA-HA- (MEDN) -NONONAte showed maximum absorption at 260nm, while MA-HA- (MEDN) did not show any specific absorption peak at 260 nm. Thus, the data demonstrate the successful synthesis of MA-HA- (MEDN) -NONONONAte.

Experimental example 2: characterization of the nanofibers

Fig. 5(a) shows SEM images of nanofibers in the dry state and confocal microscopy images of nanofibers in the swollen state. From the images, the fiber morphology, diameter and swelling behavior of the nanofibers were studied. When the content of MA-HA- (MEDN) -NOnoate in 2% (w/v) of PEO-blended precursor polymer was increased from 4% (w/v) to 10% (w/v), uniform nanofibers having various diameters depending on the concentration were successfully synthesized. In FIG. 5(B), the average diameters of the nanofibers were 240. + -.30 nm, 330. + -.50 nm and 490. + -.60 nm in the case where the MA-HA- (MEDN) -NONONONAte contents were 4% (w/v), 7% (w/v) and 10% (w/v), respectively. Thus, it HAs been found that the diameter of nanofibers produced by electrospinning increases with increasing concentration of MA-HA- (MEDN) -NONONONAte. The fiber diameter of the fluorescently labeled hydrated nanofibers was also measured by confocal microscopy. The distribution of fiber diameters became broader, and the average diameters of 4% (w/v), 7% (w/v) and 10% (w/v) MA-HA- (MEDN) -NONONONAte were increased to 670. + -. 160nm, 830. + -. 250nm and 1320. + -. 320nm, respectively. Since HA HAs water absorbing properties, the higher the content of HA in the nanofibers, the larger the diameter of the nanofibers.

Experimental example 3: nitric oxide storage and release profiles

The storage and release profile of nitric oxide from nanofibers was evaluated based on the molar ratio between MA-HA and (MEDN) -NONOate and the content of MA-HA- (MEDN) -NONOate in the electrospun polymer precursor (w/v). For example, when the molar ratio between MA-HA and (MEDN) -NONOate is 1:7 and 10% (w/v) of MA-HA- (MEDN) -NONOate, 10% (w/v) of MA-HA (MEDN) -NONOate is 1: 7.

Evaluation of t [ NO ]](total moles of NO released), t_1/2(halving time of NO Release) [ NO]_m(maximum instantaneous concentration of NO Release), t_m(to [ NO ]]_mRequired time) and t_d(duration of NO until NO release is complete). The results are summarized in table 1 below.

Fig. 6(a) and 6(B) show the released amount and total released amount of nitric oxide over time for representative examples.

TABLE 1

Referring to Table 1 and FIG. 6, the nitric oxide payload of the nanofibers is significantly affected by the total MA-HA- (MEDN) -NONONONAte concentration in the precursor polymer and the ratio of MA-HA to (MEDN) -NONONONAte in the synthesized MA-HA- (MEDN) -NONONONONAte.

Specifically, t [ NO ] at a fixed MA-HA- (MEDN) -NONONONAte concentration of 7% (w/v)]And [ NO]_mThe trend is that MA-HA (MEDN) -NONONONAte is 1:2<1:5<1:7. I.e., t [ NO ]]From 20 nmol. mg^-1Increased to 580 nmol. mg^-1，[NO]_mFrom 190ppb mg^-1Increased to 7,230ppb mg^-1。

Furthermore, increasing the concentration of MA-HA- (MEDN) -NONOate in the precursor solution from 4 to 10% (w/v) under conditions of immobilized MA-HA (MEDN) -NONOate ═ 1:7 resulted in t [ NO ],]and [ NO]_mAll increase significantly. Specifically, t [ NO ]]From 350 nmol. mg^-1Increased to 620 nmol. mg^-1，[NO]_mFrom 4,460ppb mg^-1Increased to 8,920ppb mg^-1。

Thus, if nitric oxide-releasing nanofibers are fabricated using high concentrations of MA-HA- (MEDN) -NONOate (i.e., 10% (w/v)) or high molar ratios of HA backbone polymer NONOate groups (i.e., MA-HA (MEDN) -NONOate ═ 1:7), higher concentrations of NONOate groups are included in the fibers, resulting in greater amounts of NONOate decomposition and greater NO release and extended release times.

In various embodiments of the present disclosure, the payload of nitric oxide can be controlled at 5nmol mg by adjusting the molar ratio between MA-HA and (MEDN) -NONONONOATE and the content% (w/v) of MA-HA- (MEDN) -NONONONONAte in the polymer precursor^-1To 5,000 nmol. mg^-1Within the range of (1).

Experimental example 4: biodegradability test

For in vivo applications, nanofibers according to various embodiments of the present disclosure must be biodegradable. For in vivo applications, the implanted material (suture, gauze or bandage type) should be biodegradable so that no secondary surgery is required to remove the implant. To evaluate the biodegradability of nanofibers against hyaluronidase (HAse), nanofibers were placed in PBS or HAse solution (10-1000 U.mL)^-1) And the percent weight loss was monitored. Referring to fig. 7, the degradation rate increases with increasing HAse concentration. In particular, even at an enzyme concentration of 100 U.mL similar to that of the actual biological system^-1Next, excellent biodegradability was also detected, indicating the in vivo applicability of the nanofibers of the present invention.

In addition, the measured biodegradability increases with increasing% (w/v) of MA-HA- (MEDN) -NONONONAte, i.e., with increasing HA content in the nanofibers.

Experimental example 5: cytotoxicity assays

In vivo cytotoxicity assays were performed using nanofibers prepared from 7% (w/v) MA-HA- (MEDN) -NONOate (MA-HA (MEDN) -NONOate ═ 1:2, 1:5, 1: 7). As shown in table 1, the average nitric oxide payload based on nanofibers of 7% (w/v) MA-HA (med) -NONOate ═ 1:2, 1:5, and 1:7 were 20, 140, and 580nmol mg, respectively^-1. In FIG. 8, the nanofibers are represented by [ NO ] respectively]₂₀NF、[NO]₁₄₀NF and [ NO]₅₈₀NF (carbon fiber). For example, [ NO ]]₂₀NF representsThe amount of nitric oxide released per 1mg of nanofibres was 20 nmol.

Due to their important role in wound healing, nitric oxide-releasing nanofibers were evaluated for cytotoxicity for promising therapeutic applications of NIH/3T3 fibroblasts as a model cell line. To evaluate the toxicity of the nanofibers themselves, nanofibers comprising MA-HA- (MEDN) were prepared as positive controls. As another positive control (blank), a cell monolayer without any nanofiber contact was also prepared.

As shown in the upper graph of FIG. 8(A), on the first day, a blank, a control, [ NO ] were determined]₂₀NF、[NO]₁₄₀NF and [ NO]₅₈₀The cell viability of NF was 98.1, 98.7, 98.8, 98.2 and 98.8%, respectively. In the lower panel of fig. 8(a), when fibroblasts were cultured with nitric oxide-releasing nanofibers for 3 days, live/dead images showed faster proliferation with more spindle shape than the control and blank groups. By chemically binding HA to methacrylic acid and secondary amines containing NONOate groups, NONOate and crosslinking chemicals are not only simply physically trapped in the nanofiber network, but are actually chemically immobilized in the network. Meanwhile, in a physiological environment, NONONOate decomposes and releases nitric oxide from the nanofibers, while other chemical substances are not easily separated from the nanofibers. Thus, the nitric oxide-releasing nanofibers and the chemical species forming the nanofibers are non-toxic to cells, thereby reducing potential toxicity issues for any in vivo application.

Experimental example 6: cell proliferation assay

Nitric oxide is known to be involved in wound healing. In this experimental example, cell proliferation experiments were performed under the assumption that nitric oxide-releasing nanofibers are effective for proliferation of fibroblasts. Briefly, the quantitative effect of nanofibers on fibroblast proliferation was investigated by the WST-8 assay. As shown in FIG. 8(B) [ NO]₂₀NF、[NO]₁₄₀NF and [ NO]₅₈₀The 3-day fibroblast proliferation of NF increased 5.3%, 15.2% and 18.5% respectively compared to the blank. In contrast, the control group had negligible effect on cell proliferationSlightly disregarded. These results indicate that nitric oxide released from the nanofibers can effectively provide a suitable environment for fibroblast proliferation.

Experimental example 7: cell motility assay

To investigate the therapeutic potential of nanofibers according to various embodiments of the present disclosure, an in vitro scratch test was performed. Referring to the top row of images in fig. 9(a), a single layer of fibroblasts was scraped to establish an in vitro wound healing model (single line wound site). Scraped fibroblasts were cultured for 12 hours in the presence or absence of nitric oxide-releasing nanofibers. After 12h incubation with [ NO ]]₂₀NF、[NO]₁₄₀NF and [ NO]₅₈₀The relatively scarred areas of NF-treated fibroblasts repopulate 34%, 33%, and 49%, respectively. In contrast, as shown in fig. 9(B), fibroblasts untreated (blank) or nanofiber only (control) were repopulated by only 22% and 29%. After 36h of culture, [ NO ] was applied against the original wound as shown in FIGS. 9(B) and 9(C)]₂₀NF、[NO]₁₄₀NF and [ NO]₅₈₀NF-treated fibroblasts showed significantly improved wound closure of 52, 65 and 72%. Fibroblasts untreated and nanofiber-only treatment showed lower wound closure effect, 33% and 40%, respectively. In addition, allowing a uniform monolayer of cells to be formed within 60 hours, all nitric oxide-releasing nanofibers promoted proliferation of fibroblasts. Thus, nanofibers according to various embodiments of the present disclosure may be found to drive cell movement, thereby improving wound healing. Thus, the data indicate that nanofibers have therapeutic potential in wound healing applications. In other words, various embodiments of the present disclosure find application in various areas of regenerative medicine, including burn treatment, kidney transplantation, and the like.

Experimental example 8: the nitric oxide release profile depends on the type and amount of base added to the polymer precursor

In step 5 of synthesizing nanofibers, a polymer precursor for electrospinning was prepared as follows, and nitric oxide storage and release profiles were determined.

(1) According to NH₄Determination of the molar concentration of OH the nitric oxide storage and release curves

In the preparation of the polymer precursor, NH was added at various molar concentrations₄OH is used as the base. Subsequently, the nitric oxide release profile of the electrospun nanofibers was determined.

The results are given in table 2 below and fig. 10. From the data, it follows NH₄The reduction of the OH molar concentration, the total NO release and the maximum NO flux increase.

TABLE 2

t [ NO ]: total amount of NO released

t_1/2: half time of NO

[NO]_m: maximum flux of NO

t_m: time to maximum flux

t_d: duration of NO

(2) Determination of nitric oxide storage and Release profiles based on NaOMe molarity

NaOMe was added as a base at various molar concentrations in the preparation of the polymer precursor. Subsequently, the nitric oxide release profile of the electrospun nanofibers was determined.

As can be seen from the data in Table 3 and FIG. 11, the total NO release amount peaked at 1.30. mu. mol. mg at a NaOMe molar concentration of 1.35M^-1Binh₄OH (7.87M) was about twice as high, indicating that the nitric oxide storage capacity of nanofibers from NaOMe-added polymer precursors was very high. Furthermore, it was observed that the total amount of NO released increased two-fold or more as the NaOMe molarity increased from 0.68M to 1.35M, but decreased as the NaOMe molarity increased to 2.7M and 5.4M. Meanwhile, when NaOMe was used at a molar concentration of 2.7M, the duration of nitric oxide release was 63.2hr, which was the longest. This molar concentration is believed to be useful for long duration of NO release.

TABLE 3

(3) Determination of nitric oxide storage and Release profiles based on molar concentration of NaOEt

In the preparation of the polymer precursor, NaOEt was added as a base at various molar concentrations. Subsequently, the nitric oxide release profile of the electrospun nanofibers was determined.

As can be seen from the data of Table 4 and FIG. 12, when NaOEt was used at a molar concentration of 0.67M, the peak of total NO release was 0.92. mu. mol. mg^-1. The total NO release was observed to increase as the NaOEt molarity increased from 0.34M to 0.67M, but decreased as the NaOEt molarity increased to 1.34M and 2.68M.

TABLE 4

(4) Determination of nitric oxide storage and Release profiles based on the molar concentration of NaOPr

In the preparation of the polymer precursor, NaOPr was added as a base at various molar concentrations. Subsequently, the nitric oxide release profile of the electrospun nanofibers was determined.

As can be seen from the data in Table 5 and FIG. 13, when NaOPr was used at a molar concentration of 0.53M, the peak of total nitric oxide release was 1.21. mu. mol. mg^-1. The total NO release was observed to increase two-fold or more as the NaOPr molarity increased from 0.28M to 0.53M, but decreased as the NaOPr molarity increased to 1.06M and 2.11M.

TABLE 5

The data obtained in experimental example 8 show that the base contained in the polymer precursor can be applied depending on the amount and duration of nitric oxide at the target concentration.

The features, structures, effects, and the like described in the above embodiments include at least one embodiment of the present invention, but the present invention is not limited to only one embodiment. Further, those skilled in the art may combine or modify the features, structures, effects, and the like shown in each embodiment into other embodiments. Therefore, contents related to the combination or modification should be construed to be included in the scope of the present disclosure.

Further, while the present disclosure has been described in detail with reference to exemplary embodiments, the present disclosure is not limited thereto. It will be appreciated by persons skilled in the art that various modifications and applications not shown above may be made without departing from the spirit and scope of the disclosure. For example, each component shown in the embodiments may be modified and manufactured. It is to be understood that differences relating to such modifications and applications are included in the scope of the present invention as defined in the appended claims.