Preparation method of hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-PNIPAM)

Abstract

The present invention provides a method for preparing hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM), comprising: a) preparing a mixture comprising a hyaluronic acid compound, poly(N-isopropylacrylamide), and a carbodiimide crosslinker in a water-miscible solvent; b) adjusting the pH to a range of 7.2 to 7.8 and reacting the mixture by stirring at 20 to 40° C. for 1 to 3 days; c) drying the mixture of step b), The molar ratio of hyaluronic acid, poly(N-isopropylacrylamide), and carbodiimide crosslinker ranges from 0.1:0.0025:1 to 0.2:0.0125:1.

Description

[Technical Field]

[0001] The present invention relates to the field of chemistry, and in particular to a method for preparing hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-G-PNIPAM).

[0002] Hyaluronic acid, or HA, is a naturally occurring glycosaminoglycan (GAG) consisting of a disaccharide subunit formed by the chemical combination of D-glucuronic acid and N-acetyl-D-glucosamine. These disaccharide subunits are linked via 2,4-glycosidic bonds to form long, large structures. Approximately 15 g of HA is naturally present in the skin, joints, eyes, and other tissues of a 70 kg human body. Today, HA can be naturally produced, for example, from rooster combs and from the fermentation of Staphylococcus aureus, with molecular weights ranging from 5,000 to 1,800,000 kDa.

[0003] Hyaluronic acid has excellent properties, especially its water-retaining capacity; one gram of HA has been shown to be able to hold 6 liters of water. Furthermore, HA is hygroscopic, has the appropriate viscosity to form gels, is biodegradable, and is biocompatible. Currently, HA is widely used in biomaterials and cosmetics, such as gels, creams, foams, injectables, or hydrogels for anti-inflammatory, wound healing, and tissue regeneration.

[0004] Poly(N-isopropylacrylamide), or pNIPAM, is a thermoresponsive polymer obtained from the polymerization reaction of N-isopropylacrylamide monomers. pNIPAM's lower critical solution temperature (LCST), or the temperature at which the polymer undergoes a phase transition from a flexible, solvated state to a more rigid, unsolvated state, is 32°C. At temperatures below 32°C, pNIPAM is hydrophilic and soluble. However, at temperatures above 32°C (such as body temperature), pNIPAM is hydrophobic and insoluble, and can form hydrogels with tissue-adhesive properties.

[0005] Nanogels are a type of colloid, a three-dimensional, nanosized, hydrogel-like material in the nanoscale range. Their main advantages are their water retention, ability to form emulsions that allow systemic administration to hard-to-reach organs, tissues, and cells, and rapid pharmacokinetics. Therefore, nanogels have recently attracted attention in the fields of drug delivery, diagnostics, imaging, glucose sensing, and tissue engineering.

[0006] From a literature survey, hyaluronic acid grafted poly(N-isopropylacrylamide) has been disclosed in the following documents:

[0007] US Patent Publication No. 20130171197A1 discloses a hyaluronic acid-grafted poly(N-isopropylacrylamide) hydrogel with a specific honeycomb structure, which can be thermoresponsively formed and used to enhance the efficiency of tissue engineering.

[0008] Chen et al. (Journal of Pharmaceutical Sciences, 2011, 100(2), 655-666.) developed hyaluronic acid-grafted poly(N-isopropylacrylamide) hydrogels for the drug delivery of the cancer drug cisplatin. This hydrogel has a grafting degree of over 40%, forming a network-like hydrogel.

[0009] Amenda (Wagner, A. 2019) reported the preparation of hyaluronic acid-grafted poly(N-isopropylacrylamide) nanogels using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as carbodiimide crosslinkers. These nanogels have a grafting degree of approximately 25% and are useful for hydrophobic drug delivery systems.

[0010] However, previously disclosed hyaluronic acid-grafted poly(N-isopropylacrylamide) has a high degree of grafting, resulting in high viscosity and the formation of a network-like hydrogel. Therefore, the objective of the present invention is to synthesize hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) with a low degree of grafting, which provides low viscosity while maintaining high stability and biocompatibility. The HA-g-pNIPAM of the present invention can be used in many applications, including delivery systems, wound healing, cell proliferation, anti-inflammation, antioxidants, and photoprotection. Summary of the Invention

[0011] In one embodiment, the present invention relates to a method for preparing hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM), said method comprising: a) preparing a mixture comprising a hyaluronic acid compound, poly(N-isopropylacrylamide), and a carbodiimide crosslinker in a water-miscible solvent; b) adjusting the pH to a range of 7.2 to 7.8 and stirring the mixture at 20 to 40°C for 1 to 3 days to react; c) drying the mixture of step b), The molar ratio of hyaluronic acid, poly(N-isopropylacrylamide), and carbodiimide crosslinker ranges from 0.1:0.0025:1 to 0.2:0.0125:1.

[0012] Further provided is hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) prepared by the above method. [Brief explanation of the drawings]

[0013] [Figure 1] Nuclear magnetic resonance (NMR) spectra of (a) sample 1, (b) sample 2, (c) sample 3, (d) sample 4, and (e) sample 5 are shown. [Figure 2]The chemical structure of hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) is shown. [Figure 3] Figure 1 shows the infrared (IR) spectrum of Sample 2. The grafting of HA with pNIPAM is shown as characteristic peaks at wavenumbers of 1606.4, 1406.3, and 1373.2 cm-1. [Figure 4] 1 shows the thermogravimetric analysis (TGA) spectrum of sample 3. [Figure 5] 1 shows the thermal response of Sample 3. [Figure 6] Transmission electron microscope (TEM) images of (a) 0.25% by mass of sample 1, (b) 0.25% by mass of sample 2, (c) 0.25% by mass of sample 4, and (d) 0.25% by mass of sample 5 are shown. [Figure 7] 1 shows the cell proliferation efficiency of Sample 2 in (a) keratinocytes (HaCaT cells) and (b) fibroblasts (BJ cells). [Figure 8] 1 shows the wound healing efficiency of Sample 2 in (a) keratinocytes (HaCaT cells) and (b) fibroblasts (BJ cells). [Figure 9] 1 shows the anti-inflammatory efficiency of Sample 2 in keratinocytes (HaCaT cells). [Figure 10] 1 shows the antioxidant efficiency of Sample 2 in keratinocytes (HaCaT cells), (a) in the presence or absence of hydrogen peroxide (H2O2), and (b) shows the dose-dependent efficiency. [Figure 11] 1 shows the photoprotective efficacy of Sample 2 in keratinocytes (HaCaT cells), (a) treated with 5 J / cm3 of UVA, and (b) treated with 7.5 J / cm3 of UVA. [Figure 12] 1 shows the collagenase degradation efficiency of sample 2 in keratinocytes (HaCaT). [Figure 13] Transmission electron microscope (TEM) images of curcumin encapsulated in (a) 0.25% by weight / volume of Sample 1, (b) 0.25% by weight / volume of Sample 4, and (c) 0.25% by weight / volume of Sample 5 are shown. [Figure 14]Transmission electron microscope (TEM) images of asiatic acid encapsulated in Sample 2 at (a) 0.1% by mass / volume, (b) 0.15% by mass / volume, and (c) 0.25% by mass / volume are shown. [Figure 15] Transmission electron microscope (TEM) images of poly(I:C) encapsulated in sample 2 at (a) 0.1% by volume, (b) 0.25% by volume, and (c) 0.5% by volume are shown. [Figure 16] Zeta potential and size of poly(I:C) encapsulated in sample 2 at (a) 0.1% by volume, (b) 0.25% by volume, and (c) 0.5% by volume. [Figure 17] 1 shows confocal microscope images of curcumin cellular uptake efficiency test into NIH-3T3 cells for Samples 1, 4, and 5. [Figure 18] 1 shows confocal microscope images of a test of the efficiency of curcumin cellular uptake into periodontal ligament stem cells for Sample 3. [Figure 19] Fibroblast growth factor (FGF) stability of sample 2 is shown. [Figure 20] Curcumin dissolution efficiency of (a) 0.1% by mass / volume of sample 2 and (b) 0.25% by mass / volume of samples 1, 4, and 5 is shown. [Figure 21] (a) Loading amount (mM), (b) loading efficiency (%), (c) loading capacity (%), and (e) encapsulation efficiency (%) of Sample 2 are shown. [Figure 22] (a) shows the resveratrol dissolution efficiency of Sample 2 without ethanol solution, and (b) shows the resveratrol dissolution efficiency of Sample 2 with ethanol solution. [Figure 23] 1 shows the cytotoxicity of corneal epithelial cells at concentrations of Sample 2 of 0, 0.06, 0.12, 0.25, 0.5, 1, and 2% by mass / volume. [Figure 24] This shows the cell viability of keratinocytes (HaCaT) at concentrations of 0, 0.05, and 0.5% by mass / volume of sample 2 irradiated with 7.5 J / cm2 of ultraviolet A (UVA). [Figure 25] 1 shows cell viability at 0, 0.1, 0.15, and 0.25% by weight / volume of Sample 2 by PrestoBlue™ assay. Detailed Description of the Invention

[0014] The present invention relates to a method for preparing hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) with a low degree of grafting, which provides low viscosity while maintaining high stability and biocompatibility. The HA-g-pNIPAM of the present invention can be used in many applications, including delivery systems, wound healing, cell proliferation, anti-inflammation, antioxidants, and photoprotection.

[0015] Any embodiment described herein is meant to include applications to other embodiments of the invention unless otherwise specified.

[0016] definition Technical and scientific terms used herein have definitions that are understood by those of ordinary skill in the art unless otherwise specified.

[0017] Any tool, device, method, or chemical listed herein refers to a tool, device, method, or chemical commonly used by those skilled in the art, unless it is specifically stated that the tool, device, method, or chemical is unique to the present invention.

[0018] The use of "comprising" and a singular noun or pronoun in any claim or specification means "one" and also includes "one or more," "at least one," and "one or more than one."

[0019] All compositions and / or methods disclosed in this application, as well as the claims, are intended to cover any operation, performance, modification, or adjustment that, although not specifically recited in the claims, would be apparent to one skilled in the art without extensive experimentation from the present invention in order to obtain an object having the same utility and results as the present embodiment. Therefore, objects that are alternative to or similar to the present embodiment should be construed as remaining within the spirit, scope, and concept of the invention as defined in the appended claims, including minor modifications or adjustments that would be obvious to a person skilled in the art.

[0020] Throughout this application, the term "about" means that any numerical value displayed or indicated herein may vary or deviate due to any error of the device, method, or individual using said device or method.

[0021] The following embodiments of the present invention are not intended to limit the scope of the present invention.

[0022] In an exemplary embodiment, the present invention provides a method for preparing hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM), comprising: a) preparing a mixture comprising a hyaluronic acid compound, poly(N-isopropylacrylamide), and a carbodiimide crosslinker in a water-miscible solvent; b) adjusting the pH to a range of 7.2 to 7.8 and stirring the mixture at 20 to 40°C for 1 to 3 days to react; c) drying the mixture of step b), The molar ratio of hyaluronic acid, poly(N-isopropylacrylamide), and carbodiimide crosslinker ranges from 0.1:0.0025:1 to 0.2:0.0125:1.

[0023] In preferred exemplary embodiments, the carbodiimide crosslinker is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or a mixture thereof.

[0024] In a preferred exemplary embodiment, the carbodiimide crosslinker is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of 1:0.5 to 1:1.5.

[0025] In a preferred exemplary embodiment, the weight average molecular weight of the hyaluronic acid compound ranges from 30,000 to 60,000 daltons.

[0026] In a preferred exemplary embodiment, the hyaluronic acid compound is selected from hyaluronic acid, a hyaluronate salt, or a mixture thereof.

[0027] In a preferred exemplary embodiment, the hyaluronate is selected from sodium hyaluronate, potassium hyaluronate, or a mixture thereof.

[0028] In a preferred exemplary embodiment, the hyaluronate is sodium hyaluronate.

[0029] In a preferred exemplary embodiment, the weight average molecular weight of the poly(N-isopropylacrylamide) ranges from 4,000 to 6,000 daltons.

[0030] In a preferred exemplary embodiment, the water-miscible solvent used in step a) is selected from water, phosphate buffered saline (PBS), citrate buffer, Tris buffer, potassium phosphate buffer, hydroalcoholic solution, or a mixture thereof.

[0031] In a preferred exemplary embodiment, the water-miscible solvent used in step a) is water.

[0032] In a preferred exemplary embodiment, the method further comprises, before carrying out step b), adjusting the pH of the mixture obtained from step a) to a range of 4.8 to 5.8, and stirring the mixture for 0.5 to 2 hours.

[0033] In a preferred exemplary embodiment, the pH is adjusted with a pH adjuster selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, calcium hydroxide, or mixtures thereof.

[0034] In a preferred exemplary embodiment, the pH is adjusted with a pH adjuster selected from hydrochloric acid, sulfuric acid, or a mixture thereof.

[0035] In a preferred exemplary embodiment, step c) is carried out by a process selected from freeze-drying, vacuum drying, air drying, or a combination thereof.

[0036] In a preferred exemplary embodiment, the method further comprises the step of purifying the product obtained from step b) before carrying out step c).

[0037] In a preferred exemplary embodiment, the purifying step is carried out by dialysis, cross-flow filtration, liquid-liquid extraction, or a combination thereof, before carrying out step c).

[0038] In another embodiment, the present invention relates to hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) according to the present invention.

[0039] In a preferred exemplary embodiment, the hyaluronic acid grafted poly(N-isopropylacrylamide) has a grafting degree in the range of 4 to 8%.

[0040] In another embodiment, the hyaluronic acid-grafted poly(N-isopropylacrylamide) colloid of the present invention is present in a water-miscible solvent selected from water, phosphate buffered saline (PBS), citrate buffer, or mixtures thereof at a concentration of 0.0001 to 2% by weight / volume.

[0041] To better understand the present invention, examples of hyaluronic acid-grafted poly(N-isopropylacrylamide) according to the present invention are given below. However, the following examples are intended to illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

[0042] Throughout this application, the water-miscible solvent is selected from, but not limited to, deionized water, distilled water, tap water, phosphate buffered saline (PBS), citrate buffer, cell culture medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris buffer, potassium phosphate buffer, hydroalcoholic solution, or a biocompatible organic solvent, or a mixture thereof. The biocompatible organic solvent is selected from the group of dilute methanol, dilute ethanol, or dilute acetic acid, or a mixture thereof.

[0043] Preparation of hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) (Samples 1-5) Hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) samples 1 to 5 were prepared by the following procedure.

[0044] (1) Sodium hyaluronate (NaHA) with a weight-average molecular weight of approximately 47,000 daltons was dissolved in water.

[0045] (2) To the sodium hyaluronate solution from step (1), poly(N-isopropylacrylamide) (pNIPAM) with a weight-average molecular weight of approximately 5,500 daltons was added, followed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), respectively.

[0046] (3) The pH of the solution from step (2) was adjusted to about 5.5 with sodium hydroxide, and the mixture was allowed to react at room temperature with stirring for about 1 hour.

[0047] (4) The pH of the solution from step (3) was adjusted to about 7.5 with hydrochloric acid, and the mixture was further reacted at room temperature for about 3 days with stirring.

[0048] (5) The HA-g-pNIPAM obtained in step (4) was purified by dialysis and then dried by freeze-drying.

[0049] The amounts of sodium hyaluronate (NaHA), poly(N-isopropylacrylamide) (pNIPAM), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) used in Samples 1 to 5 are shown in Table 1. TIFF0007771351000001.tif58141

[0050] Characterization of the present HA-g-pNIPAM (1) Nuclear magnetic resonance (NMR) The nuclear magnetic resonance (NMR) spectra of samples 1–5 are shown in Figure 1. Samples 1–5 exhibited peaks characteristic of hyaluronic acid and poly(N-isopropylacrylamide), with peaks at 1.10–1.65 ppm corresponding to the aliphatic protons of poly(N-isopropylacrylamide) and peaks at 1.65–2.01 ppm corresponding to the overlap of the N-acetyl groups on the D-glucosamine rings of hyaluronic acid with the protons of poly(N-isopropylacrylamide). Furthermore, the degree of grafting of samples 1–5 was calculated from the two prominent peaks in the NMR spectra. The chemical structure of hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) is shown in Figure 2. The grafting degree, average molecular weight, and mass ratio of pNIPAM and HA are shown in Table 2. TIFF0007771351000002.tif71141

[0051] (2) Infrared spectroscopy (IR) spectrum As shown in Figure 3, the infrared (IR) spectrum of sample 2 showed the characteristic peak of HA-g-pNIPAM at 1,606 cm -1 The peaks at 1,406.3 and 1,373 cm were amide bands (stretching vibration of C=O). -1 The peaks in the graph were characteristic deformation vibration peaks of the C—H bond in the methyl group of N-isopropylacrylamide. The IR results confirmed that HA was grafted onto the pNIPAM core structure.

[0052] (3) Thermogravimetric analysis (TGA) The thermal decomposition of Sample 3 was investigated by thermogravimetric analysis (TGA), and the results are shown in Figure 4. As a result, the decomposition of Sample 3 was separated into two phases, at approximately 200°C and approximately 400°C. Compared to the decomposition temperature of hyaluronic acid compounds, the decomposition of Sample 2 occurred at approximately 200°C.

[0053] (4) Particle size Sample 2 was dispersed in water to obtain colloids at concentrations ranging from 0.0001, 0.001, 0.01, 0.1, 0.15, and 0.25% by mass / volume. The resulting colloids of Sample 2 were examined by dynamic light scattering (DLS). The particle size and average particle size for each concentration of colloidal Sample 2 are shown in Table 3. The results showed that the average particle size increased with increasing colloid concentration. TIFF0007771351000003.tif57141

[0054] (5) Thermal responsiveness The thermal response of Sample 3 colloid was investigated using dynamic light scattering (DLS). The thermal response of Sample 3 is shown in Figure 5. The results showed that spherical particles of 0.5% by volume of Sample 3 colloid had particle diameters in the range of approximately 30 to 80 nm at temperatures of approximately 25 to 32°C, and when the temperature rose above 32°C, the particle diameter increased due to the lower critical solution temperature (LCST) properties of poly(N-isopropylacrylamide).

[0055] (6) Particle morphology Samples 1, 2, 4, and 5 were dispersed in water to obtain 0.25% by volume colloids, which were then observed under a transmission electron microscope (TEM).

[0056] The particle morphology is shown in Figure 6. The results showed that the colloids had well-defined spherical shapes, as noted for 0.25 wt. vol.% Sample 1 (Figure 6a), 0.25 wt. vol.% Sample 2 (Figure 6b), 0.25 wt. vol.% Sample 4 (Figure 6c), and 0.25 wt. vol.% Sample 5 (Figure 6d).

[0057] Efficiency test of HA-g-pNIPAM Below, the efficiency of HA-g-pNIPAM was investigated using colloidal samples 1, 2, 4, and 5 with concentrations ranging from 0.0001 to 2% by mass / volume.

[0058] (1) Cell proliferation Colloidal Sample 2 was tested for cell proliferation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT assay is the gold standard for measuring cell viability and proliferation. The experiment was performed as follows: HaCaT keratinocytes and BJ fibroblasts were treated with Sample 2 at concentrations of 0, 0.06, 0.12, 0.25, 0.5, 1, and 2% by weight for 24 hours. After treatment, cell proliferation was evaluated using the MTT assay.

[0059] The cell proliferation efficiency test is shown in Figure 7. The results showed that Sample 2 was able to promote skin cell proliferation, as determined by the increased cell viability of HaCaT keratinocytes (Figure 7a) and BJ fibroblasts (Figure 7b) compared to untreated cells.

[0060] (2) Wound healing The wound healing test of colloidal Sample 2 was carried out as follows: BJ fibroblasts were scratched with a razor and treated with Sample 2 at concentrations of 0.0001, 0.0005, 0.001, 0.005, 0.01, and 0.05% by weight / volume for 24 hours. The healing activity of the compounds was observed under a microscope.

[0061] The wound healing efficiency test is shown in Figure 8. The results showed that fibroblasts treated with Sample 2 were able to heal wounds faster than hyaluronic acid-treated or untreated cells (Figure 8a). The wound healing activity of Sample 2 was dose-dependent (Figure 8b). These results suggested that Sample 2 has promising wound healing activity.

[0062] (3) Anti-inflammatory effect An anti-inflammatory test of colloidal Sample 2 at a concentration of 0.1% by weight / volume was performed as follows: HaCaT keratinocytes were treated with Sample 2 for 24 hours in the presence of tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ). After treatment, the level of released TARC was measured by enzyme-linked immunosorbent assay (ELISA). TNF-α and IFN-γ are inflammation inducers, and thymus and activation-regulated chemokine (TARC) is an inflammation marker.

[0063] The anti-inflammatory efficiency test is shown in Figure 9. The results showed that exposure to Sample 2 could reduce the thymus and activation-regulated chemokine (TARC) level, suggesting that Sample 2 has anti-inflammatory activity.

[0064] (4) Antioxidant effect The antioxidant activity of colloidal Sample 2 was tested as follows. HaCaT keratinocytes were treated with Sample 2 at concentrations of 0, 0.0005, 0.0001, 0.005, 0.01, 0.05, and 0.1% by weight / volume for 24 hours. After treatment, the cells were incubated with hydrogen peroxide for 1 hour, and the oxidative stress status was evaluated using the 2'-7'-dichlorofluorescein diacetate (DCFH-DA) assay. Hydrogen peroxide was used as an oxidative stress inducer.

[0065] The antioxidant efficiency test is shown in Figure 10. The results showed that Sample 2 was able to reduce oxidative stress after hydrogen peroxide treatment, indicating that Sample 2 had antioxidant activity.

[0066] (5) Photoprotection Photoprotection testing of colloidal Sample 2 was performed as follows: HaCaT keratinocytes were treated with Sample 2 at concentrations of 0, 0.0001, 0.0005, 0.001, 0.01, and 0.05% by weight / volume for 24 hours. After treatment, the cells were exposed to ultraviolet A (UVA) at 5 J / cm. 2 (Fig. 11a) and 7.5 J / cm 2 (Fig. 11b) and cell viability was measured by MTT assay.

[0067] The photoprotection efficiency test is shown in Figure 11. The results showed that Sample 2 could protect keratinocytes from the toxicity caused by UVA irradiation, suggesting that Sample 2 has photoprotective activity.

[0068] (6) Collagenase degradation A collagenase degradation test of colloidal sample 2 was carried out as follows: HaCaT cells were treated with sample 2 for 24 hours. After treatment, the cells were exposed to 7.5 J / cm 2 The cells were irradiated with ultraviolet A (UVA) for 24 hours. The levels of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-9 (MMP-9) were measured by Western blot analysis 24 hours after irradiation.

[0069] The collagenase degradation efficiency test is shown in Figure 12. The results showed that Sample 2 could suppress the expression of MMP-1 and MMP-9 in response to UVA irradiation. This result suggested that Sample 2 could promote collagenase degradation.

[0070] (7) Delivery system The encapsulation of compounds including curcumin, asiatic acid, and polyinosinic:polycytidylic acid (poly(I:C)) was tested as follows: The compounds were encapsulated into nanoparticles using a simple incubation method.

[0071] For curcumin, an ethanolic stock solution of curcumin was added dropwise to colloidal samples 1, 4, and 5 at a volume ratio of approximately 1:10, incubated with stirring at 4°C for 24 h, and then centrifuged to remove undissolved compound.

[0072] For asiatic acid, an ethanolic stock solution of asiatic acid was added dropwise to colloidal sample 2 at a volume ratio of approximately 1:10 and incubated with stirring at 25°C for 6 h, followed by centrifugation to remove undissolved compound.

[0073] For polyinosinic acid:polycytidylic acid (poly(I:C)), an aqueous stock solution of poly(I:C) was added dropwise to colloidal sample 2 at a volume ratio of approximately 1:10 while stirring at room temperature for 30 min. Transmission electron microscopy (TEM) was used to observe the morphological shape and appearance of the encapsulated compound nanoparticles, and dynamic light scattering (DLS) was used to measure the size and zeta potential of polyinosinic acid:polycytidylic acid (poly(I:C))-encapsulated nanoparticles.

[0074] Transmission electron microscope (TEM) images of the curcumin-encapsulated nanoparticles are shown in Figure 13. The results indicated that curcumin was successfully encapsulated in colloidal form in Sample 1, Sample 4, and Sample 5 at 0.25% by mass / volume, judging from the changes in size, shape, and appearance of the nanoparticles.

[0075] Transmission electron microscope (TEM) images of the encapsulated asiatic acid nanoparticles are shown in Figure 14. The results showed that asiatic acid was successfully encapsulated in colloidal form in Sample 2 at 0.1 wt. vol. %, 0.15 wt. vol. %, and 0.25 wt. vol. %, judging from the changes in size, shape, and appearance of the nanoparticles.

[0076] Transmission electron microscopy (TEM) images of polyinosinic acid:polycytidylic acid (poly(I:C))-encapsulated nanoparticles are shown in Figure 15. The results indicated that curcumin was successfully encapsulated in colloidal form at 0.1 wt vol %, 0.25 wt vol %, and 0.5 wt vol % in Sample 2, judging from the changes in size, shape, and appearance of the nanoparticles.

[0077] The encapsulation of polyinosinic:polycytidylic acid (poly(I:C)) is shown in Figure 16. The results indicated that different concentrations (0.2 and 1 μg / ml) of polyinosinic:polycytidylic acid (poly(I:C)) were encapsulated in colloidal Sample 2, as shown by the particle size reduction of Sample 2 at 0.1% (Figure 16a) and 0.5% (Figure 16b) by weight / volume, compared to the samples encapsulating the active ingredient.

[0078] (8) Cellular uptake Cellular uptake tests of colloidal curcumin-encapsulated samples 1, 4, and 5 were performed as follows. NIH-3T3 cells were treated with samples 1, 4, and 5 for 24 hours. After treatment, the cells were stained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI) (nuclear stain) and rhodamine phalloidin (actin stain) for 15 to 30 minutes. After 24 hours of treatment, the cellular uptake levels were measured using a fluorescence microscope.

[0079] Confocal microscope images of the test for the efficiency of curcumin cellular uptake into NIH-3T3 cells are shown in Figure 17. As a result, the cellular uptake of curcumin colloid was indicated by green fluorescence intensity, indicating that curcumin colloid was taken up into NIH-3T3 cells.

[0080] The cellular uptake test for encapsulated curcumin in Sample 3 was performed as follows. Periodontal ligament stem cells were treated with Sample 3 for 24 hours. After treatment, the cells were stained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI) (nuclear stain) and rhodamine phalloidin (actin stain) for 15 to 30 minutes. After 24 hours of treatment, the cellular uptake level was measured using a fluorescence microscope.

[0081] Confocal microscope images of the curcumin cellular uptake efficiency test into periodontal ligament stem cells are shown in Figure 18. The results showed that the cellular uptake of curcumin colloid, indicated by green fluorescence intensity, was higher than that of curcumin in water.

[0082] (9) Transparency The permeability of sodium ascorbyl phosphate (SAP) and colloidally encapsulated SAP at 0.1% by weight / volume of Sample 2 was tested on pig skin using Franz diffusion cells. The results showed that SAP had a permeability of 6 μg / cm. 2 , 0.1% by mass / volume of sample 2 colloidally encapsulated SAP was 68.78 μg / cm 2 , and each penetrated porcine skin. Therefore, it was confirmed that HA-g-pNIPAM can enhance epidermal permeability.

[0083] (10) Stability of active pharmaceutical ingredients The stability of fibroblast growth factor (FGF), including physical stability factors (e.g., color, precipitates) and chemical stability factors (e.g., pH, cell proliferation induction in L-929 cells) of colloidal Sample 2, was investigated by comparing FGF with FGF encapsulated in Sample 2. Experiments were performed 0, 2, 4, 8, and 18 weeks after incorporation of FGF into colloidal Sample 2.

[0084] Cell proliferation induction in L-929 cells was analyzed using the PrestoBlue™ assay to analyze metabolic rates of L-929 cells. L-929 cells were cultured for 24 hours in Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM-F-12) containing 5% fetal bovine serum (v / v) and 150 μL of 2 mM L-glutamine (hereafter referred to as "medium") to promote cell adhesion to 96-well plates. The medium was then removed from each well and replaced with 50 μL of the test materials, FGF (control) and FGF encapsulated in Sample 2. The cells were then cultured for 24 hours in a 5% CO2, 37°C incubator. The test materials were then removed, washed with pH 7.4 phosphate-buffered saline (PBS), and replaced with 10% PrestoBlue™-containing medium. The cells were then incubated for another 1 hour in a 5% CO2, 37°C incubator. Finally, the fluorescence intensity was measured at 560 / 590 nm (excitation / emission) in a microplate reader.

[0085] The cell viability test is shown in Figure 19. The results showed that the colloid-FGF formulation maintained FGF-induced cell proliferation over an 18-week storage period, while reducing the biological activity of FGF.

[0086] (11)Solubility The solubility of colloidal sample 2 spiked with curcumin, asiatic acid, and resveratrol, and colloidal samples 1, 4, and 5 spiked with curcumin, was tested by the following procedure: ultraviolet-visible (UV-Vis) spectrophotometry for resveratrol and curcumin, and high-performance liquid chromatography for asiatic acid.

[0087] The solubilities of curcumin-loaded colloidal sample 2 at a concentration of 0.1% by weight and 0.25% by weight, and curcumin-loaded colloidal samples 1, 4, and 5 at a concentration of 0.25% by weight, are shown in Figure 20. As a result, the colloid was able to increase the solubility of curcumin by 26 times in sample 2 (Figure 20a) at a concentration of 0.1%. Samples 1, 4, and 5 were able to increase the solubility of curcumin by approximately 2 times, 3 times, and 2 times, respectively, compared to water as a control.

[0088] The solubility of colloidal asiatic acid-loaded Sample 2 at concentrations of 0, 0.1, 0.15, and 0.25% by mass / volume is shown in Figure 21 in terms of the loading amount (Figure 21a), loading efficiency (Figure 21b), loading capacity (Figure 21c), and encapsulation efficiency (Figure 21d). The results showed that when Sample 2 was added to water at concentrations of 0.1%, 0.15%, and 0.25%, the colloids were able to increase the loading amount of asiatic acid by approximately 400-fold, 370-fold, and 250-fold, respectively, compared to the water control.

[0089] The solubility of colloidal Sample 2 loaded with resveratrol was measured at concentrations of 0, 0.1, and 0.2% by mass / volume, and the results are shown in Figure 22. As a result, when Sample 2 at a concentration of 0.2% was dissolved in water or an aqueous hydroalcoholic solution, the colloid was able to increase the solubility of resveratrol by 4.5 to 5 times compared to when water was used as a control.

[0090] (12) Moisture retention test Moisture retention tests of colloidal Sample 2 at 0.1 and 2% by weight / volume concentrations were conducted on five healthy volunteers using the following procedure. Prior to treatment, participants were required to refrain from using cosmetics on the test area for one week. The samples were applied evenly to the forearm twice daily for 14 days in an environment with a temperature of 25±2°C and a humidity of 50±5%. Before and after application of Sample 2 and its control, skin moisture content was checked using a measuring capacitor pressed against the skin with constant pressure, and the measurements were evaluated using a corneometer. Alternatively, transepidermal water loss (TEWL) tests were conducted using a TEWL measurement probe to compare the results before and after application of Sample 2.

[0091] The moisturizing test of colloidal Sample 2 at 0.1 and 2% by mass / volume concentrations is shown in Table 4. The results showed that Sample 2 increased skin moisture content by 124% and 11.91% after application of 2% and 0.1% concentrations of Sample 2 in water, respectively, while unmodified HA failed to change skin moisture content at a 0.1% concentration of the control polymer. Furthermore, application of 0.1% Sample 2 to the skin of the upper arm reduced transepidermal water loss (TEWL) by 12.1%. TIFF0007771351000004.tif81141

[0092] (13) Toxicity of hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) In vitro toxicity tests of colloidal sample 2 in keratinocytes (HaCaT cells), fibroblasts (BJ cells), and corneal epithelial cells were conducted as follows: Cells were treated with 0, 0.06, 0.12, 0.25, 0.5, 1, and 2% by weight / volume for 24 hours, and then toxicity was evaluated by MTT assay.

[0093] Cytotoxicity tests in keratinocytes (HaCaT cells) and fibroblasts (BJ cells) are shown in Figure 7. These results suggest that Sample 2 does not cause toxicity to either cell line, as it did not reduce the cell viability of either skin cell line.

[0094] Furthermore, a cytotoxicity test using corneal epithelial cells is shown in Figure 23. From these results, no toxicity was observed in the cells treated with Sample 2. These results support the safety profile of Sample 2.

[0095] (14) Phototoxicity of HA-g-pNIPAM An in vitro phototoxicity test of colloidal Sample 2 was carried out as follows: HaCaT keratinocytes were treated with Sample 2 at concentrations of 0, 0.05, and 0.5% by weight / volume for 24 hours. After treatment, the cells were exposed to 7.5 J / cm 2 The cells were irradiated with ultraviolet A (UVA) and cell viability was measured by MTT assay.

[0096] The phototoxicity test is shown in Figure 24. As a result, it was found that Sample 2 can prevent toxicity caused by UVA irradiation.

[0097] (15) Side effect testing The side effect test of colloidal sample 2 at 0, 0.1, 0.15, and 0.25% w / v concentrations was investigated by considering the toxicity reduction of asiatic acid in fibroblast L929 cells by PrestoBlue™ assay.

[0098] Figure 25 shows the side effect test of colloidal sample 2 at concentrations of 0, 0.1, 0.15, and 0.25% by volume. When 12.5 μM asiatic acid dissolved in dimethyl sulfoxide (DMSO) was used in the cell culture medium, cell viability decreased to 5%. However, when 12.5 μM asiatic acid was made into a colloid at concentrations of 0.1, 0.15, and 0.25% by volume, cell viability increased to 100%. Therefore, it was confirmed that the hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) of the present invention can reduce the toxicity of asiatic acid.

[0099] (16) Skin irritation test The irritation potential of colloidal sample 2 at a concentration of 2.0% by weight / volume was investigated using a 24-hour occlusive human patch test. After obtaining informed consent and confirming the inclusion / exclusion criteria, i.e., all subjects were healthy adults aged 20–59 years and had no other exclusion criteria, a laboratory technician examined the back (paravertebral region) of each subject. Furthermore, the technician observed and photographed the test site on the upper back (paravertebral region) of each selected subject. The subject then applied the appropriate dose of the test sample using a patch test unit. After 24 hours of application, the subject removed the patch. A technician photographed the test site 1 hour (total 24 hours) and 24 hours (total 48 hours) after patch removal. Furthermore, a dermatologist assessed skin reactivity and scored the skin irritation.

[0100] As a result, none of the 24 adult subjects showed any reaction to the sample. As a result, according to Sugai's classification (Cosmetic Sciences, 1995, 19: 49-56.), the skin irritation score was 0, which is less than 5. Therefore, Sample 2 was confirmed to be a safe and non-irritating product. TIFF0007771351000005.tif96141

[0101] (17) Sensitization test The sensitization potential of colloidal sample 2 at a concentration of 2.0% by weight / volume was investigated using a human repeated challenge patch test (HRIPT). The HRIPT was broadly divided into four phases: (1) screening, (2) induction, (3) rest, and (4) challenge. During the screening phase, informed consent and subject inclusion criteria, including healthy adults aged 20–59 years and no exclusion criteria, were obtained and confirmed. Next, a laboratory technician observed and photographed the test site on the upper back (paraspinal region) of each selected subject. During the induction phase, subjects applied the appropriate dose of the test sample using a patch test unit. After 24 hours, the subjects removed the patch themselves. Before applying the next patch test unit 24 or 48 hours later, a trained laboratory technician evaluated the skin reaction and photographed the results. The same sample was then applied to the same location on each subject's back by the laboratory technician. This procedure was repeated three times a week for three consecutive weeks (a total of nine sets of application and removal). After the induction period, all subjects were given a 10-14 day rest period. Finally, during the challenge period, the same test sample was applied to normal skin near the area applied during the induction period using a patch test unit. Each patch test unit was removed from each subject 24 hours after application. Photographs of each subject's test site were taken by a laboratory technician, and skin reactions were assessed by a dermatologist 1 and 24 hours after removal of the patch test unit ("24 hours" and "48 hours", respectively).

[0102] As a result, as shown in Table 6, most of the 56 subjects showed no reaction, and only one or two subjects showed slight erythema at the test site. This indicates that Sample 2 does not cause sensitization and can be considered a hypoallergenic product under the test conditions. TIFF0007771351000006.tif137141

[0103] Best Mode for Carrying Out the Invention The best or preferred embodiment of the present invention is as set forth in the description of the invention.

Claims

1. 1. A method for preparing hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM), comprising: Step a) preparing a mixture in a water-miscible solvent comprising a hyaluronic acid compound, poly(N-isopropylacrylamide), and a carbodiimide crosslinker; Step b) adjusting the pH to a range of 7.2 to 7.8 and reacting the mixture by stirring at 20 to 40°C for 1 to 3 days; step c) drying the mixture of step b); The method wherein the molar ratio of hyaluronic acid, poly(N-isopropylacrylamide), and carbodiimide crosslinker ranges from 0.1:0.0025:1 to 0.2:0.0125:

1.

2. 2. The method of claim 1, wherein the carbodiimide crosslinker is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or a mixture thereof.

3. 3. The method of claim 2, wherein the carbodiimide crosslinker is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of 1:0.5 to 1:1.

5.

4. 2. The method of claim 1, wherein the weight average molecular weight of the hyaluronic acid compound is in the range of 30,000 to 60,000 daltons.

5. 10. The method of claim 1, wherein the hyaluronic acid compound is selected from hyaluronic acid, a hyaluronate salt, or a mixture thereof.

6. 6. The method of claim 5, wherein the hyaluronate salt is selected from sodium hyaluronate, potassium hyaluronate, or a mixture thereof.

7. 7. The method of claim 6, wherein the hyaluronic acid salt is sodium hyaluronate.

8. 10. The method of claim 1, wherein the weight average molecular weight of the poly(N-isopropylacrylamide) ranges from 4,000 to 6,000 daltons.

9. 2. The method of claim 1, wherein the water-miscible solvent used in step a) is selected from water, phosphate buffered saline (PBS), citrate buffer, Tris buffer, potassium phosphate buffer, hydroalcoholic solution, or a mixture thereof.

10. 10. The method of claim 9, wherein the water-miscible solvent used in step a) is water.

11. 10. The method of claim 1, further comprising the steps of adjusting the pH of the mixture resulting from step a) to a range of 4.8 to 5.8 and stirring the mixture for 0.5 to 2 hours before carrying out step b).

12. 10. The method of claim 1, wherein the pH is adjusted with a pH adjuster selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, calcium hydroxide, or mixtures thereof.

13. 12. The method of claim 11, wherein the pH is adjusted with a pH adjuster selected from hydrochloric acid, sulfuric acid, or a mixture thereof.

14. 10. The method of claim 1, wherein step c) is performed by a process selected from freeze-drying, vacuum drying, air drying, or a combination thereof.

15. 10. The method of claim 1, further comprising purifying the product obtained from step b) before carrying out step c).

16. 16. The method of claim 15, wherein the purifying step is carried out by dialysis, cross-flow filtration, liquid-liquid extraction, or a combination thereof, prior to carrying out step c).

17. Hyaluronic acid grafted poly(N-isopropylacrylamide) (HA-g-pNIPAM) obtainable by the method according to any one of claims 1 to 16.

18. 18. The hyaluronic acid-grafted poly(N-isopropylacrylamide) of claim 17, wherein the hyaluronic acid-grafted poly(N-isopropylacrylamide) has a degree of grafting in the range of 4 to 8%.

19. 19. The hyaluronic acid-grafted poly(N-isopropylacrylamide) colloid according to any one of claims 17 to 18, which is present in a water-miscible solvent selected from water, phosphate buffered saline (PBS), citrate buffer, or a mixture thereof, at a concentration of 0.0001 to 2% by weight / volume.

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