KR101971834B1 - Ph sensitive hydrogel comprising copolymer of carboxymethylcellulose and hydroxyethylacrylate - Google Patents

Abstract

The present invention provides a PH-sensitive hydrogel comprising a copolymer of carboxymethyl cellulose and hydroxyethyl acrylate. The hydrogel comprises a copolymer crosslinked with poly(ethylene glycol) diacrylate as a copolymer of carboxymethyl cellulose and 2-hydroxyethyl acrylate. According to the present invention, effective skin penetration of drugs can be performed.

Description

PH SENSITIVE HYDROGEL COMPRISING COPOLYMER OF CARBOXYMETHYLCELLULOSE AND HYDROXYETHYLACRYLATE CONTAINING COPOLYMERS OF CARBOXYMethylcellulose and hydroxyethyl acrylate [

The present invention relates to a PH-sensitive hydrogel comprising a copolymer of carboxymethylcellulose and hydroxyethyl acrylate.

The skin consists of three layers of epidermis, dermis and subcutaneous fat and surrounds the whole body of the body. Among them, the epidermis consists of a basal layer, an outer layer, a granular layer and a stratum corneum layer. The stratum corneum, which is the outermost layer of the skin, plays a role of a strong skin barrier to protect the skin from external stimuli, which makes it difficult to pass the drug into the stratum corneum. Therefore, various transdermal drug delivery systems (TDDS) have been developed to overcome these skin barriers and to enhance the permeation efficiency of drugs.

Hydrogels are hydrophilic polymer systems capable of absorbing large amounts of moisture in the structure, and are chemically or physically crosslinked networks. The hydrogel swelling in an aqueous solution has been applied in various fields such as tissue engineering, contact lens, drug delivery system and the like due to its unique thermodynamically stable physical properties. On the other hand, in the transdermal delivery, a patch system containing various active ingredients has been studied for enhancing absorption of drugs. Particularly, the hydrogel used as a patch has an advantage of locally sealing the skin and promoting the absorption of the drug by hydrating the stratum corneum with a large amount of water. It has been reported that the transdermal permeability of a drug-containing hydro-patch system is 3 to 4 times better than that of unsealed conditions. In particular, it has been reported that the horny layer can be hydrated so that most of the drug can be penetrated through the skin attachment such as pores and sweat glands.

Under these circumstances, the present inventors have made extensive efforts to provide a drug delivery hydrogel having transdermal permeability, and as a result, it has been found that a hydrogel made of a copolymer of carboxymethyl cellulose (CMC) and 2-hydroxyethyl acrylate has excellent transdermal permeability And exhibits pH-responsive drug release characteristics, thereby completing the present invention.

J. S. Boateng, K. H. Matthews, H. N. Stevens, and G. M. Eccleston, Wound healing dressings and drug delivery systems: a review, J. Pharm. Sci., 97 (8), 2892 (2008).

The object of the present disclosure is to provide a hydrogel capable of transdermal delivery of a drug. Specifically, it is intended to provide a pH-sensitive hydrogel, that is, a hydrogel having an excellent transdermal delivery efficiency of a drug capable of selectively releasing a drug under specific pH conditions.

In one aspect, the present invention provides a hydrogel comprising a copolymer of carboxymethylcellulose and hydroxyethyl acrylate.

Hereinafter, the present invention will be described in detail.

In drug delivery systems, hydrogels can exhibit different properties in various environmental conditions (temperature, pH, current, etc.) depending on their functional groups, and 'smart hydrogels' are developed that respond to these environmental conditions. On the other hand, the stratum corneum must perform an effective barrier function and maintain the homeostasis of the skin. For this, it is very important to maintain the pH at about 5.5. If such weak acidic conditions of the stratum corneum are destroyed, various skin diseases caused by skin barrier disturbance and bacterial propagation may be caused. Especially, atopic skin and dry skin have basicity of around pH 7 ~ 9.

Accordingly, the present disclosure is to provide a pH-sensitive hydrogel which can effectively treat skin diseases, that is, capable of effective drug release due to such a pH difference, in view of the difference in pH.

The hydrogel according to the present disclosure uses carboxymethyl cellulose (CMC), which is a derivative of cellulose, which is a biocompatible polymer material. Carboxymethylcellulose is widely used in various fields due to its high biodegradability, non-toxicity and high biocompatibility. It has a hydroxyl group (-OH) and a carboxyl group (-COOH) as a functional group, and the pKa of the carboxyl group is about 4.3. Therefore, the carboxyl group is mostly ionized under basic conditions, and its physicochemical properties are changed by the change of ionization degree according to pH. Carboxymethylcellulose exists abundantly in nature and is inexpensive, but exhibits low mechanical properties resulting in limited application.

Thus, in the present disclosure, a copolymer of acrylate or methacrylate and carboxymethylcellulose was used. The acrylate or methacrylate according to the present disclosure can undergo a copolymerization reaction with carboxymethylcellulose through a carbon double bond of a (meth) acrylate structure. In addition, such an acrylate or methacrylate may further comprise a hydroxy group (OH). Specifically, the acrylate or methacrylate may each have the following structure:

또는

.

or

.

In the above formula, n may be an integer of 1 to 10, specifically an integer of 1 to 5, more specifically 2. Specific examples of the acrylate or methacrylate include hydroxyethyl acrylate (HEA) and hydroxyethyl methacrylate (HEMA).

In one embodiment, 2-hydroxyethyl acrylate has been used, which can be used as a grafting agent to improve mechanical properties by improving adhesion and flexibility. Also, -OH, which is a hydrophilic functional group, can increase water retention. Thus, this 2-hydroxyethyl acrylate is also used as a material for contact lenses and is known as a biocompatible material. Therefore, in the hydrogel according to the present disclosure, there is provided a hydrogel which has improved physical properties by using a copolymer of carboxymethyl cellulose and hydroxyethyl cellulose, and which is highly biocompatible and can respond to pH conditions It is possible.

In the hydrogel according to the present disclosure, the copolymer may be a crosslinked copolymer. Specifically, the crosslinked copolymer may have a coupling structure due to crosslinking. The coupling structure of the copolymer is a structure in which an alkene (C = C) group of each copolymer, such as an alkenyl group of hydroxyethyl acrylate, is allowed to participate in crosslinking, and these are connected to each other through a crosslinking agent or a crosslinking agent. In one embodiment, poly (ethylene glycol) diacrylate (PEGDA) may be used as a crosslinking agent to prepare the crosslinked copolymer. In this case, the process for producing the copolymer is as follows.

The hydrogels according to the present disclosure have improved physical properties, particularly good gelation rates and adhesiveness, due to the use of such hydroxyethyl acrylates and the use of crosslinking agents.

The hydroxyethyl acrylate contained in the copolymer is used in an amount of 1 to 20 parts by weight, specifically 5 to 15 parts by weight, more specifically 6 to 12 parts by weight, more preferably 7 to 11 parts by weight, based on 1 part by weight of carboxymethylcellulose More particularly 8 to 10 parts by weight. The amount of the crosslinking agent contained in the copolymer is 0.1 to 1 part by weight, specifically 0.1 to 0.7 part by weight, more specifically 0.1 to 0.6 part by weight, more specifically 0.1 to 0.4 part by weight, more preferably 0.1 to 4 parts by weight, relative to 1 part by weight of carboxymethyl cellulose. And more specifically 0.1 to 0.3 parts by weight.

The copolymer may be prepared using a radical polymerization reaction. As the initiator of the radical polymerization reaction, any known in the art can be used, and potassium peroxosulfate (KPS) can be used as a non-limiting example.

The hydrogel according to the present disclosure may have a gelation rate of 50 to 99%, specifically 60 to 95%, 70 to 90%, and 80 to 85%. The gelation rate can be measured as disclosed in Example 1 of the present disclosure, and the gelation rate can be changed as the weight of hydroxyethyl acrylate and the weight of crosslinking agent are changed with respect to the weight of carboxymethyl cellulose. In one embodiment, a hydrogel having a gelation rate of up to about 83% was prepared, and it was confirmed that this hydrogel had excellent physical properties.

Also, in one embodiment, SEM image observation, thermogravimetric analysis (TGA), swelling ratio, rheology, and texture analysis of the hydrogel according to the present disclosure were confirmed. The three-dimensional network structure of the hydrogel was confirmed through SEM image observation. Especially, it was confirmed that the pores had a super-porous structure having a size of several tens to several hundreds of 탆 in diameter and an open cell structure Respectively. In addition, the swelling rate of the hydrogel was varied according to the pH condition, and it was confirmed that the hydrogel had a higher swelling rate than the pH condition of 5.5, which is the pH condition of the normal skin, at pH conditions (7.5 and 8.5) of weak basicity. That is, it exhibits a higher swelling rate at a specific pH and can promote the permeation of the medium in the hydrogel. This allows pH-selective drug release. Further, it was confirmed that the hydrogel had excellent physical properties such as strength, adhesiveness, cohesiveness and elasticity.

The hydrogel according to the present disclosure can be used as an excellent drug delivery agent, and thus contains a drug inside the hydrogel. The drug may be a flavonoid such as naringenin, eye siculitis, luteolin. And any other known drugs used to treat other atopic, acne, juvenile, athlete's foot, psoriatic skin or skin dryness. The drug delivery agent may comprise 0.01 to 50, 0.05 to 20, 0.1 to 10, 0.5 to 5, 0.8 to 3, 1 to 2 weight of the drug based on the total weight of the drug delivery agent.

In one embodiment, to confirm the drug delivery effect of the hydrogel of the present invention, a hydrogel containing naringenin was prepared. Naringenin is a flavonoid that is abundant in citrus fruits and is known to have antioxidant, anti-inflammatory, anti-cancer and anti-proliferative properties. In particular, according to recent studies, naringenin inhibits Th1 immune response in atopic dermatitis-induced mice induced by 2,4-dinitrofluorobenzene (DNFB), inhibition of cell migration to skin lesions, Of the total number of patients. It has also been reported that Staphylococcus aureus , which is present in more than 90% of atopic patients, has a high antimicrobial activity.

The hydrogel according to the present disclosure may be a drug delivery vehicle used to treat atopy, acne, juvenile, athlete's foot, psoriasis skin or skin dryness. Unlike normal skin condition (pH 5.5), it exhibits weak basic skin condition of pH 7-9 for atopic and skin dryness. Since the hydrogel according to the present disclosure is a pH-sensitive hydrogel and has superior drug delivery properties under such weakly basic conditions, it can be more usefully used for the treatment of diseases that form such weakly basic skin conditions. Specifically, in one embodiment, the drug release effect according to the pH of the hydrogel according to the present disclosure was confirmed, and as a result, the drug release effect was more excellent in a weakly basic condition than in a weakly acidic condition. In addition, the skin permeation ability of the hydrogel according to the present disclosure was confirmed, and as a result, an excellent drug transdermal delivery effect was exhibited. This shows that the hydrogel according to the present disclosure hydrates the skin and temporarily interrupts the skin barrier function so that it can easily penetrate the drug or the active ingredient. In addition, in one embodiment, the antimicrobial activity against Staphylococcus aureus according to each pH condition was confirmed by supporting the drug-bound neurin on a hydrogel, and as a result, the antimicrobial activity was more excellent under weakly basic conditions than in a weakly acidic condition. That is, it exhibits better antimicrobial activity under the same conditions except for pH, so that different drug release characteristics according to the pH of the hydrogel according to the present disclosure are confirmed. Therefore, the hydrogel has potential as an efficient skin carrier in the transdermal delivery of drugs since it can reduce the loss of efficacy by being able to target certain lesions, such as acne.

Thus, the present disclosure provides a pH-sensitive hydrogel, specifically a hydrogel capable of selective drug release under basic conditions, which hydrogel provides more effective drug release in skin diseases that produce weakly basic skin conditions, Permeation, i.e. transdermal delivery of the drug, is possible.

Further, as another embodiment of the present disclosure, there is provided a drug carrier comprising the hydrogel and the drug. The drug delivery vehicle is capable of selective drug release at a specific pH as described above. Specifically, it has selective drug release characteristics at pH 7-9. In addition, since the transdermal delivery efficiency of a drug is high, the drug delivery system can be particularly useful for skin diseases. The drug delivery system is excellent in efficiency of being attached to the skin surface and delivering the drug to the percutaneous tissue. The drug contained in the drug delivery vehicle may be a drug for treating a disease showing a lesion of pH 7 to 9. Since the drug delivery vehicle exhibits higher drug release characteristics in the pH range, it can specifically act on diseases that cause lesions showing such pH. Accordingly, such a drug delivery system can effectively deliver the drug and more efficiently use the amount of the drug injected into the drug delivery system. The disease can be atopic, acne, juvenile, athlete's foot psoriasis or skin dry. The drug delivery system may be provided in the form of a hydro patch, a mask pack, or the like.

In another aspect of the disclosure, there is provided a pharmaceutical composition for the treatment of the above diseases, which comprises the hydrogel and a drug for treating a disease showing a lesion at a pH of 7 to 9. Since the hydrogel is capable of selective drug release at a specific pH as described above, the drug may be present in a form incorporated in the hydrogel. In such a case, the hydrogel contained in the pharmaceutical composition may selectively release the drug at a pH of 7 to 9, so that it can act specifically on the disease. The disease can be atopic, acne, juvenile, athlete's foot, psoriatic skin or skin dry.

In another aspect of the present disclosure, there is provided a skin permeable composition comprising the hydrogel and a physiologically active ingredient as an active ingredient. The skin permeation composition may be used as cosmetics, quasi-drugs, and the like. The physiologically active ingredient may be a skin lightening agent, an anti-aging agent, an antioxidant, an antibacterial agent, an anti-inflammatory agent, a skin dryness treatment agent, a drug, a natural product extract or a flavonoid. The physiologically active component is incorporated into the hydrogel and can be released in a pH-responsive manner, as described above. In addition, transdermal delivery of physiologically active ingredients is possible due to local skin sealing and hydration of the hydrogel due to skin adhesion.

The hydrogel according to the present disclosure is a smart hydrogel in which drug release characteristics are changed according to pH conditions, and it is possible to effectively deliver drugs to lesions having specific pH conditions. In addition, drug delivery through the stratum corneum is possible, which enables effective transdermal delivery of drugs.

FIG. 1 shows FT-IR spectroscopic results of CMC, 2-HEA, PEGDA and hydrogel of Example 1 confirmed in Experimental Example 1-1.
FIG. 2 shows ¹ H NMR of CMC, 2-HEA, PEGDA and ¹ H HR-MAS NMR spectroscopy of the hydrogel of Example 1 as confirmed in Experimental Example 1-1.
Fig. 3 shows an SEM image of the hydrogel of Example 1 identified in Experimental Example 1-2, and the scale bar is 100 m. CMC-g-pHEA-3, (d) cl-CMC-g-pHEA-2, (c) g-pHEA-4, and (e) is the surface of cl-CMC-g-pHEA-5. CMC-g-pHEA-1, (g), cl-CMC-g-pHEA-2, (h) g-pHEA-4, and (j) is the intersection of cl-CMC-g-pHEA-5.
Fig. 4 shows the results of the thermogravimetric analysis TGA curve of the hydrogel of Example 1 identified in Experimental Example 1-3. Fig.
FIG. 5 shows the swelling ratios of the hydrogel of Example 1 identified in Experimental Examples 1-4, and the results are shown at pH 5.5, 7.5, and 8.5, respectively.
6 is a rheological property confirmed in Experimental Example 1-5, which shows the storage elastic modulus G 'and loss elastic modulus G "of the hydrogel of Example 1, respectively.
Figure 7 shows the results of the texture analysis of Experimental Example 1-5, showing the hardness, cohesiveness, resilience, chewiness, adhesiveness, and springiness of Example 1, Lt; / RTI > Results were expressed as mean ± SD (n = 3).
FIG. 8 shows the cell viability according to each treatment of the hydrogel determined in Experimental Example 2. FIG.
FIG. 9 shows the drug release behavior of the hydrogel over time as confirmed in Experimental Example 3-1.
Fig. 10 shows the amount of naringenin present in each region as confirmed in Experimental Example 4. Fig. Naringenin (tapes) present in the stratum corneum, naringenin (skin) present in the epidermis and dermis layers except the stratum corneum, and narinenin (transdermal) present on the receptor through the dermal layer.
Fig. 11 shows the results of an antibacterial activity test of a hydrogel as confirmed in Experimental Example 5. Fig. Fig. 11 (a) shows a case of hydrogel not carrying naringenin, and Fig. 11 (b) shows a case of hydrogel carrying naringenin.

In the following, embodiments will be described in detail with reference to the accompanying drawings. However, various modifications may be made in the embodiments, and the scope of the patent application is not limited or limited by these embodiments. It is to be understood that all changes, equivalents, and alternatives to the embodiments are included in the scope of the right.

The terms used in the examples are used for descriptive purposes only and are not to be construed as limiting. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this embodiment belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

In the following description of the present invention with reference to the accompanying drawings, the same components are denoted by the same reference numerals regardless of the reference numerals, and redundant explanations thereof will be omitted. In the following description of the embodiments, a detailed description of related arts will be omitted if it is determined that the gist of the embodiments may be unnecessarily blurred.

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Materials and equipment used

Sodium carboxymethyl cellulose (average molecular weight: 90,000), 2-hydroxyethyl acrylate, poly (ethylene glycol) diacrylate, and the like, which are used in the hydrogel synthesis, (Mn 700), potassium peroxosulfate (KPS), naringenin used as a carrier material, sodium phosphate dibasic dodecahydrate (Na ₂ HPO ₄ .12H ₂ O) were all available from Sigma-Aldrich Co. (St. Louis, Mo., USA). Sodium hydroxide (NaOH, assay = 98.0%) was purchased from OCI Co. Ltd. (Seoul, Korea), sodium dihydrogen phosphate dihydrate (NaH ₂ PO ₄ .2H ₂ O) was purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan) were used. Solvents such as 1,3-butylene glycol (1,3-BG), ethanol, and polyoxyl 60 hydrogenated castor oil (HCO-60) And distilled water was purified by using Milli-Q. The UV-visible spectrophotometer was a Cary 50 from Varian (Australia) and a pH meter from Hanna (Korea).

Example 1: Synthesis of hydrogel

The synthesis process of the hydrogel is shown below. The radical reaction can be divided into three stages: initiation, propagation, and termination. In the initiation step, KPS produces radicals in sodium carboxymethylcellulose (CMC) under thermal conditions. In the propagation phase, the radicals generated in CMC induce polymerization of 2-HEA monomers and PEGDA. Finally, after all of the radicals produced react, the radical reaction is terminated. The hydrogels were prepared with different concentrations of grafting agent and crosslinking agent.

50 mL of distilled water and 0.5 g of CMC were placed in a 250 mL three-necked round-bottomed flask, and dissolved sufficiently by stirring at 75 ° C and 400 rpm in water. N ₂ gas was injected into the dissolved CMC solution for 20 minutes to sufficiently lower the oxygen partial pressure in the flask, and 0.005 g of KPS as a radical initiator was added thereto. 20 minutes after the initiator was added, 2-HEA was added to start grafting. After the 2-HEA injection, the crosslinking agent, PEGDA, was added and the reaction proceeded for 3 hours. At the end of the reaction, the reaction mixture was cooled to room temperature. At this time, the stirring speed was controlled during synthesis to induce uniform synthesis. The compound was dialyzed in 5 L distilled water for at least 3 days to remove unreacted crosslinking agent and monomer. Thereafter, the hydrogel was dried for 72 hours using a freeze dryer (-60 ° C, 5 mm Torr; Ilshin biobase, Seoul, Korea) and weighed. The weight of the dried gel until no weight change is the weight of the dried gel (W _d ). The gelation rate was calculated from the weight (W _i ) of the polymer initially used and the weight (W _d ) of the dried gel.

······ (1)

······ (One)

Hydrogel # CMC
(w / w%) 2-HEA
(w / w%) PEGDA
(w / w%) KPS
(mg) Water (mL) Gelation rate (%) cl-CMC-g-pHEA-1 One 7 0.4 5 50 61.7 ± 2.2 cl-CMC-g-pHEA-2 One 9 0.4 5 50 81.9 ± 1.8 cl-CMC-g-pHEA-3 One 11 0.4 5 50 74.2 ± 1.9 cl-CMC-g-pHEA-4 One 9 0.2 5 50 82.6 ± 4.1 cl-CMC-g-pHEA-5 One 9 0.6 5 50 79.2 ± 3.0

Experimental Example 1: Analysis of hydrogel

Experimental Example 1-1. Spectroscopic analysis of hydrogel

The chemical structure of the synthesized material was confirmed by FT-IR and ¹ H NMR spectroscopy. FT-IR spectroscopy of the hydrogel of CMC, 2-HEA, PEGDA and dried Example 1 was obtained from an ATR-FTIR spectrometer (Travel IR, Smiths Detection, USA). It was measured in the range of 650-4000 cm ^-1 using Smart Orbit ATR accessory diamond crystal and Omnic 8.0 software. The network system of cl-CMC-g-pHEA hydrogel is shown in Fig. 1 in comparison with CMC, 2-HEA and PEGDA. 1631 and 1635 cm ^-1 of 2-HEA and PEGDA are peaks of C = C double bonds conjugated to ester (-COO-). The peaks corresponding to the C = C double bonds (1600-1650 cm ^-1 ) conjugated in the spectra of the synthesized hydrogels disappeared, indicating that the double bonds of 2-HEA and PEGDA disappeared, Respectively. As a result, it was confirmed that the hydrogel was well formed.

In addition, the broad peak at 3500 ~ 3300 cm ^-1 indicates the stretching vibration of -OH, and the peaks at 2950 cm ^-1 and 2884 cm ^-1 indicate the asymmetric and symmetric stretching vibration of methylene CH, respectively. 1723 and 1720 cm ^-1 are stretching vibration bands of ester C═O and 1396 cm ^-1 are symmetrically stretching vibration bands of carboxylate (-COO ^- ) in the CMC skeleton. Peaks at 1246 cm ^-1 and 1165 cm ^-1 represent the stretching vibration of ester CO and the peaks at lower frequencies compared to the stretching vibrations of ester CO in 2-HEA and PEGDA are 1276 cm ^-1 and 1195 cm ^-1 , , since the double bond of the? -unsaturated ester (-C = C-COO-) is lost and ester CO bond strength is weakened. 1076 cm ^-1 and 945 cm ^-1 are symmetrically stretched vibration absorption bands of ether (-COC-) CO and ether CO, including stretching vibration of secondary alcohol CO, respectively, and ether CO (1107 cm ^-1 , 952 cm ^-1 ), the weaker bond strength due to hydrogen bonding is thought to be due to the weaker absorption band at lower frequencies.

¹ H NMR spectra of CMC, 2-HEA and PEGDA were performed using an NMR Spectrometer (Model: DD2 700, Agilent Technologies-Korea, USA). ¹ H NMR spectroscopy of the synthesized hydrogel was obtained using a 700 MHz FT-NMR spectrometer (Bruker, Rheinstetten, Germany). The spin rate was 6000 Hz, the temperature was 298 K, the probe was high-resolution magic angle spinning (HR-MAS) and the recycle delay time was 4 seconds.

2 shows the ¹ H NMR spectrum of CMC, 2-HEA, PEGDA and the ¹ H HR-MAS NMR spectrum of the cl-CMC-g-pHEA hydrogel. The anomeric proton (H1) was observed at 4.576 ppm in the ¹ H NMR spectrum of CMC, and the proton (H2-H7) of the methylene protons and the sugar rings of the substituted carboxymethyl groups appeared at 3.395-4.310 ppm. For 2-HEA, chemical shifts between δ = 6.527-6.553, 6.288-6.328 and 6.076-6.091 ppm are due to the protons associated with unsaturated carbons, namely H8, H9 and H8 '. The chemical shifts of δ = 3.925-3.929 and 4.349-4.353 ppm are due to protons at the H10 and H11 positions, respectively. In the case of PEGDA, chemical shifts to the unsaturated protons of δ = 6.526-6.551, 6.289-6.330 and 6.090-6.105 ppm, H12, H13 and H12 ', respectively. While chemical shifts between δ = 3.770-3.803, 3.900 and 4.427 ppm represent protons of H14 / H15, H16 and H17, respectively. chemical shifts between δ = 4.486, 4.210-4.217, 3.455 and 3.302 ppm in the ¹ H HR-MAS NMR spectrum of the cl-CMC-g-pHEA hydrogel resulted in anomeric proton (H1) and other protons (H2-H7 ). The chemical shifts of δ = 3.593-3.599, 3.738, 3.854, 4.022 and 4.116-4.146 ppm are due to H17, H10 / H18, H8, H12 and H11 / H19 protons, respectively. The chemical shifts between δ = 1.609-1.746, 1.949-1.956, 2.384 and 2.458 ppm are due to the H13, H15, H9 / H14 and H16 protons, respectively. The NMR spectra of the synthesized hydrogels showed no chemical shift between δ = 6.076-6.553 ppm (unsaturated protons), a new chemical shift between δ = 1.609-2.458 ppm, and 2-HEA and PEGDA polymerization Respectively. Chemical shifts at δ = 3.854 ppm (H8) confirm that CMC and 2-HEA are grafted to the -OH group of CMC. On the other hand, no chemical shifts of the unsaturated protons of PEGDA were observed, and new chemical shifts at δ = 1.949-1.956 and 2.458 ppm were observed, indicating that PEGDA crosslinked the copolymers of CMC and poly (2-HEA) cl-CMC-g-pHEA hydrogel.

Experimental Example 1-2. Morphological analysis

Porosity is an important property of hydrogels because high porosity is beneficial to drug release through drug loading and diffusion. Thus, the hydrogel of Example 1 was observed morphologically. The three - dimensional network structure of the hydrogel was confirmed by SEM (Tescan Vega3, Tescan, Czecho) with 15 kV acceleration voltage. The dried hydrogel was observed after depositing Pt, and each sample was photographed at a magnification of x500 at a voltage of 1.0 to 1.5 kV during measurement and shown in Fig.

As shown in FIG. 3, the hydrogel of Example 1 all showed a porous structure. CMC-g-pHEA-1 (40 to 89 탆)> cl-CMC-g-pHEA-4 (33 to 56 탆)> cl- CMC-g-pHEA-3 (12 to 26 탆)> cl-CMC-g-pHEA-5 (12 to 22 탆). As the grafting and crosslinking agent concentrations increased, the pore size decreased. From the viewpoint of the water molecules and dissociated water molecules bound to the hydrogel, as the pore size increases, the proportion of water molecules bound to the hydrogel decreases and the proportion of dissociated water molecules increases. Therefore, as the concentration of the grafting agent and the crosslinking agent is lower, the pore size increases, so that the collection efficiency of the aqueous solution carrying the active ingredient is increased and the skin is easily transferred.

Hydrogels can be classified into four types according to the size and structure of pores. Non-porous, micro-porous with a pore size of 100-1000 Å, macroscopically porous with a diameter of 0.1-1 urn, depending on the size of the pores, And super-porous with a diameter of several tens to several hundreds of micrometers. Among them, non-porous, micro-porous, and macroporous are closed cell structures without inter-pore connection. The hydrogel of Example 1 identified in FIG. 3 is considered to be a super-porous hydrogel (SPH) having an open cell structure interconnected with inner pores with a larger diameter. SPH is a capillary system that captures moisture quickly into the pore structure and is widely used in drug delivery systems (DDS).

Experimental Examples 1-3. Thermal gravimetric analysis

Thermo Gravimetric Analysis (TGA) measures the mass change of a substance with temperature and provides useful information such as component analysis, thermal stability, and moisture content of multi-component materials. Particularly in the field of transdermal drug delivery, moisture content is an important feature. In this experiment, the TGA curve was analyzed to evaluate the water binding capacity and water evaporation rate of the hydrogel of Example 1.

Thermal gravimetric analysis of the hydrogel was performed using JP / DTG-60 (Shimadzu, Tokyo, Japan). The swollen hydrogel was washed with an excess amount of water using a filter paper, and the initial weight was weighed to about 20 mg. Under a nitrogen atmosphere at a rate of 50 mL / min and at a heating rate of 3 DEG C / min from 20 DEG C to 250 DEG C. [ As a standard material,? -Alumina powder was used and the results are shown in FIG.

All of the hydrogels showed a similar curve in which the mass decreased with temperature change from 20 ° C to 140 ° C. This phenomenon is due to water evaporation in the hydrogel. The moisture of the hydrogel began to evaporate, and the mass of the hydrogel at 140 ° C was mostly decreased. CMC-g-pHEA-1 (95.3%)> cl-CMC-g-pHEA-4 (90.3%)> cl-CMC-g-pHEA-1 when the hydrogel mass loss rate at 100 ° C was compared. CMC-g-pHEA-3 (81.5%)> cl-CMC-g-pHEA-5 (79.2%). These results are consistent with the results of the SEM observation, as shown above, as the size of the pores increases as the ratio of dissociated water molecules increases and the water-binding capacity of the hydrogel decreases to increase the water evaporation rate.

Experimental Examples 1-4. Measurement of swelling of hydrogel

Hydrogels are generally characterized by swelling when exposed to external solvents. This swelling behavior is mainly related to the osmotic force, the electrostatic force and the resilience of the viscoelasticity. Important factors for the swelling rate and swelling kinetics of hydrogels are porosity, which is affected by the size of the pores and the porous structure.

The swelling phenomenon of the hydrogel was observed according to the pH. The pH was controlled by adjusting the pH of the skin to 5.5 and the pH of the skin to 7.5 and 8.5, respectively. The dried hydrogel of Example 1 was cut to a constant weight (100 mg) and then placed in a phosphate buffered saline (PBS) buffer solution at pH 5.5, 7.5 and 8.5. After swelling for 24 hours at 37 ° C, only the water on the surface of the hydrogel was removed and the weight was measured. The swelling ratio was calculated using equation (2). The results are shown in Fig.

······ (2)

······ (2)

W _s : weight of swollen hydrogel (g)

W _d : weight of dried hydrogel (g)

The cl-CMC-g-pHEA hydrogel of all Example 1 increased swelling rate as the pH increased from 5.5 to 7.5 or 8.5. These results are due to carboxyl group ionization and proton balance of CMC. At pH 5.5, the carboxyl groups have been protonated, and strong hydrogen bonds between them cause predominant polymer-polymer interactions than polymer-water interactions. On the other hand, high pH values induced ionization of carboxyl groups, and electrostatic repulsion between them further extended the network, thereby increasing the swelling power of the hydrogel. The cl-CMC-g-pHEA hydrogel was found to follow the Henderson-Hassel-Baha equation which expresses the degree of ionization of the hydrogel in the swelling medium. However, as the pH was increased from 7.5 to 8.5, the charge of the carboxylic acid anion was blocked due to the charge blocking effect of ions such as Na ⁺ present in the medium, thereby reducing the electrostatic repulsion between the polymer chains. As a result, the swelling value was slightly decreased.

The effect of grafting agent and crosslinker concentration on the swelling value can also be seen in FIG. As the content of 2-HEA and PEGDA increased, the swelling value decreased. It is well known that the swelling rate of the hydrogel depends on the polymer chain and the cross-link density. The high polymer chains and crosslinking densities result in a dense gel structure that reduces the swelling ratio, limits interrangement repulsion and slows the permeation of the medium. These pH-reactive hydrogels can be designed to release the drug from a limited pH to the desired target.

Experimental Examples 1-5. Analysis of Mechanical Properties of Hydrogel

1) Rheological properties

The rheological properties analysis provides quick and accurate information on the elasticity and viscosity of the hydrogel. The storage elastic modulus G 'means energy stored without loss by elasticity, and the loss elastic modulus G' means energy lost by viscosity. Another useful parameter is tan?, Which represents the viscoelasticity of the material. The tan δ is represented by the value of G "/ G ', which can be regarded as an index indicating the physical properties of the produced hydrogel. Generally, when the tan δ value is greater than 1, it is close to the liquid state, while when it is smaller than 1, it is close to the solid state. tan δ becomes smaller as the viscoelastic behavior increases and the gel stiffens.

The rheological properties of the hydrogel of Example 1 were measured at 37 占 폚 using a Thermo Haake MARS rheometer (Thermo Electron GmBH, Karlsruhe, Germany) equipped with a 35 mm parallel flat plate probe. The sample was cut to the diameter of the probe to a thickness of 2 mm. The frequency sweep measurement was performed in the range of 10 ^-2 to 10 ^-1 Hz. An amplitude sweep was performed with a change of shear stress (from 10 to 1000 Pa) at 1 Hz to find the yield stress. The shear viscosity of the gel samples was measured between shear rate range 1-500 / s.

FIG. 6 (a) shows the storage elastic modulus (G ') and the loss modulus (G ") independent of the frequency of the cl-CMC-g-pHEA-4 hydrogel having the highest gelation rate. The gel is G 'independent of frequency and the storage modulus (G') is higher than the loss modulus (G "). Therefore, the cl-CMC-g-pHEA hydrogel showed the rheological properties of the gel. In Fig. 6 (b) showing the amplitude sweep measurement, G 'was maintained above G "at a certain limit of shear stress. In addition, after certain shear stresses, both G 'and G "were abruptly reduced due to the bridging network breakdown of the hydrogel. This shear stress is referred to as yield stress (). Also, FIG. 6 (c) shows a tendency that the shear viscosity continuously decreases with an increase in the shear rate, indicating the shear walling behavior of cl-CMC-g-pHEA-4.

2) Texture analysis

When a hydrogel is used as a transdermal delivery system, the skin exhibits very dynamic movement and therefore requires more intimate contact between the gel and the skin. The hardness may indicate the applicability of the hydrogel to the skin, and the adhesiveness may be an indicator of the duration of the skin application site. These properties are directly related to polymer concentration and composition. Texture analysis provides reliable information about these properties.

A texture analyzer (TA.XT Express Enhanced, Stable Micro Systems, Godalming, Surrey, UK) was used for the texture analysis of the hydrogel. The load cell used was able to measure up to 5 kg of force and a disk probe of 40 mm diameter was used. Trigger load was 5.0 g, strain was 30%, probe speed was fixed at 2.0 mm / s and compressed twice at room temperature. The hardness, cohesiveness, resilience, chewiness, adhesiveness, and springiness of the hydrogel were calculated from the resulting force-distance curves using Texture Exponent 32 software . The results are shown in Figs. 7 (a), (b), (c), (d), (e) and (f).

As the grafting ratio increased, the hardness and cohesion also increased (cl-CMC-g-pHEA-1 <cl-CMC-g-pHEA-2 <cl-CMC-g-pHEA-3). (Cl-CMC-g-pHEA-4 < cl-CMC-g-pHEA-2 < cl-CMC-g-pHEA-5) as the ratio of crosslinking increases. As the ratio of grafting and crosslinking increases, the hydrogel becomes entangled when it forms a network, resulting in a hard, agglomerated gel structure. Therefore, a loose gel structure is formed when the ratio of grafting and crosslinking is low. The elasticity and chewiness showed the tendency such as hardness and cohesion. In particular, cl-CMC-g-pHEA-1, which has the least amount of 2-HEA to improve elasticity, showed very low elasticity. The chewiness is closely related to the hardness since it is calculated as hardness x cohesion x elasticity value.

Adhesion may result from dehydration by hydrophilic functional groups such as -OH. The hydrogel of Example 1 had decreased adhesion when the polymeric polymer and cross-link density were increased. This is considered to be the result that the adhesion to the other surface is lowered as the attractive force inside the gel increases. However, in this experimental example, the adhesiveness can not be explained only by the attractive force between the gel surface and the probe surface. A small piece of the gel sticks to the probe and can be separated from the original gel sample during the drawing process. The adhesiveness is closely related not only to the adhesion to the probe surface but also to the cohesion of the gel itself. Therefore, in order to produce an optimal transdermal delivery system, the gel must have a balance of adhesion and cohesion so that the duration of the application at the hydrogel application site is prolonged. On the other hand, the elasticity (height of gel after extrusion / height of gel before extrusion) was measured to be close to 1 without any difference in all samples.

Experimental Example 2: Cytotoxicity of hydrogel

In order to use the hydrogel as a skin transporter, it was confirmed whether it was cytotoxic. Cytotoxicity was assessed using an assay of methylthiazoldiphenyl-tetrazolium bromide (MTT). The hydrogel was eluted at 1 × 1 cm in size on a cell culture medium at 37 ° C. for 3 days and 7 days to obtain an eluate. HaCaT cells were plated at a density of 1 × 10 ⁴ cells in a 96-well plate, ₂ , and 37 ° C, and the eluted solution was dispensed. The cytotoxicity was evaluated by comparing the survival rate of HaCaT cells by the sample using MTT assay. The absorbance at 570 nm was measured using an ELISA reader and the absorbance of the untreated group was determined as negative control (100%), and the cell survival rate relative to the treated group according to equation (9) Respectively. This is shown in Fig.

······ (9)

(9)

As a result, it was confirmed that all the experimental groups showed cell viability of 90% or more and did not show cytotoxicity.

Experimental Example 3. In vitro drug release of hydrogel

Experimental Example 3-1. Drug collection efficiency and release behavior

Among the hydrogels of Example 1, a cl-CMC-g-pHEA-4 hydrogel showing a high gelation rate, excellent adhesion and adequate cohesion was judged suitable as a transdermal delivery system. Therefore, drug release behavior according to pH was confirmed for the gel.

100 mg of the dried cl-CMC-g-pHEA-4 hydrogel was immersed in 20 mL of a 20% 1,3-BG solution containing 0.5 mM naringenin in PBS to measure the drug capture efficiency of the hydrogel. The hydrogel carried a solution of narinin at 37 ° C for 24 hours. The hydrogel was removed from the solution and the liquid on the surface was removed.

The collection efficiency was analyzed by using a calibration curve of Naringenin by UV / Vis spectrophotometer. Absorbance was measured at the maximum absorption wavelength of naringinin at 292 nm. The amount of drug trapped in the hydrogel was calculated by Equation (3).

······ (3)

(3)

IE: incorporating efficiency

V ₁ : Volume of initial drug solution (mL)

V ₂ : volume of remaining drug solution (mL)

C ₁ : initial drug concentration (mM)

C ₂ : concentration of remaining drug (mM)

The hydrogel collecting efficiency for naringenin calculated from this was 51.5%. This collection is due to the swelling of the hydrogel.

On the other hand, the drug release behavior was evaluated with phosphate buffered saline (PBS) buffer solutions at pH 5.5, 7.5 and 8.5. Naringenin-loaded hydrogel was immediately released into the release medium (20% 1,3-BG in PBS) and released at 37 ° C. The release medium (0.5 mL) was withdrawn at regular time intervals and a fresh medium (0.5 mL) was added to maintain a constant volume. Release studies were performed for 24 hours. The concentration of the drug was measured with a UV / Vis spectrophotometer, which is shown in FIG.

The emission amount was calculated by considering the capture efficiency of naringenin as 51.5%. After 24 hours of release, the cumulative release of the drug at pH 5.5, 7, 5 and 8.5 was 42%, 73% and 70%, respectively. The rate of drug release was slightly different depending on the pH condition, which is consistent with the degree of swelling. That is, the release was suppressed at low pH conditions and the release was promoted at high pH. This is a result suggesting that pH in the cl-CMC-g-pHEA hydrogel system may affect drug release. Thus, it was confirmed that the present invention can be applied to a transdermal drug delivery system targeting atopic skin having a high pH skin condition.

Experimental Example 3-2. Drug release rate and mechanism

As shown in FIG. 9, although there was a slight difference in the release rate depending on the pH, all of them showed early release early. The crosslinked network of porous hydrogels can not control structurally surface drug release as soon as it is exposed to the release medium. Thus allowing free elution of the drug initially. On the other hand, these drug releases did not stop at the beginning, but were found to increase continuously with time. Therefore, the drug release behavior of the cl-CMC-g-pHEA hydrogel was evaluated using a mathematical model.

The in vitro drug release mechanism of hydrogels can be explained by erosion of network structure and diffusion of naringenin. This mechanism is suitable for mathematical models such as first-order, Hixson-crowell and Higuchi equations. The equation is calculated by Polymath professional 6.1, and the best model has the highest correlation coefficient (R ² ). The drug release mechanism can be deduced based on equations including diffusion, corrosion and coupling mechanisms. The model equations are as follows.

(4)First-order model:

(4)

(5)Hixson-crowell model:

(5)

(6)Higuchi model:

(6)

Where M _t is the fraction of drug released at each time, K ₁ , K _hc, and K _h represent the first-order, Hixson-crowell, and Higuchi release rate constants, respectively.

The results are shown in Table 2 below.

Based on the correlation coefficient (R ² ), the in vitro drug release of the hydrogel was better followed by the Higuchi model, a diffusion release system, than the Hixson-crowell model, which is the erosion release system, Respectively. In order to examine more clearly the release process, Korsmeyer-Peppas and Peppas-Sahlin models were used.

Using the Korsmeyer-Peppas model, we have mechanically analyzed the mechanism by which the system dissolves when it releases the active material.

(7)Korsmeyer-Peppas model:

(7)

Where M _t is the rate of drug released at each time, K is the rate constant incorporating the structural and geometric characteristics of the system, and n is the release index indicating the mechanism of drug release. Thus, the value of n can be used to characterize the mechanism of drug release. At n ≤ 0.45, the release of drug by diffusion follows the predominant Fickian diffusion mechanism, with non-Fickian transport at 0.45 <n <0.89 and dominant drug release by corrosion at n = 0.89 case II transport, and 0.89 ≤ n are described as following the super case II transport mechanism. Drug release curves within 60% are used to determine the release index (n).

All experimental results (100% emission curve) were analyzed using another model proposed by Peppas and Sahlin. This has been proposed to explain the relaxation behavior of polymers as well as diffusion in drug release mechanisms from polymer matrices.

(8)Peppas-Sahlin model:

(8)

M _t is the ratio of released drug at each time, and the value of constant m is determined by the aspect ratio of the matrix. The K _f constant is the contribution by the Fickian diffusion, and the K _r constant represents the contribution to the polymer relaxation.

These results are shown together in Table 2 above.

As a result, the release behavior of cl-CMC-g-pHEA hydrogel showed a high correlation (R ² > 0.95) and a significant analysis. Korsmeyer-Peppas (n <0.45) equation and _{Peppas-Sahlin (K r <K} f) of these drug release according to the equation was characterized by dominant Fickian diffusion mechanism in the diffusion process. In addition, as the pH increased, the diffusion constant, K _f , increased, and the polymer relaxation constant, K _r, was lower than that of K _f . As a result, the release of naringenin in the cl-CMC-g-pHEA hydrogel is predominantly diffusion-dependent, and it is believed that the rate of release of the drug can be controlled by the pH of the skin.

Experimental Example 4. In vitro skin permeation of hydrogel

In vitro skin permeation studies of synthesized hydrogels were performed using Franz diffusion cells (Permegear, USA). Subcutaneous fat and excess tissue were removed from the skin of hairless mice (6 weeks of age, female). The experiment was carried out using a Franz diffusion cell (Permegear, USA). The skin was fixed with the stratum corneum facing up between the donor and the receptor phase. The area of the skin to which the sample was applied was 0.6362 cm ² . The receptor chamber was filled with a receptor (HCO-60: EtOH: PBS = 2: 20: 78 (v / v%)) and stirred at 150 rpm for 24 hours. The temperature was maintained at 37 캜 using a constant temperature water bath. As a control, naringenin was dissolved in PBS and PBS containing 20% 1,3-BG at the same concentration. The center hole of the donor was made to form the compartment. The skin in the compartment was treated with 0.1 mL of the same concentration of the sample, and the amount of the hydrogel was treated in consideration of the collection efficiency. After 24 hours, the receptor phase was sampled through a sampling port. To measure the amount of naringenin contained in the stratum corneum and skin, the surface of the skin was washed with PBS solution. The stratum corneum was removed three times by stripping using a 3 M scotch tape (Korea 3M), and then the tape and naringenin in the skin were dissolved by ultrasonication with 100% ethanol, respectively. The concentration of naringenin sampled was determined by UV / Vis spectrophotometer. At this time, the pH of the experiment was 7.4. The results are shown in Fig.

The results are shown in Fig. 10 by the contents of naringenin (tape) present in the stratum corneum, naringenin (skin) existing in the epidermal layer and dermal layer excluding the stratum corneum, and naringenin (transdermal) . After 24 hours, the total permeation amount of cl-CMC-g-pHEA-4 hydrogel was 58.8%, which was better than that of 1,3-BG and 20% 1,3-BG / PB solution (43.5 and 42.4%, respectively). On the other hand, the highest amount of 1,3-BG solution was found to exist in the stratum corneum. This indicates that the moisturizing agent and solvent components alone are difficult to penetrate through the skin and that naringenin can not pass through the dermal layer and remains in the stratum corneum outermost layer. However, the amount (transdermal) passed from the 20% BG / PB solution to the dermal layer slightly increased. The pathway through the pores can penetrate the most active ingredients. Therefore, it seems that permeation between pores was more easily occurred when PB was mixed than with 1,3-BG having viscosity.

The amount of drug (tapes and skin) present in the epidermis and dermis layers including the stratum corneum was slightly different from that of the control group. However, the amount of drug (transdermal) permeated to the dermis layer Respectively. This is because the hydrogel seals the surface of the skin, thereby promoting hydration of the skin, thereby further improving the diffusion of the drug. It is believed that the hydrated stratum corneum temporarily interferes with the function of the skin barrier, whereby the active ingredient carried on the hydrogel is easily permeable to the skin. In conclusion, in the 24-hour skin permeation experiment, cl-CMC-g-pHEA hydrogel showed the highest skin permeability when compared with the control group. This showed that naringenin was delivered to the skin to protect various skin diseases This study suggests that the drug delivery system can be applied as an effective drug delivery system.

Experimental Example 5. Antibacterial activity of hydrogel

Staphylococcus aureus is the predominant bacterium in atopic skin and has been selected to evaluate the antimicrobial activity of the cl-CMC-g-pHEA hydrogel loaded with naringenin. The antimicrobial activity was measured by a disc diffusion assay. The cultured strains were used in this experiment at a concentration of 1 × 10 ^ 6 to 7 CFU / mL. Each strain was plated on a plate medium with a sterile swab of 100 μl. The hydrogels were cut so that the cross section of the hydrogels became circular, and the hydrogels were adhered on a plate-coated flat plate culture medium. The hydrogels were cultured to measure the clear zone (mm) produced around the disk.

The results of antibacterial activity of cl-CMC-g-pHEA-4 carrying naringenin are shown in FIG. 11 and Table 3.

For the release of naringenin, the surface of the naringenin-loaded hydrogel was treated with pH 5.5, 7.5 and 8.5 buffer and adhered onto the medium (Fig. 11 (b)). In the control group, the surface of the non-naringenin-loaded hydrogel was treated with pH 5.5, 7.5 and 8.5 buffer to adhere onto the medium (Fig. 11 (a)). As a result, the hydrogel containing naringenin showed an inhibitory effect on S. aureus , and showed a higher antimicrobial activity especially when treated with pH 7.5 and 8.5 buffer. These results are consistent with the drug release studies and show that more antinicrobial activity is exhibited because more naringenin is released at pH 7.5 and 8.5. This tendency is expected to work effectively in acidic membrane-destructive skin diseases. On the other hand, no inhibition was observed in the control group. Thus, the cl-CMC-pHEA hydrogel may be applied as a skin delivery system for the treatment of skin diseases such as atopy.

Inhibition diameter (mm) pH 5.5 pH 7.5 pH 8.5 (a) cl-CMC-g-pHEA-4 - - - (b) Naringenin loaded cl-CMC-g-pHEA-4 16 ± 2 35 ± 3 32 ± 3

Although the embodiments have been described with reference to the drawings, various technical modifications and variations may be applied to those skilled in the art. For example, it is to be understood that the techniques described may be performed in a different order than the described methods, and / or that components of the described systems, structures, devices, circuits, Lt; / RTI &gt; or equivalents, even if it is replaced or replaced.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (5)

Carboxymethylcellulose; And a copolymer of 2-hydroxyethyl acrylate, which is crosslinked with poly (ethylene glycol) diacrylate,
Wherein the hydrogel is capable of selective drug release under the condition of pH 7 to 9 when the drug is loaded in the hydrogel, and the drug is naringenin.
delete delete A drug carrier comprising the hydrogel according to claim 1.
A pharmaceutical composition for the treatment of diseases showing a lesion of pH 7 to 9, wherein the disease is selected from the group consisting of atopy, athlete's foot, athlete's foot, psoriatic skin and dry skin, the composition comprising the hydrogel according to claim 1 and a naringenin &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof.

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