KR20210060174A - Composition including cyclodextrin-functionalized agarose for delivering drugs and method for preparing same - Google Patents

Abstract

According to the present application, cyclodextrin is introduced into agarose to lower the high gelation temperature of agarose to a low temperature of room temperature, and to induce sustained release of a drug. According to an embodiment of the present application, the present invention is to provide a method for manufacturing a composition for carrying drugs comprising: a step of preparing agarose and cyclodextrin, respectively; and a step of introducing cyclodextrin into agarose to form agarose functionalized with cyclodextrin.

Description

A composition for drug-bearing containing agarose functionalized with cyclodextrin, and a method for manufacturing the same TECHNICAL FIELD {COMPOSITION INCLUDING CYCLODEXTRIN-FUNCTIONALIZED AGAROSE FOR DELIVERING DRUGS AND METHOD FOR PREPARING SAME}

The present application relates to a drug-carrying composition comprising agarose functionalized with cyclodextrin and a method for manufacturing the same, and a functionalized agar having a low gelation temperature by functionalizing a cyclodextrin capable of supporting a hydrophobic drug and capable of sustained release It relates to a drug-carrying composition containing rose and a method of manufacturing the same.

Hydrogel is easy to use as a drug delivery agent because it has good biocompatibility and is made of hydrophilic polymer. There are two representative methods of loading a drug on a hydrogel.

The first is a method of loading a drug while the hydrogel is prepared, and is a method of loading the drug by putting the hydrogel in a solution containing the drug. However, this method is difficult to control the drug loading, and it is difficult to provide stability of the drug.

The second method is to put drugs when manufacturing the hydrogel. However, while this method is easy to control the drug loading, the hydrogel constituents may react with the drug or the constituents do not polymerize and remain toxic.

Agarose is a biocompatible polymer and has low toxicity and gelation properties at a high temperature of about 65 degrees Celsius, so it is possible to load a drug as a second method, but it is known that it is difficult to load a protein product or drug that is sensitive to temperature.

In the case of derivatives in which an alkyl group or a hydroxypropyl group is substituted for agarose, the gelation temperature is lower, so that drug loading can be performed stably. Since the mechanical strength of the prepared agarose is lowered, there is a problem that the drug in the hydrogel is easily released.

It is time to study on a sustained-release drug delivery system capable of solving these problems, low gelation temperature, excellent mechanical strength, and capable of carrying hydrophobic drugs.

Korean Patent Registration No. 10-1633137 (published on June 23, 2016)

According to the present application, by introducing cyclodextrin into agarose, it is intended to lower the high gelation temperature of agarose to a low temperature level of room temperature, and to induce sustained release of the drug.

According to an embodiment of the present application, the steps of preparing agarose and cyclodextrin, respectively; And it is intended to provide a method for preparing a composition for drug delivery comprising the step of forming agarose functionalized with cyclodextrin by introducing cyclodextrin into agarose.

According to an embodiment of the present application, it is intended to provide a composition for drug delivery comprising agarose functionalized with cyclodextrin.

According to an embodiment of the present application, it is intended to provide a drug delivery system comprising the above-described composition.

According to an exemplary embodiment of the present application, a composition for drug delivery having a low gelation temperature may be provided.

According to an exemplary embodiment of the present application, a composition for drug delivery having excellent mechanical strength may be provided.

According to an exemplary embodiment of the present application, a composition for drug delivery capable of carrying a hydrophobic drug may be provided.

According to an exemplary embodiment of the present application, a composition having low cytotoxicity may be provided.

According to an exemplary embodiment of the present application, a composition for sustained-release drug delivery may be provided.

1 is a flow chart for explaining a method of manufacturing a composition for drug delivery of the present application.
2 is a schematic diagram for explaining a method of manufacturing a composition for drug delivery of the present application.
3 is a graph showing the results of 13C NMR spectrum analysis according to an embodiment of the present application.
4 is a graph showing the results of 13C NMR spectral analysis of p-toluenesulfonyl cholide.
5 is a graph showing the results of 13C NMR spectral analysis of TOAG.
6 is a graph showing the results of 13C NMR spectral analysis of ETAG.
7 is a graph showing the results of 13C NMR spectral analysis of cyclodextrin.
8 is a graph showing the results of 13C NMR spectral analysis of CFA.
9 is an image of agarose and CFA-1 according to temperature.
10 is a graph of the results for the loss factor and storage factor of agarose, CFA-1, CFA-2 and CFA-3 gels.
11 is a graph showing the results of Differential Scanning Calorimetry of agarose, CFA-1, CFA-2, and CFA-3 gels.
12 is an image of agarose, CFA-1, CFA-2, and CFA-3 gels taken with FE-SEM.
13 is a graph of the results of thioflavin T (ThT) analysis.
14 is a graph of the results for the amount of drug release compared to 1 hour.
15 is a graph of storage coefficient and loss coefficient versus temperature of βCD/agarose gel.
16 is a graph of the drug delivery behavior of βCD/agarose gel.
17 is a graph of storage coefficient and loss coefficient versus temperature of 1% CFA-1 and 3% CFA-1 gels.
18 is a graph showing the amount of drug release for various samples.
19 is a graph showing the results of the toxicity evaluation of the hydrogel for HEk 293 cells and the toxicity evaluation of the hydrogel and DOX-loaded hydrogel for HeLa cells.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "include" or "have" are intended to designate that features, elements, etc. described in the specification exist, but one or more other features or elements may not exist or be added. It doesn't mean none.

Unless otherwise defined, 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 the present invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.

In this specification, the term "TOAG" is an abbreviation referring to tosylated agarose.

In the present specification, the term "ETAG" is an abbreviation referring to tosylated agarose.

In the present specification, the term "βCD" is an abbreviation referring to β-cyclodextrin.

In the present specification, the term "Suc-βCD" is an abbreviation for a beta-cyclodextrin substituted with a monomolecular succinate (mono succinyl β-cyclodextrin).

In this specification, the term "EDC-NHS coupling" is an abbreviation for 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling.

In the present specification, the term "CFA" is an abbreviation for a beta-cyclodextrin functionalized agarose (β-cyclodextrin functional agarose).

An embodiment of the present application provides a composition capable of supporting not only hydrophilic drugs but also hydrophobic drugs, while lowering the gelation temperature to room temperature by introducing cyclodextrin to agarose. It is possible to provide a controlled release drug delivery system.

Hereinafter, the composition for drug delivery of the present application and a method of manufacturing the same will be described in detail with reference to the accompanying drawings. However, the accompanying drawings are exemplary, and the scope of the composition for drug delivery of the present application and a method of manufacturing the same is not limited by the accompanying drawings.

First, a method of preparing a composition for drug delivery, which is an aspect of the present application, will be described.

1 is a flow chart for explaining a method of manufacturing a composition for drug delivery of the present application.

2 is a schematic diagram for explaining a method of manufacturing a composition for drug delivery of the present application.

1 and 2, agarose and cyclodextrin are prepared, respectively (S10).

Agarose is a polysaccharide and is generally derived from certain red algae. It is a linear polymer consisting of repeating units of agarobiose, a disaccharide composed of D-galactose and 3,6-anhydro-L-galactopyranose with α(1, 4) and β(1, 3) linkers. These polysaccharides form a double helix structure at low temperature to form a gel-like structure insoluble in water. Although used in a variety of applications, it is also limited due to its high stiffness and high gelling temperature. In particular, tissues or drugs that are sensitive to high temperatures are easily deactivated at high temperatures. However, in order to generate the agarose gel, heating and cooling processes are required, and gelation occurs at a high temperature, making it difficult to load heat-sensitive drugs. Therefore, as described later, by introducing cyclodextrin, the gelation temperature can be lowered.

Cyclodextrin has a ring structure through α-(1,4) bonds of glucopyranose units, and contains 6 units of α-cyclodextrin, 7 units of β-cyclodextrin, and 8 units of γ-cyclodextrin. do. In the present application, it is preferable to use beta-cyclodextrin. Since the hydroxy group is located outside the ring, the outer ring of the ring is hydrophilic, and the inside of the ring is hydrophobic. By using the hydrophobic properties inside the ring, fitting by size with the hydrophobic guest molecules occurs, forming a host-guest inclusion complex. Cage-forming properties of cyclodextrin are not formed by covalent bonds, but are formed by interactions between molecules. In particular, βCD is introduced into the hydrogel and can change the rheological and hydrophilic properties of the hydrogel. In particular, the introduction of a functional group hinders the formation of a helical structure at low temperatures, thereby lowering the gelation temperature of the agarose.

As shown in Figure 3, the step of preparing the agarose includes tosylation of at least one hydroxyl group of the agarose to form an agarose into which a tosyl group is introduced, and the tosyl At least one tosyl group of the agarose into which the group is introduced is ethylnedeamined to form an agarose into which an amine group is introduced. Through this, the agarose is substituted with a primary amine group. In each step, additional substances, for example, solvents, etc. may be added for the need or efficiency of the reaction.

The agarose into which the amine group is introduced is ethylenediamino agarose (ETAG), and the degree of substitution (DS) of the amine for the ETAG is preferably 0.3 or less. In the case of 0.3 or less, when cyclodextrin is conjugated to ETAG, an amine-substituted derivative, CFA is dissolved in water.

As shown in Figure 3, the step of preparing the cyclodextrin includes: succinating at least one hydroxyl group of cyclodextrin to form a cyclodextrin into which a succinate single molecule is introduced. do. In each step, an additional substance, for example, a solvent, or the like may be added for the need or efficiency of the reaction.

By introducing the prepared cyclodextrin to the prepared agarose, agarose functionalized with cyclodextrin is formed (S20).

As shown in Figure 3, the step of forming agarose functionalized with the cyclodextrin is: 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added, EDC/NHS coupling (1-ethyl-3 -[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling) to form agarose functionalized with cyclodextrin.

That is, a primary amine group is bonded to agarose, a carbboxyl group is bonded to a cyclodextrin, and a chemical reaction is introduced with cyclodextrin to agarose through EDC/NHS coupling. As shown in the experimental examples to be described later, the effect is different from that of physically including cyclodextrin.

In agarose gels, the diffusion properties of various substances including drugs are related to the rheological properties of the gel. The rheological properties of agarose gels are determined by the amount of agarose in the gel formulation, and agarose gels with a high storage coefficient have a low diffusion rate of a substance, whereas a low storage coefficient has a high Indicates the rate of diffusion. Therefore, the agarose derivative having a low gelation temperature can deliver a material quickly because the double helix structure is reduced and the storage coefficient is lowered during gelation.

In addition, through such synthesis, the prepared composition can provide a low gelation temperature and control the release rate of the drug delivery system, as described later.

Hereinafter, a composition for drug delivery, which is an aspect of the present application, will be described.

All the contents described in the above-described preparation method or synthesis method of the drug delivery composition may also be applied to the drug delivery composition. Therefore, in order to prevent overlapping descriptions, descriptions that can be applied identically are excluded.

The composition comprises agarose functionalized with cyclodextrin. As described above, agarose is a linear polymer composed of repeating units of agarobiose.

The cyclodextrin is bound to agarose by a linker, and the linker is a carbodiimide formed by EDC/NHS coupling (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling) (carbodiimide).

As described above, the cyclodextrin is preferably beta-cyclodextrin.

The cyclodextrin may carry some or all of a predetermined drug. As described above, by forming a hose-guest-containing complex, a predetermined drug may be included. Here, the drug is not particularly limited. Hydrophilic drugs as well as hydrophobic drugs may be included. In the following experimental examples, BSA and DOX are described as examples, but these are only examples for explanation, and the present application is not limited thereto.

Agarose is generally not soluble in water, but as an agarose derivative, CFA may be water-soluble, which can be achieved when the degree of substitution of the amine for ETAG is 0.3 or less.

In addition, the composition may be gelled at 32 °C or less. As described above, by introducing cyclodextrin, the gelation temperature can be greatly reduced.

Hereinafter, a drug delivery system which is an aspect of the present application will be described.

All the contents described in the above-described drug delivery composition, a method of manufacturing a drug delivery composition, or a synthesis method can also be applied to a drug delivery system. Therefore, in order to prevent overlapping descriptions, descriptions that can be applied identically are excluded.

The drug delivery system may include all compositions for drug delivery. In addition, all components included in order to act as a drug delivery system may be additionally included. This specification does not specifically describe these components. And, it may include all the properties of the composition.

Hereinafter, a hydrogel which is an aspect of the present application will be described.

All the contents described in the above-described drug delivery system, drug delivery composition, preparation method of drug delivery composition, or synthesis method may also be applied to hydrogels. Therefore, in order to prevent overlapping descriptions, descriptions that can be applied identically are excluded.

Hydrogels include the above-described drug delivery system and deionized water. Here, the drug delivery system may be included in a concentration of 1 to 3% (w/v). In this concentration range, the gelation temperature of the hydrogel may be formed as low as 21 °C to 32 °C. In addition, depending on the concentration of 1 to 3% (w/v), the drug release rate and the like can be controlled, and as the dose increases, the drug may be gradually released (sustained release).

The agarose derivatives prepared by substituting various groups with conventional agarose can reduce the gelation temperature by interfering with the super-helical agglomeration of agarose. However, the three-dimensional network formation of the hydrogel is reduced due to the reduced super-helical agglomeration. The intensity is lowered. Therefore, it exhibits rapid drug release characteristics as a result.

However, in the present application, the introduction of cyclodextrin into agarose prevents the formation of helical structures and super-helical aggregates of agarose, thereby inducing the formation of super-helical aggregates at a low temperature to provide a hydrogel capable of gelation. In addition, by using the hydrophobic drug of cyclodextrin and the ability to form a host-guest complex in the agarose gel, the drug can be sustained release.

Hereinafter, the present application will be described in more detail through experimental examples.

[Experimental Example 1]

Agarose (electroendosmosis (EEO) value: 0.05-0.13; sulfate, <0.15%) was purchased from BioPure (Seoul, Korea), and βCD and pyridine were purchased from Samchun Pure Chemical Co. Succinic anhydride, N-hydroxy succinimide (NHS) and bovine serum albumin (BSA) were purchased from Sigma Aldrich (St. Louis, Mo.). Dimethylsulfoxide (DMSO), p-toluene sulfonyl chloride (TsCl), ethylene diamine, 1,3-dimethyl-2-imidazolidinone (DMI) and 1-(3-dimethylaminopropyl)-3-ethylcar Bodyimide hydrochloride (EDC) was purchased from Tokyo Chemical Industry. Doxorubicin (DOX) was purchased from Pharmachemie (BV, Haarlem, The Netherlands). mono-6-sucninicyl-6-deoxy-βCD (Suc-βCD) was provided by the Microbial Carbohydrate Resource Bank of Kongu University in Korea.

In order to tosylate the prepared agarose (TOAG-1), agarose (2g, 6.53mmol) was dissolved in 100mL DMI at 140°C for 2 hours. After the mixture was cooled to 25° C. overnight, triethylamine (1.983 g, 19.59 mmol) was added to the reaction mixture followed by TsCl (1.866 g, 9.796 mmol). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was precipitated with 500 mL of ethanol. The obtained solid product was washed 3 times with ethanol. Then, the white powder was dried in vacuo. TOAG-2 and TOAG-3 were prepared similarly to TOAG-1. For TOAG-2 and TOAG-3, 39.19 mmol of trimethylamine and 19.59 mmol of TsCl were added to the agarose solution, and the mixture was stirred for 1 hour and 6 hours, respectively.

TOAGs were substituted nucleophilically by ethylene diamine. TOAG-1, TOAG-2 and TOAG-3 (1 g) were dissolved in 35 mL of anhydrous DMSO at 85° C. under nitrogen. Ethylenediamine (5 mL) was added to the mixture and stirred continuously at 85° C. for 24 hours. Ethylenediamine-modified agarose (ETAG-1, ETAG-2 and ETAG-3) was collected through ethanol precipitation. The solid product was dried under vacuum at 60°C.

Beta-cyclodextrin (βCD) functionalized agarose (βCD functionalized agarose, CFA) was prepared with Suc-βCD and ETAG using the EDC-NHS coupling method. Suc-βCD was analyzed using Bruker Autoflex Speed MALDI-TOF MASS (Bruker Daltonics, USA) and 1H NMR spectroscopy (Bruker Avance III, 500 MHz) analysis. EDC (115.02 mg, 0.3 mmol) was added to 7 mL of DMSO, followed by NHS (69.054 mg, 0.3 mmol) and Suc-βCD (251.4 mg, 0.2 mmol) and the reaction mixture was stirred at room temperature for 3.5 hours. . ETAG-1 (314.4 mg) was dissolved in 43 mL of DMSO. The Suc-βCD mixture was added to the ETAG-1 solution. The reaction mixture was stirred at 45° C. for 24 hours. CFA-1 was collected in 200 mL of ethanol. The precipitate was removed by filtration, washed three times with acetone, and then dried at 60° C. under vacuum. CFA-2 and CFA-3 were similarly synthesized using CFA-1 of ETAG-2 and ETAG-3, respectively. For the synthesis of CFA-2 and CFA-3, two and three times the amount of EDC, NHS and Suc-βCD, respectively, were used. All hydrogels were prepared at 2% (w/v) using deionized water (DW).

Since ETAG was not dissolved in water, the NHS-EDC coupling reaction between ETAG and Suc-βCD was performed in DMSO. All agarose and agarose derivatives were analyzed using elemental analysis, and the results are shown in Table 1 below. Elemental analysis was performed on a Vario-Micro Cube elemental analyzer (ELEMENTAR LTD., Germany). The amounts of C, H, N, and S of each sample were measured, and the DS value was calculated.

[Table 1]

a: DS was calculated using the N or S ratio.

b: DS of toluenesulfonyl group in anhydrous galactose of the agarose unit.

c: DS of the ethyleneamino group in the anhydrous galactose of the agarose unit.

d: DS of Suc-βCD group in anhydrous galactose of ETAG unit.

e: DS of unreacted ethyleneamino group in anhydrous galactose of the CFA unit.

In the case of ETAG, an amine-substituted derivative of agarose, only derivatives with a degree of substitution (DS) of 0.3 or less in which the amine is substituted in the galactose of agarose are substituted with Cyclodextrin afterwards using EDC/NHS coupling. It was confirmed through an experiment described below that it dissolves in water when conjugated to the resulting derivative ETAG.

In order to measure the NMR spectra of agarose and agarose derivatives, each sample was recorded on a Bruker Avance III 500 MHz spectrometer (Karlsruhe, Germany) at 25°C. Agarose and agarose derivatives were dissolved in DMSO-d6 for NMR analysis. Fig. 3 shows the results of 13 C NMR spectrum analysis.

Agarose has a hydroxyl group at the G-6 position, which can be used for various chemical modifications. As shown in Fig. 3, 13C NMR spectra of agarose and modified TOAG-1, ETAG-1 and CFA-1 are shown. In the 13C NMR spectrum (Fig. 2b), the tosyl group in TOAG-1 appears through the appearance of new peaks at 145.02 (C-7), 131.89 (C-10), 130.25 (C-8) and 127.80 ppm (C-9). Was able to confirm. Figure 4 shows the 13C NMR spectrum of pure tosyl chloride. This result indicates that the tosyl group was successfully functionalized in agarose. From Table 1 and FIG. 5, it was confirmed that the spectrum of TOAG prepared from DMI exhibited increasingly higher intensity depending on the molar ratio and reaction time.

DS values for tosylation were obtained using 13C NMR spectroscopy and elemental analyzer, and these values varied from 0.1 to 0.6 under different experimental conditions. 3B shows the 13C NMR spectrum of ETAG-1; When the tosyl group was replaced with ethylene diamine, the peak of the tosyl group found above 120 ppm disappeared. This means that referring to FIG. 6, the tosyl groups of ETAG-2 and ETAG-3 are all substituted with ethylenediamine.

The 13C NMR spectrum of CFA-1 revealed a characteristic peak of βCD observed after the NHS-EDC coupling reaction. In general, βCD peaks appear at 102.58, 72.67, 73.89, 81.94, 72.89 and 61.17 ppm.

The six peaks of cyclodextrin in CFA-1 after reaction are 101.32 (Cy-1), 81.50 (Cy-4), 73.02 (Cy-3), 72.39 (Cy-5), 72.01 (Cy-2) and 60.00 ( Cy-6) appeared at ppm. Referring to FIG. 7, it can be compared with pure beta-cyclodextrin, and it was confirmed that cyclodextrin was well introduced in CFA-1.

Although the amount of amine substituents in ETAG that can react with Suc-βCD increased, similar DS values were shown for all CFAs. Large-sized βCD may cause steric hindrance in the coupling reaction, and as a result, it was confirmed that not all βCDs were capable of reacting with the ethylenediamino group of agarose (see FIG. 8). Thus, CFA-1 and CFA-3 were replaced by βCD to a similar extent, and the agarose with the widest βCD substitution was CFA-2.

After βCD substitution in ETAG, CFA was dissolved in deionized water (DW) at 60-100 °C. In addition, CFA-1 exhibited an altered gelation temperature compared to the agarose gel. 9 is an image of agarose and CFA-1 according to temperature. As shown in Fig. 9, the initial start of gelation of agarose was approximately 50-60°C, whereas CFA-1 formed a gel state at 25°C.

[Experimental Example 2]

Rheological analysis can provide information on the mechanical properties of the hydrogel. Thus, the rheological properties, gelation temperature (Tg) and melting temperature (Tm) of the gel sample were analyzed using a DHR-2 rheometer (TA Instruments, USA) equipped with a 40-mm parallel plate. Before the experiment, each gel was prepared as a 2% (w/v) sample in 100° C. deionized water. The measured temperature range of the cooling cycle was 100° C. to 10° C., and the heating cycle was 10° C. to 100° C. with a temperature change of 1° C./min. It was set to an angular frequency of 1.0 Hz and a strain of 5%. The sample was covered with silicone oil to prevent water vapor during the measurement.

The gel state shows a storage coefficient (G') higher than the loss factor (G''), and the sol state shows the opposite result in rheological analysis. Therefore, during the cooling and heating process, rheological analysis can be used to determine the Tm and Tg values of the gel through the intersection of the G'and G'' curves. Figure 10 shows the loss factor and storage factor of agarose, CFA-1, CFA-2 and CFA-3 gels upon heating (Figures 10a, c, e and g) and during cooling (Figures 10b, d, e and h). The loss factor and storage factor of agarose, CFA-1, CFA-2 and CFA-3 gels are shown.

As shown in Fig. 10, Tm and Tg of the agarose gel were 95°C and 65°C, respectively. However, the Tm and Tg values for the CFA-1 gel were 66.1°C and 26.7°C, which is lower than the lower sol-gel transition temperature than for the agarose gel. In general, agar or agarose molecules take the shape of an arbitrary coil with a uniform distribution in water at a temperature close to the boiling point. The agarose molecule forms a double helix at low temperatures. Three of the four hydroxyl groups of the 3,6-anhydrogalactose of agarose can participate in hydrogen bonding with adjacent double helices. The helical shape allows one or more agarose molecules to participate in and maintain the gelled state at low temperatures. These agglomerated double helices are called suprafibers and ^{can contain 10 4} double helices. The stiffness or rheological properties of the gel depend on the degree to which the double helix is assembled.

Thus, the ethylenediamine and βCD molecules introduced into the CFA interfere with the hydrogen bonding of 3,6-hydrogalactose of agarose, resulting in assembly. It is smaller than the original agarose of double helix and results in a sol-gel transition that occurs at low temperatures.

In addition, CFA-2 and CFA-3 with more ethylene diamine substituents did not maintain an appropriate gel state, and in particular, in the case of CFA-3, the sol-gel transition point could not be confirmed. The ethylenediamine group having an amine group capable of hydrogen bonding is insoluble in water as the degree of agarose substitution increases.

One reason is that it can be expected that ETAG cannot effectively form random coils at high temperatures due to substitution of ethylenediamine. ETAG-1 prepared to produce CFA-1 was not dissolved in water. However, cross-linking between Suc-βCD and the primary amine group of ethylenediamine may interfere with the bonding of the amine group of ethylenediamine in hydrogen, and as a result, CFA-1 can be soluble in water.

11 is a graph of the results of differential scanning calorimetry (DSC) of agarose, CFA-1, CFA-2, and CFA-3 gels. In DSC analysis, a strong endothermic peak appeared at 140° C. in agarose, but the endothermic peak of CFA decreased. The endothermic peak represents a thermal transition in which hydrogen bonds are split between polymers, which also suggests that agarose has more hydrogen bonds and other strong bonds than CFA.

During the heating process, both agarose and CFA-1 gel maintained a gel form at 37° C., but the G'of agarose was about 100 KPa and 1600 Pa for CFA-1 (see, FIGS. 10A and C). In addition, G'of CFA-2 and CFA-3 is 3.4513 and 0.0469 Pa, respectively, and CFA-2 and CFA-3 cannot maintain a gel form at 37° C. (FIGS. 10e and g). This result also means that the stiffness strength of agarose is the highest among the hydrogels studied. Therefore, it was confirmed that the higher the DS of the ethylenediamino group and βCD, the weaker the stiffness of the agarose-based gel.

[Experimental Example 3]

Agarose, CFA-1, CFA-2 and CFA-3 (20 mg) were each dissolved in 1 mL of deionized water at 100°C. Each sample was held at 25° C. for 24 hours and then freeze-dried. Freeze-dried samples were fixed using double-sided adhesive carbon tape for FE-SEM analysis. The surfaces of the hydrogels were made electrically conductive by coating them with platinum at 30 W for 120 seconds in vacuum. The surface of the hydrogel was observed using Hitachi S-4700 (Tokyo, Japan). 12 is an image of agarose, CFA-1, CFA-2 and CFA-3 gels taken with FE-SEM.

As shown in Fig. 12, the CFA-1 gel is similar to the agarose gel, but exhibits relatively large pores in the cross section of the gel. In addition, the cross section of the agarose gel was very complex and various pore sizes were observed. In general, the pore size of the agarose gel can be determined based on the concentration and temperature.

As the concentration of agarose increased, the stiffness increased and the pore size decreased. Thus, the large pores of the CFA-1 gel indicate that the same amount of agarose was used to form the gel, but the degree of aggregation of the double-helis is smaller than that of the agarose gel. Unlike CFA-1, CFA-2 gel did not show specific pores, whereas CFA-3 showed fibrous appearance. In rheological analysis, CFA-2 and CFA-3 show a G'value greater than G'at room temperature, indicating that they do not maintain the gel state like CFA-1. Both CFAs have a functionalized βCD of CFA-1 or higher, but still exhibit different forms as there are many unreacted ethylenediamino groups attached to the agarose backbone. Many of the ethylene diamino groups substituted on agarose interfere with the formation of the double helix structure of the agarose. This FE-SEM result was confirmed to be consistent with the results of rheological studies.

[Experimental Example 4]

A total of 40 mg of each sample was dissolved in 4 mL of deionized water at 100° C. using an autoclave. BSA (400 μL, 50 mg/mL) and DOX (400 μL, 2 mg/mL) solutions were added to the sample solution. For agarose, the loading temperature was 65° C. for uniform loading conditions. The loading temperature of the other samples was 30°C. Drug-loading samples were held at 25° C. for gelation using a cold incubator. After 24 hours, it was immersed in pH 7.4 phosphate buffer (PB) at 37° C. for drug release testing. Then, 500 μl aliquots were periodically collected from the release buffer solution, after which an equal volume of fresh buffer was added simultaneously. Emitted BSA and DOX were observed using a UV-vis spectrometer (UV-2450, Shimadzu, Japan) at 280 and 480 nm, respectively. The cumulative amount of bovine serum albumin (BSA) and doxorubicin (DOX) was calculated as follows:

Where V is the volume of the release buffer, Vi is the volume of the sample collected, and Cn and Ci are the concentration of the drug in the sample collected at each time.

BSA and DOX were chosen for drug loading and release testing. BSA is an α-helical multi-domain protein that aggregates above 60 ℃, and DOX is an anticancer agent that decomposes when exposed to ultraviolet rays or heat. BSA and DOX were loaded on agarose and CFA. In order to uniformly load the hydrogel, drug loading for agarose was performed at 65°C, and for CFA at 30°C. Through thioflavin T (ThT) analysis, it was confirmed whether BSA could aggregate during the loading process. The results are shown in FIG. 13. It was confirmed that there was no aggregation. This is because the agarose gel loaded with BSA was quickly transferred to the cooling incubator and a small amount of the agarose gel can be quickly cooled through the cooling incubator. 14 is a graph of the results of the amount of drug release versus time. As shown in Figure 14, it shows the amount of release of BSA and DOX. The release rate of BSA in the hydrogel reflects the rheological properties, and the release rate is lower with a higher G'. BSA is released through diffusion in a typical hydrogel. The higher the crosslinking point of the gel, the slower the diffusion and release rate of BSA, but in the case of an agarose gel, the higher the agarose concentration, the slower the diffusion rate. Because CFA has a lower crosslinking point than agarose due to low double helix formation, BSA was almost completely released from CFA within 12 hours. CFA has a lower crosslinking point than agarose with low double helix formation. CFA-2 and CFA-3 gels disintegrated in PBS buffer within 4 hours. Since agarose gels have a solid network unlike other gels, approximately 70% of the BSA was released from the agarose gel within 12 hours. In an agarose gel, the release and diffusion rate of a solute is related to the concentration and stiffness of the agarose regardless of the size of the solute.

For DOX release, almost all DOX was released from CFA-2 and CFA-3 within 12 hours, which is similar to the release profile of BSA. Neither agarose nor CFA-1 completely released DOX within 24 hours. DOX is often released very slowly in hydrogels, i.e. over a week or longer. The release behavior of DOX in the CFA-1 gel was similar to that of the agarose gel until the first 6 hours. The storage coefficient of CFA-1 gel was 1600 Pa at 37° C. similar to 0.5% agarose gel. This sustained release of DOX is due to the formation of a complex between DOX and βCD inside the CFA-1 gel. Since the amount of βCD in the CFA-1 gel was less than the amount of DOX loaded, it showed effective sustained-release characteristics for 6 hours.

In addition, βCD was physically introduced into agarose (βCD/agarose gel). A hydrogel was prepared by dissolving βCD and agarose together, and the gelation temperature and drug delivery behavior of the βCD/agarose gel were measured, and are shown in Figs. 15 and 16, respectively. 15 and 16, βCD/agarose gel showed both gelation temperature and drug delivery behavior similar to that of the agarose gel alone. This is because when βCD is not functionalized and physically introduced into the agarose, it does not change the gelation process or network of the agarose. Additionally, while controlling the concentration of CFA-1, drug release experiments were performed for 1% CFA-1 and 3% CFA-1. A graph of the storage coefficient and loss coefficient versus temperature of the 1% CFA-1 and 3% CFA-1 gels is shown in FIG. 17. As shown in FIG. 17, the 1% CFA-1 and 3% CFA-1 gelling temperatures were 23.2°C and 31.2°C, respectively. 18 is a graph showing the amount of drug release for various samples. 18, 3% CFA-1 gel released BSA faster than agarose, but released DOX slowly. The 3% CFA-1 gel shows a high storage coefficient of approximately 3.1 KPa (data not shown) compared to the 2% CFA-1 gel, but this value is still lower than the agarose gel. Nevertheless, 3% CFA-1 released DOX more slowly than agarose gels due to the large amount of βCD in 3% CFA-1. In drug release from a hydrogel functionalized with βCD, the amount of functionalized βCD and the concentration of the hydrogel component are both important. The lower the amount of functionalized βCD, the less the formation of host-guest interactions, the faster the released hydrophobic drug, and the lower the concentration of the hydrogel composition, the denser the network structure of the hydrogel, less rigidity, and faster. Mass movement occurs. From these results, CFA-1 gel showed the potential for agarose gel-based controlled drug delivery system. This is because it can be easily gelled at low temperatures and, despite its low stiffness compared to agarose gel, it is possible to control the sustained release of the drug forming an inclusion complex with βCD.

[Experimental Example 5]

The cytotoxicity of hydrogels and drug-loaded hydrogels was evaluated using WST-applied human embryonic kidney 239 cells (HEK-293, Korea Cell Line Bank, Korea) and HeLa (cervical cancer cell line, Korea Cell Line Bank, Korea) cells. . WST-8 analysis was performed through a conventionally known method. The drug-loaded hydrogel was prepared in the same way as the DOX loading experiment. HEK-293 and HeLa cells were cultured in 24 wells at a cell density ^{of 3x10 4} cells/mL in minimal essential medium (MEM, WELGENE, Gyeongsan-si, South Korea) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Seed on a plate. 50 μg of DOX, 5 mg of hydrogel and 5 mg of DOX-loaded hydrogel (containing 50 μg of DOX) were each added at 37° C. in an atmosphere containing _{5% CO 2.} After 24 hours of incubation, cell viability was measured.

Through an in vitro cytotoxicity assay, the cytotoxicity of the hydrogel for use as a drug delivery system was evaluated. HEK 293 cells are cells commonly used in cytotoxicity tests to evaluate the effect of drug candidates, and HeLa is a cancer cell line frequently used to evaluate the activity of anticancer agents.

Toxicity evaluation of the hydrogel to HEk 293 cells and the toxicity evaluation of the hydrogel and DOX-loaded hydrogel to HeLa cells were performed, and a graph of the results is shown in FIG. 19.

DOX-loaded CFA-2 and CFA-3 gels were able to release the same amount of DOX relative to the DOX-treated control, so the cell viability of HeLa cells was almost the same as that of native DOX. For DOX-loaded agarose gels, DOX was slowly released from the agarose gel, resulting in a cytotoxicity of approximately 40% for HeLa cells. Because CFA-1 gels can accommodate large amounts of DOX release, they exhibited higher cytotoxicity against HeLa cells compared to DOX-loaded agarose gels. Through these results, it was confirmed that the released DOX maintains the same bioactivity as the natural DOX, and the CFA-1 gel can be used in a drug delivery system without cytotoxicity.

Through these experiments, it was confirmed that βCD was successfully cross-linked to the agarose main chain using EDC-NHS coupling. Introduction of βCD or ethylene diamino groups into the agarose resulted in a change in rheological properties, reducing the storage coefficient of the hydrogel. The CFA-1 gel had a lower melting and gelling temperature than the agarose gel. This means that the introduced βCD or ethylenediamino groups prevented the formation of internal double helix aggregates during gelation of the agarose. In particular, CFA-1 gel was able to easily and uniformly load drugs at 30°C. The CFA-1 gel demonstrated sustained release of DOX in contrast to BSA via a host-guest complex containing βCD in CFA-1 despite its low stiffness. DOX-loaded CFA gel retained the anticancer activity of the drug. In addition, the CFA gel did not show toxicity to HEK-293 and HeLa cells. In conclusion, CFA has lower stiffness than agarose, so it can rapidly release proteins such as BSA, which cannot form complexes with CD, but slowly releases drugs such as DOX in complexes with βCD. CFA gels with such a low gelation temperature have the potential to provide a controllable hydrogel drug delivery system for heat-sensitive chemical drugs, and due to the nature of low storage coefficient and gelation temperature, can provide an injectable hydrogel system. Was able to confirm.

Although the above has been described with reference to the preferred embodiments of the present application, those skilled in the art will be able to variously modify and change the present application within the scope not departing from the spirit and scope of the present invention described in the following claims. You will understand that you can.

Claims (16)

Preparing agarose and cyclodextrin, respectively; And
A method for preparing a composition for drug delivery comprising the step of introducing cyclodextrin to agarose to form agarose functionalized with cyclodextrin. The method of claim 1,
The cyclodextrin is beta-cyclodextrin production method. The method of claim 1,
The step of preparing the agarose is:
Tosylation of at least one hydroxyl group of the agarose to form agarose into which a tosyl group is introduced; And
A manufacturing method comprising the step of forming an agarose into which an amine group is introduced by ethylenediamination of at least one tosyl group of the agarose into which the tosyl group is introduced. The method of claim 3,
The agarose into which the amine group is introduced is ethylenediamino agarose (ETAG),
The amine for the ETAG has a degree of substitution (DS) of 0.3 or less. The method of claim 1,
Preparing the cyclodextrin is:
A method comprising the step of succinating at least one hydroxyl group of the cyclodextrin to form a cyclodextrin into which a succinate single molecule is introduced. The method of claim 1,
The step of forming agarose functionalized with the cyclodextrin is:
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added and functionalized into cyclodextrin by EDC/NHS coupling (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling). A manufacturing method that is a step of forming an agarose. A composition for drug delivery comprising agarose functionalized with cyclodextrin. The method of claim 7,
The cyclodextrin is a composition bonded to agarose by a linker. The method of claim 8,
The linker is a composition comprising a carbodiimide formed by EDC/NHS coupling (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling). The method of claim 7,
The cyclodextrin is a beta-cyclodextrin composition. The method of claim 7,
The composition is a water-soluble composition. The method of claim 7,
The cyclodextrin is a composition that supports some or all of a predetermined drug. The method of claim 7,
The composition is a composition that is gelled at 32 °C or less. A drug delivery system comprising the composition of any one of claims 7 to 13. A hydrogel comprising the drug delivery system of claim 14 and deionized water. The method of claim 15,
The drug delivery composition is a hydrogel contained in a concentration of 1 to 3% (w / v).

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