KR102312456B1 - 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.

Description

Composition for drug loading comprising agarose functionalized with cyclodextrin and method for manufacturing the same

The present application relates to a drug-loading composition comprising agarose functionalized with cyclodextrin and a method for preparing the same, wherein the functionalized agar with a low gelation temperature is functionalized with a cyclodextrin capable of supporting a hydrophobic drug and capable of sustained release by supporting a hydrophobic drug It relates to a composition for drug loading comprising rose and a method for preparing the same.

The hydrogel has good biocompatibility and is made of a hydrophilic polymer, making it easy to use as a drug delivery agent. There are typically two methods of loading a drug into a hydrogel.

The first is a method of loading a drug in a state in which the hydrogel is manufactured, and 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 a method of adding a drug when manufacturing a hydrogel. However, while this method is easy to control the drug loading, the hydrogel composition may react with the drug or the composition may remain unpolymerized, resulting in toxicity.

Agarose is a biocompatible polymer that has low toxicity and gels at a high temperature of about 65 degrees or less.

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

It is time to study a sustained-release drug delivery system capable of solving these problems, having a 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, 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, preparing agarose and cyclodextrin, respectively; and introducing cyclodextrin into agarose to form agarose functionalized with cyclodextrin.

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

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

According to an embodiment of the present application, it is possible to provide a composition for drug delivery having a low gelation temperature.

According to an embodiment of the present application, it is possible to provide a composition for drug delivery excellent in mechanical strength.

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

According to an embodiment of the present application, it is possible to provide a composition with less cytotoxicity.

According to an embodiment of the present application, it is possible to provide a composition for sustained-release drug delivery.

1 is a flowchart for explaining a method for preparing a composition for drug delivery of the present application.
2 is a schematic diagram for explaining a method of manufacturing the composition for drug delivery of the present application.
3 is a 13C NMR spectrum analysis result graph of an embodiment of the present application.
4 is a graph showing the results of 13C NMR spectrum analysis of p-toluenesulfonyl cholide.
5 is a graph of the 13C NMR spectrum analysis result of TOAG.
6 is a graph showing the results of 13C NMR spectrum analysis of ETAG.
7 is a graph showing the results of 13C NMR spectrum analysis of cyclodextrin.
8 is a graph showing the results of 13C NMR spectrum analysis of CFA.
9 is an image according to the temperature of agarose and CFA-1.
10 is a graph showing the loss coefficient and storage coefficient 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 taken by FE-SEM of agarose, CFA-1, CFA-2 and CFA-3 gels.
13 is a graph of results for Thioflavin T (ThT) assay.
14 is a graph of the results for the amount of drug release compared to 1 hour.
15 is a graph of the 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 evaluation of the toxicity of the hydrogel to HEk 293 cells and the evaluation of the toxicity of the hydrogel and DOX-loaded hydrogel to 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. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that the features, components, etc. described in the specification are present, and one or more other features or components may not be present or may be added. Doesn't mean there isn't.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

As used herein, the term “TOAG” is an abbreviation for tosylated agarose.

As used herein, the term “ETAG” is an abbreviation for tosylated agarose.

As used herein, the term "βCD" is an abbreviation for beta-cyclodextrin (β-cyclodextrin).

As used herein, the term “Suc-βCD” is an abbreviation for beta-cyclodextrin substituted with monomolecular succinate (mono succinyl β-cyclodextrin).

As used herein, the term “EDC-NHS coupling” is an abbreviation for 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling.

As used herein, the term "CFA" is an abbreviation for beta-cyclodextrin functionalized agarose (β-cyclodextrin functional agarose).

An embodiment of the present application provides a composition that introduces cyclodextrin into agarose, lowers the gelation temperature to room temperature, maintains excellent mechanical strength, and can support not only hydrophilic drugs but also hydrophobic drugs, in particular, the release rate of the drug It is possible to provide a sustained-release drug delivery system with controlled

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

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

1 is a flowchart for explaining a method for preparing a composition for drug delivery of the present application.

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

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

Agarose is a polysaccharide and is usually extracted from certain red algae. It is a linear polymer composed 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 it is used in a variety of applications, it is also limited by its high stiffness and high gelation temperature. In particular, tissues or drugs sensitive to high temperatures are easily inactivated at high temperatures. However, heating and cooling processes are required to produce agarose gel, and it is difficult to load a heat-sensitive drug because gelation occurs at a high temperature. Therefore, as described later, the gelation temperature can be lowered by introducing cyclodextrin.

Cyclodextrin has a ring structure of glucopyranose units through α-(1,4) bonds, and includes 6 units of α-cyclodextrin, 7 units of β-cyclodextrin, and 8 units of γ-cyclodextrin. do. In the present application, beta-cyclodextrin is preferably used. Since the hydroxyl group is positioned outside the ring, the outer ring has hydrophilic properties and the inside of the ring has hydrophobic properties. By using the hydrophobic property of the inside of the ring, fitting by size with hydrophobic guest molecules occurs, thereby forming a host-guest inclusion complex. The cage-forming properties of cyclodextrins are not formed by covalent bonds, but by intermolecular interactions. In particular, βCD can be introduced into the hydrogel to alter the rheological and hydrophilic properties of the hydrogel. In particular, the introduction of functional groups hinders the formation of helical structures at low temperatures, thereby lowering the gelation temperature of the agarose.

As shown in FIG. 3 , the preparing of the agarose includes tosylation of at least one hydroxyl group of the agarose to form an agarose having a tosyl group introduced thereto, and the tosyl group. and ethylenediaminated (ethylnedeamine) at least one tosyl group of the group-introduced agarose to form an amine group-introduced agarose. Through this, the primary amine group is substituted for the agarose. In each step, additional substances such as solvents and the like 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 FIG. 3 , the step of preparing the cyclodextrin includes: succination of at least one hydroxyl group of the cyclodextrin to form a cyclodextrin into which a succinate single molecule is introduced. do. In each step, additional substances, for example, solvents, etc. 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 FIG. 3 , the step of forming agarose functionalized with 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 carboxyl group is bonded to the cyclodextrin, and a chemical reaction of the cyclodextrin is introduced into agarose through EDC/NHS coupling. As shown in the experimental examples to be described later, the effect is different from that of physically including the cyclodextrin.

The diffusion properties of various substances including drugs in agarose gels are related to the rheological properties of the gels. The rheological properties of agarose gels are determined by the amount of agarose in the gel formulation. Agarose gels with a high storage modulus have a low diffusion rate of substances, whereas a low storage modulus has a high storage coefficient regardless of the material’s properties or size. indicates the rate of diffusion. Therefore, agarose derivatives with a low gelation temperature can deliver materials quickly because the double helix structure is reduced during gelation and the storage coefficient is lowered.

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

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

All contents described in the method for preparing or synthesizing the composition for drug delivery described above may also be applied to the composition for drug delivery. Accordingly, in order to prevent overlapping descriptions, descriptions that may be equally applied 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 contain some or all of a predetermined drug. As described above, by forming a hose-guest inclusion complex, a predetermined drug may be included. Here, the drug is not particularly limited. Hydrophobic drugs as well as hydrophilic drugs may be included. In the following experimental examples, BSA and DOX are described as examples, but these are only examples for description, and the present application is not limited thereto.

Agarose is generally insoluble in water, but CFA as an agarose derivative may be water-soluble, which can be achieved when the degree of substitution of the amine for the ETAG is 0.3 or less.

In addition, the composition may be gelled at 32° C. or lower. As described above, by introducing cyclodextrin, it is possible to significantly lower the gelation temperature.

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

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

The drug delivery system may include any composition for drug delivery. In addition, all components included in order to act as a drug delivery system may be additionally included. In this specification, these components are not specifically described. 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 or synthesis method of the drug delivery composition may also be applied to the hydrogel. Accordingly, in order to prevent overlapping descriptions, descriptions that may be equally applied are excluded.

The hydrogel includes the aforementioned drug carrier and deionized water. Here, the drug carrier 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 can be released gradually (sustained release).

Conventional agarose derivatives prepared by substituting various groups in agarose can lower the gelation temperature by interfering with the superhelical aggregation of agarose, but the three-dimensional network formation of the hydrogel is reduced due to the reduced superhelical aggregation, which reduces the mechanical strength is lowered. Therefore, as a result, it exhibits rapid drug release properties.

However, in the present application, a hydrogel capable of gelation is provided by introducing cyclodextrin into agarose to prevent the formation of a helical structure and superhelical aggregates of agarose, thereby inducing the formation of superhelical aggregates at a low temperature. In addition, it is possible to release the drug using the hydrophobic drug and host-guest complex formation ability of cyclodextrin in agarose gel.

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, USA). Dimethylsulfoxide (DMSO), p-toluene sulfonyl chloride (TsCl), ethylene diamine, 1,3-dimethyl-2-imidazolidinone (DMI) and 1-(3-dimethylaminopropyl)-3-ethyl car 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 Konkoo University, Korea.

To tosylate (TOAG-1) the prepared agarose, 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 h. The reaction mixture was precipitated with 500 mL of ethanol. The obtained solid product was washed 3 times with ethanol. The white powder was then vacuum dried. 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 nucleophilically substituted 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 h. Ethylenediamine-modified agarose (ETAG-1, ETAG-2 and ETAG-3) was collected through ethanol precipitation. The solid product was vacuum dried at 60 °C.

Beta-cyclodextrin (βCD) functionalized agarose (βCD functionalized agarose, CFA) was prepared from 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. To 7 mL of DMSO was added EDC (115.02 mg, 0.3 mmol) 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 h. . 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 h. 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. Two-fold and three-fold amounts of EDC, NHS and Suc-βCD were used for the synthesis of CFA-2 and CFA-3, respectively. All hydrogels were prepared at 2% (w/v) using deionized water (DW).

Since ETAG was not soluble 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 to calculate the DS value.

[Table 1]

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

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

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

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

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

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

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. The result of 13 C NMR spectrum analysis is shown in FIG. 3 .

Agarose has a hydroxyl group at the G-6 position, which is available 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 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 check 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 showed higher and higher intensity according to the molar ratio and reaction time.

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

The 13C NMR spectrum of CFA-1 showed 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.

After the reaction, the six peaks of cyclodextrin in CFA-1 were 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) 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 of ETAG that can react with Suc-βCD increased, similar DS values were found for all CFAs. A large size of βCD may cause steric hindrance in the coupling reaction, and as a result, it was confirmed that not all βCDs could react with the ethylenediamino group of agarose (see FIG. 8 ). Thus, CFA-1 and CFA-3 were replaced with βCD to a similar extent, and the agarose with the most extensive 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 altered gelation temperature compared to agarose gel. 9 is an image according to the temperature of agarose and CFA-1. As shown in Fig. 9, the onset of initial gelation of agarose was almost 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 hydrogels. Therefore, the rheological properties, gelation temperature (Tg) and melting temperature (Tm) of the gel samples were analyzed using a DHR-2 rheometer (TA Instruments, USA) equipped with a 40-mm parallel plate. Before the experiment, each gel was prepared with a 2% (w/v) sample in deionized water at 100°C. 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. An angular frequency of 1.0 Hz and a strain of 5% were set. The sample was covered with silicone oil to prevent water vapor during the measurement.

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

As shown in FIG. 10 , the 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 form of arbitrary coils with a uniform distribution in water at a temperature close to the boiling point. Agarose molecules form a double helix at low temperatures. Three of the four hydroxyl groups of 3,6-anhydrogalactose in agarose can participate in hydrogen bonding with adjacent double helices. The helical conformation allows one or more agarose molecules to participate and maintain a gelled state at low temperatures. These aggregated double helices are called suprafibers and ^{may contain 10 4} double helices. The stiffness or rheological properties of a gel depend on the degree to which the double helix is assembled.

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

In addition, CFA-2 and CFA-3 having 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. Ethylenediamine groups having amine groups capable of hydrogen bonding are 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 the substitution of ethylenediamine. ETAG-1 prepared to produce CFA-1 was not soluble in water. However, the cross-linking between Suc-βCD and the primary amine group of ethylenediamine may interfere with the intrahydrogen bonding of the amine group of ethylenediamine, and as a result, CFA-1 may be soluble in water.

11 is a graph showing the results of Differential Scanning Calorimetry (DSC) of agarose, CFA-1, CFA-2 and CFA-3 gels. DSC analysis showed a strong endothermic peak at 140 °C in agarose, but the endothermic peak of CFA decreased. The endothermic peak indicates a thermal transition where hydrogen bonds are split between the polymers, which also suggests that agarose has more hydrogen bonds and other strong bonds than CFAs.

During the heating process, both agarose and CFA-1 gel maintained the gel form at 37 °C, but the G' of agarose was about 100 KPa and 1600 Pa for CFA-1 (cf. 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 their gel form at 37°C (FIGS. 10e and g). This result also means that the stiffness strength of agarose is the highest among the studied hydrogels. Therefore, it was confirmed that the higher the DS of the ethylenediamino group and βCD, the weaker the rigidity 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 kept at 25 °C for 24 h and then lyophilized. Freeze-dried samples were fixed using double-sided adhesive carbon tape for FE-SEM analysis. The surfaces of the hydrogels were coated with platinum at 30 W for 120 s in vacuum to make them electrically conductive. The surface of the hydrogel was observed using a Hitachi S-4700 (Tokyo, Japan). 12 is an image taken by FE-SEM of agarose, CFA-1, CFA-2 and CFA-3 gels.

As shown in FIG. 12 , the CFA-1 gel was similar to the agarose gel, but showed 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 an agarose gel can be determined based on concentration and temperature.

As the concentration of agarose increased, the stiffness increased and the size of the pores 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-helices was smaller than that of the agarose gel. Unlike CFA-1, CFA-2 gel did not show specific pores, whereas CFA-3 showed fibrous form. In the rheological analysis, CFA-2 and CFA-3 show G' values greater than G' at room temperature, indicating that they do not retain the same gel state as CFA-1. Although both CFAs have functionalized βCDs greater than or equal to CFA-1, they still exhibit different morphologies due to the presence of many unreacted ethylenediamino groups attached to the agarose backbone. A number of ethylene diamino groups substituted on agarose prevent the formation of a double helix structure of agarose. It was confirmed that this FE-SEM result was consistent with the rheological study result.

[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-loaded samples were maintained at 25 °C for gelation using a cold incubator. After 24 h, they were 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, 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 amounts of bovine serum albumin (BSA) and doxorubicin (DOX) were calculated as follows:

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

BSA and DOX were chosen for drug loading and release testing. BSA is an α-helical multidomain protein that aggregates above 60 °C, and DOX is an anticancer agent that is decomposed when exposed to ultraviolet light or heat. BSA and DOX were loaded on agarose and CFA. For uniform loading on the hydrogel, drug loading on agarose was performed at 65 °C and CFA at 30 °C. Thioflavin T (ThT) analysis was used to confirm 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 BSA-loaded agarose gel was quickly transferred to the cooling incubator and a small amount of the agarose gel could be rapidly cooled through the cooling incubator. 14 is a graph of the results for the amount of drug release versus time. As shown in FIG. 14 , the amount of BSA and DOX released is shown. The release rate of BSA from the hydrogel reflects the rheological properties, and the release rate is lower with higher G'. BSA is released via diffusion in typical hydrogels. 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 h. CFA has a lower crosslinking point than agarose due to its low double helix formation. CFA-2 and CFA-3 gels disintegrated in PBS buffer within 4 h. Because the agarose gel has a solid network unlike other gels, approximately 70% of the BSA was released from the agarose gel within 12 hours. The release and diffusion rates of solutes in an agarose gel are related to the concentration and stiffness of the agarose, regardless of the size of the solute.

In the case of DOX release, almost all DOX was released from CFA-2 and CFA-3 within 12 h, which is similar to the release profile of BSA. Neither agarose nor CFA-1 completely released DOX within 24 h. DOX is often released from the hydrogel very slowly, i.e. over a week or longer. The release behavior of DOX from the CFA-1 gel was similar to that of the agarose gel until the first 6 h. The storage modulus of the CFA-1 gel was 1600 Pa at 37 °C, similar to the 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 properties 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 shown in FIGS. 15 and 16, respectively. As shown in FIGS. 15 and 16 , the βCD/agarose gel showed both gelation temperature and drug delivery behavior similar to those of the agarose gel alone. This is because βCD does not change the gelation process or network of agarose when physically introduced into the agarose without being functionalized. Additionally, while controlling the concentration of CFA-1, drug release experiments were performed on 1% CFA-1 and 3% CFA-1. A graph of the storage coefficient and loss coefficient versus temperature of 1% CFA-1 and 3% CFA-1 gels is shown in FIG. 17 . As shown in FIG. 17 , the gelation temperatures of 1% CFA-1 and 3% CFA-1 were 23.2° C. and 31.2° C., respectively. 18 is a graph showing the amount of drug release for various samples. As shown in Figure 18, the 3% CFA-1 gel released BSA faster than agarose, but released DOX slowly. Although the 3% CFA-1 gel exhibits a higher storage coefficient of approximately 3.1 KPa (data not shown) compared to the 2% CFA-1 gel, this value is still lower than that of the agarose gel. Nevertheless, 3% CFA-1 released DOX more slowly than the agarose gel due to the large amount of βCD in 3% CFA-1. In drug release from hydrogels functionalized with βCD, both the amount of functionalized βCD and the concentration of hydrogel components are 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, the less rigidity, and the faster the mass movement takes place. From these results, CFA-1 gel showed promise for agarose gel-based controlled drug delivery system. This is because it can easily gel at a low temperature and control the sustained release of a drug that forms an inclusion complex with βCD despite its low stiffness compared to agarose gel.

[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. Drug-loaded hydrogels were prepared in the same manner as for DOX loading experiments. 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. The plates were seeded. 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 hydrogels for use as a drug delivery system was evaluated. HEK 293 cells are cells commonly used in cytotoxicity tests to evaluate the effects of drug candidates, and HeLa is a cancer cell line frequently used to evaluate the activity of anticancer drugs.

The 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 .

The DOX-loaded CFA-2 and CFA-3 gels were able to release the same amount of DOX as compared to the DOX-treated control, and thus the cell viability of HeLa cells was almost identical to that of native DOX. For the DOX-loaded agarose gel, DOX was slowly released from the agarose gel, resulting in a cytotoxicity to HeLa cells of approximately 40%. Because the CFA-1 gel can accommodate large amounts of DOX release, it exhibited higher cytotoxicity against HeLa cells compared to the DOX-loaded agarose gel. Through these results, it was confirmed that the released DOX maintains the same bioactivity as native DOX, and that 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 backbone using EDC-NHS coupling. Introduction of βCD or ethylene diamino groups into the agarose resulted in changes in rheological properties, reducing the storage modulus of the hydrogel. The CFA-1 gel had a lower melting and gelation 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 agarose. In particular, the CFA-1 gel could easily and uniformly load the drug at 30 °C. The CFA-1 gel demonstrated sustained release of DOX in contrast to BSA through the host-guest complex involving βCD in CFA-1 despite its low stiffness. The DOX-loaded CFA gel retained the anticancer activity of the drug. In addition, the CFA gel showed no toxicity to HEK-293 and HeLa cells. In conclusion, since CFA has lower stiffness than agarose, it can rapidly release proteins such as BSA, which cannot be complexed with CD, but slowly release drugs such as DOX from complexes with βCD. CFA gels with such a low gelation temperature have the potential to provide a controllable hydrogel-based drug delivery system for heat-sensitive chemical drugs, and their properties of low storage coefficient and gelation temperature may 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 make various modifications and changes to the present application within the scope without departing from the spirit and scope of the present invention as set forth in the claims below. You will understand that you can.

Claims (16)

Preparing agarose (agarose) and cyclodextrin (cyclodextrin), respectively; and
introducing a cyclodextrin into the agarose to form an agarose functionalized with cyclodextrin;
In the step of forming agarose functionalized with cyclodextrin, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added, and EDC/NHS coupling (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/ A method for preparing a composition for drug delivery, which is a step of forming agarose functionalized with cyclodextrin by N-hydroxysuccinimide coupling). The method of claim 1,
wherein the cyclodextrin is beta-cyclodextrin. The method of claim 1,
The steps of preparing the agarose are:
forming agarose into which a tosyl group is introduced by tosylation of at least one hydroxyl group of the agarose; and
A manufacturing method comprising the step of ethylenediaminated (ethylnedeamine) at least one tosyl group of the agarose tosyl group is introduced, to form an agarose having an amine group (amine group) introduced. 4. The method of claim 3,
The agarose into which the amine group is introduced is ethylenediamino agarose (ETAG),
The degree of substitution (DS) of the amine for the ETAG is 0.3 or less. The method of claim 1,
The steps of preparing the cyclodextrin are:
A method for producing a cyclodextrin, comprising the step of succination of at least one hydroxyl group of a cyclodextrin to form a cyclodextrin into which a succinate monomolecule is introduced. delete agarose functionalized with cyclodextrin;
The cyclodextrin is transferred to agarose by a linker containing carbodiimide formed by EDC/NHS coupling (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling). A composition for combined drug delivery. delete delete 8. The method of claim 7,
wherein the cyclodextrin is beta-cyclodextrin. 8. The method of claim 7,
The composition is a water-soluble composition. 8. The method of claim 7,
The cyclodextrin is a composition containing a part or all of a predetermined drug. 8. The method of claim 7,
The composition is a composition that gels at 32 ℃ or less. A drug delivery system comprising the composition of any one of claims 7 and 10 to 13. A hydrogel comprising the drug carrier of claim 14 and deionized water. 16. The method of claim 15,
The composition for drug delivery is a hydrogel included in a concentration of 1 to 3% (w / v).

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