WO2017200218A1 - Self-assembled nanocomposite based on supramolecular interaction, comprising albumin, method for producing same and use thereof - Google Patents

Abstract

The present invention relates to a self-assembled nanocomposite based on a supramolecular interaction, comprising albumin and, more specifically, to a self-assembled nanocomposite based on albumin, which has excellent target orientation to anticancer sites, in vivo stability, and encapsulated drug release by using a supramolecular interaction of cyclodextrin and adamantine, a method for producing the same, and a use thereof.

Description

Self-assembled nanocomposites based on supramolecular interactions, including albumin, methods for their preparation and uses thereof

The present invention relates to self-assembled nanocomposites based on albumin but prepared using supramolecular interactions.

Until recently, anticancer drugs have been developed that have excellent therapeutic effects against cancers of various sites, and more than 50 anticancer drugs are commercially available today. These anticancer drugs have excellent therapeutic effects, but because they do not selectively act on cancer cells, they cause damage to normal cells, especially tissue cells with active cell division, resulting in various side effects such as bone marrow dysfunction, gastrointestinal mucosa damage, and hair loss. exist. Therefore, the use of the anticancer agent in the treatment of cancer is very limited situation. To solve this problem, a technique using a nanocarrier system with excellent targeting ability has been developed ( J. Control. Release 2 00: 138-157, 2015). These nanocarrier systems accumulate preferentially at the tumor location due to passive targeting by EPR effects, and thus act on all cells that are rapidly dividing or proliferating. Therefore, the accumulation of drugs occurs only in the degree of normal cell division. do. However, there is a difference in the amount of the anticancer agent encapsulated in the nanocarrier, it is possible to suppress the side effects than the conventional administration of the anticancer agent directly.

In order to improve this problem, various nanocarriers have been developed that have improved active targeting ability on tumor tissues. For example, nanoparticles using albumin, which are very safe because of their biocompatibility, biodegradability and non-immunogenicity, have been known, which have an effect of extracellular transcytosis and EPR through gp60 (clathrin). This has the advantage of selectively targeting tumor tissue.

Albumin nanoparticles, however, have been subjected to many traditional methods such as desolvation, thermal gelation, emulsification, self-assembly or nanospray drying and high-pressure homogenization. It is produced by), non-environmental, irreversible, and contains some toxic substances, there is a problem such as accompanying in vivo harmfulness.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a self-assembled nanocomposite based on albumin of a novel structure reversible, excellent target orientation, excellent stability in vivo.

In addition, another object of the present invention is to provide a manufacturing method capable of mass-producing the self-assembled nanocomposite under environmentally friendly conditions.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, including the self-assembled nanocomposite and an anticancer agent encapsulated in the self-assembled nanocomposite.

The present invention to achieve the above object, (a) albumin having an adamantane group; And (b) glycol chitosan to which cyclodextrin is bound, and provides self-assembled nanocomposites bonded by supramolecular interactions between the adamantane of (a) and the cyclodextrin of (b).

The present invention provides a method for producing the self-assembled nanocomposite comprising the following steps to achieve the above another object.

i) binding a first linker to the adamantane compound;

ii) binding albumin to a conjugate of the adamantane compound and the first linker to prepare (a) albumin having an adamantane group;

iii) binding a second linker to the cyclodextrin compound;

iv) combining glycol chitosan to a conjugate of the cyclodextrin compound and a second linker to prepare (b) a glycol chitosan to which cyclodextrin is bound;

v) slowly reacting (b) the albumin having an adamantane group prepared through step ii) with (b) cyclodextrin-linked glycol chitosan prepared through step iv); And

vi) irradiating ultrasonic waves to the reactants of step v);

The first linker and the second linker are the same as or different from each other, and each independently oxalic acid, malonic acid, malic acid, succinic acid, glutaric acid, glutamic acid, adipic acid, sebacicolic acid, suberic acid, dodecano And any biodegradable dicarboxyl linker selected from the group consisting of acid, fumaric acid, maleic acid, phthalic acid and terephthalic acid.

The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the self-assembling nanocomposite and an anticancer agent encapsulated in the self-assembling nanocomposite.

The present invention is a self-assembling nanocomposite through the supramolecular interaction with an anticancer agent can reduce the toxicity and side effects that can be caused by the cross-linking agent, and can be easily prepared in an aqueous solution for human harmful substances such as organic solvents. There is no risk of exposure, and at the same time it can maintain the target orientation to cancer cells and can control the release of the drug can be widely used for cancer treatment and prevention.

1 is a photograph showing the before and after leaving the self-assembled nanocomposites prepared from Examples 1 to 4 at room temperature for 24 hours in order to compare the stability of the solution in the long-term storage at room temperature.

2 is a diagram confirming the interaction of the molecules constituting the self-assembled nanocomposite according to the present invention:

a: diagram showing the molecular interaction between the glycochitosan modified with cyclodextrin and phenolphthalein in a UV-vis graph; And

b: UV-vis graph confirming the formation of supramolecular interactions between adamantane and cyclodextrin.

FIG. 3 shows a case where GC-CD prepared from Preparation Example 4 and albumin (HSA) were mixed, and HSA-ADA ₂₆ prepared from Preparation Example 2 and a glycol chitosan (GC) compound were mixed and from Example 4. It is a photograph of the solution of the prepared self-assembled nanocomposite.

4 is a graph showing the amount of anticancer agent released from the self-assembled nanocomposite containing the anticancer agent prepared in Example 5 with time.

Figure 5 is a graph showing the cytotoxicity of self-assembled nanocomposites loaded with anticancer agents prepared from Example 5 in lung cancer cell line (A549) or colon cancer cell line (HCT116).

FIG. 6 shows stained colorectal cancer cell line (HCT116) and colon cancer-induced mice after treatment with an anticancer agent-encapsulated self-assembled nanocomposite (labeled cy5.5) prepared in Example 5 or a negative control group or a positive control group. Fluorescence images in the model.

Hereinafter, the present invention will be described in more detail with reference to examples and the like, but the scope and contents of the present invention are not limited or interpreted by the following examples.

Preparation Examples 1 to 3. Synthesis of albumin having an adamantane group represented by Formula (Ia) (hereinafter also referred to as HSA-ADA ₁₆ , HSA-ADA ₂₆ , HSA-ADA ₄₀ )

[Formula Ia]

In Formula Ia, q is an integer of 16 to 40. In order to prepare an albumin having an adamantane group represented by the formula (Ia), it was synthesized through the process shown in Scheme 1.

Scheme 1

First, in order to bind a linker to the adamantane compound represented by Chemical Formula 1a, 107 µl of the adamantane compound represented by Chemical Formula 1a was dissolved in 5 ml of 0.3% (TEA / DMSO), and 120 mg of succinic anhydride was used. Addition and stirring for 3 hours to prepare a combination of the adamantane compound and the linker. In order to bind albumin to the combination of the adamantane compound and the linker, 250 mg of DCC (N, N'-Dicyclohexylcarbodiimide) and 140 mg of NHS (N-Hydroxysuccinimide) were added to the combination of the adamantane compound and the linker. The reaction was further performed to activate the conjugate of the adamantane compound and the linker. The activated adamantane compound and linker conjugate (20 mg / ml) was slowly added to an albumin solution (10 mg / ml) dissolved in 5 ml PBS (10 mM pH 7.4) for 25 minutes, followed by stirring at room temperature for 12 hours. It was. After the reaction was completed, the precipitate was removed, and the supernatant was concentrated to 50 mg / ml using Centricon (MWCO: 30k) to obtain an albumin having an adamantane group represented by Chemical Formula Ia. At this time, Preparation Example 1 (HSA-ADA ₁₆ ) is a 0.25 ml (ADA: HSA = 20 equivalents) of the combination of the activated adamantane compound and the linker (20 mg / ㎖) to the albumin solution, Preparation Example 2 ( HSA-ADA ₂₆ ) is a mixture of activated adamantane compound and linker (20 mg / ml) in albumin solution (0.5 mg (ADA: HSA = 40 equivalents)), and Preparation Example 3 (HSA-ADA ₄₀ ) 1 ml (ADA: HSA = 80 equivalents) of a combination of the activated adamantane compound and the linker (20 mg / ml) was added to the albumin solution.

Preparation Example 4 Synthesis of Glycol Chitosan Conjugateed to Cyclodextrin Represented by Formula (IIa) (hereinafter Also referred to as GC-CD)

[Formula IIa]

In the formula, N and M are each an integer of 10 to 3000. In order to prepare the glyco-chitosan combined with the cyclodextrin represented by the formula (IIa), it was synthesized through the process shown in Scheme 2.

Scheme 2

First, in order to bind the linker to the cyclodextrin compound represented by Formula 2a, 100 mg of the cyclodextrin compound (Formula 2a) was dissolved in 2 ml of 0.3% TEA / DMSO, and 120 mg of succinic anhydride was mixed together. Stir for hours. In order to bind glycol chitosan to the conjugate of the cyclodextrin compound and the linker, 250 mg of DCC and NHS 140 mg were added to the conjugate of the cyclodextrin compound and the linker, and reacted for further 6 hours to react the cyclodextrin compound and the linker. The conjugate was activated. 1.6 ml of the combined cyclodextrin compound and linker conjugate was diluted in 6.4 ml PBS (100 mM pH 8.0) and the resulting precipitate was removed by centrifugation. To the separated supernatant, a 22 ml glycol chitosan (hereinafter also referred to as 'GC') (formula 3a) solution (100 mg: 1/60 equivalents to cyclodextrin) was quickly added. Finally, the mixed solution was reacted at room temperature for 24 hours, and then dialysis (MWCO 10 kDa) for 3 days to remove unreacted cyclodextrin, and lyophilized to bind the cyclodextrin represented by Formula IIa in powder form. Glycol chitosan was prepared. At this time, the glycol chitosan solution was prepared by dissolving 100 mg glycol chitosan (Formula 3a) in 22 ml PBS (100 mM pH 8.0) for 6 hours.

Examples 1 to 4. Preparation of Self-Assembly Nanocomposites

0.5 ml of an albumin solution (HSA-ADA ₂₆ , 2 mg / ml) having an adamantane group prepared from Preparation Example 2 using a 1 ml syringe was prepared using a cyclodextrin-bound glycol chitosan solution prepared from Preparation Example 4 (GC -CD) slowly added dropwise to 1.5 ml for 25 minutes. At this time, the final equivalent ratio of the cyclodextrin derived from GC-CD to the adamantane group derived from HSA-ADA ₂₆ is [ADA]: [CD] = 1: 0.5, 1: 1.0, 1: 2.0, 1: 4.0 Were mixed so as to be Examples 1, 2, 3 and 4 in order. Each reaction result was placed in a separate container with ice and treated for 2 minutes using an ultrasonic syringe (Sonics & Materials Inc. New-town, CT, USA: 40%). Hereinafter, Example 1 is GC-CD / HSA-ADA 0.5, Example 2 is GC-CD / HSA-ADA 1, Example 3 is GC-CD / HSA-ADA 2, and Example 4 is GC- Sometimes referred to as CD / HSA-ADA 4.

Example 5. Preparation of Self-Assembled Nanocomposite (Anti-Cancer Composition) Enclosed with Doxorubicin

0.5 mg of doxorubicin was mixed with 0.5 ml of an albumin solution (HSA-ADA ₂₆ , 2 mg / ml) having an adamantane group prepared in Preparation Example 2, and the resultant was put into a 1 ml syringe to bind the cyclodextrin prepared from Preparation Example 4. To the prepared glycol chitosan solution (GC-CD) 1.5 ml (5.4 mg) was slowly added dropwise for 25 minutes. At this time, the final equivalent ratio of the cyclodextrin derived from GC-CD to the adamantane group derived from HSA-ADA ₂₆ was mixed such that [ADA]: [CD] = 1: 2.0. The reaction product was placed in each container containing ice and treated for 2 minutes using an ultrasonic wave filter (Sonics & Materials Inc. New-town, CT, USA: amp 40%), which contained an anticancer drug doxorubicin. An assembled nanocomposite was prepared.

Experimental Example 1. Physical properties of albumin having adamantane groups of Preparation Examples 1 to 3

Molecular masses were measured using an Ultraflex Xtreme MALDI-TOF mass spectrometer (Bruker, Conventry, UK). Spectra were recorded in linear mode at 25 kV pressurization. In order to measure the molecular weight of the albumin (HSA-ADA ₁₆ , HSA-ADA ₂₆ , HSA-ADA ₄₀ ) modified with the conventional albumin (HSA) and adamantane prepared from Preparation Examples 1 to 3, 1 in deionized water, respectively It melt | dissolves in mg / ml, and prepares the 1st solution. A second solution was prepared by preparing a saturated solution in which sinapin acid was dissolved in 0.1% trifluoroacetic acid (TFA) / deionized water-acetonitrile (ACN) (1: 1). Each of the first solution and the second solution were mixed at a volume ratio of 1: 4, and then 1 μl of each was added to the target plate, dried at room temperature to crystallize the sample, and then inserted into the device to each sample. Molecular weight and physical properties were measured for (Table 1).

sample	ADA: HSA equivalence ratio	Molecular weight (m / z)	DS
Conventional albumin (HSA)	0	66470	none
Preparation Example 1 (HSA-ADA 16 )	20	70789	16
Preparation Example 2 (HSA-ADA 26 )	40	73574	26
Preparation Example 3 (HSA-ADA 40 )	80	77451	40

Table 1 shows that in the albumin having an adamantane group prepared from Preparation Examples 1 to 3, the degree of substitution (DS) of the adamantane group for one albumin is 16, 26, and 40.

Experimental Example 2. HPLC measurement of albumin having adamantane groups of Preparation Examples 1 to 3

Albumin having an adamantane group prepared from Preparation Examples 1 to 3 was subjected to reverse phase-high pressure liquid chromatography (RP-HPLC) using a PLRP-S column (150 ×) equipped with a protective cartridge (5 × 3 mm, Agilent Technologies). 4.6 mm, Agilent Technologies). As a result, the delay time of albumin having an adamantane group prepared from Preparation Examples 1 to 3 was 6.60 minutes, 7.43 minutes, and 9.08 minutes, whereas the albumin had a delay time of 5.65 minutes.

Experimental Example 3 Nuclear Magnetic Resonance ( ¹ H NMR) Analysis of GC-CD, Pure CD and Pure GC Prepared from Preparation Example 4

GC-CD prepared from Preparation Example 4, pure CD and pure GC, respectively, dissolved in ₂ mg / ml heavy water (D ₂ O), and then ¹ H nuclear magnetic resonance spectrometer (Varian Unity-Inova 500, St, Louis, MO) Was measured. At this time, the pure cyclodextrin (CD) refers to a cyclodextrin compound (Formula 2a) and glycol chitosan (Formula 3a) before the GC-CD is prepared by mixing GC and CD in Preparation Example 4. It was confirmed that GC-CD of Preparation Example 4 exhibited nuclear magnetic resonance spectra including characteristic peaks of pure CD and pure GC. Through this, the GC-CD of Preparation Example 4 was made through the combination of pure CD and pure GC, and at the same time, it can be seen that the pure CD and pure GC were successfully bound using succinic acid as a linker.

Experimental Example 4. Average diameter of self-assembled nanocomposites prepared from Examples 1 to 4

The self-assembled nanocomposites prepared from Examples 1, 2, 3 and 4 had mean diameters of 500 to 700 nm, 300 to 400 nm, 200 to 300 nm and 350 to 450 nm, respectively. That is, as the final equivalent ratio of the GC-CD-derived cyclodextrin to [ADA]: [CD] gradually increases with respect to the adamantane group derived from the ADA-HSA, the average diameter of the self-assembled nanocomposite decreased significantly, but the equivalent ratio The mean diameter of the self-assembled nanocomposites increased rather than at some point. This confirmed that the self-assembled nanocomposite prepared in Example 3 is the most suitable average diameter to have the EPR effect at 200 to 300nm.

Experimental Example 5. Stability of Self-Assembled Nanocomposites Prepared from Examples 1 to 4

In the case of the self-assembling nanocomposite prepared from Example 3, the solution remained cloudy even after standing at room temperature for 24 hours (FIG. 1). In the structure of the self-assembling nanocomposite prepared from Example 3, the adamantane group Interaction between the albumin and cyclodextrin bound glycol chitosan still takes place, inferring that the spherical nanocomposites remained on solution for at least 24 hours without being released or degraded. Through this, the equivalent ratio of cyclodextrin derived from glycol chitosan to which (b) cyclodextrin contained in the self-assembled nanocomposite is bound to an adamantane group derived from albumin having an adamantane group is 1: It was confirmed that 1.5-2.5 is the best in terms of long-term stability. If the equivalent ratio of the cyclodextrin derived from glycol chitosan to which (a) cyclodextrin contained in the self-assembled nanocomposite is contained (a) to the adamantane group derived from albumin having an adamantane group is 1: 1.5 If less than or greater than 1: 2.5, the state of the solution is already transparent 24 hours before, such as the self-assembled nanocomposites prepared in Examples 1, 2, and 4, whereby the self-assembled nanocomposites form aggregates and precipitate. It means.

That is, it was confirmed that the self-assembled nanocomposite prepared from Example 3 of the present invention can maintain excellent colloidal stability at room temperature for at least 24 hours, preferably 1 to 30 hours, while having an average diameter suitable for the EPR effect. Could (Figure 1).

Experimental Example 6. Demonstration of supramolecular interaction between cyclodextrin and phenolphthalein-ultraviolet absorption spectrum

In FIG. 2A, the use concentrations of the cyclodextrin-bound glycol chitosan prepared from Preparation Example 4 are a: 0 mg / ml, b: 1.0 mg / ml, c: 1.5 mg / ml, and d: 3.0 mg / ml. . The UV-Vis graph measured and recorded peaks in the absorption wavelength region of 350 to 650 nm using an Infinite 200 PRO Microplate Reader (Tecan Genios, Durham, NC). As shown in FIG. 2A, when cyclodextrin-coupled glycol chitosan (Preparation Example 4) and phenolphthalein were simply mixed (in this case, the concentration of phenolphthalein was fixed and the concentration of GC-CD was increased), It can be seen that phenolphthalein interacts with each other.

Experimental Example 7. Demonstration of supramolecular interaction between adamantane and cyclodextrin-ultraviolet absorption spectrum

Peaks in the absorption wavelength region of 350-650 nm were measured and recorded using an Infinite 200 PRO Microplate Reader (Tecan Genios, Durham, NC). The concentration of the albumin solution (HSA-ASA ₂₆ ) having an adamantane group prepared from Preparation Example 2 in FIG. 2B was a: 0 mg / ml, b: 0.2 mg / ml, c: 0.4 mg / ml, and d: 1.0. Mg / ml. As shown in FIG. 2B, as the concentration of the albumin solution having an adamantane group (HSA-ASA ₂₆ ) prepared in Preparation Example 2 increases, the interaction between the adamantane group having a high binding constant and the cyclodextrin is increased. Able to know. In this case, phenolphthalein serves as an absorption stage showing UV-Vis absorption, and is not encapsulated in the self-assembled nanocomposite and competitively interacts with adamantane with respect to the cyclodextrin.

Experimental Example 8. Characterization of the self-assembled nanocomposite containing the self-assembled nanocomposite (GC-CD / HSA-ADA NPs) of Example 4 and the anticancer agent prepared from Example 5

The anticancer agent-embedded self-assembled nanocomposite prepared in Example 5 was stained three times with 10 μl of 1% phosphotungstric acid and dried, and then dried. As a result of photographing under a microscope (TEM), the surface zeta potential of the self-assembled nanocomposite containing the anticancer agent prepared in Example 5 was found to be 1 to 15 kV, preferably 5 to 15 mV, specifically 11.5 kV. The average particle diameter and zeta potential were measured using a Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, UK) with a helium-neon laser beam with a wavelength of 633 nm at a scattering angle of 90 °. As a result, it was confirmed that the surface zeta potential of the self-assembled nanocomposite prepared in Example 4 was 1 to 15 mV, preferably 5 to 15 mV, specifically 5.93 mV.

Experimental Example 9. Confirmation of the properties of HSA-ADA ₂₆ prepared from Preparation Example 2, GC-CD prepared from Preparation Example 4

The albumin having an adamantane group prepared in Preparation Example 2 had an average diameter of 4.67 nm and a surface potential of -34.0 mV with negative charge. Cyclodextrin-bound glycol chitosan (GC-CD) prepared from Preparation Example 4 had an average diameter of 15.77 nm and a zeta potential of 16.2 mV. Putting together the results of the above experimental example, the self-assembled nanocomposites of Examples 4 and 5 were surrounded by glycol chitosan (GC-CD) in which cyclodextrin was bound to albumin (HSA-ADA ₂₆ ) having an adamantane group. It can be inferred that it has a positive charge because it is formed in such a structure. Since the self-assembled nanocomposite according to the present invention has a positive charge, it has the advantage of being stably dispersed in a solution.

Experimental Example 10 When the HSA-ADA ₂₆ prepared from Preparation Example 2 and the cyclodextrin compound were mixed, the GC-CD prepared from Preparation Example 4 and albumin were mixed and the self-assembled nanocomposites prepared from Example 4 were prepared. Check interaction

As shown in FIG. 3, when the GC-CD prepared from Preparation Example 4 and albumin (HSA) were mixed (HSA + GC-CD), no change was observed and it was confirmed that the solution was transparent. When the HSA-ADA prepared from Preparation Example 2 and the cyclodextrin compound were mixed (HSA-ADA + GC), no change was observed and it was confirmed that the solution remained transparent. On the other hand, the solution of the self-assembled nanocomposite prepared from Example 4 was confirmed to be opaque. That is, the presence of cyclodextrin and adamantane does not produce a spherical nanocomposite capable of encapsulating a drug, and if neither of the adamantane molecule or the cyclodextrin molecule is present, the spherical nanocomposite is not formed. Since there is no problem, it is possible to prepare spherical nanocomposites capable of encapsulating drugs only by combining cyclodextrin-linked glycol chitosan (GC-CD) and albumin having adamantane (HSA-ADA). .

Experimental Example 11. Release pattern of anticancer agent from self-assembled nanocomposite containing anticancer agent prepared in Example 5

In order to measure the amount of anticancer agent released from the self-assembled nanocomposite containing the anticancer agent prepared from Example 5, the self-assembled nanocomposite solution containing the anticancer agent prepared from Example 5 was added to 2 ml of dialysis bag (MwCO 10kDa). Insert and seal, place it in a beaker containing 200 ml PBS, fix it and leave at 37 ° C. for 24 hours, while leaving for hourly (0, 1, 2, 3, 6, 9, 12, 18 and 24 hours) ) The amount of doxorubicin present in 2 ml of PBS outside the dialysis bag was measured at a wavelength of 480 nm, and the results are shown as Dox (NPs) of FIG. 4, and the emission pattern calculated using the relative value was shown. As a control (Dox (free)), pure doxorubicin solution (0.14 mg / ㎖) was put in the dialysis bag. Cumulative percent (%) was calculated using Equation 1 below.

[Equation 1]

{The amount of doxorubicin released outside the dialysis bag measured over time} / {The amount of doxorubicin present inside the initial dialysis bag} * 100

As shown in FIG. 4, the self-assembled nanocomposite containing the anticancer agent prepared in Example 5 starts to be released from 1 hour, but only releases up to 60% until 3 hours, and gradually releases after 3 hours. It was confirmed that only up to 80% was released by 24 hours. On the contrary, the control group had already released more than 60% in one hour, and 100% was released before three hours. When using the self-assembled nanocomposite according to the present invention, it was confirmed that the release rate of the drug enclosed therein is slowly maintained for 24 hours.

Experimental Example 12 Stability of Self-Assembled Nanocomposites Containing an Anticancer Agent Prepared from Example 5

The self-assembled nanocomposite containing the anticancer agent prepared in Example 5 was redispersed in 10 mM PBS (pH 7.4) and then left at 37 ° C. for 48 hours. At this time, the self-assembly containing the anticancer agent prepared in Example 5 using Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, UK) according to the time (0, 3, 6, 9, 12, 24 and 48 hours) The average particle diameter of the nanocomposite was measured. As a result, the self-assembled nanocomposite prepared with the anticancer agent prepared in Example 5 was periodically observed in the range of 200 to 300 nm in average particle size change for the first 0 to 12 hours, but was confirmed to be maintained after 12 hours. However, it can be seen that the structural stability does not change more than a significant difference in the degree of structural change, and has structural stability for more than 48 hours.

Experimental Example 13. Growth Inhibition Effect of Self-Assembled Nanocomposites Containing Anticancer Agents Prepared from Example 5 on Cancer Cells

Except that the amount of doxorubicin mixed in the method of Example 5 was 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 µg / ml, all of them were the same as Example 5 An assembled nanocomposite was prepared and tested for cytotoxicity. Lung cancer cells (A549) or colorectal cancer cell line (HCT116) were incubated in RPMI 1640 medium (10% (v / v) FBS with 1% penicilly / streptomycin), then the medium was removed from each well and various The self-assembled nanocomposites containing the content of doxorubicin were treated, incubated for 24 hours, and cell viability was measured by MTT assay. At this time, the control group (Dox (free)) was treated with pure doxorubicin in the cells. As a result, it can be seen that, when the anticancer agent was enclosed in the self-assembled nanocomposite according to the present invention for lung cancer cells (A549) and colon cancer cell lines (HCT116), the cell viability of cancer was much lower than that of the control group (Fig. 5).

Experimental Example 14. Cancer cell targeting of the self-assembled nanocomposite containing the anticancer agent prepared from Example 5 in vivo

Colorectal cancer cell lines (HCT116) were aliquoted onto a cover glass of 12 wells (1 × 10 ⁵ cells / well) and preincubated for 24 hours. Next, it was replaced with 900 μl of DMEM containing 1% (v / v) FBS, and the self-assembled nanocomposite (1 μg / ml of doxorubicin (anticancer agent) enclosed with the anticancer agent of Example 5) or negative control group was added. 100 μl of (control (Non-treat)) or positive control group (Dox (free) 1 μg / ml) was treated for 4 hours each. The cells were then washed with DPBS, fixed with 4% formaldehyde, and the endosomes and nuclei of the cells were Lysotracker® green DND-26 (green fluorescent dye, Molecular Probe) and DAPI, respectively. (blue fluorescent dye, Sigma). After the staining was completed, the cells were analyzed for fluorescence images using CLSM Image Browser software (Zeiss). As shown in Figure 6a, colorectal cancer cell line treated with the self-assembled nanocomposite containing the anticancer agent of Example 5 was confirmed that the doxorubicin is located inside the cytoplasm stained with DAPI. In contrast, the negative control group did not detect any doxorubicin in or out of the cytoplasm at all, and found that only a small amount was detected in the cell in the positive control group. Through these results, when the drug is encapsulated in the self-assembled nanocomposite according to the present invention, the cellular uptake of the drug can be greatly improved and the target orientation to cancer cells is excellent. It was confirmed.

Experimental Example 15 Cancer Cell Target Orientation of Self-Assembled Nanocomposites Containing Anticancer Agents Prepared in Example 5 in Vitro

Colorectal cancer cells (HCT 116) (4 × 10 ⁶ cells / mouse, 100 μl injection volume) were BALB / c nu / nu After inoculating subcutaneously with mice, a mouse model in which colon cancer was induced was prepared for 4 weeks. 50 μl of the self-assembled nanocomposite containing the anticancer agent prepared from Example 5 labeled cy5.5 was injected through the tail vein in the mouse cancer-induced mouse model. Cyx-labeled nanocomposites localized to cancer cell sites over 3, 6, 9, 12 and 24 hours post-injection by Optix MX3 in vivo imaging system (Advanced Research Technologies, Saint-Laurent, Quebec, Canada) was taken and the results are shown in Figure 6b. In this case, the self-assembled nanocomposite containing the anticancer agent prepared from Example 5 labeled with cy5.5 was Cyc5.5 NHS ester dye (GE Healthcare, Piscataway, NJ) into the self-assembled nanocomposite containing the anticancer agent prepared from Example 5. , USA) was added thereto to react for 3 hours. As shown in Figure 6b, the self-assembled nanocomposite containing the anticancer agent prepared from Example 5 labeled cy5.5 was observed in the hepatocytes and cancer cells after 6 hours after being injected into a mouse model induced colon cancer After 12 hours and 24 hours, the fluorescence intensity in the hepatocytes gradually decreased, and strong fluorescence intensity was observed only at the cancer cell site. Through these results, the self-assembled nanocomposite according to the present invention was placed in hepatocytes and cancer cells after the first injection (also at this time, the concentration of the self-assembled nanocomposites is stronger in cancer cells than hepatocytes). It can be seen that only accumulate. That is, the self-assembled nanocomposite according to the present invention was confirmed that the target orientation for cancer cells through the in vivo experiment is very excellent.

Claims (5)

1. (a) albumin having an adamantane group and

   (b) cyclodextrin bound to glycol chitosan,

   Self-assembled nanocomposite bonded by supramolecular interaction between the adamantane of (a) and the cyclodextrin of (b).
2. The method of claim 1,

   The self-assembling nanocomposite is characterized in that the average diameter of the self-assembling nanocomposite is 200 to 300nm.
3. i) binding a first linker to the adamantane compound;

   ii) binding albumin to a conjugate of the adamantane compound and the first linker to prepare (a) albumin having an adamantane group;

   iii) binding a second linker to the cyclodextrin compound;

   iv) combining glycol chitosan to a conjugate of the cyclodextrin compound and a second linker to prepare (b) a glycol chitosan to which cyclodextrin is bound;

   v) slowly reacting (b) the albumin having an adamantane group prepared through step ii) with (b) cyclodextrin-linked glycol chitosan prepared through step iv); And

   vi) irradiating ultrasonic waves to the reactants of step v).
4. The method of claim 3,

   Self-assembly, characterized in that in step ii) the conjugate of the adamantane compound and the linker; and the equivalent ratio of albumin is mixed in the range of 20 to 80 equivalents of the albumin based on 1 equivalent of the conjugate of the adamantane compound and the linker. Method for producing a nanocomposite.
5. Including a self-assembled nanocomposite according to claim 1,

   A pharmaceutical composition for preventing or treating cancer, characterized in that an anticancer agent is enclosed in the self-assembled nanocomposite.

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