KR100572725B1 - Anti-Cancer MTX-LDH Hybrid Capable of Inhibiting Proliferation of Tumor Cell - Google Patents

Anti-Cancer MTX-LDH Hybrid Capable of Inhibiting Proliferation of Tumor Cell Download PDF

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KR100572725B1
KR100572725B1 KR1020030067649A KR20030067649A KR100572725B1 KR 100572725 B1 KR100572725 B1 KR 100572725B1 KR 1020030067649 A KR1020030067649 A KR 1020030067649A KR 20030067649 A KR20030067649 A KR 20030067649A KR 100572725 B1 KR100572725 B1 KR 100572725B1
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최진호
오제민
정지선
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재단법인서울대학교산학협력재단
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Abstract

본 발명은 MTX-LDH 하이브리드를 종양 세포에 처리함으로써 종양 세포의 증식을 억제하는 방법을 제공한다. 보다 구체적으로, 본 발명은 금속 이중층 수산화물(LDH)에 이온 교환 반응을 통하여 MTX(메토트렉세이트)를 층간 삽입시켜 제조한 MTX-LDH 하이브리드를 종양 세포에 처리함으로써 MTX 단독 처리시보다 빠른 시간 내에 종양 세포의 증식을 억제하는 방법을 제공한다.  The present invention provides a method of inhibiting proliferation of tumor cells by treating MTX-LDH hybrids with tumor cells. More specifically, the present invention treats tumor cells with MTX-LDH hybrids prepared by intercalating MTX (methotrexate) through ion exchange reactions in metal bilayer hydroxide (LDH) to tumor cells in a faster time than MTX alone treatment. Provided are methods for inhibiting proliferation.

본 발명에 따르면, 약물의 부작용을 최소화하면서 약물의 효과를 극대화시키켜 종양 세포의 증식을 단시간 내에 억제할 수 있다.According to the present invention, it is possible to inhibit the proliferation of tumor cells in a short time by maximizing the effect of the drug while minimizing the side effects of the drug.

MTX-LDH 하이브리드, 종양 세포, 증식 억제, 메토트렉세이트MTX-LDH Hybrid, Tumor Cells, Proliferation Inhibitor, Methotrexate

Description

종양 세포 증식 억제능을 갖는 ＭＴＸ-ＬＤＨ 하이브리드{Anti-Cancer MTX-LDH Hybrid Capable of Inhibiting Proliferation of Tumor Cell}Anti-Cancer MTX-LDH Hybrid Capable of Inhibiting Proliferation of Tumor Cell

도 1은 본 발명에서 제조한 메토트렉세이트 (MTX)-LDH 시료에 대한 X선 회절도.1 is an X-ray diffraction diagram of a methotrexate (MTX) -LDH sample prepared in the present invention.

도 2는 본 발명에서 제조한 MTX-LDH를 골육종 세포(Saos-2 세포)에 처리후 살아남은 종양 세포의 수에 대한 그래프.Figure 2 is a graph of the number of tumor cells that survived after treating MTX-LDH prepared in the present invention to osteosarcoma cells (Saos-2 cells).

도 3은 본 발명에서 제조한 MTX-LDH를 골육종 세포(Saos-2 세포)에 적용한 후, 그 세포의 DNA를 취해 얻은 전기영동 사진.Figure 3 is applied to the MTX-LDH prepared in the present invention to osteosarcoma cells (Saos-2 cells), electrophoresis picture obtained by taking the DNA of the cells.

본 발명은 MTX-LDH 하이브리드를 종양 세포에 처리함으로써 종양 세포의 증식을 억제하는 방법을 제공한다.The present invention provides a method of inhibiting proliferation of tumor cells by treating MTX-LDH hybrids with tumor cells.

보다 구체적으로, 본 발명은 금속 이중층 수산화물(Layered Double Hydroxide, LDH)에 이온 교환 반응을 통하여 MTX를 층간 삽입시켜 제조한 MTX-LDH 하이브리드를 종양세포에 처리함으로써 MTX 단독 처리시보다 빠른 시간 내에 종양 세포의 증식을 억제하는 방법을 제공한다.More specifically, the present invention treats tumor cells with an MTX-LDH hybrid prepared by intercalating MTX into an ion exchange reaction in a layered double hydroxide (LDH), and thereby treats tumor cells in a faster time than MTX alone treatment. It provides a method of inhibiting the proliferation of.

악성 종양의 치료에서 항암 화학요법은 수술적 치료와 함께 가장 기본적인 치료방법의 하나로서, 생존 기간의 연장과 치료 성적 개선에 필수적인 수단이 되었다. 1980년대에 들어서 치료의 효과를 높이기 위하여 항암 화학요법을 보다 효과적으로 이용하기 위한 연구가 계속되어 골육종의 경우 약 60%의 생존율을 보고하고 있다. 그러나, 이같은 성적은 1980년 중반 Rosen 등이 고용량 MTX를 항암 화학 요법에 이용한 결과에 기인한 것으로 (Biochemical Pharmacology, Volume 33, Issue 17, 1984, Pages 2711-2714), 그 이후에는 뚜렷한 생존율 개선없이 답보 상태인 것이 사실이다. 이에 장기 생존율 개선을 위한 획기적인 방법을 찾기위한 노력이 요구되는 실정이다.In the treatment of malignant tumors, chemotherapy is one of the most basic treatments along with surgical treatment and has become an essential tool for prolonging survival and improving treatment outcomes. In the 1980s, studies to use chemotherapy more effectively in order to increase the effectiveness of treatment have been reported, and the survival rate of osteosarcoma is about 60%. However, these results were due to the results of Rosen et al.'S use of high-dose MTX for chemotherapy in the mid-1980s (Biochemical Pharmacology, Volume 33, Issue 17, 1984, Pages 2711-2714). It is true. Therefore, efforts to find a breakthrough method for improving long-term survival rate are required.

항암 화학요법은 현재 골육종의 치료에서 표준 치료법으로 인정받고 있으며, 대부분의 임상의사들이 시행하고 있는데, 메토트렉세이트 (Methotrexate, MTX), 아드리아마이신 (adriamycin), 이포스파미드 (ifosfamide), 시스플라티눔 (cisplatinum) 등을 고용량으로 사용하여 높은 관해율을 얻고자 노력하고 있다. 그러나, 이러한 항암제는 부작용으로 환자의 면역체계 손상이나 정상세포의 성장 저해등 신체 장기에 치명적인 손상을 유발시키는 것이 문제가 있는 것으로 지적되고 있다. Chemotherapy is now recognized as the standard of care for the treatment of osteosarcoma, and is practiced by most clinicians, including methotrexate (MTX), adriamycin, ifosfamide and cisplatinum. ) Is trying to get a high remission rate using a high dose. However, these anticancer drugs have been pointed out as a side effect of causing fatal damage to body organs such as damage to the immune system of the patient or inhibiting the growth of normal cells.

따라서, 이러한 부작용을 최소화하고 약제의 효과를 극대화시키기 위해서는 종양세포에 선택적으로 항암제를 조기에 투과시킬수 있는 방법이 모색되어야 한다.이러한 목적으로 약물 전달 체계(drug delivery system)를 개발하고 이것을 항암 화학 요법에 이용하고자 하는 노력이 계속되고 있다. Therefore, in order to minimize these side effects and maximize the effect of the drug, a method for selectively penetrating the anticancer drug to tumor cells should be sought. For this purpose, a drug delivery system has been developed and this is anticancer chemotherapy. Efforts to take advantage of this continue.

최근 나노기술의 발달로 인체 질병의 치료에 이용하고자 하는 연구가 진행되고 있는데, 이중 염색체, 핵산 및 약제 등의 세포 내 전달을 위해 금속 이중층 수산화물(LDH)을 통한 연구가 진행되었으며, 현재 시험관 내에서 LDH 하이브리드를 이용한 세포내 DNA 전달이 효과적인 방법이라는 것이 밝혀진 상태이다. Recently, the development of nanotechnology has been conducted to treat human diseases, and research through metal double layer hydroxide (LDH) for intracellular delivery of dual chromosomes, nucleic acids and drugs has been conducted. Intracellular DNA delivery using LDH hybrids has been found to be an effective method.

LDH의 독특한 음이온 교환 능력은 음전하를 띤 생체 물질이 양이온의 부루사이트(brucite) 유사층 사이에서 정전기적 상호작용 (electrostatic interaction)에 의하여 안정화를 이루게 되면 LDH와 혼성화(hybridization)에 의하여 전기적으로 중성화를 이루어 음전하를 띠고 있는 세포막에 대한 음이온의 생체 분자와의 정전기적 반발 작용 (electrostatic repulsive interaction)을 크게 감소시킴으로써 엔도사이토시스(endocytosis)에 의한 하이브리드의 세포내 진입을 촉진시킬 수 있는 가능성에 대하여 보고되었다. LDH's unique anion exchange ability allows the neutralization of negatively charged biomaterials to be electrically neutralized by hybridization with LDH once they are stabilized by electrostatic interactions between the cation's brucite-like layers. It has been reported about the possibility of promoting the cellular entry of the hybrid by endocytosis by greatly reducing the electrostatic repulsive interaction of the anions with the biomolecules on the negatively charged cell membranes. .

따라서, 본 발명의 목적은 상기한 바와 같은 LDH의 특성을 이용하여 약물의 부작용을 최소화하고 약물의 효과를 극대화시켜 종양 세포의 증식을 단시간 내에 억제하는 방법을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a method of minimizing side effects of drugs and maximizing the effects of drugs by using the properties of LDH as described above to inhibit tumor cell proliferation in a short time.

본 발명자들은 종양 치료에 많이 사용되는 MTX와 LDH 하이브리드를 합성하고, 이를 종양 세포에 처리함으로써 MTX 단독 처리시보다 빠른 시간 내에 종양 세포의 증식을 억제할 수 있음을 밝혀내어 본 발명을 완성하게 되었다.The present inventors have completed the present invention by synthesizing MTX and LDH hybrids, which are widely used in the treatment of tumors, and treating them with tumor cells, thereby inhibiting the proliferation of tumor cells in a faster time than MTX alone treatment.

따라서, 본 발명은 MTX-LDH 하이브리드를 종양 세포에 처리함으로써 종양 세포의 증식을 억제하는 방법을 제공한다.Accordingly, the present invention provides a method of inhibiting proliferation of tumor cells by treating MTX-LDH hybrids with tumor cells.

보다 구체적으로, 본 발명은 금속 이중층 수산화물에 이온 교환 반응을 통하여 MTX를 층간 삽입시켜 제조한 MTX-LDH 하이브리드를 골육종 세포에 처리함으로써 MTX 단독 처리시보다 빠른 시간 내에 종양 세포의 증식을 억제하는 방법을 제공한다. More specifically, the present invention provides a method of inhibiting the proliferation of tumor cells in a faster time than MTX alone treatment by treating MTX-LDH hybrid prepared by intercalating MTX into metal bilayer hydroxide through intercalation of MTX to osteosarcoma cells. to provide.

본 발명의 방법에 의해 그 증식이 억제되는 종양 세포는 원발성 악성 골종양 중 가장 흔한 골육종 세포로서, 예를 들어 Saos-2 세포, MG-63 세포 등을 언급할 수 있고, 이로 제한되지 않는다.Tumor cells whose proliferation is inhibited by the method of the present invention are the most common osteosarcoma cells of primary malignant bone tumors, and examples thereof include, but are not limited to, Saos-2 cells, MG-63 cells, and the like.

본 발명의 일 실시태양에 따르면, MTX-LDH 하이브리드를 합성하고, 이를 종양 세포에 처리함으로써 종양 세포의 증식을 효과적으로 억제할 수 있음이 밝혀졌다.According to one embodiment of the present invention, it has been found that by synthesizing the MTX-LDH hybrid and treating it with tumor cells, it is possible to effectively inhibit the proliferation of tumor cells.

본 발명은 하기의 실시예들 및 비교예들을 통해 더욱 상세히 설명하나, 본 발명이 이들 실시예에 의해 제한되는 것은 아니다.The present invention will be described in more detail with reference to the following examples and comparative examples, but the present invention is not limited to these examples.

[실시예 1]Example 1

Mg(II)과 Al(III)의 비가 2:1이 되도록 Mg(NO3)2와 Al(NO3)3를 혼합한 용액에 0.1 M NaOH 용액을 적가하여 공침시킴으로써 침전물을 얻고, 이를 세척하고 원심분리시켜 층간에 질산 음이온이 도입된 금속 이중층 수산화물 (MgAl(NO3)-LDH, 이하 NO3-LDH)를 합성하였다. 전 과정은 공기 중의 CO2에 의한 오염을 막기 위해 질소 분위기 하에서 수행되었다. 합성한 금속 이중층 수산화물 (NO3-LDH)의 X선 회절 분석 결과는 도 1의 (a)에 나타내었다. A precipitate was obtained by dropping and coprecipitating 0.1 M NaOH solution dropwise to a solution of Mg (NO 3 ) 2 and Al (NO 3 ) 3 so that the ratio of Mg (II) and Al (III) was 2: 1, and washed it. By centrifugation, a metal double layer hydroxide (MgAl (NO 3 ) -LDH, hereinafter NO 3 -LDH) having nitrate anions introduced therebetween was synthesized. The entire process was carried out under a nitrogen atmosphere to prevent contamination by CO 2 in the air. X-ray diffraction analysis of the synthesized metal double layer hydroxide (NO 3 -LDH) is shown in Figure 1 (a).

이렇게 얻은 금속 이중층 수산화물 현탁액 (1 mg/1 ml H2O)에, 끓인 증류수에 NaOH를 가하여 용해시킨 MTX 0.2 g을 혼합하고 60 ℃에서 72시간 반응시켰다. 이온교환반응이 끝난 후 끓인 증류수로 3회에 걸쳐 생성물을 세척하여 과량의 MTX를 완전히 제거하고 40 ℃ 오븐에서 건조시켰다. 건조 후 얻어진 생성물인 MTX-LDH 하이브리드에 대한 X-선 회절 분석 결과는 도 1의 (b)에 나타내었다. 층간거리가 MTX 크기에 상응한다. To the metal double layer hydroxide suspension (1 mg / 1 ml H 2 O) thus obtained, 0.2 g of MTX dissolved by adding NaOH to boiled distilled water was mixed and reacted at 60 ° C. for 72 hours. After the ion exchange reaction, the product was washed three times with boiled distilled water to completely remove excess MTX and dried in an oven at 40 ℃. X-ray diffraction analysis results for the MTX-LDH hybrid obtained after drying are shown in FIG. Interlayer distance corresponds to MTX size.

[실시예 2]Example 2

Saos-2 세포는 RPMI 1640 배지 (Gibco, Grand Island, N.Y.,USA)를 사용하여 75 ㎠ 플라스크에서 배양하였다. 배지는 10% 태아 소 혈청 (FBS; Gibco), 페니실린 G 나트륨 100 units/ml (Gibco)과 스트렙토마이신 술페이트 100 units/ml (Gibco)를 첨가하여 사용하였으며, 세포주는 배지에 분주 후 5% CO2, 37 ℃ 배양기에서 배양하였다. Saos-2 cells were cultured in 75 cm 2 flasks using RPMI 1640 medium (Gibco, Grand Island, NY, USA). Medium was used by adding 10% fetal bovine serum (FBS; Gibco), penicillin G sodium 100 units / ml (Gibco) and streptomycin sulfate 100 units / ml (Gibco). 2 , 37 ℃ incubator was incubated.

동결된 상태로 한국세포주 은행으로부터 분양 받은 세포주는 37℃ 온탕 수조에 넣어 급속하게 해동시킨 후 배양액으로 희석하였다. 이를 1500 rpm으로 5분간 원심분리를 시행한 후 상층액을 제거하고 세포를 배지에 부유시켜 75 ㎠ 플라스크에 분주 배양하였다.Cell lines that were frozen from the Korea Cell Line Bank were frozen and thawed rapidly in a 37 ° C. hot water bath and diluted with culture. After centrifugation at 1500 rpm for 5 minutes, the supernatant was removed, the cells were suspended in medium, and cultured in a 75 cm 2 flask.

[실시예 3]Example 3

골육종 세포가 80-90% 세포 증식도 (cellular confluence)에 도달했을 때, 단층 배양 세포를 트립신-EDTA (Gibco)를 이용하여 분리하였다. 분리한 세포는 배양액으로 희석하고 1500 rpm에서 5분간 원심분리하였다. 상층액을 제거한 후, 다시 세포를 배지로 부유하여 혈구계수기로 세포수를 측정하였다. When osteosarcoma cells reached 80-90% cellular confluence, monolayer culture cells were isolated using trypsin-EDTA (Gibco). The isolated cells were diluted with culture and centrifuged for 5 minutes at 1500 rpm. After removing the supernatant, the cells were suspended in medium again and the cell number was measured with a hemocytometer.

96-웰 플레이트의 첫번째 웰에는 2 x 106개의 세포가 들어간 배지 200 ㎕를 넣고, 다음 웰부터는 세포의 농도를 차례로 반으로 희석하여 11번째 웰에는 195개의 세포가 들어가도록 하였다. 마지막 12번째 웰에는 세포없이 배지만 넣고, 각각의 웰에는 배지가 총 100 ㎕가 되도록 만들어 5% CO2, 37 ℃ 배양기에서 4일간 배양하였다. 200 μl of medium containing 2 × 10 6 cells was added to the first well of the 96-well plate, and the concentration of cells was diluted in half from the next well so that 195 cells were put into the 11th well. In the last 12th wells, only cells without cells were added, and each well was incubated in a 5% CO 2 , 37 ° C. incubator for a total of 100 μl of medium.

배양 후 테트라졸륨-염기 화합물인 3-(4,5-디메틸티아졸-2-일)-2,5-페닐테트라졸륨 브로마이드 (MTT) 용액 10 ㎕ (g/ml)를 각각의 웰에 첨가하고 다시 4시간 동안 5% CO2, 37 ℃ 배양기에서 MTT에 의해 형성된 결정을 용해시킨 후 ELISA 판독기로 파장 540 nm에서 흡광도를 측정하였다. 측정된 흡광도로 세포주의 성장 곡선을 구하였다. 항암제에 대한 세포 증식 억제 검사를 위해 각 세포주에서 급격하게 세포수가 증가되는 점을 기준으로 적정 세포수를 산출하였다. Saos-2의 적정 세포수는 에서는 1.2 x 104개로 산출되었다.After incubation, 10 μl (g / ml) of 3- (4,5-dimethylthiazol-2-yl) -2,5-phenyltetrazolium bromide (MTT) solution of tetrazolium-base compound was added to each well, The crystals formed by MTT were dissolved in a 5% CO 2 , 37 ° C. incubator for another 4 hours, and then absorbance was measured at 540 nm with an ELISA reader. The measured curves were used to determine the growth curve of the cell line. Proper cell counts were calculated based on the rapid increase in cell number in each cell line. The optimal cell number of Saos-2 was calculated to be 1.2 x 10 4 .

[실시예 4]Example 4

96-웰 플레이트의 각 웰마다 적정 세포수의 골육종 세포를 넣고, 첫번째 컬럼에는 아무 약물도 첨가하지 않고 대조군으로 사용하였다. 두번째 컬럼에는 MTX 단독으로 최고 농도 (500 ㎍/㎖)의 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8배로 희석하여 투여하고, 세번째 컬럼에는 MTX-LDH 하이브리드를, 네번째 컬럼에는 LDH를 같은 방법으로 희석하여 투여하였다. In each well of a 96-well plate, osteosarcoma cells of the appropriate cell number were added, and no drug was added to the first column and used as a control. The second column was diluted with MTX alone in 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 folds at the highest concentration (500 ㎍ / ㎖). In the third column, MTX-LDH hybrid was administered, and in the fourth column, LDH was diluted and administered in the same manner.

동일한 조건의 96-웰 플레이트를 4개 준비하여 배양 시작 후 24시간, 48시간, 72시간, 96시간에 각각의 웰 플레이트에 대하여 MTT 검사법을 시행하여 각 웰의 분획 흡광도(fractional absorbance)를 구하였다. 최고 농도 (500 ㎍/㎖)에서 각 약물에 대한 Saos-2의 증식을 측정하여 MTX (24시간; 93% ±5%, 48시간; 76% ±2%, 72시간; 65% ±7%, 96시간; 46% ±6%)와 MTX-LDH 하이브리드 (24시간; 56% ±5%, 48시간; 56% ±3%, 72시간; 50% ±10%, 96시간; 44% ±2%)를 비교한 결과, 같은 시간대에서 대체적으로 MTX-LDH 하이브리드를 처리한 경우에 MTX만을 처리한 경우보다 큰 증식 억제 효과를 나타내었고, 특히 24시간대에서 MTX-LDH 하이브리드처리가 MTX 단독 처리시보다 37%의 더 큰 증식 억제 효과를 나타내었다. 각 웰의 분획 흡광도는 도 2에 도시하였다.Four 96-well plates with the same conditions were prepared, and fractional absorbance of each well was determined by performing MTT assay on each well plate at 24 hours, 48 hours, 72 hours, and 96 hours after the start of the culture. . The proliferation of Saos-2 for each drug at the highest concentration (500 μg / ml) was determined by MTX (24 hours; 93% ± 5%, 48 hours; 76% ± 2%, 72 hours; 65% ± 7%, 96 hours; 46% ± 6%) and MTX-LDH hybrid (24 hours; 56% ± 5%, 48 hours; 56% ± 3%, 72 hours; 50% ± 10%, 96 hours; 44% ± 2% ), MTX-LDH hybrid treatment showed greater growth inhibition effect than MTX-only treatment at the same time, especially MTX-LDH hybrid treatment at 37 hours compared with MTX alone treatment. It showed a greater growth inhibition effect of%. Fractional absorbance of each well is shown in FIG. 2.

[실시예 5]Example 5

골육종 세포에 최고 농도 (500 ㎍/㎖)의 MTX, MTX-LDH 하이브리드와 LDH를 각각 처리하여 24시간 배양한 후 원심분리하여 세포들을 모았다. 혈구계수기로 1 x 106 개의 세포수를 측정하여 용해 완충액 (10 mM Tris, 1 mM 에틸렌디아민-테트라아세트산 [EDTA] 및 0.2% Triton X-100) 400 ㎕를 첨가하여 4 ℃에서 5분 동안 용해시켰다. 용해액을 13,000 x g에서 15분 동안 원심분리한 후 상층액만 분리하여 96% 에탄올 700 ㎕와 5 M NaCl 120 ㎕를 첨가하여 -20 ℃에서 철야 정치시킨 후 13,000 x g에서 15분 동안 원심분리하였다. Osteosarcoma cells were treated with MTX, MTX-LDH hybrid and LDH at the highest concentration (500 μg / ml), respectively, and cultured for 24 hours, followed by centrifugation to collect the cells. 1 x 10 6 cells were counted with a hemocytometer, and 400 μl of lysis buffer (10 mM Tris, 1 mM ethylenediamine-tetraacetic acid [EDTA] and 0.2% Triton X-100) was added and lysed at 4 ° C. for 5 minutes. I was. The lysate was centrifuged at 13,000 xg for 15 minutes, then only the supernatant was separated, and 700 μl of 96% ethanol and 120 μl of 5 M NaCl were added and left overnight at −20 ° C., followed by centrifugation at 13,000 xg for 15 minutes.

상층액을 제거하고 펠렛을 70% 에탄올 200 ㎕로 2회 세척한 후 상온에서 건조하여 TE 완충액 (10 mM Tris 및 1 mM EDTA) 20 ㎕로 65 ℃에서 10분간 용해시켰다. RNAse (100 units/ml)와 프로테이나제 K (2 mg/ml)를 가하여 상온에서 10분간 반응시킨 후 6x 로딩 염료를 넣고 전기영동을 시행한 후 자외선을 조사하여 DNA 단편을 관찰하였다. MTX-LDH 하이브리드가 MTX보다 더 큰 역가를 갖는다는 사실을 바탕으로 같은 조건 하에서 MTX와 MTX-LDH 하이브리드의 세포사멸 (apoptosis) 정도의 차이를 DNA 래더를 사용하여 관찰하였다. The supernatant was removed and the pellet was washed twice with 200 μl of 70% ethanol, dried at room temperature and dissolved in 20 μl of TE buffer (10 mM Tris and 1 mM EDTA) at 65 ° C. for 10 minutes. RNAse (100 units / ml) and proteinase K (2 mg / ml) were added and reacted at room temperature for 10 minutes, followed by electrophoresis with 6x loading dye, followed by ultraviolet irradiation to observe DNA fragments. Based on the fact that MTX-LDH hybrids have greater titers than MTX, differences in the degree of apoptosis of MTX and MTX-LDH hybrids were observed using DNA ladders under the same conditions.

그 결과, Saos-2에서는 최고 농도 500 ㎍/㎖로 24시간 동안 약제 처리하였을 때 세포사멸의 특징을 나타내는 DNA 래더가 LDH와 MTX에서는 나타나지 않았지만, MTX-LDH 하이브리드에서는 DNA 래더가 생성되었다 (도 3 참조).As a result, in the Saos-2, the DNA ladder, which is characterized by apoptosis, was not seen in LDH and MTX when treated with the drug at the highest concentration of 500 µg / ml for 24 hours. Reference).

[실시예 6]Example 6

MG-63 세포에 대해 실시예 2 내지 5와 동일한 과정으로 처리하여 실시한 결과, Saos-2 세포와 동일한 결과를 얻을 수 있었고, LDH가 MTX를 빠르게 세포 내로 진입시켜 기존 약제보다 더 큰 역가를 보인다는 것을 알 수 있다. As a result of treatment of MG-63 cells by the same procedure as in Examples 2 to 5, the same results as Saos-2 cells were obtained, and LDH rapidly enters MTX cells and shows higher titers than conventional drugs. It can be seen that.

본 발명에 따르면, 약물의 부작용을 최소화하면서 약물의 효과를 극대화시키켜 종양 세포의 증식을 단시간 내에 억제할 수 있다.According to the present invention, it is possible to inhibit the proliferation of tumor cells in a short time by maximizing the effect of the drug while minimizing the side effects of the drug.

Claims (4)

메토트렉세이트 (MTX) 단독 처리시보다 빠른 시간 내에 골육종 세포의 증식 억제능을 갖는 메토트렉세이트-금속 이중층 수산화물 (MTX-LDH) 하이브리드.Methotrexate-metal bilayer hydroxide (MTX-LDH) hybrids having the ability to inhibit proliferation of osteosarcoma cells within a faster time than methotrexate (MTX) alone treatment. 제1항에 있어서, 골육종 세포가 Saos-2 세포인 하이브리드. The hybrid of claim 1, wherein the osteosarcoma cells are Saos-2 cells. 제1항에 있어서, 골육종 세포가 MG-63 세포인 하이브리드. The hybrid of claim 1, wherein the osteosarcoma cells are MG-63 cells. 제1항에 있어서, 메토트렉세이트-금속 이중층 수산화물 (MTX-LDH) 하이브리드가 이온 교환 반응을 통하여 메토트렉세이트 (MTX)를 금속 이중층 수산화물 (LDH)에 층간 삽입시켜 제조한 것인 하이브리드.The hybrid of claim 1, wherein the methotrexate-metal bilayer hydroxide (MTX-LDH) hybrid is prepared by intercalating methotrexate (MTX) into the metal bilayer hydroxide (LDH) via an ion exchange reaction.
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