KR102000488B1 - Nanoparticle for chemotherapy of anoikis-resistant cancer cell - Google Patents

Nanoparticle for chemotherapy of anoikis-resistant cancer cell Download PDF

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KR102000488B1
KR102000488B1 KR1020170045949A KR20170045949A KR102000488B1 KR 102000488 B1 KR102000488 B1 KR 102000488B1 KR 1020170045949 A KR1020170045949 A KR 1020170045949A KR 20170045949 A KR20170045949 A KR 20170045949A KR 102000488 B1 KR102000488 B1 KR 102000488B1
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albumin
nanoparticles
drug
cancer cells
chemotherapy
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KR20170131212A (en
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김현철
이호현
이민지
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서강대학교 산학협력단
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Abstract

본 발명은 아노이키스 내성 암세포의 화학요법을 위한 알부민 나노입자에 대한 것으로, 더욱 상세하게는 약물을 담지한 알부민 나노입자와 상기 알부민 나노입자에 결합한 CEACAM6 항체를 포함하며, 상기 알부민 나노입자는 알부민 분자 사이 디설파이드 결합에 의해 형성되고, 상기 CEACAM6 항체는 알부민 나노입자 표면의 아민 그룹과 아마이드 결합을 하여, CEACAM6 항체가 아노이키스 내성 암세포를 타켓팅하며 암세포에 섭취된 알부민 나노입자는 세포기질에 존재하는 글루타틴온에 의해 디설파이드 결합이 끊어져 약물을 방출하는 아노이키스 내성 암세포의 화학요법을 위한 알부민 나노입자에 대한 것이다.The present invention relates to albumin nanoparticles for chemotherapy of anoikis-resistant cancer cells, and more particularly include albumin nanoparticles carrying a drug and CEACAM6 antibody bound to the albumin nanoparticles, wherein the albumin nanoparticles are albumin molecules. It is formed by the disulfide bond, and the CEACAM6 antibody is amide bond with the amine group on the surface of the albumin nanoparticles, the CEACAM6 antibody targets anoikis-resistant cancer cells, the albumin nanoparticles ingested in the cancer cells gluta existing in the cell substrate The present invention relates to albumin nanoparticles for chemotherapy of anoikis-resistant cancer cells that release a drug by breaking disulfide bonds by tinons.

Description

아노이키스 내성 암세포의 화학요법을 위한 나노입자{Nanoparticle for chemotherapy of anoikis-resistant cancer cell}Nanoparticles for chemotherapy of anoikis-resistant cancer cells

본 발명은 아노이키스 내성 암세포의 화학요법을 위한 나노입자에 대한 것으로, 더욱 상세하게는 약물을 담지한 알부민 나노입자와 상기 알부민 나노입자에 결합한 CEACAM6 항체를 포함하며, 상기 알부민 나노입자는 알부민 분자 사이 디설파이드 결합에 의해 형성되고, 상기 CEACAM6 항체는 알부민 나노입자 표면의 아민 그룹과 아마이드 결합을 하여, CEACAM6 항체가 아노이키스 내성 암세포를 타켓팅하며 암세포에 섭취된 알부민 나노입자는 세포기질에 존재하는 글루타틴온에 의해 디설파이드 결합이 끊어져 약물을 방출하는 아노이키스 내성 암세포의 화학요법을 위한 나노입자에 대한 것이다.The present invention relates to nanoparticles for chemotherapy of anoikis resistant cancer cells, and more particularly include albumin nanoparticles carrying a drug and CEACAM6 antibody bound to the albumin nanoparticles, wherein the albumin nanoparticles are between albumin molecules. Formed by disulfide bonds, the CEACAM6 antibody binds to an amine group on the surface of the albumin nanoparticles, such that the CEACAM6 antibody targets anobics resistant cancer cells, and the albumin nanoparticles ingested into the cancer cells are glutathione present in the cell substrate. It is a nanoparticle for chemotherapy of an Aikis-resistant cancer cells that release the drug by breaking the disulfide bond.

암은 조직 내에서 질서를 무시하고 무제한 증식하는 미분화 세포로 구성된 종괴 또는 종양을 형성하는 병을 말하며, 궁극적으로는 주위의 정상조직이나 기관을 침윤하여 파괴시키고 원발병소에서 개체의 어떤 기관이든 전이하여 새로운 성장 장소를 만들 수 있어 개체의 생명을 빼앗아 갈 수 있는 질환군을 총칭한다. 따라서, 암의 정확한 진단 및 치료하기 기술이 널리 개발되고 있으며, 암치료를 위한 대표적인 방법으로 외과적인 수술 방법, 방사선 치료 방법 및 화학요법 등이 있다. 이중, 화학용법은 하기의 특허문헌처럼 암세포에 항암제를 전달하여 암세포를 사멸시킨다.Cancer is a disease that forms a mass or tumor that consists of undifferentiated cells that grow indefinitely and ignore order within a tissue, and ultimately infiltrate and destroy surrounding normal tissues or organs and metastasize any organ of the individual in the primary pathogen. A group of diseases that can create a new place of growth that can take an individual's life. Therefore, techniques for accurately diagnosing and treating cancer have been widely developed, and typical surgical methods for treating cancer include surgical procedures, radiation treatment methods, and chemotherapy. Of these, the chemotherapy delivers an anticancer agent to cancer cells and kills the cancer cells as in the following patent document.

<특허문헌><Patent Documents>

특허공개공보 제10-2013-0088081호(2013. 08. 07. 공개) "수난용성 약물을 내부에 포함하는 알부민 나노입자의 제조방법"Patent Publication No. 10-2013-0088081 (2013. 08. 07. published) "Method for producing albumin nanoparticles containing a poorly water-soluble drug therein"

하지만, 종래의 암을 치료하기 위한 나노입자의 전달은 EPR(enhanced permeability and retention) 효과를 이용하였는데, 혈관 또는 림프관을 따라 플로우팅(floating)하는 전이 암세포에서는 EPR 효과를 기대할 수 없어, 종래의 화학요법은 위한 나노입자는 전이 암세포를 사멸시키기 어려운 문제가 있다.However, the delivery of nanoparticles for the treatment of conventional cancers uses an enhanced permeability and retention (EPR) effect, which cannot be expected in metastatic cancer cells floating along blood vessels or lymphatic vessels. Nanoparticles for therapy have a problem that is difficult to kill metastatic cancer cells.

본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로,The present invention has been made to solve the above problems,

본 발명은 전이 암세포를 효과적으로 사멸시킬 수 있는 아노이키스 내성 암세포의 화학요법을 위한 나노입자를 제공하는데 그 목적이 있다.It is an object of the present invention to provide a nanoparticle for chemotherapy of an Aokis resistant cancer cells that can effectively kill metastatic cancer cells.

또한, 본 발명은 알부민 나노입자에 결합한 CEACAM6 항체가 아노이키스 내성 암세포를 타켓팅하여 전이 암세포에 약물을 효과적으로 전달할 수 있는 아노이키스 내성 암세포의 화학요법을 위한 나노입자를 제공하는데 그 목적이 있다.In addition, an object of the present invention is to provide a nanoparticle for chemotherapy of anoikis-resistant cancer cells that CEACAM6 antibody bound to the albumin nanoparticles can effectively deliver the drug to metastatic cancer cells by targeting an aikis-resistant cancer cells.

또한, 본 발명은 약물을 담지하는 알부민 나노입자는 디설파이드 결합에 의해 생성되므로, 암세포 내에 섭취된 알부민 나노입자는 세포기질에 존재하는 글루타틴온에 의해 쪼개져 비목적 지역에 약물을 방출을 억제할 수 있는 아노이키스 내성 암세포의 화학요법을 위한 나노입자를 제공하는데 그 목적이 있다.In addition, according to the present invention, since albumin nanoparticles carrying a drug are produced by disulfide bonds, albumin nanoparticles ingested in cancer cells may be cleaved by glutathione present in the cell substrate to inhibit release of the drug in a non-target area. The purpose of the present invention is to provide nanoparticles for chemotherapy of anoikis resistant cancer cells.

본 발병은 앞서 본 목적을 달성하기 위하여 다음과 같은 구성을 가진 실시예에 의해 구현된다.The onset is implemented by the embodiment having the following configuration in order to achieve the above object.

본 발명의 일 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자는 약물을 담지한 알부민 나노입자와, 상기 알부민 나노입자에 결합한 CEACAM6 항체를 포함하는 것을 특징으로 한다.According to one embodiment of the present invention, the nanoparticles for cancer cell chemotherapy according to the present invention are characterized in that it comprises albumin nanoparticles carrying a drug, and CEACAM6 antibody bound to the albumin nanoparticles.

본 발명의 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자에 있어서 상기 약물을 담지한 알부민 나노입자는 알부민 분자 사이의 디설파이드 결합에 의해서 형성되는 것을 특징으로 한다.According to another embodiment of the present invention, in the nanoparticles for cancer cell chemotherapy according to the present invention, the albumin nanoparticles carrying the drug are formed by disulfide bonds between albumin molecules.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자에 있어서 상기 약물을 담지한 알부민 나노입자는 구형의 형태를 가지는 것을 특징으로 한다.According to another embodiment of the present invention, the albumin nanoparticles carrying the drug in the nanoparticles for cancer cell chemotherapy according to the present invention is characterized in that it has a spherical form.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자는 150 내지 350nm의 직경을 가지는 것을 특징으로 한다.According to another embodiment of the invention, the nanoparticles for cancer cell chemotherapy according to the invention is characterized in that it has a diameter of 150 to 350nm.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자에 있어서 상기 CEACAM6 항체는 상기 약물을 담지한 알부민 나노입자 표면의 아민 그룹과 아마이드 결합하는 것을 특징으로 한다.According to another embodiment of the present invention, in the nanoparticles for cancer cell chemotherapy according to the present invention, the CEACAM6 antibody is characterized in that the amide bond with the amine group on the surface of the albumin nanoparticles carrying the drug.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자에 있어서 상기 약물은 독소루비신이 사용되는 것을 특징으로 한다.According to another embodiment of the present invention, in the nanoparticles for cancer cell chemotherapy according to the present invention, the drug is characterized in that doxorubicin is used.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자는 전이 암세포를 타켓팅하여 사멸시키는 것을 특징으로 한다.According to another embodiment of the present invention, the nanoparticles for cancer cell chemotherapy according to the present invention are characterized by targeting metastatic cancer cells and killing them.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자는 전이 암세포 내에 섭취되어 세포 기질에 존재하는 글루타티온에 의해 쪼개져 약물을 방출하는 것을 특징으로 한다.According to another embodiment of the present invention, the nanoparticles for cancer cell chemotherapy according to the present invention are characterized in that they are ingested in metastatic cancer cells and cleaved by glutathione present in the cell substrate to release the drug.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자의 제조방법은 알부민을 티올화하는 티올화단계와, 티올화된 알부민과 약물을 이용하여 이황화 결합 및 탈용매화 과정을 통해 약물이 담지된 알부민 나노입자를 형성하는 입자형성단계와, 상기 약물이 담지된 알부민 나노입자에 CEACAM6 항체를 결합시키는 항체결합단계를 포함하는 것을 특징으로 한다.According to another embodiment of the present invention, a method for preparing nanoparticles for cancer cell chemotherapy according to the present invention is a thiolation step of thiolating albumin, and a disulfide bond and desolvation process using a thiolated albumin and a drug. Particle forming step of forming a drug-supported albumin nanoparticles through, and characterized in that it comprises an antibody binding step of binding the CEACAM6 antibody to the albumin nanoparticles loaded with the drug.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자의 제조방법에 있어서 상기 티올화단계는 piperazine-N'-ethanesulfonic acid와 ethylenediaminetetraacetic acid를 함유하는 반응버퍼에 알부민을 녹이고, 2-Iminothiolane hydrochloride에 의한 티올화 과정을 통해 티올화된 알부민을 생성하는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for producing nanoparticles for cancer cell chemotherapy according to the present invention, the thiolation step dissolves albumin in a reaction buffer containing piperazine-N'-ethanesulfonic acid and ethylenediaminetetraacetic acid. In addition, thiolated albumin is produced by thiolation with 2-Iminothiolane hydrochloride.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자의 제조방법에 있어서 상기 티올화단계에서 알부민과 2-IT의 몰비율은 1 대 20 내지 30인 것을 특징으로 한다.According to another embodiment of the present invention, the molar ratio of albumin and 2-IT in the thiolation step in the method for producing nanoparticles for cancer cell chemotherapy according to the present invention is characterized in that 1 to 20 to 30. .

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자의 제조방법에 있어서 상기 입자형성단계는 HEPES 버퍼에 상기 티올화된 알부민과 약물이 혼합된 용액에 에탄올을 드롭방식으로 첨가반응시켜 약물이 담지된 알부민 나노입자를 생성하는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for preparing nanoparticles for cancer cell chemotherapy according to the present invention, the particle forming step is a method of dropping ethanol in a solution in which the thiolated albumin and a drug are mixed in a HEPES buffer. It is characterized in that the addition reaction to produce the albumin nanoparticles carrying the drug.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 암세포 화학요법을 위한 나노입자의 제조방법에 있어서 상기 항체결합단계는 N-Hydroxysuccinimide와 N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride를 포함하는 MES 반응버퍼 내에 CEACAM6 항체 용액을 혼합하여 반응시켜 NHS/EDC-활성화된 항체를 형성하고, 상기 활성화된 황체를 상기 약물이 담지된 알부민 나노입자 용액에 혼합반응시켜, 약물이 담지된 알부민 나노입자에 CEACAM6 항체를 결합시키는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for preparing nanoparticles for cancer cell chemotherapy according to the present invention, the antibody binding step includes N-Hydroxysuccinimide and N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride. Mixing and reacting the CEACAM6 antibody solution in the MES reaction buffer to form a NHS / EDC-activated antibody, the activated lutein is mixed with the albumin nanoparticle solution loaded with the drug, albumin nanoparticles loaded with the drug It is characterized by binding the CEACAM6 antibody to.

본 발명은 앞서 본 실시예에 의해 다음과 같은 효과를 얻을 수 있다.The present invention can obtain the following effects by the above embodiment.

본 발명은 전이 암세포를 효과적으로 사멸시킬 수 있는 효과가 있다.The present invention has the effect of effectively killing metastatic cancer cells.

또한, 본 발명은 알부민 나노입자에 결합한 CEACAM6 항체가 아노이키스 내성 암세포를 타켓팅하여 전이 암세포에 약물을 효과적으로 전달할 수 있는 효과가 있다.In addition, the present invention has the effect that the CEACAM6 antibody bound to the albumin nanoparticles can effectively deliver the drug to metastatic cancer cells by targeting an aikis resistant cancer cells.

또한, 본 발명은 약물을 담지하는 알부민 나노입자는 디설파이드 결합에 의해 생성되므로, 암세포 내에 섭취된 알부민 나노입자는 세포기질에 존재하는 글루타틴온에 의해 쪼개져 비목적 지역에 약물을 방출을 억제할 수 있는 효과가 있다.In addition, according to the present invention, since albumin nanoparticles carrying a drug are produced by disulfide bonds, albumin nanoparticles ingested in cancer cells may be cleaved by glutathione present in the cell substrate to inhibit release of the drug in a non-target area. It has an effect.

도 1은 tHSA-NPs의 Zetasizer 및 TEM 측정결과를 나타내는 도표.
도 2는 D-tHSA-NPs의 Zetasizer 측정결과를 나타내는 도표.
도 3은 티올화 비율에 따라 약물 담지효율을 나타내는 도표.
도 4는 글푸타티온 여부에 따라 D-tHSA-NPs의 약물 방출 양을 나타내는 도표.
도 5는 글루타티온 여부에 따라 D-tHSA-NPs의 크기 변화를 나타내는 도표.
도 6은 D-tHSA-NPs 및 C6D-tHSA-NPs의 입자 분포를 나타내는 도표.
도 7 및 8은 C6D-tHSA-NPs에서 D-tHSA-NPs의 표면에 CEACAM6가 결합한 것을 확인하기 위한 공초점 현미경 이미지.
도 9는 배양된 암세포의 CEACAM6 단백질 발현을 확인하기 위한 형광 현미경 이미지.
도 10은 배양된 암세포의 CEACAM6 유전자 발현을 확인하기 위한 RT-PCT 결과를 나타내는 도표.
도 11은 C6-tHSA-NPs의 아노이키스 내성 암세포로의 표적 능력을 확인하기 위한 공초점 현미경 이미지.
도 12는 C6D-tHSA-NPs의 아노이키스 내성 암세포의 치료 효율을 확인하기 위한 Cell viability 결과를 나타내는 도표.
도 13은 동물모델에서 C6D-tHSA-NPs의 효과를 확인하기 위한 마우스 체중 측정결과를 나타내는 도표.
도 14는 동물모델에서 C6D-tHSA-NPs의 효과를 확인하기 위한 폐 조직을 촬영한 이미지.
도 15는 동물모델에서 C6D-tHSA-NPs의 효과를 확인하기 위한 폐 조직의 전이암 면적 비율을 나타내는 도표.1 is a chart showing the results of Zetasizer and TEM of tHSA-NPs.
Figure 2 is a table showing the Zetasizer measurement results of D-tHSA-NPs.
Figure 3 is a chart showing the drug loading efficiency according to the thiolation ratio.
Figure 4 is a chart showing the amount of drug release of D-tHSA-NPs depending on whether or not glutathione.
Figure 5 is a chart showing the size change of D-tHSA-NPs depending on whether glutathione.
6 is a chart showing particle distribution of D-tHSA-NPs and C6D-tHSA-NPs.
7 and 8 are confocal microscopy images to confirm that CEACAM6 is bound to the surface of D-tHSA-NPs in C6D-tHSA-NPs.
9 is a fluorescence microscope image for confirming CEACAM6 protein expression in cultured cancer cells.
FIG. 10 is a chart showing RT-PCT results for confirming CEACAM6 gene expression in cultured cancer cells. FIG.
FIG. 11 is a confocal microscopy image to confirm the target ability of C6-tHSA-NPs to anocitic resistant cancer cells.
Figure 12 is a chart showing the results of Cell viability for confirming the therapeutic efficiency of C6D-tHSA-NPs treatment of anoikis-resistant cancer cells.
Figure 13 is a table showing the results of mouse weight measurement to confirm the effect of C6D-tHSA-NPs in animal models.
Figure 14 is an image of the lung tissue to confirm the effect of the C6D-tHSA-NPs in the animal model.
Figure 15 is a chart showing the metastatic cancer area ratio of lung tissue to confirm the effect of C6D-tHSA-NPs in animal models.

이하에서는 본 발명에 따른 아노이키스 내성 암세포의 화학요법을 위한 나노입자를 첨부된 도면을 참조하여 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 정의에 따른다. 또한, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대해 상세한 설명은 생략한다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다.Hereinafter, with reference to the accompanying drawings, the nanoparticles for chemotherapy of an Aokis resistant cancer cells according to the present invention will be described in detail. Unless otherwise defined, all terms in this specification are equivalent to the general meaning of the terms understood by those of ordinary skill in the art to which the present invention pertains and, if they conflict with the meanings of the terms used herein, Follow the definition used in the specification. In addition, detailed description of well-known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted. Throughout the specification, when a part is said to "include" a certain component, it means that it may further include other components, without excluding other components unless specifically stated otherwise.

본 발명의 일 실시예에 따른 아노이키스 내성 암세포의 화학요법을 위한 나노입자는 약물을 담지한 알부민 나노입자와, 상기 알부민 나노입자에 결합한 CEACAM6 항체를 포함하며, 상기 알부민 나노입자는 알부민 분자 사이의 디설파이드 결합(disulfide bonding)에 의해서 형성되고, 상기 CEACAM6 항체는 알부민 나노입자 표면의 아민 그룹(amine group)과 아마이드 결합(amide bonding)하여 알부민 나노입자의 표면에 결합한다. 상기 아노이키스 내성 암세포의 화학요법을 위한 나노입자는 150 내지 350nm의 직경을 가지며, 상기 알부민 나노입자는 구형의 형태를 가진다. 상기 약물은 암세포를 사멸할 수 있는 다양한 약물이 사용될 수 있으며 일 에로 독소루비신이 사용될 수 있다. 혈관 또는 림프관을 통해 이동하는 암세포(floating cancer cell)에 의한 전이(metastasis)에서, 앵커리지 의존 프로그램드 세포 사멸(anchorage dependent programmed cell death)을 의미하는 아노이키스에 대한 내성은 필수적이며, 아노이키스 내성 특성을 갖는 암세포에서는 CEACAM6(carcinoembryonic antigen-related cell adhesion molecule 6)가 과발현(overexpressing)되므로, 본 발명의 암세포의 화학요법을 위한 나노입자는 알부민 나노입자의 표면에 CEACAM6를 타켓팅하는 CEACAM6 항체가 결합하여 전이 암세포(floting(metastasis) cancer cell)를 효과적으로 타켓팅할 수 있고, 알부민 나노입자는 상대적으로 강한 디설파이드 결합에 형성되므로 약물을 효과적으로 담지할 수 있으며, 디설파이드 결합을 끊는 글루타티온(glutathione)은 세포 기질(cell cytosol)에는 존재하나 혈액 및 림프액에는 존재하지 않아 본 발명의 나노입자는 암세포에 섭취된 다음 약물을 방출하므로 비목적하는 지역이 아닌 암세포에 효과적으로 약물을 방출할 수 있어 부작용을 줄이고 암세포 사멸효과를 증가시킬 수 있다. 본 발명의 나노입자는 아노이키스 내성을 가지는 유방암, 위암, 결장암, 췌장암 등의 다양한 전이암에 적용될 수 있다.Nanoparticles for chemotherapy of anoikis-resistant cancer cells according to an embodiment of the present invention comprises albumin nanoparticles carrying a drug, CEACAM6 antibody bound to the albumin nanoparticles, the albumin nanoparticles between the albumin molecules Formed by disulfide bonding, the CEACAM6 antibody binds to the surface of the albumin nanoparticle by amide bonding with an amine group on the surface of the albumin nanoparticle. The nanoparticles for chemotherapy of the anoikis resistant cancer cells have a diameter of 150 to 350 nm, and the albumin nanoparticles have a spherical shape. As the drug, various drugs capable of killing cancer cells may be used, and one doxorubicin may be used. In metastasis by floating cancer cells, which travel through blood vessels or lymphatic vessels, resistance to annoyis, which means anchorage dependent programmed cell death, is essential and annoyis resistant. In cancer cells having CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6) is overexpressed (overexpressing), the nanoparticles for chemotherapy of cancer cells of the present invention, the CEACAM6 antibody targeting CEACAM6 to the surface of the albumin nanoparticles bind and metastasize Can effectively target the floating (metastasis) cancer cells, albumin nanoparticles are formed in the relatively strong disulfide bonds can effectively support the drug, glutathione that breaks disulfide bonds (cell cytosol) ) But not in blood and lymph Since the nanoparticles of the invention release the drug after being ingested into cancer cells, the drug can be effectively released into cancer cells instead of the undesired area, thereby reducing side effects and increasing cancer cell death effects. Nanoparticles of the present invention can be applied to a variety of metastatic cancers, such as breast cancer, stomach cancer, colon cancer, pancreatic cancer resistant to annoyis.

본 발명의 다른 실시예에 따른 아노이키스 내성 암세포의 화학요법을 위한 나노입자의 제조방법에 대한 것으로, 상기 나노입자의 제조방법은 알부민을 티올화하는 티올화단계와, 티올화된 알부민과 약물을 이용하여 이황화 결합 및 탈용매화 과정을 통해 약물이 담지된 알부민 나노입자를 형성하는 입자형성단계와, 상기 약물이 담지된 알부민 나노입자에 CEACAM6 항체를 결합시키는 항체결합단계를 포함한다.The present invention relates to a method for preparing nanoparticles for chemotherapy of anoikis-resistant cancer cells according to another embodiment of the present invention, wherein the method for preparing nanoparticles comprises a thiolation step of thiolating albumin, and a thiolated albumin and a drug. Particle forming step of forming a drug-supported albumin nanoparticles through disulfide bond and desolvation process, and antibody binding step of binding the CEACAM6 antibody to the drug-supported albumin nanoparticles.

상기 티올화단계는 알부민을 티올화하는 단계로, 구체적으로 piperazine-N'-ethanesulfonic acid(HEPES)와 ethylenediaminetetraacetic acid(EDTA)를 함유하는 반응버퍼에 알부민(human serum albumin)을 녹이고, 2-Iminothiolane hydrochloride(2-IT)에 의한 티올화 과정(thiolation process)을 통해 티올화된 알부민(thiolated albumin)을 생성한다. 상기 티올화단계에서 알부민과 2-IT의 몰비율은 1 대 20 내지 30인 것이 바람직하다.The thiolation step is to thiolate albumin, specifically, albumin (human serum albumin) is dissolved in a reaction buffer containing piperazine-N'-ethanesulfonic acid (HEPES) and ethylenediaminetetraacetic acid (EDTA), and 2-Ithiothiolane hydrochloride Thiolated albumin is produced through a thiolation process with (2-IT). In the thiolation step, the molar ratio of albumin and 2-IT is preferably 1 to 20 to 30.

상기 입자형성단계는 티올화된 알부민과 약물을 이용하여 이황화 결합 및 탈용매화 과정을 통해 약물이 담지된 알부민 나노입자를 형성하는 단계로, 구체적으로 HEPES 버퍼에 상기 티올화된 알부민과 약물이 혼합된 용액에 에탄올을 드롭방식(dropwise)으로 첨가반응시켜 약물이 담지된 알부민 나노입자를 생성한다.The particle forming step is to form a drug-supported albumin nanoparticles through disulfide bond and desolvation process using thiolated albumin and the drug, specifically, the thiolated albumin and drug are mixed in the HEPES buffer Ethanol is added dropwise to the solution to produce drug-bearing albumin nanoparticles.

상기 항체결합단계는 상기 약물이 담지된 알부민 나노입자에 CEACAM6 항체를 결합시키는 단계로, 구체적으로 N-Hydroxysuccinimide(NHS)와 N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride(EDC)를 포함하는 MES 반응버퍼 내에 CEACAM6 항체 용액을 혼합하여 반응시키고 정제하여 NHS/EDC-활성화된 항체(NHS/EDC-activated antibody)를 형성하고, 활성화된 항체와 상기 약물이 담지된 알부민 나노입자 표면의 아민 그룹(amine group) 사이에 아마이드 결합(amide bonding)을 형성하기 위해, 상기 활성화된 황체를 상기 약물이 담지된 알부민 나노입자 용액에 혼합반응시켜, 약물이 담지된 알부민 나노입자에 CEACAM6 항체가 결합시킨다.The antibody binding step is to bind the CEACAM6 antibody to the albumin nanoparticles carrying the drug, specifically N-Hydroxysuccinimide (NHS) and N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) A mixture of CEACAM6 antibodies in the MES reaction buffer is reacted and purified to form an NHS / EDC-activated antibody, and an amine group on the surface of the activated nanoparticle and the albumin nanoparticles carrying the drug ( In order to form an amide bond between amine groups, the activated corpus luteum is mixed with the albumin nanoparticle solution loaded with the drug to bind the CEACAM6 antibody to the albumin nanoparticle loaded with the drug.

이하, 실시예를 통해서 본 발명을 보다 상세히 설명하기로 한다. 하지만, 이들은 본 발명을 보다 상세하게 설명하기 위한 것일 뿐, 본 발명의 권리범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are only for explaining the present invention in more detail, the scope of the present invention is not limited thereto.

<실시예 1> 항체가 결합하고 약물이 담지된 알부민 나노입자의 제조Example 1 Preparation of Albumin Nanoparticles to Which Antibodies Bound and Drug Carryed

(1) 1mM piperazine-N'-ethanesulfonic acid(HEPES)와 0.1mM ethylenediaminetetraacetic acid(EDTA)를 함유하는 반응버퍼(pH 8.0)에 알부민(human serum albumin) 40mg을 녹이고, 몰비율 1:25로 2-Iminothiolane hydrochloride(2-IT)에 의한 티올화 과정(thiolation process)을 통해 티올화된 알부민(thiolated albumin)을 생성하였다.(1) Dissolve 40 mg of albumin (human serum albumin) in a reaction buffer (pH 8.0) containing 1 mM piperazine-N'-ethanesulfonic acid (HEPES) and 0.1 mM ethylenediaminetetraacetic acid (EDTA). Thiolated albumin was produced through a thiolation process with Iminothiolane hydrochloride (2-IT).

(2) 이황화 결합(disulfide bonding) 반응 및 탈용매화 과정(desolvation)에 의한 티올화된 알부민 분자의 나노 집합체를 만들기 위해 HEPES 버퍼에 상기 티올화된 알부민과 독소루비신(doxorubicin)이 혼합된 용액에 에탄올을 드롭방식(dropwise)으로 첨가하여 실온에서 하룻밤 동안 반응시켜 독소루비신이 담지된 알부민 나노입자(D-tHSA-NPs)를 생성하였다.(2) Ethanol was added to a solution in which the thiolated albumin and doxorubicin were mixed in a HEPES buffer in order to form nano aggregates of thiolated albumin molecules by disulfide bonding reaction and desolvation. It was added dropwise and reacted at room temperature overnight to produce doxorubicin loaded albumin nanoparticles (D-tHSA-NPs).

(3) 5mM N-Hydroxysuccinimide(NHS)와 2mM N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride(EDC)를 포함하는 MES 반응버퍼 내에 CEACAM6 항체 용액 1mg/ml를 혼합하고 실온에서 15분 반응시키고 정제하여 NHS/EDC-활성화된 항체(NHS/EDC-activated antibody)를 형성하고, 활성화된 항체와 나노입자(D-tHSA-NPs)의 표면의 아민 그룹(amine group) 사이에 아마이드 결합(amide bonding)을 형성하기 위해, 상기 활성화된 황체를 나노입자 용액(D-tHSA-NPs solution)에 혼합하여 1.5시간 반응시켜, 독소루비신이 담지된 알부민 나노입자에 CEACAM6 항체가 결합한 알부민 나노입자(C6D-tHSA-NPs)를 생성하였다.(3) 1 mg / ml of CEACAM6 antibody solution was mixed in a MES reaction buffer containing 5 mM N-Hydroxysuccinimide (NHS) and 2 mM N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), and reacted for 15 minutes at room temperature. Purification to form NHS / EDC-activated antibody and amide bonding between the activated antibody and the amine group on the surface of the nanoparticles (D-tHSA-NPs). In order to form), the activated corpus luteum is mixed with a nanoparticle solution (D-tHSA-NPs solution) and reacted for 1.5 hours, and albumin nanoparticles (C6D-tHSA-) having a CEACAM6 antibody bound to doxorubicin-containing albumin nanoparticles. NPs).

<실시예 2> 알부민 나노입자의 특성 확인Example 2 Characterization of Albumin Nanoparticles

(1) 독소루비신을 사용하지 않는 것을 제외하고는 실시예 1의 (2)의 방법과 동일하게 하여 독소루비신이 담지되지 않은 알부민 나노입자(tHSA-NPs)를 생성하고, Zetasizer(Nano-ZS, Malvern) 및 TEM으로 측정하여 그 결과를 도 1에 나타내었다. 또한, 실시예 1의 (2)에서 제조된 알부민 나노입자(D-tHSA-NPs)를 Zetasizer로 측정하여 도 2에 나타내었다. 도 1 및 2를 통해, 약물의 캡슐화 여부와 상관없이 상기 입자들은 대략 180nm대의 직경을 가짐을 알 수 있고, 도 1을 보면 상기 나노입자(tHSA-NPs)는 구형의 형태를 가짐을 알 수 있다.(1) Except for not using doxorubicin, in the same manner as in Example (2), doxorubicin-containing albumin nanoparticles (tHSA-NPs) were produced, and Zetasizer (Nano-ZS, Malvern) And measured by TEM and the results are shown in FIG. In addition, albumin nanoparticles (D-tHSA-NPs) prepared in Example (2) was measured in Zetasizer and shown in FIG. 2. 1 and 2, it can be seen that the particles have a diameter of approximately 180 nm regardless of whether or not the drug is encapsulated. Referring to FIG. 1, the nanoparticles (tHSA-NPs) have a spherical shape. .

(2) 알부민과 2-IT의 몰비율을 1:25에서 1:12.5 및 37.5로 바꾼 것을 제외하고는 실시예 1의 (2)의 방법과 동일하게 하여 알부민 나노입자들(D-tHSA-NPs(12.5), D-tHSA-NPs(37.5))를 제조하였다. 실시예 1의 (2)에서 제조된 알부민 나노입자(D-tHSA-NPs(25)) 및 상기 나노입자들(D-tHSA-NPs(12.5), D-tHSA-NPs(37.5))을 HPLC 방법에 의해 독소루비신 담지 효율을 측정하여 도 3에 나타내었다. 도 3을 보면 티올화 비율 1: 25일 때 담지 효율이 가장 큰 것을 알 수 있는데, 티올화 비율 1:12.5인 경우 내부분자의 불충분한 가교가 일어났기 때문에 담지 효율이 낮은 것으로 보이고, 위의 실험 전에 원심분리를 통해 마이크로 사이즈의 나노입자를 제거하는데 티올화 비율이 1:37.5인 경우는 과한 가교가 일어나 마이크로 사이즈의 나노입자가 많이 생성되어 담지 효율이 낮은 것으로 보인다.(2) Albumin nanoparticles (D-tHSA-NPs) in the same manner as in Example (2) except that the molar ratio of albumin and 2-IT was changed from 1:25 to 1: 12.5 and 37.5. (12.5), D-tHSA-NPs (37.5)) were prepared. Albumin nanoparticles prepared in Example (2) (D-tHSA-NPs (25)) and the nanoparticles (D-tHSA-NPs (12.5), D-tHSA-NPs (37.5)) HPLC method Doxorubicin carrying efficiency was measured and shown in FIG. 3. Referring to FIG. 3, it can be seen that the supporting efficiency is the highest when the thiolation ratio is 1:25, and when the thiolation ratio is 1: 12.5, the supporting efficiency seems to be low because insufficient crosslinking of internal molecules occurs, before the above experiment. If the thiolation ratio is 1: 37.5 to remove the micro-sized nanoparticles by centrifugation, excessive crosslinking occurs to produce a large number of micro-sized nanoparticles, which seems to be low in carrying efficiency.

(3) 나노입자(D-tHSA-NPs)로부터 독소루비신 방출 속도를 확인하기 위해서, 실시예 1의 (2)에서 제조된 나노입자(D-tHSA-NPs)는 dispersion medium 내의 dialysis membrane(molecular weight cutoff 30kDa)에 패킹되어 나노입자 내에 담지된 독소루비신은 증류수 10mL로 배출되도록 한 후, HPLC를 통해 분석하여 그 결과를 도 4에 나타내었다. 실험은 dispersion medium 내에 glutathione이 있는 경우(with GSH)와 없는 경우(w/o GSH)로 나누어 실험하였다. 도 4를 보면, glutathione이 있는 경우(6시간 내에 90% 방출) 없는 경우보다 빠른 시간 내 많은 양의 독소루비신을 방출함을 알 수 있다.(3) In order to confirm the release rate of doxorubicin from the nanoparticles (D-tHSA-NPs), the nanoparticles (D-tHSA-NPs) prepared in Example (2) were prepared in a dialysis membrane (molecular weight cutoff) in a dispersion medium. Doxorubicin packed in the nanoparticles packed in 30 kDa) was discharged into 10 mL of distilled water, and analyzed by HPLC. The experiment was divided into glutathione (with GSH) and no (w / o GSH) in dispersion medium. 4, it can be seen that glutathione releases a large amount of doxorubicin in a faster time than without (with 90% release within 6 hours).

(4) 50% serum 내에 실시예 (1)의 2에서 제조한 나노입자를 첨가하고 5mM의 glutathion이 있는 경우와 없는 경우로 나누어 입자 사이즈 측정하여 도 5에 나타내었다. 도 5를 보면, GSH가 없는 경우 24시간 동안 입자 크기의 변화가 거의 없는데, GSH가 첨가된 경우 1시간이 지난 후에 입자 크기 현저하게 커졌음을 알 수 있다. 이는 GSH가 나노입자의 이황화 결합을 끊어 나노입자의 사이즈가 커진 것으로 보인다.(4) Adding the nanoparticles prepared in Example 2 in 50% serum and dividing the particles into and without 5mM glutathion is shown in FIG. Referring to Figure 5, there is almost no change in particle size for 24 hours in the absence of GSH, it can be seen that the particle size significantly increased after 1 hour when GSH is added. It is believed that GSH breaks the disulfide bonds of nanoparticles, thereby increasing the size of the nanoparticles.

(5) 실시예 1의 (2)에서 제조된 나노입자(D-tHSA-NPs)와 실시예 1의 (3)에서 제조된 나노입자(C6D-tHSA-NPs)를 Zetasizer로 측정하여, 도 6에 나타내었다. 또한, 실시예 1의 (2)에서 제조된 나노입자(D-tHSA-NPs)와 실시예 1의 (3)에서 제조된 나노입자(C6D-tHSA-NPs) 각각에 Alexa Fluor 555-NHS ester(나노입자를 시각화하기 위해 사용) 및 goat anti-rabbit IgG(Alexa Fluor 488-conjugated)(나노입자 표면의 항체를 시각화하기 위해 사용)를 적용하고 confocal laser scanning microscopy로 측정하여 그 결과를 도 7 및 8에 나타내었다. 도 6을 보면, C6D-tHSA-NPs의 입자크기가 D-tHSA-NPs보다 큼을 알 수 있고, 도 7을 보면 항체를 시각화하는 Alexa Fluor 488에 의해 형광이미지가 형성되지 않음을 알 수 있고, 도 8을 보면 Alexa Fluor 488에 의한 형광이미지(녹색)가 나노입자를 시각화하는 Alexa Fluor 555(붉은색)과 일치하여 합친(merge) 영상이 녹색으로 표현됨을 알 수 있어, D-tHSA-NPs의 외면에 CEACAM6 항체가 결합하여 C6D-tHSA-NPs가 형성되었음을 알 수 있다.(5) The nanoparticles (D-tHSA-NPs) prepared in (2) of Example 1 and the nanoparticles (C6D-tHSA-NPs) prepared in Example (3) of Example 1 were measured by Zetasizer, and FIG. 6 Shown in Further, Alexa Fluor 555-NHS ester (D-tHSA-NPs) prepared in Example (2) and nanoparticles (C6D-tHSA-NPs) prepared in Example 1 (3), respectively, were used. Used to visualize nanoparticles) and goat anti-rabbit IgG (Alexa Fluor 488-conjugated) (used to visualize antibodies on the surface of nanoparticles) and measured by confocal laser scanning microscopy to determine the results of FIGS. 7 and 8 Shown in Referring to FIG. 6, it can be seen that the particle size of C6D-tHSA-NPs is larger than that of D-tHSA-NPs, and FIG. 7 shows that fluorescence images are not formed by Alexa Fluor 488 for visualizing antibodies. 8 shows that the fluorescence image (green) by Alexa Fluor 488 is green in combination with Alexa Fluor 555 (red), which visualizes the nanoparticles. It can be seen that the CEACAM6 antibody binds to C6D-tHSA-NPs.

<실시예 3> 세포 배양 및 세포의 CEACAM6 발현 확인Example 3 Confirmation of CEACAM6 Expression in Cell Culture and Cells

(1) MDA-MB-231 and MCF-7 human breast cancer cell이 10% fetal bovine serum(FBS)이 보충된 Dulbecco's modified Eagle's medium(DMEM)에서 5% CO2, 37℃ 환경으로 배양된다. 부착 배양(adherent culture)은 the tissue culture dishes(Falcon, San Jose, CA)에서 행하여지며, 유방암 세포의 아노이키스 내성 성질(anoikis resistant property)을 유도하기 위한 부유 배양(Suspension culture)은 10mg/ml Poly-HEMA(Poly(2-hydroxyethyl methacrylate))(Sigma, St. Louis, MO)가 고팅된 dishes에서 행하여진다. 구체적으로, 1×106 세포를 poly-HEMA가 코팅된 디쉬에 시트하고 일주일 이상 성장시키고, 3일마다 신선한 배지를 첨가하고 7일 후 trypsin-EDTA를 처리한다. 이하에서 부착 배양된 것을 adherent라 표시하고 아노이키스 내성 성질을 유도하기 위해 부유 배양된 것을 AR로 표시하고 한다.(1) MDA-MB-231 and MCF-7 human breast cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 5% CO 2 and 37 ° C. Adherent cultures are performed in the tissue culture dishes (Falcon, San Jose, Calif.), And suspension culture to induce anikis resistant properties of breast cancer cells is 10 mg / ml Poly. -HEMA (Poly (2-hydroxyethyl methacrylate)) (Sigma, St. Louis, Mo.) is performed in a dish that has been coated. Specifically, 1 × 10 6 cells are sheeted in a dish coated with poly-HEMA and grown for at least a week, fresh medium is added every 3 days and treated with trypsin-EDTA after 7 days. In the following, the adherent culture is labeled as adherent, and the suspended culture is labeled as AR in order to induce anobic acid resistance.

(2) 상기 과정에서 배양된 세포를 각각 펠릿화하고 1X PBS(500ul)에 재부유하고, 3.7 PFA 고정화한 후에 PBTA 버퍼용액에서 투과(permeability) 및 블락킹(blocking) 과정을 수행한다. 이후, 상기 세포는 rabbit anti-human CEACAM6 antibody(Sigma, Saint Louis, MO)를 가지고 실온에서 1시간 동안 인큐베이팅되고, 1X PBS로 린스되고 goat anti-rabbit IgG(FITC-conjugated)와 실온에서 30분 동안 반응시킨다. 커버 글라스에 염색된 세포를 DAPI를 가진 마운팅 배지에 마운팅된 후 형광현미경으로 측정하여 그 결과를 도 9에 나타내었다.(2) The cells cultured in the above process are pelleted and resuspended in 1X PBS (500ul), and after 3.7 PFA immobilization, permeability and blocking are performed in PBTA buffer solution. The cells are then incubated with rabbit anti-human CEACAM6 antibody (Sigma, Saint Louis, MO) for 1 hour at room temperature, rinsed with 1X PBS and for 30 minutes at room temperature with goat anti-rabbit IgG (FITC-conjugated). React. Cells stained on the cover glass were mounted on a mounting medium with DAPI and measured by fluorescence microscopy. The results are shown in FIG. 9.

(3) 또한, CEACAM6의 발현을 수량화하기 위해 adherent 세포와 AR 세포에 대한 RT-PCT를 수행하여 그 결과를 도 10에 나타내었다. 사용된 CEACAM6 primer(Bioneer,Daejeon, Korea는 다음 서열을 가지고 있다. CEACAM6 forward: 5'-TACTCAGCGTCAAAAGGAAC-3', CEACAM6 reverse: 5'-AGAGACTGTGATCATCGTGA-3'. RT-PCR gel retardation assay에서 유전자 밴드(gene bands)는 EtBr로 염색되고, Gel Doc imaging device에 의해 시각화된다.(3) RT-PCT was performed on adherent cells and AR cells to quantify the expression of CEACAM6, and the results are shown in FIG. 10. The CEACAM6 primers used (Bioneer, Daejeon, Korea) have the following sequence: CEACAM6 forward: 5'-TACTCAGCGTCAAAAGGAAC-3 ', CEACAM6 reverse: 5'-AGAGACTGTGATCATCGTGA-3'.The gene band in the RT-PCR gel retardation assay bands) are stained with EtBr and visualized by Gel Doc imaging device.

(4) 도 9를 보면, CEACAM6 단백질을 표지하는 녹색 형광이 anoikis resistant 특성을 유도한 MDA-MB-231 and MCF-7 암세포에서 높게 발현됨을 알 수 있다. 또한, 도 10을 보면, CEACAM6 유전자 역시 anoikis resistant 특성을 유도한 MDA-MB-231 and MCF-7 암세포에서 높게 발현됨을 알 수 있다. 이를 통해, 아노이키스 내성이 유도된 플로우팅 조건에서 CEACAM6가 유방암에 세포에서 과발현됨을 알 수 있다.9, it can be seen that the green fluorescence labeling CEACAM6 protein is highly expressed in MDA-MB-231 and MCF-7 cancer cells inducing anoikis resistant properties. In addition, it can be seen that the CEACAM6 gene is also highly expressed in MDA-MB-231 and MCF-7 cancer cells that induce anoikis resistant properties. Through this, it can be seen that CEACAM6 is overexpressed in the cells of breast cancer in the floating conditions induced by anikis resistance.

<실시예 4> 알부민 나노입자의 아노이키스 내성 암세포로의 표적 능력 확인Example 4 Confirmation of Target Ability of Albumin Nanoparticles into Anoikis-Resistant Cancer Cells

(1) 독소루비신을 사용하지 않는 것을 제외하고는 실시예 1의 (3)의 방법과 동일하게 하여 독소루비신이 담지되지 않은 알부민 나노입자(C6-tHSA-NPs)를 생성하였다.(1) Except for not using doxorubicin, albumin nanoparticles (C6-tHSA-NPs) without doxorubicin were produced in the same manner as in Example 1 (3).

(2) MCF-7 anoikis-resistant cell(5×105 cells/each well)을 Poly-HEMA가 코팅된 12-well cell culture dish에 시드하고 배양하였다. 상기 세포는 37℃에서 C6-tHSA-NPs, tHSA-NPs를 가지고 처리되며, 30분 이내로 배양한 후, 세포를 모으고 1X PBS로 워싱하고 3.7% PFA 용액을 이용하여 10분 동안 고정하고, 세포막을 1/40로 희석된 F-actin antibody(invitrogen, Alexa Fluor 488 Phalloidin)을 가지고 30분 동안 염색하고 PBTA buffer를 가지고 워싱한다. 이후, 상기 세포는 DAPI를 가진 마운팅 배지에 이식되며, 532nm/580nm(red color), 488nm/521nm(green color), and 358nm/461nm(blue color)의 광원 및 공초점 현미경을 이용하여 나노입자(Alexa Fluor 555), 세포막(F-actin), 세포핵(DAPI) 각각을 이미지화하여 그 결과를 도 11에 나타내었다.(2) MCF-7 anoikis-resistant cells (5 × 10 5 cells / each well) were seeded and incubated in a 12-well cell culture dish coated with Poly-HEMA. The cells were treated with C6-tHSA-NPs, tHSA-NPs at 37 ° C., incubated within 30 minutes, then the cells were collected, washed with 1 × PBS and fixed with 3.7% PFA solution for 10 minutes, and the cell membrane Stain for 30 minutes with 1 / 4-diluted F-actin antibody (Invitrogen, Alexa Fluor 488 Phalloidin) and wash with PBTA buffer. The cells are then implanted in a mounting medium with DAPI, using nanoparticles using a light source and a confocal microscope of 532 nm / 580 nm (red color), 488 nm / 521 nm (green color), and 358 nm / 461 nm (blue color). Alexa Fluor 555), the cell membrane (F-actin), each of the cell nucleus (DAPI) was imaged and the results are shown in FIG.

(3) 도 11을 보면, C6-tHSA-NPs가 아노이키스 내성 특성을 가진 MCF-7 세포 내에 많이 분포하는 것을 알 수 있어 CEACAM6 항체가 결합된 나노입자는 아노이키스 내성 특성을 가진 암 세포로 방향성을 가지고 있음을 알 수 있다.(3) In FIG. 11, it can be seen that C6-tHSA-NPs are widely distributed in MCF-7 cells having an ANOKI resistance, so that the nanoparticles bound to CEACAM6 antibodies are directed to cancer cells having ANOI resistance. It can be seen that it has.

<실시예 5> 아노이키스 암세포에서 알부민 나노입자의 치료 효율 확인Example 5 Confirmation of Therapeutic Efficiency of Albumin Nanoparticles in Anoiki Cancer Cells

(1) 정상적인(Adh.) MDA-MB-231 세포 및 아노이키스 내성 특성을 가진(AR) MDA-MB-231 세포를 6-well plates에 시드하고, 각각 tHSA-NPs, 독소루시신(100mM), D-tHSA-NPs(100mM의 독소루비신 포함), C6D-tHSA-NPs(100mM의 독소루비신 포함)을 포함하는 배지를 가지고 37℃에서 6시간 배양하며, 이후 상기 세포를 노멀 배지(normal media)를 가지고 72시간 동안 배양하여 Cell vialbilty를 수행하여 그 결과를 도 12에 나타내었다. Cell viability는 MTT assay와 trypan blue dye exclusion method를 이용하는데, Cell viability는 0.4% trypan blue dye를 가진 세포를 배양하고 Neubauer hemocytometer로 카운팅하는 것에 의해 결정된다. MTT assay에서 96-well plates와 1.5mg/ml MTT reagent(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide)가 사용되며, 15ul의 MTT reagent를 가지고 2시간 배양한 후 200μl의 DMSO가 각 well에 추가되고, Resulting culture plates는 plate reader(Bio Tek Instruments, Inc, Winooski, VT, USA)로 570nm에서 측정하였다.(1) Normal (Adh.) MDA-MB-231 cells and ADA MDA-MB-231 cells with anikis resistance properties were seeded in 6-well plates and tHSA-NPs and doxoleucine (100 mM), respectively. , Incubated at 37 ° C. for 6 hours with a medium containing D-tHSA-NPs (including 100 mM doxorubicin) and C6D-tHSA-NPs (including 100 mM doxorubicin), and then the cells were treated with normal media. Cell vialbilty was carried out by incubation for 72 hours, and the results are shown in FIG. 12. Cell viability is determined by MTT assay and trypan blue dye exclusion method. Cell viability is determined by culturing cells with 0.4% trypan blue dye and counting with a Neubauer hemocytometer. In the MTT assay, 96-well plates and 1.5mg / ml MTT reagent (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide) were used, followed by incubation for 2 hours with 15ul of MTT reagent and 200μl. DMSO was added to each well, and the resulting culture plates were measured at 570 nm with a plate reader (Bio Tek Instruments, Inc., Winooski, VT, USA).

(2) 도 12를 보면, C6D-tHSA-NPs의 세포독성(cytotoxicity)은 Adh. 및 AR 암세포 모두에 가장 효율적임을 알 수 있고, AR 암세포에서 C6D-tHSA-NPs과 D-tHSA-NPs 사이의 p값은 0.01보다 작고(p값이 0.01보다 작은 것은 통계적으로 중요한 의미를 가짐), Adh. 암세포에서 C6D-tHSA-NPs과 D-tHSA-NPs 사이의 p값은 0.05보다 크다(p값이 0.05보다 큰 것은 통계적으로 중요한 의미를 갖지 않음).(2) Referring to Figure 12, the cytotoxicity (cytotoxicity) of C6D-tHSA-NPs was Adh. And AR cancer cells, the p value between C6D-tHSA-NPs and D-tHSA-NPs in AR cancer cells is less than 0.01 (p value less than 0.01 has a statistical significance), Adh. In cancer cells, the p-value between C6D-tHSA-NPs and D-tHSA-NPs is greater than 0.05 (the p value greater than 0.05 has no statistical significance).

<실시예 6> 동물 모델에서 알부민 나노입자의 항암 효과 확인Example 6 Anticancer Effect of Albumin Nanoparticles in Animal Models

(1) BALB/c 누드 마우스의 꼬리에 정맥주사로 전이암 A549 AR 세포(마리당 2×106cells)를 각각 이식한 후, 세포 주입 즉시 및 이로부터 4일 후 각각 독소루시신(DOX), D-tHSA-NPs, C6D-tHSA-NPs를 정맥주사를 통해 주입하였다(독소루시신(DOX), D-tHSA-NPs 및 C6D-tHSA-NPs 각각에 사용된 독소루비신 양은 동일함). 이후, 2일 간격으로 14일 동안 체중을 측정하여 그 결과를 도 13에 나타내었고, 2주 후 마우스 안락사시킨 후 페 조직을 적출하고 적출된 폐 조직은 전이암 colony의 명확한 구분을 위하여 Bouin's solution에 염색하고 24시간 이후 폐 조직을 촬영하여 그 결과를 도 14에 나타내었고, 상기 조직 촬영 결과를 Image-Pro Plus software(Media Cybernetics, LP, USA)를 이용하여 전이암 면적 비율(%)을 분석하여, 그 결과를 도 15에 나타내었다. 이때, 전이암 면적 비율(%)은 전체 폐 면적 대비 전이암 면적으로 계산하였다. 도 14에서 front는 폐 조직 앞면을 촬영한 것이고 Back는 폐 조직 뒷면을 촬영한 것으로, 표면에서 특히 밝게 보이는 부분이 주입된 A549 AR 세포가 폐로 전이되어 생긴 전이암 colony를 나타낸다.(1) After injection of metastatic carcinoma A549 AR cells (2 × 10 6 cells per horse) into the tail of BALB / c nude mice, respectively, immediately after cell injection and 4 days thereafter, doxolysine (DOX), D-tHSA-NPs, C6D-tHSA-NPs were injected intravenously (the amount of doxorubicin used for each of doxolysine (DOX), D-tHSA-NPs and C6D-tHSA-NPs is the same). Then, the body weight was measured for 14 days at intervals of 2 days, and the results are shown in FIG. 13. After 2 weeks, the mice were euthanized, the lung tissues were extracted, and the extracted lung tissues were placed in Bouin's solution for clear classification of metastatic cancer colony. Lung tissue was photographed 24 hours after staining, and the results are shown in FIG. 14. The tissue imaging results were analyzed by using the Image-Pro Plus software (Media Cybernetics, LP, USA) to analyze the percentage of metastatic cancer area. The results are shown in FIG. 15. At this time, the metastatic cancer area ratio (%) was calculated as the metastatic cancer area to the total lung area. In FIG. 14, the front is taken of the front of the lung tissue and the back is taken of the back of the lung tissue, and shows the metastatic cancer colony resulting from metastasis of the A549 AR cells into which the particularly visible part of the surface is injected.

(2) 도 13을 보면 C6D-tHSA-NPs가 사용된 경우 체중감소의 부작용이 전혀 나타나지 않음을 알 수 있고, 도 14 및 15를 보면 C6D-tHSA-NPs가 사용된 경우 타 그룹에 비해 월등한 전이암 억제 효과를 보임을 알 수 있다. 즉, 독소루비신이 사용된 경우 초기 치료 이후 약간의 체중 감소가 있음을 알 수 있고, D-tHSA-NPs가 사용된 경우 표적능의 부재로 인해 매우 불규칙적이고 난류 조건을 가지는 혈류 내를 부유하고 있는 전이 암세포에 대한 약물전달이 오히려 단순 항암제(독소루비신) 주입에 비하여 비효율적임을 알 수 있는데 반해, C6D-tHSA-NPs가 사용된 경우 전이 암세포를 효과적으로 표적하여 세포 내로 도입됨으로써 담지하고 있는 항암제를 암세포에 전달하여 이를 높은 효율로 사멸시켜 전이를 억제할 수 있음을 알 수 있다.(2) Figure 13 shows that when the C6D-tHSA-NPs are used does not show any side effects of weight loss, and Figures 14 and 15 are superior to other groups when the C6D-tHSA-NPs are used. It can be seen that it shows the effect of inhibiting metastatic cancer. That is, when doxorubicin is used, there is a slight weight loss after the initial treatment. When D-tHSA-NPs are used, metastasis that floats in the blood stream with very irregular and turbulent conditions due to the lack of target ability While drug delivery to cancer cells is rather inefficient compared to simple anticancer drug injection (doxorubicin), when C6D-tHSA-NPs are used, they effectively target metastatic cancer cells to be introduced into the cells to deliver the supported anticancer drugs to the cancer cells. It can be seen that it is possible to suppress the transition by killing it with high efficiency.

이상에서, 출원인은 본 발명의 다양한 실시예들을 설명하였지만, 이와 같은 실시예들은 본 발명의 기술적 사상을 구현하는 일 실시예일 뿐이며, 본 발명의 기술적 사상을 구현하는 한 어떠한 변경예 또는 수정예도 본 발명의 범위에 속하는 것으로 해석되어야 한다.In the above, the Applicant has described various embodiments of the present invention, but these embodiments are merely one embodiment for implementing the technical idea of the present invention, and any changes or modifications may be made to the present invention as long as the technical idea of the present invention is implemented. It should be interpreted as falling within the scope of.

Claims (13)

삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 알부민을 티올화하는 티올화단계와, 티올화된 알부민과 약물을 이용하여 이황화 결합 및 탈용매화 과정을 통해 약물이 담지된 알부민 나노입자를 형성하는 입자형성단계와, 상기 약물이 담지된 알부민 나노입자에 CEACAM6 항체를 결합시키는 항체결합단계를 포함하며,
상기 약물은 독소루비신이 사용되는 것을 특징으로 하는 암세포 화학요법을 위한 나노입자의 제조방법.A thiolation step of thiolating albumin, a particle formation step of forming albumin nanoparticles carrying a drug through disulfide bonding and desolvation using thiolated albumin and a drug, and albumin nanoparticles carrying the drug An antibody binding step of binding the CEACAM6 antibody to,
The drug is a method for producing nanoparticles for cancer cell chemotherapy, characterized in that doxorubicin is used. 제9항에 있어서, 상기 티올화단계는
piperazine-N'-ethanesulfonic acid와 ethylenediaminetetraacetic acid를 함유하는 반응버퍼에 알부민을 녹이고, 2-Iminothiolane hydrochloride에 의한 티올화 과정을 통해 티올화된 알부민을 생성하는 것을 특징으로 하는 암세포 화학요법을 위한 나노입자의 제조방법.The method of claim 9, wherein the thiolation step is
Dissolution of albumin in a reaction buffer containing piperazine-N'-ethanesulfonic acid and ethylenediaminetetraacetic acid, and the formation of thiolated albumin through thiolation with 2-Iminothiolane hydrochloride to produce nanoparticles for cancer cell chemotherapy. Manufacturing method. 제10항에 있어서,
상기 티올화단계에서 알부민과 2-IT의 몰비율은 1 대 20 내지 30인 것을 특징으로 하는 암세포 화학요법을 위한 나노입자의 제조방법.The method of claim 10,
The molar ratio of albumin and 2-IT in the thiolation step is a method for producing nanoparticles for cancer cell chemotherapy, characterized in that 1 to 20 to 30. 제9항에 있어서, 상기 입자형성단계는
HEPES 버퍼에 상기 티올화된 알부민과 약물이 혼합된 용액에 에탄올을 드롭방식으로 첨가반응시켜 약물이 담지된 알부민 나노입자를 생성하는 것을 특징으로 하는 암세포 화학요법을 위한 나노입자의 제조방법.The method of claim 9, wherein the particle forming step
A method for producing nanoparticles for cancer cell chemotherapy, wherein the drug-supported albumin nanoparticles are produced by adding ethanol in a drop method to a solution in which the thiolated albumin and the drug are mixed in a HEPES buffer. 제9항에 있어서, 상기 항체결합단계는
N-Hydroxysuccinimide와 N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride를 포함하는 MES 반응버퍼 내에 CEACAM6 항체 용액을 혼합하여 반응시켜 NHS/EDC-활성화된 항체를 형성하고, 상기 활성화된 항체를 상기 약물이 담지된 알부민 나노입자 용액에 혼합반응시켜, 약물이 담지된 알부민 나노입자에 CEACAM6 항체를 결합시키는 것을 특징으로 하는 암세포 화학요법을 위한 나노입자의 제조방법.The method of claim 9, wherein the antibody binding step
Mixing and reacting a CEACAM6 antibody solution in a MES reaction buffer containing N-Hydroxysuccinimide and N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride to form an NHS / EDC-activated antibody, wherein the activated antibody is used as the drug A method for producing nanoparticles for cancer cell chemotherapeutic treatment, characterized in that the CEACAM6 antibody is bound to the albumin nanoparticles carrying the drug by mixing and reacting with the supported albumin nanoparticle solution.
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