T 세포 백혈병 치료를 위한 HDAC inhibitor 가 탑재된 표적 지향적 PLGA 나노입자에 대한 연구

Abstract

Gene regulation is closely related with packaging of histone protein and DNA chromatins. Through some modification such as post-translational acetylation and methylation, the packaging of histone complex is changed into open chromatin structure. Therefore, transcription factors have opportunity to approach DNA and gene promoter regions easily. In cancer, epigenic gene regulation mechanisms are different from those in the normal ones. High level expression of HDACs (histone deacetylases) which are related with cell proliferation, differentiation, and pathogenesis of cancer is generally discovered in cancer, acting in concert with hypoacetylation of histone proteins. Thus, histone deacetylase inhibitors can be used as one kind of anti-cancer drugs. The histone deacetylase inhibitors (HDACi) can increase sensitivity of apoptosis, obstructing activity of HDACs, increasing accessibility of transcription factors, normally re-activating repressed tumor suppressor genes. In this study, we have developed a stable, biodegradable, biocompatible, FDA approved PLGA polymer based panobinostat (LBH589, HDACi) filled T-cell specific targeting immuno-nanoparticles. Panobinostat (LBH589) is one of the HDAC inhibitors which is being tested in clinical trials. To minimize side effects as anti-cancer drug in non-tumor regions, we fabricated PLGA nanoparticles that encapsulate HDAC inhibitors. These nanoparticles were of size approximately around 200nm.To be delivered to the tumor specifically, targeting protein, anti-CD7 single chain antibody that has been previously shown as a T-cell specific targeting moiety was attached to the surface of PLGA nanoparticles through bioconjugation reactions. We found that scFvCD7-conjugated and drug-encapsulated PLGA nanoparticles (CD7-PLGA/LBH589) have significant effect on inhibition of proliferation and induction of apoptosis in cancer cells in vitro. In addition, scFvCD7 conjugated PLGA nanoparticles (CD7-PLGA) were delivered specifiaclly into leukemia cancer in vivo. Furthermore, we identified that CD7-PLGA/LBH589 inhibits tumor growth and induces apoptosis in vivo xenograft model bearing leukemia cells.|세포에서 유전자 발현의 조절은 히스톤 단백질과 DNA 크로마틴 구조의 결합의 정도와 밀접한 관련이 있다. 전사 후 아세틸화와 메틸화 과정 같은 몇 가지의 변화를 통해 히스톤 단백질 복합체의 패킹이 열린 크로마틴 구조로 변화하게 된다. 이 때, 많은 전사조절 인자들이 DNA 와 특정 유전자의 프로모터에 쉽게 접근할 수 있는 기회를 갖게 된다. 암 세포에서 후생적인 유전자 조절 메커니즘이 정상세포와 다르다. 그래서, 세포의 생장, 분화, 암의 발병과정에 기인하는 HDAC (히스톤 디아세틸화효소)의 발현이 암세포에서 두드러지게 나타나고 또한, 그와 마찬가지로 아주 적은 히스톤 단백질의 아세틸화가 발견이 된다. 그러므로 이러한 HDAC 을 저해하는 HDAC inhibitor (히스톤 디아세틸화효소 저해제)가 항암제의 한 종류로 발견이 되어 연구가 되어오고 있다. 이러한 히스톤 디아세틸화효소 저해제는 히스톤 디아세틸화 효소의 활성을 방해하면서 세포사멸의 민감도를 증가시키고, 정상세포와 같이 전자 조절 인자들의 접근을 도와주고, 억제되어있던 종양 억제 유전자의 발현을 활성화 시킨다. 본 연구에서는 히스톤 디아세틸화효소 저해제 중 하나인 panobinostat (LBH589)를 PLGA 고분자를 이용하여 표적 약물 전달 시스템을 개발 하였다. Panobinostat (LBH589)은 현재 다국적 제약사인 Norvatis 사에서 임상을 진행 중에 있고, 여기서 사용된 PLGA 고분자는 생분해성, 생체 적합성등이 우수하여 미국 FDA 로부터 승인을 받았다. 약물은 PLGA 고분자를 이용해 전달을 하면, 방출조절을 통해 약물을 전달 할 수 있고, 약물의 부식이나 분해 등을 막을 수 있다. 기존의 항암제와 같이 부작용을 갖고 있어서 이를 줄이기 위해 PLGA 나노 입자로 panobinostat 을 캡슐화 하였다. 이렇게 만들어진 나노 입자는 사이즈가 200nm 이하였고, 약물도 잘 내포하고 약물 방출 정도도 조절이 되고 있는 것을 확인하였다. 후에 T 세포로 표적 전달을 하기 위해 타깃 단백질인 CD7 을 타깃 하는 단일조각항체를 사용하였다. 이 항체는 이전에 T 세포 전달에 사용되어 이번에 PLGA 나노 입자 전달에 사용이 되었다. 생체분자결합을 통해 PLGA 표면에 CD7 타깃 단일조각항체를 붙였다. In vitro 상에서 약물을 타깃 전달하였을 때 세포의 생장이 억제되었고 세포사멸이 유도되는 것을 확인 하였다. 게다가 in vivo 상에서도 PLGA 타깃 나노입자가 leukemia 에 특이적으로 전달 되는 것을 확인 하였으며 더불어 약물을 타깃 전달 하였을 때 tumor growth 를 inhibition 하고, 세포사멸을 유도 하는 것을 확인 하였다.; Gene regulation is closely related with packaging of histone protein and DNA chromatins. Through some modification such as post-translational acetylation and methylation, the packaging of histone complex is changed into open chromatin structure. Therefore, transcription factors have opportunity to approach DNA and gene promoter regions easily. In cancer, epigenic gene regulation mechanisms are different from those in the normal ones. High level expression of HDACs (histone deacetylases) which are related with cell proliferation, differentiation, and pathogenesis of cancer is generally discovered in cancer, acting in concert with hypoacetylation of histone proteins. Thus, histone deacetylase inhibitors can be used as one kind of anti-cancer drugs. The histone deacetylase inhibitors (HDACi) can increase sensitivity of apoptosis, obstructing activity of HDACs, increasing accessibility of transcription factors, normally re-activating repressed tumor suppressor genes. In this study, we have developed a stable, biodegradable, biocompatible, FDA approved PLGA polymer based panobinostat (LBH589, HDACi) filled T-cell specific targeting immuno-nanoparticles. Panobinostat (LBH589) is one of the HDAC inhibitors which is being tested in clinical trials. To minimize side effects as anti-cancer drug in non-tumor regions, we fabricated PLGA nanoparticles that encapsulate HDAC inhibitors. These nanoparticles were of size approximately around 200nm.To be delivered to the tumor specifically, targeting protein, anti-CD7 single chain antibody that has been previously shown as a T-cell specific targeting moiety was attached to the surface of PLGA nanoparticles through bioconjugation reactions. We found that scFvCD7-conjugated and drug-encapsulated PLGA nanoparticles (CD7-PLGA/LBH589) have significant effect on inhibition of proliferation and induction of apoptosis in cancer cells in vitro. In addition, scFvCD7 conjugated PLGA nanoparticles (CD7-PLGA) were delivered specifiaclly into leukemia cancer in vivo. Furthermore, we identified that CD7-PLGA/LBH589 inhibits tumor growth and induces apoptosis in vivo xenograft model bearing leukemia cells.