KR101587386B1 - Fluorescent protein nanopaticles for in vivo imaging - Google Patents
Fluorescent protein nanopaticles for in vivo imaging Download PDFInfo
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- KR101587386B1 KR101587386B1 KR1020150149311A KR20150149311A KR101587386B1 KR 101587386 B1 KR101587386 B1 KR 101587386B1 KR 1020150149311 A KR1020150149311 A KR 1020150149311A KR 20150149311 A KR20150149311 A KR 20150149311A KR 101587386 B1 KR101587386 B1 KR 101587386B1
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- Peptides Or Proteins (AREA)
Abstract
본 발명은 높은 형광강도를 가지는 동시에 생체 적합성 및 안정성이 뛰어난 재조합 형광 단백질 나노입자 및 상기 재조합 형광 단백질 나노입자의 인 비보 이미징을 위한 용도에 관한 것이다. 본 발명에 따른 형광 단백질 나노입자는 형광단백질 단량체에 비하여 현저히 증폭된 형광 강도를 보이며, 체온에 상응하는 온도에서 형광단백질의 구조적 변성을 방지하여 형광단백질의 구조적, 기능적 안정성을 향상시킬 수 있다. 또한 표적 지향성 펩타이드가 표면 발현된 형광 단백질 나노입자는 표적 특이적으로 타겟팅이 가능하며, 증폭된 형광 강도를 통해 생체 진단 이미징이 가능하므로, 타겟팅과 이미징이 동시에 가능한 생체 진단에 적합한 신소재를 제공한다.The present invention relates to recombinant fluorescent protein nanoparticles having high fluorescence intensity, excellent biocompatibility and stability, and use for in vivo imaging of the recombinant fluorescent protein nanoparticles. The fluorescent protein nanoparticles according to the present invention exhibit significantly enhanced fluorescence intensity as compared to the fluorescent protein monomers and can prevent the structural modification of the fluorescent protein at a temperature corresponding to the body temperature, thereby improving the structural and functional stability of the fluorescent protein. In addition, fluorescent protein nanoparticles surface-expressing the target-directed peptide can be target-specifically targeted, and biomedical imaging can be performed through amplified fluorescence intensity, thus providing a new material suitable for biodiagnosis that can simultaneously perform targeting and imaging.
Description
본 발명은 높은 형광강도를 가지는 동시에 생체 적합성 및 안정성이 뛰어난 재조합 형광 단백질 나노입자 및 상기 재조합 형광 단백질 나노입자의 인 비보 이미징을 위한 용도에 관한 것이다.The present invention relates to a recombinant fluorescent protein nanoparticle having high fluorescence intensity and excellent biocompatibility and stability, and to a use of the recombinant fluorescent protein nanoparticle for in vivo imaging.
고령화 사회로 접어들면서 질병 예방 및 조기 검진의 중요성이 대두됨에 따라 질병 조기 진단 시스템의 개발이 중요시되고 있다. 혈청 및 체액을 이용한 진단 시스템에서 나아가 체내에 직접 적용하여 조직 및 기관 내 질환 발병 유무를 판단하기 위한 생체 진단 시스템의 개발이 활발히 진행되고 있다. 특히 나노 크기의 무기, 유기 소재는 독특한 구조적 특성을 기반으로 생체 진단 소재로 개발하기 위한 노력이 지속되고 있으며, 특히 형광을 띠는 나노소재는 높은 민감도와 편의성으로 인해 각광받고 있다.As the society enters an aging society, the importance of disease prevention and early screening has emerged, so the development of an early disease diagnosis system is becoming more important. In addition to the diagnosis system using serum and body fluids, the development of a biometric diagnosis system for determining the presence or absence of disease in tissues and organs by applying directly into the body is being actively progressed. In particular, efforts to develop nano-sized inorganic and organic materials as biodiagnostic materials based on their unique structural characteristics are continuing, and in particular, nanomaterials that exhibit fluorescence are in the spotlight for their high sensitivity and convenience.
생체 이미징 소재는 높은 형광 신호를 보이고, 생체 적합성이 뛰어나야 하며, 특정 조직 및 질환 특이적으로 타겟팅이 가능해야 한다. 최근 광학적 생체 이미징 소재의 기능성 향상을 위하여 1) 이미징 소재의 형광 강도 증폭 기술, 2) 생체 적합성 나노 소재 개발을 위한 표면 처리 기술 및 생체 적합성 소재 발굴, 3) 조직, 질환 특이적 타겟팅 모티프 표출 이미징 소재 개발이 활발히 진행 중이다.The bio-imaging material must exhibit a high fluorescence signal, have excellent biocompatibility, and be able to target specific tissues and diseases. In order to improve the functionality of recent optical bioimaging materials, 1) fluorescence intensity amplification technology of imaging materials, 2) surface treatment technology and biocompatibility material discovery for the development of biocompatible nanomaterials, 3) tissue and disease-specific targeting motif expression imaging materials Development is actively underway.
현재 잘 알려진 화학적 형광 물질(organic dye)인 Cy3, Cy5, 그 외 FITC 등은 단백질 및 DNA의 형광 수식 기법이 널리 알려지면서 생체 진단 소재로 개발하기 위한 노력이 계속 되어왔으나 낮은 구조적 안정성과, 낮은 광 안정성(photostability)으로 인해 효용성이 떨어질 뿐 아니라, 형광 신호 증폭 기술의 부재로 낮은 민감도를 나타내는 단점이 있다. Currently well-known chemical fluorescent substances (organic dyes) such as Cy3, Cy5, and other FITC have been made efforts to develop biodiagnostic materials as the fluorescence modification techniques of proteins and DNA are widely known, but low structural stability and low light Not only does it not be effective due to photostability, but also has a disadvantage of showing low sensitivity due to the absence of a fluorescence signal amplification technology.
최근 퀀텀닷(Quantum dot)이 높은 안정성과 형광 강도로 각광받고 있지만 중금속을 기반으로 제조되기 때문에 생체 적합성 문제나 독성문제가 해결되어야 할 과제로 남아있으며 제조 단가가 높고 생산 및 수식화에 복잡한 공정이 요구된다.Recently, quantum dots are in the spotlight for their high stability and fluorescence intensity, but since they are manufactured based on heavy metals, biocompatibility and toxicity problems remain a task to be solved, and manufacturing costs are high and complex processes are required for production and formulation. do.
반면 형광 검출 신호 물질로 널리 사용되고 있는 형광 단백질은 구조적 안정성, 높은 양자 효율, 높은 형광 밝기 등을 가지며, 대장균을 통해 쉽게 생산이 가능하고, 생체 유래의 단백질 기반의 소재이므로 이를 생체 진단에 활용하기 위한 다양한 연구가 수행되고 있다. 하지만 생체 내에서의 안정성 및 검출 가능한 수준의 형광 강도를 충족시키지는 못하는 실정이며, 이러한 제한 점을 극복하고 효용성을 증대시킬 수 있는 형광 단백질 기반의 새로운 기능성 소재 개발이 필요한 실정이다.On the other hand, a fluorescent protein widely used as a fluorescent detection signal material has structural stability, high quantum efficiency, high fluorescence brightness, etc., can be easily produced through E. coli, and is a protein-based material derived from a living body. Various studies are being conducted. However, it is a situation that does not meet the stability and detectable level of fluorescence intensity in vivo, and there is a need to develop a new functional material based on a fluorescent protein that can overcome these limitations and increase the effectiveness.
또한, 나노 크기의 무기입자, 고분자기반의 구조체에 화학 공정을 거쳐 화학적 형광 물질을 고정한 소재를 이미징 소재로 개발하기 위한 시도가 활발히 진행되고 있으나, 이들이 독성으로 인한 안전성 문제는 끊임없는 이슈가 되고 있다.In addition, attempts to develop materials in which chemical fluorescent materials are fixed through chemical processes to nano-sized inorganic particles and polymer-based structures as imaging materials are being actively progressed, but safety problems due to toxicity of these materials are constantly becoming an issue. .
재현성 있는 생체 이미징 결과를 얻기 위해서는 입자의 입도 분포 및 표면 성질이 일정해야 하는데, 기존 생체 이미징에 적용하고자 연구 개발되고 있는 무기 입자, 자성입자, 고분자 소재 기반의 나노입자는 재현성 있는 균일한 입자의 제조가 어려울 뿐 아니라, 형광 물질을 추가로 화학적 반응을 통해 표면 고정화 내지는 입자 내 캡슐화(encapsulation) 과정을 거쳐야 하는 단점이 있다. 특히 생체 이미징 소재가 최종 질환 조직까지 도달하기 위해서는, 생체 내에서 면역 세포에 의해 제거되지 않아야 하고, 신장을 통해 빠져가 나가는 신장 클리어런스(renal clearance)를 극복해야 한다. 신장 클리어런스는 30 nm 이하 입자의 경우 주로 일어난다고 알려져 있으므로 30 nm 이상의 입자를 제조하는 기술 연구가 진행되고 있으나 화학적 반응을 통해 제조되는 입자는 균일한 크기로 재현성 있게 제조하는데 한계가 있다. 또한 기존 연구되고 있는 무기소재 기반의 이미징 소재는 면역 세포에 의해 빠르게 외부 물질로 인식되어 대부분이 간에 축적되는 현상을 보이며, 이를 방지하려면 표면 개질을 추가로 수행해야 하는 단점이 있다.In order to obtain reproducible biological imaging results, the particle size distribution and surface properties of the particles must be constant. However, nanoparticles based on inorganic particles, magnetic particles, and polymer materials that are being researched and developed to apply to existing biological imaging are reproducible and uniform particles. In addition to being difficult, there is a disadvantage in that the fluorescent material has to undergo surface immobilization or encapsulation in particles through an additional chemical reaction. In particular, in order for the biological imaging material to reach the final diseased tissue, it must not be removed by immune cells in vivo, and must overcome renal clearance through the kidney. Since it is known that the elongation clearance mainly occurs in the case of particles of 30 nm or less, technical research for manufacturing particles of 30 nm or more is in progress, but there is a limit to reproducibly manufacturing particles manufactured through a chemical reaction with a uniform size. In addition, the existing researched inorganic material-based imaging materials are rapidly recognized as foreign substances by immune cells, and most of them are accumulated in the liver. To prevent this, there is a disadvantage that additional surface modification is required.
또한 생체에 적용 가능한 나노입자를 개발하고 그 응용 범위를 확대하기 위하여 다양한 크기와 형태의 나노소재 개발이 활발이 진행되고 있다. 기존 무기 나노입자의 생체 분포 및 입자의 특성을 응용한 특정 장기 및 질환의 이미징 기술이 다수 보고된 바 있으나, 아직까지 단백질 나노입자 기반의 이미징 소재 개발은 단백질 나노입자를 생체에 적용하는데 있어서 정보가 충분하지 않아 그 응용에 한계가 있는 실정이다. 즉, 기존 무기 나노입자를 대체할 수 있는 생체 유래의 단백질 나노입자 라이브러리를 확보하고, 이의 적절한 생체 적용을 위해서는 다양한 크기와 형태의 단백질 나노입자에 대한 인 비보 연구가 요구되고 있다. In addition, in order to develop nanoparticles applicable to a living body and expand the application range, the development of nanomaterials of various sizes and shapes is actively progressing. Existing biodistribution of inorganic nanoparticles and imaging techniques for specific organs and diseases that apply the characteristics of particles have been reported, but the development of imaging materials based on protein nanoparticles has yet to provide information on applying protein nanoparticles to living organisms. It is not enough, so there is a limit to its application. That is, in vivo studies on protein nanoparticles of various sizes and shapes are required to secure a library of protein nanoparticles derived from a living body that can replace the existing inorganic nanoparticles, and to apply the same in vivo.
본 발명의 목적은 형광 강도 증폭, 생체 안정성이 향상된 생체 진단용 신소재로서 형광 단백질 나노입자 및 이의 용도를 제공하는 것이다.An object of the present invention is to provide a fluorescent protein nanoparticle and its use as a new material for biodiagnostics with improved fluorescence intensity amplification and biostability.
상기 목적을 달성하기 위하여, 본 발명은 표면에 형광물질 및 표적 지향성 펩타이드를 표출하는 형광 단백질 나노입자를 제공한다.In order to achieve the above object, the present invention provides a fluorescent protein nanoparticle that expresses a fluorescent substance and a target-oriented peptide on the surface.
본 발명은 또한 본 발명에 따른 형광 단백질 나노입자; 및 약제학적으로 허용가능한 담체를 포함하는 표적 지향형 조영제 조성물을 제공한다.The present invention also provides a fluorescent protein nanoparticle according to the present invention; And it provides a target-directed contrast medium composition comprising a pharmaceutically acceptable carrier.
본 발명은 또한 본 발명에 따른 형광 단백질 나노입자; 및 약제학적으로 허용가능한 담체를 포함하는 동시 진단 또는 치료용 조영제 조성물을 제공한다.The present invention also provides a fluorescent protein nanoparticle according to the present invention; And it provides a contrast medium composition for simultaneous diagnosis or treatment comprising a pharmaceutically acceptable carrier.
본 발명은 또한 본 발명에 따른 형광 단백질 나노입자; 및 진단 프로브를 포함하고, 상기 진단 프로브는 영상 판독을 위한 표지 물질인 다중 진단 프로브를 제공한다.The present invention also provides a fluorescent protein nanoparticle according to the present invention; And a diagnostic probe, wherein the diagnostic probe provides a multiple diagnostic probe as a labeling material for image reading.
본 발명은 또한 자기조립성 단백질에 형광물질이 결합된 10 내지 50 nm 크기의 형광 단백질 나노입자; 및 약제학적으로 허용가능한 담체를 포함하고, 상기 형광 단백질 나노입자는 B형 간염 바이러스 코어 단백질, 페리틴 중쇄 단백질(Ferritin Heavy chain), 써모플라즘 애시도필룸(Thermoplasm acidophilum) 유래의 프로테아좀(tPTS) 또는 대장균 유래의 DNA 결합 단백질(eDPS)을 포함하는 자기조립성 단백질로부터 자기조립된 단백질 나노입자에 형광물질을 표면 결합시켜 제조된 것인, 생체 이미징용 조성물을 제공한다.The present invention also provides a fluorescent protein nanoparticle having a size of 10 to 50 nm in which a fluorescent material is bound to a self-assembled protein; And a pharmaceutically acceptable carrier, wherein the fluorescent protein nanoparticles include hepatitis B virus core protein, ferritin heavy chain, and thermoplasmic acidophilum. acidophilum ) derived from a proteasome (tPTS) or a self-assembled protein containing a DNA binding protein (eDPS) derived from Escherichia coli, prepared by surface-binding a fluorescent substance to protein nanoparticles self-assembled Provides.
본 발명에 따른 형광 단백질 나노입자는 형광 단백질 단량체에 비하여 현저히 증폭된 형광 강도를 보이며, 체온에 상응하는 온도에서 형광 단백질의 구조적 변성을 방지하여 형광단백질의 구조적, 기능적 안정성을 향상시킬 수 있다. 또한 표적 지향성 펩타이드가 표면 발현된 형광 단백질 나노입자는 표적 특이적으로 타겟팅이 가능하며, 증폭된 형광강도를 통해 생체 진단 이미징이 가능하므로, 타겟팅과 이미징이 동시에 가능한 생체 진단에 적합한 신소재를 제공한다.The fluorescent protein nanoparticles according to the present invention exhibit significantly amplified fluorescence intensity compared to the fluorescent protein monomer, and prevent structural denaturation of the fluorescent protein at a temperature corresponding to body temperature, thereby improving structural and functional stability of the fluorescent protein. In addition, since the fluorescent protein nanoparticles on which the target-oriented peptide is surface-expressed can be targeted specifically, and biodiagnostic imaging is possible through the amplified fluorescence intensity, it provides a new material suitable for biodiagnostics capable of targeting and imaging at the same time.
표적 지향성 펩타이드를 표출하지 않으나, 일정 크기와 구조의 특성을 갖는 형광 단백질 나노입자는 감시림프절 이미징에 사용할 수 있다.Fluorescent protein nanoparticles that do not express target-oriented peptides but have characteristics of a certain size and structure can be used for surveillance lymph node imaging.
도 1은 녹색(eGFP), 적색(DsRed) 형광단백질을 융합한 캡시드 단백질 나노입자 제조를 위한 발현 벡터의 모식도를 나타낸 것이다((A) gFCNP(w/o linker), (A) rFCNP(w/o linker), (C) gFCNP(w/ linker), (D) rFCNP(w/ linker)).
도 2는 형광단백질이 표면 표출된 구형의 단백질 나노입자의 모식도를 나타낸 것이다((A) gFCNP (w/ linker), (B) rFCNP (w/ linker)).
도 3은 대장균에서 발현 후 분리 정제한 HBV 캡시드 유래 키메릭 형광 나노입자의 구조를 보여주는 TEM 사진도이다((A) gFCNP (w/ linker), (B) rFCNP (w/ linker)).
도 4는 형광단백질 단량체(eGFP, DsRed)와 링커를 삽입하지 않은 형광 단백질 나노입자[gFFNP, gFCNP(w/o linker), rFFNP, rFCNP(w/o linker)], 링커를 삽입한 형광 단백질 나노입자[gFFNP, gFCNP(w/ linker), rFFNP, rFCNP(w/ linker)]의 형광 세기를 비교한 결과를 나타낸 것이다.
도 5는 PBS에 존재하는 형광단백질 단량체[eGFP(○), DsRed(▽)], 링커를 삽입한 형광 단백질 나노입자 [gFCNP(w/ linker)(●), rFCNP(w/ linker)(▼)]의 보관 시간에 따른 형광 세기를 비교한 결과를 나타낸 것이다.
도 6은 30%, 50% 혈청에 존재하는 형광단백질 단량체, 링커를 삽입한 형광 단백질 나노입자의 보관 시간에 따른 형광 세기를 비교한 결과를 나타낸 것이다((A) 적색형광단백질 단량체와 적색형광단백질 융합 나노입자 [30% 혈청내 rFCNP (●), 30% 혈청 내 DsRed (○), 50% 혈청 내 rFCNP (▼), 50% 혈청 내 DsRed (▽)], (B) 녹색형광단백질 단량체와 녹색형광단백질 융합 나노입자 [30% 혈청내 gFCNP (●), 30% 혈청 내 eGFP (○), 50% 혈청 내 gFCNP (▼), 50% 혈청 내 eGFP (▽)]).
도 7은 반복된 광원 노출에 대한 광안정성을 비교한 결과를 나타낸 것이다([gFCNP(●), FAM (▽), eGFP (○), Qdot655(▲), rFCNP (■), DsRed(□)]).
도 8은 동물 모델의 복부에 피하 주사한 형광단백질 단량체와 형광 단백질 나노입자의 생체 안정성을 비교한 결과를 나타낸 것이다((DsRed: 형광단백질 단량체, rFFNP: 페리틴 기반 적색형광단백질 나노입자, rFCNP: 캡시드 기반 적색형광단백질 나노입자)).
도 9는 생체진단용 표적 지향적 펩타이드(RGD)를 표출하는 본 발명에 따른 형광 단백질 나노입자 제조를 위한 발현 벡터의 모식도를 나타낸 것이다((A) RGD-rFFNP, (B) RGD-rFCNP).
도 10은 생체진단용 표적 지향성 펩타이드(RGD)를 표출하는 본 발명에 따른 형광 단백질 나노입자(RGD-rFFNP)와 형광 단백질 나노입자(rFFNP)의 암세포(U87MG) 업테이크 효율을 비교한 실험 결과이다.
도 11은 U87MG 암세포를 이식한 동물 모델에 rFFNP와 RGD-rFFNP를 각각 정맥 주사한 후 암 타겟팅 효율을 인 비보 이미징을 통해 비교한 결과를 나타낸 것이다.
도 12는 생체 진단용 표적 지향적 펩타이드(RGD)를 표출하는 본 발명에 따른 형광 단백질 나노입자를 적용한 동물 실험 후 조직별 형광 세기 비교 결과를 나타낸 것이다((A) SCC7 종양 모델, (B) U87MG 종양 모델).
도 13은 본 발명에 따른 eDPS 및 tPTS 기반 형광 단백질 나노입자와 RGD 표출 형광 단백질 나노입자의 제작을 위한 발현 벡터 모식도(A)와 각 나노입자의 크기와 형태를 보여주는 DLS 결과와 TEM 사진도(B)이다.
도 14는 본 발명에 따른 eDPS, RGD-eDPS, tPTS, RGD-tPTS 형광 단백질 나노입자를 이용한 인 비보 암 타겟팅 결과(A)와 암에 나타나는 형광 세기 측정을 통해 암 타겟팅 효율을 비교해서 보여주는 그래프(B) 이다.
도 15는 감시림프절 이미징에 적용한 본 발명에 따른 4개의 형광 단백질 나노입자의 크기와 구조를 보여주는 TEM 사진도(A)와 각 입자의 형광세기(B)를 나타낸 것이다.
도 16은 본 발명에 따른 4개의 형광 단백질 나노입자를 이용한 감시림프절 이미징 결과이다((A) eDPS, (B) tPTS, (C) FTN-h, (D) HBVcAg). 1 shows a schematic diagram of an expression vector for preparing capsid protein nanoparticles fused with green (eGFP) and red (DsRed) fluorescent proteins ((A) gFCNP (w/o linker), (A) rFCNP (w/ o linker), (C) gFCNP(w/linker), (D) rFCNP(w/linker)).
Figure 2 shows a schematic diagram of a spherical protein nanoparticles on which a fluorescent protein is surface-expressed ((A) gFCNP (w/ linker), (B) rFCNP (w/ linker)).
3 is a TEM photograph showing the structure of chimeric fluorescent nanoparticles derived from HBV capsids separated and purified after expression in E. coli ((A) gFCNP (w/ linker), (B) rFCNP (w/ linker)).
Figure 4 is a fluorescent protein monomer (eGFP, DsRed) and a fluorescent protein nanoparticles without a linker inserted [gFFNP, gFCNP (w/o linker), rFFNP, rFCNP (w/o linker)], a fluorescent protein nanoparticle inserted with a linker It shows the result of comparing the fluorescence intensity of the particles [gFFNP, gFCNP (w / linker), rFFNP, rFCNP (w / linker)].
5 is a fluorescent protein monomer present in PBS [eGFP (○), DsRed (▽)], a fluorescent protein nanoparticle inserted with a linker [gFCNP (w/ linker) (●), rFCNP (w/ linker) (▼)) ] Shows the result of comparing the fluorescence intensity according to the storage time.
6 shows the result of comparing the fluorescence intensity according to the storage time of the fluorescent protein monomer present in 30% and 50% serum, and the fluorescent protein nanoparticles inserted with a linker ((A) red fluorescent protein monomer and red fluorescent protein Fusion nanoparticles [rFCNP in 30% serum (●), DsRed in 30% serum (○), rFCNP in 50% serum (▼), DsRed in 50% serum (▽)], (B) green fluorescent protein monomer and green Fluorescent protein fusion nanoparticles [gFCNP in 30% serum (●), eGFP in 30% serum (○), gFCNP in 50% serum (▼), eGFP in 50% serum (▽)]).
Figure 7 shows the results of comparing the photostability for repeated exposure to the light source ([gFCNP(●), FAM (▽), eGFP (○), Qdot655(▲), rFCNP (■), DsRed(□))) ).
Figure 8 shows the results of comparing the biostability of the fluorescent protein monomer and the fluorescent protein nanoparticles injected subcutaneously into the abdomen of the animal model ((DsRed: fluorescent protein monomer, rFFNP: ferritin-based red fluorescent protein nanoparticles, rFCNP: capsid Based red fluorescent protein nanoparticles)).
9 shows a schematic diagram of an expression vector for preparing a fluorescent protein nanoparticle according to the present invention that expresses a target-directed peptide (RGD) for biodiagnosis ((A) RGD-rFFNP, (B) RGD-rFCNP).
10 is an experiment result comparing the uptake efficiency of cancer cells (U87MG) of fluorescent protein nanoparticles (RGD-rFFNP) and fluorescent protein nanoparticles (rFFNP) according to the present invention expressing target-directed peptide (RGD) for biodiagnosis.
FIG. 11 shows the results of comparing cancer targeting efficiency through in vivo imaging after intravenous injections of rFFNP and RGD-rFFNP into an animal model transplanted with U87MG cancer cells.
Figure 12 shows the results of comparison of the fluorescence intensity of each tissue after an animal experiment to which the fluorescent protein nanoparticles according to the present invention expressing a target-directed peptide (RGD) for biodiagnosis are applied ((A) SCC7 tumor model, (B) U87MG tumor model ).
13 is a schematic diagram of an expression vector for the production of eDPS and tPTS-based fluorescent protein nanoparticles and RGD-expressing fluorescent protein nanoparticles according to the present invention (A), and DLS results and TEM photographs showing the size and shape of each nanoparticle (B )to be.
FIG. 14 is a graph showing the comparison of cancer targeting efficiency through in vivo cancer targeting results (A) using eDPS, RGD-eDPS, tPTS, RGD-tPTS fluorescent protein nanoparticles according to the present invention and fluorescence intensity measured in cancer ( B) It is.
15 is a TEM photograph showing the size and structure of four fluorescent protein nanoparticles according to the present invention applied to surveillance lymph node imaging (A) and fluorescence intensity (B) of each particle.
16 is a result of surveillance lymph node imaging using four fluorescent protein nanoparticles according to the present invention ((A) eDPS, (B) tPTS, (C) FTN-h, (D) HBVcAg).
이하, 본 발명의 구성을 구체적으로 설명한다.Hereinafter, the configuration of the present invention will be described in detail.
본 발명은 표면에 형광물질 및 표적 지향성 펩타이드를 표출하는 형광 단백질 나노입자에 관한 것이다.The present invention relates to a fluorescent protein nanoparticle that expresses a fluorescent substance and a target-oriented peptide on the surface.
본 발명의 형광 단백질 나노입자는 나노미터의 크기의 직경을 가지는 구형의 단백질 입자로서 자기조립성 단백질에 의한 자기조립에 의해 입자를 이루고 있어 매우 균일한 입도 분포를 보이는 입자를 재현성 있게 제조할 수 있을 뿐 아니라, 생체 내에서 사용된 후 분해될 수 있는 생체친화성 물질이기 때문에 생체 이미징 후의 나노입자의 잔존으로 인한 독성의 문제가 없다. 특히 인간 유래의 자기조립성 단백질을 사용할 경우 생체 이미징에 사용 시 안전성과 독성에 전혀 문제가 없고, 면역세포에 의한 제거 또한 타 무기 소재 입자에 비해 현저히 낮은 편이다. The fluorescent protein nanoparticles of the present invention are spherical protein particles having a diameter of nanometers, and are formed by self-assembly by self-assembled proteins, so that particles having a very uniform particle size distribution can be reproducibly manufactured. In addition, since it is a biocompatible material that can be decomposed after being used in a living body, there is no problem of toxicity due to the remaining nanoparticles after bio-imaging. In particular, when using human-derived self-assembled protein, there is no problem with safety and toxicity when used for in vivo imaging, and removal by immune cells is also significantly lower than that of other inorganic material particles.
따라서, 본 발명의 형광 단백질 나노입자는 생체 적용이 가능하고 입자에 따른 표적 타겟팅 효과가 우수하여 진단 조직 또는 질환에 적합한 것을 특징으로 한다.Accordingly, the fluorescent protein nanoparticles of the present invention can be applied to a living body and have excellent target targeting effects according to the particles, and are suitable for diagnostic tissues or diseases.
본 발명의 형광 단백질 나노입자는 한 구체예에 따르면 형광단백질, 자기조립성 단백질, 및 표적 지향성 펩타이드가 융합된 단량체가 자가조립되어 하나의 구형 형광 단백질 나노입자를 이룰 수 있다. 다른 구체예에 따르면, 자기조립성 단백질에 표적 지향성 펩타이드가 결합된 키메릭 단백질로부터 단백질 나노입자가 제조되고 형광물질로 표면 수식시켜 형광 단백질 나노입자를 제조할 수도 있다.According to one embodiment, the fluorescent protein nanoparticles of the present invention may be self-assembled to form one spherical fluorescent protein nanoparticle by self-assembly of a fluorescent protein, a self-assembled protein, and a target-oriented peptide-fused monomer. According to another embodiment, protein nanoparticles may be prepared from a chimeric protein in which a target-directed peptide is bound to a self-assembled protein, and the fluorescent protein nanoparticles may be prepared by surface modification with a fluorescent material.
본 명세서에서, 용어 "자기조립성 단백질"은 단백질, 단백질의 서브유닛 또는 펩타이드가 수 개가 모였을 때 이들이 스스로 조직적인 구조 또는 패턴을 형성하며 집합체를 형성하는 단백질, 단백질의 서브유닛 또는 펩타이드를 의미한다. 이러한 자기조립성 단백질은 별도의 조작 없이도 단백질의 나노입자 형성을 가능하게 하므로 본 발명에 따른 단백질 나노입자의 제조를 위해 유리하게 사용할 수 있다. In the present specification, the term "self-assembling protein" refers to a protein, a subunit of a protein, or a peptide that forms an aggregate by forming an organizational structure or pattern by itself when several proteins, subunits or peptides of a protein are gathered. . Such self-assembled protein enables the formation of protein nanoparticles without separate manipulation, and thus can be advantageously used for the production of protein nanoparticles according to the present invention.
상기 자기조립성 단백질은 외래 단백질과 융합할 수 있고, 상기 외래 단백질은 목적에 따라 자기조립 시 내부 또는 외부에 위치시킬 수 있다. 본 발명은 단백질 나노입자를 타겟 물질의 탐지 목적으로 사용하므로 외래 단백질로 형광단백질과 표적 지향성 펩타이드를 사용할 수 있고, 상기 형광단백질과 표적 지향성 펩타이드는 자기조립성 단백질의 자기조립 시 외부에 위치하도록 조절된다. 만일 형광단백질이 자기조립성 단백질의 자기조립시 내부에 위치하도록 발현되면 형광 강도의 저하를 야기하게 된다. 그러므로, 형광단백질과 표적 지향성 펩타이드가 융합단백질의 외부에 위치할 수 있도록 융합 파트너로서 사용되게 되는 자기조립성 단백질의 종류나 융합되는 부위, 또는 단백질의 융합이나 발현의 방법 등을 적절히 선택하는 것이 바람직하다. The self-assembled protein may be fused with a foreign protein, and the foreign protein may be located inside or outside when self-assembling, depending on the purpose. In the present invention, since protein nanoparticles are used for the purpose of detection of a target substance, a fluorescent protein and a target-oriented peptide can be used as foreign proteins, and the fluorescent protein and the target-oriented peptide are controlled to be located outside when self-assembling the self-assembled protein. do. If the fluorescent protein is expressed so as to be located inside the self-assembled protein during self-assembly, the fluorescence intensity decreases. Therefore, it is desirable to appropriately select the type of self-assembled protein to be used as a fusion partner, the site to be fused, or the method of fusion or expression of the protein, so that the fluorescent protein and the target-directed peptide can be located outside the fusion protein. Do.
이러한, 자기조립성 단백질로, B형 간염 바이러스 코어 단백질(이하, 'HBV'라 함)을 사용할 수 있다.As such a self-assembled protein, a hepatitis B virus core protein (hereinafter, referred to as'HBV') may be used.
HBV 코어 단백질은 180-240개의 단량체가 자가조립되어 하나의 구형 단백질 나노입자를 이루며, 유전자 재조합 기술을 이용하여 대장균에서 대량 생산이 가능하다. 특히 스파이크 부위에 외래 단백질을 융합 발현할 경우 외래 단백질이 단백질 나노입자의 표면에 표출되는 특징이 있다. The HBV core protein is self-assembled of 180-240 monomers to form a single globular protein nanoparticle, and can be mass-produced in E. coli using genetic recombination technology. In particular, when a foreign protein is fused to the spike site, the foreign protein is expressed on the surface of the protein nanoparticle.
상기 HBV 코어 단백질은 구조상 스파이크에 해당하는 79-80번째 아미노산 서열 사이, N-말단 또는 C-말단에 외래 단백질을 융합 발현할 경우, 발현된 외래 단백질이 단백질 나노입자의 바깥 표면에 표출될 수 있다. 다만, 이러한 외래 단백질의 표면 표출은 HBV 코어 단백질의 3차 구조를 형성하는데 영향을 줄 수 있으므로 상기 3차 구조를 형성할 수 있는 범위에서 외래 단백질을 채택할 수 있다.When the HBV core protein fusion-expresses a foreign protein between the 79-80 amino acid sequence corresponding to the spike in structure, at the N-terminus or at the C-terminus, the expressed foreign protein may be expressed on the outer surface of the protein nanoparticle. . However, since the surface expression of the foreign protein may have an effect on the formation of the tertiary structure of the HBV core protein, the foreign protein may be adopted within a range capable of forming the tertiary structure.
본 발명의 일 구체예에 따르면, HBV 코어 단백질의 1-78번째 아미노산 서열 부분과 상기 코어 단백질의 81-149번째 아미노산 서열 부분 사이에 증강된 녹색형광단백질(enhanced green fluorescent protein) 또는 적색형광단백질(DsRed)을 위치시키고, 상기 형광단백질의 N-말단 부분은 헥사 히스티딘과 링커 펩타이드를, C-말단 부분은 링커 펩타이드를 결합시키며, HBV 코어 단백질의 N-말단 또는 C-말단에는 표적 지향성 펩타이드를 결합시킨 키메릭 단백질을 제조할 수 있다. 1개의 키메릭 단백질 즉 단량체 180-240개가 자가조립되어 표면에 형광단백질과 표적 지향성 펩타이드를 표출하는 형광 단백질 나노입자를 형성할 수 있다.According to an embodiment of the present invention, an enhanced green fluorescent protein or red fluorescent protein (enhanced green fluorescent protein) or a red fluorescent protein (enhanced between the 1-78 amino acid sequence portion of the HBV core protein and the 81-149 amino acid sequence portion of the core protein) ( DsRed), and the N-terminal portion of the fluorescent protein binds hexahistidine and a linker peptide, the C-terminal portion binds a linker peptide, and a target-directed peptide is bonded to the N-terminus or C-terminus of the HBV core protein. The prepared chimeric protein can be prepared. One chimeric protein, that is, 180-240 monomers can be self-assembled to form fluorescent protein nanoparticles expressing a fluorescent protein and a target-oriented peptide on the surface.
또는, 자기조립성 단백질의 아미노 말단과 카르복실 말단이 표면으로 표출되는 특징을 이용하여 형광 단백질 나노입자를 제조할 수도 있다. 예컨대, HBV 코어 단백질의 N-말단 또는 C-말단에 표적 지향성 펩타이드가 결합된 키메릭 단백질로부터 자기조립된 단백질 나노입자에 화학적 공정을 통해 형광물질을 표면 결합시켜 형광 단백질 나노입자를 형성할 수 있다.Alternatively, a fluorescent protein nanoparticle may be prepared by using the characteristic that the amino terminal and the carboxyl terminal of the self-assembled protein are expressed on the surface. For example, a fluorescent protein nanoparticle can be formed by surface-binding a fluorescent substance through a chemical process to a protein nanoparticle self-assembled from a chimeric protein having a target-directed peptide bound to the N-terminus or C-terminus of the HBV core protein. .
상기 자기조립성 단백질로, 인간 유래의 자기조립성 단백질을 사용할 수 있다. 상기 인간 유래의 자기조립성 단백질 또는 이를 포함하는 단백질 나노입자는 인간화된 자기조립성 단백질 또는 인간화된 단백질 나노입자를 또한 포함하는 것으로 간주된다. 예컨대, 상기 자기조립성 단백질은 페리틴일 수 있다. 페리틴은 중쇄와 경쇄로 구성된 24개의 동일한 단백질 서브유닛으로 구성되며, 자기조립성을 갖고 있어 생체 내에서 구형의 속이 빈 쉘 구조(hollow shell)을 형성한다. 한 구체예에서, 상기 자기조립성 단백질은 페리틴 중쇄 단백질(Ferritin Heavy Chain: 이하 'FTN-H'이라고도 함)일 수 있다.As the self-assembled protein, a human-derived self-assembled protein may be used. The human-derived self-assembled protein or protein nanoparticle comprising the same is considered to also comprise a humanized self-assembled protein or humanized protein nanoparticle. For example, the self-assembled protein may be ferritin. Ferritin is composed of 24 identical protein subunits composed of heavy and light chains, and has self-assembly to form a spherical hollow shell structure in vivo. In one embodiment, the self-assembled protein may be a ferritin heavy chain protein (hereinafter also referred to as'FTN-H').
본 발명의 일 구체예에 따르면, FTN-H은 양 말단에 링커 펩타이드에 의해 표적 지향성 펩타이드와 형광단백질이 각각 결합될 수 있다. 보다 구체적으로, 표적 지향성 펩타이드는 FTN-H의 N-말단에, 형광단백질은 C-말단에 융합될 수 있다. FTN-H과 융합되는 표적 지향성 펩타이드와 형광단백질은 단백질 나노입자의 표면에 표출됨으로써 강한 형광 강도를 제공하여 표적 물질의 탐지시 매우 유용하게 활용될 수 있다. According to an embodiment of the present invention, FTN-H may be coupled to a target-directed peptide and a fluorescent protein by linker peptides at both ends. More specifically, the target-directed peptide may be fused to the N-terminus of FTN-H, and the fluorescent protein may be fused to the C-terminus. Target-oriented peptides and fluorescent proteins that are fused with FTN-H are expressed on the surface of protein nanoparticles to provide strong fluorescence intensity, and thus can be very usefully used when detecting target substances.
또는, 자기조립성 단백질의 아미노 말단과 카르복실 말단이 표면으로 표출되는 특징을 이용하여 형광 단백질 나노입자를 제조할 수도 있다. FTN-H의 N-말단 또는 C-말단에 표적 지향성 펩타이드가 결합된 키메릭 단백질로부터 자기조립된 단백질 나노입자에 화학적 공정을 통해 형광물질을 표면 결합시켜 형광 단백질 나노입자를 형성할 수 있다.Alternatively, a fluorescent protein nanoparticle may be prepared by using the characteristic that the amino terminal and the carboxyl terminal of the self-assembled protein are expressed on the surface. Fluorescent protein nanoparticles can be formed by surface-binding a fluorescent substance to protein nanoparticles self-assembled from a chimeric protein having a target-directed peptide bound to the N-terminus or C-terminus of FTN-H through a chemical process.
상기 자기조립성 단백질로, 써모플라즘 애시도필룸(Thermoplasm acidophilum) 유래의 프로테아좀(이하, 'tPTS'라 함)을 사용할 수 있다. 상기 tPTS는 서열이 다른 14개의 알파 단백질과 14개의 베타 단백질이 자기조립에 의해 약 원기둥 형태의 단백질 나노입자를 이루며, 유전자 재조합 기술을 이용하여 대장균에서 발현이 가능하다. 이 단백질 나노입자는 매우 높은 열안정성을 보일 뿐 아니라, 알파 단백질의 아미노 말단과 카르복실 말단, 베타 단백질의 카르복실 말단이 외부로 표출되어 있어 각 부분에 외래 단백질을 융합발현할 경우 외래 단백질이 단백질 나노입자의 표면에 표출되는 특징이 있다.As the self-assembling protein, Thermoplasmic acidophilum (Thermoplasm acidophilum) derived proteasome (hereinafter referred to as'tPTS') can be used. The tPTS is composed of 14 alpha proteins and 14 beta proteins with different sequences to form a protein nanoparticle in a cylindrical shape by self-assembly, and can be expressed in E. coli using gene recombination technology. These protein nanoparticles not only exhibit very high thermal stability, but also the amino and carboxyl ends of the alpha protein, and the carboxyl ends of the beta protein are exposed to the outside. There is a characteristic that is expressed on the surface of the nanoparticles.
이에 제한되는 것은 아니나, tPTS는 일 말단에 표적 지향성 펩타이드가 결합될 수 있다. 표적 지향성 펩타이드를 표면에 표출하는 단백질 나노입자로 자기조립된 후 화학적 공정을 통해 형광물질을 표면에 결합시킴으로써 형광 단백질 나노입자가 제조되는 것이다.Although not limited thereto, tPTS may have a target-directed peptide bound to one end. Fluorescent protein nanoparticles are prepared by self-assembling a target-oriented peptide into protein nanoparticles expressing the surface and then binding a fluorescent substance to the surface through a chemical process.
상기 자기조립성 단백질로, 대장균 유래의 DNA 결합 단백질(이하 'eDPS'라 함)을 사용할 수 있다. eDPS는 12개의 동일한 단량체의 자기조립에 의해 8-10 nm의 구형 단백질 나노입자를 이룬다. 유전자 재조합 기술을 이용하여 대장균에서 대량 발현이 가능하며, 이의 아미노 말단과 카르복실 말단이 표면으로 표출되어 있어 외래 단백질의 표출을 위한 융합 발현이 가능하다.As the self-assembled protein, a DNA binding protein derived from E. coli (hereinafter referred to as'eDPS') may be used. eDPS forms 8-10 nm spherical protein nanoparticles by self-assembly of 12 identical monomers. Mass expression is possible in E. coli using gene recombination technology, and its amino terminal and carboxyl terminal are expressed on the surface, enabling fusion expression for the expression of foreign proteins.
이에 제한되는 것은 아니나, eDPS는 일 말단에 표적 지향성 펩타이드가 결합될 수 있다. 표적 지향성 펩타이드를 표면에 표출하는 단백질 나노입자로 자기조립된 후 화학적 공정을 통해 형광물질을 표면에 결합시킴으로써 형광 단백질 나노입자가 제조되는 것이다.Although not limited thereto, eDPS may have a target-directed peptide bonded to one end thereof. Fluorescent protein nanoparticles are prepared by self-assembling a target-oriented peptide into protein nanoparticles expressing the surface and then binding a fluorescent substance to the surface through a chemical process.
상기 자기조립성 단백질은 형광단백질 또는 표적 지향성 펩타이드와 링커 펩타이드를 통해 융합될 수 있다.The self-assembled protein may be fused with a fluorescent protein or a target-directed peptide through a linker peptide.
상기 링커 펩타이드는 자기조립성 단백질과 외래 단백질 간의 간격을 만들어 주는데, 자기조립성 단백질과 형광단백질 사이에 링커 펩타이드를 연결할 경우, 일반적으로 형광물질들이 1 내지 10 nm 이내로 서로 위치할 형광의 퀀칭 현상이 나타나는 것으로 알려져 있고, 링커 펩타이드는 형광단백질 간의 공간을 넓혀줌으로써 이러한 형광 ?칭을 억제하고 형광의 강도를 증가시키는 효과를 제공할 수 있다. 따라서, 링커 펩타이드는 형광단백질 간의 적절한 공간을 확보해 줄 수 있는 길이를 가질 수 있다. 예컨대, 링커 펩타이드의 길이는 형광단백질의 종류 및 크기에 따라 달라질 수 있을 것이다. 예를 들어 링커 펩타이드는 5 내지 20개, 예컨대, 5 내지 15개의 아미노산으로 이루어진 펩타이드일 수 있다. The linker peptide creates a gap between the self-assembled protein and the foreign protein. When the linker peptide is connected between the self-assembled protein and the fluorescent protein, in general, the fluorescence quenching phenomenon in which the fluorescent substances are located within 1 to 10 nm. It is known to appear, and the linker peptide can provide an effect of suppressing such fluorescence and increasing the intensity of fluorescence by expanding the space between fluorescent proteins. Therefore, the linker peptide may have a length that can secure an appropriate space between fluorescent proteins. For example, the length of the linker peptide may vary depending on the type and size of the fluorescent protein. For example, the linker peptide may be a peptide consisting of 5 to 20 amino acids, such as 5 to 15 amino acids.
본 발명의 한 구체예에서, 링커 펩타이드는 글라이신을 포함하는 것일 수 있다. 이에 제한되는 것은 아니나, 본 발명의 링커 펩타이드는 예를 들어, SEQ ID NO: 35 내지 39에 기재된 아미노산 서열 중 어느 하나로 표시될 수 있다. In one embodiment of the present invention, the linker peptide may include glycine. Although not limited thereto, the linker peptide of the present invention may be represented by any one of the amino acid sequences set forth in SEQ ID NO: 35 to 39, for example.
또한, 본 발명의 형광 단백질 나노입자는 자기조립성 단백질에 형광단백질을 연결하는 대신 형광물질을 표면 고정시켜 사용할 수 있다. 상술한 자기조립성 단백질은 모두 아미노산의 작용기가 표면에 표출되어 있으므로, 유전자 재조합 기술에 의한 외래 단백질의 표면 표출뿐 아니라 화학적 반응을 통한 기능성 소재의 표면 고정화가 가능하다. 따라서, 자기조립성 단백질의 자기조립 후 화학적 공정을 통해 형광물질을 표면에 고정할 수 있다.In addition, the fluorescent protein nanoparticles of the present invention may be used by surface-immobilizing a fluorescent material instead of linking a fluorescent protein to a self-assembled protein. Since the functional groups of amino acids are expressed on the surface of all of the above-described self-assembled proteins, it is possible not only to express the surface of foreign proteins by genetic recombination but also to immobilize the surface of functional materials through chemical reactions. Therefore, after self-assembly of the self-assembled protein, the fluorescent material can be fixed to the surface through a chemical process.
이러한 형광물질로, 플루오레신(fluorescein), 보디피(BODYPY), ATTO Dyes, 로다민(Rhodamine), 헵타메틴(Heptamethine), DyLight Fluor, 알렉사(Alexa), 시아닌(Cyanine) 또는 알로피코시아닌(allopicocyanine) 등을 사용할 수 있으나, 이에 특별히 제한하는 것은 아니다.Such fluorescent materials include fluorescein, BODYPY, ATTO Dyes, Rhodamine, Heptamethine, DyLight Fluor, Alexa, Cyanine, or allophycocyanin. (allopicocyanine) or the like may be used, but is not particularly limited thereto.
또한, 본 발명의 형광 단백질 나노입자에 사용할 수 있는 형광단백질은 녹색형광단백질(GFP), 변형된 녹색형광단백질(modified green fluorescent protein), 증강된 녹색형광단백질(enhanced green fluorescent protein; EGFP), 적색형광단백질(RFP, DSRed), 증강된 적색형광단백질(ERFP), 청색형광단백질(BFP), 증강된 청색형광단백질(EBFP), 황색형광단백질(YFP), 증강된 황색형광단백질(EYFP), 남색형광단백질(CFP), 증강된 남색형광단백질(ECFP) 등을 사용할 수 있다. 보다 구체적으로는, 증강된 녹색형광단백질 또는 적색형광단백질일 수 있다.In addition, fluorescent proteins that can be used in the fluorescent protein nanoparticles of the present invention include green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), and red. Fluorescent protein (RFP, DSRed), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo blue Fluorescent protein (CFP), enhanced indigo fluorescent protein (ECFP), and the like can be used. More specifically, it may be an enhanced green fluorescent protein or a red fluorescent protein.
한편, 본 명세서에서, 용어 "표적 지향성 펩타이드"는 본 발명에 따른 형광 단백질 나노입자에 표적 지향성을 부여할 수 있는 펩타이드를 의미한다. 생체 이미징 소재 개발에 있어서, 표적 특이적으로 타겟팅을 가능하게 하기 위하여 표적 지향성 펩타이드를 나노입자의 표면에 고정하는 기술이 활발히 연구되고 있다. 하지만 기존 무기, 고분자 기반 나노입자의 경우 화학적 결합을 통해 형광물질을 표면 수식화하므로, 동시에 표적 지향성 펩타이드를 표면에 수식화하는데 어려움이 있으며 형광물질과 표적 지향성 펩타이드의 표출 빈도를 조절하는데 어려움이 있다. 반면 본 발명에서는 유전공학적 기법을 이용하여 추가 화학적 공정 없이 표적 지향성 펩타이드와 형광단백질을 동시에 표출하는 단백질 나노입자를 제조하거나, 표적 지향성 펩타이드를 표출하는 단백질 나노입자의 표면 아민기와 형광물질의 결합을 통해 형광 단백질 나노입자를 제조할 수 있으며, 표적 지향성 펩타이드의 표면 표출 빈도 및 표출 위치를 조절할 수 있는 장점이 있다. Meanwhile, in the present specification, the term "target-directed peptide" refers to a peptide capable of imparting target directivity to the fluorescent protein nanoparticles according to the present invention. In the development of bio-imaging materials, technologies for immobilizing target-oriented peptides on the surface of nanoparticles have been actively studied in order to enable target-specific targeting. However, in the case of conventional inorganic and polymer-based nanoparticles, since the fluorescent material is surface-modified through chemical bonding, it is difficult to formulate the target-oriented peptide on the surface at the same time, and it is difficult to control the frequency of expression of the fluorescent material and the target-oriented peptide. On the other hand, in the present invention, a protein nanoparticle that simultaneously expresses a target-oriented peptide and a fluorescent protein is prepared by using a genetic engineering technique, or by combining a fluorescent substance with a surface amine group of a protein nanoparticle expressing the target-oriented peptide. Fluorescent protein nanoparticles can be prepared, and there is an advantage of being able to control the frequency and location of surface expression of the target-oriented peptide.
본 발명에 따른 표적 지향성 펩타이드는 형광 단백질 나노입자의 표면에 표출되어 있다. 따라서, 인 비보 이미징에 적용시 본 발명에 따른 형광 단백질 나노입자를 특정 타겟 부위에 전달해 주는 역할을 충실히 수행한다. The target-oriented peptide according to the present invention is expressed on the surface of the fluorescent protein nanoparticle. Therefore, when applied to in vivo imaging, it faithfully delivers the fluorescent protein nanoparticles according to the present invention to a specific target site.
본 발명에 있어서, 형광단백질, 자기조립성 단백질 및 표적 지향성 펩타이드가 융합된 단백질; 또는 자기조립성 단백질과 표적 지향성 펩타이가 융합된 단백질은 하나의 발현 벡터를 통해 발현된다.In the present invention, a fluorescent protein, a self-assembled protein, and a protein in which a target-oriented peptide is fused; Alternatively, a protein in which a self-assembled protein and a target-directed peptide are fused is expressed through a single expression vector.
따라서, 표적 지향성 펩타이드는 상기 발현 벡터에 쉽게 삽입 가능하면서, 본 발명에 따른 형광 단백질 나노입자의 형광 강도 및 나노입자의 구조에 영향을 미치지 않는 것이라면 펩타이드 서열의 길이와 관계없이 어떠한 것이든 사용할 수 있다. 특히 특정 질환 조직 및 세포에서 특이적으로 과발현되는 수용체에 실제 호르몬과 유사하게 대신 결합하여 신호 전달을 억제한다고 알려진 다수 펩타이드 형태의 수용체 길항근(receptor antagonist); 소마토스타틴(somatostatins), 어피바디(affibody), 봄베신(bombesin), 콜레시스토키닌(cholecystokinin) 또는 가스트린 유사체(gastrin analog) 등의 펩타이드 호르몬 등으로 대체 가능하다. Therefore, the target-oriented peptide can be easily inserted into the expression vector, and any one can be used regardless of the length of the peptide sequence as long as it does not affect the fluorescence intensity and structure of the nanoparticle according to the present invention. . In particular, a receptor antagonist in the form of a number of peptides known to inhibit signal transduction by binding to receptors that are specifically overexpressed in specific diseased tissues and cells in a similar manner to real hormones; Peptide hormones such as somatostatins, affibody, bombesin, cholecystokinin or gastrin analog can be substituted.
본 발명의 일 구체예에서, 표적 지향성 펩타이드는 RGD(Arginine-Glycine-Aspartic acid) 펩타이드일 수 있다. 암세포는 정상 세포에 비해 빠른 성장을 위해서 주변에 새로운 혈관 생성을 촉진하기 위한 수용체를 표출하고 있다고 알려져 있다. 특히 인테그린(integrin) αvβ3 수용체는 혈관생성(angiogenesis)에 결정적인 역할을 한다고 알려져 있으며 기존 연구를 통해 RGD(Arginine-Glycine-Aspartic acid) 서열을 가지는 특정 펩타이드와 결합한다고 알려져 있다. 이에 본 발명에서는 암세포 특이적 타겟팅을 목적으로 RGD 서열을 표면 표출하는 형광 단백질 나노입자를 제조하여 암특이적 타겟팅과 이미징이 동시에 가능하도록 하였다. In one embodiment of the present invention, the target-directed peptide may be an Arginine-Glycine-Aspartic acid (RGD) peptide. Cancer cells are known to express receptors to promote the creation of new blood vessels around them for faster growth than normal cells. In particular, the integrin αvβ3 receptor is known to play a critical role in angiogenesis, and it is known that it binds to a specific peptide having an Arginine-Glycine-Aspartic acid (RGD) sequence through previous studies. Accordingly, in the present invention, for the purpose of targeting specific to cancer cells, fluorescent protein nanoparticles expressing the surface of the RGD sequence were prepared to enable cancer specific targeting and imaging at the same time.
본 발명의 일 구체예에 따르면, 본 발명의 표적 지향성 펩타이드가 표출된 형광 단백질 나노입자는 암세포에 특이적으로 업테이크되며, 인 비보 이미징에 적용하기 위해, 인테그린 수용체 발현 빈도가 높은 U87MG 암세포를 누드 마우스에 주사하여 암을 신장시킨 동물 모델에서 2시간 이후에도 암에서 형광이 측정되었다. 또한 상대적으로 인테그린 수용체 발현 빈도가 낮은 SCC7 암세포, 인테그린 수용체 발현 빈도가 높은 U87MG 암세포를 각각 준비하여 누드 마우스에 주사하여 암을 신장시킨 동물 모델에 형광 단백질 나노입자 및 형광단백질 단량체를 정맥 주사한 후 각 조직에 남아있는 형광을 측정한 결과, SCC7, U87MG 암 모델 모두 형광 단백질 단량체는 RGD 펩타이드가 있음에도 신장을 통해 체내에서 제거되나, 본 발명에 따른 표적 지향성 펩타이드가 표출된 형광 단백질 나노입자는 상대적으로 간에 주로 축적됨을 확인하였다. 이는 단백질 나노입자 기반의 형광 나노입자는 단량체에 비해 구조적 안정성이 향상되고, 체내 순환률이 향상됨을 보여주며, 형광단백질 단량체에 비해 인 비보 이미징에 적합한 소재임을 보여준다. 또한 암세포 특이적 펩타이드를 표출하는 형광 단백질 나노입자는 암세포에 특이적으로 타겟팅이 가능하며, 동시에 높은 형광 세기를 통해 이미징이 가능함을 보여준다.According to an embodiment of the present invention, the fluorescent protein nanoparticles expressing the target-directed peptide of the present invention are specifically uptaken in cancer cells, and for application to in vivo imaging, U87MG cancer cells with high integrin receptor expression frequency are nude Fluorescence was measured in cancer even after 2 hours in an animal model in which cancer was elongated by injection into a mouse. In addition, SCC7 cancer cells with relatively low integrin receptor expression frequency and U87MG cancer cells with high integrin receptor expression frequency were prepared, respectively, and injected into nude mice to elongate cancer. After intravenous injection of fluorescent protein nanoparticles and fluorescent protein monomers into an animal model. As a result of measuring the fluorescence remaining in the tissue, the fluorescent protein monomer in both SCC7 and U87MG cancer models is removed from the body through the kidney even in the presence of the RGD peptide, but the fluorescent protein nanoparticles expressing the target-oriented peptide according to the present invention are relatively It was confirmed that it was mainly accumulated. This shows that the protein nanoparticle-based fluorescent nanoparticles have improved structural stability and improved circulation rate in the body compared to the monomers, and show that they are suitable materials for in vivo imaging compared to the fluorescent protein monomers. In addition, the fluorescent protein nanoparticles that express cancer cell-specific peptides can be specifically targeted to cancer cells, and at the same time, imaging is possible through high fluorescence intensity.
상술한 바와 같이, 본 발명에 따른 표적 지향성 펩타이드가 표출된 형광 단백질 나노입자는 높은 형광강도, 우수한 생체 안정성, 다양한 표면 수식화를 토대로 표적 타겟팅과 이미징이 가능하며, 그 효용성이 검증되었다.As described above, the fluorescent protein nanoparticles expressing the target-oriented peptide according to the present invention are capable of targeting and imaging based on high fluorescence intensity, excellent biological stability, and various surface modification, and their effectiveness has been verified.
따라서, 본 발명은 또한 본 발명에 따른 형광 단백질 나노입자; 및Accordingly, the present invention also provides a fluorescent protein nanoparticle according to the present invention; And
약제학적으로 허용가능한 담체를 포함하는 표적 지향형 조영제 조성물을 제공한다. It provides a target-directed contrast medium composition comprising a pharmaceutically acceptable carrier.
본 발명의 형광 단백질 나노입자는 표적 지향성 펩타이드가 결합되어 있어 특정 세포나 조직에서 표적 지향이 가능하므로 자기공명 및 광학영상장치를 등을 통해 표적 부위의 이미징이 가능한 조영제로 사용할 수 있다.The fluorescent protein nanoparticles of the present invention can be used as a contrast medium capable of imaging the target site through magnetic resonance and optical imaging devices, since target-oriented peptides are bound to enable target-oriented in specific cells or tissues.
상기 약제학적으로 허용가능한 담체는 의약 분야에서 통상 사용되는 담체 및 비히클을 포함하며, 구체적으로 이온 교환 수지, 알루미나, 알루미늄 스테아레이트, 레시틴, 혈청 단백질(예, 사람 혈청 알부민), 완충 물질(예, 각종 인산염, 글리신, 소르브산, 칼륨 소르베이트, 포화 식물성 지방산의 부분적인 글리세라이드 혼합물), 물, 염 또는 전해질(예, 프로타민 설페이트, 인산수소이나트륨, 인산수소캄륨, 염화나트륨 및 아연 염), 교질성 실리카, 마그네슘 트리실리케이트, 폴리비닐피롤리돈, 셀룰로즈계 기질, 폴리에틸렌 글리콜, 나트륨 카르복시메틸셀룰로즈, 폴리아릴레이트, 왁스, 폴리에틸렌 글리콜 또는 양모지 등을 포함하나 이에 제한되지 않는다. 또한, 본 발명의 조영제 조성물은 상기 성분들 이외에 윤활제, 습윤제, 유화제, 현탁제, 또는 보존제 등을 추가로 포함할 수 있다. The pharmaceutically acceptable carrier includes carriers and vehicles commonly used in the field of medicine, and specifically, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, camium hydrogen phosphate, sodium chloride and zinc salts), colloidal Silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool paper, etc., but are not limited thereto. In addition, the contrast agent composition of the present invention may further include a lubricant, a wetting agent, an emulsifying agent, a suspending agent, or a preservative in addition to the above components.
한 양태로서, 본 발명에 따른 조영제 조성물은 비경구 투여를 위한 수용성 용액으로 제조할 수 있으며, 바람직하게는 한스 용액(Hank's solution), 링거 용액(Ringer's solution) 또는 물리적으로 완충된 염수와 같은 완충 용액을 사용할 수 있다. 수용성 주입(injection) 현탁액은 소디움 카르복시메틸셀룰로즈, 솔비톨 또는 덱스트란과 같이 현탁액의 점도를 증가시킬 수 있는 기질을 첨가할 수 있다. In one embodiment, the contrast agent composition according to the present invention may be prepared as an aqueous solution for parenteral administration, and preferably, a buffer solution such as Hank's solution, Ringer's solution, or physically buffered saline Can be used. Aqueous injection suspensions may be added with a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.
본 발명의 조영제 조성물의 다른 바람직한 양태는 멸균 주사용 수성 또는 유성 현탁액의 멸균 주사용 제제의 형태일 수 있다. 이러한 현탁액은 적합한 분산제 또는 습윤제(예를 들면 트윈 80) 및 현탁화제를 사용하여 본 분야에 공지된 기술에 따라 제형화할 수 있다. Another preferred embodiment of the contrast medium composition of the present invention may be in the form of a sterile injectable preparation in an aqueous or oily suspension for sterile injection. Such suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg Tween 80) and suspending agents.
또한, 상기 멸균 주사용 제제는 무독성의 비경구적으로 허용되는 희석제 또는 용매 중의 멸균 주사 용액 또는 현탁액(예를 들면 1,3-부탄디올 중의 용액)일 수 있다. 사용될 수 있는 비히클 및 용매로는 만니톨, 물, 링거 용액 및 등장성 염화나트륨 용액이 있다. 또한, 멸균 비휘발성 오일이 통상적으로 용매 또는 현탁화 매질로서 사용된다. 이러한 목적을 위해 합성 모노 또는 디글리세라이드를 포함하여 자극성이 적은 비휘발성 오일은 그 어느 것도 사용할 수 있다.In addition, the sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (for example, a solution in 1,3-butanediol). Vehicles and solvents that can be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile nonvolatile oils are commonly used as solvents or suspending media. For this purpose, any non-volatile oil with less irritation, including synthetic mono or diglycerides, can be used.
본 발명의 조영제 조성물은 진단 대상에서 분리한 조직 또는 세포에 투여하여 형광 단백질 나노입자가 발산하는 신호를 감지하여 영상을 수득하는데 이용될 수 있다. 상기 형광 단백질 나노입자에 의해 발산되는 신호를 감지하기 위해서는 자기공명영상장치(MRI)와 광학 이미징의 이용이 바람직하다. The contrast agent composition of the present invention can be used to obtain an image by detecting a signal emitted by fluorescent protein nanoparticles by administration to tissues or cells isolated from a diagnosis subject. In order to detect the signal emitted by the fluorescent protein nanoparticles, it is preferable to use a magnetic resonance imaging apparatus (MRI) and optical imaging.
자기공명영상장치는 강력한 자기장 속에 생체를 넣고 특정 주파수의 전파를 조사하여 생체조직에 있는 수소 등의 원자핵에 에너지를 흡수시켜 에너지가 높은 상태로 만든 후, 상기 전파를 중단하여 상기 수소 등의 원자핵 에너지가 방출되게 하고 이 에너지를 신호로 변환하여 컴퓨터로 처리하여 영상화한 장치이다. 자기 또는 전파는 골에 방해를 받지 않기 때문에 단단한 골 주위 또는 뇌나 골수의 종양에 대하여 종단, 횡단, 임의의 각도에서 선명한 입체적인 단층상을 얻을 수 있다. 특히 상기 자기공명영상장치는 T2 스핀-스핀 이완 자기공명영상장치인 것이 바람직하다.The magnetic resonance imaging apparatus puts a living body in a strong magnetic field and irradiates radio waves of a specific frequency to absorb energy in an atomic nucleus such as hydrogen in a living tissue to make the energy high, and then stops the radio wave and stops the nuclear energy such as the hydrogen. It is a device that emits and converts this energy into a signal and processes it with a computer to image it. Since magnetism or propagation is not disturbed by bone, it is possible to obtain a clear three-dimensional tomographic image at a longitudinal, transverse, or arbitrary angle for a tumor around a hard bone or in the brain or bone marrow. In particular, the magnetic resonance imaging device is preferably a T2 spin-spin relaxation magnetic resonance imaging device.
본 발명은 또한 본 발명에 따른 형광 단백질 나노입자; 및 약제학적으로 허용가능한 담체를 포함하는 동시 진단 또는 치료용 조영제 조성물에 관한 것이다. The present invention also provides a fluorescent protein nanoparticle according to the present invention; And it relates to a contrast agent composition for simultaneous diagnosis or treatment comprising a pharmaceutically acceptable carrier.
본 발명의 형광 단백질 나노입자는 약제학적 활성성분과 물리 화학적으로 결합하여 표적 지향성 펩타이드가 타겟팅하는 세포 또는 조직에서 형광을 나타낼 수 있어 자기공명 및 광학영상장치 등을 통해 생체 분자의 분리, 진단 또는 치료 등의 나노 프로브 및 약물 또는 유전자 전달체(delivery vehicle)등에 이용할 수 있다. The fluorescent protein nanoparticles of the present invention are physicochemically combined with a pharmaceutical active ingredient to display fluorescence in cells or tissues targeted by the target-oriented peptide, so separation, diagnosis, or treatment of biomolecules through magnetic resonance and optical imaging devices It can be used for nano-probes such as, and drug or gene delivery vehicles.
상기 형광 단백질 나노입자를 이용한 생체 진단의 한 대표적인 예로서 분자 자기공명영상 진단 또는 자기 이완 센서(magnetic relaxation sensor)를 들 수 있다. 형광 단백질 나노입자는 그 크기가 커짐에 따라 더 큰 T2 조영효과를 나타내는데, 이러한 성질을 이용하면 생체 분자를 검출하는 센서로 사용될 수 있다. 즉, 특정한 생체 분자가 형광 단백질 나노입자의 엉김을 유도하게 되면 이에 의해 T2 자기 공명 영상 효과가 증대된다. 이러한 차이를 이용하여 생체 분자를 검출한다.As a representative example of biodiagnosis using the fluorescent protein nanoparticles, a molecular magnetic resonance imaging diagnosis or a magnetic relaxation sensor may be mentioned. Fluorescent protein nanoparticles exhibit a greater T2 contrast effect as their size increases, and if this property is used, it can be used as a sensor for detecting biomolecules. That is, when a specific biomolecule induces agglomeration of the fluorescent protein nanoparticles, the T2 magnetic resonance imaging effect is increased. Biomolecules are detected using these differences.
또한, 본 발명의 형광 단백질 나노입자는 종양과 관련된 다양한 질병, 예를 들어 편평상피세포암, 자궁암, 자궁경부암, 전립선암, 두경부암, 췌장암, 뇌종양, 유방암, 간암, 피부암, 식도암, 고환암, 신장암, 대장암, 직장암, 위암, 신장암, 방광암, 난소암, 담관암, 담낭암을 진단 및/또는 치료하는데 이용될 수 있다. 또한, 류마티스 관절염, 골관절염과 같은 염증성 질환과, 치매, 뇌졸중 등을 포함하는 난치성 질환에서 영상화하는 방법에 이용될 수 있다. 보다 구체적으로 설명하면, 종양 세포는 정상 세포에서는 거의 또는 전혀 생산되지 않는 특정 물질을 발현 및/또는 분비하는데 이들을 일반적으로 "종양 마커(tumor marker)"라고 명명한다. 그러한 종양 마커와 특이적으로 결합할 수 있는 물질을 상기 형광 단백질 나노입자에 결합시켜 만든 형광 단백질 나노입자는 종양 진단에 유용하게 이용될 수 있다. 당업계에는 다양한 종양 마커뿐만 아니라 이들과 특이적으로 결합할 수 있는 물질이 공지되어 있다. 본 발명에서는 표적 지향성 펩타이드로 종양 마커와 특이적으로 결합할 수 있는 펩타이드를 사용함으로써 종양 진단에 이용될 수 있다. In addition, the fluorescent protein nanoparticles of the present invention may be used in various diseases related to tumors, such as squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, and kidney. It can be used to diagnose and/or treat cancer, colon cancer, rectal cancer, gastric cancer, kidney cancer, bladder cancer, ovarian cancer, bile duct cancer, and gallbladder cancer. In addition, it can be used for imaging methods in inflammatory diseases such as rheumatoid arthritis and osteoarthritis, and in intractable diseases including dementia and stroke. More specifically, tumor cells express and/or secrete specific substances that are little or no production in normal cells, which are generally referred to as “tumor markers”. Fluorescent protein nanoparticles made by binding a substance capable of specifically binding to such a tumor marker to the fluorescent protein nanoparticles may be usefully used for tumor diagnosis. A variety of tumor markers, as well as substances capable of specifically binding to them, are known in the art. In the present invention, it can be used for tumor diagnosis by using a peptide capable of specifically binding to a tumor marker as a target-directed peptide.
상기 종양 마커는 작용 기작에 따라 리간드, 항원, 수용체, 및 이들을 코딩하는 핵산으로 분류할 수 있다. 종양 마커가 "리간드"인 경우에는 상기 리간드와 특이적으로 결합하는 수용체 또는 항체를 표적 지향성 펩타이드로 사용할 수 있고, 수용체의 예로는 시냅토타그민의 C2(synaptotagmin의 C2)와 포스파티딜세린, 아넥신 V(annexin V)와 포스파티딜세린, 인테그린(integrin)과 이의 수용체, VEGF(Vascular Endothelial Growth Factor)와 이의 수용체, 안지오포이에틴(angiopoietin)과 Tie2 수용체, 소마토스타틴(somatostatin)과 이의 수용체, 바소인테스티날 펩타이드(vasointestinal peptide)와 이의 수용체 등이 있지만 이에 제한되는 것은 아니다. The tumor markers can be classified into ligands, antigens, receptors, and nucleic acids encoding them according to their mechanism of action. When the tumor marker is a "ligand", a receptor or antibody that specifically binds to the ligand can be used as a target-directed peptide. Examples of the receptor include synaptotagmin C2 (synaptotagmin C2), phosphatidylserine, and annexin V. (annexin V) and phosphatidylserine, integrin and its receptor, VEGF (Vascular Endothelial Growth Factor) and its receptor, angiopoietin and Tie2 receptor, somatostatin and its receptor, vasointestinal Peptide (vasointestinal peptide) and its receptors, etc., but are not limited thereto.
본 발명은 또한 본 발명에 따른 형광 단백질 나노입자; 및 진단 프로브를 포함하고, 상기 진단 프로브는 영상 판독을 위한 표지 물질인 다중 진단 프로브에 관한 것이다. The present invention also provides a fluorescent protein nanoparticle according to the present invention; And a diagnostic probe, wherein the diagnostic probe relates to a multiple diagnostic probe that is a labeling material for image reading.
상기 진단 프로브는 T1 자기공명 영상 진단 프로브, 광학 진단 프로브, CT 진단 프로브, 또는 방사선 동위원소 등을 사용할 수 있다. The diagnostic probe may be a T1 magnetic resonance imaging probe, an optical diagnostic probe, a CT diagnostic probe, or a radioisotope.
상기 다중 진단 프로브는 예를 들면, 단백질 나노입자에 T1 자기공명 영상 진단 프로브를 결합시키면 T2 자기공명영상 및 T1 자기공명영상 진단을 동시에 진행할 수 있고, 광학 진단 프로브를 결합시키면 자기공명 영상과 광학 이미징을 동시에 할 수 있으며, CT 진단 프로브를 결합시키면 자기공명영상과 CT 진단을 동시에 할 수 있다. 또한 방사선 동위원소와 결합시키면 자기공명영상과 PET, SPECT 진단을 동시에 할 수 있다. The multi-diagnosis probe, for example, can perform T2 magnetic resonance imaging and T1 magnetic resonance imaging at the same time when the T1 magnetic resonance imaging probe is coupled to the protein nanoparticle, and when the optical diagnostic probe is coupled, magnetic resonance imaging and optical imaging Simultaneously, the magnetic resonance imaging and CT diagnosis can be performed at the same time by combining the CT diagnosis probe. In addition, when combined with radioactive isotopes, magnetic resonance imaging and PET and SPECT diagnosis can be performed at the same time.
상기 T1 자기공명 영상 진단 프로브로는 Gd 화합물, 또는 Mn 화합물 등을 포함하며, 광학 진단 프로브로는 유기 형광 염료(dye), 양자점, 또는 염료 표지(dye labelled) 무기 지지체(예 SiO2, Al2O3))를 포함하며, CT 진단 프로브로는 I(요오드) 화합물, 또는 금 나노 입자를 포함하고, 방사선 동위원소로는 In, Tc, 또는 F 등을 포함한다.The T1 magnetic resonance imaging probe includes a Gd compound or an Mn compound, and the optical diagnostic probe includes an organic fluorescent dye, quantum dot, or dye-labeled inorganic support (e.g. SiO 2 , Al 2 O 3 )), and the CT diagnostic probe includes an I (iodine) compound, or gold nanoparticles, and the radioisotope includes In, Tc, or F.
본 발명은 또한 자기조립성 단백질에 형광물질이 결합된 10 내지 50 nm 크기의 형광 단백질 나노입자; 및 약제학적으로 허용가능한 담체를 포함하고, 상기 형광 단백질 나노입자는 B형 간염 바이러스 코어 단백질, 페리틴 중쇄 단백질(Ferritin Heavy chain), 써모플라즘 애시도필룸(Thermoplasm acidophilum) 유래의 프로테아좀(tPTS) 또는 대장균 유래의 DNA 결합 단백질(eDPS)을 포함하는 자기조립성 단백질로부터 자기조립된 단백질 나노입자에 형광물질을 표면 결합시켜 제조된 것인, 생체 이미징용 조성물에 관한 것이다.The present invention also provides a fluorescent protein nanoparticle having a size of 10 to 50 nm in which a fluorescent material is bound to a self-assembled protein; And a pharmaceutically acceptable carrier, wherein the fluorescent protein nanoparticles include hepatitis B virus core protein, ferritin heavy chain, and thermoplasmic acidophilum. acidophilum ) derived from a proteasome (tPTS) or a self-assembled protein containing a DNA binding protein (eDPS) derived from Escherichia coli, prepared by surface-binding a fluorescent substance to protein nanoparticles self-assembled It is about.
표적 지향성 펩타이드를 표면 표출하지 않은 본 발명의 형광 단백질 나노입자는 그 자체의 크기와 구조의 특성만으로 특정 조직이나 질환을 타겟팅할 수 있다. 예컨대, 형광 단백질 나노입자를 이용하여 감시림프절 (Sentinel lymphnode) 이미징에 적용할 수 있다. The fluorescent protein nanoparticles of the present invention, which do not surface-express a target-oriented peptide, can target specific tissues or diseases only by the characteristics of their size and structure. For example, it can be applied to Sentinel lymphnode imaging using fluorescent protein nanoparticles.
감시림프절 생검은 유방암이나 흑색종 등의 암의 전이 여부를 판단하는데 널리 사용되는 방법이다. 감시림프절은 고형암에서 가장 가까운 림프절을 지칭하는 용어로, 감시림프절을 적출하여 그 안에 암세포의 유무를 확인함으로써 암세포의 전이 여부를 진단한다. 이때 정확한 감시림프절을 찾아내는 것이 매우 중요한데, 림프절은 주변 조직, 특히 지방과 육안으로 구분하기 어려울 뿐 아니라, 특히 여러 림프절이 림프관을 따라 존재하므로 종양에서 가장 가까이 존재하는 감시 림프절을 구분하는데 어려움이 있다. 기존에 감시림프절을 찾는데 사용되는 이소설판블루(Isosulfan blue)는 일부 환자에서 과민성 쇼크를 유발한다고 알려져 있으며 실제 감시림프절에 머무르는 시간이 짧고 다음 림프절로 쉽게 이동하는 단점이 있다. 반면 방사성 물질 테크네티움(Technetium(Tc) 99m-labeled colloid)은 시료를 주사한 부위에서 림프절로 이동하는데 오랜 시간이 소요되는 단점이 있다. Surveillance lymph node biopsy is a widely used method to determine whether cancer has metastasized, such as breast cancer or melanoma. Sentinel lymph node is a term referring to the lymph node closest to solid cancer, and diagnosis of metastasis of cancer cells is performed by extracting the sentiment lymph node and checking the presence or absence of cancer cells in it. At this time, it is very important to find an accurate sentinel lymph node, and it is difficult to distinguish the lymph node into surrounding tissues, especially fat and the naked eye, and in particular, since several lymph nodes exist along the lymphatic vessels, it is difficult to distinguish the sentinel lymph nodes that are closest to the tumor. Isosulfan blue, which is conventionally used to search for sentinel lymph nodes, is known to induce irritable shock in some patients, and has the disadvantage of short staying in the sentinel lymph node and easily moving to the next lymph node. On the other hand, the radioactive material Technetium (Tc) 99m-labeled colloid has the disadvantage that it takes a long time to move from the injection site to the lymph node.
본 발명에서는 감시림프절 이미징에 적절하다고 알려진 10 내지 50 nm 크기의 형광 단백질 나노입자를 이용하여 감시림프절 이미징에 적용하였으며 그 효용성을 검증하였다. In the present invention, fluorescent protein nanoparticles with a size of 10 to 50 nm, which are known to be suitable for surveillance lymph node imaging, were applied to the surveillance lymph node imaging, and their effectiveness was verified.
그 결과, 크기가 다른 4개의 형광 단백질 나노입자, eDPS, tPTS, FTN-h, HBVcAg 나노입자를 동물 모델의 앞발에 주사하였을 때 주사한 부위로부터 가장 가까운 림프절에 형광 단백질 나노입자가 축적되는 것을 확인하였다. 주사한 즉시 감시림프절에서 표지 신호가 특정될 뿐 아니라 1시간 이상으로 표지 신호가 유지되며 다른 림프절로 확산되지 않는 것을 통해, 형광 단백질 나노입자가 감시림프절을 타겟팅하는데 매우 효과적임을 검증하였다. 특히, tPTS 나노입자의 경우 다른 입자에 비해 가장 높은 감시림프절 타겟팅 효율을 보였으며, 이는 표적 지향성 펩타이드가 없이도 단백질 나노입자 고유의 크기나 구조적 특성이 특정 조직을 타겟팅하여 이미징하는 소재로 활용될 수 있음을 보여준다.As a result, when four fluorescent protein nanoparticles of different sizes, eDPS, tPTS, FTN-h, and HBVcAg nanoparticles were injected into the forefoot of an animal model, it was confirmed that fluorescent protein nanoparticles were accumulated in the lymph nodes closest to the injection site. I did. Immediately after injection, not only the label signal was specified in the sentinel lymph node, but also the label signal was maintained for more than 1 hour and did not spread to other lymph nodes. Thus, it was verified that the fluorescent protein nanoparticles were very effective in targeting the sentinel lymph node. In particular, tPTS nanoparticles showed the highest surveillance lymph node targeting efficiency compared to other particles, and this can be used as a material for imaging by targeting specific tissues with the inherent size or structural characteristics of protein nanoparticles without a target-oriented peptide. Show.
따라서, 표적 지향성 펩타이드를 표출하지 않으나 일정 크기나 구조적 특성을 갖는 단백질 나노입자는 감시림프절 인 비보 이미징에 적합한 소재일 수 있다. Therefore, protein nanoparticles that do not express target-oriented peptides but have a certain size or structural characteristics may be suitable materials for in vivo imaging, which is a surveillance lymph node.
상기 형광 단백질 나노입자는 입자 크기가 10 내지 50 nm가 되도록 자기조립성 단백질을 자기조립한 후 형광물질로 표면 수식하여 제조한 것일 수 있다. The fluorescent protein nanoparticles may be prepared by self-assembling a self-assembled protein to have a particle size of 10 to 50 nm, and then surface modification with a fluorescent material.
상기 자기조립성 단백질로, B형 간염 바이러스 코어 단백질, 페리틴 중쇄 단백질(Ferritin Heavy chain), 써모플라즘 애시도필룸(Thermoplasm acidophilum) 유래의 프로테아좀(tPTS) 또는 대장균 유래의 DNA 결합 단백질(eDPS) 등을 사용할 수 있다. As the self-assembled protein, hepatitis B virus core protein, ferritin heavy chain protein, thermoplasmic acidophilum acidophilum )-derived proteasome (tPTS) or E. coli-derived DNA binding protein (eDPS), and the like can be used.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.Advantages and features of the present invention, and a method of achieving them will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to the possessor, and the invention is only defined by the scope of the claims.
<실시예 1> HBV 캡시드 유래 키메릭 형광 나노입자의 제조<Example 1> Preparation of HBV capsid-derived chimeric fluorescent nanoparticles
(HBV 캡시드 유래 키메릭 형광 나노입자의 생합성을 위한 발현 벡터 제조)(Preparation of expression vector for biosynthesis of chimeric fluorescent nanoparticles derived from HBV capsid)
하기 표 1에 기재된 프라이머를 사용한 PCR 후에, HBV 코어 단백질(HBVcAg) 유전자로부터 유래한 N-NdeI-HBVcAg(1-78)-XhoI-C 및 N-BamHI-HBVcAg(81-149)-HindIII-C의 합성을 코드하는 두 유전자 클론을 얻었다. 또한, 형광단백질(enhanced green fluorescent protein; eGFP 또는 red fluorescent protein; DsRed)로 HBVcAg의 P79A80를 대체하기 위하여, 4가지의 다른 클론, 5’-XhoI-hexahistidine-EGFP-BamHI-3’, 5’-XhoI-hexahistidine-DsRed-BamHI-3’와 링커 펩타이드(G3SG3TG3SG3)가 삽입된 5’-XhoI-hexahistidine-linker-eGFP-linker-BamHI-3’와 5’-XhoI-hexahistidine-linker-DsRed-linker-BamHI-3’를 PCR 또는 extention PCR을 이용하여 제조하였다. 플라즈미드 pT7-7에 상기 유전자 클론의 일련의 라이게이션을 통하여, A) N-HBVcAg(1-78)-His6-eGFP-HBVcAg(81-149)-C, B) N-HBVcAg(1-78)-His6-DsRed-HBVcAg(81-149)-C, C) N-HBVcAg(1-78)-His6-linker-eGFP-linker-HBVcAg(81-149)-C 및 D) N-HBVcAg(1-78)-His6-linker-DsRed-linker-HBVcAg(81-149)-C의 합성을 코딩하는 4개의 플라즈미드 발현 벡터 pT7-형광단백질-HBVcAg를 구축하였다(도 1). 제작된 모든 플라즈미드 발현 벡터는 젤-정제 후 완전한 DNA 시퀀싱을 통해 서열을 확인하였다. After PCR using the primers shown in Table 1 below, N-NdeI-HBVcAg(1-78)-XhoI-C and N-BamHI-HBVcAg(81-149)-HindIII-C derived from the HBV core protein (HBVcAg) gene. Two gene clones encoding the synthesis of were obtained. In addition, in order to replace P79A80 of HBVcAg with an enhanced green fluorescent protein (eGFP or red fluorescent protein; DsRed), four different clones, 5'-XhoI-hexahistidine-EGFP-BamHI-3', 5'- XhoI-hexahistidine-DsRed-BamHI-3' and 5'-XhoI-hexahistidine-linker-eGFP-linker-BamHI-3' and 5'-XhoI-hexahistidine-linker-DsRed-linker- inserted with a linker peptide (G3SG3TG3SG3) BamHI-3' was prepared using PCR or extention PCR. Through a series of ligation of the gene clone to plasmid pT7-7, A) N-HBVcAg(1-78)-His6-eGFP-HBVcAg(81-149)-C, B) N-HBVcAg(1-78) -His6-DsRed-HBVcAg(81-149)-C, C) N-HBVcAg(1-78)-His6-linker-eGFP-linker-HBVcAg(81-149)-C and D) N-HBVcAg(1- 78)-His6-linker-DsRed-linker-HBVcAg(81-149)-C four plasmid expression vectors encoding the synthesis of pT7-fluorescent protein-HBVcAg were constructed (Fig. 1). All of the prepared plasmid expression vectors were gel-purified and then sequenced through complete DNA sequencing.
HBV 캡시드 유래 키메릭 나노입자의 제조와 관련된 프라이머 서열(표 1)과 주형에 대한 정보 및 이에 대한 좀 더 상세한 기재는 아래와 같다.Information on the primer sequence (Table 1) and template related to the preparation of chimeric nanoparticles derived from HBV capsid, and a more detailed description thereof are as follows.
번호order
number
1) 첫 번째 부분은 HBV 코어 단백질의 유전자 서열(NCBI Nucleotide accession number: AF286594 서열 중 1901-2452 서열)을 주형으로 하여 제한 효소 NdeI이 포함되어 있는 프라이머 1과 XhoI을 포함하고 있는 프라이머 2를 이용하여 PCR을 진행하였다. 그 결과 5‘-NdeI-HBV 코어 단백질(아미노산 서열 1-78)-XhoI-3' 으로 이루어진 PCR 산물을 확보하였다.1) The first part is the HBV core protein gene sequence (NCBI Nucleotide accession number: 1901-2452 of the AF286594 sequence) as a template, and
2) 두 번째 부분은 HBV 코어 단백질의 유전자 서열을 주형으로 하여 제한 효소 BamHI이 포함되어 있는 프라이머 3과 HindIII를 포함하고 있는 프라이머 4를 이용하여 PCR 을 진행하였다. 그 결과 5‘-BamHI-HBV 코어 단백질(아미노산 서열 81-149)-HindIII-3' 으로 이루어진 PCR 산물을 확보하였다.2) In the second part, PCR was performed using
3) 세 번째 부분은 eGFP(또는 DsRed)의 염기서열을 주형으로 하여(NCBI Nucleotide accession number of eGFP: AJ566337, NCBI Nucleotide accession number of DsRed: EU827527) XhoI과 hexahistidine을 포함하고 있는 프라이머 5(또는 프라이머 7), BamHI을 포함하는 프라이머 6(또는 프라이머 8)을 이용하여 PCR을 수행하였다. 5’-XhoI-His6-eGFP-BamHI-3’제작을 위해서 프라이머 5와 6을 이용하였으며, 5’-XhoI-His6-DsRed-BamHI-3’제작을 위해서 프라이머 7과 8을 이용하였다.3) The third part uses the base sequence of eGFP (or DsRed) as a template (NCBI Nucleotide accession number of eGFP: AJ566337, NCBI Nucleotide accession number of DsRed: EU827527) Primer 5 (or primer ) containing Xho I and hexahistidine. 7), PCR was performed using primer 6 (or primer 8) containing Bam HI.
4) 네 번째 부분은 eGFP(또는 DsRed)의 염기서열을 주형으로 하여 링커서열(아미노산 서열 G3SG3TG3SG3)을 포함하고 있는 프라이머 9 및 10 (eGFP) 또는 프라이머 11 및 12 (DesRed)을 이용하여 PCR을 수행한 후, 합성된 PCR 산물을 다시 주형으로 하여 프라이머 13 및 15를 이용하여 확장 PCR을 수행하였다. 합성된 PCR 산물을 주형으로 다시 프라이머 14 및 15를 이용하여 이차 확장 PCR을 수행하였으며, 그 결과 5‘-XhoI-His6-linker (G3SG3TG3SG3)-eGFP-linker(G3SG3TG3SG3)-BamHI-3', 5‘-XhoI-His6-linker(G3SG3TG3SG3)-DsRed-linker(G3SG3TG3SG3)-BamHI-3'으로 이루어진 PCR 산물을 확보하였다.4) For the fourth part, PCR was performed using
이렇게 만들어진 6개의 PCR 산물을 순차적으로 pT7-7 벡터에 삽입하여 HBV 캡시드 유래 키메릭 형광 나노입자를 합성할 수 있는 발현 벡터를 구성하였다. 또한, 형광 단백질 나노입자의 형광 강도 개선을 위하여 형광단백질과 코어 단백질 사이에 링커를 삽입한 발현벡터를 제조하였다(도 1). The resulting 6 PCR products were sequentially inserted into the pT7-7 vector to construct an expression vector capable of synthesizing chimeric fluorescent nanoparticles derived from HBV capsid. In addition, in order to improve the fluorescence intensity of the fluorescent protein nanoparticles, an expression vector having a linker inserted between the fluorescent protein and the core protein was prepared (FIG. 1).
본 발명에서 구축한 발현 벡터를 통해 제조하고자 하는 형광 단백질 나노입자의 모식도는 도 2와 같다.A schematic diagram of a fluorescent protein nanoparticle to be prepared through the expression vector constructed in the present invention is shown in FIG. 2.
(HBV 캡시드 유래 키메릭 형광 나노입자의 생합성 및 분리정제)(Biosynthesis and separation and purification of chimeric fluorescent nanoparticles derived from HBV capsid)
대장균 균주 BL21(DE3)[F-ompThsdSB(rB-mB-)]를 상기 제조된 발현 벡터로 각각 형질전환하고, 앰피실린-저항성 형질전환체를 선택하였다. 형질전환된 대장균을 50 mL의 Luria-Bertani(LB) 배지(100 mg L-앰피실린 함유)를 함유하는 플라스크(250 mL Erlenmeyer flasks, 37 ℃, 150 rpm)에서 배양하였다. 배지 탁도(O.D600)가 약 0.6-0.7에 도달할 때, IPTG(Isopropyl-β-D-thiogalactopyranosid) (0.7 mM)를 첨가하여 재조합 유전자의 발현을 유도하였다. E. coli strain BL21(DE3)[F-ompThsdSB(rB-mB-)] was transformed with the prepared expression vector, respectively, and ampicillin-resistant transformants were selected. The transformed E. coli was cultured in a flask (250 mL Erlenmeyer flasks, 37° C., 150 rpm) containing 50 mL of Luria-Bertani (LB) medium (containing 100 mg L-ampicillin). When the medium turbidity (OD 600 ) reached about 0.6-0.7, Isopropyl-β-D-thiogalactopyranosid (IPTG) (0.7 mM) was added to induce expression of the recombinant gene.
20 ℃에서 16~18 시간 배양 후, 배양한 대장균을 4,500 rpm으로 10분간 원심 분리하여 균체 침전물을 회수한 후 5 ㎖의 파쇄 용액(10 mM Tris-HCl 완충액, pH 7.5, 10 mM EDTA)에 현탁하여 초음파 파쇄기(Branson Ultrasonics Corp., Danbury, CT, USA)를 이용하여 파쇄하였다. 파쇄한 후 13,000 rpm으로 10분간 원심분리한 뒤 상등액과 불용성 응집체를 분리하였다. After incubation at 20° C. for 16-18 hours, the cultured E. coli was centrifuged at 4,500 rpm for 10 minutes to recover the cell sediment, and then suspended in 5 ml of a disruption solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA). Then, it was crushed using an ultrasonic crusher (Branson Ultrasonics Corp., Danbury, CT, USA). After crushing, centrifugation was performed at 13,000 rpm for 10 minutes, and the supernatant and insoluble aggregates were separated.
분리된 상등액에 대해 자가조립된 형광단백질이 융합된 단백질 나노입자를 정제하기 위하여 3단계의 정제 과정을 거쳐 진행하였다. In order to purify the protein nanoparticles in which the self-assembled fluorescent protein was fused to the separated supernatant, it was carried out through a three-step purification process.
먼저 1) 재조합 단백질에 융합 발현된 히스티딘과 니켈의 결합을 이용한 Ni2+-NTA 친화성 크로마토그래피(Ni2 +-NTA affinity chromatography)를 진행한 후, 2) 재조합 단백질의 자가조립 효율을 향상시키기 위하여 Ultracentrifugal filter (Amicon Ultra 100K, Millipore, Billerica, MA)를 이용하여 재조합 단백질이 포함된 수용액을 조성이 다른 자가조립용 버퍼(500mM NaCl .50mM Tris-HCl pH 7.0)로 변경해 줌과 동시에 재조합 단백질을 농축하였으며, 3) 마지막으로 자가조립된 단백질 나노입자만을 분리하기 위하여 수크로스 구배 초고속 원심분리(sucrose gradient ultracentifugation)를 진행하였다. 각 단계별 상세한 기재는 아래와 같다.First, 1) and then proceed to the Ni 2+ -NTA affinity chromatography (Ni 2 + -NTA affinity chromatography) using a combination of a fused protein expressed in a recombinant histidine and nickel, and 2) to enhance the self-assembly efficiency of the recombinant protein For this purpose, using an Ultracentrifugal filter (Amicon Ultra 100K, Millipore, Billerica, MA), the aqueous solution containing the recombinant protein was changed to a buffer for self-assembly with a different composition (500mM NaCl .50mM Tris-HCl pH 7.0). 3) Finally, sucrose gradient ultracentifugation was performed to separate only the self-assembled protein nanoparticles. Detailed description of each step is as follows.
1) Ni2+-NTA 친화성 크로마토그래피1) Ni 2+ -NTA affinity chromatography
재조합 단백질을 정제하기 위하여 상기 명시된 동일한 방법으로 배양된 대장균을 회수하여 그 세포 펠렛을 5 mL 라이시스 버퍼(pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole)에 재부유하고 초음파 파쇄기를 이용하여 세포를 파쇄하였다. 파쇄된 세포액을 13,000 rpm에서 10분간 원심분리하여 그 상등액만 분리한 후 각 재조합 단백질을 Ni2 +-NTA 컬럼(Qiagen, Hilden, Germany)을 사용하여 각각 분리하였다(세척 버퍼: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 80 mM imidazole / 용출 버퍼: pH 8.0, 50 mM sodium phosphate, 300mM NaCl, 200 mM imidazole). To purify the recombinant protein, the cultured E. coli was recovered in the same manner as specified above, and the cell pellet was resuspended in 5 mL Lysis buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole), and an ultrasonic disruptor. Cells were disrupted. The disrupted cell solution was centrifuged at 13,000 rpm for 10 minutes to separate only the supernatant, and then each recombinant protein was separated using a Ni 2 + -NTA column (Qiagen, Hilden, Germany) (wash buffer: pH 8.0, 50 mM). sodium phosphate, 300 mM NaCl, 80 mM imidazole / elution buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 200 mM imidazole).
2) 자가조립 촉진을 위한 버퍼 변경 및 농축2) Buffer change and concentration to promote self-assembly
Ni2 +-NTA 친화성 크로마토그래피를 거쳐 용출된 3 mL의 재조합 단백질을 Ultracentrifugal filter(Amicon Ultra 100K, Millipore, Billerica, MA)에 담아 5,000 g로 10분간 원심분리한 후 컬럼 상부를 자가조립용 버퍼(500 mM NaCl, 50 mM Tris-HCl pH 7.0)로 가득 채운 후 컬럼 위에 500㎕의 용액이 남을 때까지 5,000 g로 원심분리를 진행하였다. 이를 3회 반복한 후 최종 부피를 1 mL로 맞춘 후 아래 단계를 진행하였다.3 mL of recombinant protein eluted through Ni 2 + -NTA affinity chromatography was put in an Ultracentrifugal filter (Amicon Ultra 100K, Millipore, Billerica, MA), centrifuged at 5,000 g for 10 minutes, and then the upper part of the column was buffered for self-assembly. After filling with (500 mM NaCl, 50 mM Tris-HCl pH 7.0), centrifugation was performed at 5,000 g until 500 µl of the solution remained on the column. After repeating this three times, the final volume was adjusted to 1 mL, and the following steps were performed.
3) 수크로스 구배 초고속 원심 분리 3) Sucrose gradient ultrafast centrifugation
자가조립용 버퍼에 수크로스를 농도별로 각각 첨가하여 60%, 50%, 40%, 30%, 20%, 10%의 수크로스를 포함하는 용액을 각각 준비한 후 초고속 원심 분리용 튜브(ultraclear 13.2 mL tube, Beckman)에 각 농도별(60~20%) 수크로스 용액을 농도가 높은 용액부터 2 mL씩 담았다. 마지막으로 10% 수크로스 용액을 0.5 mL 담은 후 상기 준비된 자가조립용 버퍼에 존재하는 재조합 단백질 용액을 1 mL 채운 후 24,000 rpm으로 12시간 동안 20 ℃로 초고속 원심분리를 시행하였다(Ultracentrifuge L-90k, Beckman). 원심분리 후 조심스럽게 피펫을 이용하여 위 층(10~30% 수크로스 용액 부분)을 없애주고 40~60% 수크로스 용액 부분을 2)에 명시된 바와 같이 ultracentrifugal filter와 자가조립용 버퍼를 이용하여 재조합 단백질의 버퍼를 교체해 주었다. After preparing a solution containing 60%, 50%, 40%, 30%, 20%, and 10% sucrose by adding sucrose to each concentration in the self-assembly buffer, an ultra-high-speed centrifugation tube (ultraclear 13.2 mL) tube, Beckman) each concentration (60-20%) of sucrose solution was added 2 mL each from the high concentration solution. Finally, after containing 0.5 mL of 10% sucrose solution, 1 mL of the recombinant protein solution present in the prepared self-assembly buffer was filled, and ultra-high-speed centrifugation was performed at 20° C. for 12 hours at 24,000 rpm (Ultracentrifuge L-90k, Beckman). After centrifugation, carefully remove the upper layer (10~30% sucrose solution part) with a pipette and recombinate the 40~60% sucrose solution part using an ultracentrifugal filter and self-assembly buffer as specified in 2). The protein buffer was replaced.
(HBV 캡시드 유래 키메릭 형광 나노입자의 구조 분석)(Structural analysis of chimeric fluorescent nanoparticles derived from HBV capsid)
상기 과정을 거친 후 정제된 재조합 단백질 나노입자의 구조 분석을 위하여 투과전자현미경(TEM)으로 재조합 단백질 나노입자를 촬영하였다. 먼저 염색하지 않은 정제한 단백질 샘플을 탄소 코팅된 구리 전자 현미경 그리드(grids)에 올린 후 자연 건조하였다. 단백질 나노입자의 염색된 이미지를 얻기 위해, 자연 건조된 샘플을 포함하는 전자 현미경 그리드를 2%(w/v) 수성 우라닐 아세테이트 용액과 함께 10분 동안 실온에서 인큐베이션하고, 증류수로 3-4회 세척하였다. After the above process, the recombinant protein nanoparticles were photographed with a transmission electron microscope (TEM) to analyze the structure of the purified recombinant protein nanoparticles. First, a purified protein sample that was not stained was placed on a carbon-coated copper electron microscope grid and then naturally dried. To obtain stained images of protein nanoparticles, electron microscopy grids containing naturally dried samples were incubated with 2% (w/v) aqueous uranyl acetate solution for 10 minutes at room temperature and 3-4 times with distilled water. Washed.
단백질 나노입자 이미지를 Philips Technai 120 kV 전자 현미경을 이용하여 관찰한 결과 형광단백질이 표면 표출된 30 내지 35 nm의 구형 나노입자가 형성된 것을 확인하였으며 그 결과는 도 3과 같다.As a result of observing the protein nanoparticle image using a Philips Technai 120 kV electron microscope, it was confirmed that spherical nanoparticles of 30 to 35 nm in which the fluorescent protein was expressed on the surface were formed, and the result is shown in FIG. 3.
< 실험예 1> HBV 캡시드 유래 키메릭 형광 나노입자와 페리틴 유래 키메릭 형광 나노입자의 형광 측정 < Experimental Example 1> HBV Capsid-derived chimeric fluorescent nanoparticles and ferritin-derived chimeric optical measurement of fluorescent nanoparticles
상기 실시예 1에서 제조한 4개의 HBV 캡시드 유래 키메릭 형광 나노입자: Four HBV capsid-derived chimeric fluorescent nanoparticles prepared in Example 1:
gFCNP(w/o linker) : N-HBVcAg(1-78)-His6-eGFP-HBVcAg(81-149)-C, gFCNP(w/o linker): N-HBVcAg(1-78)-His6-eGFP-HBVcAg(81-149)-C,
rFCNP(w/o linker) : N-HBVcAg(1-78)-His6-DsRed-HBVcAg(81-149)-C, rFCNP(w/o linker): N-HBVcAg(1-78)-His6-DsRed-HBVcAg(81-149)-C,
gFCNP(w/ linker): N-HBVcAg(1-78)-His6-linker-eGFP-linker-HBVcAg(81-149)-C, 및gFCNP(w/linker): N-HBVcAg(1-78)-His6-linker-eGFP-linker-HBVcAg(81-149)-C, and
rFCNP(w/ linker) : N-HBVcAg(1-78)-His6-linker-DsRed-linker-HBVcAg(81-149)-CrFCNP(w/ linker): N-HBVcAg(1-78)-His6-linker-DsRed-linker-HBVcAg(81-149)-C
기존에 본 발명자가 출원한 출원번호 US13/487,921, KR2011-0128615에서 제조한 4개의 페리틴 유래 키메릭 형광 나노입자:Four ferritin-derived chimeric fluorescent nanoparticles prepared in application number US13/487,921, KR2011-0128615 previously filed by the present inventors:
gFFNP(w/o linker) : N-Ferritin-eGFP-H6-C, gFFNP(w/o linker): N-Ferritin-eGFP-H6-C,
rFFNP(w/o linker) : N-Ferritin-DsRed-H6-C, rFFNP(w/o linker): N-Ferritin-DsRed-H6-C,
gFFNP(w/ linker) : N-Ferritin-linker-eGFP-H6-C, 및gFFNP (w/ linker): N-Ferritin-linker-eGFP-H6-C, and
rFFNP(w/ linker) : N-Ferritin-linker-DsRed-H6-CrFFNP(w/ linker): N-Ferritin-linker-DsRed-H6-C
의 형광 세기를 측정 및 나노입자당 형광 세기를 비교하기 위하여 eGFP 단량체, DsRed 단량체, 페리틴 유래 키메릭 형광 나노입자, 캡시드 유래 키메릭 형광 나노입자를 각각 준비하여 블랙 96-웰 플레이트(black 96-well plate(Nunc, Roskide, Denmark))에 1 pmole(단백질 나노입자 기준) 씩 담아 형광을 측정하였다. eGFP 단량체 및 eGFP 융합 단백질 나노입자의 형광 측정은 λex = 485 nm/λem = 535 nm로, DsRed 단량체 및 DsRed 융합 단백질 나노입자의 형광 측정은 λex = 550 nm/λem = 590 nm에서 GENios (Tecan, Austria)을 이용하여 각각 3번씩 측정하였다. To measure the fluorescence intensity of and to compare the fluorescence intensity per nanoparticle, eGFP monomer, DsRed monomer, ferritin-derived chimeric fluorescent nanoparticles, and capsid-derived chimeric fluorescent nanoparticles were prepared, respectively, and prepared in a black 96-well plate (black 96-well plate). plate (Nunc, Roskide, Denmark)), each 1 pmole (based on protein nanoparticles) was measured for fluorescence. Fluorescence measurement of eGFP monomer and eGFP fusion protein nanoparticles was λex = 485 nm/λem = 535 nm, and fluorescence measurement of DsRed monomer and DsRed fusion protein nanoparticles was performed by GENios (Tecan, Austria) at λex = 550 nm/λem = 590 nm. ) Was measured three times each.
도 4에 나타난 바와 같이, 단량체나 페리틴 융합 형광단백질에 비해 HBVcAg 캡시드 융합 형광단백질이 단백질 나노입자당 표출된 형광단백질의 수의 증대로 인해 높은 형광 세기를 나타낸 것을 확인하였다. 또한 링커가 삽입된 경우 형광 세기가 증가하였는데, 이는 링커를 삽입함으로써 단백질 나노입자 표면에 고밀도로 표출된 형광 단백질 간의 ?칭(quenching) 현상이 해소되었기 때문이다. 링커를 삽입하지 않았을 때에도 형광단백질 단량체에 비해 현저히 증폭된 형광강도를 보이는 것을 확인하였으며, 링커를 삽입함으로써 최적의 형광 단백질 나노입자를 제조하였다. As shown in FIG. 4, it was confirmed that the HBVcAg capsid fusion fluorescent protein exhibited high fluorescence intensity due to an increase in the number of fluorescent proteins expressed per protein nanoparticle compared to the monomer or ferritin fused fluorescent protein. In addition, the fluorescence intensity increased when the linker was inserted, because the quenching phenomenon between the fluorescent proteins expressed at high density on the protein nanoparticle surface was eliminated by the insertion of the linker. Even when the linker was not inserted, it was confirmed that the fluorescence intensity was significantly amplified compared to that of the fluorescent protein monomer, and the optimal fluorescent protein nanoparticles were prepared by inserting the linker.
< 실험예 2> 인 비트로에서 HBV 캡시드 유래 키메릭 형광 나노입자의 안정성 검증 < Experimental Example 2> In vitro HBV Stability verification of capsid- derived chimeric fluorescent nanoparticles
상기 실시예 1에서 제조한 형광 단백질 나노입자(gFCNP, rFCNP w/ linker)의 구조적, 기능적 안정성 유지 여부를 확인하기 위하여 eGFP, DsRed 단량체와 비교 실험을 진행하였다. eGFP, DsRed, gFCNP, rFCNP를 각각 1 nM을 최종 부피가 100 ㎕이 되도록 PBS에 준비하여 37 ℃에서 30분 간격으로 3.5시간 동안 형광 강도를 측정, 비교하였다.In order to confirm the structural and functional stability of the fluorescent protein nanoparticles (gFCNP, rFCNP w/ linker) prepared in Example 1, a comparative experiment was conducted with eGFP and DsRed monomers. Each 1 nM of eGFP, DsRed, gFCNP, and rFCNP was prepared in PBS so that the final volume was 100 μl, and fluorescence intensity was measured and compared at 37° C. for 3.5 hours at 30 minute intervals.
또한 동일한 형광단백질을 5 nM이 되도록 30%, 50% 의 인간 혈청(Cat. No. H4522, Sigma-Aldrich) 100 ㎕에 혼합하여 30분 간격으로 상기 실험예 1의 방법대로 형광 세기 변화를 측정하였다. In addition, the same fluorescent protein was mixed with 100 µl of 30% and 50% human serum (Cat. No. H4522, Sigma-Aldrich) to be 5 nM, and the change in fluorescence intensity was measured according to the method of Experimental Example 1 at intervals of 30 minutes. .
도 5에 나타난 바와 같이, 형광단백질 단량체는 3.5시간 이후 초기 형광값의 20% 이하로 형광 세기가 감소했지만, 형광 단백질 나노입자의 경우 시간이 지나도 초기 형광값의 80% 이상이 유지되는 것을 확인하였다. As shown in FIG. 5, it was confirmed that the fluorescence intensity of the fluorescent protein monomer decreased to 20% or less of the initial fluorescence value after 3.5 hours, but in the case of the fluorescent protein nanoparticles, 80% or more of the initial fluorescence value was maintained over time. .
또한, 도 6에 나타난 바와 같이, 형광단백질 단량체는 혈청에 혼합 후 1시간 이내에 초기 형광의 절반 이하로 형광 세기가 감소하였으나, 형광 단백질 나노입자는 5시간 동안 60% 이상의 형광 세기를 유지하는 것을 확인하였다. In addition, as shown in FIG. 6, the fluorescence intensity of the fluorescent protein monomer decreased to less than half of the initial fluorescence within 1 hour after mixing with the serum, but it was confirmed that the fluorescent protein nanoparticles maintained a fluorescence intensity of 60% or more for 5 hours. I did.
이는 형광단백질 단량체의 경우 혈청 내에서 안정성이 낮으므로 생체 진단에 적용하기에 제약이 있으나 형광단백질을 단백질 나노입자에 융합 발현할 경우, 안정성이 향상되어 생체 진단에 적용 가능함을 보여준다.This shows that fluorescent protein monomers have low stability in serum and thus have limitations in applying them to biodiagnostics, but when fluorescent proteins are fused to protein nanoparticles, the stability is improved and thus can be applied to biodiagnostics.
또한 형광물질을 생체 진단에 이용하는데 제한점인 광원 노출에 대한 광안정성(photostability)을 비교하였다. 형광단백질이나 형광염료 등은 반복된 광원에 노출될 경우 광퇴색(photobleaching)에 의해 형광기능을 소실하는 단점이 있다. 형광물질을 생체 진단에 활용할 경우 시간별로, 조직별로 반복해서 형광 신호를 검출하므로 광원 노출에 대한 안정성이 보장되어야만 생체 진단에 적용가능하다. In addition, photostability against exposure to a light source, which is a limitation in using fluorescent materials for biodiagnosis, was compared. Fluorescent proteins or fluorescent dyes have a disadvantage of losing their fluorescence function due to photobleaching when exposed to repeated light sources. When a fluorescent material is used for biometric diagnosis, it is applicable to biometric diagnosis only when stability against exposure to a light source is ensured since the fluorescence signal is repeatedly detected by time and by tissue.
생명공학분야에서 널리 사용되고 있는 형광 물질인 FAM과 광퇴색에 대해 안정성이 뛰어나다고 알려져 있는 퀀텀닷(FAM, Genotech, Quantum dot(Qdot655/CdSe, invitrogen)을 대조군으로 하여 형광단백질 단량체와 형광 단백질 나노입자의 초기 형광 세기를 45,000 RFU (Relatuve fluorescnce unit)가 되도록 조정 후 각 시료를 10 ㎕씩 슬라이드 글라스 위에 떨어뜨린 다음, 15분간 30초 간격으로 형광 현미경 (Olympus BX51)으로 관측하여 형광 변화를 비교하였다.Quantum dot (FAM, Genotech, Quantum dot (Qdot655/CdSe, invitrogen)), which is known to have excellent stability against FAM, a fluorescent substance widely used in the biotechnology field, as a control, and fluorescent protein monomer and fluorescent protein nanoparticles After adjusting the initial fluorescence intensity of 45,000 RFU (Relatuve fluorescnce unit), each sample was dropped on the slide glass by 10 μl, and then observed with a fluorescence microscope (Olympus BX51) at 30 second intervals for 15 minutes to compare the fluorescence change.
그 결과 FAM을 비롯하여 형광단백질 단량체의 경우 광원에 노출되는 횟수가 증가할수록 형광 강도가 급격히 감소하지만, 형광 단백질 나노입자의 경우 퀀텀닷과 유사하게 형광 세기가 유지되는 것을 확인하였다(도 7). As a result, in the case of the fluorescent protein monomer including FAM, the fluorescence intensity rapidly decreased as the number of exposures to the light source increased, but in the case of the fluorescent protein nanoparticles, it was confirmed that the fluorescence intensity was maintained similarly to the quantum dot (FIG. 7).
이는 형광단백질의 아미노 말단과 카르복실 말단이 단백질 나노입자(capsid)의 스파이크 부위에 융합됨으로써 형광단백질의 베타-베럴(beta-barrel) 구조가 온전히 유지되기 때문에 형광단백질의 형광단(fluorophore)이 광원에 노출되는 것을 방지하기 때문이다.This is because the beta-barrel structure of the fluorescent protein is maintained intact by fusion of the amino and carboxyl ends of the fluorescent protein to the spike site of the protein nanoparticle (capsid), so that the fluorophore of the fluorescent protein is light source. This is because it prevents exposure to.
< 실험예 3> 페리틴 유래 키메릭 형광 나노입자, HBV 캡시드 유래 키메릭 형광 나노입자의 생체 안정성 검증 < Experimental Example 3> Ferritin-derived chimeric fluorescent nanoparticles, HBV Capsid stability in vivo verification of the resulting chimeric optical nanoparticles
형광 단백질 나노입자를 생체 진단에 적용하기 위해서는 혈청 내에서 구조적, 기능적으로 안정성을 유지해야 한다. 이를 확인하기 위하여 형광단백질 단량체와 형광 단백질 나노입자를 30%, 50% 혈청에 희석한 후 시간별로 형광 세기의 변화를 비교하였다. 이를 위해, 형광단백질 단량체와 형광 단백질 나노입자를 초기 형광 세기가 40,000 RFU가 되도록 조정하여 20 ㎕를 각각 BALB/cSlc-nude mice(female, 4wnfud, JAPAN SLC, Inc) 3 마리의 복부에 피하주사한 후 6시간 동안 피하에 남아 있는 물질의 형광 세기를 비교하였다. 복부 형광 이미지는 KODAK 12 bit CCD camera를 이용해 얻었으며 Excitation은 523-635 nm, Emission은 560-675 nm 형광 필터를 사용하였으며 최종적으로 550 nm/590nm에서 이미지를 촬영하였다. In order to apply fluorescent protein nanoparticles to biodiagnosis, it is necessary to maintain structural and functional stability in serum. To confirm this, fluorescent protein monomers and fluorescent protein nanoparticles were diluted in 30% and 50% serum, and then the changes in fluorescence intensity were compared over time. To this end, the fluorescent protein monomer and the fluorescent protein nanoparticles were adjusted so that the initial fluorescence intensity was 40,000 RFU, and 20 µl was injected subcutaneously into the abdomen of three BALB/cSlc-nude mice (female, 4wnfud, JAPAN SLC, Inc) respectively. After 6 hours, the fluorescence intensity of the substance remaining under the skin was compared. Abdominal fluorescence images were obtained using a
도 8에 나타난 바와 같이, 형광단백질 단량체는 주사 후 1.5 시간 이후 모두 형광을 소실한 반면 형광 단백질 나노입자는 6 시간 이후에도 형광을 나타내는 것을 확인하였다. As shown in FIG. 8, it was confirmed that the fluorescent protein monomers all disappeared after 1.5 hours after injection, whereas the fluorescent protein nanoparticles showed fluorescence even after 6 hours.
이는 위에서 확인한 바와 같이 형광 단백질 나노입자는 단량체에 비해 온도, 생체 조건, 광원에 대한 구조적 안정성 유지가 가능하기 때문이다. 이는 형광 단백질 나노입자의 생체 이미징 또는 약물 전달 소재로 적합함을 입증하는 자료이다.This is because, as confirmed above, fluorescent protein nanoparticles are capable of maintaining structural stability against temperature, in vivo conditions, and light sources compared to monomers. This is data that proves suitable as a material for in vivo imaging or drug delivery of fluorescent protein nanoparticles.
< 실시예 2> 생체 진단용 표적 지향성 펩타이드 표출 형광 단백질 나노입자의 제조 < Example 2> Preparation of fluorescent protein nanoparticles expressing target-oriented peptide for biodiagnosis
형광 단백질 나노입자의 인 비보 표적 타겟팅이 가능한지 검증하기 위하여 암세포 표면의 인테그린(integrin)과 특이적으로 결합한다고 알려진 RGD 펩타이드를 표출하는 형광 단백질 나노입자를 제조하였다.In order to verify whether fluorescent protein nanoparticles can be targeted in vivo, fluorescent protein nanoparticles expressing RGD peptides known to specifically bind to integrins on the surface of cancer cells were prepared.
아미노 말단에 표적 지향성 펩타이드(GRGDSG3SG3SG3SG)을 포함하는 형광 단백질 나노입자를 제조하기 위하여 출원번호 US13/487,921에 기재된 클론 6을 주형으로 프라이머 서열 16 및 17을 이용하여 PCR을 시행하고 합성된 PCR 산물을 주형으로 프라이머 서열 19, 17을 이용하여 확장 PCR을 수행하였다. 마찬가지로 실시예 1에서 제조한 N-HBVcAg(1-78)-His6-linker-DsRed-linker-HBVcAg(81-149)-C를 주형으로 프라이머 18, 4를 이용하여 PCR을 시행 후 합성된 PCR 산물을 주형으로 다시 프라이머 19, 4를 이용하여 확장 PCT을 수행하였다. In order to prepare fluorescent protein nanoparticles containing a target-directed peptide (G RGD SG3SG3SG3SG) at the amino terminal, PCR was performed using the
그 결과 5‘-NdeI-GRGDSG3SG3SG3SG-FTH-linker-DsRed-H6-HindIII-3’과 5‘-NdeI-GRGDSG3SG3SG3SG-HBVcAg(1-79)-linker-DsRed-linker-HBVcAg(81-149)-HindIII-3’로 이루어진 PCR 산물을 확보하였으며 이를 pT7-7 벡터에 삽입하여 도 9와 같이 페리틴 기반 적색 형광 나노입자의 아미노 말단, 캡시드 기반 적색 형광 나노입자의 아미노 말단에 RGD 펩타이드를 표출하는 나노입자 제조를 위한 발현 벡터를 제조하였다. 이를 실시예 1에 명시한 대로 발현, 정제하여 인 비보 이미징에 적용하기 위한 형광 단백질 나노입자를 확보하였다.As a result, 5'-NdeI-G RGD SG3SG3SG3SG-FTH-linker-DsRed-H6-HindIII-3' and 5'-NdeI-G RGD SG3SG3SG3SG-HBVcAg(1-79)-linker-DsRed-linker-HBVcAg(81- A PCR product consisting of 149)-HindIII-3' was obtained and inserted into the pT7-7 vector to express RGD peptide at the amino terminal of ferritin-based red fluorescent nanoparticles and the amino terminal of capsid-based red fluorescent nanoparticles as shown in FIG. 9 An expression vector for preparing the nanoparticles was prepared. This was expressed and purified as specified in Example 1 to obtain fluorescent protein nanoparticles for in vivo imaging.
< 실험예 4> 표적 지향성 펩타이드 표출 형광 단백질 나노입자를 이용한 생체 진단 가능성 검증 < Experimental Example 4> Verification of biodiagnosticability using fluorescent protein nanoparticles expressing target-oriented peptide
실시예 2에서 제조한 표적 지향성 펩타이드(RGD)를 표출하는 형광 단백질 나노입자가 실제 인 비보 이미징에 적합한지 검증하기 위하여 RGD가 결합한다고 알려진 인테그린 표출 암 세포를 선정하여 암 모델을 준비하였다. In order to verify whether the fluorescent protein nanoparticles expressing the target-oriented peptide (RGD) prepared in Example 2 are suitable for actual in vivo imaging, integrin-expressing cancer cells known to bind to RGD were selected to prepare a cancer model.
먼저 암세포에 표적 지향성 펩타이드 융합 형광단백질이 선택적으로 업테이크 되는지 확인하기 위하여 6-웰 플레이트에 인테그린 발현 빈도가 높은 U87MG 암세포를 한 웰당 1×105 개를 부착시킨 후 각 1 μM의 rFFNP, RGD-rFFNP를 37℃, CO2 인큐베이터에서 1시간 동안 반응시킨 후 형광현미경을 이용하여 세포내 형광을 검경하였다. 인 비보 이미징을 위해서는 U87MG 암세포를 이식한 암모델에 각 형광 단백질 나노입자를 정맥 주사하여 IVIS 이미지 분석장비를 이용하여 형광 필터 550 nm/590nm 를 이용하여 암 부위의 형광을 측정하였다. First, 1×10 5 U87MG cancer cells with high integrin expression frequency per well were attached to a 6-well plate in order to check whether the target-oriented peptide fusion fluorescent protein is selectively uptaken to cancer cells, and then 1 μM of rFFNP and RGD- After rFFNP was reacted for 1 hour in a CO 2 incubator at 37° C., intracellular fluorescence was examined using a fluorescence microscope. For in vivo imaging, each fluorescent protein nanoparticle was intravenously injected into a cancer model implanted with U87MG cancer cells, and fluorescence of the cancer site was measured using a fluorescent filter 550 nm/590 nm using an IVIS image analysis device.
도 10에 나타난 바와 같이, RGD가 융합발현된 형광 단백질 나노입자가 암세포로 업테이크 된 것을 확인하였다. 이는 RGD 펩타이드가 형광 단백질 나노입자의 표면에 효과적으로 표출되어 암세포에 특이적으로 업테이크 되는 것을 도운 것으로 생각된다. As shown in Figure 10, it was confirmed that the fluorescent protein nanoparticles fusion-expressed RGD were uptaken into cancer cells. This is thought to have helped the RGD peptide to be effectively expressed on the surface of the fluorescent protein nanoparticles and specifically uptake in cancer cells.
이후 실제 인 비보 이미징에 적용하기 위하여 인테그린 수용체 발현 빈도가 높은 U87MG 암세포를 각각 준비하여 누드 마우스에 주사 후 암을 신장 시킨 동물 모델에 형광 단백질 나노입자를 정맥 주사하였다.Afterwards, U87MG cancer cells with high integrin receptor expression frequencies were prepared for application to actual in vivo imaging, and then injected into nude mice, and then fluorescent protein nanoparticles were intravenously injected into an animal model with cancer growth.
먼저 RGD가 융합된 적색형광단백질 단량체(RGD-rFFNP), RGD가 없는 페리틴 기반 적색 형광 나노입자, RGD가 융합발현된 페리틴 기반 적색 형광 나노입자(rFFNP)를 동일한 형광값을 보이도록 농도를 맞춰 정맥 주사 후 암에서의 형광단백질의 축적량을 비교하였다. First, RGD-fused red fluorescent protein monomer (RGD-rFFNP), ferritin-based red fluorescent nanoparticles without RGD, and ferritin-based red fluorescent nanoparticles (rFFNP) with RGD fusion-expressed in a vein by adjusting the concentration to show the same fluorescence value. After injection, the amount of fluorescence protein accumulated in cancer was compared.
도 11에 나타난 바와 같이, 2시간 이후에도 RGD-rFFNP에서 암에서 형광이 측정되었고, RGD가 없는 rFFNP 또한 초기 시간에 암에서 형광이 측정되나 RGD-rFFNP에 비해 낮은 형광 세기를 보이는 것을 확인하였다.As shown in FIG. 11, it was confirmed that fluorescence was measured in the dark in RGD-rFFNP even after 2 hours, and the fluorescence was also measured in the dark in the initial time for rFFNP without RGD, but it was confirmed that the fluorescence intensity was lower than that of RGD-rFFNP.
또한 형광 재조합 단백질의 체내 분포를 분석하기 위하여 인테그린 수용체 발현 빈도가 낮다고 알려진 SCC7 암세포와 인테그린 수용체 발현 빈도가 높다고 알려진 U87MG 암세포를 준비하여 누드 마우스에 이식한 후 종양이 5-8 mm 크기가 되도록 성장시킨 후 RGD-DsRed, rFFNP, RGD-rFFNP를 동일한 형광값을 보이도록 준비하여 정맥 주사한 후 24 시간 후에 조직을 절개하여 12-bit CCD 카메라로 조직의 형광 세기를 비교하였다.In addition, in order to analyze the distribution of fluorescent recombinant proteins in the body, SCC7 cancer cells known to have a low integrin receptor expression frequency and U87MG cancer cells known to have a high integrin receptor expression frequency were prepared and transplanted into nude mice, and the tumors were grown to a size of 5-8 mm. Thereafter, RGD-DsRed, rFFNP, and RGD-rFFNP were prepared to show the same fluorescence values, and after intravenous injection, the tissue was dissected 24 hours later, and the fluorescence intensity of the tissue was compared with a 12-bit CCD camera.
도 12에 나타난 바와 같이, SCC7, U87MG 암 모델 모두 형광단백질 단량체는 RGD 펩타이드가 있음에도 신장을 통해 체내에서 제거되는 것을 확인하였으며, 페리틴 기반 형광 나노입자는 상대적으로 간에 주로 축적됨을 확인하였다. 이는 단백질 나노입자 기반의 형광 나노입자는 단량체에 비해 구조적 안정성이 향상되고, 체내 순환률이 향상됨을 보여주며, 형광단백질 단량체에 비해 인 비보 이미징에 적합한 소재임을 보여준다. 또한 페리틴 기반 형광 나노입자에 RGD를 융합발현 한 경우 RGD 펩타이드가 표출되지 않은 형광 나노입자에 비해 암세포에 축적되는 비율이 높아짐을 확인하였다. 이는 암세포 특이적 펩타이드를 표출한 형광 단백질 나노입자가 암세포를 특이적으로 타겟팅하며, 동시에 높은 형광 세기를 통해 이미징이 가능함을 보여준다.As shown in FIG. 12, it was confirmed that the fluorescent protein monomer was removed from the body through the kidney even in the presence of the RGD peptide in both SCC7 and U87MG cancer models, and it was confirmed that ferritin-based fluorescent nanoparticles were relatively mainly accumulated in the liver. This shows that the protein nanoparticle-based fluorescent nanoparticles have improved structural stability and improved circulation rate in the body compared to the monomers, and show that they are suitable materials for in vivo imaging compared to the fluorescent protein monomers. In addition, it was confirmed that when RGD was fused to ferritin-based fluorescent nanoparticles, the rate of accumulation in cancer cells increased compared to fluorescent nanoparticles in which RGD peptide was not expressed. This shows that fluorescent protein nanoparticles expressing cancer cell-specific peptides specifically target cancer cells, and at the same time, imaging is possible through high fluorescence intensity.
< 실시예 3> eDPS 및 tPTS 기반 표적 지향성 펩타이드 표출 형광 나노입자의 제조 < Example 3> Preparation of fluorescent nanoparticles expressing target-oriented peptides based on eDPS and tPTS
(eDPS 및 tPTS 기반 형광 단백질 나노입자의 생합성을 위한 발현 벡터의 제조)(Preparation of expression vector for biosynthesis of fluorescent protein nanoparticles based on eDPS and tPTS)
표적 지향성 펩타이드를 융합 발현하지 않은 크기와 형태가 다른 형광 단백질 나노입자를 제작하여 암 타겟팅이 가능한지, 가능하다면 효율이 어떻게 달라지는지 비교하였다. 또한 상기 단백질 나노입자에 표적 펩타이드를 융합 발현할 경우 암 타겟팅 효율이 향상되는지를 검증하기 위한 실험을 진행하였다.Fluorescent protein nanoparticles of different sizes and shapes without fusion expression of the target-directed peptide were fabricated to compare whether cancer targeting was possible and, if possible, how the efficiency was changed. In addition, an experiment was conducted to verify whether cancer targeting efficiency is improved when the target peptide is fused to the protein nanoparticle.
이를 위해 크기와 형태가 다른 eDPS와 tPTS 나노입자를 생산하기 위한 발현 벡터를 제작하였다. 구체적으로 설명하면, 하기 표 2에 기재된 프라이머를 사용한 PCR 후에, 대장균 유전자로부터 유래한 N-NdeI-His6-eDPS-HindIII-C 및 N-NdeI-RGD-eDPS-His6-HindIII-C의 합성을 코드하는 두 유전자 클론을 얻었다. 써모플라즘 애시도필룸(Thermoplasm acidophilum) 유전자로부터 유래한 N-NdeI-tPTSα-HindIII-C, NdeI-tPTSα-RGD-HindIII-C 및 N-NdeI-tPTSβ-His6-HindIII-C의 합성을 코드하는 세 개의 유전자 클론을 얻었다. 플라즈미드 pT7-7에 상기 유전자 클론의 라이게이션을 통하여, N-eDPS-His6-C, N-RGD-eDPS-His6 -C, N-tPTSβ-His6-C 합성을 코딩하는 3개의 플라즈미드 발현 벡터를 구축하였다. 또한 pET28a에 상기 유전자 클론의 라이게이션을 통하여 N-tPTSα-C, N-tPTSα-RGD-C 합성을 코딩하는 3개의 플라즈미드 발현 벡터를 구축하였다(도 13A). 제작된 모든 플라즈미드 발현 벡터는 젤-종제 후 완전한 DNA 시퀀싱을 통해 서열을 확인하였다. 상기 단백질 나노입자의 제조와 관련된 프라이머 서열(표 2)과 주형에 대한 정보 및 이에 대한 좀 더 상세한 기재는 아래와 같다.For this, an expression vector for producing eDPS and tPTS nanoparticles of different sizes and shapes was constructed. Specifically, after PCR using the primers shown in Table 2 below, the synthesis of N-NdeI-His6-eDPS-HindIII-C and N-NdeI-RGD-eDPS-His6-HindIII-C derived from E. coli genes is coded. Two gene clones were obtained. Thermoplasmic Acidophilum acidophilum ) three gene clones encoding the synthesis of N-NdeI-tPTSα-HindIII-C, NdeI-tPTSα-RGD-HindIII-C and N-NdeI-tPTSβ-His6-HindIII-C derived from the gene were obtained. Three plasmid expression vectors encoding the synthesis of N-eDPS-His6-C, N-RGD-eDPS-His6 -C, and N-tPTSβ-His6-C were constructed through ligation of the gene clone to plasmid pT7-7. I did. In addition, three plasmid expression vectors encoding the synthesis of N-tPTSα-C and N-tPTSα-RGD-C were constructed through ligation of the gene clone to pET28a (Fig. 13A). All of the prepared plasmid expression vectors were gel-finished and sequenced through complete DNA sequencing. Information on the primer sequence (Table 2) and the template related to the preparation of the protein nanoparticle, and a more detailed description thereof are as follows.
번호order
number
(eDPS 및 tPTS 기반 표적 지향성 펩타이드 표출 형광 단백질 나노입자의 생합성, 정제 및 형광입자 제조)(Biosynthesis and purification of fluorescent protein nanoparticles expressing target-oriented peptides based on eDPS and tPTS, and production of fluorescent particles)
eDPS, RGD-eDPS, tPTS 및 RGD-tPTS 나노입자의 생합성은 상기 실시예 1과 동일한 방법으로 수행하였다. 다만 tPTS와 RGD-tPTS 나노입자는 알파단백질과 베타 단백질을 대장균에서 동시에 발현하기 위하여 상기에서 제작한 pT7 N-tPTSβ-His6-C와 pET28a N-tPTSα-C(또는 pET28a N-tPTSα-RGD-C)를 형질전환하여 암피실린과 카나마이신을 모두 포함하는 배지에서 배양하였다. 각 단백질 나노입자의 정제는 상기 실시예 1과 동일한 방법으로 수행하였다. 수크로스 원심분리 후 정제한 단백질 나노입자의 버퍼를 ultracentrifugal filter를 이용하여 0.1 M 소듐 바이카보네이트(pH 8.5)로 바꿔준 후 단백질 나노입자 몰 농도의 4배에 해당하는 형광다이 Cy5.5를 섞어주고 상온에서 12시간 교반하여 반응한 후 다시 한 번 수크로스 초고속 원심분리를 통해 free-Cy5.5와 Cy5.5 가 결합된 단백질 나노입자를 분리하였다. 최종 제작된 형광 단백질 나노입자의 크기와 구조를 TEM을 통해 확인하였다.Biosynthesis of eDPS, RGD-eDPS, tPTS and RGD-tPTS nanoparticles was performed in the same manner as in Example 1. However, tPTS and RGD-tPTS nanoparticles are pT7 N-tPTSβ-His6-C and pET28a N-tPTSα-C (or pET28a N-tPTSα-RGD-C) prepared above to simultaneously express alpha and beta proteins in E. coli. ) Was transformed and cultured in a medium containing both ampicillin and kanamycin. Purification of each protein nanoparticle was performed in the same manner as in Example 1. After sucrose centrifugation, the buffer of the purified protein nanoparticles was changed to 0.1 M sodium bicarbonate (pH 8.5) using an ultracentrifugal filter, and then a fluorescent die corresponding to 4 times the molar concentration of the protein nanoparticles was mixed with Cy5.5. After the reaction was stirred at room temperature for 12 hours, the protein nanoparticles to which free-Cy5.5 and Cy5.5 were bound were separated again through ultrahigh-speed centrifugation with sucrose. The size and structure of the finally produced fluorescent protein nanoparticles were confirmed through TEM.
도 13B에 나타난 바와 같이, 각 형광 단백질 나노입자는 대략 9-18 nm 정도의 크기를 나타내었다.As shown in FIG. 13B, each fluorescent protein nanoparticle exhibited a size of approximately 9-18 nm.
< 실험예 5> eDPS 및 tPTS 기반 표적 지향성 펩타이드 표출 형광 단백질 나노입자를 이용한 생체 진단 가능성 검증 < Experimental Example 5> Verification of biodiagnosticability using eDPS and tPTS-based target-oriented peptide- expressing fluorescent protein nanoparticles
1 μM의 eDPS, RGD-eDPS, tPTS 및 RGD-tPTS 기반 형광 단백질 나노입자를 실험예 4와 같이 준비한 U87MG 암세포 이식 동물 모델에 정맥 주사하여 eXplore Optix System (Near-infrared fluorescence(NIRF) emission(600e700 nm)을 이용하여 시간별로 형광을 측정하였다. 또한 암에서 나타나는 형광 세기를 time-correlated single photon counting system을 이용하여 정량화한 후 그래프로 나타내어 암 타겟팅 효율을 비교하였다. 1 μM of eDPS, RGD-eDPS, tPTS, and RGD-tPTS-based fluorescent protein nanoparticles were intravenously injected into a U87MG cancer cell transplant animal model prepared as in Experimental Example 4 and eXplore Optix System (Near-infrared fluorescence (NIRF) emission (600e700 nm). Fluorescence was measured by time using) In addition, the fluorescence intensity in cancer was quantified using a time-correlated single photon counting system and then displayed as a graph to compare cancer targeting efficiency.
도 14A에 나타난 바와 같이, eDPS 나노입자는 간에 주로 축적되는 반면 tPTS 나노입자는 장에서 주로 관측되었으며 eDPS 나노입자 보다 tPTS 나노입자가 암 표적 펩타이드가 없음에도 암에 타겟팅이 더 잘 되는 것을 확인하였다. As shown in Fig. 14A, eDPS nanoparticles mainly accumulate in the liver, whereas tPTS nanoparticles are mainly observed in the intestine, and it was confirmed that tPTS nanoparticles were better targeted to cancer than eDPS nanoparticles even in the absence of a cancer targeting peptide.
또한 eDPS, tPTS 모두 단백질 나노입자 표면에 암 표적 펩타이드가 표출되었을 때 암 타겟팅 효율이 향상됨을 확인하였다(도 14B). 이는 단백질이라는 동일한 소재로 이루어진 단백질 나노입자라 할지라도 그 크기와 형태에 따라 생체 적용시 체내 분포뿐 아니라 특정 조직에 대한 타겟팅 효율이 달라짐을 보여준다. In addition, it was confirmed that the cancer targeting efficiency was improved when the cancer targeting peptide was expressed on the surface of the protein nanoparticles in both eDPS and tPTS (FIG. 14B). This shows that even protein nanoparticles made of the same material, such as proteins, have different targeting efficiency for specific tissues as well as distribution in the body when applied to a living body according to their size and shape.
< 실시예 4> 구조와 형태가 다른 형광 단백질 나노입자를 이용한 감시림프절 이미징 < Example 4> Surveillance lymph nodes using fluorescent protein nanoparticles having different structures and morphologies Imaging
크기와 형태가 다른 형광단백질 나노입자를 제조하여 기존 감시림프절(Sentinel lymphnode) 이미징에 적용하였다. Fluorescent protein nanoparticles of different sizes and shapes were prepared and applied to conventional sentinel lymphnode imaging.
이를 위해, 상기 실시예 3에서 제작한 eDPS와 tPTS 형광 단백질 나노입자와, 추가로 FTN-h, HBVcAg 기반 형광 단백질 나노입자를 제작하였다. 하기 표 3에 기재된 프라이머를 사용한 PCR 후에 N-NdeI-His6-FTN-h-HindIII-C 및 N-NdeI-His6-HBVcAg-HindIII-C의 합성을 코드하는 두 유전자 클론을 얻었다. 플라즈미드 pT7-7에 상기 유전자 클론의 라이게이션을 통하여, N-His6-FTN-h-C, N-His6-HBVcAg-C 2개의 플라즈미드 발현 벡터를 구축하였다. 또한 FTN-h, HBVcAg 기반 형광 단백질 나노입자를 제작하는 방법은 상기 실시예 3과 같다. 최종적으로 정제, 제작한 형광 단백질 나노입자의 형광량을 비교하여 동일한 형광 세기를 보이는 농도를 설정하고 tPTS 형광단백질 나노입자 1 μM의 형광 세기와 동일한 형광 세기를 보이는 농도를 준비하여 정상 누드 마우스의 앞발에 20 ㎕씩 피하주사하였다. 10분 간격으로 KODAK imaging station(12-bit-CCD camera) 을 이용하여 형광을 측정하였다.To this end, eDPS and tPTS fluorescent protein nanoparticles prepared in Example 3, and additionally FTN-h and HBVcAg-based fluorescent protein nanoparticles were prepared. After PCR using the primers shown in Table 3 below, two gene clones encoding the synthesis of N-NdeI-His6-FTN-h-HindIII-C and N-NdeI-His6-HBVcAg-HindIII-C were obtained. Two plasmid expression vectors N-His6-FTN-h-C and N-His6-HBVcAg-C were constructed by ligation of the gene clone to plasmid pT7-7. In addition, a method of preparing FTN-h and HBVcAg-based fluorescent protein nanoparticles is the same as in Example 3. Finally, compare the fluorescence amount of the purified and produced fluorescent protein nanoparticles to set the concentration showing the same fluorescence intensity, and prepare a concentration showing the same fluorescence intensity as that of 1 μM of tPTS fluorescent protein nanoparticles. 20 µl of each was injected subcutaneously. Fluorescence was measured at 10-minute intervals using a KODAK imaging station (12-bit-CCD camera).
번호order
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도 15에 나타난 바와 같이, 크기가 다른 4개의 형광단백질 나노입자를 제조하였으며, 이를 동물 모델의 앞발에 주사하였을 때 주사한 부위로부터 가장 가까운 림프절에 형광 단백질 나노입자가 축적되는 것을 확인하였다. 주사한 즉시 감시림프절에서 표지 신호가 특정될 뿐 아니라 1시간 이상으로 표지 신호가 유지되며 다른 림프절로 확산되지 않는 것을 통해, 형광 단백질 나노입자가 감시림프절을 타겟팅하는데 매우 효과적임을 검증하였다(도 16).As shown in FIG. 15, four fluorescent protein nanoparticles having different sizes were prepared, and when injected into the forefoot of an animal model, it was confirmed that fluorescent protein nanoparticles were accumulated in the lymph nodes closest to the injection site. Immediately after injection, not only the labeling signal was specified in the sentinel lymph node, but also the labeling signal was maintained for more than 1 hour and did not diffuse to other lymph nodes, thereby verifying that the fluorescent protein nanoparticles were very effective in targeting the sentinel lymph nodes (FIG. 16). .
특히, tPTS 나노입자의 경우 다른 나노입자에 비해 주사한 가장 높은 감시림프절 타겟팅 효율을 보였으며, 이는 표적 지향성 펩타이드가 없이도 단백질 나노입자 고유의 크기나 구조적 특성이 특정 조직을 타겟팅하여 이미징하는 소재로 활용될 수 있음을 보여준다.In particular, tPTS nanoparticles showed the highest surveillance lymph node targeting efficiency injected compared to other nanoparticles, and this is used as a material for imaging by targeting specific tissues with the inherent size or structural characteristics of protein nanoparticles even without target-oriented peptides. Show that it can be.
<110> Korea University Industrial & Academic Collaboration Foundation <120> Fluorescent protein nanoparticles for in vivo imaging <130> P15U13C1547 <150> KR 2012-122548 <151> 2012-10-31 <160> 39 <170> KopatentIn 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 1 <400> 1 catatggaca ttgacccgta taaaga 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 2 <400> 2 ctcgaggtct tccaaattac ttccca 26 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 3 <400> 3 ggatcctcca gggaattagt agtcagc 27 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 4 <400> 4 aagcttttaa acaacagtag tttccggaag tgt 33 <210> 5 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 5 <400> 5 ctcgagcatc accatcacca tcacgtgagc aagggcgag 39 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 6 <400> 6 ggatcccttg tacagctcgt ccatgcc 27 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 7 <400> 7 ctcgagcatc accatcacca tcacgacaac accgaggac 39 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 8 <400> 8 ggatccctgg gagccggagt ggcg 24 <210> 9 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 9 <400> 9 ggctctggtg gcggaagtgg gggtggcgtg agcaagggcg ag 42 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 10 <400> 10 agagccaccc ccacttccgc cacccttgta cagctcgtcc at 42 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 11 <400> 11 ggctctggtg gcggaagtgg gggtggcgac aacaccgagg ac 42 <210> 12 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 12 <400> 12 agagccaccc ccacttccgc caccctggga gccggagtgg cg 42 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 13 <400> 13 ggtggcggaa gtgggggtgg ctctggtggc ggaagt 36 <210> 14 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 14 <400> 14 ctcgagcatc accatcacca tcacggtggc ggaagtggg 39 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 15 <400> 15 ggatccgcca cccccacttc cgccaccaga gccaccccca ct 42 <210> 16 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 16 <400> 16 gaggcggttc gggcggaggt tctggtggcg gaacgaccgc gtccacc 47 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 17 <400> 17 aagcttgtga tggtgatgga gatgctggga gccggagtgg 40 <210> 18 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 18 <400> 18 gaggcggttc gggcggaggt tctggtggcg gagacattga cccgtataa 49 <210> 19 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 19 <400> 19 catatgggac gtggtgatag cggcggaggg tctggaggcg gttcgggcg 49 <210> 20 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 20 <400> 20 catatgcacc atcaccatca ccatagtacc gctaaattag tta 43 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 21 <400> 21 aagcttttat tcgatgttag actc 24 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 22 <400> 22 catatgagta ccgctaaatt agtta 25 <210> 23 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 23 <400> 23 catatgtgcg attgccgtgg tgattgcttc tgcctcgaga gtaccgctaa attagtta 58 <210> 24 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 24 <400> 24 aagcttttag tgatggtgat ggtgatgttc gatgttagac tcgataaac 49 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 25 <400> 25 catatgcaac agggccagat ggct 24 <210> 26 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 26 <400> 26 aagcttagag gaatttttta acttcctcct g 31 <210> 27 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 27 <400> 27 cacgtccgca atcgcaactt ccaccgccga ggaatttttt aacttcc 47 <210> 28 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 28 <400> 28 aagcttagca gaagcaatca ccacgtccgc aatcgca 37 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 29 <400> 29 catatggccg cggttggtat tacactgaaa gacg 34 <210> 30 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 30 <400> 30 aagcttaatg gtgatggtga tggtgcagaa ttaatccgag cttc 44 <210> 31 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 31 <400> 31 catatgcacc atcaccatca ccatacgacc gcgtccacct cgcaggtg 48 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 32 <400> 32 aagctttcag ctttcattat cactgtc 27 <210> 33 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 33 <400> 33 catatgcacc atcaccatca ccatgacatt gacccgtata aagaa 45 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 34 <400> 34 aagcttttaa acaacagtag tttc 24 <210> 35 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 1 <400> 35 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 2 <400> 36 Gly Gly Gly Gly Gly 1 5 <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 3 <400> 37 Gly Gly Gly Ser Gly Gly Gly Thr Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 4 <400> 38 Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr 1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 5 <400> 39 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <110> Korea University Industrial & Academic Collaboration Foundation <120> Fluorescent protein nanoparticles for in vivo imaging <130> P15U13C1547 <150> KR 2012-122548 <151> 2012-10-31 <160> 39 <170> KopatentIn 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer No. One <400> 1 catatggaca ttgacccgta taaaga 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 2 <400> 2 ctcgaggtct tccaaattac ttccca 26 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 3 <400> 3 ggatcctcca gggaattagt agtcagc 27 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 4 <400> 4 aagcttttaa acaacagtag tttccggaag tgt 33 <210> 5 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 5 <400> 5 ctcgagcatc accatcacca tcacgtgagc aagggcgag 39 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 6 <400> 6 ggatcccttg tacagctcgt ccatgcc 27 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 7 <400> 7 ctcgagcatc accatcacca tcacgacaac accgaggac 39 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 8 <400> 8 ggatccctgg gagccggagt ggcg 24 <210> 9 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 9 <400> 9 ggctctggtg gcggaagtgg gggtggcgtg agcaagggcg ag 42 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 10 <400> 10 agagccaccc ccacttccgc cacccttgta cagctcgtcc at 42 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 11 <400> 11 ggctctggtg gcggaagtgg gggtggcgac aacaccgagg ac 42 <210> 12 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 12 <400> 12 agagccaccc ccacttccgc caccctggga gccggagtgg cg 42 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 13 <400> 13 ggtggcggaa gtgggggtgg ctctggtggc ggaagt 36 <210> 14 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 14 <400> 14 ctcgagcatc accatcacca tcacggtggc ggaagtggg 39 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 15 <400> 15 ggatccgcca cccccacttc cgccaccaga gccaccccca ct 42 <210> 16 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 16 <400> 16 gaggcggttc gggcggaggt tctggtggcg gaacgaccgc gtccacc 47 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 17 <400> 17 aagcttgtga tggtgatgga gatgctggga gccggagtgg 40 <210> 18 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 18 <400> 18 gaggcggttc gggcggaggt tctggtggcg gagacattga cccgtataa 49 <210> 19 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 19 <400> 19 catatgggac gtggtgatag cggcggaggg tctggaggcg gttcgggcg 49 <210> 20 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 20 <400> 20 catatgcacc atcaccatca ccatagtacc gctaaattag tta 43 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 21 <400> 21 aagcttttat tcgatgttag actc 24 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 22 <400> 22 catatgagta ccgctaaatt agtta 25 <210> 23 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 23 <400> 23 catatgtgcg attgccgtgg tgattgcttc tgcctcgaga gtaccgctaa attagtta 58 <210> 24 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 24 <400> 24 aagcttttag tgatggtgat ggtgatgttc gatgttagac tcgataaac 49 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 25 <400> 25 catatgcaac agggccagat ggct 24 <210> 26 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 26 <400> 26 aagcttagag gaatttttta acttcctcct g 31 <210> 27 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 27 <400> 27 cacgtccgca atcgcaactt ccaccgccga ggaatttttt aacttcc 47 <210> 28 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 28 <400> 28 aagcttagca gaagcaatca ccacgtccgc aatcgca 37 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 29 <400> 29 catatggccg cggttggtat tacactgaaa gacg 34 <210> 30 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 30 <400> 30 aagcttaatg gtgatggtga tggtgcagaa ttaatccgag cttc 44 <210> 31 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 31 <400> 31 catatgcacc atcaccatca ccatacgacc gcgtccacct cgcaggtg 48 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 32 <400> 32 aagctttcag ctttcattat cactgtc 27 <210> 33 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 33 <400> 33 catatgcacc atcaccatca ccatgacatt gacccgtata aagaa 45 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer No. 34 <400> 34 aagcttttaa acaacagtag tttc 24 <210> 35 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 1 <400> 35 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 2 <400> 36 Gly Gly Gly Gly Gly 1 5 <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 3 <400> 37 Gly Gly Gly Ser Gly Gly Gly Thr Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 4 <400> 38 Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr 1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 5 <400> 39 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
Claims (8)
B형 간염 바이러스 코어 단백질의 1-78번째 아미노산 서열 부분과 상기 코어 단백질의 81-149번째 아미노산 서열 부분 사이에 형광단백질이 위치하고, 상기 형광단백질의 N-말단 부분은 헥사 히스티딘과 링커 펩타이드가, C-말단 부분은 링커 펩타이드가 결합되어 있고, 상기 표적 지향성 펩타이드는 B형 간염 바이러스 코어 단백질의 N-말단 또는 C-말단에는 결합되어 있는 키메릭 단백질로부터 형성되는 형광 단백질 나노입자.A fluorescent substance and a target-oriented peptide are expressed on the surface,
The fluorescent protein is positioned between the 1-78th amino acid sequence portion of the hepatitis B virus core protein and the 81-149th amino acid sequence portion of the core protein, and the N-terminal portion of the fluorescent protein is composed of hexa histidine and linker peptide, - a fluorescent protein nanoparticle in which the linker peptide is bound to the terminal portion and the target-directed peptide is formed from a chimeric protein bound to the N-terminal or C-terminal of the hepatitis B virus core protein.
형광단백질은 녹색형광단백질(GFP), 변형된 녹색형광단백질(modified green fluorescent protein), 증강된 녹색형광단백질(enhanced green fluorescent protein; EGFP), 적색형광단백질(RFP, DSRed), 증강된 적색형광단백질(ERFP), 청색형광단백질(BFP), 증강된 청색형광단백질(EBFP), 황색형광단백질(YFP), 증강된 황색형광단백질(EYFP), 남색형광단백질(CFP) 또는 증강된 남색형광단백질(ECFP) 중 어느 하나인, 형광 단백질 나노입자.The method according to claim 1,
Fluorescent proteins are composed of green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP, DSRed), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP) ). ≪ / RTI >
형광단백질은 증강된 녹색형광단백질(enhanced green fluorescent protein) 또는 적색형광단백질(DsRed)인, 형광 단백질 나노입자.The method according to claim 1,
The fluorescent protein is an enhanced green fluorescent protein or a red fluorescent protein (DsRed), a fluorescent protein nanoparticle.
링커 펩타이드는 SEQ ID NO: 35 내지 39에 기재된 아미노산 서열 중 어느 하나로 표시되는 형광 단백질 나노입자.The method according to claim 1,
Wherein the linker peptide is represented by any one of the amino acid sequences set forth in SEQ ID NOS: 35 to 39.
표적 지향성 펩타이드는 RGD 펩타이드, 소마토스타틴(somatostatins), 어피바디(affibody), 수용체 길항근(receptor antagonist), 봄베신(bombesin), 콜레시스토키닌(cholecystokinin) 또는 가스트린 유사체(gastrin analog)를 포함하는 형광 단백질 나노입자.The method according to claim 1,
Targeted peptides include fluorescent protein nanoparticles that include RGD peptides, somatostatins, affibodies, receptor antagonists, bombesin, cholecystokinin, or gastrin analogs.
약제학적으로 허용가능한 담체를 포함하는 표적 지향형 조영제 조성물. A fluorescent protein nanoparticle according to claim 1; And
A composition comprising a pharmaceutically acceptable carrier.
약제학적으로 허용가능한 담체를 포함하는 동시 진단 또는 치료용 조영제 조성물.A fluorescent protein nanoparticle according to claim 1; And
A contrast agent composition for simultaneous diagnosis or treatment comprising a pharmaceutically acceptable carrier.
진단 프로브를 포함하고, 상기 진단 프로브는 영상 판독을 위한 표지 물질인 다중 진단 프로브. A fluorescent protein nanoparticle according to claim 1; And
And a diagnostic probe, wherein the diagnostic probe is a marking material for image reading.
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