KR101796474B1 - Fluorescent peptide combined with a polymeric Bile acid-Chitosan for detecting cell apoptosis and the composition including thereof for the diagnosis of disease - Google Patents
Fluorescent peptide combined with a polymeric Bile acid-Chitosan for detecting cell apoptosis and the composition including thereof for the diagnosis of disease Download PDFInfo
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- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
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Abstract
Description
본 발명은 담즙산-키토산 복합체 나노입자가 도입된 사멸세포 검출용 형광 펩타이드, 이의 제조방법 및 상기 형광 펩타이드를 포함하는 암 등 질병의 조기진단을 위한 형광 조성물에 관한 것으로, 보다 상세하게는 사멸세포에 특이적으로 작용하는 펩타이드(아미노산 서열 CQRPPR)에 형광 염료를 결합시킨 형광 펩타이드에 고분자인 담즙산-키토산(HGC)을 결합시켜 형성한 형광 복합체로서, 본 형광 복합체는 사멸세포에 특이적으로 작용하고 특히 종래의 기술에 비하여 암 등의 질병의 발병부위에 안착하는 시간이 길어 생체 분자 내에서 보다 높은 형광 세기와 축적률을 가지는 형광 펩타이드 및 이의 제조방법에 관한 것이다.The present invention relates to a fluorescent composition for the early diagnosis of diseases, such as a fluorescent peptide for detecting apoptotic cells into which bile acid-chitosan complex nanoparticles are introduced, a method for producing the same, and a cancer including the fluorescent peptide. More particularly, A fluorescence complex formed by binding a polymeric cholanic acid-chitosan (HGC) to a fluorescent peptide having a fluorescent dye bound to a specifically acting peptide (amino acid sequence CQRPPR). This fluorescent complex specifically acts on apoptotic cells, The present invention relates to a fluorescent peptide having a higher fluorescence intensity and a higher accumulation rate in a biomolecule, and a method for producing the same.
최근 질병 조기 진단 분야에서 광학 분자 영상 기술의 중요성이 대두되었다. 특히, 암 조기 진단 및 치료 분야에 있어서 광학 분자 영상 기술의 중요성이 대두되고 있으며, 근적외선 투시에 의한 암 조직 영상법은 기존의 PET/CT 또는 PET/SPECT 진단기술과 비교하여, 방사성 동위원소의 불필요성, 고해상도의 영상, 저렴한 비용 등의 장점을 가지고 있다.Recently, the importance of optical molecular imaging technology has emerged in the early diagnosis of diseases. In particular, the importance of optical molecular imaging technology in the field of early diagnosis and treatment of cancer has been growing, and compared with conventional PET / CT or PET / SPECT diagnostic techniques, cancer tissue imaging by near- , High-resolution images, and low cost.
그러나 근적외선 투시에 의한 암 조직 영상법을 사용하여 암을 조기에 진단할 수 있는 가장 효율적인 방법은 근적외선 파장(650nm~900nm)의 스펙트럼 범위 내에서 흡수 및 형광 최대치를 갖고, 물에 대한 용해도가 높으며 생체 적합성이 우수한 근적외선 형광체의 개발과 더불어 이러한 근적외선 형광체가 암 조직에 선택적으로 전달 및 축적되어 암 질환 조직에서 높은 형광 세기를 나타내는 조영제를 개발하는 것이다.However, the most effective method for early diagnosis of cancer using near-infrared-visible cancer tissue imaging is to have a maximum absorption and fluorescence within the spectral range of near-infrared wavelength (650 nm ~ 900 nm), high solubility in water, In addition to the development of highly compatible near infrared ray phosphors, such near infrared ray phosphors are selectively transferred to and accumulated in cancer tissues to develop contrast agents exhibiting high fluorescence intensities in cancer diseases.
이에 본 발명자들은 암 조직의 신생 혈관의 높은 투과율에 의해서 암 조직에 선택적으로 축적이 되는 고분자와 근적외선 투시가 가능한 근적외선 형광체 및 사멸세포에 작용하는 펩타이드를 화학적으로 결합한 고분자 나노입자형 신규 화합물을 개발함으로써 본 발명을 완성하였다.Accordingly, the present inventors have developed a novel polymer nanoparticle-type compound chemically bonding a polymer capable of selectively accumulating in cancer tissues and a near infrared ray fluorescent substance capable of near infrared ray penetration and a peptide acting on apoptotic cells by high transmittance of cancer blood vessels Thus completing the present invention.
선행기술문헌Prior art literature
-특허문헌- Patent literature
(특허문헌 1) KR10-0952841(Patent Document 1) KR10-0952841
본 발명의 목적은 사멸세포에 특이적으로 작용하고 암 등 질병의 발병부위에 정착하는 시간이 연장된 신규 펩타이드-형광염료-고분자 키토산 나노입자가 결합된 복합체를 제공하여 종래의 형광 조영제가 가지는 단점인 암 조직에 대한 낮은 특이도 및 낮은 형광 강도를 개선하고자 하는 것이다.It is an object of the present invention to provide a complex in which new peptide-fluorescent dye-polymer chitosan nanoparticles are specifically bound to act as apoptotic cells and have a prolonged time for fixing to a disease site such as cancer, And to improve the low specificity and low fluorescence intensity of cancer tissue.
상기 과제를 해결하기 위하여 본 출원인은 세포사멸에 특이적으로 작용하는 펩타이드와 형광 염료가 결합된 펩타이드-형광염료 복합체에 암 등의 발병부위에 안착하는 시간이 연장된 고분자 담즙산-키토산을 결합하여 고분자-형광 펩타이드 복합체를 제조하게 되었다.In order to solve the above-mentioned problems, the applicant of the present invention has proposed a peptide-fluorescent dye conjugate in which a peptide and a fluorescent dye are combined specifically with apoptosis, and a polymer cholanic acid- - < / RTI > fluorescent peptide complex.
본 발명의 고분자-형광 펩타이드 복합체의 제조방법은 a) 키토산을 정제하는 단계, b) 담즙산-키토산 나노입자를 합성하는 단계, c) 사멸세포 검출용 형광 펩타이드를 제조하는 단계 및 d) 상기 담즙산-키토산 나노입자와 상기 형광 펩타이드를 결합시키는 단계를 포함하여 구성된다.The method for producing a polymer-fluorescent peptide complex of the present invention comprises the steps of: a) purifying chitosan, b) synthesizing cholanic acid-chitosan nanoparticles, c) preparing a fluorescent peptide for detecting apoptotic cells, and d) And binding the chitosan nanoparticles to the fluorescent peptide.
본 발명에서 제공하는 고분자-형광 펩타이드 복합체는 암 등 질병의 조기진단을 위한 조영제의 조성물로 사용할 수 있다.The polymer-fluorescent peptide complex provided in the present invention can be used as a contrast agent composition for early diagnosis of diseases such as cancer.
본 발명에서 제공하는 고분자-형광 펩타이드 복합체의 화학구조는 아래의 화학식 1에서 제시하는 바와 같다.The chemical structure of the polymer-fluorescent peptide complex provided in the present invention is as shown in the following chemical formula (1).
[화학식 1][Chemical Formula 1]
본 발명은 담즙산-키토산(HGC) 고분자 나노입자를 근적외선에 발광하는 형광물질이 결합된 사멸세포에 특이적으로 작용하는 펩타이드에 결합한 복합체에 관한 것으로서, 상기 복합체는 생체 적합성 및 생분해성이 뛰어나 생체분자의 표지에 이용 가능하며, 암 부위에 특이적으로 안착되고 장기 축적률이 낮으며, 높은 형광 강도를 가진다. The present invention relates to a complex of cholanic acid-chitosan (HGC) polymer nanoparticles bound to a peptide specifically acting on apoptotic cells to which a fluorescent substance capable of emitting light in the near-infrared light is bound. The complex is excellent in biocompatibility and biodegradability, It is specific to the cancer site, has a low accumulation rate in the long term, and has a high fluorescence intensity.
도 1은 본 발명에서 제공되는 화합물의 형광강도를 나타낸 그래프이다.
도 2는 본 발명에서 제공되는 화합물의 입자크기의 분포를 나타낸 그래프이다.
도 3은 본 발명에서 제공되는 화합물을 이용하여 사멸세포를 검출한 결과를 보여주는 사진이다.1 is a graph showing the fluorescence intensity of the compound provided in the present invention.
2 is a graph showing the distribution of particle sizes of the compounds provided in the present invention.
FIG. 3 is a photograph showing the results of detection of apoptotic cells using the compound provided in the present invention. FIG.
본 발명의 고분자-형광 펩타이드 복합체의 제조방법은 a) 키토산을 정제하는 단계, b) 담즙산-키토산 나노입자를 합성하는 단계, c) 사멸세포 검출용 형광 펩타이드를 제조하는 단계 및 d) 상기 담즙산-키토산 나노입자와 상기 형광 펩타이드를 결합시키는 단계를 포함하여 구성된다.The method for producing a polymer-fluorescent peptide complex of the present invention comprises the steps of: a) purifying chitosan, b) synthesizing cholanic acid-chitosan nanoparticles, c) preparing a fluorescent peptide for detecting apoptotic cells, and d) And binding the chitosan nanoparticles to the fluorescent peptide.
특히, 본 발명의 고분자-형광 펩타이드 복합체의 제조방법에서는, 근적외선 형광체에 키토산 유도체 고분자를 결합시키는데 최적화된 온도, 농도 및 시간 등 반응 조건을 제공하며, 이에 따라, 제조된 본 발명의 고분자-형광 펩타이드 복합체는 종래 기술에 비해 높은 형광 세기와 암 부위에 대한 높은 특이도를 나타낸다.In particular, in the method for producing a polymer-fluorescent peptide complex of the present invention, reaction conditions such as temperature, concentration and time optimized for binding a chitosan derivative polymer to a near infrared ray fluorescent substance are provided, The complexes exhibit higher fluorescence intensities and higher specificity for cancer sites than in the prior art.
더욱 상세하게는, 담즙산-키토산(HGC) 복합체의 구조는 하기 화학식 2와 같으며, 상기 담즙산-키토산(HGC) 복합체를 합성하는 과정은 키토산을 정제하는 단계와 정제된 키토산 고분자에 담즙산을 결합시키는 단계를 포함하여 이루어지며, 하기와 같이 보다 상세하게 설명될 수 있다.More specifically, the structure of the cholanic acid-chitosan (HGC) complex is as shown in the following formula (2). The process for synthesizing the cholanic acid-chitosan (HGC) complex includes a step of purifying chitosan, a step of binding bile acid to the purified chitosan polymer Step, and can be described in more detail as follows.
[화학식 2](2)
<담즙산-키토산(HGC) 나노입자의 구조><Structure of bile acid-chitosan (HGC) nanoparticles>
본 발명의 고분자-형광 펩타이드 복합체의 제조방법의 첫 번째 단계로서 먼저, 본 발명에 사용될 키토산을 정제하는 과정을 설명한다. As a first step of the method for producing a polymer-fluorescent peptide complex of the present invention, a process for purifying chitosan to be used in the present invention will be described.
글라이콜 키토산 (Glycol Chitosan, GC) (100kDa) 1 g을 250 mL 삼각플라스크에 넣고 초순수수 80 mL에 녹인 후 투석용 멤브레인(MWCO 100kDa)을 사용하여 수차례 투석하였다. 투석 완료 후, 영하 80℃에서 12시간 냉각 후 동결건조 진행하여 정제된 GC 를 얻었다( 0.571 g, 57.1 %).1 g of Glycol Chitosan (GC) (100 kDa) was dissolved in 80 mL of ultra-pure water in a 250 mL Erlenmeyer flask and dialyzed several times using dialysis membrane (
다음으로, 상기 정제된 키토산(GC) 고분자에 담즙산을 결합시키는 과정을 설명한다.Next, the process of binding bile acid to the purified chitosan (GC) polymer will be described.
정제된 GC 500 mg을 초순수수 60 mL에 완용한 후 60 mL의 메탄올을 추가로 투입하여 충분히 교반한다. 150 mg의 담즙산(5β-Cholanic acid)을 120 mL의 메탄올에 넣고 완용하였다. 150 mg N-(3-디메틸아미노프로필)-N'-에틸카보디이미드 염산염(N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), EDC)에 1000 mL의 메탄올을 넣고 교반한 후 GC용액에 투입하여 교반하였다. 72 mg의 N-하이드록시석신이미드(N-Hydroxysuccinimide, NHS)을 메탄올 1 mL를 넣고 교반한 후 담즙산 용액에 투입하여 교반하였다. 담즙산 혼합용액을 GC 혼압용액에 투입하여, 상온 일야교반 진행하였다. 반응액을 주사기필터(pore size : 80um)를 이용하여 여과한 후, 투석용 멤브레인(MWCO 12~14 kDa)에 넣고 4일 동안 투석 진행하였다. 투석 완료 후 초음파 분산기(Waterbath sonicator)에 30초 동안 분산한 후 동결건조 진행하여 HGC 나노입자를 얻었다( 0.492 g., 75.7 %).500 mg of purified GC is dissolved in 60 mL of ultrapure water, and 60 mL of methanol is further added thereto. 150 mg of 5 [beta] -cholanic acid was added to 120 mL of methanol. 1000 mL of methanol was added to 150 mg of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride) And stirred. 72 mg of N-Hydroxysuccinimide (NHS) was added to 1 mL of methanol, stirred, and then added to the bile acid solution and stirred. The mixed bile acid solution was added to the GC mixed solution, and stirred at room temperature overnight. The reaction solution was filtered using a syringe filter (pore size: 80 μm), dialyzed for 4 days in a dialysis membrane (MWCO 12-14 kDa). After completion of dialysis, HGC nanoparticles were obtained (0.492 g., 75.7%) after being dispersed in a waterbath sonicator for 30 seconds and then lyophilized.
- 1~2일차 투석용액 (메탄올/초순수수 3:1 v/v)- 1 to 2 dialysis solution (methanol / deionized water 3: 1 v / v)
- 3일차 투석용액 (메탄올/초순수수 1:1 v/v)- 3 dialysis solution (methanol / deionized water 1: 1 v / v)
- 4일차 투석용액 (초순수수)- Fourth dialysis solution (ultra pure water)
본 발명의 고분자-형광 펩타이드 복합체의 제조방법의 두 번째 단계로서 사멸세포 검출용 형광 펩타이드의 제조방법을 설명한다. 이는 본 발명에서 사용되는 세포사멸에 특이적으로 작용하는 아미노산 서열 CQRPPR로 이루어지는 Apopep-1을 본 발명에서 사용되는 형광 염료와 결합시키는 단계이다.As a second step of the method for producing a polymer-fluorescent peptide complex of the present invention, a method for producing fluorescent peptides for apoptotic cells will be described. This is a step of binding Apopep-1 comprising the amino acid sequence CQRPPR, which is specifically used for cell death, to the fluorescent dye used in the present invention.
먼저 상기 형광염료를 제조하는 설명하면, Flamma®Fluore 675 carboxylic acid 53 mg(56.1 umol, 1 eq, BioActs 社)을 디메틸포름알데히드(N,N,-Dimethylformaldehyde, DMF) 15 ml에 넣고 완용하였다. 디숙신이미딜 카보네이트(N,N'-Disuccinimidyl carbonate, DSC) 43 mg(168 umol, 3 eq, Sigma-Aldrich)를 완용액에 투입한 후, 디이소프로필에틸아민(N,N-diisopropylethylamine) 97 ul(561 umol, 10 eq, Sigma-Aldrich)을 투입한 후 40℃에서 2시간 동안 교반하였다. 반응액에 에테르(Diethyl ether, Sigma-Aldrich) 를 투입하여 입자를 생성한 후, 여과 및 건조하여 아래의 화학식 3의 구조를 갖는 형광 염료물질을 제조하였다.To prepare the fluorescent dye, 53 mg (56.1 μmol, 1 eq, BioActs) of Flamma®
[화학식 3](3)
다음으로 상기 화학식 3으로 이루어지는 화합물과 상기 Apopep-1을 결합하는 단계를 설명한다.Next, the step of binding the compound of Formula 3 with Apopep-1 will be described.
위의 단계에서 수득된 염료 화합물 58 mg(56.05 umol)을 DMF 10 ml에 완용하였다. Apopep-1 Peptide(아미노산 서열 CQRPPR)을 DMF 10 ml에 완용한 후, 상기 화합물 3의 용액에 투입하였다. 카보네이트 버퍼 용액 10 ml(Carbonate buffer, pH=9.0)을 상기 화합물 3의 혼합용액에 투입한 후 상온에서 일야 교반하였다. 반응액을 동결건조 진행한 후 얻어진 물질을, HPLC를 사용하여 정제한 후 동결건조 과정을 진행하여 아래의 화학식 4의 구조를 가지는 상기 염료 물질과 Apopep-1의 펩타이드가 결합된 순수한 화합물을 얻었다(48 mg, 50.9%). 58 mg (56.05 umol) of the dye compound obtained in the above step was dissolved in 10 ml of DMF. The Apopep-1 Peptide (amino acid sequence CQRPPR) was added to 10 ml of DMF and then added to the solution of Compound 3 above. Carbonate buffer (pH = 9.0) was added to the mixed solution of Compound 3, and stirred overnight at room temperature. After the reaction solution was freeze-dried, the obtained substance was purified using HPLC and then subjected to a freeze-drying process to obtain a pure compound in which the dye material having the structure represented by the following formula (4) and the peptide of Apopep-1 were combined 48 mg, 50.9%).
[화학식 4][Chemical Formula 4]
상기 화학식 4의 화합물의 Maldi-TOF/MS, 계산치 C73H99N15O21S5 1682.98, 측정치 1682.51Maldi-TOF / MS of the compound of Formula 4, the calculated value C 73 H 99 N 15 O 21 S 5 1682.98, the measured value 1682.51
본 발명의 복합체의 제조방법의 마지막 단계인 상기 화학식 2의 화합물인 고분자 담즙산 키토산(HGC) 나노입자와 상기 화학식 4의 화합물을 결합시키는 단계를 설명한다.The step of binding the polymer cholanic acid chitosan (HGC) nanoparticles, which is the compound of Formula 2, to the compound of Formula 4, which is the last step of the method of the present invention, will be described.
HGC 나노입자 7 mg을 100 uL 디메틸 설폭사이드(Dimethyl sulfoxide, DMSO, Sigma-Aldrich)에 완용한 후 14 mL PBS 용액(pH=7.4)에 희석하였다. 화학식 4의 화합물 1.7 mg을 100 uL DMSO에 완용한 후, HGC 용액에 투입하였다. 4-(N-말레이미도메틸)사이클로헥산카복시산 N-히드록시숙신이미드 에스터(4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester, Signa-Aldrich)를 DMSO 100 uL와 PBS 100 uL에 희석한 후, HGC 혼합용액에 투입한 후 상온에서 일야 교반 진행하였다. 반응액을 투석용 멤브레인 (MWCO 12~14 kDa)을 사용하여 5일 동안 하기 기재된 방법대로 투석 진행하였다. 투석이 완료된 화합물을 동결건조 하여 화학식 1로 표시되는 담즙산-키노산 나노입자와 형광 펩타이드가 결합된 화합물의 나노입자를 얻었다(7 mg, 100 %). 7 mg of HGC nanoparticles was dissolved in 100 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich) and diluted in 14 mL of PBS solution (pH = 7.4). 1.7 mg of the compound of Formula 4 was dissolved in 100 uL of DMSO, and then added to the HGC solution. N-maleimidomethyl cyclohexanecarboxylic acid N-hydroxysuccinimide ester (Signa-Aldrich) was diluted with 100 μL of DMSO and 100 μL of PBS. Then, the mixture was added to the HGC mixed solution, and stirred overnight at room temperature. The reaction solution was dialyzed for 5 days using the dialysis membrane (MWCO 12-14 kDa) according to the method described below. The dialyzed compound was lyophilized to obtain nanoparticles (7 mg, 100%) of a compound in which a cholanic acid-chinosanic acid nanoparticle represented by Chemical Formula 1 and a fluorescent peptide were combined.
- 1~2일차 투석용액 (정제염/초순수수 8.4 g/1 L)- 1 to 2 days dialysis solution (refined salt / deionized water 8.4 g / 1 L)
- 3~5일차 투석용액 (초순수수)- Dialysis solution (ultrapure water) for 3-5 days
<고분자-형광 펩타이드 복합체의 광학특성 분석><Optical Characterization of Polymer-Fluorescent Peptide Complex>
상기 본 발명에서 제공하는 고분자-형광 펩타이드 복합체(화학식 1)의 물질을 확인하기 위해 광학 특성을 분석하였다.In order to confirm the substance of the polymer-fluorescent peptide complex (Formula 1) provided by the present invention, optical characteristics were analyzed.
상기 제조단계를 통하여 제조된 화학식 1의 화합물을 초순수수를 사용하여 62.5㎍/㎖의 농도로 1㎖씩 제조한 후 UV 스펙트로미터(SHIMADZU, UV-2600), 형광 스펙트로미터(SCINCO, FluoroMate FS-2)를 이용하여 각각 측정하여, 그 결과를 도 1에 제시하였다. 1 ml of the compound of
측정 결과, 최대 흡수파장은 673 nm 이며, 최대 형광파장은 690.5 nm 로 측정되었으며, 이는 Flamma®Fluore 675 carboxylic acid 의 광학 특성과 거의 동일한 결과를 나타냄을 확인하였다. 형광염료와 펩타이드, HGC 나노입자를 도입하였을 경우, 형광염료 자체의 광학특성이 유지되고 있음을 확인할 수 있었다.As a result, it was confirmed that the maximum absorption wavelength was 673 nm and the maximum fluorescence wavelength was 690.5 nm, which is almost the same as the optical characteristic of
<고분자-형광 펩타이드 복합체의 입자 크기 측정>≪ Measurement of Particle Size of Polymer-Fluorescent Peptide Complex >
본 발명의 고분자-형광 펩타이드 복합체를 1 mg 분취하여 초순수수 1 mL 에 분산하여 1 mg/mL 의 농도로 만든 후, 얼음 냉각 하에 프로브 형태의 초음파 분산기 (Probe type sonicator, Sonics Vibracell VCX750, Sonics) 를 5초 간격으로 1분간 분산 진행하였다. 분산이 완료된 용액을 입도분석기용 큐벳에 담은 후 입도분석장치 (Zetasizer Nano ZS, Malvern) 을 사용하여 입자 크기를 측정하였으며, 그 결과는 도 2에 제시하였다.1 mg of the polymer-fluorescent peptide complex of the present invention was dispersed in 1 mL of ultrapure water to give a concentration of 1 mg / mL. Then, a probe-type ultrasonic disperser (Sonics Vibracell VCX750, Sonics) Dispersion was carried out at intervals of 5 seconds for 1 minute. The dispersed solution was placed in a cuvette for a particle size analyzer and the particle size was measured using a particle size analyzer (Zetasizer Nano ZS, Malvern). The results are shown in FIG.
측정 결과, 122.4 nm 의 입자 크기를 확인하였다. 이는 합성된 나노입자를 사용하여 소동물 실험 진행시, EPR 효과 (Enhanced permeability and retention effect) 를 나타내기에 알맞은 크기임을 확인하였다.As a result of measurement, a particle size of 122.4 nm was confirmed. It is confirmed that this is a suitable size for exhibiting the EPR effect (enhanced permeability and retention effect) in a small animal experiment using the synthesized nanoparticles.
<고분자-형광 펩타이드 복합체의 사멸세포 관측><Observation of apoptotic cells of polymer-fluorescent peptide complex>
본 발명의 고분자-형광 펩타이드 복합체의 사멸세포 검출 여부를 확인하기 위하여, 다음과 같은 실험을 진행하였다. 실험에 사용된 세포는 HeLa cell (Human uterine cervical cancer cell) 을 사용하였으며, 형광 관측 장비는 NiKon Eclipse Ti-U microscope 현미경을 사용하였다.In order to confirm the detection of the apoptotic cells of the polymer-fluorescent peptide complex of the present invention, the following experiment was conducted. The cells used for the experiment were HeLa cells (human uterine cervical cancer cell), and the fluorescence observation equipment was a NiKon Eclipse Ti-U microscope.
1) Confocal dish 에 HeLa cell 2X105 cell/well 로 cell seeding 한다.1) Cell seed with HeLa cell 2X105 cell / well in Confocal dish.
2) 37℃ 5% 이산화탄소 조건으로 하루 동안 배양한다.2) Incubate at 37 ℃ 5% CO2 for one day.
3) Actinomycin D 를 well 당 1 uM 로 6 시간동안 처리한다.3) Actinomycin D is treated for 6 hours at 1 uM per well.
4) 합성된 HGC-Apopep 형광 나노입자 용액 (1mg/mL) 을 사용하여 배양된 세포에 24시간 동안 흡수시킨 후 1X PBS 용액으로 2회 세척한다.4) The cells were incubated with the synthesized HGC-Apopep fluorescent nanoparticle solution (1 mg / mL) for 24 hours and washed twice with 1X PBS solution.
5) 4% Paraformaldehyde 1 mL 를 투입하여 5분 동안 처리한 후 1X PBS 용액으로 2회 세척한다.5) Add 1 mL of 4% paraformaldehyde for 5 minutes and wash twice with 1X PBS solution.
6) DAPI 용액 1mL 를 투입 후 3분 동안 염색한 후, 1X PBS 용액으로 2회 세척한다.6) Add 1 mL of DAPI solution, stain for 3 minutes, and wash twice with 1X PBS solution.
7) 형광 현미경을 사용하여 세포를 관찰한다(도 3).7) Observe the cells using a fluorescence microscope (Fig. 3).
세포사멸을 유도하지 않은 세포 (Control) 와 세포사멸을 유도한 세포 (HGC-Apopep) 에 HGC-Apopep 형광 나노입자를 각각 처리한 후 비교 관찰 한 결과, 세포사멸을 유도한 세포에서 형광이 나타남을 확인하였다. 이는, 본 발명의 고분자-형광 펩타이드 복합체는 사멸세포가 아닌 세포에서는 형광을 나타내지 않으나, 사멸세포에서는 형광이 관찰되어, 사멸세포 검출을 위한 용도로 사용될 수 있음을 확인하였다.HGC-Apopep fluorescence nanoparticles were treated with HGC-Apopep and HGC-Apopep, respectively. The results showed that fluorescence was observed in apoptotic cells. Respectively. It was confirmed that the polymer-fluorescent peptide complex of the present invention does not fluoresce in cells that are not apoptotic cells, but fluorescence is observed in apoptotic cells and can be used for apoptosis detection.
Claims (2)
[화학식 1]
[화학식 2]
[화학식 3]
[Chemical Formula 1]
(2)
(3)
[화학식 1]
[화학식 2]
[화학식 3]
[화학식 4]
[Chemical Formula 1]
(2)
(3)
[Chemical Formula 4]
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