KR101923919B1 - Aptamer-hydrophilic polymer-photosensitizer conjugates and preparation method thereof - Google Patents

Aptamer-hydrophilic polymer-photosensitizer conjugates and preparation method thereof Download PDF

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KR101923919B1
KR101923919B1 KR1020160138355A KR20160138355A KR101923919B1 KR 101923919 B1 KR101923919 B1 KR 101923919B1 KR 1020160138355 A KR1020160138355 A KR 1020160138355A KR 20160138355 A KR20160138355 A KR 20160138355A KR 101923919 B1 KR101923919 B1 KR 101923919B1
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이은성
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Abstract

본 발명은 압타머; 고분자; 및 광응답제로 이루어진 압타머-친수성 고분자-광응답제 접합체에 관한 기술로, 보다 상세하게는 상기 압타머-친수성 고분자-광응답제 접합체는 수용액 상에서 안정하게 분산되어 광응답제의 난용성 부작용을 감소시킬 수 있으며, 특정 암세포와 특이적으로 결합하는 압타머에 의해 광응답제의 암세포 표적화를 증가시켰으며, 광 조사시 암세포와 결합된 광응답제가 생성하는 일항산소로 인하여 효과적인 항암 치료가 가능하다. The present invention relates to an abatumer; Polymer; And a light-responsive agent, and more particularly, to a conjugation product of an abatumer-hydrophilic polymer-light-responsive agent conjugate, which is stably dispersed in an aqueous solution, so that an incompatible side effect of the light- And it is possible to increase the target of cancer cell targeting of the photoresponsive agent by the tympanic membrane which specifically binds to specific cancer cells and to perform effective chemotherapy by the united oxygen generated by the photoresponsive agent combined with cancer cells during the light irradiation .

Description

압타머-친수성 고분자-광응답제 접합체 및 이의 제조방법{Aptamer-hydrophilic polymer-photosensitizer conjugates and preparation method thereof}Aptamer-hydrophilic polymer-photoresponsive conjugate and method for preparing the same

본 발명은 친수성이 증가된 광응답제를 이용한 압타머-친수성 고분자-광응답제 접합체 및 이의 제조방법에 관한 것이다.The present invention relates to a plastomer-hydrophilic polymer-light-responsive conjugate using a photoresponsive agent having increased hydrophilicity and a method for producing the same.

광역학 치료(photodynamic therapy, PDT)는 각종 종양에 대한 선택성 및 광증감성이 있는 광응답제(photosensitizer, PS)를 이용하여 수술 없이 암 등의 난치병을 치료하는 기술로, 암의 진단과 치료, 자가골수이식, 항생제, AIDS 치료, 피부이식 수술 및 관절염 등의 치료에서 환자의 면역성을 높이기 위해 사용되는 등 응용 범위가 점차 확대되고 있다. Photodynamic therapy (PDT) is a technique to treat intractable diseases such as cancer by using photosensitizer (PS), which has selectivity for various tumors and sensitivity to photosensitivity. The application range is being expanded to improve immunity of patients in bone marrow transplantation, antibiotics, AIDS treatment, skin transplantation surgery and arthritis.

암을 비롯한 다양한 질병을 치료하기 위해, 상기 광응답제를 정맥주사에 의해 대상자에 투여하고, 빛을 조사하면 그로 인해 생성된 활성산소종(reactive oxygen species, ROS)이 질병 부위의 세포를 파괴 및 궤멸시키게 된다. 이러한 광역학 치료는 정상 세포를 보존하면서 질병 부위의 세포만을 선택적으로 제거할 수 있다. 광응답제에는 포르피린(porphyrin) 유도체, 클로린(chlorin), 박테리오클로린(bacteriochlorin), 프탈로시아닌(phthalocyanine) 등이 있고, 화합물의 특성상 형광이나 인광을 나타내므로 종양의 조기진단으로도 활용될 수 있다. In order to treat various diseases including cancer, the photoreactive agent is administered to a subject by intravenous injection, and a reactive oxygen species (ROS) generated by irradiation of light is destroyed and destroyed It is collapsed. These photodynamic therapies can selectively remove only cells in the diseased area while preserving normal cells. Light responsive agents include porphyrin derivatives, chlorin, bacteriochlorin, and phthalocyanine, which can be used for early diagnosis of tumors because they exhibit fluorescence or phosphorescence depending on the nature of the compound.

압타머(aptamer)는 특이적이고 안정된 삼차구조를 가지면서 표적 분자에 대하여 높은 친화성과 특이성으로 결합할 수 있는 단일가닥 DNA, RNA 또는 변형핵산을 이르는 말로, 1990년에 SELEX(Systematic Evolution of Ligands by EXponential enrichment) 기술이 개발된 이후, 이온, 약물, 펩타이드, 효소와 같은 단백질, 세포와 같은 다양한 표적분자에 결합할 수 있는 다양한 압타머가 지속적으로 발굴되고 있다. An aptamer is a term that refers to a single stranded DNA, RNA or modified nucleic acid that has a specific, stable tertiary structure and can bind with high affinity and specificity to a target molecule. In 1990, a systematic Evolution of Ligands by EXpon enrichment techniques have been developed, various aptamers that can bind to various target molecules such as ions, drugs, peptides, enzymes, proteins, and cells have been continuously discovered.

압타머는 높은 친화성과 특이성으로 표적분자에 결합할 수 있는 특성 덕분에 종종 단일 항체와 비교되는데, 항체와 달리 압타머는 염기서열만 알면 생체 밖에서 화학적으로 합성이 가능하므로 대량생산이 가능하여 'chemical antibody'라 불리며 항체를 대체할 물질로서의 높은 가능성을 보이고 있다. Aptamers are often compared to single antibodies because of their high affinity and specificity for their ability to bind to target molecules. Unlike antibodies, aptamers can be synthesized chemically outside the body by knowing the base sequence, And has a high possibility as a substitute for an antibody.

항체는 분자 구조가 커서 (~150 kDa) 생산하기 어렵고 부가적인 변형이 어렵지만, 압타머는 약 20~60 mer 길이의 핵산 올리고머로, 여러 필요한 변형이 용이하며, 진단을 위한 형광물질을 분자량이 큰 항체에 접합하는 경우, 형광물질의 접합 개수가 일정하지 않아 원하는 접합체를 분리, 정제하는 것이 쉽지 않은 반면에, 분자량이 작은 압타머와 형광물질을 접합하는 경우 1 대 1 접합이 가능하며, 분리, 정제 과정이 훨씬 용이하다. 또한, 압타머는 항체에 비해 안정성이 매우 높다. Although the antibody has a large molecular structure (~ 150 kDa) and is difficult to add and modify, the aptamer is a nucleic acid oligomer having a length of about 20 to 60 mer, and various necessary modifications are easy. , It is not easy to separate and purify the desired conjugate because the number of the fluorescent substance is not constant. On the other hand, when the fluorescent substance is conjugated with the fluorescent substance having a small molecular weight, one-to-one bonding is possible. The process is much easier. In addition, aptamers are very stable compared to antibodies.

단백질로 이루어진 항체는 우선 pH와 온도에 굉장히 민감하고, 체내에서 소수성 상호작용에 의하여 응집될 수 있으며, 실온 보관 및 운반이 불가능하지만, 압타머의 경우에는 실온 보관 및 운반이 가능하고 심지어 멸균 후에도 기능을 유지할 수 있어, 만약 변성이 되더라도 단시간 내에 재생이 가능하기 때문에 특히 장시간 또 반복사용이 요구되는 진단용으로의 응용이 매우 용이하다.Antibodies composed of proteins are very sensitive to pH and temperature. They can be aggregated by hydrophobic interaction in the body and can not be stored and transported at room temperature. However, in the case of platemer, it is possible to store and transport at room temperature and even after sterilization Therefore, even if denaturation occurs, regeneration is possible in a short period of time. Therefore, it is very easy to apply it to diagnostics, which requires repeated use for a long time.

항체는 동물이나 세포를 이용하여 생산하기 때문에 많은 시간과 비용이 요구되고, 생산된 시기에 따라 항체의 기능성이 달라질 가능성(batch to batch variation)이 있으나, 압타머는 화학적 합성방법을 이용하기 때문에 단시간에 적은 비용으로 대량생산이 가능하고, batch to batch variation이 거의 없으며, 높은 순도의 정제과정이 매우 용이하여 생산적인 측면에서 탁월한 장점을 갖고 있다.Since antibodies are produced using animals or cells, they require a lot of time and cost, and there is a possibility of batch to batch variation depending on the time of production. However, since aptamers use chemical synthesis methods, It can be mass-produced at low cost, has almost no batch to batch variation, and has a great advantage in terms of productivity because it is very easy to purify with high purity.

또한, 항체에 물질을 접하는 경우, 항체에 기본적으로 존재하는 thiol group, amine group 등의 반응기에 물질이 무작위적으로 부착되기 때문에 항원결합부위에 부착될 수 있고 균일한 결과물이 제조되기에 어려움이 있으며, 항체는 Fab 부위 (Fab region, fragment antigen-binding region)와 Fc 부위 (Fc region, fragment crystallizable region)로 이루어져 있는데, 이 중에서 Fc 부위는 체내 보체 단백질이나 세포막에 존재하는 Fc 수용체 (Fc receptor)와 상호작용하여 체내 면역반응을 활성화시키는 반면에, 압타머는 Fc 부위가 존재하지 않는 핵산 올리고머 형태이기 때문에, 생체 내에 적용하였을 때 항체나 다른 의약용 단백질의 경우 쉽게 나타나는 생체 내 면역반응이 거의 일어나지 않는 것으로 알려져 있으며, 이는 치료용으로의 개발연구에 매우 중요한 장점이다. In addition, when a substance is brought into contact with an antibody, since the substance is randomly attached to a reaction unit such as a thiol group or an amine group, which is basically present in an antibody, it can be attached to an antigen binding site and it is difficult to produce a homogeneous product , The antibody consists of a Fab region and a fragment antigen-binding region and an Fc region (Fc region, fragment crystallizable region). Among these, the Fc region includes the Fc receptor (Fc receptor) Interacts to activate the body's immune response, whereas aptamer is a nucleic acid oligomer form in which the Fc region is absent, so that the in vivo immune response, which is readily apparent in the case of antibodies or other medicinal proteins when applied in vivo, hardly occurs This is a very important advantage for development studies for therapeutic use.

최근에는 다양한 표적분자에 결합할 수 있는 다양한 압타머가 지속적으로 발굴되기 때문에 항체를 만들기 어려운 독소나 단백질 복합체, 당과 단백질 복합체에 대한 압타머를 만들 수 있다. 이처럼 다양한 장점을 가지는 압타머를 생체 내 이미징에 적용하면, 작은 크기 덕분에 항체에 비해 더 빠른 반응속도를 나타내고 더 우수한 신호-노이즈 비를 가질 수 있다. 또한 압타머와 광응답제를 접합하여 내시경에 적용하면 질병 부위를 눈으로 확인하면서 진단과 치료를 즉각적으로 할 수 있다는 장점을 가지고 있다. 면역반응의 활성화와 관련된 항체를 이용할 경우에는 감염성 질환을 중심으로 정성적 검사가 가능하지만 압타머를 활용하면 감염성 질환을 비롯한 만성질환까지 진단이 가능하며 정성적, 정량적 검사를 할 수 있다. In recent years, a variety of aptamers that can bind to various target molecules have been continuously unearthed, making it possible to produce a complex of toxins or protein complexes and sugar-protein complexes that are difficult to make antibodies. When applied to in vivo imaging, aptamer with these various advantages can result in faster reaction rates and better signal-to-noise ratios than antibodies due to its small size. In addition, when applied to an endoscope by bonding an abdominal thermometer and a light-responsive agent, it has an advantage that the diagnosis and treatment can be performed promptly while confirming the diseased part with eyes. When antibodies related to the activation of immune response are used, it is possible to perform qualitative tests focusing on infectious diseases. However, when using an urethrometer, chronic diseases including infectious diseases can be diagnosed and qualitative and quantitative tests can be performed.

따라서, 암치료제로써의 압타머는 성장인자나 성장인자 수용체를 막음으로써 암세포 성장 신호경로를 직접 억제하거나, 암세포 치료분자를 타겟세포로 운반하는 'escort aptamer'로 사용될 수 있으므로, 다른 암세포 타입이나 암세포 표면 단백질을 타겟팅하는 특이적인 압타머의 수득은 신규 암진단제 및 치료제의 개발에 도움이 된다.Therefore, aptamer as a cancer treatment agent can be used as an 'escort aptamer' that directly inhibits the cancer cell growth signal pathway by blocking growth factors or growth factor receptors, or carries a cancer cell therapy molecule to a target cell, Obtaining a specific tympanic membrane targeting a protein is helpful in the development of novel cancer diagnostic and therapeutic agents.

한국공개특허 제10-2015-0011521호(2015년 2월 2일 공개)Korean Patent Publication No. 10-2015-0011521 (published on February 2, 2015)

본 발명은 질병 부위 표지 및 질병 부위 치료를 위해 부가적으로 형광 물질을 접합하거나 항암제를 봉입하여 전달하는 기존의 광응답제 접합체의 생체안정성 및 질환 표적성 문제를 해결하기 위해, 친수성 고분자 및 압타머가 결합된 광응답제 접합체를 제조하고 이를 암 진단 및 치료제로 사용하는 기술에 관한 것이다.In order to solve the problem of biostability and disease specificity of a conventional photoreactive receptor conjugate conjugating a fluorescent substance or encapsulating an anticancer agent for the treatment of disease site markers and disease sites, the present invention provides a hydrophilic polymer and an aptamer To a conjugated photoresponsive conjugate and to a technique for using the conjugate as a diagnostic and therapeutic agent for cancer.

본 발명은 압타머, 고분자, 및 광응답제로 이루어진 압타머-친수성 고분자-광응답제 접합체를 제공한다.The present invention provides an abatumer-hydrophilic polymer-light-responsive agent conjugate consisting of an abtamer, a polymer, and a light-responsive agent.

또한, 본 발명은 광응답제를 촉매가 포함된 유기용매에 용해시키는 단계(제1단계); 친수성 고분자를 유기용매에 용해시키는 단계(제2단계); 상기 제1단계 용해물과 제2단계 용해물을 혼합하여 반응시키는 단계(제3단계); 상기 제3단계의 반응혼합물을 여과하여 친수성 고분자-광응답제 접합체를 수득하는 단계(제4단계); 상기 제4단계의 친수성 고분자-광응답제 접합체를 촉매가 포함된 증류수에 용해시키고 압타머를 첨가하여 반응시키는 단계(제5단계); 및 상기 제5단계의 반응혼합물을 여과하고 원심분리하여 압타머-친수성 고분자-광응답제 접합체를 수득하는 단계(제6단계)를 포함하는 압타머-친수성 고분자-광응답제 접합체 제조방법을 제공한다.The present invention also relates to a method of manufacturing a light-emitting device, comprising the steps of: (1) dissolving a light-responsive agent in an organic solvent containing a catalyst; Dissolving the hydrophilic polymer in an organic solvent (second step); Mixing and reacting the first-stage liquor with the second-stage liquor (third stage); Filtering the reaction mixture in the third step to obtain a hydrophilic polymer-light responsive conjugate (step 4); Dissolving the hydrophilic polymer-light-responsive agent conjugate in the fourth step in distilled water containing a catalyst, and adding an extruder to react (Step 5); And a step of filtering and centrifuging the reaction mixture in the fifth step to obtain a conjugate of hydrothermal-hydrophilic polymer-light-responsive agent (step 6). do.

본 발명은 압타머-친수성 고분자-광응답제 접합체를 유효성분으로 함유하는 암질환 치료용 광역학 조성물을 제공한다.The present invention provides a photomechanical composition for the treatment of cancer diseases, which comprises a platemaker-hydrophilic polymer-light-responsive agent conjugate as an active ingredient.

또한, 본 발명은 압타머-친수성 고분자-광응답제 접합체를 유효성분으로 함유하는 암질환 진단용 광역학 조성물을 제공한다.The present invention also provides a photomechanical composition for diagnosing cancer diseases, which comprises a platemaker-hydrophilic polymer-light-responsive agent conjugate as an active ingredient.

본 발명은 압타머와 친수성 고분자-광응답제 접합체의 1:1 반응로 균일한 압타머-친수성 고분자-광응답제 접합체를 제조할 수 있으며, 상기 압타머-친수성 고분자-광응답제 접합체는 수용액 상에서 안정하게 분산되어 광응답제의 난용성 부작용을 감소시킬 수 있으며, 특정 암세포와 특이적으로 결합하는 압타머에 의해 광응답제의 암세포 표적화를 증가시켰으며, 광 조사 시 암세포와 결합된 광응답제가 생성하는 일항산소로 인하여 효과적인 항암 치료가 가능하다. The present invention can produce a uniform platemagic-hydrophilic polymer-light responsive conjugate by a 1: 1 reaction between platemaker and a hydrophilic polymer-light responsive conjugate, wherein the platemaker-hydrophilic polymer- And it is possible to reduce the incompatible side effect of the photoresponsive agent, and it is possible to increase the targeting of cancer cells in the photoresponsive agent by aptamer that specifically binds to specific cancer cells. In addition, It is possible to have effective chemotherapy because of the uniaxial oxygen that I generate.

도 1은 본 발명의 압타머-친수성 고분자-광응답제 접합체를 제조하는 과정의 모식도이다.
도 2는 본 발명에 따라 제조된 폴리에틸렌글리콜-헤마토포르피린 접합체의 1H-NMR 결과이다.
도 3는 본 발명에 따라 제조된 폴리에틸렌글리콜-클로린 e6 접합체의 1H-NMR 결과이다.
도 4는 본 발명에 따라 제조된 폴리에틸렌글리콜-프탈로시아닌 접합체의 1H-NMR 결과이다.
도 5는 본 발명에 따라 제조된 압타머-친수성 고분자-광응답제 접합체의 UV-VIS 분석 결과이다.
도 6은 본 발명에 따라 제조된 압타머-친수성 고분자-광응답제 접합체의 제타-포텐셜(Zeta-potential) 분석 결과이다.
도 7은 본 발명에 따라 제조된 압타머-친수성 고분자-광응답제 접합체의 일항산소 생성능 분석 결과이다.
도 8은 본 발명에 따라 제조된 압타머-친수성 고분자-광응답제 접합체의 농도에 따른 정상세포와 암세포에서 세포 독성을 확인한 결과이다.
도 9는 본 발명에 따라 제조된 압타머-친수성 고분자-광응답제 접합체의 농도에 따른 정상세포와 암세포에서의 세포 내 유입을 확인한 공초점 현미경 분석 결과이다.
도 10은 정상세포와 암세포에서 본 발명에 따라 제조된 압타머-친수성 고분자-광응답제 접합체의 세포 내 유입 정도를 확인하기 위해, 유세포 분석기로 세포 내 형광강도를 정량적으로 분석 결과이다.
도 11은 본 발명에 따라 제조된 압타머-친수성 고분자-광응답제 접합체가 처리된 정상세포와 암세포에서 레이저 조사량에 따른 세포독성을 확인한 결과이다.1 is a schematic diagram of a process for producing an abatumer-hydrophilic polymer-light-responsive agent conjugate of the present invention.
2 shows the 1 H-NMR results of the polyethylene glycol-hematoporphyrin conjugate prepared according to the present invention.
3 shows the 1 H-NMR results of the polyethylene glycol-chlorine e6 conjugate prepared according to the present invention.
4 shows the 1 H-NMR results of the polyethylene glycol-phthalocyanine conjugate prepared according to the present invention.
FIG. 5 is a UV-VIS analysis result of the platemer-hydrophilic polymer-light responsive conjugate prepared according to the present invention.
FIG. 6 shows the zeta-potential analysis results of the zeta-hydrophilic polymer-light responsive conjugate prepared according to the present invention.
FIG. 7 shows the results of the analysis of the oxygen production ability of the platemer-hydrophilic polymer-light responsive conjugate prepared according to the present invention.
FIG. 8 shows cytotoxicity of normal cells and cancer cells according to the concentration of the platemaker-hydrophilic polymer-light-responsive agent conjugate prepared according to the present invention.
FIG. 9 is a result of a confocal microscope analysis showing intracellular inflow of normal cells and cancer cells according to the concentration of the platemaker-hydrophilic polymer-light-responsive agent conjugate prepared according to the present invention.
FIG. 10 is a graph showing the results of quantitative analysis of intracellular fluorescence intensity using a flow cytometer in order to confirm the degree of intracellular influx of a platameric-hydrophilic polymer-light responsive conjugate prepared according to the present invention in normal cells and cancer cells.
FIG. 11 shows the cytotoxicity of the platemaker-hydrophilic polymer-light-responsive conjugate prepared according to the present invention in the treated normal cells and cancer cells according to the laser irradiation dose.

본 발명은 압타머, 친수성 고분자, 및 광응답제로 이루어진 압타머-친수성 고분자-광응답제 접합체를 제공할 수 있다.The present invention can provide an abaptamer-hydrophilic polymer-light-responsive agent conjugate consisting of an abtamer, a hydrophilic polymer, and a light-responsive agent.

상기 친수성 고분자 및 광응답제는 에스테르 결합할 수 있으며, 상기 압타머는 친수성 고분자와 펩티드 결합할 수 있다.The hydrophilic polymer and the light-responsive agent may be ester-bonded, and the aptamer may be bonded to the hydrophilic polymer by peptides.

상기 광응답제는 포르피린계 (porphyrin) 화합물, 클로린계 (chlorin)화합물, 박테리오클로린계 (bacteriochlorin) 화합물, 프탈로시아닌계 (phthalocyanine) 화합물, 나프탈로시아닌계 (naphthalocyanine) 화합물 및 5-아미노레불린 에스테르계 (5-aminolevulinic acid ester) 화합물로 이루어진 군에서 선택된 어느 하나일 수 있다.The light responsive agent may be selected from the group consisting of a porphyrin compound, a chlorin compound, a bacteriochlorin compound, a phthalocyanine compound, a naphthalocyanine compound, and a 5- 5-aminolevulinic acid ester) compound.

보다 바람직하게는 상기 광응답제는 헤마토포르피린, 클로린 e6 및 프탈로시아닌으로 이루어진 군에서 선택된 어느 하나일 수 있으나, 이에 한정되는 것은 아니다.More preferably, the light-responsive agent may be any one selected from the group consisting of hematoporphyrin, chlorin e6, and phthalocyanine, but is not limited thereto.

상기 친수성 고분자는 폴리에틸렌글리콜, 플루로닉, 플루란, 히알루론산, 글리콜키토산, 헤파린, 콘트로이친황산, 푸코이단, 덱스트란 및 이의 유도체로 이루어진 군에서 선택된 어느 하나일 수 있으며, 보다 바람직하게는 폴리에틸렌글리콜일 수 있으나, 이에 한정되는 것은 아니다.The hydrophilic polymer may be any one selected from the group consisting of polyethylene glycol, pluronic, pluran, hyaluronic acid, glycol chitosan, heparin, control sulfuric acid, fucoidan, dextran and derivatives thereof, more preferably polyethylene Glycol, but is not limited thereto.

상기 압타머는 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종 및 피부암으로 이루어진 군에 선택된 어느 하나의 암에 특이적으로 나타나는 펩타이드, 단백질 또는 세포에 결합할 수 있다.The above-mentioned aptamer can be used for the treatment of brain tumors, benign astrocytomas, malignant astrocytomas, pituitary adenomas, meningiomas, brain lymphomas, oligodendrogliomas, intracranial lesions, epidermoid tumors, brain tumors, head and neck tumors, laryngeal cancer, Cancer, breast cancer, breast cancer, gastric cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer, small bowel cancer, pancreatic cancer, breast cancer, pancreatic cancer, cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, thoracic tumor, small cell lung cancer, non- Protein or cell that is specifically expressed in any cancer selected from the group consisting of colon cancer, colon cancer, bladder cancer, kidney cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma and skin cancer. .

상기 압타머는 양쪽 말단 중 어느 하나에 아민기를 포함할 수 있다.The aptamer may include an amine group at either end.

보다 상세하게는 종래 기술인 항체를 이용하여 물질을 접합하는 경우, 항체에 기본적으로 존재하는 티올기(thiol group), 아민기(amine group) 등의 반응기에 물질이 무작위적으로 부착되기 때문에 균일한 결과물이 제조되기에 어려운 반면, 본원발명의 압타머는 5번 말단에만 아민기가 존재하므로 친수성 고분자-광응답제 접합체와 1대 1로 반응할 수 있어 균일한 결과물을 제조할 수 있다.More specifically, when a substance is conjugated using a conventional antibody, a substance is randomly attached to a reaction unit such as a thiol group or an amine group, which is basically present in the antibody. Therefore, The aptamer of the present invention can react with the hydrophilic polymer-light-responsive agent conjugate on a one-to-one basis, since an amine group is present only at the 5'-terminal, so that a uniform result can be produced.

또한, 본 발명은 광응답제를 촉매가 포함된 유기용매에 용해시키는 단계(제1단계); 친수성 고분자를 유기용매에 용해시키는 단계(제2단계); 상기 제1단계 용해물과 제2단계 용해물을 혼합하여 반응시키는 단계(제3단계); 상기 제3단계의 반응혼합물을 여과하여 친수성 고분자-광응답제 접합체를 수득하는 단계(제4단계); 상기 제4단계의 친수성 고분자-광응답제 접합체를 촉매가 포함된 증류수에 용해시키고 압타머를 첨가하여 반응시키는 단계(제5단계); 및 상기 제5단계의 반응혼합물을 여과하고 원심분리하여 압타머-친수성 고분자-광응답제 접합체를 수득하는 단계(제6단계)를 포함하는 압타머-친수성 고분자-광응답제 접합체 제조방법을 제공할 수 있다.The present invention also relates to a method of manufacturing a light-emitting device, comprising the steps of: (1) dissolving a light-responsive agent in an organic solvent containing a catalyst; Dissolving the hydrophilic polymer in an organic solvent (second step); Mixing and reacting the first-stage liquor with the second-stage liquor (third stage); Filtering the reaction mixture in the third step to obtain a hydrophilic polymer-light responsive conjugate (step 4); Dissolving the hydrophilic polymer-light-responsive agent conjugate in the fourth step in distilled water containing a catalyst, and adding an extruder to react (Step 5); And a step of filtering and centrifuging the reaction mixture in the fifth step to obtain a conjugate of hydrothermal-hydrophilic polymer-light-responsive agent (step 6). can do.

상기 촉매는 디메틸아미노피리딘, 디사이클로헥실카보디이미드 및 N-하이드록시숙신이미드로 이루어진 군에서 선택된 어느 하나이거나 이들의 혼합물일 수 있다.The catalyst may be any one selected from the group consisting of dimethylaminopyridine, dicyclohexylcarbodiimide and N-hydroxysuccinimide, or a mixture thereof.

상기 유기용매는 디메틸설폭사이드, 포름아마이드 및 디메틸포름아마이드로 이루어진 군에서 선택된 어느 하나일 수 있다.The organic solvent may be any one selected from the group consisting of dimethylsulfoxide, formamide, and dimethylformamide.

본 발명의 일실시예에 따르면, 상기 압타머-친수성 고분자-광응답제 접합체가 암세포 특이적으로 유입되는지를 정량적으로 확인하기 위해, 정상세포인 L-929와 암세포인 MCF-7 및 HCT 116을 배양한 후 압타머-친수성 고분자-광응답제 접합체를 2시간동안 처리하고 완충용액으로 세척한 후 유세포 분석기를 이용하여 세포에 따른 유입 특성을 분석하였다.According to one embodiment of the present invention, in order to quantitatively determine whether the platelet-hydrophilic polymer-light-responsive agent conjugate is specifically infiltrated into cancer cells, L-929, a normal cell, and MCF-7 and HCT 116, cancer cells, After incubation, the platamer-hydrophilic polymer-light-responsive conjugate was treated for 2 hours, washed with buffer solution, and analyzed by flow cytometry using a flow cytometer.

그 결과, 도 10과 같이 정상세포에서는 non-treat군과 sample군의 형광강도가 유사하게 나타난 것이 확인됨에 따라, 정상세포에서는 압타머에 의한 세포 유입이 일어나지 않는 것이 확인된 반면, 암세포에서는 non-treat군에 비해서 압타머-친수성 고분자-광응답제 접합체를 처리한 군(sample군)의 형광강도가 크게 증가하는 것이 확인되었다.As a result, as shown in FIG. 10, it was confirmed that the fluorescence intensities of the non-treat group and the sample group were similar in the normal cells, and thus it was confirmed that the cells were not infiltrated by the squamous cell in the normal cells, the fluorescence intensity of the group treated with the platamer-hydrophilic polymer-light-responsive conjugate (sample group) was significantly increased as compared with the treatment group.

상기 결과로부터 본 발명의 압타머-친수성 고분자-광응답제 접합체는 암세포 특이적으로 유입되는 것을 확인할 수 있었다.From the above results, it was confirmed that the platemaker-hydrophilic polymer-light-responsive conjugate of the present invention was specifically infiltrated into cancer cells.

또한, 본 발명의 다른 일실시예에 따르면, 정상세포인 L-929와 암세포인 MCF-7 및 HCT 116 세포에 상기 압타머-친수성 고분자-광응답제 접합체를 처리한 후 레이저를 조사해주었을 때 정상세포 및 암세포에서의 세포 생존율의 변화를 분석한 결과, 도 11과 같이 정상세포에서는 조사해준 레이저의 세기에 관계없이 세포 독성이 나타나지 않은 반면, 뉴클레올린이 과 발현된 암세포에서는 압타머에 의한 세포 내 유입이 일어나기 때문에 동일한 농도의 압타머-친수성 고분자-광응답제 접합체를 처리하고 레이저 조사량을 늘릴수록 세포 독성이 증가하는 것을 확인할 수 있었다. In addition, according to another embodiment of the present invention, when the above-described platameric-hydrophilic polymer-light responsive conjugate is treated with a normal cell, L-929 and cancer cells MCF-7 and HCT 116 cells, As a result of analyzing the change of cell viability in normal cells and cancer cells, cytotoxicity was not observed regardless of the intensity of the laser irradiated in normal cells as shown in Fig. 11, whereas in cytotoxicity of necleolin and expressed cancer cells, It was confirmed that cytotoxicity was increased as the dose of platamer-hydrophilic polymer-light-responsive agent was increased and the laser dose was increased.

상기 결과들로부터 본 발명의 압타머-친수성 고분자-광응답제 접합체는 암세포 내로 효과적으로 유입되며, 레이저 조사 시 일항산소를 효과적으로 생성함으로써 암세포의 독성을 유도하는 것이 확인되었다.From the above results, it was confirmed that the platemaker-hydrophilic polymer-light-responsive agent conjugate of the present invention is effectively introduced into cancer cells and induces toxicity of cancer cells by effectively producing unilateral oxygen during laser irradiation.

따라서, 본 발명은 압타머-친수성 고분자-광응답제 접합체를 유효성분으로 함유하는 암질환 치료용 광역학 조성물을 제공한다.Accordingly, the present invention provides a photomechanical composition for the treatment of cancer diseases, which comprises a conjugate of platemer-hydrophilic polymer-light-responsive agent as an active ingredient.

또한, 본 발명은 압타머-친수성 고분자-광응답제 접합체를 유효성분으로 함유하는 암질환 진단용 광역학 조성물을 제공한다.The present invention also provides a photomechanical composition for diagnosing cancer diseases, which comprises a platemaker-hydrophilic polymer-light-responsive agent conjugate as an active ingredient.

이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<< 실시예Example 1> 친수성이  1> hydrophilic 증가된Increased 고분자- The polymer- 광응답제Light responsive agent 접합체 제조 Assembly

1. One. 폴리에틸렌글리콜Polyethylene glycol -- 헤마토포르피린Hematoporphyrin 접합체 제조 Assembly

헤마토포르피린(hematoporphyrin; HP) 0.043g, 디사이클로헥실카보디이미드 (dicyclohexylcarbodiimide; DCC) 0.022g과 디메틸아미노피리딘(4-dimethylaminopyridine; DMAP) 0.013g을 50ml 플라스크에서 디메틸설폭사이드(dimethylsulfoxid; DMSO) 5ml에 용해시키고, 폴리에틸렌글리콜(polyethylene glycol; PEG) 0.2g을 디메틸설폭사이드 5ml에 녹여서 각각 6시간 동안 교반하였다.0.022 g of hematoporphyrin (HP), 0.022 g of dicyclohexylcarbodiimide (DCC) and 0.013 g of 4-dimethylaminopyridine (DMAP) were dissolved in 5 ml of dimethylsulfoxide (DMSO) in a 50 ml flask , 0.2 g of polyethylene glycol (PEG) was dissolved in 5 ml of dimethylsulfoxide, and the mixture was stirred for 6 hours each.

상기 두 용액을 섞은 후에 상온에서 24시간 반응시켰고, 반응에 참여하지 않은 물질과 불순물을 제거하기 위하여 필터링을 한 후 투석막(molecular weight cut-off size : 1,000 Da)을 이용하여 2일 동안 1차 증류수를 이용하여 투석하였다. 투석 후 최종 반응물은 동결 건조를 통하여 건조하여 폴리에틸렌글리콜-광응답제 접합체를 수득하였다. After the two solutions were mixed, the reaction was carried out at room temperature for 24 hours. To remove unreacted substances and impurities, the filtrate was filtered and washed with distilled water (distilled water) for 2 days using a molecular weight cut- Was dialyzed. After dialysis, the final reaction product was lyophilized and dried to obtain a polyethylene glycol-light-responsive conjugate.

폴리에틸렌글리콜Polyethylene glycol -- 헤마토포르피린Hematoporphyrin 접합체  Junction 1One H-NMR 분석H-NMR analysis

상기 방법으로 제조된 폴리에틸렌글리콜-광응답제 접합체의 접합 여부를 확인하기 위하여, (a) 실시예 1, (b) 폴리에틸렌글리콜, (c) 광응답제를 각각 NMR용 디메틸설폭사이드에 분산하여 측정하였다. (A) Example 1, (b) polyethylene glycol, and (c) light-responsive agent were dispersed in dimethylsulfoxide for NMR measurement, respectively, to confirm whether or not the polyethylene glycol-light- Respectively.

그 결과 도 2와 같은 데이터를 얻었다.As a result, data as shown in FIG. 2 was obtained.

1-2. 1-2. 폴리에틸렌글리콜Polyethylene glycol -- 클로린Chlorine e6 접합체 제조 Preparation of e6 conjugates

클로린 e6(chlorin e6; Ce6) 0.047g, 디사이클로헥실카보디이미드 0.018g과 디메틸아미노피리딘 0.011g을 50ml 플라스크에서 디메틸설폭사이드 5ml에 용해시키고, 폴리에틸렌글리콜 0.2g을 디메틸설폭사이드 5ml에 녹여서 각각 6시간 동안 교반하였다.0.047 g of dicyclohexylcarbodiimide and 0.011 g of dimethylaminopyridine were dissolved in 5 ml of dimethylsulfoxide in a 50 ml flask and 0.2 g of polyethylene glycol was dissolved in 5 ml of dimethylsulfoxide to prepare 6 Lt; / RTI &gt;

상기 두 용액을 섞은 후에 상온에서 24시간 반응시키고, 반응에 참여하지 않은 물질과 불순물을 제거하기 위하여 필터링을 한 후 투석막(molecular weight cut-off size : 1,000 Da)을 이용하여 2일 동안 1차 증류수를 이용하여 투석하였다. 투석 후 최종 반응물은 동결 건조를 통하여 건조하여 폴리에틸렌글리콜-광응답제 접합체를 수득하였다. After the two solutions were mixed, the mixture was reacted at room temperature for 24 hours, filtered to remove impurities and impurities, and then filtered using a dialysis membrane (molecular weight cut-off size: 1,000 Da) Was dialyzed. After dialysis, the final reaction product was lyophilized and dried to obtain a polyethylene glycol-light-responsive conjugate.

폴리에틸렌글리콜Polyethylene glycol -- 클로린Chlorine e6  e6 1One H-NMR 분석H-NMR analysis

상기 방법으로 제조된 폴리에틸렌글리콜-광응답제 접합체의 접합 여부를 확인하기 위하여, (a) 실시예 1-2, (b) 폴리에틸렌글리콜, (c) 클로린 e6을 각 NMR용 디메틸설폭사이드에 분산하여 측정하였다. (A) Example 1-2, (b) polyethylene glycol, and (c) chlorine e6 were dispersed in dimethyl sulfoxide for each NMR to confirm whether or not the polyethylene glycol-light- Respectively.

그 결과 도 3과 같은 데이터를 얻었다.As a result, data as shown in FIG. 3 was obtained.

1-3. 폴리에틸렌글리콜-프탈로시아닌 접합체 제조1-3. Preparation of polyethylene glycol-phthalocyanine conjugate

폴리에틸렌글리콜 0.01ml, 디사이클로헥실카보디이미드 0.022g과 디메틸아미노피리딘 0.013g을 25ml 플라스크에서 디메틸설폭사이드 5ml에 용해시키고, 폴리에틸렌글리콜-프탈로시아닌(polyethylene glycol-phthalocyanine, PEG-PC) 0.2g을 디메틸설폭사이드 5ml에 녹여서 각각 6시간 동안 교반하였다.0.01 g of polyethylene glycol, 0.022 g of dicyclohexylcarbodiimide and 0.013 g of dimethylaminopyridine were dissolved in 5 ml of dimethyl sulfoxide in a 25 ml flask, and 0.2 g of polyethylene glycol-phthalocyanine (PEG-PC) Dissolved in 5 ml of the side, and stirred for 6 hours each.

상기 두 용액을 섞은 후에 상온에서 24시간 반응시키고, 반응에 참여하지 않은 물질과 불순물을 제거하기 위하여 필터링을 한 후 투석막(molecular weight cut-off size : 1,000 Da)을 이용하여 2일 동안 1차 증류수를 이용하여 투석하였다. 투석 후 최종 반응물은 동결 건조를 통하여 건조하여 폴리에틸렌글리콜-광응답제 접합체를 수득하였다.After the two solutions were mixed, the mixture was reacted at room temperature for 24 hours, filtered to remove impurities and impurities, and then filtered using a dialysis membrane (molecular weight cut-off size: 1,000 Da) Was dialyzed. After dialysis, the final reaction product was lyophilized and dried to obtain a polyethylene glycol-light-responsive conjugate.

폴리에틸렌글리콜Polyethylene glycol -- 프탈로시아닌Phthalocyanine 1One H-NMR 분석H-NMR analysis

상기 방법으로 제조된 폴리에틸렌글리콜-광응답제 접합체의 접합 여부를 확인하기 위하여, (a) 실시예 1-3, (b) 폴리에틸렌글리콜, (c) 폴리에틸렌글리콜-프탈로시아닌 을 각 NMR용 디메틸설폭사이드에 분산하여 측정하였다. (A) Examples 1-3, (b) polyethylene glycol, and (c) polyethylene glycol-phthalocyanine were dissolved in dimethylsulfoxide for each NMR to confirm whether or not the conjugation of the polyethylene glycol- .

그 결과 도 4와 같은 데이터를 얻었다.As a result, the data shown in FIG. 4 was obtained.

<< 실시예Example 2>  2> 압타머Abtamer -친수성 고분자-- Hydrophilic polymer - 광응답제Light responsive agent 접합체 제조 Assembly

2-1. 2-1. 압타머Abtamer -- 폴리에틸렌글리콜Polyethylene glycol -- 헤마토포르피린Hematoporphyrin 접합체 제조 Assembly

상기 실시예 1-1에서 수득한 폴리에틸렌글리콜-헤마토포르피린 접합체를 이용하여 하기 반응식 1과 같은 과정으로 압타머-폴리에틸렌글리콜-헤마토포르피린 접합체를 얻었다.Polyethylene glycol-hematoporphyrin conjugate was obtained by the following reaction scheme 1 using the polyethylene glycol-hematoporphyrin conjugate obtained in Example 1-1.

[반응식 1][Reaction Scheme 1]

Figure 112016103076671-pat00001
Figure 112016103076671-pat00001

폴리에틸렌글리콜-헤마토포르피린 접합체 0.015g을 핵산분해효소가 없는 증류수 0.15ml에 녹이고, 에틸디메틸아미노프로필카보디이미드(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDC) 0.012g과 N-하이드록시숙신이미드(N-Hydroxysuccinimide; NHS) 0.002g을 각각 0.2ml에 녹여서 혼합한 다음 상온에서 30분 동안 교반하였다. 압타머(5´-NH2-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3´) 0.001g을 증류수 1ml에 녹여서 활성화된 폴리에틸렌글리콜-헤마토포르피린 접합체 용액에 섞은 후 4시간 동안 반응시키고 정제 필터(molecular weight cut-off size : 10,000 Da)로 옮겨서 원심분리기를 이용하여 반응에 참여하지 않은 물질을 제거하였다.0.015 g of a polyethylene glycol-hematoporphyrin conjugate was dissolved in 0.15 ml of distilled water without nucleic acid degrading enzyme and 0.012 g of ethyldimethylaminopropyl carbodiimide (EDC) and 0.012 g of N- 0.002 g of N-Hydroxysuccinimide (NHS) was dissolved in 0.2 ml of each, and the mixture was stirred at room temperature for 30 minutes. 0.001 g of platamer (5'-NH 2 -TTGGTGGTGGTGGTTGTGGTGGTTGGTGG-3 ') was dissolved in 1 ml of distilled water and mixed with the activated polyethylene glycol-hematoporphyrin conjugate solution. The mixture was reacted for 4 hours and filtered through a molecular weight cut- Da) and removed the unincorporated material using a centrifuge.

2-2. 2-2. 압타머Abtamer -- 폴리에틸렌글리콜Polyethylene glycol -- 클로린Chlorine e6 접합체 제조 Preparation of e6 conjugates

상기 실시예 1-2에서 수득한 폴리에틸렌글리콜-클로린 e6 접합체를 이용하여 하기 반응식 2와 같은 과정으로 압타머-폴리에틸렌글리콜-클로린 e6 접합체를 얻었다.Polyethylene glycol-chlorine e6 conjugate obtained in Example 1-2 was used to obtain an eptaderm-polyethylene glycol-chlorine e6 conjugate in the same manner as in Reaction Scheme 2 below.

[반응식 2][Reaction Scheme 2]

Figure 112016103076671-pat00002
Figure 112016103076671-pat00002

폴리에틸렌글리콜-클로린 e6 접합체 0.015g을 핵산분해효소가 없는 증류수 0.15ml에 녹이고, 에틸디메틸아미노프로필카보디이미드 0.012g과 N-하이드록시숙신이미드 0.002g을 각각 0.2ml에 녹여서 혼합한 다음 상온에서 30분 동안 교반하였다. 압타머(5´-NH2-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3´) 0.001g을 증류수 1ml에 녹여서 활성화 된 폴리에틸렌글리콜-클로린 e6 접합체 용액에 섞은 후 4시간 동안 반응시키고 정제필터(molecular weight cut-off size : 10,000 Da)로 옮겨서 원심분리기를 이용하여 반응에 참여하지 않은 물질을 제거하였다.0.015 g of the polyethylene glycol-chlorine e6 conjugate was dissolved in 0.15 ml of distilled water without nucleic acid degrading enzyme, and 0.012 g of ethyldimethylaminopropylcarbodiimide and 0.002 g of N-hydroxysuccinimide were dissolved in 0.2 ml of each and mixed at room temperature And stirred for 30 minutes. 0.001 g of platamer (5'-NH 2 -TTGGTGGTGGTGGTTGTGGTGGTTGGTGG-3 ') was dissolved in 1 ml of distilled water and mixed in an activated polyethylene glycol-chlorine e6 conjugate solution. The mixture was reacted for 4 hours and filtered using a molecular weight cut- ), And the material not participating in the reaction was removed using a centrifuge.

2-3. 2-3. 압타머Abtamer -- 폴리에틸렌글리콜Polyethylene glycol -- 프탈로시아닌Phthalocyanine 접합체 제조 Assembly

상기 실시예 1-3에서 수득한 폴리에틸렌글리콜-프탈로시아닌 접합체를 이용하여 하기 반응식 3과 같은 과정으로 압타머-폴리에틸렌글리콜-프탈로시아닌 접합체를 얻었다.Polyethyleneglycol-phthalocyanine conjugate obtained in Example 1-3 was used to obtain an adipomer-polyethylene glycol-phthalocyanine conjugate in the same manner as in Reaction Scheme 3 below.

[반응식 3][Reaction Scheme 3]

Figure 112016103076671-pat00003
Figure 112016103076671-pat00003

폴리에틸렌글리콜-프탈로시아닌 접합체 0.015g을 핵산분해효소가 없는 증류수 0.15ml에 녹이고, 에틸디메틸아미노프로필카보디이미드 0.012g과 N-하이드록시숙신이미드 0.002g을 각각 0.2ml에 녹여서 혼합한 다음 상온에서 30분 동안 교반하였다. 압타머(5´-NH2-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3´) 0.001g을 증류수 1ml에 녹여서 활성화 된 폴리에틸렌글리콜-프탈로시아닌 접합체 용액에 섞은 후 4시간 동안 반응시키고 정제필터(molecular weight cut-off size : 10,000 Da)로 옮겨서 원심분리기를 이용하여 반응에 참여하지 않은 물질을 제거하였다.0.015 g of a polyethyleneglycol-phthalocyanine conjugate was dissolved in 0.15 ml of distilled water without nucleic acid degrading enzyme, and 0.012 g of ethyldimethylaminopropylcarbodiimide and 0.002 g of N-hydroxysuccinimide were dissolved in 0.2 ml of each, followed by mixing at room temperature for 30 Lt; / RTI &gt; 0.001 g of platamer (5'-NH 2 -TTGGTGGTGGTGGTTGTGGTGGTTGGTGG-3 ') was dissolved in 1 ml of distilled water and mixed with the activated polyethylene glycol-phthalocyanine conjugate solution. The mixture was reacted for 4 hours and filtered through a molecular weight cut- , And the material not participating in the reaction was removed using a centrifuge.

<< 실험예Experimental Example 1> UV- 1 > UV- VISVIS 흡광분석Absorption analysis

상기 실시예 2에서 제조된 압타머-친수성 고분자-광응답제 접합체의 접합 여부를 확인하기 위하여, 폴리에틸렌글리콜-광응답제 접합체와 압타머-폴리에틸렌글리콜-광응답제 접합체를 UV-VIS 분광광도계를 이용하여 분석하였다.In order to confirm whether or not the conjugate of the platemaker-hydrophilic polymer-light-responsive agent prepared in Example 2 was bonded, the polyethylene glycol-light-responsive conjugate and the platamer-polyethylene glycol-light-responsive conjugate were measured with a UV-VIS spectrophotometer Respectively.

그 결과, 도 5와 같이 압타머를 접합한 후, 260 nm에서 압타머의 흡광도 피크가 나타나는 것이 확인되었다.As a result, it was confirmed that the absorbance peak of the squid polymer was observed at 260 nm after bonding the squid polymer as shown in Fig.

상기 결과로부터 압타머-폴리에틸렌글리콜-광응답제 접합체가 형성된 것을 확인할 수 있었다.From the above results, it was confirmed that the abutmenter-polyethylene glycol-light-responsive conjugate was formed.

<실험예 2> 제타-포텐셜(Zeta-potential) 분석<Experimental Example 2> Zeta-potential analysis

상기 실시예 2에서 제조된 압타머-친수성 고분자-광응답제 접합체의 접합 여부를 확인하기 위하여, 폴리에틸렌글리콜-광응답제 접합체와 압타머-폴리에틸렌글리콜-광응답제 접합체를 제타-포텐셜 분석기기를 이용하여 측정하였다.In order to confirm whether or not the conjugate of the abatumer-hydrophilic polymer-light-responsive agent prepared in Example 2 was bonded, a polyethylene glycol-light-responsive conjugate and an aptamer-polyethylene glycol-light-responsive conjugate were applied to a zeta-potential analyzer .

그 결과, 도 6과 같이 압타머 접합 전과 후의 접합체의 표면 전하가 약 20 mV 차이 나는 것을 확인할 수 있었다.As a result, it was confirmed that the surface charge of the bonded body before and after the tymator bonding was about 20 mV different as shown in FIG.

상기 결과로부터 음전하를 나타내는 압타머가 폴리에틸렌글리콜-광응답제 접합체에 도입되면서 표면 전하가 더욱 강한 음전하를 띠는 방향으로 증가된 것을 확인하였다.From the above results, it was confirmed that aptamer exhibiting a negative charge was introduced into the polyethylene glycol-light-responsive agent conjugate and the surface charge was increased in a direction of stronger negative charge.

<실험예 3> 일항산소 생성능 분석&Lt; Experimental Example 3 >

상기 실시예 2에서 제조된 압타머-친수성 고분자-광응답제 접합체의 광활성 여부를 확인하기 위하여, 수상에서 광응답제와 압타머-폴리에틸렌글리콜-광응답제 접합체의 일항산소 생성능을 형광분광광도계를 이용하여 분석하였다. In order to confirm the photoactivity of the plastomer-hydrophilic polymer-light-responsive agent conjugate prepared in Example 2, the oxygen production ability of the light-responsive agent and the plastomer-polyethylene glycol-light-responsive agent conjugate in the aqueous phase was measured using a fluorescence spectrophotometer Respectively.

일항산소를 검출할 수 있는 singlet oxygen detector로 singlet oxygen sensor green(SOSG)을 2 μM 농도로 제조한 후에 샘플과 1:1 부피비로 혼합하고, 레이저를 50초 간격으로 조사하면서 형광 spectrophotometer를 이용하여 Ex 504nm, Em 525nm에서 형광을 측정하였다.The samples were mixed at 1: 1 volume ratio with a singlet oxygen detector (SOSG) at a concentration of 2 μM, and irradiated at 50-second intervals. The fluorescence spectrophotometer was used to measure Ex The fluorescence was measured at 504 nm and Em 525 nm.

그 결과, 도 7과 같이 수상에서 기존의 광응답제는 일항산소 생성능을 나타내지 못했지만 압타머-광응답제 접합체는 레이저를 5 J/cm2까지 조사하였을 때 일항산소 생성능이 각각 6배(HP), 12.6배(Ce6) 증가하는 것을 확인할 수 있었다.As a result, the conventional light-responsive agents did not show a homogeneous oxygen producing ability aptamer in water as shown in Fig. 7 - photoresponse the conjugate homogeneous oxygen-producing ability is six times (HP) respectively, when irradiated with a laser beam to 5 J / cm 2 , And 12.6 times (Ce6), respectively.

프탈로시아닌의 경우 폴리에틸렌글리콜이 접합되어있는 프탈로시아닌에 카르복실기가 있는 폴리에틸렌글리콜을 도입 후 압타머 접합체를 제조하였기 때문에 대조군으로 기존 폴리에틸렌글리콜-프탈로시아닌을 사용하였으며, 압타머-폴리에틸렌글리콜-프탈로시아닌 접합체 제조 후에도 이전과 유사한 일항산소 생성능을 보이는 것을 확인할 수 있었다.In the case of phthalocyanine, polyethylene glycol-phthalocyanine was used as a control group since polyethylene glycol with carboxyl group was introduced into phthalocyanine to which polyethylene glycol was conjugated. Thus, after the preparation of aptamer-polyethylene glycol-phthalocyanine conjugate, It was confirmed that the oxygen producing ability was observed.

<< 실험예Experimental Example 4>  4> 압타머Abtamer -친수성 고분자-- Hydrophilic polymer - 광응답제Light responsive agent 접합체 농도에 따른 세포 독성 평가 Assessment of cytotoxicity by conjugate concentration

압타머-친수성 고분자-광응답제 접합체가 세포에서 독성을 나타내지 않는 농도를 확인하기 위하여, 정상세포인 L-929(normal fibroblast)와 암세포인 MCF-7(breast cancer cell) 및 HCT 116(colon cancer cell)으로 세포독성 농도를 확인하였다. L-929 (normal fibroblast) and cancer cells MCF-7 (breast cancer cell) and HCT 116 (colon cancer) were used to confirm the concentrations of the platamater-hydrophilic polymer- cell).

세 종류의 세포 주를 96 well 플레이트에 하루 동안 배양한 후 광응답제 농도를 기준으로 하여 샘플을 0-10 ㎍/ml 로 2시간 동안 처리한 후 세포 생존율을 MTT 정량법을 수행하여 분석하였다. Three cell lines were cultured on a 96 - well plate for one day, and the cell viability was analyzed by MTT assay after treating the sample with 0-10.

MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 정량법은 물질이 세포에 얼마나 독성을 나타내는지 확인하는 실험으로, MTT를 처리하면 살아있는 세포에서는 미토콘드리아에 있는 환원효소(reductase)에 의해 MTT가 환원되어 포르마잔(formazan) 결정을 형성하게 된다. 반면에, 죽은 세포에서는 미토콘드리아와 환원효소가 제 기능을 하지 못하므로 포르마잔도 형성될 수 없어 세포생존율에 따라 생성되는 포르마잔양이 달라지는데 이를 정량하여 세포의 생존율을 분석하였다.MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay is an experiment to determine the extent of toxicity of a substance to a cell. MTT treatment reduces the mitochondrial reduction MTT is reduced by an enzyme (reductase) to form formazan crystals. On the other hand, in dead cells, mitochondria and reductase do not function, and therefore, formazan can not be formed. Therefore, the size of forma generated by cell viability varies.

그 결과, 도 8과 같이, 광응답제 농도 기준 1 ㎍/ml보다 높은 농도에서는 압타머-친수성 고분자-광응답제 접합체에 의한 세포 독성이 나타나는 것이 확인되었다.As a result, as shown in FIG. 8, it was confirmed that the cytotoxicity of the plastomer-hydrophilic polymer-light responsive conjugate was exhibited at a concentration higher than the photoresponsive concentration of 1 ㎍ / ml.

<실험예 5> 압타머-친수성 고분자-광응답제 접합체의 세포 내 위치 확인<Experimental Example 5> Determination of intracellular localization of plastomer-hydrophilic polymer-light-responsive conjugate

본 발명에 따른 압타머-친수성 고분자-광응답제 접합체가 암세포 특이적인 세포유입을 나타내는지 확인하였다.It was confirmed that the platemaker-hydrophilic polymer-light-responsive conjugate according to the present invention exhibited cancer cell-specific cell inflow.

먼저, 정상세포인 L-929와 암세포인 MCF-7 및 HCT 116을 배양한 후, 본 발명에 따른 압타머-친수성 고분자-광응답제 접합체를 0-10 ㎍/ml의 농도로 세포에 2시간 동안 처리한 후 완충용액으로 세척하고 4% 파라포름알데히드로 고정하였다. 압타머-친수성 고분자-광응답제 접합체의 농도에 따른 세포 내 분포는 공초점 현미경을 이용하여 시각적으로 확인하였다. First, the normal cell, L-929, and cancer cells MCF-7 and HCT 116 were cultured. Then, the platemaker-hydrophilic polymer-light responsive conjugate according to the present invention was added to cells at a concentration of 0-10 / / ml for 2 hours , Washed with buffer solution and fixed with 4% paraformaldehyde. The intracellular distribution according to the concentration of the platamer-hydrophilic polymer-light-responsive conjugate was visually confirmed using a confocal microscope.

그 결과, 도 9와 같이 정상세포인 L-929에서는 가장 높은 농도인 10 ㎍/ml에서만 약한 형광이 관찰되었고, 암세포인 MCF-7 및 HCT 116에서는 0.1 ㎍/ml 이상의 농도에서 광응답제에 의한 형광이 나타나는 것을 확인할 수 있었다.As a result, weak fluorescence was observed only at the highest concentration of 10 μg / ml in the normal cell L-929 as shown in FIG. 9, and in the cancer cells MCF-7 and HCT 116 at the concentration of 0.1 μg / Fluorescence was observed.

<실험예 6> 압타머-친수성 고분자-광응답제 접합체의 세포 내 유입 확인Experimental Example 6 Confirmation of intracellular inflow of plastomer-hydrophilic polymer-light-responsive conjugate

상기 실험예 4와 실험예 5의 압타머-친수성 고분자-광응답제 접합체의 농도 최적화 결과로부터 세포 처리 농도를 1 ㎍/ml으로 설정하고, 본 발명에 따른 압타머-친수성 고분자-광응답제 접합체가 암세포 특이적으로 유입되는지 정량적으로 확인하기 위해, 정상세포인 L-929와 암세포인 MCF-7 및 HCT 116을 배양한 후 압타머-친수성 고분자-광응답제 접합체를 2시간 동안 처리하고 완충용액으로 세척한 후 유세포 분석기를 이용하여 세포에 따른 유입 특성을 분석하였다.The cell treatment concentration was set to 1 쨉 g / ml from the concentration optimization result of the conjugate of hydrothermal-hydrophilic polymer-light-responsive agent of Experimental Example 4 and Experimental Example 5, and the adherent-hydrophilic polymer- L-929 and MCF-7 and HCT 116, which are cancer cells, were cultured for 2 hrs. After the incubation for 2 hours, the platelet-hydrophilic polymer-light- And analyzed by flow cytometry using a flow cytometer.

그 결과, 도 10과 같이 정상세포에서는 non-treat군과 sample군의 형광강도가 유사하게 나타난 반면, 암세포에서는 non-treat군에 비해서 압타머-친수성 고분자-광응답제 접합체를 처리한 군(sample군)의 형광강도가 크게 증가하는 것을 확인할 수 있었다.As a result, the fluorescence intensities of the non-treat group and the sample group were similar in the normal cells as shown in FIG. 10, while the fluorescence intensities of the cancer cells in the non-treat group The fluorescence intensities of the cells were significantly increased.

상기 결과로부터 정상세포에서는 압타머에 의한 세포 유입이 일어나지 않는 것이 확인되었다.From the above results, it was confirmed that cell infiltration by the platemer did not occur in normal cells.

<실험예 7> 레이저 조사량에 따른 세포 독성 평가<Experimental Example 7> Evaluation of cytotoxicity according to laser dose

앞선 실험결과로부터 압타머-친수성 고분자-광응답제 접합체의 암세포 선택적 분포 양상을 확인하였으며, 추가적으로 레이저를 조사해주었을 때 정상세포 및 암세포에서의 세포 생존율의 변화를 분석하였다. From the results of the previous experiments, we confirmed the selective distribution of cancer cells in the platelet - hydrophilic polymer - light responsive conjugate, and analyzed the changes in cell viability in normal and cancer cells when irradiated with laser.

먼저, 정상세포인 L-929와 암세포인 MCF-7 및 HCT 116을 배양한 후 압타머-친수성 고분자-광응답제 접합체를 2시간 동안 처리하고 레이저를 0-5 J/cm2 세기로 조사한 후 다시 배양기에서 하루 동안 배양시켰다. First, L-929, a cancer cell, and MCF-7 and HCT 116, cancer cells, were cultured and treated with a platamer-hydrophilic polymer-light responsive conjugate for 2 hours and irradiated with 0-5 J / cm 2 laser And cultured for another day in an incubator.

배양이 끝난 세포에 MTT 시약을 처리하여 3~4시간 동안 배양시킨 후 배양액과 MTT 시약 등을 모두 제거하고 디메틸설폭사이드를 가하여 세포에 형성된 포르마잔을 용해시켰다. 그 후 570nm에서 흡광도를 측정하여 형성된 포르마잔 양을 비교하여 세포의 생존율 및 압타머-친수성 고분자-광응답제 접합체의 세포 독성을 분석하였다.After culturing, cells were treated with MTT reagent and cultured for 3 to 4 hours. Then, the culture solution and MTT reagent were all removed, and dimethylsulfoxide was added to dissolve the formazan formed in the cells. Then, the absorbance at 570 nm was measured to compare the amount of formazan formed, and the cell viability and cytotoxicity of the plastomer-hydrophilic polymer-light responsive conjugate were analyzed.

그 결과, 도 11과 같이 정상세포와 암세포 모두에서 레이저를 조사하지 않은 군에서는 세포 독성이 나타나지 않았으며, 정상세포에서는 조사해준 레이저의 세기에 관계없이 세포 독성이 나타나지 않았다. 반면, 뉴클레올린이 과 발현된 암세포에서는 압타머에 의한 세포 내 유입이 일어나기 때문에 동일한 농도의 압타머-친수성 고분자-광응답제 접합체를 처리하고 레이저 조사량을 늘릴수록 세포 독성이 증가하는 것이 확인되었다. As a result, as shown in Fig. 11, no cytotoxicity was observed in the non-irradiated group in both normal cells and cancer cells, and no cytotoxicity was observed in normal cells regardless of the intensity of the irradiated laser. On the other hand, it was confirmed that cytotoxicity was increased with treatment of the same concentration of platamer-hydrophilic polymer-light-responsive conjugate and increase of laser dose, since intracellular infiltration by necleolin and expressed cancer cells occurred.

상기 결과로부터 본 발명의 압타머-친수성 고분자-광응답제 접합체가 암세포 내에 효과적으로 유입되며, 이를 통하여 레이저 조사 시 효과적으로 일항산소가 생성되어 암세포에 독성을 유도함으로써 암을 치료할 수 있다는 사실이 확인되었다.From the above results, it has been confirmed that the platemaker-hydrophilic polymer-light-responsive agent conjugate of the present invention is efficiently introduced into cancer cells, and thus, an anoxic oxygen is effectively generated upon laser irradiation to induce toxicity to cancer cells.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (12)

삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 클로린 e6를 촉매가 포함된 유기용매에 용해시키는 단계(제1단계);
폴리에틸렌글리콜을 유기용매에 용해시키는 단계(제2단계);
상기 제1단계 용해물과 제2단계 용해물을 혼합하여 반응시키는 단계(제3단계);
상기 제3단계의 반응혼합물을 여과하여 폴리에틸렌글리콜-클로린 e6 접합체를 수득하는 단계(제4단계);
상기 제4단계의 폴리에틸렌글리콜-클로린 e6 접합체를 촉매가 포함된 증류수에 용해시키고 압타머를 첨가하여 반응시키는 단계(제5단계); 및
상기 제5단계의 반응혼합물을 여과하고 원심분리하여 압타머-폴리에틸렌글리콜-클로린 e6 접합체를 수득하는 단계(제6단계)를 포함하는 압타머-폴리에틸렌글리콜-클로린 e6 접합체 제조방법.Dissolving chlorin e6 in an organic solvent containing a catalyst (first step);
Dissolving polyethylene glycol in an organic solvent (second step);
Mixing and reacting the first-stage liquor with the second-stage liquor (third stage);
Filtering the reaction mixture of the third step to obtain a polyethylene glycol-chlorine e6 conjugate (step 4);
Dissolving the polyethylene glycol-chlorine e6 conjugate of the fourth step in distilled water containing a catalyst and adding an extruder to react (step 5); And
Polyethylene glycol-chlorine e6 conjugate (step 6) by filtration and centrifugation of the reaction mixture in the fifth step to obtain an eptaderm-polyethylene glycol-chlorine e6 conjugate (step 6). 청구항 8에 있어서, 상기 촉매는 디메틸아미노피리딘 및 디사이클로헥실카보디이미드로 이루어진 군에서 선택된 어느 하나이거나 이들의 혼합물인 것을 특징으로 하는 압타머-폴리에틸렌글리콜-클로린 e6 접합체 제조방법.[Claim 8] The method according to claim 8, wherein the catalyst is any one selected from the group consisting of dimethylaminopyridine and dicyclohexylcarbodiimide, or a mixture thereof. 청구항 8에 있어서, 상기 유기용매는 디메틸설폭사이드, 포름아마이드 및 디메틸포름아마이드로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 압타머-폴리에틸렌글리콜-클로린 e6 접합체 제조방법.[Claim 9] The method according to claim 8, wherein the organic solvent is any one selected from the group consisting of dimethylsulfoxide, formamide, and dimethylformamide. 삭제delete 삭제delete
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