KR102629203B1 - Nanoparticle conjugated with astrocyte-selective peptide and uses thereof - Google Patents
Nanoparticle conjugated with astrocyte-selective peptide and uses thereof Download PDFInfo
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- KR102629203B1 KR102629203B1 KR1020210069959A KR20210069959A KR102629203B1 KR 102629203 B1 KR102629203 B1 KR 102629203B1 KR 1020210069959 A KR1020210069959 A KR 1020210069959A KR 20210069959 A KR20210069959 A KR 20210069959A KR 102629203 B1 KR102629203 B1 KR 102629203B1
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- astrocyte
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- peptide
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Abstract
본 발명은 별아교세포 선택적 펩타이드가 결합된 나노입자에 관한 것으로, 보다 상세하게는 상기 나노입자를 포함하는 허혈성 뇌질환 예방, 개선 또는 치료용도에 관한 것이다. 본 발명에 따른 별아교세포 선택적 펩타이드가 결합된 나노입자는 생체적합성 및 생분해성이 우수하며, 동물 수준에서 허혈성 뇌손상을 치료하는 효과가 있음을 확인하였다. 이는 본 발명의 나노입자가 허혈성 뇌질환의 치료제로서 이용될 수 있음을 의미하는바, 허혈성 뇌질환의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.The present invention relates to nanoparticles bound with an astrocyte-selective peptide, and more specifically, to the use of nanoparticles for preventing, improving, or treating ischemic brain disease. It was confirmed that the nanoparticles combined with the astrocyte-selective peptide according to the present invention have excellent biocompatibility and biodegradability, and are effective in treating ischemic brain damage at the animal level. This means that the nanoparticles of the present invention can be used as a treatment for ischemic brain disease, and can be used in a variety of fields in the fields of prevention, improvement, and treatment of ischemic brain disease.
Description
본 발명은 별아교세포 선택적 펩타이드가 결합된 나노입자에 관한 것으로, 보다 상세하게는 상기 나노입자를 포함하는 허혈성 뇌질환 예방, 개선 또는 치료용도에 관한 것이다.The present invention relates to nanoparticles bound with an astrocyte-selective peptide, and more specifically, to the use of nanoparticles for preventing, improving, or treating ischemic brain disease.
허혈성 뇌졸중(ischemic stroke)은 주로 혈전 형성에 의해 대뇌 동맥이 막힐 때 발생한다. 이는 산소와 포도당의 공급을 심각하게 낮추어 뉴런 세포의 대사 에너지를 빼앗아 결국 뉴런의 죽음을 초래한다. 뇌졸중 환자의 대부분은 영구적인 뇌 허혈성 경색을 앓고 있으며, 2% 미만의 환자만 시기 적절한 치료를 받는다. 혈전 경로 및 염증 경로는 모두 허혈성 뇌손상의 중요한 병태생리학적 원인이라는 것이 밝혀졌다. 급성 허혈성 뇌졸중에 대한 입증된 치료법에 대한 임상 시험이 진행되었으나, 효과적인 치료 방법은 미비한 실정이다. 최근 허혈성 뇌졸중 병변에 치료제인 별아교세포의 표적 전달을 통한 치료 방법이 대두되고 있다.Ischemic stroke usually occurs when a cerebral artery is blocked by the formation of a blood clot. This severely reduces the supply of oxygen and glucose, depriving neuron cells of metabolic energy and ultimately leading to neuron death. Most stroke patients suffer from permanent cerebral ischemic infarction, and less than 2% of patients receive timely treatment. Both thrombotic and inflammatory pathways have been shown to be important pathophysiological causes of ischemic brain injury. Clinical trials have been conducted on proven treatments for acute ischemic stroke, but effective treatment methods are lacking. Recently, a treatment method through targeted delivery of astrocytes, a therapeutic agent for ischemic stroke lesions, is emerging.
별아교세포는 허혈성 뇌손상 중에 형태학적 및 기능적 변화를 겪는다. 신경교세포가 심각한 손상이나 퇴행에 대응하기 위해 세포 생존을 촉진할 수 있기 때문에 반응성있는 별아교세포증(reactive astrogliosis)이 신경보호적이라는 것이 널리 알려져있다. 반면에 최근 관찰에 따르면 반응성있는 별아교세포증은 '신경 독성'과 '신경 보호' '반응성 상태를 모두 가질 수 있다. 따라서 손상이 유발되는 동안 별아교세포의 메커니즘과 기능은 불분명하다.Astrocytes undergo morphological and functional changes during ischemic brain injury. It is widely known that reactive astrogliosis is neuroprotective because glial cells can promote cell survival to respond to severe damage or degeneration. On the other hand, recent observations suggest that reactive astrogliosis can have both ‘neurotoxic’ and ‘neuroprotective’ ‘reactive states. Therefore, the mechanisms and functions of astrocytes during injury are unclear.
한편, SOX9는 고도로 보존된 전사 인자 계열에 속하며 배아 및 성인 중추 신경계에서 신경줄기세포 풀을 유지하는 데 중요한 것으로 밝혀졌다. SOX9는 심실 영역을 따라 특별히 발현되며, 소뇌를 발달시키고 신경교 형성 및 신경 전구 특성을 확립하는 데 기여한다. 또한, SOX9는 발달 과정에서 별아교세포와 희소 돌기 세포의 계통별 분화를 주도하며, 많은 병리학적 조건에서 반응성 별아교세포에 의해 상향 조절되는 것으로 나타났다. 따라서 SOX9는 발달과 병인을 조절하는 전사 캐스케이드의 핵심 역할을 하지만, 성인 뇌 손상에서의 역할은 잘 알려져 있지 않다.Meanwhile, SOX9 belongs to a highly conserved family of transcription factors and has been shown to be important in maintaining the neural stem cell pool in the embryonic and adult central nervous system. SOX9 is specifically expressed along the ventricular zone and contributes to developing cerebellum and establishing glial formation and neuroprogenitor properties. Additionally, SOX9 drives lineage-specific differentiation of astrocytes and oligodendrocytes during development and has been shown to be upregulated by reactive astrocytes in many pathological conditions. Therefore, SOX9 is a key player in the transcriptional cascade that regulates development and pathogenesis, but its role in adult brain injury is not well understood.
이에 본 발명자들은 (i) 별아교세포에 특이적인 SOX9 유전자 발현을 위한 플라스미스가 캡슐화되고, (ii) 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자를 개발하고, 이의 허혈성 뇌손상 치료 효과를 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors developed PLGA nanoparticles in which (i) a plasmid for expression of the astrocyte-specific SOX9 gene was encapsulated and (ii) an astrocyte-selective peptide was combined, and confirmed its effectiveness in treating ischemic brain damage. The invention was completed.
따라서 본 발명의 목적은, 서열번호 1의 아미노산 서열로 표시되는 별아교세포 선택적 펩타이드를 포함하는, 별아교세포 표적용 나노입자를 제공하는 것이다.Therefore, the purpose of the present invention is to provide nanoparticles for targeting astrocytes, comprising an astrocyte-selective peptide represented by the amino acid sequence of SEQ ID NO: 1.
본 발명의 다른 목적은, 상기 별아교세포 표적용 나노입자를 포함하는 허혈성 뇌질환 예방, 개선 또는 치료용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for preventing, improving or treating ischemic brain disease containing the astrocyte-targeting nanoparticles.
본 발명의 또 다른 목적은, 허혈성 뇌질환 예방 또는 치료용 약학적 조성물을 인간을 제외한 개체에 투여하는 단계;를 포함하는 허혈성 뇌질환 치료방법을 제공하는 것이다.Another object of the present invention is to provide a method of treating ischemic brain disease, comprising administering a pharmaceutical composition for preventing or treating ischemic brain disease to an entity other than a human.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 별아교세포 선택적 펩타이드를 포함하는, 별아교세포 표적용 나노입자를 제공한다.In order to achieve the above object, the present invention provides nanoparticles for targeting astrocytes, comprising an astrocyte-selective peptide represented by the amino acid sequence of SEQ ID NO: 1.
또한 본 발명은 상기 별아교세포 표적용 나노입자를 포함하는 허혈성 뇌질환 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating ischemic brain disease containing the astrocyte-targeting nanoparticles.
또한 본 발명은 상기 별아교세포 표적용 나노입자를 포함하는 허혈성 뇌질환 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving ischemic brain disease containing the astrocyte-targeting nanoparticles.
또한 본 발명은 상기 허혈성 뇌질환 예방 또는 치료용 약학적 조성물을 인간을 제외한 개체에 투여하는 단계;를 포함하는 허혈성 뇌질환 치료방법을 제공한다.The present invention also provides a method of treating ischemic brain disease, comprising administering the pharmaceutical composition for preventing or treating ischemic brain disease to an entity other than a human.
본 발명에 따른 별아교세포 선택적 펩타이드가 결합된 나노입자는 생체적합성 및 생분해성이 우수하며, 동물 수준에서 허혈성 뇌손상을 치료하는 효과가 있음을 확인하였다. 이는 본 발명의 나노입자가 허혈성 뇌질환의 치료제로서 이용될 수 있음을 의미하는바, 허혈성 뇌질환의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.It was confirmed that the nanoparticles combined with the astrocyte-selective peptide according to the present invention have excellent biocompatibility and biodegradability, and are effective in treating ischemic brain damage at the animal level. This means that the nanoparticles of the present invention can be used as a treatment for ischemic brain disease, and can be used in a variety of fields in the fields of prevention, improvement, and treatment of ischemic brain disease.
도 1a는 FT-IR을 통해 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자의 특성을 분석한 결과를 나타낸 도이다.
도 1b는 zetasizer를 이용하여 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자의 특성을 분석한 결과를 나타낸 도이다.
도 1c는 주사전자현미경을 이용하여 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자의 특성을 분석한 결과를 나타낸 도이다.
도 1d는 1H NMR을 이용하여 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자의 특성을 분석한 결과를 나타낸 도이다.
도 1e 및 1f는 면역염색을 통해 세포 유형에 따른 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자의 흡수 여부를 분석한 결과를 나타낸 도이다.
도 2a는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자를 제조하기 위한 SOX9 발현 플라스미드의 개열지도를 나타낸 도이다.
도 2b는 SOX9 발현 플라스미드를 형질감염시킨 1차 별아교세포에서 tdTomato의 발현을 관찰한 결과를 나타낸 도이다.
도 2c는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자의 입자 크기 및 제타 전위를 분석한 결과를 나타낸 도이다.
도 2d는 FT-IR을 통해 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자의 특성을 분석한 결과를 나타낸 도이다(파란색: 유전자발현이 없는 공벡터 플라스미드 캡슐화 NP, 주황색: SOX9 발현 플라스미드 캡슐화 NP, 빨간색 : 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP, 초록색: 별아교세포 선택적 펩타이드가 결합되고 SOX9 발현 플라스미드 캡슐화 NP).
도 2e는 주사전자현미경을 통해 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자의 특성을 분석한 결과를 나타낸 도이다.
도 3a는 행잉테스트를 통해 광혈전성 허혈 마우스 모델의 운동장애 발생 여부를 확인한 결과를 나타낸 도이다.
도 3b는 광혈전성 혀혈 마우스 모델의 경색 부위를 육안으로 관찰한 결과를 나타낸 도이다.
도 3c는 광혈전성 허혈 마우스 모델의 경색 부피를 측정한 결과를 나타낸 도이다.
도 3d는 면역염색을 통해 M2 및 cg1 영역에서 GFAP의 발현을 분석한 결과를 나타낸 도이다.
도 3e는 이중면역염색을 통해 반음영 부위에서 별 GFAP의 발현을 분석한 결과를 나타낸 도이다.
도 4a는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자의 신경 보호 효과를 확인하기 위한 실험 스케줄을 나타낸 도이다.
도 4b는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자를 투여한 마우스 모델의 행잉 테스트 결과를 나타낸 도이다.
도 4c 및 4d는 TTC 염색을 통해 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자 투여에 따른 경색 부피를 측정한 결과를 나타낸 도이다.
도 5a 및 5b는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자 투여에 따른 반음영 부위에서 SOX9 발현을 분석한 결과를 나타낸 도이다.
도 5c는 웨스턴 블롯팅을 통해 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자 투여에 따른 SOX9 단백질 발현을 분석한 결과를 나타낸 도이다.
도 5d는 웨스턴 블롯팅을 통해 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자 투여에 따른 프로스타글란딘 D2 합성 효소의 발현을 분석한 결과를 나타낸 도이다.
도 5e는 웨스턴 블롯팅을 통해 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 PLGA 나노입자 투여에 따른 프로스타글란딘 D2의 발현을 분석한 결과를 나타낸 도이다.Figure 1a is a diagram showing the results of analyzing the characteristics of PLGA nanoparticles bound with an astrocyte-selective peptide through FT-IR.
Figure 1b is a diagram showing the results of analyzing the characteristics of PLGA nanoparticles bound with an astrocyte-selective peptide using a zetasizer.
Figure 1c is a diagram showing the results of analyzing the characteristics of PLGA nanoparticles bound with an astrocyte-selective peptide using a scanning electron microscope.
Figure 1d is a diagram showing the results of analyzing the characteristics of PLGA nanoparticles bound with an astrocyte-selective peptide using 1 H NMR.
Figures 1e and 1f show the results of analyzing the absorption of PLGA nanoparticles bound to astrocyte-selective peptide according to cell type through immunostaining.
Figure 2a is a diagram showing a cleavage map of the SOX9 expression plasmid for producing plasmid-encapsulated PLGA nanoparticles bound to an astrocyte-selective peptide.
Figure 2b is a diagram showing the results of observing the expression of tdTomato in primary astrocytes transfected with the SOX9 expression plasmid.
Figure 2c is a diagram showing the results of analyzing the particle size and zeta potential of plasmid-encapsulated PLGA nanoparticles bound with an astrocyte-selective peptide.
Figure 2d is a diagram showing the results of analyzing the characteristics of plasmid-encapsulated PLGA nanoparticles bound with an astrocyte-selective peptide through FT-IR (blue: empty vector plasmid-encapsulated NP without gene expression, orange: SOX9 expression plasmid-encapsulated NP) , red: plasmid-encapsulated NPs conjugated with astrocyte-selective peptide, green: NPs conjugated with astrocyte-selective peptides and encapsulated SOX9 expression plasmid).
Figure 2e is a diagram showing the results of analyzing the characteristics of plasmid-encapsulated PLGA nanoparticles bound with an astrocyte-selective peptide through a scanning electron microscope.
Figure 3a is a diagram showing the results of confirming whether movement disorders occur in a photothrombotic ischemia mouse model through a hanging test.
Figure 3b is a diagram showing the results of visual observation of the infarction area of the photothrombotic tongue blood mouse model.
Figure 3c is a diagram showing the results of measuring the infarct volume of a photothrombotic ischemia mouse model.
Figure 3d is a diagram showing the results of analyzing the expression of GFAP in the M2 and cg1 regions through immunostaining.
Figure 3e is a diagram showing the results of analyzing the expression of star GFAP in the penumbra region through double immunostaining.
Figure 4a is a diagram showing an experimental schedule to confirm the neuroprotective effect of plasmid-encapsulated PLGA nanoparticles conjugated with an astrocyte-selective peptide.
Figure 4b is a diagram showing the results of a hanging test in a mouse model administered with plasmid-encapsulated PLGA nanoparticles conjugated with an astrocyte-selective peptide.
Figures 4c and 4d show the results of measuring the infarct volume following administration of plasmid-encapsulated PLGA nanoparticles conjugated with an astrocyte-selective peptide through TTC staining.
Figures 5a and 5b show the results of analyzing SOX9 expression in the penumbra region following administration of plasmid-encapsulated PLGA nanoparticles conjugated with an astrocyte-selective peptide.
Figure 5c is a diagram showing the results of analyzing SOX9 protein expression following administration of plasmid-encapsulated PLGA nanoparticles conjugated with an astrocyte-selective peptide through Western blotting.
Figure 5d is a diagram showing the results of analyzing the expression of prostaglandin D2 synthetase following administration of plasmid-encapsulated PLGA nanoparticles to which astrocyte-selective peptide was conjugated through Western blotting.
Figure 5e is a diagram showing the results of analyzing the expression of prostaglandin D2 following administration of plasmid-encapsulated PLGA nanoparticles conjugated with an astrocyte-selective peptide through Western blotting.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 별아교세포 선택적 펩타이드를 포함하는, 별아교세포 표적용 나노입자를 제공한다.According to an aspect of the present invention, the present invention provides nanoparticles for targeting astrocytes, comprising an astrocyte-selective peptide represented by the amino acid sequence of SEQ ID NO: 1.
본 발명의 구체예에서, 본 발명의 별아교세포 선택적 펩타이드는 서열번호 1로 표시되는 아미노산 서열로 이루어질 수 있고, 상기 단백질의 기능적 동등물을 포함한다. In an embodiment of the present invention, the astrocyte-selective peptide of the present invention may consist of the amino acid sequence represented by SEQ ID NO: 1, and includes a functional equivalent of the above protein.
상기 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1로 표시되는 아미노산 서열과 적어도 80％ 이상의, 바람직하게는 90％, 더욱 바람직하게는 95％이상의 서열 상동성(즉, 동일성)을 갖는 것으로 예를 들면, 80％, 81％, 82％, 83％, 84％, 85％, 86％, 87％, 88％, 89％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％, 100％의 서열 상동성을 갖는 것을 포함하며, 서열번호 1로 표시되는 펩타이드와 실질적으로 동질의 생리활성을 나타내는 펩타이드를 말한다. 또한, 본 발명의 서열번호 1로 표시되는 펩타이드에는 이의 천연형 아미노산 서열을 갖는 단백질뿐만 아니라 이의 아미노산 서열 변이체가 또한 본 발명의 범위에 포함된다. 변이체란, 서열번호 1로 표시되는 펩타이드의 천연 아미노산 서열과 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 단백질을 의미한다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 상기 서열번호 1로 표시되는 펩타이드 또는 이의 변이체는 천연에서 추출하거나 합성(Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963) 또는 DNA 서열을 기본으로 하는 유전자 재조합 방법에 의해 제조될 수 있다(Sambrook et al, Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, 2판, 1989).The "functional equivalent" refers to a sequence homology of at least 80%, preferably 90%, more preferably 95% or more with the amino acid sequence shown in SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids (i.e. , identity), for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology, and a peptide that exhibits substantially the same physiological activity as the peptide represented by SEQ ID NO: 1. says In addition, the peptide represented by SEQ ID NO: 1 of the present invention includes not only proteins having their native amino acid sequences but also amino acid sequence variants thereof within the scope of the present invention. A variant refers to a protein having a sequence that is different from the natural amino acid sequence of the peptide represented by SEQ ID NO: 1 due to deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof. Amino acid exchanges in proteins and peptides that do not overall alter the activity of the molecule are known in the art. The peptide represented by SEQ ID NO: 1 or a variant thereof can be extracted from nature, synthesized (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963), or manufactured by a genetic recombination method based on DNA sequence. (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).
또한 상기 서열 상동성은 단백질을 구성하는 아미노산 서열의 유사한 부분을 비교하기 위해 사용되는 일반적인 표준 방법에 의해 결정할 수 있다. BLAST 또는 FASTA와 같은 컴퓨터 프로그램은 두 개 이상의 단백질을 각각 구성하는 아미노산이 최적으로 매칭(matching) 되도록 정렬한다(하나 또는 두 서열의 전장서열을 따라 또는 하나 또는 두 서열의 예측된 부분을 따라). 상기 프로그램은 디펄트 오프닝 패널티(default opening penalty) 및 디펄트 갭 페널티(default gap penalty)를 제공하며 컴퓨터 프로그램과 함께 연계되어 사용될 수 있는 PAM250(표준 스코링 매트릭스; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp. 3, 1978)와 같은 스코링 매트릭스를 제공한다. 예를 들어, 백분율로 표시되는 서열 상동성은 다음과 같이 계산할 수 있다: 일치하는 서열(indentical matches)의 총 수에 100을 곱한 다음 대응되는 스팬(machted span) 내의 보다 긴 서열의 길이와두 서열을 정렬하기 위해 보다 긴 서열내로 도입된 갭(gaps)의 수의 합으로 나눈다.Additionally, the sequence homology can be determined by a general standard method used to compare similar portions of amino acid sequences constituting proteins. Computer programs such as BLAST or FASTA align the amino acids that make up two or more proteins to optimally match (along the full length of one or two sequences or along a predicted portion of one or two sequences). The program provides a default opening penalty and a default gap penalty and can be used in conjunction with a computer program called PAM250 (standard scoring matrix; Dayhoff et al., in Atlas of Protein). It provides a scoring matrix such as Sequence and Structure, vol 5, supp. 3, 1978). For example, sequence homology expressed as a percentage can be calculated as follows: multiply the total number of identical matches by 100 and then multiply the length of the longer sequence in the corresponding span by the two sequences. To align, divide by the sum of the number of gaps introduced into the longer sequence.
상기에서 "실질적으로 동질의 생리활성"이란 허혈성 뇌질환의 개선, 예방 또는 치료 활성을 의미한다. In the above, “substantially homogeneous physiological activity” means activity in improving, preventing or treating ischemic brain disease.
또한 상기 기능적 동등물의 범위에는 서열번호 1의 아미노산 서열로 표시되는 별아교세포 선택적 펩타이드의 기본골격; 및 허혈성 질환의 개선, 예방 또는 치료 활성을 유지하면서 구성하는 아미노산의 일부 화학 구조가 변형된 유도체;가 포함된다. 예를 들어 단백질의 안정성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한 구조변경이 이에 포함된다.In addition, the scope of the functional equivalent includes the basic framework of the astrocyte-selective peptide represented by the amino acid sequence of SEQ ID NO: 1; and derivatives in which some chemical structures of constituting amino acids have been modified while maintaining the activity of improving, preventing or treating ischemic diseases. For example, this includes structural changes to change the stability, storage, volatility or solubility of the protein.
본 발명의 구체예에서, 상기 별아교세포 선택적 펩타이드는 PLGA 나노입자의 표면에 결합된 것이 바람직하다.In an embodiment of the present invention, the astrocyte-selective peptide is preferably bound to the surface of PLGA nanoparticles.
본 발명의 구체예에서, 상기 PLGA 나노입자는 SOX9 또는 이를 코딩하는 유전자를 더 포함하는 것이 바람직하다.In an embodiment of the present invention, the PLGA nanoparticles preferably further include SOX9 or a gene encoding it.
본 발명의 바람직한 구체예에서, 상기 SOX9를 코딩하는 유전자는 세포 내 전달을 위한 발현 벡터에 포함된 것이 바람직하다. 상기 SOX9를 코딩하는 유전자는 DNA 및 DEAE-덱스트란의 복합체, DNA 및 핵 단백질의 복합체, DNA 및 지질의 복합체 등의 다양한 형질전환 기술을 이용하여 세포 내로 도입시킬 수 있는데, 이를 위해 상기 SOX9를 코딩하는 유전자는 세포 내로의 효율적인 도입을 가능하게 하는 전달체 내에 포함된 형태일 수 있다. 상기 전달체는 바이러스 벡터 및 비바이러스 벡터 모두 사용할 수 있으며, 상기 벡터의 종류는 플라스미드, 파지, 코즈미드 등이 있다.In a preferred embodiment of the present invention, the gene encoding SOX9 is preferably included in an expression vector for intracellular delivery. The gene encoding the SOX9 can be introduced into cells using various transformation techniques such as a complex of DNA and DEAE-dextran, a complex of DNA and nuclear protein, and a complex of DNA and lipids. For this, the gene encoding the SOX9 can be introduced into cells. The gene may be contained in a carrier that enables efficient introduction into cells. Both viral vectors and non-viral vectors can be used as the carrier, and types of vectors include plasmids, phages, and cosmids.
본 발명에 있어서, 나노입자는 약물을 중심으로 하여 고분자의 코팅층이 형성되어 이루어진 입자를 의미하는 것으로서, 편의상 "고분자의 종류 나노입자"의 형태로 표기할 수 있다. 상기 약물을 둘러싸는(즉, 코팅하는) 고분자로는 당업계에 공지된 생분해성 고분자(biodegradable polymer)가 이용될 수 있다. In the present invention, nanoparticles refer to particles formed by forming a polymer coating layer around a drug, and for convenience, they can be expressed in the form of “polymer type nanoparticles.” As a polymer surrounding (i.e., coating) the drug, a biodegradable polymer known in the art may be used.
본 발명의 구체예에서, 상기 나노입자는 폴리락티드-코-글리콜라이드, 폴리스티렌-코-에틸렌글리콜, 폴리에틸렌이민-코-에틸렌글리콜, 폴리포스파겐-코-에틸렌글리콜, 폴리락타이드-코-에틸렌글리콜, 폴리락티드-코-글리콜라이드-코-에틸렌글리콜, 폴리카프로락톤-코-에틸렌글리콜, 폴리안하이드라이드-코-에틸렌글리콜, 폴리말릭산-코-에틸렌글리콜 및 이의 유도체, 폴리알킬시아노아크릴레이트-코-에틸렌글리콜, 폴리하이드로옥시부틸레이트-코-에틸렌글리콜, 폴리카르보네이트-코-에틸렌글리콜 및 폴리오르소에스테르-코-에틸렌글리콜, 폴리에틸렌글리콜, 폴리-L-라이신-코-에틸렌글리콜, 폴리글리콜라이드-코-에틸렌글리콜, 폴리메틸메타아크릴레이트-코-에틸렌글리콜, 폴리비닐피롤리돈-코-에틸렌글리콜 및 이의 공중합체로 이루어진 군으로부터 선택되는 1종 이상인 것이 바람직하며, 더 바람직하게는 폴리락티드-코-글리콜라이드(PLGA, (poly(lactic-co-glycolic acid)일 수 있다. 상기 고분자들은 낮은 독성뿐만 아니라 탁월한 생체 적합성을 보여주는 것으로 알려져 있다[A. J. Domb, et al. Handbook of biodegradable polymers, Harwood academic publishers, USA, 1997).In an embodiment of the present invention, the nanoparticles include polylactide-co-glycolide, polystyrene-co-ethylene glycol, polyethyleneimine-co-ethylene glycol, polyphosphagen-co-ethylene glycol, polylactide-co- Ethylene glycol, polylactide-co-glycolide-co-ethylene glycol, polycaprolactone-co-ethylene glycol, polyanhydride-co-ethylene glycol, polymalic acid-co-ethylene glycol and its derivatives, polyalkyl Cyanoacrylate-co-ethylene glycol, polyhydroxybutylate-co-ethylene glycol, polycarbonate-co-ethylene glycol and polyorthoester-co-ethylene glycol, polyethylene glycol, poly-L-lysine- It is preferable that it is at least one selected from the group consisting of co-ethylene glycol, polyglycolide-co-ethylene glycol, polymethyl methacrylate-co-ethylene glycol, polyvinylpyrrolidone-co-ethylene glycol, and copolymers thereof. And, more preferably, it may be poly(lactic-co-glycolic acid) (PLGA). These polymers are known to show not only low toxicity but also excellent biocompatibility [A. J. Domb, et al. Handbook of biodegradable polymers, Harwood academic publishers, USA, 1997).
본 발명의 다른 양태에 따르면, 상기 별아교세포 표적용 나노입자를 포함하는 허혈성 뇌질환 예방, 개선 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, a composition for preventing, improving, or treating ischemic brain disease comprising the astrocyte-targeting nanoparticles is provided.
본 발명에 있어서, 예방은 본 발명의 조성물에 의해 병인을 제거하거나 조기 발견하여 해당질환을 막는 모든 행위를 의미한다.In the present invention, prevention refers to all actions to prevent the disease by removing or early detecting the etiological agent using the composition of the present invention.
본 발명에 있어서, 치료는 본 발명의 조성물에 의해 질환의 증세가 호전되는 모든 행위를 의미한다.In the present invention, treatment refers to all actions in which disease symptoms are improved by the composition of the present invention.
본 발명에 있어서, 개선은 본 발명의 조성물에 의해 생체 대사, 세포 또는 기관의 기능이 이롭게 변경되는 모든 행위를 의미한다.In the present invention, improvement refers to any action in which biological metabolism, cell or organ function is beneficially changed by the composition of the present invention.
본 발명의 조성물은 상기 별아교세포 표적용 나노입자와 함께 허혈성 뇌질환의 예방, 치료 또는 개선 효과를 갖는 공지의 유효성분을 1종 이상 더 함유할 수 있다.The composition of the present invention may further contain one or more known active ingredients that have the effect of preventing, treating or improving ischemic brain disease along with the astrocyte-targeting nanoparticles.
본 발명에 따른 조성물은 허혈성 뇌질환 예방 또는 치료용 약학적 조성물; 또는 허혈성 뇌질환 예방 또는 개선용 식품 조성물;일 수 있다.The composition according to the present invention is a pharmaceutical composition for preventing or treating ischemic brain disease; Or it may be a food composition for preventing or improving ischemic brain disease.
본 발명의 조성물이 약학적 조성물인 경우, 본 발명의 약학적 조성물은 투여를 위하여 약학적으로 허용 가능한 담체를 1종 이상 포함할 수 있다. 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 이상의 성분을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화 할 수 있다. 더 나아가 당 분야의 적정한 방법으로 각 질환에 따라 또는 성분에 따라 제제화 할 수 있다.When the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers for administration. Pharmaceutically acceptable carriers can be used in combination with saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients, and if necessary, antioxidants and buffer solutions. Other common additives such as bacteriostatic agents can be added. Additionally, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Furthermore, it can be formulated according to each disease or ingredient using an appropriate method in the art.
상기 약학적 조성물은 추가적인 성분을 더 포함할 수 있다. 상기 추가적인 성분의 예로는 유당, 탈크, 전분, 스테아린산 마그네슘, 결정성 셀룰로오스, 락토오스, 젤라틴, 만니톨, 과옥소산, 이성화당, 폴리비닐알코올, 붕산 및 탄산나트륨이 있다.The pharmaceutical composition may further include additional ingredients. Examples of such additional ingredients include lactose, talc, starch, magnesium stearate, crystalline cellulose, lactose, gelatin, mannitol, peroxoacid, isomerized sugar, polyvinyl alcohol, boric acid and sodium carbonate.
상기 약학적 조성물은 경구 투여 또는 비경구 투여할 수 있으며, 신속한 효과를 위하여 비경구 투여하는 것이 바람직하며, 척수강에 척수관 주사되는 것이 더 바람직하다. 비경구 투여를 하는 경우, 정맥내 주입, 근육내 주입, 관절내(intra-articular) 주입, 활액내(intra-synovial) 주입, 수망강내 주입, 간내(intrahepatic) 주입, 병변내(intralesional) 주입 또는 두 개강내(intracranial) 주입 등으로 투여할 수 있다. 상기 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다.The pharmaceutical composition can be administered orally or parenterally, and is preferably administered parenterally for rapid effect, and is more preferably injected into the spinal canal. When administered parenterally, intravenous injection, intramuscular injection, intra-articular injection, intra-synovial injection, intrathecal injection, intrahepatic injection, intralesional injection, or It can be administered by intracranial injection. The appropriate dosage of the pharmaceutical composition can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. there is.
본 발명의 조성물이 식품 조성물인 경우, 본 발명의 식품 조성물은 건강기능식품, 식품 첨가제 또는 식이보조제일 수 있다. 본 발명의 조성물이 식품 첨가제인 경우, 별아교세포 표적용 나노입자를 그대로 첨가하거나, 다른 식품 또는 식품 성분과 함께 혼합하여 사용되는 등 통상적인 방법에 따라 적절하게 사용될 수 있다.When the composition of the present invention is a food composition, the food composition of the present invention may be a health functional food, food additive, or dietary supplement. When the composition of the present invention is a food additive, it can be appropriately used according to conventional methods, such as adding astrocyte-targeting nanoparticles as is or mixing it with other foods or food ingredients.
구체적인 예로, 식품 또는 음료의 제조 시에는 본 발명의 별아교세포 표적용 나노입자는 원료에 대하여 15중량% 이하, 바람직하게는 10중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하여 장기간 섭취할 경우에는 상기 범위 이하의 양으로 첨가될 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. 상기 식품의 종류에는 특별한 제한은 없으나, 본 발명의 별아교세포 표적용 나노입자를 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료, 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.As a specific example, when manufacturing a food or beverage, the astrocyte-targeting nanoparticles of the present invention are added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, when consumed for a long period of time for health and hygiene purposes or health control, it can be added in an amount below the above range. Since there is no problem in terms of safety, the active ingredient can be used in an amount above the above range. there is. There is no particular limitation on the type of food, but examples of food to which astrocyte-targeting nanoparticles of the present invention can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, and gum. , dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the conventional sense.
본 발명의 식품 조성물이 음료로 제조될 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등의 추가 성분을 포함할 수 있다. 상기 천연 탄수화물로는 포도당, 과당 등의 모노사카라이드; 말토오스, 수크로오스 등의 디사카라이드; 덱스트린, 사이클로덱스트린 등의 천연 감미제; 사카린, 아스파르탐 등의 합성 감미제 등이 사용될 수 있다. 상기 천연 탄수화물은 본 발명의 식품 조성물 총 중량에 대하여 0.01 내지 10중량%, 바람직하게는 0.01 내지 0.1중량%로 포함된다.When the food composition of the present invention is manufactured into a beverage, it may contain additional ingredients such as various flavoring agents or natural carbohydrates, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is included in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.
본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 포함할 수 있으며, 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있으나 이에 제한되지 않는다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 상기의 첨가제 비율은 크게 제한되지는 않으나, 본 발명의 식품 조성물 총 중량에 대하여 0.01 내지 0.1중량% 범위내로 포함되는 것이 바람직하다.The food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may include carbonating agents used in beverages, and may include pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages, but is not limited thereto. These ingredients can be used independently or in combination. The ratio of the above additives is not greatly limited, but is preferably contained within the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.
건강 및 위생을 목적으로 하거나 건강 조절을 목적으로 하는 장기간의 섭취인 경우, 본 발명의 식품 조성물은 안전성 면에서 아무런 문제가 없기 때문에 장기간 복용이 가능하다.In the case of long-term intake for the purpose of health and hygiene or health control, the food composition of the present invention can be taken for a long period of time because there is no problem in terms of safety.
본 발명의 또 다른 양태에 따르면, 상기 허혈성 뇌질환 예방 또는 치료용 약학적 조성물을 인간을 제외한 개체에 투여하는 단계;를 포함하는 허혈성 뇌질환 치료방법을 제공한다.According to another aspect of the present invention, there is provided a method of treating ischemic brain disease comprising the step of administering the pharmaceutical composition for preventing or treating ischemic brain disease to an entity other than a human.
본 발명의 바람직한 구체예에 따르면, 상기 약학적 조성물은 개체의 척수에 주입되는 것이 바람직하나, 이에 제한되지 않는다.According to a preferred embodiment of the present invention, the pharmaceutical composition is preferably injected into the spinal cord of an individual, but is not limited thereto.
본 발명에 따른 치료방법은 별아교세포 표적용 나노입자가 척수 내 세포의 SOX9의 발현을 증가시킴으로써, 허혈성 뇌질환을 치료할 수 있다.The treatment method according to the present invention can treat ischemic brain disease by using astrocyte-targeting nanoparticles to increase the expression of SOX9 in cells in the spinal cord.
중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention pertains.
이하, 실험예 및 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실험예 및 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실험예 및 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through experimental examples and examples. These experimental examples and examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as limited by these experimental examples and examples. will be.
[실험예][Experimental example]
본 실험에 사용한 보는 시약은 분석용 등급(analytical grade)을 사용하였다. PLGA-PEG-COOH(poly(lactide-co-glycolide)-b-poly(ethylene glycol)-carboxylic acid endcap; AI078)는 mPEG-PLGA(Methoxy poly(ethylene glycol)-b-(poly(lactide-co-glycolide); AK037; PolySciTech, AkiNA, Inc; West Lafayette, USA)을 가진 PLGA의 변형을 통해 수득하였다.The viewing reagent used in this experiment was of analytical grade. PLGA-PEG-COOH(poly(lactide-co-glycolide)-b-poly(ethylene glycol)-carboxylic acid endcap; AI078) is mPEG-PLGA(Methoxy poly(ethylene glycol)-b-(poly(lactide-co- glycolide); AK037; PolySciTech, AkiNA, Inc; West Lafayette, USA).
실험예 1. 플라스미드 캡슐화 PLGA 나노입자(PLGA NP)의 제조Experimental Example 1. Preparation of plasmid-encapsulated PLGA nanoparticles (PLGA NPs)
GFAP-SOX9-tdRomato-pZac2.1 벡터와 대조 벡터는 조은혜(아주대학교 의과대학, 수원)으로부터 받았다. 플라스미드 캡슐화 PLGA NP는 약간의 변형을 가하여 제조하였다. 플라스미드 캡슐화 PLGA NP를 제조하기 위하여, 200 μL의 TE 8.0 완충액에 담긴 플라스미드 100 μg를 PLGA-PEG-COOH 2.5 mg 및 초음파 처리(10%, 30 sec; SFX 550; Branson Ultrasonics, Sterling Heights, MI, USA)에 의해 유화된 mPEG-PLGA 22.5 mg를 함유하는 1.0 ml의 디클로로메탄(dichloromethane, DCM)에 적가하였다(1차 W/O 에멀젼). 1% PVA1500(w/v; Thermo Fisher Scientific) 2 mL을 1차 에멀젼에 직접 첨가하였고, 1분 동안 초음파 처리 및 유화시켜 W/O/W 이중 에멀젼을 제조하였다. 제조된 W/O/W 이중 에멀젼을 6 mL의 1 % PVA1500(w/v)으로 희석하고 실온에서 3시간 동안 후드에서 교반하여 디클로로메탄을 증발시켰다. PLGA NP를 4℃에서 10분 동안 13,000 RPM으로 원심분리(한일 과학사, 대전)하여 수집하고, 탈이온수로 2회 세척 및 동결건조하여 플라스미드 캡슐화 PLGA NP를 수득하였다.The GFAP-SOX9-tdRomato-pZac2.1 vector and control vector were received from Eunhye Cho (Ajou University College of Medicine, Suwon). Plasmid-encapsulated PLGA NPs were prepared with slight modifications. To prepare plasmid-encapsulated PLGA NPs, 100 μg of plasmid in 200 μL of TE 8.0 buffer was mixed with 2.5 mg of PLGA-PEG-COOH and sonicated (10%, 30 sec; SFX 550; Branson Ultrasonics, Sterling Heights, MI, USA). ) was added dropwise to 1.0 ml of dichloromethane (DCM) containing 22.5 mg of mPEG-PLGA emulsified by (primary W/O emulsion). 2 mL of 1% PVA1500 (w/v; Thermo Fisher Scientific) was added directly to the primary emulsion, sonicated for 1 min and emulsified to prepare a W/O/W double emulsion. The prepared W/O/W double emulsion was diluted with 6 mL of 1% PVA1500 (w/v) and stirred in a hood for 3 h at room temperature to evaporate dichloromethane. PLGA NPs were collected by centrifugation at 13,000 RPM for 10 minutes at 4°C (Hanil Science Co., Ltd., Daejeon), washed twice with deionized water, and lyophilized to obtain plasmid-encapsulated PLGA NPs.
실험예 2. 펩티드 결합 나노입자의 표면 개질Experimental Example 2. Surface modification of peptide-binding nanoparticles
실험에 사용된 펩타이드는 Peptron(대한민국, 대전)에서 합성했다. PLGA 나노입자는 펩타이드 2 mg와 함께 배양하였다. 펩티드 결합 반응은 환원 시약의 존재하에 실온에서 4시간 동안 EDC/Sulfo-NHS 공유 결합 프로토콜(EDC 22980; Sulfo-NHS 24510; Thermo Scientific, Massachusetts, USA)로 처리하였다. 결합되지 않은 펩타이드는 4℃에서 10분 동안 13,000 RPM으로 원심분리하여 제거하고, 펩티드 결합된 PLGA 나노입자를 수집하여 동결건조하였다.The peptides used in the experiment were synthesized by Peptron (Daejeon, Korea). PLGA nanoparticles were incubated with 2 mg of peptide. Peptide coupling reactions were treated with the EDC/Sulfo-NHS covalent coupling protocol (EDC 22980; Sulfo-NHS 24510; Thermo Scientific, Massachusetts, USA) for 4 h at room temperature in the presence of reducing reagents. Unbound peptides were removed by centrifugation at 13,000 RPM for 10 minutes at 4°C, and peptide-bound PLGA nanoparticles were collected and lyophilized.
실험예 3. 플라스미드 캡슐화 PLGA 나노입자의 특징Experimental Example 3. Characteristics of plasmid-encapsulated PLGA nanoparticles
제조된 PLGA 나노입자의 크기와 제타 전위는 Zetasizer Nano ZS (Malvern Instruments, Malvern, UK)를 사용하여 동적광산란(dynamic light scattering, DLS)으로 측정하였다. 제조된 PLGA 나노입자의 직경 및 형태는 SEM(SNE-4500 M; SEC Co., Ltd., Suwon, Republic of Korea)을 사용하여 평가하였다. SEM 관찰을 위하여, 나노입자 샘플을 금으로 음성 염색하였다. FT-IR 스펙트럼은 ALPHA II 분광계(Bruker)에서 투과 모드로 기록하였다. 64개의 스캔은 실온에서 3 cm-1의 해상도로 기록하였다. 나노입자용 투명 필름은 각 분말을 캐스팅하여 준비하였다. 또한 중수소로 치환된 용매(DMSO-d6)에서 나노입자의 1H NMR 스펙트럼을 획득하였다.The size and zeta potential of the prepared PLGA nanoparticles were measured by dynamic light scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The diameter and shape of the prepared PLGA nanoparticles were evaluated using SEM (SNE-4500 M; SEC Co., Ltd., Suwon, Republic of Korea). For SEM observation, nanoparticle samples were negatively stained with gold. FT-IR spectra were recorded in transmission mode on an ALPHA II spectrometer (Bruker). 64 scans were recorded with a resolution of 3 cm -1 at room temperature. A transparent film for nanoparticles was prepared by casting each powder. Additionally, 1 H NMR spectra of the nanoparticles were obtained in a deuterium-substituted solvent (DMSO-d 6 ).
실험예 4. 동물 및 나노입자 투여Experimental Example 4. Animal and nanoparticle administration
수컷 C57BL/6 마우스(20-25g)는 다물 사이언스(대한민국, 대전)에서 구입하여 실험 전 1주일 동안 적응시켰다. 동물 연구는 충남대학교 동물 관리 및 사용위원회(CNUH-019-A0065)의 승인을 받았으며 국립 보건원과 국제뇌졸중동물연구협회의 윤리 지침을 따랐다. 마우스는 암흑과 빛의 12주기를 번갈아 가며 22℃에 수용하고, 상업적인 식단을 공급하고 물을 제공하였다. 각 그룹은 마우스 6마리로 구성하였고, 모든 투여는 보정된 기화기(calibrated vaporizer)를 이용하여 이소플루란 마취 후 수행하였다. 나노입자는 두개골과 C1 척추 사이의 환추후두막(atlanto-occipital membrane)을 통해 대조(cisterna magna)의 CSF에 직접 투여하였다. 해밀턴 주사기(26 게이지)를 사용하여 나노입자(in PBS) 20 μL를 주입하고, 바늘을 제거하기 전에 추가 1분 동안 제자리에 두었다.Male C57BL/6 mice (20–25 g) were purchased from Damul Science (Daejeon, Korea) and acclimatized for 1 week before experiments. The animal study was approved by the Chungnam National University Animal Care and Use Committee (CNUH-019-A0065) and followed the ethical guidelines of the National Institutes of Health and the International Association for Stroke Animal Research. Mice were housed at 22°C with 12 alternating cycles of dark and light, fed a commercial diet, and provided with water. Each group consisted of 6 mice, and all administrations were performed after isoflurane anesthesia using a calibrated vaporizer. Nanoparticles were administered directly into the CSF of the control cisterna magna through the atlanto-occipital membrane between the skull and C1 vertebra. Inject 20 μL of nanoparticles (in PBS) using a Hamilton syringe (26 gauge) and leave the needle in place for an additional 1 min before removing it.
실험예 5. 광혈전성 피질 허혈(Photothrombotic cortical ischemia)Experimental Example 5. Photothrombotic cortical ischemia
광혈전성 피질 허혈을 유도하기 위하여, 마우스를 공기 중 1% 이소플루 란을 사용하여 마취하였다. 마우스의 두피 주위 조직 절개를 위해 중간선 두피를 절개하였고, 브레그마(bregma) 및 람다 점(lambda point)을 확인하였다. 미세조작장치(micromanipulator)를 사용하여 CL 6000 LED 냉광원(Carl Zeiss, Gottingen, Germany) 및 4mm 조리개가 있는 광섬유 번들을 브레그마에서 2.0 mm 측면 중앙에 배치하였다. 감광성 로즈벵갈(Sigma-Aldrich; St. Louis, MO, USA) 염료를 멸균 식염수(10 mg/mL)에 용해시키고 로즈벵갈 0.1 mL을 빛을 조사하기 30분 전에 각 마우스에 복강 내 주사하였다. 노출된 부분 중 손상되지 않은 두개골에 6분 동안 빛을 조사하였다. 마지막으로 수술 상처를 봉합하고 마우스를 마취에서 회복시켰다. 냉광원을 제외하고 대조군 그룹(sham)의 마우스에서 동일한 절차를 수행하였다.To induce photothrombotic cortical ischemia, mice were anesthetized using 1% isoflurane in air. To incise tissue around the mouse's scalp, the midline scalp was incised, and bregma and lambda point were identified. A CL 6000 LED cold light source (Carl Zeiss, Gottingen, Germany) and a fiber optic bundle with a 4 mm aperture were placed centered 2.0 mm lateral from the bregma using a micromanipulator. Photosensitive rose bengal (Sigma-Aldrich; St. Louis, MO, USA) dye was dissolved in sterile saline (10 mg/mL), and 0.1 mL of rose bengal was injected intraperitoneally into each mouse 30 min before light irradiation. Among the exposed parts, the undamaged skull was irradiated with light for 6 minutes. Finally, the surgical wound was sutured and the mouse recovered from anesthesia. The same procedure was performed on mice in the control group (sham), except for the cold light source.
실험예 6. 행잉 테스트 및 경색 부피 측정Experimental Example 6. Hanging test and infarct volume measurement
신경학적 결손은 행잉 테스트(hanging test)로 평가하였다. 뇌졸중 전에 블라인드 관찰자가 행동 테스트를 수행하였다. 마우스를 지면에서 50 내지 60 cm 높이의 두 기둥 사이에 뻗어있는 단일 와이어에 매달리도록 훈련시켰다. 동물의 운동 변화를 평가하기 위해 신경학적 평가가 수행되었고, 같은 관찰자가 광혈전성 피질 허혈 유발 2일 후에 행동 테스트를 수행하였다. 마우스를 와이어 위에 놓고 두 앞다리로 매달린 후 떨어질 때까지의 시간을 측정하였다. Neurological deficits were assessed by the hanging test. Behavioral tests were performed by a blinded observer before stroke. Mice were trained to hang on a single wire stretched between two poles 50 to 60 cm above the ground. Neurological evaluations were performed to assess motor changes in the animals, and behavioral tests were performed by the same observer 2 days after photothrombotic cortical ischemia induction. The mouse was placed on the wire, hung with both front legs, and the time until it fell was measured.
경색 부피를 측정하기 위하여, 뇌의 앞쪽 끝에서 2 mm 두께로 컷팅하여 절편 4개를 얻었다. 상기 절편 4개를 멸균된 증류수에 희석된 1% 2,3,5-트리페닐테트라졸륨클로라이드(2,3,5-triphenyltetrazolium chloride, TTC; T8877, Sigma-Aldrich; St. Louis; MO, USA)에 담궜고, 실온에서 30분 동안 4% PFA로 고정하였다. 고정된 절편을 카메라로 촬영하고 경색 부피를 이미지 분석 소프트웨어 Image J(NIH, Bethesda, MD, USA)을 활용하여 계산하였다. 접힘 경색 부피는 이전에 설명한 공식에 따라 계산하였다.To measure the infarct volume, four 2-mm-thick slices were obtained from the anterior end of the brain. The four sections were treated with 1% 2,3,5-triphenyltetrazolium chloride (TTC; T8877, Sigma-Aldrich; St. Louis; MO, USA) diluted in sterilized distilled water. and fixed with 4% PFA for 30 minutes at room temperature. Fixed sections were photographed with a camera, and the infarct volume was calculated using image analysis software Image J (NIH, Bethesda, MD, USA). Fold infarct volume was calculated according to the previously described formula.
실험예 7. 1차 별아교세포 배양Experimental Example 7. Primary astrocyte culture
1차 대뇌 별아교세포는 마우스의 새끼에서 정제하였다. 구체적으로, 50 마리의 Sprague-Dawley 마우스의 새끼(생후 1일, P1)(다물 사이언스, 한국)로부터 대뇌 피질을 채취한 후 해부 배지에서 해리하였다. 원심분리 후, 세포를 폴리-L-라이신이 코팅된 T75 플라스크에 접종하고, 이를 최소 필수 배지(Minimum Essential Medium, MEM) 기반의 성장 배지에서 유지하였다. 14일 후, 플라스크를 오비탈 쉐이커로 교반하여 모든 비부착 희소돌기교세포 및 미세교세포를 제거하였다. 그 후 부착성 별아교세포를 트립신으로 분리하고, DMEM(Dulbecco‘s Modified Eagle Medium) 기반 별아교세포 배지에서 증식시켰다.Primary cerebral astrocytes were purified from mouse pups. Specifically, cerebral cortex was collected from 50 Sprague-Dawley mouse pups (1 day after birth, P1) (Damul Science, Korea) and dissociated in dissection medium. After centrifugation, the cells were inoculated into a T75 flask coated with poly-L-lysine and maintained in a growth medium based on Minimum Essential Medium (MEM). After 14 days, the flask was shaken with an orbital shaker to remove all non-adherent oligodendrocytes and microglia. Afterwards, adherent astrocytes were separated with trypsin and grown in DMEM (Dulbecco's Modified Eagle Medium)-based astrocyte medium.
실험예 8. 면역조직화학(Immuonohistochemistry) 및 이중 면역형광(double immunofluorescence)Experimental Example 8. Immunohistochemistry and double immunofluorescence
내인성 퍼옥시다제의 활성을 차단하기 위하여, 떠있는(free-floating) 뇌 절편에 1% H2O2(in PBS)를 처리한 후, 블로킹 버퍼(0.3% Triton X-100 및 1% chicken serum in PBS)을 처리하여 30분 동안 반응시켰다. 절편에 1차 항-GFAP 항체(1:200; cat no. MAB360, Millipore), 항-IBA-1 항체(1:200; catalog no. 019-19741, Wako) 및 항-NeuN 항체(1:200; catalog no. 324307s, Cell Signaling)을 처리한 후 하룻밤동안 배양하였다. To block the activity of endogenous peroxidase, free-floating brain slices were treated with 1% H 2 O 2 (in PBS), followed by blocking buffer (0.3% Triton X-100 and 1% chicken serum). in PBS) and reacted for 30 minutes. Sections were incubated with primary anti-GFAP antibody (1:200; cat no. MAB360, Millipore), anti-IBA-1 antibody (1:200; catalog no. 019-19741, Wako), and anti-NeuN antibody (1:200). ; catalog no. 324307s, Cell Signaling) and cultured overnight.
이중 면역형광 분석은 Cy3-conjugated anti-rabbit IgG 항체(Amersham, UK) 및 Cy2-conjugated anti-rabbit IgG 항체(Amersham Pharmacia Biotech)를 사용하여 수행하였다. 세포 핵은 4',6-diamidino-2-phenylindole(DAPI)로 염색하였다. 이중염색된 절편은 AxioPhot 현미경(Leica, TCS SP8, Wetzlar, Germany)을 사용하여 분석하였다.Double immunofluorescence analysis was performed using Cy3-conjugated anti-rabbit IgG antibody (Amersham, UK) and Cy2-conjugated anti-rabbit IgG antibody (Amersham Pharmacia Biotech). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Double-stained sections were analyzed using an AxioPhot microscope (Leica, TCS SP8, Wetzlar, Germany).
실험예 9. 웨스턴 블롯팅Experimental Example 9. Western blotting
마우스 뇌 조직을 해부하고 차가운 PBS 완충액에서 균질화시켰다. 균질화 된 조직 샘플을 1200 RPM에서 3분 동안 원심 분리하고 상층액을 제거하였다. 단백질 추출 완충액(PRO-PERP, iNtRON Biotechnology, 대전, 대한민국)과 1X 프로테아제 억제제를 조직 샘플에 첨가한 후 균질화시켰다. 균질화 후 원심분리하여 세포파편을 제거하여, 단백질 샘플을 얻었다. 5X 로딩 버퍼를 단백질 샘플과 혼합하고, 100℃에서 10분 동안 끓였다. 추출된 단백질 샘플을 12% SDS-PAGE로 분리하고 멤브레인에 블롯팅하였다. 멤브레인에 SOX9 항체(1:1000; catalog no. sc-20095, Santa Cruz biotechnology), Prostaglandin D synthase 항체(1:1000; catalog no. 160013, Cayman)에 대한 적절한 1차 항체를 1,000배 희석하여 4℃에서 밤새 배양하였다. 멤브레인 세척 후 HRP(horseradish peroxidase)-결합된 2차 항체(anti-rabbit-HRP 또는 anti-mouse-HRP; AbFrontier, Korea)를 처리하여 배양하였다. 배양 후 enhanced chemiluminescence solution(Bio-Rad, 170-5061)과 함께 ChemiDoc Touch 이미징 시스템(Bio-Rad)을 사용하여 단백질 발현을 분석하였다. 발현 수준은 ImageJ 소프트웨어를 사용하여 베타 액틴의 수준으로 정규화하였다.Mouse brain tissue was dissected and homogenized in cold PBS buffer. Homogenized tissue samples were centrifuged at 1200 RPM for 3 minutes and the supernatant was removed. Protein extraction buffer (PRO-PERP, iNtRON Biotechnology, Daejeon, Korea) and 1X protease inhibitor were added to the tissue samples and then homogenized. After homogenization, cell debris was removed by centrifugation to obtain a protein sample. 5X loading buffer was mixed with the protein sample and boiled at 100°C for 10 minutes. Extracted protein samples were separated by 12% SDS-PAGE and blotted onto membrane. The membrane was incubated with 1,000-fold dilution of appropriate primary antibodies against SOX9 antibody (1:1000; catalog no. sc-20095, Santa Cruz biotechnology) and Prostaglandin D synthase antibody (1:1000; catalog no. 160013, Cayman) at 4°C. was cultured overnight. After washing the membrane, it was incubated with HRP (horseradish peroxidase)-conjugated secondary antibody (anti-rabbit-HRP or anti-mouse-HRP; AbFrontier, Korea). After incubation, protein expression was analyzed using the ChemiDoc Touch imaging system (Bio-Rad) with enhanced chemiluminescence solution (Bio-Rad, 170-5061). Expression levels were normalized to that of beta actin using ImageJ software.
실험예 10. ELISA (Enzyme-Linked Immunosorbent Assay)Experimental Example 10. ELISA (Enzyme-Linked Immunosorbent Assay)
뇌 조직에서 프로스타글란딘 D2의 농도는 ELISA 키트(Prostaglandin D2 kit, 512031, Cayman)를 사용하여 분석하였다. 구체적으로, 50 μg의 전체 세포용해물을 각 웰에 분주하고, 제조사의 매뉴얼에 따라 실험을 수행하였다.The concentration of prostaglandin D 2 in brain tissue was analyzed using an ELISA kit (Prostaglandin D2 kit, 512031, Cayman). Specifically, 50 μg of total cell lysate was dispensed into each well, and the experiment was performed according to the manufacturer's manual.
[통계분석][Statistical analysis]
모든 통계 분석은 GraphPad Prism 6(GraphPad Software Inc., San Diego, CA, USA)을 사용하여 수행하였다. 모든 데이터는 평균 ± SEM으로 표현되었으며, Tukey의 사후 검정 다중 비교 테스트와 함께 일원 분산 분석(ANOVA)을 사용하였다. 그룹 간의 분석은 짝을 이루지 않은 Student's t-test를 사용하여 수행하였다. 또한 P-value < 0.05는 통계적으로 유의한 결과를 나타내는 것으로 간주하였다.All statistical analyzes were performed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). All data were expressed as mean ± SEM and one-way analysis of variance (ANOVA) with Tukey's post hoc multiple comparison test was used. Analysis between groups was performed using unpaired Student's t-test. Additionally, P-value <0.05 was considered to indicate statistically significant results.
[실시예][Example]
실시예 1. 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자의 제조 및 특성화Example 1. Preparation and characterization of PLGA nanoparticles coupled with astrocyte-selective peptide
별아교세포 선택적 펩타이드(K-C-LNSSQPS-C, AstroLa, 서열번호 1)를 결합시키기 위하여, 두 가지 유형의 PLGA를 이용한 W/O/W 에멀젼을 사용하여 나노입자를 얻었다. 기능화된 나노입자는 PLGA-polyethylene glycol(PEG)-COOH(10% v/v) 및 metyl-PEG-PLGA(90% v/v)를 혼합하여 제조하였다. PLGA-PEG-COOH의 카르복실기는 원하는 아미노산과 펩티드 결합을 생성하는데 필요하다. To bind astrocyte-selective peptide (K-C-LNSSQPS-C, AstroLa, SEQ ID NO: 1), nanoparticles were obtained using W/O/W emulsion using two types of PLGA. Functionalized nanoparticles were prepared by mixing PLGA-polyethylene glycol (PEG)-COOH (10% v/v) and metyl-PEG-PLGA (90% v/v). The carboxyl group of PLGA-PEG-COOH is required to form a peptide bond with the desired amino acid.
별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자의 특성을 분석하기 위하여, 공중합체(copolymer)는 FT-IR, zetasizer, 주사전자현미경(SEM) 및 1H NMR로 분석하였다. 펩타이드가 없는 나노입자(NP); 및 펩타이드가 결합된 NP;의 FTIR 스펙트럼을 측정 및 분석하였다. FT-IR, zetasizer, 주사전자현미경(SEM) 및 1H NMR 분석 결과는 각각 도 1a 내지 1d에 나타내었다.To analyze the properties of PLGA nanoparticles bound with astrocyte-selective peptide, the copolymer was analyzed by FT-IR, zetasizer, scanning electron microscopy (SEM), and 1 H NMR. Peptide-free nanoparticles (NPs); and NP to which the peptide was bound; the FTIR spectrum was measured and analyzed. FT-IR, zetasizer, scanning electron microscopy (SEM), and 1 H NMR analysis results are shown in FIGS. 1A to 1D, respectively.
도 1a에 나타낸 바와 같이, FT-IR 스펙트럼에서, 펩타이드가 결합된 NP(빨간색 화살표)는 1760 cm-1에서 에서 carbonyl 그룹의 강한 신호가 검출되었다.As shown in Figure 1a, in the FT-IR spectrum, a strong signal of the carbonyl group was detected in the peptide-bound NP (red arrow) at 1760 cm -1 .
도 1b에 나타낸 바와 같이, NP 및 펩타이드가 결합된 NP의 입자 크기 및 제타 전위를 측정한 결과, 직경이 약 270 nm인 두 종류의 나노입자 크기는 유의한 차이가 없었다. 그러나 NP의 펩타이드 결합은 펩타이드가 없는 NP에 비해 제타 전위를 증가시키는 것을 관찰하였다. 구체적으로, NP는 제타 전위가 -30 mV에 가까웠으나, 펩타이드가 결합된 NP의 제타 전위 값은 펩타이드 내 아미노 그룹의 양전하로 인해 -0.175 mV임을 확인하였다.As shown in Figure 1b, as a result of measuring the particle size and zeta potential of NP and peptide-bound NP, there was no significant difference in the size of the two types of nanoparticles with a diameter of about 270 nm. However, it was observed that the peptide binding of NPs increased the zeta potential compared to NPs without peptides. Specifically, the zeta potential of NP was close to -30 mV, but the zeta potential value of NP bound to the peptide was confirmed to be -0.175 mV due to the positive charge of the amino group in the peptide.
도 1c에 나타낸 바와 같이, SEM 이미지에서 NP 및 펩타이드가 결합된 NP는 모두 표면이 매끄럽고 응집이 없는 구형이며, 이들 간의 유의한 형태학적 차이는 없는 것을 확인하였다.As shown in Figure 1c, in the SEM image, it was confirmed that both NPs and peptide-bound NPs were spherical with smooth surfaces and no aggregation, and there was no significant morphological difference between them.
도 1d에 나타낸 바와 같이, 별아교세포 선택적 펩타이드, NP 및 펩타이드가 결합된 NP의 1H NMR 스펙트럼으로부터 나노입자에 별아교세포 선택적 펩타이드가 잘 결합된 것을 확인하였다.As shown in Figure 1d, it was confirmed that the astrocyte-selective peptide was well bound to the nanoparticles from the 1 H NMR spectrum of the astrocyte-selective peptide, NP, and NP to which the peptide was bound.
아데노-관련 바이러스(adeno-associated virus, AAV)-mCherry 발현 벡터 시스템을 사용하여, 뇌의 어떤 세포 유형이 NP 및 펩타이드가 결합된 NP를 흡수하는지 평가하였다. 이를 위해 마우스의 두개골과 C1 척추 사이의 환추후두막(atlanto-occipital membrane)을 통해 대조(cisterna magna)의 CSF에 NP 및 펩타이드가 결합된 NP를 직접 투여하였다. 또한 뇌 조직 절편은 세포 유형을 구분하기 위해 면역염색하였다. 면역염색 결과 및 NP 흡수 여부를 분석한 결과는 각각 도 1e 및 1f에 나타내었다.Using the adeno-associated virus (AAV)-mCherry expression vector system, we evaluated which cell types in the brain uptake NPs and peptide-conjugated NPs. For this purpose, NPs and peptide-bound NPs were directly administered to the CSF of the control cisterna magna through the atlanto-occipital membrane between the mouse skull and C1 vertebra. Additionally, brain tissue sections were immunostained to distinguish cell types. The results of immunostaining and analysis of NP absorption are shown in Figures 1e and 1f, respectively.
도 1e 및 1f에 나타낸 바와 같이, 뇌 조직 절편은 세포 유형에 특이적인 마커로 면역염색하였다: 별아교세포의 경우 GFAP, 미세아교세포의 경우 Iba-1, 뉴런의 경우 NeuN. mCherry와 상기 세포 유형에 특이적인 마커의 공동 발현을 분석한 결과, 펩타이드가 결합된 NP가 주로 별아교세포로 흡수되는 것을 확인하였다. 보다 상세하게는, NP는 모든 유형의 세포에서 관찰되었으나, 다른 세포에 비해 미세아교세포에서 더 높았다. 반면에, 펩타이드가 결합된 NP는 모든 유형의 세포에서 관찰되었으나, 대뇌 피질의 별아교세포에서 2배 이상 높은 수준으로 검출되었다. As shown in Figures 1E and 1F, brain tissue sections were immunostained with cell type-specific markers: GFAP for astrocytes, Iba-1 for microglia, and NeuN for neurons. As a result of analyzing the co-expression of mCherry and markers specific to the above cell types, it was confirmed that peptide-conjugated NPs were mainly absorbed into astrocytes. More specifically, NP was observed in all cell types, but was higher in microglia compared to other cells. On the other hand, peptide-conjugated NP was observed in all types of cells, but was detected at a level more than two times higher in astrocytes of the cerebral cortex.
상기 결과는 별아교세포 선택적 펩타이드가 결합된 나노입자는 미세아교세포 및 뉴런보다 별아교세포에 흡수 및 결합된다는 것을 의미한다.The above results mean that nanoparticles bound with astrocyte-selective peptide are absorbed and bound to astrocytes rather than microglia and neurons.
실시예 2. 별아교세포에서 특이적으로 GFAP:SOX9:tdTOM을 발현하는 PLGA 나노입자의 생산 및 분석Example 2. Production and analysis of PLGA nanoparticles specifically expressing GFAP:SOX9:tdTOM in astrocytes
본 실시예를 통해, (i) 별아교세포에 특이적인 SOX9 유전자 발현을 위한 플라스미스가 캡슐화되고, (ii) 별아교세포 선택적 펩타이드가 결합된 NP를 생산하였다.Through this example, (i) a plasmid for expression of the astrocyte-specific SOX9 gene was encapsulated, and (ii) NPs to which an astrocyte-selective peptide was bound were produced.
2-1. 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP 제조2-1. Preparation of plasmid-encapsulated NPs conjugated with astrocyte-selective peptides
SOX9 과발현이 광혈전성 피질 허혈에서 별아교세포증(astrogliosis)의 영향을 역전시키는지 여부를 조사하기 위하여, SOX9 발현 플라스미드 캡슐화 NP를 합성하였다. 또한 상기 SOX9 발현 플라스미드 캡슐화 NP에 별아교세포 선택적 펩타이드(서열번호 1)를 결합시켰다. SOX9 발현 플라스미드는 도 2a와 같이 별아교세포 선택적 GFAP 프로모터 및 tdTomato 발현 리포터 시스템을 포함한다. To investigate whether SOX9 overexpression reverses the effects of astrogliosis in photothrombotic cortical ischemia, SOX9 expression plasmid-encapsulated NPs were synthesized. Additionally, an astrocyte-selective peptide (SEQ ID NO: 1) was conjugated to the SOX9 expression plasmid-encapsulated NP. The SOX9 expression plasmid contains an astrocyte-selective GFAP promoter and a tdTomato expression reporter system, as shown in Figure 2A.
2-2. 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP의 특성분석2-2. Characterization of plasmid-encapsulated NPs conjugated with astrocyte-selective peptides
세포에서 tdTomato의 발현 관찰Observation of expression of tdTomato in cells
1차 별아교세포에서 SOX9 및 tdTomato의 발현을 확인하기 위해, (i) 음성 대조군(pNC); 또는 (ii) pSOX9를 형질감염시킨 세포(pSOX9);에서 24시간 동안 tdTomato 발현을 관찰하였다. 세포에서 tdTomato의 발현을 관찰한 결과는 도 2b에 나타내었다.To determine the expression of SOX9 and tdTomato in primary astrocytes, (i) negative control (pNC); or (ii) cells transfected with pSOX9 (pSOX9); tdTomato expression was observed for 24 hours. The results of observing the expression of tdTomato in cells are shown in Figure 2b.
도 2b에 나타낸 바와 같이, pNC-tdtomato 및 pSOX9-tdTomato는 모두 1차 별아교세포에서 발현되는 것을 확인하였다.As shown in Figure 2b, both pNC-tdtomato and pSOX9-tdTomato were confirmed to be expressed in primary astrocytes.
입자 크기 및 제타 전위 분석Particle size and zeta potential analysis
별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP를 준비하였다. 준비된 NP는 입자 크기 및 제타 전위를 분석하였으며, 그 결과는 도 2c에 나타내었다.Plasmid-encapsulated NPs conjugated with astrocyte-selective peptide were prepared. The prepared NPs were analyzed for particle size and zeta potential, and the results are shown in Figure 2c.
도 2c에 나타낸 바와 같이, 펩타이드 결합 여부에 관계없이 NP의 크기는 유사한 것을 확인하였다. 그러나 펩타이드가 결합된 NP는 펩타이드가 결합되지 않은 NP에 비해 제타 전위가 증가한 것을 확인하였다.As shown in Figure 2c, it was confirmed that the size of the NPs was similar regardless of whether or not the peptide was bound. However, it was confirmed that the zeta potential of the NPs to which the peptide was bound was increased compared to the NPs to which the peptide was not bound.
FT-IR 분석FT-IR analysis
별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP의 FT-IR 분석을 실시하였고, 그 결과는 도 2d에 나타내었다.FT-IR analysis of plasmid-encapsulated NPs conjugated with astrocyte-selective peptide was performed, and the results are shown in Figure 2d.
도 2d에 나타낸 바와 같이, FT-IR 스펙트럼에서, 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP는 1760 cm-1에서 carbonyl 그룹의 강한 신호(빨간색 및 초록색 데이터, 빨간색 화살표)가 검출되었다.As shown in Figure 2d, in the FT-IR spectrum, a strong signal of the carbonyl group (red and green data, red arrow) was detected at 1760 cm -1 for the plasmid-encapsulated NP conjugated with the astrocyte-selective peptide.
주사전자현미경 분석Scanning electron microscopy analysis
SEM을 통해 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP의 표면을 분석하였다. SEM 이미지는 도 2e에 나타내었다.The surface of plasmid-encapsulated NPs to which astrocyte-selective peptide was conjugated was analyzed using SEM. The SEM image is shown in Figure 2e.
도 2e에 나타낸 바와 같이, 펩타이드 결합 여부에 관계없이 NP의 모양 및 표면은 유사한 것을 확인하였다.As shown in Figure 2e, it was confirmed that the shape and surface of the NPs were similar regardless of whether or not the peptide was bound.
실시예 3. 반음영 부위(penumbra)에서 별아교세포의 GFAP 면역 반응성 분석Example 3. Analysis of GFAP immunoreactivity of astrocytes in the penumbra
3-1. 행잉 테스트(hanging test)3-1. hanging test
생체 내 SOX9의 신경 보호 효과를 확인하기 위하여, 로즈벵갈(Rose Bengal, RB)을 복강 내 주사한 광혈전성 허혈 마우스 모델을 제조하였다. 제조된 광혈전성 허혈 유도 2일째에 행잉 테스트(hanging test)를 수행하였으며, 그 결과는 도 3a에 나타내었다.To confirm the neuroprotective effect of SOX9 in vivo, a photothrombotic ischemia mouse model was prepared by intraperitoneal injection of Rose Bengal (RB). A hanging test was performed on the second day of photothrombotic ischemia induction, and the results are shown in Figure 3a.
도 3a에 나타낸 바와 같이, 와이어 걸이 시간은 대조군(n=5, 134.4 ± 19.5)보다 로즈벵갈 투여 그룹(n=7, 15.1±3.2)에서 더 짧았으며, 이는 광혈전성 허혈 유도 후 광혈전성 허혈 마우스 모델에서 운동장애가 발생하였다는 것을 의미한다.As shown in Figure 3A, the wire hanging time was shorter in the rose bengal-administered group (n = 7, 15.1 ± 3.2) than in the control group (n = 5, 134.4 ± 19.5), which was observed in mice with photothrombotic ischemia after induction of photothrombotic ischemia. It means that movement disorder has occurred in the model.
행잉 테스트 후 마우스를 희생시켜 뇌의 경색 부위를 관찰하였다. 관찰 부위는 도 3b에 나타내었다.After the hanging test, the mouse was sacrificed and the infarcted area of the brain was observed. The observation area is shown in Figure 3b.
도 3b에 나타낸 바와 같이, 로즈벵갈 투여 그룹은 광혈전으로 인한 허혈이 육안으로 관찰되었다. As shown in Figure 3b, ischemia due to photothrombosis was visually observed in the rose bengal administered group.
3-2. 경색 부피 측정3-2. Infarct volume measurements
TTC(2,3,5-triphenyl tertrazoium chloride, TTC) 염색을 통해 경색 부피를 측정하였다. 경색 부피를 측정한 결과는 도 3c에 나타내었다.Infarct volume was measured using 2,3,5-triphenyl tertrazoium chloride (TTC) staining. The results of measuring the infarct volume are shown in Figure 3c.
도 3c에 나타낸 바와 같이, 로즈벵갈 투여 그룹(즉, 광혈전 허혈 유도 그룹)은 반음영 부위(penumbra)와 허혈성 코어 영역 사이에서 허혈 부위가 관찰되었다.As shown in Figure 3c, in the rose bengal administration group (i.e., photothrombotic ischemia induction group), an ischemic area was observed between the penumbra and the ischemic core area.
3-3. GFAP 발현 확인3-3. Confirmation of GFAP expression
GFAP 발현 확인에 앞서, GFAP 면역 반응성 별아교세포는 광혈전성 허혈 유도 후 대뇌 영역에 따라 다른 패턴으로 관찰되는 것을 확인하였다. 이를 기반으로, M2(secondary motor cortex) 영역 및 cg1(cingulated area 1) 영역에 초점을 맞추어 GFAP의 발현을 분석하였다. GFAP의 발현을 분석한 결과는 도 3d 및 3e에 나타내었다.Prior to confirming GFAP expression, it was confirmed that GFAP-immunoreactive astrocytes were observed in different patterns depending on the cerebral region after photothrombotic ischemia induction. Based on this, the expression of GFAP was analyzed by focusing on the M2 (secondary motor cortex) region and cg1 (cingulated area 1) region. The results of analyzing the expression of GFAP are shown in Figures 3d and 3e.
도 3d에 나타낸 바와 같이, M2 영역의 GFAP 활성은 정상 상태에서 거의 관찰되지 않았다. cg1 영역은 전체 피질에서 GFAP의 발현이 높았다. 그러나 M2 영역의 GFAP 발현은 로즈벵갈에 의한 허혈 상태에서 48시간 후 훨씬 높아진 것을 확인하였다.As shown in Figure 3d, little GFAP activity in the M2 region was observed at steady state. The cg1 region had high expression of GFAP in the entire cortex. However, GFAP expression in the M2 region was confirmed to be significantly higher after 48 hours in rose bengal-induced ischemia.
도 3e에 나타낸 바와 같이, 내부 반음영 부위(penumbra)의 별아교세포는 손상된 코어를 향한 확장과 함께 극성화된 형태인 것을 확인하였다. 이와 대조적으로, 외부 반음영 부위의 별아교세포는 비대성이지만 극성화되지 않은 형태인 것을 확인하였다.As shown in Figure 3e, it was confirmed that the astrocytes in the inner penumbra were in a polarized form with expansion toward the damaged core. In contrast, astrocytes in the outer penumbra region were confirmed to be hypertrophic but not polarized.
상기 결과는 광혈전성 허혈 후 반음영 부위의 별아교세포를 표적할 때 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP를 사용하는 것이 적합하다는 것을 의미한다.The above results mean that it is appropriate to use plasmid-encapsulated NPs conjugated with an astrocyte-selective peptide when targeting astrocytes in the penumbra area after photothrombotic ischemia.
실시예 4. 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP의 허혈성 뇌졸중 치료 효과Example 4. Ischemic stroke treatment effect of plasmid-encapsulated NPs conjugated with astrocyte-selective peptide
SOX9-과발현의 신경 보호 효과를 확인하기 위한 실험을 도 4a와 같이 수행하였다. 구체적으로, 행잉 테스트 및 NP 주입은 로즈벵갈 투여 1일 전에 동시에 수행하였다. 상기 NP 주입은 NP 20 μL(in 250 μL PBS)을 마우스의 대조(cisterna magna)에 투여하였다. 광혈전성 허혈 유도 2일 후 행잉 테스트를 수행하고, 마우스를 희생시켰다. 희생된 마우스의 뇌를 채취하여 TTC 염색하였다. 상기 행잉 테스트 결과는 도 4b에 나타내었고, TTC 염색결과는 도 4c 및 4d에 나타내었다.An experiment to confirm the neuroprotective effect of SOX9-overexpression was performed as shown in Figure 4a. Specifically, the hanging test and NP injection were performed simultaneously 1 day before rose bengal administration. For the NP injection, 20 μL (in 250 μL PBS) of NP was administered to the control (cisterna magna) of the mouse. A hanging test was performed 2 days after photothrombotic ischemia induction, and the mice were sacrificed. The brains of sacrificed mice were collected and stained with TTC. The hanging test results are shown in Figure 4b, and the TTC staining results are shown in Figures 4c and 4d.
도 4b에 나타낸 바와 같이, 대조군(NPs-pNC 그룹, AstroLa-NPs-pNP 그룹)은 광혈전성 허혈 유도에 따라 운동장애가 발생한 것을 확인하였다. 이와 달리, pSOX9 플라스미드 캡슐화 NP 처리 그룹은 대조군에 비해 운동장애가 개선된 것을 확인하였다. 특히, 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP 처리 그룹(AstroLa-NPs-pSOX9)은 별아교세포 선택적 펩타이드가 결합되지 않은 NP 처리 그룹(NPs-pSOX9)에 비해 현저한 운동장애 개선 효과가 있는 것을 확인하였다.As shown in Figure 4b, it was confirmed that movement disorders occurred in the control group (NPs-pNC group, AstroLa-NPs-pNP group) due to photothrombotic ischemia induction. In contrast, the pSOX9 plasmid encapsulated NP treatment group was confirmed to have improved motor dysfunction compared to the control group. In particular, it was confirmed that the plasmid-encapsulated NP treatment group (AstroLa-NPs-pSOX9) to which the astrocyte-selective peptide was bound had a significant improvement in motor dysfunction compared to the NP treatment group (NPs-pSOX9) to which the astrocyte-selective peptide was not bound. .
도 4c 및 4d에 나타낸 바와 같이, 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP 처리 그룹(AstroLa-NPs-pSOX9)은 다른 실험 그룹 및 대조군에 비해 경색 부피가 현저히 감소한 것을 확인하였다.As shown in Figures 4c and 4d, the group treated with plasmid-encapsulated NPs conjugated with an astrocyte-selective peptide (AstroLa-NPs-pSOX9) showed a significant decrease in infarct volume compared to other experimental groups and the control group.
상기 결과는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP가 로즈벵갈로 유도된 광혈전성 허혈에 따른 허혈성 뇌 손상을 효과적으로 치료할 수 있음을 의미한다.The above results mean that plasmid-encapsulated NPs conjugated with astrocyte-selective peptide can effectively treat ischemic brain damage caused by photothrombotic ischemia induced by rose bengal.
실시예 5. SOX9 과발현에 따른 PGD2 경로 분석Example 5. PGD2 pathway analysis according to SOX9 overexpression
5-1. 면역염색을 통한 SOX9 발현 조사5-1. Investigation of SOX9 expression through immunostaining
SOX9가 로즈벵갈로 유도된 뇌졸중 모델에서 뇌 손상을 줄이는 메커니즘을 조사하였다. 구체적으로, 로즈벵갈 주입 전에 플라스미드 캡슐화 NP(pSOX9 NP) 또는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP(AstroLa-pSOX9 NP)를 주입하였다. 광혈전성 허혈 유도 후 반음영 부위에서 SOX9 발현을 조사하였다. SOX9의 발현을 조사한 결과는 도 5a 및 5b에 나타내었다.The mechanism by which SOX9 reduces brain damage in a rose bengal-induced stroke model was investigated. Specifically, plasmid-encapsulated NPs (pSOX9 NPs) or plasmid-encapsulated NPs conjugated with an astrocyte-selective peptide (AstroLa-pSOX9 NPs) were injected before rose bengal injection. After photothrombotic ischemia induction, SOX9 expression was examined in the penumbra region. The results of examining the expression of SOX9 are shown in Figures 5a and 5b.
도 5a 및 5b에 나타낸 바와 같이, 플라스미드 캡슐화 NP(pSOX9 NP) 또는 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP(AstroLa-pSOX9 NP)는 모두 허혈성 반음영 부위의 별아교세포에 국한된 것을 확인하였다. 또한 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP(AstroLa-pSOX9 NP) 처리 그룹의 반음영 부위에서 SOX9의 발현은 플라스미드 캡슐화 NP(pSOX9 NP)에 비해 유의하게 증가하였다. As shown in Figures 5a and 5b, it was confirmed that both plasmid-encapsulated NP (pSOX9 NP) or plasmid-encapsulated NP conjugated with astrocyte-selective peptide (AstroLa-pSOX9 NP) were localized to astrocytes in the ischemic penumbra area. In addition, the expression of SOX9 in the penumbra region of the group treated with plasmid-encapsulated NPs (AstroLa-pSOX9 NPs) conjugated with astrocyte-selective peptide was significantly increased compared to plasmid-encapsulated NPs (pSOX9 NPs).
5-2. 웨스턴 블롯팅을 통한 SOX9 발현 조사5-2. Investigation of SOX9 expression through Western blotting
상기 실시예 5-1의 결과를 바탕으로 각 그룹의 SOX9 관련 단백질 발현을 웨스턴 블롯팅을 통해 분석하였다. 웨스턴 블롯팅 결과는 도 5c에 나타내었다.Based on the results of Example 5-1, the expression of SOX9-related proteins in each group was analyzed through Western blotting. The Western blotting results are shown in Figure 5c.
도 5c에 나타낸 바와 같이, 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP(AstroLa-pSOX9 NP) 처리 그룹에서 SOX9의 발현이 가장 높은 것을 확인하였다.As shown in Figure 5c, it was confirmed that the expression of SOX9 was highest in the group treated with plasmid-encapsulated NP (AstroLa-pSOX9 NP) conjugated with astrocyte-selective peptide.
5-3. 웨스턴 블롯팅을 통한 프로스타글란딘 D2의 발현 조사5-3. Investigation of the expression of prostaglandin D2 through Western blotting
SOX9 발현의 특이적인 효과를 확인하기 위하여, SOX9-다운스트림 유전자인 프로스타글란딘 D2 합성 효소(prostaglandin D2 synthase, PTGDS)의 발현을 웨스턴 블롯팅을 통해 조사하였다. 웨스턴 블롯팅 결과는 도 5d에 나타내었다.To confirm the specific effect of SOX9 expression, the expression of prostaglandin D2 synthase (PTGDS), a SOX9-downstream gene, was examined through Western blotting. The Western blotting results are shown in Figure 5d.
도 5d에 나타낸 바와 같이, 별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP(AstroLa-pSOX9 NP) 처리 그룹은 플라스미드 캡슐화 NP(pSOX9 NP)에 비해 PTGDS의 발현이 유의하게 증가한 것을 확인하였다. As shown in Figure 5d, the group treated with plasmid-encapsulated NPs (AstroLa-pSOX9 NPs) conjugated with an astrocyte-selective peptide showed a significant increase in the expression of PTGDS compared to the plasmid-encapsulated NPs (pSOX9 NPs).
당업계에서, PTGDS의 산물인 프로스타글란딘 D2(prostaglandin D2, PGD2)가 DP2 수용체를 통해 HO-1의 발현을 유도하는 것으로 알려져 있다. PTGDS 발현에 따라 PGD2의 발현량을 ELISA를 통해 분석하였고, 그 결과는 도 5e에 나타내었다.In the art, it is known that prostaglandin D2 (PGD2), a product of PTGDS, induces the expression of HO-1 through the DP2 receptor. The expression level of PGD2 was analyzed through ELISA according to PTGDS expression, and the results are shown in Figure 5e.
도 5e에 나타낸 바와 같이, 플라스미드 캡슐화 NP(NPs-pSOX9) 처리 그룹은 발현된 PGD2의 양이 30.6 pg/ml이고, 플라스미드가 캡슐화되지 않은 NP(NPs-pNP) 처리 그룹은 22.7 pg/ml인 것을 확인하였다. As shown in Figure 5e, the amount of expressed PGD2 in the plasmid-encapsulated NPs (NPs-pSOX9) treatment group was 30.6 pg/ml, and the amount of expressed PGD2 in the plasmid-encapsulated NPs (NPs-pNP) treatment group was 22.7 pg/ml. Confirmed.
별아교세포 선택적 펩타이드가 결합된 플라스미드 캡슐화 NP(AstroLa-pSOX9 NP) 처리 그룹은 PGD2의 양이 43.2 pg/ml로, 가장 높은 것을 확인하였다.The group treated with plasmid-encapsulated NPs (AstroLa-pSOX9 NP) coupled with an astrocyte-selective peptide had the highest amount of PGD2 at 43.2 pg/ml.
상기 결과는 광혈전성 허혈 유도 후 SOX9 발현 증가가 별아교세포에서 PTGDS 및 PGD2의 유도에 의해 신경 보호를 조절한다는 것을 의미한다.The above results indicate that increased SOX9 expression after induction of photothrombotic ischemia regulates neuroprotection by induction of PTGDS and PGD2 in astrocytes.
종합적으로 본 발명자들은 (i) 별아교세포에 특이적인 SOX9 유전자 발현을 위한 플라스미스가 캡슐화되고, (ii) 별아교세포 선택적 펩타이드가 결합된 PLGA 나노입자를 개발하고, 개발된 PLGA 나노입자가 생체적합성 및 생분해성이 우수하며, 동물 수준에서 허혈성 뇌손상을 치료하는 효과가 있음을 확인하였다. 이는 본 발명의 PLGA 나노입자가 허혈성 뇌질환의 치료제로서 이용될 수 있음을 의미하는바, 허혈성 뇌질환의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.Overall, the present inventors developed PLGA nanoparticles in which (i) a plasmid for expression of the astrocyte-specific SOX9 gene was encapsulated and (ii) conjugated with an astrocyte-selective peptide, and the developed PLGA nanoparticles were biocompatible and biocompatible. It has excellent biodegradability and has been confirmed to be effective in treating ischemic brain damage at the animal level. This means that the PLGA nanoparticles of the present invention can be used as a treatment for ischemic brain disease, and can be used in a variety of fields in the field of prevention, improvement, and treatment of ischemic brain disease.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 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 의해 정의된다고 할 것이다.As above, specific parts of the present invention have been described in detail, and it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> The Industry & Academic Cooperation in Chungnam National University (IAC) Nanoglia <120> Nanoparticle conjugated with astrocyte-selective peptide and uses thereof <130> 1-77 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> astrocyte-targeting peptide, AstroLa <400> 1 Lys Cys Leu Asn Ser Ser Gln Pro Ser Cys 1 5 10 <110> The Industry & Academic Cooperation in Chungnam National University (IAC) Nanoglia <120> Nanoparticle conjugated with astrocyte-selective peptide and uses of that <130> 1-77 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> astrocyte-targeting peptide, AstroLa <400> 1 Lys Cys Leu Asn Ser Ser Gln Pro Ser Cys 1 5 10
Claims (8)
A pharmaceutical composition for preventing or treating ischemic brain disease comprising PLGA nanoparticles bound to an astrocyte-selective peptide represented by the amino acid sequence of SEQ ID NO: 1, wherein the PLGA nanoparticles include a plasmid containing a gene encoding SOX9. A pharmaceutical composition for preventing or treating ischemic brain disease.
A food composition for preventing or improving ischemic brain disease comprising PLGA nanoparticles bound to an astrocyte-selective peptide represented by the amino acid sequence of SEQ ID NO: 1, wherein the PLGA nanoparticles contain a plasmid containing a gene encoding SOX9. A food composition for preventing or improving ischemic brain disease.
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| KR1020210069959A KR102629203B1 (en) | 2021-05-31 | 2021-05-31 | Nanoparticle conjugated with astrocyte-selective peptide and uses thereof |
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Non-Patent Citations (3)
| Title |
|---|
| Biomaterials. 2014. Vol.35, pp.8236-8248.* |
| Glia. 2018. Vol.66, No.2, pp.445-458.* |
| Molecular Therapy: Nucleic Acids. 2018. Vol.11, pp.203-215.* |
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