KR20140094964A - Calcium phosphate nano-aggregates using hydrophilic polymer modified orgarnic compound containing catechol group and preparation method therof - Google Patents
Calcium phosphate nano-aggregates using hydrophilic polymer modified orgarnic compound containing catechol group and preparation method therof Download PDFInfo
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- KR20140094964A KR20140094964A KR1020130007675A KR20130007675A KR20140094964A KR 20140094964 A KR20140094964 A KR 20140094964A KR 1020130007675 A KR1020130007675 A KR 1020130007675A KR 20130007675 A KR20130007675 A KR 20130007675A KR 20140094964 A KR20140094964 A KR 20140094964A
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- 239000001506 calcium phosphate Substances 0.000 title claims abstract description 139
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 title claims abstract description 139
- 229910000389 calcium phosphate Inorganic materials 0.000 title claims abstract description 137
- 235000011010 calcium phosphates Nutrition 0.000 title claims abstract description 137
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K91/00—Lines
- A01K91/03—Connecting devices
- A01K91/04—Connecting devices for connecting lines to hooks or lures
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K91/00—Lines
- A01K91/03—Connecting devices
- A01K91/053—Fishing booms, i.e. connecting devices spreading out the leaders, e.g. to avoid tangling thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K91/00—Lines
- A01K91/03—Connecting devices
- A01K91/047—Connecting devices for connecting lines to lines
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Marine Sciences & Fisheries (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
본 발명은 칼슘 포스페이트, 전달 유전자, 및 카테콜기를 포함하는 유기화합물로 개질된 친수성 고분자를 포함하는 유전자 전달용 나노 복합체 및 이의 제조방법에 대한 것이다.The present invention relates to a gene transfer nanocomposite comprising a hydrophilic polymer modified with an organic compound containing calcium phosphate, a transfer gene, and a catechol group, and a method for producing the same.
칼슘 포스페이트(CAP)는 가장 널리 사용되는 경조직(hard tissue) 대체제이다. 이는 인체의 천연 무기 성분과 매우 유사하며 높은 생체 적합성을 갖는다(Laurencin et al., 1999; Petite et al., 2000). 생체의료용으로 적합하게 사용하기 위해, 칼슘 포스페이트 나노상(phase)은 적절한 모폴로지 및 좁은 크기 분포를 가져야 한다(Ginebra et al., 2004). 또한 CaP는 시험관 내 유전자 형질감염에 효과적인 non-viral vector로 사용되어 왔다(Bisht et al., 2005; Roy et al., 2003). Calcium phosphate (CAP) is the most widely used hard tissue substitute. (Laurencin et al., 1999; Petite et al., 2000), which is very similar to the natural inorganic constituents of humans and has high biocompatibility. For use in biomedical applications, the calcium phosphate nano phase should have adequate morphology and narrow size distribution (Ginebra et al., 2004). CaP has also been used as a non-viral vector for in vitro gene transfection (Bisht et al., 2005; Roy et al., 2003).
CAP에 기초한 유전자 형질감염에서 가장 빈번히 사용되는 기술은 칼슘 포스페이트-유전자-공침법이다(Khosravi-Darani et al., 2010). 이는 CAP의 칼슘 이온과 유전자의 인산기의 상호작용을 통해 CAP-유전자 복합체를 형성하는 방법이다. 그러나 CAP-유전자 복합체 제조에 있어서 주요한 단점은 샘플의 농도, pH 및 온도와 같은 실험 조건의 변화에 따라 재현가능성이 떨어진다는 점이다. 따라서 나노 단위의 불규칙한 입자 성장 없이, 크기가 잘 조절된 CAP를 제조하는 것이 중요하다. CAP의 크기를 조절하고 바람직한 형질감염 효율을 확보하기 위한 다양한 접근들이 있어왔다. 최근 지질이 코팅된 CAP가 제조되었으며 이는 LCP(lipid coating CAP)라고 불린다. 이는 water-in-oil nanoemulsion 방법을 이용하여 제조된다. siRNA를 포함하는 LCP는 siRNA의 세포 내 전달을 성공적으로 매개하고, 효과적으로 타겟 유전자를 사일런싱(silencing)시킨다(Li et al., 2010). 추가적으로 LCP를 PEG로 표면 개질하는 경우, 고형 암에서 타겟 유전자 사일런싱이 더욱 효과적으로 일어나며 전신투여 후 암 축적 프로파일이 개선된다(Li et al., 2012). 그러나, 상기 에멀젼에 기초한 제법들은 대개 제조에 여러 단계가 필요하며, 후에 제거되어야 하는 유기용매의 사용을 요구하기도 한다. The most frequently used technique for CAP-based gene transfection is calcium phosphate-gene-co-injection (Khosravi-Darani et al., 2010). This is a method of forming a CAP-gene complex through the interaction of the calcium ion of CAP with the phosphate group of the gene. However, the main disadvantages in the preparation of CAP-gene complexes are that they are less reproducible with changes in experimental conditions such as concentration, pH and temperature of the sample. Therefore, it is important to produce CAPs with well-controlled size without irregular particle growth in the nanometer scale. There have been various approaches for controlling the size of CAPs and ensuring desirable transfection efficiency. Recent lipid-coated CAPs have been produced and are called LCP (lipid coating CAP). It is prepared using the water-in-oil nanoemulsion method. LCPs containing siRNA successfully mediate intracellular delivery of siRNAs and effectively silence target genes (Li et al., 2010). In addition, when LCP is surface modified with PEG, target gene silencing occurs more efficiently in solid cancers and improves cancer accumulation profiles after systemic administration (Li et al., 2012). However, the above-mentioned emulsion-based formulations usually require several steps in their manufacture and require the use of organic solvents which must be removed later.
최근, 다양한 생체재료에서 3,4-디하이드록시-L-페닐알라닌(dopa)의 접착 성질을 이용하려는 시도들이 있어왔다(Bae et al., 2011; Lee et al., 2010; Lee et al., 2009). Dopa는 홍합(Mytilus edulis)에 의해 분비되는 특이한 화합물이다. 이는 다양한 유기 또는 무기 표면에 홍합을 강하게 부착시키는 중요한 역할을 한다(Lee et al., 2007; Waite et al., 1981). 이러한 독특한 접착 성질은 dopa를 이용한 다양한 유기 또는 무기 재료(예, 금속 산화물, 금속, 고분자)의 표면 개질을 가능하게 하였다. dopa 구조 중 카테콜 모이어티는 철 및 칼슘을 포함하는 다양한 금속 이온에 대해 강한 친화력을 가진다(Holten-Andersen et al., 2009; Harrington et al., 2010). 상기 금속-dopa 복합체는 다양한 실용적 목적으로 널리 사용되어 왔다. 이러한 사용의 예로는 금속-매개의 가역적 생체고분자 네트워크(하이드로겔)의 제조 및 폴리도파민 보조의 다목적 재료의 생체모방성 보편적 생광물화(biomimetic universal biomineralization)가 있다. 후자의 예에서, 폴리도파민의 카테콜 모이어티는 simulated body fluid(SBF)의 칼슘이온을 농축시키며, 그 결과 칼슘 포스페이트의 핵 생성 및 생광물화를 유도한다. Recently, attempts have been made to utilize the adhesion properties of 3,4-dihydroxy-L-phenylalanine (dopa) in various biomaterials (Bae et al., 2011; Lee et al., 2010; Lee et al. 2009). Dopa is a unique compound secreted by mussels (Mytilus edulis). This plays an important role in strongly attaching mussels to various organic or inorganic surfaces (Lee et al., 2007; Waite et al., 1981). This unique adhesion property enables surface modification of various organic or inorganic materials (eg, metal oxides, metals, polymers) using dopa. Among the dopa structures, catechol moieties have strong affinity for various metal ions including iron and calcium (Holten-Andersen et al., 2009; Harrington et al., 2010). The metal-dopa composites have been widely used for various practical purposes. Examples of such use include the preparation of metal-mediated reversible biomolecule networks (hydrogels) and the biomimetic universal biomineralization of polydopamine assisted multipurpose materials. In the latter example, the catechol moiety of polydodamine concentrates the calcium ions of the simulated body fluid (SBF), resulting in nucleation and mineralization of calcium phosphate.
이에 본 발명자들은 칼슘 포스페이트에 기초한 새로운 유전자 전달체에 대해 연구하던 중, 카테콜기를 포함하는 유기화합물, 그중에서도 특히 dopa가 접합된 친수성의 고분자 및 칼슘 포스페이트로 이루어진 복합체가 칼슘 포스페이트 나노입자의 성장을 억제함과 동시에 입자를 안정화 시키며 훌륭한 유전자 전달체로 기능한다는 사실을 확인하고 본 발명을 완성하였다. Therefore, the present inventors investigated a new gene carrier based on calcium phosphate, and found that an organic compound including a catechol group, particularly, a complex composed of a hydrophilic polymer doped with dopa and a calcium phosphate inhibits the growth of calcium phosphate nanoparticles And at the same time stabilizes the particles and functions as a good gene transporter, thus completing the present invention.
본 발명의 목적은 칼슘 포스페이트, 전달 유전자, 및 카테콜기(1,2-dihydroxybenzene)를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자를 포함하는 유전자 전달용 나노 복합체를 제공하는 것이다.It is an object of the present invention to provide a gene delivery nanocomposite comprising calcium phosphate, a transfer gene, and a hydrophilic polymer modified with a monomolecular or polymer containing a catechol (1,2-dihydroxybenzene).
본 발명의 또 다른 목적은 1) 카테콜기(1,2-dihydroxybenzene)를 포함하는 단분자 또는 중합체를 친수성 고분자와 반응시켜 친수성 고분자에 카테콜기를 포함하는 단분자 또는 중합체를 개질하는 단계, 2) 칼슘포스페이트와 전달유전자를 혼합하여 칼슘포스페이트/전달유전자 복합체를 제조하는 단계, 3) 상기 단계 1)에서 제조한 카테콜기를 포함하는 단분자 또는 중합체가 개질된 친수성 고분자와 2)에서 제조한 칼슘포스페이트/전달유전자 복합체를 혼합하여 칼슘포스페이트/전달유전자/카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자 복합체를 제조하는 단계를 포함하는 유전자 전달용 나노 복합체의 제조방법을 제공하는 것이다.
It is still another object of the present invention to provide a method for producing a hydrophilic polymer, comprising the steps of: 1) reacting a monomolecule or polymer containing a 1,2-dihydroxybenzene with a hydrophilic polymer to modify a monomolecule or a polymer containing a catechol group in the hydrophilic polymer; 2) Preparing a calcium phosphate / transfer gene complex by mixing calcium phosphate and a transferring gene; 3) preparing a mono-or polymer-modified hydrophilic polymer containing the catechol group prepared in the above step 1) and calcium phosphate / Transgene complex to prepare a hydrophilic polymer complex modified with a single molecule or a polymer containing a calcium phosphate / transcription gene / catechol group. The present invention also provides a method for producing a gene delivery nanocomposite.
상기 과제를 해결하기 위하여 본 발명의 제1양태는 칼슘 포스페이트, 전달 유전자, 및 카테콜기(1,2-dihydroxybenzene)를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자를 포함하는 유전자 전달용 나노 복합체를 제공한다.
In order to solve the above-described problems, a first aspect of the present invention is to provide a gene transfer nanocomposite comprising a hydrophilic polymer modified with a monomolecular or polymer modified with calcium phosphate, a transfer gene, and a catechol (1,2-dihydroxybenzene) to provide.
본 발명에서 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자는 칼슘 포스페이트 나노 입자 외부를 감싸고 있다. 카테콜(catechol)기는 수용액 상의 칼슘이온(Ca2+)을 킬레이팅(chelating)하여 칼슘 포스페이트 결정화를 위한 핵점(nucleation point)으로 작용한다. 상기 핵점을 중심으로 칼슘 포스페이트 결정의 성장이 일어난다. 이때 포스페이트 주사슬을 지닌 전달유전자 또한 입자에 포합되어 나노크기의 복합체를 형성하며, 친수성 고분자는 칼슘 포스페이트 입자 가장 바깥쪽 표면에 위치하여 입자의 수용액상 안정성을 증진시킨다.
In the present invention, a monomolecular or polymer modified hydrophilic polymer containing a catechol group surrounds the outside of calcium phosphate nanoparticles. The catechol group acts as a nucleation point for calcium phosphate crystallization by chelating calcium ions (Ca 2+ ) in aqueous solution. The growth of calcium phosphate crystals takes place around the nuclei. At this time, a transfer gene having a phosphate main chain is also incorporated into the particles to form a nanoscale complex, and the hydrophilic polymer is located on the outermost surface of the calcium phosphate particle to enhance the aqueous liquid phase stability of the particles.
본 발명에서 친수성 고분자는 키토산, 히알루론산, 셀룰로스 등의 고분자당 (polysaccharides), 폴리펩타이드, 친수성단백질, 폴리도파민(polydopamine), 폴리노르에피네프린(poly(norepinephrin)), 폴리에틸렌이민(polyethylenimine), 폴리옥사졸린(polyoxazolin), 폴리비닐피롤리돈(polyvinylpyrolidone), 폴리아크릴아미드(polyacrylamide), 폴리비닐알콜(polyvinylalcohol), 폴리히드록시에틸메타아크릴레이트(poly(2-hydroxymethylmethacrylate)), 폴리메틸메타아크릴레이트-아크릴릭산 공중합체(poly(methylmethacrylate-co-acrylic acid)), 폴리메틸메타아크릴레이트 (poly(methylmethacrylate)) 또는 폴리아크릴릭산(poly(acrylic acid))으로 이루어진 군에서 선택되는 하나 이상일 수 있다.
In the present invention, the hydrophilic polymer may be at least one selected from the group consisting of polysaccharides such as chitosan, hyaluronic acid and cellulose, polypeptides, hydrophilic proteins, polydopamine, poly (norepinephrine), polyethylenimine, But are not limited to, polyoxazoline, polyvinylpyrolidone, polyacrylamide, polyvinylalcohol, poly (2-hydroxymethylmethacrylate), polymethylmethacrylate- Acrylic acid copolymers such as poly (methylmethacrylate-co-acrylic acid), poly (methylmethacrylate), and polyacrylic acid.
본 발명에서, 카테콜기를 포함하는 단분자 또는 중합체는 3,4-디하이드록시-L-페닐알라닌일 수 있다. 3,4-디하이드록시-페닐알라닌은 홍합의 접착성에 관여하는 물질로서, DOPA라고도 한다. 상기 4-디하이드록시-페닐알라닌은 칼슘에 대한 친화성을 가짐과 동시에 칼슘 포스페이트 핵 형성 및 크리스탈 형성을 촉진한다. 친수성 고분자로 키토산을 사용한 경우, 3,4-디하이드록시-L-페닐알라닌이 개질된 키토산의 구조는 하기와 같다.In the present invention, the monomolecular or polymer comprising the catechol group may be 3,4-dihydroxy-L-phenylalanine. 3,4-Dihydroxy-phenylalanine is a substance involved in the adhesion of mussels and is also referred to as DOPA. The 4-dihydroxy-phenylalanine has affinity for calcium and promotes calcium phosphate nucleation and crystal formation. When chitosan is used as the hydrophilic polymer, the structure of chitosan modified with 3,4-dihydroxy-L-phenylalanine is as follows.
본 발명에서 사용되는 용어 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자는 친수성 고분자의 일부가 카테콜기를 포함하는 단분자 또는 중합체로 치환된 고분자를 의미한다. 본 발명에서 카테콜기를 포함하는 단분자 또는 중합체는 상기 친수성 고분자에 대하여 0.5 내지 50%의 치환도를 가질 수 있다. 친수성 고분자로 키토산을 사용하는 경우에는, 1 내지 10%의 치환도가 더욱 바람직하다. 본 발명에서 사용되는 용어 '치환도'는, 친수성 고분자 총 반복단위에 대한 카테콜기를 포함하는 단분자 또는 중합체로 개질된 고분자 단위의 비를 의미하며, 상기 그림에서 [a]+[b]의 총합에 대한 [b]의 비율을 의미한다.
As used herein, the term " hydrophilic polymer modified with a monomolecular or polymer containing a catechol group " means a polymer in which a part of the hydrophilic polymer is substituted with a monomolecule or a polymer containing a catechol group. In the present invention, a monomolecular or polymer comprising a catechol group may have a degree of substitution of 0.5 to 50% with respect to the hydrophilic polymer. When chitosan is used as the hydrophilic polymer, a degree of substitution of 1 to 10% is more preferable. The term "degree of substitution" used in the present invention means a ratio of a polymer unit modified with a single molecule or a polymer containing a catechol group to the total number of repeating units of hydrophilic polymer. In the figure, [a] + [b] Means the ratio of [b] to the total.
상기 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자는 칼슘 포스페이트 나노 입자의 성장을 억제하고, 입자를 안정화시킨다. 칼슘 포스페이트는 생체 내 전달체로 많이 사용되어 왔으나, 체내에서 입자가 계속 성장하여 균일한 크기의 안정성을 확보하기 어려운 문제점이 있었다. 그러나 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자가 본 발명의 나노 복합체 표면에 위치함으로써, 입자의 성장을 효과적으로 억제한다.
The monomolecular or polymer modified hydrophilic polymer comprising the catechol group inhibits the growth of calcium phosphate nanoparticles and stabilizes the particles. Calcium phosphate has been widely used as a delivery agent in vivo, but there has been a problem that it is difficult to secure uniform size stability since particles continuously grow in the body. However, the hydrophilic polymer modified with a monomolecular or polymer modified with a catechol group is located on the surface of the nanocomposite of the present invention, thereby effectively suppressing the growth of the particles.
본 발명의 나노 복합체는, 전달 유전자를 포함하고 있으며 세포와의 접촉에 의해 유전자를 세포 내로 전달한다. 본 발명에서 사용되는 용어 '전달 유전자'는 나노 복합체에 포함되어 세포 내로 전달되는 유전자를 의미한다. 본 발명에서 전달 유전자는 재조합 DNA, pDNA(plasmid DNA), siRNA(smallinterfering RNA), miRNA(microRNA) 또는 안티센스뉴클레오티드로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다.
The nanocomposite of the present invention contains a transfer gene and transfers the gene into the cell by contact with the cell. As used herein, the term " transgene " refers to a gene that is incorporated into a nanocomposite and delivered into a cell. In the present invention, the transfer gene may be any one selected from the group consisting of recombinant DNA, pDNA (plasmid DNA), siRNA (small interfering RNA), miRNA (microRNA) or antisense nucleotide.
본 발명에서 전달 유전자에 대한 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자의 중량비는 2.5 내지 100 사이인 것이 바람직하며, 보다 바람직하게는 10 내지 25 사이이다. 중량비가 2.5 보다 작은 경우, 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자에 의한 입자 성장 억제 및 안정화 효과가 충분히 나타나지 않아 크기가 큰 나노입자가 형성될 수 있다.
In the present invention, the weight ratio of the monomolecular or polymer modified hydrophilic polymer containing the catechol group to the transfer gene is preferably 2.5 to 100, more preferably 10 to 25. When the weight ratio is less than 2.5, the effect of inhibiting and stabilizing the growth of the particles due to the hydrophilic polymer modified with a single molecule or a polymer including a catechol group is not sufficiently exhibited, and thus large nanoparticles can be formed.
본 발명에서 유전자 전달용 나노 복합체의 직경은 50 nm 내지 200 nm 인 것이 바람직하다. 상기 크기는 카테콜기를 포함하는 단분자 또는 중합체로 개질되지 않은 친수성 고분자를 사용했을 때에 비하여 작은 수치이다. 즉, 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자는 칼슘 포스페이트의 입자 성장을 억제하여, 보다 작은 크기의 나노 복합체를 형성할 수 있게 한다.
In the present invention, the diameter of the gene transfer nanocomposite is preferably 50 nm to 200 nm. This size is smaller than when a hydrophilic polymer not modified with a monomolecular or polymer containing a catechol group is used. That is, a hydrophilic polymer modified with a monomolecular or polymer containing a catechol group inhibits the particle growth of calcium phosphate, so that a nanocomposite of a smaller size can be formed.
또한, 본 발명의 제2양태는 1) 카테콜기를 포함하는 단분자 또는 중합체를 친수성 고분자와 반응시켜 친수성 고분자에 카테콜기를 포함하는 단분자 또는 중합체를 개질하는 단계, 2) 칼슘포스페이트와 전달유전자를 혼합하여 칼슘포스페이트/전달유전자 복합체를 제조하는 단계, 3) 상기 단계 1)에서 제조한 카테콜기를 포함하는 단분자 또는 중합체가 개질된 친수성 고분자와 2)에서 제조한 칼슘포스페이트/전달유전자 복합체를 혼합하여 칼슘포스페이트/전달유전자/카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자 복합체를 제조하는 단계를 포함하는 유전자 전달용 나노 복합체의 제조방법을 제공하는 것이다.
The second aspect of the present invention also relates to a method for producing a protein, comprising the steps of: 1) reacting a monomolecule or a polymer containing a catechol group with a hydrophilic polymer to modify a monomolecular or polymer containing a catechol group in the hydrophilic polymer; 2) To prepare a calcium phosphate / transfer gene complex; 3) a step of hydrolyzing a monomolecular or polymer modified hydrophilic polymer comprising the catechol group prepared in the above step 1) and a calcium phosphate / transgene complex prepared in 2) And preparing a hydrophilic polymer complex modified with a single molecule or polymer modified with a calcium phosphate / transfer gene / catechol group by mixing.
상기 제조방법에서, 카테콜기를 포함하는 단분자 또는 중합체는 3,4-디하이드록시-L-페닐알라닌일 수 있다.
In the above production method, the monomolecular or polymer comprising the catechol group may be 3,4-dihydroxy-L-phenylalanine.
그 외 본 발명의 제2양태에서 사용되는 용어는 본 발명의 제1양태에서 사용되는 용어와 동일 또는 유사하다.
Other terms used in the second aspect of the present invention are the same as or similar to those used in the first aspect of the present invention.
본 발명에 따른 칼슘 포스페이트, 전달 유전자, 및 카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자를 포함하는 유전자 전달용 나노 복합체는 입자의 성장을 억제함과 동시에 입자를 안정화시켜 훌륭한 유전자 전달체로 기능한다. 본 발명의 유전자 전달용 나노 복합체는 생체 안정성이 우수하며, 세포독성이 없고 유전자 형질감염(transfection) 효율이 우수하다. The gene transfer nanocomposite comprising a hydrophilic polymer modified with a monomolecular or polymer containing calcium phosphate, a transfer gene and a catechol group according to the present invention inhibits the growth of particles and stabilizes the particles, Function. The gene transfer nanocomposite of the present invention has excellent biostability, is free from cytotoxicity, and has excellent gene transfection efficiency.
도 1은 3,4-디하이드록시-L-페닐알라닌(dopa) 개질된 키토산의 구조 및 NMR 데이터이다.
도 2a는 dopa-Chi의 첨가량에 따라 생성된 나노 복합체의 크기를 나타낸 것이며, 도 2b는 pDNA에 대한 dopa-키토산의 중량비가 25일때 CAP/pDNA/dopa-Chi의 크기 분포를 나타낸 것이다.
도 3a는 CAP/pDNA/Chi 및 CAP/pDNA/dopa-Chi의 TEM 이미지, 도 3b는 CAP/pDNA/dopa-Chi의 XRD 패턴, 도 3c는 CAP/pDNA/dopa-Chi, CAP/pDNA 및 CAP의 FT-IR 스펙트라이다.
도 4는 pDNA, CAP/pDNA 및 CAP/pDNA/dopa-Chi의 혈청 안정성을 분석한 결과이다.
도 5a는 CAP/pDNA/Chi의 유전자 농축 효율(gene condensation efficiency), 도 5b는 CAP/pDNA/dopa-Chi의 세포 유입 효율, 도 5c는 CAP/pDNA/dopa-Chi를 이용한 pDNA의 형질감염을 나타낸 것이다.
도 6a는 CAP/siRNA/Chi 및 CAP/siRNA/dopa-chi의 TEM 이미지, 도 6b는 CAP/GFP siRNA/dopa-chi의 유전자 사일런싱 효과를 나타낸 것이다.
도 7은 CAP/pDNA/dopa-Chi 및 CAP/siRNA/dopa-chi의 세포 독성 분석 결과를 나타낸 것이다.
도 8은 본 발명 나노복합체의 제조 과정 및 구조를 나타낸 모식도이다.
도 9는 CAP/siRNA 및 CAP/siRNA/dopa-HA의 TEM 이미지 이다.
도 10은 CAP/siRNA/dopa-HA 복합체의 luciferase 지속발현세포주(established cell line, HT29-Luc)에서의 luciferase 단백질 발현 억제 정도와 히알루론산(HA)을 함께 처리했을 때의, CD44+ 세포(HT-29-Luc)에 대한 표적 지향 효과를 나타낸 것이다.
도 11은 dopa-HA와 HA의 세포 독성 분석 결과를 나타낸 것이다.1 shows the structure and NMR data of 3,4-dihydroxy-L-phenylalanine (dopa) modified chitosan.
FIG. 2A shows the size of the nanocomposite produced according to the amount of dopa-Chi added, and FIG. 2B shows the size distribution of CAP / pDNA / dopa-Chi when the weight ratio of dopa-chitosan to pDNA is 25.
FIG. 3A shows the TEM image of CAP / pDNA / Chi and CAP / pDNA / dopa-Chi, FIG. 3B shows the XRD pattern of CAP / pDNA / dopa-Chi, FIG. 3C shows CAP / pDNA / dopa-Chi, CAP / Of the FT-IR spectra.
FIG. 4 shows the results of analysis of serum stability of pDNA, CAP / pDNA and CAP / pDNA / dopa-Chi.
FIG. 5A shows the gene condensation efficiency of CAP / pDNA / Chi, FIG. 5B shows the cell infusion efficiency of CAP / pDNA / dopa-Chi, FIG. 5C shows the transfection of pDNA using CAP / pDNA / .
FIG. 6A shows TEM images of CAP / siRNA / Chi and CAP / siRNA / dopa-chi, and FIG. 6B shows the gene silencing effect of CAP / GFP siRNA / dopa-chi.
Fig. 7 shows the results of cytotoxicity analysis of CAP / pDNA / dopa-Chi and CAP / siRNA / dopa-chi.
8 is a schematic view showing a process and structure of the nanocomposite of the present invention.
Figure 9 is a TEM image of CAP / siRNA and CAP / siRNA / dopa-HA.
10 is a graph showing the effect of inhibition of luciferase protein expression on hyaluronic acid (HA) in the luciferase sustained expression line (HT29-Luc) of the CAP / siRNA / 29-Luc).
Fig. 11 shows the results of cytotoxicity analysis of dopa-HA and HA.
이하, 실시예를 통해 본 발명을 보다 상세히 설명한다. 그러나 하기의 실시예는 오로지 본 발명을 설명하기 위한 것으로 이들 실시예에 의해 본 발명의 범위가 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
물질matter
키토산-10(탈아세틸화도 78%)은 Wako Pure Chemical Industries Ltd(오사카, 일본)사로부터 구입하였다. 키토산-10의 점도 평균 분자량은 Mv = 2×105이었다. 칼슘 클로라이드(CaCl2), 인산수소이나트륨(Na2HPO4), 아세트산, 가교된 폴리에틸렌이민(PEI, Mw 25000), 하이드로카페인산(hydrocaffeic acid), N-(3-디메틸아미노프로필)-N'-에틸카르보디이미드 하이드로클로라이드(EDC), N-모폴리노에탄 술폰산(MES) 및 4',6-디아미디노-2-페닐인돌(DAPI)은 Sigma Chemical Company(St. Louis, MO)로부터 구입하였다. 본 발명에서 사용한 cell counting kit-8(CCK-8)은 Dojindo(Kumamoto, Japan)사로부터 구입하였다. 모든 다른 화학 시약은 분석용 품위(analytical grade)였다. 형질감염된 E. coli로부터 GFP encoding DNA (pEGFP-C1)를 추출하고 정제하기 위한 Maxiprep kit Qiagen(Valencia, CA)사로부터 구입하였다. Luciferase 단백질의 발현 정도를 확인하기 위한 Luciferase assay kit는 Promega(Madison, WI)사로부터 구입하였다. GFP-silencing siRNA(sense: GCAUCAAGGUGAACUUCAAUU; antisense: AAUUGAAGUUCACCUUGAUGC)와 Luciferase-silencing siRNA는 Bioneer(Daejeon, Korea)에 의해 합성되었다.
Chitosan-10 (deacetylation degree 78%) was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). The viscosity average molecular weight of chitosan-10 was Mv = 2 × 10 5 . Calcium chloride (CaCl 2), phosphate susoyi sodium (Na 2 HPO 4), acetic acid, cross-linked polyethylene imine (PEI, Mw 25000), hydro caffeic acid (hydrocaffeic acid), N- (3- dimethylaminopropyl) -N ' -Ethyl carbodiimide hydrochloride (EDC), N-morpholinoethanesulfonic acid (MES) and 4 ', 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Chemical Company Respectively. The cell counting kit-8 (CCK-8) used in the present invention was purchased from Dojindo (Kumamoto, Japan). All other chemical reagents were analytical grade. The kit was purchased from Maxiprep kit Qiagen (Valencia, Calif.) For the extraction and purification of GFP encoding DNA (pEGFP-C1) from transfected E. coli . Luciferase assay kit was purchased from Promega (Madison, Wis.) To confirm the expression level of Luciferase protein. GFP-silencing siRNA (sense: GCAUCAAGGUGAACUUCAAUU; antisense: AAUUGAAGUUCACCUUGAUGC) and Luciferase-silencing siRNA were synthesized by Bioneer (Daejeon, Korea).
실시예Example 1: One: dopadopa -키토산 복합체의 제조- Preparation of chitosan complex
MES 완충용액(pH 5)에 키토산 10을 용해시켰다. EtOH:MES의 1:1 완충용액에 EDC(chitosan N:EDC molar ratio = 1:5) 및 하이드로카페인산(chitosan N:hydrocaffeic acid molar ratio = 1:0.5)을 15분 동안 반응시켰다. MES에 녹아있는 키토산 10의 용액에 상기 반응용액을 첨가하고 6시간 동안 유지시켰다. 과량의 차가운 아세톤을 첨가하여 dopa-개질된 키토산(이하, dopa-Chi)을 침전시켰다. 상기 침전물을 필터 페이퍼를 이용하여 정제하였다. 상기 정제된 dopa-Chi를 하루 동안 진공건조시켰다. dopa-Chi의 결합 효율을 1H NMR을 이용하여 측정하고 이를 도 1에 나타내었다. 그 결과, 키토산과 dopa의 결합 효율은 2%임을 확인할 수 있었다. 1H NMR 스펙트라 중 δ 6.0 - 7.0 ppm에서 나타나는 신호는 dopa의 방향족 프로톤의 공명에 의한 것으로, dopa와 키토산이 성공적으로 결합했음을 나타낸다.
실시예Example 2: 2: CAPCAP // pDNApDNA // dopadopa -키토산 복합체의 제조- Preparation of chitosan complex
18 mM의 CaCl2 용액(pH 9) 50 ㎕를 11.5 mM의 Na2HPO4 용액(pH 9) 50 ㎕와 30초 동안 반응시켰다. 그 후 100 마이크로리터의 pDNA를 첨가하고 30초 동안 반응시켰다. 상기 반응 용액에 1% 아세트산 용액에 녹아있는 다양한 양의 dopa-키토산을 첨가하고 20분 동안 반응시켰다. 이때 pDNA에 대한 dopa-키토산의 중량비는 2.5 내지 25 사이였다. 최종 반응 용액의 pH는 대략 7이었다. 상기 공정을 통해 칼슘 포스페이트/pDNA/dopa로 개질된 키토산의 복합체(이하, CAP/pDNA/dopa-chi)가 제조되었다. dopa로 개질되지 않은 키토산 10(이하, Chi)을 음성 대조군으로 사용하였다.
50 μl of 18 mM CaCl 2 solution (pH 9) was reacted with 50 μl of 11.5 mM Na 2 HPO 4 solution (pH 9) for 30 seconds. Then 100 microliters of pDNA was added and reacted for 30 seconds. Various amounts of dopa-chitosan dissolved in 1% acetic acid solution were added to the reaction solution and reacted for 20 minutes. The weight ratio of dopa-chitosan to pDNA was between 2.5 and 25. The pH of the final reaction solution was approximately 7. Through this process, a complex of chitosan modified with calcium phosphate / pDNA / dopa (hereinafter, CAP / pDNA / dopa-chi) was prepared. chitosan 10 (hereinafter Chi) not modified with dopa was used as a negative control.
생성된 입자의 크기를 광분산법을 이용하여 측정하고 이를 도 2a에 나타내었δ다. 그 결과, dopa와 결합되지 않은 대조군 칼슘포스페이트/pDNA/키토산 복합체(이하, CAP/pDNA/Chi)의 경우 키토산의 농도가 증가할수록 입자의 크기가 작아지는 것을 확인할 수 있었다. 그러나, dopa-Chi를 사용한 경우 고분자의 농도에 관계없이 균일하고 안정한 나노 복합체를 얻을 수 있었다. 제조된 CAP/pDNA/dopa-Chi(중량비=25)의 사이즈 분포를 도 2b에 나타내었다. 입자의 분포는 131.0 ± 2.1 nm 로, 좁은 분포를 보이고 있음을 확인할 수 있었다. The size of the generated particles was measured using a light scattering method and is shown in Fig. 2a. As a result, the particle size of the control calcium phosphate / pDNA / chitosan complex (hereinafter referred to as CAP / pDNA / Chi) which was not conjugated with dopa decreased with increasing concentration of chitosan. However, when dopa-Chi was used, a homogeneous and stable nanocomposite could be obtained regardless of the concentration of the polymer. The size distribution of the prepared CAP / pDNA / dopa-Chi (weight ratio = 25) is shown in FIG. 2B. The distribution of the particles was 131.0 ± 2.1 nm, indicating a narrow distribution.
수용액에서, dopa-키토산은 dopa 모이어티간의 약한 소수성 상호작용을 통해, 약 573 ± 31.2 nm 크기의 미셀 유사구조를 형성하였다. 또한, 양이온성 dopa-키토산은 음 전하를 띠는 pDNA와의 정전기적 인력에 의해 고분자전해질 복합체(PEC)를 형성하였다. dopa-Chi/pDNA PEC의 크기는 약 409.2 ± 195.1 nm로, dopa-Chi 미셀 보다 약간 작았다. 이는 dopa-Chi의 첨가가 CAP/pDNA 입자의 표면에서 칼슘 포스페이트의 추가적인 축적을 막는다는 것을 의미한다. In aqueous solution, dopa-chitosan formed a micelle-like structure with a size of about 573 ± 31.2 nm through weak hydrophobic interactions between the dopa moieties. In addition, cationic dopa-chitosan formed a polymer electrolyte complex (PEC) by electrostatic attraction with negatively charged pDNA. The size of the dopa-Chi / pDNA PEC was about 409.2 ± 195.1 nm, slightly smaller than the dopa-Chi micelle. This means that the addition of dopa-Chi prevents further accumulation of calcium phosphate on the surface of the CAP / pDNA particles.
카테콜기의 칼슘 이온에 대한 특징적인 결합특성을 고려해 볼 때, 가장 설득력있는 매커니즘은 dopa와 칼슘의 상호작용을 통해 CAP/pDNA 입자 표면에 dopa-Chi가 결합하였고, dopa-Chi의 표면 안정화로 인하여 CAP의 입자 성장이 억제되었다는 것이다.
Considering the characteristic binding characteristics of catechol to calcium ions, the most convincing mechanism is that dopa-Chi bonds to the surface of CAP / pDNA particles through the interaction of dopa and calcium, and due to the surface stabilization of dopa-Chi The particle growth of CAP was suppressed.
입자의 모폴로지를 TEM을 통해 확인하고 이를 도 3a에 나타내었다. CAP/pDNA/dopa-Chi 복합체의 경우 좁은 크기 분포를 가지며 잘 분산된 구형의 구조가 관찰되었다. 그러나 CAP/pDNA/Chi의 경우 입자의 크기가 훨씬 크고 그 분포가 일정하지 않았다. 이를 통해, CAP/pDNA/dopa-Chi 복합체가 입자의 크기 성장을 억제하며 균일한 분포를 가짐을 확인할 수 있었다.
The morphology of the particles is confirmed by TEM and is shown in Fig. 3A. In the CAP / pDNA / dopa-Chi complex, a well-dispersed spherical structure with a narrow size distribution was observed. However, in the case of CAP / pDNA / Chi, the particle size was much larger and its distribution was not constant. It was confirmed that the CAP / pDNA / dopa-Chi complex inhibited the size growth of the particles and had a uniform distribution.
CAP/pDNA/dopa-Chi의 XRD 패턴을 도 3b에 나타내었다. XRD 패턴에서 결정화된 CAP에 의한 회절 피크를 확인할 수 있었다. 또한, 회절 피크를 통해 CAP/pDNA/dopa-chi의 대부분이 칼슘 포스페이트, 트리칼슘 포스페이트(TCP) 및 하이드록시아파타이트(HA)로 이루어졌다는 것을 확인할 수 있었다. dopa-Chi나 Chi의 첨가량에 관계없이, 회절 패턴은 거의 동일하였다. 이는 키토산과 dopa 분자 모두 CAP의 결정 성장에 영향을 미치지 않는다는 것을 의미한다.
The XRD pattern of CAP / pDNA / dopa-Chi is shown in Figure 3B. The diffraction peaks due to the crystallized CAP in the XRD pattern can be confirmed. Also, it was confirmed through the diffraction peak that most of CAP / pDNA / dopa-chi was composed of calcium phosphate, tricalcium phosphate (TCP) and hydroxyapatite (HA). Regardless of the addition of dopa-Chi or Chi, the diffraction pattern was almost identical. This means that both chitosan and dopa molecules do not affect the crystal growth of CAP.
CAP/pDNA/dopa-Chi, CAP/pDNA 및 CAP의 FT-IR 스펙트럼을 도 3c에 나타내었다. 세 가지 샘플 모두에서 포스페이트의 흡수 밴드가 나타났다. 이를 통해 CAP/pDNA/dopa-Chi의 결정 구조를 확인할 수 있었다.
FT-IR spectra of CAP / pDNA / dopa-Chi, CAP / pDNA and CAP are shown in FIG. In all three samples, the absorption band of phosphate appeared. This confirmed the crystal structure of CAP / pDNA / dopa-Chi.
실시예Example 3: 혈청에서의 3: Serum CAPCAP // pDNApDNA // dopadopa -- ChiChi 의 안정성 측정Stability measurement
혈청 안정성 시험을 위해, 50%의 fetal bovine serum(FBS)을 포함하는 PBS 용액에 pDNA, CAP/pDNA 및 CAP/pDNA/dopa-Chi를 넣은 후, 37℃에서 보관하였다. 특정 시간(1,2,3,8,16,24시간)마다 샘플을 채취한 후 1% 아가로스젤로 겔 전기영동하여 분석하였다. 겔 전기영동에 앞서, CAP/pDNA/dopa-Chi를 산성 조건에서 헤파린 소듐 염 수용액(50 mg/mL)을 이용하여 30분동안 전처리하여, 복합체로부터 pDNA를 분리시켰다. For serum stability testing, pDNA, CAP / pDNA and CAP / pDNA / dopa-Chi were added to a PBS solution containing 50% fetal bovine serum (FBS) and stored at 37 ° C. Samples were taken at specific times (1, 2, 3, 8, 16, and 24 hours) and analyzed by gel electrophoresis on 1% agarose gel. Prior to gel electrophoresis, CAP / pDNA / dopa-Chi was pretreated with an aqueous solution of heparin sodium salt (50 mg / mL) under acidic conditions for 30 minutes to isolate pDNA from the complex.
일반적으로 CAP가 플라스미드 DNA를 보호하며, 혈청 내에서 기능을 유지하는 것을 도와준다고 알려져 있다. dopa-Chi는 CAP/pDNA 복합체를 둘러 감쌈으로써 pDNA를 보호한다. 따라서, CAP/pDNA/dopa-Chi는 CAP/pDNA와 비교하였을때 혈청 내에서 유전자를 더욱 잘 안정화하고 구조를 유지시킬 것이라고 예측하였다. It is generally known that CAP protects plasmid DNA and helps maintain its function in serum. dopa-Chi protects the pDNA by wrapping around the CAP / pDNA complex. Thus, CAP / pDNA / dopa-Chi predicted that the gene would be better stabilized and maintained in the serum when compared to CAP / pDNA.
상기 안정성 시험 결과를 도 4에 나타내었다. 그 결과, CAP/pDNA/dopa-Chi는 24시간이 지난 후에도 pDNA의 구조를 유지하고 있음을 확인할 수 있었다. 반면, pDNA나 CAP/pDNA는 1 내지 4시간 경과 후 구조를 완전히 상실했다. CAP/pDNA는 pDNA에 비하여 조금 개선된 혈청 안정성을 보여주었으나, 4시간 후, pDNA의 분해를 의미하는 스미어 밴드가 관측되었다. 반면, CAP/pDNA/dopa-Chi는 24시간 후에도 pDNA의 구조적 완전함을 유지하였고, 이를 통해 dopa-Chi가 serum-driven nuclease의 공격으로부터 pDNA를 효과적으로 보호함을 확인할 수 있었다.
The stability test results are shown in Fig. As a result, it was confirmed that CAP / pDNA / dopa-Chi retained the pDNA structure even after 24 hours. On the other hand, pDNA or CAP / pDNA completely lost its structure after 1 to 4 hours. CAP / pDNA showed slightly improved serum stability as compared to pDNA, but after 4 hours, a smear band, which means the degradation of pDNA, was observed. On the other hand, CAP / pDNA / dopa-Chi maintained structural integrity of pDNA after 24 hours, confirming that dopa-Chi effectively protected pDNA from attack of serum-driven nuclease.
실시예Example 4: 4: CAPCAP // pDNApDNA // dopadopa -- ChiChi 의 세포 내로의 유입확인Of cells into the cell
COS-7 세포를 4-well culture plate에 1×105cells/well의 농도로 위치시켰다. 하루의 pre-incubation 후, 상기 세포를 플루오레세인 라벨된 CAP/pDNA/dopa-Chi(1 ㎍ pDNA/well)로, DMEM medium에서 2.5시간 동안 처리하였다. 처리된 세포를 PBS 용액으로 세 번 세척하고, 1%(w/v)의 포름알데히드 용액으로 고정시켰다. 푸른색을 띠는 DAPI로 핵을 염색하고 LSM510 공초점 현미경(Carl Zeiss, Germany)을 이용하여 세포를 확인하였다. COS-7 cells were placed in a 4-well culture plate at a concentration of 1 × 10 5 cells / well. After one day pre-incubation, the cells were treated with fluorescein-labeled CAP / pDNA / dopa-Chi (1 ug pDNA / well) for 2.5 hours in DMEM medium. The treated cells were washed three times with PBS solution and fixed with 1% (w / v) formaldehyde solution. Nuclei were stained with blue DAPI and cells were identified using an LSM510 confocal microscope (Carl Zeiss, Germany).
상기 결과를 도 5b에 나타내었다. 처리시간이 30분 일때, 세포의 세포질에서 녹색의 플루오로세인이 관찰되었다. 처리시간이 2시간이 되자, 녹색의 플루오로세인은 세포 전체를 통해 고르게 퍼져있었다. 이를 통해 CAP/pDNA/dopa-Chi 나노 복합체가 효과적으로 세포 내 유입을 매개한다는 것을 확인할 수 있었다. 이러한 효과적인 세포 유입은, 입자의 나노 크기 구조와 양전하를 띄는 표면에 기인한 것으로 판단된다.
The results are shown in FIG. 5B. When the treatment time was 30 minutes, green fluorescein was observed in the cytoplasm of the cells. When the treatment time was two hours, the green fluorescein spread evenly throughout the cell. This confirms that the CAP / pDNA / dopa-Chi nanocomposite mediates intracellular influx. These effective cell inflows are attributed to the nanoscale structure of the particles and the positively charged surface.
실시예Example 5: 5: CAPCAP // pDNApDNA // dopadopa -- ChiChi 의 유전자 형질감염 효율 측정Gene transfection efficiency
플라스미드 DNA의 유전자 형질감염을 조사하기 위해, COS-7 세포를 12 Well culture plate에 1×105cells/well의 농도로 위치시켰다. 하루의 pre-incubation 후, 실시예 2와 동일한 방법으로 제조된, CAP/pDNA/Chi와 CAP/pDNA/dopa-chi 복합체를 제조하여 혈청이 첨가되지 않은 media로 교체된 well에 첨가한다. 이 때 복합체의 제조를 위해 2 ㎍의 GFP 플라스미드 DNA가 사용되었으며, 1% 아세트산에 용해된 키토산 또는 dopa-키토산을 25 중량비로 pDNA와 함께 20분 동안 배양시켰다. 5시간 후, 혈청이 첨가된 media로 교환하고 48시간 동안 배양하였다. 처리된 세포를 1%(w/v) Triton X-100 treatment를 이용하여 용해시키고, 각각의 용해물에서 GFP 플루오로세인의 강도를 측정하였다.To investigate the gene transfection of plasmid DNA, COS-7 cells were placed in a 12 well culture plate at a concentration of 1 × 10 5 cells / well. After one day pre-incubation, CAP / pDNA / Chi and CAP / pDNA / dopa-chi complexes prepared in the same manner as in Example 2 were prepared and added to the wells replaced with serum-free media. At this time, 2 μg of GFP plasmid DNA was used for the preparation of the complex, and chitosan or dopa-chitosan dissolved in 1% acetic acid was incubated with pDNA for 20 minutes at a weight ratio of 25. After 5 hours, serum was added to the medium and cultured for 48 hours. Treated cells were lysed using a 1% (w / v) Triton X-100 treatment and the intensity of GFP fluorocaine was measured in each lysate.
상기 측정된 강도를 도 5c에 나타내었다. 또한 비교를 위해 Chi/pDNA, dopa-Chi/pDNA PEC, 및 CAP/pDNA/Chi에서 측정된 강도도 나타내었다. 모든 샘플에서, pDNA에 대한 키토산의 중량비는 25이다. 비교 결과, Chi/pDNA, dopa-Chi/pDNA 및 CAP/pDNA/Chi 모두 낮은 형질감염 효율을 보인 반면, CAP/pDNA-dopa-Chi는 대조군들에 비하여 훨씬 높은 유전자 형질감염 효율을 나타내었다. The measured intensity is shown in Figure 5c. Also for comparison, the intensities measured in Chi / pDNA, dopa-Chi / pDNA PEC, and CAP / pDNA / Chi were also shown. In all samples, the weight ratio of chitosan to pDNA is 25. In comparison, Chi / pDNA, dopa-Chi / pDNA and CAP / pDNA / Chi showed low transfection efficiency, whereas CAP / pDNA-dopa-Chi showed much higher transfection efficiency than control.
가교된 폴리에틸렌이민(PEI 25k, MW 25000 DA)을 양성 대조군으로 사용하였다. 다양한 동물 세포에 대한 지속적 형질감염 능력 때문에, PEI 25k는 non-viral transfection에 있어서 gold standard로 널리 사용된다. 그러나, PEI 25k의 뛰어난 세포 형질감염 능력에도 불구하고, PEI 25k는 세포독성 및 생분해성의 부족으로 인해 의학적 용도로 사용되기 어렵다. 도 5c를 통해 비교하여 보면, CAP/pDNA-dopa-Chi는 PEI 25k와 유사한 형질감염 효율을 나타내어 그 우수함을 확인할 수 있었다.
Crosslinked polyethyleneimine (
실시예Example 6: 6: CAPCAP // siRNAsiRNA // dopadopa -- ChiChi 의 합성 및 유전자 형질감염 효율 측정And measurement of gene transfection efficiency
pDNA 대신 siRNA를 사용하는 것을 제외하고는, 실시예 1 내지 2 와 동일한 방법으로 CAP/siRNA/dopa-Chi 나노 복합체를 제조하였다. 이때 siRNA에 대한 dopa-Chi의 중량비는 50으로 하였다. 이는 pDNA 제조시 중량비가 10이었던 것에 비해 다소 높은 수치인데, 이는 siRNA가 pDNA에 비해 짧고 단단한 구조를 가지기 때문이다. 따라서 CAP/pDNA/dopa-Chi보다 CAP/siRNA/dopa-Chi의 경우, 입자 성장 억제 및 표면 안정화에 있어서 dopa-Chi의 역할이 더 중요하다. CAP / siRNA / dopa-Chi nanocomposite was prepared in the same manner as in Examples 1 and 2 except that siRNA was used instead of pDNA. At this time, the weight ratio of dopa-Chi to siRNA was 50. This is somewhat higher than that of pDNA at a weight ratio of 10 because siRNA has a shorter and more rigid structure than pDNA. Therefore, in the case of CAP / siRNA / dopa-Chi than CAP / pDNA / dopa-Chi, the role of dopa-Chi in particle growth inhibition and surface stabilization is more important.
제조된 CAP/siRNA/dopa-Chi 및 CAP/siRNA/Chi 입자의 TEM 이미지를 도 6a에 나타내었다. 그 결과, dopa-Chi를 이용하지 않은 CAP/siRNA/Chi 입자는 현저히 큰 크기를 가지며 뭉침 현상이 일어나는 것을 확인할 수 있었다. 그러나 CAP/siRNA/dopa-Chi는 작은 입자 크기를 가지며 고루 분산되어 있는 것을 확인할 수 있었다.
TEM images of the prepared CAP / siRNA / dopa-Chi and CAP / siRNA / Chi particles are shown in FIG. 6A. As a result, it was confirmed that the CAP / siRNA / Chi particles not using dopa-Chi had a remarkably large size and aggregation phenomenon occurred. However, it was confirmed that CAP / siRNA / dopa-Chi has a small particle size and is uniformly dispersed.
siRNA의 유전자 inhibition assay를 위하여, MDA-MB-435-GFP(stably expressed GFP) cell line을 12-well plate에 1×105cells/well의 농도로 위치시켰다. 세포 부착을 위해, CAP/siRNA/Chi 및 CAP/siRNA/dopa-Chi(각 well마다 1 ㎍의 siRNA)를 5시간 동안 배양하였다. 상기 media를 media를 포함하는 신선한 혈청으로 교환하고, 추가로 36시간 동안 배양시켰다. 세포 용해 후, GFP 플루오로세인의 상대적 세기를 측정하였다. 상기 측정결과를 도 6b에 나타내었다. 그 결과, CAP/siRNA/dopa-Chi가 처리된 세포는 약 44.2±1.1% 정도의 높은 사일런싱 효율을 나타내었다. 그러나, CAP/siRNA/Chi가 처리된 세포의 경우 사일런싱이 관찰되지 않았다.
For gene inhibition assay of siRNA, MDA-MB-435-GFP (stably expressed GFP) cell line was placed at a concentration of 1 × 10 5 cells / well in a 12-well plate. For cell attachment, CAP / siRNA / Chi and CAP / siRNA / dopa-Chi (1 ug siRNA per well) were incubated for 5 hours. The media was exchanged with fresh serum containing media and incubated for an additional 36 hours. After cell lysis, the relative intensities of GFP fluorocaine were measured. The measurement results are shown in Fig. 6B. As a result, cells treated with CAP / siRNA / dopa-Chi showed high silencing efficiency of about 44.2 ± 1.1%. However, no silencing was observed for cells treated with CAP / siRNA / Chi.
실시예Example 7: 7: CAPCAP // pRNApRNA // dopadopa -- ChiChi 및 And CAPCAP // siRNAsiRNA // dopadopa -- ChiChi 의 세포독성 측정 Cytotoxicity measurement
세포 독성 측정을 위해, Cos-7 세포를 96-well plate에 5×103 cells/well의 농도로 위치시켰다. 하루의 pre-incubation 후, 세포를 다양한 농도의 CAP/siRNA/dopa-Chi 및 CAP/pDNA/dopa-Chi에 각각 5시간 동안 배양시켰다. 5시간 후에 세포를 fresh cultuer media에 위치시킨 후, 48시간 동안 배양시켰다. 제조사의 가이드라인에 따라 CCK-8 assay를 이용하여 cell viability를 측정하였다. 측정 결과를 도 7에 나타내었다. 그 결과, CAP/siRNA/dopa-Chi 및 CAP/pDNA/dopa-Chi 모두 중량비가 100에 이를 때까지 세포 독성이 관측되지 않았다. 따라서 제조된 나노 복합체들이 생체 이용에 적합함을 확인할 수 있었다.
For cytotoxicity measurement, Cos-7 cells were placed in a 96-well plate at a concentration of 5 × 10 3 cells / well. After one day pre-incubation, cells were incubated for 5 h at various concentrations of CAP / siRNA / dopa-Chi and CAP / pDNA / dopa-Chi, respectively. After 5 hours, the cells were placed in fresh culturing media and cultured for 48 hours. Cell viability was measured using the CCK-8 assay according to the manufacturer's guidelines. The measurement results are shown in Fig. As a result, no cytotoxicity was observed until the weight ratio of CAP / siRNA / dopa-Chi and CAP / pDNA / dopa-Chi reached 100. Therefore, it was confirmed that the prepared nanocomposites were suitable for bioavailability.
실시예Example 8: 8: CAPCAP // siRNAsiRNA // dopadopa -- HAHA 복합체의 제조 Manufacture of Composites
2.5 M의 CaCl2 용액 44.6㎕를 10uM의 siRNA 용액 65㎕와 30초 동안 반응시켰다. 그 후에 11.8mM의 Na2HPO4 용액 44.6㎕를 첨가하여 30초 동안 반응시켰다. 상기 반응 용액에 물에 녹아있는 다양한 양의 dopa-HA를 첨가하고, 25분 동안 반응시켰다. 상기 공정을 통해 칼슘 포스페이트/siRNA/dopa로 개질된 히알루론산(HA)의 복합체(이하, CAP/siRNA/dopa-HA)가 제조되었다.
2.5 M CaCl22 44.6 μl of the solution was reacted with 65 μl of 10 uM siRNA solution for 30 seconds. After that, 11.8 mM Na2HPO4 And the reaction was carried out for 30 seconds. Various amounts of dopa-HA dissolved in water were added to the reaction solution and reacted for 25 minutes. A complex of hyaluronic acid (HA) modified with calcium phosphate / siRNA / dopa (hereinafter referred to as CAP / siRNA / dopa-HA) was prepared through the above process.
입자의 모폴로지를 TEM을 통해 확인하고 이를 도 9에 나타내었다. CAP/siRNA/dopa-HA 복합체의 경우 좁은 크기 분포를 가지며 잘 분산된 구형의 구조가 관찰되었다. 그러나, CAP/siRNA의 경우 입자가 성장되어 있는 형태의 구조가 관찰되었다. 이를 통해, CAP/siRNA/dopa-HA 복합체가 입자의 크기 성장을 억제하며 균일한 분포를 가짐을 확인할 수 있었다.
The morphology of the particles was confirmed by TEM and is shown in Fig. In the CAP / siRNA / dopa-HA complex, a well-dispersed spherical structure with a narrow size distribution was observed. However, in the case of CAP / siRNA, the structure in which the particles are grown is observed. It was confirmed that the CAP / siRNA / dopa-HA complex suppressed the size growth of the particles and had a uniform distribution.
실시예Example 9: 9: CAPCAP // siRNAsiRNA // dopadopa -- HAHA 의 유전자 형질감염 효율 측정, CAP/siRNA/dopa-HA의 표적 지향 효과 측정, The target-directed effect of CAP / siRNA / dopa-HA
Luc siRNA의 유전자 inhibition assay를 위하여, luciferase를 발현하는 결장선암 세포주인 HT-29-luc 세포를 12 well culture plate에 1.5×105cells/well의 농도로 위치시켰다. 실시예 8과 동일한 방법으로 CAP/siRNA/dopa-HA 복합체를 제조하여, 혈청이 첨가되지 않은 media로 교환된 12 well에 첨가하였다. 4시간 후에, 혈청이 첨가된 media로 교환하고, 24시간 동안 배양하였다. 처리된 세포를 Cell lysis 버퍼를 이용하여 용해시키고, luciferase assay kit를 이용하여, 각각의 용해물에서 luciferase 단백질의 양을 측정하였다.
For Luc inhibition assay, HT-29-luc cells, a colon adenocarcinoma cell line expressing luciferase, were placed in a 12-well culture plate at a concentration of 1.5 × 10 5 cells / well. The CAP / siRNA / dopa-HA complex was prepared in the same manner as in Example 8 and added to 12 wells exchanged with serum-free media. After 4 hours, serum was added to the medium and cultured for 24 hours. The treated cells were lysed with Cell lysis buffer and the amount of luciferase protein was measured in each lysate using luciferase assay kit.
표적 지향 효과를 측정하기 위하여, 히알루론산(HA)을 실시예 8과 동일한 방법으로 제조된 CAP/siRNA/dopa-HA 복합체의 첨가에 1시간 앞서서 첨가하였다. 1시간 뒤에 CAP/siRNA/dopa-HA 복합체를 혈청이 첨가되지 않은 media로 교환된 12 well에 첨가하였다. 4시간 후에, 혈청이 첨가된 media로 교환하고, 24시간 동안 배양하였다. 처리된 세포를 Cell lysis 버퍼를 이용하여 용해시키고, 제조사의 가이드 라인에 따라 luciferase assay kit를 이용하여, 각각의 용해물에서 luciferase 단백질의 양을 측정하였다.
To measure the target-directed effect, hyaluronic acid (HA) was added 1 hour prior to the addition of the CAP / siRNA / dopa-HA complex prepared in the same manner as in Example 8. After one hour, the CAP / siRNA / dopa-HA complex was added to 12 wells exchanged with serum-free media. After 4 hours, serum was added to the medium and cultured for 24 hours. The treated cells were lysed using Cell lysis buffer and the amount of luciferase protein was measured in each lysate using the luciferase assay kit according to the manufacturer's guidelines.
상기 측정된 luciferase 단백질의 양을 도 10에 나타내었다. 또한 비교를 위해 CAP/siRNA, HA/siRNA 및 dopa-HA/siRNA에 측정된 단백질의 양도 나타내었다. 비교 결과, CAP/siRNA/dopa-HA 복합체에 의한 luciferase 단백질의 억제 효율이 대조군들에 비하여 훨씬 높게 나타내었다. 또한, 양성 대조군으로 사용된 가교된 폴리에틸렌이민(PEI 25k, MW 25,000 Da)과 유사한 luciferase 단백질 억제 효율을 나타내었다.
The measured amount of luciferase protein is shown in Fig. Also shown for comparison is the amount of protein measured in CAP / siRNA, HA / siRNA and dopa-HA / siRNA. As a result, the inhibition efficiency of luciferase protein by CAP / siRNA / dopa-HA complex was much higher than that of the control group. In addition, luciferase protein inhibition efficiency was similar to that of crosslinked polyethyleneimine (
표적 지향 효과를 측정하기 위해 HA를 처리했을 때, CAP/siRNA/dopa-HA의 경우, 선처리된 HA에 의해 영향을 받지 않는 PEI 25k와 CAP/siRNA와 비교했을 때, luciferase 단백질의 억제 효율이 크게 감소했음을 알 수 있었다. 이 결과는, HA에 대한 리셉터인 CD44를 발현하는 HT29-luc세포에 HA를 CAP/siRNA/dopa-HA 복합체의 처리에 앞서 처리 했을 때, 선처리된 HA가 HT29-luc세포의 CD44에 먼저 위치하게 되어서, CAP/siRNA/dopa-HA 복합체의 HA 부분의 리셉터 결합을 막기 때문이다. 이를 통해서, CAP/siRNA/dopa-HA 복합체의 표적 지향 효과를 확인할 수 있었다.
In the case of CAP / siRNA / dopa-HA when treated with HA to measure the target-directed effect, the inhibition efficiency of luciferase protein was higher than that of
실시예Example 10: 10: DopaDopa -- HAHA 의 세포독성 실험Cytotoxicity experiment
세포 독성 측정을 위해, HT29-luc 세포를 96-well plate에 5×103 cells/well의 농도로 위치시켰다. 하루의 pre-incubation 후, 세포에 다양한 농도의 dopa-HA와 HA를 처리하여 24시간 동안 배양하였다. 제조사의 가이드 라인에 따라 MTT assay를 이용하여 cell viability를 측정하였다. 측정결과를 도 11에 나타내었다. 그 결과, dopa-HA의 cell viability가 500 ㎍/㎖에 이를 때까지 세포 독성이 관측되지 않았다. 따라서, 생체 이용에 적합함을 확인할 수 있었다.For cytotoxicity measurement, HT29-luc cells were placed in a 96-well plate at a concentration of 5 × 10 3 cells / well. After pre-incubation for a day, the cells were treated with various concentrations of dopa-HA and HA for 24 hours. Cell viability was measured using the MTT assay according to the manufacturer's guidelines. The measurement results are shown in Fig. As a result, cytotoxicity was not observed until the cell viability of dopa-HA reached 500 / / ml. Therefore, it was confirmed that it is suitable for bioavailability.
Claims (10)
1. A gene delivery nanocomposite comprising a hydrophilic polymer modified with a monomolecular or polymer containing calcium phosphate, a transfer gene, and a 1,2-dihydroxybenzene.
2. The gene delivery nanocomposite according to claim 1, wherein the hydrophilic polymer modified with a monomolecular or polymer containing a catechol group surrounds the calcium phosphate nanoparticle.
2. The gene delivery nanocomposite according to claim 1, wherein the weight ratio of the hydrophilic polymer modified with a single molecule or polymer modified with a catechol group to the transfer gene is 2.5 to 100.
The method of claim 1, wherein the hydrophilic polymer is selected from the group consisting of polysaccharides such as chitosan, hyaluronic acid and cellulose, polypeptides, hydrophilic proteins, polydopamine, poly (norepinephrine), polyethyleneimine ), Polyoxazoline, polyvinylpyrolidone, polyacrylamide, polyvinylalcohol, poly (2-hydroxymethylmethacrylate), polymethylmethacrylate, polymethylmethacrylate, Wherein the polymer is at least one member selected from the group consisting of poly (methylmethacrylate-co-acrylic acid), polymethylmethacrylate, and polyacrylic acid. Transfer nanocomposites.
2. The gene delivery nanocomposite according to claim 1, wherein the monomolecular or polymer comprising the catechol group is 3,4-dihydroxy-L-phenylalanine.
The gene delivery nanocomposite according to claim 1, wherein the monomolecular or polymer comprising the catechol group has a degree of substitution of 0.5 to 50% with respect to the hydrophilic polymer.
2. The gene transfer kit according to claim 1, wherein the transfer gene is at least one selected from the group consisting of recombinant DNA, plasmid DNA, siRNA (small interfering RNA), miRNA (microRNA), or antisense nucleotide. Complex.
The gene delivery nanocomposite according to claim 1, wherein the gene transfer nanocomposite has a diameter of 50 nm to 200 nm.
2) 칼슘포스페이트와 전달유전자를 혼합하여 칼슘포스페이트/전달유전자 복합체를 제조하는 단계;
3) 상기 단계 1)에서 제조한 카테콜기를 포함하는 단분자 또는 중합체가 개질된 친수성 고분자와 2)에서 제조한 칼슘포스페이트/전달유전자 복합체를 혼합하여 칼슘포스페이트/전달유전자/카테콜기를 포함하는 단분자 또는 중합체로 개질된 친수성 고분자 복합체를 제조하는 단계;
를 포함하는 제1항 내지 제8항 중 어느 한 항의 유전자 전달용 나노 복합체의 제조방법.
1) reacting a monomolecule or a polymer containing a 1,2-dihydroxybenzene with a hydrophilic polymer to modify a monomolecule or a polymer containing a catechol group in the hydrophilic polymer;
2) preparing a calcium phosphate / transfer gene complex by mixing calcium phosphate and a transfer gene;
3) A single molecule or polymer modified hydrophilic polymer containing the catechol group prepared in step 1) and a calcium phosphate / transfer gene complex prepared in 2) are mixed to prepare a calcium phosphate / transfer gene / Preparing a hydrophilic polymer complex modified with a molecule or a polymer;
9. The method of producing a gene complex according to any one of claims 1 to 8,
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| CN109908339A (en) * | 2019-04-24 | 2019-06-21 | 中国医学科学院生物医学工程研究所 | A kind of sheet calcium phosphate vaccine adjuvant and preparation method thereof |
| CN113876601A (en) * | 2021-09-30 | 2022-01-04 | 宁波爱诗化妆品有限公司 | Modified amorphous tricalcium phosphate for cosmetics and preparation method thereof |
| CN117467708A (en) * | 2023-10-17 | 2024-01-30 | 中山大学孙逸仙纪念医院 | Nucleic acid delivery complex and preparation method and application thereof |
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| CN109908339A (en) * | 2019-04-24 | 2019-06-21 | 中国医学科学院生物医学工程研究所 | A kind of sheet calcium phosphate vaccine adjuvant and preparation method thereof |
| CN109908339B (en) * | 2019-04-24 | 2022-07-26 | 中国医学科学院生物医学工程研究所 | Flaky calcium phosphate vaccine adjuvant and preparation method thereof |
| CN113876601A (en) * | 2021-09-30 | 2022-01-04 | 宁波爱诗化妆品有限公司 | Modified amorphous tricalcium phosphate for cosmetics and preparation method thereof |
| CN113876601B (en) * | 2021-09-30 | 2023-09-29 | 宁波爱诗化妆品有限公司 | Modified amorphous tricalcium phosphate for cosmetics and preparation method thereof |
| CN117467708A (en) * | 2023-10-17 | 2024-01-30 | 中山大学孙逸仙纪念医院 | Nucleic acid delivery complex and preparation method and application thereof |
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