JP2011010549A - Organic-inorganic hybrid nano particle composed of nucleic acid conjugate having polyethylene glycol bound thereto and calcium phosphate - Google Patents
Organic-inorganic hybrid nano particle composed of nucleic acid conjugate having polyethylene glycol bound thereto and calcium phosphate Download PDFInfo
- Publication number
- JP2011010549A JP2011010549A JP2007280803A JP2007280803A JP2011010549A JP 2011010549 A JP2011010549 A JP 2011010549A JP 2007280803 A JP2007280803 A JP 2007280803A JP 2007280803 A JP2007280803 A JP 2007280803A JP 2011010549 A JP2011010549 A JP 2011010549A
- Authority
- JP
- Japan
- Prior art keywords
- nucleic acid
- peg
- sirna
- conjugate
- oligo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5115—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Nanotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Public Health (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Inorganic Chemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Optics & Photonics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Medical Informatics (AREA)
- Immunology (AREA)
- Ceramic Engineering (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
本発明は、核酸を標的に送達するための有機−無機ハイブリッド型のナノ粒子、ならびにその製造方法および送達用の使用に関する。 The present invention relates to organic-inorganic hybrid-type nanoparticles for delivering nucleic acids to a target, and a method for producing the same and use for delivery.
ｓｍａｌｌ ｉｎｔｅｒｆｅｒｉｎｇ ＲＮＡ（ｓｉＲＮＡ）は、転写後レベルでのジーン・サイレンシングに関する細胞過程を制御するための魅惑的な研究手段である。ｓｉＲＮＡの哺乳類細胞内への導入はｍＲＮＡレベルで高度の配列特異的な遺伝子の発現阻害をもたらすので、この手法はアンチセンスの使用よりも有意に優れた効能を示すことが知られている（非特許文献１参照）。数年前の哺乳類細胞内でのＲＮＡ干渉（ＲＮＡｉ）が媒介するジーン・サイレンシングの発見から、ｓｉＲＮＡを標的遺伝子のノックダウンに利用する膨大な数の研究活動が存在する（非特許文献２、３および４参照）。ウイルス疾患や癌などの各種遺伝子疾患の治療におけるｓｉＲＮＡの使用についての潜在力は極めて高いとはいえ、ヌクレアーゼに対して本質的に不安定であり、生体利用能も低いためｓｉＲＮＡの実用的な用途は限定されている（非特許文献５参照）。臨床に適する療法に組み入れる上での主要な障壁の一つは効率のよい遺伝子送達ベクターの開発にあることは衆目の一致するところである。 Small interfering RNA (siRNA) is a fascinating research tool for controlling cellular processes related to gene silencing at the post-transcriptional level. Since the introduction of siRNA into mammalian cells results in a high degree of sequence-specific gene expression inhibition at the mRNA level, this approach is known to show significantly better efficacy than the use of antisense (non- Patent Document 1). From the discovery of gene silencing mediated by RNA interference (RNAi) in mammalian cells several years ago, there is a vast number of research activities utilizing siRNA for knockdown of target genes (Non-Patent Document 2, 3 and 4). Although the potential for the use of siRNA in the treatment of various genetic diseases such as viral diseases and cancer is extremely high, practical use of siRNA due to its inherent instability to nucleases and low bioavailability Is limited (see Non-Patent Document 5). There is a consensus that one of the major barriers to incorporation into clinically relevant therapies is the development of efficient gene delivery vectors.
ウイルスベクターは、有用な遺伝子送達ベクターであることが明らかにされている一方で、それらの免疫原性、発癌性および高い製造コスト、等により臨床上の使用が制限されている（例えば、非特許文献６および７参照）。非ウイルス性ベクターは見込みのあるウイルスベクターの代替物としての考えが高まっている。非ウイルスベクターの中でも、カチオン性リピッドおよびポリマーをベースとする送達系に強い興味が集まっている。しかし、遺伝子療法でそれらの有用性を拡大するためには、血清耐容性、毒性の低減、ｉｎ ｖｉｖｏでの使用効率の向上およびコストの低下が解決すべき課題として残存する。 While viral vectors have been shown to be useful gene delivery vectors, their clinical use is limited by their immunogenicity, carcinogenicity and high manufacturing costs (eg, non-patented). References 6 and 7). Non-viral vectors are increasingly considered as promising alternatives to viral vectors. Among non-viral vectors, there is a strong interest in delivery systems based on cationic lipids and polymers. However, in order to expand their usefulness in gene therapy, serum tolerance, reduced toxicity, improved in vivo use efficiency and reduced cost remain issues to be solved.
リン酸カルシウムの結晶形態物（以下、ＣａＰと略記する）であるハイドロキシアパタイト（ＨＡｐ）がＤＮＡに対して結合親和性を示すことは既に報告されている（非特許文献８参照）。ナノサイズのＣａＰ粒子はＤＮＡについての効率のよいキャリアーであることが証明されてきた（例えば、非特許文献９および１０参照）。また、ＣａＰナノ粒子のサイズが効率のよいトランスフェクションを達成する上で重要な役割を担うことも見出されていたが、一方で、ＣａＰ結晶の迅速な成長が大きな沈殿を生成し、トランスフェクション効率を劇的に低下させることも知られていた（非特許文献１１参照）。 It has already been reported that hydroxyapatite (HAp), which is a crystalline form of calcium phosphate (hereinafter abbreviated as CaP), exhibits binding affinity for DNA (see Non-Patent Document 8). Nano-sized CaP particles have been proven to be efficient carriers for DNA (see, for example, Non-Patent Documents 9 and 10). It has also been found that the size of CaP nanoparticles plays an important role in achieving efficient transfection, while rapid growth of CaP crystals produces large precipitates, It was also known to dramatically reduce the efficiency (see Non-Patent Document 11).
遺伝子送達用の新規なキャリアーの開発に向けた本発明者等のここ数年に亙る連続する研究を通じて、本発明者等はＤＮＡおよびｓｉＲＮＡの送達用にＣａＰをベースとするハイブリッドナノベクターを至適化すべ鋭意研究してきた（例えば、それぞれ、非特許文献１２および非特許文献１３参照）。先に、本発明者等の一部は、ポリ（エチレングリコール）−ｂ−ポリ（メタクリル酸）（ＰＥＧ−ＰＭＡ）を用いてＰＥＧ−ＰＭＡ／ＣａＰ／ｓｉＲＮＡハイブリッド型ナノ粒子を作製した（非特許文献１４および特許文献１参照）。培養細胞株では、かなり高濃度のＰＥＧ−ＰＭＡ（すなわち、４５０−５５０μｇ／ｍｌ）で有意なジーン・サイレンシングが達成された。しかし、単分散ナノ粒子の形成にとって高いＰＥＧ−ＰＭＡ濃度が必要であり、これが効率的なジーン・サイレンシングおよびハイブリッドナノ粒子中へのｓｉＲＮＡの取り込み効率に悪影響を及ぼすことから、可能ならば、さらに効果的な遺伝子または核酸の送達系を提供することが必要である。 Through our years of continuous research towards the development of new carriers for gene delivery, we have optimized CaP-based hybrid nanovectors for DNA and siRNA delivery We have intensively studied (see, for example, Non-Patent Document 12 and Non-Patent Document 13, respectively). Previously, some of the inventors prepared PEG-PMA / CaP / siRNA hybrid nanoparticles using poly (ethylene glycol) -b-poly (methacrylic acid) (PEG-PMA) (non-patented). Reference 14 and Patent Document 1). In cultured cell lines, significant gene silencing was achieved with fairly high concentrations of PEG-PMA (ie, 450-550 μg / ml). However, high PEG-PMA concentrations are required for monodisperse nanoparticle formation, which adversely affects efficient gene silencing and siRNA incorporation efficiency into hybrid nanoparticles, if possible There is a need to provide effective gene or nucleic acid delivery systems.
上記ＰＥＧ−ＰＭＡ／ＣａＰハイブリッド型ナノ粒子内へｓｉＲＮＡが内包される現象について、本発明者等は、次の推測を行った。アニオン種、つまりＰＭＡおよびｓｉＲＮＡの存在が、アニオン性ＰＭＡ／ｓｉＲＮＡとＣａＰ表面上の陽電荷の間の結合の際にかならず競合を起こす。言い換えれば、このような競合の結果として、ＣａＰへのｓｉＲＮＡの取り込み効率は低下する。本発明者等は、こうような競合を避ける手段として、ＰＥＧ−ＰＭＡを使用することなく、ｓｉＲＮＡにポリエチレングリコール鎖の共有結合したコンジュゲートを上記系と組み合わせると前記問題点が本質的に解決できる可能性があると推測した。こうして、ｓｉＲＮＡにポリエチレングリコール鎖の共有結合したコンジュゲートを生成し、得られたコンジュゲートをＣａＰの形成系で使用してみたところ、現に、効率よくｓｉＲＮＡをＣａＰに取り込むことができることのみならず、容易に安定な単分散ナノ粒子を取得できることを見出した。そして、このようなナノ粒子は、標的細胞に効率よく取り込まれ、細胞内で該コンジュゲートを放出し、核酸の機能が効率よく発揮されることが確認された。また、短鎖のオリゴヌクレオチドもしくは小型の分子であるｓｉＲＮＡが効率よくＣａＰに取り込むことができることから理解できるように、他の一定の核酸も同様にこの新規な系に取り込み得ることも見出した。 The present inventors have made the following estimation regarding the phenomenon in which siRNA is encapsulated in the PEG-PMA / CaP hybrid type nanoparticles. The presence of anionic species, ie PMA and siRNA, always causes competition in the binding between the anionic PMA / siRNA and the positive charge on the CaP surface. In other words, the efficiency of siRNA incorporation into CaP decreases as a result of such competition. As a means of avoiding such competition, the present inventors can essentially solve the above problem by combining a conjugate of the above-mentioned system with a covalently bound polyethylene glycol chain to siRNA without using PEG-PMA. I guessed it was possible. Thus, when a conjugate in which a polyethylene glycol chain was covalently bonded to siRNA was generated and the resulting conjugate was used in a CaP formation system, not only was siRNA capable of being efficiently incorporated into CaP, It was found that stable monodisperse nanoparticles can be easily obtained. It was confirmed that such nanoparticles were efficiently taken up by the target cells, released the conjugate in the cells, and the nucleic acid function was efficiently exhibited. It has also been found that certain other nucleic acids can be incorporated into this novel system as well, as can be understood from the fact that siRNA, a short oligonucleotide or small molecule, can be efficiently incorporated into CaP.
こうして、本発明によれば、核酸の３’または５’末端にポリエチレングリコールの共有結合したコンジュゲートと、カルシウムイオン（Ｃａ２＋）およびリン酸イオン（ＰＯ４ ３−）を含んでなる有機−無機ハイブリッド型ナノ粒子が提供される。 Thus, according to the present invention, an organic-inorganic comprising a conjugate of polyethylene glycol covalently bonded to the 3 ′ or 5 ′ end of a nucleic acid, calcium ion (Ca 2+ ) and phosphate ion (PO 4 3− ). Hybrid type nanoparticles are provided.
好適な態様のナノ粒子では、前記コンジュゲートが、一般式Ｉ
Ａ−ＰＥＧ−Ｌ−核酸 （Ｉ）
式中、
ＰＥＧはポリエチレングリコール鎖を表し、
ＡはＰＥＧの末端基または末端部分を表し、
ＬはＰＥＧのＡ結合末端の別の末端と核酸の３’または５’末端を共有結合するリンカーを表し、そして
核酸は、オリゴもしくはポリ二本鎖ＲＮＡ、オリゴもしくはポリ二本鎖ＤＮＡ、オリゴもしくはポリ一本鎖ＲＮＡおよびオリゴもしくはポリ一本鎖ＤＮＡからなる群より選ばれ、かつ、カルシウムイオンがリン酸イオンより過剰当量存在する。
In a preferred embodiment of the nanoparticle, the conjugate is of the general formula I
A-PEG-L-nucleic acid (I)
Where
PEG represents a polyethylene glycol chain,
A represents a terminal group or terminal portion of PEG,
L represents a linker that covalently joins the other end of the A-binding end of PEG and the 3 ′ or 5 ′ end of the nucleic acid, and the nucleic acid is oligo or poly double stranded RNA, oligo or poly double stranded DNA, It is selected from the group consisting of poly single stranded RNA and oligo or poly single stranded DNA, and calcium ions are present in an excess equivalent amount to phosphate ions.
別の態様の本発明としては、核酸の３’または５’末端にポリエチレングリコール鎖が共有結合したコンジュゲートおよびＣａ２＋を含んでなる水溶液と、ＰＯ４ ３−を含んでなる水溶液を、リン酸カルシウムと前記コンジュゲートがナノ粒子を形成しうる条件下で混合する工程を含んでなる、有機−無機ハイブリッド型ナノ粒子の製造方法、が提供される。 In another embodiment of the present invention, an aqueous solution comprising a conjugate of a polyethylene glycol chain covalently bonded to the 3 ′ or 5 ′ end of a nucleic acid and Ca 2+ , an aqueous solution comprising PO 4 3− , calcium phosphate, There is provided a method for producing an organic-inorganic hybrid type nanoparticle comprising a step of mixing under a condition in which the conjugate can form a nanoparticle.
また、こうして提供されるナノ粒子は、哺乳類細胞に効率よく取り込まれるので、核酸を哺乳類細胞に送達するための研究用または医療用のツールとしての用途も提供される。 The nanoparticles thus provided are also efficiently incorporated into mammalian cells, thus providing use as a research or medical tool for delivering nucleic acids to mammalian cells.
＜発明の詳細な記述＞
本発明にいう、核酸は、１より多くのヌクレオチドが５’→３’の方向に結合した核酸鎖の分子であって、動物または植物、特に、哺乳動物の生命現象に関与し得る分子を意味する。このような核酸鎖には、一本鎖または二本鎖のＲＮＡまたはＤＮＡが包含され、二本鎖は、ＤＮＡ／ＤＮＡ、ＲＮＡ／ＲＮＡまたはＤＮＡ／ＲＮＡであることができる。ヌクレオチドは、アデノシン（Ａ）、グアノシン（Ｇ）、ウリジン（Ｕ）、シチジン（Ｃ）、チミジン（Ｔ）から選ばれるが、前記核酸には、本発明の目的に悪影響を及ぼさない範囲内で天然に存在する修飾ヌクレオチドまたは動物または植物、特に、哺乳動物細胞で複製でき、転写・翻訳の可能な非天然型塩基を有するヌクレオチドが含まれていてもよい。
<Detailed Description of the Invention>
Nucleic acid as used in the present invention means a molecule of a nucleic acid chain in which more than one nucleotide is bound in the 5 ′ → 3 ′ direction and can be involved in the life phenomenon of animals or plants, particularly mammals. To do. Such nucleic acid strands include single stranded or double stranded RNA or DNA, which can be DNA / DNA, RNA / RNA or DNA / RNA. The nucleotide is selected from adenosine (A), guanosine (G), uridine (U), cytidine (C), and thymidine (T). The nucleic acid is naturally present within the range that does not adversely affect the object of the present invention. Or a nucleotide having a non-natural base that can be replicated in animals or plants, particularly mammalian cells, and can be transcribed and translated.
生命現象に関与し得る核酸分子は、欠損することにより何らかの疾患をもたらす遺伝子であることができる。また、特定の細胞内で、例えば、ＲＮＡ干渉（ＲＮＡｉ）や標的ＤＮＡとの結合等を介して究極的には遺伝子発現を調節し得る分子をいう。本発明に従えば、比較的短い（オリゴ概念に包含される）ヌクレオチド鎖の、例えば、約４００までのヌクレオチドを含んでなる短鎖もしくは小型のオリゴヌクレオチドを有機−無機ハイブリッド型ナノ粒子内へ効率よく安定に内包せしめ得るので、それ自体公知のＲＮＡもしくはＤＮＡアプタマーの概念に包含される核酸分子、およびＲＮＡｉに利用できる、例えば、ｓｍａｌｌ（またはｓｈｏｒｔ）ｉｎｔｅｒｆｅｒｉｎｇ ＲＮＡ（ｓｉＲＮＡ）の概念に包含される核酸分子に本発明を都合よく適用することができる。 Nucleic acid molecules that can participate in biological phenomena can be genes that cause some disease by being deficient. Also, it refers to a molecule that can ultimately regulate gene expression in a specific cell, for example, via RNA interference (RNAi) or binding to a target DNA. In accordance with the present invention, a short or small oligonucleotide comprising a relatively short nucleotide chain (included in the oligo concept), eg, up to about 400 nucleotides, can be efficiently incorporated into organic-inorganic hybrid nanoparticles. Nucleic acid molecules included in the concept of RNA or DNA aptamers known per se and can be used for RNAi, for example, nucleic acids included in the concept of small (or short) interfering RNA (siRNA) The present invention can be conveniently applied to molecules.
限定されるものではないが、本発明で好適、かつ、効果的に使用できる核酸分子のサイズは、二本鎖核酸である場合には、約１６〜約４００のヌクレオチド鎖長であり、一本鎖核酸である場合には約４０〜約４００のヌクレオチド鎖であることができる。特に、本発明で好適に使用できる核酸分子は、３’末端に２、３個のヌクレオチドのオーバーハングを有していてもよい、一本鎖に約１９〜３０、好ましくは、約１９〜２３のヌクレオチドを有するｓｉＲＮＡ、および約５０〜１４０のヌクレオチドを有するＲＮＡもしくはＤＮＡアプタマーを挙げることができる。限定されるものでないが、ｓｉＲＮＡの具体例は、遺伝子療法の対象となりうる遺伝子を参照して設計でき、このような遺伝子の例としては、非小細胞肺癌などに関係のあるＰＫＣα、悪性黒色腫などに関係のあるＢＣＬ−２、ク
ローン病に関係のあるＩＣＡＭ−１、Ｃ型肝炎に関係のあるＨＣＶ、関節リウマチもしくは乾癬に関係のあるＴＮＦα、喘息に関係のあるアデノシンＡＩ受容体、卵巣がんなどに関係のあるｃ−ｒａｆ ｋｉｎａｓｅ、膵臓癌などに関係のあるＨ−ｒａｓ、冠動脈疾患に関係のあるｃ−ｍｙｃ、大腸癌に関係のあるＰＫＡ Ｒｉα、エイズに関係のあるＨＩＶ、固形癌に関係のあるＤＮＡメチルトランスフェラーゼ、癌に関係のあるＶＥＧＦ受容体、腎臓癌に関係のあるリボヌクレオチド還元酵素、ＣＭＶ性網膜炎に関係のあるＣＭＶ
ＩＥ２、前立腺癌に関係のあるＭＭＰ−９、悪性グリオーマに関係のあるＴＧＦβ２、多発性硬化症に関係のあるＣＤ４９ｄ、糖尿病に関係のあるＰＴＰ−１Ｂ、癌に関係のあるｃ−ｍｙｂ、乳癌などに関係のあるＥＧＦＲ、癌に関係のあるｍｄｒ１、ａｕｔｏｔａｘｉｎおよびＧＬＵＴ−１の遺伝子を挙げることができる。
Without limitation, the size of a nucleic acid molecule that is suitable and can be used effectively in the present invention is a nucleotide chain length of about 16 to about 400 when it is a double-stranded nucleic acid. In the case of a strand nucleic acid, it can be about 40 to about 400 nucleotide strands. In particular, nucleic acid molecules that can be suitably used in the present invention may have an overhang of 2 or 3 nucleotides at the 3 ′ end and are about 19-30, preferably about 19-23, on a single strand. SiRNA having the nucleotides of RNA, and RNA or DNA aptamers having about 50-140 nucleotides. Although not limited, specific examples of siRNA can be designed with reference to genes that can be targeted for gene therapy. Examples of such genes include PKCα and malignant melanoma related to non-small cell lung cancer and the like. BCL-2 related to the above, ICAM-1 related to Crohn's disease, HCV related to hepatitis C, TNFα related to rheumatoid arthritis or psoriasis, adenosine AI receptor related to asthma, ovary C-raf kinase related to cancer, H-ras related to pancreatic cancer, c-myc related to coronary artery disease, PKA Riα related to colorectal cancer, HIV related to AIDS, solid cancer DNA methyltransferase related to cancer, VEGF receptor related to cancer, ribonucleotide reductase related to kidney cancer, CMV retinitis CMV with engaging
IE2, MMP-9 related to prostate cancer, TGFβ2 related to malignant glioma, CD49d related to multiple sclerosis, PTP-1B related to diabetes, c-myb related to cancer, breast cancer, etc. And EGFR related to cancer, mdr1, autotaxin and GLUT-1 related to cancer.
核酸３’または５’末端にポリエチレングリコール鎖の共有結合したコンジュゲートは、上記の核酸分子とポリエチレングリコールを公知の連結方法で共有結合して生成できるコンジュゲートであって（例えば、ＷＯ ２００６／０２５４１９、ＷＯ ２００７／０２１１４２参照）、カルシウムイオン（Ｃａ２＋）およびリン酸イオン（ＰＯ４ ３−）の存在する水性溶液中で有機−無機ハイブリッド型ナノ粒子を形成できるいかなるコンジュゲートをも包含する。このようなコンジュゲートの典型的なものとしては、下記一般式Ｉで表されるものを挙げることができる：
Ａ−ＰＥＧ−Ｌ−核酸 （Ｉ）
式中、
ＰＥＧはポリエチレングリコール鎖を表し、
ＡはＰＥＧの末端基または末端部分を表し、
ＬはＰＥＧのＡ結合末端の別の末端と核酸の３’または５’末端を共有結合するリンカーを表し、そして
核酸は、上述したオリゴもしくはポリ二本鎖ＲＮＡ、オリゴもしくはポリ二本鎖ＤＮＡ、オリゴもしくはポリ一本鎖ＲＮＡおよびオリゴもしくはポリ一本鎖ＤＮＡからなる群より選ばれる。
A conjugate in which a polyethylene glycol chain is covalently bonded to a nucleic acid 3 ′ or 5 ′ end is a conjugate that can be produced by covalently binding the nucleic acid molecule and polyethylene glycol by a known linking method (for example, WO 2006/025419). , WO 2007/021142), any conjugate capable of forming organic-inorganic hybrid nanoparticles in an aqueous solution in the presence of calcium ions (Ca 2+ ) and phosphate ions (PO 4 3− ). Typical examples of such conjugates include those represented by the following general formula I:
A-PEG-L-nucleic acid (I)
Where
PEG represents a polyethylene glycol chain,
A represents a terminal group or terminal portion of PEG,
L represents a linker that covalently joins the other end of the A-bonded end of PEG to the 3 ′ or 5 ′ end of the nucleic acid, and the nucleic acid comprises the oligo or poly double stranded RNA, oligo or poly double stranded DNA described above, It is selected from the group consisting of oligo or poly single stranded RNA and oligo or poly single stranded DNA.
ＰＥＧの分子量は、本発明の有機−無機ハイブリッド型ナノ粒子を形成できるものであれば限定されないが、約６０００Ｄａ〜約５００００Ｄａ、好ましくは約７０００Ｄａ〜約２５０００Ｄａ、より好ましくは約１００００Ｄａ〜約２５０００Ｄａの範囲内にあるものであることができる。 The molecular weight of PEG is not limited as long as it can form the organic-inorganic hybrid nanoparticles of the present invention, but it is in the range of about 6000 Da to about 50000 Da, preferably about 7000 Da to about 25000 Da, more preferably about 10000 Da to about 25000 Da. It can be what is inside.
ＰＥＧの末端基または末端部分であるＡは、水素原子、Ｃ１−１０の直鎖もしくは分岐したアルキルもしくはアルケニル基（例、メチル、エチル、プロピル、イソプロピル、ヘキシルまたはアリル等）、アラルキル基（例、ベンジル、フェネチル等）、ヒドロキシ基、Ｃ１−１０の直鎖もしくは分岐したアルコキシ基、アルデヒド基（またはホルミル：−ＣＨＯ）、アセタール化ホルミル基〔−ＣＨ（ＯＲ１）（ＯＲ２）、ここで、Ｒ１およびＲ２は相互に独立して、Ｃ１−４アルキルであるか、または一緒になってＣ１−４アルキレン鎖であることもできる〕、アミノ基、カルボキシル基、マレイミド基、保護されたアミノ基および保護されたカルボキシル基（ここで、保護されたと称する場合の保護基は、ペプチド合成で慣用されているアミノ基またはカルボキシル基についての保護基を意味する。）からなる群より選ばれる基もしくは官能基であることができ、また、細胞表面受容体に結合しうる前記官能基を介して結合したリガンド（例、糖、ペプチド、等）または抗体等の機能性もしくは結合性部分であることができる。 A, which is a terminal group or terminal portion of PEG, is a hydrogen atom, a C 1-10 linear or branched alkyl or alkenyl group (eg, methyl, ethyl, propyl, isopropyl, hexyl or allyl), an aralkyl group (eg, , Benzyl, phenethyl, etc.), hydroxy group, C 1-10 linear or branched alkoxy group, aldehyde group (or formyl: —CHO), acetalized formyl group [—CH (OR 1 ) (OR 2 ), R 1 and R 2 independently of one another can be C 1-4 alkyl, or together can be a C 1-4 alkylene chain], an amino group, a carboxyl group, a maleimide group, A protected amino group and a protected carboxyl group (where the protecting group, when referred to as protected, is commonly used in peptide synthesis) Or a functional group selected from the group consisting of amino group and carboxyl group), and bonded via the functional group capable of binding to a cell surface receptor. It can be a functional or binding moiety such as a ligand (eg, sugar, peptide, etc.) or antibody.
リンカーＬは、二本鎖核酸に結合する場合には、センス鎖またはアンチセンス鎖のいずれか一方の３’または５’末端に、また一本鎖核酸の場合も、その３’または５’末端のいずれかにリン酸エステル結合等の当該技術分野で公知のいずれかの結合様式で共有結合し、ＰＥＧ鎖のＡに対してもう一方の末端に炭素―炭素結合、エーテル結合、チオエーテ
ル結合、エステル結合、チオエステル結合、アミド結合、ウレイド結合、尿素結合等を介して、共有結合する連結基である。Ｌは、上記のような両端の結合以外に、酸素原子もしくは硫黄原子１もしくは２以上の箇所で中断されていてもよい総原子数３〜３０のアルキレン基を含むことができる。このようなアルキレン鎖としては、限定されるものでないが、例えば、−ＣＨ２ＣＨ２ＣＨ２−、−ＣＨ２ＣＨ２−Ｏ−ＣＨ２ＣＨ２−、−ＣＨ２ＣＨ２−（Ｏ−ＣＨ２ＣＨ２）２−、−ＣＨ２ＣＨ２−Ｓ−ＣＨ２ＣＨ２−およびＣＨ２ＣＨ２−Ｓ−（ＣＨ２）６−等を挙げることができる。本発明では、このようなアルキレン鎖中に生理的条件下で開裂しうる結合、例えば、エンドソームにおける低ｐＨ（５．０〜６．０）において開裂しうるエステル結合または還元条件下もしくは還元剤として作用しうる物質の存在下で開裂し得るジスルフィド結合を含めることが好ましい。このような結合を含むアルキレン鎖の例としては、限定されるものでないが、−ＣＨ２ＣＨ２ＯＣＯＣＨ２−、−ＣＨ２ＣＨ２ＳＳＣＨ２−、−ＣＨ２ＣＨ２ＣＨ２−ＣＯＯ−ＣＨ２−および−ＣＨ２ＣＨ２ＯＣＨ２ＣＨ２ＳＳＣＨ２ＣＨ２−を挙げることができる。
The linker L is attached to the 3 ′ or 5 ′ end of either the sense strand or the antisense strand when binding to a double-stranded nucleic acid, and also to the 3 ′ or 5 ′ end of a single-stranded nucleic acid. Is covalently bonded to any one of the bonding methods known in the art such as a phosphate ester bond, and a carbon-carbon bond, an ether bond, a thioether bond, an ester at the other end with respect to A of the PEG chain. A linking group that is covalently bonded via a bond, thioester bond, amide bond, ureido bond, urea bond, or the like. In addition to the bonds at both ends as described above, L can include an alkylene group having 3 to 30 total atoms which may be interrupted at one or more sites of oxygen atoms or sulfur atoms. Such alkylene chains, but it is not limited to, for example, -CH 2 CH 2 CH 2 - , - CH 2 CH 2 -O-CH 2 CH 2 -, - CH 2 CH 2 - (O-CH 2 CH 2) 2 -, - CH 2 CH 2 -S-CH 2 CH 2 - and CH 2 CH 2 -S- (CH 2 ) 6 - , and the like. In the present invention, a bond that can be cleaved under physiological conditions in such an alkylene chain, for example, an ester bond that can be cleaved at low pH (5.0 to 6.0) in endosomes or under reducing conditions or as a reducing agent It is preferred to include a disulfide bond that can be cleaved in the presence of an agent that can act. Examples of alkylene chains comprising such binding, but are not limited to, -CH 2 CH 2 OCOCH 2 - , - CH 2 CH 2 SSCH 2 -, - CH 2 CH 2 CH 2 -COO-CH 2 - and -CH 2 CH 2 OCH 2 CH 2 SSCH 2 CH 2 - and the like.
本発明では、理論に拘束されるものではないが、上記のＡ−ＰＥＧ−Ｌ−核酸コンジュゲートが、アニオン荷電性の核酸部分を介して、カルシウムイオン（Ｃａ２＋）およびリン酸イオン（ＰＯ４ ３−）を特定の比率で含んでなるリン酸カルシウム（ＣａＰ）粒子のカチオン性表面および／または微小粒子内部に固定されまたは取り込まれた無機ハイブリッド型のナノ粒子が提供される。そのため、該ナノ粒子における、ＣａＰでは、カルシウムイオンがリン酸イオンより過剰当量存在し、好ましくは、ＣａＰのカルシウムイオン対リン酸イオンのモル比率が、２０〜５００、好ましくは３０〜３００、より好ましくは５０〜２００にある。こうして、該ナノ粒子では、ＣａＰにおける過剰のカルシウムイオンと前記コンジュゲートの核酸に由来するアニオン荷電性のリン酸部分がイオン−イオン相互作用をすることにより、ＣａＰ中に該コンジュゲート中の核酸部分を部分的にかまたは全体的に内包した形態にあるものと推測される。後述する当該ナノ粒子の透過型電子顕微鏡写真を参照されたい。該ナノ粒子では、ＣａＰとコンジュゲートが、上述した過剰のカルシウムイオンにコンジュゲートの各分子内のリン酸部が少なくとも部分的に相互作用できるような比率で含まれておればよい。しかし、限定されるものでないが、核酸におけるリン酸部と過剰のカルシウムイオンが、モル比で、０．００１〜０．０５対１、好ましくは０．００２〜０．０１対１になるように選ばれる。また、カルシウムイオンは、別の多価カチオン、例えば、Ｍｇ２＋で部分的に置き換えられていてもよい。 In the present invention, the A-PEG-L-nucleic acid conjugate is not limited by theory, and the calcium ion (Ca 2+ ) and phosphate ion (PO 4 ) are exchanged via an anionically charged nucleic acid moiety. 3- ) Inorganic hybrid-type nanoparticles immobilized or incorporated within the cationic surface of calcium phosphate (CaP) particles and / or the interior of microparticles comprising a specific ratio are provided. Therefore, CaP in the nanoparticles has an excess equivalent of calcium ions than phosphate ions, and preferably the molar ratio of CaP calcium ions to phosphate ions is 20 to 500, preferably 30 to 300, more preferably. Is between 50 and 200. Thus, in the nanoparticle, an excess of calcium ions in CaP and an anionically charged phosphate moiety derived from the nucleic acid of the conjugate cause an ion-ion interaction, whereby the nucleic acid portion in the conjugate in CaP. Is presumed to be partially or wholly encapsulated. Please refer to the transmission electron micrograph of the nanoparticles described below. In the nanoparticle, the CaP and conjugate may be contained in such a ratio that the phosphate portion in each molecule of the conjugate can at least partially interact with the above-described excess calcium ions. However, it is not limited so that the phosphoric acid part and the excess calcium ion in the nucleic acid are in a molar ratio of 0.001 to 0.05 to 1, preferably 0.002 to 0.01 to 1. To be elected. Calcium ions may be partially replaced with another polyvalent cation, for example, Mg 2+ .
本願発明にいう、ナノ粒子は、キュムラント平均粒子径がナノオーダー、限定されるのもでないが、３０ｎｍ〜１０００ｎｍ、好ましくは、５０ｎｍ〜３００ｎｍであるが、数ミクロンの平均粒径を有する粒子も包含し得る概念として使用してきる。 Nanoparticles referred to in the present invention have a cumulant average particle size of nano-order, which is not limited, but includes 30 nm to 1000 nm, preferably 50 nm to 300 nm, but also includes particles having an average particle diameter of several microns. Use it as a possible concept.
このような有機−無機ハイブリッド型ナノ粒子は、前記コンジュゲートおよびＣａ２＋を含んでなる水溶液と、ＰＯ４ ３−を含んでなる水溶液をリン酸カルシウムと前記コンジュゲートがナノ粒子を形成しうる条件下で混合することにより製造できる。この水溶液の混合条件は、核酸に悪影響を及ぼさない条件下、例えば、１０℃〜５０℃で、１時間〜４０時間混合液を静置すればよい。混合後の水溶液中のコンジュゲートの濃度は、コンジュゲートが溶解できる濃度であればよいが、通常、ヌクレオチドを基準に０．１ｍＭ〜２０ｍＭ、好ましくは、０．２ｍＭ〜５ｍＭであり、カルシウムイオン濃度は、３０ｍＭ〜５００ｍＭ、好ましくは５０ｍＭ〜３００ｍＭであり、ＣａＰを形成するためのリン酸イオンの濃度は、０．１ｍＭ〜２０ｍＭ、好ましくは０．２ｍＭ〜１０ｍＭに設定できる。また、カルシウムイオン以外の多価カチオン、例えば、Ｍｇ２＋は、Ｃａ２＋に対して０．０１〜１当量含めることができる。 Such an organic-inorganic hybrid type nanoparticle is obtained by subjecting an aqueous solution containing the conjugate and Ca 2+ to an aqueous solution containing PO 4 3− under conditions where the conjugate can form nanoparticles with calcium phosphate. It can be manufactured by mixing. The mixing conditions of this aqueous solution may be such that the mixed solution is allowed to stand at 10 ° C. to 50 ° C. for 1 hour to 40 hours under conditions that do not adversely affect the nucleic acid. The concentration of the conjugate in the aqueous solution after mixing may be a concentration at which the conjugate can be dissolved, but is usually 0.1 mM to 20 mM, preferably 0.2 mM to 5 mM based on nucleotides, and the calcium ion concentration Is 30 mM to 500 mM, preferably 50 mM to 300 mM, and the concentration of phosphate ions for forming CaP can be set to 0.1 mM to 20 mM, preferably 0.2 mM to 10 mM. Moreover, 0.01-1 equivalent of polyvalent cations other than calcium ion, for example, Mg < 2+ >, can be included with respect to Ca <2+ >.
こうして、本発明により提供できる有機−無機ハイブリッド型のナノ粒子は、生理的条件下で動物または植物細胞、特に哺乳類細胞と接触させることにより、これらの細胞に効
率よく取り込まれ、こうして取り込まれた粒子は、通常、カルシウムイオン濃度の低い細胞内で徐々に崩壊してＡ−ＰＥＧ−Ｌ−核酸コンジュゲートを放出することができる。また、コンジュゲートのリガンド中に生理的条件下で開裂し得る結合を有する場合には、細胞内でコンジュゲートから核酸を開放し得る。カルシウムイオンおよびリン酸イオンは、本質的に細胞の生理および核酸の機能に悪影響を及ぼさないので、例えば、細胞内でカチオン性ポリマーが放出されることが予測される、ＷＯ ２００６／０２５４１９またはＷＯ ２００７／０２１１４２に記載されているｓｉＲＮＡ−ＰＥＧコンジュゲートとカチオン性ポリマーのイオンコンプレックスに比べて安全に使用できる。
Thus, the organic-inorganic hybrid-type nanoparticles that can be provided by the present invention are efficiently incorporated into these cells by contacting them with animal or plant cells, particularly mammalian cells, under physiological conditions, and thus the incorporated particles. Can normally decay slowly in cells with low calcium ion concentrations to release A-PEG-L-nucleic acid conjugates. In addition, when the ligand of the conjugate has a bond that can be cleaved under physiological conditions, the nucleic acid can be released from the conjugate within the cell. For example, WO 2006/025419 or WO 2007 where calcium and phosphate ions are expected to release a cationic polymer in the cell because they essentially do not adversely affect cell physiology and nucleic acid function. It can be used safely as compared with the ion complex of siRNA-PEG conjugate and cationic polymer described in / 021142.
以下、本発明をより具体的に説明するが、本発明はこれらの例に限定されることは意図されていない。 Hereinafter, the present invention will be described more specifically, but the present invention is not intended to be limited to these examples.
＜製造例１＞ ＰＥＧ（１２ｋＤａ）−ＳＳ−ｐｙｌの合成
α−メトキシ−ω−メルカプト−ポリエチレングリコール（日本油脂、ＳＵＮＢＲＩＧＨＴ ＳＨ、重量平均分子量Ｍｗ＝１２０００）１００ｍｇをＴＨＦ１５ｍｌに溶解させ、さらに２，２’−ジピリジルジスルフィド（Ａｌｄｒｉｃｈ）１８４ｍｇ及びｎ−プロピルアミン１．７５ｍｌを加えた。反応液を室温で３時間撹拌した後、ジエチルエーテル５００ｍｌ中に滴下することにより白色沈殿を得た。この沈殿物を減圧ろ過により回収した後、ベンゼン３０ｍｌに溶解させ、凍結乾燥を行うことにより表題の化合物を得た。 100 mg of α-methoxy-ω-mercapto-polyethylene glycol (Nippon Yushi, SUNBRIGHT SH, weight average molecular weight Mw = 12000) was dissolved in 15 ml of THF, and 184 mg of 2,2′-dipyridyl disulfide (Aldrich) and 1. 75 ml was added. The reaction solution was stirred at room temperature for 3 hours and then added dropwise to 500 ml of diethyl ether to obtain a white precipitate. The precipitate was collected by filtration under reduced pressure, dissolved in 30 ml of benzene, and freeze-dried to obtain the title compound.
＜製造例２＞ ＰＥＧ（１５ｋＤａ）−ＳＳ−ｐｙｌの合成
α−メトキシ−ω−メルカプト−ポリエチレングリコール（日本油脂、ＳＵＮＢＲＩＧＨＴ ＳＨ、重量平均分子量Ｍｗ＝１５０００）１２５ｍｇをＴＨＦ１５ｍｌに溶解させ、さらに２，２’−ジピリジルジスルフィド（Ａｌｄｒｉｃｈ）１８４ｍｇ及びｎ−プロピルアミン１．７５ｍｌを加えた。反応液を室温で３時間撹拌した後、ジエチルエーテル５００ｍｌ中に滴下することにより白色沈殿を得た。この沈殿物を減圧ろ過により回収した後、ベンゼン３０ｍｌに溶解させ、凍結乾燥を行うことにより表題の化合物を得た。 125 mg of α-methoxy-ω-mercapto-polyethylene glycol (Nippon Yushi, SUNBRIGHT SH, weight average molecular weight Mw = 15000) was dissolved in 15 ml of THF, and 184 mg of 2,2′-dipyridyl disulfide (Aldrich) and 1. 75 ml was added. The reaction solution was stirred at room temperature for 3 hours and then added dropwise to 500 ml of diethyl ether to obtain a white precipitate. The precipitate was collected by filtration under reduced pressure, dissolved in 30 ml of benzene, and freeze-dried to obtain the title compound.
＜製造例３＞ ＰＥＧ（５ｋＤａ）−ＳＳ−ｐｙｌの合成
α−メトキシ−ω−メルカプト−ポリエチレングリコール（日本油脂、ＳＵＮＢＲＩＧＨＴ ＳＨ、重量平均分子量Ｍｗ＝５０００）４１．７ｍｇをＴＨＦ１５ｍｌに溶解させ、さらに２，２’−ジピリジルジスルフィド（Ａｌｄｒｉｃｈ）１８４ｍｇ及びｎ−プロピルアミン１．７５ｍｌを加えた。反応液を室温で３時間撹拌した後、ジエチルエーテル５００ｍｌ中に滴下することにより白色沈殿を得た。この沈殿物を減圧ろ過により回収した後、ベンゼン３０ｍｌに溶解させ、凍結乾燥を行うことにより表題の化合物を得た。 41.7 mg of α-methoxy-ω-mercapto-polyethylene glycol (Nippon Yushi, SUNBRIGHT SH, weight average molecular weight Mw = 5000) was dissolved in 15 ml of THF, and further 184 mg of 2,2′-dipyridyl disulfide (Aldrich) and n-propylamine 1.75 ml was added. The reaction solution was stirred at room temperature for 3 hours and then added dropwise to 500 ml of diethyl ether to obtain a white precipitate. The precipitate was collected by filtration under reduced pressure, dissolved in 30 ml of benzene, and freeze-dried to obtain the title compound.
＜製造例４＞ ＰＥＧ（１２ｋＤａ）−ＳＳ−ｓｉＲＮＡの調製
本実施例で用いるＳＨ−ｓｉＲＮＡは、センス鎖として５’−ＣＵＵＡＣＧＣＵＧＡＧＵＡＣＵＵＣＧＡｄＴｄＴ−３’、アンチセンス鎖として：５’−ＵＣＧＡＡＧＵＡＣＵＣＡＧＣＧＵＡＡＧｄＴｄＴ−３’を用い、２本鎖を形成させたものであり、かつセンス鎖の５’末端にＳＨ修飾（Ｃ６ Ｓ−Ｓ ｍｏｄｉｆｉｅｒ、Ｇｌｅｎ Ｒｅｓｅｒｃｈ）がなされたものである。
<Production Example 4> Preparation of PEG (12 kDa) -SS-siRNA The SH-siRNA used in this example uses 5'-CUUACGCUGAGUACUCUCGAdTdT-3 'as the sense strand and 5'-UCGAAGUAUCUCAGCGUAAGdTdT-3' as the antisense strand. A double strand is formed, and the 5 ′ end of the sense strand is SH-modified (C6 S-S modifier, Glen Research).
ＳＨ−ｓｉＲＮＡ（０．１μｍｏｌ）を、０．０５Ｍジチオスレイトールを含有する１０ｍＭ Ｔｒｉｓ ｂｕｆｆｅｒ（ｐＨ７．４）の１．０ｍｌに溶解させ、室温で６時間置いた後にＮＡＰ−５カラム（ＧＥヘルスケアバイオサイエンス）を用いてジチオスレイトールを除去した。このＳＨ−ｓｉＲＮＡに対しＰＥＧ（１２ｋＤａ）−ＳＳ−ｐｙｌ（１２ｍｇ）を加え、室温で２４時間反応させた。反応物は逆相ＨＰＬＣで分取することにより精製を行った。逆相ＨＰＬＣにはカラムとしてＴＳＫｇｅｌ Ｏｌｉｇｏ‐ＤＮＡ ＲＰ（東ソー）を用い、溶離液として５％アセトニトリル含有０．１Ｍ酢酸アンモニウム溶液（溶離液Ａ）および７０％アセトニトリル含有０．１Ｍ酢酸アンモニウム溶液（溶離液Ｂ）を用い、溶離液Ａから溶離液Ｂへのリニアグラジエント（４０分、流速毎分１ｍｌ）により溶出を行った。検出にはＵＶ（検出波長２６０ｎｍ）を用いた。ＰＥＧ（１２ｋ）−ＳＳ−ｓｉＲＮＡを含有するフラクションは、遠心エバポレーターにより濃縮した後にＮＡＰ−５カラムによって処理し、３３μＭのＰＥＧ（１２ｋ）−ＳＳ−ｓｉＲＮＡを含有する１ｍＭ Ｔｒｉｓ ｂｕｆｆｅｒ溶液（ｐＨ７．４）を得た。 SH-siRNA (0.1 μmol) was dissolved in 1.0 ml of 10 mM Tris buffer (pH 7.4) containing 0.05 M dithiothreitol and placed at room temperature for 6 hours before NAP-5 column (GE Healthcare Dithiothreitol was removed using Bioscience. PEG (12 kDa) -SS-pyl (12 mg) was added to this SH-siRNA and reacted at room temperature for 24 hours. The reaction product was purified by fractionation by reverse phase HPLC. For reverse-phase HPLC, TSKgel Oligo-DNA RP (Tosoh) was used as a column, and 0.1M ammonium acetate solution containing 5% acetonitrile (eluent A) and 0.1M ammonium acetate solution containing 70% acetonitrile (eluent) as eluents. B) was used to elute with a linear gradient from eluent A to eluent B (40 minutes, flow rate 1 ml / min). UV (detection wavelength 260 nm) was used for detection. The fraction containing PEG (12k) -SS-siRNA was concentrated by a centrifugal evaporator and then treated with a NAP-5 column, and 1 mM Tris buffer solution (pH 7.4) containing 33 μM PEG (12k) -SS-siRNA. Got.
また、上記のＰＥＧ（１２ｋＤａ）−ＳＳ−ｐｙｌに代え、それぞれ、ＰＥＧ（５ｋＤａ）−ＳＳ−ｐｙｌおよびＰＥＧ（１５ｋＤａ）−ＳＳ−ｐｙｌを用いて相当するＰＥＧ（５ｋＤａ）−ＳＳ−ｓｉＲＮＡおよびＰＥＧ（１５ｋＤａ）−ＳＳ−ｓｉＲＮＡを得た。 Moreover, it replaces with said PEG (12 kDa) -SS-pyl, respectively, and PEG (5 kDa) -SS-siRNA and PEG (PEG (5 kDa) -SS-pyl and PEG (15 kDa) -SS-pyl and PEG ( 15 kDa) -SS-siRNA was obtained.
＜実施例１＞ リン酸カルシウム粒子の調製
以下の溶液を調製した。
溶液Ａ
ＰＥＧ−ＳＳ−ｓｉＲＮＡ ３０μＭ
ＣａＣｌ２ ２５０ｍＭ
ＭｇＣｌ２
Ｔｒｉｓ ｈｙｄｒｏｃｈｌｏｒｉｄｅ １ｍＭ
溶液ＡのｐＨ、ＰＥＧの分子量及びＭｇＣｌ２の濃度については表１に示した。
<Example 1> Preparation of calcium phosphate particles The following solutions were prepared.
Solution A
PEG-SS-siRNA 30 μM
CaCl 2 250 mM
MgCl 2
Tris hydrochloride 1 mM
The pH of the solution A, the molecular weight of PEG, and the concentration of MgCl 2 are shown in Table 1.
溶液Ｂ（ｐＨ ７．５）
Ｎａ２ＨＰＯ４ １．５ｍＭ
ＮａＣｌ １４０ｍＭ
Ｈｅｐｅｓ ｓｏｄｉｕｍ ｓａｌｔ ５０ｍＭ
表１に示した各組成の溶液Ａ２００μｌに対し、溶液Ｂを２００μｌ混合した後、２５℃で２４時間静置することで、粒子（１）〜（８）及び（Ｃ−１）の溶液を得た。
Solution B (pH 7.5)
Na2HPO4 1.5 mM
NaCl 140 mM
Hepes sodium salt 50 mM
After 200 μl of solution B is mixed with 200 μl of solution A having each composition shown in Table 1, the solution of particles (1) to (8) and (C-1) is obtained by allowing to stand at 25 ° C. for 24 hours. It was.
＜実施例２＞ リン酸カルシウム粒子（Ｃ−２）の調製
以下の溶液を調製した。
溶液Ａ：
ｓｉＲＮＡ（センス鎖として５’−ＣＵＵＡＣＧＣＵＧＡＧＵＡＣＵＵＣＧＡｄＴｄＴ−３’、アンチセンス鎖として５’−ＵＣＧＡＡＧＵＡＣＵＣＡＧＣＧＵＡＡＧｄＴｄＴ−３’を用い、２本鎖を形成させたもの） ７０μｇ／ｍｌ塩化カルシウム ２５０ｍＭ
以上の組成の溶液を、１ｍＭ Ｔｒｉｓ ｂｕｆｆｅｒ（ｐＨ７．６）を用いて調製した。
<Example 2> Preparation of calcium phosphate particles (C-2) The following solutions were prepared.
Solution A:
siRNA (5′-CUUACGCUGAGUACUCUCGAdTdT-3 ′ as sense strand and 5′-UCGAAGUAUCUCAGCGUAAGdTdT-3 ′ as antisense strand to form a double strand) 70 μg / ml calcium chloride 250 mM
A solution having the above composition was prepared using 1 mM Tris buffer (pH 7.6).
溶液Ｂ：
リン酸一水素二ナトリウム １．５ｍＭ
ＮａＣｌ １４０ｍＭ
ＰＥＧ−ポリ（アスパラギン酸） （ＰＥＧ分子量１２０００、ポリアスパラギン酸の重合度２４） ３００μｇ／ｍｌ
以上の組成の溶液を、５０ｍＭ ＨＥＰＥＳ ｂｕｆｆｅｒ（ｐＨ７．６）を用いて調製した。
Solution B:
Disodium monohydrogen phosphate 1.5 mM
NaCl 140 mM
PEG-poly (aspartic acid) (PEG molecular weight 12000, degree of polymerization of polyaspartic acid 24) 300 μg / ml
A solution having the above composition was prepared using 50 mM HEPES buffer (pH 7.6).
溶液Ａ２００μｌと溶液Ｂ２００μｌを混合し、２５℃で２４時間静置することで、粒子（Ｃ−２）の溶液を得た。 A solution of particles (C-2) was obtained by mixing 200 μl of solution A and 200 μl of solution B and allowing to stand at 25 ° C. for 24 hours.
＜実施例３＞ ＰＥＧ−ＳＳ−ｓｉＲＮＡの調製の確認
ＰＥＧ−ＳＳ−ｓｉＲＮＡの生成を確認するため、２０％アクリルアミドゲルを用いた電気泳動により分析を行った。泳動後のゲルはＳＹＢＲ Ｇｒｅｅｎ ＩＩ ＲＮＡ ｇｅｌ ｓｔａｉｎ（Ｉｎｖｉｔｒｏｇｅｎ）により染色した。結果を図１に示す。ｓｉＲＮＡ（レーン３）にくらべ、ＰＥＧ−ＳＳ−ｓｉＲＮＡ（レーン１及び２）では泳動度が小さくなり、ＰＥＧの付加により高分子量化が起きたことを確認できた。また、ＳＳ結合の還元環境応答性を確認するためＰＥＧ−ＳＳ−ｓｉＲＮＡに１０ｍＭジチオスレイトールを加え、３７℃で２時間置いた後のサンプルについても分析を行ったところ、元のｓｉＲＮＡと同じ位置にバンドが現れ（レーン４及び５）、ＰＥＧの切断が確認できた。
<Example 3> Confirmation of preparation of PEG-SS-siRNA In order to confirm the production of PEG-SS-siRNA, analysis was performed by electrophoresis using a 20% acrylamide gel. The gel after electrophoresis was stained with SYBR Green II RNA gel stain (Invitrogen). The results are shown in FIG. Compared with siRNA (lane 3), PEG-SS-siRNA (lanes 1 and 2) had a lower mobility and it was confirmed that the addition of PEG resulted in a higher molecular weight. In addition, in order to confirm the reduction environment responsiveness of SS bond, a sample after adding 10 mM dithiothreitol to PEG-SS-siRNA and leaving at 37 ° C. for 2 hours was analyzed, and the same position as the original siRNA. A band appeared (lanes 4 and 5), and PEG cleavage was confirmed.
＜実施例４＞ リン酸カルシウム粒子に含有されたｓｉＲＮＡ濃度の評価
各リン酸カルシウム粒子溶液４００μｌをＡｍｉｃｏｎ Ｕｌｔｒａ−４ １０ｋ（Ｍｉｌｌｉｐｏｒｅ）を用いて２５００×ｇで１０分間遠心ろ過し、ろ液の２６０ｎｍにおける吸光度をＮＤ−１０００（Ｎａｎｏｄｒｏｐ Ｔｅｃｈｎｏｌｏｇｉｅｓ）により測定した。リン酸カルシウム粒子に含有されたｓｉＲＮＡの濃度は、粒子の調製時に添加したｓｉＲＮＡの濃度と、ろ液中のｓｉＲＮＡの濃度の差から算出した。
<Example 4> Evaluation of siRNA concentration contained in calcium phosphate particles 400 μl of each calcium phosphate particle solution was centrifuged at 2500 × g for 10 minutes using Amicon Ultra-4 10k (Millipore), and the absorbance at 260 nm of the filtrate was determined to be ND. -1000 (Nanodrop Technologies). The concentration of siRNA contained in the calcium phosphate particles was calculated from the difference between the concentration of siRNA added during particle preparation and the concentration of siRNA in the filtrate.
その結果、粒子（１）において粒子に含有されたｓｉＲＮＡの量は６７μｇであり、良好な結果であった。これに対し、粒子（Ｃ−２）において粒子に含有されたｓｉＲＮＡの量は８．１μｇと低い値であった。 As a result, the amount of siRNA contained in the particles (1) was 67 μg, which was a good result. In contrast, the amount of siRNA contained in the particle (C-2) was a low value of 8.1 μg.
＜実施例５＞ 粒子径と粒子径分布の測定
粒子（１）〜（８）、（Ｃ−１）及び（Ｃ−２）について、キュムラント平均粒径及び分散度（ＰＤＩ）の測定をＺｅｔａｓｉｚｅｒ Ｎａｎｏ ＺＳ（Ｍａｌｖｅｒｎ Ｉｎｓｔｒｕｍｅｎｔｓ）を用いて行った。結果を表１に示す。粒子（１）から（８）及び粒子（Ｃ−２）のリン酸カルシウム粒子については分散度の狭い単分散な粒子が生成された。これに対し、粒子（Ｃ−１）のでは粒子径１μｍ以上の沈殿が生成された。この粒子においては、ＰＥＧの分子量が５０００と小さいためＰＥＧによる凝集防止効果が弱く、沈殿が生成したと考えられる。実施例１のサンプル（１）の粒子溶液について、粒子径分布のヒストグラムを図２（Ａ）に、そして透過型電子顕微鏡で撮影した顕微鏡写真（バーの寸法：１００ｎｍ）図２（Ｂ）に示した。
<Example 5> Measurement of particle size and particle size distribution For particles (1) to (8), (C-1) and (C-2), measurement of cumulant average particle size and dispersity (PDI) was performed on Zetasizer Nano. This was performed using ZS (Malvern Instruments). The results are shown in Table 1. With respect to the calcium phosphate particles of particles (1) to (8) and particles (C-2), monodisperse particles having a narrow degree of dispersion were produced. On the other hand, in the case of the particles (C-1), a precipitate having a particle diameter of 1 μm or more was generated. In these particles, since the molecular weight of PEG is as small as 5000, the effect of preventing aggregation by PEG is weak, and it is considered that precipitation occurred. The particle size distribution histogram of the particle solution of sample (1) of Example 1 is shown in FIG. 2 (A), and a micrograph taken with a transmission electron microscope (bar size: 100 nm) is shown in FIG. 2 (B). It was.
＜実施例６＞ ｉｎ ｖｉｔｒｏ活性の評価
本発明のｓｉＲＮＡ内包粒子について、ｉｎ ｖｉｔｒｏにおけるホタルルシフェラーゼ遺伝子に対する発現抑制活性を評価した。
<Example 6> Evaluation of in vitro activity About the siRNA inclusion particle | grains of this invention, the expression suppression activity with respect to the firefly luciferase gene in vitro was evaluated.
２４穴ポリスチレン製細胞培養プレート（ＢＤ Ｆａｌｃｏｎ）に、ヒト肝癌由来細胞株（Ｈｕｈ７ ｃｅｌｌ）を１ｘ１０４ ｃｅｌｌ／ｗｅｌｌ 播種し２４時間培養した後、ホタルルシフェラーゼベクターｐＧＬ３−ｃｏｎｔｒｏｌ（０．３６μｇ／ｗｅｌｌ，Ｐｒｏｍｅｇａ）とウミシイタケルシフェラーゼベクターｐＲＬ−ＣＭＶ（０．０４μｇ／ｗｅｌｌ，Ｐｒｏｍｅｇａ）の各レポーター遺伝子を、Ｌｉｐｏｆｅｃｔ ＡＭＩＮＥ ２０００（１μｌ／ｗｅｌｌ，Ｉｎｖｉｔｒｏｇｅｎ）を用い、製品の添付書に従って細胞に導入した。４時間インキュベートした後に培地を交換し、各粒子の溶液を加えた後４８時間細胞と接触させた。培地中でのｓｉＲＮＡの最終濃度は１００ｎＭとなるように各粒子を添加した。４８時間培養後に細胞を回収し、両レポーター遺伝子の発現量をＤｕａｌ Ｌｕｃｉｆｅｒａｓｅ Ｒｅｐｏｒｔｅｒ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ）により測定した。ウミシイタケルシフェラーゼ発現量に対するホタルルシフェラーゼ発現量の相対値（ｐＧＬ３／ｐＲＬ）により抑制活性を評価した（Ｎ＝３）。結果を図３に示す。 A 24-well polystyrene cell culture plate (BD Falcon) was seeded with 1 × 10 4 cells / well of a human liver cancer cell line (Huh7 cell) and cultured for 24 hours, and then firefly luciferase vector pGL3-control (0.36 μg / well, Promega). ) And the Renilla luciferase vector pRL-CMV (0.04 μg / well, Promega) were introduced into cells using Lipofect AMINE 2000 (1 μl / well, Invitrogen) according to the product attachment. After incubation for 4 hours, the medium was changed, and the solution of each particle was added and then contacted with the cells for 48 hours. Each particle was added so that the final concentration of siRNA in the medium was 100 nM. After 48 hours of culture, the cells were collected, and the expression levels of both reporter genes were measured by Dual Luciferase Reporter System System (Promega). The inhibitory activity was evaluated based on the relative value (pGL3 / pRL) of the firefly luciferase expression level relative to the Renilla luciferase expression level (N = 3). The results are shown in FIG.
図３のグラフから、本発明のｓｉＲＮＡ内包粒子は、効率よく癌細胞に取り込まれ、該細胞における標的遺伝子の発現を有意に抑制することが分る。 From the graph of FIG. 3, it can be seen that the siRNA-encapsulated particles of the present invention are efficiently taken into cancer cells and significantly suppress the expression of target genes in the cells.
＜実施例７＞ 血清中での安定性評価
粒子（１）の溶液１００μｌに対し、１０％牛胎児血清（ＩＣＮ Ｂｉｏｍｅｄｉｃａｌｓ）を含有するＤｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ ｍｅｄｉｕｍ（Ｓｉｇｍａ Ａｌｄｒｉｃｈ）１００μｌを加え、混合した。この溶液を３７℃の恒温槽中に静置し、一定時間毎に、前記溶液中の粒子の３７℃におけるキュムラント平均粒径をＺｅｔａｓｉｚｅｒ Ｎａｎｏ ＺＳ（Ｍａｌｖｅｒｎ Ｉｎｓｔｒｕｍｅｎｔｓ）を用いて測定した。その結果を図４に示す。１１時間後まで平均粒径に大きな変化は見られず、この粒子は血清存在下でも安定であることが示された。
Example 7 Stability Evaluation in Serum 100 μl of Dulbecco's modified Eagle's medium (Sigma Aldrich) containing 10% fetal calf serum (ICN Biomedicals) was added to 100 μl of the solution of particles (1), Mixed. This solution was allowed to stand in a 37 ° C. thermostat, and the cumulant average particle size at 37 ° C. of the particles in the solution was measured using Zetasizer Nano ZS (Malvern Instruments) at regular time intervals. The result is shown in FIG. There was no significant change in average particle size until after 11 hours, indicating that the particles were stable in the presence of serum.
Claims (13)
（Ｃａ２＋）およびリン酸イオン（ＰＯ４ ３−）を含んでなる有機−無機ハイブリッド型のナノ粒子。 An organic-inorganic hybrid type nanoparticle comprising a conjugate of polyethylene glycol covalently bonded to the 3 ′ or 5 ′ end of a nucleic acid, and calcium ions (Ca 2+ ) and phosphate ions (PO 4 3− ).
Ａ−ＰＥＧ−Ｌ−核酸 （Ｉ）
式中、
ＰＥＧはポリエチレングリコール鎖を表し、
ＡはＰＥＧの末端基または末端部分を表し、
ＬはＰＥＧのＡ結合末端の別の末端と核酸の３’または５’末端を共有結合するリンカーを表し、そして
核酸は、オリゴもしくはポリ二本鎖ＲＮＡ、オリゴもしくはポリ二本鎖ＤＮＡ、オリゴもしくはポリ一本鎖ＲＮＡおよびオリゴもしくはポリ一本鎖ＤＮＡからなる群より選ばれ、かつ、
カルシウムイオンがリン酸イオンより過剰当量存在する、
請求項１記載のナノ粒子。 The conjugate is of general formula I
A-PEG-L-nucleic acid (I)
Where
PEG represents a polyethylene glycol chain,
A represents a terminal group or terminal portion of PEG,
L represents a linker that covalently joins the other end of the A-binding end of PEG and the 3 ′ or 5 ′ end of the nucleic acid, and the nucleic acid is oligo or poly double stranded RNA, oligo or poly double stranded DNA, oligo or Selected from the group consisting of poly single stranded RNA and oligo or poly single stranded DNA, and
Calcium ions are present in excess equivalents than phosphate ions,
The nanoparticle according to claim 1.
核酸が約１６〜約４００のヌクレオチド鎖長の二本鎖または約４０〜約４００のヌクレオチド鎖を含んでなるＲＮＡまたはＤＮＡであり、リンカー中に生体内で開裂しうる結合が含まれており、かつ
カルシウムイオン対リン酸イオンのモル比率が、５０〜２００にあり、そして
核酸におけるリン酸イオンの少なくとも一部と過剰のカルシウムイオンが、イオン結合した状態にある、請求項２記載のナノ粒子。 PEG in the conjugate is in the range of molecular weight 7000 Da to 25000 Da,
The nucleic acid is a RNA or DNA comprising a double strand of about 16 to about 400 nucleotides in length or about 40 to about 400 nucleotides, wherein the linker contains a bond that can be cleaved in vivo; The nanoparticle according to claim 2, wherein the molar ratio of calcium ion to phosphate ion is 50 to 200, and at least a part of the phosphate ion in the nucleic acid and the excess calcium ion are in an ion-bonded state.
Ａ−ＰＥＧ−Ｌ−核酸 （Ｉ）
式中、
ＰＥＧはポリエチレングリコール鎖を表し、
ＡはＰＥＧ末端基または末端部分を表し、
ＬはＰＥＧのＡ結合末端の別の末端と核酸の３’または５’末端を共有結合するリンカーを表し、そして
核酸は、オリゴもしくはポリ二本鎖ＲＮＡ、オリゴもしくはポリ二本鎖ＤＮＡ、オリゴもしくはポリ一本鎖ＲＮＡおよびオリゴもしくはポリ一本鎖ＤＮＡからなる群より選ばれ、かつ、Ｃａ２＋を含んでなる水溶液がＣａＣｌ２を用いて調製され、ＰＯ４ ３−を含んでなる水溶液がＮａ２ＨＰＯ４を用いて調製される請求項１０に記載の製造方法。 The conjugate is of general formula I
A-PEG-L-nucleic acid (I)
Where
PEG represents a polyethylene glycol chain,
A represents a PEG end group or terminal moiety,
L represents a linker that covalently joins the other end of the A-binding end of PEG and the 3 ′ or 5 ′ end of the nucleic acid, and the nucleic acid is oligo or poly double stranded RNA, oligo or poly double stranded DNA, oligo or An aqueous solution selected from the group consisting of poly single stranded RNA and oligo or poly single stranded DNA and containing Ca 2+ is prepared using CaCl 2 , and an aqueous solution containing PO 4 3− is Na 2. The production method according to claim 10, which is prepared using HPO 4 .
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007280803A JP2011010549A (en) | 2007-10-29 | 2007-10-29 | Organic-inorganic hybrid nano particle composed of nucleic acid conjugate having polyethylene glycol bound thereto and calcium phosphate |
| PCT/JP2008/070154 WO2009057812A1 (en) | 2007-10-29 | 2008-10-29 | Organic-inorganic hybrid nano particle composed of nucleic acid conjugate having polyethylene glycol bound thereto and calcium phosphate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007280803A JP2011010549A (en) | 2007-10-29 | 2007-10-29 | Organic-inorganic hybrid nano particle composed of nucleic acid conjugate having polyethylene glycol bound thereto and calcium phosphate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2011010549A true JP2011010549A (en) | 2011-01-20 |
Family
ID=40591186
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2007280803A Pending JP2011010549A (en) | 2007-10-29 | 2007-10-29 | Organic-inorganic hybrid nano particle composed of nucleic acid conjugate having polyethylene glycol bound thereto and calcium phosphate |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2011010549A (en) |
| WO (1) | WO2009057812A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10046065B2 (en) | 2013-08-06 | 2018-08-14 | Japan Science And Technology Agency | Nucleic acid-encapsulating polymer micelle complex and method for producing same |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8450282B2 (en) | 2009-07-23 | 2013-05-28 | The University Of Tokyo | Anionic polymer, polyion complex and ternary polymer composite using anionic polymer, and pharmaceutical composition |
| US9750687B2 (en) | 2010-05-21 | 2017-09-05 | Japan Science And Technology Agency | Substance-encapsulating vesicle and process for producing the same |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3472970B2 (en) * | 1993-12-10 | 2003-12-02 | 株式会社アドバンス | Method for producing bioimplant material |
| JP2003523544A (en) * | 2000-02-17 | 2003-08-05 | レックスマーク・インターナショナル・インコーポレーテツド | Composition containing fine particles for supporting biologically active substance thereon or having the same supported thereon and method for preparing these |
| US6436386B1 (en) * | 2000-11-14 | 2002-08-20 | Shearwater Corporation | Hydroxyapatite-targeting poly (ethylene glycol) and related polymers |
| US7247288B2 (en) * | 2002-04-18 | 2007-07-24 | Carnegie Mellon University | Method of manufacturing hydroxyapatite and uses therefor in delivery of nucleic acids |
| AU2006280600B2 (en) * | 2005-08-17 | 2012-01-19 | Bioneer Corporation | Sirna-hydrophilic polymer conjugates for intracellular delivery of siRNA and method thereof |
-
2007
- 2007-10-29 JP JP2007280803A patent/JP2011010549A/en active Pending
-
2008
- 2008-10-29 WO PCT/JP2008/070154 patent/WO2009057812A1/en active Application Filing
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10046065B2 (en) | 2013-08-06 | 2018-08-14 | Japan Science And Technology Agency | Nucleic acid-encapsulating polymer micelle complex and method for producing same |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2009057812A1 (en) | 2009-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cavallaro et al. | Polymeric nanoparticles for siRNA delivery: Production and applications | |
| Shatsberg et al. | Functionalized nanogels carrying an anticancer microRNA for glioblastoma therapy | |
| Kargaard et al. | Polymeric siRNA gene delivery–transfection efficiency versus cytotoxicity | |
| CN108026527B (en) | Defined multiconjugated oligonucleotides | |
| Kim et al. | Efficient siRNA delivery using water soluble lipopolymer for anti-angiogenic gene therapy | |
| US9567430B2 (en) | Enzymatic synthesis of poly(amine-co-esters) and methods of use thereof for gene delivery | |
| US9895451B2 (en) | Formulations for targeted release of agents to low pH tissue environments or cellular compartments and methods of use thereof | |
| KR101564796B1 (en) | Copolymer including uncharged hydrophilic block and cationic polyamino acid block having lateral chain to which hydrophobic radical is partially introduced, and use of copolymer | |
| Pun et al. | Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles | |
| Du et al. | Intelligent nucleic acid delivery systems based on stimuli-responsive polymers | |
| EP2087912B1 (en) | Sirna carrier using disulfide-bridged polymeric micelle | |
| WO2013082529A1 (en) | Enzymatic synthesis of poly(amine-co-esters) and methods of use thereof for gene delivery | |
| KR20140127299A (en) | Branched Polymers | |
| CN110248665A (en) | The polymer oligonucleotides removed with reduced kidney | |
| WO2021257262A1 (en) | Poly(amine-co-ester) polymers with modified end groups and enhanced pulmonary delivery | |
| JP2020172534A (en) | Engineering synthetic brain penetrating gene vectors | |
| Zhu et al. | Nucleic acid-based artificial nanocarriers for gene therapy | |
| Fakhoury et al. | Antisense precision polymer micelles require less poly (ethylenimine) for efficient gene knockdown | |
| JP2011010549A (en) | Organic-inorganic hybrid nano particle composed of nucleic acid conjugate having polyethylene glycol bound thereto and calcium phosphate | |
| Fröhlich et al. | Peptide-and polymer-based delivery of therapeutic RNA | |
| JP2011511776A (en) | Cationic siRNA for RNA interference, synthesis and use | |
| Cun et al. | Polymeric nanocarriers for siRNA delivery: challenges and future prospects | |
| WO2016081621A1 (en) | Formulations for targeted release of agents under low ph conditions and methods of use thereof | |
| Salcher et al. | Chemically programmed polymers for targeted DNA and siRNA transfection | |
| US10682422B2 (en) | Formulations for targeted release of agents under low pH conditions and methods of use thereof |