JP2017014229A - ポリヌクレオチド剤を含む細胞標的化ナノ粒子およびその使用 - Google Patents
ポリヌクレオチド剤を含む細胞標的化ナノ粒子およびその使用 Download PDFInfo
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Abstract
Description
(a)ポリヌクレオチドを、カチオン性分子を含む組成物と接触させるステップであって、カチオン性分子は静電的相互作用によりポリヌクレオチドを凝縮して複合体を生成し、カチオン性分子はリポソームに含まれないステップと、
(b)約4.5以下のpHで、複合体を標的化部分に共有結合させて、標的細胞にポリヌクレオチド剤を送達するための粒子を生成するステップと
を含む、標的細胞にポリヌクレオチドを送達するための粒子を生成する方法が提供される。
(a)ポリヌクレオチド剤を、カチオン性分子を含む組成物と接触させるステップであって、カチオン性分子は静電的相互作用によりポリヌクレオチド剤を凝縮して複合体を生成し、カチオン性分子はリポソームに含まれないステップと、
(b)約4.5未満のpHで、複合体を標的化部分に共有結合させて、標的細胞にポリヌクレオチド剤を送達するためのナノ粒子を生成するステップと
を含む、標的細胞にポリヌクレオチドを送達するためのナノ粒子を生成する方法が提供される。
siRNAおよびカチオン性分子の核、ならびに標的化部分(例えば、HA)を含む殻を含む粒子であって、
粒子の集団の粒子の各々は、約−40mVのゼータ電位を含み、粒子の集団の粒子の各々は、溶液中の場合、直径が約100〜300nmである。
miRNAおよびカチオン性分子の核、ならびに標的化部分(例えば、HA)を含む殻を含む粒子であって、
粒子の集団の粒子の各々は、溶液中の場合、直径が約30〜50nmである。
実施例
実施例1
DOTAPをベースとする粒子
材料および方法
siRNAおよびmiRNA模倣体(アンタゴmir)の調製:
siRNAをカプセル化したH−mer(ヒアルロン酸被覆ナノ粒子)の調製−方法1
siRNAをカプセル化したH−mer(ヒアルロン酸被覆ナノ粒子)の調製−方法2
ヒトサイクリンD1(CCND1)
順方向:TGCTCCTGGTGAACAAGCTCAAGT(配列番号1)
逆方向:TGATCTGTTTGTTCTCCTCCGCCT(配列番号2)
GAPDH
順方向:GACCCCTTCATTGACCTCAAC(配列番号3)
逆方向:CTTCTCCATGGTGGTGAAGA(配列番号4)
STAT1
順方向:GTGCATCATGGGCTTCATCAGCAA(配列番号5)
逆方向:TAGGGTTCAACCGCATGGAAGTCA(配列番号6)
結果
siRNAを封入したヒアルロン酸被覆ナノ粒子(H−mer)の調製および構造評価
最良の製剤をスクリーニングするためのCD44発現細胞への形質移入
H−merは、T−IC中のリファレンス遺伝子を選択的にノックダウンすることができる。
いくつかの発がん遺伝子に対するsiRNAの混合物を封入したH−merは、T−ICを選択的に根絶する。
siRNAを封入したH−merは、望まない免疫応答を誘導しない。
miRNAを封入したH−mer
アンタゴmirは、Dharmaconから購入した。アンタゴmir分子の数は、リボグリーン(Invitrogen)を使用して測定した。
実施例2
DLPE:DLPGをベースとする粒子
材料および方法
結果
siRNAを封入したヒアルマーの調製および構造評価
実施例3
本発明の実施形態の粒子は、AML初代細胞およびヒト卵巣腺癌細胞を標的化することができる。
材料および方法
結果
Claims (21)
- 核及び殻を含有する水不溶性脂質化グリコサミノグリカンの粒子であって、
前記核は、カチオン性脂質、一級アミノ酸残基を有する脂質及びポリヌクレオチドを含有し、
前記殻は、前記グリコサミノグリカンを含有し、且つ
前記粒子は、リポソームではない、
ことを特徴とする、水不溶性脂質化グリコサミノグリカンの粒子。 - 前記粒子は、約−25〜−50mVのゼータ電位を含み、及び/又は、
溶液中で約30〜300nmの粒径で存在する、請求項1に記載の粒子。 - 前記ポリヌクレオチドを標的細胞に対して送達する、請求項1に記載の粒子。
- 前記ポリヌクレオチドがRNAサイレンシング剤である、請求項1に記載の粒子。
- 前記RNAサイレンシング剤は、siRNA、miRNA、アンチセンスオリゴヌクレオチド及びリボザイムから成る群から選択される、請求項4に記載の粒子。
- 前記カチオン性脂質は、1,2−ジラウロイル−sn−グリセロ−3−ホスホエタノールアミン(DLPE)、1,2−ジラウロイル−sn−グリセロ−3−グリセロール(DLPG)、18:1 TAP(DOTAP)1,2−ジオレオイル−3−トリメチルアンモニウムプロパン、14:0 TAP(1,2−ジミリストイル−3−トリメチルアンモニウムプロパン)、16:0 TAP(1,2−ジパルミトイル−3−トリメチルアンモニウムプロパン)、18:0 TAP(1,2−ステアロイル−3−トリメチルアンモニウム−プロパン)、N−[1−(2,3−ジオレイロキシ)プロピル]−N,N,N−トリメチルアンモニウムクロリド(DOTMA)、ジメチルジオクタデシルアンモニウム(DDAB)、及び1,2−ジ−(9Z−オクタデセノイル)−3−ジメチルアンモニウム−プロパン 3β−[N−(N´,N´−ジメチルアミノエタン)−カルバモイル]から成る群から選択される、請求項1に記載の粒子。
- 前記一級アミノ酸残基を有する脂質は、ホスファチジルエタノールアミンを含有する、請求項1に記載の粒子。
- 中性脂質、アニオン性リン脂質、合成ポリマー又はコレステロールのうち1つ以上を更に含む、請求項1に記載の粒子。
- 前記中性脂質がジオレイルホスファチジルエタノールアミン(DOPE)を含む、請求項8に記載の粒子。
- 前記アニオン性リン脂質は、ホスファチジルセリン、ホスファチジン酸、ホスファチジルコリン及びホスファチジルグリセロールからなる群から選択される、請求項8に記載の粒子。
- 前記合成ポリマーは、ポリエチレンイミン(PEI)又はポリ−L−リジンを含む、請求項8に記載の粒子。
- 前記グリコサミノグリカンは、ヒアルロン酸(HA)、ケラタン硫酸、コンドロイチン硫酸、ヘパリン硫酸、へパラン硫酸、デルマチン硫酸、これらの塩及び混合物からなる群から選択される、請求項1に記載の粒子。
- 抗体、抗体断片、受容体リガンド及びアプタマーからなる群から選択される、少なくとも1つの追加的な標的化部分を更に含む、請求項1に記載の粒子。
- 帯電しているか、或いは中性である、請求項1に記載の粒子。
- 約6000個以上のポリヌクレオチド分子を含む、請求項1に記載の粒子。
- 1,2−ジラウロイル−sn−グリセロ−3−ホスホエタノールアミン(DLPE)及び1,2−ジラウロイル−sn−グリセロ−3−グリセロール(DLPG)を含む、請求項1に記載の粒子。
- コレステロール及びジオレオイルトリメチルアンモニウムプロパン(DOTAP)を含有する、請求項1に記載の粒子。
- 実質的に均一な複数の請求項1に記載の粒子を含む、組成物。
- CD44を発現している標的細胞内の目的とする遺伝子を下方制御するための医薬の製造用の、請求項1に記載の粒子の使用。
- 前記目的とする遺伝子は、癌遺伝子及び/又は生存率に関連する遺伝子であることを特徴とする、請求項19に記載の使用。
- それを必要とする対象における癌の治療用医薬の製造用の、請求項1に記載の粒子の使用。
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CA2767976A1 (en) | 2011-02-03 |
AU2016250495A1 (en) | 2016-11-17 |
CN104491872A (zh) | 2015-04-08 |
JP2013500960A (ja) | 2013-01-10 |
WO2011013130A2 (en) | 2011-02-03 |
EP2459724A2 (en) | 2012-06-06 |
KR20120059491A (ko) | 2012-06-08 |
AU2010277166B2 (en) | 2016-08-04 |
CN102482685A (zh) | 2012-05-30 |
US9574210B2 (en) | 2017-02-21 |
US20120129916A1 (en) | 2012-05-24 |
KR101755049B1 (ko) | 2017-07-06 |
CN102482685B (zh) | 2015-01-07 |
US10179113B2 (en) | 2019-01-15 |
GB0913442D0 (en) | 2009-09-16 |
EP2459724B1 (en) | 2019-09-25 |
WO2011013130A3 (en) | 2011-03-24 |
US20170143641A1 (en) | 2017-05-25 |
AU2010277166A1 (en) | 2012-02-23 |
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