JP2019536003A - ヒト挙動のリアルタイムバイオセンシングのための触媒基質としてのナノ粒子ならびに診断および治療方法 - Google Patents
ヒト挙動のリアルタイムバイオセンシングのための触媒基質としてのナノ粒子ならびに診断および治療方法 Download PDFInfo
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Abstract
Description
本出願は、2016年9月15日に出願された米国仮出願番号第62/395,245号に基づく米国特許法第119条(e)項の下の利益を主張しており、その全体の内容は、本明細書中に参考として援用される。
本発明は、米空軍資材コマンド法務局(AFMCLO/JAZ)によって授与されたFA8650−15−2−5518の下、政府支援でなされた。政府は本発明において特定の権利を有する。
本発明は、概して、代謝マーカーおよび酵素マーカーの検出ならびに関連する製品および方法に関する。
いくつかの試験は運動の間接的な態様を測定するが、現在、リアルタイムでの運動の生理的効果の正確かつ高感度の追跡を可能にする試験は存在しない。
本発明は、一部の態様では、筐体と、直接または間接のいずれかで少なくとも部分的に筐体の外側部分と接続された血液抽出要素と、筐体の少なくとも一部分内でLCAT活性化因子と結合することができるナノ構造とを備えるデバイスである。一部の実施形態では、デバイスはウェアラブルまたはポータブルデバイスである。
本発明は、一部の態様では、心血管疾患および他の健康状態のヒトのリスクならびに血清タンパク質レベルの変化およびコレステロール代謝に関与する酵素の活性に起因する運動の効果を測定することができる迅速な血液試験の発見を含む。アッセイは、例えば、急および長期的な運動に応答する心血管の健康の追跡を可能にするのに有用である。標的タンパク質および酵素の活性は、ベースラインレベルで存在し、次いで酵素活性は運動後に増加することが報告されている。本発明の方法は、分析のための血液(針で刺すことにより得られる)などの少量の生体試料のみを必要とする。方法は、タンパク質吸着および酵素活性をリアルタイムで正確に測定するための手段として合成脂質官能化ナノ粒子を使用して達成される。
酵素レシチン:コレステロールアシルトランスフェラーゼ(LCAT)についての基質として、前もって開発した合成高密度ナノ粒子(HDL−NP)を使用したアッセイを行った。HDL−NPを、市販の5nm AuNPを使用して形成した。アポAIを、5倍モル過剰量で5nm AuNPの水溶液に加え、室温で穏やかにかき混ぜながら1時間ナノ粒子表面に結合させた。このインキュベーション期間の後、エタノール(20%、v/v)および脂質の混合物を、金ナノ粒子の濃度に対して250倍モル過剰量で溶液に加えて、AuNPコアの周りに脂質二重層を形成させた。使用した脂質は、a)1,2−ジパルミトイル−sn−グリセロ−3−ホスホチオエタノール(DPPTE)、AuNP32の表面に共有結合して、内葉を形成するチオール化脂質、b)アシル鎖(例えば、TopFluor PC、1−パルミトイル−2−(二フッ素化ジピロメテンボロン)ウンデカノイル−sn−グリセロ−3−ホスホコリン、Avanti Polar Lipids,Inc.)のsn2位にコンジュゲートしたフルオロフォアを有するホスファチジルコリン(PC)脂質、最後に、c)HDL−NP脂質二重層の外葉の大部分を形成する1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(DPPC)であった。脂質二重層を、穏やかに振とうして室温で終夜、形成させた。終夜インキュベートした後、HDL−NPを、15,000×gで50分間遠心分離することにより精製し、未反応の表面構成成分およびエタノールの除去を保証するために3回繰り返した。より大型のバッチのナノ粒子(初回5nm AuNP溶液>10ml)の場合、遠心分離ステップは、省略してもよく、ナノ粒子を、タンジェント流濾過システムを使用して処理して、HDL−NPの最終溶液を濾過し濃縮してもよい。
コレステロール流出アッセイと相関する、アポリポタンパク質AI吸着およびレシチン:コレステロールアシルトランスフェラーゼ活性によってHDL機能を測定するためのナノ粒子に基づいたアッセイ
序文
材料および方法
結果
考察
参照文献
Claims (71)
- 筐体と、
直接または間接のいずれかで少なくとも部分的に前記筐体の外側部分と接続された血液抽出要素と、
コア、および前記筐体の少なくとも一部分内でレシチン:コレステロールアシルトランスフェラーゼ(LCAT)活性化因子と結合することができるリン脂質シェルを含むナノ構造と
を備えるデバイス。 - ウェアラブルまたはポータブルデバイスである、請求項1に記載のデバイス。
- 前記ナノ構造が固体のコアを含む、請求項1または2に記載のデバイス。
- 前記ナノ構造が、固体のコアと、脂質層とを含む、請求項1または2に記載のデバイス。
- 前記ナノ構造が前記LCAT活性化因子と結合している、請求項1または2に記載のデバイス。
- 前記LCAT活性化因子がアポリポタンパク質であり、前記LCAT活性化因子が直接検出され得る、請求項1から3のいずれか一項に記載のデバイス。
- 前記ナノ構造が、金のコアと、脂質二重層または単層とを有する、請求項6に記載のデバイス。
- 運動および疾患リスク関連酵素を迅速に検出する方法であって、
生体試料を、固体のコアナノ粒子と、任意選択でレシチン:コレステロールアシルトランスフェラーゼ(LCAT)活性化因子と結合することができる脂質二重層または脂質単層と接触させるステップと、前記ナノ粒子が前記LCAT活性化因子と結合することができるように少なくとも15分間、前記固体のコアナノ粒子を前記生体試料とインキュベートするステップと、前記生体試料中の運動、代謝または疾患関連酵素の存在の指標としてLCAT活性化を測定するステップと
を含む、方法。 - 前記生体試料が、血液、血液マトリックス、血清、血漿、痰、脳脊髄液、呼気凝縮液、唾液、尿および涙からなる群から選択される、請求項8に記載の方法。
- 前記LCAT活性化因子がアポリポタンパク質である、請求項8または9に記載の方法。
- 標識が蛍光標識である、請求項8から10のいずれか一項に記載の方法。
- 前記蛍光標識が、ナノ構造におけるホスファチジルコリン上にある、請求項11に記載の方法。
- in vitroで実施される、請求項8から12のいずれか一項に記載の方法。
- 前記生体試料が対象から単離され、ウェアラブルまたはポータブルデバイスを使用することによって実施される、請求項8から12のいずれか一項に記載の方法。
- ナノ構造が、無機材料を含むナノ構造コアと、前記ナノ構造コアを取り囲み、それに付着している脂質層を含み、内面および外面を有するシェルとを含む、請求項1から6のいずれか一項に記載のデバイスまたは請求項6から12のいずれか一項に記載の方法。
- 前記脂質層が脂質二重層である、請求項15に記載のデバイス。
- 前記シェルにおける前記脂質が、1,2−ジパルミトイル−sn−グリセロ−3−ホスホチオエタノール(DPPTE)、ホスファチジルコリン(PC)および1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(DPPC)から構成されている、請求項15から16のいずれか一項に記載のデバイス。
- 無機コアと、リン脂質シェルとを有する脂質官能化ナノ粒子を含む組成物であって、前記脂質官能化ナノ粒子は、リン脂質、コレステロール脂質、およびリンを含有しないジアシル脂質を含む、蛍光標識された脂質を含む、組成物。
- 高密度リポタンパク質(HDL)機能を測定するための方法アッセイであって、
無機材料を含むナノ構造コアと、前記ナノ構造コアを取り囲み、それに付着しており、シェルが脂質の単層または二重層を有する、脂質層とから構成されているナノ粒子の溶液を、アポリポタンパク質を有する溶液と接触させるステップを含む、方法アッセイ。 - 前記アポリポタンパク質がアポリポタンパク質A−Iである、請求項19に記載の方法。
- 前記溶液が、レシチン:コレステロールアシルトランスフェラーゼも含有する、請求項19に記載の方法。
- 対象において心血管疾患または状態を発症するリスクを決定する方法であって、
(a)生体試料を前記対象から得るステップと、
(b)前記生体試料をナノ構造と接触させるステップであって、
前記ナノ構造は、無機材料を含むナノ構造コアと、前記ナノ構造コアを取り囲み、それに付着している脂質層を含み、内面および外面を有するシェルとを含む、ステップと、
(c)1種または複数のアポリポタンパク質を前記生体試料から隔離するのに十分な時間、前記ナノ構造を前記生体試料とインキュベートするステップと、
(d)形成されたコレステリルエステルの量を検出するステップまたは前記ナノ構造と結合しているLCATの量を検出するステップと、
(e)前記生体試料中の形成されたコレステリルエステルの量または前記ナノ構造と結合しているLCATの量を所定の値と比較するステップであって、前記所定の値は、心血管疾患または状態のいくらかのリスクを有する対象における形成されたコレステリルエステルのレベルまたはLCATの量を表す、ステップと、
(f)前記生体試料中の形成されたコレステリルエステルの量もしくは結合しているLCATの量が前記所定の値であるか、もしくはそれより多い場合、前記対象が、前記心血管疾患もしくは状態を発症するリスクが低いこと、または前記生体試料中の形成されたコレステリルエステルの量もしくは結合しているLCATの量が前記所定の値未満である場合、前記対象が、前記心血管疾患もしくは状態を発症するリスクが高いことを決定するステップと
を含む、方法。 - 対象における心血管疾患または状態の改善に対する1つまたは複数の介入の効果を評価する方法であって、
(a)生体試料を前記対象から得るステップと、
(b)前記生体試料をナノ構造と接触させるステップであって、
前記ナノ構造は、無機材料を含むナノ構造コアと、前記ナノ構造コアを取り囲み、それに付着している脂質層を含み、内面および外面を有するシェルとを含む、ステップと、
(c)1種または複数のアポリポタンパク質を前記生体試料から隔離するのに十分な時間、前記ナノ構造を前記生体試料とインキュベートするステップと、
(d)形成されたコレステリルエステルのレベルを検出するステップと、
(e)前記対象を1つまたは複数の介入にさらし、ステップ(a)〜(d)を反復するステップと、
(f)ステップ(e)において形成されたコレステリルエステルのレベルを、ステップ(d)において形成されたコレステリルエステルのレベルと比較するステップと、
(g)ステップ(e)において形成されたコレステリルエステルの量が、ステップ(d)において形成されたコレステリルエステルのレベルより多い場合、前記1つもしくは複数の介入が前記心血管疾患もしくは状態を改善したこと、またはステップ(e)において形成されたコレステリルエステルのレベルが、ステップ(d)において形成されたコレステリルエステルのレベルであるか、もしくはそれ未満である場合、前記1つもしくは複数の介入が前記心血管疾患もしくは状態を改善しなかったことを決定するステップと
を含む、方法。 - 前記脂質層が脂質二重層である、請求項19、22または23のいずれか一項に記載の方法。
- 前記シェルにおける前記脂質が、1,2−ジパルミトイル−sn−グリセロ−3−ホスホチオエタノール(DPPTE)、ホスファチジルコリン(PC)および1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(DPPC)から構成されている、請求項19、22または23から24のいずれか一項に記載の方法。
- 前記コアが金のコアである、請求項19、22または23から25のいずれか一項に記載の方法。
- 前記金のコアが5〜6nmの直径である、請求項26に記載の方法。
- 前記アポリポタンパク質がアポリポタンパク質AI(アポ−AI)である、請求項19、22または23から27のいずれか一項に記載の方法。
- 前記ナノ構造上に2〜4個のアポ−AI分子が存在する、請求項28に記載の方法。
- 前記シェルの外面に71〜95個の脂質が存在する、請求項19、22または23から29のいずれか一項に記載の方法。
- 前記脂質がリン脂質である、請求項19、22または23から30のいずれか一項に記載の方法。
- 前記ナノ構造が、約1時間、前記生体試料とインキュベートされる、請求項19、22または23から31のいずれか一項に記載の方法。
- 溶液がリン酸緩衝食塩水(PBS)溶液である、請求項19または23から32に記載の方法。
- 溶液が血清である、請求項19または23から33に記載の方法。
- 前記血清が、0.1%、0.5%、1%もしくは10%の濃度に希釈されるか、または全く希釈されない、請求項34に記載の方法。
- 前記血清が1%の濃度に希釈される、請求項34に記載の方法。
- 前記血清がアポBを枯渇させてある、請求項34から36のいずれか一項に記載の方法。
- 前記血清がPEG8000を使用してアポBを枯渇させてある、請求項37に記載の方法。
- 前記対象が哺乳動物である、請求項22から38のいずれか一項に記載の方法。
- 前記対象がヒトである、請求項22から39のいずれか一項に記載の方法。
- ナノ粒子を単離して前記コレステリルエステルのレベルを測定するステップをさらに含む、請求項22から40のいずれか一項に記載の方法。
- 前記コレステリルエステルが比色アッセイによって測定され、前記コレステリルエステルのレベルが前記生体試料中のアポAIと直接相関している、請求項41に記載の方法。
- 前記介入が治療介入である、請求項23から42のいずれか一項に記載の方法。
- 前記介入が運動である、請求項23から42のいずれか一項に記載の方法。
- 前記介入が食事の改善である、請求項23から42のいずれか一項に記載の方法。
- 前記生体試料が血清である、請求項22から45のいずれか一項に記載の方法。
- 前記血清が、0.1%、0.5%、1%、10%の濃度に希釈されるか、または全く希釈されない、請求項46に記載の方法。
- 前記血清が1%の濃度に希釈される、請求項46に記載の方法。
- 前記ナノ構造がLCATをさらに含む、請求項23から48のいずれか一項に記載の方法。
- 前記ナノ構造が1種または複数のコレステロール分子をさらに含む、請求項23から49のいずれか一項に記載の方法。
- in situでナノ構造を合成する方法であって、無機材料を含むナノ構造コアと、前記ナノ構造コアを取り囲み、それに付着している脂質層を含み、内面および外面を有するシェルとを含むナノ構造を、1種または複数のアポリポタンパク質を生体試料から隔離するのに十分な時間、前記生体試料とインキュベートするステップを含む、方法。
- 前記脂質層が脂質二重層である、請求項51に記載の方法。
- 高密度リポタンパク質(HDL)機能を測定するためのキットであって、無機材料を含むナノ構造コアと、前記ナノ構造コアを取り囲み、それに付着している脂質層を含むシェルとを含むナノ構造を含み、前記シェルは、1種または複数のアポリポタンパク質を生体試料から隔離するのに十分な時間、前記生体試料とインキュベートされる内面を有する、キット。
- in situでナノ構造を合成する方法であって、無機コアと、前記無機コアを取り囲み、それに付着しており、内面および/または外面を有する脂質シェルとを含むナノ構造を、生体試料中に存在する1種または複数のアポリポタンパク質を隔離するのに十分な時間、前記生体試料とインキュベートするステップを含む、方法。
- 前記ナノ構造がコレステロールを隔離する、請求項54に記載の方法。
- 治療として前記生体試料を対象に投与するステップをさらに含む、請求項54に記載の方法。
- 対象におけるコレステロールを隔離する方法であって、
無機コアと、前記無機コアを取り囲み、それに付着しており、内面および/または外面を有する脂質シェルとから本質的になるナノ構造を対象に投与するステップであって、前記ナノ構造はin vivoでアポリポタンパク質を隔離することができ、アポリポタンパク質はコレステロールを隔離する、ステップ
を含む、方法。 - 前記脂質シェルがリン脂質から構成されている、請求項57に記載の方法。
- 前記対象が、高コレステロールと関連する疾患を有する、請求項57に記載の方法。
- 高コレステロールと関連する前記疾患が、心血管疾患、アテローム性動脈硬化症、脂質異常症、がん、炎症、タンパク質貯蔵疾患、止血疾患、リウマチ性疾患、または神経疾患からなる群から選択される、請求項59に記載の方法。
- 無機コアと、前記無機コアを取り囲み、それに付着している脂質シェルとから本質的になるナノ構造を含み、ナノ粒子が薬学的に許容される担体中で製剤化されている、治療用または診断用組成物。
- 前記脂質シェルが脂質二重層である、請求項61に記載の組成物。
- 前記脂質シェルが脂質単層である、請求項61に記載の組成物。
- 前記シェルにおける脂質が、1,2−ジパルミトイル−sn−グリセロ−3−ホスホチオエタノール(DPPTE)、ホスファチジルコリン(PC)および1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(DPPC)から構成されている、請求項62または63に記載の組成物。
- 前記コアが金のコアである、請求項61から64のいずれか一項に記載の組成物。
- 前記金のコアが5〜6nmの直径である、請求項65に記載の組成物。
- アポリポタンパク質がアポリポタンパク質AI(アポ−AI)である、請求項61から66のいずれか一項に記載の組成物。
- 前記ナノ構造が、2〜4個のアポ−AI分子を隔離するように構築され、配置される、請求項61から67のいずれか一項に記載の組成物。
- 前記シェルに71〜95個の脂質が存在する、請求項61から68のいずれか一項に記載の組成物。
- 前記脂質がリン脂質である、請求項61から69のいずれか一項に記載の組成物。
- 生体試料中の代謝産物のレベルを評価するためのアッセイであって、
生体試料を、無機材料を含むナノ構造コアと、前記ナノ構造コアを取り囲み、それに付着している脂質層と、アポリポタンパク質を有する生体試料との、脂質の単層または二重層を有するシェルとから構成されているナノ粒子と接触させるステップと、前記ナノ構造を単離するステップと、生体試料中の前記代謝産物の尺度として前記ナノ構造と結合しているアポリポタンパク質の存在を検出するステップとを含む、アッセイ。
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