著者らは、財政支援のために米陸軍医学研究および資材コマンド（＃W81XWH-10-1から0806を付与）と国立衛生研究所（グラント＃U19 AI091031-01とグラント＃1R01GM090280）に感謝したいと思います。 BNは化学と生物工学のBalloun教授職を認識しNLBPは、学際的な工学のウィルキンソン教授職を認めるものです。我々は、ナノ粒子の機能化実験を行うには彼女の援助のためにジュリア·ベラに感謝します。

1。ハイスループット糖鎖合成<ol><li>前のdimannosideの自動合成に、適切に保護された糖供与体、通常はトリクロロと、アクセプターは、主にアルケニルフルオラスアルコールは、ベンチの上に合成される。 </li><li>プログラムはdimannosideの自動合成のために書かれています。基本的な自動化手順の概略図を図2に示されています。プログラムでは、プロモーターを添加する前に、ドナーとアクセプターの混合物を少なくとも30分間攪拌することが保証される。 </li><li>合成ドナー、アクセプターとtrimethylsilyltrifluoromethanesulfonateの溶液をジクロロメタンで作られています。トルエン、ジクロロメタンは、グリコシル化反応のために最も頻繁に使用されています。 </li><li>また、80％メタノール、100％メタノールで一時的な保護基の脱保護するための試薬の溶液を調製する。 </li><li>プログラムの開始前に、確実にそのrelat客室内にIVEの湿度が自動化チャンバ内の30％以上低くなっています。高湿度では、グリコシル化反応のために有害である。 </li><li>プログラムが開始されると、ロボットアームを順次反応バイアルにドナーとアクセプターのソリューションを転送します。その後、混合物を30分間撹拌する。 </li><li> -20のような低い温度は、°Cを達成することができる一般的に混合物にロボットアームの転送trimethylsilyltrifluoromethanesulfonateの0.2から0.3当量を、室温で次が。反応混合物を30分間撹拌する。 </li><li> 30分後、反応を停止し、小アリコートは、反応の進行状況を監視するために削除されます。完了していない場合は、反応を継続することができ、最終的に必要な時間は変更することができます。 </li><li>反応が完了したら、反応混合物を精製するためのC 8 F 17で修飾されたシリカゲルを含むフルオラス固相抽出（FSPE）カートリッジに転送されます。 </li><li>カート尾根​​は、最初の非フルオラス分を取り除くために80％メタノール - 水混合物（8 mL）で洗浄されています。 </li><li>その後、カートリッジが望ましいフルオラス·タグ付きの製品を得るために100％メタノールで洗浄されています。追加の精製が必要な場合は、マシンが停止し、反応生成物（s）は、追加の手段によって精製するために削除することができます。 </li><li>精製サイクルの後、ロボットアームを反応バイアルにナトリウムメトキシドを分配します。反応は、2時間撹拌した。完了していない場合は、この反応は、再び長い期間継続することができ、最終的に必要なプログラムされた時間を変更することができます。 </li><li>反応終了後、製品はFSPEにより精製された後、残留水を除去するために蒸発させ、無水トルエンに溶解に供した。 </li><li>所望の鎖長が標的分子について得られるまで、このサイクルは、（ステップ6から13から）が繰り返されます。 </li><li>自動化から得られた保護された製品は番目です。本発明は、さらに精製し、完全にそのような核磁気共鳴（NMR）分光法などの技法を特徴とする。それは、通常、爆発性の水素ガスとパラジウムが含まれるため、最終的な標的分子の完全な脱保護（残りのすべての保護基の除去）は、ルールとしてオートメーションプラットフォームの外で完了します。最後の脱保護工程は、オートメーション·プラットフォームの外にベンチ上で行った。最初のステップは、カルボン酸の生成したアルデヒドの酸化に続いてフルオラスタグの二重結合のオゾン分解であった。生成物をカラムクロマトグラフィーにより精製した。最後のステップでは、パラジウム触媒の水素化によってベンジルエーテル基の脱保護した。製品は、純粋な最終製品を得るためにパラジウムを取り除くためにセライトパッドに通した。 </li></ol> 2。ハイスループットナノ粒子表面の機能化<ol><li>ハイスループットポリマーの合成とナノ粒子の製造は同じPR以下の実施されotocolとロボットは、最大ピーターセンら 19で説明した設定します。粒子の製造に使用される共重合体システムは、セバシン酸（SA）と1,6 -ビス（ パラ -カルボキシフェノキシ）ヘキサン（CPH）、1,8 -ビス（に基づいています。 パラ -カルボキシフェノキシ）-3,6 -ジオキサオクタン（CPTEG）とCPH。利用ロボットの蒸着装置の概略図は図1bに示されています。 </li><li>ナノ粒子の作製に続いて​​、ナノ粒子のライブラリを使用してチューブを含むホルダはリニアアクチュエータ、ステージに再接続されています。 </li><li>無水物粒子の表面に炭水化物の添付ファイルでは、2つの連続した反応から成るアミン-カルボン酸カップリング反応20が実行されます。 </li><li>最初の反応は、最初のプログラマブルシリンジポンプのシリンジが10当量の（式）（粒子表面の平均モルのカルボン酸濃度の当量）で満たされている1 - エチル-3 - （3 - ジメチルアミノプロピル）-CArbodiimide塩酸塩（EDC）と10当量。水溶液中でエチレンジアミン、第プログラマブルシリンジポンプのシリンジは、12式がロードされている。水溶液中でN-ヒドロキシスクシンイミド（NHS）の。 </li><li> LabVIEWプログラムを使用して、試薬の懸濁液は、ナノ粒子のライブラリ*に堆積される。 </li><li>次に、各サンプルは超音波処理されている（40 Hzで30秒）と、チューブホルダーは、ロボットプラットフォームから切り離されます。 </li><li>ナノ粒子懸濁液を4℃で一定の回転で9時間**をインキュベートする</li><li>反応時間が完了した後、チューブを遠心（5分間12000×gで）と二つの洗浄手順を実行するロボットステーションに返されます。 </li><li>第2シリンジポンプのシリンジを冷たい水で満たされている間に洗浄するために、注射器は空になり、最初のプログラマブルシリンジポンプにロードされたままになります。各チューブの上清は、空の注射器と第2ポンプ預金冷たい水の中に撤回されています。 </li><li>ナノの均質化ステップ2.6で説明したように粒子懸濁液が実行されます。チューブを遠心分離されている（5分間12000×g）で、ステップ2.9で説明されているように第二洗浄工程が実行されます。 </li><li>第二の反応は、2つの成膜工程が使用されます。第一の蒸着工程においては、12式。 EDCの一つのポンプと12式で読み込まれます。 NHSの第二のポンプがロードされます。 </li><li>第2の堆積工程は、10当量が含まれています。第一および第二ポンプ（すなわち、ガラクトース、ラクトースまたはジ-マンノース）***と10当量の第3ポンプ上の特定の糖の。のグリコール酸（コントロールとして使用****）。 </li><li> 4℃ステップ2.6で記述され、一定の回転で9時間インキュベートしたとして、ナノ粒子懸濁液を均質化される</li><li>反応時間が完了した後、手順2.8、2.9および2.10で説明したように、洗浄工程が実行されます。 </li><li>官能ナノ粒子のライブラリは、少なくとも2時間乾燥させ、真空チャンバ内に配置されています。 </li><li>官能性ナノ粒子は、その後の文字です。X線光電子分光法は、それぞれ表面組成と糖の濃度を決定するためのハイスループットフェノール硫酸アッセイによって特徴づけ。電子顕微鏡法と動的光散乱をスキャンすると粒子サイズ、サイズ分布、表面電荷を決定するために利用されています。 </li></ol>注：*沈着量が各チューブに含まれるナノ粒子の質量によって異なります。 第一および第二反応の**反応時間は、最終的な糖濃度を調整するために変更することができます。 ***各糖は、目的のグループに応じて試験管に堆積される。 脱糖は既にナノ粒子表面にさらに添付ファイルを可能にする共有結合し、この分子を持っているので、****炭水化物の結合のため、本研究で用いられる特定の反応については、グリコール酸は、リンカーの制御として使用されます。 3。代表的な結果 FUL図2に示すように、LY保護dimannosideは、自動化プラットフォームを用いて合成した。合成した化合物は、溶媒としてCDCl 3を用いてVXR 400 MHzの分光計で1 H NMRによって特徴付けられた。 NMRスペクトルを図3に示します。 無水物のハイスループットナノ粒子の作製と機能化を利用すると、本明細書に記載のナノ粒子は、dimannose、ラクトースおよびガラクトースの添付ファイルが正常に10、11に行われている。このセットを使用して、最適な反応条件（すなわち、反応温度と時間）は、所望のナノ粒子の機能化と形態を達成するために、同定された。反応は4℃で行った時°Cの代わりに室温で、ナノ粒子凝集の低下をSEMで観察した（データは示さず）。表1は、官能50:50 CPTEGの特性の代表的な結果を示しています。ジ-マンノースのいずれかでCPHナノ粒子または4で合成された乳糖、℃データは、官能基による平均粒子径の小さな増加を示している。非官能化ナノ粒子は約負のゼータ電位を有した。 -20 mVの、官能粒子は、ナノ粒子表面の正常な機能化を示す、正のゼータ電位値を示した。乳糖およびジ-マンノースは、両方の中性糖であるが、エチレンから遊離アミングループは、糖類を添付するために使用リンカは正のゼータ電位の責任があるかもしれませんジアミン。 反応時間は、ナノ粒子の最終的な形態と達成された砂糖の添付ファイルの程度の両方に影響を与える可能性があり、別の変数です。反応時間を調整することにより、ナノ粒子表面に付着し、最終的な糖濃度は、 図4Aに示すように制御することができます。予想通り、50:50 CPTEGの表面上のdimannoseの濃度：CPHは、ナノ粒子と共に増加反応の合計時間および18時間後に最大に達した。 24時間の総反応時間で官能ナノ粒子がマウス骨髄由来の樹状細胞（DC）上のCLRを対象とする彼らの能力を評価するために使用された。フローサイトメトリーは、2 CL受容体（すなわち、CIRE（CD209、DC-SIGN）とマンノース受容体（CD206））非官能で刺激した後、乳糖およびジ-マンノース官能性ナノ粒子（ 図4B）の発現を評価するために使用されていました。細胞は、乳糖およびジ-マンノースの官能ナノ粒子の両方を刺激したときに効果的なターゲティングを示している両方の受容体の高い発現は、得られた。しかし、ジ-マンノース官能粒子が研究された受容体は、このリガンドの特異性を示す発現の高いレベルを示した。 <table border="1"><tbody><tr><td> ナノ粒子の種類 </td><td> 平均粒子径（nm）の </td><td> アベニュー激怒粒子ζ-電位（mV） </td></tr><tr><td>非官能</td><td> 162±43 </td><td> -20±0.6 </td></tr><tr><td>乳糖</td><td> 235±34 </td><td> 26±2.4 </td></tr><tr><td>ジ-マンノース</td><td> 243±32 </td><td> 30±4.2 </td></tr></tbody></table> 表1。ナノ粒子のキャラクタリゼーション。非官能、官能は、準弾性光散乱とゼータ電位の測定によって特徴付けられた。粒度のデータは平均値±3つの独立した実験で収集された動的光散乱データの標準偏差（SD）を表す。ゼータ電位のデータは平均値±3の独立した測定値のSDを表しています。 CPHナノ粒子表面：ゼータ電位の符号の変化は、砂糖が50:50 CPTEGに効率的に結合させたを示しています。 <img alt="図1" src<html> = &quot;/ files/ftp_upload/3967/3967fig1.jpg&quot; /&gt; 図1糖無水物ナノ粒子の機能化と説明したハイスループットなアプローチで設計することができる官能基ナノ粒子のライブラリの例を追求したアプローチの（A）グラフィカルな表現。 （B）自動化された蒸着装置の模式図は、（i）3 NE千ポンプで構成されて粒子の機能化、のために利用し、（ii）ロボットのステージでは、2つのアクチュエータ（Zaber）によって統合された：x方向の運動のためのものと他のy方向の移動のための、3アクチュエータから成る二つの隣接するラック（チューブやキュベットに適した）、各方向に1つ（x、y、およびz）と（ⅲ）第二段階のロボット。ポンプとアクチュエータ5の合計は、直列に接続されています。アクチュエータとポンプは、LabVIEWソフトウェアを使用してコンピュータによって運営されています。この図は、スケールにはありません。arge.jpg &quot;ターゲット=&quot; _blank &quot;&gt;拡大図を表示するには、ここをクリックしてください。 <img alt="図2" src="/files/ftp_upload/3967/3967fig2.jpg" /> 図2の例としてマンノースを使用して、炭水化物の自動反復合成のグラフィカル表現です。 <img alt="図3" src="/files/ftp_upload/3967/3967fig3.jpg" /> 図3。保護dimannosideの1 H NMR。 <img alt="図4" src="/files/ftp_upload/3967/3967fig4.jpg" /> 図4（A） 糖鎖のナノ粒子表面濃度の反応時間の影響。示されたデータでは、50:50 CPTEG：CPHナノ粒子は、異なる反応時間でdimannoseで官能され、反応は4℃で行った二つの独立した官能の実験の平均と標準誤差が示されています。 （B）乳糖およびジ-マンノース官能性ナノ粒子得られた発現と比較してCPHナノ粒子：骨の官能50:50 CPTEGによる刺激後にこれらの2つのマーカーの発現増強によって示されるように骨髄由来樹状細胞上で効果的にターゲットDC-SIGN（CIRE、CD209）とマンノース受容体（CD206）非官能化粒子と。

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+dendritic+cells+with+biomaterials:+developing+the+next+generation+of+vaccines.">Targeting dendritic cells with biomaterials: developing the next generation of vaccines.</a> Trends Immunol. 27, 573-580 (2006).</li><li>Higashi, N., Fujioka, K., Denda-Nagai, K., Hashimoto, S., Nagai, S., Sato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+C-type+lectin+specific+for+galactose/N-acetylgalactosamine+is+an+endocytic+receptor+expressed+on+monocyte-derived+immature+dendritic+cells.">The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.</a> J. Biol. Chem. 277, 20686 (2002).</li><li>Geijtenbeek, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+through+C-type+lectin+receptors:+shaping+immune+responses.">Signalling through C-type lectin receptors: shaping immune responses.</a> Nat. Rev. Immunol. 9, 465-479 (2009).</li><li>Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+oligosaccharide+synthesis.">Automated oligosaccharide synthesis.</a> Chem. Soc. Rev. 37, 19-28 (2008).</li><li>Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Carbohydrate+Synthesis+as+Platform+to+Address+Fundamental+Aspects+of+Glycobiology-Current+Status+and+Future+Challenges.">Automated Carbohydrate Synthesis as Platform to Address Fundamental Aspects of Glycobiology-Current Status and Future Challenges.</a> Carb. Res. 343, 1889-1896 (2008).</li><li>Jaipuri, F. A., Pohl, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+solution-phase+automated+iterative+synthesis:+fluorous-tag+assisted+solution-phase+synthesis+of+linear+and+branched+mannose+oligomers.">Toward solution-phase automated iterative synthesis: fluorous-tag assisted solution-phase synthesis of linear and branched mannose oligomers.</a> Org. Biomol. Chem. 6, 2686-2691 (2008).</li><li>Petersen, L. K., Chavez-Santoscoy, A., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+synthesis+of+and+high-throughput+protein+release+from+polymer+film+and+nanoparticle+libraries.">Combinatorial synthesis of and high-throughput protein release from polymer film and nanoparticle libraries.</a> J. Vis. Exp. , (2011).</li><li>Song, E. -. H., Osanya, A. O., Petersen, C. A., Pohl, N. L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+multivalent+tuberculosis+and+Leishmania-associated+capping+carbohydrates+reveals+structure-dependent+responses+allowing+immune+evasion.">Synthesis of multivalent tuberculosis and Leishmania-associated capping carbohydrates reveals structure-dependent responses allowing immune evasion.</a> J. Am. Chem. Soc. 132, 11428-11430 (2010).</li><li>Hakamori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+glycosylation+in+tumor+and+tumor+associated+carbohydrate+antigens.">Aberrant glycosylation in tumor and tumor associated carbohydrate antigens.</a> Adv. Cancer Res. 59, 257-331 (1989).</li><li>Atherton, T., Sheppard, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid-phase peptide synthesis: a practical approach. , (1999).</li><li>Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+synthesis+machines:+DNA+chemistry+and+the+uses.">Gene synthesis machines: DNA chemistry and the uses.</a> Science. 230, 281-285 (1985).</li><li>Plante, O. J., Palmacci, E. R., Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+solid-+phase+synthesis+of+oligosaccharides.">Automated solid- phase synthesis of oligosaccharides.</a> Science. 291, 1523-1527 (2001).</li><li>Ko, K. -. S., Park, G., Yu, Y., Pohl, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+group-based+colorimetric+monitoring+of+fluorous-phase+and+solid-phase+synthesis+of+oligoglucosamines.">Protecting group-based colorimetric monitoring of fluorous-phase and solid-phase synthesis of oligoglucosamines.</a> Org. Lett. 10, 5381-5384 (2008).</li><li>Pohl, N. L., Chen, X. H. R., Wang, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+solution-phase+oligosaccharide+synthesis+and+carbohydrate+microarrays:+development+of+fluorous-based+tools+for+glycomics.">Automated solution-phase oligosaccharide synthesis and carbohydrate microarrays: development of fluorous-based tools for glycomics.</a> Chemical Glycobiology. , 272-287 (2008).</li></ol>

NLBPは、共同設立者であり、炭水化物会社LuCellaバイオサイエンス株式会社の株式を保持している

免疫細胞に直接ナノ粒子の相互作用に薬剤を標的として炭水化物の効果は以前に10、11に実証されている。私たちの研究室でこれまでの研究では、それによってさらにT細胞の活性化10、11のために重要であるかもしれない免疫細胞の活性を高め、無水物ナノ粒子に接続された特定の糖鎖は抗原提示細胞（APC）上で異なるのCLRをターゲットにすることができることが示されている...

<table border="1"><tbody><tr><td> 名 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>電動XYZステージ：3倍T-LSM050A、軸ごとに50ミリメートルの旅行</td><td> Zaberテクノロジーズ</td><td> T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000シングルシリンジポンプ</td><td>新しい時代のポンプシステム</td><td> NE-1000 </td></tr><tr><td>パイレックス* Vista *のリムレス再利用可能なガラスの培養試験管</td><td>コー​​ニング</td><td> 07-250-125 </td></tr><tr><td> ASW 1000年</td><td> Chemspeedテクノロジーズ</td><td></td></tr><tr><td> LabVIEWの</td><td>ナショナルインスツルメンツ</td><td> 776671から35 </td></tr><tr><td> SGEガスタイトシリンジ、ルアーのLoc </td><td>シグマアルドリッチ</td><td> 509507 </td></tr><tr><td> XL-2000ソニケーター</td><td> Qsonica </td><td> Q55 </td></tr><tr><td>ミニチューブローテーター</td><td>フィッシャー·サイエンティフィック</td><td> 05-450-127 </td></tr></tbody></table>

high-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles

そのような材料設計、ナノテクノロジー、化学、免疫学などの分野を含む学際的なアプローチが合理的に有効なワクチンキャリアを設計するために使用する必要があります。ナノ粒子ベースのプラットフォームでは、ワクチンの免疫原性1を向上させることがワクチン抗原の持続性を延長することができます。いくつかの生分解性ポリマーは、ワクチンの送達媒体1として研究されている、特に、無水物粒子は、安定したタンパク質抗原の徐放性を提供し、抗原提示細胞を活性化し、免疫応答の2-12を変調する能力を実証した。 これらのワクチンキャリアの分子設計、ポリマー特性の合理的な選択と同様に適切な標的剤の取り込みを統合する必要があります。リガンドとの官能粒子を標的とのハイスループット自動化された製造は、広いrを勉強する能力を向上させる強力なツールです。プロパティのアンジュと再現性のワクチン送達デバイスの設計につながるだろう。 10,11,13 C型レクチン受容体（のCLR）が上に存在する炭水化物を認識するパターン認識受容体（PRRS）は、免疫細胞上の特定の受容体により認識されることができる標的リガンドを加え調節することが示されており、合わせて免疫応答病原体の表面。のCLRを介した免疫細胞の刺激は、抗原、さらにT細胞の活性化14,15の後続のプレゼンテーションの強化された内在化が可能になります。したがって、炭水化物分子が免疫応答の研究に重要な役割を果たしているが、これらの生体分子の使用は、しばしば構造的によく定義されており、純粋な炭水化物の可用性の欠如に苦しんでいる。液相反応を反復に基づいて自動化プラットフォームでは、有意に低いbを使用して、これらの合成に挑戦分子の迅速かつ制御された合成を有効にすることができます従来の固相方法16,17よりもブロックの数量をuilding。 ここに我々はこのような中間精製用フルオラス固相抽出とマンノースベースの標的リガンドとして糖鎖の自動化された液相合成のためのプロトコルを報告します。炭水化物ベースの標的化剤を作るための自動化手法の開発後、我々は以前に10を説明したようにLabVIEWで動作し、自動ロボットのセットを採用した無水物ナノ粒子の表面に、その添付ファイルの方法について説明します。炭水化物と表面官能はのCLRの10,11をターゲットに 、マルチパラメトリックシステムに関連付けられている複雑さが大きな価値（ 図1a）になります発掘に製造方法のスループットを増加させる効果を示している。

Watch this Scientific Journal Video about High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles at JoVE.com

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

この記事では、ハイスループット法は、抗原提示細胞上の特定の受容体を標的のさらなる使用のための無水物ナノ粒子の表面に糖鎖とその添付ファイルの合成のために提示されます。

無水物ナノ粒子のハイスループット糖鎖の合成と機能化

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

high-throughput-synthesis-carbohydrates-functionalization

Research

JoVE Journal

Bioengineering

11.3K ビュー.  Iowa State University. この記事では、ハイスループット法は、抗原提示細胞上の特定の受容体を標的のさらなる使用のための無水物ナノ粒子の表面に糖鎖とその添付ファイルの合成のために提示されます。

ビデオ: High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

バイオマテリアルのハイスループット検出用高分子マイクロアレイ

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

生物工学

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

組合せの合成と高分子膜とナノ粒子のライブラリからのハイスループットタンパクリリース

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

マウス肺胞マクロファージを採取し、無水物ナノ粒子により誘導される細胞活性化を評価する

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

ハイスループットマイクロカプセル化シングルセルと複数セル

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

ナノ粒子トラッキング分析とハイスループットフローサイトメトリーによる高分子遺伝子デリバリーナノ粒子の評価

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

アテローム性動脈硬化症の高速検出のための酸化鉄ナノ粒子のマイクロ波駆動型の合成

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

ハイスループットシングルセルの単離および培養のためのマイクロ流体プラットフォーム

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医療機器のための細菌撥ポリマーのハイスループット同定

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

注射の再生療法と高スループットの薬剤のスクリーニングのための 3 D への放射能

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学の活用方法を使用して E 型肝炎ウイルスのナノ粒子の表面化学

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...