著者らは、財政支援のためのONR-MURI賞（NN00014-06-1-1176）と米陸軍医学研究および資材コマンド（グラントNo W81XWH-10-1-0806）を認める。この材料はグラントNo EEC 0552584と0851519の下で国立科学財団によってサポートされた仕事に基づいています。

1。コンビナトリアルライブラリー合成ポリマーは、（高分子化学に変化させる） -ロボットのセットアップについては図1を参照してください。 <ol><li> （濃度= 25 mg / ml）を、適当な溶媒中で、各モノマーを溶解し、10ccのガスタイトシリンジにそれぞれロードします。 </li><li>各注射器の最後に溶媒耐性ルアーロックキャピラリーチューブを取り付けます。 </li><li>シリンジポンプ（新時代プログラマブルシリンジポンプ）に注射器を所定の位置にロックします。 </li><li>開始位置にリニアアクチュエータ（Zaber）を設定します。 </li><li>リングスタンドのクランプを使用して、モノマーの堆積の開始バイアル/ウェルに両方のキャピラリーチューブの端を置きます。 </li><li>そのよく所望の共重合体の組成に応じてそれぞれにポンプ各モノマーの可変ボリュームをLabVIEWプログラムを開始します。これは、目的のボリュームを分配するためにバイアル/ウェルとし、各ポンプにキャピラリーチューブを下げるためにZ軸アクチュエータを指示することにより、プログラムの中で達成されます。ねーXTは、プログラムは次のバイアル/ウェルの位置に移動するために、そのホームポジションとX軸とYアクチュエータに戻り、Zアクチュエータに指示します。各ウェルは、モノマーの所望の体積がそれに振り込まれるまで、これが行われている。 </li><li>モノマーの堆積後、マルチウェルまたはマルチバイアルモノマーライブラリは、予備加熱真空オーブンに転送され、縮重合反応の持続時間のために真空下でインキュベートした。 CPHの場合：SAの合成反応は、180℃で実施する1.5時間のためのC、0.3トルが、これらの反応条件が異なるポリマーシステム間で異なります。 </li></ol> 2。コンビナトリアルブランクおよびタンパク質-ロードされたポリマーナノ粒子及びフィルムライブラリーの作製-ロボットのセットアップについては図1を参照してください。 <ol><li>最初のプログラマブルシリンジポンプ、シリンジ、溶媒（空白のライブラリ）で満たされているか、またはタンパク質を用いた溶媒は、第二注射器で注射しながら（タンパク質 - ロードされたライブラリ）に分散ポンプが空のままになっている。隣接したサンプルホルダー内のナノ粒子製造用のチューブ（非溶媒を別の溶媒の比率は1から100です）、非溶媒で満たされている。映画製作のための空のマルチウェルプレートは、隣接する試料ホルダーにチューブの代わりに利用されています。 </li><li> LabVIEWプログラムを使用し、溶媒は、ライブラリのすべてのポリマーバイアル/ウェルに堆積（濃度= 20 mg / ml）を、1〜5分間インキュベートする。オプションの超音波処理ステップ（40 Hzで30秒）が完了したポリマー溶解を確実に導入することができる。 </li><li>次に、個別のLabVIEWプログラムを開始することによって、サンプルが空の注射器内に引き込まれており、隣接する試料ホルダーに非溶媒（ナノ粒子）または空の井戸（フィルム）のそれに対応する管に堆積した。 </li><li>このプロセスは、個別のポリマーライブラリの各組成物について行われている。 </li><li>ナノ粒子またはフィルム·ライブラリーは、次に溶媒と非溶媒除去（ナノ天秤座のために真空チャンバ内に配置されているRYも）真空ろ過を使用して回復できます。 </li></ol> 3。ハイスループットなタンパク質の放出動態<ol><li> 、ポリマーライブラリーからハイスループットタンパク質の放出動態を調査するための96（2ミリリットル/ウェル）ディープかなって、ポリプロピレン板が変更されているように、上部隣接する列の井戸の壁の1/3（例：AとB、 CとD、EとF、G及びH）をウェルに隣接して削除されます。タンパク質にロードされたフィルムはで作製またはナノ粒子は、高スループットの放出動態の研究を実行するために、このプレートに移さなければなりません。タンパク質にロードされたナノ粒子またはフィルムサンプルは唯一の隣接する列（A、C、E、およびG EX）の1つのウェルに配置する必要があります。詳細については、 図4を参照してください。 </li><li> 96深いかなっリリースプレートにナノ粒子の移転後、粒子を沈降させています。 PBS緩衝液（0.1mMの液、pH7.4）を徐々に（列A、C、EとG）の各サンプルウェルに添加し、その後、それぞれの隣接ウェルに（列A、B、D、F、およびH）の井戸がいっぱいになると、バッファが隣接している井戸の間を流れる解放されるまで。ナノ粒子の場合、このプロセスでは、粒子がサンプルウェルの底部（列A、C、EとG）のままとよく隣接リリースに移す（列A、B、D、Fのないことを確認するために余分な注意を払って行う必要がある、およびH）。いくつかのケースでは、遠心分離（カラムA、C、EとG）サンプルウェルの底部にナノ粒子のサンプルを定位させることが求​​められる。 </li><li>次に、リリースプレートは蓋で密閉し、実験期間中に撹拌しながら所望の温度（ すなわち 37℃）に配置されます。 </li><li>増分の時点（ すなわち 0.04、1、2、3、5、7、9、12、15、19、24、30日）にリリースさ蛍光標識タンパク質の量は、9400台風フラット蛍光スキャナーを用いて定量化されている（ GEヘルスケア）。リリースプレートは、スキャナの表面上に配置され、適切な励起レーザーや発光filteを使用してスキャンRS、およびリリースウェル（列A、B、D、FおよびH）でリリースされたタンパク質の蛍光強度（イメージクワント）定量化される。これは、既知の濃度のタンパク質標準は蛍光消光のためのタンパク質量とアカウンティングを計算するためのウェルプレートに含まれていることが示唆された。 </li></ol> 4。代表的な結果ポリマーライブラリの製作時には、キャラクタリゼーションはこのコンビナトリアル手法1,7,8,11を検証するために、1 H NMR、GPC、およびFTIRを用いて行われています。 10,000〜20,000の分子量の範囲は、g / molで、多分散指数の範囲は1.5から3.0で、化学組成は、正確かつ無水物の合成12月15日 、従来の方法との合意であることが示されている。同様に、ナノ粒子のライブラリのSEM画像は、従来から製造されたナノ粒子のその2と同様の表面形態、サイズ、サイズ分布を示した。タンパク質リリースkineti無水物ナノ粒子またはフィルムからのCSは、以前説明したように修正された1ウェルプレート中で行われる。結果は、タンパク質のローディングおよび高分子化学（ 図2及び図3）1,12,14,16依存バーストの有無にかかわらず近似ゼロ次の放出を明らかにした。 <img alt="図1" src="/files/ftp_upload/3882/3882fig1.jpg" /> 図1コンビナトリアル高分子膜とナノ粒子の製造装置。 <img alt="図2" src="/files/ftp_upload/3882/3882fig2.jpg" /> 図2 CPHからアルブミンテキサスレッドウシ血清（TRBSA）のハイスループットリリース：SAのポリマーナノ粒子のライブラリ。 CPH-リッチポリマーの化学的性質は最も遅いを離すのに対し、SA-リッチポリマーの化学的性質は、最も急速にTRBSAカプセル化解除してください。エラーバーは標準偏差を表すると、n = 4である。ピーターセンらの許可を得て転載。ら 1。著作権2010 American Chemical Societyの。 <img alt="図3" src="/files/ftp_upload/3882/3882fig3.jpg" /> 図3 CPTEGからアルブミンテキサスレッドウシ血清のハイスループットリリース（TRBSA）：CPHポリマーフィルムライブラリ。 CPH-リッチポリマーの化学的性質は最も遅いを離す一方CPTEG富む高分子化学は、最も急速にTRBSAカプセル化解除してください。エラーバーは標準偏差、n = 3の場合を表しています。 <img alt="図4" src="/files/ftp_upload/3882/3882fig4.jpg" /> 図4： &quot;の前に&quot;隣接する二つの井戸を示す画像と96ディープかなっポリプロピレン板で&quot;の後に&quot;修正。右側の &quot;後&quot;修正画像も、カプセル化された蛍光分子を有するポリマーフィルム（左の井戸の底）を添加することを示して緩衝溶液中に2ウェル間で放出される。放出された蛍光分子は、その後よく右側で検出される。

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特別な利害関係は宣言されません。

必要な合成条件と合成されるポリマーのガラス転移温度（Tg s）の知識は、ライブラリの製作のために不可欠です。 T gはsが室温以下である場合には、ナノ粒子の製造工程は、ポリマーの Tg以下に制御された温度環境で実施する必要があるかもしれません。さらに、注意が高温や溶剤に接触するすべての機器がそれらの条件を処理するために適合しなければならないこ?...

<table border="1"><tbody><tr><td> 名前 </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、軸当たり50ミリメートル旅行</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>新時代のヒートポンプシステム</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培養管</td><td>コー​​ニング</td><td> 07-250-125 </td></tr><tr><td>ガラスキュベット</td><td>科学的な戦略</td><td> G102 </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のガスタイトシリンジ、ルアーLocの</td><td>シグマアルドリッチ</td><td> 509507 </td></tr><tr><td> U96ディープウェルプレート1.3ミリリットルと2.0ミリリットル</td><td>サーモサイエンティフィック：ヌンク</td><td> 278743 </td></tr><tr><td>まあキャップマット</td><td>サーモサイエンティフィック：ヌンク</td><td> 276000 </td></tr><tr><td>台風9400 </td><td> GEヘルスケア</td><td> 63-0055-79 </td></tr><tr><td>ワットマングレード50円90ミリメートル</td><td>ワットマン</td><td> 1450-090 </td></tr></tbody></table>

combinatorial synthesis of and high-throughput protein release from polymer film and nanoparticle libraries

無水物は、優れた生体適合性および薬物送達機能を備えた生体材料の一種である。彼らは従来のワンサンプルを1つずつ合成技術で広く研究されてきたが、より最近のハイスループットアプローチがポリアンヒドリド1の大きなライブラリーの合成および試験を可能に開発されました。これにより、より効率的な最適化と薬剤およびワクチン送達用途のためのこれらの生体材料の設計プロセスを容易にするでしょう。この作品の方法は、生分解性ポリ無水物膜およびナノ粒子のライブラリーのコンビナトリアル合成し、これらのライブラリーからのタンパク質放出のハイスループット検出について説明します。このロボット操作方法（ 図1）では、リニアアクチュエータ、シリンジポンプは、ユーザーエラーを排除し、ハンズフリーで自動化されたプロトコルを使用可能には、LabVIEW、によって制御されます。さらに、この方法は、赤、ミクロスケールのポリマーライブラリーの迅速な製造を可能に多変量ポリマー系の創出をもたらしながら、バッチサイズをucing。ポリマー合成にこのコンビナトリアルアプローチは、それが従来の1ポリマーを合成するのにかかる時間に相当する量で、15の異なるポリマーまで​​の合成を促進します。また、コンビナトリアル高分子ライブラリ（ナノ粒子）の非溶媒にまたは真空乾燥（フィルム用）による溶媒や降水量のフィルムやポリマーライブラリが解散した場合においてナノ粒子を含む空白またはタンパク質にロードされた幾何学的形状に加工することができます。以前に1記載の高分子ライブラリに蛍光標識タンパク質をロードする際、タンパク質の放出動態を蛍光ベースの検出方法（ 図2および図3）を用いた高スループットで評価することができる。この組み合わせのプラットフォームは、従来の方法で検証されています2、ポリ無水物膜およびナノ粒子のライブラリはと特徴づけられている<sup&gt; 1のH-NMRとFTIR。 インビトロ細胞毒性、サイトカイン産生、表面マーカーの発現、接着、増殖、分化に ;、ライブラリはタンパク質放出動態、安定性および抗原性のためにスクリーニングされており、 生体内で生体内分布と粘膜付着1〜11インチここに開発されたコンビナトリアル手法は、順番に、生体材料の性能を最適化するために、in vitroおよび in vivo でスクリーニングすることができるタンパク質にロードされたナノ粒子及びフィルム·ライブラリーのハイスループットポリマー合成および製造を可能にします。

Watch this Scientific Journal Video about Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries at JoVE.com

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

この方法では、生分解性ポリ無水物膜およびナノ粒子のライブラリのコンビナトリアル合成し、これらのライブラリーからのタンパク質放出のハイスループット検出について説明します。

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 ...

combinatorial-synthesis-high-throughput-protein-release-from-polymer

Research

JoVE Journal

Bioengineering

10.3K ビュー.  Iowa State University. この方法では、生分解性ポリ無水物膜およびナノ粒子のライブラリのコンビナトリアル合成し、これらのライブラリーからのタンパク質放出のハイスループット検出について説明します。

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

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

合成、組立、および単分子膜のキャラクタリゼーションは、タンパク質単分子層の電気化学のための金ナノ粒子膜を保護

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

生物工学

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

キトサンベース、レーザー活性薄膜外科用接着剤、 &#39;SurgiLux&#39;：準備とデモ

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

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 ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

マイクロ流体プラットフォームを使用した高スループットのタンパク質発現ジェネレータ

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

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 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 ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

パルスレーザー励起時の金ナノ粒子の統合フォト応答性リポソームとそのマイクロバブルキャビテーションの測定の合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

のフィルム押出<em&gt;ハマナabyssinica</em&gt; /小麦グルテンブレンド

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

磁気および感熱性ポリ（<em&gt; N</em&gt; - イソプロピルアクリルアミド）ベースのミクロゲル

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...