The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

1. PCPS粒子の準備<ol><li> 2時間、95℃での過酸化水素の30％溶液中の非官能化多孔質シリコン粒子を酸化する。緩やかに攪拌しながら65℃で2日間、イソプロピルアルコール2％（3-アミノプロピル）トリエトキシシランの溶液中で酸化された粒子をアミノ化。 </li><li>ペレットを懸濁する短時間の超音波処理を使用して、18,800×gで30分間溶液を遠心し、イソプロピルアルコールとエタノールで3回二粒子を洗浄する。ステップ1.3および1.4を実行する際にエタノール溶液中の粒子のままにしておきます。 </li><li>アイソトン希釈液10mlに粒子懸濁液（ 例えば 、10μl）を既知の容量を追加し、ストック溶液中の粒子の濃度を決定するために、粒子計数分析装置の粒子を数える。 </li><li> （3-ジメチルアミノプロピル） - - N &#39;エチルカルボジイミド塩酸塩（EDC、0.1ナノモル）/ N NとL-アルギニン（0.1ナノモル）の酸基を活性</em&gt;穏やかに撹拌しながら室温で2時間、エタノール20ml中のヒドロキシスクシンイミド（NHS、0.1ナノモル）。 </li><li>簡単に言えば、粒子原液を超音波処理し、L-アルギニン溶液に10億粒子を添加し、緩やかに攪拌しながら室温で18時間反応を残す。 </li><li>で4時間エタノール20mlにEDC（0.1ナノモル）/ NHS（0.1ナノモル）と（tert-ブトキシカルボニル）-L-アスパラギン酸カルボニル（Boc-ASP-OH、1ナノモル） - Nの最初のアスパラギン酸グループをアクティブ化緩やかに攪拌しながら4℃。 </li><li> 10ミリリットルのエタノール中50 mgのポリエチレンイミンを溶解及びBoc-ASP-OH混合物にソリューションを追加します。反応を穏やかに撹拌しながら室温で24時間進行しましょう​​。 </li><li>緩やかに攪拌しながら6時間4℃でEDC（0.1ナノモル）/ NHS（0.1ナノモル）とのBoc-ASP-OH / PEI溶液の第二アスパラギン酸基をアクティブにします。 </li><li> PCPS粒子を得るためには、ステップ1.8からのBoc-ASP-OH / PEI溶液にステップ1.5から粒子溶液を追加。反応は穏やかST RTで18時間進行させる<html> irring。 </li><li>ペレットを懸濁する短時間の超音波処理を使用して、18,800×gで30分間溶液を遠心し、エタノールで粒子溶液を3回洗浄する。 </li></ol> 2. PCPS粒子特性評価<ol><li>走査型電子顕微鏡（SEM）を用いて、粒子のサイズを測定する。 <ol><li>きれいなシリカのSEMサンプルスタブ上の粒子懸濁液を一滴（エタノール中万個/μl）を配置し、真空下、室温で乾燥させてください。 </li><li>レンズ内検出器を用いての3-5mm作業距離8 15kVでSEM画像を測定する。 </li></ol></li><li>粒子アナライザーシステムを用いて、粒子のゼータ電位を測定する。 <ol><li> 1ミリリットルの10mMリン酸緩衝液（pH7.4）で、粒子懸濁液10μl（エタノール中万個/μl）を混ぜる。 </li><li>折り畳まれた毛細管細胞にサンプルをロードし、製造者の指示に従ってゼータ電位を測定する。 </li></ol></li></ol>ル &quot;&gt; 3。PCPS粒子へのsiRNAのロード中<ol><li>真空O / Nの下（PCPS粒子作製工程1.9から）PCPS粒子を乾燥させます。 </li><li>乾燥したPCPS粒子と超音波処理を簡単にヌクレアーゼフリー水（20μL）中のsiRNA（4μgの）を追加します。 siRNAの比率に次粒子を使用してください：2×10 5個/ 0.2μgのsiRNAを、4×10 5個/ 0.2μgのsiRNAを、6×10 5個/ 0.2μgのsiRNAを、8×10 5個/ 0.2μgのsiRNAを、10× 10 5個/ 0.2μgのsiRNAおよび12×10 5個/ 0.2μgのsiRNAを。 </li><li> siRNAは、粒子への結合を可能にするためにシェーカー上で4℃で3時間（1,000 rpm）でインキュベートする。 </li></ol>のsiRNA / PCPSパーティクル比の4の最適化<ol><li> siRNAの比が異なる粒子（PCPS粒子へのsiRNAのロードを参照）PCPS /コントロールsiRNA粒子の20μlにDNAローディング色素を追加します。 </li><li> SAMのロードDNAゲル染色を含む2％アガロースゲルに公正妥当。 </li><li>ランニング緩衝液中で20分間120 Vの定電圧で電気泳動を行います。 </li><li>画像取得と解析ソフトウェアでゲルを分析します。 </li></ol> PCPS粒子からのsiRNAの5リリース<ol><li>ドデシル硫酸ナトリウム（SDS、2％）中のsiRNA比（ステップ3を参照）とは異なる粒子とPCPS /対照siRNA粒子の20μLを混合し、室温で1時間放置する。 </li><li>サンプルにDNAローディング色素を追加します。 </li><li> DNAゲル染色を含む2％アガロースゲルにロードしたサンプル。 </li><li> DNAの電気泳動装置と電源を用いてランニング緩衝液中で20分間、120 Vの定電圧で電気泳動を行う。 </li><li>画像取得と解析ソフトウェアでゲルを分析します。 </li></ol> PCPS粒子の6。共焦点顕微鏡<ol><li> PCPS /蛍光コントロールsiRNA粒子（10×10 5個/ 0の5μlを添加する。ガラスカバースリップへの2μgのsiRNA / 20μL）。 </li><li>共焦点顕微鏡により粒子層を可視化。 </li></ol>のArg-PEI /コントロールsiRNAナノ粒子の7キャラ<ol><li>シリコン材料を分解およびArg-PEI /対照siRNAナノ粒子を形成するために、リン酸緩衝生理食塩水100μlにPCPS / siRNAの粒子（10×10 6 PCPS粒子/2μgのsiRNA）を追加し、37℃で（1,000 rpm）で振る2日。 </li><li> 18800×gで30分間サンプルを遠心し、上清を収集します。 </li><li>動的光散乱（DLS）による形成のArg-PEI / siRNAのナノ粒子のサイズを測定する。 <ol><li> 1ミリリットルの10mMリン酸緩衝液（pH7.4）で10μlの上清を混合し、プラスチック製キュベットに配置します。 </li><li>製造業者の説明書に従って粒子アナライザーシステムを用いて粒子のサイズを測定する。 </li></ol></li><li>形成されたのArg / PEI-siRNAのサイズと形態を決定します原子間力顕微鏡のナノ粒子。 <ol><li>シリコンウエハー上に上清と場所の10μlのを取る。 </li><li>原子間力顕微鏡（AFM）を有する粒子を可視化する。 </li></ol></li></ol> 8.細胞培養<ol><li>細胞培養培地中で培養MDA-MB-231ヒト乳癌細胞を、10％ウシ胎児血清および1％ペニシリン-ストレプトマイシン、5％CO 2、湿度95％及び37℃で補充した。 </li></ol> PCPS粒子との生細胞の9共焦点顕微鏡<ol><li> 2ウェル培養プレート中で細胞を24時間/ウェルの3×10 5細胞の播種密度でスライドする。 </li><li>細胞に蛍光対照siRNAを搭載しPCPS粒子（10×10 5個/ siRNAの比に0.2μgの粒子、50 nMのsiRNA）を追加します。 </li><li> 12時間後の粒子博覧会のために（チャンバに付属、5％CO 2、湿度95％、37℃）、共焦点顕微鏡で細胞の映画を記録確認してください。 </li></ol> PCPS粒子との固定された細胞の10共焦点顕微鏡<ol><li> 2ウェル培養プレート中で細胞を24時間/ウェルの3×10 5細胞の播種密度でスライドする。 </li><li>細胞に蛍光対照siRNA（10×10 5個/ siRNAの比に0.2μgの粒子、50 nMのsiRNA）をを搭載しPCPS粒子を追加し、1日、7日間と10日間インキュベートする。 </li><li>リン酸緩衝生理食塩水で細胞を2回洗浄し、その後、10分間、4％パラホルムアルデヒド溶液で固定します。 </li><li>リン酸緩衝生理食塩水で細胞を洗浄。 </li><li> 10分間0.1％のオクチルフェノールエトキシレートと細胞を透過した後、それらをリン酸緩衝生理食塩水で三回洗浄する。 </li><li>穏やかに攪拌しながら室温で10分間、リン酸緩衝生理食塩水、ウシ血清（10 mg / ml）でのアルブミンで細胞をブロックする。 </li><li>繊維状アクチンを可視化するために、蛍光標識したファロイジン（1μL/ 40μlの細胞をインキュベート穏やかに攪拌しながら室温で20分間この溶液）をブロックした後、リン酸緩衝生理食塩水で洗う。 </li><li>フレームからスライドを削除し、核を可視化するために4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）で抗退色試薬を加える。 </li><li>上にカバーガラスを追加し、共焦点顕微鏡を用いた細胞の画像を撮影する。 </li></ol> PCPS /蛍光コントロールsiRNA粒子を用いた細胞の11。フローサイトメトリー<ol><li> 24時間/ウェルの3×10 5細胞の播種密度で、6ウェルプレート中の細胞をプレート。 </li><li>細胞に蛍光対照siRNAを搭載しPCPS粒子（10×10 5個/ siRNAの比に0.2μgの粒子、50 nMのsiRNA）を追加し、24時間インキュベートする。 </li><li>リン酸緩衝生理食塩水で細胞を洗浄し、細胞スクレーパーでそれらをこすり。 </li><li>分析前に、2％ウシ胎児血清を含むリン酸緩衝生理食塩水で細胞を保存する。ネガティブコントロールとして未処理細胞を使用してください。 </li><li>流れCYを実行しますtometry。 </li></ol> PCPS粒子およびPCPS /コントロールsiRNA粒子を用いた細胞の12細胞の生存率<ol><li> 24時間/ウェルの3×10 3個の細胞密度で96ウェルプレート中の細胞をプレート。 </li><li> PCPS粒子で細胞を処理（1.5×10 5 /ウェルおよび6×10 5 /ウェル）またはPCPS /コントロールsiRNA粒子48（10×10 5個/ siRNAの比に0.2μgの粒子、10および100nMのsiRNA）時間と72時間。対照としてリン酸緩衝生理食塩水（追加粒子と同じ体積）で処理した未処理細胞および細胞を使用する。三連で各サンプルをアッセイ。 </li><li>製造業者の説明書に従って細胞増殖アッセイを行う。 </li><li>平均±標準偏差としてデータを表す。 </li></ol> PCPS / ATM変異のsiRNA粒子を用いた細胞の13ウエスタンブロット<ol><li>細胞密度で6ウェルプレート中の細胞をプレートに2×10 5細胞/ワット24時間のエル。 </li><li> PCPS /コントロールsiRNA（50 nM）の粒子またはPCPS / ATM siRNAを72時間（50 nM）の粒子で細胞をインキュベートする。コントロールとして未処理細胞を使用してください。 </li><li>溶解プロテアーゼ阻害剤カクテルを補充したタンパク質抽出試薬を用いて細胞。 </li><li> 14000×gで10分間の細胞溶解物を遠心し、上清を回収する。 </li><li>製造業者の説明書に従ってタンパク質定量アッセイを用いてタンパク質濃度を決定する。 </li><li> （5μlの2-メルカプトエタノール/ mlのバッファーで）サンプルローディングバッファーをサンプルに添加し、99℃で6分間、それらを加熱する。 </li><li>ランニング緩衝液中で12％SDSポリアクリルアミドゲル中のタンパク質サンプル（20μgの/μl）をロードし、電気泳動装置と電源を用いてポリアクリルアミドゲル電気泳動（1時間、120 V）を行う。 </li><li>電気泳動装置を用いてニトロセルロース膜（1時間、100 V）を（20％メタノール）転写バッファーでゲルを転送し電源。 </li><li> 1時間5％ドライミルクで膜をブロックします。 </li><li>千希釈O / N：1で（ステップ13.9）からブロッキング溶液中で（ウサギから）ATM一次抗体で膜をインキュベートする。 </li><li> 1時間2500希釈：1、0.1％ポリエチレングリコールソルビタンモノラウレートを含有するリン酸緩衝生理食塩水でメンブレンを洗浄した後（ステップ13.9）から、ブロッキング溶液中で二次抗体（抗ウサギ）と一緒にインキュベートする。 </li><li> 0.1％ポリエチレングリコールソルビタンモノラウレートを含有するリン酸緩衝生理食塩水でメンブレンを洗浄し、画像取得及び解析ソフトウェアを用いてウェスタンブロット検出試薬を有するタンパク質バンドを検出する。 </li><li>二次抗体（抗マウス1：4000希釈）：ローディングコントロールのために、膜と繰り返し（10,000希釈マウスから、1）βアクチン一次抗体を使用して13.9から13.12ステップ洗う。 </li></ol>

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The authors declare that they have no competing financial interests.

このプロトコルは、細胞へのsiRNAの配信の成功とトランスフェクションのための方法を説明します。具体的には、siRNAの送達は、カチオン官能化されたpSi粒子からなる多機能プラットフォームを使用することによって達成される。様々な癌遺伝子は高い特異性で標的化することができるようにするsiRNA療法の使用は、大きな可能性、 例えば 、癌治療を有する。したがって、siRNAの治療の...

低分子干渉RNA（siRNA）は、遺伝子の発現を抑制することができる二本鎖RNA分子である。近年では、siRNAは、臨床応用1-5将来の使用のための治療の可能性を示してbiodrugsの新世代として開発されている。しかし、siRNAの治療の成功の実装が原因ヌクレアーゼ、貧しい細胞内取り込み、低トランスフェクション効率およびエンドソーム/リソソーム5から非効率的なリリースによる分解に、かなりの課題である。これらの障害の多くは、安全かつ効率的に病変組織にsiRNAを送達することができ、配信プラットフォームの開発によって克服することができる。ウイルスのキャリアと比較して、非ウイルスプラットフォームは、安全性、低コスト、調整の容易さなどのいくつかの利点を提供する。特に、このようなポリマーおよび脂質などの陽イオン性ナノ粒子は、siRNA送達のために有用3が証明されている。 以前、我々は、円盤状のDRを開発したUG送達システム、多段ベクトル（MSV）と呼ばれる。このプラットフォームは、1つの車両が別のから解放されているシーケンシャルな段階に基づいている。第二段目の車両が薬物または造影剤6,7を装填したナノ粒子である一方、第段階車両は、生分解性の多孔質シリコン（のpSi）から作られたマイクロ粒子である。 Siは8を分解するのpSi材料に埋め込 ​​まれたナノ粒子は、徐々に放出される。 Si粒子を使用する利点は、形態および表面特性を容易に最適な生体内分布および薬物放出を達成するように調整することができることである。最近、腫瘍組織へのsiRNAのリポソームの送達のためのMSVプラットホームの使用の成功は、卵巣癌および乳癌のマウスモデル9、10に示した。 本研究では、MSVプラットフォームのプリンシパルに基づいてsiRNAのための普遍的なデリバリーシステムを作製した。この送達システムの有効性は、以前に我々を実証されている異なるsiRNA分子11をる。システムは、ポリエチレンイミン（PEI）およびアルギニン（Argで）でグラフトされたpSiからなる、ポリカチオン官能化多孔質シリコン（PCPS）担体である。以前に11 demonstartedとしてArgおよびのpSiが、PEIの毒性を減少するために役立つことができるが、PEIは、siRNAとの静電相互作用を形成するのを助けることができる。のpSi微粒子がsiRNAの保護と持続的な放出を可能にしながら、また、PEIの存在は、細胞内取り込みとエンドソーム脱出を支援することができます。のpSi粒子が徐々に異なる形態および狭いサイズ分布11を有しているのArg-PEI / siRNAのナノ粒子（ 図1）の形成をもたらす、生理学的条件下で分解する。 PCPS / siRNAのシステムの安定性に関する詳細については、シェンらの研究を参照してください。11。第二段階のナノ粒子は最初にpresaの複数形ではないので、このPCPSプラットフォームは、従来のMSVとは異なるntのキャリアであるが、第一段階キャリヤ11は、12を劣化させるような時間をかけて形成されている。PCPSシステムのsiRNA積載効率、細胞毒性および遺伝子サイレンシング効率は、 インビトロで評価されている。トランスフェクション効率は、DNA修復10に関与する突然変異した毛細血管拡張性運動失調（ATM）癌遺伝子に対するsiRNAを用いて測定した。以前に、ATMの抑制は乳癌モデル10における腫瘍増殖を減少させることが示されている。

<table border="0" cellpadding="0" cellspacing="0" width="1812">
	<tbody>
		<tr height="55">
			<td height="55" style="height:55px;width:605px;">Name of the Material/Equipment</td>
			<td style="width:421px;">Company/Institution</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:653px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Polyethylenimine (PEI), branched</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408727</td>
			<td style="width:653px;">Average molecular weight ~25,000 Da</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">L-Arginine</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A5006</td>
			<td style="width:653px;">Reagent grade, &#8805;98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Boc-Asp-OH</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408-468</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">(3-&#8203;Aminopropyl)&#8203;triethoxysilane</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">440140</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Hydrogen peroxide solution</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">216763</td>
			<td style="width:653px;">30&#160;wt. % in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Sulfuric acid</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">339741</td>
			<td style="width:653px;">100.00%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Isopropyl alcohol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">W292907</td>
			<td style="width:653px;">&#8805;99.7%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Ethanol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">459844</td>
			<td>&#8805;99.5%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>03449</td>
			<td>&#8805;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-10G</td>
			<td>Blocking agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Ataxia-telangiectasia mutated siRNA&#160;</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Designed in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Tris Acetate-EDTA buffer</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">T9650</td>
			<td style="width:653px;">For DNA agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Penicillin-Streptomycin</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P4333</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fetal Bovine Serum</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>F2442</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">TWEEN 20</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:653px;">Polyethylene glycol sorbitan monolaurate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">2-Mercaptoethanol</td>
			<td style="width:421px;">Sigma Aldrich</td>
			<td>M6250</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium dodecyl sulfate</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>L3771</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate monobasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71496</td>
			<td style="width:653px;">For making phosphate buffer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate dibasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71640</td>
			<td style="width:653px;">For making phosphate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A4416</td>
			<td style="width:653px;">Secondary antibody (anti-mouse) for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">N-Hydroxysuccinimide (NHS)</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">130672</td>
			<td style="width:653px;">98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid</td>
			<td style="width:421px;">Greiner Bio-One</td>
			<td style="width:133px;">655182</td>
			<td style="width:653px;">96-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">10X Tris-Glycine Liquid</td>
			<td style="width:421px;">Li-Cor&#160;</td>
			<td style="width:133px;">928-40010</td>
			<td style="width:653px;">Transfer buffer for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Paraformaldehyde solution 4% in PBS&#160;</td>
			<td style="width:421px;">Santa Cruz</td>
			<td>Sc-281692</td>
			<td style="width:653px;">Fixation of cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)</td>
			<td style="width:421px;">Promega</td>
			<td style="width:133px;">G5421</td>
			<td style="width:653px;">Proliferation assay</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Phosphate buffered saline</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP399-500</td>
			<td style="width:653px;">10 X Solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Corning cellgro Dulbecco&#39;s Modification of Eagle&#39;s (Mod.)</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">MT-15-017-CM</td>
			<td style="width:653px;">Cell culture media, 1X solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Triton X-100</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>AC21568-2500</td>
			<td style="width:653px;">Octyl phenol ethoxylate, permeabilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Cover glass</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>12-530C&#160;</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Methanol</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td>A412-1</td>
			<td style="width:653px;">For Western blot transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Plastic Cuvettes</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">14-377-010&#160;</td>
			<td style="width:653px;">For size measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Molecular BioProducts RNase AWAY Surface Decontaminant</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">14-754-34</td>
			<td style="width:653px;">Spray for removing RNAse contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Agarose</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP165-25</td>
			<td style="width:653px;">Low melting point, for running RNA samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI&#160;</td>
			<td style="width:421px;">Invitrogen</td>
			<td>P36935</td>
			<td style="width:653px;">Antifade reagent with DAPI, nucelus detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">A12379</td>
			<td style="width:653px;">Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">S33102</td>
			<td style="width:653px;">Visualization of RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Negative Control siRNA</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1022076</td>
			<td style="width:653px;">Control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">AllStars Neg. siRNA AF 555</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1027294</td>
			<td style="width:653px;">Fluorescent control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Cell scraper</td>
			<td style="width:421px;">Celltreat</td>
			<td>229310</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite Multidishes and Microwell Plates</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>130184</td>
			<td style="width:653px;">6-well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce LDS Sample Loading Buffer (4X)&#160;</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>84788</td>
			<td style="width:653px;">Sample loading buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Pierce BCS Protein Assay Kit</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">23227</td>
			<td style="width:653px;">Protein quantification assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Halt Protease Inhibitor Single-Use Cocktails (100X)</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">78430</td>
			<td style="width:653px;">Protease inhibitor cocktail, use at 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-PER Mammalian Protein Extraction Reagent</td>
			<td style="width:421px;">Thermo Scientific&#160;</td>
			<td style="width:133px;">78501</td>
			<td style="width:653px;">Protein extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sorvall Legend Micro 21R</td>
			<td>Thermo Scientific</td>
			<td>75002440</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Beta Actin Antibody&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>MA1-91399</td>
			<td>&#946;-actin primary antibody (from mouse) for western blor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6X TriTrack DNA Loading Dye</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>R1161</td>
			<td style="width:653px;">DNA loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-Free Water&#160;</td>
			<td style="width:421px;">Life Technologies</td>
			<td>AM9938</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Fat Dry Milk</td>
			<td style="width:421px;">Lab Scientific</td>
			<td>M0841</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well BD Falcon culture slides&#160;</td>
			<td style="width:421px;">BD Biosciences</td>
			<td>354102</td>
			<td style="width:653px;">2-well culture slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Western blot detection reagent.&#160;</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td style="width:133px;">RPN2106</td>
			<td style="width:653px;">Western blot detection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BA Membranes</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td>10402096</td>
			<td style="width:653px;">Nitrocellulose membrane for Wester blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATM (D2E2) Rabbit mAb</td>
			<td style="width:421px;">Cell Signaling</td>
			<td>2873S</td>
			<td style="width:653px;">ATM primary antibody (from rabbit) for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:421px;">Cell Signaling&#160;</td>
			<td>7074</td>
			<td style="width:653px;">Secondary antibody (anti-rabbit) for Western blot&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Folded capillary cells</td>
			<td style="width:421px;">Malvern&#160;</td>
			<td style="width:133px;">DTS 1061</td>
			<td style="width:653px;">For zeta potentail measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">MDA-MB-231 cell line</td>
			<td style="width:421px;">ATCC</td>
			<td style="width:133px;">HTB-26</td>
			<td style="width:653px;">Mammary Gland/Breast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">12% Mini-PROTEAN TGX Gel</td>
			<td>Bio-rad</td>
			<td>456-1043&#160;</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biorad PowerPac HC</td>
			<td>Bio-rad</td>
			<td>164-5052</td>
			<td>Power supply for electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Tris/Glycine/SDS Buffer</td>
			<td>Bio-rad</td>
			<td>161-0732</td>
			<td>Running buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wide Mini-Sub Cell GT Cell</td>
			<td>Bio-rad</td>
			<td>170-4405</td>
			<td>Electrophoresis equipment for DNA agarose gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra cell</td>
			<td>Bio-rad</td>
			<td>165-8000</td>
			<td>Electrophoresis equipment for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChemiDoc XRS+ System with Image Lab Software</td>
			<td>Bio-rad</td>
			<td>170-8265</td>
			<td>Image acquisition and analysis software for gels and blots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#34; (10cm) dia., 5x7mm diced Silicon Wafer</td>
			<td>Ted Pella&#160;</td>
			<td>16007</td>
			<td>Silicon waferfor scanning electron microscopy and atomic force microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer R</td>
			<td>Eppendorf</td>
			<td>22670107</td>
			<td>Shaker&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoton II diluent</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td>Isoton diluent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td>Particle counting analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Non-functionalized porous silicon particles&#160;</td>
			<td style="width:421px;">Microelectronics Research Center, University of Texas at Austin</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Dicoidal shape. 2.6 &#956;m (diameter) x 0.7 &#956;m (hight), provided in isopropyl alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Zetasizer Nano ZS</td>
			<td style="width:421px;">Malvern</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle analyzer system for size and zeta potential&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Scanning Electron Microscope</td>
			<td style="width:421px;">FEI</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Atomic Force Microscope</td>
			<td style="width:421px;">Bruker</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fluo ViewTM 1000 Confocal Microscope</td>
			<td style="width:421px;">Olympus</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Visualization of fixed and live cells</td>
		</tr>
	</tbody>
</table>

porous silicon microparticles for delivery of sirna therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

このプロトコルは、安全で効率的なsiRNAトランスフェクションのための非ウイルス送達システムの使用を記載する。 SEMの結果は、PCPS粒子の形状は円筒形であり、2.6ミクロン（ 図2A）の直径を有することが明らかになった。粒子は、正にそれによって負に帯電したヌクレオチドと結合静電が可能約8.21のゼータ電位（ 図2B）が充電される。 PCPS粒子の異なる層の共焦点?...

Watch this Scientific Journal Video about Porous Silicon Microparticles for Delivery of siRNA Therapeutics at JoVE.com

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

配信は、低分子干渉RNA（siRNA）の治療的実施のための主要な課題である。このプロトコルは、アルギニン及びポリエチレンイミングラフト化多孔質シリコン微粒子からなる多機能性および生体適合性siRNA送達プラットフォームの使用を含む。

siRNAの治療を送達するための多孔質シリコンマイクロ粒子

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

porous-silicon-microparticles-for-delivery-of-sirna-therapeutics

Research

JoVE Journal

Bioengineering

10.8K ビュー.  Houston Methodist Research Institute. 配信は、低分子干渉RNA（siRNA）の治療的実施のための主要な課題である。このプロトコルは、アルギニン及びポリエチレンイミングラフト化多孔質シリコン微粒子からなる多機能性および生体適合性siRNA送達プラットフォームの使用を含む。 

ビデオ: Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

ソフトリソグラフィ機能化およびパターニング酸化物フリーシリコンとゲルマニウム

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

生物工学

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3次元細胞培養のための自己申告足場

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

化学とバイオセンシングアプリケーション用のシリコンナノワイヤ電界効果トランジスタの作製

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

組織工学のためのアルギン酸微粒子と熱ゲルポリ（N-イソプロピルアクリルアミド） - グラフト - コンドロイチン硫酸複合材料の合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

カチオン性およびアニオン性リポソームを用いてCNSへの治療用のsiRNAの送達

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

神経成長因子のキャリアとしてポーラス シリコン膜の設計

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3 D プリントされた多孔性セルロース ナノコンポジット型ヒドロゲル足場

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

細胞質 siRNA 送達のための、中立的充電された pH 応答性高分子ナノ粒子の調製

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

シリコンナノワイヤと細胞間電気結合の研究のための光刺激

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation durations. A substantial contributor to the observed decline is the damage to the neural tissue proximal to the implant and subsequent neuroinflammatory response. Efforts to improve device longevity include chemical modifications or coating applications to the device surface to improve the tissue response. Development of such surface treatments is typically completed using non-functional "dummy" probes that lack the electrical components required for the intended application. Translation to functional devices requires additional consideration given the fragility of intracortical microelectrode arrays. Handling tools greatly facilitate surface treatments to assembled devices, particularly for modifications that require long procedural times. The handling tools described here are used for surface treatments applied via gas-phase deposition and aqueous solution exposure. Characterization of the coating is performed using ellipsometry and x-ray photoelectron spectroscopy. A comparison of electrical impedance spectroscopy recordings before and after the coating procedure on functional devices confirmed device integrity following modification. The described tools can be readily adapted for alternative electrode devices and treatment methods that maintain chemical compatibility.

The present protocol describes tools for handling silicon planar intracortical microelectrodes during treatments for surface modification via gas deposition and aqueous solution reactions. The assembly of the components used to handle the devices throughout the procedure is explained in detail.

シリコン平面皮質内微小電極の表面処理用ツール

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation ...