We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.

すべてのマウスは、コロラド州立大学の施設内動物管理使用委員会によって承認されたプロトコルに従って、ラボ動物ケア・インターナショナルの評価と認定協会の認定を受け、研究室動物資源で飼育し、維持しました。 LSPCsの調製<ol><li> 1 DOTAP（1,2-ジオレオイル-3-トリメチルアンモニウム - プロパン）：LSPCsのためのコレステロール比1を使用してください。 1クロロホルム：フラスコ内のメタノール溶液4ナノモルのリポソーム製剤については、DOTAPの2ナノモルと1の10ミリリットルへのコレステロールの2ナノモルを混ぜます。 </li><li>ドラフト内でN 2ガ ​​スを用いたメタノール溶液：クロロホルムの9ミリリットルを蒸発させます。ガスなしで一晩換気フード内の溶媒の最後の1ミリリットルを蒸発させます。フラスコの底に薄い脂質フィルムを観察します。 </li><li>熱、55℃に10％ショ糖溶液10mlを。 </li><li> GEながら、 すなわち、1ml /分、徐々に薄い脂質フィルム上に加熱された10％スクロース溶液を注ぎntlyフラスコを旋回。脂質分子はゲル - 液相に残るように55℃での脂質と10％のショ糖、フラスコを維持します。多重膜小胞（MLV）の形成での再水和をもたらします。 </li><li>脂質がリポソームをサイジングする前に、少なくとも1時間、55℃で再水和することができます。 </li><li> 1.0μmのフィルターを製造者の指示に従って、押出機を組み立てたり、サイジングのために1.0μmのシリンジフィルターを使用しています。 </li><li>押出機での注射器のいずれかに加熱されたリポソーム懸濁液の1ミリリットルを追加し、2つのシリンジ11時間の間のサスペンションを渡します。懸濁液を11回通過した後に開始し、注射器よりも反対注射器であることを確認します。 </li><li>サイズその後、0.45μmの0.2μmのフィルターを通して再びリポソームは、大型単層小胞（LUVを）を生成します。簡単にサイズ調整のために加熱し、リポソーム懸濁液を保管してください。 </li><li> 20μM（4200μlで4ナノモルリポソーム懸濁液（200μM）の20μLをインキュベート室温で10分間ナノモル合計）株式のsiRNA溶液。 </li><li> RTで10分間のsiRNA /リポソーム溶液と500μM（40ナノモル合計）株式RVG-9R液80μLをインキュベートします。 LSPCsはできるだけ早く使用されるべきであるが、調製後数時間まで使用することができます。調製後、数時間のために4℃で保存しLSPCs。 </li></ol> PALETSの調製<ol><li> 40：：DSP​​E（1,2-ジステアロイル-sn-グリセロ-3-ホスホエタノールアミン）のいずれかの5のモル比：コレステロール：DSPE-PEGまたはDOTAP：コレステロール：DSPE-PEG PALETSのための55を使用してください。 1クロロホルム：フラスコ内のメタノール溶液4ナノモルのリポソーム製剤については、2の10ミリリットルにDSPEまたはDOTAPのいずれかの2.2ナノモル、コレステロールの1.6ナノモル、及びDSPE-PEGの0.2ナノモルを混ぜます。 </li><li>ステップ1.1以上1.2で説明したように正確にリポソーム懸濁液を準備します。 1×PBS 10ml中DSPEリポソームを再水和を1ml /分を追加して、75℃に加熱しました。脂質は時のために75℃で再水和することを許可しますサイジングの前に少なくとも1時間。 </li><li>サイズステップ1.6から1.8に記載とおりにリポソーム懸濁液。 </li><li> 4ナノモルのそれぞれのピペットを20μl（200μM）リポソーム懸濁液の単一使用のアリコートへのDOTAPおよびDSPEリポソームはサイズされた後、およびベンチトップ凍結乾燥機（ 図2）を使用して、30分間凍結乾燥。 </li><li> DSPE PALETSリポソーム中に封入されるsiRNAをRTで10分間、硫酸プロタミンの26.6μM溶液を1.4μlの20μM（4ナノモル合計）株式のsiRNA溶液200μlをインキュベートします。 </li><li> siRNAの4ナノモルストック溶液の200μlでDOTAP PALETSリポソームを再水和またはsiRNA /プロタミン溶液を201.4μlのDSPE PALETSリポソームを再水和。 RTで10分間リポソーム/ siRNA溶液をインキュベートします。 </li><li> 60mMの1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩（EDC）の10μLをDOTAPとDSPE PALETS両方用リポソーム/ siRNA溶液の溶液を加えます。 </li><li>両方のDOTAPおよびDSPE PALETS用リポソーム/ siRNA溶液に150mMのN-ヒドロキシ（スルホNHS）溶液10μlを加えます。 </li><li> RTで2時間のsiRNA /リポソーム懸濁液とEDCおよびスルホNHSをインキュベートします。 </li><li> RTで10分間この溶液を架橋するsiRNA /リポソームと500μM（40ナノモル合計）株式RVG-9R液80μLをインキュベートします。 注：EDC /スルホNHSはPEG脂質に共有結合にRVG-9Rを可能にします。調製後、数時間以内にPALETSを使用してください。調製後、数時間のために4℃で保存しPALETS。 </li><li> PALETSのsiRNA封入効率を決定するには： <ol><li> 260nmで設定し、分光光度計を使用してプロタミン及びリポソームの添加前および後のsiRNAの濃度を測定します。 </li><li> 50kDaの遠心フィルターを使用してリポソーム/カプセル化されたsiRNAの懸濁液から封入されていないのsiRNAをフィルタリングします。 20分1,000×gで遠心分離します。 </li><li>の濃度を測定260nmで分光光度計を使用して、ろ液および保持液中のカプセル化されたsiRNAの濃度でカプセル化されていないのsiRNA。 </li></ol></li></ol> 3. LSPCsまたはPALETSをマウスに注射<ol><li>血管系を拡張するために熱ランプに5〜10分間、マウスを公開します。 </li><li> 5〜10分間注入前、および注入中に2から3パーセントイソフルオラン/酸素流量でマウスを麻酔。動物がもはや通院で、呼吸がつま先のピンチを実行して、減速しているときに麻酔を確認していません。尾静脈にアクセスするには、その背面または側面にマウスを置きます。麻酔下ながら乾燥を防ぐために、マウスの目に獣医軟膏を使用してください。 </li><li>ワイプ/ 70％エタノールで尾をスプレーし、26 Gインスリン注射器を用いて、マウスの尾静脈にLSPCsまたはPALETSの300μLを注入します。尾静脈に遠位に注入開始し、静脈が崩壊または破裂場合近位に移動します。 </li><li>それは胸骨横臥位を維持するまで、クリーンなケージにマウスを置きます。 MOを置かないでくださいそれは完全に回復するまで、他のケージの仲間と一緒に使用。マウスは、5〜15分で回復する必要があります。彼らは、回復に時間がかかる場合には、体温を維持するために加温パッド上又は加熱ランプの下に配置することができます。マウスは、過熱から保護するために注意深く監視する必要があります。 </li><li>麻酔が切れる確実にするために数時間注入後に動物を監視し、治療の全く悪影響はありません。 LSPCsまたはPALETSは、少なくとも24時間循環させます。 </li></ol>フローサイトメトリーを介して、タンパク質発現の4解析<ol><li> 15分間、20％CO 2の流量でマウスを安楽死させます。 </li><li>ハサミを用いてマウスの皮膚と頭蓋骨を切り取ることにより、脳の半分の半球を解剖。ピール離れて半分離れトングやハサミの先端を使って頭蓋骨から脳の半球。 </li><li>プレス半分のFACS緩衝液2.5ミリリットルを使用して、40μmのメッシュセルストレーナーを介して、各マウスからの脳の半球（1倍、シリンジのプランジャーを有するペトリ皿にPBS、1％ウシ胎児血清、10mMのEDTA）。 </li><li> FACS緩衝液のさらに2.5 mlの細胞ストレーナーとペトリ皿をすすぎます。氷上で15ミリリットルコニカルチューブに単一細胞懸濁液を入れてください。 </li><li>ピペットで350×gで5分間1.5 mlのマイクロ遠心チューブと遠心分離機に5ミリリットル単一細胞懸濁液100μl。上清を捨て、FACS緩衝液1ml中に細胞ペレットを再懸濁します。 5分間、350×gでスピン。 FACS緩衝液の別の1ミリリットルで繰り返します。氷上で細胞懸濁液を保管してください。 </li><li> 1100μlの細胞をインキュベート：FACS緩衝液中の0.5mg / mlのラット抗マウスFcブロックの100倍希釈を氷上で30〜60分間。ペレット化し、ステップ4.5のように細胞を洗浄します。氷上で細胞懸濁液を保管してください。 </li><li> 30〜60分間氷上でFACS緩衝液中で7％マウス血清中のマウスのPrP Cに対する20 / mlの蛍光抗体を100μlで細胞をインキュベートします。ペレット化し、ステップ4.5のように細胞を洗浄します。細胞懸濁液を保ちます冷やして。 </li><li>ペレットおよび1分RBC溶解緩衝液（1×PBS、155 mMのNH 4 Cl を 、12 mMの炭酸水素ナトリウム、0.1mMのEDTA）1ml中に再懸濁細胞。ステップ4.5のようにペレットとは、FACS緩衝液1mlに細胞を懸濁します。氷上で細胞懸濁液を保管してください。 </li><li>以前10記載されているように、フローサイトメーターを使用してのPrP Cの発現を解析。ゲートは、全細胞集団から細胞を生きています。ゲートのPrP C + MFI（蛍光強度中央値）を決定するために、生細胞集団由来の細胞。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+can+infect+primary+cultured+neurons+and+astrocytes+and+promote+neuronal+cell+death.">Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death.</a> Proc Natl Acad Sci. 101 (33), 12271-12276 (2004).</li><li>Diedrich, J., Bendheim, P., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie-associated+prion+protein+accumulates+in+astrocytes+during+scrapie+infection.">Scrapie-associated prion protein accumulates in astrocytes during scrapie infection.</a> Proc Natl Acad Sci. 88 (2), 375-379 (1991).</li></ol>

The authors declare that they have no competing financial interests.

このレポートでは、効率的にCNSに対するsiRNAをトランスポート2の標的送達システムを作成するためのプロトコルについて説明します。 CNSに対するsiRNAを送達する従来の方法は、脳、標的としたsiRNAの静脈内注射、または非標的リポソームのsiRNA複合体の静脈内注射に直接のsiRNA / shRNAのベクトルを注入含まれています。 CNSへのsiRNA / shRNAのベクターの注入は、標的タンパク質の発現レベルの減少...

プリオンは、CNSに影響を与える深刻な神経変性疾患です。プリオン病は、PrP RESと呼ばれる感染性異性体によって、正常な細胞プリオンタンパク質、のPrP Cのミスフォールディングに起因します。これらの疾患は、人間1-3でシカで羊、慢性消耗病におけるスクレイピー、牛における牛海綿状脳症を含む種の多種多様な影響を及ぼし、およびクロイツフェルト・ヤコブ病。プリオンは、シナプスの損失で始まる神経変性を引き起こし、空胞化、グリオーシス、ニューロン損失、およびプラーク沈着に進行します。結局、4個々の動物/の死に至ります。数十年にわたって、研究者は、プリオン病の進行を遅らせるまたは停止することを意味する化合物を調査しました。しかし、研究者は、成功した治療や効果的な全身送達ビヒクルのいずれかを見つけていません。 内因性のPrP Cの発現は、プリオン病の発症のために必要とされる5 </s&gt;アップ。したがって、減少または排除するのPrP Cの発現が遅延または疾患の改善をもたらすことができます。いくつかのグループは、プリオン病でのPrP C発現レベルの役割を調べるために、マウス脳組織に直接shRNAを発現のPrP Cの減少したレベルまたは注入レンチベクターを用いてトランスジェニックマウスを作成しました。これらの研究者は、プリオン病の進行性の神経病理の停止をもたらし、動物6-9の寿命を延長し、ニューロンのPrP Cの量を低減することがわかりました。我々は、マウス神経芽腫細胞10内のPrP RESのクリアランスのことのPrP C siRNA処理の結果を報告しています。これらの研究は療法の使用は、低分子干渉RNA（siRNA）、切断するmRNAのように、のPrP Cの発現レベルを減少させるために十分なプリオン病の進行を遅らせることができることを示唆しています。しかし、プリオン病について調査し、最も治療法は実用的ではない方法で配信されていました臨床設定インチこのために、siRNA療法は、CNSに静脈内に送達し、標的とする全身性送達システムを必要とします。 研究者らは、遺伝子治療製品の送達ビヒクルとしてのリポソームの使用を研究しました。カチオン性およびアニオン性脂質は、両方の、リポソームの形成に使用されます。カチオン性脂質とDNA / RNA間の電荷の差が効率的なパッケージングを可能にするためのカチオン性脂質は、より広くアニオン性脂質よりも使用されています。カチオン性脂質の他の利点は、他の脂質11-14より容易に細胞膜を通過することです。しかし、カチオン性脂質はアニオン性脂質13,14よりも免疫原性です。そのため、研究者らは、リポソーム中のアニオン性脂質にカチオンを使用してからシフトし始めています。遺伝子治療製品を効率的にDNA / RNA分子15-19を凝縮正に荷電したペプチドの硫酸プロタミンを用いてアニオン性リポソーム中にパッケージングすることができます。アニオン性LIPIので、DSは、彼らが循環時間を増加していることがあり、より多くの動物モデル13,14に許容され得るカチオン性脂質よりも免疫原性が低いです。リポソームは、リポソームに結合しているペプチドを標的に使用して特定の組織を標的とします。ニコチン性アセチルコリン受容体に結合するRVG-9R神経ペプチドは、CNS 17-20にsiRNAおよびリポソームを標的化するために使用されてきました。 このレポートは、3 siRNA送達車を生産するための、および神経細胞（ 図1）に対するsiRNAをパッケージ化して提供するためのプロトコルの概要を説明します。リポソームのsiRNAペプチド複合体（LSPCs）は、siRNAおよび静電リポソームの外表面に付着したRVG-9Rターゲティングペプチドを有するリポソームから構成されています。ペプチドは、RVG-9Rが共有脂質、PEG基に結合して、siRNAおよびリポソーム内に封入されたプロタミンで構成されている治療のsiRNA（PALETS）をカプセル化されたリポソームを取り上げました。 LSPCsとPALETを生成するには、以下の方法を使用してSは、のPrP C siRNAは、治癒または、実質的にプリオン病の病態の発症を遅延させるための途方もない約束を保持している神経細胞で90％、最大のPrP Cの発現を低下させます。

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DOTAP lipid</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">890890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">700000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">850715</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-PEG</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">880125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">AC268320010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:104px;">EMD Millipore</td>
			<td style="width:119px;">113351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2 Gas</td>
			<td style="width:104px;">AirGas</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">S5-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extruder</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.0, 0.4, and 0.2um filters</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610010, 610007, 610006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS</td>
			<td style="width:104px;">Life Technologies</td>
			<td style="width:119px;">70011-044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">ICN10275205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDC</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">22980</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sulfo-NHS</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">24510</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40um Cell Strainer</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rat anti-mouse CD16/CD32 Fc block</td>
			<td style="width:104px;">BD Pharmigen</td>
			<td style="width:119px;">553141</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-PrP antibody (PRC5)</td>
			<td style="width:104px;">Proprietary - PRC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

アニオンPALETS内のsiRNAのカプセル化の効率を高めるために、siRNAは、プロタミンと混合しました。 1（ 図3A）：1〜2：siRNAのための最高のプロタミン濃度を決定するために、siRNAは、1から、プロタミンの異なる濃度で混合しました。プロタミンを使用せずに、アニオン性リポソームで60〜65%のsiRNA封入効率がありました。プロタミンとサンプル：siRNAのモル比?...

Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

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

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 ...

delivery-therapeutic-sirna-to-cns-using-cationic-anionic

Research

JoVE Journal

Bioengineering

10.4K ビュー.  Colorado State University. 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. 

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

生物工学

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

上皮成長因子受容体をターゲットとしたゼラチンベースのエンジニアNanovectorsを使用してヒト膵臓癌細胞における遺伝子デリバリーとトランスフェクション

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

微小電極アレイを用いたsiRNAの付着細胞への高効率、サイト固有のトランス体（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

液体射出成形用の3Dプリント金型を用いた医療機器を迅速かつ低コストプロトタイピング

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

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.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

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 ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

ウサギの乳腺への管内配達

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

準備、精製、および脂肪酸含有リポソームを用いる

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

3 つの補完的な手法を使用して胎盤をターゲットとした薬剤投与の安全性の総合評価

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

体外の効率的な配信のカチオン性 Nanoliposomes の生成は、メッセンジャー RNA を転写

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...