Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

SLNsの1処理<ol><li> SLNsの合成 注：サブ20 nmのSLNs 1を準備するために 、PIT法1,17,20を使用します。 <ol><li>無菌技術、バイオ試薬や細胞培養グレードの試薬 ​​、および滅菌材料（ すなわち 、ピペット、ヘラ、バイアルなど）を使用します。 </li><li> SLNsの合成（ 図2）のための安全キャビネットを使用してください。 </li><li>蛍光染料の0.6 mgの組み合わせバイアル（15ml）中にヘネイコサン（脂質、5重量/重量％の脂質濃度）0.10gを、で共溶融（ナイルレッド（NIR）を、近赤外濃度は、約0.3 mg / mlです） 90℃、および攪拌。 </li><li> 0.11グラムのポリオキシエチレン（10）オレイルエーテル（界面活性剤、5.5重量/重量％の界面活性剤濃度）を追加します。 90℃、攪拌で得られた混合物を共溶融。 </li><li>混合物に滅菌水1.79グラムを追加し、90℃に加熱し、透明なナノエマルジョンが形成されるまで攪拌します。 注意：継続的な攪拌および制御冷却、SLの下でNSが作成されます。これらの条件は、水中油型エマルションに油中水型エマルジョンの反転を引き起こします。このような脂肪滴へのNIRパーティションと親油性分子。 </li><li>対照試料として機能するためには、蛍光色素を加えることなく、並行してナノエマルジョンを準備します。 0.10ヘネイコサンのGおよび0.11グラムのポリオキシエチレン（10）オレイルエーテル、バイアル（15 ml）を90℃で共溶融し、攪拌中に結合します。 <ol><li>混合物に滅菌水1.79グラムを追加し、90℃に加熱し、透明なナノエマルジョンが形成されるまで攪拌します。 </li></ol></li><li>さらに、滅菌0.2μmのフィルターを使用して、各ナノエマルジョンを殺菌。 </li></ol><img alt="図2" src="/files/ftp_upload/53102/53102fig2.jpg" /> SLNsの合成の図2の回路図。 <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> </li><li>粒子サイズとポルydispersity <ol><li>ガラスキュベット及び粒径を測定するために設計された装置を用いてSLNs 1の粒子サイズおよび多分散性を測定するための動的光散乱（DLS）を使用します。 </li><li>サンプルの測定の前に、標準の準備と測定のための製造業者のプロトコルに従って二つの既知の規格の粒子サイズを測定します。 </li><li>標準溶液の1.5 mlをガラスキュベットに充填し、試料と同じ測定条件を用いて、標準溶液を測定します。 注意：前のステップでは、各標準のために繰り返しました。 </li><li>調製したサンプルを測定します。それぞれについて、以下の実験のパラメータを使用します。100秒、水の屈折率（1.33）、水の粘度の実行時間を20°C（1.002 MPA・秒）で。 注：この方法は、周波数を使用してナノ粒子のサイズを測定するために光をシフトしました。 </li><li>パルティの平均粒径および多分散性を報告CLEのサイズ分布。 </li></ol></li><li>融点および融解潜熱<ol><li> SLNs 1の熱挙動を調査するために、オートサンプラーおよび液体窒素冷却源を備えた示差走査熱量計（DSC）を使用します。 </li><li>約25 mgのの質量と40μlのアルミニウムパンに調製されたままのSLNsをピペット気密DSC走査中に水分損失を最小限に抑えるために、ユニバーサルクリンパプレスを使用して、パンを密封します。 </li><li> 5〜80°Cから各ナノエマルションを測定します。 </li><li>融点と谷が融点および材料の量で割ったDSCプロットの曲線下面積の積分を表し、DSCプロットから、融解潜熱が融解潜熱を表して報告します。 </li></ol></li></ol>線維芽細胞と樹状細胞との2 SLNsの相互作用<ol><li>初代ヒト線維芽細胞の培養<ol><li>文化初代ヒトFiの低血清成長サプリメント（LSGS）キットを補充したヒト皮膚線維芽細胞（媒体106）、培養用液体培地中で、製造業者の指示に従ってbroblasts。 80％コンフルエンスに達したときに、37℃、5％CO 2および継代培養の加湿雰囲気中で細胞を維持します。 </li><li> MTTおよび画像解析の両方のために、96ウェルプレートで、種子細胞、ウェルあたり2×10 4細胞の密度で、前SLN暴露に37°のCO / Nで平衡することができます。 </li><li>時間ゼロで、脂質濃度に関して、示された（ 図4）のように、完全培地中でSLNsを希釈し、それぞれにウェルあたり10μLを追加し、96ウェルプレートでよく複製します。 </li><li>インキュベーション期間中、37°C、5％CO 2で細胞を維持します。 </li></ol></li><li> MTT毒性アッセイ<ol><li> SLNsとのインキュベーションの24時間後に、製造業者のプロトコールに従って、MTTアッセイを用いて細胞生存率を決定します。&lt;/ LI&gt; <li>細胞を1回洗浄し、各ウェル（100μl/ウェル）に新鮮な培地を追加します。新鮮な培地で交換する前に、10分間、70％のメタノール溶液中でインキュベートすることにより、コントロールウェルを殺します。 </li><li>マイクロプレートの各ウェルにMTT試薬（ウェルあたり10μl）を加え、37℃でO / Nインキュベートします。 </li><li> O / Nインキュベートした後、製造業者のプロトコルに従って提供される界面活性剤溶液（ウェル当たり100μl）を用いて細胞内のホルマジン結晶を可溶化します。 </li><li> RTでのインキュベーションの3時間後、マイクロプレートリーダーを用いて570nmの測定波長での吸光度の値を得ます。 注：3-（4,5- dimethylthiazolyl-2）-2、5-​​ジフェニルテトラゾリウムブロミド（w / v）の1％未満であるべきで使用MTT試薬。 </li></ol></li><li>蛍光顕微鏡<ol><li> SLNと線維芽細胞の投与後の特定の時点で、滅菌リン酸緩衝食塩水（PBS）で2回、線維芽細胞を洗浄し、冷70％メタノールで10分間固定します。 </li><li> 10分後PBSでメタノールを交換し、バンドパス（BP）50分の690 nmのフィルターセットを使用して、倒立顕微鏡を用いて画像をキャプチャします。 </li></ol></li><li>マウス骨髄樹状細胞の培養<ol><li>以前23,24に記載されているように 、マウス骨髄由来樹状細胞（BMDC）を誘導します。簡単に述べると、組換えマウス顆粒球-マクロファージコロニー刺激因子（GM-CSF、300 U / ml）および組換えマウスIL-4で2×10 6個/プレートで100 mmのペトリ皿中のC57BL / 10骨髄のプレートの単一細胞懸濁液（B細胞刺激因子、200 U / ml）を。 3日おきにメディアを変更します。 </li><li> 7日目に、0.5および5.0 / mlの脂質の最終濃度で皿に近赤外ロードSLNsを追加し、37℃、暴露の所望の長さのために、5％のCO 2インキュベーター内で細胞を維持します。 </li><li>緩く広告を取り除くために（プレートを徹底的に洗浄し、50mlチューブにそれを収集、プレート内のすべてのメディアを吸引することにより、皿から細胞を回収4 mMのエチレンジアミン四酢酸（EDTA）を含むリン酸緩衝生理食塩水5mlの（PBS）とエラン細胞）。同じ50mlチューブで得られた細胞懸濁液を収集します。 </li></ol></li><li>フローサイトメトリーを介したSLNs定款の評価<ol><li>ポイント2.4.3で得られた細胞懸濁液の濃度を決定し、12ミリメートルX 75ミリメートルの丸底FACSチューブに3×10 5細胞を配布します。 </li><li> 1mlのPBSを添加し、次に4℃の温度で、400×gで5分間チューブを遠心分離して細胞を洗浄します。 </li><li>上清を捨て、PBS / 2mMのEDTA /抗CD11cの-FITCモノクローナル抗体（ml最終0.25μgの/）を含む0.1％のウシ血清アルブミン（BSA）（染色緩衝液）100μlでペレットを再懸濁。暗所で氷上で30分間インキュベートします。 </li><li> 1mlのPBSで細胞を洗浄した後、4℃の温度で、400×gで5分間チューブを遠心します。 </li><li>上清を捨て、それぞれを再懸濁染色緩衝液の400μlのペレット。細胞は現在、（おそらく青と赤のレーザーを装備した）フローサイトメーターでサンプルを取得するための準備ができています。 </li><li>解析ソフトウェアフローサイトメトリーを用いて、細胞（FITC陽性）のCD11c +の集団に対してゲーティングし、（PerCPを-のCy5.5またはAPCチャネルで測定）SLNs関連蛍光の強度を比較することによって、DCによってSLNsの取り込みのレベルを評価します。 </li></ol></li></ol>

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J., Souto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+lipid+nanoparticles+as+a+drug+delivery+system+for+peptides+and+proteins.">Solid lipid nanoparticles as a drug delivery system for peptides and proteins.</a> Adv Drug Deliv Rev. 59, 478-490 (2007).</li><li>Pardeshi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+lipid+based+nanocarriers:+an+overview.">Solid lipid based nanocarriers: an overview.</a> Acta Pharm. 62, 433-472 (2012).</li><li>Attama, A. A., Momoh, M. A., Builders, P. F., Sezer, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Nanoparticulate+Drug+Delivery+Systems:+A+Revolution+in+Dosage+Form+Design+and+Development.">Lipid Nanoparticulate Drug Delivery Systems: A Revolution in Dosage Form Design and Development.</a> Recent Advances in Novel Drug Carrier Systems. , 107-140 (2012).</li></ol>

The authors have nothing to disclose.

本研究では、SLNsの合成および細胞内標的化適用のためのそれらの適用が検討されました。これらの生体適合性ナノ粒子は、薬物送達、遺伝子サイレンシング、およびワクチン技術25-30を含む複数の用途のための送達媒体として有望であることが示されています。超小型SLNsは容易なプロセスを使用して合成し、そして一次皮膚細胞および初代免疫細胞との相互作用を調べました。 SLNs?...

ナノ粒子ベースの送達ビヒクルは、特に細胞のシグナル伝達および遺伝子発現を変化させるメカニズムを提供する、細胞内標的化用途のための大きな期待を示しています。これらの車両は、細胞応答に影響を与え、標的組織内の所望の効果を達成するように設計された薬物、タンパク質、および核酸を装填することができます。ナノキャリアの多くの種類は、脂質、ポリマー、シリコン、及び磁性材料を含む、治療的および診断的利益のために検討されています。これらのシステムは、ローカライズによる薬物送達のためのそれらの潜在的に魅力的であり、全身毒性の低減と、標的組織に治療濃度を増加させました。 固体脂質ナノ粒子（SLNs）は近年、有望な薬物送達ビヒクルとして浮上しているナノ粒子送達システムのよく研究例です。 SLNsは、容易にバイオセンシング1、化粧品2、およびTを含む複数の用途のために処方することができますherapeutic配信3-7。それらの有用性は、それらが生体適合性を高め、その結果、吸収性、非毒性脂質から完全に構成されているという事実から生じます。合成中に、親油性薬剤は、それによって、非経口投与のための薬剤の溶解性および適合性を増大させる、SLN車両に組み込むことができます。 SLN車両はまたそれらの分解およびクリアランスを減少させ、治療効果を最大化し、カプセル化された治療薬を安定化させるのを助けます。これらの車両は、体温3,4,8,9での安定性のために、制御放出製剤、長時間作用のために特に適しています。重要なことには、脂質ナノ粒子中の薬物のカプセル化は、薬物分子の固有の薬物動態プロファイルを変化させます。これは、狭い治療指数を有する薬物の制御放出を可能にすることによって潜在的な利点を提供します。 SLN内蔵治療薬の放出速度は、脂質の分解速度または中の薬物の拡散速度に基づいて調整することができます脂質マトリックス。 SLNsは、しばしば、特定の標的組織に蓄積するように操作されています。例えば、そのサイズ（通常は10を超えるnm）は、腫瘍組織の漏れやすい血管系は、堆積を促進する循環に保持を増強します。また、粒子の投与経路は、リンパ節10,11のような特定の生理学的構造を標的とする可能性のある生体内分布を変化させることが示されています。標的組織における沈着すると、ナノ粒子の適切な細胞の相互作用および最終的な内在化を達成することは、選択的に、セル12のうち、イオンおよび分子の流れを制御するために、細胞膜の能力に困難です。細胞取り込みを容易にするために、ペプチド、小分子、およびモノクローナル抗体13,14を含む特異的なリガンドを持つナノキャリアを変更することが可能です。受動浸透とナノ粒子の能動輸送の両方を含むいくつかのメカニズム細胞膜を横切って、以前3,12,15に記載されています 。一般的に、細胞-ナノ粒子相互作用は、細胞型または細胞周期相12とセル固有のパラメータに加えて、サイズ、形状、表面電荷及び表面化学を含むナノ粒子の物理化学的特性に影響されることが実証されています。 以前の研究は、転相温度（PIT）法17を用いて、16の局所的バイオマーカー検出アプリケーション1のためのサブ10 nmのSLNsの合成を実証しました。これは、温度を徐々に変化させながら2組成が一定のままで穏やかな合成方法です。加熱した溶液を連続的に攪拌しながら、それはナノエマルジョンで結果を室温に冷却します。このプロセスよりも小さい粒径を有する1 SLNsの合成の結果は、以前に、脂質ナンの合成のための種々の方法を使用して報告しますoparticles 17-22。得られたサイズスケール、20nm未満では、増加による表面積および増強された細胞の相互作用のための潜在的に細胞内標的用途のための利点を提供します。 蛍光色素または治療薬を送達するように設計さSLNsの概略図は、 図 1に示されている。SLNsは、脂質内部（例えば、直鎖アルカン）、親油性化合物（例えば、染料または治療剤）の取り込みを可能にし、界面活性剤の外部から成り水に囲まれた（例えば、線形非イオン性界面活性剤）。本研究では、SLNsは、蛍光色素を負荷した、粒子 - 細胞相互作用を研究するためのモデルとして使用します。初代ヒト皮膚線維芽細胞およびマウス樹状細胞は、毒性及び粒子の取り込みに対する相互作用を特徴付けるために、経時SLNをロードされた色素に曝露しました。 3-（4,5-ジメチルチアゾール-2-イル）-2,5- diphenylphenyltetrazoliumブロミド（MTT）アッセイはutiliました適切な投薬レベルを確立するために、ZED。蛍光顕微鏡およびフローサイトメトリーは 、in vitroでの粒子の取り込みを調べるために用いられる二つの方法でした。 <img alt="図1" src="/files/ftp_upload/53102/53102fig1.jpg" /> 主要な構成要素を示すSLNの図1の回路図。 <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Nile Red (NiR)</td>
			<td>Sigma</td>
			<td>19123</td>
			<td>BioReagent, suitable for fluorescence, &#8805;98.0%</td>
		</tr>
		<tr>
			<td>Heneicosane</td>
			<td>Aldrich</td>
			<td>286052</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Brij O10</td>
			<td>Sigma</td>
			<td>P6136</td>
			<td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>Sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.2 &#181;m Supor Membrane Low Protein Binding</td>
			<td>Life Sciences</td>
			<td>PN4612</td>
			<td>Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Nanotrac Ultra&#160;</td>
			<td>Microtrac</td>
			<td>serial number U1985IS</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Differential Scanning Calorimeter</td>
			<td>Mettlet-Toledo</td>
			<td>----</td>
			<td>Instument</td>
		</tr>
		<tr>
			<td>Primary human fibroblasts&#160;</td>
			<td>Life Technologies</td>
			<td>C-004-5C</td>
			<td>Neonatal (HDFn)</td>
		</tr>
		<tr>
			<td>Medium 106</td>
			<td>Life Technologies</td>
			<td>M-106-500</td>
			<td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Life Technologies</td>
			<td>S-003-K</td>
			<td>All the components of complete LSGS</td>
		</tr>
		<tr>
			<td>MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-025-K</td>
			<td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td>
		</tr>
		<tr>
			<td>Safire2 microplate reader</td>
			<td>Tecan</td>
			<td>----</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>For molecular biology</td>
		</tr>
		<tr>
			<td>Recombinant murine GM-CSF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>415</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-4&#160;</td>
			<td>R&#38;D Systems</td>
			<td>404</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
	</tbody>
</table>

solid lipid nanoparticles (slns) for intracellular targeting applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

PIT法はSLNsを合成するために使用され、転相温度は、水浴を用いて決定しました。溶液が透明に見えるまで、サンプルをゆっくりと加熱し、穏やかに撹拌しました。ヘネイコサン脂質を使用して行わSLNsための転相温度は45℃である。 表1は、粒子サイズ、多分散性、融点およびSLNsの融解潜熱を要約します。上述した処理条件を用いて合成SLNsは、18.59と16.87ナノメートルの平均粒径?...

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

細胞内ターゲティングアプリケーションのための固体脂質ナノ粒子（SLNs）

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

solid-lipid-nanoparticles-slns-for-intracellular-targeting

Research

JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.8K ビュー.  The Johns Hopkins Applied Physics Laboratory. In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. 

ビデオ: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

のためのウイルス粒子<emインビボ&gt;</em&gt;腫瘍イメージング

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

生物工学

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

堅牢、マイクロ流体細胞内送達プラットフォームとしての細胞絞る

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

回生研究ハーモニックナノ粒子

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生産と一価量子ドットの標的

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

マルチモーダル分子イメージングアプリケーションのためのBiofunctionalizedプルシアンブルーナノ粒子

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

細胞標識および超常磁性酸化鉄ナノ粒子とターゲティング

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

シルクナノ粒子の製造および薬物送達アプリケーション

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

ヒト間葉系幹細胞によって Falcarindiol を含む新しい脂質被覆ナノ粒子の取り込み

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

癌細胞培養から採取した培地における液体クロマトグラフィー-質量分析法による選択キヌレニンの同時定量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

マイクロ流体混合プラットフォームを用いた遺伝子導入のための脂質ナノ粒子の作製と特性化

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...