Name: solid lipid nanoparticles (slns) for intracellular targeting applications
Uploaded: 2015-11-17T14:00:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

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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	<li>Calderon-Colon, X., 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=Synthesis+of+sub-10+nm+solid+lipid+nanoparticles+for+topical+and+biomarker+detection+applications.">Synthesis of sub-10 nm solid lipid nanoparticles for topical and biomarker detection applications.</a> J Nanopart Res. 16 (2252), 1-10 (2014).</li><li>Patwekar, S., 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=Review+on+nanoparticles+used+in+cosmetics+and+dermal+products.">Review on nanoparticles used in cosmetics and dermal products.</a> World Journal of Pharmacy and Pharmaceutical Sciences. 3 (8), 1407-1421 (2014).</li><li>Martins, S., 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+nanoparticles+as+intracellular+drug+transporters:+An+investigation+of+the+uptake+mechanism+and+pathway.">Solid lipid nanoparticles as intracellular drug transporters: An investigation of the uptake mechanism and pathway.</a> International Journal of Pharmaceutics. 430, 216-227 (2012).</li><li>Yadav, N., Khatak, S., Sara, U. V. S. <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+-+A+Review.">Solid Lipid Nanoparticles - A Review.</a> International Journal of Applied Pharmaceuticals. 5 (2), 8-18 (2013).</li><li>Weber, S., Zimmer, A., Solid Pardeike, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Nanoparticles+(SLN)+and+Nanostructured+Lipid+Carriers+(NLC)+for+pulmonary+application:+a+review+of+the+state+of+the+art.">Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) for pulmonary application: a review of the state of the art.</a> Eur J Pharm Biopharm. 86 (1), 7-22 (2014).</li><li>Mahajan, A., Kaur, S., Grewal, N. K., Kaur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+Lipd+Nanoparticles+(SLNs)+-+As+Novel+Lipd+based+Nanocarriers+for+Drugs.">Solid Lipd Nanoparticles (SLNs) - As Novel Lipd based Nanocarriers for Drugs.</a> International Journal of Advanced Research. 2 (1), 433-441 (2014).</li><li>Buse, J., El-Aneed, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties,+engineering+and+applications+of+lipid-based+nanoparticle+drug-delivery+systems:+current+research+and+advances.">Properties, engineering and applications of lipid-based nanoparticle drug-delivery systems: current research and advances.</a> Nanomedicine (Lond). 5, 1237-1260 (2010).</li><li>Malam, Y., Loizidou, M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+and+nanoparticles:+nanosized+vehicles+for+drug+delivery+in+cancer.">Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer.</a> Trends Pharmacol Sci. 30 (11), 592-599 (2009).</li><li>Lim, S. B., Banerjee, A., Onyuksel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+drug+safety+by+the+use+of+lipid-based+nanocarriers.">Improvement of drug safety by the use of lipid-based nanocarriers.</a> Journal of controlled release : official journal of the Controlled Release Society. 163, 34-45 (2012).</li><li>Ali Khan, A., Mudassir, J., Mohtar, N., Darwis, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+drug+delivery+to+the+lymphatic+system:+lipid-based+nanoformulations.">Advanced drug delivery to the lymphatic system: lipid-based nanoformulations.</a> International journal of nanomedicine. 8, 2733-2744 (2013).</li><li>Oussoren, C., Storm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+to+target+the+lymphatics+by+subcutaneous+administration.">Liposomes to target the lymphatics by subcutaneous administration.</a> Advanced drug delivery reviews. 50, 143-156 (2001).</li><li>Shang, L., Nienhaus, K., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+nanoparticles+interacting+with+cells:+size+matters.">Engineered nanoparticles interacting with cells: size matters.</a> J Nanobiotechnology. 12, 5 (2014).</li><li>Joshi, M. D., Muller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+nanoparticles+for+parenteral+delivery+of+actives.">Lipid nanoparticles for parenteral delivery of actives.</a> European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V. 71, 161-172 (2009).</li><li>Torchilin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micellar+nanocarriers:+pharmaceutical+perspectives.">Micellar nanocarriers: pharmaceutical perspectives.</a> Pharmaceutical research. 24, 1-16 (2007).</li><li>Ashley, C. E., 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=The+targeted+delivery+of+multicomponent+cargos+to+cancer+cells+by+nanoporous+particle-supported+lipid+bilayers.">The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.</a> Nature materials. 10, 389-397 (2011).</li><li>Patchan, M., 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=Nanotech;+Nanotechnology+2013:+Bio+Sensors+Instruments,+Medical,+Environment+and+Energy;+Chapter+3:+Materials+for+Dru.,+and+Gene+Delivery.">Nanotech; Nanotechnology 2013: Bio Sensors Instruments, Medical, Environment and Energy; Chapter 3: Materials for Dru., and Gene Delivery.</a> Nanobandage for controlled release of topical therapeutics. 3, 255-258 (2013).</li><li>Forgiarini, A., Esquena, J., Gonzalez, C., C, S., Koutsoukos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+stability+of+nano-emulsions+in+mixed+nonionic+surfactant+systems.+Trends+in+colloid+and+interface+science+XV.">Formation and stability of nano-emulsions in mixed nonionic surfactant systems. Trends in colloid and interface science XV.</a> Progress in Colloid and Polymer Science. 118, 184-189 (2001).</li><li>Nantarat, T., Chansakaow, S., Leelapornpisid, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization,+characterization+and+stability+of+essential+oils+blend+loaded+nanoemulsions+by+PIC+technique+for+anti-tyrosinase+activity.">Optimization, characterization and stability of essential oils blend loaded nanoemulsions by PIC technique for anti-tyrosinase activity.</a> International Journal of Pharmacy and Pharmaceutical Sciences. 7, 308-312 (2015).</li><li>Sevcikova, P., Vltavska, P., Kasparkova, V., Formation Krejci, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+Stability+of+Nanoemulsions+Prepared+by+Phase+Inversion.">Characterization and Stability of Nanoemulsions Prepared by Phase Inversion.</a> , 132-137 (2011).</li><li>Forgiarini, A., Esquena, J., Gonzalez, C., Solans, C., Buckin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+relation+between+phase+behavior+and+emulsification+methods+with+nanoemulsion+formation.+Trends+in+colloid+and+interface+science+XIV.">Studies of the relation between phase behavior and emulsification methods with nanoemulsion formation. Trends in colloid and interface science XIV.</a> Progress in Colloid and Polymer Science. 115, 36-39 (2000).</li><li>Forgiarini, A., Esquena, J., Gonzalez, C., Solans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+nano-emulsions+by+low-energy+emulsification+methods+at+constant+temperature.">Formation of nano-emulsions by low-energy emulsification methods at constant temperature.</a> Langmuir. 17 (7), 2076-2083 (2001).</li><li>Cabone, C., Tomasello, B., Ruozi, B., Renis, M., Puglisi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+optimization+of+PIT+solid+lipid+nanoparticles+via+statistical+factorial+design.">Preparation and optimization of PIT solid lipid nanoparticles via statistical factorial design.</a> Eur J Med Chem. 49, 110-117 (2012).</li><li>Raimondi, G., 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=Mammalian+Target+of+Rapamycin+Inhibition+and+Alloantigen-Specific+Regulatory+T+Cells+Synergize+To+Promote+Long-Term+Graft+Survival+in+Immunocompetent+Recipients.">Mammalian Target of Rapamycin Inhibition and Alloantigen-Specific Regulatory T Cells Synergize To Promote Long-Term Graft Survival in Immunocompetent Recipients.</a> J Immunol. 184, 624-636 (2010).</li><li>Jhunjhunwala, S., Raimondi, G., Thomson, A. W., Little, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+rapamycin+to+dendritic+cells+using+degradable+microparticles.">Delivery of rapamycin to dendritic cells using degradable microparticles.</a> J Control Release. 133 (13), 191-197 (2009).</li><li>Kapse-Mistry, S., Govender, T., Srivastava, R., Yergeri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodrug+delivery+in+reversing+multidrug+resistance+in+cancer+cells..">Nanodrug delivery in reversing multidrug resistance in cancer cells..</a> Front Pharmacol. 5 (159), 1-22 (2014).</li><li>Musacchio, T., Torchilin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+lipid-based+pharmaceutical+nanocarriers.">Recent developments in lipid-based pharmaceutical nanocarriers.</a> Front Biosci (Landmark Ed). 1 (16), 1388-1412 (2011).</li><li>Cerpnjak, K., Zvonar, A., Ga&#353;perlin, M., Vre&#269;er, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-based+systems+as+a+promising+approach+for+enhancing+the+bioavailability+of+poorly+water-soluble+drugs).">Lipid-based systems as a promising approach for enhancing the bioavailability of poorly water-soluble drugs).</a> Acta Pharm. 63 (4), 27-445 (2013).</li><li>Rodr&#236;guez-Gasc&#242;n, A., Pozo-Rodr&#236;guez, A., Solin&#236;s, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+nucleic+acid+vaccines:+use+of+self-amplifying+RNA+in+lipid+nanoparticles.">Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles.</a> Int J Nanomedicine. 9, 1833-1843 (2014).</li><li>Almeida, A. 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><tbody><tr><td>Nile Red (NiR)</td><td>Sigma</td><td>19123</td><td>BioReagent, suitable for fluorescence, &amp;ge;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 &amp;micro;m Supor Membrane Low Protein Binding</td><td>Life Sciences</td><td>PN4612</td><td>Non-Pyrogenic</td></tr><tr><td>Nanotrac Ultra&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>P5493</td><td>For molecular biology</td></tr><tr><td>Recombinant murine GM-CSF&amp;nbsp;</td><td>Peprotech</td><td>315-03</td><td>&gt;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td></tr><tr><td>Recombinant murine IL-4&amp;nbsp;</td><td>Peprotech</td><td>214-14</td><td>&gt;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

17.0K 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

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

カーボンナノチューブにより安定化内部自己組織化脂質粒子の簡便な調製

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

化学

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 ...

生物工学

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

液晶ナノ粒子キャリアにおける疎水性貨物カプセル化の標的細胞膜配達

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

ナノディスクを含む生理活性物質の製剤化とキャラクタリゼーション

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

生化学

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 ...

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 ...

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 ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生体活性親水性球状タンパク質の捕捉に対するナノリポソームの調製と特性解析

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

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 ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

カチオン性リポソームを介した特異的siRNAの再配達によるマウスの肺におけるIntersectin-1Sの長期サイレン。電子顕微鏡によるノックダウン効果の評価

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...