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 1. İşleme <ol><li> SLNs sentezi Not: alt 20 nm SLNs 1 hazırlamak için PIT yöntemi 1,17,20 kullanın. <ol><li> Aseptik teknik, bioreagents veya hücre kültürü sınıf reaktifler ve steril malzemeler (yani, pipetler, spatula, şişeler, vs.) kullanın. </li><li> SLNs sentezinde (Şekil 2) için bir biyogüvenlik kabini kullanın. </li><li> Floresan boya, 0.6 mg birleştirin ve bir viyal (15 ml) içine Heneicosane (Lipid, 5 ağırlık / ağırlık olarak% lipid konsantrasyonu), 0.10 g, en ortak eriyik (Nil Kırmızısı (NIR), NIR konsantrasyonu yaklaşık 0,3 mg / ml&#39;dir) 90 ° C ve heyecan. </li><li> 0,11 g polioksietilen (10) oleil eter (yüzey aktif madde, 5,5 ağırlık / ağırlık olarak% sürfaktan konsantrasyonu) eklenir. 90 ° C ve karıştırın de elde edilen karışımın Co-eritebilir. </li><li> 90 ° C&#39;ye kadar karışıma ısı steril su 1,79 g ekleyin ve şeffaf nanoemülsiyondur oluşana dek karıştırın. Not: sürekli karıştırma ve kontrollü bir soğutma, SL altındaNs oluşturulur. Bu koşullar, bir yağ-içinde-su emülsiyonu su içinde yağ emülsiyonu, bir inversiyon neden olur. Böyle lipid damlacıkları Nir bölüm olarak lipofilik moleküller. </li><li> Bir kontrol numunesi olarak hizmet vermek üzere, floresan boya eklemeden paralel nanoemülziyon hazırlayın. 90 ° C ve kızgın bir şişeye (15 mi) birlikte eriyik halinde Heneicosane 0.10 g ve 0.11 g polioksietilen (10) oleil eter birleştirin. <ol><li> 90 ° C&#39;ye kadar karışıma ısı steril su 1,79 g ekleyin ve şeffaf nanoemülsiyondur oluşana dek karıştırın. </li></ol></li><li> Dahası steril 0.2 mikron filtre kullanarak her nanoemülziyon sterilize edin. </li></ol><img alt="Şekil 2," src="/files/ftp_upload/53102/53102fig2.jpg" /> Şekil SLNs sentezi 2. şematik. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> </li><li> Partikül Boyutu ve Polydispersity <ol><li> Parçacık boyutu ve bir çoklu-dağınıklığa cam küvet kullanıldığında SLNs 1 ve tanecik boyutunu ölçmek için tasarlanmış bir cihaz ölçmek için dinamik ışık saçılımı (DLS) kullanın. </li><li> Numunelerin ölçümleri önce, standart hazırlanması ve ölçümü için, imalatçının protokolü takip eden iki bilinen standartların parçacık boyutlarına ölçer. </li><li> Standart çözeltinin 1,5 ml &#39;si ile, cam küvet doldurun ve örnekleri gibi aynı ölçüm koşulları kullanılarak standart solüsyonu ölçer. Not: Önceki adım, her standart için tekrarlandı. </li><li> Hazırlanan numuneler ölçün. Her biri için, aşağıdaki deney parametreleri kullanın: 100 saniye, suyun kırılma indeksi (1.33), su viskozitesi bir çalışma süresi 20 ° C (1,002 mPa saniye). Not: Bu teknik frekans nanopartiküllerin boyutunu ölçmek için ışık kaymıştır kullanır. </li><li> Partiküller ortalama parçacık çapı ve poli dağılım raporcle boyut dağılımı. </li></ol></li><li> Erime Noktası ve erime gizli ısısı <ol><li> SLNs 1 termal davranışını incelemek için bir otomatik numune alıcı ve sıvı nitrojen soğutma kaynağı ile donatılmış bir diferansiyel taramalı kalorimetre (DSC) kullanarak. </li><li> Yaklaşık 25 mg değerinde bir kütle ile 40 ul alüminyum tavaya olarak hazırlanmış SLNs pipet ve hava geçirmez bir şekilde DSC taraması sırasında nem kaybını en aza indirmek için evrensel bir kıvırma presi kullanılarak pan mühür. </li><li> 5-80 ° C arasında, her nanoemülziyon ölçün. </li><li> Erime noktası ve vadi erime noktasına ve malzeme miktarına bölünmesiyle DSC arsa eğri altında kalan alan integralini temsil DSC arsa gelen erime latent ısı erime için gerekli gizli ısının temsil bildirin. </li></ol></li></ol> Fibroblastları ve dendritik hücrelerle 2. SLNs Etkileşim <ol><li> Primer insanlara özgü fibroblastlar Kültür <ol><li> Kültür primer insan fibroblasts düşük serum büyüme ek (LSGS) kiti ile desteklenmiş (106 ortamı), insan dermal fibroblastlar, kültürü için sıvı ortam içinde üreticinin talimatlarına göre aktifleştirilir. % 80 kaynaşmaya eriştikten sonra 37 ° C&#39;de,% 5 CO2 ve alt-kültür nemlendirilmiş bir atmosferde hücreleri koruyun. </li><li> MTT ve görüntüleme oyuk başına 2 x 10 4 hücre yoğunluğunda analizi bir 96-çukurlu plaka içinde tohum hücrelerinde, ve 37 ° CO / N önceden SLN maruz en dengelenmeye olanak her ikisi için. </li><li> Lipit konsantrasyonuna göre (Şekil 4) belirtilmiştir, ve her bir oyuk başına 10 ul olarak Sıfır zamanında, tam bir ortamda SLNs seyreltik 96 oyuklu plakanın üzerindeki çukurların çoğaltırlar. </li><li> Kuluçka süresi boyunca, 37 ° C&#39;de,% 5 CO2 hücreleri koruyun. </li></ol></li><li> MTT Deneyi Toksisite <ol><li> SLNs inkübasyonu 24 saat sonra, üreticinin protokolüne uygun olarak, MTT deneyi kullanılarak hücre canlılığı belirler. &lt;/ li&gt; <li> Bir kez hücre yıkayın ve her bir oyuğa (100 ul / çukur) taze ortam ilave edin. Taze ortam ile değiştirmeden önce 10 dakika boyunca% 70&#39;lik bir metanol çözeltisi içinde inkübe edilerek kontrol kuyuları öldürmek. </li><li> Mikrolevhanın her çukuruna MTT ayıracı (oyuk başına 10 ul) ilave edin ve 37 ° C &#39;de O / N inkübe edin. </li><li> O / N inkübasyondan sonra, üreticinin protokolüne göre elde edilen bir deterjan çözeltisi ile hücre içi formazin kristaller (oyuk başına 100 ul) çözünür. </li><li> Oda sıcaklığında kuluçkalamadan 3 saat sonra, bir mikroplaka okuyucu kullanılarak 570 nm&#39;lik bir dalga boyunda ölçülmesi absorbans değerlerini elde. Not: 3- (4,5-Dimetiltiazolil-2) -2, 5-difenil-tetrazolyum bromid (ağırlık / hacim)% 1&#39;den daha az olmalıdır kullanılan MTT ayıracı. </li></ol></li><li> Floresan Mikroskopi <ol><li> SLN olan fibroblastların uygulamayı takiben belirli bir zaman noktalarında, steril fosfat tamponlu salin (PBS) ile iki kez yıkama fibroblastlar ve soğuk% 70 metanol ile 10 dakika boyunca sabitlemek. </li><li> 10 dakika sonraBir grup geçiş (BP) 690/50 nm filtre seti kullanarak, ters bir mikroskop kullanılarak PBS ve yakalama görüntüleri ile metanol değiştirin. </li></ol></li><li> Fare Kemik İliği Hücrelerinin Kültürü Dendritik <ol><li> Indükleyin fare kemik iliğinden elde edilen dendritik hücreler (BMDC) halinde, daha önce tarif edildiği 23,24. Kısaca, yeniden birleştirici sıçangil granülosit-makrofaj koloni stimüle edici faktör (GM-CSF, 300 U / ml) ve rekombinant fare IL-4 ile 2 x 10 6 / plaka 100 mm Petri tabaklarda C57BL / 10 kemik iliği levhası tek hücre süspansiyonları (B hücre faktörü, 200 U / ml uyarıcı). 3 günde ortamı değiştirmek. </li><li> 7. günde, 37 ° C hücreleri 0,5 ve 5,0 ug / ml lipid nihai konsantrasyonda yemekleri NIR yüklü SLNs eklemek ve korumak, maruz kalma istenen uzunluğu için,% 5 CO2 kuluçka makinesine kondu. </li><li> Plakasının ayrıntılı yıkanır, 50 ml&#39;lik bir tüp içinde toplanacak şekilde plakanın tüm ortamı aspire yemekler hücreleri toplamak (gevşek reklam çıkarmak için4 mM etilendiamintetraasetik asit (EDTA) içeren fosfat tamponlu tuzlu su, 5 ml (PBS) herent hücreleri) içerir. Aynı 50 ml bir tüp içinde elde edilen hücre süspansiyonu toplanır. </li></ol></li><li> Akım Sitometrisi aracılığıyla SLNs Kuruluş Değerlendirilmesi <ol><li> Alanına 2.4.3 &#39;de elde edilen hücre süspansiyonu konsantrasyonunu belirlemek ve 12 mm x 75 mm yuvarlak tabanlı tüp içine FACS 3x10 5 hücreleri dağıtın. </li><li> 1 ml PBS ilave edilmesi ve daha sonra 4 ° C arasındaki bir sıcaklıkta, 400 x g&#39;de 5 dakika boyunca santrifüje tabi tüpler hücreleri yıkayın. </li><li> Süpernatant atılır ve anti-CD11c-FITC monoklonal antikoru (0.25 ug / ml nihai) ihtiva eden PBS / 2 mM EDTA /% 0.1 sığır serum albümini, 100 ul (BSA) (boyama tamponu) pelletini. Karanlıkta buz üzerinde 30 dakika süreyle inkübe edilir. </li><li> 1 ml PBS ile hücreleri yıkanır ve daha sonra 4 ° C arasındaki bir sıcaklıkta, 400 x g&#39;de 5 dakika boyunca santrifüj tüpleri. </li><li> Süpernatantı atın ve her tekrar süspansiyonlekeleme tampon maddesi içinde 400 ul pelet. Hücreler artık bir akış sitometresinin (muhtemelen mavi ve kırmızı lazer ile donatılmış) örneklerin edinimi için hazırız. </li><li> Analiz yazılımı kullanılarak akış sitometrisi, CD11c + hücreleri (FITC pozitif) nüfusu üzerindeki yolluk ve (PerCP Cy5.5-veya APC kanalları olarak ölçülür) SLNs ilişkili floresans yoğunluğunu karşılaştırarak DC ile SLNs dahil edilme düzeyini değerlendirmek. </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.

Bu çalışmada, SLNs sentezi ve hücre içi uygulamalar için hedefleme uygulanabilirlikleri incelenmiştir. Bu biyouyumlu nanopartiküller ilaç dağıtımı, gen susturma ve aşı teknolojilerine 25-30 olmak üzere birden fazla uygulama için dağıtım araçları olarak söz göstermiştir. Ultra küçük SLNs bir uyduruk işlemi kullanılarak sentezlendi ve primer deri hücreleri ve primer immün hücreleri ile etkileşimleri incelenmiştir. SLNs model terapötik kargo olarak görev yapan bir floresan bo...

Nanoparçacık tabanlı dağıtım araçları, özellikle hücresel sinyalizasyon ve gen ifadesini değiştirmek için bir mekanizma sağlayarak, hücre içi hedefleme uygulamalar için büyük umut göstermiştir. Bu araçlar, ilaç, proteinler, hem de hücresel yanıtları etkileyecek ve hedef dokularda arzu edilen bir etkiyi elde etmek için tasarlanmış bir nükleik asit ile yüklenebilir. Nanocarriers birçok tipi lipidler, polimerler, silisyum ve manyetik malzemeler de dahil olmak üzere terapötik ve diagnostik yararı için incelenmiştir. Bu sistemler bir terapötik hedef dokularda konsantrasyonu, ve sistemik toksisite azalmasına sebep lokalize ilaç dağıtımı için potansiyeli, için çekicidir. Katı lipid nanoparçacıklarının (SLNs) son yıllarda umut verici bir ilaç verme vasıtası olarak ortaya çıkan bir nanopartikül verme sisteminin iyi çalışılmış örnektir. SLNs hali hazırda biyo-algılayıcı 1, kozmetik 2 ve t de dahil olmak üzere çok sayıda uygulama için formüle edilebilirherapeutic teslimat 3-7. Onların yarar onlar gelişmiş biyouyumluluk sonuçlanan emilebilir, toksik olmayan lipitlerin tamamen oluşmaktadır gerçeğinden kaynaklanmaktadır. Sentez sırasında, lipofilik ilaçlar böylece parenteral uygulama için ilacın çözünürlüğünü ve uygunluğu, artan SLN araç içine dahil edilebilir. SLN araçlar aynı zamanda bozulması ve açıklığını azaltarak ve terapötik etkisini maksimize kapsüllü terapötikler stabilize etmek için yardımcı olur. Bu araçlar sayesinde vücut sıcaklığı 3,4,8,9 de stabiliteleri özellikle uzun etkili, kontrollü salım preparasyonlar için uygundur. Önemli olarak, lipid nanoparçacıklarının ilaç kapsülleme ilaç moleküllerin içsel farmakokinetik profiller değiştirir. Bu dar bir terapötik indeksi olan ilaçların kontrollü bir salınıma izin veren, bir potansiyel bir avantaj sağlar. SLN-dahil terapötik salım hızı ayarlanmış lipid bozunum hızı ya da ilaç yayılma hızına dayalı olabilirlipit matris. SLNs genellikle belirli hedef dokularda birikir tasarlanmıştır. Örneğin, kendi boyutu (tipik olarak daha fazla 10 nm), tümör dokusunun sızdıran damar birikimi kolaylaştıran dolaşım içinde tutma güçlendirir. Buna ek olarak, partikül uygulama yolu gibi lenf düğümleri 10,11 gibi özel fizyolojik yapılar hedef potansiyeli olan bir biyo dağılıma değiştirmek için gösterilmiştir. Hedef dokularda birikimi, uygun hücresel etkileşimi sağlamak ve nanopartiküllerin nihai bunun üzerine bağlı seçici hücre içine ve 12 üzerinden iyon ve moleküllerin akışını kontrol etmek için hücre zarlarının yeteneği nedeniyle zordur. Hücresel alımı kolaylaştırmak için, peptidler, küçük moleküller, ve monoklonal antikorlar da dahil olmak üzere 13,14 spesifik ligandlarla nanocarriers değiştirmek mümkündür. Pasif penetrasyon ve nanopartiküllerin aktif ulaşım de dahil olmak çeşitli mekanizmalarhücre zarı boyunca, daha önce 3,12,15 tarif edilmiştir. Genel olarak, bu, hücre-nanopartikül etkileşimleri, hücre tipi veya hücre döngüsü faz 12 olarak hücreye özel parametrelere ek olarak, boyut, şekil, yüzey yükü ve yüzey kimyası da dahil olmak üzere nanopartiküller fizikokimyasal özellikleri tarafından etkilendiği gösterilmiştir. Bir önceki soruşturma aşaması inversiyon sıcaklığı (PIT) yöntemini kullanarak 17 16 topikal ve biyomarker algılama uygulamaları 1 için alt 10 nm SLNs sentezini gösterdi. Bu sıcaklık yavaş yavaş değişir ise bileşimi aynı kalır 2 hafif bir sentez yöntemidir. Isıtılmış çözeltinin sürekli karıştırma, gibi nanoemülziyon RT sonuçlarına soğur. Daha küçük bir partikül boyutuna sahip 1 SLNs sentezinde Bu işlem, daha önce sonuçlar lipid nan sentezi için çeşitli yöntemler kullanılarak raporoparticles 17-22. Elde edilen boyut ölçeği, en az 20 nm, artan yüzey alanı ve gelişmiş bir hücresel etkileşimler için potansiyeli, hücre içi hedef uygulamalar için avantaj sağlamaktadır. Bir floresan boya veya terapötik sağlamak üzere tasarlanmıştır SLNs şematik, Şekil 1 &#39;de gösterilmiştir. SLNs bir lipit iç (örneğin, lineer alkan) lipofilik bileşiklerin (örneğin, boyalar veya terapötik) dahil edilmesine izin veren ve bir yüzey aktif dış oluşmaktadır su ile çevrili (örneğin, doğrusal noniyonik yüzey aktif madde). Bu çalışmada, SLNs bir floresan boya ile yüklenir ve partikül-hücre etkileşimleri incelemek için bir model olarak kullanılmıştır. Primer insanlara özgü dermal fibroblastlar ve fare dendritik hücreler toksisite ve parçacık alımı ile ilgili etkileşimler karakterize etmek amacıyla zaman SLN yüklü boyamaktadır maruz bırakıldı. A: 3- (4,5-dimetiltiazol-2-il) -2,5-diphenylphenyltetrazolium bromid (MTT) deneyi utili olduUygun doz seviyelerinin kurmak amacıyla zed. Floresan mikroskobu ve akım sitometri in vitro parçacık alımını incelemek için kullanılan iki yöntem vardı. <img alt="figür 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> Önemli bileşenlerini gösteren SLN Şekil 1. şematik. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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 yöntemi SLNs sentezlemek için kullanılmış ve faz tersinmesi sıcaklık bir su banyosu kullanılarak belirlenmiştir. Numuneler yavaş yavaş ısıtılır ve çözelti berrak göründü kadar yavaşça ajite edildi. Heneicosane lipid kullanılarak yapılan SLNs için faz tersinmesi sıcaklığı 45 ° C. Tablo 1 parçacık boyutuna, polidispersiteye, erime noktası ve SLNs erimeye latent ısı özetlemektedir. Yukarıda tarif edilen işlem koşulları kullanılarak sentezlenmiştir SLNs 18,59 ve...

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.

Katı Lipit nanopartiküller (SLNs) Hücre içi Hedefleme için başvurular

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 Görüntüleme.  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. 

Video: 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.

Viral Nanopartiküller<em&gt; In vivo</em&gt; Tümör Görüntüleme

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

Biyomühendislik

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.

Sağlam, mikroakışkan Hücre içi Delivery Platform olarak hücre Sıkma

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.

Rejeneratif Araştırma harmonik nanopartiküller

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.

Üretim ve Monovalent Kuantum Noktalarının Hedefleme

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.

Modlu Moleküler Görüntüleme Uygulamaları için Biofunctionalized Prusya Mavisi Nanopartikülleri

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.

Hücre Etiketleme ve Süperparamanyetik Demir Oksit Nanopartiküller ile Hedefleme

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.

İpek Nanopartiküller imalatı ve İlaç Dağıtım Uygulamaları

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.

Falcarindiol tarafından insan mezenkimal kök hücre içeren yeni Lipid kaplı nano tanecikleri alımını

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.

Kanser Hücre Kültürlerinden Toplanan Ortamda Sıvı Kromatografi-Kütle Spektrometresi ile Analiz Edilen Seçilmiş Kynureninlerin Eşzamanlı Nicelemesi

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.

Mikroakışkan Karıştırma Platformu Kullanarak Gen Teslimatı için Lipid Nanopartiküllerinin Formülei ve Karakterizasyonsu

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