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.

1. Traitement des SLN <ol><li> Synthèse de SLN Remarque: Utilisez la méthode PIT 1,17,20 pour préparer les sous-SLN 1 20 nm. <ol><li> Utiliser une technique aseptique, bioréactifs ou culture cellulaire réactifs de qualité, et les matériaux stériles (ie, pipettes, spatules, flacons, etc.). </li><li> Utilisez une armoire de biosécurité pour la synthèse de SLN (Figure 2). </li><li> Moissonneuse 0,6 mg de colorant fluorescent (rouge Nil (NIR), la concentration NiR est d&#39;environ 0,3 mg / ml) et 0,10 g de heneicosane (lipides, 5% poids / poids concentration des lipides) dans un flacon (15 ml), co-fusion à 90 ° C, et remuer. </li><li> Ajouter 0,11 g de polyoxyéthylène (10) oléyl éther (tensioactif, 5,5% poids / poids tensioactif concentration). Co-fondre le mélange obtenu à 90 ° C et remuer. </li><li> Ajouter 1,79 g d&#39;eau stérile pour le mélange, la chaleur à 90 ° C, et remuer jusqu&#39;à une nanoémulsion transparente est formé. Remarque: Sous agitation continue et refroidissement contrôlé, SLNs sont créés. Ces conditions provoquent une inversion de l&#39;émulsion eau-dans-huile à une émulsion huile-dans-eau. Les molécules lipophiles telles que la partition NiR aux gouttelettes lipidiques. </li><li> Préparer une nanoémulsion en parallèle sans addition du colorant fluorescent, afin de servir comme un échantillon de contrôle. Mélanger 0,10 g de heneicosane et 0,11 g de polyoxyéthylène (10) oléyléther dans un flacon (15 ml) co-fusion à 90 ° C, et remuer. <ol><li> Ajouter 1,79 g d&#39;eau stérile pour le mélange, la chaleur à 90 ° C, et remuer jusqu&#39;à une nanoémulsion transparente est formé. </li></ol></li><li> Stériliser plus, chaque nanoémulsion l&#39;aide d&#39;un filtre de 0,2 um stérile. </li></ol><img alt="Figure 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> Figure 2. Schéma de synthèse de SLN. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> </li><li> Taille des particules et Polydispersity <ol><li> Utilisez diffusion de lumière dynamique (DLS) pour mesurer la taille des particules et la polydispersité des SLN 1 en utilisant une cuvette de verre et un instrument conçu pour mesurer la taille des particules. </li><li> Avant les mesures des échantillons, mesurer les tailles de particules de deux normes connues suivant le protocole du fabricant pour la préparation des normes et de la mesure. </li><li> Remplir la cuve en verre avec 1,5 ml de la solution étalon et de mesurer la solution standard en utilisant les mêmes conditions de mesure que les échantillons. Remarque: l&#39;étape précédente a été répétée pour chaque norme. </li><li> Mesurer les échantillons tel que préparé. Pour chacun, utiliser les paramètres expérimentaux suivants: un temps de fonctionnement de 100 s, indice de réfraction de l&#39;eau (1.33), la viscosité de l&#39;eau à 20 ° C (1.002 mPa.s). Remarque: Cette technique utilise une fréquence décalée lumière pour mesurer la taille des nanoparticules. </li><li> Signaler le diamètre de particule moyen et la polydispersité de partidistribution de taille de cle. </li></ol></li><li> Point de fusion et la chaleur latente de fusion <ol><li> Utiliser un calorimètre à balayage différentiel (DSC) équipé d&#39;un auto-échantillonneur et la source de refroidissement à l&#39;azote liquide à étudier le comportement thermique de 1 SLN. </li><li> Introduire à la pipette les SLN comme préparés dans une casserole en aluminium de 40 pi avec une masse d&#39;environ 25 mg et fermer hermétiquement la casserole à l&#39;aide d&#39;une presse à sertir universelle pour minimiser la perte d&#39;humidité lors de l&#39;analyse de DSC. </li><li> Mesurer chaque nanoémulsion de 5 à 80 ° C. </li><li> Déclarez le point de fusion et la chaleur latente de fusion de la parcelle de DSC, où la vallée représente le point de fusion et de l&#39;intégrale de l&#39;aire sous la courbe dans la parcelle de DSC divisée par la quantité de matière représente la chaleur latente de fusion. </li></ol></li></ol> 2. SLN Interaction avec fibroblastes et les cellules dendritiques <ol><li> Culture de fibroblastes humains primaires <ol><li> Culture fi humaine primairefibroblastes selon les instructions du fabricant en milieu liquide pour la culture de fibroblastes dermiques humains (moyenne 106), complétés avec le kit supplément faible croissance de sérum (LSGS). Maintenir les cellules dans une atmosphère humidifiée à 37 ° C, 5% CO 2 et de sous-culture après avoir atteint 80% de confluence. </li><li> Pour les deux MTT et l&#39;analyse de l&#39;imagerie, les cellules de semences dans une plaque à 96 puits, à une densité de 2 x 10 4 cellules par puits, et ramener à 37 ° CO / N avant l&#39;exposition SLN. </li><li> Au temps zéro, diluer SLN dans un milieu complet comme indiqué (Figure 4), par rapport à la concentration de lipides, et ajouter 10 ul par puits à chaque répliquer bien dans la plaque de 96 puits. </li><li> Maintenir les cellules à 37 ° C, 5% CO 2, au cours de la période d&#39;incubation. </li></ol></li><li> MTT Essai de toxicité <ol><li> Après 24 h d&#39;incubation avec SLN, déterminer la viabilité cellulaire en utilisant le test MTT, selon le protocole du fabricant. &lt;/ li&gt; <li> Laver les cellules une fois et ajouter du milieu frais à chaque puits (100 pl / puits). Tuer puits témoins par incubation dans une solution de methanol à 70% pendant 10 min avant de le remplacer par du milieu frais. </li><li> Ajouter le réactif MTT (10 pi par puits) à chaque puits de la microplaque et incuber O / N à 37 ° C. </li><li> Après O / N incubation, solubiliser cristaux de formazin intracellulaires avec la solution de détergent fourni (100 pi par puits) selon le protocole du fabricant. </li><li> Après 3 h d&#39;incubation à TA, obtenir des valeurs d&#39;absorbance à une longueur d&#39;onde de mesure de 570 nm en utilisant un lecteur de microplaque. Réactif MTT utilisée doit être inférieure à 1% (p / v) 3- (4,5-dimethylthiazolyl-2) -2, le bromure de 5-diphényl-tétrazolium: Remarque. </li></ol></li><li> Microscopie à fluorescence <ol><li> A des moments spécifiques suivants dosage des fibroblastes avec la SLN, laver les fibroblastes deux fois avec un tampon phosphate salin stérile (PBS), et fixer pendant 10 min avec 70% de méthanol froid. </li><li> Après 10 min, Remplacer le méthanol avec du PBS et de capturer des images à l&#39;aide d&#39;un microscope inversé, en utilisant un passe-bande (BP) 690/50 nm de jeu de filtres. </li></ol></li><li> Culture de cellules dendritiques de moelle osseuse de souris <ol><li> Induire osseuse de souris des cellules dendritiques dérivées de moelle (BMDC) tels que décrits précédemment 23,24. Brièvement, suspensions plaque monocellulaires de C57BL / 10 moelle osseuse à 100 mm boîtes de Petri à 2 x 10 6 / plaque avec murin facteur stimulant les colonies de granulocytes-macrophages recombinant (GM-CSF, 300 U / ml) et murine IL-4 recombinante (cellules B facteur de stimulation, 200 U / ml). Changer de support tous les 3 jours. </li><li> Au jour 7, ajouter SLN NIR chargés aux plats à une concentration finale de 0,5 et 5,0 ug / ml de lipide et de maintenir les cellules dans un 37 ° C, 5% de CO 2 incubateur pendant toute la durée de l&#39;exposition désiré. </li><li> Recueillir des cellules de la vaisselle en aspirant tous les médias de la plaque, en la recueillant dans un tube de 50 ml, suivi par un lavage soigneux de la plaque (pour déloger annonce lâcheHerent cellules) avec 5 ml de solution saline tamponnée au phosphate (PBS) contenant 4 mM d&#39;acide éthylènediaminetétraacétique (EDTA). Recueillir la suspension cellulaire obtenue dans le même tube de 50 ml. </li></ol></li><li> Évaluation de la SLN Incorporation par cytométrie en flux <ol><li> Déterminer la concentration de la suspension cellulaire obtenue au point 2.4.3 et distribuer 3x10 5 cellules dans un 12 mm x 75 mm à fond rond FACS tube. </li><li> Laver les cellules en ajoutant 1 ml de PBS, puis centrifuger les tubes pendant 5 min à 400 x g, à une température de 4 ° C. </li><li> Jeter le surnageant et remettre en suspension le culot avec 100 ul d&#39;EDTA / 0,1% d&#39;albumine de sérum bovin PBS / 2 (BSA) (tampon de coloration) contenant des anticorps anti-CD11c monoclonal FITC (0,25 ug / ml final). Incuber pendant 30 min sur de la glace dans l&#39;obscurité. </li><li> Laver les cellules avec 1 ml de PBS, puis centrifuger les tubes pendant 5 min à 400 x g, à une température de 4 ° C. </li><li> Jeter le surnageant et remettre chaqueun culot avec 400 ul de tampon de coloration. Les cellules sont maintenant prêts pour l&#39;acquisition des échantillons avec un cytomètre de flux (peut-être équipé avec des lasers bleus et rouges). </li><li> Utilisation de la cytométrie de flux logiciel d&#39;analyse, d&#39;évaluer le niveau d&#39;incorporation de SLN par DC par gating sur la population de CD11c + cellules (FITC positif) et en comparant l&#39;intensité de la SLN-associés fluorescence (mesurée dans les canaux PerCP-Cy5.5 ou APC). </li></ol></li></ol>

<ol>
	<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.

Dans cette étude, la synthèse de la SLN et de leur applicabilité pour les applications ciblage intracellulaire ont été explorées. Ces nanoparticules biocompatibles ont montré des résultats prometteurs en tant que véhicules de livraison pour de multiples applications, y compris l&#39;administration de médicaments, le silençage génique, et technologies vaccinales 25-30. Ultra-petits SLN ont été synthétisés en utilisant un processus facile, et leurs interactions avec les cellules de la peau et de...

Véhicules de livraison à base de nanoparticules ont montré de grandes promesses pour les applications ciblage intracellulaire, fournissant un mécanisme de modifier spécifiquement la signalisation cellulaire et l&#39;expression des gènes. Ces véhicules peuvent être chargés avec des médicaments, des protéines et des acides nucléiques destinés à influer sur les réponses cellulaires et obtenir un effet souhaité dans les tissus cibles. De nombreux types de nanocarriers ont été explorées pour un bénéfice thérapeutique et de diagnostic, y compris les lipides, les polymères, le silicium, et les matières magnétiques. Ces systèmes sont attrayants en raison de leur potentiel pour la livraison localisée de médicaments, augmentation de la concentration thérapeutique dans les tissus cibles, et la réduction de la toxicité systémique. Nanoparticules lipidiques solides (SLN) en sont un exemple bien étudié d&#39;un système de livraison de nanoparticules qui a émergé comme un véhicule de livraison de médicament prometteur au cours des dernières années. SLN peuvent être facilement formulés pour de multiples applications, y compris la bio-détection 1, cosmétiques 2, et tlivraison herapeutic 3-7. Leur utilité réside dans le fait qu&#39;ils sont constitués exclusivement d&#39;résorbables, des lipides non toxiques, ce qui entraîne une meilleure biocompatibilité. Au cours de la synthèse, les médicaments lipophiles peuvent être incorporées dans des véhicules SLN, ce qui augmente la solubilité du médicament et de l&#39;aptitude à l&#39;administration parentérale. SLN véhicules contribuent également à stabiliser thérapeutiques encapsulés, la réduction de leur dégradation et de dégagement, et de maximiser l&#39;action thérapeutique. Ces véhicules sont particulièrement bien adaptés pour longue durée d&#39;action, à libération contrôlée de préparations en raison de leur stabilité à 3,4,8,9 de la température du corps. Fait important, l&#39;encapsulation de médicaments dans des nanoparticules lipidiques modifie les profils pharmacocinétiques intrinsèques des molécules de médicament. Ceci permet d&#39;obtenir un avantage potentiel en permettant la libération contrôlée de médicaments ayant un indice thérapeutique étroit. Le taux de la thérapeutique de la SLN incorporée libération peut être réglé en fonction du taux de dégradation des lipides ou de la vitesse de diffusion de la drogue dans lematrice lipidique. SLN sont souvent conçus pour accumuler dans les tissus cibles spécifiques. Par exemple, leur taille (typiquement supérieure à 10 nm) potentialise la rétention dans la circulation, où le système vasculaire qui fuit de tissu tumoral facilite le dépôt. En outre, la voie d&#39;administration de particules a été montré pour modifier la biodistribution avec le potentiel de cibler des structures physiologiques spécifiques tels que les ganglions lymphatiques 10,11. Après dépôt dans les tissus cibles, la réalisation des interactions cellulaires appropriées et internalisation éventuelle des nanoparticules est difficile en raison de la capacité de la membrane cellulaire pour commander sélectivement le flux d&#39;ions et de molécules dans et hors de la cellule 12. Afin de faciliter l&#39;absorption cellulaire, il est possible de modifier nanocarriers avec des ligands spécifiques, y compris des peptides, des petites molécules et des anticorps monoclonaux, 13,14. Plusieurs mécanismes, y compris à la fois la pénétration passive et le transport actif de nanoparticulesà travers la membrane cellulaire ont été précédemment décrite 3,12,15. En général, il a été démontré que les interactions cellule-nanoparticules sont influencées par les propriétés physico-chimiques des nanoparticules, y compris la taille, la forme, la charge de surface et de chimie de surface, en plus des paramètres spécifiques de cellules telles que le type de cellule ou cellule phase du cycle 12. Une enquête précédente a démontré la synthèse de sous-SLN 10 nm pour les topiques 16 et biomarqueurs applications de détection 1 selon la méthode 17 température d&#39;inversion de phase (PIT). Ceci est un procédé de synthèse doux 2 ​​où la composition reste constante tandis que la température est progressivement modifiée. Sous agitation continue de la solution chauffée, lors de son refroidissement à RT résultats dans une nanoémulsion. Ce procédé conduit à la synthèse de la SLN de plus petite taille de particule de 1 que précédemment rapporté en utilisant divers procédés pour la synthèse de lipides nanoparticles 17-22. L&#39;échelle de taille résultante, inférieure à 20 nm, fournit un avantage pour les applications de ciblage intracellulaire due à l&#39;augmentation de la surface et le potentiel d&#39;interactions cellulaires améliorées. Un schéma de SLN, conçu pour fournir un colorant fluorescent ou thérapeutique, est représenté sur la Figure 1. Les SLN sont constitués d&#39;un intérieur (par exemple alcane linéaire) qui permet l&#39;incorporation de composés lipophiles (par exemple, des colorants ou des agents thérapeutiques) et un extérieur de tensioactif lipidique (tensioactif non ionique, par exemple, linéaire) entourée par l&#39;eau. Dans cette étude, SLN ont été chargées avec un colorant fluorescent et utilisées comme modèle pour étudier les interactions de particules cellulaires. Fibroblastes dermiques humains primaires et les cellules dendritiques de souris ont été exposées à colorant chargé SLN au fil du temps afin de caractériser les interactions par rapport à la toxicité et l&#39;absorption de particules. Un dosage 3- (4,5-diméthylthiazol-2-yl) -2,5-de diphenylphenyltetrazolium (MTT) est utilized afin d&#39;établir les niveaux de dosage appropriées. La microscopie à fluorescence et la cytométrie en flux, deux méthodes employées pour étudier l&#39;absorption de particules in vitro. <img alt="Figure 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> Figure 1. Schéma de la SLN montrant les principaux constituants. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</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.

La méthode de PIT a été utilisé pour synthétiser les SLN et la température d&#39;inversion de phase a été déterminée en utilisant un bain d&#39;eau. Les échantillons ont été chauffés lentement et doucement agitée jusqu&#39;à ce que la solution est apparue clairement. La température d&#39;inversion de phase pour les SLN faites en utilisant des lipides heneicosane est de 45 ° C. Le tableau 1 résume la taille des particules, polydispersité, point de fusion et la chaleur latente de la fo...

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.

Solide Lipid nanoparticules (SLN) Applications pour le ciblage intracellulaire

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 vues.  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. 

Vidéo: 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.

Les nanoparticules virales pour<em&gt; In vivo</em&gt; Imagerie des tumeurs

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

Recherche

Bio-ingénierie

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.

Presser cellule comme, microfluidique Delivery Platform intracellulaire robuste

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.

Harmonique nanoparticules régénératrice recherche

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.

La production et le ciblage des monovalent Quantum Dots

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.

Biofunctionalized bleu de Prusse nanoparticules pour l&#39;imagerie moléculaire multimodale Applications

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.

Cellule étiquetage et le ciblage des nanoparticules d&#39;oxyde de fer Superparamagnetic

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.

Fabrication and Drug Delivery Applications de soie Nanoparticules

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.

Absorption de nouvelles nanoparticules lipides enduit contenant Falcarindiol par des cellules souches mésenchymateuses humaines

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.

Quantification simultanée de kynurenines sélectionnées analysées par chromatographie liquide-spectrométrie de masse dans le milieu recueilli à partir des cultures de cellules cancéreuses

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.

Formulation et caractérisation de nanoparticules lipidiques pour la livraison de gènes à l’aide d’une plate-forme de mélange microfluidique

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