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. Il trattamento di linfonodi sentinella <ol><li> Sintesi di linfonodi sentinella Nota: Utilizzare il metodo PIT 1,17,20 per preparare i sub-20 nm linfonodi sentinella 1. <ol><li> Utilizzare una tecnica asettica, bioreagents o reagenti di colture cellulari di qualità e materiali sterili (cioè, pipette, spatole, fiale, ecc.) </li><li> Utilizzare un armadio biosicurezza per la sintesi dei linfonodi sentinella (Figura 2). </li><li> Combinare 0,6 mg di colorante fluorescente (Nilo Red (NIR), concentrazione NiR è di circa 0,3 mg / ml) e 0,10 g di Heneicosane (Lipid, 5 peso / peso% concentrazione di lipidi) in una fiala (15 ml), co-fonde a 90 ° C, e mescolate. </li><li> Aggiungere 0,11 g di poliossietilene (10) oleil etere (tensioattivo, 5.5 wt / wt% concentrazione di tensioattivo). Co-melt la miscela risultante a 90 ° C e mescolare. </li><li> Aggiungere 1,79 g di acqua sterile alla miscela, riscaldare a 90 ° C, e mescolare fino ad avere una nanoemulsione trasparente. Nota: In continua agitazione e raffreddamento controllato, SLNs vengono creati. Queste condizioni provocano un&#39;inversione della emulsione acqua-in-olio ad una emulsione olio-in-acqua. Molecole lipofile quali NiR partizione per le gocce lipidiche. </li><li> Preparare una nanoemulsione in parallelo senza aggiungere il colorante fluorescente, per servire come campione di controllo. Combinare 0,10 g di Heneicosane e 0,11 g di poliossietilene (10) oleil etere in un flacone (15 ml) co-fuso a 90 ° C, e mescolate. <ol><li> Aggiungere 1,79 g di acqua sterile alla miscela, riscaldare a 90 ° C, e mescolare fino ad avere una nanoemulsione trasparente. </li></ol></li><li> Ulteriori sterilizzare ogni nanoemulsione utilizzando una sterile 0,2 micron filtro. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> Figura 2. Schema di sintesi di linfonodi sentinella. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> </li><li> Dimensione delle particelle e Polydispersity <ol><li> Utilizzare light scattering dinamico (DLS) per misurare la dimensione delle particelle e polidispersità dei linfonodi sentinella 1 utilizzando una cuvetta di vetro e uno strumento progettato per misurare la dimensione delle particelle. </li><li> Prima le misure dei campioni, misurare le dimensioni delle particelle di due standard noti seguendo il protocollo del produttore per la preparazione e la misura standard. </li><li> Riempire la provetta di vetro con 1,5 ml di soluzione standard e misurare la soluzione standard con le stesse condizioni di misura come i campioni. Nota: passo precedente è stato ripetuto per ogni standard. </li><li> Misurare i campioni così come predisposto. Per ciascuna, utilizzare i seguenti parametri sperimentali: un tempo di esecuzione di 100 sec, indice di rifrazione di acqua (1.33), viscosità dell&#39;acqua a 20 ° C (1.002 mPa · sec). Nota: Questa tecnica utilizza frequenza spostata luce per misurare le dimensioni delle nanoparticelle. </li><li> Riportare il diametro delle particelle media e polidispersità di partidistribuzione delle dimensioni cle. </li></ol></li><li> Punto di fusione e di calore latente di fusione <ol><li> Utilizzare un calorimetro a scansione differenziale (DSC) equipaggiato con un autocampionatore e la sorgente di raffreddamento azoto liquido per studiare il comportamento termico dei linfonodi sentinella 1. </li><li> Pipettare i linfonodi sentinella come preparati in una vaschetta di alluminio 40 microlitri con una massa di circa 25 mg e sigillare ermeticamente la padella con una pressa piegatore universale per minimizzare la perdita di umidità durante la scansione DSC. </li><li> Misurare ogni nanoemulsione da 5 a 80 ° C. </li><li> Riportano il punto di fusione e il calore latente di fusione DSC dal terreno, dove la valle rappresenta il punto di fusione e l&#39;integrale della area sotto la curva nel grafico DSC diviso per la quantità di materiale rappresenta il calore latente di fusione. </li></ol></li></ol> 2. linfonodi sentinella Interazione con fibroblasti e cellule dendritiche <ol><li> Cultura di primari fibroblasti umani <ol><li> Cultura primario fi umanofibroblasti secondo le istruzioni del produttore in mezzo liquido per la coltura di fibroblasti dermici umani (media 106), integrati con il kit supplemento bassa crescita nel siero (LSGS). Mantenere celle in atmosfera umidificata a 37 ° C, 5% CO 2 e subcultura raggiungimento 80% di confluenza. </li><li> Per entrambi MTT e analisi di immagini, alveoli in una piastra a 96 pozzetti, ad una densità di 2 x 10 4 cellule per pozzetto, e lasciar equilibrare a 37 ° CO / N prima dell&#39;esposizione SLN. </li><li> Al tempo zero, diluire linfonodi sentinella in terreno completo come indicato (figura 4), ​​rispetto alla concentrazione di lipidi, e aggiungere 10 ml per pozzetto a ciascun replicarsi bene nella piastra a 96 pozzetti. </li><li> Mantenere cellule a 37 ° C, 5% CO 2, durante il periodo di incubazione. </li></ol></li><li> MTT Tossicità Assay <ol><li> Dopo 24 ore di incubazione con linfonodi sentinella, determinare la vitalità cellulare con il saggio MTT, secondo il protocollo del produttore. &lt;/ li&gt; <li> Lavare le cellule una volta e aggiungere mezzo fresco a ciascun pozzetto (100 l / pozzetto). Uccidi pozzetti di controllo incubando in una soluzione di metanolo al 70% per 10 minuti prima di sostituire con terreno fresco. </li><li> Aggiungere il reagente MTT (10 ml per pozzetto) in ciascun pozzetto della micropiastra e incubare O / N a 37 ° C. </li><li> Dopo O / N incubazione, solubilizzare cristalli formazina intracellulari con la soluzione detergente fornito (100 pl per pozzetto) secondo il protocollo del produttore. </li><li> Dopo 3 ore di incubazione a RT, ottenere valori di assorbanza alla lunghezza d&#39;onda di misura di 570 nm con un lettore di micropiastre. Reagente MTT utilizzato deve essere inferiore a 1% (w / v) 3- (4,5-dimethylthiazolyl-2) -2, bromuro di 5-difenil-tetrazolio: Note. </li></ol></li><li> Microscopia a fluorescenza <ol><li> In punti temporali specifici dopo somministrazione di fibroblasti con SLN, lavare i fibroblasti due volte con sterile tampone fosfato salino (PBS) e fissare per 10 minuti con il freddo il 70% di metanolo. </li><li> Dopo 10 min, Sostituire il metanolo con PBS e catturare le immagini utilizzando un microscopio invertito, con una banda passante (BP) 690/50 nm set di filtri. </li></ol></li><li> Cultura di cellule di topo midollo osseo dendritiche <ol><li> Indurre le cellule derivate dal midollo osseo di topo dendritiche (BMDC) come precedentemente descritto 23,24. In breve, le sospensioni piastra singola cellula del C57BL / 10 midollo osseo a 100 mm piastre di Petri a 2 x 10 6 / piastra con il fattore ricombinante murino granulociti-macrofagi stimolante le colonie (GM-CSF, 300 U / ml) e ricombinante murino IL-4 (cellule B fattore, 200 U / ml stimolante). Modificare i media ogni 3 giorni. </li><li> Il giorno 7, aggiungere linfonodi sentinella NiR caricati ai piatti ad una concentrazione finale di 0,5 e 5,0 mg / ml di lipidi e mantenere le cellule a 37 ° C, 5% CO 2 incubatore per la durata di esposizione desiderato. </li><li> Raccogliere cellule dalle stoviglie aspirando tutti i media nel piatto, raccogliendola in un tubo da 50 ml, seguito da lavaggio accurato della piastra (per rimuovere annuncio liberamentecellule Herent) con 5 ml di tampone fosfato salino (PBS) contenente 4 mM di acido etilendiamminotetraacetico (EDTA). Raccogliere la sospensione cellulare ottenuta nello stesso tubo 50 ml. </li></ol></li><li> La valutazione di linfonodi sentinella incorporazione tramite Citometria a Flusso <ol><li> Determinare la concentrazione della sospensione cellulare ottenuta al punto 2.4.3 e distribuire 3x10 5 cellule in un fondo rotondo tubo FACS 12 mm x 75 mm. </li><li> Lavare le cellule aggiungendo 1 ml di PBS e quindi i tubi centrifugazione per 5 minuti a 400 xg, ad una temperatura di 4 ° C. </li><li> Eliminare il surnatante e risospendere il pellet con 100 ml di PBS / 2 mM EDTA / 0,1% di albumina sierica bovina (BSA) (tampone colorazione) contenenti anticorpo monoclonale anti-CD11c-FITC (0,25 mg / ml finale). Incubare per 30 min in ghiaccio al buio. </li><li> Lavare le cellule con 1 ml di PBS e centrifugare le provette per 5 minuti a 400 xg, ad una temperatura di 4 ° C. </li><li> Scartare il surnatante e risospendere ognipellet con 400 ml di buffer di colorazione. Le cellule sono ora pronti per l&#39;acquisizione dei campioni con un citometro a flusso (eventualmente dotato di laser blu e rosso). </li><li> Utilizzando il software di analisi di citometria a flusso, valutare il livello di incorporazione di linfonodi sentinella da DC dal gating sulla popolazione di CD11c + cellule (FITC positivo) e confrontando l&#39;intensità di linfonodi sentinella-associati fluorescenza (misurata nei canali PerCP-Cy5.5 o APC). </li></ol></li></ol>

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The authors have nothing to disclose.

In questo studio, la sintesi di linfonodi sentinella e loro applicabilità per le applicazioni destinate intracellulari sono state esplorate. Queste nanoparticelle biocompatibili hanno mostrato risultati promettenti come veicoli di consegna per più applicazioni, tra cui la somministrazione di farmaci, silenziamento genico, e le tecnologie di vaccini 25-30. Ultra-piccoli linfonodi sentinella sono stati sintetizzati utilizzando un processo facile, e sono state esplorate le loro interazioni con le cellule della...

Veicoli di consegna basata nanoparticelle hanno mostrato una grande promessa per le applicazioni destinate intracellulari, fornendo un meccanismo per alterare specificamente segnalazione cellulare e l&#39;espressione genica. Questi veicoli possono essere caricati con farmaci, proteine ​​e acidi nucleici progettati per influenzare la risposta cellulare e ottenere un effetto desiderato nei tessuti bersaglio. Molti tipi di nanovettori sono stati esplorati per beneficio terapeutico e diagnostica compresi lipidi, polimeri, silicio e materiali magnetici. Questi sistemi sono interessanti per la loro potenziale per la consegna della droga localizzato, aumento della concentrazione terapeutica nei tessuti bersaglio, e la riduzione della tossicità sistemica. Nanoparticelle lipidiche solide (linfonodi sentinella) sono un esempio ben studiato di un sistema di erogazione di nanoparticelle che è emerso come un veicolo di consegna promettente farmaco negli ultimi anni. Linfonodi sentinella possono essere facilmente formulati per molteplici applicazioni tra cui bio-sensing 1, cosmetici 2, e tconsegna herapeutic 3-7. La loro utilità deriva dal fatto che essi sono costituiti interamente da riassorbibili, lipidi non tossici, con conseguente maggiore biocompatibilità. Durante la sintesi, farmaci lipofili possono essere incorporati in veicoli SLN, aumentando così la solubilità e l&#39;idoneità farmaco per somministrazione parenterale. Veicoli SLN anche aiutare a stabilizzare terapeutica incapsulati, riducendo la loro degradazione e di liquidazione, e massimizzando azione terapeutica. Questi veicoli sono particolarmente adatti per lunga durata d&#39;azione, le preparazioni a rilascio controllato per la loro stabilità alle 3,4,8,9 temperatura corporea. È importante sottolineare che l&#39;incapsulamento dei farmaci in nanoparticelle lipidiche altera i profili farmacocinetici intrinseche delle molecole di droga. Ciò fornisce un vantaggio potenziale permettendo il rilascio controllato di farmaci con un ristretto indice terapeutico. Il tasso di rilascio di terapie SLN-incorporato può essere sintonizzato sulla base del tasso di degradazione dei lipidi o il tasso di diffusione della droga nelmatrice lipidica. Linfonodi sentinella sono spesso progettati ad accumularsi nei tessuti bersaglio specifici. Ad esempio, le loro dimensioni (tipicamente maggiore di 10 nm) potenzia ritenzione nella circolazione, dove la vascolarizzazione del tessuto tumorale leaky facilita la deposizione. Inoltre, la via di somministrazione particella ha dimostrato di alterare biodistribuzione con la possibilità di indirizzare specifiche strutture fisiologiche quali linfonodi 10,11. Dopo la deposizione nei tessuti bersaglio, ottenendo adeguate interazioni cellulari ed eventuale internalizzazione delle nanoparticelle è difficile a causa della capacità delle membrane cellulari per controllare selettivamente il flusso di ioni e molecole dentro e fuori della cella 12. Per facilitare assorbimento cellulare, è possibile modificare nanovettori con leganti specifici inclusi peptidi, piccole molecole, e anticorpi monoclonali 13,14. Diversi meccanismi compresi sia penetrazione passivo e trasporto attivo di nanoparticelleattraverso la membrana cellulare precedentemente descritte 3,12,15. In generale, è stato dimostrato che le interazioni cellula-nanoparticelle sono influenzati dalle proprietà fisico-chimiche delle nanoparticelle tra cui le dimensioni, la forma, carica superficiale e chimica di superficie, oltre ai parametri specifici delle cellule, quali tipo di cellula o cella fase del ciclo 12. Una precedente inchiesta ha dimostrato la sintesi di sub-10 nm linfonodi sentinella per uso topico 16 e biomarcatori applicazioni di rilevamento 1 con il metodo del 17 temperatura di inversione di fase (PIT). Questo è un metodo di sintesi in cui delicato 2 la composizione rimane costante mentre la temperatura viene gradualmente cambiata. Continua agitazione della soluzione riscaldata, mentre si raffredda a risultati RT in una nanoemulsione. Questo processo determina la sintesi di linfonodi sentinella con granulometria inferiore 1 di quella precedentemente riportata utilizzando vari metodi per la sintesi di lipidi nanoparticles 17-22. La scala di dimensioni risulta, a meno di 20 nm, offre un vantaggio per le applicazioni di targeting intracellulare a causa di una maggiore superficie e il potenziale di interazioni cellulari avanzati. Uno schema di linfonodi sentinella, destinata a fornire un colorante fluorescente o terapeutico, è mostrata in Figura 1. I linfonodi sentinella costituiti da un lipide interni (ad esempio, alcano lineare) permettendo l&#39;incorporazione di composti lipofili (ad esempio, coloranti o terapeutici) e un esterno tensioattivo (tensioattivo non ionico ad esempio, lineare) circondato da acqua. In questo studio, linfonodi sentinella sono stati caricati con un colorante fluorescente e usati come modello per studiare le interazioni delle particelle-cellula. Fibroblasti dermici umani primari e le cellule dendritiche di topo sono state esposte per tingere-caricato SLN nel tempo al fine di caratterizzare le interazioni in termini di tossicità e l&#39;assorbimento delle particelle. Un test 3- (4,5-dimetiltiazol-2-il) -2,5-diphenylphenyltetrazolium bromuro (MTT) era Utilized al fine di stabilire adeguati livelli di dosaggio. Microscopia a fluorescenza e citometria a flusso sono stati due metodi utilizzati per esaminare l&#39;assorbimento delle particelle in vitro. <img alt="Figura 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> Figura 1. Schema del linfonodo sentinella che mostra i principali componenti. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</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.

Il metodo è stato utilizzato PIT per sintetizzare i linfonodi sentinella e la temperatura di inversione di fase è stato determinato utilizzando un bagno di acqua. I campioni erano riscaldata lentamente e delicatamente agitata finché la soluzione appariva chiaro. La temperatura inversione di fase per i linfonodi sentinella effettuate utilizzando lipidi heneicosane è di 45 ° C. La tabella 1 riassume la dimensione delle particelle, polidispersità, punto di fusione e calore latente di fusione per i li...

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.

Solid Lipid nanoparticelle (linfonodi sentinella) Applicazioni per intracellulare targeting

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

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JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.8K Views.  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.

Nanoparticelle virali per<em&gt; In vivo</em&gt; Imaging Tumore

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

Ricerca

Bioingegneria

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.

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

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.

Harmonic Nanoparticelle for Regenerative ricerca

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.

Produzione e Targeting di monovalente 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 Prussian Blue Nanoparticelle per applicazioni di imaging multimodale molecolari

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.

Etichettatura delle cellule e Targeting con superparamagnetico ossido di ferro nanoparticelle

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.

Fabbricazione e consegna della droga Applicazioni di seta nanoparticelle

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.

Assorbimento di nuove nanoparticelle rivestite con lipidi contenenti Falcarindiol di cellule staminali mesenchimali umane

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.

Quantificazione simultanea di kynurenine selezionate analizzate dalla cromatografia liquida-spettrometria di massa in mezzo raccolto da colture cellulari tumorali

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.

Formulazione e caratterizzazione di nanoparticelle lipidiche per la somministrazione genica utilizzando una piattaforma di miscelazione microfluidica

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

Methods Article

Solid Lipid nanoparticelle (linfonodi sentinella) Applicazioni per intracellulare targeting