This work was supported by the following grants: NIH-SC3 grant, NSF-PREM grant, Hampton University Faculty Research Grant. We would like to thank Mrs. Michele A. Cochran at Virginia Institute of Marine Science (VIMS) for the use of the particle size analyzer. We would also like to thank Mrs. Corinne R. Ramaley for reviewing the manuscript.

1. Sintesi di DOX-PA <ol><li> Pesare 390 mg di doxorubicina e 243 mg di acido palmitico idrazide, e versare in un pallone a fondo tondo. </li><li> Aggiungere 150 ml di metanolo anidro al pallone con una siringa di vetro. Aggiungere 39 ml di acido trifluoroacetico (TFA) con una pipetta. Utilizzando un agitatore magnetico, agitare la miscela di reazione per 18 ore a RT al buio. NOTA: Le quantità di materiali di reazione possono essere scalati verso l&#39;alto o verso il basso per ottenere diverse quantità di DOX-PA. Il rapporto dei reagenti dovrebbe essere mantenuto nelle stesse proporzioni. Reazioni che utilizzano quantitativi DOX nell&#39;intervallo 78 mg a 1.170 mg possono essere eseguite in un laboratorio chimico regolare. </li><li> Purificazione di DOX-PA usando una colonna di gel di silice. 17 <ol><li> Rimuovere il solvente nella miscela di reazione con un evaporatore rotante. Aggiungere 3 g di gel di silice dopo il volume della miscela si riduce a circa 20 ml. Continuare evaporatore ruotante per produrre polveri secche e permettere tegli adsorbimento di prodotti sul gel di silice. Mantenere il campione sotto vuoto per altri 30 minuti dopo si formano le polveri secche. </li><li> Confezione 50 g di gel di silice in una colonna diclorometano come solvente. Aggiungere con cautela il campione di gel di silice contenente prodotto adsorbito alla colonna. </li><li> Eluire la colonna con una miscela di diclorometano e metanolo, aumentando gradualmente la percentuale di metanolo, aumentando così la polarità del solvente (Tabella 1). </li><li> Raccogliere frazioni di eluente in provette (25 ml / tubo) e monitorare i progressi mediante cromatografia su strato sottile (TLC). </li><li> Combinare tutte le frazioni contenenti puro DOX-PA e rimuovere il solvente usando un evaporatore rotante fino alla formazione di polvere secca. Ulteriori essiccare il prodotto sotto vuoto O / N. </li></ol></li><li> Analisi di DOX-PA da TLC. <ol><li> Tagliare una sezione di quattro centimetri x 8 cm della piastra TLC. soluzioni campione Spot 0,5 cm dal fondo del piatto con TLC capillari avvistamento utilizzando soddisfatteHANOL come solvente. </li><li> Porre la piastra TLC in una camera di sviluppo contenente una miscela di diclorometano e metanolo (3/1, v / v). La profondità del solvente deve essere appena inferiore a 0,5 cm. </li><li> Rimuovere la placca dalla camera di sviluppo quando il fronte del solvente raggiunge la parte superiore della piastra. Segnare la posizione del fronte del solvente con una matita e lasciare la piastra si asciughi. Porre la piastra TLC in una camera di colorazione contenente saturo vapori di iodio per visualizzare campioni. </li></ol></li><li> Analisi di DOX-PA con 1 H-nucleare spettroscopia di risonanza magnetica (1 H-NMR). 18 <ol><li> Sciogliere 15 mg di DOX-PA in 1 ml di metil solfossido-d6 (DMSO) e trasferire il campione in un tubo NMR. </li><li> Inserire il tubo NMR nel magnete dello strumento NMR. Misurare lo spettro protonico, selezionando DMSO come solvente. Rimuovere il tubo NMR dal magnete. Analizzare il risultato NMR 18. </li></ol></li></ol> 2. Preparazione di DOX-PA Le micelle con il metodo Film-dispersione <ol><li> Sciogliere DSPE-PEG (40 mg) e DOX-PA (4 mg) con 2 ml di metanolo in una fiala di vetro da 10 ml. </li><li> Rimuovere il solvente organico sotto vuoto utilizzando un evaporatore rotante fino forma una sottile pellicola nel flacone. NOTA: In alternativa, evaporare i solventi organici sotto gas inerte (per esempio, argon o azoto) per formare una pellicola e mantenere la fiala in un essiccatore a vuoto per rimuovere ulteriormente solvente residuo. </li><li> Trasferimento 2 ml di tampone fosfato di Dulbecco (pH 7,4, DPBS) nella fiala di vetro. </li><li> Posizionare il flacone in un bagno ad ultrasuoni per 3 minuti a temperatura ambiente per generare micelle. NOTA: il potere ad ultrasuoni varia tra i diversi modelli di bagni ad ultrasuoni. Selezionare un&#39;unità in grado di generare energia sufficiente ad ultrasuoni per disperdere la sottile pellicola di polimero / farmaco. La potenza di uscita del bagno a ultrasuoni utilizzati in questo protocollo è di 110 W. </li><li> Mantenere micelle a 4 ° C per una conservazione a breve termine e -20 ° C a lungostoccaggio -term. NOTA: In alternativa, le micelle possono anche essere liofilizzati e ricostituiti con acqua prima dell&#39;uso. Di solito, non crioprotettore o lyoprotectant è necessario per questa formulazione. </li></ol> 3. Caratterizzazione di DOX-PA Le micelle <ol><li> Determinazione della concentrazione DOX-PA in micelle e l&#39;efficienza di incapsulamento farmaco <ol><li> Sciogliere DOX-PA sintetizzato nelle precedenti fasi di DMSO per preparare le soluzioni DOX-PA di cinque diverse concentrazioni: 1 mg / ml, 5 mg / ml, 20 mg / ml, 50 mg / ml, e 100 mg / ml. Misurare l&#39;assorbimento delle soluzioni DOX-PA con un UV-VIS spettrometro a 490 nm. Generare una curva standard in base alle concentrazioni di farmaco DOX-PA e il loro assorbimento corrispondente a 490 nm. </li><li> Diluire 25 ml di micelle di droga-caricato con 500 ml di DMSO. Misurare l&#39;assorbimento a 490 nm con uno spettrofotometro UV-VIS. Calcolare le concentrazioni di farmaco con la curva standard generata in 3.1.1. </li><li> Calcolare efficienza incapsulamento utilizzando la seguente equazione: Encapsulation Drug Efficienza (%) = (quantità di farmaci in micelle) / (quantità di farmaco aggiunto) × 100% </li></ol></li><li> Caratterizzazione della dimensione delle particelle con dispersione della luce dinamica (DLS) <ol><li> Diluire micelle con DPBS (pH 7,4) ad una concentrazione finale DSPE-PEG di 1 mg / ml. Analizzare un campione 2 ml con un analizzatore di dimensione delle particelle per ottenere le dimensioni e polidispersità indice Z-medio (PDI). </li></ol></li><li> Valutazione in vitro dell&#39;attività antitumorale NOTA: utilizzare appropriati tecnica sterile ed operare all&#39;interno di un armadio biosicurezza. <ol><li> Rimuovere il terreno di coltura cellulare da un pallone di coltura cellulare (ad esempio, T25) contenente DU-145 cellule cancerose della prostata umana e lavare le cellule con 2 ml di DPBS (pH 7,4). </li><li> Aspirare DPBS, aggiungere 1 ml di soluzione di tripsina (0,25%), e incubare per 2 minuti a 37 ° C per staccare le cellule. </li><li> Aggiungere 10 ml di terreno di coltura cellulare (RPMI 1640 + 10% siero fetale bovino + 1% Antibiotico-antimicotica), quando la maggior parte delle cellule si staccano dal pallone. Trasferire le cellule in una provetta da centrifuga da 15 ml e centrifugare le cellule a 1.000 xg per 5 min. </li><li> Risospendere il pellet cellulare con 5 ml di mezzo di coltura cellulare e rimuovere un campione per contare il numero di cellule con un emocitometro. Diluire le cellule con mezzo di coltura cellulare ad una densità di 50.000 cellule / ml. Aggiungere sospensioni diluito di cellule in una piastra di coltura cellulare a 96 pozzetti (100 ul / pozzetto). Incubare le cellule in un incubatore di coltura cellulare (37 ° C, 5% CO 2) per 18 ore per permettere l&#39;adesione cellulare. </li><li> Diluire DOX dimetilsolfossido soluzione (DMSO) e la soluzione DOX-PA DMSO con terreno di coltura cellulare per ottenere concentrazioni finali di droga di 0,1 mM, 0,5 mM, 2 mM, 5 mM e 10 mM, rispettivamente. Mantenere concentrazione DMSO finale in tutti i campioni superiori allo 0,5%. Diluire DOX-PA micelle con terreno di coltura cellulare per ottenere finale co di drogancentrations di 0,1 mM, 0,5 mM, 2 mM, 5 mM e 10 mM, rispettivamente. Utilizzare il mezzo di coltura cellulare in bianco un controllo. </li><li> Rimuovere la piastra di coltura cellulare a 96 pozzetti dal termostato e sostituire il mezzo di coltura cellulare con 100 ml di mezzo contenente diversi agenti di trattamento previste al punto 3.3.5 (n = 4 per ciascun gruppo). Incubare le cellule nella coltura cellulare incubatore (37 ° C, 5% CO 2) per un ulteriore 72 hr. </li><li> Aspirare il mezzo e aggiungere 100 ml di mezzo contenente 0,5 mg / ml di 3- (4,5-dimetil-tiazol-2-il) -2,5-difenil tetrazolio bromuro (MTT). </li><li> Incubare le cellule in incubatore di coltura cellulare per ulteriori 2 ore. Rimuovere con attenzione il supporto e aggiungere 100 ml di DMSO per sciogliere i cristalli formazano. </li><li> Misurare assorbanza con lo spettrofotometro per micropiastre alla lunghezza d&#39;onda di 570 nm e una lunghezza d&#39;onda di riferimento di 670 nm. </li><li> Calcolare la vitalità cellulare utilizzando la seguente equazione: <strong&gt; vitalità cellulare (%) = (A Test / Un controllo) × 100% NOTA: Confrontare la vitalità cellulare tra i diversi gruppi utilizzando una analisi della varianza ad una via (ANOVA) test statistico. Calcolare il IC50 sulla base di vitalità cellulare contro dati di concentrazione di droga. </li></ol></li></ol>

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The authors have nothing to disclose.

In questo lavoro, si descrive un metodo pellicola dispersione rapida semplice per la preparazione di micelle. Questo metodo utilizza le proprietà auto-assemblaggio di un polimero amfifilico (ad esempio, DSPE-PEG) per formare core-shell micelle strutturati in un ambiente acquoso. Questo metodo di preparazione micelle ha diversi vantaggi. 1. Si tratta di un semplice processo di formulazione, che evita l&#39;uso di fasi dimensioni riduzione complicate (come estrusione o omogeneizzazione) comunemente utilizzati ne...

La chemioterapia è comunemente usato per trattare varie forme di cancro. La maggior parte, se non tutti, i farmaci chemioterapici hanno effetti collaterali tossici che possono variare da condizioni minori gestibili, come nausea e diarrea, a condizioni che minacciano la vita più. Poiché la maggior parte farmaci antitumorali sono tossici, l&#39;esposizione non selettivo di questi farmaci al tessuto normale provoca inevitabilmente la tossicità. Pertanto, vi è una grande necessità di un approccio terapeutico che può trasportare selettivamente farmaci nelle cellule tumorali. Un&#39;altra sfida con la somministrazione di farmaci antitumorali è la loro scarsa solubilità in acqua. Di solito, sono necessari agenti solubilizzanti per formulare questi farmaci scarsamente solubili. Tuttavia, la maggior parte agenti solubilizzanti, come dimetil solfossido (DMSO), Cremophor EL, e polisorbato 80 (Tween 80) possono causare tossicità epatica e renale, emolisi, reazioni di ipersensibilità acuta e neuropatie periferiche. 1 Perciò, formulazioni sicure e biocompatibili sono necessari per l&#39;uso clinico di poverifarmaci antitumorali mente solubili. Nanovettori sono promettenti sistemi di drug delivery per affrontare le sfide di cui sopra. Questi nanovettori includono liposomi, nanoparticelle 2, 3 micelle, 4-7 coniugati polimero-farmaco, 8 e materiali inorganici. 9 Diversi prodotti nanomedicina (ad esempio, Doxil, Abraxane, e Genexol) sono stati approvati dalle agenzie di regolamentazione per il trattamento di pazienti affetti da cancro. 10 Micelle polimeriche sono promettenti vettori di consegna di droga nano-scala, che sono stati utilizzati con successo per la consegna dei farmaci antitumorali. 4-7,11,12 tipici micelle polimeriche sono preparati da polimeri anfifilici attraverso un processo di auto-assemblaggio. Il core-shell strutturati micelle polimeriche includono un guscio idrofilo e un nucleo idrofobico. Il guscio idrofilo può stabilizzare stericamente micelle e prolungare la loro circolazione nel flusso sanguigno. Il nucleo idrofobico può incapsulare in modo efficace idrofoba dtappeti. A causa delle piccole dimensioni delle micelle (tipicamente inferiori a 200 nm) e proprietà di lunga circolazione, micelle polimeriche sono creduti per conseguire targeting tumorale attraverso una maggiore permeabilità ed effetti di ritenzione (EPR) (tumore passiva targeting). la stabilità della droga di carico è fondamentale per il tumore mira capacità di micelle. Per ottenere il targeting tumorale ottimale, micelle dovrebbero avere perdite di droga minimo prima di raggiungere il sito del tumore, ma rilasciare rapidamente il farmaco dopo aver inserito le cellule tumorali. Inoltre, la stabilità formulazione è anche un requisito essenziale per lo sviluppo dei prodotti, perché la stabilità formulazione determina la fattibilità di sviluppo del prodotto, così come la shelf-life di prodotti sviluppati. Recentemente, sono stati fatti molti sforzi per migliorare il carico di droga in vettori di consegna. Il lipofila approccio pro-farmaco è una strategia che è stata esplorata per migliorare carico di droga in nanoparticelle lipidiche ed emulsioni. 13,14 La conjugation dei lipidi con farmaci in grado di migliorare significativamente la loro lipofilia e migliorare il carico e la conservazione delle componenti lipofile di nanovettori. Qui, descriviamo un protocollo per la preparazione di lipofila doxorubicina pro-farmaco micelle caricati. In primo luogo, la procedura di sintesi per doxorubicina lipofila pro-farmaco è descritta. Poi, viene introdotto un protocollo per la generazione di micelle con un metodo film dispersione. Questo metodo è stato utilizzato con successo nei nostri studi precedenti. 5 DSPE-PEG è stato scelto come materiale di supporto per la preparazione di micelle perché è stato usato con successo per la consegna micelle farmaco. 15,16 Infine, si descrivono diversi saggi in vitro utilizzati per caratterizzare micelle formulazioni e per valutare l&#39;attività antitumorale.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">DSPE-PEG2K</td>
			<td style="width:191px;">Cordenpharm</td>
			<td style="width:119px;">LP-R4-039</td>
			<td style="width:167px;">&#62;95%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Doxorubicin</td>
			<td style="width:191px;">LC Laboratories</td>
			<td style="width:119px;">D-4000</td>
			<td>&#62;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Palmitic Acid Hydrazide</td>
			<td style="width:191px;">TCI AMERICA&#160;&#160;</td>
			<td style="width:119px;">P000425G</td>
			<td>&#62;98.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">610981000</td>
			<td>Anhydrous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene chloride&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">D151-4</td>
			<td>99.90%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl sulfoxide-d6</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC320760075</td>
			<td>NMR solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic Acid&#160;</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC293811000</td>
			<td>99.50%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silica Gel</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">L-7446</td>
			<td>230-400 mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BAKER FLEX TLC PLATES&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">NC9990129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPBS</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DU 145&#160; Prostate Cancer Cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:119px;">HTB-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">158990050</td>
			<td>98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium</td>
			<td style="width:191px;">MEDIATECH INC&#160;</td>
			<td style="width:119px;">10041CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotic-Antimycotic&#160;</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">15240062</td>
			<td>100x stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">10437077</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nuclear Magnetic Resonance Spectroscopy</td>
			<td style="width:191px;">Varian, Inc</td>
			<td style="width:119px;"></td>
			<td>300 NMR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B&#252;chi R-3 Rotavapor</td>
			<td style="width:191px;">Buchi</td>
			<td style="width:119px;">1103022V1&#160;</td>
			<td>Rotary evaporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasonic Bath</td>
			<td style="width:191px;">BRANSON ULTRASONICS CORPORATION&#160;</td>
			<td style="width:119px;">CPX952318R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV-VIS spectrometer Biomate 3</td>
			<td style="width:191px;">Thermo Spectronic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zetasizer Nano ZS90&#160;</td>
			<td style="width:191px;">Malvern Instruments</td>
			<td style="width:119px;"></td>
			<td>Particle Size Analyer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Spectrophotometer&#160;</td>
			<td style="width:191px;">Rio-Rad</td>
			<td style="width:119px;">Benchmark Plus&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:191px;">Napco</td>
			<td style="width:119px;">CO2 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biological Safety Cabinet</td>
			<td style="width:191px;">Nuaire</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SigmaPlot&#160;</td>
			<td style="width:191px;">Systat Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>Analytical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-Well Cell Culture Plate</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin&#160; 0.25%</td>
			<td style="width:191px;">Corning Cellgro</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and characterization of lipophilic doxorubicin pro-drug micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

La Figura 1 mostra lo schema di sintesi di DOX-PA. DOX-PA è stato sintetizzato dalla coniugazione di acido palmitico con doxorubicina attraverso un legame idrazone sensibile al pH. Un leggero eccesso di idrazide dell&#39;acido palmitico è stato utilizzato per agevolare il completamento della reazione. Questo metodo di reazione ha un rendimento molto elevato e solo una piccola quantità di doxorubicina rimasto dopo una reazione 18 ore (Figura 2). La res...

Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Un protocollo per la preparazione e caratterizzazione di lipofila doxorubicina pro-farmaco caricata 1,2-distearoyl- SN -glycero-3-phosphoethanolamine- N - [amino (polietilene glicole) -2000] (DSPE-PEG) micelle è descritto.

Preparazione e caratterizzazione di lipofila doxorubicina micelle pro-farmaco

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

preparation-characterization-lipophilic-doxorubicin-pro-drug

Research

JoVE Journal

Bioengineering

14.8K Views.  Hampton University.  Un protocollo per la preparazione e caratterizzazione di lipofila doxorubicina pro-farmaco caricata 1,2-distearoyl- SN -glycero-3-phosphoethanolamine- N - [amino (polietilene glicole) -2000] (DSPE-PEG) micelle è descritto. 

Video: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Costruzione e caratterizzazione di un Fold bioreattore Novel Vocal

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Ricerca

Bioingegneria

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Preparazione e caratterizzazione di novel nanoparticelle che imitano HDL per l&#39;incapsulamento del fattore di crescita del nervo

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Sintesi del monocito-targeting Peptide Amphiphile micelle per l'Imaging di aterosclerosi

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabbricazione e caratterizzazione dei fantasmi di tessuto ottico contenente macrostruttura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparazione, purificazione e l'uso dei liposomi contenenti acidi grassi

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Rilevamento dell'endotossina in Nano-formulazioni utilizzando dosaggi di Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Crescita e caratterizzazione di organoidi irradiati da ghiandole mammarie

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

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

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

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

Preparazione e caratterizzazione dei nanoliposomi per l'entratura delle proteine globulari bioattive

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

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

Focalizzata la litografia del fascio ionico per esacire di nano-architetture nei microelettrodi

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Generazione e caratterizzazione quantitativa di cisti epiteliali biliari funzionali e polarizzate

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...