This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1. Photomask Fabrication <ol><li> Pulire un wafer 4 &quot;vetro con soluzione Piranha (H 2 SO 4 / H 2 O 2 in rapporto 2: 1) per 20 minuti a 120 ° C per immersione in una vasca di quarzo. </li><li> Depositare uno strato di cromo / oro (30 nm di Cr / 1 micron di Au) strato sul wafer di vetro usando un e-beam evaporatore 7 (Figura 1A). <ol><li> Posizionare i wafer in un evaporatore e-beam. Una volta che il vuoto raggiunge 5 x 10 -6 Torr, accendere la sorgente ad alta tensione (10 kV). Controllare lo spessore dalla centrale del monitor. </li><li> Pre-pulita ogni materiale per 30 secondi utilizzando la pistola e-beam, mantenendo l&#39;otturatore &quot;OFF&quot; (per evitare la deposizione sui wafer). </li></ol></li><li> Generare un Cr / Au strato di resina fotosensibile di mascheratura per una profonda incisione umido del vetro. <ol><li> Applicare un photoresist spessore 2 micron filando 5 ml di soluzione per 30 secondi a 3000 rpm utilizzando un sistema di rivestimento spin-spruzzo.Precottura fotoresist su una piastra riscaldante a 100 ° C per 1,5 min. </li><li> Esporre e duro cuocere il fotoresist a 120 ° C per 30 minuti su una piastra calda. È fondamentale per generare una superficie idrofobica e una forte adesione del fotoresist allo strato metallico. Motivo dello strato di Cr / Au utilizzando Cr e Au soluzioni di attacco attraverso il photoresist maschera 8,9. </li></ol></li><li> Per la protezione della superficie del vetro non-modellato, legare temporaneamente il wafer vetro per un wafer di silicio manichino. 9 <ol><li> Posizionare il wafer di vetro su una piastra riscaldante a 110 ° C e sciogliere la cera sul lato opposto del wafer di vetro (in modo tale che l&#39;intera superficie del wafer è coperto di cera). </li><li> Collocare un wafer di silicio manichino in contatto con il wafer di vetro e premere per rimuovere l&#39;eccesso di cera. Per evitare fuoriuscite di cera, posizionare una carta velina camera pulita sulla piastra. </li></ol></li><li> Eseguire attacco isotropo della lente utilizzando acido fluoridrico ottimizzato (49% v / v)soluzione di acido cloridrico (37% v / v) (in un rapporto volumetrico di 10: 1). con un agitatore magnetico per 10 min 8.5 Presenza di HCl è critico per il raggiungimento di una buona qualità superficiale di lenti generati. <ol><li> Assicurarsi che la velocità di attacco è di 7 micron / min; utilizzando un volume totale di 200 ml di soluzione di attacco. Eseguire incisione in un contenitore di plastica e prendere precauzioni di sicurezza per questa fase di lavorazione. </li><li> Pulire il wafer in (DI) acqua deionizzata da risciacquo e asciugatura ulteriormente a temperatura ambiente. </li></ol></li><li> Dopo il completamento della procedura, separare il wafer di vetro dal wafer di silicio manichino e riscaldare la cera con una piastra calda a 100 ° C per 15 sec. Come la cera si scioglie a questa temperatura, staccare il wafer di vetro dal wafer di silicio manichino. </li><li> Rimuovere rimanente cera, il photoresist e gli strati sovrastanti Cr / Au ai bordi delle lenti con ultrasuoni per 1 ora usando N-metil-2-pirrolidone come solvente a 80 ° C in un bagno di ultrasuoni.</li><li> Creare uno stampo replica PDMS delle microlenti fabbricati sui fotomaschere 11. </li><li> Caratterizzare le dimensioni fotomaschere (lunghezza e larghezza) e lo stampo microlenti PDMS (profondità e diametro) repliche utilizzando rispettivamente un microscopio elettronico a scansione e stereomicroscopio. 12-14 </li></ol> 2. Alberi MN Fabrication <ol><li> Creare una cavità di 2,5 cm × 0,9 cm utilizzando i vetrini montate su entrambi i lati di un vetro. Il numero dei vetrini impilati su ciascun lato determinerà l&#39;altezza della cavità noto come lo spessore del distanziale (Figura 1B). </li><li> Fissare ogni strato del vetrino applicando uno strato sottile della soluzione prepolimero contenente poli (glicole etilenico) diacrilato (PEGDA, MW = 258 Da) con 0,5% w / w 2-idrossi-2-metil-propiofenone (HMP) sulla vetrino seguito da irradiazione della luce impostato con l&#39;ultravioletta ad alta intensità (UV) per 2 sec. </li><li> Posizionare il photomask (precedentemente fabbricato) con le superfici Cr / Au rivestite rivolta verso l&#39;interno della cavità. Assicurarsi che i lati delle pareti della cavità non sono oscurando le lenti incorporate nel fotomaschere. </li><li> Riempire la cavità con la soluzione di prepolimero finché la superficie rivestita Cr / Au è in contatto con la soluzione senza bolle visibili. </li><li> Irradiare la configurazione con luce UV ad alta intensità di intensità desiderata per 1 sec ad una distanza di 3,5 cm dalla sorgente UV tramite la stazione di trattamento UV con una gamma filtro UV di 320-500 nm. Utilizzare un adattatore di collimazione con la sonda luce UV. </li><li> Misurare l&#39;intensità della luce UV utilizzata con un radiometro. </li><li> A seguito di esposizione ai raggi UV, rimuovere il fotomaschera con l&#39;array di MNs. Versare la soluzione di prepolimero eccesso che non polimerizzate nel processo nel suo recipiente originale per il riutilizzo. </li><li> Quantificare la lunghezza e punta il diametro delle MNs utilizzando uno stereomicroscopio secondo le istruzioni del produttore. </li></ol><pclass = &quot;jove_title&quot;&gt; 3. MN Backing strato Fabrication <ol><li> Con pinze, collocare i MNs (precedentemente fabbricato) attaccati sopra fotomaschera in un pozzetto di una piastra da 24 pozzetti, come mostrato nella Figura 1C. </li><li> Aggiungere un volume specificato (300 - 400 ml) della soluzione di prepolimero nel pozzo fino gli aghi sono sommersi all&#39;altezza desiderata. Questo volume determina lo spessore dello strato di supporto risultante. </li><li> Irradiare la configurazione con luce ad alta intensità UV (15,1 W / cm 2), 10,5 cm di distanza dalla sorgente UV per una durata di 1 sec. </li><li> Separare lo strato di supporto sulla matrice MN dalla fotomaschera utilizzando una lama affilata. </li><li> Quantificare la lunghezza, diametro punta e base diametro delle MNs con lo strato di supporto utilizzando uno stereomicroscopio secondo le istruzioni del produttore. </li></ol>

<ol>
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Y., Kang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+microneedle+array+fabrication+for+transdermal+drug+delivery.">A simple method of microneedle array fabrication for transdermal drug delivery.</a> Drug development and industrial pharmacy. 39, 299-309 (2013).</li><li>Kochhar, J. S., Zou, S., Chan, S. Y., Kang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+encapsulation+in+polymeric+microneedles+by+photolithography.">Protein encapsulation in polymeric microneedles by photolithography.</a> International journal of nanomedicine. 7, 3143-3154 (2012).</li><li>Tay, F. E. 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E. H., Miao, J., Sun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+masking+layers+for+deep+wet+etching+of+glass+in+an+improved+HF/HCl+solution.">Characterization of masking layers for deep wet etching of glass in an improved HF/HCl solution.</a> Surface and Coatings Technology. 198, 314-318 (2005).</li><li>Pan, J., 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=Fabrication+of+a+3D+hair+follicle-like+hydrogel+by+soft+lithography.">Fabrication of a 3D hair follicle-like hydrogel by soft lithography.</a> Journal of biomedical materials research. Part A. 101, 3159-3169 (2013).</li><li>Jay, T. R., Stern, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preshaping+photoresist+for+refractive+microlens+fabrication.">Preshaping photoresist for refractive microlens fabrication.</a> P Soc Photo-Opt Ins. 1992, 275-282 (1993).</li><li>Friedman, G. B., Sandhu, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+Spherical+Aberration+of+a+Thin+Lens.">Longitudinal Spherical Aberration of a Thin Lens.</a> Am J Phys. 35, 628 (1967).</li><li>Xu, Q. A., Li, J., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collimated+the+laser+diode+beam+by+the+focus+lens.">Collimated the laser diode beam by the focus lens.</a> Semiconductor Lasers and Applications IV. 7844, (2010).</li><li>Lin, T. W., Chen, C. F., Yang, J. J., Liao, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual-directional+light-control+film+with+a+high-sag+and+high-asymmetrical-shape+microlens+array+fabricated+by+a+UV+imprinting+process.">A dual-directional light-control film with a high-sag and high-asymmetrical-shape microlens array fabricated by a UV imprinting process.</a> J Micromech Microeng. 18, (2008).</li><li>Dunne, S. M., Millar, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+distance+from+curing+light+tip+to+restoration+surface+on+depth+of+cure+of+composite+resin.">Effect of distance from curing light tip to restoration surface on depth of cure of composite resin.</a> Prim Dent Care. 15, 147-152 (2008).</li><li>Kochhar, J. 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The authors declare no conflict of Interest.

Il protocollo descritto sopra per la fabbricazione della matrice MNs è stata presentata per fabbricare la matrice MNs di ~ 1 cm 2. Gli array possono essere scalati da creare una grande cavità dimensioni e utilizzando un fotomaschere grande. La maggiore dimensione della cavità può essere creata aumentando la larghezza tra i distanziatori su entrambi i lati. Anche se ogni passo per fabbricare gli array MN nel protocollo era importante, i passi più importanti sono stati: il posizionamento fotomaschera, il r...

Somministrazione di farmaci transdermica offre un approccio alternativo attraente per la somministrazione del farmaco, soprattutto per le biomolecole, che sono gestiti quasi esclusivamente da iniezioni ipodermiche. Tuttavia, la pelle, in particolare lo strato superiore (strato corneo), è una formidabile barriera impedendo molecole esogene di entrare nel corpo umano. Di recente, i dispositivi MN sono emersi come consentire strumenti per fornire farmaci attraverso la pelle. I dispositivi MN creano pori temporanei all&#39;interno dello strato corneo per consentire il passaggio di molecole di farmaco per ottenere l&#39;attività fisiologica desiderata con migliorata compliance del paziente e convenienza 1-3. Vari metodi di fabbricazione sono stati adottati per fabbricare MNs polimerici 4. Tuttavia, di solito coinvolgono processi step complicati e più che richiedono tempi lunghi e / o alte temperature per fabbricare array MNS. 4 Per semplificare il processo di fabbricazione, un unico processo senza muffa passo conun photomask stato sviluppato recentemente 5,6. Tuttavia, con questo metodo, fabbricato MNs avevano punte smussate aghi, come era alcun meccanismo in atto per modificare il percorso ultravioletta (UV) coinvolti nella fotolitografia. In questo studio, microlenti incorporati nella fotomaschera sono stati proposti per definire la geometria delle MNs. Il protocollo per fabbricare fotomaschere costituiti da microlenti integrate e successivamente Mn fabbricazione con punte affilate utilizzando fotomaschera sono segnalati.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td>
			<td>SIGMA&#160;</td>
			<td>475629-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-hydroxy-2-methyl-propiophenone (HMP)</td>
			<td>SIGMA&#160;</td>
			<td>405655-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine collagen type 1, FITC conjugate&#160;</td>
			<td>SIGMA&#160;</td>
			<td>C4361</td>
			<td></td>
		</tr>
		<tr>
			<td>UV curing station&#160;&#160;&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>OmniCure S200-XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Adaptor&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>EXFO 810-00042</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Nikon SMZ 1500 stereomicroscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Dillon GL-500 digital force gauge&#160;</td>
			<td>Dillon, USA</td>
			<td></td>
			<td></td>
		</tr>
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			<td>A-1R confocal microscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
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</table>

polymeric microneedle array fabrication by photolithography

Questo manoscritto descrive la fabbricazione di microago polimerico (MN) array per fotolitografia. Si tratta di un semplice processo senza stampo utilizzando una fotomaschera costituito da incorporati microlenti. Embedded microlenti sono stati trovati per influenzare MN geometria (nitidezza). Array MN Robusti con diametri compresi tra 41,5 punta micron ± 8.4 micron e 71,6 micron ± 13,7 micron, con due diverse lunghezze (1336 micron ± 193 micron e 957 micron ± 171 micron) sono stati fabbricati. Questi array MN possono fornire potenziali applicazioni nella fornitura di agenti terapeutici a basso molecolare e macromolecolare attraverso la pelle.

La geometria delle MNs può essere significativamente influenzata dalle caratteristiche fotomaschere e microlenti integrate. Il grado di rifrazione influenza il percorso di trasmissione dei raggi UV, che hanno influenzato la geometria MN (Figura 2A). Ogni microlenti è stato trovato ad avere un diametro di 350 micron, a 130 micron appiattite superficie convessa, e un 62,3 micron di profondità (Figura 2B-D). Utilizzando il teorema di Pitagora, il raggio di curvatura della prima superfic...

Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

Polimerici Microneedle Array Fabrication per Fotolitografia

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

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Bioengineering

11.9K Views.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

Video: Polymeric Microneedle Array Fabrication by Photolithography

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

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Bioingegneria

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Formulazione di nanoparticelle polimeriche diblock attraverso Tecnica Nanoprecipitation

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Hollow microago-based sensore per Multiplexed transdermico elettrochimica Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Valutazione della consegna del gene polimerici Nanoparticelle per l&#39;analisi delle nanoparticelle Monitoraggio e High-throughput Citometria a Flusso

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabbricazione microfluidica di fibre polimeriche e bioibride con dimensioni e forma predesignate

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication dal solvente assistita doppio strato lipidico (SALB) Metodo

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Realizzazione superidrofobiche Materiali Polimerici per applicazioni biomediche

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Multi-step altezza variabile fotolitografia per Valvolati multistrato microfluidici Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabbricazione di una matrice di multiplex artificiale microambiente cellulare

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

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.

Methods Article

Polimerici Microneedle Array Fabrication per Fotolitografia