Questo lavoro è stato finanziato dalla Golfers contro il cancro, Carolina del Centro Pilota per concedere Nanotechnology Excellence, University Cancer Research Fund e Istituto Superiore di Sanità K-12 Career Development Award.

1. Sintesi di PLGA-b-PEG polimero <ol><li> Poli (D, L-lattide-co-glicolico) (PLGA) con terminale gruppi carbossilato (PLGA-carbossilato) è disciolto in un solvente per PLGA (come indicato nella sezione materiali) ad una concentrazione di 5 mm. PLGA può essere sciolto a questa concentrazione con delicata agitazione. </li><li> Entrambi NHS (peso molecolare 115,09) e EDC (peso molecolare 191,7) si sciolgono nella soluzione PLGA ad una concentrazione di 25 mm. (Sia EDC e NHS sono aggiunti in un eccesso stechiometrico di 5 volte rispetto al PLGA). PLGA-carbossilato viene convertito in PLGA-SSN con l&#39;aggiunta di EDC e NHS a PLGA-carbossilato soluzione con delicata agitazione per circa 1 ora. </li><li> Il prodotto di reazione PLGA-SSN è precipitato con l&#39;aggiunta del metanolo soluzione di lavaggio. Circa 10 volte l&#39;eccesso di volume di metanolo viene aggiunto alla soluzione. La soluzione è centrifugata a 2000 xg per precipitare il PLGA-NHS e scartare il surnatante (rimuove le tracce di CED e Servizio Sanitario Nazionale. La procedura di lavaggio con metanolo viene ripetuto almeno tre volte. </li><li> Il PLGA-NHS pellet viene essiccato sotto vuoto per 30 minuti per rimuovere ogni traccia della soluzione di lavaggio. </li><li> Il PLGA-NHS pellet viene nuovamente sciolto nello stesso solvente alla stessa concentrazione che è stato utilizzato inizialmente per sciogliere PLGA. Il PEG heterobifunctional (ammine PEG-carbossilato) viene poi aggiunto alla soluzione PLGA ad una concentrazione di 5 mm (rapporto stechiometrico di 1:1). La soluzione miscela è incubata per 24 ore mescolando costantemente. </li><li> Dopo 24 ore, il prodotto di reazione PLGA-b-PEG copolimero a blocchi è precipitato con l&#39;aggiunta del metanolo soluzione di lavaggio in eccesso. Ripetere il processo di lavaggio e centrifugazione, come già detto tre volte. Questo rimuoverà tutti i PEG in eccesso non reagito. </li><li> Il PLGA-b-PEG copolimero a blocchi è essiccato sotto vuoto. </li></ol> 2. PLGA-b-PEG nanoparticelle preparazione Nanoparticelle con anima PLGA coperto con PEG in superficie possono essere preparati con questi copolimeri diblock. Una varietà di diversi farmaci idrofobici possono essere incapsulati in nanoparticelle del genere. Composti fluorescenti possono essere incapsulati in nanoparticelle o può essere coniugato con PLGA, e quindi queste nanoparticelle possono essere utilizzate per l&#39;imaging di fluorescenza. Nanoprecipitation metodo è usato per fare nanoparticelle soprattutto quando il carico voluto essere incapsulato è altamente idrofoba in natura. <ol><li> Il PLGA-b-PEG copolimero a blocchi e la droga / carico (per essere incapsulato) sono disciolti in qualsiasi soluzione che si scioglie PLGA. PLGA può essere sciolto da molti solventi comuni, tra cui acetonitrile, DCM, tetraidrofurano, acetone o acetato di etile. La scelta del solvente è fondamentale, in quanto influenza le proprietà delle nanoparticelle. Quindi, opportuno solvente, deve essere usato in questo passaggio. </li><li> La miscela di polimero / farmaco viene quindi aggiunta goccia a goccia a 3-5 volumi di acqua mescolando dando una concentrazione finale polimero di circa 3 mg / ml. (Fig. 2) </li><li> L&#39;agitazione è continuata per 2 ore a pressione ridotta per consentire le nanoparticelle di formare da self-assembly e rimuovere le tracce del solvente organico. </li><li> La raccolta e la depurazione: Le nanoparticelle vengono poi concentrati per centrifugazione a 2.700 x g per 10 minuti utilizzando un filtro Amicon (MWCO 20KDa), lavato, e ricostituito in PBS. Questo elimina tutti i carichi non-farmaco intrappolato /. Di base caratterizzazioni biofisici, come le dimensioni, carica di superficie, l&#39;efficienza e il caricamento della droga possono essere eseguite per comprendere meglio le proprietà delle nanoparticelle. </li></ol> 3. Immagazzinamento Liofilizzazione è un metodo comunemente utilizzato per memorizzare nanoparticelle 11. Liofilizzazione manterrà le caratteristiche fisiche e chimiche delle nanoparticelle per la stabilità a lungo termine 12. Il processo di liofilizzazione può causare stress sulle particelle e destabilizzare la formulazione, in modo da crio-protettori (protezione dallo stress congelamento) e lyo-protettori (protezione dallo stress asciugatura) sono comunemente usati. La scelta di questi protettori è determinata dalla lunghezza desiderata del tempo di conservazione 13. <ol><li> Nella liofilizzazione, c&#39;è solidificazione totale del campione congelamento di sotto del suo Tg. </li><li> Nella fase di essiccazione, il ghiaccio viene rimosso per sublimazione. Temperatura e la pressione deve essere ottimizzata per ottenere un efficiente processo di liofilizzazione. </li></ol> 4. Rappresentante dei risultati: Caratterizzazione di PLGA-b-PEG Di-copolimero a blocchi Diverse tecniche possono essere utilizzate per confermare la riuscita coniugazione di polimeri. La composizione del PLGA-b-PEG può essere caratterizzato con un 400 MHz 1H risonanza magnetica nucleare (NMR). Peso molecolare del prodotto formato (PLGA-b-PEG) possono essere verificate da cromatografia a permeazione di gel (GPC). Il PLGA-b - PEG peso molecolare distributiocurva n ed il tempo di eluizione dovrebbe essere diverso da PLGA e PEG solo. La combinazione di queste tecniche dovrebbero caratterizzare il prodotto formato e determinare se la reazione di coniugazione ha avuto successo. Caratterizzazione di PLGA-b-PEG nanoparticelle Dimensione delle particelle e la distribuzione delle dimensioni può essere misurata con dispersione della luce dinamica. Parametri diversi nel processo nanoprecipitation influisce sulla dimensione delle particelle. Pesi molecolari dei polimeri utilizzati inizialmente (sia PLGA e PEG) anche l&#39;effetto della distribuzione granulometrica. Microscopia elettronica di transizione (TEM) può anche essere usato per confermare la distribuzione delle dimensioni e della struttura delle nanoparticelle come si vede in figura 3. La gamma di dimensione delle particelle è generalmente nella gamma nm. Dimensioni delle particelle di grandi dimensioni con distribuzione delle dimensioni irregolari potrebbe indicare sia un errore nella reazione di coniugazione o il metodo nanoprecipitation esigenze di ottimizzazione. Inoltre, la superficie potenziale zeta può essere misurata con ZetaPALS. Il farmaco / carico l&#39;efficienza di carico può essere quantificato con lo standard HPLC. Le particelle sono sciolti in solvente organico e HPLC può essere effettuata per misurare l&#39;assorbimento del farmaco / carico (Fig. 4). Lo studio rilascio del farmaco cinetica può essere fatto se noti quantità fisse delle nanoparticelle sono dializzati in 30 Slide-A-Lyzer unità di dialisi MINI. Ad intervalli di tempo fisso, il contenuto nell&#39;unità di dialisi sono raccolti e volume uguale di solvente organico viene aggiunto per sciogliere le nanoparticelle. HPLC è fatto su questi campioni di quantificare il farmaco / carico di contenuti. <img alt="Figura 1" src="/files/ftp_upload/3398/3398fig1.jpg" /> Figura 1. EDC / NHS chimica <img alt="Figura 2" src="/files/ftp_upload/3398/3398fig2.jpg" /> Figura 2. Nanoprecipitation metodo per la preparazione di nanoparticelle polimeriche. La soluzione di un solvente organico (acetonitrile o DCM) che contiene il PEG-PLGA diblock e la droga o merci da caricare nella particella viene aggiunta goccia a goccia a 3-5 ml di mescolare H 2 O. <img alt="Figura 3" src="/files/ftp_upload/3398/3398fig3.jpg" /> Figura 3. Microscopia elettronica a trasmissione di nanopartices. Un&#39;immagine TEM di PEG-PLGA contenenti nanoparticelle wortamin. Acido fosfotungstico è stato usato come agente di contrasto. <img alt="Figura 4" src="/files/ftp_upload/3398/3398fig4.jpg" /> Figura 4. Rilascio controllato di farmaci da nanoparticelle. Rilascio di nanoparticelle di Paclitaxel dopo la dialisi in PBS. Al tempo notato, le particelle sono state rimosse da cassette dialisi e solublized in acetonitrile. La soluzione è stata misurata mediante HPLC. Due lotti separati di nanoparticelle sono stati confrontati. <div id="myEventWatcherDiv"></div><div id="myEventWatcherDiv"></div>

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Nessun conflitto di interessi dichiarati.

Il metodo nanoprecipitation utilizzando diblock copolimeri rappresenta un metodo semplice e veloce per progettare nanoparticelle polimeriche. Le nanoparticelle risultanti sono composte da un nucleo idrofobico che può essere utilizzata per l&#39;erogazione di composti scarsamente solubili. Lo strato superficiale idrofilo consente un&#39;eccellente solubilità in acqua, fornendo un residuo potenziale per coniugazione a seguito di una ligando targeting. Ci sono piattaforme di nanoparticelle, t...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> EDC </td><td> Thermo Scientific </td><td> 22980 </td><td> Coniugazione dei reagenti </td></tr><tr><td> SSN </td><td> Thermo Scientific </td><td> 24500 </td><td> Coniugazione dei reagenti </td></tr><tr><td> amine-PEG-carbossilato </td><td> Laysan Bio Inc. </td><td> NH2-PEG-CM-5000 </td><td> Polimero (può usare qualsiasi MW PEG, 5000 è elencato qui) </td></tr><tr><td> PLGA-carbxylate </td><td> Lactel </td><td> B6013-2 </td><td> Polimero </td></tr><tr><td> Diclorometano (DCM) </td><td> Sigma-Aldrich </td><td> 34856 </td><td> Solvente </td></tr><tr><td> Acetonitrile&gt; 99% di purezza </td><td> Sigma-Aldrich </td><td> 34851 </td><td> Solvente </td></tr><tr><td> Metanolo&gt; 99% di purezza </td><td> Sigma-Aldrich </td><td> 34860 </td><td> Lavare </td></tr></tbody></table>

formulation of diblock polymeric nanoparticles through nanoprecipitation technique

La nanotecnologia è una branca relativamente nuova della scienza che coinvolge sfruttando le proprietà uniche di particelle che sono nanometri di scala (nanoparticelle). Le nanoparticelle possono essere progettate in modo preciso in cui la loro chimica dimensione, composizione e superficie può essere attentamente controllato. Ciò permette una libertà senza precedenti di modificare alcune delle proprietà fondamentali del loro carico, come ad esempio la solubilità, diffusività, distribuzione biologica, caratteristiche di rilascio e l&#39;immunogenicità. Fin dalla loro creazione, le nanoparticelle sono stati utilizzati in molti settori della scienza e della medicina, tra cui la somministrazione di farmaci, l&#39;imaging, e la biologia delle cellule 1-4. Tuttavia, non è stato utilizzato completamente al di fuori dei &quot;laboratori di nanotecnologia&quot; a causa della percezione ostacolo tecnico. In questo articolo, si descrive un metodo semplice per sintetizzare una piattaforma basata nanoparticella polimerica che ha una vasta gamma di applicazioni potenziali. Il primo passo è quello di sintetizzare un diblock co-polimero che ha sia un dominio idrofobico e il dominio idrofilo. Utilizzando PLGA e PEG come polimeri modello, abbiamo descritto una reazione di coniugazione con EDC / NHS chimica 5 (Fig. 1). Discutiamo anche il processo di purificazione del polimero. Il diblock sintetizzato co-polimero può auto-assemblano in nanoparticelle nel processo nanoprecipitation attraverso interazioni idrofobiche-idrofilo. La nanoparticella polimerica descritto è molto versatile. Il nucleo idrofobico della nanoparticella può essere utilizzato per trasportare farmaci a bassa solubilità per la somministrazione di farmaci experiments6. Inoltre, le nanoparticelle possono superare il problema di solventi tossici per scarsamente solubili reagenti di biologia molecolare, come wortmannina, che richiede un solvente come il DMSO. Tuttavia, DMSO può essere tossico per le cellule e di interferire con l&#39;esperimento. Questi farmaci scarsamente solubili e reagenti possono essere efficacemente forniti utilizzando nanoparticelle polimeriche con tossicità minima. Nanoparticelle polimeriche può essere caricato anche con colorante fluorescente e utilizzati per gli studi di traffico intracellulare. Infine, queste nanoparticelle polimeriche può essere coniugato con targeting ligandi attraverso PEG superficie. Tali nanoparticelle mirata possono essere utilizzati per etichettare epitopi specifici su o in celle 7-10.

Watch this Scientific Journal Video about Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique at JoVE.com

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Questo articolo descrive un metodo per sintetizzare nanoprecipitation polimero a base di nanoparticelle utilizzando diblock co-polimeri. Discuteremo la sintesi di copolimeri diblock, la tecnica nanoprecipitation e potenziali applicazioni.

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 ...

formulation-diblock-polymeric-nanoparticles-through-nanoprecipitation

Research

JoVE Journal

Bioengineering

37.1K Views.  University of North Carolina School of Medicine. Questo articolo descrive un metodo per sintetizzare nanoprecipitation polimero a base di nanoparticelle utilizzando diblock co-polimeri. Discuteremo la sintesi di copolimeri diblock, la tecnica nanoprecipitation e potenziali applicazioni.

Video: Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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 ...

Ricerca

Bioingegneria

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Cellule staminali per la programmazione terapeutica angiogenesi Uso biodegradabili polimerico Nanoparticelle

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

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 ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

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 ...

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 ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

Predizione del silenziamento del gene attraverso il controllo spatiotemporale della siRNA rilasciata da nanocarrier polimerici sensibili alla foto

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Montaggio veloce e scalabile e caricamento di proteine bioattive e immunostimolanti in nanovettori diversi sintetici tramite Flash nanoprecipitazione

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Proteina chinasi C-delta inibitore Peptide formulazione usando nanoparticelle d'oro

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

Flash nanoprecipitazione per l'incapsulamento di composti idrofobi e idrofili in nanoparticelle polimeriche

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

Preparazione di nanoparticelle polimeriche neutralmente cariche e sensibili al pH per la consegna del siRNA citosolico

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...