Bu çalışma, Kanser, Carolina Nanoteknoloji Mükemmeliyet Pilot hibe Üniversitesi Kanser Araştırma Fonu ve Ulusal Sağlık Enstitüsü K-12 Kariyer Geliştirme Ödülü Merkezi Karşı Golfçüler tarafından finanse edildi.

1. PLGA-b-PEG polimerin sentezi <ol><li> Terminali karboksilat grupları ile poli (D, L-lactide-co-glycolide) (PLGA) (PLGA-karboksilat) 5mM bir konsantrasyon PLGA (malzeme bölümünde belirtildiği gibi) için herhangi bir çözücü içinde çözülür. PLGA nazik karıştırma bu konsantrasyonda çözünmüş olabilir. </li><li> NHS (moleküler ağırlığı 115,09) ve EDC (molekül ağırlığı 191,7) 25mm bir konsantrasyon PLGA çözelti içinde çözülür. (EDC ve NHS PLGA kıyasla 5 kat aşan bir stokiyometrik eklenir). PLGA-karboksilat EDC ve NHS ekleyerek yaklaşık 1 saat için nazik karıştırma PLGA-karboksilat çözüm için PLGA-NHS dönüştürülür. </li><li> Reaksiyon ürünü PLGA-NHS yıkama solüsyonu metanol eklenerek çöktürülür. Metanol yaklaşık 10 kat fazla hacim çözüm eklenir. Çözüm çökelti PLGA-NHS ve supernatant atın (EDC ve NHS izlerini ortadan kaldırır. Metanol ile yıkama Bu işlem en az üç kez tekrarlanır xg 2000 santrifüj edilir. </li><li> PLGA-NHS pelet yıkama solüsyonu tüm izlerini silmek için 30 dakika süreyle vakum altında kurutulur. </li><li> PLGA-NHS pelet PLGA çözmek için başlangıçta kullanılan aynı konsantrasyonda aynı çözücü yeniden çözülür. Heterobifunctional PEG (amin-PEG-karboksilat) sonra 5mM bir konsantrasyon PLGA çözüm eklenir (1:1 stokiyometrik oranı). Karışımı bir çözüm sürekli karıştırarak 24 saat inkübe edilir. </li><li> 24 saat sonra, reaksiyon ürünü PLGA-b-PEG blok kopolimer aşırı yıkama solüsyonu metanol eklenerek çöktürülür. Üç kez Yukarıda da belirtildiği gibi yıkama ve santrifüj işlemi tekrarlayın. Bu, tüm aşırı girmemiş PEG kaldıracaktır. </li><li> PLGA-b-PEG blok kopolimer, vakum altında kurutulur. </li></ol> 2. PLGA-b-PEG nanoparçacık hazırlık Yüzeyde PEG ile kaplı PLGA çekirdekli Nanopartiküller bu diblock kopolimerler hazırlanabilir. Bir dizi farklı hidrofobik ilaçlar gibi nanopartiküller kapsüllü olabilir. Floresan bileşikler nanopartiküller kapsüllü olabilir veya PLGA konjuge ve böylece bu nanopartiküller floresan görüntüleme için kullanılabilir olabilir. Nanoprecipitation yöntem özellikle kapsüllü olması istenen kargo son derece doğal hidrofobik nanopartiküller yapmak için kullanılır. <ol><li> PLGA-b-PEG blok kopolimeri ve uyuşturucu / kargo (kapsüllü) PLGA çözünür olan herhangi bir çözücü içinde çözülür. PLGA asetonitril, DKMP, tetrahidrofuran, aseton veya etil asetat da dahil olmak üzere pek çok ortak çözücüler tarafından çözülebilir. Nanoparçacık özelliklerini etkiler olarak solvent seçimi kritik öneme sahiptir. Bu nedenle, uygun bir çözücü bu adımda kullanılmalıdır. </li><li> Polimer / ilaç karışımı daha sonra yaklaşık 3 mg / ml son bir polimer konsantrasyonu vererek su karıştırarak 3-5 birimlere damla damla eklenir. (Şekil 2) </li><li> Karıştırarak nanopartiküller self-assembly oluşturmak için izin ve organik çözücü izlerini kaldırmak için düşük basınç altında 2 saat boyunca devam eder. </li><li> Hasat ve saflaştırma: nanopartiküller sonra yıkanmış bir Amicon filtresi (MWCO 20KDa) kullanarak 10 dakika için 2.700 x g santrifüj konsantre ve PBS içinde sulandırılmış. Bu tüm un-sürüklenen bir ilaç / kargo kaldırır. Temel biyofiziksel boyut gibi nitelendirmeler, yüzey yükü ve ilaç yükleme verimliliğini nanoparçacıkların özelliklerini daha iyi anlamak için yapılabilir. </li></ol> 3. Depolama Dondurma-kurutma nanopartiküller 11 depolamak için yaygın olarak kullanılan bir yöntemdir. Dondurma-kurutma, uzun vadeli istikrar için 12 nanopartiküller fiziksel ve kimyasal özellikleri koruyacaktır . Dondurarak kurutma işlemi parçacıklar üzerinde strese neden ve formülasyon istikrarsızlaştırmak, cryo-koruyucu (donma stresinden koruma) ve LYO-koruyucu (kurutma stres koruma) yaygın olarak kullanılmaktadır. Bu koruyucu seçim saklama süresi 13 istenilen uzunluğa göre belirlenir. <ol><li> Dondurarak kurutma, toplam katılaşma, Tg donma noktasının altına örnek vardır. </li><li> Kuruma adım, buz süblimasyonu kaldırılır. Sıcaklık ve basınç etkili bir dondurarak kurutma işlemi ulaşmak için optimize edilmelidir. </li></ol> 4. Temsilcisi sonuçları: PLGA-b-PEG Di-blok kopolimeri Karakterizasyonu Polimerlerin başarılı konjugasyon onaylamak için farklı teknikler kullanılabilir. PLGA-b-PEG kompozisyon, 400 MHz 1H nükleer manyetik rezonans (NMR) ile karakterize edilebilir. Jel geçirgenlik kromatografisi (GPC) oluşan ürün Moleküler ağırlığı (PLGA-b-PEG) tarafından kontrol edilebilir. PLGA-b-PEG molekül ağırlığı distribution eğrisi ve elüsyon zaman PLGA ve PEG tek başına farklı olmalıdır. Birlikte, bu tekniklerin oluşan ürün karakterize ve konjugasyon reaksiyon başarılı olup olmadığını belirlemek gerekir. PLGA-b-PEG Karakterizasyonu nanopartiküller Partikül boyut ve boyut dağılımı, dinamik ışık saçılması ile ölçülebilir. Nanoprecipitation süreç içinde farklı parametreler partiküllerin boyutunu etkileyebilir. Başlangıçta kullanılan polimerler (PLGA ve PEG) Moleküler ağırlıkları da tanecik boyut dağılımı etkisi. Geçiş elektron mikroskobu (TEM) de Şekil 3&#39;te görüldüğü gibi nanopartiküllerin boyut dağılımı ve yapısını onaylamak için kullanılabilir. Partikül boyutu aralığı nm aralığında genellikle. Düzensiz boyut dağılımı ile büyük parçacık boyutları ya nanoprecipitation yöntemi ihtiyaçlarını optimizasyonu konjugasyon reaksiyon veya bir hata olduğu anlamına gelebilir. Buna ek olarak, yüzey zeta potansiyel ZetaPALS tarafından ölçülebilir. Uyuşturucu / kargo yükleme verimliliği standart HPLC ile belirlenebilir. Parçacıklar organik çözücü ve HPLC çözülür ilaç / kargo (Şekil 4) absorbansı ölçmek için yapılabilir. Nanopartiküller sabit miktarları 30 Slayt-A-Lyzer MINI Diyaliz üniteleri diyalize bilinen yerlerde ilaç salım kinetik çalışmada yapılabilir. Sabit zaman aralıklarında içerik, diyaliz ünitesinde toplanan ve eşit miktarda organik çözücü nanopartiküller çözmek için eklenir. HPLC, uyuşturucu / kargo içeriği ölçmek için bu numuneler üzerinde yapılır. <img alt="Şekil 1" src="/files/ftp_upload/3398/3398fig1.jpg" /> Şekil 1. EDC / NHS kimya <img alt="Şekil 2" src="/files/ftp_upload/3398/3398fig2.jpg" /> Şekil 2. Polimerik nanopartiküller hazırlamak için Nanoprecipitation yöntemi. PEG-PLGA diblock ve parçacık yüklü olması uyuşturucu ya da kargo içeren (acetonitrile veya DCM) bir çözücü organik çözelti 3-5 mL H 2 O karıştırarak damla damla eklenir <img alt="Şekil 3" src="/files/ftp_upload/3398/3398fig3.jpg" /> Şekil 3. Nanopartices Transmisyon Elektron Mikroskobu PEG-PLGA TEM görüntü wortamin içeren nanopartiküller. Fosfotungstik asit, bir kontrast madde olarak kullanılmıştır. <img alt="Şekil 4" src="/files/ftp_upload/3398/3398fig4.jpg" /> Şekil 4. PBS içinde diyaliz sonra nanopartiküller Paklitakselin nanoparçacık ilaç kontrollü salım Release. Kaydetti zamanda parçacıklar diyaliz kaset çıkarıldı asetonitril&#39;de solublized. Çözüm HPLC ile ölçüldü. İki ayrı nanoparçacıkların birçok karşılaştırıldı. <div id="myEventWatcherDiv"></div><div id="myEventWatcherDiv"></div>

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Çıkar çatışması ilan etti.

Diblock co-polimerler kullanarak nanoprecipitation yöntem polimer nanopartiküller mühendisi basit ve hızlı bir yöntem temsil eder. Nanopartiküller az çözünebilir bileşiklerin teslimat için kullanılabilir bir hidrofobik çekirdeğin oluşmaktadır. Bir hedefleme ligand potansiyeli daha fazla konjugasyon için benzer parçaları sağlarken hidrofilik yüzey tabakası mükemmel sulu çözünürlük sağlar. Lipozomlar dahil olmak üzere birçok nanoparçacık platformları, polimer...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> EDC </td><td> Thermo Scientific </td><td> 22980 </td><td> Konjugasyon Reaktif </td></tr><tr><td> NHS </td><td> Thermo Scientific </td><td> 24500 </td><td> Konjugasyon Reaktif </td></tr><tr><td> amin-PEG-karboksilat </td><td> Laysan Bio Inc. </td><td> NH2-PEG-CM-5000 </td><td> Polimer (herhangi bir PEG MW, 5000 kullanabilir miyim burada listelenir) </td></tr><tr><td> PLGA-carbxylate </td><td> Lactel </td><td> B6013-2 </td><td> Polimer </td></tr><tr><td> Diklorometan (DKMP) </td><td> Sigma-Aldrich </td><td> 34856 </td><td> Çözücü </td></tr><tr><td> Asetonitril&gt;% 99 saflıkta </td><td> Sigma-Aldrich </td><td> 34851 </td><td> Çözücü </td></tr><tr><td> Metanol&gt;% 99 saflıkta </td><td> Sigma-Aldrich </td><td> 34860 </td><td> Yıkama </td></tr></tbody></table>

formulation of diblock polymeric nanoparticles through nanoprecipitation technique

Nanoteknoloji harnessing ölçekte nanometre (nanopartiküller) parçacıkların eşsiz özelliklerini içerir nispeten yeni bir bilim dalıdır. Nanopartiküller boyutları, bileşimi ve yüzey kimyası dikkatli bir şekilde kontrol edilebilir kesin bir biçimde tasarlanmış olabilir. Bu onların kargo temel özellikleri, çözünürlük, yayınım, biodistribution, sürüm özellikleri ve immünojenite gibi bazı değiştirmek için benzeri görülmemiş bir özgürlük sağlar. , Kuruluşundan bu yana, nanoparçacıkların, ilaç dağıtım, görüntüleme ve hücre biyolojisi 1-4 da dahil olmak üzere birçok alanda, bilim ve tıp kullanılmıştır. Ancak, tam olarak algılanan teknik bir engel nedeniyle &quot;nanoteknoloji laboratuvarları dışında kullanılmıştır değildir. Bu makalede, potansiyel uygulamaları çok geniş bir yelpazesi olan bir polimer esaslı nanoparçacık platformu sentezlemek için basit bir yöntem açıklanmaktadır. Hidrofobik bir etki alanı ve hidrofilik etki alanı hem de bir diblock co-polimer sentezlemek için ilk adım. PLGA ve PEG model polimerler olarak kullanarak, EDC / NHS kimya 5 (Şekil 1) kullanarak bir konjugasyon tepki nitelendirdi. Ayrıca polimer arınma süreci tartışacağız. Sentezlenmiş diblock co-polimer hidrofilik-hidrofobik etkileşimler yoluyla nanoprecipitation sürecinde nanopartiküller içine kendi kendine bir araya getirebilirsiniz. Açıklanan polimer nanoparçacık çok yönlü. Nanoparçacık hidrofobik çekirdek ilaç dağıtım experiments6 için az çözünen ilaç taşımak için kullanılabilir. Ayrıca, nanopartiküller, DMSO gibi bir çözücü gerektirir wortmannin az çözünür moleküler biyoloji reaktifleri, toksik solventlerin sorunun üstesinden gelebilir. Bununla birlikte, DMSO hücreleri için toksik ve deney ile müdahale edebilirsiniz. Bu kötü çözünen ilaçlar ve reaktifler etkili minimal toksisite ile polimer nanopartiküller kullanılarak teslim edilebilir. Polimer nanoparçacıkların da floresan boya ile yüklenir ve hücre içi ticareti çalışmaları için kullanılmaktadır. Son olarak, bu polimer nanopartiküller yüzey PEG yoluyla ligandlar hedefleyen konjuge olabilir. Bu hedef nanopartiküller 7-10 veya hücreleri belirli epitoplar etiket kullanılabilir.

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

Bu makale diblock co-polimerler kullanarak polimer bazlı nanopartiküller sentezlemek için nanoprecipitation yöntem açıklanır. Biz diblock sentezi co-polimerler, nanoprecipitation tekniği ve potansiyel uygulamalar ele alınacaktır.

Nanoprecipitation Tekniği Diblock Polimerik Nanopartiküller formülasyonu

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 Görüntüleme.  University of North Carolina School of Medicine. Bu makale diblock co-polimerler kullanarak polimer bazlı nanopartiküller sentezlemek için nanoprecipitation yöntem açıklanır. Biz diblock sentezi co-polimerler, nanoprecipitation tekniği ve potansiyel uygulamalar ele alınacaktır.

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.

Polimerik Gen Taşıyıcı Değerlendirilmesi Nanoparçacık İzleme Analizi ve Yüksek throughput Flow Sitometri tarafından Nanopartiküller

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

Biyomühendislik

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.

Biyobozunur polimerik nano partiküllerin kullanılması Tedavi Angiogenezis Programlama Kök Hücreler

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.

Önceden Tasarlanmış Boyut ve Şekle Sahip Polimerik ve Biyohidred Elyafların Mikroakışkan İmalatı

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.

Fotolitografiyle polimerik Microneedle Dizi Fabrikasyon

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.

Biyomedikal Uygulamalar için Süperhidrofobik Polimer Malzemeler imalatı

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.

Fotoğrafa Duyarlı Polimerik Nanokarbonlardan SiRNA Salınımının Spatiotemporal Kontrolü ile Gen Silenceresinin Öngörülmesi

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.

Ölçeklenebilir, Hızlı montaj ve biyoaktif proteinler ve Immunostimulants yükleme yolu ile birden parlamak Nanoprecipitation çeşitli sentetik Nanocarriers içine

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.

Protein kinaz C-delta inhibitör peptid formülasyonu altın nano tanecikleri kullanarak

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.

Hidrofobik ve hidrofilik bileşikler polimer nano tanecikleri Kapsülleme için NanoPrecipitation flash

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.

Sitosolik siRNA teslim için Nötrsel olarak şarj edilmiş, pH duyarlı polimerik nanopartiküller hazırlanması

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