Bu çalışma, Ulusal Kanser Enstitüsü Kanser Kanser Nanoteknoloji Mükemmeliyet Merkezi (CCNE) hibe U54-CA151881 Nanoteknoloji Alliance tarafından desteklenmiştir.

<ol>
	<li>Vimalachandran, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+prevention+of+pancreatic+cancer.">Genetics and prevention of pancreatic cancer.</a> Cancer. Control. 11, 6-14 (2004).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+thiol-modified+gelatin+nanoparticles+for+intracellular+DNA+delivery+in+response+to+glutathione.">Preparation and evaluation of thiol-modified gelatin nanoparticles for intracellular DNA delivery in response to glutathione.</a> Bioconjug. Chem. 16, 1423-1432 (2005).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+glycol)-modified+thiolated+gelatin+nanoparticles+for+glutathione-responsive+intracellular+DNA+delivery.">Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery.</a> Nanomedicine. 3, 32-42 (2007).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenic+gene+therapy+with+systemically+administered+sFlt-1+plasmid+DNA+in+engineered+gelatin-based+nanovectors.">Antiangiogenic gene therapy with systemically administered sFlt-1 plasmid DNA in engineered gelatin-based nanovectors.</a> Cancer. Gene. Ther. 14, 488-498 (2007).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+and+pharmacokinetic+analysis+of+long-circulating+thiolated+gelatin+nanoparticles+following+systemic+administration+in+breast+cancer-bearing+mice.">Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice.</a> J. Pharm. Sci. 96, 397-407 (2007).</li><li>Kaul, G., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-targeted+gene+delivery+using+poly(ethylene+glycol)-modified+gelatin+nanoparticles:+in+vitro+and+in+vivo+studies.">Tumor-targeted gene delivery using poly(ethylene glycol)-modified gelatin nanoparticles: in vitro and in vivo studies.</a> Pharm. Res. 22, 951-961 (2005).</li><li>Bardeesy, N., DePinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+biology+and+genetics.">Pancreatic cancer biology and genetics.</a> Nat. Rev. Cancer. 2, 897-909 (2002).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+peptide+ligand+of+epidermal+growth+factor+receptor+for+targeted+delivery+of+therapeutics.">Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics.</a> FASEB. J. 19, 1978-1985 (2005).</li><li>McCormick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+gene+therapy:+fringe+or+cutting+edge.">Cancer gene therapy: fringe or cutting edge.</a> Nat. Rev. Cancer. 1, 130-141 (2001).</li><li>Green, D. R., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+functions+of+the+tumour+suppressor+p53.">Cytoplasmic functions of the tumour suppressor p53.</a> Nature. 458, 1127-1130 (2009).</li><li>Barton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+of+the+p53+tumour+suppressor+gene+in+human+pancreatic+cancer.">Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer.</a> Br. J. Cancer. 64, 1076-1082 (1991).</li></ol>

Çıkar çatışması ilan etti.

Kontrol ve EGFR hedef thiolated jelatin nanopartiküller verimli DNA kapsülleme ve istikrar ile hazırlanmıştır. Tüm bu sistemlerin parçacık boyutları, çapı 150-250 nm aralığında. Zeta potansiyeli, bu sistemi biraz daha negatif bir sistem olduğunu kanıtladı. SEM analizi ile, nanoparçacıkların boyutları Zetasizer sonuç ile aynıydı. ESCA analizi, PEG ve peptid yüzey modifikasyonu onaylamak. Western Blot analizi Panc-1 hücreleri yüksek EGFR ekspresyon düzeyleri vardı ve bu hücre hattında in vitro çalışmalar için kullanılmış olduğunu gösterdi. Panc-1 hücreleri ve kontrol yüzeyi modifiye nanopartiküller PEI ile karşılaştırıldığında göreceli olarak daha az sitotoksik edildi. Hücre ticareti çalışmaları hızlı alımı ve EGFR-hedefli nanoparçacıkların Panc-1 hücrelerinde plazmid sürümü gösterdi. EGFR-hedefli nanopartiküller ifade muhabiri plazmid DNA Teslim en yüksek seviyelerindeLipofectin-complexed DNA da dahil olmak üzere diğer denetimleri, GFP ifade. Wt-p53 plazmid ile aynı sistem ile transfeksiyon mansap apoptotik yolağı ve Panc-1 hücreleri kaynaklı hızlı apoptozu tetikledi. Bu ön sonuçlar EGFR-hedefli thiolated jelatin nanopartiküller, pankreas kanseri için bir tedavi olarak gen tedavisi için güvenli ve verimli bir DNA dağıtım sistemi olarak hizmet verebilir öneririz.

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> B Tipi jelatin, Bloom 225 </td><td> Sigma-Aldrich </td><td> G9391 </td><td></td></tr><tr><td> 2-iminothiolane hidroklorür </td><td> Sigma-Aldrich </td><td> I6256 </td><td></td></tr><tr><td> plazmid pEGFP-N1 </td><td> Elim Biopharm </td><td> N / A </td><td></td></tr><tr><td> pORF-hp53 E. coli </td><td> Invivogen </td><td> porf-hp53 </td><td></td></tr><tr><td> Glyoxal çözümü (H 2 O 40wt.%) </td><td> Sigma-Aldrich </td><td> 128465 </td><td></td></tr><tr><td> Glisin </td><td> Sigma-Aldrich </td><td> 410.225-250G </td><td></td></tr><tr><td> QIA filtre Plazmid Mega kiti </td><td> Qiagen </td><td> 12281 </td><td></td></tr><tr><td> Beckman LE 80K ultrasantrifüjdeki </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> FreeZone 6 Litre Konsol Kuru Sistemleri Freeze </td><td> Labconco </td><td> 7753020 </td><td></td></tr><tr><td> MPEG-SCM, MW 2.000 Da </td><td> Laysan </td><td> MPEG-SCM-2K-1g </td><td></td></tr><tr><td> MAL-PEG-SCM, 2.000 MW Da </td><td> Jenkem Teknoloji </td><td> A5001-1 </td><td></td></tr><tr><td> Zetasizer Nano </td><td> Malvern </td><td> Zetasizer Nano ZS </td><td></td></tr><tr><td> Hitachi 4800 alan emisyonu taramalı elektron mikroskobu </td><td> Hitachi </td><td> S-4800 UHR FE-SEM </td><td></td></tr><tr><td> Quant-iT PicoGreen dsDNA Reaktif ve Kitleri </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> Lipofectin Transfeksiyon Reaktif </td><td> Invitrogen </td><td> 18292011 </td><td></td></tr><tr><td> DMEM </td><td> Mediatech cellgro </td><td> 10 013 CM </td><td></td></tr><tr><td> RPMI </td><td> Mediatech cellgro </td><td> 50 020 PB </td><td></td></tr><tr><td> Pierce BCA Protein Testi Kiti </td><td> Thermo Scientific </td><td> 23225 </td><td></td></tr><tr><td> iBlot Kuru Blot Sistemi </td><td> Invitrogen </td><td> IB1001 </td><td></td></tr><tr><td> XCell SureLock Mini-Hücre ve XCell II Blot Modülü Kit CE İşareti </td><td> Invitrogen </td><td> EI0002 </td><td></td></tr><tr><td> Pierce ECL Batı Yüzey Blotting </td><td> Thermo Scientific </td><td> 32109 </td><td></td></tr><tr><td> Kodak Dijital X-ray Örnek (DXS) Sistemi </td><td> Kodak </td><td> N / A </td><td>; </td></tr><tr><td> CellTiter 96 AQ ueous Tek Çözüm Hücre Çoğalması Assay (MTS) </td><td> Promega </td><td> G3580 </td><td></td></tr><tr><td> BioTek SynergyHT plaka okuyucu </td><td> BioTek </td><td> N / A </td><td></td></tr><tr><td> Nanodrop 2000 </td><td> Thermo Scientific </td><td> N / A </td><td></td></tr><tr><td> Tek aşamalı RT-PCR kiti </td><td> Qiagen </td><td> 210212 </td><td></td></tr><tr><td> Kromatin Yoğunlaşma / Membran Geçirgenlik / Ölü Hücre Apoptoz Kiti </td><td> Invitrogen </td><td> V23201 </td><td></td></tr><tr><td> Apo-ONE Homojen Kaspaz-3 / 7 Assay kiti </td><td> Promega </td><td> G7790 </td><td></td></tr><tr><td> Hybaid PCR Sprint Thermal Cycler </td><td> Thermo Scientific </td><td> N / A </td><td></td></tr><tr><td> EGF Reseptör Antikor </td><td> Hücresinyalizasyon </td><td> 2232 </td><td></td></tr><tr><td> β-Aktin Antikor </td><td> Hücre içi sinyal </td><td> 4967 </td><td></td></tr><tr><td> Anti-tavşan IgG, HRP-bağlantılı Antikor </td><td> Hücre içi sinyal </td><td> 7074 </td><td></td></tr><tr><td> Fare Monoklonal GFP Antikor </td><td> Novus Biyolojik </td><td> NB600-597 </td><td></td></tr><tr><td> Keçi Poliklonal GFP antikor (Alkalin Fosfataz) </td><td> Novus Biyolojik </td><td> NB600-1502 </td><td></td></tr><tr><td> Fosfataz Yüzey Kiti </td><td> Thermo Scientific </td><td> 37620 </td><td></td></tr></tbody></table>

therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles

32.000 &#39;den fazla hasta yılda Amerika Birleşik Devletleri, pankreas kanseri tanısı ve hastalığın, çok yüksek mortalite 1 ile ilişkilidir. Acil ihtiyaç, pankreas kanseri hastalarının kasvetli hayatta kalma istatistiklerine artırabilir roman klinik olarak çevrilebilir tedavi stratejileri geliştirmek için vardır. Kanser gen tedavisi muazzam bir söz gösterilmiş olsa da, en büyük zorluk, sürekli transgen ekspresyonu yol açabilir, güvenli ve etkili bir dağıtım sistemi gelişimi. Jelatin, gıda ve ilaç ürünlerinde yaygın olarak kullanılan çok yönlü doğal biopolimer biridir. Laboratuvarımızda Önceki çalışmalar, B tipi jelatin fiziksel DNA, hücre içi teslimat üzerine plazmid ve geliştirilmiş transfeksiyon verimlilik supercoiled yapısı korunarak kapsülleyen olduğunu göstermiştir. Jelatin thiolation sülfidril grupları polimer ve f. Olabilirnanopartiküller, tüm kompleks ve bir kez disülfit bağ dengeler içinde orm disülfit bağ cytosolda glutatyon varlığı nedeniyle bozuldu, yükü 2-5 serbest olacaktır. Poli (etilen glikol) (PEG) modifiye gens, sistemik dolaşıma uygulanan, uzun tirajlı kez sağlar ve gelişmiş geçirgenlik ve saklama etkisi 6 neovaskülarizasyon hiper geçirgenliği nedeniyle tümör kitlesi tercihen hedeflemektedir. Çalışmalar Panc-1 insan pankreatik adenokarsinoma hücreleri 7 epidermal büyüme faktörü reseptörü (EGFR)-ifade göstermiştir. Pankreas kanseri hücre hattı aktif olarak hedef için, EGFR spesifik peptid PEG ayırıcı üzerinden parçacık yüzey üzerine konjuge 8 Çoğu anti-tümör gen terapileri, vahşi tip p53 (wt-P53) olarak 9 hücreleri pro-apoptotik fonksiyonu geri tümör baskılayıcı genler, yönetim üzerinde duruldu . </&gt; destek. Kritik bir sinyal apoptozis düzenleyen hücre büyümesi yolağı, hücre döngüsü tutuklama, metabolizma ve diğer işlemler 10 olarak p53 mekanizması fonksiyonları. Pankreas kanseri, en hücrelerin apoptotik aktivite kaybına neden, p53 proteini mutasyonlar var. Wt-p53 giriş apoptozis tamir edilebilir ve kanser hücrelerini 11 daha fazla hücre ölümü tetikler . Yukarıdaki mantığı dayanarak, EGFR wt-p53 geni teslimat ve Panc-1 hücreleri değerlendirilir teslim verimlilik ve transfeksiyon peptid modifiye thiolated jelatin nanopartiküller yönelik tasarladık.

Watch this Scientific Journal Video about Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles at JoVE.com

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

B Tipi jelatin tabanlı mühendislik nanovectors sistemi (gens), pankreas kanseri tedavisi sistemik gen teslimat ve transfeksiyon için geliştirilmiştir. Epidermal büyüme faktörü reseptörü (EGFR) nanparticles yüzey üzerinde belirli peptid değişiklik, EGFR reseptör hedef olabilir ve plazmid gibi yüksek intrasellüler glutatyon konsantrasyonları olarak azaltılması, çevre altında serbest.

İnsan Pankreas Kanseri Hücrelerinde Epidermal Büyüme Faktörü kullanarak Gen Teslimat ve Transfeksiyon Jelatin Tabanlı Engineered Nanovectors Reseptör-hedefli

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

therapeutic-gene-delivery-transfection-human-pancreatic-cancer-cells

Research

JoVE Journal

Bioengineering

27.9K Görüntüleme.  Northeastern University. B Tipi jelatin tabanlı mühendislik nanovectors sistemi (gens), pankreas kanseri tedavisi sistemik gen teslimat ve transfeksiyon için geliştirilmiştir. Epidermal büyüme faktörü reseptörü (EGFR) nanparticles yüzey üzerinde belirli peptid değişiklik, EGFR reseptör hedef olabilir ve plazmid gibi yüksek intrasellüler glutatyon konsantrasyonları olarak azaltılması, çevre altında serbest.

Video: Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

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

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Kas Yenilenmesinin Fare Modellerinde İndüklenmiş Pluripotent Kök Hücre türevi Mezoangioblast benzeri Miyojenik Progenitörlerin Nakli

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

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, ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Lifli iskelelerinin içine Microspheres ibra Elektroeğirme Büyüme Faktörü

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Korelatif Mikroskopisi kullanılarak sağlam hücrelerde Epidermal Büyüme Faktörü Alıcısının stokiyometrisinden incelenmesi

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Katyonik ve Anyonik lipozomlar kullanılarak merkezi sinir sistemine Terapötik siRNA teslimi

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

Heparin Binding Domain osteojenik Büyüme Faktörü Teslim polielektrolit Kompleksi

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

İpek Nanopartiküller imalatı ve İlaç Dağıtım Uygulamaları

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

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

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

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

Sinir Büyümesi Faktörü Kapsüllemesi için Yeni HDL&#39;yi taklit eden Nanopartiküllerin Hazırlanması ve Karakterizasyonu

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

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Beyin kanseri ve nörodejeneratif hastalıkların Incelenmesi için insan nöral Organoidler

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...