この研究はがんナノエクセレンス（CCNE）助成U54 - CA151881のためのがんのセンターのナノテクノロジーの国立がん研究所のア​​ライアンスによってサポートされていました。

<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>

利害の衝突は宣言されません。

制御とEGFR標的とチオール化ゼラ​​チンナノ粒子は、効率的なDNAのカプセル化と安定性を用いて調製した。これらのシステムのすべての粒子サイズは直径150〜250 nmの範囲であった。ゼータ電位は、このシステムはわずかに負のシステムであることを証明しています。 SEMの分析では、ナノ粒子のサイズはゼータサイザーの結果と同じであった。 ESCA分析では、PEGとペプチド表面改質を確認することができた。 ウェスタンブロット分析は、PANC - 1細胞は、高いEGFRの発現のレベルを持っていたし、この細胞株はin vitro試験のために使用されることを示した。 PEIと比較して、コントロールと表面修飾ナノ粒子の両方がPANC - 1細胞​​では比較的少ない細胞毒性であった。 細胞の人身売買の研究は急速な取り込みとPANC - 1細胞​​におけるEGFR標的ナノ粒子のプラスミド放出を示した。 EGFR標的ナノ粒子で表現するレポータープラスミドDNAの配信は、最高レベルの結果リポフェクチン-複合DNAを含む他のコントロールに対する相対的なGFP発現。同じシステムで、WT - p53のプラスミドでトランスフェクションは、下流のアポトーシス経路およびPANC - 1細胞​​において誘導急速にアポトーシスを誘発した。 これらの予備的な結果は、EGFRを標的とチオール化ゼラ​​チンナノ粒子は、膵臓癌の治療として遺伝子治療のための安全かつ効率的なDNAの配信システムとして提供できることを示唆している。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>タイプBゼラチン、ブルーム225 </td><td>シグマアルドリッチ</td><td> G9391 </td><td></td></tr><tr><td> 2 - イミノチオラン塩酸塩</td><td>シグマアルドリッチ</td><td> I6256 </td><td></td></tr><tr><td> pEGFPを- N1プラスミド</td><td> ELIMバイオファーム</td><td> N / </td><td></td></tr><tr><td> PORF - hp53大腸菌</td><td> Invivogen </td><td> PORF - hp53 </td><td></td></tr><tr><td>グリオキサール溶液（H 2 Oで40wt.％） </td><td>シグマアルドリッチ</td><td> 128465 </td><td></td></tr><tr><td>グリシン</td><td>シグマアルドリッチ</td><td> 410225 - 250G </td><td></td></tr><tr><td> QIAフィルタープラスミドメガキット</td><td> QiageN </td><td> 12281 </td><td></td></tr><tr><td>ベックマンLE 80K超遠心機</td><td>ベックマン</td><td> N / </td><td></td></tr><tr><td>フリーゾーン6リットルコンソールのフリーズドライシステム</td><td> Labconco </td><td> 7753020 </td><td></td></tr><tr><td> MPEG - SCM、MW 2000ダ</td><td>ライサン島</td><td> MPEG - SCM - 2K - 1G </td><td></td></tr><tr><td> MAL - PEG - SCM、MW 2000ダ</td><td> Jenkemテクノロジー</td><td> A5001 - 1 </td><td></td></tr><tr><td>ゼータサイザーナノ</td><td>マルバーン</td><td>ゼータサイザーナノZS </td><td></td></tr><tr><td>日立4800電界放出走査型電子顕微鏡</td><td>日立</td><td> S - 4800 UHR FE - SEM </td><td></td></tr><tr><td>クワント- IT PicoGreen 二本鎖DNAの試薬とキット</td><td>インビトロジェン</td><td> P7589 </td><td></td></tr><tr><td>リポフェクチントランスフェクション試薬</td><td>インビトロジェン</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>ピアースBCAタンパク質アッセイキット</td><td>サーモサイエンティフィック</td><td> 23225 </td><td></td></tr><tr><td> iBlotドライブロッティングシステム</td><td>インビトロジェン</td><td> IB1001 </td><td></td></tr><tr><td> X細胞SureLockミニセルとX細胞IIブロットモジュールキットCEマーク</td><td>インビトロジェン</td><td> EI0002 </td><td></td></tr><tr><td>ピアースECLウェスタンブロッティング基質</td><td>サーモサイエンティフィック</td><td> 32109 </td><td></td></tr><tr><td>コダックデジタルX線検体（DXS）システム</td><td>コダック</td><td> N / </td><td>; </td></tr><tr><td> CellTiter 96 AQ ueous Oneソリューションの細胞増殖アッセイ（MTS） </td><td>プロメガ</td><td> G3580 </td><td></td></tr><tr><td> BioTekのSynergyHTプレートリーダー</td><td> BioTekの</td><td> N / </td><td></td></tr><tr><td>光度計2000 </td><td>サーモサイエンティフィック</td><td> N / </td><td></td></tr><tr><td>ワンステップRT - PCRキット</td><td>キアゲン</td><td> 210212 </td><td></td></tr><tr><td>クロマチン凝縮/膜透過性/死細胞アポトーシスキット</td><td>インビトロジェン</td><td> V23201 </td><td></td></tr><tr><td>アポ- ONE均質なカスパーゼ-3 / 7アッセイキット</td><td>プロメガ</td><td> G7790 </td><td></td></tr><tr><td> Hybaid PCRスプリントサーマルサイクラー</td><td>サーモサイエンティフィック</td><td> N / </td><td></td></tr><tr><td> EGF受容体抗体</td><td>セルシグナル伝達</td><td> 2232 </td><td></td></tr><tr><td> β-アクチン抗体</td><td>細胞シグナリング</td><td> 4967 </td><td></td></tr><tr><td>抗ウサギIgG、HRP結合抗体</td><td>細胞シグナリング</td><td> 7074 </td><td></td></tr><tr><td>マウスモノクローナルGFP抗体</td><td>ノーバスバイオ</td><td> NB600 - 597 </td><td></td></tr><tr><td>ヤギポリクローナルGFP抗体（アルカリホスファターゼ） </td><td>ノーバスバイオ</td><td> NB600 - 1502 </td><td></td></tr><tr><td>ホスファターゼ基質キット</td><td>サーモサイエンティフィック</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人以上の患者が年間米国で膵臓癌と診断され、この病気は非常に高い死亡率1に関連付けられています。緊急の必要性は、膵臓癌患者の悲惨な生存統計で改善できる新たな臨床的に翻訳可能な治療戦略を開発するために存在します。癌の遺伝子治療は、途方もない約束を示しているものの、主要な課題は、持続的な導入遺伝子の発現につながることができる安全で効果的なデリバリーシステムの開発中です。 ゼラチンは、広く食品や医薬品に使用される最も汎用性の高い天然の生体高分子の一つである。我々の研究室からのこれまでの研究では、タイプBゼラチンは物理的な細胞内送達時にプラスミドと改良されたトランスフェクション効率のスーパーコイル構造を維持したDNAを、カプセル化することができることが示されている。ゼラチンのチオール化により、スルフヒドリル基は、ポリマーとのとFに導入することができます全体の複雑度ジスルフィド結合を安定化ナノ粒子、内ORMのジスルフィド結合は、細胞質ゾルのグルタチオンの存在のために壊れている、ペイロードは2-5を放出される。ポリ（エチレングリコール）（PEG）修飾GENS、全身循環に投与、長期循環時間を提供し、優先的に強化された透過性と保持効果6により新生血管の超透過性に起因する腫瘍塊の対象とする。研究は7 PANC - 1ヒト膵臓腺癌細胞の上皮成長因子受容体（EGFR）の発現を介して示している。積極的に膵臓癌細胞株を標的とするためには、EGFR特異的ペプチドは、PEGのスペーサーを介して粒子表面上に結合させた8。 ほとんどの抗腫瘍遺伝子治療は、9セルのアポトーシス機能を復元するために、そのような野生型p53の腫瘍抑制遺伝子、（WT - P53）の管理に焦点を当てている</&gt;のSup。アポトーシス、細胞周期の停止、代謝および他のプロセス10を制御する細胞増殖、の重要なシグナル伝達経路としてのp53機構機能。膵臓癌では、ほとんどの細胞がアポトーシス活性の損失を引き起こし、p53タンパク質に変異を持っている。 WT - p53の導入により、アポトーシスが修復される可能性があり、さらに癌細胞11に細胞死を誘発する。 上記の理論的根拠に基づいて、我々は、EGFRがWT - p53遺伝子のデリバリーとPANC - 1細胞​​で評価した配信の効率化とトランスフェクションのためにペプチドで修飾されたチオール化ゼラ​​チンナノ粒子をターゲットに設計されている。

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

タイプBゼラチンベースの設計nanovectorsシステム（GENS）が膵臓癌の治療における全身遺伝子デリバリーとトランスフェクションのために開発されました。上皮成長因子受容体（EGFR）nanparticlesの表面上の特定のペプチドによる修飾により、彼らは、EGFR受容体をターゲットに可能性があり、そのような高い細胞内のグルタチオン濃度として、環境を還元条件下でプラスミド離します。

上皮成長因子受容体をターゲットとしたゼラチンベースのエンジニアNanovectorsを使用してヒト膵臓癌細胞における遺伝子デリバリーとトランスフェクション

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 ビュー.  Northeastern University. タイプBゼラチンベースの設計nanovectorsシステム（GENS）が膵臓癌の治療における全身遺伝子デリバリーとトランスフェクションのために開発されました。上皮成長因子受容体（EGFR）nanparticlesの表面上の特定のペプチドによる修飾により、彼らは、EGFR受容体をターゲットに可能性があり、そのような高い細胞内のグルタチオン濃度として、環境を還元条件?...

ビデオ: 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.

ナノ粒子トラッキング分析とハイスループットフローサイトメトリーによる高分子遺伝子デリバリーナノ粒子の評価

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

生物工学

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.

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

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.

生分解性ポリマーナノ粒子を用いた治療的血管形成のためのプログラミング幹細胞

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.

繊維状足場に微小球を離すエレクトロ成長因子

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.

相関顕微鏡を用いて、無傷の細胞における上皮成長因子受容体の化学量論を学びます

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.

カチオン性およびアニオン性リポソームを用いてCNSへの治療用のsiRNAの送達

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.

ヘパリン結合ドメイン骨形成成長因子の送達のための高分子電解質複合体

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.

シルクナノ粒子の製造および薬物送達アプリケーション

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.

神経成長因子カプセル化のための新規HDL模倣ナノ粒子の調製とキャラクタリゼーション

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.

脳癌と神経変性疾患を研究するためのヒト神経オルガノイド

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