这项研究是由美国国家癌症研究所的纳米技术联盟，为癌症的癌症纳米技术卓越中心（CCNE）授予U54 - CA151881的支持。

<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>

没有利益冲突的声明。

控制和表皮生长因子受体有针对性的硫醇盐明胶纳米粒子的制备与高效的DNA封装和稳定。所有这些系统的粒径在150-250 nm的范围内。 Zeta电位已经证明，这个系统是一个稍微负的系统。纳米粒子的大小随着扫描电镜分析，激光粒度仪的结果相同。 ESCA分析，可以确认PEG和肽表面改性。 Western blot分析表明，PANC - 1细胞有高EGFR表达水平，此细胞株在体外研究中使用。在控制和表面改性的纳米粒子在PANC - 1细胞相对较少的细胞毒性相比，爱德华王子岛。 细胞贩运研究表明针对性的纳米粒子在PANC - 1细胞表皮生长因子受体的质粒快速吸收和释放。报告质粒DNA与表皮生长因子受体针对性的纳米粒子表达的交付导致最高水平的GFP的表达相对其他控制系统，包括脂质体复合物的DNA。与同一个系统，与野生型p53质粒转染引发下游的细胞凋亡途径和诱导PANC - 1细胞快速凋亡。 这些初步结果表明，表皮生长因子受体有针对性的硫醇盐明胶纳米粒子可以作为一个安全，高效的DNA传递系统治疗胰腺癌的基因治疗。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> B型明胶，布卢姆225 </td><td> Sigma - Aldrich公司</td><td> G9391 </td><td></td></tr><tr><td> 2 iminothiolane盐酸</td><td> Sigma - Aldrich公司</td><td> I6256 </td><td></td></tr><tr><td>以pEGFP - N1质粒</td><td>琳生物制药</td><td> N / A </td><td></td></tr><tr><td> PORF - hp53大肠杆菌</td><td> Invivogen </td><td> PORF - hp53 </td><td></td></tr><tr><td>乙二醛溶液（40wt.％H 2 O） </td><td> Sigma - Aldrich公司</td><td> 128465 </td><td></td></tr><tr><td>甘氨酸</td><td> Sigma - Aldrich公司</td><td> 410225 - 250G </td><td></td></tr><tr><td> QIA过滤质粒兆丰套件</td><td> Qiageñ </td><td> 12281 </td><td></td></tr><tr><td>贝克曼乐80K超速离心机</td><td>贝克曼</td><td> N / A </td><td></td></tr><tr><td>自由贸易区6升控制台冷冻干燥系统</td><td> Labconco </td><td> 7753020 </td><td></td></tr><tr><td> MPEG - SCM，MW 2000大</td><td> Laysan </td><td> MPEG - SCM - 2K - 1G </td><td></td></tr><tr><td> MAL - PEG - SCM，分子量2000大</td><td> Jenkem技术</td><td> A5001 - 1 </td><td></td></tr><tr><td>纳米激光粒度仪</td><td>马尔文</td><td>纳米激光粒度仪安芷生</td><td></td></tr><tr><td>日立4800场发射扫描电子显微镜</td><td>日立</td><td> S - 4800 UHR FE - SEM </td><td></td></tr><tr><td> QUANT - IT PicoGreen dsDNA抗体试剂和试剂盒</td><td> Invitrogen公司</td><td> P7589 </td><td></td></tr><tr><td>脂质体转染试剂</td><td> Invitrogen公司</td><td> 18292011 </td><td></td></tr><tr><td> DMEM培养液</td><td> Mediatech cellgro </td><td> 10 013厘米</td><td></td></tr><tr><td>的RPMI </td><td> Mediatech cellgro </td><td> 50 020 PB </td><td></td></tr><tr><td>皮尔斯BCA蛋白检测试剂盒</td><td> Thermo Scientific的</td><td> 23225 </td><td></td></tr><tr><td> iBlot干杂交系统</td><td> Invitrogen公司</td><td> IB1001 </td><td></td></tr><tr><td> XCell SureLock迷你细胞和XCell第二杂交模块套件CE标志</td><td> Invitrogen公司</td><td> EI0002 </td><td></td></tr><tr><td>皮尔斯的ECL西方印迹基板</td><td> Thermo Scientific的</td><td> 32109 </td><td></td></tr><tr><td>柯达数字化X射线标本（DXS）系统</td><td>柯达</td><td> N / A </td><td>; </td></tr><tr><td> CellTiter 96 ueous AQ的一个解决方案细胞增殖实验（MTS） </td><td> Promega公司</td><td> G3580 </td><td></td></tr><tr><td>百特SynergyHT酶标仪</td><td>百特</td><td> N / A </td><td></td></tr><tr><td> Nanodrop 2000 </td><td> Thermo Scientific的</td><td> N / A </td><td></td></tr><tr><td>一步法RT - PCR试剂盒</td><td> Qiagen公司</td><td> 210212 </td><td></td></tr><tr><td>染色质凝聚/膜通透性/死细胞凋亡试剂盒</td><td> Invitrogen公司</td><td> V23201 </td><td></td></tr><tr><td> APO - ONE均质的caspase - 3 / 7检测试剂盒</td><td> Promega公司</td><td> G7790 </td><td></td></tr><tr><td> Hybaid PCR扩增Sprint的热循环仪</td><td> Thermo Scientific的</td><td> N / A </td><td></td></tr><tr><td>表皮生长因子受体抗体</td><td>细胞信号</td><td> 2232 </td><td></td></tr><tr><td> β-肌动蛋白抗体</td><td>细胞信号转导</td><td> 4967 </td><td></td></tr><tr><td>抗兔IgG，HRP联抗体</td><td>细胞信号转导</td><td> 7074 </td><td></td></tr><tr><td>鼠单克隆GFP抗体</td><td> Novus公司生物制品</td><td> NB600 - 597 </td><td></td></tr><tr><td>山羊多克隆GFP抗体（碱性磷酸酶） </td><td> Novus公司生物制品</td><td> NB600 - 1502 </td><td></td></tr><tr><td>磷酸酶底物包</td><td> Thermo Scientific的</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

在美国每年超过32000例患者被诊断出患有胰腺癌和相关的疾病是具有非常高的死亡率 1的。存在迫切需要制定小说翻译临床治疗策略，可以提高胰腺癌患者的惨淡生存统计。虽然在癌症基因治疗已经显示出了巨大的承诺，面临的主要挑战是在安全和有效的传递系统，从而导致持续的转基因表达的发展。 明胶是一个最多才多艺的天然生物高分子，广泛应用于食品和医药产品。从我们的实验室以往的研究表明，B型明胶的物理封装的DNA，保存后，细胞内交货的质粒和提高转染效率的超螺旋结构。明胶硫醇，巯基可以被引入到聚合物中，并会fORM纳米粒子，从而稳定整个复杂，一旦二硫键内二硫键被打破，由于在细胞质中的谷胱甘肽存在，有效载荷将发布2-5。聚乙二醇（PEG）的修改氏族，进入体循环，提供长期流通的时间和优先目标，以肿块，由于超的新生血管通透性增强通透性和保留的效果6。有研究表明过度表达的表皮生长因子受体（EGFR）的PANC - 1人类胰腺癌细胞7。为了积极目标胰腺癌细胞株，表皮生长因子受体（EGFR）特异性多肽通过PEG间隔的粒子表面上的共轭。 大多数管理抑癌基因，如野生型p53（WT - P53），恢复在细胞 9的促凋亡的功能都集中在抗肿瘤基因疗法</SUP&gt;。 p53作为一个关键的信号通路在细胞生长，调节细胞凋亡，细胞周期阻滞，新陈代谢和其他进程 10的机制功能。在胰腺癌，大多数细胞p53蛋白的基因突变，从而导致凋亡活性的损失。引进野生型p53，细胞凋亡可被修复，并进一步触发癌细胞的细胞死亡 11 。 基于上述理由，我们设计了野生型p53基因传递和评估交付效率和PANC - 1细胞的转染表皮生长因子受体靶向肽修饰硫醇盐明胶纳米粒子。

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

系统性基因传递和转染治疗胰腺癌的B型明胶为基础的设计nanovectors系统（氏族）的开发。他们可以通过与表皮生长因子受体（EGFR）的特异性多肽nanparticles表面改性，表皮生长因子受体受体目标和下降低细胞内谷胱甘肽的浓度高，如环境，释放质粒。

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

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 次观看.  Northeastern University. 系统性基因传递和转染治疗胰腺癌的B型明胶为基础的设计nanovectors系统（氏族）的开发。他们可以通过与表皮生长因子受体（EGFR）的特异性多肽nanparticles表面改性，表皮生长因子受体受体目标和下降低细胞内谷胱甘肽的浓度高，如环境，释放质粒。

视频: 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.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

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

科研

生物工程

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.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

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.

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

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.

静电生长因子释放到微纤维支架

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.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

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.

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

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.

聚电解质复合物肝素结合域成骨生长因子交货

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.

纳米丝绸制造和药物输送应用

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.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

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.

研究脑癌和神经退行性疾病的人类神经器官

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