이 연구는 암 나노기술 우수에 대한 암 센터 (CCNE) 부여 U54 - CA151881에 대한 나노기술의 국립 암 연구소의 연합에 의해 지원되었다.

<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>

관심 없음 충돌 선언하지 않습니다.

제어 및 EGFR 대상 thiolated 젤라틴 nanoparticles는 효율적인 DNA의 캡슐화 및 안정성과 함께 준비했다. 이러한 시스템의 모든 입자 크기는 직경 150-250 nm의 범위에 있었다. 제타 전위는이 시스템이 약간 부정적인 시스템입니다 것을 증명했다. SEM 분석, nanoparticles의 크기는 Zetasizer 결과와 동일한되었습니다. ESCA 분석 PEG와 펩타이드 표면 수정을 확인 수 있습니다. 서양 얼룩 분석 Panc - 1 세포가 높은 EGFR 발현 수준을했고이 셀 라인은 체외 연구에서 사용되었다 것으로 나타났다. PEI에 비해 제어 및 표면 수정 nanoparticles 모두 Panc - 1 세포에서 상대적으로 덜 세포 독성되었습니다. 세포 밀매 연구 빠른 이해와 Panc - 1 세포에서 EGFR 타겟 nanoparticles의 플라스미드 릴리스를 보여주었다. EGFR 타겟 nanoparticles로 표현 기자 plasmids의 DNA의 배달의 최고 수준의 결과Lipofectin - complexed DNA 등 다른 컨트롤에 상대적으로 GFP의 표현. 동일한 시스템으로 wt - p53의 플라스미드와 함께 transfection은 하류 apoptotic 통로 및 Panc - 1 세포에서 유도된 apoptosis 빠른 울렸어요. 이러한 예비 결과는 EGFR 타겟 thiolated 젤라틴 nanoparticles는 췌장암에 대한 치료로 유전자 치료를위한 안전하고 효율적인 유전자 전달 시스템으로 사용할 수있는 것이 좋습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 타입 B 젤라틴, 블룸 225 </td><td> 시그마 - 올드 리치 </td><td> G9391 </td><td></td></tr><tr><td> 2 iminothiolane 하이드로 클로라이드 </td><td> 시그마 - 올드 리치 </td><td> I6256 </td><td></td></tr><tr><td> pEGFP - N1 플라스미드 </td><td> Elim Biopharm </td><td> N / A </td><td></td></tr><tr><td> pORF - hp53 E. 대장균 </td><td> Invivogen </td><td> porf - hp53 </td><td></td></tr><tr><td> Glyoxal 솔루션 (H 2 O에 40wt. %) </td><td> 시그마 - 올드 리치 </td><td> 128,465 </td><td></td></tr><tr><td> 글리신 </td><td> 시그마 - 올드 리치 </td><td> 410225 - 250G </td><td></td></tr><tr><td> QIA 필터 플라스미드 메가 키트 </td><td> QiageN </td><td> 12,281 </td><td></td></tr><tr><td> 베크 만 LE 80K의 초원 심 분리기 </td><td> 베크 만 </td><td> N / A </td><td></td></tr><tr><td> FreeZone 6 리터 콘솔 드라이 시스템을 고정 </td><td> Labconco </td><td> 7,753,020 </td><td></td></tr><tr><td> MPEG - SCM, MW 2000 다 </td><td> Laysan </td><td> MPEG - SCM - 2K - 1g </td><td></td></tr><tr><td> 말 - PEG - SCM, MW 2000 다 </td><td> Jenkem 기술 </td><td> A5001 - 1 </td><td></td></tr><tr><td> Zetasizer 나노 </td><td> Malvern </td><td> Zetasizer 나노 ZS </td><td></td></tr><tr><td> 전자 현미경 스캐닝 히타치 4800 전계 방출 </td><td> 히타치 </td><td> S - 4800 UHR FE - SEM </td><td></td></tr><tr><td> 양의 - IT PicoGreen dsDNA 시약 및 키트 </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> Lipofectin Transfection 시약 </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> 피어스 BCA 단백질 분석 키트 </td><td> 써모 과학 </td><td> 23,225 </td><td></td></tr><tr><td> iBlot 드라이 Blotting 시스템 </td><td> Invitrogen </td><td> IB1001 </td><td></td></tr><tr><td> XCell SureLock 미니 셀 및 XCell II 얼룩 모듈 키트 CE 마크 </td><td> Invitrogen </td><td> EI0002 </td><td></td></tr><tr><td> 피어스 ECL 웨스턴 기판 Blotting </td><td> 써모 과학 </td><td> 32,109 </td><td></td></tr><tr><td> 코닥 디지털 X - 선 표본 (DXS) 시스템 </td><td> 코닥 </td><td> N / A </td><td>; </td></tr><tr><td> CellTiter 96 AQ ueous 하나의 솔루션 세포 증식 분석 (MTS) </td><td> Promega </td><td> G3580 </td><td></td></tr><tr><td> BioTek SynergyHT 플레이트 리더 </td><td> BioTek </td><td> N / A </td><td></td></tr><tr><td> Nanodrop 2000 </td><td> 써모 과학 </td><td> N / A </td><td></td></tr><tr><td> 한 단계 RT - PCR 키트 </td><td> Qiagen </td><td> 210,212 </td><td></td></tr><tr><td> 염색질은 응축 / 분리막 투과율 / 죽은 세포 Apoptosis 키트 </td><td> Invitrogen </td><td> V23201 </td><td></td></tr><tr><td> APO - ONE 동차 Caspase - 7분의 3 분석 키트 </td><td> Promega </td><td> G7790 </td><td></td></tr><tr><td> Hybaid PCR 스프린트 열 자전거 타는 사람 </td><td> 써모 과학 </td><td> N / A </td><td></td></tr><tr><td> EGF 수용체 항체 </td><td> 세포신호 </td><td> 2232 </td><td></td></tr><tr><td> β - 굴지의 항체 </td><td> 세포 신호 </td><td> 4967 </td><td></td></tr><tr><td> 안티 - 토끼 IgG, HRP - 연결된 항체 </td><td> 세포 신호 </td><td> 7074 </td><td></td></tr><tr><td> 마우스 단클론 항체 GFP </td><td> Novus 체액 </td><td> NB600 - 597 </td><td></td></tr><tr><td> 염소 Polyclonal 항체 GFP (알카라인 인산 가수 분해 효소) </td><td> Novus 체액 </td><td> NB600 - 1502 </td><td></td></tr><tr><td> 인산 가수 분해 효소 기판 키트 </td><td> 써모 과학 </td><td> 37,620 </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 명 이상의 환자가 매년 미국에서 췌장암 진단을하고 있으며이 질병은 매우 높은 사망률 1 연결되어 있습니다. 긴급 필요 췌장암 환자의 음침한 생존 통계에 향상시킬 수있는 소설 임상 - 번역 치료 전략을 개발하기 위해 존재합니다. 암의 유전자 치료는 엄청난 약속을 보여주었다 있지만, 가장 큰 문제는 지속적인 transgene 발현으로 이어질 수있는 안전하고 효과적인 전달 시스템의 개발에 있습니다. 젤라틴은 널리 음식과 의약품에 사용되는 가장 다용도 천연 생체 고분자 중 하나입니다. 저희 연구실에서 이전 연구는 B 타입 젤라틴 실제 세포내 전달시 플라스미드 향상된 transfection 효율의 supercoiled 구조를 보존 DNA를 캡슐 수있는 것으로 나타났습니다. 젤라틴의 thiolation으로 sulfhydryl 그룹은 고분자에 도입 F 것이 될 수전체 복잡하고 한번 이황화 결합을 안정화 nanoparticles 이내 orm의 이황화 결합은 cytosol에서 글루타티온의 존재로 인해 고장, 페이로드는 2-5 출시 것입니다. 폴리 (에틸렌 글리콜) (PEG) - 수정 GENS, 체계 유통 관리, 긴 순환 시간을 제공하며 우선적으로 향상된 투자율 및 유지 효과 6 neovasculature의 하이퍼 투자율에 의한 종양 미사를 대상으로하고 있습니다. 연구 Panc - 1 인간의 췌장 선암 세포 7 표피 성장 인자 수용체 (EGFR)의 표현을 통해 보여주었다. 적극적으로 췌장암 세포주를 대상으로하기 위해서는 EGFR 특정 펩타이드가 PEG 스페이서를 통해 입자 표면에 복합되었습니다 8. 대부분의 안티 종양 유전자 요법은 9 세포에서 프로 apoptotic 기능을 복원하는 등 야생 유형 p53의 (wt - p53의)와 같은 종양 억제 유전자,의 관리에 초점을 맞추고있다 </&gt;를 한모금. 중요한 신호 apoptosis를 조절 세포 성장 경로, 세포주기 체포, 신진 대사 및 기타 프로세스 10 p53의 메커니즘 기능. 췌장암에서 대부분의 세포가 apoptotic 활동의 손실을 초래하는 p53 단백질에 돌연변이 있습니다. wt - p53의 도입과 함께, apoptosis는 수리 수 있으며 추가로 암 세포를 11 세포 죽음을 트리거합니다. 위의 근거를 바탕으로, 우리는 EGFR이 wt - p53의 유전자 전달 및 Panc - 1 세포에서 평가 배달 효율성 및 transfection을위한 펩타이드 - 수정 thiolated 젤라틴 nanoparticles을 대상으로 설계했습니다.

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 타입 젤라틴 기반 공학 nanovectors 시스템 (GENS)은 췌장암의 치료 체계 유전자 전달 및 transfection을 위해 개발되었습니다. 표피 성장 인자 수용체 (EGFR) nanparticles의 표면에 특정 펩타이드와 수정함으로써, 그들은 EGFR 수용체에서 대상 수와 같은 높은 세포내 글루타티온의 농도로, 환경을 감소 밑에 플라스미드 놓습니다.

표피 성장 인자를 사용하여 인간의 췌장 암 세포의 유전자 전달 및 Transfection은 다행 기반 엔지니어링된 Nanovectors를 수용체 타겟

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

28.0K 조회수.  Northeastern University. B 타입 젤라틴 기반 공학 nanovectors 시스템 (GENS)은 췌장암의 치료 체계 유전자 전달 및 transfection을 위해 개발되었습니다. 표피 성장 인자 수용체 (EGFR) nanparticles의 표면에 특정 펩타이드와 수정함으로써, 그들은 EGFR 수용체에서 대상 수와 같은 높은 세포내 글루타티온의 농도로, 환경을 감소 밑에 플라스미드 놓습니다.

비디오: 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.

고분자 유전자 배달에 대한 평가는 Nanoparticle 추적 분석 및 높은 처리량 유동 세포 계측법에 의해 나노 입자

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

연구

생체공학

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.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

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.

생분해 성 고분자 나노 입자를 사용하여 치료 혈관 신생에 대한 프로그래밍 줄기 세포

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.

섬유 공사장 공중 발판으로 마이크로 스피어를 해제 전기 방사의 성장 인자

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.

상호 현미경을 사용하여 본래 세포의 표피 성장 인자 수용체의 화학 양론을 공부

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.

양이온과 음이온 리포좀을 사용 CNS에 치료의 siRNA 배달

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.

헤파린 결합 도메인 골 형성 성장 인자 전달을위한 고분자 전해질 복합

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.

실크 나노 입자의 제조 및 약물 전달 응용 프로그램

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.

신경 성장 인자 인 캡슐 레이션을위한 새로운 HDL 모방 나노 입자의 제조 및 특성 규명

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

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

PDMS를 이용한 높은 처리량 견인력 현미경 검사법은 상피-중간엽 전이에 대한 성장 인자-β 변환의 용량 의존적 효과를 밝혀냅니다.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...