저자는 금융에 대한 ONR-MURI 수상 (NN00014-06-1-1176)과 미군 의료 연구 및 Materiel 명령을 (부여 번호 W81XWH-09-1-0386 및 W81XWH-10-1-0806) 감사드립니다 지원합니다.

<ol>
	<li>Ulery, B. D., Kumar, D., Ramer-Tait, A. E., Metzger, D. W., Wannemuehler, M. J., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+protective+single-dose+intranasal+nanoparticle-based+vaccine+platform+for+respiratory+infectious+diseases.">Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases.</a> PLoS One. 6, e17642 (2011).</li><li>Kasturi, S. P., Skountzou, I., Albrecht, R. A., Koutsonanos, D., Hua, T., Nakaya, H. I., Ravindran, R., Stewart, S., Alam, M., Kwissa, M., Villinger, F., Murthy, N., Steel, J., Jacob, J., Hogan, R. J., Garc&#237;a-Sastre, A., Compans, R., Pulendran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+the+magnitude+and+persistence+of+antibody+responses+with+innate+immunity.">Programming the magnitude and persistence of antibody responses with innate immunity.</a> Nature. 470, 543-547 (2011).</li><li>Rice-Ficht, A. C., Arenas-Gamboa, A. M., Kahl-McDonagh, M. M., Ficht, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+particles+in+vaccine+delivery.">Polymeric particles in vaccine delivery.</a> Curr. Opin. Microbiol. 13, 106-112 (2010).</li><li>Jain, J. P., Chitkara, D., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyanhydrides+as+localized+drug+delivery+carrier:+an+update.">Polyanhydrides as localized drug delivery carrier: an update.</a> Expert. Opin. Drug. Deliv. 5, 889-907 (2008).</li><li>Pfeifer, B. A., Burdick, J. A., Little, S. R., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ester-anhydride):poly(beta-amino+ester)+micro-+and+nanospheres:+DNA+encapsulation+and+cellular+transfection.">Poly(ester-anhydride):poly(beta-amino ester) micro- and nanospheres: DNA encapsulation and cellular transfection.</a> Int. J. Pharm. 304, 210-219 (2005).</li><li>Ahmed, F., Friend, S., George, T. C., Barteneva, N., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numbers+matter:+quantitative+and+dynamic+analysis+of+the+formation+of+an+immunological+synapse+using+imaging+flow+cytometry.">Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry.</a> J. Immunol. Methods. 347, 79-86 (2009).</li><li>Hampton, M. B., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+phagocytosis+and+bacterial+killing+by+human+neutrophils.">Methods for quantifying phagocytosis and bacterial killing by human neutrophils.</a> J. Immunol. Methods. 232, 15-22 (1999).</li><li>Rieger, A. M., Hall, B. E., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+differentially+modulates+particle+binding,+phagocytosis+and+downstream+antimicrobial+mechanisms.">Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms.</a> Dev. Comp. Immunol. 34, 1144-1159 (2010).</li><li>Murphy, K. C., Campellone, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lambda+Red-mediated+recombinogenic+engineering+of+enterohemorrhagic+and+enteropathogenic+E.+coli.">Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli.</a> BMC. Mol. Biol. 4, 11 (2003).</li><li>Karsi, A., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+host+range+fluorescence+and+bioluminescence+expression+vectors+for+Gram-negative+bacteria.">Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria.</a> Plasmid. 57, 286-295 (2007).</li><li>Ulery, B. D., Phanse, Y., Sinha, A., Wannemuehler, M. J., Narasimhan, B., Bellaire, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+chemistry+influences+monocytic+uptake+of+polyanhydride+nanospheres.">Polymer chemistry influences monocytic uptake of polyanhydride nanospheres.</a> Pharm. Res. 26, 683-690 (2009).</li><li>Doherty, G. J., McMahon, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+endocytosis.">Mechanisms of endocytosis.</a> Annu. Rev. Biochem. 78, 857-902 (2009).</li><li>Vercauteren, D., Vandenbroucke, R. E., Jones, A. T., Rejman, J., Demeester, J., De Smedt, S. C., Sanders, N. N., Braeckmans, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+inhibitors+to+study+endocytic+pathways+of+gene+carriers:+optimization+and+pitfalls.">The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.</a> Mol. Ther. 18, 561-569 (2010).</li><li>Di Marzio, L., Marianecci, C., Cinque, B., Nazzarri, M., Cimini, A. M., Cristiano, L., Cifone, M. G., Alhaique, F., Carafa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-sensitive+non-phospholipid+vesicle+and+macrophage-like+cells:+binding,+uptake+and+endocytotic+pathway.">pH-sensitive non-phospholipid vesicle and macrophage-like cells: binding, uptake and endocytotic pathway.</a> Biochim. Biophys. Acta. 1778, 2749-2756 (2008).</li><li>Torres, M. P., Vogel, B. M., Narasimhan, B., Mallapragada, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+novel+polyanhydrides+with+tailored+erosion+mechanisms.">Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms.</a> J. Biomed. Mater. Res. A. 76, 102-110 (2006).</li></ol>

Sherree L. 친구는 ImageStream의 X 시스템을 제조 Amnis 회사에 의해 고용된다.

연구는 폴리 (락트 - 공동 glycolic 산 (PLGA) 또는 polyanhydrides을 기반으로 생분해성 nanoparticles는 대상 세포를 캡슐 항원이나 약물을 전달하는 데 사용할 수있는 것으로 나타났습니다. phagocytic 세포에 의해 이러한 nanoparticles의 이해 이렇게 양적으로 만들어 그들의 효과를 위해 중요하다 . 소설 nanoparticle 전달 시스템을 설계에서 중요한 내면화의 분석이 방법을 사용함으로써는 다양한 세포 유형별로 nano...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> RAW 264.7 세포주 </td><td> 미국 유형 문화 수집 (ATCC) </td><td> 티비 - 71 </td><td></td></tr><tr><td> Dulbecco의 수정일 독수리 중간 (DMEM) </td><td> Cellgro </td><td> 10-013 - 이력서 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> 애틀랜타 체액 </td><td> S 11,150 </td><td> 프리미엄 학년 </td></tr><tr><td> Glutamax </td><td> Gibco </td><td> 35050-061 </td><td></td></tr><tr><td> HEPES </td><td> Gibco </td><td> 15630-080 </td><td></td></tr><tr><td> 24 잘 플레이트</td><td> TPP </td><td> 92,024 </td><td></td></tr><tr><td> 세포 배양 Flasks </td><td> TPP </td><td> 90,151 </td><td></td></tr><tr><td> 셀 스크레이퍼 </td><td> TPP </td><td> 99,002 </td><td> 24cm </td></tr><tr><td> 살모넬라 enterica serovar Typhimurium </td><td> ATCC </td><td> 14,028 </td><td></td></tr><tr><td> BTX ECM630 전기 세포 조작하는 사람 </td><td> BTX 하버드 장치 </td><td></td><td></td></tr><tr><td> 맙스 </td><td> 피셔 과학 </td><td> BP308 </td><td></td></tr><tr><td> 인산염은 식염수 (PBS)를 버퍼 </td><td> Cellgro </td><td> 21-040 - 이력서 </td><td></td></tr><tr><td> 초음파 액체 프로세서 </td><td> MISONIX </td><td> S-4000 </td><td></td></tr><tr><td> Cytochalasin-D </td><td> 시그마 - 올드 리치, </td><td> C8273 </td><td></td></tr><tr><td> 포름 알데히드 </td><td> Polysciences </td><td> 04,018 </td><td></td></tr><tr><td> 버퍼를 씻으십시오 </td><td> 2퍼센트 열 inactivated FBS, PBS에 0.​​1 %의 나트륨 azide. </td><td></td><td></td></tr><tr><td> 버퍼를 씻고 / 파마 </td><td> BD Biosciences </td><td> 554,714 </td><td></td></tr><tr><td> 스냅 캡 microtubes를 클리어 뷰 </td><td> 시그마 </td><td> T4816 </td><td></td></tr><tr><td> 알렉사 형석 phalloidin 660 </td><td> Invitrogen </td><td> A22285 </td><td></td></tr><tr><td> ImageStream X </td><td> Amnis Corpora기 </td><td> 100,200 </td><td> 옵션 : 658nm 레이저, autosampler </td></tr><tr><td> 나트륨 azide </td><td> 피셔 과학 </td><td> S 227I-500 </td><td></td></tr></tbody></table>

analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry

Nanoparticulate 시스템은 항원 제시 세포를 1-5으로, 효율적으로 단백질을 포함하여화물을 전달하는 능력을 통해 백신 전달에 유용한 도구로 등장했습니다. 항원 제시 세포에 의해 nanoparticles의 내면화 (NP)는 캡슐화된 항원에 대한 효과적인 면역 반응을 생성에 중요한 단계입니다. nanoparticle 제제 영향을 미치는 기능에 어떻게 변경 사항을 확인하려면, 우리는 internalized nanoparticles뿐만 아니라 박테리아를 검출과 호환였다 높은 처리량, 양적 실험 프로토콜을 개발하기 위해 모색하고 있습니다. 현재까지, 두 개의 독립적인 기법, 현미경 및 유동세포계측법는 nanoparticles의 phagocytosis를 연구하는 데 사용되는 방법 왔습니다. 유동세포계측법의 높은 처리량 본질은 강력한 통계 데이터를 생성합니다. 그러나, 낮은 해상도로 인해, 그것은 정확하게 셀 바운드 nanoparticles 비해 internalized 수치 실패합니다. 현미경은 높은 공간적 해상도로 이미지를 생성합니다; However, 그건 시간이 소요되며 작은 샘플 크기에게 6-8을 포함한다. 멀티 스펙트럼 이미징 유동세포계측법 (MIFC)는 판상 코어를 통해 동시에 멀티 컬러 스펙트럼 형광 및 명시야 현미경 이미징을 수행하고 유동세포계측법 양쪽 측면을 통합함으로써 새로운 기술입니다. 이 기능은 형광 신호 강도와 다양한 구조와 고속 세포 기능 사이의 공간적 관계의 정확한 분석을 제공합니다. 여기, 우리는 polyanhydride의 nanoparticles이나 살모넬라 enterica serovar Typhimurium을 internalized 않은 세포 인구를 특성화하기 위해 MIFC을 활용한 방법을 설명합니다. 우리는 또한 nanoparticle의 현탁액, 세포 라벨, ImageStream X 시스템의 취득 및 아이디어 응용 프로그램을 사용하여 데이터 분석 준비에 대해 설명합니다. 또한 국제화 P를 차별화하는 데 사용할 수있는 기술의 응용을 설명actin - 중재의 phagocytosis의 억제제로서 cytochalasin-D를 사용하여 nanoparticles와 박테리아 athways.

Watch this Scientific Journal Video about Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry at JoVE.com

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

이 문서에서는 RAW 264.7 세포에 의한 polyanhydride의 nanoparticles이나 박테리아의 내면화를 수치 멀티 스펙트럼 이미징 유동세포계측법을 활용한 방법을 설명합니다.

멀티 스펙트럼 이미징 유동세포계측법 의해 Nanoparticles과 박테리아의 세포 내면화 분석

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

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Research

JoVE Journal

Bioengineering

16.2K 조회수.  Iowa State University. 이 문서에서는 RAW 264.7 세포에 의한 polyanhydride의 nanoparticles이나 박테리아의 내면화를 수치 멀티 스펙트럼 이미징 유동세포계측법을 활용한 방법을 설명합니다.

비디오: Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

원자 힘 현미경에 의해 영상에 대한 세균 고정

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

연구

생체공학

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Polyanhydride의 Nanoparticles에 의해 유도된 Murine Macrophages 폐포 수확 및 평가 세포 활성화

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

에 대한 바이러스 나노 입자<em&gt; 생체에</em&gt; 종양 이미징

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

휴대 전화에 폭 넓은 분야 형광 현미경과 형광 이미징 유동 세포 계측법

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

멀티 스펙트럼 이미징 유동 세포 계측법과 세포의 현지화 및 탄소 나노 튜브의 상대 정량

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

유동 세포 계측법에 의한 일차 망막 신경절 세포 (RGCs)의 분리

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

간염 E 바이러스 나노 화학 활용 방법을 사용 하 여의 표면 기능화

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

광음향 유동 세포측정을 이용한 난소암 검출

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

현탁액에서 금 나노 입자의 크기 조정을위한 비대칭 유동 필드 흐름 분획

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...