الكتاب أشكر MSCRF TEDCO (2009-MSCRFE-0098-00) وNIH R21CA152473 حصول على الدعم.

<ol>
	<li>Putnam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymers+for+gene+delivery+across+length+scales.">Polymers for gene delivery across length scales.</a> Nature Materials. 5, 439-451 (2006).</li><li>Sheyn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+cells+in+regenerative+medicine+and+tissue+engineering.">Genetically modified cells in regenerative medicine and tissue engineering.</a> Advanced Drug Delivery Reviews. 62, 683-698 (2010).</li><li>Boussif, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301 (1995).</li><li>Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rita+Schaffer+Lecture:+Nanoparticles+for+Intracellular+Nucleic+Acid+Delivery.">Rita Schaffer Lecture: Nanoparticles for Intracellular Nucleic Acid Delivery.</a> Ann. Biomed. Eng. 40, 1408-1418 (2011).</li><li>Lynn, D. M., 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=Degradable+poly(beta-amino+esters):+Synthesis,+characterization,+and+self-assembly+with+plasmid+DNA.">Degradable poly(beta-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA.</a> J. Am. Chem. Soc. 122, 10761-10768 (2000).</li><li>Sunshine, J. C., Peng, D. Y., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+transfection+with+polymeric+nanoparticles+are+dependent+on+polymer+end-group+structure,+but+largely+independent+of+nanoparticle+physical+and+chemical+properties.">Uptake and transfection with polymeric nanoparticles are dependent on polymer end-group structure, but largely independent of nanoparticle physical and chemical properties.</a> Mol. Pharm. , (2012).</li><li>Shmueli, R. B., Sunshine, J. C., Xu, Z., Duh, E. J., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+nanoparticles+specific+for+human+microvasculature+and+macrovasculature.">Gene delivery nanoparticles specific for human microvasculature and macrovasculature.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. , (2012).</li><li>Bhise, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+terminal+functionalization+and+molecular+weight+of+a+gene+delivery+polymer+and+transfection+efficacy+in+mammary+epithelial+2-D+cultures+and+3-D+organotypic+cultures.">The relationship between terminal functionalization and molecular weight of a gene delivery polymer and transfection efficacy in mammary epithelial 2-D cultures and 3-D organotypic cultures.</a> Biomaterials. 31, 8088-8096 (2010).</li><li>Tzeng, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+gene+delivery+nanoparticles+based+on+poly(beta-amino+esters)+for+treatment+of+glioblastoma.">Non-viral gene delivery nanoparticles based on poly(beta-amino esters) for treatment of glioblastoma.</a> Biomaterials. 32, 5402-5410 (2011).</li><li>Sunshine, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Molecule+End-Groups+of+Linear+Polymer+Determine+Cell-Type+Gene-Delivery+Efficacy.">Small-Molecule End-Groups of Linear Polymer Determine Cell-Type Gene-Delivery Efficacy.</a> Adv. Mater. 21, 4947 (2009).</li><li>Bhise, N. S., Shmueli, R. B., Gonzalez, J., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+quantifying+the+number+of+plasmids+encapsulated+by+polymer+nanoparticles.">A novel assay for quantifying the number of plasmids encapsulated by polymer nanoparticles.</a> Small. 8, 367-373 (2012).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+intracellular+stability+and+unpacking+of+DNA+nanocomplexes+by+quantum+dots-FRET.">Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.</a> Journal of Controlled Release: Official journal of the Controlled Release Society. 116, 83-89 (2006).</li><li>Kreiss, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid+DNA+size+does+not+affect+the+physicochemical+properties+of+lipoplexes+but+modulates+gene+transfer+efficiency.">Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.</a> Nucleic Acids Research. 27, 3792-3798 (1999).</li><li>Pitard, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-sized+self-assembling+lamellar+complexes+between+plasmid+DNA+and+cationic+micelles+promote+gene+transfer.">Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 14412-14417 (1997).</li><li>Collins, L., Kaszuba, M., Fabre, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+solution+of+(Lys)(16)-containing+bifunctional+synthetic+peptide/DNA+nanoparticles+for+gene+delivery.">Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery.</a> Biochimica et Biophysica Acta. 1672, 12-20 (2004).</li><li>Green, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polymeric+vectors+for+gene+delivery+to+human+endothelial+cells.">Biodegradable polymeric vectors for gene delivery to human endothelial cells.</a> Bioconjug. Chem. 17, 1162-1169 (2006).</li></ol>

واضعي التقرير أي تضارب في المصالح.

البروتوكولات أعلاه تصف أساليب تقييم فعالية ترنسفكأيشن من الصيغ جسيمات متناهية الصغر، وكذلك وسيلة لتوصيف حجم الجسيمات والتحميل DNA من الجسيمات النانوية. عدد البلازميدات في الجسيمات هي العوامل الهامة التي يمكن أن تساعد في التنبؤ فعالية من الجسيمات ويمكن أن تستخدم أيض...

تحديد عدد البلازميدات المعقد في جسيمات متناهية الصغر من المهم أن التصميم الفعال القائم على جسيمات متناهية الصغر لتوصيل الجينات الاستراتيجيات، وخاصة للتسليم المشترك من البلازميدات متعددة إلى نفس الخلية المستهدفة، كما هو مطلوب في كثير من الأحيان الخلايا الجذعية إعادة برمجة الدراسات 12. وقد وصفت بعض النهج لحساب عدد من البلازميدات المرتبطة جسيمات متناهية الصغر واحدة، ولكل النهج عيوب في التقنيات المستخدمة لتقدير 13-16. وقد استخدم الكم نقطة (QD) جنبا إلى جنب مع وضع العلامات TEM لتقدير البلازميدات في الجسيمات في الشيتوزان القائمة على الجسيمات النانوية. معقد تقدير مع هذه التقنية QD بسبب الحاجة لتسمية DNA، والتي قد تغير في خصائص التجميع الذاتي، واحتمال أن لا يتم الكشف عن الحمض النووي غير المسماة مغلفة مباشرة؛ البلازميدات يحتمل أن تكون متداخلة وQDs في الصور TEM 2D من الجسيمات، و الافتراضات الأخرى تبسيط 13. نهج بديل هو أنpplicable عندما microdomains أمر موجود في جزيئات وقد استخدمت لدراسة الحمض النووي المجمعات Lipopolyamine عبر المجهر الإلكتروني انتقال البرد (البرد TEM)، وتشتت الأشعة السينية، وديناميكية تشتت الضوء (DLS) 14،15. للأسف، مثل المواد النانوية البوليمرية التحقيق هنا لا تنطبق مع هذا الأسلوب. وفي دراسة أخرى، وتستخدم كولينز وآخرون تدفق الجسيمات صورة تقنية التحليل لدراسة (ليز) التي تحتوي على الببتيد 16 / DNA المجمعات، إلا أنه يمكن تقييم طريقتهم فقط أكبر، ميكرون الحجم الجسيمات 16. وهكذا، فإننا وضعت مؤخرا فحص الرواية ومرنة لتحديد عدد من جسيمات متناهية الصغر في البلازميدات 11.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td></td><td></td><td> كاشف </td></tr><tr><td> التخزين المؤقت المالحة الفوسفات، 1X (PBS) </td><td> إينفيتروجن </td><td> 10010 </td><td></td></tr><tr><td> EGM-2MV BulletKit </td><td> Lonza </td><td> CC-3202 </td><td></td></tr><tr><td> التربسين </td><td> إينفيتروجن </td><td> 25300 </td><td></td></tr><tr><td> خلات الصوديوم العازلة </td><td> سيغما الدريخ </td><td> S7899 </td><td> تمييع الى 25mm في الماء منزوع الأيونات </td></tr><tr><td> ثنائي ميثيل سلفوكسيد </td><td> سيغما الدريخ </td><td> 276855 </td><td></td></tr><tr><td> pEGFP DNA </td><td> المستحضرات الصيدلانية البيولوجية ايليم </td><td> NA </td><td></td></tr><tr><td> DsRed DNA </td><td> Addgene </td><td> 21718 </td><td></td></tr><tr><td> PEI، تشعبت </td><td> سيغما الدريخ </td><td> 408727 </td> &lt;TD&gt; </td></tr><tr><td> CellTiter 96 مائي واحد </td><td> Promega </td><td> G3580 </td><td></td></tr><tr><td></td><td></td><td></td><td> المواد </td></tr><tr><td> مسطحة القاع واضحة بشكل جيد 96-وحة، عقيمة </td><td> Sarstedt </td><td> 82.1581.001 </td><td></td></tr><tr><td> جولة القاع 96-اضحة جيدا لوحة، ومعقمة </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> 12-قناة Finnpipette </td><td> الحرارية العلمية </td><td> NA </td><td> 5-50 ميكرولتر و50-300 </td></tr><tr><td> مضان المجهر </td><td> زايس </td><td> NA </td><td> موديل: AX10 </td></tr><tr><td> C6 عداد الكريات Accuri تدفق </td><td> BD العلوم البيولوجية </td><td> NA </td><td></td></tr><tr><td> HyperCyt المرفقات </td><td> Intellicyt </td><td> NA </td><td></td></tr><tr><td> NS500</td><td> Nanosight </td><td> NA </td><td></td></tr></tbody></table>

evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry

وقد برزت غير الفيروسية توصيل الجينات باستخدام الجسيمات النانوية البوليمرية كنهج جاذبية للعلاج بالجينات لعلاج الأمراض الوراثية (1) وبوصفها تكنولوجيا للطب التجديدي 2. على عكس الفيروسات، والتي لها قضايا السلامة كبيرة، ويمكن تصميم النانوية البوليمرية أن تكون غير سامة، غير مناعة، غير مطفرة وأسهل لتجميع، وتنوعا كيميائيا، قادرة على حمل أكبر البضائع القابلة للتحلل الحمض النووي وو / أو استجابة للبيئة. البوليمرات الموجبة الشحنة السالبة مع DNA عن طريق التفاعل الكهربائي لتشكيل مجمعات بناء على أمر من 100 نانومتر التي تسمى عادة النانوية البوليمرية النفس التجمع. أمثلة على المواد الحيوية المستخدمة لتشكيل البنى النانوية لتوصيل الجينات polycationic تشمل متعدد الليزين، polyphosphoesters، وبولي (amidoamines) ق وpolyethylenimine (PEI)، وهو غير القابلة للتحلل قبالة الجاهزة للاستخدام البوليمر الموجبة تستخدم عادة لتسليم الحمض النووي 1،3. بولي (بيتا الأمينيةاستر) ق (PBAEs) هي أحدث فئة من البوليمرات الموجبة 4 أن للتفكك وهيدروليكيا 5،6 وقد ثبت أن تكون فعالة في إيصال الجينات إلى أنواع الخلايا التي يصعب بالنقل مثل خلايا الشبكية البطانية الإنسان (HRECs) 7، الماوس الخلايا الظهارية الثديية 8، خلايا سرطان الدماغ 9 و الأوعية الكبيرة (الإنسان الوريد السري، HUVECs) الخلايا البطانية 10. ووصف بروتوكول جديد لتوصيف جزيئات البوليمر باستخدام تحليل جسيمات متناهية الصغر تتبع (NTA). في هذا النهج، ويتم الحصول على كل حجم الجسيمات توزيع وتوزيع عدد من الجسيمات في البلازميدات 11. وبالإضافة إلى ذلك، يتم تقديم الإنتاجية العالية ترنسفكأيشن 96-وحة جيدا الفحص للكشف السريع للفعالية ترنسفكأيشن من الجسيمات النانوية البوليمرية. وتستخدم في هذا البروتوكول، وتستخدم بولي (بيتا الأمينية استر) ق (PBAEs) والبوليمرات ونموذج الإنسان الخلايا البطانية الشبكية (HRECs) كما موديل الخلايا البشرية. يمكن هذا البروتوكول تكيف بسهولة مع أي جسيمات متناهية الصغر البوليمرية تقييم وأي نوع من الخلايا في المصالح في شكل لوحة متعددة أيضا.

ويظهر الشكل 1 صورة من مضان المجهري مثال على نجاح HRECs ترنسفكأيشن مع البلازميد EGFP. الصورة brightfield مفيد لضمان الحفاظ على التشكل أن الخلايا المعتادة. بالإضافة إلى ذلك، يمكن استخدام المقايسات بقاء الخلية، مثل MTS أو المقايسات مماثلة، لتقييم سمية جسيمات متناهية الصغ...

Watch this Scientific Journal Video about Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry at JoVE.com

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

يوصف بروتوكول للتحليل تتبع جسيمات متناهية الصغر (NTA) والإنتاجية العالية التدفق الخلوي لتقييم البوليمرية النانوية لتوصيل الجينات. ويستخدم لوصف NTA حجم الجسيمات جسيمات متناهية الصغر وتوزيع البلازميد توزيع الجسيمات في. الإنتاجية العالية التدفق الخلوي تمكن الكمي تقييم فعالية ترنسفكأيشن للمكتبة من المواد الحيوية لتوصيل الجينات.

تقييم توصيل الجينات البوليمرية الجسيمات النانوية من تحليل تتبع جسيمات متناهية الصغر وعالية الإنتاجية قياس التدفق الخلوي

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

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Bioengineering

15.7K Views.  Johns Hopkins University School of Medicine. يوصف بروتوكول للتحليل تتبع جسيمات متناهية الصغر (NTA) والإنتاجية العالية التدفق الخلوي لتقييم البوليمرية النانوية لتوصيل الجينات. ويستخدم لوصف NTA حجم الجسيمات جسيمات متناهية الصغر وتوزيع البلازميد توزيع الجسيمات في. الإنتاجية العالية التدفق الخلوي تمكن الكمي ت...

Video: Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

صياغة النانوية Diblock البوليمر من خلال تقنيات Nanoprecipitation

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

[ميكروأرس] البوليمر لاكتشاف إنتاجية عالية من المواد الحيوية

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

اندماجي تجميع وعالية الإنتاجية الإصدار البروتين من فيلم البوليمر ومكتبات جسيمات متناهية الصغر

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

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

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

تحليل التطبع الخلوي من الجزيئات الدقيقة والبكتيريا التي متعددة الأطياف التصوير التدفق الخلوي

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

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

الإنتاجية العالية لتخليق الكربوهيدرات وFunctionalization النانوية Polyanhydride

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

البوليمر تصنيع صفيف إبرة مجهرية من قبل الطباعة التصويرية

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

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.

تطبيقات تصنيع وتسليم المخدرات من النانوية الحرير

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

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.

عزل الخلايا العقدة الشبكية الفئران الأولية (رغس) عن طريق التدفق الخلوي

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

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

نظام ميكروبيوريكتور لإنتاج IgG1 النموذجي في الخلايا شو الآلي استخدام الفائق

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

فلاش نانوبريسيبيتيشن لتغليف مركبات مسعور وماء في الجسيمات النانوية البوليمرية

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...