המחברים מבקשים להודות צבא ארה&quot;ב למחקר רפואי ואמצעי פיקוד (במדבר גרנט W81XWH-09-1-0386 ו W81XWH-10-1-0806) עבור תמיכה כספית ד&quot;ר שון ריגבי מ מתקן מדינת איווה אוניברסיטת cytometry זרימה עבור מומחה סיוע טכני שלו.

<ol>
	<li>Mallapragada, S. K., 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=Immunomodulatory+biomaterials.">Immunomodulatory biomaterials.</a> Int. J. Pharm. 364, 265-271 (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><li>Lopac, S. K., Torres, M. P., Wilson-Welder, J. H., Wannemuehler, M. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+innate+immune+responses+in+a+pathogen-mimicking+manner+by+amphiphilic+polyanhydride+nanoparticle+adjuvants.">Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants.</a> Biomaterials. 32, 6815-6822 (2011).</li><li>Torres, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyanhydride+microparticles+enhance+dendritic+cell+antigen+presentation+and+activation.">Polyanhydride microparticles enhance dendritic cell antigen presentation and activation.</a> Acta. Biomater. 7, 2857-2864 (2011).</li><li>Kirby, A. C., Coles, M. C., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+transport+pathogens+to+lung+draining+lymph+nodes.">Alveolar macrophages transport pathogens to lung draining lymph nodes.</a> J. Immunol. 183, 1983-1989 (2009).</li><li>Barletta, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+compartments+in+the+mouse+lung:+Distinguishing+between+marginated,+interstitial,+and+alveolar+cells+in+response+to+injury.">Leukocyte compartments in the mouse lung: Distinguishing between marginated, interstitial, and alveolar cells in response to injury.</a> J. Immunol. Methods. , (2011).</li><li>Rubins, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages:+wielding+the+double-edged+sword+of+inflammation.">Alveolar macrophages: wielding the double-edged sword of inflammation.</a> Am. J. Respir. Crit. Care Med. 167, 103-104 (2003).</li><li>Sun, K., Gan, Y., Metzger, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Murine+Genetic+Predisposition+to+Pneumococcal+Infection+Reveals+a+Critical+Role+of+Alveolar+Macrophages+in+Maintaining+the+Sterility+of+the+Lower+Respiratory+Tract.">Analysis of Murine Genetic Predisposition to Pneumococcal Infection Reveals a Critical Role of Alveolar Macrophages in Maintaining the Sterility of the Lower Respiratory Tract.</a> Infect. Immun. 79, 1842-1847 (2011).</li><li>Morimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+Macrophages+that+Phagocytose+Apoptotic+Neutrophils+Produce+Hepatocyte+Growth+Factor+during+Bacterial+Pneumonia+in+Mice.">Alveolar Macrophages that Phagocytose Apoptotic Neutrophils Produce Hepatocyte Growth Factor during Bacterial Pneumonia in Mice.</a> Am. J. Respir. Cell Mol. Biol. 24, 608-615 (2001).</li><li>Gordon, S., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+and+macrophage+heterogeneity.">Monocyte and macrophage heterogeneity.</a> Nat. Rev. Immunol. 5, 953-964 (2005).</li><li>Ulery, B. D. <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>Petersen, L. K., Sackett, C. K., 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=High-throughput+analysis+of+protein+stability+in+polyanhydride+nanoparticles.">High-throughput analysis of protein stability in polyanhydride nanoparticles.</a> Acta Biomater. 6, 3873-3881 (2010).</li><li>Irvin, C. G., Bates, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+lung+function+in+the+mouse:+the+challenge+of+size.">Measuring the lung function in the mouse: the challenge of size.</a> Respir. Res. 4, 4 (2003).</li></ol>

אין ניגוד עניינים הצהיר.

Nanoparticle Polyanhydride פלטפורמות החיסונים הראו יעילות כאשר מנוהל intranasally ב משטרי מינון יחיד 5. מדידת ההפעלה של אוכלוסיות תאים מקומיים phagocytic של הריאות הנגרמת על ידי אספקת פלטפורמה זו מאפשרת חיסון הערכת היכולת הפוטנציאלית שלה בסופו של דבר לקדם את המערכת החיסונית אדפטיבי?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> CAM מדיה </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> 50 מ&quot;מ 2-mercaptoethanol </td><td> סיגמא </td><td> M3148-25ML </td><td></td></tr><tr><td> פניצילין / סטרפטומיצין 10,000 מיקרוגרם / מ&quot;ל ​​פתרון </td><td> Cellgro </td><td> 30-002-CI </td><td></td></tr><tr><td> בסרום שור עוברית </td><td> אטלנטה ביולוגיות </td><td> S11150 </td><td></td></tr><tr><td> מאגר FACS </td><td></td><td></td><td></td></tr><tr><td> נתרן כלורי </td><td> פישר סיינטיפיק </td><td> S671-500 </td><td></td></tr><tr><td> סודיום פוספט </td><td> פישר סיינטיפיק </td><td> MK7868500 </td><td></td></tr><tr><td> אשלגן כלורי </td><td> פישר סיינטיפיק </td><td> P217500 </td><td></td></tr><tr><td> אשלגן זרחתי </td><td> פישר סיינטיפיק </td><td> P288-200 </td><td></td></tr><tr><td> BSA (שור אלבומין סרום) </td><td> סיגמא </td><td> A7888 </td><td></td></tr><tr><td> אזיד הנתרן </td><td> סיגמא </td><td> S2002 </td><td></td></tr><tr><td> נוגדנים </td><td></td><td></td><td></td></tr><tr><td> עכברוש IgG </td><td> סיגמא </td><td> I4341 </td><td></td></tr><tr><td> Anti-MS CD16/32 </td><td> eBioscience </td><td> 16-0161 </td><td></td></tr><tr><td> Anti-MS MHC II haplotype IA / IE, שיבוט M5/114.15.2, מצומדות כדי isothiocyanate fluorescein (FITC) </td><td> eBioscience </td><td> 11-5321 </td><td></td></tr><tr><td> Anti-CD86 העכבר, שיבוט GL-1, מצומדות כדי allophycocyanin (APC), Cy7 </td><td> Biolegend </td><td> 105030 </td><td></td></tr><tr><td> Anti-CD40 העכבר, שיבוט 1C10, מצומדות כדי APC </td><td> eBioscience </td><td> 17-0401 </td><td></td></tr><tr><td> אנטי עכבר CD209, שיבוט 5H10, מצומדות כדי ביוטין </td><td> eBioscience </td><td> 13-2091 </td><td></td></tr><tr><td> נגד העכבר CD11b, שיבוט M1/70, מצומדות כדי Alexa פלואוריד 700 </td><td> eBioscience </td><td> 56-0112 </td><td></td></tr><tr><td> נגד העכבר F4/80, שיבוט BM8, מצומדות כדי phycoerythrin (PE)-Cy7 </td><td> eBioscience </td><td> 25-4801 </td><td></td></tr><tr><td> PE-טקסס אדום מצומדות streptavidin </td><td> BD Biosciences </td><td> 551487 </td><td></td></tr><tr><td> מתכלים אחרים ריאגנטים </td><td></td><td></td><td></td></tr><tr><td> אתנול </td><td> פישר סיינטיפיק </td><td> A405-20 </td><td> משמש% 70 (V / V) </td></tr><tr><td> דחוס CO 2 </td><td> Linweld </td><td> 16000060 </td><td></td></tr><tr><td> 1 מ&quot;ל מזרק </td><td> BD Biosciences </td><td> 309659 </td><td></td></tr><tr><td> ריבונית ½ 3 &quot;Catcatheter האב טום </td><td> קנדל </td><td> 703021 </td><td></td></tr><tr><td> בטיחות ביולוגית הממשלה </td><td> NUAIRE </td><td> סדרה 22 </td><td></td></tr><tr><td> דיסקציה מספריים </td><td> דיג </td><td> 138082 </td><td></td></tr><tr><td> מצבטים </td><td> Roboz </td><td> RS-8254 </td><td></td></tr><tr><td> PBS, 1X ללא סידן ומגנזיום </td><td> Cellgro </td><td> 21-040-CM </td><td></td></tr><tr><td> 15 צנטריפוגות צינורות מ&quot;ל עם שווי בורג </td><td> VWR הבינלאומי </td><td> 21008-216 </td><td></td></tr><tr><td> ששת גם בתרבית רקמה המטופלים צלחות </td><td> שחקן המשנה </td><td> 3516 </td><td></td></tr><tr><td> תחתית Racks פלסטיק </td><td> Nalgene </td><td> 5970 </td><td></td></tr><tr><td> מגרד תא 24 ס&quot;מ </td><td> TPP </td><td> 99002 </td><td></td></tr><tr><td> 5 מ&quot;ל פוליסטירן מסביב התחתונה Tube </td><td> בז </td><td> 352008 </td><td></td></tr><tr><td> Pipet, סיוע XL </td><td> דראמונד </td><td> 4-000-105 </td><td></td></tr><tr><td> 10, 5, ו - 2 מ&quot;ל טפטפות </td><td> דיג </td><td> 13-675 </td><td></td></tr><tr><td> 200 ו 10 micropipettors μL </td><td> Gilson Pipetman </td><td> F123601 </td><td></td></tr><tr><td> 200 ו 10 פיפטה טיפים μL </td><td> דיג </td><td> 02-707 </td><td></td></tr><tr><td> BD ייצובמיצב </td><td> BD Biosciences </td><td> 338036 </td><td></td></tr><tr><td> Isoton II diluent </td><td> Beckman Coulter- </td><td> 8546719 </td><td></td></tr><tr><td> זאפ ב-oglobin מגיב ממוסס </td><td> Beckman Coulter- </td><td> 7546138 </td><td></td></tr><tr><td> קולטר בקבוקוני פלסטיק פוליסטירן מונה </td><td> Beckman Coulter- </td><td> 14310-684 </td><td></td></tr><tr><td> מבחנות </td><td> BD Biosciences </td><td> 352008 </td><td></td></tr><tr><td> ציוד </td><td></td><td></td><td></td></tr><tr><td> בקירור צנטריפוגה </td><td> Labnet </td><td> 50075040 </td><td></td></tr><tr><td> החממה Humidified CO 2 </td><td> Nuaire </td><td> דגם Autoflow 8500 </td><td></td></tr><tr><td> FACSCanto תזרים Cytometer </td><td> BD Biosciences </td><td> 338960 </td><td></td></tr><tr><td> קולטר החלקיקים מונה Z1 </td><td> Beckman Coulter- </td><td> WS-Z1DUALPC </td><td></td></tr><tr><td> נוזלי Sonicator ציוד לעיבוד עם Microtip </td><td> Misonix </td><td> מס &#39;דגם S-4000 </td><td></td></tr></tbody></table>

harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles

חלקיקים מתכלה צמחו כפלטפורמה צדדי על התכנון והיישום של אפי חיסונים חדשים כנגד מחלות זיהומיות בדרכי הנשימה. באופן ספציפי, polyanhydride חלקיקים מורכב חומצה sebacic aliphatic (SA), ארומטי 1,6-bis (p-carboxyphenoxy) הקסאן (CPH), או amphiphilic 1,8-bis (p-carboxyphenoxy) -3,6-dioxaoctane (CPTEG) להציג בתפזורת ייחודי סחף קרקע קינטיקה 1,2 וניתן לנצל לשחרר באיטיות ביומולקולות תפקודית (למשל, אנטיגנים, נוגדנים חלבון וכד &#39;) in vivo 3,4,5. אלו חלקיקים גם בעלי פעילות משלים מהותי, מה שהופך אותם בחירה מצוינת עבור פלטפורמת אספקת החיסון 6,7,8. על מנת להבהיר את המנגנונים השולטים ההפעלה של חסינות מולדת בעקבות החיסון הרירית אפי, יש להעריך את התגובות מולקולרית תאית של p אנטיגןתאים כועס (נגמ&quot;שים) אחראית ליזום התגובה החיסונית. התאים הדנדריטים הם נגמ&quot;שים העיקריים שנמצאו בניהול דרכי הנשימה, בעוד מקרופאגים מכתשיים (AMɸ) שולטים parenchyma ריאות 9,10,11. AMɸ הם היעילה ביותר בניקוי הריאות של פתוגנים חיידקים ופסולת תא 12,13. בנוסף, זה סוג תא יש תפקיד חשוב בהובלה של אנטיגנים של חיידקים אל קשרי הלימפה וניקוז, וזה צעד ראשון חשוב בייזום של תגובה חיסונית אדפטיבית 9. AMɸ גם מביעים רמות גבוהות של זיהוי תבניות מולדות קולטנים זבל, מפרישים Pro דלקת מתווכים, וראש ותאי T נאיבי 12,14. האוכלוסייה טהורה יחסית של AMɸ (למשל, יותר מ 80%) יכול בקלות להיות מושגת באמצעות שטיפה ריאות למחקר במעבדה. תושב שנקטפו AMɸ מבעלי חיים המוסמכות החיסונית לספק הפנוטיפ מייצג של מקרופאגים אשר encounter חיסון החלקיקים מבוסס in vivo. במסמך זה אנו מתארים את הפרוטוקולים המשמשים הקציר ותרבות AMɸ מעכברים ולבדוק את הפנוטיפ הפעלה של מקרופאגים לאחר הטיפול עם חלקיקים polyanhydride במבחנה.

Watch this Scientific Journal Video about Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles at JoVE.com

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

במסמך זה אנו מתארים פרוטוקולים קצירת מקרופאגים מכתשיים Murine, אשר תושבי תאים חיסוניים מולדים בריאה, ובדיקת ההפעלה שלהם כתגובה לתרבות בשיתוף עם חלקיקים polyanhydride.

הפעלת נייד קציר המקרופאגים מכתשיים Murine והערכת בהשפעת חלקיקים Polyanhydride

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

harvesting-murine-alveolar-macrophages-evaluating-cellular-activation

Research

JoVE Journal

Bioengineering

19.7K Views.  Iowa State University. במסמך זה אנו מתארים פרוטוקולים קצירת מקרופאגים מכתשיים Murine, אשר תושבי תאים חיסוניים מולדים בריאה, ובדיקת ההפעלה שלהם כתגובה לתרבות בשיתוף עם חלקיקים polyanhydride.

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

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.

ניתוח הפנמה נייד של חלקיקים וחיידקים על ידי Cytometry Multi-Flow דימות ספקטרלי

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.

תפוקה גבוהה סינתזה של פחמימות functionalization של חלקיקים Polyanhydride

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

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.

הערכת משלוח גן פולימרית חלקיקים על ידי Nanoparticle מעקב ניתוח וזרימת cytometry תפוקה גבוהה

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

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

שימוש בעכבת Cell-מצע והדמיה של תא חי כדי למדוד שינויים בזמן אמת בסלולרית הידבקות ודה-הידבקות הנגרמים על ידי מטריקס שינוי

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

מיקרוביולוגי מושרה קלציט רטיבות בתיווך<em&gt; Sporosarcina pasteurii</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

ייצור של חלקיקי מתכת על ידי פעימה לייזר ablation בנוזלים: כלי לחקר מאפיינים אנטיבקטריאליים של חלקיקים

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

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.

Functionalization פני השטח של חלקיקי וירוס הפטיטיס E בשיטות כימיות ההטיה

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

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

הדחקת שעתוק גנים על ידי ניתוב מחדש מכונות הסלולר עם המגבילים Epigenetic כימי

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

ניקוי קטליטי של הצמח חמצן תגובתי מינים In Vivo על ידי חלקיקי תחמוצת צריום Anionic

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...