Name: harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles
Uploaded: 2012-06-08T12:00:00
Description: Watch this Scientific Journal Video about Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles at JoVE.com

The authors would like to thank the U.S. Army Medical Research and Materiel Command (Grant Numbers W81XWH-09-1-0386 and W81XWH-10-1-0806) for financial support and Dr. Shawn Rigby from the Iowa State University Flow Cytometry Facility for his expert technical assistance.

1. Harvesting Alveolar Macrophages (AM&#632;) from the Mouse using Lung Lavage
<ol>
 <li>Wear appropriate personal protective equipment (PPE) such as a lab coat, disposable gloves, and proper eye protection.</li>
 <li>Prepare complete AM&#632; (cAM&#632;) medium before initiation of harvest. Add 2.5 mL of penicillin/streptomycin (pen/strep) solution, 250 &mu;L of &beta;-mercaptoethanol (2-mercaptoethanol), and 25 mL of heat inactivated fetal bovine serum (FBS) to 222.25 mL of Dulbelcco's Modified Eagle Medium (DMEM). F...

<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. 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=Effect+of+polymer+chemistry+and+fabrication+method+on+protein+release+and+stability+from+polyanhydride+microspheres.">Effect of polymer chemistry and fabrication method on protein release and stability from polyanhydride microspheres.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 91, 938-947 (2009).</li><li>Torres, M. P., Determan, A. S., Anderson, G. L., 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=Amphiphilic+polyanhydrides+for+protein+stabilization+and+release.">Amphiphilic polyanhydrides for protein stabilization and release.</a> Biomaterials. 28, 108-116 (2007).</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=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>Petersen, L. K., Xue, L., Wannemuehler, M. J., Rajan, 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=The+simultaneous+effect+of+polymer+chemistry+and+device+geometry+on+the+in+vitro+activation+of+murine+dendritic+cells.">The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells.</a> Biomaterials. 30, 5131-5142 (2009).</li><li>Petersen, L. 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>

Polyanhydride nanoparticle vaccine platforms have shown efficacy when administered intranasally in single dose regimens5. Measuring the activation of the resident phagocytic cell populations in the lungs induced by this vaccine delivery platform permits evaluation of its potential capability to ultimately promote adaptive immune responses.
	Specifically, harvesting alveolar macrophages from lung lavage fluid and treating them with different formulations of the nanoparticles pr...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>cAM Media</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 mM 2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148-25ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10,000 &mu;g/ mL Solution</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>FACS Buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S671-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>Fisher Scientific</td>
			<td>MK7868500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher Scientific</td>
			<td>P217500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Fisher Scientific</td>
			<td>P288-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA (Bovine Serum Albumin)</td>
			<td>Sigma</td>
			<td>A7888</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Rat IgG</td>
			<td>Sigma</td>
			<td>I4341</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Ms CD16/32</td>
			<td>eBioscience</td>
			<td>16-0161</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Ms MHC II haplotype I-A/I-E, clone M5/114.15.2, conjugated to fluorescein isothiocyanate (FITC)</td>
			<td>eBioscience</td>
			<td>11-5321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD86, clone GL-1, conjugated to allophycocyanin (APC)-Cy7</td>
			<td>Biolegend</td>
			<td>105030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40, clone 1C10, conjugated to APC</td>
			<td>eBioscience</td>
			<td>17-0401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD209, clone 5H10, conjugated to Biotin</td>
			<td>eBioscience</td>
			<td>13-2091</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b, clone M1/70, conjugated to Alexa Fluor 700</td>
			<td>eBioscience</td>
			<td>56-0112</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80, clone BM8, conjugated to phycoerythrin (PE)-Cy7</td>
			<td>eBioscience</td>
			<td>25-4801</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PE-Texas red conjugated Streptavidin</td>
			<td>BD Biosciences</td>
			<td>551487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Other Supplies and Reagents</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A405-20</td>
			<td>Used as 70% (v/v)</td>
		</tr>
		<tr>
			<td>Compressed CO2</td>
			<td>Linweld</td>
			<td>16000060</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD Biosciences</td>
			<td>309659</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sovereign 3 &frac12;" Fr Tom Catcatheter</td>
			<td>Kendall</td>
			<td>703021</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>NUAIRE</td>
			<td>Series 22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection Scissors</td>
			<td>Fisher</td>
			<td>138082</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS, 1X without calcium and magnesium</td>
			<td>Cellgro</td>
			<td>21-040-CM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tubes with Screw Cap</td>
			<td>VWR International</td>
			<td>21008-216</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Six-well Tissue Culture Treated Plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic Tube Racks</td>
			<td>Nalgene</td>
			<td>5970</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Scraper 24 cm</td>
			<td>TPP</td>
			<td>99002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube</td>
			<td>Falcon</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipet-aid XL</td>
			<td>Drummond</td>
			<td>4-000-105</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10, 5, and 2 mL Pipettes</td>
			<td>Fisher</td>
			<td>13-675</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 and 10 &mu;L micropipettors</td>
			<td>Gilson Pipetman</td>
			<td>F123601</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 and 10 &mu;L pipette tips</td>
			<td>Fisher</td>
			<td>02-707</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Stabilizing Fixative</td>
			<td>BD Biosciences</td>
			<td>338036</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoton II Diluent</td>
			<td>Beckman-Coulter</td>
			<td>8546719</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zap-oglobin II Lytic Reagent</td>
			<td>Beckman-Coulter</td>
			<td>7546138</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coulter Counter Polystyrene Vials</td>
			<td>Beckman-Coulter</td>
			<td>14310-684</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Test Tubes</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Labnet</td>
			<td>50075040</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Humidified Incubator CO2</td>
			<td>Nuaire</td>
			<td>Model Autoflow 8500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACSCanto Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>338960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coulter Particle Counter Z1</td>
			<td>Beckman-Coulter</td>
			<td>WS-Z1DUALPC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sonicator Liquid Processing Equipment with Microtip</td>
			<td>Misonix</td>
			<td>Model No. S-4000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

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

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.

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

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.

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.

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.

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.

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.

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

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

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.

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.

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.

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