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). Filter-sterilize cAM&#632; medium using a 0.2 &mu;m pore size bottle top filter under vacuum.</li>
 <li>Euthanize a healthy mouse by CO2 asphyxiation in an euthanization chamber. Although a C57BL/6 mouse (6-8 weeks old) was used for the purposes of this project, this lung lavage technique can be performed on any strain of mouse. Using a compressed gas cylinder, let gas flow into the chamber for 1 min prior to placing the mouse in the chamber to assure that the chamber is fully charged with CO2. Place the mouse in the chamber for at least one minute to euthanize the mouse. In addition, exsanguinate each mouse via cardiac puncture as a physical method to confirm mouse death as described in 1.4.</li>
 <li>Lay mouse on its back (dorsal side), spray with 70% ethanol, locate the sternum and, at a 30 degree angle from the abdomen, insert a 23 gauge needle (attached to a 1 mL syringe) about 50 mm into the thoracic cavity. With proper placement of the needle, it should be possible to collect between 0.4 to 1 mL of blood depending on the size of the mouse. Examine the animal for cessation of vital signs.</li>
 <li>Place mouse on lab bench so that it is lying on its abdomen (ventral side). Spray mouse with 70% ethanol to sterilize the working environment. Pinch skin about halfway down the spine of the mouse. Using dissection scissors that have been sterilized with 70% ethanol, make a small incision through the skin.</li>
 <li>Grab the skin on both sides of the incision and pull the skin in opposite directions along the cranial-caudal axis of the mouse. When done correctly, the skin will be removed from the underlying carcass.</li>
 <li>The neck of the mouse should now show exposed muscles and glands. Carefully cut that tissue and pull it towards the anterior of the mouse using forceps, thereby exposing the larynx, trachea, and esophagus of the mouse. Avoid cutting the jugular vein or carotid artery.</li>
 <li>The trachea is now visible and is identifiable as the tissue with cartilaginous rings around it. Delicately hold the trachea with forceps and gently lift and separate the trachea from the esophagus, which is located on the dorsal side.</li>
 <li>Make a small incision anterior to where one holds the trachea but posterior to the larynx. The incision should be made at approximately a 45&deg; degree angle. Also, do not completely transect the trachea, as this will prevent it from retracting into the body cavity.</li>
 <li>Fill a 1 cc syringe fitted with a Tom Cat catheter with sterile PBS in preparation to insert the catheter into the lumen of the trachea. Hold the trachea using forceps and with your hand hold the catheter close to the tip to increase control and gently insert it into the tracheal incision site. Gently insert catheter into trachea about 20 mm. Grip the trachea that is around the catheter tube, freeing the other hand.</li>
 <li>Using a 1 mL syringe fitted with the catheter, gently infuse 0.75 mL of room temperature, sterile PBS into the lungs and then gently aspirate the PBS back into syringe. Repeat this process three times while externally massaging the chest. Disconnect the syringe from the catheter and dispense the lavage fluid into a 15 mL centrifuge tube. After filling the syringe with 0.75 mL of fresh PBS and reconnecting the syringe to the catheter, repeat steps 1.9 and 1.10 twice for each mouse. Fluid recovery may be partial, obtaining less than the volume infused. If collecting cells from more than one mouse, place the 15 mL tube with the harvested cells on ice until all lung lavages have been performed.</li>
</ol>
2. Processing of Lung Lavage AM&#632; Harvest
<ol>
 <li>Centrifuge lung lavage fluid at 250 x g for 10 min at 4 &deg;C.</li>
 <li>Using sterile technique, decant supernatant into appropriate waste container inside a biosafety cabinet. Resuspend the cell pellet by gently raking the centrifuge tube across the top of a test tube rack. Add 1 mL of cAM&#632; medium to resuspend the cells.</li>
 <li>Enumerate the cells using a Coulter Particle Counter Z1 by pipetting 20 &mu;L of cells into 10 mL of Isoton II Diluent in Coulter counter vial. Add 3 to 4 drops of Zap-oglobin II lytic reagent into vial, gently invert 3 to 4 times and then load the sample vial onto the counter to obtain a cell concentration. Be sure to program the counter with a dilution factor of 2 x 104 to display the cell concentration as the number of cells per mL.</li>
 <li>Dilute cells to 2.5 x 105 per mL using cAM&#632; medium.</li>
 <li>Dispense 2 mL of 2.5 x 105 cells/mL into each well of a 6-well tissue culture dish.</li>
 <li>To enrich for AM&#632;, incubate culture dishes inside a 37 &deg;C humidified incubator with a 5% CO2 atmosphere for 6 h to allow cells to adhere to the bottom of the well. Gently aspirate the supernatant as to not disrupt adherent cells.</li>
 <li>Rinse the wells using sterile PBS (calcium and magnesium free) to remove non-adherent cells and debris. Add 2 mL of cAM&#632; medium to each well.</li>
 <li>Incubate cells overnight inside a 37 &deg;C humidified incubator with a 5% CO2 atmosphere prior to addition of stimulants. After this enrichment step, approximately 87% of the cells are positive for the macrophage markers CD11b and F4/80 (Figure 2c).</li>
</ol>
3. Addition of Polyanhydride Nanoparticles and Control Treatments
<ol>
 <li>Fabricate nanoparticles via polyanhydride anti-solvent nanoencapsulation15. In this process, the polymer is dissolved in methylene chloride (4 &deg;C at a concentration of 25 mg/mL) and precipitated in pentane (-20 &deg;C at a ratio 1:200 methylene chloride:pentane).</li>
 <li>Weigh out polyanhydride nanoparticles using sterilized weigh paper on a balance that can accurately measure microgram amounts. Add particles to a 1.5 mL microcentrifuge tube.</li>
 <li>Resuspend the nanoparticles in ice cold cAM&#632;. Calculations are made such that no more than 0.3 mL of medium is added to each well to obtain the desired concentration of nanoparticles. Keep the resuspended particles on ice until the nanoparticles are added to the culture well.</li>
 <li>Prior to adding the nanoparticles to the AM&#632; cultures, sonicate particles using a bench top sonicator with a microtip. Sterilize microtip by spraying with 70% ethanol and wiping with a sterile Kimwipe prior to sonication. Sonicate at 10-20 joules for 30 to 45 s while keeping the tube on ice. Observe nanoparticle suspension macroscopically. If particles are not sufficiently dispersed, allow the suspension to set on ice for 1 to 2 min to ensure that the nanoparticles are below the glass transition temperature of the polymer before sonicating again for 30 to 45 sec. The intent of this sonication step is to adequately disperse the nanoparticles, as the 50:50 CPTEG:CPH nanoparticles tend to aggregate when exposed to an aqueous environment. However, some small aggregates of nanoparticles may still remain in the suspension.</li>
 <li>Nanoparticle suspension (adequately dispersed) should be kept on ice and be used immediately to prevent premature surface erosion or aggregation.</li>
 <li>Remove the culture dishes containing the AM&#632; from incubator and place in the biosafety cabinet. Tilt plate at approximately a 45 degree angle and pipette off the amount of medium that will be added to the nanoparticle suspension (it should not exceed 0.3 mL). Do not disturb the adherent cells when removing the medium.</li>
 <li>Vortex the polyanhydride nanoparticle suspension briefly prior to adding it to the appropriate wells. Pipette the amount of nanoparticle suspension (no more than 0.3 mL) into the specific well(s); however, do not pipette up and down to mix. This action could detach the adherent AM&#632; from the well. The particles will settle to the bottom of the well and contact the cells. For the studies outlined in this report, the final concentration of nanoparticles co-cultured with the AM&#632; was 125 &mu;g/mL. Proceed with subsequent wells and desired treatment groups. Include positive and negative control groups, such as medium only and 200 ng/mL lipopolysaccharide (LPS) to designated wells.</li>
 <li>Return the culture dishes to the 37 &deg;C humidified incubator with a 5% CO2 atmosphere for 48 h. Kinetic studies in our laboratory have demonstrated 48 hours to be the optimal culture period for evaluating both cell-surface marker expression and cytokine secretion as it permits adequate time for transcription and protein synthesis to occur. Although greater levels of cytokines accumulate during an extended culture period (i.e., 72 hr), evaluating cell surface marker expression at this time point can be confounded by the contribution of the elevated levels of cytokines. The changes in surface marker expression observed may not be directly attributable to stimulation with the nanoparticles themselves.</li>
</ol>
4. Evaluating AM&#632; Activation using Flow Cytometry
<ol>
 <li>Prepare fluorescence-activated cell sorting (FACS) buffer (0.1 % sodium azide and 0.1 % bovine serum albumin (BSA)) in 0.1 M phosphate buffered saline (pH 7.4) prior to harvesting the plated macrophages that have been co-cultured with nanoparticles or stimulants.</li>
 <li>Prepare working solutions of the blocking buffer and the monoclonal antibodies. To prepare the block buffer, dilute rat IgG (Sigma) and anti-CD16/32 (eBioscience) to 1 mg/mL and 10 &mu;g/mL, respectively, in FACS buffer. Dilute surface marker antibody panel in FACS buffer to either the manufacturer recommended concentration or the user optimized concentration. For the results presented in this work, the surface marker panel consists of antibodies against MHC II, CD86, CD40 and CIRE (CD209). Anti-mouse antibodies against the macrophage markers F4/80 and CD11b are also included in the panel to positively identify the macrophage phenotype during data analysis. Care must be taken when selecting antibodies against MHC II molecules, as inbred mouse strains often differ in their MHC II haplotypes. The antibodies selected must react with the specific MHC alloantigens expressed by your mouse strain of interest.</li>
 <li>After 48 h of incubation with nanoparticles and/or stimulants, place cell culture dishes on ice for 20 min. To remove the adherent macrophages from the plates, gently scrape wells with a 24 cm cell scraper.</li>
 <li>Using a 5 mL serological pipette, gently aspirate the cells and medium up and down five times to generate a single cell suspension. Pipette the contents of individual wells into separate 5 mL polystyrene tubes so as not to mix together cells from different experimental treatments.</li>
 <li>Add 2 mL of FACS buffer to each tube.</li>
 <li>Centrifuge tubes of cells at 250 x g at 4 &deg;C for 10 min.</li>
 <li>Decant supernatant and gently resuspend cell pellet in residual fluid by raking the tubes across the top of a test tube rack.</li>
 <li>In order to prevent non-specific binding of the monoclonal antibodies, add 10 &mu;L of blocking buffer (prepared in 4.2) to each tube of cells. Vortex tubes and incubate on ice for 30 min.</li>
 <li>Add appropriate volume of diluted monoclonal antibodies to each tube and incubate in the dark on ice for 15 min. Tubes that contain samples to be analyzed should receive the combination of antibodies of the polycromatic panel (previous optimization of combination of antibodies should be performed by individual labs). Additional control groups should be included to correctly set the acquisition parameters on the flow cytometer, including a tube of cells that were not labeled, and compensation tubes that correspond to labeled cells using each single color separately. To correctly set the gates for flow cytometric analysis, the Fluorescence Minus One (FMO) controls should be included during acquisition. FMO controls are cells labeled with all the fluorochrome-conjugated antibodies that are used on the panel except one that is replaced by its respective isotype control. Positive and negative populations (i.e., separate aliquots of cells treated with a positive control stimulant and medium alone) should also be included in FMO and compensation control tubes.</li>
 <li>Add 2 mL of FACS buffer to each tube, vortex and centrifuge at 250 x g at 4 &deg;C for 10 min.</li>
 <li>Decant supernatant and resuspend cell pellet in residual fluid by raking the tubes across the top of a test tube rack.</li>
 <li>If the antibody panel consists of an antibody not directly conjugated to a fluorochrome but is instead conjugated to biotin, a fluorochrome-conjugated streptavidin is needed to visualize the surface marker. In this step, add an appropriate volume of the diluted fluorochrome-conjugated streptavidin and incubate in the dark for 15 min on ice. As in 4.2, the streptavidin will be diluted to a concentration recommended by the manufacturer or to one optimized by the user. This step is only needed for panels containing biotinylated primary antibodies.</li>
 <li>Perform a wash step as described in 4.10.</li>
 <li>Dilute an appropriate volume of BD stabilizing fixative 1:3 in deionized water. Add 100 &mu;L of diluted fixative to each tube while gently vortexing to prevent clumping of cells. Labeled and fixed cells can be stably stored in the dark at 4 &deg;C for approximately one week prior to acquisition on a flow cytometer.</li>
</ol>
5. Representative Data
Nanoparticles fabricated in step 3.1 had an average diameter of 163 &plusmn; 24 nm and a morphology consistent with that obtained in previous studies5,16. Nanoparticles in solution prior to and after sonication are shown in Figure 1 to demonstrate the need for sonication to ensure adequate dispersal of the particles. Flow cytometric analysis of harvested AM&#632;s obtained via lung lavage is shown in Figure 2. Labeling cells with a combination of anti-mouse CD11b and anti-mouse F4/80 antibodies as well as the corresponding FMO controls allows for establishing background labeling, identifying AM&#632;s and gating for further analysis. Treatment of alveolar macrophages with polyanhydride nanoparticles enhances activation as shown by the increased mean fluorescence intensity of MHC II, CD40, CD86, and CIRE (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/3883/3883fig1.jpg" /> 
Figure 1. 50:50 CPTEG:CPH nanoparticles prior to (A) and after (B) 30 s of sonication.
<img alt="Figure 2" src="/files/ftp_upload/3883/3883fig2.jpg" /> 
Figure 2. Flow cytometric analysis of harvested alveolar macrophages labeled with (A) Alexa Fluor 700 anti-CD11b and the PE-Cy7 FMO Control, (B) Alexa Fluor 700 FMO Control and the PE-Cy7 anti-F4/80 and (C) Alexa Fluor 700 anti-CD11b and PE-Cy7 anti-F4/80. The number in the top right corner represents the percentage of double-positive cells.
<img alt="Figure 3" src="/files/ftp_upload/3883/3883fig3.jpg" /> 
Figure 3. Histograms demonstrating an increase in the fluorescence intensity for surface expression of MHC II (A), CD40 (B), CD86 (C) and CIRE (D) after co-culture of alveolar macrophages with 50:50 CPTEG:CPH polyanhydride nanoparticles for 48 hr. Histograms depict results for AM&#632; labeled as FMO controls (<img alt="Graph Line 1" src="/files/ftp_upload/3883/3883line1.jpg" />), untreated AM&#632; labeled with antibodies against all cell surface markers (<img alt="Graph Line 3" src="/files/ftp_upload/3883/3883line3.jpg" />) and AM&#632; cultured with nanoparticles and labeled with antibodies against all cell surface markers (<img alt="Graph Line 2" src="/files/ftp_upload/3883/3883line2.jpg" />).

<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>

No conflicts of interest declared.

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

19.7K Views.  Iowa State University. 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.

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.

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 ...

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 ...

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.

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 ...