The authors thank the TEDCO MSCRF (2009-MSCRFE-0098-00) and NIH R21CA152473 for support.

1. Cell Seeding
<ol>
 <li>Do not allow cells to grow to overconfluency. Use early passage cells when transfecting primary cells.</li>
 <li>Twenty-four hours prior to transfection, trypsinize the cells, count the cells using a hemocytometer, and dilute the cell suspension with media to achieve the desired cell density (cells/volume). Seed cells into clear tissue culture-treated flat-bottom 96-well plates using a reservoir and multichannel pipettes. The chosen density should give 70-80% confluency on the day of transfection. For example, as displayed in Table 1, cells were diluted to 25 to 50 cells/&mu;l for the transfection data shown here.</li>
</ol>
2. Cell Transfection
<ol>
 <li>Dilution of polymer and DNA stocks. Thaw polymer and DNA stock solutions at room temperature (RT). Dilute polymer stock solution and DNA stock solution, separately in clear 96-well plates using a twelve-channel pipette, with the appropriate solvent to concentrations required to obtain the desired polymer weight to DNA weight ratios (wt/wt). In this case, the chosen solvent is 25 mM sodium acetate buffer (pH=5.2).
	 <ol>
		 <li>DNA dilution. Typically, DNA stored at 1 mg/ml is diluted in sodium acetate buffer to a concentration of 0.03 to 0.06 mg/ml in a clear non-tissue culture-treated 96-well plate (one well for a single formulation). Table 2 shows the typical DNA dilution protocol for a single formulation used to transfect four replicate wells in a 96-well plate seeded with cells from Step 1.</li>
		 <li>Polymer dilution. The 100 mg/ml polymer/DMSO solution is diluted in sodium acetate buffer according to the concentration required to obtain the desired polymer to DNA wt/wt ratio. The range of wt/wt ratios typically used for gene delivery with poly(beta-amino ester)s (PBAEs) is 20 to 100. PBAE polymers are first diluted to 10 mg/ml, followed by the dilution protocol as shown in Table 3. The polymer dilutions can be performed in a clear non-tissue culture-treated 96-well plate that matches the sample orientation of the DNA dilution plate.</li>
	 </ol>
	</li>
	<li>Nanoparticle formation. Add the PBAE solution to an equal volume of the plasmid DNA solution using a twelve-channel pipette and mix vigorously. Let the mixture incubate at RT for 10 min to allow self-assembly.</li>
 <li>Nanoparticle transfection. Following self-assembly, 20 &mu;l nanoparticles are added per well to the culture medium dropwise using the twelve-channel pipette. Replicate wells are left untreated or are transfected with commercially available reagents as controls. The transfected cells are incubated at 37 &deg;C for two to four hours and then the wells are replaced with fresh media (100 &mu;l/well). The choice of incubation time will depend on the cell line, culture conditions, and transfection system. The difference in transfection efficiency and cell toxicity as a result of varying incubation period will be quantified by the analysis protocol described in Step 3.</li>
</ol>
3. Analysis of Transfection Efficiency and Cell Toxicity
Transfection efficiency is analyzed visually with a fluorescence microscope and quantified using a flow cytometer forty-eight hours post-transfection. Cell toxicity is analyzed visually with a fluorescence microscope and quantified using the CellTiter 96 AQueous One assay twenty-four hours post-transfection.
<ol>
 <li>Fluorescence microscopy. Visually analyze the wells for the expression of the transfected reporter gene (for example, EGFP or DsRed) using the appropriate fluorescence channel. Acquire images for each well, choosing fields of view that appropriately represent the transfection efficiency for the particular formulations (Figure 1). Make a note of any cell toxicity observed visually.</li>
 <li>Flow Cytometry.
	 <ol>
		 <li>Use multichannel pipettes to prepare the 96-well plate for flow cytometry. Wash with PBS, trypsinize with 30 &mu;l/well of trypsin/EDTA, neutralize with 170 &mu;l FACS buffer (PBS + 2% FBS), pipette up and down in each well, including around edges, and transfer the entire 200-&mu;l volume of each well into corresponding wells in a round-bottom or V-bottom 96-well plate.</li>
		 <li>Centrifuge the plate at 130 x g for 5 min at 4 &deg;C.</li>
	 <li>Remove 170 &mu;l media from each well and pipette to resuspend cells while avoiding bubbles. To use a viability stain like propidium iodide (PI), add 10 &mu;l of FACS buffer+PI (50:1 FACS buffer:PI dilution; PI stock concentration 1 mg/ml) to each well. Immediately place on ice and cover from light.</li>
	 <li>Start the C6 Accuri flow cytometer, the HyperCyt 96-well attachment, and the HyperView software. Use HyperView Design and Protocol tabs to choose the appropriate plate type, plate layout, and other settings such as shake/sip/rinse/shake time. For the above protocol, the following parameters were used, a pre-plate prime for one minute, and a pre-plate shake for 30 sec at 2,400 rpm. Sip time was set for 15 sec at 15 rpm, a probe rinse every well for 2 sec, and an inter-well shake every 12 wells for 12 sec at 2,400 rpm. The inter-well shake is important to keep the cells dispersed in the media, as well as for improved ability to process the data by including timed breaks between groups of wells (in this case 12 sec).</li>
	 <li>Use HyperView Well Identification tab to process the data and separate into the appropriate well (Figure 2). In order to identify the individual wells, it can help to use the software's noise filter, as well as including a base even filter under the advanced settings.</li>
	 <li>Quantify percentage of positively transfected live cells by appropriate gating to separate different populations (Figure 3). First view the flow data on a FSC vs. SSC scatter plot in order to separate cells from debris (Figure 3A). If propidium iodide (PI) was used, gate the individual cell population and view that data on a FSC vs. FL3 plot in order to separate live cells from dead cells and gate the live cells. In order to quantify the number of transfected cells, view the live cell population on the appropriate channels, for example, GFP can be seen in FL1. Using the untreated population, gate the untreated cells in order to identify the background signal (Figure 3B). The positively transfected cells can then be isolated using this gate and by viewing the transfected populations using both scatter and contour plots (Figure 3C and 3D). There may be a continuum of positively transfected cells, as different cells will be transfected to different degrees of fluorescence.</li>
	 </ol>
	 </li>
	 <li>CellTiter 96 AQueous One assay
	 <ol>
		 <li>Transfect another plate in exactly the same manner for cell toxicity measurements.</li>
		 <li>Premix 1 ml of the CellTiter 96 AQueous One assay solution with 10 ml fresh media. Remove media from cells and use a multichannel pipette to add 110 &mu;l of the premixed solution+media per well.</li>
	 <li>Measure the absorbance at 490 nm every one hour interval from 1 to 4 hr until the absorbance from the well with untreated condition is in the linear range of the assay. Include four control wells with only solution+media for a background absorbance reading.</li>
	 <li>Average the absorbance values from the four replicate wells per condition and subtract the average background absorbance from each average. Normalize the corrected average for each treated condition to the corrected average of the untreated condition to determine the percent cell viability.</li>
	 </ol>
	 </li>
</ol>
4. Nanoparticle Sizing with NTA and Plasmid Per Particle Calculations
<ol>
 <li>Prime the fluidics system of the Nanosight NS500 by running the diluent pump forward at approximately 1/5th the max speed and the sample pump backward at approximately 1/10th the max speed. Continue until the fluidics system is flushed with diluent.</li>
 <li>Prepare the nanoparticles to be analyzed. In the case of PBAEs, separately dilute the stock plasmid DNA (1 mg/ml) and stock PBAE (100 mg/ml) in sodium acetate buffer (25 mM, pH 5) in Eppendorf tubes.</li>
 <li>Dilute the plasmid DNA concentration to 0.06 mg/ml. Polymer concentration can vary depending on the required weight-weight ratio of polymer to DNA (for example, for 60 wt/wt particles, polymer is diluted to 3.6 mg/ml).</li>
 <li>Add the polymer solution to an equal volume of the plasmid DNA solution and mix vigorously. Incubate the mixture at room temperature for 10 min to allow self-assembly.</li>
 <li>Dilute the nanoparticle solution 100-fold into PBS in order for the nanoparticle concentration to be in the appropriate range for NTA and to obtain a final volume of at least 500 &mu;l.</li>
 <li>Load the sample into the Nanosight by placing the loading tube into the sample Eppendorf tube (Figure 4A). Make sure not to introduce air bubbles.</li>
 <li>In capture mode, increase the camera level until particles can be seen. Adjust the focus so that the particles look smooth (Figure 4A and 4B).</li>
 <li>While in standard mode, adjust the camera level past the point that all particles can be seen on screen. Then decrease the camera level to the lowest level such that the particles can all still be observed. If an intermediate camera level is needed, go into advanced mode and modify the camera shutter and gain to the appropriate levels.</li>
 <li>Visually check to make sure there are between 20 - 100 particles on the screen. An ideal number for nanoparticle tracking is approximately 50 particles. If there are too many or too few, flush the NS500, adjust the dilution into PBS, and re-load the sample.</li>
 <li>Adjust the capture duration according to the standard mode table. Typically 30 - 60 sec is an appropriate capture length (Figure 4B).</li>
 <li>To load the next sample, flush the system, then re-load the new sample.</li>
 <li>Once videos are captured, proceed to the processing stage by opening a video file.</li>
 <li>There are a number of parameters that can be tuned in order to best process a video (Figure 4C). The goal is to select parameters that best capture each particle on the screen, as indicated by a red cross mark on the screen over each particle (Figure 5).</li>
 <li>Increase the screen gain to better see the particles. Under the standard or advanced mode, select the auto adjust for the parameters by clicking the appropriate boxes. In the case that the auto settings do not adequately select particles, unclick the box and manually set the parameters (Figure 4C).</li>
 <li>Once all the particles are picked on screen, click the process button of the software to process the video file (Figure 4C). This provides the particle size distribution, size averages, as well as particle concentration.</li>
 <li>In order to make certain that the particle concentration is accurate, change the PBS dilution, such as by increasing dilution by 2x, and repeat the above procedure. Measuring multiple dilutions of the same sample is recommended to ensure that the concentration measurement of the sample is accurate. A good range for measurement is 107-109 particles/ml.</li>
 <li>Verify that all plasmids are incorporated into nanoparticles by running gel electrophoresis of the nanoparticles and evaluating whether any free DNA is present. If not all plasmids are encapsulated, use standard techniques to measure DNA absorbance in order to quantify total encapsulated plasmids.</li>
 <li>To calculate the average number of plasmids per particle, divide the total encapsulated plasmid concentration by the NTA measured particle concentration. In order to estimate the plasmid per particle sample distribution, first use the NTA particle size histogram to calculate the volume fraction of each 1-nm bin of particle. Multiply this volume fraction distribution by the total plasmid amount to obtain the number of plasmids in each bin. Divide these numbers by the number of particles in each bin to obtain the number of plasmids-per-particle for each particle size (Figure 6).</li>
</ol>

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

The authors report no conflicts of interest.

The protocols above describe methods of evaluating the transfection efficacy of nanoparticle formulations, as well as a way to characterize the particle size and DNA loading of the nanoparticles. The number of plasmids per particle is an important parameter that can help predict the effectiveness of the particle and can also be used for dose determination. Nanoparticle tracking analysis can be performed in a range of different aqueous solutions, such as those differing in salt concentration. Often this characteriz...

The determination of the number of plasmids complexed per nanoparticle is important to design effective nanoparticle-based gene delivery strategies, particularly for co-delivery of multiple plasmids to the same cell target, as often is required in stem cell reprogramming studies12. Few approaches to calculate the number of plasmids associated with a single nanoparticle have been described, and each approach has drawbacks in the techniques used for estimation13-16. Quantum dot (QD) labeling combined with TEM has been used to estimate plasmids per particle in chitosan-based nanoparticles. Estimation with this QD technique is complicated due to the need to label the DNA, which may alter its self-assembly properties; the possibility that encapsulated unlabeled DNA is not directly detected; potentially overlapping plasmids and QDs in the 2D TEM images of particles; and other simplifying assumptions13. An alternative approach that is applicable when ordered microdomains exist in the particles has been used to study Lipopolyamine-DNA complexes via cryo-transmission electron microscopy (cryo-TEM), X-ray scattering, and dynamic light scattering (DLS)14,15 . Unfortunately, materials such as the polymeric nanoparticles investigated here are not applicable with this method. In another study, Collins et al. used a flow particle image analysis technique to study (Lys)16-containing peptide/DNA complexes; however, their method can only evaluate larger, micron-sized particles16. Thus, we recently developed a novel and flexible assay to quantify the number of plasmids per nanoparticle11.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline, 1x (PBS)</td>
 <td>Invitrogen</td>
 <td>10010</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2MV BulletKit</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Invitrogen</td>
 <td>25300</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium acetate buffer</td>
 <td>Sigma-Aldrich</td>
 <td>S7899</td>
 <td>Dilute to 25mM in deionized water</td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide</td>
 <td>Sigma-Aldrich</td>
 <td>276855</td>
 <td></td>
 </tr>
 <tr>
 <td>pEGFP DNA</td>
 <td>Elim Biopharmaceuticals</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>DsRed DNA</td>
 <td>Addgene</td>
 <td>21718</td>
 <td></td>
 </tr>
 <tr>
 <td>PEI, branched </td>
 <td>Sigma-Aldrich</td>
 <td>408727</td>
 <td></td>
 </tr>
 <tr>
 <td>CellTiter 96 AQueous One</td>
 <td>Promega</td>
 <td>G3580</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Clear flat bottom 96-well plate, sterile</td>
 <td>Sarstedt</td>
 <td>82.1581.001</td>
 <td></td>
 </tr>
 <tr>
 <td>Clear round bottom 96-well plate, sterile</td>
 <td>Sarstedt</td>
 <td>82.1582.001</td>
 <td></td>
 </tr>
 <tr>
 <td>12-channel Finnpipette</td>
 <td>Thermo Scientific</td>
 <td>NA</td>
 <td>5-50 and 50-300 &mu;l</td>
 </tr>
 <tr>
 <td>Fluorescence Microscope</td>
 <td>Zeiss</td>
 <td>NA</td>
 <td>Model number: AX10</td>
 </tr>
 <tr>
 <td>C6 Accuri flow cytometer</td>
 <td>BD Biosciences</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>HyperCyt attachment</td>
 <td>Intellicyt</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>NS500</td>
 <td>Nanosight</td>
 <td>NA</td>
 <td></td>
 </tr>
 </tbody>
</table>

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

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.

Figure 1 shows a fluorescence microscopy image of an example of a successful transfection of HRECs with the EGFP plasmid. The brightfield image is helpful to ensure that cells maintain their usual morphology. Additionally, cell viability assays, such as MTS or similar assays, can be used to assess the nanoparticle toxicity7. Flow cytometry, as described, can be used to quantify the transfection efficiency. When using the HyperCyt multi-well plate attachment, the data will need to be processed ...

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

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

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 ...

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15.8K ビュー.  Johns Hopkins University School of Medicine. ナノ粒子トラッキング解析（NTA）とポリマー遺伝子送達ナノ粒子を評価するためのハイスループットフローサイトメトリーのためのプロトコルが記載されている。国税庁は、ナノ粒子の粒度分布およびプラスミド当たり粒子分布を特徴付けるために利用される。ハイスループットフローサイトメトリーは、遺伝子送達バイオマテリアルのライブラリの定?...

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

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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

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

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

Polymer Microarrays for High Throughput Discovery of Biomaterials

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

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

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

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

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

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

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

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

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

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

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 ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

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

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

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

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

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

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

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

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

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

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

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...