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Bioengineering

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

Published: March 1st, 2013

DOI:

10.3791/50176

1Biomedical Engineering Department, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, 3Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Institute for Nanobiotechnology, Johns Hopkins University School of Medicine

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 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.

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 ....

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1. Cell Seeding

  1. Do not allow cells to grow to overconfluency. Use early passage cells when transfecting primary cells.
  2. 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 da.......

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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 .......

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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.......

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

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

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  2. Sheyn, D., et al. Genetically modified cells in regenerative medicine and tissue engineering. Advanced Drug Delivery Reviews. 62, 683-698 (2010).
  3. Boussif, O., et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301 (1995).
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  11. Bhise, N. S., Shmueli, R. B., Gonzalez, J., Green, J. J. A novel assay for quantifying the number of plasmids encapsulated by polymer nanoparticles. Small. 8, 367-373 (2012).
  12. Yu, J., et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 324, 797-801 (2009).
  13. Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET. Journal of Controlled Release: Official journal of the Controlled Release Society. 116, 83-89 (2006).
  14. Kreiss, P., et al. Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Research. 27, 3792-3798 (1999).
  15. Pitard, B., et al. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer. Proc. Natl. Acad. Sci. U.S.A. 94, 14412-14417 (1997).
  16. Collins, L., Kaszuba, M., Fabre, J. W. Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery. Biochimica et Biophysica Acta. 1672, 12-20 (2004).
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