Polyethylenimine-based polyplexes are promising nonviral gene delivery systems for preclinical and clinical applications. Pipette-based polyplexing is associated with several disadvantages, such as batch-to-batch variability, restriction to smaller volumes, and variable gene delivery results. The present protocol describes syringe-pump-mediated upscaled synthesis of well-defined gene delivery nanoparticles capable of efficient in vitro and in vivo gene delivery. Syringe-pump-based synthesis ensures controlled mixing, upscaling, and reproducible gene delivery. Nanoparticle tracking analysis of the upscaled formulations involved single nanoparticle tracking, thereby generating highly resolved biophysical characterization. Gene delivery performance was investigated by luciferase gene expression in cells and three-dimensional bioluminescence imaging in mice.
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References
1.
GehrigS, SamiH, OgrisM. Gene therapy and imaging in preclinical and clinical oncology: Recent developments in therapy and theranostics. Ther Deliv, 2014; 5:1275–1296.
2.
TalsmaH, CherngJ, LehrmannH, et al.Stabilization of gene delivery systems by freeze-drying. Int J Pharm, 1997; 157:233–238.
KasperJC, SchaffertD, OgrisM, et al.The establishment of an up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined plasmid/LPEI polyplexes. Eur J Pharm Biopharm, 2011; 77:182–185.
5.
HirotaS, de IlarduyaCT, BarronLG, et al.Simple mixing device to reproducibly prepare cationic lipid-DNA complexes (lipoplexes). Biotechniques, 1999; 27:286–90.
6.
FilipeV, HaweA, JiskootW. Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res, 2010; 27:796–810.
7.
WightmanL, KircheisR, RosslerV, et al.Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med, 2001; 3:362–372.
8.
GoulaD, BenoistC, ManteroS, et al.Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther, 1998; 5:1291–1295.
9.
FerrariS, MoroE, PettenazzoA, et al.ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther, 1997; 4:1100–1106.
10.
GoulaD, RemyJS, ErbacherP, et al.Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther, 1998; 5:712–717.
11.
Di GioiaS, ConeseM. Polyethylenimine-mediated gene delivery to the lung and therapeutic applications. Drug Des Devel Ther, 2009; 2:163–188.
12.
KhalilIA, KogureK, AkitaH, et al.Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev, 2006; 58:32–45.
13.
BoeckleS, von GersdorffK, van der PiepenS, et al.Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J Gene Med, 2004; 6:1102–1111.
14.
von GersdorffK, SandersNN, VandenbrouckeR, et al.The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type. Mol Ther, 2006; 14:745–753.
15.
GoulaD, BeckerN, LemkineGF, et al.Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes 965. Gene Ther, 2000; 7:499–504.
16.
RödlW, SchaffertD, WagnerE, et al.Synthesis of polyethylenimine-based nanocarriers for systemic tumor targeting of nucleic acids. Methods Mol Biol, 2013; 948:105–120.
17.
UngaroF, De RosaG, MiroA, et al.Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations. J Pharm Biomed Anal, 2003; 31:143–149.
18.
TannousBA. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat Protoc, 2009; 4:582–591.
19.
TannousBA, KimDE, FernandezJL, et al.Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther, 2005; 11:435–443.
20.
LenterMC, GaridelP, PelisekJ, et al.Stabilized nonviral formulations for the delivery of MCP-1 gene into cells of the vasculoendothelial system. Pharm Res, 2004; 21:683–691.
21.
KoppF, SchnoedtM, HaaseR, et al.De-targeting by miR-143 decreases unwanted transgene expression in non-tumorigenic cells. Gene Ther, 2013; 20:1104–1109.
