Abstract
Nanoparticles of defined size can be easily obtained by simply mixing Protamine, a pharmaceutical drug that is used to neutralize heparin after surgery, and RNA in the form of oligonucleotides or messenger RNA. Depending on the concentrations of the two reagents and their salt contents, homogenous nanoparticles with a mean diameter of 50 to more than 1000 nm can spontaneously be generated. RNA is a danger signal because it is an agonist of for example TLR-3, -7, and -8; therefore, Protamine–RNA nanoparticles are immunostimulating. We and others have shown in vitro that nanoparticle size and interferon-alpha production by human peripheral blood mononuclear cells (PBMCs) are inversely correlated. Conversely, nanoparticle size and TNF-alpha production by PBMCs are positively correlated (Rettig et al., Blood 115:4533–4541, 2010). Particles of less than 450 nm are most frequently used for research and clinical applications because they are very stable, remain polydispersed and induce interferon-alpha proteins, which are a natural antiviral and anticancer protein family with 12 members in humans. Herein, we describe a method to generate 130 nm nanoparticles as well as some of their physical and biological characteristics.
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Acknowledgement
The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 601939 (MERIT).
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Supplementary Fig. 1
(a) Influence of reagent dilutions: Particle size. Protamine and RNA were diluted in water at 0.1, 1, or 2 mg/ml before being mixed together (mass ratio 1:1). The graphs report “Intensity-Weighted Gaussian Distribution Analysis” for solid particles. Average particle sizes are 85 nm for 0.1 mg/ml, 146 nm for 1 mg/ml, and 192 nm for 2 mg/ml. Thus, particle size can be finely tuned by adjusting the Protamine and RNA ratio. The greater the dilution, the smaller the particles. (b) Influence of reagent dilutions: Induction of alpha interferons. The graph reports the calculated values of alpha-interferon contents in supernatants from PBMCs alone (“PBMCs”) or those incubated with Protamine–RNA nanoparticles made with reagents at 0.1 mg/ml (“PR01”), 1 mg/ml (“PR1”), or 2 mg/ml (“PR2”). Each bar represents the average value of three data points (three culture wells) and the standard deviation. The three types of particles induce similar amounts of alpha interferons (PDF 66 kb)
Supplementary Fig. 2
(a) Influence of reagent ratio: Particle size. Protamine and RNA were diluted in water at 0.5 mg/ml before being mixed together at mass ratios of 1:2 (twofold more RNA than Protamine “PR12”), 1:1 (equivalent mass amounts of RNA and Protamine “PR11”), or 2:1 (twofold more Protamine than RNA “PR21”). The graphs report “Intensity-Weighted Gaussian Distribution Analysis” for solid particles. Average particle size is 174.7 nm for the 1:2 ratio, 131.6 nm for the 1:1 ratio, and 154.7 nm for the 2:1 ratio. Thus, particle sizes are similar when using Protamine–RNA ratios from 1:2 to 2:1. (b) Influence of reagent ratio: Induction of alpha interferons. The graph reports the calculated values of alpha-interferon content in supernatants from PBMCs alone (“PBMCs”) or those incubated with Protamine–RNA nanoparticles made with reagents at a 1:2 mass ratio (“PR12”), 1:1 mass ratio (“PR11”) or 2:1 mass ratio (“PR21”). Each bar represents the average value of three data points (three culture wells) and the standard deviation. The three types of particles induce similar amounts of alpha interferons (PDF 66 kb)
Supplementary Fig. 3
Stability of particles: Induction of alpha interferons. The graph reports the calculated values of alpha-interferon content in supernatants from PBMCs alone (“PBMCs”) or those incubated with Protamine–RNA nanoparticles made with reagents at a 1:1 mass ratio and used immediately (“PR11 fresh”) as well as after storage at room temperature for 1 week (“PR11 7 days RT”) or 4 °C for 10 days, (“PR11 10 days 4 °C”) or after having been frozen and thawed once (“PR11 −20 °C”). Each bar represents the average value of three data points (three culture wells) and the standard deviation. The particles are very stable when kept in liquid but partly deactivated by freezing/thawing (PDF 66 kb)
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Tusup, M., Pascolo, S. (2017). Generation of Immunostimulating 130 nm Protamine–RNA nanoparticles. In: Kramps, T., Elbers, K. (eds) RNA Vaccines. Methods in Molecular Biology, vol 1499. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6481-9_9
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DOI: https://doi.org/10.1007/978-1-4939-6481-9_9
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