Abstract
Immune responses have to be tightly controlled to guarantee maintenance of immunological tolerance and efficient clearance of pathogens and tumorigenic cells without induction of unspecific side effects. CD4+ CD25+ regulatory T cells (Tregs) play an important role in these processes due to their immunosuppressive function. Genetic modification of Tregs would be helpful to understand which molecules and pathways are involved in their function, but currently available methods are limited by time, costs or efficacy. Here, we made use of biofunctionalized gold nanoparticles as non-viral carriers to transport genetic information into murine Tregs. Confocal microscopy and transmission electron microscopy revealed an efficient uptake of the bioconjugates by Tregs. Most importantly, coupling eGFP-siRNA to those particles resulted in a dose and time dependent reduction of up to 50% of eGFP expression in Tregs isolated from Foxp3eGFP reporter mice. Thus, gold particles represent a suitable carrier for efficient import of nucleic acids into murine CD4+ CD25+ Tregs, superior to electroporation.
Highlights
Generate nanobiconjugates with defined surface structures is pulsed laser ablation in liquids (PLAL)
The gold nanoparticle-oligonulcotide-peptide conjugates that were used within these experiments carried 7.9 ± 1.7 locked nucleic acid (LNA) and 29.7 ± 0.1 nuclear localization signaling sequence (NLS) per AuNP which leads to stable bioconjugates with a gold concentration of 51.2 ± 0.9 μg/mL and a hydrodynamic diameter of 16.6 ± 4.0 nm (Fig. 1B)
The effect of stabilization using LNA can be seen in the Primary Particle Index (PPI), which gives a high value for low agglomeration and is determined by UV-Vis spectrometry[20,21]
Summary
Generate nanobiconjugates with defined surface structures is pulsed laser ablation in liquids (PLAL). Since laser ablation in water yields nanoparticles with broad size distributions, low salinity solutions of e.g. sodium phosphate buffer or sodium chloride can be used[20] for size quenching and reduction of polydispersity. This allows the generation of monodisperse gold nanoparticles at particle sizes < 10 nm[21,22]. Since the combination of positively charged peptides and negatively charged nanoparticles leads to charge compensation and to the formation of agglomerated and presumably unstable conjugates[21], oligonucleotides were used for pre-stabilization These nanobioconjugates were examined for their potential to efficiently transfer siRNA into unstimulated murine CD4+ CD25+ Tregs
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