Ultrabright Fluorescent Polymeric Nanoparticles with a Stealth Pluronic Shell for Live Tracking in the Mouse Brain

Abstract

The inability to visualize single organic nanoparticles (NPs) in vivo is an unsolved issue in the field of nanomedicine. To enable high single particle fluorescence, we loaded polymer poly(methyl methacrylate)-sulfonate (PMMA-SO3H) NPs with octadecyl rhodamine B together with hydrophobic counter-ions as a fluorophore insulator to prevent aggregation-induced quenching. To create NPs with stealth properties we used the amphiphilic block copolymers pluronic F-127 and F-68. Fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) showed that pluronics remain at the NP surface after dialysis (density: one amphiphile per 5.5 nm 2) and protected NPs from degradation by serum proteins and surfactants. Single particle brightness was increased 150-fold as compared to commercially available preparations by increasing dye loading to 20 wt% and optimizing particle size. Added to primary cultured neurons, these NPs were stable and interacted with dendrites and axons. After intravenous injection in mice, NPs could be tracked in cerebral vessels for at least 1h by in vivo two-photon microscopy. Following brain injury or neuroinflammation, NPs extravasated from meningeal vessels, were taken up by meningeal macrophages, and entered the brain parenchyma. In summary, we developed biocompatible NPs suitable for drug delivery with in vitro and in vivo stealth properties and extremely bright fluorescence. Thus, individual NPs could be tracked in mouse brain and their dynamics visualized with subcellular resolution. Using superbright NPs may thus open new avenues for the investigation of NPs in living organisms.

Dynamic tracing using ultra-bright labelling and multi-photon microscopy identifies endothelial uptake of poloxamer 188 coated poly(lactic-co-glycolic acid) nano-carriersin vivo

“…At this concentration about 100 pluronics per NP remained on the surface after dialysis. This corresponds to about one PEG chain per 10 nm 2 , a value well in line with previous results (21, 26). Adsorption of pluronic PF-127 or PF-68 onto the surface of NP increased the particle size by 10 and 4 nm, respectively (Figure 1B) .…”

“…per 10 nm², a value well in line with previous results (21,26). Adsorption of pluronic PF-127 or PF-68 onto the surface of NP increased the particle size by 10 and 4 nm, respectively ( Figure 1B).…”

“…Internal organs. Free floating 50 µm sections of brain, liver, kidney, spleen and heart were prepared as previously described (21). The sections were permeabilized for 30 minutes in PBS Tween 20, blocked for 60 minutes in 10% goat serum in PBS and then stained with the primary antibody for 12 hours at 4°C.…”

“…For the moment, poly(methyl methacrylate) (PMMA)-based NPs were already used for this purpose being loaded with high amounts of the salt of a rhodamine B derivative (R18) with a bulky hydrophobic counter-ion, fluorinated tetraphenylborate (F5-TPB), thereby reaching exceptional brightness (20). Recently, we tracked such NPs individually in the murine cerebral microcirculation and demonstrated extravasation following brain injury (21). So far, we used PMMA NPs as an experimental tool to maximize particle brightness and to perform in vitro experiments, however, since PMMA is not biodegradable, its use in a living organism is questionable.…”

Size‐Dependent Electroporation of Dye‐Loaded Polymer Nanoparticles for Efficient and Safe Intracellular Delivery

“…[ 11,40–42 ] At the same time, their size can be tuned over a wide range from less than 10 to over 50 nm, and they can be endowed with stealth properties through the chemistry of the polymer and the conditions used for particle assembly. [ 11,12,41–44 ] Such particles have already found various applications, including multicolor labeling of cells in vitro and in vivo, [ 45 ] single‐particle tracking in cells [ 11,12 ] and mice brain, [ 46 ] and oxygen sensing, [ 47 ] as well as the detection of DNA and RNA down to the single‐particle and single‐molecule level. [ 36,42,48 ]…”