Cellular Activity Modulation Mediated by Near Infrared-Irradiated Polydopamine Nanoparticles: In Vitro and Ex Vivo Investigation

Abstract

The precise control of cell activity is crucial for understanding and potentially treating many disorders. Focusing on neurons and myotubes, recent advancements in nanotechnology have introduced photoresponsive nanoparticles as an alternative tool for modulating cell function with high spatial and temporal resolution. This approach offers a noninvasive alternative to traditional stimulation techniques, reducing potential tissue damage and improving the specificity of cell activation. Here, we introduce an approach envisioning fully organic polydopamine nanoparticles (PDNPs) to remotely modulate the activity of differentiated SH-SY5Y cells and differentiated C2C12 cells, via near-infrared (NIR) laser stimulation. Confocal microscopy imaging revealed the possibility of thermally activating individual neuron-like cells, eliciting a significant cellular response characterized by the generation of calcium transients and the subsequent release of the neurotransmitter acetylcholine. Similarly, we demonstrated the possibility of precisely triggering the muscle contraction of single myotubes. Additionally, we investigated the antioxidant properties of PDNPs, demonstrating their capacity to prevent an increase in oxidative stress levels related to an increase in intracellular temperature. Moreover, proteomic analysis revealed that a PDNP treatment could positively affect neuronal plasticity and nervous system maturation, besides promoting muscle growth and preserving its functional integrity, underscoring its potential to support both neural and musculoskeletal development. Eventually, the effect of the NIR laser irradiation in the presence of PDNPs in neuron-like cells was successfully evaluated ex vivo on brains of Drosophila melanogaster, genetically modified to express the fluorescent calcium indicator jGCaMP7c.

Keywords: Drosophila melanogaster; acetylcholine release; cell activity modulation; photothermal stimulation; polydopamine nanoparticles.

Figure 1

PDNP characterization and their interaction with SH-SY5Y and C2C12 cells. Representative (a) SEM and (b) TEM images. (c) Hydrodynamic diameter distribution and (d) ζ-potential analysis. (e) NIR absorption spectrum. (f) Trolox antioxidant activity standard curve, with highlighted a Trolox-equivalent antioxidant capacity of 1 μg of PDNP (gray dot). (g) PicoGreen assay on differentiated SH-SY5Y cells (n = 6, ***p < 0.001). (h) Flow cytometry analysis of PDNP internalization by differentiated SH-SY5Y cells. (i) PicoGreen assay on differentiated C2C12 cells (n = 6, *p < 0.05). (j) flow cytometry analysis of PDNP internalization by differentiated C2C12 cells.

Figure 2

Neuronal stimulation analysis. (a) Schematization of the irradiation setup. (b) Representative acquisitions highlighting the thermosensitive dye fluorescence decrement during NIR laser irradiation of differentiated SH-SY5Y cells treated with PDNPs. (c) Temperature increment during the NIR stimulation.

Figure 3

Myotubes stimulation analysis. (a) Schematization of the irradiation setup. (b) Representative acquisitions highlighting thermosensitive dye fluorescence decrement during NIR laser irradiation of differentiated C2C12 cells treated with PDNPs. (c) Temperature increment during the NIR stimulation.

Figure 4

Calcium imaging on SH-SY5Y cells subjected to repeated 50 ms on/100 ms off NIR laser stimulation, for 20 times. (a) Representative time frames images. (b) Time course of the variation of cell fluorescence levels, indicative of calcium concentration (n = 10).

Figure 5

Acetylcholine release analysis in differentiated SH-SY5Y cells. (a) Representative confocal acquisitions of acetylcholine content along the plasma membrane (acetylcholine biosensor in green, DiI-PDNPs in red). (b) Time course of the variation of cell fluorescence levels, indicative of acetylcholine release, during NIR laser stimulation.

Figure 6

Myotube contraction analysis. (a) Representative images of C2C12 myotubes treated with PDNPs (cytoplasm in red, DiO-PDNPs in green). (b) Temperature increments provided by 10 s NIR laser stimulation in the presence of PDNPs (red dashes representing laser on). (c) Kymograph related to the analyzed segment, as shown in a), in the absence or presence of blebbistatin (red dashes representing laser on). (d) Quantitative analysis of the displacement induced by the NIR + PDNP treatment.

Figure 7

Oxidative stress level analyses of differentiated neurons and myotubes exposed to NIR irradiation. Representative flow cytometry plots of (a) differentiated SH-SY5Y and (b) differentiated C2C12 cells (in dark gray ROS-negative cells, in light gray ROS-positive cells). Oxidative stress level quantification for (c) differentiated SH-SY5Y and (d) differentiated C2C12 cells (in dark gray ROS-negative cells, in light gray ROS-positive cells; n = 3, ***p < 0.001). Time-course variation of fluorescence levels indicative of intracellular ROS of (e) differentiated SH-SY5Y and (f) differentiated C2C12 cells (n = 10).

Figure 8

Analysis following Drosophila brain stimulation. (a) Representative time frames acquisition of calcium imaging performed on Drosophila brains (scale bar 30 μm); ROIs selected for the analysis are highlighted in red. Time course of the variation of cell fluorescence levels, indicative of calcium concentration: (b) control, (c) PDNPs treatment, (d) PDNPs + donepezil (100 nM) treatment, and (e) PDNPs + α-BTX (5 μM) treatment.