Long-term delivery of brain-derived neurotrophic factor (BDNF) from nanoporous silica nanoparticles improves the survival of spiral ganglion neurons in vitro.

Sensorineural hearing loss (SNHL) can be overcome by electrical stimulation of spiral ganglion neurons (SGNs) via a cochlear implant (CI). Restricted CI performance results from the spatial gap between the SGNs and the electrode, but the efficacy of CI is also limited by the degeneration of SGNs as one consequence of SHNL. In the healthy cochlea, the survival of SGNs is assured by endogenous neurotrophic support. Several applications of exogenous neurotrophic supply have been shown to reduce SGN degeneration in vitro and in vivo. In the present study, nanoporous silica nanoparticles (NPSNPs), with an approximate diameter of <100 nm, were loaded with the brain-derived neurotrophic factor (BDNF) to test their efficacy as long-term delivery system for neurotrophins. The neurotrophic factor was released constantly from the NPSNPs over a release period of 80 days when the surface of the nanoparticles had been modified with amino groups. Cell culture investigations with NIH3T3 fibroblasts attest a good general cytocompatibility of the NPSNPs. In vitro experiments with SGNs indicate a significantly higher survival rate of SGNs in cell cultures that contained BDNF-loaded nanoparticles compared to the control culture with unloaded NPSNPs (p<0.001). Importantly, also the amounts of BDNF released up to a time period of 39 days increased the survival rate of SGNs. Thus, NPSNPs carrying BDNF are suitable for the treatment of inner ear disease and for the protection and the support of SGNs. Their nanoscale nature and the fact that a direct contact of the nanoparticles and the SGNs is not necessary for neuroprotective effects, should allow for the facile preparation of nanocomposites, e.g., with biocompatible polymers, to install coatings on implants for the realization of implant-based growth factor delivery systems

Survival rates of spiral ganglion neurons after a cultivation of two days.

<p>Cells were cultivated in presence of the supernatants from the release experiments of BDNF-loaded amino-modified NPSNPs (NPSNP-NH₂-BDNF) or of amino-modified NPSNPs (NPSNP-NH₂) as control experiment. Values are given as mean ± standard error of the mean (<i>N</i> = 3, <i>n</i> = 3). Statistical assessment was performed using one-way ANOVA with Bonferroni´s multiple comparison test (n.s. = not significant, *p<0.05; **p<0.01; ***p<0.001). Asterisks over the bars indicate the significance of the survival rates of different conditions compared to the medium control (serum-free SGC medium). Asterisks between two bars indicate the significance between the different conditions.</p

Quantification of the amount of immobilized BDNF on unmodified and amino-modified NPSNPs which were incubated in a solution containing 1 μg mL^-1 BDNF.

<p>Quantification of the amount of immobilized BDNF on unmodified and amino-modified NPSNPs which were incubated in a solution containing 1 μg mL^-1 BDNF.</p

Relative cell viabilities of NIH3T3 fibroblasts in the presence of unmodified and amino-modified nanoparticles in different concentrations determined by the NRU assay after an incubation for four days.

<p>Values are given as mean ± standard error of the mean (<i>n</i> = 3).</p

BDNF release profile of unmodified NPSNPs in PBS (0.1% BSA) over 60 days at 37°C.

<p>The left axis represents the cumulative BDNF release with regard to 1 mg of nanoparticles and the right axis shows the cumulative release of BDNF referred to 1 mL release medium.</p

BDNF release profile of amino-modified NPSNPs in PBS (0.1% BSA) over 80 days at 37°C.

<p>The left axis represents the cumulative BDNF release with regard to 1 mg of nanoparticles and the right axis shows the cumulative release of BDNF referred to 1 mL release medium.</p

Scheme of the possible interactions between BDNF with (left) unmodified NPSNPs and (right) amino-modified NPSNPs.

<p>With unmodified NPSNPs, BDNF can interact via hydrogen bonds and electrostatic interactions, and with amino-modified NPSNPs via hydrogen bonding, electrostatic interactions and via hydrophobic effects. This schematic view does not show the nanoparticles, atoms and the protein with their real sizes.</p

Comparison of the survival rates of spiral ganglion neurons after 48 h hof cultivation.

<p>Cells were cultivated in the presence of amino-modified nanoparticles (NPSNP-NH₂; light grey), BDNF-loaded amino-modified nanoparticles (dark grey) and amino-modified nanoparticles with an exogenous addition of 50 ng mL^-1 BDNF (light grey, striped). Values are given as mean ± standard error of the mean (<i>N</i> = 2, <i>n</i> = 3). Statistical assessment was performed using one-way ANOVA with Bonferroni´s multiple comparison test (n.s. = not significant, *p<0.05; **p<0.01; ***p<0.001). Asterisks over the bars indicate the significance of the survival rates of different conditions compared to the negative control (serum-free SGC medium). Asterisks between two bars indicate the significance between the respective conditions.</p

Representative microscopic images of spiral ganglion cell cultures cultivated for 48 h.

<p>The cells were cultivated in the presence of amino-modified nanoparticles (w/o BDNF), BDNF-loaded amino-modified nanoparticles (immobilized BDNF) and amino-modified nanoparticles with an exogenous addition of 50 ng mL^-1 BDNF (exogenous BDNF) in two different concentrations. For comparison, SGCs were also cultivated in serum-free SGC medium (medium) and in serum-free medium supplemented with BDNF (50 ng mL^-1 BDNF) as well as in a serum-free medium/PBS solution (1:1) (PBS (0.1% BSA)).</p

Zeta potential titration curves for the unmodified NPSNPs in comparison to the amino-modified NPSNPs.

<p>Zeta potential titration curves for the unmodified NPSNPs in comparison to the amino-modified NPSNPs.</p