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.
Citation: Schmidt N, Schulze J, Warwas DP, Ehlert N, Lenarz T, Warnecke A, et al. (2018) Long-term delivery of brain-derived neurotrophic factor (BDNF) from nanoporous silica nanoparticles improves the survival of spiral ganglion neurons in vitro. PLoS ONE 13(3): e0194778. https://doi.org/10.1371/journal.pone.0194778
Editor: Hélder A. Santos, Helsingin Yliopisto, FINLAND
Received: November 17, 2017; Accepted: March 11, 2018; Published: March 27, 2018
Copyright: © 2018 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by DFG Cluster of Excellence EXC 1077/1 “Hearing4all”, http://hearing4all.eu/, NS JS NE.
Competing interests: The authors have declared that no competing interests exist.
According to the World Health Organization (February 2017), 360 million people worldwide suffer from hearing loss. From genetic defects and infectious diseases to excessive noise and aging, hearing loss has many causes and presents a heavy burden for the affected individuals . One common form of deafness is the sensorineural hearing loss (SNHL) caused by damage of the hair cells within the cochlea [2–4]. For almost over 30 years now, a standard therapy of patients suffering from profound or severe SNHL is the cochlear implant, a complex bionic device. Cochlear implants can replace the function of hair cells by direct electrical stimulation of primary auditory neurons (spiral ganglion neurons, SGNs) [5,6]. However, the efficacy of cochlear implants is limited by the anatomical gap between the electrode array inserted into the scala tympani and the SGNs situated in the Rosenthal´s canal leading to an unspecific stimulation of relatively large groups of neurons [7,8]. Moreover, the performance of cochlear implants strongly depends on the number of surviving neurons and their functionality, i.e., their excitability [3,9]. As many studies reported, progressive degeneration of the SGNs is one consequence of SNHL [6,10] and this was not only observed in animal models , but also in humans . A primary cause of SGN degeneration can be the missing neurotrophic support with growth factors by sensory hair cells and cochlear-supporting cells [13–17]. Growth factors like brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are produced by these cells and regulate neuronal survival, differentiation of neurons and axonal growth [18,19]. Therefore, neurotrophic factors are promising as therapeutic agents to inhibit or delay degenerative processes in SGNs. In addition to supporting SGN survival, neurotrophic factors have been shown to stimulate the neurite outgrowth from SGNs [20–22], which is important for an improved electro-neural interface. Due to the limitations of the cochlear implant, strategies to improve the survival and the growth of SGNs with simultaneous electrical stimulation from cochlear implants have received much interest. Despite the fact that electrical stimulation itself may induce neurotrophic signaling pathways via depolarization [6,23,24], additional treatment with neurotrophins effectively increases the survival of SGNs [25–27]. In comparison to the historical treatment of inner ear diseases with systemic therapies, one of the most common application method for exogenous local supply with neurotrophic factors are osmotic pumps [25,28–31]. But both methods have their limitations. Systemic drug delivery on the one hand is limited by restricted blood flow to the inner ear and poor penetration of the blood-cochlea barrier [2,32]. On the other hand, the neurotrophic supply with pump-based devices is finite, because the reservoir has to be refilled periodically, and the presence of a pump offers the possibility for infection of the inner ear .
An alternative to pump-based neurotrophin administration is a local delivery of neurotrophins performed by cell-based and gene therapy. For example, inoculation of a viral vector containing the BDNF gene leads to expression of BDNF which results in higher SGN survival [33,34]. Nevertheless, several safety concerns, like the cell toxicity caused by certain viral vectors and the non-controllable neurotrophic expression, have to be solved before considering clinical applications [6,13,35]. Thus, current research has focused on the development of neurotrophin delivery systems that provide a safe and effective neurotrophic supply for the long term, especially if they can be combined directly with the implant (implant-associated drug delivery). Promising approaches are electrode coating materials [36–38] and carrier systems like hydrogels [39–41], microspheres  and nanoparticles [43–46]. Even though each carrier system has advantages and disadvantages, nanoparticular systems for growth factor delivery have recently attracted increasing interest [35,44,47,48].
In the present study we describe the use of nanoporous silica nanoparticles (NPSNPs) as a special delivery platform for neurotrophic factors. BDNF is chosen as model growth factor for this application. NPSNPs offer a great potential as delivery platforms due to their advantageous properties. These properties include a high surface area, a large pore volume (up to 50%), the amenability for surface modification as well as a general biocompatibility [49–53]. Recently, a first in-human study of these particles was reported . By reaction with the surface silanol groups, the surface chemistry characteristics can be adjusted by decorating with different functional groups for an envisaged immobilization of different bioactive molecules. Moreover, via such modifications, targeted delivery can be performed by anchoring specific ligands for receptor recognition .
The NPSNPs used in this study have already proved to be suitable for growth factor immobilization. For example, our previous studies have shown that BMP2-loaded NPSNPs supported osteogenic differentiation of human mesenchymal stem cells . Moreover, current research has presented the efficacy of BDNF-loaded supraparticles (≈ 500 μm) of mesoporous silica to improve SGN survival in vivo [10,43]. However, these particles are considerably larger than those used here, and to the best of our knowledge, the efficacy of NPSNPs with an approximate diameter of <100 nm as carrier systems for BDNF has not been investigated so far. The group of Praetorius has already shown that silica nanoparticles with 20 nm in size are potentially safe for use in the inner ear . Nanoparticles with diameters <100 nm are amenable to the incorporation into different matrices, e.g. polymers, and are therefore useful for the construction of cochlear implant-associated delivery systems, e.g. in coatings of the electrode contacts or attached to the silicone surface. For example, we have shown in a previous study that it was possible to incorporate NPSNPs into dental composite materials . When a cochlear implant is placed anyway, the incorporation of a delivery system seems very appropriate. Released BDNF can then act as a neuroprotective factor and might guide neurite outgrowth towards the cochlear electrode [37,45,57].
The main purpose of the present study is to establish the general suitability of BDNF-loaded NPSNPs for the construction of cochlear implant-associated BDNF delivery systems. Thus, apart from testing the cytocompatibility of the nanoparticles with SGNs, the question whether BDNF which is released from the nanoparticles has a positive effect on the survival of SGNs in vitro (without the necessity for a direct contact between nanoparticles and SGNs) is most important. In view of the fact that growth factor-based engineering of cellular behavior should aim at long-term effects, a specific focus is posed on the question whether this is also true for the amounts delivered after long release periods, which is why we studied the release for 80 days.
Synthesis and modification of nanoporous silica nanoparticles
All chemicals, except absolute ethanol, were obtained commercially from Sigma Aldrich (Munich, Germany) and used without further purification. Absolute ethanol was purchased from Merck (Darmstadt, Germany).
NPSNPs were prepared by adding 3.16 g cetyltrimethylammonium bromide (CTAB) and 0.23 g diethanolamine (DEA) to a solution of 75 mL ultrapure water and 13.4 mL absolute ethanol. The mixture was heated to 40°C and stirred. After 30 min, 8.56 mL tetraethoxysilane (TEOS) were added and the reaction mixture was stirred for additional 2 h. The product was centrifuged (30 min at 18000 g) and washed twice with water and once with ethanol. Afterwards, the particles were dried overnight at 60°C. To remove the structure-directing agent, a calcination for 5 h at 550°C (heating rate: 1 K min-1) followed .
The amino modification of the silica surface was performed via post-grafting by dispersing 500 mg of NPSNPs in 20 mL toluene. To this dispersion 75 μL 1,8-diaza-bicyclo[5.4.0]undec-7-ene (DBU) and 95 μL 3-aminopropyltrimethoxysilane (APTMS) were added. Afterwards, the solution was stirred for 2 h at 80°C. The modified nanoparticles were collected by centrifugation, washed three times with ethanol and dried at 60°C .
Immobilization and release of BDNF
BDNF immobilization took place in sterile solutions with a concentration of 1 μg mL-1 in phosphate-buffered saline (PBS, Sigma Aldrich, Munich, Germany), which contained 0.1% bovine serum albumin (BSA, Sigma Aldrich, Munich, Germany). BSA acted as stabilizer of BDNF and as filler protein to prevent protein adhesion to reaction tubes . Recombinant BDNF was purchased from Life Technologies (Darmstadt, Germany). It was produced in Escherichia coli and had a purity higher than 98%.
5 mg of the nanoparticles, unmodified or amino-modified, were sterilized by shining UV light on the sample. Afterwards, the nanoparticles were incubated in 1 mL of the sterile protein solution for 24 h at 4°C in a Thermomixer (Biozym Scientific, Hessisch Oldendorf, Germany) under constant shaking of 1000 rpm. After the incubation, the samples were centrifuged and washed once with PBS (0.1% BSA). All incubation and washing solutions were kept frozen at -20°C to prevent any leakage of protein during storage for later analysis by ELISA. For control experiments 5 mg nanoparticles were treated under similar conditions, but without BDNF additive.
After incubation and washing, the release was started by giving 1 mL PBS (0.1% BSA) to the samples. PBS was chosen as release medium because it is an often used standard release medium to simulate physiological conditions and has a pH value of 7.4 similar to the cochlear fluid . The samples were kept at 37°C. At various time intervals, the samples were centrifuged, the supernatants were removed and 1 mL fresh PBS (0.1% BSA) was added to continue the release. Similar to the incubation and washing solutions, all supernatants were frozen to be later analyzed by ELISA.
For the characterization of NPSNPs transmission electron microscopy (TEM) was performed. TEM images were taken on a FEI Tecnai G2 F20 TMP instrument (Hillsboro, USA) operated with 200 kV. For the preparation ethanolic or aqueous dispersions were dropped on Cu grids with a 400 mesh and dried overnight.
Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZSP from Malvern Instruments (Worcestershire, UK). The nanoparticles were redispersed in water with a concentration of 0.5 mg mL-1 by ultrasonification. Afterwards, the suspensions were transferred to a polystyrene cuvette.
Zeta potential titration curves were measured with a Zetasizer Nano ZSP and a MPT2 Autotitrator from Malvern Instruments (Worcestershire, UK). For each measurement disposable folded capillary cells (DTS1070) were used. Each sample was dispersed in water with a concentration of 0.5 mg mL-1. The curves were recorded on proceeding from basic to acidic pH by addition of HCl (0.2 M). At each pH value, the zeta potential was measured three times. For data analysis, Malvern Zetasizer Software Version 7.11 was used.
Nitrogen sorption measurements were performed at 77°K on a Quantachrome Autosorb-3 instrument (Boynton Beach, USA). The unmodified nanoparticles were outgassed in vacuum at 100°C and the amino-modified nanoparticles at 80°C for 24 h prior to the sorption measurements. To evaluate the data, the ASiQWin 2.0 software was used. Surface areas were estimated by applying the Brunauer-Emmett-Teller (BET) equation. The pore size distribution was calculated using non-linear density-functional theory (NLDFT) and fitting of the Quantachrome Kernel “N2 at 77 K on silica for cylinder pores, NLDFT equilibrium model” to the experimental data. Values for total pore volumes were estimated by the single point method at p/p0 = 0.92 to exclude interparticular volume.
For the quantification of the amount of immobilized and released BDNF an enzyme-linked immunosorbent assay (ELISA) kit against human BDNF (Boster Biological Technology Co., Ltd., Pleasanton, USA) was applied. The BDNF-ELISA kit was used in accordance to the manufacturer´s recommendations. In brief, a monoclonal antibody for BDNF had been precoated onto the 96-well plate. Standards and samples were diluted in sample dilution buffer and were added to the wells. After an incubation period of 90 min, the plate content was discarded and a working solution of a biotinylated anti-human BDNF antibody was added for 60 min. This was followed by three washing steps with PBS and by the incubation with a working solution containing an avidin-biotin-peroxidase complex. Then, the unbound conjugates were washed off with PBS. Finally, the detection was performed with 3,3´,5,5´-tetramethylbenzidine and stopped with 2 M sulphuric acid. All incubation steps were performed at 37°C. Absorbance was read at 450 nm on an EON spectrophotometer (Biotek, Winooski, USA).
Cell culture investigations
The cell culture investigations were performed according to the investigations of Williams et al. . The murine fibroblast cell line NIH3T3 (ATCC-Number: CRL-165) was used for the initial cytocompatibility tests. The nanoparticles were sterilized under UV light. For cultivation, nanoparticle stock solutions with a concentration of 1000 μg mL-1 were produced with sterile water. From these stock solutions the other tested concentrations of 10 μg mL-1, 100 μg mL-1, 250 μg mL-1 and 500 μg mL-1 were prepared by dilution with sterile water.
The NIH3T3 fibroblasts were cultivated in high glucose Dulbecco´s Modified Eagle´s Medium (DMEM, Biochrom, Berlin, Germany) with supplements like 10% fetal calf serum (FCS), 1% penicillin and streptomycin (Biochrom, Berlin, Germany). Cells were seeded with a density of 1 x 104 cells per well in a 96-well plate (TPP, Trasadingen, Switzerland). The cultivation was performed for three days at 37°C in a humidified atmosphere (5% CO2) for expansion. After removing the medium, 50 μL fresh medium and 50 μL aqueous nanoparticle dispersion were given to the wells. So, the final concentrations which were tested are 5 μg mL-1, 50 μg mL-1, 125 μg mL-1, 250 μg mL-1 and 500 μg mL-1. Incubation took place for four days and every day the morphology and proliferation of the fibroblasts were checked with a transmission light microscope (CKX41, Olympus, Hamburg, Germany) with a CCD-camera (Colorview III, SIS, Olympus).
Neutral red uptake assay.
After the incubation, the cell viability was determined by the neutral red uptake (NRU) assay. Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride, Merck, Darmstadt, Germany) stock solution was prepared by dissolving 40 mg neutral red dye in 10 mL purified water (4 mg mL-1). The stock solution was diluted 1:50 in pre-heated (37°C) DMEM. The medium was removed and 100 μL of the neutral red medium were added per well. After an incubation of 3 h at 37°C and 5% CO2, the neutral red medium was discarded. Afterwards, the cells were washed and fixed by adding solution I (1% calcium chloride, 0.5% formaldehyde in purified water). After 5 min incubation time solution I was removed. Now, 100 μL of a neutral red destaining solution (1% acetic acid, 50% ethanol (95%) in purified water) were added. The plate was shaken and incubated for 10 min at 4°C. The absorption of the neutral red extract was measured at 570 nm using a microplate reader (Multiskan Ascent, Thermo Scientific Inc., Waltham, USA).
Ethics statement for isolation of SGCs from neonatal rats.
The experiments and analysis of this study were conducted from October 2015 to September 2017. All experiments were carried out in accordance with the institutional guidelines for animal welfare of the Hannover Medical School following the standards described by the German ´Law on Protecting Animals´ (Tierschutzgesetz) and with the European Directive 2010/63/EU for protection of animals used for experimental purposes. For our in vitro experiments an euthanasia was used, which is registered (no.:2013/44) with the local authorities (Zentrales Tierlaboratorium, Laboratory Animal Science, Hannover Medical School, including an institutional animal care and use committee) and reported on a regular basis as demanded by law. For exclusive sacrifice of animals for tissue analysis in research, no further approval is needed if no other treatment is applied beforehand (§4). The rats were bred and born for research study purposes. A breeding stock was supplied by Charles River (Charles River, Wilmington, USA) and housed with their litters in the facilities of the licensed Institution of Laboratory Animal Science of the Hannover Medical School. To minimize the stress level for the neonatal rats, they were rapidly decapitated prior to any experimentation by a licensed person.
Spiral ganglion cells.
In the cell culture investigations with spiral ganglion cells (SGCs), the neuroprotective action of amino-modified nanoparticles with immobilized BDNF and of released BDNF amounts was investigated. SGCs, obtained by dissociation of the spiral ganglion, provide mixed cultures containing neurons, fibroblasts and glial cells. The primary SGCs were isolated from neonatal Sprague-Dawley rats (postnatal day 3–5). Rats were sacrificed by rapid decapitation. The dissection of the cochleae and the enzymatic and mechanical dissociation of the spiral ganglia were performed according to a previously described protocol [61,62]. Afterwards, the number of viable cells was determined using a Neubauer chamber (Brand, Wertheim, Germany) and the trypan blue staining (Sigma Aldrich, Munich, Germany). The dissociated cells were seeded at a density of 1 x 104 cells per well in a 96-well plate (TPP). Prior to cell seeding, the used plates were coated with poly-D/L-ornithine (0.1 mg mL-1, Sigma Aldrich, Munich, Germany) and laminin (0.01 mg mL-1, Life Technologies, Carlsbad, USA). 50 μL of the tested samples (nanoparticle dispersion or released supernatants) and 50 μL fresh medium were given to the wells. The incubation was performed for 48 h in serum-free medium (Panserin 401, PAN Biotech, Aidenbach, Germany), which was supplemented with HEPES (25 mM, Life Technologies, Carlsbad, USA), glucose (6 mg mL-1, Braun AG, Melsungen, Germany), penicillin (30 U mL-1, Grünenthal GmbH, Aachen, Germany), N2-supplement (3 μg mL-1, Life Technologies, Carlsbad, USA) and insulin (5 μg mL-1, Sigma Aldrich, Munich, Germany). After the incubation, the SGCs were fixed with a 1:1 methanol (Carl Roth, Karlsruhe, Germany) and acetone (J.T. Baker, Arnhem, Netherlands) solution and washed with PBS (Gibco® by Life Technologies, Carlsbad, USA). In all experiments, we included a negative control (SGCs cultivated in serum-free medium), a positive control (SGCs in