Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.
Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites’ length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.
NGF is essential for the development and maintenance of neurons in the peripheral nervous system (PNS)1 and plays a crucial role in the survival and function of basal forebrain cholinergic neurons in the central nervous system (CNS)2. Its high pharmacological potential for treating central neurodegenerative diseases, such as Alzheimer's and Parkinson's, has been widely demonstrated, with clinical trials currently in progress3,4,5,6. The greatest challenge in the delivery of NGF to the CNS resides in its inability to cross the blood brain barrier (BBB), when systemically administered7. Moreover, NGF susceptibility to rapid enzymatic degradation renders its short half-life and significantly limits its therapeutic use8,9. Therefore, there is an unmet challenge to design delivery systems which allow for a prolonged and controlled release of NGF in a safe manner. Various NGF delivery systems, including polymer-based systems, have been studied10,11,12,13,14,15,16,17. The release profiles of these systems were often characterized by a distinct initial burst followed by a slow continuous release, where in the latter stage the release rate was significantly low in comparison to the initial burst11,18,19. Furthermore, inactivation of the protein by the acidic degradation products of the polymers (e.g., poly(lactic-co-glycolic) acid) or loss of NGF bioactivity during the encapsulation process were observed with this systems20.
Nanostructured PSi is characterized by several appealing properties, including its high surface area, large porous volume, biocompatibility, and tunable degradability in bodily fluids, predestining it for a promising drug delivery platform21,22,23,24,25,26,27,28. Proper selection of its anodization conditions allows to easily adjust the PSi structural properties (e.g., porosity and pore size) for tailoring drug loading and release kinetics21,27. Moreover, various convenient chemical routes allow to modify the surface of the PSi and by that further tune the dissolution rate of the Si scaffold under physiological conditions and the release rates of the drug22,24,29,30.
This work focuses on designing a PSi-based delivery system for prolonged controlled release of NGF. The effect of the NGF-PSi carriers on neuronal differentiation and outgrowth is examined using PC12 cells and dissociated DRG neurons. We demonstrate that the loaded NGF has retained its bioactivity by inducing neurite outgrowth and profound differentiation throughout a 1-month release period within a single administration.
Degradable nanostructured PSiO2 films are fabricated and employed as carriers for NGF, allowing for its continuous and prolonged release, whilst retaining its biological activity. The potential of the PSiO2 to serve as a delivery system for NGF is demonstrated in vitro by demonstrating their ability to release sufficient NGF dosage to induce neuronal differentiation and promote outgrowth of PC12 cells and DRG neurons. The engineered films can be used as long-term reservoirs of NGF for future treatment in vivo.
The structural properties of the fabricated PSi films were tailored specifically for NGF payload; the current density of the electrochemical etching process was adjusted to obtain pore size of approximately 40 nm that would easily accommodate the NGF, a protein with a molecular weight of 26.5 kDa32 and a characteristic diameter of ~4 nm33, within the porous matrix. Moreover, thermal oxidation of the porous scaffold was performed to enable physical adsorption of NGF by electrostatic attraction of the positively charged protein to the negatively charged oxidized PSi surface. The surface chemistry of PSi exerts a major effect on the loading efficacy and can be easily tuned in order to better control the interactions between the payload and the porous matrix. These interactions subsequently dictate the structure of adsorbed protein molecules and their bioactivity34,35,36. In conclusion, the system was adjusted to obtain optimal loading of NGF by carefully selecting the appropriate pore size, surface characteristics and the ideal loading solvent and the resulting effect of the mentioned parameters dictates the protein loading efficacy. Therefore, any change in the fabrication parameters (e.g., current density, etching time, type and concentration of dopant or electrolyte), surface chemistry or loading solution composition can affect the loading efficacy and bioactivity of the loaded protein.
The release rate of a payload from the PSi orPSiO2host is generally dictated by a combination of two simultaneous mechanisms, out-diffusion of the payload molecules and the degradation of the Si scaffold37. The erosion and subsequent dissolution rate are affected by the implantation site, its pathology and disease state28,38,39. It was established in previous work that if a different release rate is required for a certain therapeutic application, the release profile can be modified and prolonged by changing the surface chemistry of the PSi surface38,40,41. Various chemical modifications, such as thermal oxidation, thermal carbonization and hydrosilylation techniques, have been shown to stabilize the PSi surface and affect its degradation and consequent payload release35,42,43,44,45. Moreover, loading of NGF into the carriers by covalent attachment of the protein molecules to the Si scaffold via various surface chemistry routes should result in a more prolonged release because the payload is only released when the covalent bonds are broken or the supporting Si matrix is degraded21.
Furthermore, following its fabrication process, PSi can be rendered into various configurations besides thin films, such as microparticles46, nanoparticles47 or free-standing membranes26, which can also be employed as carriers for NGF and meet specific application needs.
In order to be clinically relevant, the NGF content within the PSiO2 carriers should reach the range of therapeutic doses. In the method described in the protocol, the NGF-loaded PSiO2 carriers are introduced into a consistent volume of 2 mL of cell media or PBS buffer and thus, the concentration of the loading solution and the respective NGF mass loaded were adjusted to yield a released NGF concentration that is relevant for the tested in vitro system. When utilizing this method for different systems, such as ex vivo or in vivo environments, the concentration of the NGF loading solution should be increased and adjusted according to the needed dose. Alternatively, higher NGF content can be obtained by introducing multiple carriers per tested area or by using larger areas of PSiO2 samples.
Moreover, it should be noted that in later time points along the release period, the released NGF concentrations are much lower than in earlier time points. The fact that the NGF flux is not constant over time must be taken into consideration when designing the system according to application needs.
Numerous NGF delivery systems have been developed and reported in the literature, most of them are polymer-based systems, comprising of synthetic or natural polymer conjugates10,11,12,15,16,17. These systems have shown effective sustained release profiles, however, the release period spanned over a period of several days with a significant burst effect. Some of these delivery platforms suffer from critical limitations such as loss of bioactivity upon the encapsulation process, requiring usage of different stabilizing agents18,48, as well as sophisticated and complex fabrication techniques16. One of the greatest challenges in designing delivery systems for proteins is the ability to preserve the bioactivity of the molecules upon entrapment within the carrier system. Proteins or peptides can be loaded into PSi/PSiO2 at RT or even at lower temperatures without using strong organic solvents, which are both important factors when loading these sensitive biomolecules. Previous studies have demonstrated that PSi/PSiO2 surface chemistry plays a crucial role in minimizing possible denaturation of the loaded proteins35,36. Therefore, PSi/PSiO2 is an advantageous nanomaterial for developing delivery systems for growth factors in general and NGF in particular.
The current work is focused on utilizing this method as a new therapeutic approach for direct administration of NGF into the CNS for potential treatment of neurodegenerative diseases. The NGF-loaded PSiO2 carriers can be implanted in mice brains and the efficacy of the platform as long-term implants is studied in vivo. Furthermore, combining this promising carriers with non-invasive biolistics49,50 may enable one to administer the NGF-loaded PSiO2 particles in a highly spatial resolution to a localized area using a novel pneumatic capillary gun for treating neurodegenerative disorders, where a spatiotemporal drug administration is required. Moreover, NGF can direct neuronal growth in a chemical gradient manner51, similar to axon guidance molecules. Thus, the loaded PSiO2 carriers can serve as attractant hot spots to NGF, to direct growth, complementary to other directing cues52,53. In addition, the PSiO2carriers can be specifically tailored to sustain the delivery of NGF for a much-extended time period of up to several months by further tuning the PSiO2 nanostructure and its surface chemistry.
The authors have nothing to disclose.
MS and ES acknowledge the core services and support from the Lorry I. Lokey Center for Life Science and Engineering and the financial support of the Russell Berrie Nanotechnology Institute at the Technion.
Acetone | Gadot | 830101375 | |
Amphotericin | Biological Industries | 03-028-1B | |
Aqueous HF (48%) | Merck | 101513 | |
AZ4533 photoresist | Metal Chem, Inc. | AZ4533 | |
BSA fraction v | MP biomedicals | 0216006950 | |
BSA solution (10%) | Biological Industries | 03-010-1B | |
Collagen type l | Corning Inc. | 354236 | |
Collagenase | Enco | LS004176 | |
Collagen-coated plastic coverslips | NUNC Thermanox | 1059846 | |
D-(+)-glucose | Sigma-Aldrich Chemicals | G8170 | |
Dispase-II | Sigma-Aldrich Chemicals | 4942078001 | |
Donkey anti mouse IgG H&L conjugated Alexa Fluor 488 | Abcam | ab150073 | |
Ethanol absolute (99.9%) | Merck | 818760 | |
FBS | Biological Industries | 04-121-1A | |
Formaldehyde/glutaraldehyde (2.5%) in 0.1 M sodium cacodylate | Electron Microscopy Sciences | 15949 | |
Freon | Sigma-Aldrich Chemicals | 613894 | |
Guanidine-HCl | Sigma-Aldrich Chemicals | G7294 | |
Ham's F-12 nutrient mixture | Thermo Scientific | 11765054 | |
HBSS | Thermo Scientific | 14185-045 | |
HEPES (1M) | Thermo Scientific | 15630-056 | |
HS | Biological Industries | 04-124-1A | |
Human β-NGF ELISA Development Kit | Peprotech | 900-K60 | |
Immumount solution | Thermo Scientific | 9990402 | |
L-15 medium | Sigma-Aldrich Chemicals | L5520 | |
Laminin | Thermo Scientific | 23017015 | |
L-glutamine | Biological Industries | 03-020-1A | |
Mouse anti neurofilament H (NF-H) (phosphorylated antibody) antibody | BiolLegend | SMI31P | |
Murine β-NGF | Peprotech | 450-34-20 | |
Normal donkey serum (NDS) | Sigma-Aldrich Chemicals | G9023 | |
Papain | Sigma-Aldrich Chemicals | p-4762 | |
Paraformaldehyde 16% solution | Electron Microscopy Sciences | BN15710 | |
PBS (pH 7.4) | prepared by dissolving 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl in double-distilled water (ddH2O, 18 MΩ). |
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PBS X10 | Biological Industries | 02-020-1A | |
PC12 cell line | ATCC | CRL-1721 | |
Penicillin–streptomycin | Biological Industries | 03-032-1B | |
Percoll | Sigma-Aldrich Chemicals | p1644 | |
Poly-L-lysine | Sigma-Aldrich Chemicals | P4832 | |
PrestoBlue reagent | Thermo Scientific | A13261 | |
RPMI medium | Biological Industries | 01-100-1A | |
Si wafer | Siltronix Corp. | Highly-B-doped, p-type, 0.00095 Ω-cm resistivity, <100> oriented | |
Sodium azide | Sigma-Aldrich Chemicals | S2002 | |
Sodium hydroxide (NaOH) | Sigma-Aldrich Chemicals | S8045 | |
Tannic acid | Sigma-Aldrich Chemicals | 403040 | |
Triton X-100 | Chem-Impex International Inc. | 1279 |