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.

All methods have been approved by the Ethics Committee of Bar Ilan University.
1. Fabrication of Oxidized PSi (PSiO2) Carriers
<ol>
	<li>Cut a Si wafer (single side polished on the &#60;100&#62; face and heavily Boron-doped, p-type, 0.95 m&#937;&#183;cm) into 1.5 cm &#215; 1.5 cm samples using a diamond-tipped pen.</li>
	<li>Oxidize the Si samples in a tube furnace at 400 &#176;C for 2 h in ambient air (heating rate: 25 &#176;C/min, natural cooling).</li>
	<li>Immerse the Si samples in a solution of aqueous hydrofluoric acid (HF) (48%), ddH2O and ethanol (99.9%) (1:1:3 v/v/v) for 5 min; then rinse the samples with ethanol three times and dry under a nitrogen stream. 
	NOTE: Prepare and store HF solution in plasticware only, as HF dissolves glass. 
	CAUTION: HF is a highly corrosive liquid, and it should be handled with extreme care. In case of exposure, rinse thoroughly with water and treat the affected area with HF antidote gel; seek for medical care immediately.</li>
	<li>Mount the Si sample in a polytetrafluoroethylene etching cell, using a strip of aluminum foil as a back-contact and a platinum coil as the counter electrode.</li>
	<li>Etch a sacrificial layer in a 3:1 (v/v) solution of aqueous HF and ethanol (99.9%) for 30 s at a constant current density of 250 mA/cm2; then rinse the surface of the resulting PSi film with ethanol three times and dry under a nitrogen stream.</li>
	<li>Dissolve the freshly-etched porous layer in an aqueous NaOH solution (0.1 M) for 2 min. Then rinse with ethanol three times and dry under a nitrogen stream.</li>
	<li>Immerse the sample in a solution of aqueous HF (48%), ddH2O and ethanol (99.9%) (1:1:3 v/v) for 2 min. Then rinse with ethanol three times and dry under a nitrogen stream.</li>
	<li>Electrochemically etch the Si sample in a 3:1 (v/v) solution of aqueous HF and ethanol (99.9%) for 20 s at a constant current density of 250 mA/cm2; then rinse the surface of the resulting PSi film with ethanol three times and dry under a nitrogen stream.</li>
	<li>Thermally oxidize the freshly-etched PSi samples in a tube furnace at 800 &#176;C for 1 h in ambient air (heating rate: 25 &#176;C/min, natural cooling) to form a porous SiO2 (PSiO2) scaffold.</li>
	<li>Spin-coat the PSiO2 samples with a positive thick photoresist at 4,000 rpm for 1 min; then bake the coated samples at 90 &#176;C for 2 min (heating rate: 5 &#176;C/min, natural cooling).</li>
	<li>Dice the PSiO2 samples into 8 mm &#215; 8 mm samples using a dicing saw.</li>
	<li>To remove the photoresist, soak the diced samples in acetone for 3 h; then thoroughly rinse with ethanol and dry under a nitrogen stream.</li>
</ol>
2. Loading PSiO2 with NGF
<ol>
	<li>To prepare the NGF loading solution, dissolve 20 &#181;g of murine &#946;-NGF in 400 &#181;L of 1:1 (v/v) solution of 0.01 M phosphate-buffered saline (PBS) and ddH2O.</li>
	<li>Add 52 &#181;L of the loading solution on top of the PSiO2 sample and incubate for 2 h at RT in a capped dish. 
	NOTE: Maintain high humidity in the dish to prevent drying up of the solution during incubation.</li>
	<li>Collect the solution on the top of the sample for subsequent quantification of NGF content within the PSiO2 carrier. 
	NOTE: NGF loading into PSiO2 should be performed immediately before the intended use, the protocol cannot be paused here due to the risk of drying and denaturation of the protein.</li>
</ol>
3. Quantification of NGF loading by NGF ELISA
<ol>
	<li>To prepare the ELISA plate, add 100 &#181;L of 0.5 &#181;g/mL capture antibody (rabbit anti-h&#946;-NGF) to each well, seal the plate and incubate overnight at RT.</li>
	<li>Aspirate the wells to remove liquid and wash the plate four times using 300 &#181;L of wash buffer (0.05% Tween-20 in PBS) per well; after the last wash invert the plate to remove residual buffer and blot on a paper towel.</li>
	<li>Add 300 &#181;L of block buffer (1% bovine serum albumin (BSA) in PBS) to each well and incubate for at least 1 h at RT. Then aspirate and wash the plate four times as described in step 3.2.</li>
	<li>To prepare a calibration curve, dilute NGF loading solution (see step 2.1) in diluent (0.05% Tween-20, 0.1% BSA in PBS) to 1 ng/mL. Then perform 2-fold serial dilutions from 1 ng/mL to zero to obtain the calibration curve samples.</li>
	<li>To prepare the samples, dilute the NGF loading solution (from step 2.1) and the collected post-loading solution (from step 2.3) in diluent 1:100,000 and 1:10,000 respectively, to reach the concentrations range of the ELISA kit in use (16-1,000 pg/mL).</li>
	<li>Add 100 &#181;L of the diluted samples and calibration curve samples to each well in triplicate and incubate at RT for 2 h; then aspirate and wash the plate four times as described in step 3.2.</li>
	<li>Add 100 &#181;L of 1 &#181;g/mL detection antibody (biotinylated rabbit anti-h&#946;-NGF) to each well and incubate at RT for 2 h. Then aspirate and wash the plate four times as described in step 3.2.</li>
	<li>Dilute Avidin-Horseradish Peroxidase (HRP) 1:2,000 in diluent, add 100 &#181;L per well and incubate for 30 min at RT. Now aspirate and wash the plate four times as described in step 3.2.</li>
	<li>Add 100 &#181;L of substrate solution to each well and incubate 25 min at RT for color development. Measure the absorbance at 405 nm with wavelength correction set at 650 nm using a microplate reader.</li>
	<li>Determine NGF concentration in both the loading solution and the collected post-loading solution based on the calibration curve.</li>
	<li>To calculate NGF mass loaded within PSiO2 carrier, subtract NGF mass in the collected post-loading solution from the NGF mass in loading solution. NGF loading efficacy is calculated by the following equation: 
	<img alt="Equation" src="/files/ftp_upload/58982/58982eq1.jpg" /></li>
</ol>
4. In vitro NGF Release from PSiO2
<ol>
	<li>Incubate the NGF-loaded PSiO2 carriers in 2 mL of 0.01 M PBS containing 1% (w/v) BSA and 0.02% (w/v) sodium azide at 37 &#176;C and under orbital agitation of 100 rpm.</li>
	<li>Every 2 days collect the solution and replace it with 2 mL of fresh PBS. Freeze the collected release samples in liquid nitrogen and store at -20 &#176;C for further analyses.</li>
	<li>Use the commercially available NGF ELISA assay to quantify NGF content in the release samples as described in steps 3.1-3.9. 
	NOTE: When preparing the release samples for ELISA quantification, different dilutions should be performed for different time points along the release period (i.e., earlier time points should be diluted much more than later time points). Moreover, for each release sample, at least two different dilutions should be performed and quantified to ensure reaching the concentrations range of the ELISA kit.</li>
	<li>Determine NGF concentration in the release samples based on the calibration curve and plot a graph of accumulative NGF release over time.</li>
</ol>
5. Quantification of in vitro Si Erosion by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)
<ol>
	<li>Dilute 1 mL of release samples 1:10 in release buffer (0.01 M PBS containing 1% (w/v) BSA and 0.02% (w/v) sodium azide) for a total volume of 10 mL.</li>
	<li>Monitor Si atomic emission peaks at 212.4, 251.6 and 288.2 nm for the diluted samples.</li>
	<li>Determine Si concentration in the samples based on a calibration curve.</li>
	<li>To establish the Si degradation profile, express Si content at each time point as a percentage of the total Si content of the carriers (i.e., the cumulative Si content along the release period). 
	NOTE: To ensure accurate quantification of the total Si content of the carriers, release samples are sampled 1 month following the end-point of the release study and analyzed for Si concentration using ICP-AES. Summing the cumulative Si content along the release period and the Si content in the post-release samples provides the 100% of Si content.</li>
</ol>
6. Cell Viability and Growth in the Presence of NGF-Loaded Psio2 Carriers
<ol>
	<li>Rat pheochromocytoma (PC12) cell culture
	<ol>
		<li>Prepare the basic growth medium by adding 10% horse serum (HS), 5% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin streptomycin and 0.2% amphotericin to Roselle Park Medical Institute (RPMI) medium.</li>
		<li>Prepare differentiation medium by adding 1% HS, 1% L-glutamine, 1% penicillin streptomycin and 0.2% amphotericin to RPMI medium.</li>
		<li>Grow cell suspension (106 cells) in a 75 cm2 culture flask with 10 mL of basic growth medium for 8 days; every 2 days add 10 mL of basic growth medium to the flask.</li>
		<li>To generate differentiated PC12 cell culture, transfer the cell suspension to a centrifuge tube; centrifuge cells for 8 min at 200 x g and RT. Discard the supernatant.</li>
		<li>Suspend the cells in 5 mL of fresh basic growth medium and re-centrifuge the cells for 5 min at 200 x g and RT; discard the supernatant and resuspend the cell pellet in 3 mL of basic growth medium.</li>
		<li>To separate cell clusters, aspirate the cells ten times using a 23 G syringe.</li>
		<li>Count the cells using a hemocytometer cell counter and seed 104 cells/cm2 working area on collagen type l coated plates in the presence of differentiation medium.</li>
		<li>After 24 h, add fresh murine &#946;-NGF (50 ng/mL) or NGF-loaded PSiO2 carrier per plate. 
		NOTE: Higher NGF concentrations (&#62;50 ng/mL) possess the exact effect as the specified concentration.</li>
		<li>Renew the differentiation medium every 2 days.</li>
		<li>To evaluate cell viability, add 10% (v/v) of the viability indicator solution (resazurin-based) at representative time points and incubate for 5 h at 37 &#176;C; measure the absorbance at 490 nm using a spectrophotometer.</li>
	</ol>
	</li>
	<li>Mice Dorsal Root Ganglia (DRG) Cell Culture
	<ol>
		<li>To isolate DRGs from two 7-week-old C57bl mice, first douse the mice with 70% ethanol and make an incision to remove the skin.</li>
		<li>Cut the base of the skull and the abdominal wall muscles till the spinal cord is exposed.</li>
		<li>Remove the spinal column by making a cut at the level of the femurs and clean the surrounding tissues.</li>
		<li>Cut the column into three pieces; then cut each piece in the midline to generate two halves and peel out the spinal cord.</li>
		<li>Under the binocular, extract all DRG ganglia and immerse them in a cold Hank&#39;s balanced salt solution (HBSS).</li>
		<li>Clean the surrounding connective tissues by pining the DRGs on a petri dish and gently remove residual meninges under the binocular.</li>
		<li>Dissociate the DRG cells by incubating them for 20 min in 1,000 U of papain.</li>
		<li>Centrifuge the cells for 4 min at 400 x g. Discard the supernatant and keep the cell pellet.</li>
		<li>Resuspend the pellet in a 10 mg/mL collagenase and 12 mg/mL dispase-II solution and incubate for 20 min at 37 &#176;C.</li>
		<li>Centrifuge the cells for 2 min at 400 x g; discard the supernatant and keep the cell pellet.</li>
		<li>Triturate the cell pellet in 1 mL of HBSS, 10 mM glucose and 5 mM HEPES (pH 7.35) by repeated passage through a constricted Pasteur pipette.</li>
		<li>Gently add the dissociated cells to L-15 medium containing 20% of silica-based colloidal medium for cell separation by density gradient centrifugation; centrifuge for 8 min at 1,000 x g. Aspirate the supernatant and keep the cell pellet. 
		NOTE: The aim of this step is to separate and purify the neuronal cells from axonal debris, Schwann cells and fibroblasts.</li>
		<li>Resuspend the cell pellet in 2 mL of F-12 medium; centrifuge for 2 min at 1,000 x g; discard the supernatant and resuspend the pellet in 1 mL of F-12 medium.</li>
		<li>Count the cells and seed 2 &#215; 104 cells on poly-L-lysine and laminin coated glass bottom 35 mm culture dishes, in a F-12 antibiotic-free medium supplemented with 10% FBS. 
		NOTE: Initially, seed the cells in a small volume of medium (150-200 &#181;L); following 2 h incubation at 37 &#176;C, add medium for a final volume of 2 mL.</li>
		<li>Gently add the NGF-loaded PSiO2 carrier or fresh murine &#946;-NGF (50 ng/mL) per plate.</li>
		<li>After 2 days, fix the cell culture by incubating it with a 4% paraformaldehyde (PFA) solution for 15 min at RT; immunostain the cell culture using a common immunofluorescence staining procedure31.</li>
		<li>Image the neuronal cell culture using a confocal microscope.</li>
	</ol>
	</li>
</ol>
7. Cell Differentiation Analysis
<ol>
	<li>PC12 cells differentiation percentage in the presence of NGF-loaded PSiO2 carriers
	<ol>
		<li>Incubate the NGF-loaded PSiO2 carriers in 2 mL of differentiation medium, in a humidified incubator at 37 &#176;C containing 5% CO2.</li>
		<li>Every 2 days, collect the solution and replace it with 2 mL of fresh medium. Freeze the collected release samples in liquid nitrogen and store at -20 &#176;C for further analyses.</li>
		<li>Seed PC12 cells in 12-well collagen-coated plates as instructed in section 4.1 (4.1.3-4.1.6).</li>
		<li>After 24 h, introduce the release samples of representative time points (from section 5.1.2) to the different wells; for control wells, add fresh murine &#946;-NGF (50 ng/mL).</li>
		<li>After 24 h, image the cells using a light microscope.</li>
		<li>To determine the percentage of neurite-bearing cells, manually count cells that had outgrown neurites out of the total number of cells in the images (n = 5 frames for each well); plot a graph of percentage of PC12 cells with neurite outgrowth over time. 
		NOTE: Undifferentiated PC12 cells appeared to have rounded structure without neurites, while differentiated cells can be identified by outgrowth of neurites (at least one of minimum length equal to the cell soma diameter) or morphological changes.</li>
	</ol>
	</li>
	<li>Morphometric analysis of differentiated PC12 cells
	<ol>
		<li>For image processing analysis, acquire phase images of cultured cells up to 3 days after treatment with NGF (as free reagent or as the release product of the NGF-loaded PSiO2 carriers). 
		NOTE: At later time points (beyond day 5) the cells develop highly complex networks, preventing morphometric measurements at a single cell resolution.</li>
		<li>Download the image processing program NeuronJ, an ImageJ plug-in, which enables a semi-automatic neurite tracing and length measuring.</li>
		<li>Convert the image file to an 8-bit format and measure pixel to micrometer ratio using the image scale bar.</li>
		<li>Set the scale using the Analyze | Set Scale command and measure the length of each neurite.</li>
		<li>Manually count the number of branching points and the number of neurites originating from each cell soma. 
		NOTE: An average neurite length 1 day after NGF treatment is approximately 30 &#181;m. The measured neurite lengths may be widely distributed.</li>
	</ol>
	</li>
</ol>

The authors declare no competing financial interests.

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.&#160;Moreover,&#160;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.

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&#39;s and Parkinson&#39;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.

<table><tbody><tr><td>Acetone</td><td>Gadot</td><td>830101375</td><td></td></tr><tr><td>Amphotericin</td><td>Biological Industries</td><td>03-028-1B</td><td></td></tr><tr><td>Aqueous HF (48%)</td><td>Merck</td><td>101513</td><td></td></tr><tr><td>AZ4533 photoresist</td><td>Metal Chem, Inc.</td><td>AZ4533</td><td></td></tr><tr><td>BSA fraction v</td><td>MP biomedicals</td><td>0216006950</td><td></td></tr><tr><td>BSA solution (10%)</td><td>Biological Industries</td><td>03-010-1B</td><td></td></tr><tr><td>Collagen type l</td><td>Corning Inc.</td><td>354236</td><td></td></tr><tr><td>Collagenase</td><td>Enco</td><td>LS004176</td><td></td></tr><tr><td>Collagen-coated plastic coverslips</td><td>NUNC Thermanox</td><td>1059846</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Sigma-Aldrich Chemicals</td><td>G8170</td><td></td></tr><tr><td>Dispase-II</td><td>Sigma-Aldrich Chemicals</td><td>4942078001</td><td></td></tr><tr><td>Donkey anti mouse IgG H&amp;amp;L conjugated Alexa Fluor 488</td><td>Abcam</td><td>ab150073</td><td></td></tr><tr><td>Ethanol absolute (99.9%)</td><td>Merck</td><td>818760</td><td></td></tr><tr><td>FBS</td><td>Biological Industries</td><td>04-121-1A</td><td></td></tr><tr><td>Formaldehyde/glutaraldehyde (2.5%) in 0.1 M sodium cacodylate</td><td>Electron Microscopy Sciences</td><td>15949</td><td></td></tr><tr><td>Freon</td><td>Sigma-Aldrich Chemicals</td><td>613894</td><td></td></tr><tr><td>Guanidine-HCl</td><td>Sigma-Aldrich Chemicals</td><td>G7294</td><td></td></tr><tr><td>Ham&#039;s F-12 nutrient mixture</td><td>Thermo Scientific</td><td>11765054</td><td></td></tr><tr><td>HBSS</td><td>Thermo Scientific</td><td>14185-045</td><td></td></tr><tr><td>HEPES (1M)</td><td>Thermo Scientific</td><td>15630-056</td><td></td></tr><tr><td>HS</td><td>Biological Industries</td><td>04-124-1A</td><td></td></tr><tr><td>Human &amp;beta;-NGF ELISA Development Kit</td><td>Peprotech</td><td>900-K60</td><td></td></tr><tr><td>Immumount solution</td><td>Thermo Scientific</td><td>9990402</td><td></td></tr><tr><td>L-15 medium</td><td>Sigma-Aldrich Chemicals</td><td>L5520</td><td></td></tr><tr><td>Laminin</td><td>Thermo Scientific</td><td>23017015</td><td></td></tr><tr><td>L-glutamine</td><td>Biological Industries</td><td>03-020-1A</td><td></td></tr><tr><td>Mouse anti neurofilament H (NF-H) (phosphorylated antibody) antibody</td><td>BiolLegend</td><td>SMI31P</td><td></td></tr><tr><td>Murine &amp;beta;-NGF</td><td>Peprotech</td><td>450-34-20</td><td></td></tr><tr><td>Normal donkey serum (NDS)</td><td>Sigma-Aldrich Chemicals</td><td>G9023</td><td></td></tr><tr><td>Papain</td><td>Sigma-Aldrich Chemicals</td><td>p-4762</td><td></td></tr><tr><td>Paraformaldehyde 16% solution</td><td>Electron Microscopy Sciences</td><td>BN15710</td><td></td></tr><tr><td>PBS (pH 7.4)</td><td></td><td></td><td>prepared by dissolving 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM&lt;br/&gt; NaCl and 2.7 mM KCl in double-distilled water (ddH2O, 18 M&amp;Omega;).</td></tr><tr><td>PBS X10</td><td>Biological Industries</td><td>02-020-1A</td><td></td></tr><tr><td>PC12 cell line</td><td>ATCC</td><td>CRL-1721</td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Biological Industries</td><td>03-032-1B</td><td></td></tr><tr><td>Percoll</td><td>Sigma-Aldrich Chemicals</td><td>p1644</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich Chemicals</td><td>P4832</td><td></td></tr><tr><td>PrestoBlue reagent</td><td>Thermo Scientific</td><td>A13261</td><td></td></tr><tr><td>RPMI medium</td><td>Biological Industries</td><td>01-100-1A</td><td></td></tr><tr><td>Si wafer</td><td>Siltronix Corp.</td><td></td><td>Highly-B-doped, p-type, 0.00095 &amp;Omega;-cm resistivity, &amp;lt;100&gt; oriented</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich Chemicals</td><td>S2002</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich Chemicals</td><td>S8045</td><td></td></tr><tr><td>Tannic acid</td><td>Sigma-Aldrich Chemicals</td><td>403040</td><td></td></tr><tr><td>Triton X-100</td><td>Chem-Impex International Inc.</td><td>1279</td><td></td></tr></tbody></table>

designing porous silicon films as carriers of nerve growth factor

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.

Oxidized PSi films are fabricated as described in the protocol. The Si wafer is subjected to electrochemical etching for 20 s at 250 mA/cm2 (Figure 1ai), followed by thermal oxidation at 800 &#176;C (Figure 1aii) to produce a PSiO2 scaffold. High-resolution scanning electron microscopy (HR-SEM) images of the resulting PSiO2 film are shown in Figure 1b, c. Top-view micrograph of the film (Figure 1b) depicts its highly porous nature with pores of approximately 40 nm in diameter. Cross-sectional micrograph of a cleaved film (Figure 1c) reveals a porous layer thickness of 2.9 &#181;m, characterized by interconnecting cylindrical pores.
The PSiO2 films are loaded with the NGF loading solution as illustrated in Figure 1aiii. NGF loading is quantified using NGF ELISA by measuring the NGF concentrations in the loading solution before and after incubation with the PSiO2 films. The average mass of the loaded NGF per PSiO2 film is determined as 2.8 &#177; 0.2 &#181;g, which corresponds to a high loading efficacy of 90% (w/w) (data not shown31). In order to establish the NGF release profile from the PSiO2 carriers, the loaded films are incubated in PBS at 37 &#176;C and every 2 days aliquots are sampled for quantification of the released protein concentration using NGF ELISA. In addition, the Si content in the release samples is quantified using ICP-AES and the Si erosion kinetics of the PSiO2 carriers is established. Figure 2 depicts the NGF release and the corresponding Si degradation profiles of the porous carriers. A sustained release of NGF, without burst effect, is attained for a period of 1 month. NGF release in the first week is faster (slope = 0.442) compared to a much slower release in later days along the release period (slope 0.043). It should be noted that throughout the entire 1-month release period, the amount of NGF released is sufficient for inducing profound differentiation of PC12 cells, as will be discussed later. The accumulative NGF released is found to be in a good correlation (R2 = 0.971) with the remaining Si content, as shown in the inset of Figure 2.
Next, the bioactivity of the NGF-loaded PSiO2 carriers is characterized in vitro, PC12 cells and DRG neurons are used as models for neuronal differentiation and outgrowth. PC12 cells and dissociated DRG neurons are seeded as described in the protocol. First, cell viability in the presence of the PSiO2 carriers is examined by incubating the cells with neat (not loaded) or NGF-loaded PSiO2; control plates and cells treated with neat PSiO2 are supplemented with free NGF every 2 days. No cytotoxicity is observed upon exposure of the cells to the neat or NGF-loaded PSiO2 carriers (Figure 3a), demonstrating that PSiO2 is biocompatible with this cell line. Figure 3b shows representative HR-SEM micrographs of PC12 cells treated with the NGF-loaded PSiO2 carriers. The observed cells extract neurites and form characteristic branched neuronal networks. Similar results are obtained when seeding dissociated DRG neuronal cells in the presence of the NGF-loaded PSiO2 carriers. Confocal images of immunostained DRG cells cultured with the NGF-loaded PSiO2 carriers (Figure 3e) show an extensive neurite initiation and elongation, similar to the positive control (i.e., neurons supplemented with free NGF, see Figure 3d), while the negative control (i.e., untreated neurons, see Figure 3c), exhibits a poor extent of neurite outgrowth.
To evaluate PC12 cells differentiation percentage in the presence of NGF released from the PSiO2 carriers, differentiation is quantified by counting the cells with neurite outgrowth out of the total cell population. Figure 4 summarizes the results in terms of the percentage of differentiated cells at different time points over a 26-day period. Significant differentiation percentage values are attained throughout the studied duration. Notably, after 26 days, the released NGF has induced differentiation of above 50%, a differentiation percentage which is significantly higher compared to previous studies with polymer-based carriers16. These results indicate that NGF entrapment within the porous host preserved the biological activity of the protein for inducing profound differentiation for a period of ~1 month.
To further examine the effect of NGF-PSiO2 carriers on neuronal differentiation, three characteristic morphological parameters of the differentiated PC12 cells are measured at the single cell level: (i) the number of neurites extracting from the soma (ii) the total neurites&#39; length and (iii) the number of branching points. The cells are treated with NGF-loaded PSiO2 carriers or with repetitive administration of free NGF (every 2 days), as a control. Figure 5a-c presents the morphometric analysis of the cell population at days 1 and 3. The PC12 cells treated with the NGF-loaded PSiO2 show morphometric values which are similar to the control treatment for all three tested parameters. After 3 days, the values of all morphological parameters are observed to increase at the same rate for both the NGF-loaded PSiO2 carriers and the control treatment of repetitive administration of free NGF.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58982/58982fig1.jpg" /> 
Figure 1: Fabrication of PSiO2 films. (a) Fabrication scheme of the PSiO2 carriers: (i) Silicon wafer is subjected to electrochemical etching for 20 s at 250 mA/cm2, followed by (ii) thermal oxidation at 800 &#176;C to produce a PSiO2 scaffold, and (iii) NGF loading by physical adsorption (schematics are not drawn to scale).(b-c) Top-view and cross-section electron micrographs of a characteristic PSiO2 film. <a href="https://www.jove.com/files/ftp_upload/58982/58982fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58982/58982fig2.jpg" /> 
Figure 2: NGF release and Si erosion profiles of NGF-loaded PSiO2 carriers. The extent of degradation of the PSiO2 carriers is presented as the fraction of the remaining Si content at each time point out of the total Si content of the porous film. The inset shows the correlation between the accumulative NGF released and remaining Si content. Error bars represent SD, n=3. <a href="https://www.jove.com/files/ftp_upload/58982/58982fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58982/58982fig3.jpg" /> 
Figure 3: Cell viability and growth in the presence of NGF-loaded PSiO2 carriers. (a) In vitro cytotoxicity characterization. The PC12 cells viability is measured on 1, 3 and 7 days following their exposure to neat (not loaded) PSiO2 carriers, NGF-loaded PSiO2 carriers or free NGF (50 ng/mL every 2 days, control plates). (b) Electron micrographs of PC12 cells cultured with NGF-loaded PSiO2 carriers for 9 days. Left panel: a zoom-in, depicting neurite outgrowth from the cell soma; right panel: a zoom-out, indicating the highly complex neuronal network formed. (c-e) Confocal microscopy images of immunostained DRG neuronal cell culture (2 days after seeding): (c) untreated cells; (d) cells supplemented with free NGF (50 ng/mL); (e) NGF-PSiO2 carriers. Immunofluorescent staining of neurofilament H enables imaging of the cell soma and the outgrowing neurites. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58982/58982fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58982/58982fig4.jpg" /> 
Figure 4: PC12 cells differentiation percentage values upon exposure to NGF released from NGF-loaded PSiO2 carriers at representative time points. Differentiation percentage is expressed as the number of cells with neurite outgrowth out of the total cell population. Control treatment refers to cells supplemented with free NGF (50 ng/mL). Representative light microscopy images of the cells at the different time points are depicted along the x-axis; scale bar = 25 &#181;m&#160;for micrographs of days 2, 8, 14, 26 and 50 &#181;m for control micrograph. Error bars represent SD, n=3. <a href="https://www.jove.com/files/ftp_upload/58982/58982fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58982/58982fig5.jpg" /> 
Figure 5: Morphometric analysis of differentiated PC12 cells. Three morphological parameters of differentiated PC12 cells are measured, at the single cell level, at day 1 and 3 following exposure to NGF-loaded PSiO2 carriers or repetitive administration of free NGF every 2 days (control); (a) total neurites&#39; length (b) number of neurites extracting from the cell soma and (c) number of branching points. Error bars represent SD, n=3. <a href="https://www.jove.com/files/ftp_upload/58982/58982fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Designing Porous Silicon Films as Carriers of Nerve Growth Factor at JoVE.com

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

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 ...

designing-porous-silicon-films-as-carriers-of-nerve-growth-factor

Research

JoVE Journal

Bioengineering

9.7K Views.  Technion - Israel Institute of Technology. NGF geleverd aan de hersenen is een uitdaging. Hier presenteren we een nieuw type drager die een veilige en langdurige afgifte van het bioactieve eiwit mogelijk maakt. Deze techniek maakt een eenvoudige en efficiënte belasting van NGF in poreuze silicium dragers.Het wordt uitgevoerd bij kamertemperatuur en zonder gebruik te maken van sterke organische oplosmiddelen. Aangezien de poreuze silicium drager ontwerp is tunable de dragers kunnen dienen als NGF reservoir implantaten voor een ...

Video: Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biology

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle&#8217;s size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.

We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...

Engineering

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Neuroscience

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation durations. A substantial contributor to the observed decline is the damage to the neural tissue proximal to the implant and subsequent neuroinflammatory response. Efforts to improve device longevity include chemical modifications or coating applications to the device surface to improve the tissue response. Development of such surface treatments is typically completed using non-functional "dummy" probes that lack the electrical components required for the intended application. Translation to functional devices requires additional consideration given the fragility of intracortical microelectrode arrays. Handling tools greatly facilitate surface treatments to assembled devices, particularly for modifications that require long procedural times. The handling tools described here are used for surface treatments applied via gas-phase deposition and aqueous solution exposure. Characterization of the coating is performed using ellipsometry and x-ray photoelectron spectroscopy. A comparison of electrical impedance spectroscopy recordings before and after the coating procedure on functional devices confirmed device integrity following modification. The described tools can be readily adapted for alternative electrode devices and treatment methods that maintain chemical compatibility.

The present protocol describes tools for handling silicon planar intracortical microelectrodes during treatments for surface modification via gas deposition and aqueous solution reactions. The assembly of the components used to handle the devices throughout the procedure is explained in detail.

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

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