NGF delivered to the brain is challenging. Here we present a new type of carrier that allows a safe and prolonged release of the bioactive protein. This technique allows a simple and efficient loading of NGF into porous silicon carriers.
It is carried out at room temperature and without using strong organic solvents. As the porous silicon carrier design is tunable the carriers can serve as NGF reservoir implants for a long term release but can also be modified to carry other factors and drugs. For engineers and chemists, the fabrication process may be easier to perform.
While for life scientists and clinicians, the cell culture procedures should be relatively straightforward to reproduce. Begin by mounting a one point five by one point five centimeter silicon wafer with a well characterized resistivity into a polytetrafluro ethaline etching cell using a strip of aluminum foil as a back contact and an o-ring to seal the cell. The fabric is represistable for use silicon wafers with similar resistivity values each time.
Electrochemically etch the silicon sample in a three to one solution of akleus hydrofluoric acid and ethanol for 20 seconds at a constant current density of 250 milliamps per centimeter squared. Then rinse the surface of the resulting porous silicon film with ethanol three times before drying under a nitrogen stream. When the sample is dry, thermally oxidize the freshly etched porous silicon sample in the tube furnace at 800 degrees Celsius for one hour in ambient air to form an oxidized porous silicon scaffold.
When the sample has cooled, use a dicing saw to cut it into an eight by eight millimeter sample. To load the samples with NGF, dispense 52 microliters of freshly prepared NGF loading solution on top of each sample and incubate the samples for two hours at room temperature in a covered dish under high humidity conditions. After two hours, collect the solution on top of the samples and dilute NGF loading solution and the collected post loading solution samples to the appropriate concentration range for the iliza kit.
When all of the samples have been diluted, add 100 microliters of the diluted samples in calibration curve samples to each well of an NGF capture antibody coated 96 well iliza plate for a two hour incubation at room temperature. At the end of the incubation, wash the plate four times and add one hundred microliters of one microgram per milliliter of biotentilated detection antibody to each well for a two hour incubation at room temperature. After four washes, as demonstrated add 100 microliters of Avidin-Horseradish Peroxidace to each well for a 30 minute incubation at room temperature followed by four washes in wash buffer as demonstrated.
After the last wash, add 100 microliters of substrate solution to each well for a 25 minute color development incubation at room temperature. Then use a microplate reader to measure the absorbance at 405 nanometers with the wave length correction set at 650 nanometers and determine the NGF concentration in both the loading solution and the collected post loading solution based on the calibration curve. For In Vitro NGF release, incubate the NGF loaded oxidized porous silicon carriers in two milliliters of zero point zero one molar PBS supplemented with one percent boyvine serum albaman in zero point zero two percent sodium azide at 37 degrees Celsius and 100 rotations per minute of orbital adjatation.
Collect the solution every two days replacing it with two milliliters of fresh PBS after each aspiration. Then freeze the collected release samples in liquid nitrogen for minus 20 degrees Celsius storage until NGF analysis by iliza as just demonstrated. To generate a differentiated feo cromo sitoma 12 or PC 12, cell culture, collect the cell suspension by sentripugation and re suspend the cells in five milliliters of fresh basic growth medium.
After a second sentribugation, re suspend the palate in three milliliters of basic growth medium and use a 23 gauge syringe to aspirate the cells 10 times to separate any cell clusters. After counting, seed one times 10 to the fourth cells per centimeter squared working area on a colligen type one coated plates in the presence of differentiation medium. Place the plates in the incubator.
After 24 hours at 37 degrees Celsius add fresh murine beta NGF or NGF loaded porous silicon carrier to each plate and return the plates to the incubator. To evaluate the cell viability, incubate the cultures with 10 percent of an appropriate viability indicator solution at representative time points for five hours at 37 degrees Celsius and measure the absorbance on a spectropetometer at 490 nanometers with the wave length correction set at 630 nanometers. To assess PC 12 cell differentiation in the presence of NGF loaded porous silicon carriers, seed the PC 12 cells in 12 low collagen coated plates for 24 hours in a cell culture incubator and introduce the previously obtained In Vitro released samples to the appropriate experimental wells.
After a 24 hour incubation, image the cells by light microscopy and manually count the number of cells with alkronurites in each well. For morphrometric analysis of the differentiated PC 12 cells acquire phase images of the cultured cells up to three days after treatment with NGF and open the files in neuron J.Convert the image file to an eight bit format and use the image scale bar to measure the pixel to micrometer ratio. Use analyze and set scale to set the scale and to measure the length of each neurite.
Then manually count the number of branching points and the number of nourites originating from each cell soma. High resolution scanning electron microscopy images of the resulting oxidized porous silicon film are shown. A top view micrograph of the film reveals its highly porous nature with pores of approximately 40 nanometers in diameter.
A cross sectional micrograph of a cleaved film reveals a porous layer thickness of two point nine micrometers that is characterized by interconnecting cylindrical pores. A sustained release of NGF without burst effect is attained for a period of one month with a faster NGF release during the first week compared to a much slower release rate in the later days of the release period. Treatment with NGF loaded porous silicon carriers induces the formation of neurites in characteristic branched neuronal networks in PC 12 cell cultures.
Note that even after 26 days, the released NGF induces cell differentiation of above 50 percent indicating that NGF entrapment within the porous host preserves the biological activity of the protein for a period of about one month. Morphometric analysis of the NGF loaded porous silicon treated cell populations at days one and three reveals that PC 12 cells treated with NGF loaded porous silicon exhibit morphometric values similar to those observed in the controlled treatment cultures for all three parameters tested. Following and fulfilling adequately the presented steps will provide you with an advantageous NGF delivery system capable of sustaining the survival and differentiation of neuronal cells.
We are currently investigating the use of NGF porous silicon carriers as long term releasing implants in the brain to study their normal protective effect and know degenerative diseases such as Alzheimer's disease. Following this procedure, the effect of the NGF loaded porous silicon carriers on a normal growth of dosal wood gangrene cells can also be studied. Porous silicon is a promising nano material that can be utilized for a wide range of applications.
Other than the one presented here, including biological or chemical sensors, diagnostics, and imaging. To rule out any potential toxicity of the porous silicon carriers, towards your cell line, be sure to perform severability essay and sterilize your porous silicon samples.
