The overall goal of the following experiment is to produce pH responsive nanosensors and self-reporting scaffolds that can be monitored in real time and in situ in order to assist with the understanding of conditions that influence cellular growth. This is achieved by first preparing biocompatible analyte responsive nanosensors, which can monitor pH through fluorescence outputs. As a second step, pH responsive nanosensors are incorporated into polymeric electro spun scaffolds forming a 3D environment upon which cells can be cultured and monitored.
Next mammalian cells are seated on the electros spun scaffolds and nanosensors are also delivered into their intracellular environment in order to monitor intracellular pH. During experimentation results are obtained that show the ability of intracellular nanosensors and self-reporting scaffolds to monitor pH within tissue engineered environments based on fluorescence ratios acquired by confocal microscopy. The main advantage of this technique over existing methods, such as the use of electronic pH probes, is that measurements can be performed in situ and in real time without having to disturb cellular growth.
To begin prepare the fluorophores by dissolving 1.5 milligrams of fam SE into one milliliter of dimethylformamide known as DMF in a round bottom. Flask also dissolve 1.5 milligrams of Tamara SE into one milliliter of DMF in a second flask. Then add 1.5 milliliters of three amino propyl triethyl to each flask and stir continuously in the dark under a dry nitrogen atmosphere at 21 degrees Celsius for 24 hours.
Next, add 250 microliters of both dye solutions to a mixture containing six milliliters of ethanol and four milliliters of ammonium hydroxide contained in a round bottom flask. Stir this new mixture at 21 degrees Celsius after one hour. Slowly add 0.5 milliliters of tetraethyl orthos silicate to the mixture, and continue stirring it for another two hours.
During this time, expect the mixture to turn cloudy, then collect the nanosensors by rotary evaporation and store them in a glass vial at four degrees Celsius for future use. Next, add the dried pH nanosensors at five milligrams per milliliter in sorenson's phosphate buffers, ranging from pH 5.5 to pH 7.5 in increments of pH 0.5. Resuspend the nanosensors by vortexing the samples for three minutes and then sonicate them for five minutes or until the solution becomes cloudy.
Collect calibration data by measuring the samples at an excitation wavelength of 488 nanometers for FAM and 568 nanometers. For Tamara, collect the emissions at 500 to 530 nanometers for FAM and 558 to 580 nanometers for Tamara, and calculate the ratio of emission maxima produced at each pH value. Then prepare the sample for SEM imaging by placing a sample of nanosensors onto a carbon coated electron microscope, stub and sputter coating the particles with gold for five minutes under an argon atmosphere.
Once coated, place the samples into the electron microscope and adjust the working distance, voltage, and magnification to minimize electron charging and produce the best images in a fume hood. Make a 20%PLGA solution in di chloro methane with 1%perineum formate. Load the solution into a 10 milliliter syringe with an 18 gauge blunt fill needle and securely fit it on a syringe pump.
This solution will be used to make control PLGA scaffolds distance the needle tip 20 centimeters away from a 20 by 15 centimeter aluminum collecting plate, and attach the electrode of a high voltage power supply to the tip of the syringe and ground the wire to the aluminum collecting plate. Next, turn on the power supply and adjust the voltage to 12 kilovolts. Then turn on the syringe pump and deliver the solution using a constant flow rate of 3.5 milliliters per hour for two hours.
This will produce a scaffold depth of approximately 60 microns. Once finished, turn the equipment off and leave the scaffold in the fume hood for 24 hours to allow the solvent residue to evaporate. To prepare scaffolds incorporated with pH responsive nanosensors, prepare the PLGA solution as before, but this time also add five milligrams per milliliter of the nanosensors.
Then electro spin the pH responsive using the same settings as before. Next, calibrate the pH responsiveness of the scaffold by placing small samples of the dried scaffold with incorporated nanosensors into 35 millimeter culture plates and immersing them in two milliliters of sorensen phosphate buffers between pH 5.5 and 7.5 in increments of 0.5 on a confocal microscope. Use a 488 nanometer argon laser to excite FAM and a 568 nanometer krypton laser to excite Tamara within the nanosensors.
Observe the emission at 500 to 530 nanometers for FAM and 558 to 580 nanometers for Tamara, and then calculate the ratio of emission maxima produced at each pH value. First, sterilize the surfaces of the scaffolds with a UV light source at a distance of eight centimeters for 15 minutes on each side. Then sterilely transfer the scaffolds to a 12 well culture plate and place a steel ring with an inner diameter of one centimeter and outer diameter of two centimeters over the scaffold.
Next, add PBS with 5%pen strip to the inside and outside of the steel ring and incubate the samples overnight. The next day. Remove the PBS with 5%pen strep and wash the scaffolds with PBS.
Then add 500 microliters of cell culture media to the inside and outside of the steel ring and prewarm the plate in an incubator. Next, harvest the cells of interest and prepare a cell suspension of one times 10 to the six cells per milliliter. When the cells are ready, remove the media from the well and add one milliliter of fresh media to the outside of the steel ring.
Then add 300 microliters of the cell suspension to the inside of the steel ring and gently rock the plate to evenly distribute the cells. Place the plate into a 37 degree Celsius incubator with 5%CO2, and incubate the cells for as long as required. Refreshing the media every two to three days seed cells onto the scaffolds as described in the previous section, and allow them to grow uninterrupted for 72 hours.
Then rinse the cells with PBS and add 500 microliters of serum free media to the outside of the ring and 300 microliters inside the ring. Incubate the plate for one hour at 37 degrees Celsius during incubation. Prepare solution A by adding five milligrams of the nanosensors with 50 microliters of optimum and briefly fornicating.
Then mixed solution B by adding five microliters of lipectomy 2000 to 45 microliters of Optum, and allow the mixture to incubate at room temperature for five minutes. Next, add solution A to solution B mix and then incubate at room temperature for 20 minutes to form solution C, which is the nanosensor liposome complex. Remove the plate from the incubator and add 100 microliters of the complex solution to the inside of the steel ring.
Then place the plate back into the incubator and incubate the cells with the lipectomy reagent complexes for three hours following incubation. Aspirate the media and wash the cells twice with warmed PBS before immersing the cell seated scaffold into buffers of different phs to monitor the response of the nanosensors. Now inside the cells, image the cells using fluorescence confocal microscopy to track the location of the nanosensors within mammalian cells.
First, prepare and deliver only fam containing nanosensors to cells seated on PLGA scaffolds. Then add two microliters of 50 nano molar lyo tracker red to 300 microliters of media and incubate for one hour at 37 degrees Celsius following the incubation period. Remove the media and wash the cells twice with PBS.
Then add either two microliters of PBS or other phenol red free media to cover the cells during confocal imaging. Next, add five micromolar of the nuclear stain, drac five to the PBS and incubate with the cells for three minutes at 37 degrees Celsius. After three minutes, remove the plate from the incubator and place it onto the confocal stage for imaging image, the nanosensors acidic organelles and nucleus using various excitation and emission wavelengths as described in the accompanying text protocol while using sequential scanning to avoid collection of excitation wavelengths, the size distribution of the prepared pH responsive nanosensors was characterized using SEM where the population of nanosensors imaged were measured and found to have nanometer dimensions in the range of 240 to 470 nanometers.
Shown on the left is an image of electros spun PLGA fibers, and on the right are electros spun PLGA fibers with nanosensors. The SEM micrographs of the fibers provide evidence of nanosensor association on the surface of the fibers. However, they may also be incorporated within the scaffold fibers.
Once electros spun, the nanosensors retain their ability to remain optically and chemically active and are shown here associated with the scaffold fibers. On the left is the fam dye shown in green, and on the right is the Tamara dye shown in red. The fluorescent intensity ratio produces a calibration curve as shown here.
Nanosensors assessed alone and when incorporated into electros spun PLGA scaffolds mirrored each other, demonstrating that the nanosensors retain their optical activity following incorporation into the scaffolds. The incorporated nanosensors also reversibly respond well to changing pH values as the buffer was alternated between a pH of 5.5 and 7.5. The fam Tamra ratio predictably responded each time shown.
Here are confocal images of fibroblasts that had nanosensors delivered intracellularly via transfection reagent. The punctate colocalization of the fam and Tamara dyes suggests that the dyes remained entrapped in the nanosensor matrix. Intracellular delivery of the nanosensors can be confirmed by measuring the fam Tamara ratio of cells placed into various buffers.
As you can see here, the ratio increases when measuring the scaffold based nanosensors, but stays steady when measuring the sensors within the cells. After the development of this technique, researchers in the field of tissue engineering may investigate in situ and in real time. Microenvironmental intracellular pH changes within 3D cellular constructs without disturbing the ongoing experiment.
