The overall goal of this procedure is to synthesize functionalized polymer-coated gold nanospheres that are 10 nanometers in diameter, biocompatible, have extended circulation time and can be imaged with fluorescence microscopy. This method can help answer key questions in cardiology about endothelium permeability under diseased conditions. The main advantage of this technique is it creates biocompatible ultra-small particles that have functional groups available for further customization.
First, in a micro centrifuge tube, dilute 12 microliters of 80%THPC with one milliliter of ultra pure water. Clamp a 100 milliliter round bottom flask over a magnetic stir plate. Add to the flask 45 milliliters of water and a small egg-shaped stir bar.
Start stirring the water at 300RPM. Add to the flask 0.5 milliliter of a one molar sodium hydroxide solution, and one milliliter of the dilute THPC solution. Continue vigorously stirring the reaction mixture for five minutes.
Then, add two milliliters of a 25 millimolar chloroauric acid solution to the reaction mixture while stirring. Confirm that the yellow reaction mixture becomes dark brown within seconds, indicating the formation of THPC capped, negatively charged gold nanoparticles. Continue stirring the mixture for 15 minutes.
While the mixture stirs, prepare aqueous solution of heterobifunctional amine PEG thiol, carboxymethyl PEG thiol, and monofunctional methoxy PEG thiol. Once the gold nanoparticle reaction mixture has stirred for 15 minutes, reduce the stirring speed to 100 RPM. Add the PEG solutions drop wise to the mixture while stirring.
Continue gently stirring the mixture overnight at room temperature. Then, close one end of a 12-14 kilodalton MWCO cellulose membrane with a dialysis clip. Use a pipette to transfer the reaction mixture to the membrane.
Close the other end of the membrane with a second dialysis clip. Dialyze the nanoparticle mixture against four liters of water stirred at 60RPM for 72 hours. Change the water every two hours for the first six hours, and every six to 12 hours thereafter.
Then, filter the semi purified pegylated gold nanoparticle mixture through a 0.2 micrometer syringe filter, into a 50 milliliter conical tube. Freeze the purified nanoparticle solution at negative 80 degrees Celsius for about five hours. lyophilize the purified nanoparticles using a freeze dryer.
To begin preparing the fluorescent pegylated gold nanoparticles, clamp a 100 milliliter round bottom flask over a magnetic stir plate. Add 18 milliliters of ultra pure water and a small egg-shaped stir bar to the flask, and begin stirring at 100RPM. In a four milliliter conical tube, combine two milligrams of lyophilized pegylated gold nanoparticles with two milliliters of water.
Then, transfer the nanoparticle solution to the round bottom flask for further reconstitution. Add one milliliter of a pH 8.8, 0.1 molar carbonate buffer to the reconstituted nanoparticles while stirring. Then, add five microliters of a 10 milligram per milliliter solution of a fluorescent anhydroxysuccinimide ester in dimethyl sulphoxide.
Wrap the flask in foil to completely exclude light. Continue stirring the fluorescent nanoparticle mixture overnight at room temperature. Then, dialyze the mixture against four liters of water for 72 hours using the procedure described in the preparation of the pegylated gold nanoparticles.
Keep the container covered with foil throughout the dialysis. Set aside one milliliter of the purified fluorescent pegylated nanoparticles for characterization. Freeze, lyophilize and store the remaining fluorescent nanoparticles at four degrees celsius.
To visualize the gold nanoparticle core with transmission electron microscopy, first place 10 microliters of the purified fluorescent nanoparticle solution on a carbon-coated mesh TEM grid. Allow the drop to sit for five minutes. Then, use filter paper to carefully wick away excess fluid from the edge of the TEM grid, until only a film of the fluorescent nanoparticles remains on the grid.
Image the nanoparticles at 80 kilovolts, with a magnification of 50, 000 to 150, 000. To measure the hydrodynamic nanoparticle size, place one milligram of lyophilized THPC coated gold nanoparticles in a micro centrifuge tube. Add one milliliter of water to each tube, and transfer the solutions to one milliliter clear plastic cuvettes.
Evaluate each solution with dynamic light scattering, and measure the spherical hydrodynamic diameters of the particles. Then, transfer the cuvette containing the fluorescent pegylated gold nanoparticles to a spectrofluorometer. Set the exotation wavelength to 633 nanometers, and read the emission signals from 650 to 800 nanometers.
Next, to assess biocompatibility at 7 nanoparticle concentrations, first pipette 100 microliters of a rat fat pad endothelial cell suspension in DMEM into each of 21 wells of a 96 well clear bottom sterile tissue culture treated plate. Incubate the cells until confluency's achieved, then, obtain seven solutions of fluorescent nanoparticles in DMEM with concentrations ranging from zero to 1000 micrograms per milliliter. Aspirate the DMEM from the wells.
Place 100 microliters of each of the seven nanoparticle solutions in each of the separate wells in triplicate. Allow the cells and nanoparticles to co incubate for 16 hours. Then, aspirate the DMEM and suspended or loosely attached nanoparticles from the wells.
Add 100 microliters of fresh DMEM containing 20 microliters of an MTS/PES solution to each well. Incubate the cells at 37 degrees Celsius for two hours. Then, assess cell viability by chlorometric analysis at 492 nanometers with the plate reader.
Next, seed rat fat pad endothelial cells onto sterile 12 milliliter glass cover slips at 10, 000 cells per square centimeter. Feed the cells with DMEM supplemented with 10%FBS and 1%penicillin streptomycin. Incubate the cells until full confluency is achieved.
Modify the endothelial glycocalyx if desired, then, incubate the cells with 550 micrograms per milliliter of fluorescent gold nanoparticles for 16 hours. Prepare the cells for immunofluorescence microscopy. Mount each cover slip on a microscope slide with anti-fade mounting medium containing DAPI.
Seal the cover slip edges with nail polish. Image the immuno stained endothelial cells with confocal microscopy. Visualize the nuclei, the heparin sulfate glycocalyx, and the nanoparticles with blue, green and far red channels, respectively.
TEM of THPC coated gold nanoparticles, showed particle diameters of two to three nanometers. TEM of the pegylted nanoparticles could only confirm the presence of dispersed particles, as PEG is not visible in TEM images. DLS of the THPC coated and pegylated nanoparticles showed a shift in the peak particle diameter from 2.5 nanometers to 10.5 nanometers.
The fluorescent emission had a maximum between 660 and 672 nanometers when excited with 633 nanometer light, which was consistent with the expected maximum emission at 665 nanometers for the fluorescent probe used. Rat fat pad endothelial cells showed similar levels of viability after 16 hours of co-incubation with fluorescent pegolated gold nanoparticles in concentrations of up to one milligram per milliliter, indicating no significant toxicity from the nanoparticles. Minimal nanoparticle uptake was observed into rat fat pad endothelial cells with healthy glycocalyxes.
Significantly greater uptake occurred in cells with degraded glycocalyxes as shown by the increase in red fluorescent signals from the nanoparticles. Following this procedure, further modification such as conjugation onto the open functional groups can be done to modify the particles for specific targeting of an endothelium component or delivery of a drug.
