This protocol is significant because it demonstrates, how to prepare Polymeric nanoparticles, encapsulated nucleic acids in one simple step. The main advantages of this technique, are versatility, as it can be applied to different nucleic acids and cell types. The use of simple procedures for particle preparation, such as pipetting up and down, and the possibility to extend the stability of the nanoparticles and drum conditions by lyophilization.
Demonstrating the procedure will be Laura Olmo, Coral Garcia-Fernandez, Maria Stampa Lopez-Pinto and Maria Navalon-Lopez, phd student of our laboratory and Marta Diaz-Caballero, a post doc for our laboratory. For polyplexes formation, thaw the previously prepared polymers, C6 peptide, poly beta amino esters, and vortex the solution. After pipetting the polymer mix up and down, prepare a 12.5 millimolar solution in sodium acetate.
Vortex the solution and wait for 10 minutes. Next, prepare mRNA at 0.5 milligrams per milliliter and mixed by pipetting. Vortex the polymer solution again, then mix the genetic material solution in the polymer solution in a one-to-one ratio in a micro centrifuge tube and incubate the tube at 25 degrees Celsius for 30 minutes in a Thermoblock.
After incubation precipitate the mixture with one to two RNase free water by adding the sample to a micro centrifuge tube containing the water, then to include the excipients, add a 20 millimolar heaps and 4%sucrose solution in the same volume as the mixture of mRNA and poly beta amino esters, and mixed by pipetting. For Lyophilization after freezing the polyplex solution at minus 80 degrees Celsius for one hour, perform the primary drying, then immediately store the polyplexes at minus 20 degrees Celsius to avoid rehydration. For polyplex resuspension, remove the lyophilized nano-particles from the minus 20 degrees Celsius freezer and rapidly add the corresponding amount of deep hydrogenated water to re-disperse the solid and achieved the desired concentration.
Pipette gently until total resuspension and once dissolved, pipette up and down vigorously avoiding bubbles. After 24 hours of transfection for qualitative assessment by fluorescence microscopy, place the 96 well plate containing the transfected Hela cells onto the microscope and start visualizing using the 10 times objective. First, apply a white balance to create a background reference for the software, then acquire an image to overlay it with the fluorescence image during the analysis.
Change the microscope mode to reflection mode and move the filter wheel to the blue laser to visualize EGFP. Then acquire images for all the conditions or wells employing the same exposure time. For quantitative assessment by flow cytometry, wash the Hela cells transfected in a 96 well plate using 100 microliters of PBS per well.
After aspirating the PBS at 25 microliters of trypsin per well and incubate at 37 degrees Celsius for five minutes. Once the cells are detached, add the previously recovered media to inactivate the trypsin, then fix the cells by adding 31.25 microliters of 10%formalin per well and incubating for 20 minutes. Next turn on the flow cytometer and the software, then set up the proper conditions for the experiment, including the type of plate sample volume and other parameters such as shaking and rinsing between samples.
Set up the appropriate parameters to quantify the percentage of positively transfected cells by first viewing the flow data on forward scattered light versus side scattered light to distinguish the cells from the debris. And then plotting another scatterplot, comparing the amplitude versus height to gate and discriminate individual cells. One day before transfection, plate 10, 000 JAWS II cells per well in a 96 well plate for overnight incubation.
The following day transfect the cells with 0.6 micrograms mRNA per well and incubate the plate for 24 hours in a dry air incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, wash the cells remaining in the well with 25 microliters of PBS. After aspirating the PBS at 25 microliters of trypsin and incubate the plate for five minutes at 37 degrees Celsius to detach the cells.
Stop the trypsin reaction by adding the previously recovered media onto the correspondent well. Then centrifuge the plate, aspirate the media, and fix the cells by adding 50 microliters of PBS and 2.5%formalin per well followed by incubating the plate at four degrees Celsius for 20 minutes, centrifuge the plate. Then add 50 microliters of PBS and 3%BSA per well and incubator for 30 minutes at four degrees Celsius.
After incubation centrifuge the plate again, then add 50 microliters of the mouse ovalbumin antibody in PBS and 3%BSA and incubator for 30 minutes at four degrees Celsius. Repeat centrifugation step, then wash the cells with 50 microliters of PBS. After aspirating the PBS, add the secondary antibodies and incubate for one hour at four degrees Celsius.
After incubation, repeat the centrifugation and washing steps. Then resuspend the cells and 100 microliters of PBS and 2.5%formalin for flow cytometry analysis. Physiochemical characterization of fresh and lyophilized GFP mRNA encapsulating nanoparticles show no significant differences in size and poly dispersity index.
Transmission electron microscopy of the polyplexes confirms the formation of spherical and mono dispersed nanoparticles of approximately 50 nanometers in diameter. Encapsulation efficiency data of a Lego peptide and modified poly beta amino ester polyplexes encapsulating GFP, or ovalbumin show no significant differences depending on the type of mRNA. A qualitative analysis of EGFP expression, 24 hours post-transaction in the JAWS II cell line is shown here.
Quantitatively compared to the negative control. The percent GFP positive cells is significantly higher and comparable to a positive control performed with a classical transfection reagent. The ovalbumin mRNA loaded oligopeptide and modified poly beta amino ester nanoparticles can activate dendritic cells as seen by the significantly higher expression of CD11b and CD86 membrane markers in transfected cells compared to non-transfected cells.
Our vaccination platform based on the use of mRNA as the active principle, as well as our proprietary polymers can be adapted to be used in prophylaxis as well as for cancer therapeutics. We can use our technology to make a highlight that contribution in cancer immunotherapeutics. since we can encapsulate tumor-associated antigens in our nanoparticles and make a shift in cancer treatments.
