The overall goal of this procedure is to perform and evaluate gene delivery experiments using non-viral polymeric nanoparticles. This is accomplished by first transecting cells with gene delivery particles. The transfection efficiency is then evaluated by microscopy and high throughput flow cytometry analysis.
Next, the nanoparticle size distribution concentration, and plasmids per particle are determined using a nanoparticle tracking analysis instrument. Ultimately, this method allows for nanoparticle size characterization, and transfection efficacy using nanoparticles. The main advantage of using biodegradable polymeric nanoparticles for gene delivery is that they can be safer and more effective than other gene transfer methods.
The primary advantage of nanoparticle tracking analysis over existing methods like dynamic light scattering is that nanoparticle tracking analysis directly gives the number average distribution and particle concentration. This method would help answer key questions in the non-viral gene delivery field, such as the number of plasmids per particle, which is important for co-expression studies. The following protocol is demonstrated using human retinal endothelial cells or hre as model cells and poly beta amino esters or PBA AEs as model polymers.
Note that culture conditions and solvents may need to be adjusted depending on the cells and polymers used 24 hours before.Transfection. Use a multi-channel pipette to seed hre into clear tissue culture treated flat. Bottom 96 well plates at a concentration of 25 to 50 cells per microliter.
This will give 70 to 80%co fluency on the day of transfection, prepare and label clear non tissue culture treated. 96 well plates to serve as master plates to prepare and dilute the PBA and DNA. Two identical plates will be prepared.
One for evaluating toxicity and one for evaluating transfection efficiency. Follow the poly beta amino ester or PVAE polymer and P-E-G-F-P-N 1D NA stock solutions at room temperature. Once hawed, use a 12 channel pipette to prepare a well containing 50 microliters of 0.06 milligrams per milliliter DNA in 25 millimolar sodium acetate buffer for each condition.
Next on the same plate, dilute the PBAE polymers to 10 milligrams per milliliter in sodium acetate buffer. Then further dilute the PBAE polymers into polymer weight to DNA weight ratios of 30 and 60 and 50 microliters of sodium acetate buffer. For nanoparticle formation, use a 12 channel pipette to combine the 50 microliters of diluted PBA with the 50 microliters of diluted plasma DNA mixed vigorously by pipetting up and down.
Then let the mixture incubate at room temperature for 10 minutes to allow self-assembly. Following self-assembly, use a 12 channel pipette to add 20 microliters of the nanoparticle solution dropwise to each well of the seeded cells containing medium transfect four wells per condition as shown in this diagram. For each plate, reserve at least four wells for each of the negative and positive controls.
Negative control consists of untreated cells and positive controls, consist of cells transfected with a commercially available reagent such as lipo 2000 or Fuge hd. Incubate the plates with the transfected cells at 37 degrees Celsius for four hours After the incubation, use the 12 channel pipette to remove the medium from the cells and replace it with 100 microliters per well of fresh hre. Medium incubate for an additional 24 to 48 hours.
The next day, remove one plate from the incubator. Evaluate cell transfection and toxicity by fluorescence microscopy, and quantify the cell toxicity using the cell titer 96 aqueous one assay as described in the accompanying text. After 48 hours, remove the other plate from the incubator.
Assess transfection efficiency by fluorescence, microscopy, and flow cytometry as described in the accompanying text. Use the toxicity and transfection data acquired here to determine the optimal concentrations of PBAs to use for future experiments. The nano site NS 500 is a nanoparticle tracking analysis instrument that can provide information on the size distribution of a nanoparticle sample by tracking the diffusion of individual nanoparticles prior to use prime the fluidic system of the nano site in a 0.5 milliliter micro centrifuge tube.
Prepare the PBA and plasma DNA for analysis as before at the same concentrations used in the previous section of this video. Following self-assembly, dilute the nanoparticle solution 100 fold in PBS in a new 1.5 milliliter tube to obtain a final volume of at least 500 microliters. This will yield a nanoparticle concentration in the appropriate range for nanoparticle tracking analysis.
Next, load the sample into the nano site. Be sure not to introduce air bubbles visually checked to make sure there are between 20 to 100 particles on the screen. An ideal number for nanoparticle tracking is approximately 50 particles.
If there are too many or too few, flush the NS 500, adjust the dilution into PBS and reload the sample. If the number of particles on the screen is between 20 to 100 capture videos. Once the videos are captured, proceed to the processing stage by opening a video file in the same software.
Then once the file is open, increase the screen gain. Select the auto adjust for the image parameters by clicking the appropriate boxes. If a particle on the screen is recognized by the software, it will be marked with a red cross.
Once all the particles are marked, click the process button in the software to process the video file. The particle size, distribution size averages and particle concentration will then be displayed to verify that the particle count is accurate. Repeat this procedure using the same sample diluted by two x and four.
Forex in PBS apre were transfected with the E-G-F-P-P-B-A-E nanoparticles as shown in this video. To visually assess transfection efficiency fluorescence microscopy imaging was performed. These images show the fluorescence brightfield and merged images of the transfected cells as seen in the merged image, a majority of the cells express EGFP and maintain normal morphology.
To quantify the transfection efficiency flow cytometry was performed as can be seen in this figure after gating on the live population. The unresected population did not contain EGFP positive cells. However, 71.6%of the treated cells were positive for EGFP nano site Nanoparticle tracking analysis was then used to determine the nanoparticle size distribution and concentration.
For this analysis, it is important that the particle size distribution is mono disperse. Using this information, the average number of plasmids per particle can be calculated by dividing the concentration of plasmids in solution by the particle concentration determined by the NS 500. The number of plasmids per particle is a distribution that reflects the particle size distribution.
Within the sample, there will be larger particles with the plasmids per particle value greater than the average, as well as smaller particles with the lower plasmids per particle value than the average. While attempting this procedure, it is important to remember not to allow degradable polymers to stay in aqueous solvent too long before adding nanoparticles to cells for using ncy, adjust the dilution appropriate for sizing. After watching this video, you should have a good understanding of how to transfect adherence cell types with non-viral gene delivery polymers, as well as measure transfection efficacy, as well as particle size distribution.
Don't forget that working with human cells is biohazardous. Always wear personal protective equipment and work in a biosafety cabinet while performing this procedure.
