This video article demonstrates a procedure for synthesizing, a targeting polymeric nanoparticle system for delivery of plasmid to cancer cells. To begin folated gelatin polymers are synthesized and DNA plasmids encoding EGFP are encapsulated. Next, the folated nanoparticles are pegylated and EGFR specific peptides are conjugated to the surface.
The nanoparticles are then applied to the cells of interest and incubated to allow protein expression analysis by fluorescence. Microscopy reveals efficient expression of nanoparticle delivered plasmids as soon as 48 hours after transfection. The main advantage of this technique over existing methods like delivery of genetic materials using ca vectors, is that gelatin nanoparticles are much less cytotoxic.
The implication of this technique extend towards cancer therapy, but touting on the overexpressed receptors on cancer cells is easy to deliver genetic materials into target cells. Those s can provide insight into plasmid delivery. It can also be used in other systems such as srna and microRNA.
Begin this procedure by generating folated gelatin. First, dissolve one gram of gelatin in 100 milliliters of deionized water and incubate with 20 milligrams of two immuno hydrochloride at room temperature for 15 hours. Next, to remove non-reactive reagent, transfer the solution to a dialysis bag and submerge it in one liter of five millimolar hydrochloric acid solution.
After three hours, transfer the bag to one liter of fresh five millimolar hydrochloric acid. Then after another three hours, transfer the bag to one liter of one millimolar hydrochloric acid for three hours following dialysis. Transfer the solution to four 50 milliliter conical tubes and place them at minus 80 degrees Celsius once frozen.
Place them in a lyophilizer for two days. Store the samples at four degrees Celsius until needed for the next step. The preparation of DNA containing nanoparticles is the most difficult aspect of this.
To ensure success is important to follow the next few steps of this particle ly, the pH must be properly adjusted. In the addition of ethno should be performed precisely as showing. To prepare DNA containing nanoparticles dissolve 200 milligrams of folated, gelatin and water.
Adjust the pH of the solution to seven by adding 0.2 molar sodium hydroxide. Then add one milligram of EGFP and one plasmid DNA and mix gently add a stir bar to the solution, and while stirring the solution at a high speed, slowly add chilled ethanol to the mixture. Once the composition reaches 75%hydro alcoholic solution, nanoparticles will form and the solution becomes opaque.
To further stabilize the nanoparticles slowly add 0.1 milliliters of 8%glyoxal solution to cross-link amino groups in the nanoparticles. Then to quench the unreactive reagents, add 0.5 milliliters of 0.2 molar glycine. Transfer the solution to an ultracentrifuge tube and spin at 16, 000 RPM for 30 minutes.
Following the spin, rinse the pellets gently with deionized water twice. If not proceeding immediately, the sample can be freeze dried as before and stored at four degrees Celsius. Next to modify the surface of the nanoparticles, resuspend the pellets in 0.1 molar phosphate buffer at a concentration of 10 milligrams per milliliter.
Then add two times the weight of mal peg SCM and incubate for two hours at room temperature with slow stirring. After the incubation centrifuge at 16, 000 RPM for 30 minutes to collect the pegylated nanoparticles. Then wash the pellets with deionized water twice.
Freeze dry the nanoparticles and store them at four degrees Celsius resuspend the EG SCM modified nanoparticles in 0.1 molar phosphate buffer at a concentration of 10 milligrams per milliliter. Then incubate with 10%weight of EGFR specific peptide for six hours at room temperature with slow stirring after six hours. Collect the peptide modified nanoparticles by ultracentrifugation at 16, 000 RPM for 30 minutes.
Then wash the pellets twice and freeze dried the purified nanoparticles store at four degrees Celsius. The EGFR targeted nanoparticles should then be characterized by scanning electron microscopy, electros spectroscopy for chemical analysis gel electrophoresis and western blotting as described in the accompanying written. Once the nanoparticles have been characterized, they should be assessed for transfection efficiency.
The plasmid used in this example encodes a fluorescent protein, so transfection efficiency can be assessed by fluorescent microscopy. Expression of non fluorescent proteins should be assessed by other methods such as western blotting or Eliza. First, add two milliliters of two milligram per milliliter, P-E-G-F-P-N one plasma nanoparticles in DMEM to each of a six well plate containing pan.
One cells grown on glass cover slips as a positive control combined 20 micrograms of plasmid with 20 microliters of lipofection, a onic lipid transfection reagent, and added to the cells. A well containing untreated cells is used as a negative control. Incubate the plate for six hours at 37 degrees Celsius.
Then replace the transfection medium with culture medium, and incubate for an additional 24, 48, 72, or 96 hours. After the incubation, replace the medium with culture medium containing one microgram per milliliter of hooks, 3, 3, 3, 4, 2, and incubate for 15 minutes at room temperature. After mounting the cover slips on microscope slides assess the expression of EGFP in the cells by differential interference, contrast and fluorescence microscopy.
Transfected cells will appear green as shown in this image of pan one cells 48 hours after transfection. The blue color represents hooks staining of the nucleus. The results should then be verified by Eliza.
Once the transfection efficiency has been determined, the nanoparticles can be used to deliver therapeutic plasmid. Nanoparticles containing wild type P 53 plasmid were generated as described in this video, transfection was verified by reverse transcriptase PCR to determine therapeutic efficiency. Transfected pan one cells were stained 24 48, 72, and 96 hours post transfection to evaluate proa activity.
Expression was then assayed using an ISYS imaging cytometer as can be seen here. Cells treated with EGFR targeted, violated gelatin. Nanoparticles had the greatest amount of apoptotic activity.
These results indicate that the targeting system sh gel peg peptide has the highest transfection efficiency and can successfully induce apoptosis and pancreatic cancer cells. After watching this video, you should have a good understanding of how to synthesize targeted jet genetic particles for plasmid delivery. Don't forget that working with glaso or other cross linkers can be extremely hazardous.
Lab coats and gloves should always be worn while performing. This p.
