Editing MRNA Transfection (section)

== <br>Complex Formation <br>  ==

1. Dilute three micrograms of heat-denatured mRNA (10 min at 65°C) and 6—10 μg of DOTAP&nbsp; separately in 100 μL DMEM media (pH 7.5). 2. Incubate for 15 min.<br> 

3. Add DOTAP to the RNA solution.<br> 

4. Incubate for 15 min at room temperature. <br> 

5. Add 4 m M CaCl2 and 0 to 50 μg/mL fibronectin solution and then&nbsp; adjust final volume to 1 mL 

6. Incubate for 30 min at 37°C. 

7. Add transfection complexes to cells (medium with serum). 

8. Incubate at 37°C in a 5% CO2 humidified environment for 2—4 h. 

9. Remove the transfection mix and replace with complete medium (with serum )<br><br> 

Lipid-coated nanoparticles with a poly-1 core were synthesized via two different processes:<br>a double emulsion/solvent evaporation approach or a solvent diffusion/nanoprecipitation<br>strategy. For double emulsion synthesis, we adapted a previous approach we used for<br>preparing lipid-enveloped PLGA particles37: 30 mg of poly-1 (or PLGA for pH-insensitive<br>control particles) and 2 mg of the phospholipids DOPC, DOTAP, and DSPE-PEG in a 7:2:1<br>molar ratio were co-dissolved in 1 ml of dicholoromethane (DCM). PBS (200 μl) was then<br>added to the mixture on ice during a 1 min sonication step at 7 W using a probe tip sonicator<br>(Misonix XL2000, Farmingdale, NY) to form a first emulsion. The primary emulsion was<br>then dispersed into 6 ml of distilled, deionized nuclease-free water with sonication at 12 W<br>for 5 min, before leaving on a shaker for 18 h at 25 °C to evaporate the organic solvent. For<br>nanoprecipitation synthesis, the same quantities of DOPC, DOTAP, and polymer were co-<br>dissolved in 4 ml of ethanol and added drop-wise to 40 ml of distilled, deionized nuclease-<br>free water, followed by gentle stirring for 5 h to evaporate ethanol. For particles prepared by<br>nanoprecipitation, we found that adding DSPE-PEG in the organic phase together with<br>DOPC, DOTAP and polymer reduced particle yield significantly, so DSPE-PEG was<br>introduced into the lipid coating via a post-insertion process: DSPE-PEG lipid was added at<br>1 mM to 0.5 mg/ml particles in distilled, deionized nuclease-free water and the suspension<br>was stirred for 16 h at 25 °C. The particles were collected and washed once via<br>centrifugation, resuspended in fresh water and stored at 4 °C until use. Lipid-free PVA-<br>stabilized particles were prepared by similar processes except the organic emulsion or<br>solution containing polymer only was dispersed into a 2% w/vol PVA aqueous solution.<br>A fraction of each particle batch was dried in a vacuum oven to determine the particle<br>concentration (mg/ml) by measuring the dry mass. Dynamic light scattering (DLS) and zeta<br>potential measurements were used to determine the particle size and surface charge using a<br>ZetaPALS dynamic light scattering detector (Brookhaven Instruments). To estimate the<br>percentage of PEG-lipid incorporated and the resultant mol% of PEG-lipid present in the<br>lipid surface coating, lipid-enveloped particles were prepared by nanoprecipitation as<br>described above with 1 mol% DOPE-rhodamine added as a tracer for measuring the total<br>amount of lipid incorporated into the particles, before the post-insertion of a DSPE-PEG<br>lipid labeled with carboxyfluorescein (DSPE-PEG-CF). The lipids were then stripped from<br>the particle surface by treatment with 2% triton X-100 for 15 min and the supernatant was<br>measured for both rhodamine and fluorescein signals to quantitate the amount of PEG lipid<br>incorporated.<br><br> 

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