The overall goal of this experimental procedure is to use a liquid crystal based nanoparticle as a delivery platform for the controlled delivery of a hydrophobic dicargo to the lipophilic portion of the plasma membrane of living mammalian cells. This method can help answer key questions in the drug delivery field, such as how to efficiently enable the delivery and cellular uptake of poorly water soluble hydrophobic cargos. The main advantage of this technique is that it allows more specific delivery of membrane targeted hydrophobic cargos, to the plasma membrane, coupled with their reduced cytotoxicity.
Demonstrating the procedure will be Dr.Okhil Nag, a postdoc from my laboratory. Begin this procedure with preparation of DiO liquid crystal nanoparticle as described in the text protocol. Then, add 20 microliters of freshly prepared working solution of NHSS/EDC to 1.0 millileters of the DiO liquid crystal nanoparticle in HEPES buffer.
And stir for five minutes. Next, add 20 microleters of a stock solution of cholesterol PEG amine to this mixture and stir for two hours. After two hours, briefly centrifuge the reaction mixture at maximum speed for 30 seconds using a tabletop mini centrifuge.
Then pass the supernatant through a PD 10 size exclusion chromatography column equilibrated with Dulbecco's phosphate buffered saline. To confirm the successful covalent conjugation of PEG cholesterol to the DiO liquid crystal nanoparticle surface by gel electrophoresis, prepare an agarose gel as described in the text protocol. Once the gel has solidified, add enough TBE running buffer to submerge the gel in the chamber.
Then, add 35 microleters of DiO liquid crystal nanoparticle sample to the wells of the gel and run for 20 minutes at a voltage of 110 volts. Image the gel using a gel imaging system with an excitation filter at 488 nanometers and emission filters of 500 to 550 nanometers. Prepare 35 millimeter diameter tissue culture dishes fitted with 14 millimeter number one cover glass inserts by coating them with bovine fibronectin in DPBS for two hours at 37 degrees Celsius.
After two hours, remove the fibronectin coating solution from the culture dish. Harvest HEK 293 T17 cells from the T25 flask by first washing the cell monolayer with three milliliters of DPBS, and then adding two milliliters of trypsin EDTA. Incubate the flask at 37 degrees Celsius for approximately three minutes.
Once cells are detached from the flask, neutralize the trypsin by adding four milliliters of complete medium to the flask. Determine the cell concentration in the suspension by counting them in a cell counter. Then adjust the cell concentration to approximately 80, 000 cells per milliliter with growth medium.
Add three milliliters of the cell suspension to the dish, and culture in the incubator overnight. The next day, the cells should be at approximately 70 percent confluency and ready for use. Adequate cell density is critical for robust and efficient labeling of a high percentage of cells.
Prepare one milliliter solutions of DiO, DiO liquid crystal nanoparticle, and DiO liquid crystal nanoparticle PEG cholesterol, in delivery medium by diluting the corresponding stock solutions. Remove the growth medium from the culture dishes using a serological pipette, and wash the cell monolayers two times with HEPES DMEM. Perform the washes by using a pipette to gently add HEPES DMEM to the edge of the dishes, and then removing it.
Add 0.2 milliliters of the prepared DiO free or DiO liquid crystal nanoparticle delivery solutions to the center of the culture dishes. Then return the dishes to the incubator for an appropriate period of time. Longer incubation times lead to nonspecific cellular labeling of non membraneous areas.
After the incubation period, remove the delivery solutions using a serological pipette. Wash the cell monolayers two times with DPBS using two milliliters for each wash. Fix the cell monolayers using four percent paraformaldehyde for 15 minutes at room temperature.
Remove the paraformaldehyde solution using a pipette and gently wash the cells one time with two milliliters of DPBS. Now the fixed cells are ready to be imaged for the presence of a membraneous fluorescent signal, using confocal laser scanning microscopy. On a microscope stage equipped with a heated incubation chamber, image the live cell sample using a confocal microscope to assess membrane labeling.
If the imaging is not performed immediately, replace the medium with DPBS containing 0.05 percent sodium azide and store the dishes at four degrees Celsius. In this method, cells are colabeled with DiO as a FRET donor, and DiI as a FRET acceptor. Label the sells sequentially with DiO liquid crystal nanoparticle PEG cholesterol, and DiI free as before.
After staining, wash the cells one time with two millileters of DPBS using a serological pipette and replace with two milliliters of the live cell imaging solution. On a microscope stage equipped with a heated incubation chamber, image the live cell sample using a confocal microscope with a FRET imaging. Configure the experiment for 30 minute intervals over a four hour period by exciting the DiO donor at 488 nanometers, and collecting full emission spectra of both the DiO donor and the DiI acceptor from 490 to 700 nanometers, with a 32 channel spectral detector.
Then, determine the time resolved emission intensity of both the DiO donor and the DiI acceptor from cells stained with DiO liquid crystal nanoparticle PEG cholesterol, and DiI. Finally, calculate the time resolved acceptor donor FRET ratio for the images as described in the text protocol. Free DiO accumulates nonspecifically in the cell interior, rather than the plasma membranes of the cells which were counterstained with a dilabeled membrane phospholipid.
This can be visualized with the merged images. Conversely, the DiO liquid crystal nanoparticle PEG cholesterol specifically labels just the plasma membrane of the cell, as visualized by colocalization with the dilabeled membrane phospholipid. FRET confirms diopartitioning from the liquid crystal nanoparticle into the membrane lipid bilayer through an observed increase in energy transfer from the DiO donor to the DiI acceptor.
FRET between DiO and DiI, as well as DiO and DiI emission changes, confirm increased FRET from DiO to DiI as a function of time. The DiO liquid crystal nanoparticle PEG cholesterol nanoparticles exhibit minimal cytotoxicity at 90 percent viability, as compared to significant toxicity for free DiO at approximately 50 percent viability. After watching this video, you should have a good understanding of how to synthesize cargo loaded liquid crystal nanoparticles, biofunctionalize them for cellular targeting, and image the cargo loaded cells for successful cellular delivery.
Generally, individuals new to this method will struggle because the specific delivery of DiO from aqueous solution to the plasma membrane is a significant challenge. We first had the idea for this method after we used the liquid crystal nanoparticle platform for the cellular delivery of the anticancer drug doxorubicin, and saw its ability to modulate the drug's cellular uptake kinetics. Following this procedure, this approach can be implemented for the cellular delivery of a wide array of hydrophobic cargos that pose a particular challenge with respect of their delivery to cells from aqueous media.
This approach can be utilized to achieve more efficient delivery of membrane targeted drugs and bioactive molecules, such as voltage sensitive dyes for brain imaging, or photodynamic drugs for photonic assisted drug therapy.
