Bioengineering
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Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells
Chapters
Summary February 9th, 2019
This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.
Transcript
The fabrication of lipid-coated nanoparticles with the rapid injection method provides a stable drug delivery system for cancer therapy. This technique is a simple, fast, scalable, and reproducible technique for nanoparticle fabrication that encapsulates hydrophobic drugs. This technique is applicable for fabricating diagnostic and therapeutic nanoparticles from hydrphobic materials that can be effectively used in several disease conditions.
This fabrication technique, along with DLS, can be used to study the nucleation and growth of hydrophobic materials, such as lipids, proteins, and polymers. Knowing the physical chemical properties of your drug substance is critical before fabricating the nanoparticles. Also, be sure to use the correct concentrations of coating lipids.
Visual demonstration of this method is critical to show the simplicity of the fabrication technique and to show the successful delivery of the drug as nanoparticles to the cells. Precaution needs to be taken when working with chloroform and formaldehyde, and working under a fume hood is required. To begin, dispense 2.4 milliliters of 250 micromolar falcarindiol stock solution dissolved in 70%ethanol in a scintillation vial.
Using a sample concentrator, evaporate the liquid fraction for approximately four hours to obtain dry falcarindiol. Using the sample concentrator, deliver gas over the sample at room temperature to concentrate the sample. Once dried, add the components shown here to the scintillation vial in a fume hood, making sure to clean the syringe with chloroform after adding each component to avoid cross contamination.
Then immediately close the vials containing the lipids so that the solvent does not evaporate and thereby modify the concentration. Wrap the vial with aluminum foil to protect DiI from light and leave the sample overnight in the desiccator to evaporate the chloroform. The next day, dissolve the desiccated sample in absolute ethanol to make a final volume of 1.2 milliliters.
This solution represents the organic phase. Take a 12 milliliter glass vial and fill it with nine milliliters of purified water. Then add a magnetic flea into the vial containing nine milliliters of water.
Place the vial on a magnetic stirrer and stir at 500 rpm. Next attach a one milliliter glass syringe to the dispensing system and slowly pull chloroform in and out of the glass syringe at least 10 times. Dispense the chloroform into its waste collector each time.
Cleaning the syringe with chloroform helps to avoid any contamination. Now prime the syringe with ethanol. Priming replaces the old solvent as well as removes any air bubbles.
Using the syringe, aspirate one milliliter of the organic phase, and insert the syringe into the glass vial up to the middle of the nine milliliter water mark. Maintain it steady in the middle of the vial, and inject the solution at 833 microliters per second by pressing the dispense button on the dispensing system. This generates 10 microliters of 50 micromolar lipid coated nanoparticles of falcarindiol in 10%ethanol.
This injection speed has been found to achieve finest nanoparticles with narrow particle size distribution. While injecting, it is important to make sure that the needle is inserted in the center, steady and straight. Immediately after the injection, remove the vial from the stirrer and transfer the sample to a 50 milliliter round bottom flask.
Attack the flask to the rotary evaporator, and evaporate one milliliter of the organic solvent at room temperature. Be cautious to avoid excess bubble formation. Transfer the nanoparticle suspension from the flask to another 12 milliliter glass vial.
Ensure that the volume is nine milliliters, and then split the sample in two 12 milliliter glass vials. Then add 0.5 milliliters of ultra pure water in one of the vials and 0.5 milliliter of 10X phosphate buffered saline in the other vial. Turn on the digital light scattering instrument, and set the desired temperature to 20 degrees Celsius until it stabilizes.
Then set the instrument parameters as shown here. Fill the plastic cuvette with one milliliter of nanoparticle suspension, and start the measurement. Report the measured size depending on the solvent that was used.
Seed approximately 50, 000 cells on sterile 1.5 cover slips placed in six-well plate to obtain a cell density of approximately 30%Then add culture medium in order to have a final volume of three milliliters in each well. Incubate the cells for 24 hours under standard culture conditions. The next day, add three microliters of the nanoparticle solution for a final falcarindiol concentration of five micromolar.
Then return the cells to the incubator for 24 hours. After 24 hours of treatment, wash the cells twice with PBS, and then fix them in 4%formaldehyde for 10 minutes at room temperature. Following fixation, permeabilize the cells with 0.1%Triton X-100 for 30 minutes.
Then wash them twice with PBS, and stain them with 250 microliters of 300 nanomolar DAPI for five minutes, protected from light. Place the cover slip onto a slide using a drop of PBS. Turn to a 150x subjective, and transfer the slide to the stage of a wide-field fluorescence microscope equipped with an electron multiplied CCD camera.
Image cells using the GFP-LP channel. Additionally, verify the uptake of the nanoparticles into the cells by taking confocal microscopy images using a 63x oil objective with a numerical aperture of 1.4. Image DiI using an argon laser at 514 nanometers and DAPI using a two-photon laser at 780 nanometers.
Uncoated nanoparticles of falcarindiol formed in water and measured in PBS showed high polydispersity, with PD index of 0.571. This value indicates broad and nonuniform distribution of particle sizes. In contrast, lipid coated nanoparticles of falcarindiol fabricated in this video were of 74.1 nanometers with a polydispersity index of 0.182, indicated a relatively monodisperse and uniform distribution of particle sizes.
As a first observation of the nanoparticles inside the cells, epifluorescence microscopy images were acquired after 24 hours of treatment. The nanoparticles were visualized as white, bright dots, and it could be hypothesized that nanoparticles were located inside the cells surrounding the nucleus. To verify that falcarindiol nanoparticles had entered the cells, confocal microscopy was performed on the cells as well.
A large number of nanoparticles are shown here, scattered in the cytoplasm in every cell. These results show that nanoparticles act as a stable drug delivery system for falcarindiol. While attempting this procedure, it's important to inject the solution at the right injection speed, keeping the seeding steady, in the center, to ensure proper mixing.
This procedure can be used for any hydrophobic drug to formulate therapeutic nanoparticles or for imaging agents to formulate diagnostic nanoparticles that can be used in various disease conditions, such as cancer. This technique enabled more research to be conducted on the mechanism by which the nanoparticles get taken up by cells, as well as its live cell imaging and on studying the anticancer effect of the drug.
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