Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

The overall goal of this procedure is to synthesize ultra small populations of biocompatible nanoparticles, characterize their physical properties and investigate particle cell interactions. This is accomplished by first using the phase inversion temperature method to synthesize the lipid nanoparticles. During this process, the lipid and surfactant are initially co melted.

After adding a specific amount of water, the mixture is heated until phase separation occurs. The mixture is then stirred continuously until a nano emulsion is created and the synthesis of solid lipid nanoparticles or sln is complete. Next, dynamic light scattering or DLS is used to measure particle size and poly dispersity.

Differential scanning calorimetry or DSC is an additional technique used to determine the melting point and latent heat of melting in order to further characterize the particles, ultimately fluorescence microscopy and flow cytometry can be used to investigate the particle cell interactions. The main advantage of this technique over existing high energy methods such as ultra sonication microfluid and high pressure homogenization, is that this is a comparatively gentle synthesis technique, which is both simple and scalable. To synthesize the LNS combine 0.6 milligrams of fluorescent dye or other lipophilic compound and 0.10 grams of the linear alcan or lipid.

In a 15 milliliter vial coremelt the components at 90 degrees Celsius and stir add 0.11 grams of a linear non ionic surfactant. Then mlt the resulting mixture at 90 degrees Celsius and stir. Next, add 1.79 grams of sterile water to the mixture heat to 90 degrees Celsius and visually monitor the solution until two phases are observed.

Now stir the mixture until a transparent nano emulsion is formed. Prepare a separate nano emulsion in parallel without adding the fluorescent dye in order to serve as a control sample. Further sterilize each nano emulsion using a sterile 0.2 micron filter.

Employ DLS to measure the particle size and poly dispersity of the S lns using a glass in an instrument design to measure particle size prior to the measurement of the samples, the particle size of two known standards should be measured following the manufacturer's protocol for the standards preparation and measurement. Next, measure the samples as prepared for each. Use a runtime of 100 seconds, the refractive index of water and the viscosity of water at 20 degrees Celsius.

Report the average particle diameter and poly dispersity of particle size distribution to investigate the thermal behavior of the S lns using DSC first pipette the S LNS into a 40 microliter aluminum pan with a mass of approximately 25 milligrams. Hermetically seal the pan using a universal crimp press to minimize moisture loss during the DSC scan. Before measuring each nano emulsion from five to 80 degrees Celsius Report the melting point and the latent heat of melting from the DSC plot where the valley represents the melting point and the integral of the area under the curve.

In the DSC plot, divided by the amount of material represents the latent heat of melting culture. Primary human fibroblasts according to the manufacturer's instructions in liquid medium for the culture of human dermal fibroblasts. Supplemented with the low serum growth supplement kit, maintain the cells in a humidified atmosphere at 37 degrees Celsius, 5%carbon dioxide and subculture upon reaching 80%confluence for both MTT and imaging analysis seed cells.

In a 96 well plate at a density of 20, 000 cells per well. Allow the cells to equilibrate at 37 degrees Celsius overnight prior to SLN exposure at time zero dilute s SLMs in complete medium to achieve the desired lipid concentration and add 10 microliters per well to each replicate well in the 96 well plate after 24 hours of incubation with S lns, determine cellular viability using the MTT assay according to the manufacturer's protocol. Wash the cells once and add 100 microliters of fresh medium to each.

Well kill the control wells by incubating in a 70%methanol solution for 10 minutes before replacing with fresh medium. Add the MTT reagent at 10 microliters per well to each well of the microplate and incubate overnight at 37 degrees Celsius after overnight incubation, solubilize intracellular form and crystals with 100 microliters of the provided detergent solution. According to manufacturer's protocol, incubate the cells in the detergent solution for three hours at room temperature before obtaining absorbance values.

Using a microplate reader to prepare for microscopy. Wash the fibroblasts twice with sterile phosphate puffed saline. Fix the cells for 10 minutes with cold, 70%methanol at specific time points following dosing of fibroblasts with lns.

After 10 minutes, replace the methanol with phosphate buffered saline or PBS before capturing images using an inverted microscope. The S SLN synthesized resulted in control S LNS with an average particle diameter of 18.59 and with a poly dispersity of 5.83 and Nile red loaded SLMs with an average particle diameter of 16.87 nanometers and with a poly dispersity of 4.47 using an MTT assay. The dose response effect on cellular metabolism was measured where increasing particle concentration resulted in a decrease in cellular viability.

There was no observed difference in toxicity between cells exposed to SLN alone versus Nile red loaded.SLN. In parallel cells were visually examined for adherence to the tissue culture. Polystyrene and images were taken to demonstrate both representative cellular morphology and the uptake of fluorescent particles over time, using a dose equal to five micrograms per milliliter.

Lipid particle uptake was observed by two hours post-exposure. The level of nanoparticle incorporation was determined by measuring the intensity of Nile red fluorescence in murine bone marrow derived dendritic cells, or B MDCs via flow cytometry. A direct correlation between the concentration of SNS used and the amount of fluorescence assessed in B MDCs was observed.

After watching this video, you should have a good understanding of how to synthesize ultra small populations of biocompatible nanoparticles, characterize their physical properties and investigate particle cell interactions.