Liposome Encapsulation Protocols for Hydrophilic and Hydrophobic Drugs

The thin-film method is one of the most widely used liposome encapsulation methods. It is based on the generation of a thin film of lipids, formed on the inner wall of the rotary evaporator flask. The film is then hydrated with water. Before the hydration, it is integral that the lipid film is preheated above the lipid's transitional temperature to enable a smoother creation of the bilayer, along with vigorous shaking. This allows the film to peel off the flask and form liposomes. The liposomes generated are multilamellar vesicles of different sizes. The encapsulating substance can be added with the lipids before the formation of the thin film (hydrophobic compounds) or with the water (hydrophilic compounds). The advantage of this method is its high reproducibility even when working with small quantities of compounds. This protocol describes the liposome encapsulation of hydrophobic and hydrophilic drugs using the thin-film dispersed hydration method. The extrusion method with a polycarbonate membrane is used to make liposomes of suitable sizes that can be easily internalized by mammalian cells.

Liposome encapsulation

• Dissolve 7 mmol of the lipid (DSPC), 3 mmol of cholesterol, and 1:20 (w/w) Ursolic acid (lipophilic compound) in 5 ml chloroform in a round bottom flask.
• Stir the mixture for 15 min at above Tc of the lipid (60°C).
• Dry the lipid using a rotary evaporator at 40°C.
• Further dry overnight by incubating above the Tc of the lipid (60°C) in a vacuum oven.
• Re-hydrate lipid nanoparticles in 5 ml ultrapure water.
• Liposome encapsulation of hydrophilic compounds is added after dehydration. Dissolve lipophilic compounds 1:20 (w/w) in 5 ml of ultrapure water. Re-hydrate lipid nanoparticles by adding the dissolved lipophilic compound in ultrapure water.
• Stir the liposome-encapsulated compound at a temperature above the Tc of the lipid (60°C) for 30 min.
• Vortex for 2 min.
• Extrude at a temperature above the Tc of the lipid for approximately 22 passes. Do 11 passes with a 100 nm pore size PC membrane and then another 11 passes using the 50 nm pore size PC membrane. This is done to have liposomes of between 50-100 nm in size.

Liposome characterization

• Turn on and warm up the Zetasizer for 1 hour for the laser to stabilize.
• Open Zetasizer software and set it to the desired measurement.

Particle size

• Measure the particle size of liposome samples by setting a manual SOP.
• Set the sample material to polystyrene latex. Set the dispersant of water to a temperature setting of 25°C. Set the viscosity to 0.8872 cP. Set the refracted index to 1.330. Set the dielectric constant to 78.5. Set the equilibration time to 120 seconds. Set the measurement to 173° Backscatter.
• Fill the cuvette with the sample to a depth of approximately 1 cm. Place the cuvette with the sample in the cell of the Zetasizer and analyze.

Zeta potential

• Measure the zeta potential of liposome samples by setting a manual SOP.
• Set the sample material to polystyrene latex. Set the dispersant of water to a temperature setting of 25°C. Set the viscosity to 0.8872 cP. Set the refracted index to 1.330. Set the dielectric constant to 78.5. Set the equilibration time to 120 seconds. Set the measurement to 173° Backscatter and 100 zetas run.
• Fill the cuvette with the sample to a depth of approximately 1 cm. Place the cuvette with the sample in the cell of the Zetasizer and analyze.

Creative Bioarray Relevant Recommendations

• Creative Bioarray provides various in vitro ADME/PK services, including high-throughput ADME screening, in vitro binding, in vitro metabolism, in vitro permeability, and transporter assays.
• We also provide in vivo drug metabolism and pharmacokinetic (DMPK) services to support drug development studies of in vivo absorption, distribution, metabolism, and excretion of drug candidates. Our in vivo DMPK services cover a comprehensive range of different animal studies in several species.

• Liposome composition. Choose lipids based on the desired properties of the liposomes (e.g., phosphatidylcholine, cholesterol). Optimize the molar ratio of lipids to achieve the desired size, stability, and encapsulation efficiency.
• Hydration method. Use appropriate hydration methods (e.g., film hydration, reverse phase evaporation) for the specific drug type. Maintain optimal temperatures during hydration to ensure proper liposome formation.
• Encapsulation efficiency. Determine the encapsulation efficiency by comparing the concentration of the drug before and after encapsulation. Adjust formulation parameters (e.g., lipid concentration, hydration time) to improve encapsulation efficiency.

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