We are at Mibelle involved in the Oleaf4Value project in possible encapsulations and formulations of the different extracts, focusing in the generation for liposomes towards market applications.

A liposome is a small vesicle composed of a lipid double layer. It is formed through a process called self-assembly, where the lipids arrange themselves in a specific manner in an aqueous environment. The formation of liposomes can occur spontaneously under certain conditions. Here’s a step-by-step explanation of how liposomes are formed.

Source: istockphoto. Author: Matike

The formation of liposomes can be achieved in our case using a high-pressure homogenization device that applies shear forces to efficiently mix and disrupt lipid bilayers.

Using a microfluidizer allows for precise control over the size, uniformity, and encapsulation efficiency of liposomes. The intense shear forces generated by the microfluidizer disrupt the lipid bilayers more efficiently compared to traditional methods, resulting in smaller and more homogeneous liposomes. This technique is particularly useful to produce liposomes with a narrow size distribution, which is important for various applications, including drug delivery and nanomedicine.

Here’s an explanation of how liposomes are formed using a high-pressure homogenizer.

Lipid selection and preparation

Lipids used for liposome formation are typically amphiphilic, meaning they have both hydrophilic (water-loving) and hydrophobic (water-fearing) regions. Phospholipids are commonly used due to their natural abundance in biological systems. These lipids have a hydrophilic head and hydrophobic tail.

Is desired that the active ingredient should be soluble in one of the phases. Depending on the solubility in the lipophilic phase or the hydrophobic phase, some steps would change. As a matter of simplicity, we would assume that our active is soluble in the water-soluble phase.

Dispersion in Aqueous Solution

The lipid solution is mixed with an aqueous solution or buffer containing the desired encapsulated substance, such as drugs or biomolecules. The lipid and aqueous solutions are typically kept separate initially and then they are then dispersed in an aqueous solution, typically by vortexing or sonication.

Formation of Lipid Bilayer

As the lipids are dispersed in the aqueous solution, the hydrophobic tails come together to form the interior of the bilayer, shielding themselves from water. The hydrophilic heads face outward, interacting with the surrounding water molecules.

Homogenization process

The lipid and aqueous solutions are pumped at high pressure into the microfluidizer, where they meet in a confined space or interaction chamber. The high-pressure flow creates intense shear forces and turbulence within the chamber.

Impact and disruption

As the lipid and aqueous solutions pass through the microfluidic chamber, they experience intense shearing and collisions. These forces lead to the disruption of the lipid bilayers and the formation of small, unilamellar or multilamellar liposomes.

Picture 1: Microfluidizer at laboratory scale showing refrigeration (left) and the microfluidizer chamber (middle).

Repeated cycles

The liposome-containing mixture is continuously recirculated through the high-pressure homogenizer for several cycles. Each cycle further enhances the disruption and size reduction of liposomes, resulting in a more uniform and smaller liposome population until a plateau is reached.

Picture 2: Microfluidizer at laboratory scale showing the mixture recipient (left), metal vase with refrigeration (middle) and the final product (right).

Post-processing

After the desired number of cycles, the liposome suspension is collected. If necessary, the suspension can undergo additional steps, such as filtration or centrifugation, to remove any larger debris or unencapsulated substances. A particle size measurement is mandatory.

Figure 1: Normal distribution with all the particle sizes are similar.

Figure 2: Particle size distribution were there the distribution is not concentrated in one size. This could lead to possible instabilities.

Stability studies and trials

Once the previous steps are fulfilled, the liposomes are treated for stability trials as well as quality controls. Each one of the samples will be tested against temperature, sun irradiation to check possible color changes or phase separation. Aspect and appearance for the next 3 months in order to procced with a decision.