Liposomes via Reverse-Phase Evaporation Method using Sonication

Liposomes are versatile nanocarriers used in drug delivery due to their biocompatibility and ability to encapsulate both hydrophilic and hydrophobic drugs. The reverse-phase evaporation method (also known as emulsification method or a solvent-evaporation method) is a prominent technique for liposome preparation, offering high encapsulation efficiency. This article focuses on the preparation of liposomes via the reverse-phase evaporation method enhanced by probe-type sonication, highlighting the procedural steps, benefits, and potential applications in drug delivery systems.

Liposomes are vesicular structures consisting of one or more lipid bilayers, capable of encapsulating hydrophilic and hydrophobic compounds. Their unique structure makes them ideal for targeted drug delivery. The reverse-phase evaporation method (a.k.a. emulsification or a solvent-evaporation method) is a well-established technique for liposome preparation. In the following paragraphs, we introduce you to the benefits of the reverse-phase evaporation method using probe-type sonication, which is known to improve emulsification, encapsulation efficiency and liposomal size reduction.

Methodology of Liposome Preparation via Reverse-Phase Evaporation Method

Liposome formation via the reverse evaporation method using sonication involves dissolving lipids in an organic solvent mixture of chloroform and methanol (2:1 v/v), which favors the formation of inverted micelles. An aqueous buffer is then added to this mixture. The combined solution is sonicated, for example, using a probe-type sonicator like the UP400ST, to create a water-in-oil microemulsion. The organic solvent is then evaporated using a rotary evaporator, resulting in a viscous gel that eventually collapses to form liposomes. The large aqueous core of the microemulsion bubbles promotes the entrapment of hydrophilic molecules, leading to liposomal gels that exhibit controlled release and a good permeation profile. Finally, the liposomes are downsized to a uniform size using the sonicator.

Liposome Formation via Reverse Evaporation Method using Sonication

Protocol / Step-by-Step Instructions:

1. Weigh and Dissolve Lipids:
   Accurately weigh a total of 40 mg of L-α-phosphatidylcholine and cholesterol in a mass ratio of either 4:1 or 7:3.
   Dissolve the weighed lipids in 10 mL of a chloroform/methanol mixture (4:1 v/v) in a round-bottom flask.
2. Form Lipid Film:
   Attach the round-bottom flask to a rotary evaporator.
   Rotate the flask at 8 x g at 40°C under vacuum conditions until a thin lipid film forms on the flask walls.
3. Remove Solvent Fumes:
   Evacuate the remaining fumes of the solvent mixture by flushing the flask with nitrogen gas.
4. Redissolve Lipid Film:
   Redissolve the lipid film in 10 mL of diethyl ether to form reversed-phase vesicles.
5. Prepare Aqueous Phase:
   Mix 5 mL of PBS buffer (0.1 M, pH 7.4) containing the active ingredient for encapsulation and 20 mg of Tween 80 with the organic phase (diethyl ether with dissolved lipids).
6. Sonicate the Emulsion:
   Place the w/o emulsion in an ice bath.
   Sonicate the emulsion using a probe-type sonicator UP200Ht at 26 kHz and 50% pulse mode (0.5 cycles = 30 sec ON / 30 sec OFF) and 50% amplitude for 1 minute.
7. Evaporate to Form Gel:
   Return the sonicated emulsion to the rotary evaporator.
   Evaporate under atmospheric pressure at 40°C until a gel is obtained.
8. Form Liposomes:
   Further evaporate the gel, breaking it into a semi-transparent liquid, indicating the formation of liposomes.
9. Final Liposome Suspension:
   Add another 5 mL of PBS buffer (0.1 M, pH 7.4) to the liposome suspension.
   Gently vortex the mixture.
   Evacuate the remaining fumes of diethyl ether using nitrogen gas.
10. Storage:
    Store the final liposome suspension at 4°C in a refrigerator until required.

These instructions outline the step-by-step process for preparing liposomes using the reverse-phase evaporation method with ultrasonic homogenization, ensuring high internal aqueous loading and efficient encapsulation of the active ingredient.
The reverse-phase evaporation method, particularly using probe-type sonication, is a widely adopted technique for the preparation of liposomes, especially when aiming for high internal aqueous loading. This method is advantageous over the traditional thin film hydration method due to its ability to incorporate a greater amount of aqueous phase within the liposomes.

Advantages of Probe-Type Sonication for Liposome Formation

• Enhanced Homogeneity: Probe-type sonication provides consistent energy input, resulting in a more uniform size distribution of liposomes.
• Improved Encapsulation: The mechanical forces during sonication enhance drug encapsulation, particularly for hydrophilic compounds.
• Scalability: The methods are easily scalable, making them suitable for large-scale liposome production.

Applications of Liposomes in Drug Delivery

Liposomes prepared via the emulsification and solvent-evaporation methods with probe-type sonication are suitable for various drug delivery applications, including:

1. Targeted Drug Delivery: Functionalization of liposomes with specific ligands allows for targeted delivery to particular tissues or cells.
2. Controlled Release: The lipid bilayer structure permits controlled drug release, enhancing therapeutic efficacy.
3. Versatility: These methods accommodate a wide range of therapeutic agents, from small molecules to larger biomolecules like proteins and nucleic acids.

The reverse-phase evaporation method is particularly noted for its higher internal aqueous loading compared to the thin film hydration method. This characteristic is beneficial for applications requiring substantial encapsulation of hydrophilic drugs or other therapeutic agents.
The reverse-phase evaporation method using probe-type sonication is a robust and efficient technique for liposome preparation. Its ability to achieve higher internal aqueous loading makes it a preferred method in pharmaceutical applications where maximizing the encapsulation of hydrophilic substances is crucial. The careful control of solvent evaporation and the use of sonication are key to the success of this method, leading to the production of high-quality liposomes suitable for various therapeutic purposes.

Find the Right Sonicator for Your Liposome Production

Hielscher Ultrasonics offers a wide product range of probe-type sonicators for the efficient liposome production resulting in a high entrapment efficient and high loading capacity of bioactive molecules.
You can use Hielscher sonicators for various liposome preparation routes such as the here described reverse-phase evaporation technique, the emulsification method and the thin-film method.
Read more about ultrasonic liposome preparation via thin-film method!
Read more about ultrasonically prepared liposomes encapsulating

• vitamin C
• DHA and EPA (fish oil)
• allicin

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