Ultrasonic Liposome Preparation

Ultrasonically produced liposomes show a very high entrapment efficiency, high loading capacity and uniformly small spherical size. Thereby, ultrasonic liposomes offer excellent bioavailability. Hielscher Ultrasonics offers ultrasonicators for the reliable production of pharma-grade liposomes in batch and continuous mode.

Advantages of Ultrasonic Liposome Production

Ultrasonic liposome encapsulation is a technique used to encapsulate drugs or other therapeutic agents within liposomes using ultrasonic energy. When compared with other methods for liposome encapsulation, ultrasonic encapsulation has several advantages that make it the superior production technique.

• High loading, high entrapment efficiency: Ultrasonic liposome production is well known to produce liposomes with a high loading of active ingredients, e.g. vitamin C, drug molecules etc. At the same time, the sonication method shows a high entrapment efficiency. This means that a high percentage of the active substance is encapsulated by ultrasonication. In conclusion, this makes ultrasonication a highly efficient method for liposome production.
• Uniformly small liposomes: One advantage of ultrasonic liposome encapsulation is its ability to produce highly uniform liposomes with a narrow size distribution. Ultrasonic energy can be used to break up larger liposomes or lipid aggregates into smaller, more uniform liposomes. This leads to greater consistency in the size and shape of the liposomes, which can be important for drug delivery applications where the size of the particles can impact their pharmacokinetics and efficacy.
• Applicable to any molecules: Another advantage of ultrasonic liposome encapsulation is its ability to encapsulate a wide range of drugs and other therapeutic agents. The technique can be used to encapsulate both hydrophilic and hydrophobic drugs, which can be difficult to do with other methods. Additionally, ultrasonic energy can be used to encapsulate macromolecules and nanoparticles, which may be too large to encapsulate with other methods.
• Quick and reliable: Ultrasonic liposome encapsulation is also a relatively simple and quick process. It does not require the use of harsh chemicals or high temperatures, which can be detrimental to the therapeutic agents being encapsulated.
• Scale-up: Additionally, the technique can be easily scaled up for large-scale production, making it a cost-effective option for drug delivery applications.

In summary, ultrasonic liposome encapsulation is a superior technique for liposome encapsulation due to its ability to produce uniform liposomes with a narrow size distribution, encapsulate a wide range of therapeutic agents, and its simplicity and scalability.

After the formation of a lipidic film subsequent rehydration, sonication is used to promote the entrapment of active ingredients in the liposome. Additionally, sonication achieves the desired liposome size.

Ultrasonic Liposome Preparation for Pharmaceuticals and Cosmetics

Liposomes (lipid based vesicles), transferosomes (ultradeformable liposomes), ethosomes (ultradeformable vesicles with high alcohol content), and niosomes (synthetic vesicles) are microscopic vesicles, which can be artificially prepared as globular carriers into which active molecules can be encapsulated. These vesicles with diameters between 25 and 5000 nm are often used as drug carriers in the pharmaceutical and cosmetic industry, such as oral or topical drug delivery, genetherapy, and immunization. Ultrasonication is a scientifically proven method for highly efficient liposome production. Hielscher ultrasonicators produce liposomes with high loadings of active ingredients and superior bioavailability.

Liposomes and Liposomal Formulation

Liposomes are unilamellar, oligolamellar or multilamellar vesicular systems and are composed of the same material as a cell membrane (lipid bilayer). Regarding to their composition and size, liposomes are differentiated as follows:

• multi-lamellar vesicles (MLV, 0.1-10μm)
• small unilamellar vesicles (SUV, <100 nm)
• large unilamellar vesicles (LUV, 100–500 nm)
• giant unilamellar vesicles (GUV, ≥1 μm)

The main structure of liposomes consists of phospholipids. Phospholipids have a hydrophilic head group and a hydrophobic tail group, which consists of a long hydrocarbon chain.
The liposome membrane has a very similar composition as the skin barrier, so that they can be easily integrated into the human skin. As the liposomes fusionate with the skin, they can unload the entrapped agents directly to the destination, where the actives can fulfill their functions. Thus, the liposomes create an enhancement of skin penetrability/ permeability for the entrapped pharmaceutical and cosmetical agents. Also liposomes without encapsulated agents, the vacant vesicles, are potent actives for the skin, as the phosphatidylcholin incorporates two essentials, which the human organism cannot produce by itself: linoleic acid and choline.
Liposomes are used as biocompatible carriers of drugs, peptides, proteins, plasmic DNA, antisense oligonucleotides or ribozymes, for pharmaceutical, cosmetic, and biochemical purposes. The enormous versatility in particle size and in physical parameters of the lipids affords an attractive potential for constructing tailor-made vehicles for a wide range of applications. (Ulrich 2002)

Ultrasonic Liposomes Formation

Liposomes can be formed by the use of ultrasonics. The basic material for liposome preparation are amphilic molecules derived or based on biological membrane lipids. For the formation of small unilamellar vesicles (SUV), the lipid dispersion is sonicated gently – e.g. with the handheld ultrasonic device UP50H (50W, 30kHz), the VialTweeter or the ultrasonic reactor CupHorn – in an ice bath. The duration of such an ultrasonic treatment lasts approx. 5 – 15 minutes. Another method to produce small unilamellar vesicles is the sonication of the multi-lamellar vesicles liposomes.
Dinu-Pirvu et al. (2010) reports the obtaining of transferosomes by sonicating MLVs at room temperature.
Hielscher Ultrasonics offers various ultrasonic devices, sonotrodes and accessories and can thereby provide the most suitable ultrasonic setup for a highly efficient liposome encapsulation at any scale.

Ultrasonic Encapsulation of Active Substances into Liposomes

Liposomes works as carriers for active ingredients such as vitamins, therapeutic molecules, peptides etc. Ultrasound is an effective tool to prepare and form liposomes for the entrapment of active agents. Simultaneously, sonication assists the encapsulation and entrapment process so that liposomes with a high loading of active ingredients are produced. Before encapsulation, the liposomes tend to form clusters due to the surface charge-charge interaction of phospholipid polar heads (cf. Míckova et al. 2008), furthermore they have to be opened. By way of example, Zhu et al. (2003) describe the encapsulation of biotin powder in liposomes by ultrasonication. As the biotin powder was added into the vesicle suspension solution, the solution has been sonicated. After this treatment, biotin was entrapped in the liposomes.

Liposomal Emulsions with Ultrasonication

To enhance the nurturing effect of moisturizing or anti-aging cremes, lotions, gels and other cosmeceutical formulations, emulsifier are added to the liposomal dispersions to stabilize higher amounts of lipids. But investigations had shown that the capability of liposomes is generally limited. With the addition of emulsifiers, this effect will appear earlier and the additional emulsifiers cause a weakening on the barrier affinity of phosphatidylcholine. Nanoparticles – composed of phosphatidylcholine and lipids – are the answer to this problem. These nanoparticles are formed by an oil droplet which is covered by a monolayer of phosphatidylcholine. The use of nanoparticles allows formulations which are capable to absorb more lipids and remain stable, so that additional emulsifiers are not needed.
Ultrasonication is a proven method for the production of nanoemulsions and nanodispersions. Highly intensive ultrasound supplies the power needed to disperse a liquid phase (dispersed phase) in small droplets in a second phase (continuous phase). In the dispersing zone, imploding cavitation bubbles cause intensive shock waves in the surrounding liquid and result in the formation of liquid jets of high liquid velocity. In order to stabilize the newly formed droplets of the disperse phase against coalescence, emulsifiers (surface active substances, surfactants) and stabilizers are added to the emulsion. As coalescence of the droplets after disruption influences the final droplet size distribution, efficiently stabilizing emulsifiers are used to maintain the final droplet size distribution at a level that is equal to the distribution immediately after the droplet disruption in the ultrasonic dispersing zone.

Liposomal Dispersions using Ultrasonication

Liposomal dispersions, which are based on unsaturated phosphatidylchlorine, lack in stability against oxidation. The stabilization of the dispersion can be achieved by antioxidants, such as by a complex of vitamins C and E.
 

Liposome Formation via Reverse Phase Evaporation Method using Sonication

 
Ortan et al. (2002) achieved in their study concerning the ultrasonic preparation of Anethum graveolens essential oil in liposomes good results. After sonication, the dimension of liposomes were between 70-150 nm, and for MLV between 230-475 nm; these values were approximately constant also after 2 month, but inceased after 12 month, especially in SUV dispersion (see histograms below). The stability measurement, concerning essential oil loss and size distribution, also showed that liposomal dispersions maintained the content of volatile oil. This suggests that the entrapment of the essential oil in liposomes increased the oil stability.

Ortan et al. (2009): Stability of MLV and SUV dispersions after 1 year. Liposomal formulations were stored at 4±1 ºC.

Hielscher ultrasonic processors are the ideal devices for applications in the cosmetic and pharmaceutical industry. Systems consisting of several ultrasonic processors of up to 16,000 watts each, provide the capacity needed to translate this lab application into an efficient production method to obtain finely dispersed emulsions in continuous flow or in a batch – achieving results comparable to that of todays best high-pressure homogenizers available, such as orifice valves. In addition to this high efficiency in the continuous emulsification, Hielscher ultrasonic devices require very low maintenance and are very easy to operate and to clean. The ultrasound does actually support the cleaning and rinsing. The ultrasonic power is adjustable and can be adapted to particular products and emulsification requirements. Special flow cell reactors meeting the advanced CIP (clean-in-place) and SIP (sterilize-in-place) requirements are available, too.

Frequently Asked Questions on Liposomes

What Types of Liposomes are differentiated?

Liposomes are classified into different types based on their size and the number of bilayers they contain. These categories include:

• Small Unilamellar Vesicles (SUV): These are the smallest liposomes with a single lipid bilayer.
• Large Unilamellar Vesicles (LUV): Larger than SUVs, these also have a single bilayer.
• Multilamellar Vesicles (MLV): These contain multiple concentric bilayers.
• Multivesicular Vesicles (MVV): These are composed of multiple smaller vesicles within a larger vesicle.

 
Other specialized types include:

• PEGylated Liposomes: Liposomes modified with polyethylene glycol (PEG) to enhance stability and circulation time.
• Nanoliposomes: Very small liposomes, typically used for targeted drug delivery.

What Vesicle Structures can Liposomes exhibit?

Liposomes are further categorized based on their vesicle structure into seven main types:

• Multilamellar Large Vesicles (MLV): Contain multiple bilayers.
• Oligolamellar Vesicles (OLV): Have a few bilayers.
• Small Unilamellar Vesicles (SUV): Smallest with a single bilayer.
• Medium-sized Unilamellar Vesicles (MUV): Intermediate size with a single bilayer.
• Large Unilamellar Vesicles (LUV): Larger with a single bilayer.
• Giant Unilamellar Vesicles (GUV): Very large with a single bilayer.
• Multivesicular Vesicles (MVV): Multiple vesicles within a single large vesicle.

What are the Differences between Liposomes and Niosomes?

Liposomes and niosomes differ mainly in their composition:
Liposomes: Made from double-chain phospholipids, which can be either neutral or charged.
Niosomes: Made from uncharged single-chain surfactants and cholesterol.
Both structures are formed through sonication, which promotes the assembly of the bilayered vesicles.

What is the Ideal Size of a Liposome?

For therapeutic delivery, the ideal size of a liposome is theoretically between 50 and 200 nanometers in diameter. This size range optimizes stability and bioavailability. Sonication is commonly used to reduce the vesicle to the desired size.

Can Liposomes Carry Hydrophilic Drugs?

Yes, liposomes can carry hydrophilic drugs. They are valued in biomedical applications for their ability to encapsulate both hydrophobic and hydrophilic agents. Additionally, they offer high biocompatibility and biodegradability, making them effective delivery systems.

How to Make Liposomes?

The most common techniques of liposome preparation are the Thin-film method and the Reverse Phase evaporation method.
Thin Film Hydration Method:

1. Dissolve lipids in an organic solvent.
2. Evaporate the solvent to form a thin lipid film.
3. Hydrate the film with an aqueous solution using sonication in order to form multilamellar vesicles.

Reverse Phase Evaporation Method:

1. Dissolve lipids in water and ethanol.
2. Sonicate the solution at 60°C for about 10 minutes to create a lipid paste.
3. Cool the lipid slurry and add water or buffer dropwise while stirring.
4. Hydrate the suspension for 1 hour to form multilamellar vesicles.
5. Reduce the liposome size through further sonication.

What are Archaeosomes?

Archaeosomes are liposomes made from archaeal lipids, which are known for their stability and resistance to extreme conditions. These properties make archaeosomes particularly useful for drug delivery and vaccine development in challenging environments.

How are Archaeosomes prepared?

Sonication procedure according to Pise (2022): Archaeosomes may be made from the polar lipid fraction “PLF” of Sulfolobussolfataricus by sonication at 60°C without the need for external lipid replenishment. At 0°C, polar lipids from Sulfolobusacidocaldarius were effectively sonicated to form archaeosomes. BMD-loaded archaeosomes and conventional liposomes, as well as archaeal lipids isolated from Archaea H. salinarum and enriched with phosphatidylcholine, were made using sonication techniques. Sonicated vesicles were created for topical delivery by sonicating MLV dispersions at 80 percent amplitude for 4 minutes using a Hielscher UP50H probe-type sonicator (see picture on the left).

Literature/References