Ultrasonic Liposome Formation: Methodology and Advantages

June 20, 2024, Kathrin Hielscher, published in Hielscher News

Liposomes are spherical vesicles composed of lipid bilayers, widely used in drug delivery, cosmetics, and food industries due to their biocompatibility and ability to encapsulate both hydrophilic and hydrophobic substances. Using high-intensity ultrasound for liposome formation is one of the most common techniques for liposomal encapsulation. Known for its efficiency, scalability, and ability to produce liposomes with controlled size and a high encapsulation efficiency, sonication offers numerous additional advantages when compared to alternative methods of liposome production. This article introduces you into the methodology of ultrasonic liposome formation, its advantages, and diverse applications in supplements, pharmaceuticals, therapeutics, and functional foods.

Sonication for Liposome Formation

Probe-type sonicators are an essential tool when it comes to the production of liposomes loaded with active ingredients. Here, we give you an introduction into how liposomes are formed and loaded using the ultrasound-assisted method.

  1. Preparation of Lipid Solution:
    The process begins with the preparation of a lipid solution. Common lipids used include phosphatidylcholine, cholesterol, and other phospholipids. These lipids are dissolved in an organic solvent such as chloroform or ethanol.
  2. Formation of Lipid Film:
    The lipid solution is then evaporated under reduced pressure (vacuum) using a rotary evaporator to form a thin lipid film on the walls of a round-bottom flask. This step ensures the removal of organic solvents, leaving behind a dry lipid film.
  3. Hydration of Lipid Film:
    The dried lipid film is hydrated with an aqueous solution, which may contain the active substance to be encapsulated. This step results in the formation of multilamellar vesicles (MLVs). The hydration process typically involves vortexing or gentle agitation at a temperature above the lipid transition temperature.
  4. Sonication:
    The MLVs are then subjected to ultrasound using a probe-type sonicator. The ultrasonic waves induce cavitation, creating microbubbles that collapse and generate shear forces. This process causes sonoporation so that the liposomes are efficiently loaded resulting in a high entrapment efficiency (EE%). The increased permeability due to sonoporation facilitates the diffusion of encapsulants into the liposomes. Once the sonication process stops, the lipid bilayers quickly reassemble, trapping the encapsulated substances inside.
    Additionally, sonication breaks down the MLVs into smaller unilamellar vesicles (ULVs) or small unilamellar vesicles (SUVs), with sizes typically ranging from 20 to 200 nm. Parameters such as sonication time, power, and temperature are optimized to achieve the desired liposome size and encapsulation efficiency.
  5. Purification and Characterization:
    Following sonication, the liposome suspension is often filtered or centrifuged to remove unencapsulated material and larger vesicles. The resulting liposomes are characterized using techniques such as dynamic light scattering (DLS) for size distribution, zeta potential analysis for surface charge, and transmission electron microscopy (TEM) for morphology.
The ultrasonic method ensures the formation of liposomes with specific features by promoting the encapsulation of active ingredients and by adjusting their size and lamellarity through controlled processing steps. Hielscher sonicators are renowned for best results in liposome formation.

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 probes use the forces of acoustic cavitation to encapsulate bioactive compounds into nanoliposomes.

Probe-type sonicator UP400St for ultrasonic liposome encapsulation.

Scientifically Proven

Structure of a liposome: amphiphilic bilayer with hydrophilic and hydrophobic tails and its aqueous core containing the encapsulated bioactive molecules. Probe-type sonicators have been quickly adopted as reliable technique for liposome preparation and is nowadays widely used in liposome production in research and commercial production. The efficiency and reliability of ultrasonic liposome formation and the loading of liposomes with active ingredients was demonstrated in research studies for many formulations. Below are two short overviews over the liposomal encapsulation using probe-type sonication.
 
Hadian et al. (2014) investigated the efficiency of sonication encapsulating omega-3 fatty acids from fish oil (DHA and EPA) in liposomes. In order to evaluate the efficiency and quality of entrapment, they compared the ultrasonic liposome preparation method with liposome extrusion. Using the Hielscher probe-type sonicator UP200S, the researchers find that probe-type sonication “of pre-formed liposomes facilitates a significant loading of DHA and EPA into the nanoliposomal membrane. Probe sonication technique outperformed other methods.” Liposomes prepared by probe-type sonication were spherical in shape and maintain high structural integrity.
 
Paini et al. (2015) developed a simple, yet highly efficient method using sonication in order to prepare apigenin loaded liposomes with food-grade rapeseed lecithin in an aqueous medium without utilizing any organic solvent. Using the 400 watts probe-type sonicator model UP400S (Hielscher Ultrasonics), an encapsulation efficiency of more than 92% was achieved. Liposome size can be precisely controlled by adjusting sonication amplitude and process time. Analysis showed that liposomal apigenin structures had a high Zeta potential, a good polydispersity index and maintained stable after encapsulation process.

Advantages of Ultrasonic Liposomal Encapsulation

Liposome preparation techniques vary widely, each with its own set of advantages and limitations. Ultrasonic liposome preparation stands out for several reasons as it provides a very high entrapment efficiency (EE%), excellent control over liposome size, its reliability when it comes to reproducible results, as well as the linear scalability to larger volumes.

  1. Enhanced Encapsulation Efficiency:
    Ultrasonication offers high encapsulation efficiency for both hydrophilic and hydrophobic compounds. The intense shear forces and cavitation facilitate the uniform distribution of the encapsulant within the liposomal bilayer or aqueous core.
  2. Controlled Size Distribution:
    The ability to precisely control sonication parameters allows for the production of liposomes with narrow size distributions, essential for consistent drug delivery and bioavailability.
  3. Scalability and Reproducibility:
    Ultrasonic liposome formation is highly scalable, making it suitable for both laboratory-scale and industrial-scale production. The reproducibility of the process ensures consistent quality across batches.
  4. Minimal Use of Organic Solvents:
    Compared to other liposome preparation methods, ultrasonication requires significantly less organic solvents, reducing potential toxicity and environmental impact.
  5. Versatility:
    This technique is versatile, accommodating a wide range of lipids and encapsulants, thus expanding its applicability across various industries.
Ultrasound is a reliable technique to prepare liposomes and nano-liposomes with a high entrapment efficiency and stability

Ultrasonic glass flow cell for liposomal encapsulation of bioactive substances

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Applications in Supplements, Pharmaceuticals, Therapeutics, and Functional Foods

Hielscher sonicators are used in research and in commercial production to produce liposomes in food- and pharma-grade quality. Ultrasonically produced liposomes offer high bioavailability, can carry high loads of active ingredients, high encapsulation efficiency (EE%) and stability. Additionally, sonication leads to a uniform size distribution. Fulfilling all these quality criteria, ultrasonically formulated liposomes are ideal carrier for active pharmaceutical ingredients (APIs) and phytochemicals in medicines, therapeutics, dietary supplements, functional foods and even cosmetics.

  1. Supplements:
    Ultrasonic liposomal encapsulation is used to enhance the bioavailability of dietary supplements and nutraceuticals. Vitamins, minerals, and herbal extracts encapsulated in liposomes show improved absorption and stability, leading to better efficacy. For instance, liposomal vitamin C and curcumin supplements are popular for their enhanced therapeutic benefits.
  2. Pharmaceuticals:
    In the pharmaceutical industry, liposomes serve as carriers for drug delivery, improving the solubility, stability, and targeting of drugs. Ultrasonically prepared liposomal formulations are employed for the delivery of chemotherapeutic agents, antibiotics, and vaccines. Liposomal doxorubicin, for example, reduces the cardiotoxicity associated with conventional doxorubicin therapy.
  3. Therapeutics:
    Therapeutics benefit from liposomal encapsulation by achieving controlled release and targeted delivery. Liposomes can cross biological barriers, such as the blood-brain barrier, enabling the delivery of drugs to specific tissues or cells. Ultrasonically produced nano-liposomes have a very high bioavailability as their nano-size allows them to enter targeted tissues and cells. This is particularly advantageous in treating neurological disorders and cancers.
  4. Functional Foods:
    In the functional food industry, liposomes enhance the delivery of bioactive compounds, such as omega-3 fatty acids, probiotics, and antioxidants. These encapsulated bioactives exhibit improved stability and bioavailability, contributing to better health outcomes. For example, ultrasonically-assisted liposomal encapsulation of polyphenols in beverages helps preserve their antioxidant properties.
  5. Cosmetics:
    Cosmetic formulations, also are referred to as cosmeceuticals, benefit from liposomal encapsulation technique, as liposomes enhance the encapsulation efficiency of anti-aging substances such as antioxidants, providing better protection against oxidative stress. The bilayer structure shields sensitive compounds from environmental factors, such as UV radiation and pollution, which can degrade antioxidants. The enhanced encapsulating performance of sonicated liposomes allows for the stable incorporation of volatile and sensitive compounds, which are otherwise challenging to deliver effectively.

 

Ultrasonic liposome formation is a robust and versatile technique with significant advantages in encapsulation efficiency, size control, scalability, and environmental sustainability. Its application spans various industries, from enhancing the bioavailability of supplements to improving the delivery and efficacy of pharmaceuticals and therapeutics. As research and technology advance, the potential for ultrasonic liposomal encapsulation to innovate and improve product formulations continues to expand, promising exciting developments in the fields of health, medicine, nutrition and cosmetics.

Nanocarriers Formulated by Sonication

Besides liposomes, sonication is also successfully used for the formulation and loading of various other nano-carrier forms such solid-lipid nanoparticles, nano-structured lipid carriers, and nanoemulsions. Hielscher sonicators promote the efficient formation and loading of these nano-carriers with bioactive ingredients. Known for their state-of-the-art technology, Hielscher sonicators are used worldwide in food, pharmaceutical and cosmetic production.

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