Ultrasonically-Assisted Thin Film Hydration for Liposome Preparation

Liposomes are spherical vesicles composed of one or more phospholipid bilayers enclosing an aqueous core. Due to their ability to encapsulate hydrophilic, lipophilic, and amphiphilic compounds, liposomes are widely used in pharmaceutical, nutraceutical, cosmetic, and food applications. Their biodegradability, biocompatibility, and non-immunogenic nature make them particularly attractive delivery systems.
Among the various liposome fabrication methods, thin film hydration (TFH) remains one of the most established and versatile techniques. When combined with controlled sonication, this method enables the production of nano-sized liposomes with improved homogeneity, encapsulation efficiency, and stability.

Role of Ultrasound in Thin Film Hydration

Thin film hydration initially yields predominantly multilamellar vesicles (MLVs) with relatively large diameters and broad size distributions. Due to their size, these vesicles exhibit limited cellular uptake and reduced bioavailability, particularly in applications requiring efficient membrane interaction or tissue penetration. Therefore, probe sonication is applied as a controlled size-reduction step to convert MLVs into small unilamellar or nano-sized liposomes with a narrower polydispersity index. Ultrasonically downsized nano-liposomes provide a substantially higher surface-area-to-volume ratio, improved colloidal stability, and enhanced cellular internalization, resulting in superior bioavailability and delivery performance.

1. Mechanism of Action
   High-intensity ultrasound generates acoustic cavitation – microscopic bubble formation and collapse – producing localized shear forces, microstreaming and transient high pressure gradients.

   These effects result in:

   • Disruption of large multilamellar vesicles
   • Bilayer fragmentation and reorganization
   • Formation of small unilamellar vesicles (SUVs)
   • Narrower particle size distribution

   In nano-liposomal systems prepared via thin film + ultrasound dispersion, average particle diameters in the range of ~80 nm with low polydispersity are reliably achieved.

2. Process Advantages
   Ultrasonically-assisted TFH provides:
   • Reduced particle size (nanometer range)
   • Lower polydispersity index (PDI)
   • Increased encapsulation efficiency
   • Improved colloidal stability
   • Enhanced thermal and oxidative stability of encapsulated compounds

   These improvements are critical for stabilizing oxidation-sensitive bioactives such as polyunsaturated fatty acids.

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

General Instruction: Ultrasonically-Assisted Thin Film Hydration Liposome Preparation

1. Materials and formulation
   1. Select phospholipid (e.g., lecithin / phosphatidylcholine) and optionally cholesterol.
   2. Choose lipid ratio based on stability needs.

   A lecithin/cholesterol ratio around 40:20 produced stable nanoliposomes with high zeta potential and low PDI in a food-related system.

2. Lipid dissolution (organic phase)
   1. Dissolve phospholipid + cholesterol in a volatile organic solvent (e.g., chloroform).
   2. If functional additives require a co-solvent, dissolve them separately (e.g., methanol) and combine.

   This approach is explicitly described for thin-film hydration liposomes using chloroform (lipids) and methanol (additive).

3. Thin film formation
   1. Transfer lipid solution into a round-bottom flask.
   2. Remove solvents using rotary evaporation at moderate temperature (e.g., 40 °C) under reduced pressure until a dry homogeneous film forms.
4. Hydration of lipid film
   1. Hydrate the lipid film with an aqueous phase containing the active (or buffer if producing empty liposomes).
   2. Hydrate under stirring and elevated temperature (above lipid phase transition if needed).

   Example from a biomedical formulation: hydration with HEPES buffer (pH 7.4) and stirring at 60°C for 6 h.

5. Ultrasonic size reduction
   After hydration, the dispersion typically contains multilamellar vesicles and must be downsized.

   General recommedations for the sonication step:

   • Use probe-type ultrasonication.
   • Apply pulsed sonication to control temperature.
   • Keep the sample in an ice bath or use external cooling.

   Exemplary sonication protocol with the Hielscher sonicator UP200Ht:
   15 min total, 10 s ON / 5 s OFF, 100 W, 100% amplitude. (cf. Truszkowska et al., 2025 and Ahmadi et al, 2021)

6. Optional post-processing
   Depending on the application:
   • Remove unencapsulated compound by centrifugation/filtration.
   • Wash and resuspend in fresh buffer.

   Example: unbound material removal by centrifugation and resuspension is described for liposome purification.

7. Characterization
   The attached studies consistently treat the following as essential quality metrics:
   • Particle size (nm)
   • Poly Dispersity Index (PDI)
   • Zeta potential
   • Encapsulation efficiency
   • Morphology (TEM/SEM)
   • Stability during storage

   Example results demonstrating good nano-liposome quality include:
   Size ≈82 nm, PDI ≈0.06, zeta potential ≈−56 mV, encapsulation efficiency ≈76.5%.

   Practical Notes
   Cholesterol improves stability. The studies explicitly indicate that adding cholesterol can improve liposome stability by inhibiting phospholipid phase transition.

Advantages of Hielscher Ultrasonic Systems for Liposome Production

Hielscher Ultrasonics offers advanced ultrasonic processors specifically engineered for laboratory, pilot, and full-scale industrial liposome production. These systems are particularly well suited for ultrasonically assisted thin film hydration, where precise control of acoustic energy is critical for achieving defined particle size, narrow size distribution, and reproducible product quality.
At laboratory scale, Hielscher sonicators provide precise amplitude control, reproducible energy input, programmable pulse operation, and integrated temperature monitoring. This level of control ensures consistent cavitation intensity and highly reproducible nano-liposome size with excellent batch-to-batch uniformity. Such process stability is essential during formulation development, especially when optimizing key parameters such as sonication time, acoustic power, lipid composition, and encapsulation efficiency.

Industrial Liposome Production

For industrial manufacturing, Hielscher systems are designed for linear scalability, allowing process parameters established at lab scale to be transferred to pilot and production units while maintaining equivalent energy density and process conditions. Continuous-flow ultrasonic reactors enable high-throughput liposome production with uniform energy distribution and reduced processing times. The systems are built for robust, 24/7 operation with high energy efficiency and low maintenance requirements. Furthermore, they can be integrated into GMP-compliant production environments, including closed-loop systems, automated process lines, and cleanroom installations. These characteristics make Hielscher ultrasonic technology suitable for pharmaceutical, nutraceutical, and food-grade liposome manufacturing at industrial scale.

Ultrasonically-assisted thin film hydration represents a highly efficient, controllable, and scalable method for nano-liposome production. Scientific evidence demonstrates that combining TFH with ultrasound significantly improves particle size distribution, encapsulation efficiency, and stability of sensitive bioactive compounds.
Hielscher sonicators provide:

• Precise energy control
• Reproducible nanoscale results
• Seamless scale-up from lab to industrial production
• Continuous processing capability
• Robust and GMP-compatible operation

For research laboratories developing advanced liposomal formulations and for industrial manufacturers producing high-value pharmaceutical or nutraceutical products, Hielscher ultrasonic systems offer a technically superior and economically scalable solution for ultrasonically-assisted thin film hydration liposome preparation.

The table below gives you an indication of the approximate processing capacity of our ultrasonicators:

Comparison to Other Ultrasonically-Assisted Liposome Methods

Although thin film hydration is widely used – especially in lab and R&D-, ultrasound also enhances several other liposome preparation methods:

• Reverse Phase Evaporation – Ultrasound improves emulsification and vesicle formation.
• Ethanol Injection Methods – Sonication refines particle size and reduces aggregation.
• Direct Lecithin Dispersion – Ultrasound enables rapid liposome formation from phospholipid suspensions.
• Sonoporation of Pre-Formed Liposomes – Acoustic energy temporarily increases membrane permeability for active loading.
• Post-Formation Size Reduction – Sonication is routinely applied to reduce multilamellar vesicles to nanoscale systems.

Thus, ultrasound is not merely an auxiliary step but a central enabling technology across liposomal production strategies.

Design, Manufacturing and Consulting – Sonicators Made in Germany

Hielscher ultrasonicators are widely recognized for their high quality, robust engineering, and advanced design standards. Their durability and user-friendly operation enable smooth integration into industrial production facilities. Even under harsh operating conditions and demanding environments, Hielscher ultrasonicators deliver reliable performance and consistent results.

Hielscher Ultrasonics is an ISO-certified company and places strong emphasis on manufacturing high-performance ultrasonic systems that combine state-of-the-art technology with practical usability. All Hielscher ultrasonicators are CE compliant and additionally meet the requirements of UL, CSA, and RoHS.