In recent years, liposomes, which are self-assembled and spherical bilayer structures, have been considered as one of the most outstanding drug delivery systems. Due to the amphiphilic characteristics of phospholipids (forming the bilayer), liposomes show many advantages. They are easy to modify, have a high capacity for carrying drugs, have a low toxicity, and are target-oriented.

CryoTEM has been used as one of the most important analytical tools to better characterize the properties of liposomes in terms of size distribution, circularity, lamellarity, packaging, stability, and bilayer thickness.

Percentage filled and empty liposomes

Encapsulant percentage (packaging) affects the efficiency of drug release. CryoTEM enables visualization of the encapsulant (e.g., Doxorubicin) inside the liposome (see Figure 1).

Figure 1. filled and emptyFigure 1. Morphology of an empty liposome and a liposome containing a drug crystal inside.

 

Our proprietary VAS software is used in combination with cryoTEM images to accurately provide statistical data on the distinction between filled and empty liposomes (see Figure 2).

Figure 2_Packaging analysis

Figure 2. A representative histogram (left) displaying the liposome packaging, as determined from semi-automated detection. The corresponding image (right) shows the detected and classified particles overlaid with blue/green/red outlines.

The percentage of encapsulation can be correlated to the efficiency of the drug release. This data is provided in the analytical report.

Download an example EM Services report here.

Lamellarity assessment

Lamellarity is a key factor of drug release efficiency and plays an important role in the mechanism of drug release kinetics. This is a vital feature that must be monitored during the manufacturing process. By using cryoTEM the distinction of liposomes based on their lamellarity is possible. Liposomes consisting of one lipid bilayer are considered as unilamellar liposomes, whereas liposomes composed of several lipid bilayers are defined as multilamellar. Liposomes encapsulating several smaller liposomes within a larger one, are defined as multiparticle (see Figure 3).

Figure 3. Lamellarity morphologyFigure 3. cryoTEM images displaying the morphology of liposomes with different lamellarities.

Figure 4. Lamellarity

Figure 4. A representative histogram (left) displaying the various lamellarities, as determined from semi-automated detection. The corresponding image (right) shows the detected and classified particles overlaid with red/yellow/green outlines.

Size distribution and internal volume 

The native conformation of a particle is conserved by using cryoTEM. Hence, cryoTEM is one of the few techniques that can reliably measure the size and the internal volume of liposomes.

Figure 5. size _ volume

Figure 5. Representative histograms displaying the size distribution (left) and internal volume (right), as determined from semi-automated detection of ca. 1,500 particles.

Circularity distribution

The circularity of the liposomes affects the efficiency of drug release in the final product. As the native conformation of particles is conserved in cryoTEM, the circularity can be measured precisely using our proprietary VAS software.

Figure 6. Circularity

Figure 6. A representative histogram (left) displaying the circularity of liposomes containing Doxorubicin, as measured using our proprietary VAS software. The corresponding image (right) shows a representative cryoTEM image on which the analysis was performed.

Bilayer thickness measurement

The thickness of the lipid bilayer affects the efficiency and efficacy of liposomal products. Therefore, it is an important parameter to monitor in the design of liposome-based drug carriers. By using both cryoTEM and our proprietary VAS software, the thickness of the lipid bilayer can be precisely measured.

Figure 7. membrane thickness

Figure 7. A representative histogram (left) displaying the liposomal bilayer thickness distribution, as measured using our proprietary VAS software. The corresponding cryoTEM images of a representative liposome are displayed at low magnification (middle) and high magnification (right).

FAQ

Can Vironova provide analysis on filled and empty liposomes?

Yes, Vironova's EM Services provides filled and empty analysis of liposomes by using cryoTEM. CryoTEM allows visualization of the encapsulant (e.g., Doxorubicin) inside the liposome. In the analytical report, we provide morphological observations, as well as statistical results (e.g., ratio of filled and empty liposomes).

Can Vironova perform the stability study of liposomes?

Yes, Vironova can perform the stability study of liposomes. The preferred types of analysis can be discussed with our senior scientists at EM services, please contact us for more information.

What is the volume and particle concentration of the liposomal product required to perform cryoTEM analyzes?

We require a minimum volume of 50 µL of sample and 2 mL of buffer, in case dilutions are needed. The sample concentration is dependent on different factors, particularly sample composition. Liposome samples require a lipid concentration of ca. 4 mg/mL for cryoTEM analysis.

At which temperatures can Vironova store liposome samples?

At our facilities, we can store your samples at four different temperatures: -86°C, -20°C, +4 °C, and room temperature.

 

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