Summary of Assays for Liposome Encapsulation Ratio

What is Liposome Encapsulation Ratio?

Encapsulation ratio plays a crucial role in the quality control of liposomes and is defined as the amount of drug encapsulated in the lipid bilayer as a percentage of the overall drug delivery. This percentage can accurately reflect the level of drug encapsulation in liposomes, which in turn guides the further optimization of the preparation process. The determination of encapsulation ratio is critical to ensure that liposomes exhibit optimal stability and efficacy during drug delivery. Through in-depth understanding and monitoring of the encapsulation percentage, fine tuning of the preparation process can be realized to meet the specific needs of drug delivery systems and enhance their potential for applications in medicine and biology.

Determination of Liposome Encapsulation Ratio

Due to variations in the drug properties being encapsulated and the diversity of liposome membrane materials, the optimal method for determining the encapsulation efficiency of each liposome often requires experimental investigation. The key to encapsulation efficiency determination lies in the separation of encapsulated drugs from the free, unencapsulated drugs, followed by the use of analytical techniques such as spectroscopy and chromatography to detect the concentrations of encapsulated or free drugs. The commonly employed methods for encapsulation efficiency determination are as follows.

• Centrifugation Method

Based on the different centrifugation speeds, the centrifugation method is divided into low-speed centrifugation and high-speed centrifugation. The low-speed centrifugation is suitable for lipophilic drugs. Its working principle is that lipophilic free drugs are insoluble in the aqueous phase medium required for dissolving liposomes. Instead, they remain suspended in the system. By using relatively low centrifugal force and shorter centrifugation time, these undissolved free drugs settle due to the centrifugal force, while the liposomes remain in the supernatant, achieving separation. High-speed centrifugation requires a centrifugation speed greater than 20,000 r/min, with a centrifugation time generally longer than 30 minutes. Heat is generated during the centrifugation process due to air friction, necessitating the use of a temperature-controlled centrifuge. In contrast to low-speed centrifugation, liposomes in high-speed centrifugation end up in the precipitate. As liposomes and undissolved free drugs sediment together, preventing their separation, this method is suitable for the determination of drugs with good water solubility, ensuring that free drugs remain in the supernatant. Additionally, it is important to note that the strong centrifugal force may lead to particle aggregation, disrupting the bilayer structure of liposomes and causing drug leakage.

• Ultrafiltration Centrifugation Method

The ultrafiltration centrifugation method involves placing liposomes into ultrafiltration tubes equipped with ultrafiltration membranes and subjecting them to centrifugation at an appropriate speed. Under the influence of centrifugal force, free drugs can pass through the ultrafiltration membrane, while liposomes are retained, achieving their separation. The ultrafiltration centrifugation method is commonly used to determine the encapsulation efficiency of liposomes for water-soluble drugs. However, the presence of concentration polarization limits the application of ultrafiltration. Concentration polarization occurs because, during the ultrafiltration process, solvents and small molecular solutes can permeate through the ultrafiltration membrane, while large molecular solutes are trapped within the membrane. This leads to an increase in the concentration of large molecular solutes on the surface of the ultrafiltration membrane, causing an elevation in the osmotic pressure near the membrane. This impedes the continued diffusion of the solution towards the ultrafiltration membrane, subsequently reducing the membrane permeability for solvents and small molecules.

• Pectin Gel Column Method

This is an approach that utilizes the gel structure formed by pectin particles to achieve substance separation. When pectin particles swell, they create a gel with internal pores of specific sizes. Small molecules of free drugs can enter these pores, thereby being retained to a certain extent within the gel. In contrast, the particle size of liposomes is larger than the gel pore size, preventing them from passing through these openings. By exploiting the differential retention effects of pectin gel on liposomes and free drugs in a column, effective separation of the two can be achieved. The key to this method lies in leveraging the characteristics of pectin gel, coupled with skillful control of elution conditions. This ensures that free drugs and liposomes exhibit distinct migration behaviors in the column, facilitating their separation and purification.

• Microcolumn Centrifugation Method

In comparison to the Pectin Gel Column Method, the Microcolumn Centrifugation Method significantly reduces the volume of elution fluid, thereby avoiding leakage of liposomes due to dilution effects. In this method, well-swollen pectin gel or pre-treated ion exchange resin is packed into a syringe, equilibrated and centrifuged repeatedly to form a dried microcolumn. Subsequently, a liposome suspension is added to the top of the column, followed by a few minutes of static incubation. Afterward, elution fluid is introduced, and by setting an appropriate centrifugation speed, liposomes are washed out. This method may not be suitable if the gel strongly adsorbs liposomes. Care should be taken in selecting the centrifugation speed during the experiment, as both excessive and insufficient speeds can lead to the rupture of the gel column, and excessively high speeds may also result in the elution of free drugs.

• Dialysis and Reverse Dialysis Method

Dialysis involves placing liposomes in a selectively permeable bag, using water or PBS buffer as the medium. Free drugs move into the medium due to concentration differences, while liposomes, being larger, remain inside, achieving separation. However, lipophilic drugs may aggregate on the membrane surface, blocking pores. The need for a large dialysis medium in experiments leads to system dilution, disrupting liposome equilibrium and potentially causing leakage. Prolonged dialysis further challenges liposome stability. Reverse dialysis addresses these issues by placing liposomes outside the bag with a reduced medium volume, preventing dilution-induced leakage.

• Ichthyosin Coagulation

Ichthyosin is a basic protein, positively charged, which can combine with negatively charged or neutral liposomes to form a polymer with increased density. After centrifugation, the liposome-ichthyosin polymer is precipitated, thus separating it from the free drug.

• Solid Phase Extraction

Using the principle of chromatographic adsorption, the free drug is adsorbed on the stationary phase of the SPE column, which has similar polarity, while the liposomes are not retained on the SPE column, and a small amount of water can be used to elute them. Solid phase extraction provides a high degree of separation between liposomes and free drug because it relies on the difference in adsorption capacity of a more characteristic and stable adsorbent to achieve the separation. However, the method is complex, and the strength of the adsorption between the drug and the stationary phase of the SPE column is affected by a variety of factors, requiring extensive experimentation to figure out the optimal experimental conditions.

• Fluorescence Method

Most of the encapsulation ratio determination methods currently used require the separation of liposomes and free drug, while the fluorescence method does not require separation, as long as the use of encapsulated drug and free drug with different fluorescence characteristics, and compare the change in fluorescence intensity before and after the emulsion breakage of liposomes, the encapsulation ratio can be calculated.

How to Choose the Encapsulation Ratio Measurement Method?

• Low-speed centrifugation is suitable for lipid-soluble drugs, the operation is simple, and it is not easy to destroy the structure of the liposome membrane, but the liposome and the free drug often can not be completely separated.
• Ultracentrifugation is mostly used for water-soluble drugs, and requires a certain degree of hardness of the liposome membrane structure, able to withstand the effects of high speed. The ultrafiltration centrifugation method is more widely applicable, and there are various types of ultrafiltration membrane materials and molecular weight cut-offs to meet the multiple needs of testers. The disadvantage is that concentration polarization may occur, which can be avoided by diluting the liposome solution to be tested to some extent.
• Dextran gel column method and microcolumn centrifugation method both utilize the principle of molecular exclusion to separate, but if the drug is not retained on the gel column or adsorbed too strongly, these two methods cannot be used. Dextran gel column method may lead to liposome leakage due to dilution of the liposome system by addition of large amount of eluent. However, the microcolumn centrifugation method, with its small column volume and small volume of eluent added, does not cause liposome leakage, but the centrifugal speed needs to be examined to obtain the best separation.
• Dialysis, as a common means to determine the encapsulation rate, is suitable for water-soluble drugs, but the dialysis time is generally longer than 36 h, which requires high liposome stability. In addition, the addition of a large amount of dialysis medium will also dilute the liposome system and may lead to liposome leakage.
• The reverse dialysis method greatly reduces the amount of dialysis medium and better avoids liposome leakage due to dilution.
• Ichthyosin coagulation is only suitable for negatively charged and neutral liposomes.
• Although the solid phase extraction method has good results in separating liposomes and free drugs, the experimental method is complicated and it is not easy to figure out the optimal separation conditions.
• Fluorescence method can be used to determine the encapsulation rate under the coexistence of liposome and free drug, without the requirement of separating the two, but generally need to introduce fluorescent indicator, some kind of fluorescence reaction to calculate the encapsulation rate.