Active Drug Loading Technology for Liposomes

What is Active Drug Loading Technology?

The neutral molecular forms of weak acids or weak bases can traverse lipid bilayer membranes, whereas their dissociated forms cannot. Early studies by Jacobs et al. on the permeability of red blood cell membranes, as well as research by Chappel and Crofts on mitochondrial and chloroplast membranes, demonstrated that when there is a transmembrane pH gradient, carboxylic acids, acetates, amines, and other neutral molecular forms can rapidly traverse the phospholipid bilayer, facilitating the formation and maintenance of transmembrane concentration gradients. In 1976, Nichols and Deamer, among others, first utilized the pH gradient method to prepare catecholamine liposomes using active drug loading technology. In 1989, Gabizon employed the ammonium sulfate gradient method to prepare liposomes of two different membrane materials loaded with doxorubicin hydrochloride using active drug loading technology. Subsequently, researchers have utilized a series of ion gradients such as Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺, etc., for active drug loading technology to prepare liposomes.

Liposome for Active Drug Loading

Due to the excellent biocompatibility, biodegradability, and tumor targeting properties of liposomes, they are increasingly being regarded as ideal carriers for targeted drug delivery systems. However, in the current industrialization process, the main obstacles affecting liposome drug delivery systems are inadequate stability, insufficient drug loading capacity, and drug leakage. Amphiphilic drugs encapsulated through active loading typically achieve encapsulation rates of >80%, thus eliminating the need to remove free drugs. This method is suitable for amphiphilic drugs with certain lipid solubility under physiological pH conditions, with their pKa values falling within the physiological pH range to achieve high encapsulation rates. Transmembrane gradient active loading methods mainly include pH gradient method, ammonium sulfate gradient method, calcium acetate gradient method, and ion carrier gradient method. With further research and extensive application of liposome active loading technology (Remote/Active loading), this technology can produce liposomes with high drug loading capacity and high encapsulation rates, thereby improving liposome stability and reducing drug leakage. Therefore, the superiority of liposome active loading technology has attracted increasing attention and has become the preferred method for liposome preparation.

Methods for Active Drug Loading

pH Gradient Method

The pH gradient method involves adjusting the acidity or alkalinity of the internal and external aqueous phases of liposomes to establish a specific transmembrane pH gradient. This method takes advantage of the difference in dissociation states of weak acids or weak bases in different pH environments, allowing the drug to exist in a molecular form of low polarity in the external aqueous phase. When liposomes with established pH gradients approach or exceed the phase transition temperature, the phospholipid bilayer transitions from an ordered "gel crystalline phase" to a disordered "liquid crystalline phase". During this transition, the "all-trans conformation" carbon-carbon bonds in the phospholipid fatty chains convert to "neighbouring cross conformation", significantly increasing the rotational radius of the fatty chains. This leads to increased fluidity and permeability of the liposome membrane, facilitating the penetration of molecular drugs through the phospholipid bilayer. Meanwhile, highly polar ionic drugs are stably encapsulated within the inner aqueous phase of the liposome.

Ammonium Sulfate Gradient Method

The Ammonium Sulfate Gradient Method is suitable for amphiphilic weak basic drugs, particularly anthracycline antitumor drugs such as doxorubicin, epirubicin, idarubicin, and mitoxantrone. The mechanism of action of the Ammonium Sulfate Gradient Method is complex and can be explained by two main theories:

• Chemical equilibrium-driven mechanism: Ammonium sulfate ionizes and undergoes hydrolysis in water, resulting in molecules and ions with different permeability coefficients through the phospholipid bilayer. Ammonium ions have a larger permeability coefficient compared to hydrogen ions (H⁺), thus allowing them to diffuse rapidly into the external aqueous phase, while H⁺ ions are largely unable to pass through the phospholipid bilayer and remain in the inner aqueous phase. By reducing the concentration of NH₃ in the external aqueous phase, the NH₃ continuously spills out from the inner aqueous phase, leading to the accumulation of H⁺ ions and indirectly forming a "proton gradient" across the membrane, controlling the gradient size by adjusting the [NH₄⁺]out/[NH₄]n ratio. When amphiphilic weak basic drugs enter the inner aqueous phase of the liposome, the ionization process of the weak basic drugs consumes a large amount of H⁺, leading to an increase in the pH of the inner aqueous phase and causing more NH₃ to spill out continuously, thus driving the drugs into the liposome until the NH₄⁺ in the inner aqueous phase is depleted.
• Diffusion potential-driven mechanism: With the rapid overflow of NH₃ in the inner aqueous phase, H⁺ ions also tend to diffuse into the external aqueous phase, forming an "inner positive, outer negative" transmembrane diffusion potential, driving the weak basic drugs to cross the membrane and accumulate in the inner aqueous phase. This partially explains why the kinetics of anthraquinone drugs aggregating into liposomes using the Ammonium Sulfate Gradient Method is slower than simply acidifying the inner aqueous phase of liposomes (pH gradient method).

Calcium Acetate Gradient Method

The Calcium Acetate Gradient Method involves removing calcium acetate from the external aqueous phase through processes such as dialysis, ultrafiltration, or ion exchange, thereby establishing a transmembrane gradient of calcium acetate concentration. This method utilizes the ionization and hydrolysis of calcium acetate to facilitate the diffusion of a large number of acetate molecules (HAc) from the higher concentration inner aqueous phase of liposomes to the lower concentration external aqueous phase, raising the pH of the inner aqueous phase of liposomes and indirectly generating a transmembrane pH gradient. The permeation parameter of HAc is approximately 26.4 million times higher than that of Ca²⁺. Therefore, compared to HAc, Ca²⁺ molecules are nearly incapable of traversing the phospholipid bilayer and remain inside the liposome, while HAc participates in proton transport. The establishment of a transmembrane concentration gradient of calcium acetate (with higher concentration inside) results in HAc carrying a large number of protons from the inside of the liposome to the outside, creating an internal alkaline and external acidic pH environment. This pH environment provides efficient driving force for the loading and aggregation of weak acid drugs.

Factors Affecting Active Drug Loading

Transmembrane Gradient

According to the Henderson-Hasselbalch theory, each unit change in pH results in a tenfold change in the ratio of molecular to ionized drug concentration. Therefore, a three-unit transmembrane pH gradient theoretically maintains a 1000-fold difference in drug concentration. This applies similarly to ammonium sulfate and calcium acetate gradients. Lowering the pH of the internal buffer solution can increase the pH gradient, but excessive acidity may lead to hydrolysis of drugs and phospholipids, causing instability in liposomes.

Incubation Temperature and Time

While the Arrhenius equation is commonly used to explain chemical kinetic processes, it also applies to the process of active drug loading in liposomes. For a given liposome formulation and drug loading, the activation energy (Ea) across the membrane remains constant. With increasing temperature (T), the drug loading rate (k) increases, reducing the loading time, and liposomes reach equilibrium faster. When the incubation temperature exceeds the phase transition temperature of the liposomes, the permeability of the phospholipid bilayer increases, facilitating drug loading. However, increased permeability may disrupt the pH gradient during drug loading.

Drug-to-Lipid Ratio

The drug-to-lipid ratio is closely related to the encapsulation efficiency of liposomes. Generally, as the drug-to-lipid ratio decreases, the amount of drug loaded per liposome decreases, making it easier for the drug to be encapsulated within the liposome.

External Aqueous Phase pH Regulator

External aqueous phase pH regulators neutralize weak acids or weak bases in the external aqueous phase to maintain neutrality. Since ester bonds in phospholipid molecules are sensitive to alkaline conditions, the external aqueous phase should not be adjusted to alkaline pH levels.

Internal Aqueous Phase Buffering Capacity

As the active drug loading process progresses, the transmembrane ion gradient is gradually depleted. Therefore, the buffering capacity of the internal aqueous phase plays a crucial role in maintaining the transmembrane ion gradient during the drug loading process and significantly affects the encapsulation efficiency of the drug.

Drug LogP Value and pKa Value

In the context of determining liposome formulations and preparation processes, the properties of the drug significantly impact the encapsulation efficiency of liposomes. These properties include: (1) the partition coefficient (LogP), indicating the solubility of the drug in lipid-water phases; (2) the dissociation constant (pKa), indicating the drug's ability to dissociate and its polarity.