Optimizing Liposomes/LNPs: From Lipid Solvent Screening to Formulation Stability in PK Studies

Liposomes, as a flexible and efficient drug delivery system, have been widely used and developed in crucial areas such as cancer treatment and vaccine development (Figure 1).In recent years, two LNP-mRNA vaccines developed to combat COVID-19, Pfizer-BioNTech and Moderna vaccines-utilized liposomal delivery to achieve rapid approval and market availability, further advancing liposome research to new heights.

Through surface modifications, liposomes can be equipped with specific targeting ligands, enabling them to recognize and bind to specific cell receptors, much like the precise strikes of guided missiles. Liposomes can simultaneously deliver both lipophilic and hydrophilic molecules, achieving a combined drug therapeutic effect. Furthermore, liposomes can effectively deliver DNA and RNA as gene carriers. This article provides an overview of the components and classification of liposomes and the successful application of liposome drugs in preclinical research, with a focus on the challenges and strategies in pharmacokinetic studies.

Figure 1. Different research areas of liposomes ^[1,2]

Innovation and Development of Liposome Applications

Lipid-based nanocarrier systems(Figure 2), particularly liposomes and lipid nanoparticles (LNPs), are among the most successful nanocarrier systems to date. LNPs are an innovation of liposome technology, structurally similar to traditional liposomes but featuring multi-layer cores formed by interactions between negatively charged nucleic acids and cationic lipids, widely used for nucleic acid delivery and recently becoming a research hotspot in drug delivery.

Figure 2. Classification of lipid-based nanocarrier systems: 1. Liposomes 2. Lipid nanoparticles (LNPs) 3. Solid lipid nanoparticles (SLNs) 4. Hybrid lipid nanoparticles ^[2]

Liposomes are spherical vesicles composed of one or more bilayers of amphiphilic phospholipid molecules surrounding an aqueous core. As delivery systems, liposomes have achieved significant success in the pharmaceutical industry, especially in developing anticancer drugs and vaccines. Since 1995, more than 20 liposome drugs have been approved and marketed. As shown in Figure 3, breakthrough liposome products have promoted research in broader areas such as cancer, fungal and bacterial infections, eye diseases, pain relief, diagnostics, and immune suppression in organ transplantation. Moreover, with ongoing research, more liposome-related product pipelines are emerging in preclinical and clinical development stages, demonstrating the broad prospects of liposomes in drug delivery.

Figure 3. Breakthrough liposome products on the market ^[3]

Classification and Composition of Liposome Delivery Systems

Liposomes can generally be classified into conventional liposomes and specialized liposomes. The specialized liposomes, such as pH-sensitive liposomes, immunoliposomes, and cationic liposomes, are optimized to meet specific application needs.

Table 1. Classification of liposome delivery systems

The key components of liposomes mainly include phospholipids and cholesterol. Phospholipids, the primary components of biological cell membranes, exhibit good biocompatibility and degradability. Moreover, they are endogenous substances in vivo, typically non-toxic and non-immunogenic. The final formulation generally uses only 1 to 2 types of phospholipids to simplify the characterization and scale-up production of liposomal therapeutic products.

Phospholipids

As the core components of liposomes, the structure and properties of phospholipids directly influence the function and applications of liposomes. The structures of phospholipids have common features: hydrophilic polar head groups and hydrophobic tails. Hydrophobic fatty acid chains self-assemble in aqueous phases to form bilayers, constituting the basic structure of liposomes. Based on different phospholipid headgroups, phospholipids can be classified into positively charged phospholipids (lecithin (PC) and phosphatidylethanolamine (PE)), negatively charged phospholipids (phosphatidylglycerol and phosphatidylserine), and neutral phospholipids. The surface charge of liposomes, determined by the charge properties of their constituent phospholipids, influences their behavior in vivo studies, including drug delivery efficiency, cellular uptake, and interactions with other biological components.

Figure 4. Phospholipid structure^[4]

Helper Lipids and mRNA-LNPs

Helper lipids are non-primary phospholipid components used to enhance liposome performance, stability, and drug delivery capacity. Helper lipids typically work in conjunction with primary phospholipids to improve liposome characteristics. Helper lipids include PEGylated lipids and cholesterol, which have unique functional properties (Table 2).

Table 2. Composition of liposomes

LNPs are specialized liposomes for mRNA delivery, widely used in vaccine development and gene therapy. In LNPs, helper lipids play a crucial role, significantly improving LNP stability, efficacy, and biocompatibility. As shown in Figure 5, three marketed mRNA-LNPs primarily consist of ionizable cationic lipids, phospholipids, and helper lipids (cholesterol and PEGylated lipids).

Figure 5. Composition of three approved mRNA-LNPs ^[5]

Challenges and Optimization Strategies of Liposomes in Pharmacokinetic Studies

Liposomes hold broad application potential in preclinical formulation screening but face several challenges. The selection of lipid components is a critical step in preclinical liposome development, directly influencing liposome physicochemical properties, drug loading capacity, biocompatibility, stability, and therapeutic efficacy. During the early stages of liposome development, thorough safety evaluations of selected lipid components (such as anionic phospholipids, cationic lipids, helper lipids, etc.) are necessary to ensure that these components do not cause toxicity at anticipated usage levels.

Screening Solvents for Poorly Soluble Lipid Components in PK studies

The solubility of lipid components is primarily influenced by their chemical structure and composition. Due to the hydrophilic head and hydrophobic tail structure of phospholipids, their solubility in aqueous phases is typically low. In animal dosing experiments, addressing lipid solubility issues and considering solvent component tolerance ranges within animals are crucial. Additionally, the administration method (e.g., intravenous injection and subcutaneous injection) must meet in vivo formulation requirements, including osmotic pressure, stability, and precipitation post-intravenous administration.

Table 3. Common lipid components and solvents

For poorly soluble lipid components, solubility can be improved by adding solubilizers and surfactants (e.g., ethanol, Tween 80, etc.). It is essential to ensure that selected solvents meet the tolerance ranges within animals to ensure the safe and effective administration of lipid components during vehicle screening.

Storage stability Regulation of liposomes/LNP in Pharmacokinetic studies

Ensuring liposome physical and chemical stability before dosing in in vivo pharmacokinetic (PK) studies directly affects liposome drug release and bioavailability. Using inert gas protection during storage, such as dry ice, is common, but during liposome freeze-thaw processes, CO2 dissolution in solutions can lower pH, destabilizing liposomes. Air displacement methods to remove CO2 before preparation are needed. Moreover, temperature, pH, and light exposure are key environmental factors that affect the storage stability of liposomes.

• Temperature: Low-temperature storage generally improves liposome stability, but excessively low temperatures can cause liposome phase changes.

• pH: Liposome stability is affected by pH, typically controlled between 7-8. Appropriate buffers, such as Tris buffer, help maintain stable pH environments.

• Light exposure: Light exposure can degrade liposome components; hence, light protection measures or avoiding light exposure are necessary.

Pharmacokinetic studies of liposomes/LNP: Pre-administration assessment

Due to liposomes' instability, assessing liposome quality before dosing in preclinical formulation studies is crucial. Ensuring study validity and reliability involves the following steps:

• Physicochemical property assessment: Before dosing, check liposome formulation appearance, ensuring no precipitation, bubbles, or discoloration. Use dynamic light scattering (DLS) and other techniques to measure liposome size distribution and zeta potential, assessing corresponding physicochemical properties to ensure liposome stability.

• Drug concentration confirmation: Accurately measure drug concentration in liposomes to ensure it meets experimental design requirements.

• Lipid component identification and quantification: Identify and quantify individual lipid components in liposomes to ensure composition accuracy and consistency.

Common lipid components lack significant chromophores, making traditional UV detectors ineffective. Currently, high-performance liquid chromatography with charged aerosol detection (HPLC-CAD) and LC-MS are mainly used for lipid component detection. The United States Pharmacopeia (USP) draft specified HPLC-CAD for identifying and quantifying lipid component content in LNPs ^[8]. WuXi AppTec DMPK purchased four commercial lipids: hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), and cholesterol (CHO), the four most widely used lipids. We developed an HPLC-CAD method to detect individual lipid components and liposomes (Figure 6).

Figure 6. Chromatograms of blank solution and liposome sample solution

Liposome development in preclinical animal formulation faces multiple challenges, but by evaluating and optimizing lipid components, enhancing stability, and establishing a robust quality assessment system, liposome pharmacokinetics (PK) research efficiency and success rates can be effectively improved. Preclinical liposome PK experiments should particularly focus on liposome targeting characteristics, as most liposomes accumulate in liver tissue. Understanding drug clearance processes and obtaining liver drug concentrations are crucial. Relevant research can be referenced in: Application and Best Practices of Liver Biopsy in Non-Rodents.

Summary

WuXi AppTec DMPK has rich experience in solvent screening for lipid components with different properties, quickly identifying suitable solvents for lipid component administration. By strictly controlling liposome storage conditions, we ensure liposome stability before dosing. Our comprehensive detection and analysis platform ensures the accuracy of drug concentration in the liposome. To guarantee high-quality products before dosing (Figure 7).

In conclusion, WuXi AppTec DMPK has achieved systematic PK research, encompassing high-standard control of animal formulation quality, development of various dosing methods in animal experiments, acquisition and processing of bio-samples, and construction of bio-sample analysis platforms, providing strong support and assurance for the rapid advancement of liposomes and LNPs.

Figure 7. Testing platform for liposome animal formulation screening

Authors: Yifan Yang, Yu Chen, Chen Ning, Chao Zhang, Shoutao Liu

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