In Vivo Drug Delivery Process of Lipid Nanoparticles (LNPs)

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Lipid Nanoparticles (LNPs) for Drug Delivery

Lipid-based nanodrug is one of the classic drug delivery systems, characterized by good biocompatibility and biodegradability. In clinical settings, it can effectively reduce adverse reactions of anti-tumor and anti-infective drugs. However, it has yet to fully meet clinical demands for enhancing therapeutic effects, and its clinical application remains highly limited. The intracellular processes of lipid-based nanodrug are complex, and after interacting with the body, the carrier itself can induce biological effects of unknown mechanisms, leading to drug performance inside the body deviating from the intended design and severely hindering clinical translation. In-depth research on the intrinsic properties of lipid-based nanodrug, intracellular delivery processes, and body regulation mechanisms not only provides guidance for rational carrier design but also promotes the clinical translation and precise medication of novel lipid-based nanodrug.

Schematic diagram of lipid nanoparticlesSchematic diagram of lipid nanoparticles

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Lipid Nanodrug Intracellular Delivery Process

Absorption into Blood

Apart from IV infusion and IV push, lipid nanodrug's versatility as a drug carrier has been extensively explored through oral, intramuscular (subcutaneous, intradermal, intramuscular), nasal inhalation, and ocular instillation routes. Regardless of the route, it targets tissues via bloodstream or lymphatic circulation. Orally, it adheres to intestinal mucosa, crosses the mucous layer, and is taken up by Peyer's patch M cells, entering the bloodstream via the lymphatic system. Intramuscularly, some particles enter the bloodstream through capillaries, while most are absorbed by lymphatic vessels. Mucosal routes primarily act locally, with a small fraction possibly entering the bloodstream through nasal mucosa or retina.

Blood Circulation

After entering the bloodstream, lipid nanodrug particles exhibit a high surface Gibbs free energy, leading to the adsorption of biomolecules like proteins, lipids, and carbohydrates from the plasma, forming a biomolecular corona. Proteins, being the most abundant solid component in plasma, primarily constitute this corona. Within 30s, high-abundance plasma proteins bind to the particle surfaces, gradually replaced by proteins with higher affinity over time. Some particles are engulfed by circulating leukocytes and transported to other areas during cell migration, while the rest continue circulating throughout the body via the bloodstream.

Vascular Permeation

Lipid nanodrug can extravasate from the bloodstream through various pathways: passive transport, active transport, and leukocyte-mediated transport. Passive transport relies on the size of nanoparticle relative to gaps in organ capillaries. Active transport involves modification of nanodrug with ligands or plasma proteins, facilitating receptor-mediated endocytosis by endothelial cells. Leukocytes, with their phagocytic and chemotactic abilities, can directly capture nanoparticles and transport them into tissues. Vascular permeation is crucial for therapeutic efficacy but can lead to adverse reactions in non-target sites.

Interstitial Transport

After exiting blood vessels, lipid nanodrug encounters the extracellular matrix (ECM), a complex network of macromolecules like collagen and glycosaminoglycans. This mesh-like structure limits further penetration of large nanoparticles and their contact with cells.

Cellular Binding

Lipid nanodrug, resembling cell membranes, can fuse directly with cells to release encapsulated drugs or genes. Surface modifications enable entry via receptor-mediated phagocytosis, triggering therapeutic effects or immune responses.

Intracellular Transport

Lipid nanodrug internalized via different endocytic pathways undergo distinct intracellular routes. Those internalized through clathrin-mediated endocytosis pass through early endosomes and eventually fuse with lysosomes. Caveolin-dependent endocytosis leads to entry into subcellular compartments, bypassing lysosomes and reaching the Golgi apparatus and endoplasmic reticulum. Macropinocytosis results in two pathways: a small portion enters recycling compartments and is secreted, while the majority travels to late endosomes for degradation or exits via the endoplasmic reticulum-Golgi or exocytic pathways.

In vivo Clearance

Lipid nanodrug in peripheral blood is primarily eliminated through renal and hepatic-biliary pathways. Some particles are degraded and excreted via urine, while others are phagocytosed by liver cells, degraded in lysosomes, and excreted into the intestine through bile.

Factors Influencing the Intracellular Delivery Process of Lipid Nanoparticles

Protein Corona

The protein corona is a key factor influencing the interaction between lipid nanoparticles and biological systems. It affects the intracellular delivery process of drugs through pathways such as altering the dispersion state, structure, conformation, and activity of proteins adsorbed on the surface of drugs, as well as regulating immune responses. The composition of the protein corona is influenced by the characteristics of nanoparticles and the physiological environment, thereby regulating the delivery effectiveness of lipid nanoparticles.

Lipid Nanoparticle Intrinsic Properties

The particle size of lipid nanoparticles is one of their important properties, directly affecting their tissue distribution and lysosomal escape. Lipid particles with a diameter smaller than 400nm can enter tumor tissues through the fenestrations in tumor capillaries, while those with a diameter of 100nm are retained in tumor tissues due to spatial constraints. However, lipid particles smaller than 30 nm, although enhancing penetration after intravenous administration, exhibit poor lysosomal escape capability and gene expression efficiency. Particle size also affects protein corona adsorption, indirectly influencing blood circulation time and in vivo clearance rate. Lipid nanoparticles of different sizes have varying impacts on their fate in vivo.

The shape of lipid nanoparticles significantly affects their blood circulation time, vascular permeability, and cellular internalization. Spherical particles have difficulty in approaching the blood vessel wall and contacting endothelial cells, while disc-shaped nanoparticles are influenced by torque forces, increasing the chances of contact with endothelial cells and providing a larger attachment surface area, which is conducive to firm adhesion to the blood vessel wall.

The surface potential and lipid composition of lipid nanocarriers directly influence their interaction with cells and drug delivery efficiency. Cationic lipids have the ability to electrostatically attract cell membranes and endosomal membranes, enhancing cellular uptake and drug release, particularly suitable for nucleic acid drug delivery. Additionally, cationic lipids can electrostatically attract biomolecules on the surface of endothelial cells in pathological tissues, promoting drug accumulation in lesion areas.

In Vivo Environment

The immune system, composed of immune organs, immune cells, and immune-active substances, plays a crucial role in monitoring, defending against, and regulating immune responses in the body. The intracellular delivery of lipid nanoparticles is tightly regulated by the immune system. Immune organs comprise various immune cells such as macrophages and dendritic cells, which have phagocytic and antigen-presenting functions. Immune-active substances such as antibodies and complement components have significant impacts on the intracellular delivery of lipid nanoparticles, potentially leading to drug clearance or adverse reactions.

In pathological states such as tumors or infections, changes in the physiological environment directly affect the delivery process of lipid nanoparticles. Increased tumor vascular density and permeability allow larger-sized nanoparticles to extravasate into tumor tissues, while inadequate lymphatic drainage leads to prolonged retention of drugs in the tumor interstitium. Infections induce inflammation, promoting the accumulation of nanoparticles in infected areas. However, solid tumors exhibit high tissue hydraulic pressure, collapsed blood vessels, and insufficient blood flow, limiting drug penetration.

Regulation of Lipid Nanoparticle Intracellular Delivery Processes

PEG Modification

PEG modification is one of the primary methods to regulate the fate of lipid nanoparticles (LNPs) within the body. By encapsulating lipid nanoparticles to evade recognition by the mononuclear phagocyte system (MPS), PEGylated materials can prolong the circulation time of particles in the bloodstream. However, this modification may also decrease the binding and uptake of nano-carriers by target cells, as well as their fusion ability with cell membranes and lysosomal membranes. To address these issues, pH-responsive PEG materials have been designed to rupture under acidic and reducing environments, promoting the binding of nano-carriers with cells and facilitating lysosomal escape. In this process, LNPs represent a promising lipid nanodelivery system for targeted nucleic acid therapy. Unlike liposomes, PEG lipids are anchored on the surface of LNPs through short chains (C14) and rapidly detach during circulation in the bloodstream, enhancing the uptake of drugs by hepatic parenchymal cells and the efficiency of intracellular delivery of nucleic acids.

Targeting Molecule Modification

Targeting molecule modification is an active targeting delivery strategy aimed at increasing the distribution of drugs at disease sites by mutual recognition between targeting molecules on the surface of lipid nanoparticles and specific cell surface receptors. Targeting molecules have been developed in various forms such as peptides, nucleic acid aptamers, proteins, antibodies and fragments thereof, and small molecules. Compared to conventional lipid nanoparticles, targeted modified drugs possess selective cell binding capability, which is expected to improve therapeutic efficacy and reduce systemic toxicity. However, targeting molecule modification alters the surface properties of the delivery system, thereby affecting its interactions with the body's internal environment, such as plasma proteins, interstitial fluid, biological membranes, and intracellular fluids, leading to discrepancies between in vivo performance and design expectations. The protein corona is one of the major barriers affecting active targeted lipid nanoparticle intracellular delivery, which may affect targeting delivery through receptor shielding, immune system activation, and accelerated blood clearance. Therefore, recruiting specific proteins in vivo to form functionalized protein coronas to enhance the targeting of lipid nanoparticles has become a new strategy.

Changes in Lipid Materials

By altering lipid materials, selective delivery to organs and cells can be achieved. Researchers have developed selective organ-targeting strategies by utilizing cationic lipids with quaternary ammonium headgroups, anionic lipids, or ionizable cationic lipids to regulate the adsorption of the protein corona, thereby precisely delivering gene drugs to the lungs, spleen, and liver. Furthermore, altering the structure of lipid nanoparticles can also achieve targeting of different types of lung cells. Changes in lipid materials can also affect the endosomal escape ability of lipid nanoparticles; for example, hydrophobic tail modification of ionizable cationic lipids can accelerate fusion with endosomal membranes, promoting drug release.

The intracellular delivery of lipid nanoparticles is a process influenced by multiple factors. While significant influencing factors such as the protein corona have been identified, many factors remain elusive. Currently, research on the intracellular delivery of lipid nanoparticles mainly focuses on processes such as blood circulation and tissue distribution, while studies on aspects such as vascular leakage, interstitial transport, and intracellular translocation mostly concentrate on metallic/inorganic nanoparticles, due to the lack of analytical methods resulting from the similarity of lipid components to cell membranes, which is a fatal flaw in this field. Currently, lipid nanoparticles are mainly represented by lipid components labeled with fluorescent dyes or radioactive markers. However, biological media and mechanical forces (such as blood flow shear stress and collisions with cells) can cause labeled lipids to detach and transfer, leading to misunderstandings about the intracellular fate of lipid nanoparticles and resulting in confusion in the field. Therefore, more efforts are needed, such as utilizing lanthanide-doped inorganic upconversion particles to label lipid nanocarriers and employing techniques based on single nanoparticle imaging and orthogonal biological imaging to accurately track the intracellular delivery process of lipid nanoparticles. This will help reveal molecular regulatory mechanisms, improve understanding of the intracellular fate of lipid nanoparticles with different intrinsic properties, and ultimately achieve better design and evaluation of nanodrugs.

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