Critical quality attributes of lipid nanoparticles and in vivo fate

Abstract

Lipid nanoparticles (LNPs) have emerged as versatile carriers for the delivery of genetic medicines and small-molecule drugs, offering desired benefits for therapeutic applications. Optimization of the treatment efficacy of nanocarriers necessitates a thorough understanding of the connection between pharmacokinetics and physicochemical properties. This review consolidates scientific efforts to elucidate how LNP’s physicochemical attributes influence their in vivo fate, emphasizing particle size and shape, surface electric potential and ligand-binding chemistry. By examining the interplay between LNPs and biological barriers across various administration routes, this review provides insights into tailoring LNP properties for optimal delivery and reduced off-target effects. Recommendations for future research are provided to advance the study of LNP in vivo behaviors and offer a practical framework for optimizing in vivo performance through product design parameters.

Graphical abstract

1. Introduction

Lipid nanoparticles (LNPs) are recognized as a pivotal component in the realm of drug delivery carriers, serving as a critical platform in enhancing treatment efficacy, safeguarding active ingredients and facilitating site-specific drug delivery [[1], [2], [3]]. LNPs have shown the capability of encapsulating various types of compounds, ranging from small molecules such as hydrophilic and hydrophobic drugs to biomacromolecules such as nucleic acids and proteins [4]. Commonly used lipid components include phospholipids, stabilizing agents such as cholesterol, and surface coating materials including PEG-lipid conjugates [4]. Additional functional lipids may be incorporated depending on the encapsulated active pharmaceutical ingredient (API). For instance, solid lipids combined with surfactants have been shown to enhance physical stability and provide sustained release, which is particularly relevant within the cosmetic industry [4,5]. Furthermore, the incorporation of ionizable cationic lipids is crucial for binding and releasing negatively charged nucleic acids to ensure efficient delivery [[6], [7], [8]]. This multifunctionality underscores the adaptability and applicability of LNPs in diverse therapeutic applications.

Over the years, LNPs have witnessed a remarkable upsurge in publications. Since the introduction of liposomes in the 1960s, the number of publications has exceeded 6,000 annually, as shown in Fig. 1. LNPs, whether containing small molecular compounds or encapsulating nucleic acid drugs, have achieved translation from conceptualization to practical implementation in clinical applications [4,5]. A significant advantage contributing to this success is that LNPs markedly enhance the pharmacokinetic properties of APIs. For instance, one early product, Doxil®, a PEGylated doxorubicin liposomal formulation, demonstrated several advantages over free doxorubicin, including prolonged plasma retention and passive tumor targeting through the enhanced permeability and retention (EPR) effect, thereby reducing cardiotoxic side effects [9]. In more recent developments, LNPs have become the preferred carriers for delivering nucleic acids, shielding fragile DNA/RNA from degradation, and facilitating uptake by host cells [4,[10], [11], [12]]. Drug carriers are developed by optimizing their pharmacokinetic behaviors at the whole body, tissue and cellular levels. According to publicly available data from the Web of Science, in the past 20 years, publications related to the pharmacokinetics (PK) of LNPs have accounted for approximately 40% of the total publications in the LNP field (Fig. 1).

Fig. 1.

Trend in publications on “lipid nanoparticles or liposomes” collected with Web of Science since the1960s, with the percentage of publications in PK over the past two decades (inset).

Despite many studies reporting that the effective delivery of APIs to target sites remains below 1% [13,14], LNPs have attained significant investment in both academic and commercial fields. Undoubtedly, their full therapeutic potential is yet to be realized. A major focus is on enhancing the site-specific delivery of LNPs in vivo [5,15]. Optimizing the fate of LNPs within the body requires a deep understanding of the biological systems the nanoparticles interact with and the various physiological barriers the delivery systems need to overcome [16,17]. It is thus crucial to study how the physicochemical properties of LNPs influence their pharmacokinetic behaviors. This will benefit the design of LNP products and lead to a better therapeutic index.

Herein, we resort to fundamental principles for optimizing LNP delivery systems, emphasizing the importance of studying the PK of LNP carriers. We begin by providing an overview of the ADME processes of LNPs in vivo, followed by a systematic discussion of the biological barriers associated with different administration routes and the corresponding formulation design strategies. We then explore the critical quality attributes of LNPs in relation to cell interactions. Additionally, we emphasize the unique aspects of LNP PK. We hope this review can provide readers with a practical framework for optimizing the physicochemical properties of LNPs to achieve the rational design of LNP products.

2. Navigating the PK of LNPs

Lipid-based drug delivery systems are often categorized by various names depending on the API and lipid formulation. Terms such as liposomes, LNPs, solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) are commonly used and sometimes interchangeably. While these delivery systems differ in their construct and lipid composition, their key physicochemical characteristics—such as size, charge and shape—often exert similar effects on their in vivo behavior. Therefore, in this review, we will adopt a broad interpretation of the term “LNPs” to encompass these lipid-based delivery systems collectively.

The in vivo fate of a drug delivery system encompasses its PK—specifically its adsorption, distribution, metabolism and excretion (ADME) performance within the body [18,19]. It is crucial to understand that the in vivo fate of LNPs presents a level of complexity that far exceeds that of administered small-molecule drugs. LNPs are self-assembled systems that integrate both drug and carrier materials. Traversing through the body, LNPs undergo dynamic changes, leading to drug-loaded nanoparticles, blank nanoparticles (particles that have released their payload), released free drugs, and degraded carrier components. In this review, unless indicated otherwise, the term “LNP PK” collectively refers to the overall in vivo behaviors of an LNP system, encompassing those of both intact LNPs and any blank nanoparticles. A thorough exploration of this complexity is provided in Section 5.

Absorption of LNPs refers to the process by which they enter systemic circulation. Among the factors influencing absorption, the route of administration is particularly critical (Fig. 2). In the following section of this review, we will examine the in vivo behavior of LNPs based on their administration routes. Once absorbed, the distribution of LNPs within tissues is largely determined by their inherent physicochemical characteristics, including particle size, surface charge, morphology and surface modifications [1,20]. Certain properties, such as size, charge, shape and PEGylation, primarily affect tissue distribution by influencing the formation of the protein corona (which is detailed in Section 3.5). In contrast, other modifications, such as ligand alterations, have a more pronounced effect on cellular uptake and intracellular trafficking, as further discussed in Section 4.

Fig. 2.

Key factors influencing the in vivo fate of LNPs at various levels.

The metabolism of LNPs is broadly defined as any alteration of their initial composition and/or physicochemical properties [21]. This can occur through phagocytosis by macrophages, followed by degradation in lysosomes, or via hydrolysis in the aqueous environments of biological systems [22]. LNP metabolism primarily involves the chemical breakdown of lipid components and the encapsulated drug [23]. The lipid components of LNPs, including phospholipids and cholesterol, undergo metabolism through endogenous lipid pathways, resulting in non-toxic or low-toxicity metabolites. Encapsulated nucleic acids, such as mRNA and siRNA, are degraded by intracellular nucleases once endocytosed, generating harmless nucleotide fragments. Small-molecule drugs encapsulated in LNPs are metabolized and cleared enzymatically. Conversely, LNPs can influence the function of cytochrome enzymes, potentially leading to drug-drug interactions and introducing safety concerns when utilized in combination. Thorough evaluation of metabolic pathways and drug interactions is essential for the development and application of LNP-based therapies.

Excretion is closely associated with clearance, circulation time and tissue deposition, affecting the treatment efficacy and occurrence of adverse reactions. The liver and kidneys serve as the primary organs responsible for the excretion of LNPs. Particle size is a major determining factor. Nanoparticles smaller than 10 nm can be efficiently removed through renal filtration or extravasation [24,25]. In contrast, larger LNPs may follow different excretion pathways. Nanoparticles processed by hepatic cells, or their metabolites, are excreted via bile. Other organs, including the lungs, mammary glands and sweat glands, may contribute to additional excretion pathways. Intact LNPs are typically not excreted through these routes; most undergo disintegration, and the drug and carrier materials are then degraded before being primarily eliminated through the kidneys [23,26]. The charge of nanoparticles also plays a role in their excretion through bile; positively charged nanoparticles are more readily absorbed by hepatic cells compared to their negatively charged counterparts [27]. It is worth noting that the metabolites of carrier materials may accumulate in the kidneys, potentially leading to renal damage and dysfunction, as seen with polyethylene glycol (PEG) and cyclodextrin derivatives [28]. Following the LNP administration, it is critical to assess the degradation pathways and rates of excretion of the active drug substance and lipids. Given the unique characteristics of different carrier materials, excretion studies should be customized to reflect those specific properties.

3. In vivo fate of LNPs via different administration routes

The in vivo fate, in a broad sense, primarily involves four processes: absorption, distribution, metabolism and clearance. Different routes of administration significantly influence the absorption phase, as each route encounters unique sets of barriers (Fig. 2). These include mechanical barriers (e.g., mucociliary clearance in the respiratory tract, tear turnover and nasolacrimal drainage), biochemical barriers (e.g., pH, enzymes and surfactants), immunological barriers (e.g., alveolar macrophages and dendritic cells), and permeability-related barriers (e.g., mucus/epithelial layers) [[29], [30], [31], [32]]. Once the LNPs overcome the absorption barrier and enter the bloodstream, their distribution, clearance and metabolic processes are similar to those of intravenously injected LNPs [2,33,34]. Here, we systematically outline the physiological environments and barriers associated with different administration routes, their advantages for achieving organ-level targeted therapy, and how to optimize LNP quality parameters to overcome these barriers Fig. 3.

Fig. 3.

Biological barriers and target regions by various administration routes of LNPs.

3.1. Oral administration

Oral administration is a widely used and convenient drug delivery route, known for its high patient compliance [35]. Upon gastrointestinal administration, nanocarriers can follow two possible trajectories: they can be absorbed into the systemic circulation or excreted through feces or other pathways [23]. Some absorption occurs in the gastrointestinal tract through paracellular transport, endocytosis, and uptake by immune cells [19,29]. However, the absorption of drug-loaded nanocarriers encounters notable barriers, including low pH, various digestive enzymes, and the gastrointestinal epithelium and mucosal layers, which potentially compromise the stability and absorption of drugs. Most LNPs maintain their structural integrity in the stomach [23,36], where the limited presence of lipase results in minimal absorption [37]. After gastric emptying, LNPs are transported to the small intestine, where they undergo lipase-mediated breakdown [36].

Research suggests that nanocarriers within the size range of 50 nm to 200 nm can be effectively absorbed through Peyer's patches in the small intestine, effectively bypassing systemic metabolism [38]. Additionally, nanocarriers might be taken up by intestinal epithelial cells [39]. In a broader context, nanocarriers have demonstrated the potential to enhance adhesion to the gastrointestinal mucosa, thereby optimizing the treatment index for gastrointestinal diseases [40]. For example, Song et al. designed LNP loaded with IL-22 encoding mRNA for inflammatory bowel disease (IBD) [41]. Comprising phosphatidic acid, monogalactosyldiacylglycerol and digalactosyldiacylglycerol in a molar ratio of 5:2:3, these LNPs exhibited a diameter of around 200 nm and a surface charge of −18 mV (Table 1). The oral administration of IL-22 mRNA-loaded LNP led to a significant increase in IL-22 expression within the colonic mucosa of mouse models. These results suggest that oral administration of LNPs enhances drug utilization and serves as an effective carrier targeting the gastrointestinal tract.

Table 1.

Examples of LNPs for Different Tissue Targeting.

Target organ/site	Administration route	Size (nm)	Zeta potential	Morphology	PEG	Ref.
Gastrointestinal	Oral	200	−18 mV	Spherical	0	[41]
Liver	i.v.	<100	ζ <10 mV	Spherical	3%−5%	[23,25,[62], [63], [64], [65]]
Spleen	i.v.	100–200	ζ <10 mV	Spherical	3%−5%
Tumor	Intratumor injection; i.v.	100–200	ζ ±10 mV	Spherical	3%−5%
Lung	Inhalation	40	ζ >10 mV	Spherical	55%	[32]
Immune system	i.d., i.m., s.c.	60–100	ζ ±10 mV	Spherical	3.5%	[7,57,92]
Kidney	i.v.	80–150	ζ ±10 mV	Disk-like Rod-like	3%−5%	[93]
Skin	Transdermal	20∼200	ζ ±10 mV	Spherical	1%−10%	[94]

3.2. Inhalation

LNPs have attracted attention as potential inhalation formulations, with their capacity to stabilize the drug and facilitate effective drug accumulation in the lungs, showing potential for addressing respiratory diseases [42,43].During inhalation exposure, nanoscale particles face two competing processes: non-absorptive clearance and absorption [44]. The expansion of the alveolar surface area supports particle absorption, typically occurring after endocytosis. Nanoparticles absorbed from the alveoli can easily access the vessel and lymphatic systems [45,46]. LNPs that remain in the upper respiratory tract may be eliminated through mucociliary clearance. Additionally, it is important to consider the shear forces in the nebulizer, which may lead to the destruction of the nanoparticle structure and payloads.

Developing LNPs for inhalation demands careful consideration of factors such as particle size, charge and surface properties to optimize their behavior within the respiratory system, thus enhancing pulmonary targeting, minimizing systemic side effects, and amplifying therapeutic outcomes [47]. Dahlman and colleagues reported an optimal LNP composition for aerosolized mRNA delivery [32]. They prepared LNPs loaded with mRNA, consisting of modified PEI compound 7C1, cholesterol, DMG-PEG 2000 and cationic lipid DOTAP. A higher proportion of DMG-PEG 2000 (55%) enhanced the pulmonary delivery efficacy of LNPs, surpassing clinically employed LNPs (Table 1). The versatility of LNPs and their potential to encapsulate various types of drugs make them a potent tool for improving the treatment of respiratory diseases via the inhalation administration route.

3.3. Transdermal administration

LNPs also show promising potential in transdermal administration, a route that involves delivering drugs through the skin for systemic absorption. This approach offers distinct advantages, including non-invasiveness, sustained release and bypassing first-pass metabolism. LNPs have garnered attention for their ability to enhance transdermal drug delivery by encapsulating both hydrophilic and hydrophobic compounds, facilitating their penetration through the skin's lipid-rich stratum corneum [48].

Transdermal absorption must penetrate the stratum corneum, epidermis and dermis. Nanoparticles are absorbed through the lymphatic system and lymph nodes in the skin [46]. Nanoparticles intended for transdermal absorption typically comprise biocompatible components such as PLGA and phospholipids, allowing for interactions with the skin's lipids. LNPs, especially those with sizes below 600 nm, are considered capable of penetrating the skin [49,50]. LNPs with diameters below 200 nm usually form a monolayer, preventing the loss of skin surface moisture, which results in the relaxation of stratum corneum cells and facilitates deeper drug penetration [51,52]. Reports suggest that nanoparticles of approximately 20 nm accumulate in deeper follicular areas, while particles as large as 200 nm exhibit time-dependent follicular permeability [53]. These findings highlight the potential of LNPs to increase transdermal drug delivery by leveraging their ability to penetrate the skin barrier and reach deeper layers, thereby being widely used in various cosmetics and skin diseases, improving drug absorption and therapy.

3.4. Local injection

Local injection refers to the administration of drugs into a specific localized area of the body, allowing for exerting their effects locally or having the potential to diffuse or enter the bloodstream, producing systemic therapeutic outcomes. LNPs via local injection are primarily absorbed through the lymphatic system (mainly via local lymph nodes) before entering the systemic circulation [54]. For example, intradermal (i.d.), intramuscular (i.m.) and subcutaneous (s.c.) injections are commonly utilized for LNP vaccine administration [55,56]. These routes capitalize resident and recruited antigen-presenting cells present in the skin and muscles, which can uptake and handle antigens-encoded mRNA [55,56]. Moreover, the presence of blood vessels and lymphatic channels in these tissues helps guide mRNA vaccines to the lymph nodes, thereby stimulating T-cell immune responses. The COVID-19 vaccine serves as a typical illustration of a vaccine that exerts systemic effects following i.m. injection [57]. Other local injections may include intratumoral, subretinal and similar methods, all aimed at directly targeting the specific organ or tissue of interest with minimal off-target effects. Intratumoral LNP administration has shown promise in boosting intratumoral immune responses by stimulating T cell proliferation and differentiation while avoiding systemic toxicity [58]. Siddharth Patel et al. reported the potential of using LNPs to treat monogenic retinal degenerative disorders through direct subretinal delivery [59]. LNPs that contain ionizable lipids with low pKa values and unsaturated hydrocarbon chains show the highest transfection efficiency in the retina.

For local injections, the size of LNPs also impacts lymphatic absorption. Our previous studies indicated that after i.m. injection of LNPs with sizes of 100 nm, 200 nm and 300 nm, smaller nanoparticles were more readily absorbed into the systemic circulation [54]. Additionally, Oussoren et al. reported that s.c. injections of liposomes with sizes of 400 nm, 170 nm, 70 nm and 40 nm showed a negative correlation between particle size and lymphatic uptake. Smaller liposomes (∼40 nm) demonstrated comparatively high lymphatic uptake (76%) compared to larger liposomes, which were trapped predominantly at the injection site [60]. In conclusion, localized injection routes provide a targeted approach for drug delivery, allowing drugs to act locally while potentially yielding systemic effects, which depends on LNP design schemes.

3.5. Intravenous injection

Intravenous injection (i.v.) of LNP formulations involves administering these nanoparticles directly into the bloodstream through a vein. This route of administration allows for rapid systemic distribution of the encapsulated drugs or therapeutic agents. The in vivo tropism of LNPs primarily depends on their apparent properties, such as particle size, surface characteristics and morphology.

The size of LNPs is a vital parameter that determines drug circulation half-life. It primarily influences the phenomenon of opsonization, protein adsorption amount [61] and interactions with the mononuclear phagocyte system (MPS), which subsequently affect distribution [1]. Nanoparticles measuring approximately 100 nm exhibit prolonged circulation in the bloodstream and relatively reduced uptake rates by the MPS [23,62]. Researchers administered radiolabeled liposomes of various sizes (30–400 nm) intravenously to mice and studied their distribution in the blood, liver, spleen and tumors 4 h after injection. They reported that liposomes within the range of 100–200 nm were detected in the systemic circulation at ∼60%, whereas nanoparticles smaller than 50 nm or larger than 250 nm accounted for only 20% of the total plasma count. Nanoparticles smaller than 50 nm accumulated more in the liver, which is smaller than the liver fenestration size (100 nm), indicating that they can easily penetrate endothelial barriers.

The zeta potential of LNPs is determined by their lipid headgroup moieties, which can be positive, negative, or amphiphilic. This charge density on the surface influences protein adsorption and interactions with cells, correlating closely with their distribution kinetics [25,[63], [64], [65]]. Particles with positive surface charges (ζ > 10 mV) preferentially adsorb proteins with an isoelectric point below 5.5 (pI < 5.5), like albumin [66,67]. Conversely, particles with negative charges (ζ < −10 mV), tend to bind to those proteins with pI > 5.5, like IgG [68]. The interaction of opsonins, including IgG, fibrinogen and complement factors, can facilitate phagocytosis and eventual particle clearance by the reticuloendothelial system (RES) [69,70]. These negative particles often get rapidly sequestered in MPS organs and accumulate in the liver and spleen [71]. Neutral-charged particles (ζ ± 10 mV) experience reduced MPS uptake, thereby prolonging their blood circulation time [72]. Positively charged particles typically underwent a rapid blood clearance phase and accumulated extensively in the lungs and liver at high doses. This is because positively charged particles tend to adsorb negatively charged serum proteins, leading to their propensity for aggregation [73,74]. These aggregates are often large and can result in transient pulmonary capillary occlusion when dissociated, followed by redistribution to the liver [74].

The hydrophobicity of the nanoparticle surface affects the quantity and the characteristics of the bound proteins [75,76], thus affecting their circulation kinetics [77,78]. To minimize the phenomenon of opsonization and clearance, surface modification with PEG is a widely adopted strategy. PEG acts as a steric hindrance, reducing protein binding on the surface of NPs due to its relatively inert hydrophilic nature, reducing MPS uptake and prolonging the circulation time [79]. Compared to non-PEGylated liposomes, PEGylated liposomes show over a three-fold increase in tumor uptake of encapsulated drugs. PEGylated formulations show improved tumor accumulation and greater anti-tumor efficacy compared to free drugs or non-PEGylated liposomal delivery systems, according to multiple studies [71,80,81]. Despite the development of alternative materials to mimic the effects of PEG and reduce opsonization [[82], [83], [84]], PEGylation remains the most used method. It is well-known that excessive PEGylation can alter the balance of whole particles, often leading to unstable particles [85]. Typically, a 3%–5% PEG modification on the particle surface can lower the uptake by the RES while maintaining the stability of lipid formulations [85]. Adjusting the length of the lipid anchor will alert the whole particle distribution profiles. After i.v., liposomes modified with PEG-S-dimyristoylglycerol (C14), PEG-S-dipalmitoylglycerol (C16), or PEG-S-distearoylglycerol (C18) exhibited half-lives of 1, 6 or 15 h, respectively. Specifically, after i.v. injection, liposomes containing PEG-S-C14 primarily accumulated in the RES organs, with lower levels of accumulation in tumors. However, when the acyl chain length increased to C16 or C18, the blood circulation time extended, and subsequent tumor accumulation increased. Nonetheless, the shorter lipid anchor can lead to a faster dissociation of PEG from the surface, resulting in higher transfection efficacy compared to PEG-lipid conjugates with longer acyl chains. Therefore, the selection of PEG-derived lipids must consider indications, routes of administration, and strike a balance between circulation time and target cell interactions [86]. However, there has been increasing concern about the limitations of PEG modification, primarily due to its immunogenicity after multiple administrations [87]. Studies show a significant decrease in the circulation time of subsequent liposomes after systemic administration of PEG liposomes for four weeks, attributed to the generation of anti-PEG IgM antibodies, complement system activation, and increased uptake by macrophage [88,89]. The development of PEG derivatives aims to balance the extension of blood retention time, reduction of immunogenicity and enhancement of transfection efficiency.

Morphology also influences the biological distribution of particles. Generally, any shape that differs from spherical particles tends to increase the residence time in vivo [72]. In one study, researchers reported that worm-like micelles circulated for up to 7 d, while PEGylated spherical vehicles were eliminated within 2 d [72,90]. Worm-shaped copolymers developed by Discher and colleagues demonstrated an extremely long circulation half-life in mouse blood, lasting for 5 d [91]. This is because the worm-shaped copolymers faced strong resistance from the flow medium, preventing macrophages from phagocytosing them before being carried away by the fluid. Of course, applying the mechanism to design and development of non-spherical LNPs may still be a challenging task, but it holds promise as a potential mechanism underlying the extension of circulation time.

We have examined the in vivo fate of LNPs based on their administration routes. Each route has unique physiological barriers and benefits for targeting specific tissues. Selecting the proper route for the medical indication and optimizing LNP design can greatly enhance drug delivery efficiency and therapeutic outcomes. By carefully controlling LNP size, surface modification and other characteristics, it is possible to overcome physiological barriers, achieve targeted therapy, and ultimately improve patient treatment experiences and outcomes.

4. LNP-cell interaction and critical particle properties

In the previous discussion, we outlined the PK of LNPs from a tissue perspective across different administration routes. To maximize therapeutic efficacy while minimizing off-target toxicity, LNPs should deliver the API to the target tissue, then to the target cells, and finally to the intracellular target organelles. Therefore, a thorough understanding of LNP quality attributes and cell interactions is also crucial for achieving precise targeted delivery [95].

The composition and ratios of lipids can indeed influence the preferential distribution of LNPs at the cellular level. Dahlem’s research group investigated the impact of various types of cholesterol on the cell-targeting capabilities of LNPs [96]. They find that LNPs with oxidized cholesterol tend to deliver mRNA to the hepatic microenvironment, specifically hepatic endothelial cells and Kupffer cells, which is distinguished from conventional LNPs primarily targeting hepatocytes [97]. As for PEG lipids, when the ratio increased from 1.0% to 3.0%, more LNPs were uptake by hepatocytes, while the interaction with liver sinusoidal endothelial cells (LSEC) and Kupffer cells decreased [98]. Furthermore, if a fraction of PEG lipid is replaced with mannose-modified lipids, the LNPs specifically target LSEC, indicating that cell-specific transport can be attained by altering the proportion and structure of PEG lipids. For LNPs used in gene delivery, their transfection efficiency significantly depends on the acid dissociation constant (pKa) of ionizable cationic lipids. It has been reported that the optimal value is around pKa ∼6.4. Deviations from this pKa by as little as 0.5 units can lead to a reduction in efficiency by 100-fold or more [99]. This optimal pKa value may reflect a balance required between the low surface charge of LNPs to avoid quick clearance in systemic circulation and the positive ionization, allowing them to escape endosomes after entering the cell.

More and more reports note that coating targeting ligands onto nanoparticles can enhance intracellular delivery efficacy [100,101]. Park and colleagues demonstrated that non-targeted liposomes and immunoliposomes (liposomes modified with antibodies) had no significant difference in PK and tissue distribution, but immunoliposomes significantly enhanced intracellular drug delivery [102]. Compared to non-targeted liposomes, immune liposomes exhibited further improved anti-tumor efficacy [103]. However, using the targeting ligand approach still presents some unresolved challenges. Firstly, ligand-modified nanoparticles exhibit slow internalization upon interaction with target cells, which hinders subsequent extravasation and binding of more targeted nanoparticles [104]. Secondly, antibody-conjugated nanoparticles tend to have higher clearance rates from the bloodstream [105,106]. Thirdly, receptor-mediated endocytosis often occurs via endosomal/lysosomal pathways, where a significant portion of drugs are trapped in organelles or degraded [104]. Therefore, compared to non-targeted nanoparticles, targeted nanoparticles don't always exhibit significantly improved pharmacodynamics, and this challenge can also affect the success of translation in clinical applications [104].

5. Unique aspects of LNP PK

The PK of conventional single drugs mainly focuses on studying the changes in total drug concentration in blood and other body fluids and tissues. However, after administration of LNPs, there are complex dynamic processes involving various forms such as “drug-loaded NP,” “blank NP,” “free drugs,” and “carrier materials” in each PK process of ADME. Currently, the understanding of the in vivo disposition processes of LNPs is limited and unclear release mechanisms, uncertain potential toxicity, and incomplete pharmacokinetic behaviors may be among the reasons why the full potential of LNPs has not yet been fully realized [2,27].

LNP must reach the target site and release free drugs to exert their pharmacological effects. Therefore, merely measuring the total drug concentration cannot truly elucidate their release and distribution characteristics at the systemic level, target tissues and cells, nor can it provide comprehensive pharmacokinetic data support for the nonclinical efficacy and safety studies of nanomedicines. Premature dissociation, leakage and degradation of LNPs in vivo could result in unloaded carrier materials reaching the target site, leading to reduced efficacy and increased adverse effects [107]. Therefore, in nonclinical pharmacokinetic studies of LNP drugs, it is necessary to separately measure the concentrations of free drugs and loaded drugs in blood/tissue to further obtain information on the dynamics of drug release and carrier disintegration/degradation kinetics. However, establishing appropriate analytical methods for those various drug/carrier forms to accurately reflect the concentrations of LNP in biological samples remains a technical challenge.

For active drugs encapsulated in the carrier, standard analytical methods include HPLC, LC-MS/MS, fluorescence labeling, radiolabeling and enzyme-linked immunosorbent assay (ELISA) [108]. After administration, these active drugs typically exist in both free and encapsulated forms. Effective separation of these forms is crucial during pharmacokinetic studies and can be achieved using methods such as equilibrium dialysis, ultracentrifugation, ultrafiltration, solid-phase extraction, size-exclusion chromatography and column-switching chromatography [108]. Ensuring that the states of drug-loaded particles, free drugs and disaggregated materials remain unchanged during sample processing is critical for accurate analysis. To comprehensively understand the in vivo behavior of intact particles, labeling with fluorescent or radioactive substances is employed [27]. Techniques such as small animal live fluorescence imaging, single-photon emission computed tomography (SPECT), and whole-body autoradiography are used for tracking and semi-quantitative analysis based on imaging signals [27,109]. Environmental response probes, such as near-infrared fluorescent probes based on aggregation-caused quenching (ACQ), Förster resonance energy transfer (FRET) and aggregation-induced emission (AIE) effects, can be utilized for quantitative or semi-quantitative in vivo analysis of drug-loaded particles [108,110]. However, it is important to consider that various labels may influence the in vivo behavior of these particles, potentially affecting the accuracy of mechanistic studies.

In summary, only by systematically and accurately describing the pharmacokinetic processes of LNP in vivo can we advance targeted research on LNP, assess its efficacy and safety, improve clinical translation success rates, and promote the development of lipid-based delivery systems.

6. Conclusion

The emergence of LNPs as a prominent subject in drug delivery research necessitates a comprehensive understanding of their various physicochemical properties for improved therapeutic outcomes. This review examines the in vivo barriers encountered with different delivery routes and the corresponding design strategies. Throughout, we emphasize how different physicochemical characteristics (such as particle size, surface charge, morphology and surface modifications) affect LNP in vivo behavior, including tissue distribution and cellular interactions, and discuss the particularities of LNP PK methodologies.

However, LNPs vary in size, shape and material composition, leading to numerous combinations and attributes. This diversity complicates direct comparisons between studies and makes compiling information from thousands of nanoparticle experiments reported in the literature challenging, further hindering rational formulation design. High throughput methods and computer-based data analysis, including PBPK modeling, meta-analysis and artificial intelligence (AI), are proving invaluable in addressing this issue. High throughput methods can significantly reduce experimental costs, increase screening efficiency, and minimize experimental variability. Incorporating the influence of physicochemical parameters such as size, shape and material into a covariate framework within a physiologically based pharmacokinetic (PBPK) platform model enables the characterization and prediction of the biodistribution of nanoparticles with differing physicochemical properties, thereby handling extensive data consistently. PK-driven drug design aims to optimize formulation design from a delivery perspective, facilitating their discovery, development and translation from preclinical to clinical stages.

This review offers a fresh LNP design perspective, emphasizing the pharmacokinetic-driven approach. By understanding the intricate relationship between LNP quality attributes and their fate in vivo, we can engage in the rational and scientific engineering of LNPs to enhance their targeting precision, bioavailability and therapeutic efficacy.

Conflicts of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.