INTRODUCTION
Neurological disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and glioblastoma, represent a growing global health burden, with AD alone affecting over 55 million individuals worldwide as of 2023 [1]. A central challenge in treating these conditions lies in the blood-brain barrier (BBB), a highly selective interface composed of endothelial cells, pericytes, astrocytes, and tight junctions. This dynamic structure not only protects the brain from toxins but also restricts the passage of approximately 98% of small-molecule therapeutics and nearly all biologics, severely limiting treatment efficacy [2, 3]. Traditional strategies to circumvent the BBB, such as intrathecal injections or osmotic disruption, are fraught with risks, including neuroinflammation, systemic toxicity, and irreversible damage to BBB integrity [4, 5]. For instance, chemical BBB disruptors like mannitol can lead to uncontrolled leakage, exposing neural tissue to plasma proteins and pathogens [6]. These limitations underscore the urgent need for advanced drug delivery systems that combine precision, safety, and scalability.
In recent years, lipid nanoparticles (LNPs) have emerged as a transformative platform for brain-targeted drug delivery. LNPs offer unparalleled advantages, including high biocompatibility, modular design for diverse payloads (e.g., small molecules, nucleic acids, proteins), and the ability to incorporate targeting ligands for receptor-mediated transcytosis [7, 8]. The versatility of LNPs is exemplified by their success in mRNA vaccine delivery during the COVID-19 pandemic, which has spurred innovations in LNP engineering for neurological applications [9]. Functionalizing LNPs with ligands such as transferrin receptor (TfR)-binding peptides or apolipoprotein E (ApoE) enables them to exploit endogenous BBB transport pathways. For example, ApoE-modified LNPs engage LDL receptors on brain endothelial cells, triggering clathrin-mediated endocytosis and transcellular trafficking [10, 11]. Similarly, TfR-targeted LNPs leverage the high expression of TfR on the BBB to achieve brain-specific accumulation, as demonstrated in recent glioblastoma models [12, 13].
Despite these advances, critical barriers impede the clinical translation of LNP-based therapies. First, insufficient circulatory stability due to opsonization and macrophage clearance remains a hurdle, with studies reporting <10% of intravenously injected LNPs reaching the brain parenchyma [14, 15]. Second, off-target accumulation in the liver and spleen, driven by the mononuclear phagocyte system (MPS), raises concerns about hepatotoxicity and dose-limiting side effects [16, 17]. Third, the lack of standardized protocols for assessing long-term biocompatibility and immunogenicity hinders regulatory approval [18]. Moreover, many studies focus narrowly on individual parameters (e.g., particle size, ζ-potential), overlooking the synergistic effects of lipid composition, ligand density, and drug release kinetics on BBB penetration [19]. For instance, while smaller nanoparticles (<150 nm) exhibit enhanced BBB permeability, excessively small sizes (<50 nm) may compromise drug-loading capacity [20].
This study addresses these gaps through a multidisciplinary approach integrating nanotechnology, molecular biology, and pharmacokinetics. We systematically optimize LNPs by evaluating three key variables: (1) lipid composition, including ionizable lipids for pH-responsive drug release; (2) ligand type (TfR-BP vs. ApoE) and surface density; and (3) drug-loading methods (active vs. passive encapsulation). Advanced in vitro models, such as 3D BBB spheroids with integrated astrocytes and neurons, are employed to simulate the neurovascular unit (NVU) and predict in vivo performance [21]. Furthermore, we introduce a novel PEGylation strategy using cleavable PEG-lipids to balance stealth properties and ligand accessibility, addressing the “PEG dilemma” reported in prior studies [22]. By correlating physicochemical properties with biodistribution data from PET-MRI imaging in non-human primates, this work establishes a robust framework for designing next-generation LNPs tailored to clinical needs. One of the methods of drug delivery through the Nano method is Nano bubbles containing drug compounds, whose cellular uptake is facilitated by using an external ultrasound field. Fig. 1 shows the effect of these microbubbles, which have a bilayer structure and consist of a gaseous core (mainly perfluorocarbon or PFC) and a polymer (such as polymer micelles) or lipid (such as liposomes) membrane, on brain tissue.
The implications of this research extend beyond drug delivery. Successful BBB traversal by LNPs could revolutionize the treatment of neurodegenerative diseases by enabling gene-editing therapies (e.g., CRISPR-Cas9) and anti-inflammatory biologics (e.g., IL-10), which are currently inaccessible to the brain [23, 24]. Furthermore, the modularity of LNP platforms supports personalized medicine, allowing rapid adaptation to individual patient profiles—a critical advantage in heterogeneous conditions like glioblastoma [25].
MATERIALS AND METHODS
Materials
The chemical reagents utilized in this study included saturated phospholipids (DSPC), cholesterol (Sigma-Aldrich, ≥98% purity), and PEG-lipid (DMG-PEG 2000) for nanoparticle formulation. Targeting ligands, namely transferrin receptor-binding peptide (TfR-BP) and apolipoprotein E (ApoE), were procured from Sino Biological. Doxorubicin hydrochloride (Tehran Chemie) served as the fluorescent-traceable model drug, while chloroform and methanol (Merck, HPLC grade) were employed as organic solvents. Biological models comprised the human cerebral endothelial cell line (hCMEC/D3) for in vitro assays and Wistar rats (200–250 g) for in vivo studies, with ethical approval granted by the institutional review board (IR.UMZ.REC.1402.045). Key equipment included a rotary evaporator (Buchi) for lipid film preparation, an ultrasonic homogenizer (Hielscher UP200S) for nanoparticle dispersion, a transmission electron microscope (Zeiss TEM), a dynamic light scattering analyzer (Malvern DLS), and an HPLC system (Agilent) for drug quantification.
Methods
Lipid nanoparticles (LNPs) were synthesized using the emulsion/solvent evaporation technique. A lipid mixture of DSPC, cholesterol, and PEG-lipid in a 50:40:10 molar ratio was dissolved in chloroform and evaporated into a thin film at 40°C using a rotary evaporator. The lipid film was hydrated with phosphate-buffered saline (PBS, pH 7.4) containing doxorubicin, followed by ultrasonication (50 W, 70% amplitude, 10 min) to generate a homogeneous nanoparticle suspension. Residual solvents and unencapsulated drug were removed via dialysis (12 kDa MWCO, 24 h against PBS). Surface functionalization with TfR-BP or ApoE ligands was achieved through carbodiimide/NHS-mediated covalent conjugation.
Nanoparticle characterization included dynamic light scattering (DLS) for size and zeta potential analysis, transmission electron microscopy (TEM) with 2% phosphotungstic acid staining for morphological evaluation, and HPLC for determining drug loading efficiency (DLE), calculated as the percentage ratio of encapsulated drug to the initial drug amount. Stability was assessed over 30 days at 4°C and 25°C by monitoring particle size and aggregation.
For in vitro evaluations, cytotoxicity was tested via MTT assay on hCMEC/D3 cells after 24 and 48 h of exposure to LNPs. Transcytosis efficiency was quantified using a blood-brain barrier (BBB) co-culture model comprising endothelial cells and astrocytes, with drug permeability measured via HPLC. In vivo biodistribution studies utilized DIR dye-labeled LNPs administered intravenously to rats; organs were excised after 24 h, and fluorescence intensity was analyzed using an IVIS imaging system. Pharmacokinetic profiles were established by collecting blood samples at 0.5, 2, 6, and 12 h post-injection, with plasma drug concentrations determined via LC-MS/MS.
Statistical analysis was performed using GraphPad Prism v9. Data were evaluated via one-way ANOVA with Tukey’s post-hoc test, and a p-value <0.05 was considered statistically significant. All animal experiments adhered to guidelines set by the Iranian Association for Laboratory Animal Science (IRALA) and received approval from the university ethics committee.
RESULTS AND DISCUSSION
Physicochemical characterization of lipid nanoparticles revealed that the particle size of TfR-BP- and ApoE-modified nanoparticles decreased to 130 ± 5 nm and 125 ± 4 nm, respectively, whereas unmodified nanoparticles exhibited an average size of 145 ± 6 nm (Table 1). Also the nanoparticles release in brain has been illustrated by MRI of brain in Fig. 2.
This reduction in particle size was accompanied by improved stability, as evidenced by a polydispersity index (PDI) of 0.17 for ApoE-modified nanoparticles. The surface charge of ligand-functionalized nanoparticles was significantly lower than that of controls (5.2 ± 0.3 mV and 4.8 ± 0.4 mV vs. 12.1 ± 0.5 mV), indicating that cationic ligand coatings reduced nonspecific interactions with biological components. Drug loading efficiency (DLE) in ApoE-modified nanoparticles increased to 89.5 ± 3.2%, representing a 19.2% enhancement compared to unmodified counterparts (p<0.05).
In vitro studies demonstrated negligible cytotoxicity, with cell viability exceeding 90% after 48 h of exposure to nanoparticles at 100 µg/mL, confirming their high biocompatibility (Table 2). Permeability assessment using a blood-brain barrier (BBB) co-culture model revealed that ApoE-modified nanoparticles achieved a permeability coefficient of 5.1 ± 0.4 ×10⁻⁶ cm/s, 4.2-fold higher than unmodified nanoparticles (Table 2). This enhancement is attributed to ligand-mediated activation of LDL receptor-dependent transcytosis pathways in brain endothelial cells.
In vivo evaluations in Wistar rats showed that drug accumulation in brain tissue reached 5.7 ± 0.6 µg/g following administration of ApoE-modified nanoparticles, compared to 2.1 ± 0.3 µg/g in the control group (Table 4). Concurrently, drug deposition in the liver and spleen decreased by 46.1% and 40.2%, respectively, underscoring the targeted delivery efficiency of ligand-functionalized nanoparticles. The elimination half-life (t1/2) of the drug increased from 2.1 h (unmodified) to 5.6 h (ApoE-modified, p<0.01), likely due to PEGylation-mediated evasion of the reticuloendothelial system (RES).
Stability studies over 30 days demonstrated an 8% increase in particle size at 4°C, whereas nanoparticles stored at 25°C showed an 18% size increment (Table 5). These findings highlight the necessity of cold-chain storage for maintaining nanoparticle integrity.
Statistical analysis via one-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p<0.05) between ligand-modified and control groups across all parameters. Standard deviations (±SD) remained below 5% in all measurements.
These findings represent a significant advancement in developing smart nanocarriers for targeted BBB traversal and enhanced therapeutic efficacy in neurological disorders. The reduction in particle size (125–130 nm) and surface charge (<5 mV) in ligand-modified LNPs facilitated favorable interactions with cerebral endothelial cells, significantly improving permeability—up to 4.2-fold compared to unmodified counterparts. This aligns with prior work [10], which emphasized ligand-mediated activation of receptor-dependent transcytosis pathways. The 19.2% enhancement in drug loading efficiency (DLE) observed here surpasses earlier benchmarks [7], likely due to optimized lipid ratios and covalent conjugation methods.
The in vitro BBB co-culture model revealed that ApoE-modified LNPs achieved a permeability coefficient of 5.1 ×10⁻⁶ cm/s, consistent with [12], who reported success in using ApoE for CRISPR/Cas9 delivery. However, unlike polymeric carriers highlighted by [4], lipid-based LNPs in this study demonstrated superior biocompatibility (<10% cytotoxicity at 100 µg/mL), underscoring their clinical appeal.
In vivo results further validated the targeted delivery paradigm: ApoE-modified LNPs enhanced brain drug accumulation by 2.7-fold while reducing off-target deposition in the liver (46.1%) and spleen (40.2%). These outcomes resonate with [16], who advocated for multidisciplinary approaches to optimize nanocarriers. The prolonged elimination half-life (t1/2 = 5.6 h) observed here, attributed to PEGylation-mediated RES evasion, corroborates [14] findings on lipid nanocarrier optimization.
Despite these advances, limitations warrant consideration. First, stability assessments were confined to in vitro conditions; long-term metabolic impacts in in vivo models remain unexplored. Second, while the BBB co-culture model effectively mimics physiological conditions, inherent disparities between in vitro and in vivo systems may affect translational predictability. Third, the focus on doxorubicin as a model drug necessitates further validation with neurotherapeutic agents (e.g., proteins or nucleic acids).
Future studies should explore dual-ligand strategies (e.g., TfR-BP + ApoE) to synergize multiple transcytosis pathways. Integrating advanced molecular imaging (e.g., PET-MRI) could enable real-time tracking of nanoparticle biodistribution. From a translational perspective, scaling up synthesis protocols while maintaining cost efficiency is critical for industrial adoption.
CONCLUSION
This study compellingly demonstrates that lipid nanoparticles (LNPs) functionalized with TfR-BP and ApoE ligands serve as an efficient and biocompatible system for targeted blood-brain barrier (BBB) traversal and drug delivery to brain tissue. The reduction in particle size (125–130 nm) and surface charge (<5 mV) in ligand-modified LNPs facilitated favorable interactions with cerebral endothelial cells, enhancing drug permeability by 4.2-fold. The significant improvement in drug loading efficiency (DLE ≈90%) and minimized off-target accumulation in peripheral organs (liver and spleen) underscore the superiority of these nanocarriers over conventional drug delivery systems. The prolonged elimination half-life (t1/2 ≈5.6 hours) further highlights their potential to reduce dosing frequency in clinical settings. Building on prior research, this study advances beyond initial proof-of-concept by optimizing lipid ratios and ligand conjugation methods, addressing critical challenges such as nanoparticle stability and cytotoxicity. While earlier work by [7] focused on standardizing synthesis protocols, this research provides comprehensive in vivo data to bridge the gap toward clinical application. Moreover, unlike polymeric carriers emphasized in studies such as [4], the lipid-based LNPs here exhibited superior biocompatibility and BBB penetration. However, translating these achievements into practical therapies requires overcoming existing limitations. First, long-term metabolic effects of LNPs must be evaluated in advanced in vivo models (e.g., non-human primates). Second, developing 3D co-culture systems incorporating neurons and astrocytes could enhance the predictive accuracy of in vitro models. Third, the generalizability of this technology to other neurotherapeutics (e.g., anti-inflammatory agents or proteins) warrants further investigation. Future research should explore dual-ligand strategies (e.g., combining TfR-BP and ApoE) to synergize multiple transcytosis pathways. Integrating molecular imaging techniques (e.g., PET-MRI) for real-time nanoparticle tracking could offer deeper insights into biodistribution dynamics. From an industrial perspective, optimizing synthesis protocols for scalability and cost efficiency is crucial for commercialization. This study marks a significant stride toward targeted therapies for neurological disorders such as Alzheimer’s, glioblastoma, and Parkinson’s. By mimicking natural biological mechanisms, ligand-engineered LNPs present a transformative approach to overcoming BBB challenges. Their successful clinical translation, however, hinges on interdisciplinary collaboration across nanotechnology, neuroscience, and clinical medicine, alongside sustained investment in translational research. In conclusion, this study positions ligand-engineered LNPs as a promising platform for overcoming BBB challenges in neurological disorders. Their clinical translation, however, hinges on addressing long-term safety, refining predictive models, and fostering interdisciplinary collaboration across nanotechnology, neuroscience, and clinical medicine.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.