Nanoparticle Uptake by Human Corneal Epithelial Cells

Topical ocular medicines are convenient, but their effectiveness is constrained by rapid precorneal clearance and the limited permeability of the ocular surface. The reference study by Marjan Azadi and Allan E. David addresses a central question in ocular nanomedicine: how do nanoparticle size and surface chemistry alter interactions with human corneal epithelial cells (HCECs), and which endocytic routes account for cellular internalization?

Study Background and Research Question

The tear film and cornea form a highly effective barrier against foreign materials. The tear film combines a superficial lipid layer with a mucoaqueous layer containing mucins, which can entrap particles through physicochemical interactions and promote their clearance through continuous secretion and shedding. Beneath this interface, the cornea contains five tissue layers with distinct physicochemical properties. Although the epithelial layer represents only about 10% of total corneal thickness, it contributes approximately 90% of the corneal barrier function, according to the reference study.

Conventional strategies for increasing ocular residence or penetration include viscosity enhancers, penetration enhancers, ointments, prodrugs, and in situ gels. These approaches can improve exposure, but they may also introduce irritation, inflammation, blurred vision, or systemic complications. Polymeric nanoparticles offer a different design framework: they can prolong surface residence, protect unstable cargo, control release, and potentially improve interaction with ocular tissues. Poly(lactic-co-glycolic acid), or PLGA, is particularly useful because its formulation properties can be adjusted across a range of particle sizes and surface chemistries.

The unresolved issue was not simply whether PLGA nanoparticles enter corneal cells, but how specific physicochemical features determine the mechanism and extent of uptake. This distinction matters because increasing total cell-associated fluorescence does not necessarily identify productive internalization, intracellular trafficking, or suitability for therapeutic delivery.

Key Innovation from the Reference Study

The study’s main innovation is its integrated comparison of nanoparticle size and surface modification within a human corneal epithelial model that includes a simulated mucosal environment. The investigators produced PLGA nanoparticles and modified their surfaces with alginate and chitosan, selected for mucoadhesive behavior, or polyethylene glycol, selected for mucopenetrative behavior. This design allowed the authors to examine how particle–mucus interactions and particle–cell interactions work together rather than evaluating uptake in a simplified cell suspension alone.

The work also connects uptake magnitude with mechanism. By combining nanoparticle characterization, cell-viability testing, a HCEC monolayer model, and inhibitor-based pathway analysis, the authors moved beyond a formulation-screening study. The resulting framework helps distinguish particles that remain associated with the extracellular mucosal interface from those that are internalized through defined energy-dependent processes.

Methods and Experimental Design Insights

PLGA nanoparticles were synthesized using the emulsion–solvent evaporation method. The resulting formulations were surface-modified with alginate, chitosan, or polyethylene glycol to create different interfacial properties. The particles were characterized for morphology, size distribution, and zeta potential. Across the tested formulations, the particles were spherical and monodisperse, with a polydispersity index below 0.2. Their hydrodynamic sizes ranged from approximately 100 to 250 nm, while zeta potentials ranged from −25 to +15 mV, as reported in the published article.

Before interpreting uptake, the investigators assessed cytotoxicity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, or MTT, assay. HCECs exposed to nanoparticles for 24 hours at concentrations up to 100 μg/mL generally retained 70–100% viability, indicating mild toxicity under the reported conditions. This control is important because reduced metabolic activity can otherwise be mistaken for reduced nanoparticle uptake or altered endocytosis.

Cellular internalization was evaluated using a HCEC monolayer integrated with a simulated mucosal solution. This arrangement better represents the sequential barriers encountered by a topical formulation: diffusion through a mucin-containing interface followed by contact with a stratified epithelial-like cellular barrier. Uptake experiments were then performed in the presence of mechanistic inhibitors. The authors used the resulting changes in nanoparticle association to assess the contributions of energy-dependent endocytosis, macropinocytosis, caveolae-mediated uptake, clathrin-mediated uptake, and phagocytosis.

Protocol Parameters

• Nanoparticle platform: PLGA particles prepared by emulsion–solvent evaporation and modified with alginate, chitosan, or polyethylene glycol; these are literature-backed features of the reference design.
• Particle range: Approximately 100–250 nm with zeta potentials from −25 to +15 mV and a polydispersity index below 0.2, according to the reference study.
• Cell model: Use a HCEC monolayer together with a simulated mucosal solution when the objective is to assess uptake under a more physiologically relevant surface barrier.
• Viability control: Include an MTT or equivalent viability assay before interpreting uptake data; the reported study tested 24-hour exposure and concentrations up to 100 μg/mL.
• Mechanistic interpretation: Treat inhibitor experiments as pathway-supporting evidence rather than absolute proof, because pharmacological inhibitors can have overlapping or nonspecific effects.

Core Findings and Why They Matter

Energy-dependent endocytosis was the principal route by which the tested nanoparticles entered HCECs. Among the formulations, unmodified 100 nm PLGA nanoparticles and PEG-PLGA nanoparticles with a nominal size of 150 nm showed the highest uptake. The result suggests that smaller size can favor epithelial internalization, but surface chemistry remains equally important: PEG modification may improve movement through the simulated mucosal environment even when the particle is not the smallest formulation.

Inhibitor studies indicated that macropinocytosis and caveolae-mediated endocytosis were the dominant pathways under the experimental conditions. Clathrin-mediated endocytosis also contributed, but only partially. Phagocytosis did not appear to play a meaningful role within the tested size and surface-chemistry ranges. This pathway profile is useful for formulation development because it shows that uptake is not governed by one universal mechanism. Particle dimensions, interfacial charge, polymer coating, and the surrounding mucosal environment jointly influence how HCECs process nanoparticles.

The findings have two practical implications. First, optimization should measure both nanoparticle passage through the mucosal interface and intracellular uptake; a strongly mucoadhesive coating may increase residence but could also limit mobility toward epithelial cells. Second, a formulation that produces high uptake in a basic monolayer assay may behave differently when mucin and dynamic clearance are included. The study therefore supports a staged screening strategy that starts with physicochemical characterization, confirms tolerability, and then evaluates uptake and pathway dependence in a barrier-relevant model.

Comparison with Existing Internal Articles

The internal article Nanoparticle Uptake Mechanisms in Human Corneal Epithelial Cells reaches a compatible interpretation: energy-dependent endocytosis, particularly macropinocytosis and caveolae-mediated uptake, is central to nanoparticle internalization. Its value is explanatory and application-oriented, whereas the Azadi and David study provides the experimental backbone linking those pathways to defined PLGA sizes and surface modifications in a mucosal HCEC model.

Viewed together, the resources support a mechanism-first approach to ocular nanoparticle design. The reference paper, however, should remain the source for the reported particle ranges, viability results, and comparative uptake findings.

Limitations and Transferability

The model improves on a simple two-dimensional uptake assay but does not reproduce the full human ocular surface. A HCEC monolayer with simulated mucosal solution lacks the complete tear-film composition, blinking, epithelial stratification, stromal architecture, immune interactions, and vascular or lymphatic clearance present in vivo. Consequently, the observed uptake hierarchy should not be interpreted as a direct prediction of corneal penetration or clinical bioavailability.

The tested physicochemical space was also limited. The conclusions apply most directly to spherical PLGA nanoparticles within the reported size and surface-potential ranges. They may not transfer to substantially larger or irregular particles, other biodegradable polymers, different ligand densities, or formulations carrying cargos that alter surface charge and aggregation. Uptake measurements likewise do not establish whether cargo is released in the cytosol, trafficked to a desired compartment, or retained long enough to produce a therapeutic effect.

Finally, inhibitor-based pathway assignment requires caution. A decrease in uptake after inhibition is consistent with pathway involvement, but it does not prove exclusive dependence. Complementary approaches such as genetic perturbation, imaging of intracellular trafficking, and transport studies across more complex corneal models would strengthen mechanistic conclusions. These limitations do not weaken the study’s central contribution; rather, they define the experiments needed before translating the design rules to animal or human ocular systems.

Why this cross-domain matters, maturity, and limitations

Actin dynamics are relevant to endocytic remodeling, so an actin polymerization inhibitor can serve as a supporting perturbation when researchers test whether nanoparticle uptake depends on cytoskeletal organization. This is a mechanistic bridge, not a result reported by Azadi and David. Cytochalasin D should therefore be used to probe actin dependence alongside pathway controls, not to assign macropinocytosis or caveolae-mediated uptake by itself. Its broader effects on cell shape, adhesion, cytokinesis, and transport can complicate interpretation, particularly in corneal epithelial cells.

Research Support Resources

For related uptake experiments, researchers can use Cytochalasin D (SKU B6645) as an actin polymerization inhibitor in carefully controlled mechanistic workflows. The product information reports an IC50 of 25 nM and lists typical cell-culture concentrations of 0.2–0.5 μg/mL; these are product-level specifications rather than parameters established in the ocular nanoparticle paper. Solutions should be prepared and used promptly rather than stored long term. Separate product information also describes cell cycle arrest at G1-S transition, tumor cell proliferation inhibition, and apoptosis induction in cancer cells; those contexts should not be transferred to HCECs or presented as findings from the reference study.