Nanoparticle Uptake in Human Corneal Epithelial Cells

Topical ophthalmic formulations are convenient, but their performance is constrained by rapid tear-film clearance and the corneal epithelium. These barriers limit residence time, tissue penetration, and ultimately ocular bioavailability. The reference article, Enhancing Ocular Drug Delivery: The Effect of Physicochemical Properties of Nanoparticles on the Mechanism of Their Uptake by Human Cornea Epithelial Cells, addresses a central formulation question: how do nanoparticle size and surface chemistry influence the way particles interact with human corneal epithelial cells?

Published in ACS Biomaterials Science & Engineering, the study is valuable because it moves beyond asking whether nanoparticles enter corneal cells. Instead, it compares uptake across a deliberately varied PLGA platform and tests which endocytic routes are involved. The findings provide a mechanistic basis for designing topical ocular nanomedicines rather than relying only on empirical optimization.

Study Background and Research Question

The ocular surface presents several sequential barriers. The tear film contains a superficial lipid layer and a mucoaqueous layer enriched in mucins toward the epithelium. Mucus can retain foreign materials through physicochemical interactions, while secretion and shedding continuously remove material from the surface. Beneath this layer, the corneal epithelium forms a tightly packed, stratified barrier with junctional complexes between cells. The reference study emphasizes that although the epithelial layer represents only about 10% of total corneal thickness, it contributes approximately 90% of corneal barrier function.

Nanoparticles may help address these limitations by extending precorneal residence, protecting unstable payloads, enabling controlled release, and improving contact with ocular tissues. PLGA is particularly useful because its formulation can be adjusted across a range of sizes and surface characteristics while supporting biocompatible and biodegradable delivery systems. However, prior studies had not fully resolved how these physicochemical variables determine the cellular entry mechanism in a corneal epithelial model.

The research question was therefore both formulation-oriented and mechanistic: when PLGA nanoparticles differ in size or in their interaction with mucus, do they show different uptake levels in human corneal epithelial cells, and which forms of endocytosis account for that uptake?

Key Innovation from the Reference Study

The main innovation was the use of a controlled nanoparticle library that separated two design variables often confounded in delivery studies: particle size and surface chemistry. The authors prepared PLGA nanoparticles and modified their surfaces with alginate, chitosan, or polyethylene glycol. Alginate and chitosan were used as mucoadhesive coatings, whereas PEG represented a mucopenetrative surface strategy. This design allowed the study to examine not only whether particles reached cells, but also how interactions with simulated mucus might affect access to the epithelial surface.

The particles were reported to be spherical and monodisperse, with a polydispersity index below 0.2. Their sizes ranged from 100 to 250 nm, and their zeta potentials ranged from −25 to +15 mV, according to the published particle characterization. Keeping the materials within a defined physicochemical range strengthened the comparison between formulations. It also made the resulting uptake data more interpretable than data obtained from broadly heterogeneous particle preparations.

A second innovation was the use of a human corneal epithelial cell monolayer integrated with a simulated mucosal solution. This configuration was more informative than a conventional cell suspension assay because it introduced a relevant extracellular barrier before particles encountered the cells. The resulting model connected nanoparticle transport through a mucus-like environment with cellular internalization.

Methods and Experimental Design Insights

PLGA nanoparticles were synthesized using the emulsion-solvent evaporation method. Surface modification generated alginate-PLGA, chitosan-PLGA, and PEG-PLGA formulations alongside unmodified PLGA controls. The resulting particles were examined for morphology, size distribution, and surface charge. The combination of spherical morphology and low polydispersity reduced the possibility that a small number of oversized aggregates would dominate the uptake results.

Human corneal epithelial cells were cultured as a monolayer and evaluated in the presence of simulated mucosal solution. Cytocompatibility was assessed using the MTT assay after 24-hour incubation. At concentrations up to 100 μg/mL, the formulations produced only mild toxicity, with reported viability values between 70% and 100% in the reference paper. This preliminary safety assessment was important because apparent differences in particle uptake can be misleading if one formulation causes substantial loss of metabolic activity or cell number.

Cellular uptake was then measured across the nanoparticle panel. The investigators also used mechanistic inhibitors to distinguish energy-dependent uptake from passive association and to evaluate the contribution of macropinocytosis, caveolae-mediated endocytosis, clathrin-mediated endocytosis, and phagocytosis. This inhibitor-based strategy does not provide absolute pathway assignment, because pharmacological inhibitors can have off-target effects and may alter membrane physiology. Nevertheless, it offers a practical first-level map of uptake mechanisms when interpreted together with particle characterization and viability data.

Protocol Parameters

• Particle design: The literature-backed study compared spherical PLGA nanoparticles spanning 100–250 nm, with surface charge from −25 to +15 mV and a polydispersity index below 0.2. These values describe the investigated formulations, not universal requirements for ocular delivery.
• Cell model: Use a confluent human corneal epithelial monolayer with a simulated mucosal environment when the experimental objective is to approximate the epithelial and mucus-facing interface.
• Exposure assessment: Include a viability measurement alongside uptake measurements. The published study used a 24-hour MTT assessment at concentrations up to 100 μg/mL; new formulations should be requalified rather than assumed to have the same tolerance.
• Mechanistic testing: Compare untreated and vehicle controls with pathway-inhibitor conditions, and interpret uptake changes alongside cell viability and nanoparticle recovery. Inhibitor experiments should be viewed as pathway-supporting evidence rather than definitive molecular proof.

Core Findings and Why They Matter

Energy-dependent endocytosis was the primary route by which the tested nanoparticles entered human corneal epithelial cells. This observation argues against simple passive diffusion as the dominant explanation for intracellular particle accumulation. It also means that nanoparticle design must be considered in relation to cellular membrane trafficking, not only mucus retention or diffusion through the tear film.

Among the tested formulations, 100 nm PLGA nanoparticles and PEG-PLGA-150 nanoparticles showed the highest uptake levels. The result is informative for two reasons. First, it suggests that smaller particles within the tested range can be efficiently internalized. Second, it indicates that PEG surface modification does not necessarily reduce epithelial uptake when evaluated in a mucus-containing model. A mucopenetrative surface may help particles traverse the mucosal layer while preserving access to endocytic machinery at the cell surface.

Inhibitor studies identified macropinocytosis and caveolae-mediated endocytosis as the dominant pathways under the experimental conditions. Clathrin-mediated endocytosis contributed partially, whereas phagocytosis did not appear to play a significant role across the studied particle sizes and surface chemistries. These findings provide a more specific mechanistic interpretation than a general statement that nanoparticles are taken up by epithelial cells.

The results also have a practical implication for formulation screening. High uptake should not be treated as an isolated success criterion. A particle that enters cells efficiently but produces epithelial toxicity, disrupts barrier integrity, or fails to release its payload at the intended site may not improve therapy. The study’s combination of particle characterization, MTT viability testing, a mucosal interface, and pathway analysis is therefore a useful framework for balancing uptake with biological compatibility.

Comparison with Existing Internal Articles

The internal article Nanoparticle Uptake by Human Corneal Epithelial Cells provides a concise overview of the same 2024 study, emphasizing the controlled PLGA platform and the dominance of macropinocytosis and caveolae-mediated uptake. It is useful as an entry point, whereas the present analysis places greater emphasis on the experimental logic, the role of the mucus-containing model, and the limits of inhibitor interpretation.

A related resource, Nanoparticle Uptake in Corneal Cells: Role of Size and Surface Chemistry, focuses on how particle dimensions and surface properties shape epithelial uptake. Its emphasis complements the reference study’s mechanistic contribution. Together, these resources can help researchers connect formulation variables with uptake pathways, but neither should be treated as an independent replication of the ACS study.

Limitations and Transferability

The study provides a controlled in vitro mechanism map, not a complete prediction of ocular pharmacokinetics. A corneal epithelial monolayer with simulated mucosal solution cannot reproduce the full tear film, blinking, mucus turnover, corneal stroma, limbal biology, immune surveillance, or clearance through the nasolacrimal system. Uptake in cultured cells may therefore overestimate the fraction of a topical dose that reaches intracellular targets in vivo.

The conclusions are also bounded by the tested PLGA formulations, size range, surface chemistries, exposure conditions, and cell model. The absence of a detectable role for phagocytosis should not be generalized to all ocular cell types or to substantially larger, aggregated, or irregular particles. Similarly, inhibitor-based pathway assignments should be confirmed with orthogonal methods such as genetic perturbation, imaging of intracellular trafficking, colocalization with compartment markers, or temperature and energy-depletion controls.

For translational development, the next step is not simply to maximize uptake. Researchers should determine whether the internalized particles protect their payload, release it in a therapeutically useful compartment, preserve epithelial barrier function, and improve ocular exposure in more physiologically complete models. The reference findings are best viewed as design guidance for these subsequent studies.

Why this cross-domain matters, maturity, and limitations

Actin-dependent membrane remodeling is relevant to many forms of endocytosis, so cytoskeletal perturbation can be used as a complementary test of whether uptake depends on an intact actin network. However, the reference study did not test an actin polymerization inhibitor, and its conclusions should not be presented as direct evidence for actin dependence. Extending the work into cancer or viral models would represent a separate research question requiring its own controls, endpoints, and literature support. The mature conclusion is therefore limited: the ACS study establishes uptake pathways in a corneal epithelial model, while cytoskeletal perturbation can be used cautiously to probe mechanism in follow-up experiments.

Research Support Resources

Researchers can use Cytochalasin D (SKU B6645), an actin polymerization inhibitor, to support a mechanistic comparator workflow when testing whether nanoparticle uptake changes after actin-network disruption. The product information reports an IC50 of 25 nM and typical cell-culture concentrations of 0.2–0.5 μg/mL; these values should be treated as starting points rather than validated conditions for human corneal epithelial cells. A vehicle control, viability-matched exposure, and nanoparticle-only control are essential.

Protocol Parameters

• Comparator setup: Establish baseline nanoparticle uptake first, then compare untreated, vehicle-treated, and Cytochalasin D-treated cells under matched exposure conditions.
• Concentration planning: Begin with a small concentration range around the product information’s suggested cell-culture window and determine cytotoxicity and barrier effects independently in the selected HCEC model.
• Handling: The product information describes the compound as DMSO-soluble at greater than 10 mM and recommends desiccated storage at −20 °C. Prepare solutions close to use and avoid relying on long-term storage of diluted material.
• Interpretation: A reduction in uptake is mechanistically informative only when cell viability, morphology, and nanoparticle exposure remain adequately controlled.

The same product information describes broader observations including cell cycle arrest at G1-S transition, tumor cell proliferation inhibition, apoptosis induction in cancer cells, and viral transcription inhibition in other experimental systems. These secondary applications are not findings of the ocular nanoparticle study and should be evaluated as separate, model-specific hypotheses.