Nanoparticle Uptake in Human Corneal Epithelial Cells

Topical ophthalmic formulations are widely used, but their effectiveness is limited by rapid precorneal clearance and the restrictive properties of the tear film and corneal epithelium. The 2024 study by Marjan Azadi and Allan E. David addressed a specific unresolved question: how do nanoparticle size and surface chemistry influence interactions with human corneal epithelial cells (HCECs), and which endocytic pathways account for uptake? The full study is available through the ACS Biomaterials Science & Engineering publication.

Study Background and Research Question

The ocular surface presents several sequential barriers to drug delivery. Tear-film components can entrap foreign materials through mucin interactions, while continuous secretion and shedding promote clearance. Beneath this fluid interface, the corneal epithelium provides most of the cornea’s barrier function through tightly packed, junctionally connected cells. These features help protect the eye but also restrict penetration of conventional drug formulations.

Nanoparticles may improve ocular delivery by increasing surface residence, protecting unstable payloads, enabling sustained release, and facilitating interaction with epithelial tissues. PLGA is particularly useful because its formulation properties can be adjusted across broad ranges of particle size and surface characteristics. However, simply demonstrating that nanoparticles enter corneal cells does not explain why some formulations are taken up more efficiently than others. The reference study therefore focused on the relationship between physicochemical design and cellular internalization rather than treating uptake as a single, nonspecific process.

Key Innovation from the Reference Study

The central innovation was the use of a comparatively controlled PLGA nanoparticle series in which size and surface modification could be examined alongside uptake mechanism. The investigators compared unmodified PLGA particles with particles modified by alginate (ALG), chitosan (CHS), or polyethylene glycol (PEG). ALG and CHS were selected for mucoadhesive behavior, whereas PEG represented a mucopenetrative surface strategy.

This design created a useful contrast between formulations intended to interact more strongly with mucosal components and those intended to move through them more readily. Rather than reporting only total cellular uptake, the study combined uptake measurements with pharmacological pathway inhibition. That combination allowed the authors to distinguish energy-dependent internalization from specific contributions of macropinocytosis, caveolae-mediated endocytosis, clathrin-mediated endocytosis, and phagocytosis.

Methods and Experimental Design Insights

PLGA nanoparticles were produced using an emulsion-solvent evaporation method and then surface-modified with ALG, CHS, or PEG. The resulting particles were characterized for morphology, size distribution, and zeta potential. The formulations were described as spherical and monodisperse, providing a more interpretable comparison than a highly heterogeneous nanoparticle preparation.

For biological testing, the researchers used an in vitro HCEC monolayer integrated with a simulated mucosal solution. This model is more relevant to topical ocular delivery than a cell culture system that omits the mucosal interface, although it remains simpler than an intact, stratified cornea. Cytocompatibility was evaluated with the MTT assay before uptake experiments. Cellular internalization was then assessed across the different nanoparticle formulations, followed by inhibitor studies designed to probe pathway dependence.

Protocol Parameters

The following parameters are reported characteristics of the reference study. They should be treated as replication information rather than universal operating conditions.

• Nanoparticle platform: PLGA particles prepared by emulsion-solvent evaporation and modified with ALG, CHS, or PEG, as described in the reference study.
• Particle profile: spherical, monodisperse particles with a polydispersity index below 0.2, sizes spanning approximately 100–250 nm, and zeta potentials from about −25 to +15 mV, according to the published characterization.
• Cytotoxicity assessment: HCECs were incubated with nanoparticles for 24 hours at concentrations up to 100 µg/mL; the reported MTT results corresponded to approximately 70–100% cell viability under the tested conditions.
• Cellular model: uptake was evaluated in an HCEC monolayer combined with a simulated mucosal solution rather than in isolated cells without a mucosal interface.
• Mechanistic analysis: use pathway inhibitors as comparative perturbations, but interpret them as pharmacological evidence with possible off-target effects rather than definitive pathway-specific proof.
• Workflow suggestion: compare particle size and surface chemistry within the same cell batch, and report particle characterization together with uptake data so that biological differences can be separated from formulation variability.

Core Findings and Why They Matter

The first important result was that the tested formulations were generally well tolerated in the in vitro model. The MTT measurements indicated only mild toxicity across the reported exposure range. This supports the use of the platform for uptake studies, but it does not establish long-term ocular safety, effects on epithelial barrier integrity, or compatibility with repeated dosing.

Energy-dependent endocytosis was the principal uptake mode. Among the tested formulations, 100 nm PLGA nanoparticles and PEG-PLGA particles with a nominal size of 150 nm showed the highest uptake by HCECs. These observations suggest that smaller particles may be internalized efficiently, but the result should not be reduced to a simple rule that smaller is always better. Surface chemistry altered the interaction landscape, and PEG modification produced strong uptake in at least one intermediate-size formulation.

Inhibitor experiments provided the main mechanistic advance. Macropinocytosis and caveolae-mediated endocytosis appeared to make the largest contributions under the study conditions. Clathrin-mediated endocytosis also contributed, but only partially, while phagocytosis did not appear to account for uptake within the tested size and surface-chemistry range. The findings therefore support a distributed mechanism in which multiple endocytic routes operate simultaneously, with their relative importance affected by particle design and the epithelial model.

For ocular drug delivery, the practical implication is that nanoparticle optimization should not rely solely on retention or nominal penetration. A formulation that remains on the ocular surface may still show limited therapeutic value if it is poorly internalized by relevant epithelial cells, whereas a formulation with high uptake may not achieve useful transcorneal transport or intracellular release. The study connects these stages by showing how measurable physical properties can influence the first cellular interaction.

Comparison with Existing Internal Articles

The internal article Nanoparticle Uptake by Human Corneal Epithelial Cells provides a concise overview of the same study’s principal conclusion: energy-dependent uptake dominates, with macropinocytosis and caveolae-associated mechanisms contributing strongly. The reference paper adds the experimental context needed to interpret that summary, including particle preparation, surface modifications, cytocompatibility testing, and the comparison of size classes.

A second related resource, Physicochemical Properties Drive Nanoparticle Uptake in HCECs, emphasizes the design implications of the work. Its focus on size and surface chemistry complements the reference article’s pathway analysis. Together, the resources support a formulation strategy in which particle characterization and mechanistic uptake measurements are considered together rather than as separate stages.

Limitations and Transferability

The study’s conclusions are strongest for the tested PLGA formulations, HCEC monolayer model, simulated mucosal environment, and selected size and surface-chemistry range. An in vitro monolayer cannot reproduce the full architecture of the stratified corneal epithelium, intact tight-junction organization, tear-film dynamics, blinking, immune surveillance, or clearance by drainage. Consequently, increased cellular uptake should not be interpreted automatically as improved delivery across the entire cornea or as evidence of therapeutic benefit in vivo.

Pharmacological inhibitors also require cautious interpretation. Inhibitor sensitivity can indicate pathway involvement, but incomplete selectivity, concentration-dependent toxicity, and compensatory changes in membrane trafficking may influence the result. The study did not establish that one pathway is exclusively responsible for a given formulation’s uptake. In addition, particle uptake does not by itself reveal intracellular trafficking, degradation, endosomal escape, payload release, or the relationship between internalization and pharmacodynamic activity.

Future studies can build on this framework by combining pathway perturbation with barrier-integrity measurements, live-cell trafficking analysis, payload-specific assays, and more physiologically complex corneal models. Such extensions would test whether the uptake patterns observed here remain predictive when mucus turnover, epithelial stratification, and tissue-level transport are included.

Research Support Resources

Why this cross-domain matters, maturity, and limitations

The reference study concerns nanoparticle uptake in corneal epithelial cells; it does not test an actin-directed compound or establish that actin remodeling is responsible for the reported endocytic pattern. In a separate mechanistic workflow, researchers may use an actin polymerization inhibitor as a perturbation to examine whether cytoskeletal disruption changes nanoparticle internalization, but such experiments require vehicle, viability, barrier-integrity, and pathway-specific controls. This is a hypothesis-testing extension, not a finding of the ocular study.

The product information for Cytochalasin D (SKU B6645) describes it as an actin polymerization inhibitor suitable for supporting related cytoskeletal and uptake workflows. The same information discusses cell cycle arrest at G1-S transition, tumor cell proliferation inhibition, apoptosis induction in cancer cells, and viral transcription inhibition in separate biological contexts; those effects should not be inferred for HCECs or PLGA nanoparticle uptake without direct validation.