Nanoparticle Uptake in Human Corneal Epithelial Cells

Topical eye drops are widely used, but their effectiveness is constrained by rapid tear-film clearance and the highly organized corneal epithelium. The study by Marjan Azadi and Allan E. David addresses a central formulation question: how do nanoparticle size and surface chemistry determine interactions with human cornea epithelial cells (HCECs)? Published in ACS Biomaterials Science & Engineering, the work provides a mechanistic framework for designing polymeric carriers that must first navigate the ocular surface and then enter corneal cells.

Study Background and Research Question

The ocular surface presents several sequential barriers to topical delivery. The tear film contains a superficial lipid layer and a mucoaqueous layer enriched with mucins toward the epithelial surface. Mucin can entrap foreign materials through its heterogeneous network and functional groups, while continuous secretion and shedding promote clearance. Beneath this layer, the cornea contains multiple tissue layers with distinct physicochemical properties.

The epithelium is particularly important because its tightly packed, stratified cells and junctional complexes account for most of the corneal barrier function despite representing only a fraction of total corneal thickness. Conventional strategies, including viscosity enhancers, penetration enhancers, ointments, prodrugs, and in situ gels, may increase residence time or permeability but can also introduce irritation, inflammation, blurred vision, or systemic exposure concerns.

Polymeric nanoparticles offer a different approach. They can potentially prolong precorneal retention, protect unstable payloads, provide sustained release, and alter the balance between mucus adhesion and mucus penetration. PLGA is especially useful because its formulation properties can be adjusted across broad ranges. However, the field still lacked a clear understanding of how particle size and surface modification affect uptake by corneal epithelial cells. The reference study directly investigates that gap.

Key Innovation from the Reference Study

The study’s main innovation is its controlled comparison of PLGA nanoparticles that differ in both dimensions and interfacial chemistry while using a human corneal epithelial model containing a simulated mucosal environment. Rather than treating nanoparticle uptake as a single outcome, the authors connect uptake magnitude with likely cellular entry pathways.

Surface modifications were selected to represent different interactions with the mucosal barrier. Alginate and chitosan were used as mucoadhesive polymers, whereas polyethylene glycol (PEG) was used to produce a more mucopenetrative interface. This design allows the study to ask whether stronger interaction with mucus necessarily improves epithelial uptake, or whether a particle that moves more readily through the mucosal layer may reach cells more effectively.

The work also separates energy-dependent internalization from passive association. Inhibitor experiments were used to examine the contribution of macropinocytosis, caveolae-mediated endocytosis, clathrin-mediated endocytosis, and phagocytosis. This mechanistic resolution is valuable because two formulations with similar uptake levels may enter cells through different routes, leading to different intracellular trafficking and drug-release behavior. The reference study therefore contributes both formulation data and a way to interpret nanoparticle–cell interactions.

Methods and Experimental Design Insights

PLGA nanoparticles were produced using an emulsion–solvent evaporation method. The resulting particles were surface modified with alginate, chitosan, or PEG. Characterization showed spherical, relatively uniform populations. The study then assessed cytocompatibility with an MTT assay and evaluated cellular uptake using an HCEC monolayer combined with simulated mucosal solution.

This design is useful because it places the cells behind a simplified representation of the ocular mucus barrier rather than exposing them only to nanoparticles in an unprotected culture medium. The approach does not reproduce the complete human eye, but it provides a more physiologically relevant first screen for formulations intended for topical delivery.

To investigate mechanism, uptake was measured in the presence of pathway-selective inhibitors. The results were interpreted alongside particle size, zeta potential, and surface chemistry. Importantly, the study evaluates these variables as a set: particle dimensions influence cellular contact and internalization, while surface charge and polymer identity influence interactions with mucosal components and the plasma membrane.

Protocol Parameters

• Nanoparticle platform: PLGA particles were prepared by emulsion–solvent evaporation and modified with alginate, chitosan, or PEG, as reported in the reference study.
• Particle-property window: The investigated particles were approximately 100–250 nm, with zeta potentials ranging from −25 to +15 mV and polydispersity indices below 0.2.
• Cytotoxicity assessment: HCECs were incubated with nanoparticles for 24 hours at concentrations up to 100 μg/mL, followed by MTT-based viability evaluation; reported viability remained within approximately 70–100%.
• Cellular model: Uptake was assessed in an HCEC monolayer integrated with simulated mucosal solution rather than in a cell-only suspension assay.
• Mechanistic analysis: Uptake inhibitors were used to compare macropinocytosis, caveolae-mediated endocytosis, clathrin-mediated endocytosis, and phagocytosis.

These are literature-reported parameters, not universal operating conditions. Researchers adapting the model should independently optimize nanoparticle concentration, exposure time, mucosal composition, fluorescent labeling or quantification procedures, and inhibitor controls for their own formulation.

Core Findings and Why They Matter

The nanoparticles were monodisperse and spherical across the tested formulations, supporting a relatively clean comparison of size and surface chemistry. The MTT results indicated only mild toxicity under the reported exposure conditions. This is important for uptake studies because severe loss of viability could produce misleading changes in apparent internalization or membrane permeability. The reported viability data support the use of this particle range for exploratory HCEC experiments, while not eliminating the need for formulation-specific toxicity testing.

Uptake was primarily energy dependent, indicating that the particles entered cells mainly through endocytic processes rather than by simple passive diffusion. Among the tested formulations, 100 nm PLGA nanoparticles and PEG-PLGA particles of approximately 150 nm showed the highest uptake by HCECs. This finding argues against a simplistic assumption that either the smallest particle or every mucoadhesive coating will always perform best.

The inhibitor experiments further suggested that macropinocytosis and caveolae-mediated endocytosis were the dominant uptake routes. Clathrin-mediated endocytosis contributed partially, whereas phagocytosis did not appear to play a meaningful role within the studied size and surface-chemistry ranges. These observations matter for payload design. Endocytic pathways can influence whether a particle remains in vesicular compartments, reaches other intracellular locations, or releases its payload before degradation.

The study also highlights a practical distinction between mucosal transport and cellular entry. A surface that promotes mucus interaction may improve retention but could hinder movement through the mucosal layer. Conversely, PEG may reduce nonspecific interactions and facilitate access to epithelial cells, but the optimal outcome depends on particle size and the local biological environment. The strongest formulations therefore cannot be selected from surface chemistry alone; size, charge, polymer coating, and exposure conditions must be evaluated together.

Comparison with Existing Internal Articles

The internal article Clathrin-Mediated Entry of Grass Carp Reovirus offers a useful conceptual contrast. That article describes clathrin-mediated endocytosis as a primary entry route for a specific aquatic virus, whereas Azadi and David found macropinocytosis and caveolae-mediated uptake to be more prominent for PLGA nanoparticles in HCECs, with clathrin contributing only partially. The comparison reinforces that endocytic mechanisms are highly dependent on cargo structure, cell type, receptor context, and experimental conditions.

The two studies should not be treated as interchangeable mechanistic evidence. Viral entry involves specialized pathogen–host interactions, while the reference paper examines synthetic polymeric particles crossing a corneal epithelial interface. Nevertheless, placing them side by side helps prevent the common error of assigning one universal uptake pathway to all nanoscale materials.

Why this cross-domain matters, maturity, and limitations

The cross-domain comparison is mature enough to support a methodological lesson, not a direct formulation prediction: pathway assignments must be experimentally tested in the target cells. The reference study provides that target-cell evidence for HCECs, but it does not establish how the same particles would behave in intact human cornea, inflamed ocular tissue, or in the presence of a complete tear film.

Limitations and Transferability

The model is an important intermediate step between basic nanoparticle characterization and animal testing, but it remains an in vitro approximation. A cultured HCEC monolayer does not fully reproduce the stratified architecture, junctional organization, tear turnover, blinking, immune surveillance, or regional differences of the human ocular surface. Simulated mucosal solution also cannot capture the complete biochemical diversity of native tears and mucins.

Uptake inhibitors are informative but not perfectly pathway specific. Changes in cellular metabolism, membrane organization, or vesicle trafficking can affect several routes simultaneously. Consequently, the conclusion that macropinocytosis and caveolae-mediated endocytosis dominate should be understood as a result of the inhibitor-supported experimental model, not as a universal rule.

Transferability is also limited by formulation details. Small changes in PLGA molecular characteristics, residual solvent, coating density, aggregation state, payload loading, or surface charge can alter uptake and toxicity. The study focuses on nanoparticle–cell interactions rather than drug release, intracellular fate, ocular residence time, or therapeutic efficacy. Follow-up work should therefore combine uptake measurements with barrier integrity, release kinetics, tissue distribution, and in vivo tolerability.

Even with these limitations, the study offers a practical decision framework. A formulation should be screened for physicochemical uniformity, tested under a mucosal condition, evaluated for cell viability, and then examined with complementary mechanistic assays. This sequence is more informative than optimizing uptake alone.

Research Support Resources

The reference paper is most directly useful for researchers developing nanoparticle-based ocular delivery systems and selecting particle sizes or surface chemistries for HCEC studies. It does not test antifungal payloads, and its findings should not be interpreted as evidence for clinical ocular use of any specific antibiotic.

Why this cross-domain matters, maturity, and limitations

For antifungal formulation researchers, related questions may include inhibition of Candida albicans adhesion, liposomal Nystatin for Aspergillus infection, vulvovaginal candidiasis treatment, and antifungal resistance in non-albicans Candida. Those applications belong to different biological and therapeutic contexts, so the ocular nanoparticle findings provide a delivery-study framework rather than efficacy evidence. Any cross-domain formulation would require independent testing of loading, release, epithelial compatibility, fungal susceptibility, and tissue exposure.

Researchers can use Nystatin (Fungicidin) (SKU B1993) as an antifungal payload candidate in related laboratory workflows. Its ergosterol-binding membrane-disruption mechanism makes it suitable for controlled studies of formulation, cytotoxicity, and antifungal activity; preparation and ocular suitability should be optimized experimentally, and the material is intended for research use only.