Understanding Nanoparticle Uptake in Ocular Drug Delivery: Insights from Human Corneal Epithelial Cells

Study Background and Research Question

Ocular diseases present a persistent challenge for researchers and clinicians, largely due to the unique anatomical and physicochemical barriers of the eye. Conventional topical formulations—such as eye drops—remain the mainstay of ophthalmic therapy, yet suffer from low bioavailability and poor penetration into ocular tissues. The complex tear film and the multi-layered structure of the cornea, especially the tightly packed epithelium, act as formidable barriers that limit drug delivery and therapeutic efficacy. Researchers thus seek innovative strategies to overcome these hurdles and achieve targeted, sustained, and efficient drug delivery to the eye.

Polymeric nanoparticles, particularly those based on polylactic-co-glycolic acid (PLGA), have emerged as promising vehicles for ocular drug delivery. However, despite extensive research into their utility, a critical knowledge gap remains: how do the physicochemical properties of nanoparticles—namely, size and surface chemistry—influence their interaction with and uptake by corneal epithelial cells? The reference study by Marjan Azadi and Allan E. David (ACS Biomater. Sci. Eng. 2024, 10, 429−441) addresses this question with a focus on the mechanisms underlying nanoparticle uptake in human cornea epithelial cells (HCECs).

Key Innovation from the Reference Study

The central innovation of this work lies in its systematic analysis of how nanoparticle size and surface modifications affect cellular uptake mechanisms in an in vitro human cornea model. By synthesizing a spectrum of PLGA nanoparticles—bare and surface-modified with mucoadhesive (alginate, chitosan) and mucopenetrative (polyethylene glycol, PEG) polymers—the researchers dissected the interplay between nanoparticle physicochemical properties and epithelial barrier interactions. Importantly, the study not only quantifies uptake but also elucidates the endocytic pathways responsible, providing actionable insights for the rational design of ocular therapeutics.

Methods and Experimental Design Insights

The authors employed the emulsion-solvent evaporation technique to fabricate monodisperse PLGA nanoparticles, with polydispersity indices below 0.2 and sizes ranging from 100 to 250 nm. Surface chemistry was tuned using alginate (ALG), chitosan (CHS), and PEG, producing particles with zeta potentials between −25 and +15 mV. Cytotoxicity was assessed using the MTT assay, confirming high cell viability (70–100%) at concentrations up to 100 μg/mL after 24-hour incubation.

To model the corneal epithelial barrier, a monolayer of HCECs was integrated with a simulated mucosal solution, recapitulating the heterogeneity and functional complexity of the tear film and corneal surface. Uptake studies were performed across the nanoparticle library, both in the presence and absence of specific endocytosis inhibitors, allowing pathway-specific investigations.

Core Findings and Why They Matter

The study's primary findings can be summarized as follows:

• Size and Surface Chemistry Dictate Uptake: 100 nm PLGA nanoparticles, especially those modified with PEG (PEG-PLGA-150), exhibited the highest uptake by HCECs. In contrast, larger or differently modified particles showed reduced internalization.
• Energy-Dependent Endocytosis Dominates: Cellular uptake was primarily mediated by energy-dependent processes, as evidenced by marked reduction at low temperatures and in the presence of metabolic inhibitors.
• Pathway Specificity: Macropinocytosis and caveolae-mediated endocytosis were identified as the dominant uptake routes, with partial contribution from clathrin-mediated endocytosis. Phagocytosis and cholesterol-dependent pathways were not involved within the tested size and chemistry range.
• Safety Profile: The nanoparticles were generally well-tolerated, with minimal cytotoxicity at practical concentrations.

These findings are significant for the rational engineering of ocular nanomedicines. Enhanced uptake via optimized size (∼100 nm) and PEGylation may enable improved delivery of antifungal, anti-inflammatory, or antimicrobial agents directly to corneal tissues, addressing the limitations of conventional eye drops—such as rapid clearance and poor penetration. The identification of key endocytic pathways further enables researchers to tailor nanoparticle design for maximal efficacy and minimal off-target effects.

Comparison with Existing Internal Articles

Mechanistic insights from the reference study echo findings in other cellular systems. For example, studies on microbial entry into Drosophila S2 cells—such as Spiroplasma eriocheiris—also highlight the pivotal roles of clathrin-mediated endocytosis and macropinocytosis in cellular uptake. However, the ocular context introduces additional challenges, including the dynamic tear film and stratified epithelial architecture, underscoring the necessity for cornea-specific investigations.

In the antifungal field, Nystatin (Fungicidin): Mechanism, Efficacy & Workflow Evidence discusses how effective inhibition of Candida albicans adhesion and resistance in non-albicans species depend on drug delivery and membrane interactions. The improved nanoparticle penetration described in the current study offers a potential strategy to enhance the delivery of antifungal agents, such as Nystatin, directly to ocular tissues where conventional formulations are often inadequate. Translational perspectives are further explored in Translational Antifungal Innovation: Mechanistic Insights, which advocates for nanoparticle and liposomal formulations to improve antifungal efficacy and overcome resistance.

Limitations and Transferability

While the in vitro corneal model provides high experimental control and mechanistic clarity, it cannot fully replicate the complexity of the in vivo ocular surface, where tear dynamics, immune responses, and systemic absorption may influence outcomes. Additionally, the study focused exclusively on PLGA-based nanoparticles and a narrow size/surface chemistry spectrum. As such, extrapolation to other polymer systems or larger/smaller particles should be done cautiously.

The identification of macropinocytosis and caveolae-mediated endocytosis as dominant uptake routes may also differ depending on cell type, disease state, or presence of specific bioactive agents. Notably, the study did not assess functional delivery of therapeutic payloads or long-term tissue responses, both of which are critical for clinical translation.

Protocol Parameters

• PLGA nanoparticle synthesis: Emulsion-solvent evaporation; size range 100–250 nm; polydispersity index (PDI) < 0.2.
• Surface modification: ALG, CHS, or PEG coatings; zeta potential tuning from −25 to +15 mV.
• Cytotoxicity testing: MTT assay; up to 100 μg/mL; 24-hour incubation; cell viability ≥70% considered acceptable.
• In vitro uptake studies: HCEC monolayer with simulated mucosal solution; uptake quantified with and without endocytosis inhibitors.
• Pathway inhibition: Use of temperature shift and specific inhibitors to dissect macropinocytosis, caveolae-mediated, and clathrin-mediated endocytosis.

Research Support Resources

For researchers aiming to translate these mechanistic insights into antifungal or antimicrobial delivery workflows, benchmark agents such as Nystatin (Fungicidin) (SKU B1993) are widely used to study inhibition of Candida albicans adhesion and resistance mechanisms. According to the product information, Nystatin demonstrates potent antifungal activity and is compatible with nanoparticle and liposomal delivery strategies, supporting investigation of novel ocular and mucosal infection models. Stock solutions should be prepared in DMSO, warmed and/or sonicated for optimal solubility, and stored at −20°C for stability. As always, this reagent is intended for research use only and not for diagnostic or therapeutic applications.