Pharmaceutical Development and Technology (Informa Healthcare)2014ResearchNon-viral Gene Delivery

Formulation and optimization of celecoxib-loaded PLGA nanoparticles by the Taguchi design and their in vitro cytotoxicity for lung cancer therapy

Jaber Emami, Aida Pourmashhadi, Hojat Sadeghi, Jaleh Varshosaz, Hamed Hamishehkar29 September 2026DOI 10.3109/10837450.2014.920360

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Summary

COX-2 is overexpressed in non-small cell lung cancer (NSCLC) and is associated with poor prognosis. Celecoxib (Cxb), a COX-2 inhibitor, has antiproliferative and proapoptotic effects, but systemic administration carries cardiovascular risks. Pulmonary inhalation delivery of Cxb encapsulated in biodegradable PLGA nanoparticles could provide local targeting to lung tumours, controlled release, and reduced systemic toxicity. Particle size: 153–192 nm across formulations; optimised formulation 165 ± 10.2 nm. PLGA content, PVA concentration, and organic phase volume significantly affected size (p < 0.05). - Zeta potential: −4.5 to −8.6 mV;.

Keywords

PLGA Nanoparticles Polymeric Controlled release Biodistribution Nanocarriers Gene delivery

Purpose: COX-2 is overexpressed in non-small cell lung cancer (NSCLC) and is associated with poor prognosis. Celecoxib (Cxb), a COX-2 inhibitor, has antiproliferative and proapoptotic effects, but systemic administration carries cardiovascular risks. Pulmonary inhalation delivery of Cxb encapsulated in biodegradable PLGA nanoparticles could provide local targeting to lung tumours, controlled release, and reduced systemic toxicity.

Hypothesis: If Cxb is encapsulated in PLGA nanoparticles optimised by a Taguchi L9 design, then the formulation will achieve high entrapment efficiency, suitable particle size for alveolar deposition, controlled release, and enhanced in vitro cytotoxicity against A549 lung cancer cells—especially when co-spray dried with lactose for dry powder inhalation.

Aims: Primary aim: Develop and optimise Cxb-loaded PLGA nanoparticles by emulsion solvent diffusion/evaporation using a Taguchi L9 orthogonal array.

Secondary aim 1: Evaluate the effects of PLGA content, dichloromethane (DCM) volume, PVA concentration, and homogenisation rate on particle size, zeta potential, entrapment efficiency, and release behaviour.

Secondary aim 2: Characterise the optimised formulation by SEM, DSC, and FTIR.

Secondary aim 3: Co-spray dry the optimised NPs with lactose or mannitol to produce hybrid microparticles for dry powder inhalation (DPI) and assess aerodynamic properties.

Secondary aim 4: Assess in vitro cytotoxicity of Cxb-loaded PLGA NPs versus free Cxb on A549 NSCLC cells.

Delivery system:

Component: Polymer; Description: PLGA RG504H (50:50 lactide:glycolide), Boehringer Ingelheim

Component: Nanoparticle Type; Description: Polymeric nanoparticles prepared by emulsion solvent diffusion and evaporation (o/w)

Component: Payload; Description: Celecoxib (Cxb), 5 mg

Component: Targeting Ligand; Description: None

Component: Surfactant / Stabiliser; Description: Poly(vinyl alcohol) (PVA), 80% hydrolysed, MW 9,000–10,000

Component: Organic Solvent; Description: Dichloromethane (DCM)

Component: Optimised Formulation; Description: 5 mg Cxb, 25 mg PLGA, 0.5% PVA, 2.5 mL DCM, 15,000 rpm

Component: Particle Size; Description: Range 153–192 nm; optimised 165 ± 10.2 nm

Component: Zeta Potential; Description: −4.5 to −8.6 mV; optimised −7.2 ± 0.4 mV

Component: Entrapment Efficiency; Description: >80% for all formulations; optimised 90 ± 4.3%

Component: Loading Percentage; Description: 8.2–24.7%; optimised 14.9 ± 1.1%

Component: Release; Description: >95% released within 24–30 h; optimised mean dissolution time 594 min

Component: Final DPI Form; Description: Optimised NPs co-spray dried with lactose at 1:2 ratio

Approach: In vitro model: Human A549 NSCLC cell line. - No in vivo studies were performed. - Experimental design: Taguchi L9 orthogonal array with four factors at three levels: PLGA content (12.5, 25, 50 mg), DCM volume (1, 2.5, 5 mL), PVA concentration (0.1%, 0.25%, 0.5%), homogenisation rate (10,000, 15,000, 20,000 rpm). All experiments in triplicate. - Cytotoxicity: MTT assay with free Cxb, Cxb-loaded NPs, and empty NPs at 0, 25, 50, 75, 100, 150, and 200 µg/mL; n = 6 wells per concentration; three independent experiments. - Aerodynamic testing: Next Generation Pharmaceutical Impactor (NGI) with Aerolizer DPI at 60 L/min for 4 s; lactose and mannitol carriers at 1:1, 1:2, 1:3 ratios.

Key methods:

Technique: Taguchi L9 orthogonal array + Design Expert; Purpose: Optimise formulation variables and determine factor contributions

Technique: Photon correlation spectroscopy (PCS, Zetasizer 3000); Purpose: Measure particle size, polydispersity index, and zeta potential

Technique: UV spectrophotometry at 254 nm; Purpose: Quantify Cxb entrapment efficiency, loading, and release

Technique: Dialysis method (MWCO 12,000 Da); Purpose: In vitro release in PBS pH 7.4 + 0.1% Tween 80 at 37°C

Technique: Scanning electron microscopy (SEM); Purpose: Particle morphology and size

Technique: Differential scanning calorimetry (DSC); Purpose: Drug–polymer compatibility and crystalline state

Technique: Fourier transform infrared spectroscopy (FTIR); Purpose: Chemical interactions between drug and polymer

Technique: Spray drying (Buchi B-191); Purpose: Produce hybrid microparticles with lactose or mannitol

Technique: Next Generation Pharmaceutical Impactor (NGI); Purpose: Determine FPF, MMAD, and GSD

Technique: MTT assay; Purpose: In vitro cytotoxicity on A549 cells

Key results: Particle size: 153–192 nm across formulations; optimised formulation 165 ± 10.2 nm. PLGA content, PVA concentration, and organic phase volume significantly affected size (p < 0.05). - Zeta potential: −4.5 to −8.6 mV; PLGA content and DCM volume most influential. Low zeta potential suggests liquid suspension instability, favouring dry powder storage. - Entrapment efficiency: >80% for all formulations; optimised 90 ± 4.3%. PLGA content was the most significant factor on EE and release. - Release: >95% Cxb released within 24–30 h; higher PLGA content slowed release. Optimised formulation had MDT of 594 ± 19.3 min. - Optimised formulation validation: Predicted vs actual error <7.4% for PS, ZP, EE, and MDT, confirming model reliability. - DSC: No crystalline Cxb endothermic peak in loaded NPs, indicating amorphous or molecular dispersion. - FTIR: No chemical interaction between Cxb and PLGA; spectra were superpositions. - Aerodynamic properties: Hybrid microparticles with lactose 1:2 gave best results: FPF = 70.3%, MMAD = 1.46 µm, GSD = 3.38. - Cytotoxicity: Cxb-loaded PLGA NPs were more effective than free Cxb at 75 µg/mL in arresting A549 cell growth; empty NPs did not affect cell growth.

Interpretation: The authors conclude that Cxb-loaded PLGA NPs prepared by emulsion solvent evaporation can be successfully optimised using Taguchi design, yielding high entrapment efficiency, controlled release, and suitable morphology for pulmonary administration. Co-spray drying with lactose produces hybrid microparticles with desirable aerodynamic properties for DPI. The formulation shows enhanced in vitro cytotoxicity against A549 lung cancer cells, supporting its potential for localised inhaled lung cancer therapy with reduced cardiovascular risk compared to systemic Cxb.

Limitations: No in vivo efficacy, biodistribution, or safety data; only in vitro A549 cytotoxicity. - Low zeta potential (−7.2 mV) indicates poor colloidal stability in liquid suspension; storage as dry powder is required. - No comparison with normal lung epithelial cells or other pulmonary delivery systems. - No long-term stability data for the optimised formulation or spray-dried microparticles. - Cytotoxicity was evaluated only at 24 h; longer exposure and apoptosis mechanisms were not studied. - No active targeting ligand; delivery relies on passive accumulation and inhalation. - Aerosol performance was tested with a specific DPI device (Aerolizer) and may vary with other inhalers.

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