Translating hybrid CuS-indocyanine green-polymer nanoparticle production into flow: Multistage synthesis, purification, and photodynamic biocidal activity

Hybrid polymeric nanoparticles encapsulating indocyanine green (ICG) and decorated with copper sulfide (Cu₂–ₓS) were synthesized using semi-continuous micro- and millifluidic reactors (RS-PLGA-ICG@μCuS and RS-PLGA-ICG@mCuS). By leveraging both components as NIR-active photosensitizers, these nanoparticles were developed for enhanced antimicrobial photodynamic therapy. Continuous microfluidic processing enabled higher ICG loading than batch synthesis while maintaining tight control over particle size, ζ-potential, and copper content. Computational fluid dynamics modelling of the micromixer revealed the critical role of flow patterns and local shear stresses in achieving homogeneous nanoprecipitation. Continuous downstream processing was implemented using hollow-fiber tangential flow filtration, enabling solvent removal and nanoparticle conditioning under flow. A millifluidic reactor was further introduced as a scale-out strategy, increasing production throughput while preserving reproducibility of nanoparticle properties. Photodynamic performance was assessed using DHR 123 and SOSG probes, confirming efficient reactive oxygen species generation, predominantly via type I pathways, with 50 ppm ICG identified as the optimal concentration. Both micro- and millifluidic formulations showed reproducible activity. Bactericidal assays demonstrated significant efficacy against planktonic and biofilm-forming Staphylococcus aureus ATCC 25923, achieving higher log reductions than batch-produced nanoparticles due to improved photosensitizer encapsulation. Cytotoxicity studies with human dermal fibroblasts confirmed viability above ISO 10993-5 thresholds (>70%), with continuous-flow formulations exhibiting reduced copper-related toxicity. The semi-continuous strategy developed represents a scalable and intensified platform for manufacturing multifunctional antibacterial nanovectors with enhanced performance and biocompatibility. • Continuous-flow synthesis of hybrid CuS-ICG nanovectors • Improved ICG and CuS loading via microfluidic process intensification • Controlled band gap tuning by temperature and residence time • Enhanced bactericidal activity vs batch synthesis • Improved stability under physiologically relevant conditions