Antimicrobial resistance (AMR) has emerged as a formidable global public health challenge, contributing to over 700,000 annual deaths due to infections caused by resistant bacteria and other pathogenic microorganisms. The World Health Organization (WHO) projects that, without intervention, AMR could result in over 10 million deaths annually and trillions in economic losses by 2050 (World Health Organization, 2021-a). While inherently occurring, the accelerated spread of AMR is driven by factor such as the indiscriminate use of antibiotics, the transfer of resistant bacteria from animals to humans, and contamination of water sources with antimicrobial drugs (World Health Organization, 2020-b). In response, the WHO has called for the exploration of alternative treatments for infectious diseases (World Health Organization, 2023-c).

Photodynamic therapy (PDT) stands out as a promising antibiotic-free antimicrobial treatment. PDT employs molecules known as photosensitizers (PSs) along with light of a specific wavelength to generate reactive oxygen species (ROS). These ROS have irreversible cytotoxic effects on pathogenic microorganisms, and importantly, they exhibit low susceptibility to resistance development making PDT a valuable alternative for combating AMR (Li et al., 2023). Beyond healthcare, PDT has found application as a sanitization method in the food industry (Brovko et al., 2009). However, the broader adoption of PDT is constrained by the inherent limitations of PSs, including short half-lives, limited cell membrane penetration, and reduced quantum yields due to self-aggregation(Sztandera et al., 2021).

Among the various families of PSs, halogenated fluoresceins, a class of xanthene dyes, have garnered attention for their high photoactivity. These PSs have been studied as molecular probes (Vodolazkaya et al., 2013) in the development of optical biosensors (Rajasekar, 2021) and cancer treatment (Kim et al., 2011). Phloxine B (PhB) is an FDA-approved dye used in the pharmaceutical and cosmetic industries (FDA, 2023), is of particular interest due to rapid photodegradation and low environmental risk (Wang et al., 2008).

In addition to the industrial use described, PhB has shown potential application as an antimicrobial agent. Rasooly and Weisz reported that PhB and other halogenated fluorescein derivatives have antibacterial activity against Staphylococcus aureus and found that under standard environmental lighting, particularly an aqueous solution of PhB at 100 μg/mL killed 99 % of cultures and reduced the CFU of S. aureus by a factor of 104. However, no inhibitory activity was observed against Gram-negative bacteria (A. Rasooly & Weisz, 2002)This limitation arises from structural differences in bacterial cell walls. For example, while Gram-positive bacteria only have a thick peptidoglycan layer (20 to 80 nm thick) in their wall, Gram-negative bacteria have a peptidoglycan layer of 2 nm and an outermost layer of lipopolysaccharides, lipoproteins, and lipids (Prescott & Klein, 2002). These properties may influence the interaction and limit the antibacterial activity of PhB. In other work, Rassoly et al. tried to improve the bactericidal effect of PhB on Gram-negative bacteria by adding EDTA at a concentration of 15 mM and illuminating the samples (R. Rasooly, 2005). In this sense, EDTA acts as a permeabilizer of the bacterial wall, resulting in a broad-spectrum bactericidal effect. However, studies have noted that EDTA exerts cytotoxic effects on eukaryotic cells, even at concentrations lower than those evaluated by Rassoly, thereby limiting its clinical application (Amaral et al., 2007, Ballal et al., 2009).

Alternative approaches, such as conjugating PhB and other xanthene dyes with different polymeric matrices can help overcome the limitations of these PS molecules. For instance, the conjugation of PhB and Rose Bengal (RB) with poly(vinylamine) has yielded an enhanced bactericidal effect against both Gram-negative and Gram-positive strains (Brovko et al., 2009). Additionally, researchers have reported the antimicrobial activity of PhB in hybrid composites. For example, Dadi, N. et al. and Skoura, E. et al. prepared thin films containing PhB with saponite and polycaprolactone, respectively, which exhibited antimicrobial properties on S. aureus strains by irradiation with green light (Dadi et al., 2021, Skoura et al., 2023). Similarly, the complex formation by non-covalent interactions between xanthene dyes and polyamide-amine (PAMAM) dendrimers have proven to be an excellent strategy for improved catalytic, pharmacodynamic, and photochemical properties of these PSs.

PAMAM dendrimers, a family of highly branched synthetic polymers, have also been explored as sensitizing systems that work under environmentally friendly conditions with water-soluble xanthene dyes (Paula Militello et al., 2023) and as catalysts for synthesizing new pigments (Sheikh et al., 2020). The affinity and compatibility of PAMAMs with xanthene dyes such as Eosin-Y (Eos), Erythrosin-B (Ery), and RB have been tested in 1:1 dendrimer-dye complexes by studying the supramolecular interaction in neutral and basic media to understand better the role of pH and dendrimer generation (G0 − G3) in the binding strength (Arbeloa et al., 2018, Barraza et al., 2018). In this sense, authors report an excellent dendrimer-dye affinity, increasing as the pH decreases to neutrality conditions and the dendrimer generation increases. In the same way, it has been observed that non-covalent interactions between PAMAM dendrimers with a controlled excess of RB can result in nanometer-sized particles. These nanoparticles can promote cellular uptake and improving the stability, photocatalytic, and photodynamic activity of isolated RB (Kutz and Gröhn, 2017, Sztandera et al., 2021).

In recent years, the development of PAMAM dendrimer-based nanosystems has gained attention due to their versatility, biocompatibility, and antimicrobial potential. Among these, several studies have reported supramolecular assemblies or hybrid nanoparticle systems formed between PAMAM dendrimers and bioactive agents, demonstrating significant antibacterial activity. For example, Jiang et al. developed dual-conjugated PAMAM dendrimers with vancomycin and silver nanoparticles that effectively killed vancomycin-resistant Staphylococcus aureus without inducing resistance in susceptible strains (Jiang et al., 2021). Similarly, Padnya et al. described PAMAM-calix-lysozyme nanoparticle systems that showed synergistic antibacterial effects with low cytotoxicity (Padnya et al., 2023). Moreover, amphiphilic PAMAM-based dendrimers have demonstrated potent antibacterial and antibiofilm activity through dynamic self-assembly and membrane disruption (Dhumal et al., 2022). However, to the best of our knowledge, no studies have explored supramolecular self-assembly between PAMAM G4 dendrimers and xanthene dyes such as Phloxine B (PhB), particularly for applications in antimicrobial photodynamic therapy (aPDT).

Building on these insights and in response to the WHO's urgent call for innovative strategies to combat infectious diseases, this study presents a novel self-assembled nanoparticle formulation bases on a fourth-generation PAMAM dendrimer (G4) and Phloxine B (Fig. 1). We introduce a systematic assessment of these nanoparticles hereinafter called G4-PhB as a platform for aPDT applications. To achieve this, we meticulously determined the optimal dendrimer:dye molar ratio, ensuring the creation of a monodisperse nanomaterial with reproducible morphological and photochemical properties. Additionally, we assessed the ROS production capacity under green light exposure. Comprehensive evaluations examined its cytotoxicity and bactericidal properties under both dark and irradiated conditions, targeting Gram-negative and Gram-positive strains. This innovative nanoparticle formulation aims to overcome the inherent limitations of traditional PS molecules and provides a roadmap for the development of new nanomaterials to combat AMR. The findings herein offer a promising alternative for topical treatments of infectious diseases caused by pathogenic microorganisms.