A novel neutral red derivative loaded into polymeric nanoparticles: study of its photoinactivation against Staphylococcus aureus

Abstract

We report the synthesis, characterization, and evaluation of the physicochemical and antimicrobial properties of a new derivative of neutral red named neutral red acid (NRAc). Relevant pharmaceutical and photochemical parameters – including pK_a, stability, photostability, and reactive oxygen species (ROS) generation – were assessed. Additionally, NRAc was loaded into polyacrylamide nanoparticles (PAA-NPs), and its biological activity was evaluated. The compound was synthesized via a rapid, straightforward method without requiring purification, achieving a relative purity of 93%. Compared to the parent molecule, NRAc exhibited enhanced physicochemical properties and showed promising biocompatibility; at a concentration of 40 µM, it caused no hemolysis after 15 min of light exposure, and less than 1% after 30 min. Although it displayed limited solubility in aqueous media, encapsulation in PAA-NPs enabled effective antimicrobial performance. The nanoparticle-loaded photosensitizer significantly reduced the survival of methicillin-resistant Staphylococcus aureus (MRSA) to below 10% following photodynamic treatment. These results highlight the potential of this novel compound as a promising third-generation photosensitizer for application in antimicrobial photodynamic therapy (aPDT).

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1. Introduction

The increasing bacterial resistance to antiseptics and antibiotics is a global health concern that complicates medical care, prolongs treatment duration, raises healthcare costs, and heightens the risk of spreading resistant infections. Over the past decades, antimicrobial resistance-related deaths have risen worldwide, with Staphylococcus aureus (S. aureus) showing the greatest increase: from 103 000 attributable deaths in 1990 to 196 000 in 2021.¹

Methicillin-resistant S. aureus (MRSA) was first identified in hospitalized patients, and by the 1990s, it had begun spreading within the community. In addition, MRSA has been linked to epidemic waves, with regional variants periodically replacing previously dominant strains.² Today, MRSA remains a major public health threat, contributing significantly to healthcare-associated infections while also driving the rising incidence of community-acquired infections. Given its rapid evolution and widespread prevalence, the development of novel, highly effective, and low-toxicity antimicrobials is critical. Addressing MRSA requires innovative therapeutic strategies to overcome resistance mechanisms and improve treatment outcomes.

As a novel photochemotherapeutic approach, antimicrobial photodynamic therapy (aPDT) holds significant promise for infection management without fostering microbial resistance.^3–5 In this method, a photosensitizer (PS) undergoes photophysical and photochemical reactions when it is activated by a light source of appropriate wavelength in the presence of oxygen. Reactive oxygen species (ROS) generated by the irradiated PS are primarily responsible for microbial inactivation in the targeted area.

aPDT is a non-invasive therapeutic method that offers multiple advantages over conventional antimicrobial agents. Among its most notable benefits are the lack of need for continuous dosing, high selectivity at the target site, reduced incidence of adverse effects, and the effective elimination of pathogenic microorganisms without promoting resistance development. Notably, the antimicrobial efficacy of aPDT is independent of the resistance mechanisms typically exhibited by microbial populations.³

When the PS is exposed to light irradiation, it can transition from the ground singlet state to the excited singlet state. Subsequently, through the intersystem crossing process, it is transferred to the excited triplet state. In this state, the PS can react via electron transfer with biological molecules (Type I reactions) or undergo direct energy transfer with molecular oxygen (Type II reactions) generating various ROS. These include a hydroxyl radical, superoxide anion radical and singlet oxygen, the latter being highly efficient in photodynamic treatments.⁶

The efficacy of photosensitization in aPDT is highly dependent on the physicochemical properties of the PS employed. Over time, different generations of PSs have been developed to overcome limitations such as poor aqueous solubility, low photostability, and limited targeting efficiency. The majority of traditional PSs belong to the tetrapyrrole family, including porphyrins,⁷ chlorins,⁸ and phthalocyanines,⁹ which have shown notable success in various photodynamic applications. To date, all clinically approved agents for PDT have been porphyrin- or chlorin-based derivatives, such as porfimer sodium (Photofrin®), temoporfin (Foscan®), motexafin lutetium (Lu-Tex®), palladium bacteriopheophorbide (Tookad®), rostaporfin (Purlytin®), verteporfin (Visudyne®), and talaporfin (Laserphyrin®).

Non-porphyrinic compounds represent a less explored yet highly promising branch in the development of PSs. Their structural diversity and chemical tunability enable the rational design of molecules with optimized physicochemical properties, such as increased solubility, improved light absorption profiles, and specific affinity for selected cellular targets. Unlike complex formulations such as Photofrin®, these compounds can be synthesized through well-defined chemical routes, ensuring greater reproducibility and precise compositional control.

Among them, second-generation non-porphyrin dyes – such as phenazines, phenothiazines, xanthenes, cyanines, anthraquinones, and BODIPY derivatives – have shown encouraging results in preclinical and clinical research.^10,11 However, phenazine-based PSs, despite their advantageous red-light absorption and intrinsic redox activity, often display limited water solubility, suboptimal ROS quantum yield, and poor photostability, which restrict their broader application in aPDT. In this context, the rational modification of the phenazine scaffold offers an opportunity to overcome these drawbacks. Recent studies have demonstrated that appropriate functionalization of the phenazine core can significantly improve its photophysical properties and enhance ROS generation upon irradiation.^12,13

To address these limitations, a phenazine derivative was used as a starting point to explore new chemical modifications through the introduction of electron-withdrawing functional groups, aimed to improve its photochemical behavior, physicochemical properties, and biological activity. Accordingly, this study aimed to design and synthesize a new second-generation PS, neutral red acid (NRAc), derived from neutral red hydrochloride (NR) with enhanced photochemical and biological performance. NRAc was obtained using NR (IUPAC name: hydrochloride of 3-amino-6-dimethylamino-2-methylphenazine) as the precursor and oxalyl chloride (OC) as the acylation agent. NR is a phenazine-based compound widely employed as a vital stain in microbiological applications and as a pH-sensitive indicator in analytical procedures,¹⁴ while OC is one of the most versatile and reactive organic acid chlorides.¹⁵ The molecular structure of NRAc was thoroughly characterized by various spectroscopic techniques. The evaluation was completed by determining the photodynamic and physicochemical properties. The reaction involved in the synthesis of NRAc, as well as the chemical structure of this compound, is shown in Fig. 1.

Fig. 1 Synthesis of NRAc.

The therapeutic efficacy of aPDT can be compromised by second-generation PSs that present low aqueous solubility, chemical instability, or insufficient microbial uptake.^3,5,16 Nevertheless, optimizing physicochemical properties of PSs and designing specific delivery systems are expected to improve the microbiological activity. In this context, nanoparticles (NPs) have been prepared to create a system that overcomes these limitations. When nanotechnology is combined with aPDT, its potential to eradicate microorganisms is significantly enhanced.^17,18 Its effectiveness and selectivity are closely related to the characteristics of the therapeutic agent loaded into the NPs.³

In the context of aPDT, metallic, lipid-based, and polymeric nanoparticles have been extensively investigated as delivery systems to enhance photodynamic efficacy and mitigate photosensitizer aggregation. Third-generation photosensitizers are defined by the functionalization of second-generation PSs with carrier systems such as liposomes, micelles, and polymeric nanoparticles, aiming to enhance their accumulation at specific target sites.^18–20

Polyacrylamide nanoparticles (PAA-NPs) are nanosystems suitable for biological applications, with great potential in drug delivery due to their easy preparation, low cost, biocompatibility, and chemical flexibility.²¹ The research group has developed several PAA-NPs by combining acrylamide (AA) with different monomers such as 2-hydroxyethyl methacrylate (HEMA), N-isopropylacrylamide (NIPA) and 3-(aminopropyl)methacrylamide (APMA). (+) N,N-diallyl-L-tartardiamide (DAT) and 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AHM) were used as crosslinkers. These NPs presented optimal particle diameters (z-average) and acceptable polydispersity indices for pharmaceutical applications. Previously, these systems improved various properties of NR and its monobrominated derivative, as well as enhanced their photochemical reactivity against Gram-positive bacteria.^4,22

Considering that aPDT associated with nanotechnology has shown enhancements in the photodynamic activity of PSs in terms of delivery to infected sites and therapeutic selectivity, we incorporated the newly synthesized neutral red acid (NRAc) into PAA-NPs developed and characterized in our working group.^4,22

In summary, this study addresses a relevant gap in aPDT research by developing a novel phenazine-based photosensitizer (NRAc) designed to improve photochemical properties of existing phenazine derivatives and by evaluating its performance when encapsulated in PAA-NPs. This dual approach aims to expand the repertoire of effective PSs for aPDT and supports the development of alternative therapeutic approaches for combating resistant bacterial infections.

2. Materials and methods

2.1. Chemicals and reagents

All reagents were purchased from Sigma-Aldrich and used as supplied. Solvents were of analytical grade from Sintorgan. Buffer solutions (final volume 10 mL) were prepared with different proportions of citric acid (0.1 M aqueous solution) and dibasic sodium phosphate (0.2 M aqueous solution), obtaining pH values of 2.2, 3.0, 3.8, 4.6, 5.5, 6.2, 7.0, 7.4, and 8.0. pH was determined by using an Altronix Model EZDO-PC pH meter with a combined glass electrode. A phosphate-buffered saline (PBS, 0.1 M, pH 7.4) solution was prepared using sodium chloride, potassium chloride, sodium hydrogen phosphate dihydrate and potassium dihydrogen phosphate. All the chemicals were of the highest purity commercially available (Cicarelli), and the solutions were prepared using ultrapure water from a Milli-Q® purification system.

Chemical reactions were monitored by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). HPLC was developed using methanol/aqueous solution of triethylamine phosphate (TEAP 40 mM; 70/30 v/v) as a mobile phase. The column temperature was set at 25 °C and the flow rate at 1.0 mL min⁻¹. TLC was performed on precoated silica gel plates with fluorescent indicator UV₂₅₄ (Macherey-Nagel) using a mobile phase of chloroform/methanol/ethyl acetate (5 : 3 : 2 v/v). Purification by dialysis was performed using cellulose ester membranes (MWCO 50 kDa; Spectrum Labs).

2.2. Equipment

Ultraviolet spectrophotometric (UV) studies were carried out using a Cary 60 UV-visible spectrophotometer (Agilent Technologies) with 1 cm quartz cells. Each experiment was conducted in duplicate or more, yielding reproducible results.

Regarding the experiences with light, the samples were irradiated at a distance of 5 cm with a Parathom® LED lamp (OSRAM 10W). The irradiance measured with a Tes-1332 Digital Lux Meter was 12.47 mW cm⁻². It should be noted that the total dose of light is determined by the irradiance applied and the duration of irradiation.²³

HPLC analyses were carried out using an Agilent Series 1100 chromatograph equipped with an autosampler, a column thermostat, a UV-vis detector and a Phenomenex® Gemini C18 column (5 µm particle size, 250 mm length and 4.6 mm internal diameter). The mobile phase and the samples prepared in the mobile phase were filtered through a Millipore® Type FH filter (0.45 µm pore size). Data were acquired using a Peak Simple Chromatography Data System (version 2.86).

NMR experiments were performed on a Bruker Advance II 400 MHz ultra-shield TM spectrometer at 400.16 MHz (¹H) and 100.62 MHz (¹³C) equipped with a multinuclear inverse detection probe, digital resolution capabilities and a variable temperature unit. Chemical shift values are reported in ppm, relative to tetramethylsilane (TMS) as an internal standard. The multiplicities of the signals are indicated as s (singlet), d (doublet) and dd (double of doublets) and were acquired in DMSO-d₆ (99.8%, Merck) as a solvent. Coupling constants (J) are in Hz.

Mass spectroscopy experiments were acquired using a Xevo TQ-S Micro triple quadrupole mass spectrometer (Waters Corporation) equipped with an electrospray ion source (ESI).

The average particle size and polydispersity indexes (PDI) of the NPs were evaluated using dynamic light scattering (DLS) (Zetasizer Delsa Nano Version 2.20, Beckman Coulter Inc.) with a He–Ne laser (633 nm), a scattering angle of 165°, a viscosity of 0.8878 Pa s and a refractive index of 1.3328. All measurements were carried out in triplicate at 25 °C. The suspension of the NPs was appropriately diluted with Milli-Q water to obtain suitable concentrations for analysis. The diameter and PDI of particle sizes were estimated using the CONTIN algorithm analysis through inverse Laplace transformation of the autocorrelation function.

The statistical procedures were performed using the OriginPro v.8.0 and Microsoft Excel 2013 software.

2.3. Synthesis of neutral red acid

Several parameters such as reaction time, temperature, medium and percentages of reactants were assessed to optimize the formation of the new derivative. Once these conditions were experimentally determined, neutral red acid (NRAc) was synthesized (Fig. 1). Briefly, NR (10 mg; 34.5 µmol) was individually dissolved in anhydrous acetonitrile (ACN) (20 mL, previously dried over 4 Å molecular sieves), in a dry 50 mL reaction flask, it was mixed thoroughly under constant magnetic stirring and protected from light. To this solution, OC (30 µL; 346 µmol) was added dropwise. The mixture was left to react for 30 min at 40 °C. Then, the solvent was evaporated under reduced pressure, and the product was washed with cold water. The resulting suspension was filtered, giving the pure product (no purification was needed).

Dark purple solid (90% yield); R_f TLC (n = 6) 0.44 ± 0.06; HPLC retention time (t_R) (n = 7) 4.20 ± 0.05 min; ¹H NMR (DMSO-d₆): 2.5 (s, 3H, H-15); 3.3 (s, 6H, H-13 and H-14); 6.8 (d, 1H, H-9, J_meta = 2.6); 7.9 (dd, 1H, H-11, J_ortho = 9.8 and J_meta = 2.6); 8.0 (d, 1H, H-12, J_ortho = 9.8); 8.1 (s, 1H, H-6); 8.5 (s, 1H, H-3); 10.2 (s, 1H, H-21) and 11.3 (s,1H, H-18); ¹³C NMR (DMSO-d₆): 18.1 (C-15); 41.0 (C-13 and C-14); 94.8 (C-9); 112.5 (C-3); 124.6 (C-11); 130.5 (C-6); 131.7 (C-12); 132.8 (C-1); 136.1 (C-7); 137.6 (C-5); 138.5 (C-4); 140.8 (C-2); 141.8 (C-8); 154.2 (C-10); 157.9 (C-16) and 162.1 (C-17); MS (ESI) calculated for C₁₇H₁₇N₄O₃⁺ (M+) m/z: 325.13; found: 325.08 and UV (methanol : TEAP 70 : 30 v/v) λ_max 535 nm. Spectral data that allowed the identification and characterization of NRAc are presented in the SI (Fig. S1–S5).

2.4. pK_a determination of neutral red acid

Spectral profiles were obtained using various buffer solutions (pH values of 2.2, 3.0, 3.8, 4.6, 5.5, 6.2, 7.0, 7.4, and 8.0). The samples were prepared from stock solutions of NR and NRAc in dimethylformamide (DMF) and diluted in the tested buffer solutions. Finally, the samples were analyzed in duplicate by UV-vis spectrophotometry within the 350–750 nm wavelength range. The pK_a values were subsequently determined from the plot of the degree of ionization versus pH. The degree of ionization (DI) percentage for each PS was calculated using eqn (1), based on the absorbance ratio (A) at the wavelengths corresponding to the maximum absorption of the cationic and neutral species (A_λ acid/A_λ base) across different pH values.²⁴

It is essential to note that this methodology relies on the complete protonation of both PSs at the lowest pH value and their full deprotonation at the highest pH assessed.

2.5. Chemical stability studies of neutral red and neutral red acid

The stock solutions of each compound were prepared in DMF prior to use. Then, 100 µL of stock solution of PS were added to a vial containing 1000 µL of buffer of pH 5.5 or 7.4 to obtain the working solutions (the final absorbance value of the mixture was ∼0.5). Afterward, the vials containing the samples were placed in a water bath at 37 °C throughout the experiment. Aliquots were taken at regular intervals of 1 h. The study was conducted for 24 h, and the absorbance values of each aliquot were measured at the maximum absorption wavelength of each PS in each medium [λ_max NR = 535 nm (pH 5.5) and 460 nm (pH 7.4); λ_max NRAc = 525 nm (pH 5.5) and 400 nm (pH 7.4)]. All stability assays were performed in duplicate. HPLC was employed to analyze samples collected at the beginning and end of the study, in order to verify the absence of degradation products.²⁵

2.6. Photochemical stability studies of neutral red and neutral red acid

Photostability studies of NR and NRAc were conducted in buffer solution (pH 7.4) using quartz cuvettes with a 1 cm optical path length. The samples were irradiated for 90 min, corresponding to a total light dose of 67.4 J cm⁻², as detailed in Section 2.2. The PS solutions were monitored over time by UV-vis spectrophotometry.^26,27 Additionally, samples collected at the beginning and end of the irradiation period were analyzed by HPLC, as previously described in the chemical stability assays in Section 2.5. All photostability experiments were performed in duplicate.

2.7. Steady state photolysis

2.7.1. Photooxidation of anthracene derivative. Solutions of NR and NRAc in DMF were irradiated using a Parathom® LED lamp (OSRAM 10 W) placed at a distance of 5 cm. Irradiation was performed in 1 cm path length quartz cuvettes containing 9,10-dimethylanthracene (DMA) (absorbance ∼0.3 at 378 nm) as a chemical probe for singlet oxygen. To guarantee equivalent photon absorption by both PSs under the experimental setup, their concentrations were adjusted so that the absorbance at 455 nm – the peak emission wavelength of the light source – was approximately 0.20 (NR = 0.211; NRAc = 0.186).

The progression of DMA photooxidation was tracked by observing the reduction in absorbance at 378 nm. The observed rate constants (k_obs) were determined by a linear least-squares fit of the semilogarithmic plot of ln(A₀/A) vs. time.²⁸

The singlet oxygen quantum yields (Φ_Δ) were calculated relative to the parent compound NR (Φ_Δ = 1) using eqn (2),²⁹. In this expression, PS refers to NRAc, Ref to NR, and Abs₀ to the initial absorbance values of each PS at 455 nm, corresponding to the maximum emission wavelength of the irradiation source. The emission spectrum of the Parathom® LED lamp is shown in Fig. S6 of the SI.

2.7.2. Decomposition of nitro blue tetrazolium. The nitro blue tetrazolium (NBT) assay was performed using 0.2 mM NBT, 0.5 mM nicotinamide adenine dinucleotide (NADH), and the PS (adjusted to an absorbance of ∼0.1 at 455 nm) in 2 mL of a DMF/water mixture (90 : 10 v/v). Control experiments were conducted by omitting either NBT, NADH or the PS. Samples were irradiated under aerobic conditions in 1 cm quartz cuvettes, positioned 5 cm from a Parathom® LED lamp (OSRAM, 10 W) for a total duration of 360 s. The decomposition of NBT was tracked by monitoring the rise in absorbance at 560 nm, indicative of formazan formation.³⁰

2.8. Photohemolysis assay test

Red blood cell (RBC) samples were prepared from human blood donated by healthy volunteers, according to procedures approved by Comité Institucional de Ética de la Investigación en Salud (CIEIS), Facultad de Ciencias Médicas-UNC (CIEIS-HNC 11-16-2018), as previously described.³¹ The donors were fully informed regarding the purposes of the study. All experiments were conducted in accordance with the principles expressed in the Declaration of Helsinki, the Guidelines for research in humans (Res. No. 1480/2011 Ministerio de Salud, Argentina). Furthermore, this test was carried out following the guidelines established in the Biosafety Manual that regulates the use and handling of work equipment, chemical substances and biological materials in the laboratories of the Facultad de Ciencias Químicas-UNC (Res. HCS 454/2003).

The EDTA-stabilized whole blood sample was centrifuged at 3000 rpm for 5 min to remove the plasma and leukocyte layer. The cells were then washed with a PBS solution and centrifuged again. The separated RBCs were diluted with PBS (1 : 10) to obtain a concentration of approximately 10⁷ cells per mL.³²

Next, 1 mL of the RBC suspension was mixed with appropriate aliquots of a fresh solution of NRAc in DMF. Distilled water and fresh PBS were used as positive and negative controls, respectively. All the samples were incubated at 37 °C for 30 min and then irradiated for 15, 30, 45, and 60 min, corresponding to total light doses of 11.2, 22.4, 33.6, and 44.8 J cm⁻², respectively, following the protocol previously described. Samples containing the PS in the absence of light (−L, +PS) or exposed to light without PS (+L, −PS) served as additional negative controls.

The samples were maintained at 37 °C for 24 h. Finally, the samples were centrifuged, and 100 µL of the resulting supernatants were diluted in 1 mL of distilled water. The absorbance of the released hemoglobin was determined at 413 nm. The results were expressed as a percentage of photohemolysis, using eqn (3). The degree of photohemolysis was measured in duplicate for each sample. An unpaired T-test was employed to assess the statistical significance of differences between groups. Differences were considered significant at a 95% confidence level (p < 0.05). Results are expressed as the mean ± standard deviation for each group.

2.9. Synthesis of polyacrylamide nanoparticles: general procedure

The NPs employed in this study were synthesized via inverse microemulsion polymerization, following the procedure previously described by our research group.^4,22 Appropriate amounts of the surfactants dioctyl sulfosuccinate (AOT) and polyethylene glycol dodecyl ether (Brij 30) were added to a reaction flask containing hexane under a nitrogen atmosphere. The aqueous solution composed of the relevant monomers and crosslinkers (Section 2.9.1) for each type of NPs was then introduced into the reaction mixture. Polymerization was initiated by adding ammonium persulfate (APS) and NNN′N′-tetramethylethylenediamine (TEMED). The reaction mixture was stirred at room temperature for 24 h. Upon completion, hexane was removed by rotary evaporation, and the resulting particles were resuspended in ethanol. The samples were purified by dialysis and stored at 4 °C. DLS was used to determinate the size of NPs in aqueous solution.^4,22

2.9.1. Preparation of monomer and crosslinker solutions. PAA–HEMA–AHM (NP1): AA (monomer, 237 mg), HEMA (monomer, 20 µL), and AHM (crosslinker, 143 mg) were dissolved in PBS (0.67 mL) in a glass vial by sonication to obtain a uniform solution.⁴

PAA–NIPA–AHM (NP2): AA (monomer, 237 mg), NIPA (monomer, 19 mg), and AHM (crosslinker, 143 mg) were dissolved in PBS (0.67 mL) in a glass vial by sonication to obtain a uniform solution.²²

PAA–APMA–DAT (NP3): AA (monomer, 237 mg), APMA (monomer, 29.7 mg), and DAT (crosslinker, 152 mg) were dissolved in PBS (3.0 mL) in a glass vial by sonication to obtain a uniform solution.⁴

2.10. Loading of the photosensitizer into nanoparticles

The loading of NRAc into NPs 1–3 was carried out through a post-synthesis procedure. In order to perform this, 13.7 µL of Tween 80 was added to 100 µL of each NP solution diluted in 1 mL of Milli-Q water. Then, aliquots of 1 mL of the NRAc solution (0.3 mM) in DMF were added to the different NP solutions and stirred magnetically at a constant rotation speed for a minimum of 2 h. The products were named NRAc-NP1; NRAc-NP2 and NRAc-NP3.

To assess free NRAc, the same aliquot (1 mL) of the PS in DMF was diluted in 1 mL of Milli-Q water.

2.11. Microorganisms and preparation of cultures

The bacterium used in this study was methicillin resistant S. aureus ATCC 43300 (MRSA). The organism was maintained by weekly subculture on TSA. An overnight bacterial culture was resuspended in PBS and standardized to a concentration of approximately 10⁸ CFU mL⁻¹, corresponding to 0.5 on the McFarland turbidity scale. Finally, a 1/100 dilution of the McFarland-standardized suspension (resulting in a cell density of 10⁶ CFU mL⁻¹) was prepared and utilized for the photodynamic inactivation assays.³³

The impact of PS concentration (30 and 40 µM) of free NRAc and that loaded into NPs 1–3 and irradiation time (15 and 30 min, corresponding to total light doses of 11.2 and 22.4 J cm⁻², respectively) on the photosensitizing effect was investigated. To assess the free form of NRAc, a fresh solution in DMF/water was prepared. Additionally, the NPs were loaded with NRAc as described in Section 2.10. Finally, each solution was diluted in PBS to a final volume of 1 mL, adjusting the final concentration of PS, and transferred to a test tube containing bacterial suspension (1 mL). The tubes were exposed to the corresponding light dose, after which the samples were removed, serially diluted 10-fold in PBS, cultured in Petri dishes containing TSA, and incubated overnight at 37 °C. The CFUs were counted, and survival fractions were determined by triplicate using the drop-plate technique for bacterial enumeration according to Naghili et al.³⁴ The same experimental setup was applied for the control samples, omitting LED light irradiation.

The results were expressed as the percentage of MRSA that survived after photosensitization with different NRAc concentrations followed by 15 and 30 min of irradiation.

2.12. Controls and statistical analysis

All experiments were performed in duplicate. For the photohemolysis and photoinactivation assays, control experiments were conducted under the following conditions: (i) without irradiation and without PS, with the corresponding aliquot of DMF, (ii) without irradiation but with PS, and (iii) with irradiation but without PS. These controls enabled the differentiation of effects due to the cosolvent, the intrinsic toxicity of the PS in the dark, the light exposure alone, and the specific response resulting from the combination of PS and light, attributed to the photodynamic mechanism.

Statistical analysis was performed using the unpaired t-test to assess differences between two groups, and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test when comparing more than two groups. Differences were considered statistically significant at a confidence level of 95% (p < 0.05). In all cases, data are presented as the mean ± standard deviation (SD).

3. Results and discussion

3.1. Synthesis of neutral red acid

The synthesis of NRAc was carried out via an acylation reaction and the conditions were selected based on references for acylation reactions using OC.^35–38 As mentioned above, several parameters, such as reaction time, amount of NR and OC, and different solvents, were tested in order to obtain the desired product, NRAc, in good yield. When methylene chloride was used as the reaction medium, the results indicated the formation of a mixture of two products. In contrast, using ACN as the reaction medium resulted in the production of a single new compound.

Regarding the temperature of the reaction, 25 and 40 °C were tested. According to the assessed conditions, it is possible to state that variations in temperature from 25 to 40 °C significantly impacted the kinetics of the reaction. Specifically, at 40 °C, the reaction time decreased from 5 h to 30 min. Additionally, the amount of OC required was considerably reduced, with the NR/OC ratio dropping from 1/200 to 1/10. Under the experimental conditions compared (using ACN), NRAc was obtained as the only product, with a relative purity percentage greater than 90% (Fig. S1). Subsequently, it underwent characterization and evaluation.

These results agree with the recent publication of Jian-Qiang Chen et al., who demonstrated that ACN is one of the best solvents for the alkoxycarbonylation of alkenes with alkyloxalyl chlorides. In addition, the authors point out that temperature leads to an increase in reaction yield.³⁹

In summary, as mentioned above, the best reaction conditions to synthesize NRAc were rationally NR/OC 1 : 10, at 40 °C, for 30 min, in ACN as the solvent. The new derivative was obtained in high purity and was unequivocally characterized by NMR and MS (SI) (Fig. S2–S5).

3.2. pK_a determination of neutral red acid

To understand the behavior of NRAc in an aqueous solution, its ionization equilibrium was studied as a function of pH and the value of the ionization constant was determined. The pK_a value of a compound is a relevant property for pharmaceuticals, as it influences their biopharmaceutical characteristics such as aqueous solubility, membrane permeability and electrostatic interactions with biological targets. Drug absorption, distribution, metabolism, excretion and toxicity are strongly affected by the charge state of compounds under varied pH conditions.⁴⁰

The acid–base behavior of NRAc involves deprotonation of the carboxyl group and the equilibrium between the protonated and deprotonated forms of the amino group, which govern the overall charge of the molecule as pH increases. Spectrophotometric analysis identified two distinct species. Under acidic conditions, the compound predominantly exists as a zwitterion (neutral charge), whereas at higher pH values, the anionic form predominates.

The absorption spectrum of NRAc displayed two distinct bands: one in the 350–450 nm range and another between 450 and 550 nm, likely associated with different electronic transitions. Band analysis as a function of pH indicated independent behavior of each band (Fig. S7). Observed changes in the lower-energy band revealed that NRAc coexists in solution in both neutral and ionized forms, consistent with the behavior of neutral red hydrochloride. In acidic media, corresponding to the zwitterionic form, the absorption maximum was observed at λ_max = 563 nm. As the pH increased, a progressive shift from the protonated to the anionic form occurred, resulting in a hypsochromic shift of the absorption maximum to λ_max = 510 nm, corresponding to the fully ionized species. The ionization constant of NRAc was determined in duplicate from plots of the degree of ionization versus pH, yielding a pK_a of 4.81 ± 0.01, corresponding to the point at which 50% of the compound is ionized (Fig. 2).

Fig. 2 Determination of the pK_a value of NRAc as a function of pH.

To verify the effectiveness and validity of the method, the pK_a value of NR was determined to be 6.45 ± 0.06. It should be noted that this value is within the range previously published by Urrutia et al. (7.0 ± 0.8).⁴¹ The similarity between these values lends credibility to the results obtained and confirms that the tested methodology is suitable for these families of compounds.

In addition, various software tools were applied to calculate the theoretical pK_a value of NRAc. MARVIN-CHEMAXON determined a value of 4.08 for this constant, while 4.7 was the result using ACD/LABS. These computational studies confirmed the proposed ionization equilibrium and predicted a pK_a value similar to that experimentally obtained. As expected, the inclusion of an acidic functional group in the NR structure produced a decrease in the pK_a value.

3.3. Stability studies of neutral red and neutral red acid

Stability tests are commonly used to confirm the quality of an active pharmaceutical ingredient or drug product over time, under the influence of a variety of environmental factors, such as temperature, humidity, and light. Chemical degradation may lead to toxic substances and reduce the bioavailability of the active molecule. Numerous PSs, either already approved or in advanced stages of research, have the disadvantage of being susceptible to degradation due to various factors, including enzymatic activity, light intensity, water, acid, oxygen, temperature, humidity, and a combination of these factors.^42,43 The degradation of PSs in simple solutions and in complex environments can be monitored by a decrease of their intensity of absorption and/or fluorescence intensities, which directly impacts their photochemical reactivity.⁴⁴ To evaluate whether the incorporation of a carbonyl group into the NR structure alters these properties, both the chemical stability and photochemical stability of NR and its new derivative were assessed under physiological conditions.

To evaluate the chemical stability of NR and NRAc, the absorbance was plotted (at the λ_max of each PS) as a function of time at different pH values. Separately, the photochemical stability was evaluated by subjecting the samples to white light irradiation for 90 min, while carefully monitoring that no alterations occurred in their absorbance profiles over time. Under these experimental conditions, no degradation products were detected. Additionally, chromatograms obtained at 0 and 24 h showed a single peak for each PS, with retention times of 3.80 ± 0.02 min for NR and 4.20 ± 0.05 min for NRAc, confirming both their chemical and photochemical stability.

These results are consistent with previous reports indicating that minimal structural modifications do not significantly affect the chemical stability of photosensitizers.^31,45

3.4. Photooxidation of anthracene derivative

The generation of singlet oxygen was investigated by the photolysis of the anthracene derivative DMA in the presence of the PSs, under stationary-state and aerobic conditions. The photooxidation of DMA in DMF was monitored spectroscopically by observing the decrease in absorbance at 378 nm (Fig. S8).

The observed rate constants (k_obs) were obtained and the Φ_Δ values were calculated using eqn (2). According to the results obtained (Table 1), the new derivative produced a 15% increase in the value of Φ_Δ compared to its precursor, NR. This phenomenon may be attributed to the carbonyl group, which acts as a triplet energy transfer agent and enhances the photosensitizing effect.⁴⁶

Table 1 Kinetic parameters for the photooxidation and ϕ_Δ of NR and NRAc

PS	Absorbance^a	−kobs^a (×10^−3; s^−1)	ϕ Δ
a Values obtained after the analysis of three determinations in DMF.
NR	0.211 ± 0.005	1.83 ± 0.03	1.00
NRAc	0.186 ± 0.001	1.85 ± 0.01	1.15

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3.5. Decomposition of nitro blue tetrazolium

The generation of superoxide anion radicals was investigated by the reaction of NBT in the presence of NADH sensitized by NR and NRAc. The decomposition of NBT to formazan was monitored through the colorimetric change in the absorption spectrum at λ = 560 nm. The kinetics of NBT reduction were studied in DMF/water (90 : 10 v/v) under aerobic conditions upon irradiation with a Parathom® LED lamp (OSRAM, 10 W) for a total time of 360 s. Notably, the reduction of NBT was not detected in the photoirradiated samples containing PSs and NBT but lacking NADH (data not shown). On the other hand, the results showed that both PSs were capable of producing superoxide anion radicals, and the amount generated by NRAc was slightly higher than that generated by NR (Fig. 3). The control sample (NADH + NBT without PS) did not exhibit the presence of formazan, confirming that the superoxide anion radical generation originated from the PSs. Therefore, there is an important contribution of Type I mechanism to the photodynamic activity sensitized by NRAc and NR in the presence of NADH as a reducing agent in a homogeneous medium. It can be highlighted that other authors reported that azine family compounds interact with various substrates in Type I reactions.^47,48

Fig. 3 Time course of superoxide anion radical generation detected by the NBT method at 560 nm in DMF/water (90 : 10 v/v).

As previously mentioned, these results confirm that the carbonyl group present in NRAc facilitates the formation of the PS excited triplet state and consequently enhances the production of both ¹O₂ and superoxide radical anion.⁴⁶ In other words, this derivatization promotes photochemical reactivity via both Type I and Type II mechanisms. Both reactions contribute to the therapeutic effects of PDT by producing cell death through different, potentially simultaneous, pathways.⁴⁹

3.6. Photohemolysis

Biocompatibility is one of the primary requirements for pharmaceuticals intended for in vivo use. To assess the biocompatibility of a compound, a hemolysis assay is frequently employed as a standard measure of safety.⁵⁰ Consequently, the effect of NRAc and light treatment on erythrocyte membranes was investigated. RBCs were exposed to varying concentrations of the PS and different light doses. The irradiation periods selected for the study were carefully chosen to reflect the typical duration of a clinical protocol to which a patient would be exposed during an actual treatment.

According to the data presented in Fig. 4, no adverse effects were observed on RBC membranes treated with NRAc at a concentration of 20 µM when exposed to white light for 15, 30, and 45 min. Furthermore, when the irradiation duration was increased to 60 min, the photohemolysis percentage was 2%, which is classified as non-hemolytic.⁵¹

Fig. 4 Photohemolysis of erythrocytes caused by different concentrations of NRAc exposed to LED light for 15, 30, 45, and 60 min (n/d: not detectable).

The extent of photohemolysis increased with higher NRAc concentrations. At 30 µM, the formulation remained non-hemolytic when irradiated for 15 and 30 min, although it induced approximately 5 and 6% of photohemolysis after 45 and 60 min of irradiation, respectively. A concentration of 40 µM caused no hemolysis after 15 min of irradiation, and it was less than 1% after 30 min. However, extending the exposure time to 45 and 60 min resulted in photohemolysis levels of 4.8% and 12%, respectively.

Notably, all samples exposed to NRAc and 15 or 30 min of irradiation remained within non-photohemolytic levels. Based on these findings, these exposure periods were selected for further studies, as they demonstrated biocompatibility across the tested concentrations of NRAc. This combination of PS and light dose was therefore considered safe for continuing the research, ensuring that the potential for photohemolysis remains minimal.

3.7. Loading of photosensitizer into nanoparticles

In order to evaluate the antimicrobial activity of the new PS, it was necessary to load this compound into different NPs previously developed by our research group.^4,22 The second-generation PS synthesized showed limited solubility, especially in an aqueous medium at the concentrations necessary for the biological activity test. As solubility is crucial in aPDT applications, the new compound was incorporated into three previously synthesized NPs. The use of NPs as PS nanocarriers proved to be a valuable tool to enhance the antimicrobial efficacy of different PSs and protect them from possible degradation under physiological conditions.^5,52

The PS was loaded into the NPs using a post-loading approach, following established protocols. Previous studies have demonstrated that PAA-NPs can encapsulate NR and its monobrominated derivative (NRBr) with an efficiency of 80–99%, corresponding to a mass of 2.2–2.5 mg, when using aliquots of 800 µL of 20 mM stock solutions. Notably, these nanoparticle systems, previously developed and extensively characterized by our research group, support the assumption that the total amount of PS employed in the present study was successfully loaded into the nanoparticles.^4,22

The new systems NRAc-NPs 1–3 were studied by DLS. All sizes were in the range of 17.7–126.2 nm, with PDI values around 0.17–0.47 (Table 2 and Fig. S9). According to these results, it can be said that the new third-generation PSs are optimal and present good size and acceptable PDI. On the other hand, when NRAc is loaded into NP1, the size increases, while it decreases when it is loaded into NPs 2–3.

Table 2 Particle size and PDI values for NPs 1–3, free and loaded with NRAc

Characteristics	Free NPs			NRAc loaded into NPs
	NP 1	NP 2	NP 3	NRAc-NP1	NRAc-NP2	NRAc-NP3
The values correspond to the average size ± SD and PDI ± SD, obtained after the analysis of three determinations.
Size (nm)	72.0 ± 0.3	81.67 ± 0.03	77 ± 1	126 ± 8	17.7 ± 0.8	31 ± 7
PDI	0.302 ± 0.004	0.4 ± 0.1	0.31 ± 0.01	0.47 ± 0.05	0.37 ± 0.08	0.17 ± 0.2

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The increase or decrease in the size of NPs that generally occurs when a drug is loaded into polymeric NPs is influenced by the type of interactions between the drug and the chemical structure of the NPs, as well as the localization of the PS within the nanosystem.^53–55

3.8. Photoinactivation of MRSA in vitro

The photodynamic inactivation of MRSA developed by the free NRAc derivative and that loaded into the NPs in aqueous solution was carried out to evaluate the impact of vehiculization on the therapeutic properties of this PS. Solutions of NRAc at concentrations of 30 and 40 µM, either free or loaded into the NPs, were employed for the study. The irradiation time was set at 15 and 30 min.

As shown in Fig. 5, it is possible to affirm that free NRAc does not produce photoinactivation against the microorganism under the conditions described. However, the third-generation PS managed to reduce the percentage of the microorganism that survived, when irradiated. It is important to mention that all samples kept in the dark for 15 or 30 min did not cause any effect on the viability of the bacteria (data not shown). According to the results, it is possible to state that when a concentration of 30 µM of PS and 15 min of irradiation were used, a survival rate of approximately 60% was observed for third-generation PSs. On the other hand, when using 40 µM of PS and 15 min of irradiation, the survival percentages were 60, 30 and 50% for NRAc-NP1, NRAc-NP2 and NRAc-NP3, respectively.

Fig. 5 Photoinactivation of methicillin resistant S. aureus by NRAc (free) and NRAc loaded into NPs at different concentrations: (a) 30 µM and (b) 40 µM. The characters above the boxes indicate the statistics and data are presented as mean + SD (n = 3). The paired columns presenting the same characters indicate the absence of a statistical difference, all the other comparisons presented significant difference by the ANOVA test and the Tukey post hoc test (p <0.05).

Doubling the irradiation dose to 30 min enhanced the photodynamic efficiency of all third-generation PSs, as evidenced by a statistically significant decrease in survival. As can be seen in Fig. 5a, using a concentration of 30 µM and 30 min of irradiation, the microbial survival for NRAc-NP1 and NRAc-NP3 was 34 and 20%, respectively. Likewise, when 40 µM was used and irradiated for 30 min, the survival was 17 and 22%, respectively (Fig. 5b). Notably, NRAc-NP2 exhibited a trend toward higher photodynamic efficiency, with S. aureus survival below 10% after treatment with both PS concentrations (30 and 40 µM) and 30 min of light exposure, though the differences were not statistically significant. It is relevant to point out that the unloaded NPs were proven to be non-toxic and did not exhibit photodynamic inactivation against MRSA under the tested conditions.²²

In conclusion, the development of third-generation PS enhanced the phototoxic activity of NRAc against MRSA. Among the tested systems, NRAc-NP2 proved to be the most effective in inactivating this bacterial strain. These findings are consistent with recent advances in the field, which emphasize improving therapeutic performance through carrier systems capable of specifically targeting bacteria. Such strategies aim to increase photodynamic inactivation efficiency against drug-resistant strains, enhance photosensitizer bioavailability, protect it from degradation, and reduce the required drug dosage to minimize systemic toxicity.⁵⁶ In particular, recent reviews have shown that nanocarriers can overcome key limitations of PDT, such as the poor solubility and stability of photosensitizers in biological media.⁵ Our system contributes to this goal by demonstrating efficacy against a clinically relevant pathogen, through a platform that improves PS delivery and toxicity under light activation.

4. Conclusions

The synthesis strategy for NRAc was described, and the compound was thoroughly characterized using various spectroscopic techniques. The results revealed that this derivative exhibits physicochemical and photochemical properties similar to those of NR. However, the acid derivative showed a higher capacity for generating singlet oxygen and superoxide anion radicals compared to its precursor, while also demonstrating chemical stability, photostability, and biocompatibility.

Despite these advantages, NRAc has low solubility in aqueous media. To overcome this limitation, the compound was loaded into NPs developed by our research group, which exhibited favorable characteristics. Based on the results obtained, the NRAc-loaded NPs can enhance the performance of this second-generation PSs, representing a significant therapeutic advancement against MRSA.

The novel phenazine-based PS, with improved physicochemical properties and higher ¹O₂ quantum yields, holds promise for future applications in aPDT, although an appropriate carrier system is required for its administration in biological environments. The findings of this study contribute to the development of synthetic strategies for novel photosensitizers with high singlet oxygen generation efficiency.

Author contributions

Conceptualization and design: M. S. G., C. A. I. and C. O. Methodology, investigations and analysis: M. S. G., L. P., V. A. and J. V. Writing original draft preparation: M. S. G. and L. P. Review and editing: M. S. G., C. A. I. and C. O. Supervision: C. A. I. and C. O.

Conflicts of interest

There are no conflicts to declare.

Data availability

All data supporting the findings of this study are included within the manuscript.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d5nj03273c.

Acknowledgements

This work was supported by grants from Secretaría de Ciencia y Técnica (SeCyT) Res. No. 2020-233-E-UNC-SECYT and Fondo para la Investigación Científica y Tecnológica (FonCyT) PICT 2020 Serie A 01955. L. P. gratefully acknowledges receipt of a fellowship from CONICET. M. S. G., C. A. I., V. A., and J. V. are career members of CONICET.

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