Cluster and Kill: the Use of Clustering-Triggered Emission Materials for Singlet Oxygen Photosensitization in Antimicrobial Photodynamic Therapy

Cluster and Conquer: The Use of Clustering-Triggered Emission Materials in Photodynamic Therapy Authors: Karina Dueñas,1,+ Oscar Gulias,1,+ Montserrat Agut,1 Felipe de la Cruz-Martínez,2 Agustín LaraSánchez,2 José A. Castro-Osma,2 Juan F. García-Reyes,3 Antonio Sánchez-Ruiz,4,5 Cristina Martín,6* Santi Nonell,1* Roger Bresolí-Obach1* +These two authors contributed the same. [1] Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017, Barcelona, Spain. [2] Universidad de Castilla-la Mancha, Departamento de Química Inorgánica, Orgánica y Bioquímica-Centro de Innovación en Química Avanzada (ORFEO-CINQA), Facultad de Ciencias y Tecnologías Químicas, Avda. Camilo José Cela, 10, Ciudad Real 13071, Spain [3] Universidad de Jaén, Analytical Chemistry Research Group (FQM 323), Department of Physical and Analytical Chemistry, Campus Las Lagunillas edif. B3, Jaén 23071, Spain [4] Universidad de Castilla-la Mancha, Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Farmacia, Avda. Dr. José María Sánchez Ibáñez, s/n, Albacete 02071, Spain [5] Universidad de Castilla-la Mancha, Instituto de Biomedicina. Complejo de la Facultad de Medicina de Albacete, C/Almansa 14, Albacete 02008, Spain [6] Universidad de Castilla-la Mancha, Departamento de Química Física, Facultad de Farmacia, Avda. Dr. José María Sánchez Ibáñez, s/n, Albacete 02071, Spain Corresponding authors: C.M. ([email protected]) S.N. ([email protected]) R.B.-O. (roger.bres[email protected]) Abstract Today, there is no doubt that light is transforming modern medicine and healthcare through its ability to accurately diagnose and treat disease in complex biological systems facilitated by a wide range of luminescent agents and imaging techniques. Although there are inevitable limitations, including biocompatibility issues, the ability to tune spectral properties, and the ease and affordability of synthesis, circumventing aggregation-induced quenching at high concentrations or in aqueous physiological conditions remains a significant challenge. In this context, ClusteringTriggered Emission (CTE), in which atomic cluster formation induces light absorption and the luminescence of unconventional chromophores, offers an all-in-one solution to all the identified challenges. If the luminescent properties of CTE materials include attributes once thought to be exclusive to conventional chromophores, it seems reasonable that new capabilities, such as the formation of highly oxidative reactive oxygen species (ROS) from CTE excited states for their use in photodynamic therapy, might also be possible. A previously described CTE carvone-based polymer was used to explore this idea. The results show that not only is it possible to transfer the excess energy from the long-lived excited states to molecular oxygen to produce singlet oxygen (one of the most relevant ROS), but more importantly, under violet light irradiation, over 99.9% of Staphylococcus aureus cells are eradicated using fluences comparable to those used in traditional systems. Uncovering these photophysical properties of CTE opens the door to a revolutionary breakthrough that promises to disrupt conventional photodynamic therapy and usher in a new era of CTE-based photosensitisers. 1. Introduction Health has been a crucial aspect of human progress since ancient times. Serious diseases, particularly infectious ones, can lead to pandemic scenarios that significantly impact human civilisation's economic, political, and social aspects.[1] In a world with an ageing and infectionprone population, tackling infectious diseases and the hurdle of antimicrobial resistance is imperative.[2] That's why, in 2017, the World Health Organization (WHO) published its first list of 'priority pathogens' for antibiotic research and development.[3] The aim is to develop new antimicrobial agents that effectively combat infections while minimising resistance to them.[4,5] In this battle, photodynamic therapy (PDT) has been proposed as an alternative therapy to overcome antibiotic resistance. PDT is a non-invasive form of phototherapy [6,7] that uses harmless light to activate non or low-toxic photosensitive chemicals, known as photosensitizers (PS), to generate cytotoxic oxidant Reactive Oxygen Species (ROS) by energy or electron transfer from a long-lived excited state.[8] PDT has a multitarget mode of action for eradicating malignant cells, derived from the broad reactivity of ROS against a wide range of cell components (i.e. lipids, DNA, proteins or carbohydrates), which hinders the emergence of bacterial resistance, making PDT an ideal alternative to conventional antibacterial treatment.[9,10] However, the photosensitizing ability of traditional PSs is severely inhibited by aggregation-induced quenching (ACQ) after PS accumulation at bacteria, leading to minor ROS production.[11] Therefore, if aggregation causes an efficiency penalty, Aggregated Induced Emission (AIE) appears as the solution to overcome this problem. Upon aggregation, AIE materials' (AIEgens) emissive and photosensitizing properties are enhanced.[12-14] In fact, these materials could be rationally designed to undergo aggregation under specific physiological conditions of the bacterial cell, increasing the efficiency and the selectivity of the PDT treatment.[15-19] Although there have been some studies of AIEgens with great potential for future preclinical and clinical translation (especially as photoanticancer agents),[20,21] their structure, based on a conjugated (aromatic) system, is usually associated with low stability, mutagenicity, and biocompatibility.[22,23] In this context, several independent laboratories have reported the emission from non-conjugated unconventional chromophores such as polyamides, dendrimers or aminoacids.[24-26] The phenomenon is explained in terms of Clustering-Triggered Emission (CTE), where the aggregation of heteroatoms (N, O, S and P) and/or unsaturated bonds (C=O, C=C and C≡N) leads to the formation of emissive clusters,[27-29] due to the intramolecular through-space interaction (TSI) between the n and  electrons close in proximity.[30-32] This concept disrupts the traditional luminescence paradigm by attributing light absorption and emission to the different clouds of electrons resulting from a cluster formed by intraand/or inter-interaction in the system [27,29] rather than the typical electron conjugation through bonds. Hence, the considerable amount of research proving the similarity between excited states CTE and those of traditional chromophores and/or quantum dots suggests the possibility of additional phenomena occurring,[33] and it is not too much of a stretch to think that some CTEgens materials could generate long-lived excited states capable of generating ROS. To validate this hypothesis, a CTE system, specifically a carvone-based biomass polymer, was selected in this study. The results are a significant milestone in the production of PSs, as they provide the first conclusive evidence that CTE excited states can contribute to the generation of ROS. The principle has not only been validated spectroscopically but it has also been used as a photo antimicrobial agent against Gram-positive Staphylococcus aureus bacteria. Finally, the results undoubtedly demonstrated that while this carvone-based polymer is not toxic in the absence of light, its photoantimicrobial effect on the bacteria is significantly enhanced. It is clear, therefore, that CTEgens can undertake processes previously thought to be the exclusive domain of traditional chromophores, suggesting that applications previously limited to those chromophores, such as photosensitisation, could now be developed using CTEgens. 2. Results and discussion 2.1 Synthesis The synthesis of the polymer was developed according to a previously reported procedure (Figure 1).[26] It should be noted that the reagents (carvone and phthalic anhydride) and solvents (toluene and dichloromethane) used in the synthesis process were sublimated and/or distilled on more than three occasions to ensure their purity. However, it is of the utmost importance to identify the presence of any impurity, even at the trace level, in CTE systems, as any such impurity may affect the photophysical properties of these systems, consequently, their luminescence mechanism.[29,34] Therefore, a highly sensitive method based on liquid chromatography–highresolution mass spectrometry (LC–HRMS) was employed for this purpose. Figure S1 shows the comparison between the TICs for the polymer mixture and the blank control samples. Peaks between 13 and 19 minutes in the LC run were assigned to oligo(PA-alt-COExo)-related species; data from table S1 shows the gradual incorporation of (PA-COExo) units to COExo, starting with one unit (m/z 521.2177) and reaching up to seven units (m/z 2405.9100) (Figure S2). Molecular ions in the detected peaks were a mixture of proton (M+H+) and sodium (M+Na+) adducts, with the latter being the most abundant one. Being the foundation upon which the higher-level oligomers were built, the structures of COExo-(PA-COExo)1 and COExo-(PA-COExo)2 were studied in detail through MS/MS experiments (figure S3), with the proposed structures for the observed fragments shown in table S2 and confirming the identity of the associated species. Since no COExo peak was observed in the TIC, and the rest of the peaks were related to these oligomeric structures with no presence of extraneous species, the LC-MS analysis provided strong evidence that the source of the observed light-interacting mechanism (absorption, emission, or other relaxation processes), are the oligo(PA-alt-COExo)-related species. Figure 1. Synthesis of poly(PA-alt-COExo) according to reference.[26] DMAP = 4dimethylaminopyridine. 2.2. Photophysical and Photochemical Properties A dilute solution of carvone polymer in tetrahydrofuran (THF, a good solvent) appears colourless to the naked eye, showing neither absorption nor emission. However, when the concentration of the polymer is increased to over 0.1 g/L, the UV absorption undergoes a subtle change, exhibiting a tail extending into the visible region and becoming emissive in the blue/green region of the spectrum (Figure 2A). As previously reported, the phenomenon observed for this polymer is ascribed to the n, and ,* electronic transitions that arise from the formation of different clouds of electrons of the through-space interaction generated clusters,[26] and is exacerbated by the presence of water (Figure 2B). When the water content exceeds 50% of v/v, the polymer starts to aggregate, changing the structure of the cluster. Under these conditions, a turbid suspension with a much brighter emission is obtained due to the more crowded structure of the aggregated polymer compared to neat THF (Figure 2C). Figure 2. Absorption (red solid lines) and emission (blue dashed lines; Exc = 355 nm) spectra of the polymer in THF (A), H2O (B) and THF:H2O mixtures (C). One question that can be addressed is whether these excited clusters can interact with oxygen to produce ROS. One of the simplest methods for investigating this phenomenon is to monitor the production of singlet oxygen (1O2) by recording its weak NIR phosphorescence at 1275 nm (see Figure S4 for a detailed NIR spectral analysis).[35,36] Figure 3 shows the typical time-resolved emission of 1O2 after nanosecond pulsed excitation of the polymer in THF and in H2O (panels A,B respectively), where the 1O2 production increases non-linearly with the polymer concentration (Figure S5). This process is highly efficient, as evidenced by the high 1O2 production quantum yield (   = 0.45; Figure S6), comparable to well-established clinically used PSs.[37,38] It is also noteworthy that this pathway is an order of magnitude more favourable than the emission of a photon by the cluster (  F < 2·10-2), highlighting the significant preference of this CTEgen material for the generation of long-lived excited states capable of generating ROS. The time-resolved 1O2 phosphorescence signals provide additional insight into the formation and fate of this ROS. In THF, the kinetic traces of 1O2 show rise and decay lifetimes of 0.3 and 20 s, assigned to the lifetimes of the 1O2 precursor, namely the triplet state of the cluster (  T), and of 1O2 (  ), respectively (Figure 3A). Indeed, the long-lived CTE excited state formation was confirmed by transient absorption experiments in which the ground-state cluster absorption recovers within a lifetime of 40 s in deoxygenated samples and 0.3 s in air-saturated solutions (Figure 3A inset; Figure S7). Two main observations can be made from here: a) the  T is slightly longer than established conventional photosensitizers (typically around  T of 0.2 s)[39] with a bimolecular quenching rate larger than 1x109 M-1·s-1 (Figure S7), and b) the   rises upon increasing the polymer concentration (Figure S8). The initial observation is related to the fact that the oxygen accessibility to the cluster is slightly hindered, as well as the occurrence of both dynamic and static quenching, as evidenced by the non-linearity of the Stern-Volmer plot. The latter may be attributed to the preferential diffusion of the generated 1O2 can preferentially diffuse along the polymer chain before its escape from the polymer matrix. These two effects become more apparent when the polymer structure collapses and forms aggregates upon increasing the water concentration. Figure 3. Singlet oxygen (1O2) phosphorescence kinetic traces of the polymer in THF (A) and H2O (B), where the polymer concentration range was varied from 0 to 0.5 g/L in panels A,B. The insets in both figures depict the transient absorption traces of ground state bleaching recovery (exc = 355 nm; lobs = 380 nm) at a polymer concentration of 0.5 g/L. Comparison of 1O2 (tT (E) and t (F)) with increasing the water content in the THF:H2O solvent mixtures. To verify this hypothesis, the kinetics of the polymer in different THF:H2O (v/v) mixtures were measured (Figure 3C,D). At low water concentration,   decreases with increasing the water content, as expected from the shorter   in neat water than in neat THF (  ;H2O = 3.3 s;  ;THF = 20 s).[39] However, this observation is reverted (i.e.,   increases), when the water content exceeds a certain threshold. This fact is consistent with the observation that water induces polymer aggregation and, consequently, a stronger and more tightly-packed clusterization.[26] It is, therefore, not surprising that the kinetic profile of 1O2 is altered, with both lifetimes being enlarged (Figure 3E,F). Tight packing in the clusters hinders the diffusion of O2 into the cluster and the escape of 1O2 into the external aqueous environment. Thus, the decay rate of the cluster’s source): spray voltage, 3.0 kV for ESI− and 3.5 kV for ESI+; sheath gas flow rate, 40 arb; auxiliary gas flow rate, 10 arb; sweep gas flow rate, 0 arb; capillary temperature, 320 °C; s-lens RF level, 50; and auxiliary gas heater temperature, 350 °C. A data-dependent scan (dd-MS2) was employed to obtain high-quality MS2 data. The five most intense precursors were automatically selected for MS/MS fragmentation by HCD. The parameters of the dd-MS2 method were as follows: resolution, 17,500; AGC target, 2 × 10⁵; maximum IT, 100 ms; loop count, 5; isolation window, 1 m/z. The Normalised Collision Energy (NCE) was set at 15 and 30 V. 4.3 Photophysical and photochemical characterization All the solvents used for the photophysical, and photochemical characterization have spectroscopic grade or MiliQ water. UV-Vis absorption spectra were recorded using a Varian Cary 6000i spectrometer (Varian, Palo Alto, CA, USA). Fluorescence emission spectra were recorded using a Spex Fluoromax-4 spectrofluorometer (Horiba Jobin-Ybon, Edison, NJ, USA). 1O2 generation was studied by time-resolved near-infrared phosphorescence using a customised Fluotime 200 time-resolved spectrophotometer (PicoQuant, Berlin, Germany).[35,36] A diodepumped Nd:YAG laser (FTSS355-Q, Crystal Laser, Berlin, Germany) working at a 1-kHz repetition rate (0.5 J per pulse) was used for excitation at 355 nm. A 1064 nm rugate notch filter (Edmund Optics) and an uncoated SKG-5 filter (CVI Laser Corporation) were placed in the laser path to remove any residual NIR emission. The 1O2 phosphorescence emitted by the sample was filtered with a 1100 nm long-pass filter (Edmund Optics) and later by a narrow bandpass filter at 1275 nm (BK-1270-70-B, bk Interferenzoptik). A thermoelectric-cooled NIR-sensitive photomultiplier tube assembly (H10330C-45-C3, Hamamatsu Photonics, Hamamatsu, Japan) was used as detector. Photon counting was achieved with a multichannel scaler (NanoHarp 250, PicoQuant, Berlin, Germany). The time dependence of the 1O2 phosphorescence with the signal intensity S(t) is described by Equation 1, in which  T and  Δ are the lifetimes of the photosensitizer triplet state and of 1O2 respectively, and S(0) is a preexponential parameter proportional to the 1O2 formation quantum yield (  ). For the methodology for determining  , please check Figure S4. The used reference photosensitizer is phenalenone (   = 1).[53-55] 𝑆(𝑡)= 𝑆(0) 𝑥 ( 𝜏𝛥 𝜏𝛥−𝜏𝑇) 𝑥 (𝑒−𝑡 𝜏𝛥 ⁄−𝑒−𝑡 𝜏𝑇 ⁄) Eq. 1 Transient absorption kinetics (Abs) were acquired using a home-built nanosecond laser flash photolysis system.[56] Briefly, the sample was excited by a 355 nm Q-switched Nd:YAG Laser (Surelite I-10, Continuum) with right-angle geometry. The analysing beam is composed by a Xe lamp (PTI, 75 W) in combination with a dual-grating monochromator (mod. 101, PTI) coupled to a UV-Vis radiation detector (PTI 710). The observation wavelength was selected to 380 nm which corresponds to the ground state bleaching of the generated molecular clusters. The signal was fed to a Lecroy WaveSurfer 454 oscilloscope for digitizing and averaging (10 shots) and finally transferred to a PC for data storage and analysis. 4.4 Photoantimicrobial studies Three different strains, one gram-positive, Staphylococcus aureus (S. aureus, ATCC 29213) and two gram-negative, Escherichia coli (E. coli, ATCC 35218) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) were used in this study. Briefly, the culture media used for growing the cells was Tryptic Soy Broth (TSB). The PDT treatment protocol was adapted from Ref.[57]. The strains were grown overnight at 37±1 oC in an orbital shaker at 60 rpm under aerobic conditions. Then, 100 µL of the culture were grown in 10 mL of fresh TSB until reaching an optical density of 0.18, measured at 600 nm. These OD600 values corresponds to bacterial concentration of 107 CFU/mL for S. aureus, and 108 CFU/mL for E. coli and P. aeruginosa. After that, bacteria were harvested by centrifugation (4000 rpm, 10 min) and resuspended in the same volume of sterile PBS or deuterated-PBS. Then, the adequate concentration of the polymer dissolved in DMSO (always ≤1%) was added to the bacterial suspension and let incubate for 60 minutes in dark conditions. After, the bacterial suspension was irradiated using a violet LED (Figure S9) until reach the adequate light fluence (15.2 mW/cm2). 1 mL of the bacterial suspension was kept unirradiated and used as dark control. After the PDT treatment, the bacterial suspensions were diluted by a factor of 10, until reaching a 10-6 dilution factor. Finally, 10 µL of every dilution were spread on Tryptic Soy Agar and were incubated aerobically at 37±1 oC. The colony forming units (CFU) were counted after 24 hours of incubation and the dilution factor was considered. Acknowledgements The authors thank the MICIU/AEI projects refs. grant number PID2020-115801RB-C22, PID2022137569NA-C44, PID2021-12861OA-C22, PID2020-117788RB-I00, RYC2021-032773-I, CNS2022136052 and RED2022-134287-T (A.L.-S.)) funded by MCIN/AEI/10.13039/501100011033 and FEDER, UE. This research was also supported by SBPLY/21/180501/000132, SBPLY/21/180501/000127 and SBPLY/23/180225/000094 funded by JCCM and the EU through “Fondo Europeo de Desarrollo Regional” (FEDER). This project has also received funding from the the European Innovation Council (HORIZON EIC Grants; project number 101130615), by AGAUR (2020BP00066) and Universidad de Castilla-La Mancha (Grant 2021-GRIN-31240 (A.L.-S)). S.N. thanks the Departament de Recerca i Universitats de la Generalitat de Catalunya for the support given to our research group (2021 SGR 01023) and the ICREA—Catalan Institution for Research and Advanced Studies for grant No. Ac2232308. The authors would also like to acknowledge the scientific support provided by Servicios Centrales de Apoyo a la Investigación (SCAI-UJAEN) from the University of Jaén and Dr. David Moreno González (University of Jaén) for this research project. Author contributions C.M. and R.B.-O. conceived the research and initiated the project. F. de la C.-M., A.L.-S. and J.A.C.O. synthetized and purified the polymer. J.F.G.-R. assessed the purity of the polymer. K.D., R.B.-O. and S.N. performed the photophysical and photochemical characterization and processed the data. K.D., O.G., and M.A. performed the photoantimicrobial studies. C.M. and R.B.-O. prepared the manuscript and all the other authors contributed to the manuscript. C.M., S.N., and R.B.-O. supervised the project. Bibliography [1] MMWR Morb, Control of infectious diseases. Mortal Wkly Rep. 1999, 48, 621. 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