Spectroscopic Insights into BSA-Mediated Deaggregation of m-THPC

Spectroscopic Insights into BSA-Mediated Deaggregation of m-THPC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Spectroscopic Insights into BSA-Mediated Deaggregation of m-THPC Aleksander Kolman, Tomasz Pedzinski, Anna Lewandowska-Andralojc This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4564342/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Meta-tetra(hydroxyphenyl)chlorin ( m -THPC) is among the most potent photosensitizers, known for its high singlet oxygen generation efficiency. However, its clinical effectiveness in photodynamic therapy (PDT) is compromised by its propensity to aggregate in aqueous solutions, adversely affecting its photophysical properties and therapeutic potential. A series of spectroscopic techniques, including UV-Vis absorption, fluorescence spectroscopy, and laser flash photolysis, revealed that m -THPC exhibits significant aggregation, particularly in MeOH-PBS mixtures with MeOH content below 30%. This aggregation adversely affects its photophysical properties leading to reduced fluorescence quantum yield and most importantly reducing its singlet oxygen quantum yield. This study introduces the use of bovine serum albumin (BSA) to counteract the aggregation of m -THPC, aiming to enhance its solubility, stability, and efficacy in physiological settings. Through advanced spectroscopic analyses we demonstrated that the m -THPC@BSA complex exhibits improved photophysical characteristics, essential for effective PDT. Notably, the complex showed a significant restoration of the singlet oxygen quantum yield (Φ Δ = 0.21) compared to aggregated m -THPC. These results underscore the potential of BSA to preserve the monomeric form of m -THPC, mitigating aggregation-induced losses in singlet oxygen production. Our findings suggest that BSA-mediated delivery systems could play a crucial role in optimizing the clinical utility of hydrophobic photosensitizers like m -THPC. Biological sciences/Chemical biology Physical sciences/Chemistry photodynamic therapy singlet oxygen aggregation time-resolved spectroscopy chlorin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction One promising treatment for several types of cancer is photodynamic therapy (PDT), which is based on the action of light on a photoactive drug (photosensitizer, PS), which in the presence of oxygen induces cytotoxicity due to the generation of reactive oxygen species (ROS) such as singlet oxygen 1 O 2 . Despite its potential, the high cost and lengthy approval process for new drugs present significant economic challenges for the pharmaceutical industry [ 1 ]. An effective strategy to circumvent these obstacles involves enhancing the efficacy of existing drugs through optimized delivery systems. Meta-tetra(hydroxyphenyl)chlorin ( m -THPC or Foscan®) is one of the most effective photosensitizers now utilized in clinics. Foscan®, a photosensitizer from the second generation, has been demonstrated to cause significant cell damage by producing singlet oxygen at low drug concentrations and low light doses [ 2 , 3 ]. Its formulation, comprising ethanol and polyethylene glycol (PEG), facilitates the delivery of m -THPC, even at low concentrations and light doses, to produce substantial singlet oxygen for cell destruction [ 4 ]. However, m -THPC's hydrophobic nature necessitates organic solvents for dissolution, complicating its biological application due to a pronounced tendency to aggregate. Such aggregation significantly impacts the physicochemical and photophysical properties of chlorins, altering their spectral characteristics and reducing their efficacy in applications like PDT. Aggregation usually occurs as a result of non-covalent intermolecular interactions depending on their electronic and steric properties, also can be influenced by several factors such as the type of charge, salts [ 5 ], the nature of peripheral groups [ 5 , 6 ], pH [ 7 ] and solvent composition [ 8 ]. As a result of aggregation, chlorins show interesting photochemical characteristics that are different from monomers. In particular formation of aggregates changes their absorption spectra, emission quantum yield, lifetime of singlet and triplet states and, reduces production of singlet molecular oxygen, compromise PDT efficacy [ 9 – 12 ]. Although prior studies have explored the photophysical behavior of m -THPC in monomeric form within organic solvents, there remains a gap in understanding the influence of environmental factors on the photosensitizing ability of m -THPC. Information concerning excited state dynamics of aggregated forms is important to clarify how the aggregation modifies the photophysical characteristics of the photosensitizer monomer. This information is extremely valuable for potential applications, including drug delivery systems, photodynamic therapy [ 13 ], electronic and optoelectronic devices [ 14 , 15 ]. Aiming at a better characterization of solvent effect on the aggregation, the initial section of the paper concentrates on examining the photophysical properties of m -THPC across varying water content levels. Given the challenges posed by m -THPC aggregation for PDT applications, there is a growing interest in enhancing its solubility and stability in physiological environments through innovative delivery systems, including polymers, liposomes, nanoparticles, and proteins. Proteins, essential for various biological processes, can facilitate the transport and localization of hydrophobic molecules, potentially serving as effective drug delivery vehicles or supramolecular hosts. The binding and movement of hydrophobic molecules inside cells and throughout the body is one of these functions. Therefore, proteins can serve as drug delivery mechanisms or supramolecular hosts [ 16 – 18 ], giving hydrophobic PS solubility in physiological media [ 19 – 22 ]. A photosensitizer that possesses protein binding ability may have significant advantages in light of cancer cell damage and cancer cell targeting. At first binding of the photosensitizer to the protein may limit its aggregation under physiological environment and therefore restrain their favorable for PDT photophysical properties. 1 O 2 has a short lifetime in biological systems (0.04 µs) and therefore, the diffusion range is very limited in tumor cells (0.02 mm), implying the importance of the binding ability of the photosensitizer towards proteins which ensures 1 O 2 generation in close proximity to the targets. This study focuses on exploiting proteins, specifically bovine serum albumin (BSA), to address m -THPC's aggregation issue. By binding to BSA, m -THPC's aggregation could be mitigated, preserving its photophysical properties conducive to effective PDT. In this paper we explored the efficiency of a new m -THPC formulation that uses BSA, a typical protein for targeting delivery of phototherapeutic sensitizers [ 23 – 25 ] to disperse the photosensitizer in purely aqueous solution ( m -THPC@BSA). Our research aims to assess the degree to which BSA can disaggregate m -THPC, proposing a novel formulation ( m -THPC@BSA) for improved phototherapeutic application. Through comprehensive spectroscopic analysis in various solvent mixtures and the presence of BSA, this paper elucidates the efficacy of m -THPC@BSA in maintaining stability, preventing aggregation, and facilitating high singlet oxygen generation as a monomolecular form within a physiological environment. In our investigation, we utilized advanced spectroscopic techniques such as femtosecond and nanosecond flash photolysis, time correlated single photon counting measurements. These methods allowed us to precisely characterize the photophysical properties of m -THPC, both in varying water content environments and when interacting with BSA, offering critical insights into the deaggregation process and its implications for photodynamic therapy applications. 2. Materials and methods 2.1 Materials m -THPC and m -tetraphenylporphyrin (TPP) were purchased from Porphyrin Systems. BSA, D 2 O, methylene blue (MB), PBS tablets and D-Tube™ Dialyzer Maxi, MWCO 12–14 kDa were purchased from Merck. Methanol was purchased from VWR Chemicals. Solutions were prepared with Millipore distilled water (18 MΩ cm). 2.2 Synthesis m -THPC@BSA Complex The m -THPC@BSA complex was synthesized by mixing m -THPC with BSA in a stoichiometric ratio of 1:1. Solution A: BSA was first dissolved in PBS, then MeOH was slowly added to the solution to obtain a final concentration of 200 µM BSA dissolved in the MeOH/PBS mixture (3/5 v/v). Solution B: First m -THPC was dissolved in MeOH. Subsequently it was diluted to a final concentration of 200 µM m -THPC in MeOH/PBS mixture (3/5 v/v) just before being added to the BSA solution. A volume of 500 µl of solution B was slowly added to a solution A, stirring gently to obtain a final solution in which the concentration of both components was 100 µM. The mixture was then incubated overnight at 25°C with continuous shaking at 700 rpm. After incubation, the solution was dialyzed extensively against PBS, using dialysis tubes with a cellulose membrane with a cutoff value of 14 kDa, to remove MeOH and remaining unbound m -THPC. 2.3 Experimental apparatus Cary 100 UV-Vis two-beam spectrometer (Agilent) was used to record UV-Vis absorption spectra in the range from 800 to 200 nm with 1 nm step for these measurements, quartz cuvettes with optical paths of 10 mm, 2 mm, and 1 mm were employed. Fluorescence and excitation spectra were evaluated using a JASCO FP-8550 spectrofluorometer. Fluorescence spectra were recorded in the range of 600−760 nm for diluted solutions with an absorbance at excitation wavelength (λ exc = 416 nm) lower than 0.1. A quartz cuvette with an optical path of 10 mm was used for the study. Fluorescence quantum yield ( \({\varphi }_{f}^{x})\) was determined using a standard substance. TPP has been used as standard in experiments to determine fluorescence quantum yield [ 26 ]. The following equation have been applied to determine the quantum yield of fluorescence: $${\varphi }_{f}^{x}=\frac{{S}_{x}{A}_{st}^{\lambda }{n}_{x}^{2}}{{S}_{st}{A}_{x}^{\lambda }{n}_{st}^{2}}{\varphi }_{f}^{st}$$ 1 Where: \({\varphi }_{f}^{x}\) - fluorescence quantum yield of a sample x; S x , S st - integrated fluorescence intensity (area under the spectrum) for sample x and standard sample, respectively; \({A}_{x}^{\lambda }, {A}_{st}^{\lambda }\) – absorbance at the excitation wavelength (λ) for sample x and standard sample, respectively; \({\varphi }_{f}^{st}\) - fluorescence quantum yield of the standard sample ( \({\varphi }_{f}^{st}\) = 0.15 in ethanol); n x , n st – refractive index for sample x solvent’s and standard sample solvent’s, respectively [ 27 ]. The fluorescence lifetimes were measured on a Fluorescence Lifetime Spectrometer (FluoTime 300 from PicoQuant) with a detection system based on time-correlated single-photon counting (TCSPC). The emission decay lifetimes were acquired using 405 nm diode laser as the excitation source. In addition, an instrument response function (IRF) was obtained using Ludox solution (colloidal silica). Quantum yields of singlet oxygen production (Ф Δ ) generated by m -THPC were calculated using the results of steady-state measurements. Measurements were recorded on the PicoQuant FluoTime 300 fluorescence lifetime spectrometer with a Hamamatsu H10330B-45 NIR-PMT module, which is sensitive in the 900 to 1450 nm NIR range with excitation using picosecond laser diode (LDH-640, λ exc = 640 nm, PicoQuant) using methylene blue (MB) as standard (Φ Δ = 0.52, λ exc = 640 nm) [ 28 ]. Air-equilibrated solutions of the m -THPC were optically matched at the excitation wavelength (640 nm) to a standard reference solution. The total area under the emission spectrum was calculated separately for each substance. Finally, the quantum yield of singlet oxygen (Φ Δ ) was calculated by comparing the total area under the emission spectrum of m -THPC and MB. For time-resolved measurements the samples were using a high repetition rate 40 MHz picosecond laser diode (LDH-640 nm, PicoQuant). The decay traces at λ = 1270 nm were collected using a so-called “burst mode”, in which the sample is first excited using multiple pulses of the laser to build up the population of singlet oxygen and then left to decay in the 60 µs time window. Femtosecond transient absorption measurements were carried out using a Solstice Ti:sapphire regenerative amplifier from Spectra Physics and an optical detection system provided by Ultrafast Systems (Helios) [ 29 ]. The 800 nm laser beam was split into two: pump (95%) and probe (5%). The pump beam was directed to a Light Conversion TOPAS-Prime automatic optical parametric amplifier to obtain the desired excitation wavelength in the 290–2600 nm range. The probe beam was directed to a TA pump-probe Helios spectrometer from Ultrafast Systems LLC with an optical delay line allowing delays of up to 3 ns between pump and probe. A white-light continuum was used for transient detection, which was generated from 5% of the primary beam by passing it through a sapphire or calcium fluoride crystal. The laser flash photolysis (LFP) setup employed in this study utilizes a tunable high-end Optical Parametric Oscillator (GWU, model primoScan, Germany) pumped by the third harmonic (355 nm) of a Nd:YAG laser (Quantel, model Q-smart 450, USA), with a pulse duration of 6–8 ns to excite the samples (typically at an excitation wavelength of 420 nm). The excitation pulse energy was maintained at approximately 3 mJ/pulse to prevent undesired multiphotonic processes. The monitoring beam of the system includes a 150 W pulsed Xe lamp (Hamamatsu, model E7536, Japan), a single monochromator (Princeton Instruments, model Spectra Pro SP-2155, USA), and a photomultiplier (Hamamatsu, model R955, Japan) powered by a PS-310 power supply (Stanford Research System, USA). Data processing was carried out in real time using a digital oscilloscope (Tektronix, model MDO 3024, 350 MHz, USA), which was triggered by a fast photodiode (Thorlabs, model DET10M, USA). The oscilloscope data were transferred to a PC with custom software based on LabView 8.0 (National Instruments, Austin, TX, USA), which controlled the timing and acquisition functions of the system. Kinetic traces were recorded at intervals of 10 nm between 300 and 700 nm. To deoxygenate the sample solutions, high-purity argon was bubbled through them for at least 20 minutes prior to measurements. All experiments were conducted in 1 cm × 1 cm quartz cells. 3. Results and discussion 3.1 Aggregation of m -THPC Absorption and fluorescence measurements, along with time-resolved spectroscopy, were employed in this study to explore how aggregation influences the photophysical characteristics of the ground state and excited states (singlet and triplet) of m -THPC. Various range of parameters is available to induce aggregation on m -THPC but in our study we have selected solvent-mixtures due to relevance to the biological environment. Aggregation of m -THPC was studied spectrophotometrically over a range of methanol-buffer solutions (PBS) (0–95%) and it was of interest to determine at what water content aggregation occurs. Absorption spectra of m -THPC in different MeOH-PBS mixtures were recorded at constant concentrations of m -THPC (4 µM). In the absence of PBS, the spectrum is typical of a chlorine-type molecule [ 30 ] and is characterized by a strong absorbance with a maximum at 650 nm (ε = 5.3×10 4 M −1 cm −1 ) and a broad Soret band with a maximum at 416 nm (ε = 2.1×10 5 M −1 cm −1 ) (Fig. 1 A). In methanol, Beer-Lambert absorbance plots as a function of dye concentration were linear in the range up to 150 µM indicating it remains in a monomeric form even at higher concentration (Fig. S1 A). The band width at half height ( W 1/2 ) for the Soret band was 33 nm at 416 nm over the entire concentration range studied. Based on the above observations it can be concluded that the same species, i.e. the monomer, is present throughout. As the proportion of PBS increases in MeOH-PBS mixtures, a broadening of all absorption bands is observed with a strong decrease in their intensities, as shown in Fig. 1 A. The apparent molar absorbance coefficient of the Soret band fell markedly at high water content as shown in Fig. 1 A, where all spectra shown refers to 4 µM solutions. The λ max of the Soret band did not shift from 10% water to 60% water: above 70% of water it became concentration dependent and varied with solvent composition. W 1/2 also remained almost constant up to 60% water, but then increased markedly ( W 1/2 : 70% − 50 nm, W 1/2 : 95% − 53 nm). These data indicate that aggregation becomes significant for 4 µM solutions of m -THPC in aqueous methanol when the water content exceeds 70%. The changes in the aggregation state were graphically monitored through a plot of methanol content versus absorbance at 416 nm (Fig. 1 B). With a fixed concentration of m -THPC, an increase in PBS concentration was accompanied by a pronounced decrease in absorbance at 416 nm, particularly noticeable at 70% of PBS. The plot of absorbance at 416 nm against methanol content revealed that the absorbance remained relatively constant within the 100% to 40% methanol range, followed by a significant decrease in 30% methanol. Subsequent addition of PBS was accompanied by a slight decrease in the absorbance at the Soret band. These changes in the UV-Vis spectra were also reflected in the distinct color change of the m -THPC solution when the PBS content exceeded 70% (Fig. 1 B). A complication exists in the behavior of the more concentrated solutions with high water content: for MeOH content lower than 30%, a clear negative deviation from Beer-Lambert's law, attributed to the aggregation of m -THPC, is observed (Fig. S1 B). Moreover, it was noticed that solutions containing aggregates are unstable, as evidenced by a decrease in their absorbance to one-fifth of the initial value, after 48 hours of room-temperature storage (Fig. S2). In the presence of PBS alone, the absorbance associated with THPC is not discernible on UV-vis spectra due to its low concentration. Our data indicate that aggregation becomes significant for 4 µM solutions of m -THPC in aqueous methanol when the water content exceeds 70%. When porphyrins aggregate, they form a supramolecular structure which exhibits unique chemical and photophysical properties, including changes in their electronic absorption spectra. For H-type aggregates, the spectral shift is hypsochromic, meaning there is a shift towards a shorter wavelength, often perceived as a blue shift, compared to the spectrum of an individual monomer [ 31 , 32 ]. This shift results from the monomers arranging themselves in a stacked, face-to-face orientation. Conversely, J-type aggregates display a batochromic shift in their Soret band, known as a red shift, which indicates a longer wavelength compared to the monomer spectrum. This type of aggregation occurs when porphyrin monomers line up in a head-to-tail configuration [ 31 ]. Due to the broadening of the peak and an indistinct spectral shift, it was challenging to determine the type of aggregates formed at high PBS concentrations. Therefore, a deconvolution of the absorption spectra was performed for water concentrations ranging from 70–95%, where aggregation is appreciably observed by spectrophotometry. Some examples are shown in Fig. 2 , which enabled the detection of monomers and two distinct aggregate species: an H-dimer (H) with a broad absorption peak around 402 nm, and a J-dimer (J) that absorbs around 434 nm. Considering that the molar absorption coefficient of the monomer remains constant regardless of the solvent composition, it was estimated from the deconvolution procedure that the concentration of the monomer decreases from 4 µM in pure methanol to 1.5 µM in 30% methanol, and further to 0.5 µM in 5% methanol. Based on the Lambert-Beer additivity law, spectrum deconvolution, and the total concentration of m- THPC in the sample, the proportions of monomer and aggregates (H and J) in solutions containing 30% methanol and 5% methanol were determined (Table 1 ). This analysis reveals that the monomer constitutes only 12.5% of the 5% methanol solution, with the H-type aggregate being the predominant species, accounting for 70%. Table 1 Concentration and percentage contribution of the m -THPC monomer and aggregates in high water content. Methanol content % Monomer J-aggregate H-aggregate 30% 1.5 µM (37.5%) 0.7 µM (17.5%) 1.8 µM (45%) 5% 0.5 µM (12.5%) 0.7 µM (17.5%) 2.8 µM (70%) In methanol, when excited at 427 nm, m -THPC exhibited a fluorescence spectrum spanning from 620 to 680 nm, featuring a single emission band with a peak at 650 nm (Fig. 3 A). In the literature the emission quantum yield of m -THPC varies significantly in different solvent, from 0.089 in EtOH [ 33 ] to 0.441 in DMF [ 34 ]. The fluorescence quantum yield was determined by the method described by Williams et al. [ 35 ], which involves the use of well characterized standard samples with known fluorescence quantum yield ( \({\varphi }_{f}^{})\) values and a measurement of emission spectra for a series of concentrations of m -THPC. The fluorescence quantum yields ( \({\varphi }_{f}^{}\) ) also varied with changes in solvent composition. Under air-saturated conditions, \({\varphi }_{f}^{}\) decreased from 0.41 in pure methanol to 0.20 in 30% methanol. It was found that \({\varphi }_{f}^{}\) for 30% MeOH in PBS was dependent on the excitation wavelength (Table S1 ) which further confirms that at this solvent composition mixture m -THPC exists in various states: monomer and aggregates. This is in agreement with the results described by UV-Vis data analyses. The highest quantum yield of fluorescence was obtained with the excitation at 416 nm where the absorbance from the monomeric form was the most profound. Moreover, the fluorescence excitation spectrum recorded for the m -THPC solution of 30% MeOH in PBS matched the absorption spectrum of monomeric m -THPC (Fig. S3). These results discussed above, demonstrate that monomeric m -THPC is an emissive species and that the observed fluorescence in Fig. 3 A originates solely from monomeric m -THPC present in various solvent composition mixtures. For mixtures containing less than 30% methanol, the fluorescence intensity was substantially lowered up to only 0.02 in 5% MeOH in PBS. This is in agreement with the decrease of the content of them monomeric form in the solution with 5% MeOH. The fluorescence lifetime of m -THPC was determined using the time-correlated single photon counting (TCSPC) technique. For m -THPC in methanol, the calculated fluorescence lifetime from the monoexponential emission decay was 8.4 ns (Fig. 3 B). Increasing the percentage of PBS in the mixture did not significantly alter the emission lifetimes, further confirming that the emissions occur exclusively from the monomeric form of m -THPC. The absence of detectable fluorescence from m -THPC aggregates is consistent with their propensity to undergo fast non-radiative decay processes [ 36 – 38 ]. To check the behavior of m- THPC in the triplet state, nanosecond transient absorption experiments were performed. Figure 4 A shows the transient absorption difference spectra of a solution of m -THPC in argon-saturated MeOH following excitation with the OPO laser at 420 nm. There are three negative peaks with maximum at 410 nm that correspond to the bleach of the Soret band and peaks at 520 nm and 650 nm that matches the position of the Q bands in UV-Vis ground state spectra (Fig. 1 A). Moreover, the band with positive signals in the range of 300–390 nm, 430–460 nm, and low intensity band within the range 660–750 nm were observed on the transient spectra, which were attributed to the m -THPC triplet. The spectra did not show any shape evolution in time and all kinetic profile decayed to zero after 500 µs. The triplet lifetime for m -THPC was obtained from monoexponential fit of the bleach recovery at 410 nm and transient decay 450 nm (Fig. 4 B, Table 2 ). Both kinetic traces were characterized by the same time constant within experimental error. m -THPC triplet lifetime in MeOH was found to be 88 µs for argon-saturated solutions. The follow up experiment involved studying the kinetics of the quenching of the m -THPC triplet state in MeOH under two different conditions: in the presence of oxygen (which represents standard atmospheric conditions) and in an oxygen-free atmosphere. The goal was to understand the effect of oxygen on the triplet state quenching process and possible changes in the reaction mechanism. The triplet lifetime of m -THPC was strongly reduced in the presence of oxygen under ambient conditions (190 ns) (Fig S4). The bimolecular quenching rate constants of m -THPC by oxygen ( k q ) in air-saturated solutions were obtained from Eq. ( 2 ): $$\frac{{\tau }_{o}}{\tau }=1+{k}_{q}{\tau }_{o}\left[{O}_{2}\right]$$ 2 where \({\tau }_{o}\) is the lifetime of triplet states in degassed solutions and \(\tau\) is the lifetime of triplet state in air-saturated solutions. Assuming [O 2 ] concentration is 9.4 mM (in MeOH) [ 39 ] we calculated k q based on Eq. ( 2 ) (Table 2 ). The bimolecular rate constant k q for the triplet state of m -THPC quenching by oxygen was found to be 5.6×10 8 M −1 s − 1 . At higher methanol contents, the transient absorption spectrum closely resembled that observed in pure methanol, showing an absorption maximum at 450 nm and a bleach at 410 nm, 520 nm, and 650 nm. For different methanol-PBS mixtures, decay profiles at 410 nm and 450 nm for m -THPC registered at constant absorbance at the excitation wavelength were compared (Fig. S5). Up to 50% methanol, the signal intensity at 410 nm and 450 nm remained almost unchanged. The magnitude of triplet state absorption at 450 nm decreased significantly between samples with 50% and 30% methanol (absorbance at the excitation wavelength was kept constant), although the spectral shape remained unaltered. For the mixture with 30% methanol, the transient absorption signal at 410 nm and 450 nm decreased by half compared to pure methanol, without noticeable changes in transient absorption spectra and kinetic profiles (Fig. S6). This suggests that the decrease in absorbance at 450 nm indicates a higher contribution of aggregates in the solution at higher methanol percentages, as the monomer is the only species capable of forming a triplet state. The observation that the triplet state signal decreased by a factor of two at 30% methanol aligns with a similar decrease in fluorescence quantum yield for the same solvent composition. A complete disappearance of the signal was observed between 30% and 20% methanol, indicating a substantial increase in aggregate concentration (Fig. S6). Negligible signal was observed at either 410 nm or 450 nm probe wavelengths, which would be expected if the triplet state of m -THPC had formed. Therefore, given the detection limit of the apparatus, it can be stated the triplet state of m -THPC was not detectable at methanol concentrations of 20% or less. This unambiguously demonstrates that intersystem crossing in m -THPC aggregates is suppressed, completely preventing triplet state formation. The absence of a detectable triplet state indicates that the formation of m -THPC aggregates suppresses the generation of singlet oxygen ( vide infra ). Table 2 Comparison of the photophysical properties of m -THPC in MeOH and m -THPC@BSA in PBS. Compound λ abs [nm] ε [M −1 cm −1 ] Φ Fl a τ S [ns] τ T (argon) [µs] τ T(air) [µs] k q [M −1 s -1 ] Φ ∆ m -THPC in MeOH 416 650 2.1×10 5 5.3×10 4 0.41 8.5 88 0.19 5.6×10 8 0.42 b m -THPC@BSA in PBS 420 652 1.5×10 5 3.4×10 4 0.2 3.9 (35%) 10.1 (65%) 444 14.0 5.7×10 7 0.21 a determined with excitation λ ex = 427 a [ 52 ] To fully understand the excited state dynamics of the m -THPC, ultrafast spectroscopy experiments were conducted on m -THPC monomers in methanol and m -THPC aggregates in 5% MeOH. Immediately after the excitation of m -THPC in MeOH with a 420 nm laser pulse, the transient absorption spectra revealed a prominent and wide bleach centered around 650 nm. It was interesting to see that in the excited state of m -THPC shows structured absorption which consists of absorption peaks centered at 490, 530, 570, 620, and 680 nm which could be attributed to the presence of the singlet excited state (S 1 -S n transition) (Fig. 5 A). To further probe the relaxation dynamics of the m -THPC monomer, we monitored the transient decay kinetics. (Fig. 5 B). The excitation wavelength 420 nm used in the present work corresponds to a transition from S 0 to a vibrationally excited level of the S 2 state of chlorins. It is well-known that the S 2 → S 1 internal conversion (IC) in tetrapyrroles is generally very fast (on the order of a few hundreds of femtoseconds) [ 40 ]. The analysis of transient absorbance kinetics at 490 nm at short delays revealed a fast component with time constant of only 8.0 ps, which can be presumably attributed to vibrational cooling inside the Q-band. Unfortunately, the singlet excited state lifetime of m -THPC in methanol could not be determined due to the experiment's short time window of 3 ns, consistent with TCSPC findings that recorded a lifetime of 8.5 ns. Notably, throughout the experiment's time window, the decay of the singlet excited state signal was not accompanied by any distinct spectral evolution. Femtosecond time-resolved transient absorption spectroscopy is valuable for studying exciton dynamics in aggregates. We explored excited-state dynamics of m -THPC aggregates and compared them with those of the monomeric dye to elucidate the photophysical changes induced by aggregation. Figure 5 C shows the transient absorption spectra of aggregates at different time delays after 420 nm laser excitation. The transient spectra show a bleach peaks at 525 nm, 550 nm, 595 nm and 650 nm. It is interesting to see that the bleach peaks appear exactly at the positions of Soret- and Q-exciton bands in steady state optical absorption spectra (Fig. 1 A). A direct comparison of the aggregate TA spectra (Fig. 5 C) with that of the monomer (Fig. 5 A) shows that although the spectral shape similarity the decay of the aggregate is much faster than that of the monomer. The faster dynamics is most likely due to new nonradiative energy relaxation channels that become effective upon aggregation. This observation is consistent with both steady-state and time-resolved fluorescence studies reported by various authors, which note a significant reduction in fluorescence quantum yield and excited-state lifetime upon aggregation [ 41 , 42 ]. To study exciton relaxation at different wavelengths in the entire spectral region we have monitored the transient decay kinetics at 480, 575, 650 nm and 670 nm (Fig. 5 D and Fig. S7). The kinetic data can be fitted multiexponentially with: τ 1 = 4.8 ps (59%), τ 2 = 26.8 ps (21%), τ 3 = 204.8 ps (20%), at 480 nm, τ 1 = 4.8 ps (21%), τ 2 = 37.0 ps (41%), τ 3 = 302.0 ps (38%), at 650 nm; τ 1 = 46.0 ps (21%), τ 2 = 207.9 ps (40%), τ 3 = 1034 ps (39%), at 670 nm and finally τ 1 = 4.0 ps (61%), τ 2 = 63.8 ps (40%) at 575 nm. Such fast dynamics is in accordance with literature where it is reported that photoexcited aggregated tetrapyrroles undergo exciton relaxation dynamics at an ultrashort time scale [ 43 – 47 ]. Complex decay kinetics can reflect presence of various aggregate types in the solution. However, it can be assumed, following Kano and Kobayashi [ 48 ], that the time constants of around 4 ps and few tens of ps are attributed to intra-aggregate and aggregate-solvent vibrational energy redistribution, respectively; and around 200–300 ps component is attributed to S 1 lifetime. No residual absorbance appeared at longer time delays in any of the recorded positive traces, further supporting the absence of an excited triplet state in aggregated m -THPC, as already demonstrated in nanosecond flash photolysis experiments. However, the presence of residual bleach absorbance at 650 nm suggests photoinstability of the aggregates. 3.2 Complexation to bovine serum albumin In respect to PDT applications, our findings indicate that m -THPC is predominantly monomeric in methanol. However, at high PBS concentration at physiological pH, it aggregates and exhibits very low solubility. Considering the use of m -THPC as a photosensitizing drug, we focused on systems that were fully aqueous. Our objective was to determine whether binding m -THPC to BSA could lead to disaggregation and restore the photophysical properties of its monomeric form. The influence of BSA on the photophysics of m -THPC in aqueous solutions was studied using the same techniques previously applied to examine aggregation in methanol-PBS mixtures. Here we synthesized a m -THPC@BSA complex, with a well-defined 1:1 stoichiometry, using a PBS/MeOH mixed solvent system, with overnight incubation and purification by dialysis (removal of MeOH and unbound m -THPC) (Scheme 1 ). The UV-Vis spectrum of m -THPC@BSA in pure PBS showed that m -THPC was successfully bound to BSA (Fig. 6 ). The spectrum strongly resembles the spectrum for the m -THPC monomer in MeOH and small shift in the Soret band can be related to the solvent effect or the contribution of the aggregated m -THPC still presence in the solution. The band width at half height ( W 1/2 ) for the Soret band was 39 nm at 420 nm, which is only slightly broader than the Soret band of m -THPC in MeOH. The solution of m -THPC@BSA is stable in time, as no decrease in absorbance was observed over a 96-hour period (Fig. S8). Subsequently, emission studies were conducted to probe the excited state properties of m -THPC@BSA (Fig. 7 ). The fluorescence emission spectrum of m -THPC@BSA in PBS exhibits a peak at 653 nm (Fig. 7 A). The fluorescence quantum yield of m -THPC@BSA in PBS was determined to be 0.2 at λ exc = 427 nm, which is half that of the purely monomeric form in MeOH but notably higher than the fluorescence quantum yield observed at high PBS concentrations in the absence of BSA (Table 2 ). The excitation spectra of m -THPC@BSA perfectly align with the absorption spectra of the monomeric form of m -THPC, with an absorption peak at 415 nm (Fig. S9). This observation further confirm successful deaggregation of m -THPC upon binding to BSA. The emission lifetime for m -THPC@BSA in PBS is biexponential with the time constants of 10.05 ns and 3.9 ns (Fig. 7 B). Subsequently ultrafast spectroscopy experiments were performed on m -THPC@BSA in PBS. Right after the m -THPC@BSA in PBS was excited with a 420 nm laser pulse, the transient absorption spectra displayed a pronounced bleach centered around 650 nm (Fig. 8 ). Notably, the excited state of m -THPC exhibited structured absorption, featuring peaks at 480, 530, 570, 620, and 680 nm, likely indicative of the singlet excited state. Importantly, a direct comparison of the m -THPC@BSA TA spectra (Fig. 8 A,B) with that of the monomer (Fig. 5 A,B) reveals that both the shapes and the dynamics of the spectra are very similar. The transient signals observed at 480 nm and 650 nm exhibit only partial decay within the experimental timeframe of 3 ns, as shown in Fig. 8 B. This observation aligns with the extended fluorescence lifetimes of 3.9 ns and 10.1 ns, respectively, determined using the TCSPC technique. To probe triplet excited state properties of m -THPC bounded to BSA nanosecond absorption spectra were recorded after laser excitation at 420 nm (Fig. 9 ). The transient absorption spectrum of m -THPC@BSA in PBS includes bands from photobleaching at 420 nm and 650 nm. The triplet absorption maximum occurs at 460 nm. The spectrum of the complex is very similar to that of the monomeric form (Fig .4A). Figure 9 B shows the effect of BSA addition on the decay profile of m -THPC triplet states in argon saturated solutions. For the dynamic behavior of m -THPC triplet states the biexponential decay is observed which can also be associated with the presence of different BSA conformers in solution. Under conditions in which it can be considered that all the dye is bound to the protein the transient lifetimes values were found to be 444 µs in argon-saturated solutions. Much longer triplet lifetime compared to m -THPC in MeOH can be explained in terms of suppressed self-quenching of the m -THPC bound to BSA. Under aerobic conditions, both bleaching and triplet transient signals were shortened (Fig. S10). The influence of the protein nanoenvironment on the interaction of triplet excited state of THPC@BSA with dissolved molecular oxygen O 2 can be evaluated by applying Eq. ( 2 ). Considering a homogenous distribution of O 2 concentration in air-saturated protein solutions, i.e. [O 2 ] = 1.22 mM [ 49 ], and decrease of the triplet excited state to 14 µs the quenching rate constant of triplet excited state of THPC@BSA by O 2 was found to be 5.7×10 7 M −1 s − 1 . This value is one order of magnitude smaller in comparison to the quenching rate constant of triplet excited state of m -THPC by O 2 in MeOH. This phenomenon has been observed previously for other dyes bound to protein [ 50 ]. The local rigidity of the binding site of m -THPC can impose a barrier to the free diffusion of O 2 from the bulk buffer. Many dye molecules can be accommodated on a single protein in two principal binding sites (at the interface and deeper within the protein structure, away from the interface) [ 51 ]. Comparison of the oxygen quenching rate constant by m -THPC in MeOH and m -THPC@BSA in PBS, values given in Table 2 shows the extent of protection given to the triplet state of m -THPC by binding to the protein. At molar ratios 1:1 of BSA to THPC most of the dye resides at a site that provides restricted quenching by O 2 and must be located deeper to the interface. Taking into account that reactivity with oxygen and formation of the singlet oxygen ( 1 O 2 ) is crucial for the potential application of the as prepared THPC@BSA toward PDT it was of importance to verify whether sterically hindered THPC still enables formation of the 1 O 2 . It was found that the quenching of triplet excited state of m -THPC bound to BSA by O 2 generates singlet molecular oxygen 1 O 2 , as directly detected by the transient luminescence at 1270 nm after laser excitation of the dye at 640 nm in air-saturated deuterated buffer solutions, Fig. 10 . As compared with the 1 O 2 luminescence signal obtained for the reference methylene blue (Φ ∆ = 0.52 ) [ 28 ]. It was calculated that the quantum yields of singlet oxygen generation was 0.21 m -THPC bound to BSA (Table 2 ). High value of singlet oxygen quantum yield for m -THPC bound to BSA form suggest that it might find application in PDT. It was also demonstrated that the m -THPC in 95% of PBS in the absence of BSA does not generate 1 O 2 (Fig. 10 ). For comparison the quantum yields of singlet oxygen generation for m -THPC in MeOH was reported to be 0.42 [ 33 ] Figure 10 B displays 1 O 2 phosphorescence profile at 1270 nm, which was obtained following 640 nm diode laser excitation of m -THPC@BSA in deuterated buffer solutions. The 1 O 2 lifetime was reduced to 38 µs in comparison to 68 µs lifetime of 1 O 2 in D 2 O reported in the literature. [ 52 ]. Shortening of the 1 O 2 lifetime in the presence of BSA indicates dynamic quenching of 1 O 2 by BSA, which is a known phenomena [53,54]. The singlet oxygen molecules produced by m -THPC@BSA complex will diffuse through the surrounding medium. The radial distance traveled by 1 O 2 during a travel time t can be approximately calculated as d = (6Dt) 1/2 [ 55 ] where D is the diffusion coefficient of 1 O 2 in the media. Given that D = 2 × 10 −5 cm 2 /s in water at room temperature[ 56 ] it can be estimated that d ≈ 600 nm, a distance several orders of magnitude larger that the protein size. This allows the free diffusion of 1 O 2 generated into a protein that bears a m -THPC molecule to the bulk buffer and its interaction during its lifetime with target structures in cells. Our results show that m -THPC undergo marked changes in photophysical properties upon binding to BSA in comparison to m -THPC in high water content in. This is due to the ability of the protein to monomerize aggregates of these dyes present in aqueous solution. These results underscore the potential of BSA to preserve the monomeric form of m -THPC, mitigating aggregation-induced losses in singlet oxygen production. Our findings suggest that BSA-mediated delivery systems could play a crucial role in optimizing the clinical utility of hydrophobic photosensitizers like m -THPC. 4. Conclusion Comprehensive spectroscopic analyses, including UV-Vis absorption, fluorescence spectroscopy, and laser flash photolysis, provided in-depth insights into the photophysical properties of m -THPC both in its aggregated form and when complexed with BSA. The research underscores the significant impact of environmental factors, such as solvent composition and water content, on the aggregation behavior of m -THPC. It highlights the importance of understanding these effects to develop more effective PDT drugs and delivery systems. The study conclusively demonstrates that BSA effectively mitigates the aggregation issues of m -THPC, a crucial photosensitizer in PDT. The m -THPC@BSA complex exhibited significantly enhanced photophysical properties compared to aggregated m -THPC. This includes a higher quantum yield of fluorescence and a longer triplet state lifetime, indicative of the complex's ability to generate singlet oxygen more efficiently. The restoration of these properties in a physiological environment highlights the potential of BSA to serve as an effective delivery vehicle for m -THPC in PDT. These conclusions emphasize the significance of the study in improving the understanding and application of m -THPC in photodynamic therapy, highlighting the potential benefits of using BSA for enhanced drug delivery and treatment efficacy. Declarations Acknowledgements The authors would like to thank prof. Marek Sikorski for providing access to his OPO LFP setup. A.K thanks for funding to program under the project at AMU "Initiative of Excellence - Research University" (proposal no. 054/13/SNŚ/0024). CRediT authorship contribution statement A.Kolman: Writing – original draft, Visualization, Methodology, Investigation, T. Pedzinski: Methodology, Writing – review & editing A. 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Schematic illustration of synthesis of m-THPC@BSA Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 02 Jul, 2024 Reviews received at journal 28 Jun, 2024 Reviews received at journal 25 Jun, 2024 Reviewers agreed at journal 17 Jun, 2024 Reviewers agreed at journal 17 Jun, 2024 Reviewers invited by journal 17 Jun, 2024 Editor assigned by journal 17 Jun, 2024 Editor invited by journal 13 Jun, 2024 Submission checks completed at journal 12 Jun, 2024 First submitted to journal 11 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4564342","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":318833010,"identity":"51bfbe61-8f42-43c4-9046-b5f815d6d7cd","order_by":0,"name":"Aleksander Kolman","email":"","orcid":"","institution":"Adam Mickiewicz University","correspondingAuthor":false,"prefix":"","firstName":"Aleksander","middleName":"","lastName":"Kolman","suffix":""},{"id":318833011,"identity":"6e8668c7-c733-4720-be79-25c7629bb4c1","order_by":1,"name":"Tomasz Pedzinski","email":"","orcid":"","institution":"Adam Mickiewicz University","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Pedzinski","suffix":""},{"id":318833012,"identity":"46fc4991-30f4-448a-824e-03a82b01288a","order_by":2,"name":"Anna Lewandowska-Andralojc","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACdjDJLAfhscFF8ABmCGmM0MJMpJbEBqK18DczH5P4ucM6vX/aGQOGD2WHGcwJaZE4zJYm2XsmPXfG7RwDxhnnDjNYNhNy2GEesxu8bYdzG4BamIEMBoPDBHTIH+b/dvNv2+F0eZCWv8RoMTjMw3YbaHiCAUgLIzFaDA+zmf+WbUs33Hg7reBgz7l0HoJ+kTve/NjwbZu1vNzt5I0PfpRZy5mzNxDQgwwOADGPAQkaoIAMLaNgFIyCUTDMAQCINT/NrnyvnwAAAABJRU5ErkJggg==","orcid":"","institution":"Adam Mickiewicz University","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"","lastName":"Lewandowska-Andralojc","suffix":""}],"badges":[],"createdAt":"2024-06-11 13:06:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4564342/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4564342/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-73266-2","type":"published","date":"2024-09-27T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59180331,"identity":"81c09e35-e8ef-4d31-b611-316f428108ae","added_by":"auto","created_at":"2024-06-27 10:29:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144907,"visible":true,"origin":"","legend":"\u003cp\u003eA) Ground-state electronic absorption spectrum of 4 µM\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003em\u003c/em\u003e-THPC in various MeOH-water mixtures; B) change in absorbance intensity at 416 nm as a function of MeOH percentage. Inset: photographs of the \u003cem\u003em\u003c/em\u003e-THPC solutions for various MeOH percentage.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/8e4063f021fa975bcbed2cda.png"},{"id":59181309,"identity":"6681c6f1-d4b7-4cfb-ab99-c0e8356b54c6","added_by":"auto","created_at":"2024-06-27 10:45:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59298,"visible":true,"origin":"","legend":"\u003cp\u003eDeconvolution of absorption spectra for \u003cem\u003em\u003c/em\u003e-THPC (4 µM) in MeOH/PBS solutions with different amount of MeOH %, dotted line: fitting of the deconvolution study.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/841c2d43c03b948410c2e57f.png"},{"id":59180337,"identity":"6269ff2d-6850-40f4-b6f4-a5e04a96b215","added_by":"auto","created_at":"2024-06-27 10:29:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42217,"visible":true,"origin":"","legend":"\u003cp\u003eA) Emission spectra of \u003cem\u003em\u003c/em\u003e-THPC in solutions with different % methanol content; B)\u003cstrong\u003e \u003c/strong\u003edecay of \u003cem\u003em\u003c/em\u003e-THPC fluorescence recorded at different %MeOH; λ\u003csub\u003eex \u003c/sub\u003e= 437 nm, λ\u003csub\u003eem \u003c/sub\u003e= 650 nm; cyan line show monoexponential decay fit.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/cdfd3f6ba5a7fb4bcd6fdf7a.png"},{"id":59180857,"identity":"81c728d3-9b61-4462-85b8-b92b9043178f","added_by":"auto","created_at":"2024-06-27 10:37:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37722,"visible":true,"origin":"","legend":"\u003cp\u003eA) Transient absorption spectra obtained during laser flash photolysis (with excitation at 420 nm) of deoxygenated solutions of \u003cem\u003em\u003c/em\u003e-THPC (2.3 µM) in methanol; at time delay after flash from 30 µs to 100 µs B) Decay profiles monitored at 410 nm and 450 nm; (red lines show the monoexponential decay fits).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/0a3f460783468a791dd03123.png"},{"id":59180333,"identity":"1d716b6a-c089-4136-8b54-ee07ee2a0f35","added_by":"auto","created_at":"2024-06-27 10:29:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74459,"visible":true,"origin":"","legend":"\u003cp\u003eTransient absorption spectra acquired at various time delays for A) \u003cem\u003em\u003c/em\u003e-THPC (20 µM) in MeOH and C) \u003cem\u003em\u003c/em\u003e-THPC (20 µM) in 5% MeOH after 420 nm laser excitation; B) DA time profiles at 470 nm (red curve), 650 nm (green curve) registered for the \u003cem\u003em\u003c/em\u003e-THPC in MeOH; D) DA time profiles at 470 nm (red curve), 650 nm (green curve) registered for \u003cem\u003em\u003c/em\u003e-THPC in 5% MeOH (black lines shows the exponential decay fits).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/78f2e7c806ff9bf6a515946e.png"},{"id":59180338,"identity":"b494e760-fb26-443c-aee1-5a284f3ebd78","added_by":"auto","created_at":"2024-06-27 10:29:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36117,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis absorption spectra of \u003cem\u003em\u003c/em\u003e-THPC@BSA (100 μM) and BSA in PBS. Spectra were recorded in the cuvette with 1 mm optical path.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/5b69b2b9c09d922fe0891c6e.png"},{"id":59180334,"identity":"a49f03ff-beda-4f46-9b31-9ea286228a16","added_by":"auto","created_at":"2024-06-27 10:29:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":30672,"visible":true,"origin":"","legend":"\u003cp\u003eA) Emission spectra of \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS; λ\u003csub\u003eexc\u003c/sub\u003e = 427 nm B)\u003cstrong\u003e \u003c/strong\u003edecay of \u003cem\u003em\u003c/em\u003e-THPC@BSA fluorescence recorded in PBS; λ\u003csub\u003eex \u003c/sub\u003e= 437 nm, λ\u003csub\u003eem \u003c/sub\u003e= 653 nm \u0026nbsp;(black line shows the biexponential decay fit).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/804d5eb33864c2bb355b5790.png"},{"id":59180340,"identity":"2289078d-56e1-43f5-b719-f53e82374f94","added_by":"auto","created_at":"2024-06-27 10:29:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":44881,"visible":true,"origin":"","legend":"\u003cp\u003eA) Transient absorption spectra registered at various time delays for \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS after laser excitation at 420 nm; B) DA time profiles at 480 nm (blue curve), 650 nm (orange curve) registered for \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/3828144b8b82ed54e5b3248a.png"},{"id":59180861,"identity":"f5badee5-359f-4bed-9033-c67f932506a8","added_by":"auto","created_at":"2024-06-27 10:37:46","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":36740,"visible":true,"origin":"","legend":"\u003cp\u003eA) Transient absorption spectra obtained during laser flash photolysis (with excitation at 420 nm) of deoxygenated solutions of \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS; at time delay after flash from 90 µs to 800 µs; B) decay profiles monitored at 460 nm; (red line shows the biexponential decay fit).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/55fb8caa881bd8488b604295.png"},{"id":59180859,"identity":"aefd2c2a-833f-445e-9614-0a283498ce33","added_by":"auto","created_at":"2024-06-27 10:37:46","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":37966,"visible":true,"origin":"","legend":"\u003cp\u003eA)\u003cstrong\u003e \u003c/strong\u003eSinglet oxygen emission spectrum obtained with 640 nm LED diode excitation of 4 µM \u003cem\u003em\u003c/em\u003e-THPC bound to BSA in D\u003csub\u003e2\u003c/sub\u003eO (blue line), \u003cem\u003em\u003c/em\u003e-THPC in D\u003csub\u003e2\u003c/sub\u003eO/MeOD ( 5% of MeOD) (red line) and MB in D\u003csub\u003e2\u003c/sub\u003eO (black line); B) decay curve of the singlet oxygen generated by\u003cem\u003e m\u003c/em\u003e-THPC bound to BSA in D\u003csub\u003e2\u003c/sub\u003eO (λ\u003csub\u003eexc\u003c/sub\u003e = 640 nm, λ\u003csub\u003eem\u003c/sub\u003e= 1270 nm, collection time: 60 min).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/465e008218e89ec0daa20404.png"},{"id":65627282,"identity":"7cfe901e-d680-4e90-80f5-7cb91c6d6727","added_by":"auto","created_at":"2024-09-30 16:14:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1101493,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/c8f5b968-9f93-4228-acba-bd243084a600.pdf"},{"id":59180342,"identity":"77b9feeb-d643-4f8d-ac29-9d9638b780a5","added_by":"auto","created_at":"2024-06-27 10:29:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2789102,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationv3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/1edb3f2aa03c4fdcd6b15b32.docx"},{"id":59182166,"identity":"fde2a0bf-4a0e-48c4-b607-726bb699feec","added_by":"auto","created_at":"2024-06-27 10:53:46","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":280157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Schematic illustration of synthesis of m-THPC@BSA\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4564342/v1/ff4cf33c784dacae28c6da47.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spectroscopic Insights into BSA-Mediated Deaggregation of m-THPC","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOne promising treatment for several types of cancer is photodynamic therapy (PDT), which is based on the action of light on a photoactive drug (photosensitizer, PS), which in the presence of oxygen induces cytotoxicity due to the generation of reactive oxygen species (ROS) such as singlet oxygen \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. Despite its potential, the high cost and lengthy approval process for new drugs present significant economic challenges for the pharmaceutical industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. An effective strategy to circumvent these obstacles involves enhancing the efficacy of existing drugs through optimized delivery systems.\u003c/p\u003e \u003cp\u003eMeta-tetra(hydroxyphenyl)chlorin (\u003cem\u003em\u003c/em\u003e-THPC or Foscan\u0026reg;) is one of the most effective photosensitizers now utilized in clinics. Foscan\u0026reg;, a photosensitizer from the second generation, has been demonstrated to cause significant cell damage by producing singlet oxygen at low drug concentrations and low light doses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Its formulation, comprising ethanol and polyethylene glycol (PEG), facilitates the delivery of \u003cem\u003em\u003c/em\u003e-THPC, even at low concentrations and light doses, to produce substantial singlet oxygen for cell destruction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, \u003cem\u003em\u003c/em\u003e-THPC's hydrophobic nature necessitates organic solvents for dissolution, complicating its biological application due to a pronounced tendency to aggregate. Such aggregation significantly impacts the physicochemical and photophysical properties of chlorins, altering their spectral characteristics and reducing their efficacy in applications like PDT.\u003c/p\u003e \u003cp\u003eAggregation usually occurs as a result of non-covalent intermolecular interactions depending on their electronic and steric properties, also can be influenced by several factors such as the type of charge, salts [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], the nature of peripheral groups [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], pH [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and solvent composition [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As a result of aggregation, chlorins show interesting photochemical characteristics that are different from monomers. In particular formation of aggregates changes their absorption spectra, emission quantum yield, lifetime of singlet and triplet states and, reduces production of singlet molecular oxygen, compromise PDT efficacy [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although prior studies have explored the photophysical behavior of \u003cem\u003em\u003c/em\u003e-THPC in monomeric form within organic solvents, there remains a gap in understanding the influence of environmental factors on the photosensitizing ability of \u003cem\u003em\u003c/em\u003e-THPC. Information concerning excited state dynamics of aggregated forms is important to clarify how the aggregation modifies the photophysical characteristics of the photosensitizer monomer. This information is extremely valuable for potential applications, including drug delivery systems, photodynamic therapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], electronic and optoelectronic devices [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Aiming at a better characterization of solvent effect on the aggregation, the initial section of the paper concentrates on examining the photophysical properties of \u003cem\u003em\u003c/em\u003e-THPC across varying water content levels.\u003c/p\u003e \u003cp\u003eGiven the challenges posed by \u003cem\u003em\u003c/em\u003e-THPC aggregation for PDT applications, there is a growing interest in enhancing its solubility and stability in physiological environments through innovative delivery systems, including polymers, liposomes, nanoparticles, and proteins. Proteins, essential for various biological processes, can facilitate the transport and localization of hydrophobic molecules, potentially serving as effective drug delivery vehicles or supramolecular hosts. The binding and movement of hydrophobic molecules inside cells and throughout the body is one of these functions. Therefore, proteins can serve as drug delivery mechanisms or supramolecular hosts [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], giving hydrophobic PS solubility in physiological media [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA photosensitizer that possesses protein binding ability may have significant advantages in light of cancer cell damage and cancer cell targeting. At first binding of the photosensitizer to the protein may limit its aggregation under physiological environment and therefore restrain their favorable for PDT photophysical properties. \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e has a short lifetime in biological systems (0.04 \u0026micro;s) and therefore, the diffusion range is very limited in tumor cells (0.02 mm), implying the importance of the binding ability of the photosensitizer towards proteins which ensures \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation in close proximity to the targets.\u003c/p\u003e \u003cp\u003eThis study focuses on exploiting proteins, specifically bovine serum albumin (BSA), to address \u003cem\u003em\u003c/em\u003e-THPC's aggregation issue. By binding to BSA, \u003cem\u003em\u003c/em\u003e-THPC's aggregation could be mitigated, preserving its photophysical properties conducive to effective PDT. In this paper we explored the efficiency of a new \u003cem\u003em\u003c/em\u003e-THPC formulation that uses BSA, a typical protein for targeting delivery of phototherapeutic sensitizers [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] to disperse the photosensitizer in purely aqueous solution (\u003cem\u003em\u003c/em\u003e-THPC@BSA).\u003c/p\u003e \u003cp\u003eOur research aims to assess the degree to which BSA can disaggregate \u003cem\u003em\u003c/em\u003e-THPC, proposing a novel formulation (\u003cem\u003em\u003c/em\u003e-THPC@BSA) for improved phototherapeutic application. Through comprehensive spectroscopic analysis in various solvent mixtures and the presence of BSA, this paper elucidates the efficacy of \u003cem\u003em\u003c/em\u003e-THPC@BSA in maintaining stability, preventing aggregation, and facilitating high singlet oxygen generation as a monomolecular form within a physiological environment.\u003c/p\u003e \u003cp\u003eIn our investigation, we utilized advanced spectroscopic techniques such as femtosecond and nanosecond flash photolysis, time correlated single photon counting measurements. These methods allowed us to precisely characterize the photophysical properties of \u003cem\u003em\u003c/em\u003e-THPC, both in varying water content environments and when interacting with BSA, offering critical insights into the deaggregation process and its implications for photodynamic therapy applications.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003e \u003cem\u003em\u003c/em\u003e-THPC and \u003cem\u003em\u003c/em\u003e-tetraphenylporphyrin (TPP) were purchased from Porphyrin Systems. BSA, D\u003csub\u003e2\u003c/sub\u003eO, methylene blue (MB), PBS tablets and D-Tube\u0026trade; Dialyzer Maxi, MWCO 12\u0026ndash;14 kDa were purchased from Merck. Methanol was purchased from VWR Chemicals. Solutions were prepared with Millipore distilled water (18 MΩ cm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis \u003cem\u003em\u003c/em\u003e-THPC@BSA Complex\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe \u003cem\u003em\u003c/em\u003e-THPC@BSA complex was synthesized by mixing \u003cem\u003em\u003c/em\u003e-THPC with BSA in a stoichiometric ratio of 1:1. Solution A: BSA was first dissolved in PBS, then MeOH was slowly added to the solution to obtain a final concentration of 200 \u0026micro;M BSA dissolved in the MeOH/PBS mixture (3/5 v/v). Solution B: First \u003cem\u003em\u003c/em\u003e-THPC was dissolved in MeOH. Subsequently it was diluted to a final concentration of 200 \u0026micro;M \u003cem\u003em\u003c/em\u003e-THPC in MeOH/PBS mixture (3/5 v/v) just before being added to the BSA solution. A volume of 500 \u0026micro;l of solution B was slowly added to a solution A, stirring gently to obtain a final solution in which the concentration of both components was 100 \u0026micro;M. The mixture was then incubated overnight at 25\u0026deg;C with continuous shaking at 700 rpm. After incubation, the solution was dialyzed extensively against PBS, using dialysis tubes with a cellulose membrane with a cutoff value of 14 kDa, to remove MeOH and remaining unbound \u003cem\u003em\u003c/em\u003e-THPC.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental apparatus\u003c/h2\u003e \u003cp\u003eCary 100 UV-Vis two-beam spectrometer (Agilent) was used to record UV-Vis absorption spectra in the range from 800 to 200 nm with 1 nm step for these measurements, quartz cuvettes with optical paths of 10 mm, 2 mm, and 1 mm were employed. Fluorescence and excitation spectra were evaluated using a JASCO FP-8550 spectrofluorometer.\u003c/p\u003e \u003cp\u003eFluorescence spectra were recorded in the range of 600\u0026minus;760 nm for diluted solutions with an absorbance at excitation wavelength (λ\u003csub\u003eexc\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;416 nm) lower than 0.1. A quartz cuvette with an optical path of 10 mm was used for the study. Fluorescence quantum yield (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{x})\\)\u003c/span\u003e\u003c/span\u003e was determined using a standard substance. TPP has been used as standard in experiments to determine fluorescence quantum yield [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The following equation have been applied to determine the quantum yield of fluorescence:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${\\varphi }_{f}^{x}=\\frac{{S}_{x}{A}_{st}^{\\lambda }{n}_{x}^{2}}{{S}_{st}{A}_{x}^{\\lambda }{n}_{st}^{2}}{\\varphi }_{f}^{st}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{x}\\)\u003c/span\u003e\u003c/span\u003e- fluorescence quantum yield of a sample x; S\u003csub\u003ex\u003c/sub\u003e, S\u003csub\u003est\u003c/sub\u003e- integrated fluorescence intensity (area under the spectrum) for sample x and standard sample, respectively; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({A}_{x}^{\\lambda }, {A}_{st}^{\\lambda }\\)\u003c/span\u003e\u003c/span\u003e \u0026ndash; absorbance at the excitation wavelength (λ) for sample x and standard sample, respectively; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{st}\\)\u003c/span\u003e\u003c/span\u003e- fluorescence quantum yield of the standard sample (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{st}\\)\u003c/span\u003e\u003c/span\u003e= 0.15 in ethanol); n\u003csub\u003ex\u003c/sub\u003e, n\u003csub\u003est\u003c/sub\u003e \u0026ndash; refractive index for sample x solvent\u0026rsquo;s and standard sample solvent\u0026rsquo;s, respectively [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe fluorescence lifetimes were measured on a Fluorescence Lifetime Spectrometer (FluoTime 300 from PicoQuant) with a detection system based on time-correlated single-photon counting (TCSPC). The emission decay lifetimes were acquired using 405 nm diode laser as the excitation source. In addition, an instrument response function (IRF) was obtained using Ludox solution (colloidal silica). Quantum yields of singlet oxygen production (Ф\u003csub\u003eΔ\u003c/sub\u003e) generated by \u003cem\u003em\u003c/em\u003e-THPC were calculated using the results of steady-state measurements. Measurements were recorded on the PicoQuant FluoTime 300 fluorescence lifetime spectrometer with a Hamamatsu H10330B-45 NIR-PMT module, which is sensitive in the 900 to 1450 nm NIR range with excitation using picosecond laser diode (LDH-640, λ\u003csub\u003eexc\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;640 nm, PicoQuant) using methylene blue (MB) as standard (Φ\u003csub\u003eΔ\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.52, λ\u003csub\u003eexc\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;640 nm) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Air-equilibrated solutions of the \u003cem\u003em\u003c/em\u003e-THPC were optically matched at the excitation wavelength (640 nm) to a standard reference solution. The total area under the emission spectrum was calculated separately for each substance. Finally, the quantum yield of singlet oxygen (Φ\u003csub\u003eΔ\u003c/sub\u003e) was calculated by comparing the total area under the emission spectrum of \u003cem\u003em\u003c/em\u003e-THPC and MB. For time-resolved measurements the samples were using a high repetition rate 40 MHz picosecond laser diode (LDH-640 nm, PicoQuant). The decay traces at λ\u0026thinsp;=\u0026thinsp;1270 nm were collected using a so-called \u0026ldquo;burst mode\u0026rdquo;, in which the sample is first excited using multiple pulses of the laser to build up the population of singlet oxygen and then left to decay in the 60 \u0026micro;s time window.\u003c/p\u003e \u003cp\u003eFemtosecond transient absorption measurements were carried out using a Solstice Ti:sapphire regenerative amplifier from Spectra Physics and an optical detection system provided by Ultrafast Systems (Helios) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The 800 nm laser beam was split into two: pump (95%) and probe (5%). The pump beam was directed to a Light Conversion TOPAS-Prime automatic optical parametric amplifier to obtain the desired excitation wavelength in the 290\u0026ndash;2600 nm range. The probe beam was directed to a TA pump-probe Helios spectrometer from Ultrafast Systems LLC with an optical delay line allowing delays of up to 3 ns between pump and probe. A white-light continuum was used for transient detection, which was generated from 5% of the primary beam by passing it through a sapphire or calcium fluoride crystal.\u003c/p\u003e \u003cp\u003eThe laser flash photolysis (LFP) setup employed in this study utilizes a tunable high-end Optical Parametric Oscillator (GWU, model primoScan, Germany) pumped by the third harmonic (355 nm) of a Nd:YAG laser (Quantel, model Q-smart 450, USA), with a pulse duration of 6\u0026ndash;8 ns to excite the samples (typically at an excitation wavelength of 420 nm). The excitation pulse energy was maintained at approximately 3 mJ/pulse to prevent undesired multiphotonic processes. The monitoring beam of the system includes a 150 W pulsed Xe lamp (Hamamatsu, model E7536, Japan), a single monochromator (Princeton Instruments, model Spectra Pro SP-2155, USA), and a photomultiplier (Hamamatsu, model R955, Japan) powered by a PS-310 power supply (Stanford Research System, USA). Data processing was carried out in real time using a digital oscilloscope (Tektronix, model MDO 3024, 350 MHz, USA), which was triggered by a fast photodiode (Thorlabs, model DET10M, USA). The oscilloscope data were transferred to a PC with custom software based on LabView 8.0 (National Instruments, Austin, TX, USA), which controlled the timing and acquisition functions of the system. Kinetic traces were recorded at intervals of 10 nm between 300 and 700 nm. To deoxygenate the sample solutions, high-purity argon was bubbled through them for at least 20 minutes prior to measurements. All experiments were conducted in 1 cm \u0026times; 1 cm quartz cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Aggregation of \u003cem\u003em\u003c/em\u003e-THPC\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAbsorption and fluorescence measurements, along with time-resolved spectroscopy, were employed in this study to explore how aggregation influences the photophysical characteristics of the ground state and excited states (singlet and triplet) of \u003cem\u003em\u003c/em\u003e-THPC. Various range of parameters is available to induce aggregation on \u003cem\u003em\u003c/em\u003e-THPC but in our study we have selected solvent-mixtures due to relevance to the biological environment. Aggregation of \u003cem\u003em\u003c/em\u003e-THPC was studied spectrophotometrically over a range of methanol-buffer solutions (PBS) (0\u0026ndash;95%) and it was of interest to determine at what water content aggregation occurs. Absorption spectra of \u003cem\u003em\u003c/em\u003e-THPC in different MeOH-PBS mixtures were recorded at constant concentrations of \u003cem\u003em\u003c/em\u003e-THPC (4 \u0026micro;M). In the absence of PBS, the spectrum is typical of a chlorine-type molecule [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and is characterized by a strong absorbance with a maximum at 650 nm (ε\u0026thinsp;=\u0026thinsp;5.3\u0026times;10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e) and a broad Soret band with a maximum at 416 nm (ε\u0026thinsp;=\u0026thinsp;2.1\u0026times;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In methanol, Beer-Lambert absorbance plots as a function of dye concentration were linear in the range up to 150 \u0026micro;M indicating it remains in a monomeric form even at higher concentration (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The band width at half height (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e) for the Soret band was 33 nm at 416 nm over the entire concentration range studied. Based on the above observations it can be concluded that the same species, i.e. the monomer, is present throughout.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs the proportion of PBS increases in MeOH-PBS mixtures, a broadening of all absorption bands is observed with a strong decrease in their intensities, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The apparent molar absorbance coefficient of the Soret band fell markedly at high water content as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, where all spectra shown refers to 4 \u0026micro;M solutions. The λ\u003csub\u003emax\u003c/sub\u003e of the Soret band did not shift from 10% water to 60% water: above 70% of water it became concentration dependent and varied with solvent composition. \u003cem\u003eW\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e also remained almost constant up to 60% water, but then increased markedly (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e: 70% \u0026minus;\u0026thinsp;50 nm, \u003cem\u003eW\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e: 95% \u0026minus;\u0026thinsp;53 nm). These data indicate that aggregation becomes significant for 4 \u0026micro;M solutions of \u003cem\u003em\u003c/em\u003e-THPC in aqueous methanol when the water content exceeds 70%. The changes in the aggregation state were graphically monitored through a plot of methanol content versus absorbance at 416 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). With a fixed concentration of \u003cem\u003em\u003c/em\u003e-THPC, an increase in PBS concentration was accompanied by a pronounced decrease in absorbance at 416 nm, particularly noticeable at 70% of PBS. The plot of absorbance at 416 nm against methanol content revealed that the absorbance remained relatively constant within the 100% to 40% methanol range, followed by a significant decrease in 30% methanol. Subsequent addition of PBS was accompanied by a slight decrease in the absorbance at the Soret band. These changes in the UV-Vis spectra were also reflected in the distinct color change of the \u003cem\u003em\u003c/em\u003e-THPC solution when the PBS content exceeded 70% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A complication exists in the behavior of the more concentrated solutions with high water content: for MeOH content lower than 30%, a clear negative deviation from Beer-Lambert's law, attributed to the aggregation of \u003cem\u003em\u003c/em\u003e-THPC, is observed (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Moreover, it was noticed that solutions containing aggregates are unstable, as evidenced by a decrease in their absorbance to one-fifth of the initial value, after 48 hours of room-temperature storage (Fig. S2). In the presence of PBS alone, the absorbance associated with THPC is not discernible on UV-vis spectra due to its low concentration.\u003c/p\u003e \u003cp\u003eOur data indicate that aggregation becomes significant for 4 \u0026micro;M solutions of \u003cem\u003em\u003c/em\u003e-THPC in aqueous methanol when the water content exceeds 70%. When porphyrins aggregate, they form a supramolecular structure which exhibits unique chemical and photophysical properties, including changes in their electronic absorption spectra. For H-type aggregates, the spectral shift is hypsochromic, meaning there is a shift towards a shorter wavelength, often perceived as a blue shift, compared to the spectrum of an individual monomer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This shift results from the monomers arranging themselves in a stacked, face-to-face orientation. Conversely, J-type aggregates display a batochromic shift in their Soret band, known as a red shift, which indicates a longer wavelength compared to the monomer spectrum. This type of aggregation occurs when porphyrin monomers line up in a head-to-tail configuration [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Due to the broadening of the peak and an indistinct spectral shift, it was challenging to determine the type of aggregates formed at high PBS concentrations. Therefore, a deconvolution of the absorption spectra was performed for water concentrations ranging from 70\u0026ndash;95%, where aggregation is appreciably observed by spectrophotometry. Some examples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, which enabled the detection of monomers and two distinct aggregate species: an H-dimer (H) with a broad absorption peak around 402 nm, and a J-dimer (J) that absorbs around 434 nm. Considering that the molar absorption coefficient of the monomer remains constant regardless of the solvent composition, it was estimated from the deconvolution procedure that the concentration of the monomer decreases from 4 \u0026micro;M in pure methanol to 1.5 \u0026micro;M in 30% methanol, and further to 0.5 \u0026micro;M in 5% methanol.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBased on the Lambert-Beer additivity law, spectrum deconvolution, and the total concentration of \u003cem\u003em-\u003c/em\u003eTHPC in the sample, the proportions of monomer and aggregates (H and J) in solutions containing 30% methanol and 5% methanol were determined (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This analysis reveals that the monomer constitutes only 12.5% of the 5% methanol solution, with the H-type aggregate being the predominant species, accounting for 70%.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConcentration and percentage contribution of the \u003cem\u003em\u003c/em\u003e-THPC monomer and aggregates in high water content.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethanol content %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonomer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJ-aggregate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-aggregate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5 \u0026micro;M (37.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.7 \u0026micro;M (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8 \u0026micro;M (45%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 \u0026micro;M (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.7 \u0026micro;M (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.8 \u0026micro;M (70%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn methanol, when excited at 427 nm, \u003cem\u003em\u003c/em\u003e-THPC exhibited a fluorescence spectrum spanning from 620 to 680 nm, featuring a single emission band with a peak at 650 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the literature the emission quantum yield of \u003cem\u003em\u003c/em\u003e-THPC varies significantly in different solvent, from 0.089 in EtOH [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] to 0.441 in DMF [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The fluorescence quantum yield was determined by the method described by Williams et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which involves the use of well characterized standard samples with known fluorescence quantum yield (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{})\\)\u003c/span\u003e\u003c/span\u003e values and a measurement of emission spectra for a series of concentrations of \u003cem\u003em\u003c/em\u003e-THPC. The fluorescence quantum yields (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{}\\)\u003c/span\u003e\u003c/span\u003e) also varied with changes in solvent composition. Under air-saturated conditions, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{}\\)\u003c/span\u003e\u003c/span\u003e decreased from 0.41 in pure methanol to 0.20 in 30% methanol. It was found that \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\varphi }_{f}^{}\\)\u003c/span\u003e\u003c/span\u003e for 30% MeOH in PBS was dependent on the excitation wavelength (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) which further confirms that at this solvent composition mixture \u003cem\u003em\u003c/em\u003e-THPC exists in various states: monomer and aggregates. This is in agreement with the results described by UV-Vis data analyses. The highest quantum yield of fluorescence was obtained with the excitation at 416 nm where the absorbance from the monomeric form was the most profound. Moreover, the fluorescence excitation spectrum recorded for the \u003cem\u003em\u003c/em\u003e-THPC solution of 30% MeOH in PBS matched the absorption spectrum of monomeric \u003cem\u003em\u003c/em\u003e-THPC (Fig. S3). These results discussed above, demonstrate that monomeric \u003cem\u003em\u003c/em\u003e-THPC is an emissive species and that the observed fluorescence in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA originates solely from monomeric \u003cem\u003em\u003c/em\u003e-THPC present in various solvent composition mixtures. For mixtures containing less than 30% methanol, the fluorescence intensity was substantially lowered up to only 0.02 in 5% MeOH in PBS. This is in agreement with the decrease of the content of them monomeric form in the solution with 5% MeOH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fluorescence lifetime of \u003cem\u003em\u003c/em\u003e-THPC was determined using the time-correlated single photon counting (TCSPC) technique. For \u003cem\u003em\u003c/em\u003e-THPC in methanol, the calculated fluorescence lifetime from the monoexponential emission decay was 8.4 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Increasing the percentage of PBS in the mixture did not significantly alter the emission lifetimes, further confirming that the emissions occur exclusively from the monomeric form of \u003cem\u003em\u003c/em\u003e-THPC. The absence of detectable fluorescence from \u003cem\u003em\u003c/em\u003e-THPC aggregates is consistent with their propensity to undergo fast non-radiative decay processes [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo check the behavior of \u003cem\u003em-\u003c/em\u003eTHPC in the triplet state, nanosecond transient absorption experiments were performed. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows the transient absorption difference spectra of a solution of \u003cem\u003em\u003c/em\u003e-THPC in argon-saturated MeOH following excitation with the OPO laser at 420 nm. There are three negative peaks with maximum at 410 nm that correspond to the bleach of the Soret band and peaks at 520 nm and 650 nm that matches the position of the Q bands in UV-Vis ground state spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Moreover, the band with positive signals in the range of 300\u0026ndash;390 nm, 430\u0026ndash;460 nm, and low intensity band within the range 660\u0026ndash;750 nm were observed on the transient spectra, which were attributed to the \u003cem\u003em\u003c/em\u003e-THPC triplet. The spectra did not show any shape evolution in time and all kinetic profile decayed to zero after 500 \u0026micro;s. The triplet lifetime for \u003cem\u003em\u003c/em\u003e-THPC was obtained from monoexponential fit of the bleach recovery at 410 nm and transient decay 450 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Both kinetic traces were characterized by the same time constant within experimental error. \u003cem\u003em\u003c/em\u003e-THPC triplet lifetime in MeOH was found to be 88 \u0026micro;s for argon-saturated solutions. The follow up experiment involved studying the kinetics of the quenching of the \u003cem\u003em\u003c/em\u003e-THPC triplet state in MeOH under two different conditions: in the presence of oxygen (which represents standard atmospheric conditions) and in an oxygen-free atmosphere. The goal was to understand the effect of oxygen on the triplet state quenching process and possible changes in the reaction mechanism. The triplet lifetime of \u003cem\u003em\u003c/em\u003e-THPC was strongly reduced in the presence of oxygen under ambient conditions (190 ns) (Fig S4). The bimolecular quenching rate constants of \u003cem\u003em\u003c/em\u003e-THPC by oxygen (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eq\u003c/sub\u003e) in air-saturated solutions were obtained from Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e):\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\frac{{\\tau }_{o}}{\\tau }=1+{k}_{q}{\\tau }_{o}\\left[{O}_{2}\\right]$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\tau }_{o}\\)\u003c/span\u003e\u003c/span\u003e is the lifetime of triplet states in degassed solutions and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\tau\\)\u003c/span\u003e\u003c/span\u003e is the lifetime of triplet state in air-saturated solutions. Assuming [O\u003csub\u003e2\u003c/sub\u003e] concentration is 9.4 mM (in MeOH) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] we calculated \u003cem\u003ek\u003c/em\u003e\u003csub\u003eq\u003c/sub\u003e based on Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The bimolecular rate constant \u003cem\u003ek\u003c/em\u003e\u003csub\u003eq\u003c/sub\u003e for the triplet state of \u003cem\u003em\u003c/em\u003e-THPC quenching by oxygen was found to be 5.6\u0026times;10\u003csup\u003e8\u003c/sup\u003e M\u003csup\u003e\u0026minus;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt higher methanol contents, the transient absorption spectrum closely resembled that observed in pure methanol, showing an absorption maximum at 450 nm and a bleach at 410 nm, 520 nm, and 650 nm. For different methanol-PBS mixtures, decay profiles at 410 nm and 450 nm for \u003cem\u003em\u003c/em\u003e-THPC registered at constant absorbance at the excitation wavelength were compared (Fig. S5). Up to 50% methanol, the signal intensity at 410 nm and 450 nm remained almost unchanged. The magnitude of triplet state absorption at 450 nm decreased significantly between samples with 50% and 30% methanol (absorbance at the excitation wavelength was kept constant), although the spectral shape remained unaltered. For the mixture with 30% methanol, the transient absorption signal at 410 nm and 450 nm decreased by half compared to pure methanol, without noticeable changes in transient absorption spectra and kinetic profiles (Fig. S6). This suggests that the decrease in absorbance at 450 nm indicates a higher contribution of aggregates in the solution at higher methanol percentages, as the monomer is the only species capable of forming a triplet state. The observation that the triplet state signal decreased by a factor of two at 30% methanol aligns with a similar decrease in fluorescence quantum yield for the same solvent composition. A complete disappearance of the signal was observed between 30% and 20% methanol, indicating a substantial increase in aggregate concentration (Fig. S6). Negligible signal was observed at either 410 nm or 450 nm probe wavelengths, which would be expected if the triplet state of \u003cem\u003em\u003c/em\u003e-THPC had formed. Therefore, given the detection limit of the apparatus, it can be stated the triplet state of \u003cem\u003em\u003c/em\u003e-THPC was not detectable at methanol concentrations of 20% or less. This unambiguously demonstrates that intersystem crossing in \u003cem\u003em\u003c/em\u003e-THPC aggregates is suppressed, completely preventing triplet state formation. The absence of a detectable triplet state indicates that the formation of \u003cem\u003em\u003c/em\u003e-THPC aggregates suppresses the generation of singlet oxygen (\u003cem\u003evide infra\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the photophysical properties of \u003cem\u003em\u003c/em\u003e-THPC in MeOH and \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003csub\u003eabs\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[nm]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eε\u003c/p\u003e \u003cp\u003e[M\u003csup\u003e\u0026minus;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΦ\u003csub\u003eFl\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eτ\u003csub\u003eS\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[ns]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eτ\u003csub\u003eT (argon)\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[\u0026micro;s]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eτ\u003csub\u003eT(air)\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[\u0026micro;s]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ek\u003csub\u003eq\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[M\u003csup\u003e\u0026minus;1\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eΦ\u003csub\u003e∆\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003em\u003c/em\u003e-THPC\u003c/p\u003e \u003cp\u003ein MeOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e416\u003c/p\u003e \u003cp\u003e650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026times;10\u003csup\u003e5\u003c/sup\u003e 5.3\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c8\"\u003e \u003cp\u003e5.6\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e420\u003c/p\u003e \u003cp\u003e652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.4\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.9 (35%)\u003c/p\u003e \u003cp\u003e10.1 (65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c8\"\u003e \u003cp\u003e5.7\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ea\u003c/sup\u003e determined with excitation λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;427\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ea\u003c/sup\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo fully understand the excited state dynamics of the \u003cem\u003em\u003c/em\u003e-THPC, ultrafast spectroscopy experiments were conducted on \u003cem\u003em\u003c/em\u003e-THPC monomers in methanol and \u003cem\u003em\u003c/em\u003e-THPC aggregates in 5% MeOH. Immediately after the excitation of \u003cem\u003em\u003c/em\u003e-THPC in MeOH with a 420 nm laser pulse, the transient absorption spectra revealed a prominent and wide bleach centered around 650 nm. It was interesting to see that in the excited state of \u003cem\u003em\u003c/em\u003e-THPC shows structured absorption which consists of absorption peaks centered at 490, 530, 570, 620, and 680 nm which could be attributed to the presence of the singlet excited state (S\u003csub\u003e1\u003c/sub\u003e-S\u003csub\u003en\u003c/sub\u003e transition) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To further probe the relaxation dynamics of the \u003cem\u003em\u003c/em\u003e-THPC monomer, we monitored the transient decay kinetics. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The excitation wavelength 420 nm used in the present work corresponds to a transition from S\u003csub\u003e0\u003c/sub\u003e to a vibrationally excited level of the S\u003csub\u003e2\u003c/sub\u003e state of chlorins. It is well-known that the S\u003csub\u003e2\u003c/sub\u003e \u0026rarr; S\u003csub\u003e1\u003c/sub\u003e internal conversion (IC) in tetrapyrroles is generally very fast (on the order of a few hundreds of femtoseconds) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The analysis of transient absorbance kinetics at 490 nm at short delays revealed a fast component with time constant of only 8.0 ps, which can be presumably attributed to vibrational cooling inside the Q-band. Unfortunately, the singlet excited state lifetime of \u003cem\u003em\u003c/em\u003e-THPC in methanol could not be determined due to the experiment's short time window of 3 ns, consistent with TCSPC findings that recorded a lifetime of 8.5 ns. Notably, throughout the experiment's time window, the decay of the singlet excited state signal was not accompanied by any distinct spectral evolution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFemtosecond time-resolved transient absorption spectroscopy is valuable for studying exciton dynamics in aggregates. We explored excited-state dynamics of \u003cem\u003em\u003c/em\u003e-THPC aggregates and compared them with those of the monomeric dye to elucidate the photophysical changes induced by aggregation. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC shows the transient absorption spectra of aggregates at different time delays after 420 nm laser excitation. The transient spectra show a bleach peaks at 525 nm, 550 nm, 595 nm and 650 nm. It is interesting to see that the bleach peaks appear exactly at the positions of Soret- and Q-exciton bands in steady state optical absorption spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eA direct comparison of the aggregate TA spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) with that of the monomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) shows that although the spectral shape similarity the decay of the aggregate is much faster than that of the monomer. The faster dynamics is most likely due to new nonradiative energy relaxation channels that become effective upon aggregation. This observation is consistent with both steady-state and time-resolved fluorescence studies reported by various authors, which note a significant reduction in fluorescence quantum yield and excited-state lifetime upon aggregation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To study exciton relaxation at different wavelengths in the entire spectral region we have monitored the transient decay kinetics at 480, 575, 650 nm and 670 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Fig. S7). The kinetic data can be fitted multiexponentially with: τ\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.8 ps (59%), τ\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;26.8 ps (21%), τ\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;204.8 ps (20%), at 480 nm, τ\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.8 ps (21%), τ\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;37.0 ps (41%), τ\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;302.0 ps (38%), at 650 nm; τ\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;46.0 ps (21%), τ\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;207.9 ps (40%), τ\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1034 ps (39%), at 670 nm and finally τ\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.0 ps (61%), τ\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;63.8 ps (40%) at 575 nm. Such fast dynamics is in accordance with literature where it is reported that photoexcited aggregated tetrapyrroles undergo exciton relaxation dynamics at an ultrashort time scale [\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Complex decay kinetics can reflect presence of various aggregate types in the solution. However, it can be assumed, following Kano and Kobayashi [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], that the time constants of around 4 ps and few tens of ps are attributed to intra-aggregate and aggregate-solvent vibrational energy redistribution, respectively; and around 200\u0026ndash;300 ps component is attributed to S\u003csub\u003e1\u003c/sub\u003e lifetime. No residual absorbance appeared at longer time delays in any of the recorded positive traces, further supporting the absence of an excited triplet state in aggregated \u003cem\u003em\u003c/em\u003e-THPC, as already demonstrated in nanosecond flash photolysis experiments. However, the presence of residual bleach absorbance at 650 nm suggests photoinstability of the aggregates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Complexation to bovine serum albumin\u003c/h2\u003e \u003cp\u003eIn respect to PDT applications, our findings indicate that \u003cem\u003em\u003c/em\u003e-THPC is predominantly monomeric in methanol. However, at high PBS concentration at physiological pH, it aggregates and exhibits very low solubility. Considering the use of \u003cem\u003em\u003c/em\u003e-THPC as a photosensitizing drug, we focused on systems that were fully aqueous. Our objective was to determine whether binding \u003cem\u003em\u003c/em\u003e-THPC to BSA could lead to disaggregation and restore the photophysical properties of its monomeric form. The influence of BSA on the photophysics of \u003cem\u003em\u003c/em\u003e-THPC in aqueous solutions was studied using the same techniques previously applied to examine aggregation in methanol-PBS mixtures. Here we synthesized a \u003cem\u003em\u003c/em\u003e-THPC@BSA complex, with a well-defined 1:1 stoichiometry, using a PBS/MeOH mixed solvent system, with overnight incubation and purification by dialysis (removal of MeOH and unbound \u003cem\u003em\u003c/em\u003e-THPC) (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe UV-Vis spectrum of \u003cem\u003em\u003c/em\u003e-THPC@BSA in pure PBS showed that \u003cem\u003em\u003c/em\u003e-THPC was successfully bound to BSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The spectrum strongly resembles the spectrum for the \u003cem\u003em\u003c/em\u003e-THPC monomer in MeOH and small shift in the Soret band can be related to the solvent effect or the contribution of the aggregated \u003cem\u003em\u003c/em\u003e-THPC still presence in the solution. The band width at half height (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e) for the Soret band was 39 nm at 420 nm, which is only slightly broader than the Soret band of \u003cem\u003em\u003c/em\u003e-THPC in MeOH. The solution of \u003cem\u003em\u003c/em\u003e-THPC@BSA is stable in time, as no decrease in absorbance was observed over a 96-hour period (Fig. S8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, emission studies were conducted to probe the excited state properties of \u003cem\u003em\u003c/em\u003e-THPC@BSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The fluorescence emission spectrum of \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS exhibits a peak at 653 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The fluorescence quantum yield of \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS was determined to be 0.2 at λ\u003csub\u003eexc\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;427 nm, which is half that of the purely monomeric form in MeOH but notably higher than the fluorescence quantum yield observed at high PBS concentrations in the absence of BSA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The excitation spectra of \u003cem\u003em\u003c/em\u003e-THPC@BSA perfectly align with the absorption spectra of the monomeric form of \u003cem\u003em\u003c/em\u003e-THPC, with an absorption peak at 415 nm (Fig. S9). This observation further confirm successful deaggregation of \u003cem\u003em\u003c/em\u003e-THPC upon binding to BSA. The emission lifetime for \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS is biexponential with the time constants of 10.05 ns and 3.9 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSubsequently ultrafast spectroscopy experiments were performed on \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS. Right after the \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS was excited with a 420 nm laser pulse, the transient absorption spectra displayed a pronounced bleach centered around 650 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Notably, the excited state of \u003cem\u003em\u003c/em\u003e-THPC exhibited structured absorption, featuring peaks at 480, 530, 570, 620, and 680 nm, likely indicative of the singlet excited state.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eImportantly, a direct comparison of the \u003cem\u003em\u003c/em\u003e-THPC@BSA TA spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,B) with that of the monomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B) reveals that both the shapes and the dynamics of the spectra are very similar. The transient signals observed at 480 nm and 650 nm exhibit only partial decay within the experimental timeframe of 3 ns, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB. This observation aligns with the extended fluorescence lifetimes of 3.9 ns and 10.1 ns, respectively, determined using the TCSPC technique. To probe triplet excited state properties of \u003cem\u003em\u003c/em\u003e-THPC bounded to BSA nanosecond absorption spectra were recorded after laser excitation at 420 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The transient absorption spectrum of \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS includes bands from photobleaching at 420 nm and 650 nm. The triplet absorption maximum occurs at 460 nm. The spectrum of the complex is very similar to that of the monomeric form (Fig .4A). Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB shows the effect of BSA addition on the decay profile of \u003cem\u003em\u003c/em\u003e-THPC triplet states in argon saturated solutions. For the dynamic behavior of \u003cem\u003em\u003c/em\u003e-THPC triplet states the biexponential decay is observed which can also be associated with the presence of different BSA conformers in solution. Under conditions in which it can be considered that all the dye is bound to the protein the transient lifetimes values were found to be 444 \u0026micro;s in argon-saturated solutions. Much longer triplet lifetime compared to \u003cem\u003em\u003c/em\u003e-THPC in MeOH can be explained in terms of suppressed self-quenching of the \u003cem\u003em\u003c/em\u003e-THPC bound to BSA.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder aerobic conditions, both bleaching and triplet transient signals were shortened (Fig. S10). The influence of the protein nanoenvironment on the interaction of triplet excited state of THPC@BSA with dissolved molecular oxygen O\u003csub\u003e2\u003c/sub\u003e can be evaluated by applying Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Considering a homogenous distribution of O\u003csub\u003e2\u003c/sub\u003e concentration in air-saturated protein solutions, i.e. [O\u003csub\u003e2\u003c/sub\u003e]\u0026thinsp;=\u0026thinsp;1.22 mM [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and decrease of the triplet excited state to 14 \u0026micro;s the quenching rate constant of triplet excited state of THPC@BSA by O\u003csub\u003e2\u003c/sub\u003e was found to be 5.7\u0026times;10\u003csup\u003e7\u003c/sup\u003e M\u003csup\u003e\u0026minus;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This value is one order of magnitude smaller in comparison to the quenching rate constant of triplet excited state of \u003cem\u003em\u003c/em\u003e-THPC by O\u003csub\u003e2\u003c/sub\u003e in MeOH. This phenomenon has been observed previously for other dyes bound to protein [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The local rigidity of the binding site of \u003cem\u003em\u003c/em\u003e-THPC can impose a barrier to the free diffusion of O\u003csub\u003e2\u003c/sub\u003e from the bulk buffer. Many dye molecules can be accommodated on a single protein in two principal binding sites (at the interface and deeper within the protein structure, away from the interface) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Comparison of the oxygen quenching rate constant by \u003cem\u003em\u003c/em\u003e-THPC in MeOH and \u003cem\u003em\u003c/em\u003e-THPC@BSA in PBS, values given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the extent of protection given to the triplet state of \u003cem\u003em\u003c/em\u003e-THPC by binding to the protein. At molar ratios 1:1 of BSA to THPC most of the dye resides at a site that provides restricted quenching by O\u003csub\u003e2\u003c/sub\u003e and must be located deeper to the interface. Taking into account that reactivity with oxygen and formation of the singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) is crucial for the potential application of the as prepared THPC@BSA toward PDT it was of importance to verify whether sterically hindered THPC still enables formation of the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIt was found that the quenching of triplet excited state of \u003cem\u003em\u003c/em\u003e-THPC bound to BSA by O\u003csub\u003e2\u003c/sub\u003e generates singlet molecular oxygen \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, as directly detected by the transient luminescence at 1270 nm after laser excitation of the dye at 640 nm in air-saturated deuterated buffer solutions, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAs compared with the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e luminescence signal obtained for the reference methylene blue (Φ\u003csub\u003e∆\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.52 ) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It was calculated that the quantum yields of singlet oxygen generation was 0.21 \u003cem\u003em\u003c/em\u003e-THPC bound to BSA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). High value of singlet oxygen quantum yield for \u003cem\u003em\u003c/em\u003e-THPC bound to BSA form suggest that it might find application in PDT. It was also demonstrated that the \u003cem\u003em\u003c/em\u003e-THPC in 95% of PBS in the absence of BSA does not generate \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). For comparison the quantum yields of singlet oxygen generation for \u003cem\u003em\u003c/em\u003e-THPC in MeOH was reported to be 0.42 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB displays \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e phosphorescence profile at 1270 nm, which was obtained following 640 nm diode laser excitation of \u003cem\u003em\u003c/em\u003e-THPC@BSA in deuterated buffer solutions. The \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e lifetime was reduced to 38 \u0026micro;s in comparison to 68 \u0026micro;s lifetime of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e in D\u003csub\u003e2\u003c/sub\u003eO reported in the literature. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Shortening of the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e lifetime in the presence of BSA indicates dynamic quenching of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e by BSA, which is a known phenomena [53,54]. The singlet oxygen molecules produced by \u003cem\u003em\u003c/em\u003e-THPC@BSA complex will diffuse through the surrounding medium. The radial distance traveled by \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e during a travel time \u003cem\u003et\u003c/em\u003e can be approximately calculated as \u003cem\u003ed = (6Dt)\u003c/em\u003e\u003csup\u003e\u003cem\u003e1/2\u003c/em\u003e\u003c/sup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] where \u003cem\u003eD\u003c/em\u003e is the diffusion coefficient of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e in the media. Given that D\u0026thinsp;=\u0026thinsp;2 \u0026times; 10 \u003csup\u003e\u0026minus;5\u003c/sup\u003e cm\u003csup\u003e2\u003c/sup\u003e/s in water at room temperature[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] it can be estimated that \u003cem\u003ed\u003c/em\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;600 nm, a distance several orders of magnitude larger that the protein size. This allows the free diffusion of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generated into a protein that bears a \u003cem\u003em\u003c/em\u003e-THPC molecule to the bulk buffer and its interaction during its lifetime with target structures in cells.\u003c/p\u003e \u003cp\u003eOur results show that \u003cem\u003em\u003c/em\u003e-THPC undergo marked changes in photophysical properties upon binding to BSA in comparison to \u003cem\u003em\u003c/em\u003e-THPC in high water content in. This is due to the ability of the protein to monomerize aggregates of these dyes present in aqueous solution. These results underscore the potential of BSA to preserve the monomeric form of \u003cem\u003em\u003c/em\u003e-THPC, mitigating aggregation-induced losses in singlet oxygen production. Our findings suggest that BSA-mediated delivery systems could play a crucial role in optimizing the clinical utility of hydrophobic photosensitizers like \u003cem\u003em\u003c/em\u003e-THPC.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eComprehensive spectroscopic analyses, including UV-Vis absorption, fluorescence spectroscopy, and laser flash photolysis, provided in-depth insights into the photophysical properties of \u003cem\u003em\u003c/em\u003e-THPC both in its aggregated form and when complexed with BSA. The research underscores the significant impact of environmental factors, such as solvent composition and water content, on the aggregation behavior of \u003cem\u003em\u003c/em\u003e-THPC. It highlights the importance of understanding these effects to develop more effective PDT drugs and delivery systems. The study conclusively demonstrates that BSA effectively mitigates the aggregation issues of \u003cem\u003em\u003c/em\u003e-THPC, a crucial photosensitizer in PDT. The \u003cem\u003em\u003c/em\u003e-THPC@BSA complex exhibited significantly enhanced photophysical properties compared to aggregated \u003cem\u003em\u003c/em\u003e-THPC. This includes a higher quantum yield of fluorescence and a longer triplet state lifetime, indicative of the complex's ability to generate singlet oxygen more efficiently. The restoration of these properties in a physiological environment highlights the potential of BSA to serve as an effective delivery vehicle for \u003cem\u003em\u003c/em\u003e-THPC in PDT. These conclusions emphasize the significance of the study in improving the understanding and application of \u003cem\u003em\u003c/em\u003e-THPC in photodynamic therapy, highlighting the potential benefits of using BSA for enhanced drug delivery and treatment efficacy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank prof. Marek Sikorski for providing access to his OPO LFP setup. A.K thanks for funding to program under \u003cstrong\u003ethe project at AMU \u0026quot;Initiative of Excellence - Research University\u0026quot; (proposal no.\u003c/strong\u003e 054/13/SNŚ/0024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.Kolman:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; original draft, Visualization, Methodology, Investigation, \u003cstrong\u003eT. Pedzinski: \u0026nbsp;\u003c/strong\u003eMethodology, Writing \u0026ndash; review \u0026amp; editing\u003cstrong\u003e\u0026nbsp;A. Lewandowska-Andralojc:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing, Validation, Supervision, Methodology, Conceptualization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eT. Reuters, The Changing Role of ChemisTRy in DRug DisCoveRy, (n.d.).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.D. Klein, H. Walt, C. Richter, Photosensitization of isolated rat liver mitochondria by tetra(m- hydroxyphenyl)chlorin, Arch. Biochem. Biophys. 348 (1997) 313\u0026ndash;319.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI.P.J. Van Geel, H. Oppelaar, J.P.A. 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B\u0026auml;umler, Time-resolved investigations of singlet oxygen luminescence in water, in phosphatidylcholine, and in aqueous suspensions of phosphatidylcholine or HT29 cells, Journal of Physical Chemistry B 109 (2005)3041\u0026ndash;3046.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"photodynamic therapy, singlet oxygen, aggregation, time-resolved spectroscopy, chlorin","lastPublishedDoi":"10.21203/rs.3.rs-4564342/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4564342/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMeta-tetra(hydroxyphenyl)chlorin (\u003cem\u003em\u003c/em\u003e-THPC) is among the most potent photosensitizers, known for its high singlet oxygen generation efficiency. However, its clinical effectiveness in photodynamic therapy (PDT) is compromised by its propensity to aggregate in aqueous solutions, adversely affecting its photophysical properties and therapeutic potential. A series of spectroscopic techniques, including UV-Vis absorption, fluorescence spectroscopy, and laser flash photolysis, revealed that \u003cem\u003em\u003c/em\u003e-THPC exhibits significant aggregation, particularly in MeOH-PBS mixtures with MeOH content below 30%. This aggregation adversely affects its photophysical properties leading to reduced fluorescence quantum yield and most importantly reducing its singlet oxygen quantum yield. This study introduces the use of bovine serum albumin (BSA) to counteract the aggregation of \u003cem\u003em\u003c/em\u003e-THPC, aiming to enhance its solubility, stability, and efficacy in physiological settings. Through advanced spectroscopic analyses we demonstrated that the \u003cem\u003em\u003c/em\u003e-THPC@BSA complex exhibits improved photophysical characteristics, essential for effective PDT. Notably, the complex showed a significant restoration of the singlet oxygen quantum yield (Φ\u003csub\u003eΔ\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.21) compared to aggregated \u003cem\u003em\u003c/em\u003e-THPC. These results underscore the potential of BSA to preserve the monomeric form of \u003cem\u003em\u003c/em\u003e-THPC, mitigating aggregation-induced losses in singlet oxygen production. Our findings suggest that BSA-mediated delivery systems could play a crucial role in optimizing the clinical utility of hydrophobic photosensitizers like \u003cem\u003em\u003c/em\u003e-THPC.\u003c/p\u003e","manuscriptTitle":"Spectroscopic Insights into BSA-Mediated Deaggregation of m-THPC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 10:29:41","doi":"10.21203/rs.3.rs-4564342/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-02T04:39:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-28T14:56:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-25T12:29:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54591771896057470520232635938945697677","date":"2024-06-17T11:47:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42178800977105865096668316992271853763","date":"2024-06-17T09:37:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-17T09:27:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-17T09:07:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-13T06:06:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-12T11:14:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-11T13:04:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7769ed68-9345-455f-9cb7-869bd8b0092b","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33713278,"name":"Biological sciences/Chemical biology"},{"id":33713279,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2024-09-30T16:04:36+00:00","versionOfRecord":{"articleIdentity":"rs-4564342","link":"https://doi.org/10.1038/s41598-024-73266-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-27 15:57:45","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-06-27 10:29:41","video":"","vorDoi":"10.1038/s41598-024-73266-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-73266-2","workflowStages":[]},"version":"v1","identity":"rs-4564342","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4564342","identity":"rs-4564342","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}