Photophysical Properties of Coumarin 343 and Structural Changes in Reline–Pluronic Systems With Varying Block Composition

ABSTRACT

Experimental and simulation studies reported in the literature largely addressed the role of water as a secondary hydrogen‐bond donor (HBD) or acceptor (HBA) in reline (a Type III deep eutectic solvent). In this work, the effect of reline acting as a ternary component on Pluronic assemblies with varying hydrophilic–lipophilic balances (P123 and F108) was investigated using coumarin 343 as a fluorescent probe through spectroscopic techniques. Through differential scanning calorimetry studies, a decrease in the critical micelle temperature values of Pluronics upon reline loading was observed. A red shift accompanied by a decrease in fluorescence intensity of negatively charged coumarin 343 (C343⁻) was observed upon reline loading in Pluronic systems, via UV–visible absorption and steady‐state fluorescence studies. Differences in time‐resolved fluorescence emission decay profiles between water‐reline and Pluronic‐reline mixtures confirmed the localization of C343⁻ within the micellar systems. A decrease in the fluorescence anisotropy decay times for P123 and an increase for F108 were observed upon reline loading. Fourier transform infrared spectroscopy, dynamic light scattering, and small‐angle neutron scattering measurements were also carried out to characterize the micellar systems. Ultimately, this work facilitates the rational design of hybrid nanocarriers for advanced biomedical applications.

This graphical abstract illustrates the structural and microenvironmental modulations of Pluronic (P123/F108) micelles upon reline loading. FT‐IR confirms preferential ChCl‐PEO hydrogen bonding while DSC reveals critical micellization temperature's' reduction. C343⁻ probing confirms increased micellar polarity through fluorescence redshifts. Persistent biexponential fluorescence anisotropy decays indicate preserved microheterogeneity even at low reline content.

1. Introduction

Deep eutectic solvents (DESs) have gained considerable attention as tunable and sustainable solvent alternatives to conventional solvents [1]. When a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA) combine, typically in specific molar ratios, the intermolecular strong hydrogen bonding along with charge delocalization lowers the melting point far below that of the individual components, thereby forming deep eutectic solvents [2]. This simple yet versatile design enables the preparation of liquids with highly adaptable physicochemical properties such as viscosity, conductivity, and melting behavior which can be tuned by varying the HBD, HBA, and their molar ratios [3]. The term “deep eutectic solvent” was first coined for Type III systems—typically composed of a quaternary ammonium salt and a neutral HBD—but has since been used more broadly [4]. For example, choline chloride–urea [5] (commonly referred to as “reline”) which is a Type III DES extensively studied owing to its low cost, wide availability, straightforward solvent‐free synthesis, and environmentally benign properties [6]—such as biodegradability, low toxicity, and reduced volatility and flammability compared to conventional volatile organic compounds (VOCs) [7]—makes DESs highly attractive for greener chemical processes. Variations in the HBD structure, such as substituting urea with thiourea or glycerol [8], further modulate solvent organization and viscosity, highlighting the delicate balance of intermolecular interactions in these systems. Furthermore, the bulk properties of the resulting DES depend upon the molecular symmetry and size of the cation [5, 9], akin to the trends observed in ILs. Unlike ionic liquids (ILs), which are composed of discrete anions and cations, particularly imidazolium‐based systems, which suffer from high biotoxicity and poor biodegradability, limiting their scalability and environmental compatibility. DESs arise from eutectic mixtures of Lewis or Brønsted acids and bases and can incorporate both ionic and molecular species [4]. Owing to these advantages, DESs have found applications in various fields, such as biomass processing [10], electrochemistry [11, 12], metal extraction [13, 14], CO₂ capture [15], micellar chemistry [16, 17], and biocatalysis [18, 19]. Despite their versatility, understanding the relationship between DES composition, molecular dynamics, and bulk properties remains challenging.

DESs are known to display dynamic heterogeneity, where the microenvironments experienced by solutes do not always correlate with bulk properties such as viscosity or temperature [20, 21]. Fluorescence probe studies reveal that this decoupling is more pronounced with negatively charged probes, owing to a combination of electrostatic interactions and hydrogen bonding. The structure of the HBD may also influence the extent of microheterogeneity. For example, in ethylene glycol (EG), 1,3‐propanediol (1,3‐PD), and glycerol, variations in the number and arrangement of hydroxyl groups directly impact solvent organization and viscosity, which increases with hydrogen‐bonding complexity (EG: 18 cP < 1,3‐PD: 53 cP < glycerol: 1400 cP) [7]. This pronounced sensitivity to composition highlights the need to investigate how DESs interact with structured amphiphilic systems such as block copolymers.

In this context, reline—a Type III DES composed of choline chloride and urea—offers a particularly interesting case owing to its rich hydrogen‐bonding network and notable drug‐solubilizing capacity. While most previous studies [2, 22, 23, 24, 25] have focused on DES–water mixtures to examine solvation and polarity effects, comparatively little is known about how DESs influence organized amphiphilic assemblies such as Pluronic micelles. To address this gap, we investigated Pluronics (or Poloxamers) a class of amphiphilic macromolecular triblock copolymers composed of a central hydrophobic block of poly(propylene oxide) (PPO) positioned between two hydrophilic blocks of poly(ethylene oxide) (PEO). Their amphiphilic architecture enables them to self‐assembly into micelles, gels, and mesophases depending on concentration, temperature, and solvent environment [26]. This dual hydrophilic–hydrophobic character underlies their widespread use in drug delivery, solubilization, emulsification, and formulation sciences [27]. Incorporating DESs within Pluronics offers an attractive strategy to design hybrid systems that exploit the tunable solvent properties of DESs and the self‐assembly behavior of block copolymers. Such integrations can significantly modulate micellization, solute partitioning, and photophysical properties in a way that is not predictable from either component alone.

Several recent studies have already demonstrated the potential of Pluronic–DES systems for diverse applications. For instance, Pluronic 10R5 and L35 with choline chloride and different HBDs have been employed for the extraction of C‐phycocyanin [28]. Pluronic F127 with a choline chloride–glycerol DES has enabled kaempferol hydrogel formulations for anti‐inflammatory treatment of psoriasis‐like skin inflammation [29]. A broader set of Pluronics and poly(propylene glycol), in combination with choline chloride–lactic acid natural deep eutectic solvents (NADES), were applied to construct biphasic aqueous systems for extracting parthenolide (PAR) [30]. Similarly, a novel ethanolic two‐phase system (ETPS) composed of Pluronic L‐64 and various DESs was reported for the separation of structurally similar flavonoids, naringin (Nar) and neohesperidin (Neo), from pomelo peel [31]. Pluronic F127 with DESs such as D, L‐menthol, and HBDs, including acetic, pyruvic, lauric, and lactic acids, has been investigated in the formulation of high internal phase emulsions (HIPEs) [32]. In another study, aqueous two‐phase systems (ATPS) based on ethylene oxide–propylene oxide (EOPO) copolymers and DESs were developed for the extraction and preliminary purification of polysaccharides from Camellia oleifera seed cake [33]. Furthermore, Pluronic L35, Pluronic 10R5, and UCON, in combination with DESs composed of choline chloride (HBA) and HBDs such as ethylene glycol, 1,2‐propanediol, or glycerol, were employed to form ATPSs for studying the partitioning behavior of penicillin G, highlighting the impact of DES composition on binodal curves and partition coefficients [34].

Building upon this background, the present work focuses on the effect of reline a type III (choline chloride–urea) DES on the photophysical behavior of coumarin 343 (C343) individually in hydrophilic and hydrophobic Pluronic block copolymers, P123 and F108, respectively through UV–visible absorption, steady‐state, and time‐resolved fluorescence studies. Firstly, critical micellization temperature (CMT) of the aforementioned Pluronics with and without reline was deduced through differential scanning calorimetry (DSC). Then, a Fourier‐transform infrared spectroscopy (FT‐IR) investigation unravelled how DES interacts with the Pluronics. Additionally, to understand the effect of DES on the micellar size of Pluronics, we have also carried out dynamic light scattering (DLS) and small‐angle neutron scattering (SANS) measurements. This work builds upon previous findings and extends the application of DESs to polymer‐based hybrid systems, offering insight into their potential for tunable, environmentally friendly material design.

2. Materials and Methods

2.1. Materials

Choline chloride (purity ≥ 99%), Coumarin 343 (C343) (quality level: 200), Pluronics, P123 (molecular weight ~5,800 Da), and F108 (molecular weight ~14,600 Da) were purchased from Sigma Aldrich. We have purchased urea (purity: 99.9%) from Central Drug House Private Limited (CDH). Deuterium oxide, D₂O (99.85%, pure), was purchased from Sigma Aldrich. Chemical structures of all the chemicals are shown in Scheme 1 and were used as received. For photophysical experiments, a methanolic stock solution of C343 was prepared. An appropriate aliquot from this stock was transferred into a quartz cuvette to obtain a final C343 concentration of 5 µM in a total volume of 2 mL. The methanol was then evaporated from the cuvette. Subsequently, 2 mL of 5% w/v P123 or 5% w/v F108 solution was added separately to the cuvette. The 5% w/v poly(ethylene oxide)‐block‐poly(propylene oxide)‐block‐poly(ethylene oxide) was prepared by dissolving an adequate amount in triple‐distilled water. Reline, a deep eutectic solvent composed of choline chloride and urea in a 1:2 molar ratio, was prepared by stirring at 70 °C–80 °C. It was then added to the Pluronic solutions in varying amounts [9]. Specifically, 0.4, 0.8, 1.2, and 1.6 g of reline were introduced into 2 mL of the Pluronic solutions (with reline density [25] of 1.197 g/mL at 25 °C and 1.194 g/mL at 30 °C). This resulted in reline mole fraction (x _reline) of 0, 0.04, 0.08, 0.11, 0.14 corresponding to weights, 0, 0.4, 0.8, 1.2, and 1.6 g, respectively. Detailed calculations for these compositions are provided under Section S1 and Table S1.

SCHEME 1.

The chemical structures of (A) choline chloride, (B) urea, (C) coumarin 343 (C343), (D) Pluronic P123, and (E) Pluronic F108.

Additionally, the images in Figure 1 demonstrate that even at high reline loading, the systems maintain macroscopic homogeneity and optical clarity, with no observable turbidity or phase separation.

FIGURE 1.

Digital photographs of representative samples in quartz cuvettes: (A) 5% w/v P123 with x _reline = 0, (B) 5% w/v P123 with x _reline = 0.14, (C) 5% w/v F108 with x _reline = 0, and (D) 5% w/v F108 with x _reline = 0.14, demonstrating the macroscopic appearance and phase stability of Pluronic‐reline systems.

For a SANS study, as the contrast varies between hydrogenous and deuterated components, the hydrogenous solute (Pluronic + reline in the present case) prepared in a deuterated solvent (D₂O in the present case) will give the best scattering data (high intensity and high signal to background ratio). In general, reline preparation does not require an external solvent, so we prepared the reline using choline chloride and urea in a 1:2 molar ratio as mentioned above. For the polymeric solution, Pluronic was dissolved in deuterated water (D₂O) rather than H₂O to obtain good contrast. The resulting Pluronic‐D₂O solution was refrigerated at 4 °C to ensure complete dissolution. Subsequently, a high concentration of reline (1.6 g or x _reline = 0.14) was loaded into the Pluronic system to achieve the final experimental composition for the neutron experiments, and all measurements were carried out at a fixed temperature of 30 °C.

2.2. Methods

We performed absorption studies using a UV–visible spectrophotometer (model: UV‐2550, Shimadzu) at a constant temperature of 30 °C, maintained using a refrigerated bath circulator (JeioTech, model: RW0525G). The steady‐state excitation and emission spectra were recorded using a Fluoromax‐4 spectrofluorometer (Horiba Jobin Yvon). Time‐resolved fluorescence emission decays were measured with an Edinburgh instrument (model: LifeSpec II, UK) using F‐900 as the decay analysis software. Time‐correlated single‐photon counting (TCSPC) measurements were performed to collect time‐resolved fluorescence data using a Hamamatsu MCP‐PMT (3809U) detector at the magic angle of 54.7°. A constant temperature of 30 °C was maintained using a Peltier‐controlled cuvette holder (model: TCL50, Quantum Northwest), and excitation was provided by a 405 nm picosecond diode laser source. The bulk viscosity of the sample was measured using a Brookfield Viscometer II at a temperature of 30 °C. The refractive index was measured using a refractometer from Mettler Toledo (model: Refracto/30PX). The DSC study was performed using a PerkinElmer instrument (DSC 6000), with the temperature varied from 5 °C to 50 °C at a heating rate of 2 °C per min. The FT‐IR study was performed using a Bruker instrument (VERTEX 80v vacuum spectrometer). DLS experiments were carried out to measure the hydrodynamic diameter using a Malvern instrument (model: ZSU3305, Zetasizer Ultra) at 30 °C.

SANS experiments were performed using a SANS diffractometer installed at the Guide Tube Laboratory, Dhruva Reactor, Bhabha Atomic Research Centre, Mumbai, India. SANS involves the measurement of the coherent differential scattering cross‐section per unit volume (d∑/dΩ) as a function of wavevector transfer Q (= 4πsinθ/λ), where λ is the wavelength of the incident neutron, and 2θ is the scattering angle. The neutron velocity selector provided a monochromatized beam with a mean wavelength of 5.2 Å and a wavelength spread (Δλ/λ) of approximately 15%. The angular distribution of scattered neutrons was captured by several 1‐m‐long 1D helium position‐sensitive detectors (PSDs) arranged in a cross geometry, covering a Q range from 0.015 to 0.3 Å⁻¹.

3. Results and Discussion

3.1. Determination of Critical Micellization Temperature (CMT) in Pluronic and Reline‐Containing Systems

The critical micellization temperature (CMT) is an important parameter that highlights the role of temperature in micelle formation. It is defined as the temperature at which micelles begin to form. Alexandridis et al. [35] reported that the micellization process is entropy‐driven and characterized by an endothermic enthalpy of micellization. The hydrophobic poly(propylene oxide) (PPO) segments of Pluronics are primarily responsible for driving micellization, which can be attributed to the progressive weakening of hydrogen bonding between water and PPO with increasing temperature. In the present study, DSC was chosen to determine the CMT of Pluronics P123 and F108 in the absence and presence of the maximum amount of reline (x _reline = 0.14).

From Figure 2, the CMT values of Pluronic 5% w/v P123 and 5% w/v F108 were found to be 14.1 °C and 22.6 °C, respectively, which are in close agreement with the reported values in the literature [41], which were determined via the dye solubilization method. We know that the CMT values are inversely proportional to the PPO/ PEO ratio. Thus, due to a higher PPO/PEO ratio of 1.75 (P123) than that of 0.18 (F108), the CMT value is lower for P123 than that of F108. For Pluronic solutions with reline of x _reline = 0.14, the CMT values decreased significantly to 8.5 °C for 5% w/v P123 and 17 °C for 5% w/v F108, relative to those of the corresponding neat Pluronic solutions.

FIGURE 2.

Differential scanning calorimetric (DSC) thermograms shwoing heat flow (mW) as a function of temperature (°C) at a heating rate of 2 °C per min for (A) 5% w/v P123, (B) 5% w/v P123 with x _reline = 0.14, (C) 5% w/v F108, and (D) 5% w/v F108 with x _reline = 0.14, respectively.

The decrease in the CMT values of Pluronics on reline loading (x _reline = 0.14) indicates that reline enhances the driving force for micellization. In general, the PPO blocks of Pluronics are typically surrounded by structured hydration shells formed by water molecules (however, due to the presence of methyl groups in PPO, it is less hydrated than the PEO segments), and the hydrogen bonding between PPO and water stabilizes these hydrophobic segments while maintaining its solubility. As the temperature increases and approaches the CMT, PPO begins to dehydrate. In the presence of reline, composed of hydrophilic components such as choline chloride and urea, strong hydrogen‐bonding and electrostatic interactions with water molecules occur. These interactions sequester a significant portion of water, thereby reducing the availability of hydration water for PPO. Consequently, the stabilizing hydration cage around PPO weakens, facilitating their dehydration at lower temperatures and leading to the onset of micellization earlier than in neat Pluronic solutions. After considering the observed changes in CMT values of Pluronics in the absence and presence of reline, all experiments were conducted at 30 °C, thereby ensuring complete micellization.

3.2. Fourier Transform Infrared (FT‐IR) Spectroscopic Investigation in Pluronic and Reline‐Containing Systems

FT‐IR is an advanced analytical technique in which infrared radiation excites the vibrational energy levels of molecules present in adequate samples, generating the spectra that can be utilized for a wide range of applications [36].

In this study, FT‐IR spectral data were employed to investigate the interactions between Pluronics and reline. However, before assessing the influence of reline on Pluronic in depth, it is important to first examine the characteristic peaks of urea and choline chloride form pure reline, are altered upon its addition to water or Pluronic solutions.

The characteristic peaks of pure reline were identified, as shown in Figure 3, by referring to the experimental peak frequencies reported by Hayyan et al. [37]. The peaks arising from urea and choline chloride within the reline were assigned accordingly, with their specific origins indicated in the figure caption and numbered from (i) to (vii). Notably, systematic shifts in several key vibrational modes were observed upon loading of reline in water or Pluronics.

FIGURE 3.

FT‐IR spectra of (A) pure reline (1:2 molar ratio of choline chloride: urea), (B) x _reline = 0.14 in water, (C) 5% w/v P123 with x _reline = 0.14, and (D) 5% w/v F108 with x _reline = 0.14. Contributions of peaks from the constituents of reline, urea (vivid cyan color), and choline chloride (very soft red color) are represented where (i) symmetric NH₂ stretching; (ii) N─H bond stretching or O─H bond stretching; (iii) C═O stretching for amide and 1° amide N─H scissoring band; (iv) CH₂ bond bending; (v) C─N bond stretching; (vi) C─N⁺ symmetric stretching; (vii) N─H bond out‐of‐plane bending band.

The (i) symmetric NH₂ stretching band of urea, located at 3368 cm⁻¹ for pure reline, shifts to lower frequencies (3261–3262 cm⁻¹) in the presence of water or Pluronics. A similar downshift is seen for the (ii) N–H/O–H stretching bands (3259 cm⁻¹ → 3150–3153 cm⁻¹). Such red‐shifts are indicative of stronger hydrogen bonding, where the N–H/O–H groups engage in new intermolecular interactions with the surrounding medium, thereby weakening the vibrational force constant of the bond. The additional broad, noisy absorption in the 3500–4000 cm⁻¹ region, as shown in Figure 3B–D, further reflects the enhanced hydrogen‐bonding network facilitated by water molecules.

In the mid‐frequency region, pure reline displays distinct bands at 1661 and 1603 cm⁻¹, with a dip at 1638 cm⁻¹, assigned to (iii) C═O stretching of the amide and primary amide N–H scissoring bands, respectively. Upon introducing reline into water (x _reline = 0.14) and x _reline = 0.14 in Pluronic (5% w/v P123 or F108) solutions, the distinction between these bands diminishes. This merging suggests that the C═O stretching of the amide and the primary amide N–H scissoring modes are no longer isolated but rather embedded in an altered hydrogen‐bonding environment, where their vibrational identities overlap due to interaction with surrounding water and polymeric oxyethylene units.

In the lower‐frequency region, (iv) CH₂ bending (1477 cm⁻¹) and (v) C–N stretching (1430 cm⁻¹) modes of pure reline merge and shift to 1466 cm⁻¹ for x _reline = 0.14 in water or Pluronics (5% w/v P123 and F108), implying subtle perturbations of the alkyl and amine moieties through noncovalent interactions. In contrast, the (vi) C–N⁺ symmetric stretching and (vii) N–H out‐of‐plane bending bands remain largely unchanged, suggesting that the quaternary ammonium group of choline chloride retains its local environment. Thus, the x _reline = 0.14 in water and Pluronic solutions introduces a competitive interaction with water and Pluronic ether oxygens, which account for the observed red shifts in stretching modes, diminished amide band resolution, and selective perturbation of functional groups. Consequently, reline transforms from a compact, self‐associated eutectic structure into a delocalized hydrogen‐bonded network stabilized by the surrounding solvent and polymer environment.

Now, let us understand the effect of reline or the way the reline is interacting with Pluronics through Figure 4. The spectral region of 1000–1410 cm⁻¹ was selected, as it encompasses the characteristic peaks of PPO and PEO units in Pluronics [36, 38, 39]. Initially, a baseline spectrum of water was recorded, followed by the acquisition of FT‐IR spectra for 5% w/v Pluronics, P123 and F108. The FT‐IR spectrum of reline was then obtained for x _reline = 0.14 in water. Finally, spectra were collected for 5% w/v Pluronics, P123 and F108 with x _reline = 0.14. The C–O–C stretching vibration of PEO region is observed for 5% w/v Pluronic P123 and F108 at 1100 cm⁻¹ and 1084 cm⁻¹, respectively.

FIGURE 4.

Fourier transform infrared (FT‐IR) spectra of 5% (w/v) Pluronic (P123, F108) and also with the reline.

The peak frequency of the PPO region (C‐O stretching, C‐H bending, C‐H bending in the CH₃ group) in 5% w/v Pluronics, P123, and F108 are located at (1252, 1299, and 1349) cm⁻¹ and (1252, 1301, and 1350) cm⁻¹, respectively. In comparison, pure reline exhibits a characteristic band in the 1100–1000 cm⁻¹ region, assigned to C–N⁺ symmetric stretching of choline chloride. Notably, the spectra of x _reline = 0.14 in water do not completely overlap with those obtained in the presence of Pluronics, indicating that the C–N⁺ symmetric stretching of choline chloride interacts with the PEO units of the polymer chains.

Through all the discussions, it was found that reline loading of x _reline = 0.14 perturbs the vibrational features of Pluronic micelles, with a distinct effect on the PEO and PPO segments. Although urea does not contribute directly within the 1000–1410 cm⁻¹ window, it indirectly reorganizes the hydrogen‐bond network by engaging with water molecules and the ether oxygens of Pluronics. This reorganization weakens the intrinsic urea–choline chloride associations of pure reline, thereby increasing the availability of choline chloride for interaction. The location of the C–N⁺ symmetric stretching band of choline chloride in the same spectral window as the PEO C–O–C stretching, together with the deviations observed upon reline loading (x _reline = 0.14), provides compelling evidence for preferential choline chloride–PEO interactions. In contrast, the PPO‐associated modes (C–O stretching and C–H bending) remain largely unaltered, indicating negligible perturbation of the hydrophobic block. Collectively, these results establish that reline selectively perturbs the PEO segments of Pluronics through choline chloride–ether oxygen interactions. Urea acts as a mediator by destabilizing the native DES framework, leaving the PPO domains essentially unaffected.

3.3. Dynamic Light Scattering (DLS) Studies

DLS was employed to investigate the changes in the hydrodynamic diameter of Pluronic micelles at 30 °C in the presence of reline (x _reline = 0.14) as shown in Figure 5A,C. For 5% w/v P123 solution, the micelles exhibited a hydrodynamic diameter of 18.5 nm. However, the hydrodynamic diameter of micelles increased to 42.9 nm upon high reline (x _reline = 0.14) loading in 5% w/v P123. In contrast, 5% w/v F108 initially exhibits two populations: one at 5.9 nm corresponding to unimers and another at 59.8 nm, which can be assigned to impurity‐associated micellar aggregates as reported in our previous work [38]. On high reline loading (x _reline = 0.14) in 5% w/v F108, a single micellar peak arises at 50.2 nm. Thus, the hydrodynamic diameter of P123 micelles upon high reline loading (x _reline = 0.14) increases, whereas we found a stable single peak in F108 micelles in the presence of reline (x _reline = 0.14). The appearance of single peak of F108 micelles on high reline loading behavior arises because the CMT of F108 decreases significantly upon reline incorporation (as deduced by DSC); therefore, at the fixed measurement temperature of 30 °C used for DLS, the reline‐loaded (x _reline = 0.14) F108 system exists well above its CMT compared to the F108 system whose CMT lies close to the temperature used for DLS measurement at 30 °C. As a result, micellization is more pronounced, leading to the formation of a single, well‐defined micellar population with negligible contribution from unimers. Collectively, these observations confirm that reline not only interacts with Pluronic micelles but is also effectively incorporated within them. In addition to these intensity distribution plots in DLS, the correlation coefficient (g₁) as a function of time (µs) plots for Pluronics and with reline loaded (x _reline = 0.14) micellar systems are also shown in Figure 5B,D. Furthermore, the polydispersity index (PDI), along with associated error limits to assess data reliability, are tabulated in Table 1.

FIGURE 5.

The intensity (%) versus size (nm) plot and correlation coefficient versus time (µs) plots are shown for (A, B) 5% w/v P123 with and without x _reline = 0.14, (C, D) 5% w/v F108 with and without x _reline = 0.14 through dynamic light scattering study.

TABLE 1.

Hydrodynamic diameter (nm) and polydispersity index of each Pluronic micellar system with and without reline, where x _reline = 0.14 is listed below.

S. no.	Sample	Hydrodynamic diameter (nm)	Polydispersity index
01.	5% w/v P123	18.5 ± 0.1	0.014 ± 0.008
02.	5% w/v P123 with x reline = 0.14	42.9 ± 0.9	0.297 ± 0.007
03.	5% w/v F108	5.9 ± 0.1, 59.8 ± 1.1	0.603 ± 0.004
04.	5% w/v F108 with x reline = 0.14	50.2 ± 1.5	0.186 ± 0.010

3.4. Photophysical Investigation of C343 in Ternary Aqueous Pluronic‐Reline Systems

In this section, we first examine the behavior of C343 in the ground and excited states through UV–visible absorption and steady‐state experiments. Then, we move on to the time‐resolved experimental results and discuss the localization and rotation of C343 in the microenvironment tuned by reline in the Pluronic systems. Next, we collected the SANS data to understand the structure, including the shape and size, of the Pluronic systems upon reline loading. Finally, we provide an integrated mechanistic insight, where we discuss our work by comparing it to the previously reported binary water‐reline systems.

3.4.1. UV–Visible Absorption and Steady‐State Excitation Studies

Coumarin 343 (C343) is a pH‐sensitive dye with similar ground‐state, pK_a , and excited‐state, pK_a * values of 4.65 [40]. In acidic pH solutions (pH < 4), C343 exists in the neutral (protonated) form, while in basic/ neutral pH solutions, C343 exists in the anionic (deprotonated) form. Three sites on C343 are available to accept a proton; they are: (i) the carboxylic acid group, (ii) the cyclic ester, and (iii) the cyclic amine moieties. Using Coumarin 153, which has two sites (ii, iii) similar to those in C343, a comparative spectroscopic study confirmed that these two sites are not involved in protonation in acidic solutions [40]. Therefore, the remaining carboxylic acid group is the main active site involved in the protonation‐deprotonation behavior of C343 with the variation of the pH of the medium.

In our work, we have conducted a UV‐visible absorption study of C343 in different Pluronic micelles upon loading of reline with varying compositions above their CMT values at 30 °C as shown in Figure 6. Figures S1 and S2 show the controlled experiments conducted by using C343 in Pluronics and in water each with reline loading at x _reline = 0 and x _reline = 0.14. The absorption maxima of C343 in 5% w/v P123 and 5% w/v F108 were observed at 423 nm and 433 nm, respectively. The absorption maximum of C343 in water was observed at 436 nm which is similar to the one reported by Dana et al. [41] at 435 nm for the pH value of water ∼ 6. Thus, C343 exists in the neutral pH category in aqueous Pluronic solution. In addition to this, reline also exists in water with pH of ∼ 8.6 [42]. Thus, we may confirm that C343 will exist in the deprotonated anionic form (C343⁻) in water systems as well as in Pluronic systems upon reline loading at varying mole fractions in the ground state. The blue shifts in the absorption maxima of C343 in Pluronic systems relative to the water system at both reline loadings of x _reline = 0 and x _reline = 0.14, prove that C343 exists in micellar systems rather than in bulk water. The full‐width at half maxima (FWHM) of these absorption spectra is narrower, similar to the observations shown by Pant et al. [43] through surface spectrum measurements of C343 at the water/1,2‐dichloroethane interface using optical second harmonic generation (SHG) for the concentrations of C343 below 30 µM, this narrowness is due to the absence of any aggregate forms (J or H) even though Pant et al. reported that C343 may also exist in aggregate (J or H) forms whether protonated or deprotonated. Due to the low concentration of C343 (5 µM), in addition to the narrower absorption spectra in Pluronic systems and in water with high reline loading of varying compositions, the probability of any aggregate forms (J or H) is eliminated. Thus, C343 will exist in the deprotonated anionic (C343⁻) form in Pluronic micellar systems at varying compositions of reline in the ground state. Stettler et al. provides strong validation for the present work in micellar systems with reline loading to utilize the C343 charged dye because reline based environments exhibits enhanced sensitivity toward charged probes over neutral probes [7].

FIGURE 6.

UV‐visible absorption spectra of C343⁻ in (A) 5% w/v P123, and (B) 5% w/v F108, with varying mole fraction of reline, x _reline, from 0 to 0.14, (C) steady‐state excitation spectra of C343⁻ in 5% w/v P123, and (D) 5% w/v F108 with varying mole fraction of reline, x _reline, from 0 to 0.14 (λ_em = 500 nm) (compositional details in ternary system are given in SI) at 30 °C.

As shown in Figure 6A, the absorption maximum of C343⁻ in 5% w/v P123 systems red shifts from 423 nm to ~428 nm upon reline loading of varying compositions, x _reline, (0.04, 0.08, 0.11, and 0.14) at 30 °C. In contrast, Figure 6B shows that the absorption maximum of C343⁻ revealed no significant shifts in 5% w/v F108 (433 nm) upon reline loading of varying compositions, x _reline, (0.04, 0.08, 0.11, 0.14) at 30 °C. This trend was further confirmed by steady‐state excitation spectra. In Figure 6C, an excitation maximum of C343⁻ in 5% w/v P123 appeared at 427 nm which red shifts progressively to ~432 nm upon reline loading of varying compositions (λ_em = 500 nm) at 30 °C. Meanwhile, the excitation maximum of C343⁻ in 5% w/v F108, was observed at 432 nm, showed a minor red‐shift upon reline loading of varying compositions, x _reline, (0.04, 0.08, 0.11, 0.14) to 435 nm (λ_em = 500 nm) at 30 °C as shown in Figure 6D. These red shifts in the absorption or excitation maxima of C343⁻ in Pluronic systems upon varying compositions of reline loading reveal that C343⁻ is experiencing a more polar microenvironment due to the reline [9] (hydrophilic/ ionic Type III deep eutectic solvent (DES)) loading in Pluronic micellar systems in the ground state. Because the PPO/PEO ratio of Pluronics decreases from 1.75 in P123 to 0.18 in F108, and the overall hydrophilicity of the micellar environment of F108 increases, leading to the higher absorption or excitation maximum of C343⁻ in Pluronic F108. Furthermore, upon reline loading, the larger red shifts observed in P123 compared to the negligible shifts in F108 indicate that reline induces greater changes in the polarity of the microenvironment surrounding C343⁻ in P123 micelles than in F108 micelles.

3.4.2. Steady‐State and Time‐Resolved Fluorescence Emission Studies

As mentioned above, the pK_a and pK_a* values are similar in both the ground and excited states; thus, only one form, whether protonated or deprotonated, generally exists in both states. UV‐visible absorption and steady‐state excitation studies revealed that Coumarin 343 exists in its anionic form in Pluronic systems, even under high reline loading. Therefore, coumarin 343 exists in the anionic form (C343⁻) in the excited state within these systems. The absorption and excitation maxima of C343⁻ were obtained at ~420 nm, consequently, we excited these molecules at λ_ex = 420 nm (Figure 7) and λ_ex = 405 nm for fluorescence lifetime measurements. Emission spectra were also recorded at λ_ex = 405 nm as shown in Figure S3. In steady‐state fluorescence emission measurements, upon excitation of C343⁻ in Pluronic samples at 420 nm, emission maxima at 483 nm (5% w/v P123) and 486 nm (5% w/v F108), were observed at 30 °C. Correa et al. [44] have reported the effect of solvents on the emission spectrum of C343⁻ by exciting the molecules of C343⁻ in various solvents at 372 nm. We found that the emission maximum of C343⁻ in 5% w/v P123 lies between the formamide and propylene glycol emission maxima of 482.8 nm and 484 nm, respectively. While in 5% w/v F108, the emission maximum lies between the emission maxima of propylene glycol and methanol at 484 nm and 487 nm, respectively [44].

FIGURE 7.

Steady‐state fluorescence emission spectra of C343⁻ in (A) 5% w/v P123, and (B) 5% w/v F108 with the varying mole fraction of reline, x _reline = 0, 0.04, 0.08, 0.11, and 0.14 at (λ_ex = 420 nm) at 30 °C.

Formamide, propylene glycol, and methanol are polar protic solvents which have dielectric constants of 109, 32, and ∼ 33, respectively. As Coumarin 343 is a solvatochromic probe, this means its emission wavelength must shift on the basis of the polarity and viscosity of the microenvironment. Through the E_T (30) scales of these solvents and the α, β, π* parameters, as reported by Marcus [45] using the Kamlet‐Taft equations, we can quantify the nature of the solvents during solute‐solvent interactions. Thus, the α values follow the order, methanol > propylene glycol > formamide, suggesting methanol as a strong HBD; the β values, formamide > methanol > propylene glycol, indicate the high polarizability of formamide and its better suitability in stabilizing dipoles, while propylene glycol has balanced π* (0.71) and β (0.78) values of ∼ 0.7. But the π* value of propylene glycol is higher than that of methanol due to the longer carbon chains in its structure, signifying a friction effect due to its boundary characteristics between a pure polar and slightly hydrophobic nature.

C343⁻ in 5% w/v P123 occupies the polar regions where its dipoles are properly stabilized (like in formamide) while C343⁻ in 5% w/v F108 is in more polar regions (like in methanol) than in 5% w/v P123. C343⁻ is experiencing a more crowded microenvironment in 5% w/v P123 micellar systems as the C343⁻ emission maxima are approximately close to that of propylene glycol which lies in between a purely polar and hydrophobic nature. Also, due to more PEO content in F108 systems, it has more water molecules providing less crowded regions than P123 micellar systems. On reline loading at varying compositions, x _reline, from 0 to 0.14, small red shift in the emission maxima (λ_ex = 420 nm) of C343⁻ is observed from 483 to 488 nm in 5% w/v P123 micellar systems at 30 °C. Furthermore, a red shift up to 488 nm from 486 nm in the emission maxima (λ_ex = 420 nm) of C343⁻ is observed in 5% w/v F108 on reline loading at varying compositions, x _reline, from 0 to 0.14, at 30 °C. These red shifts reveal an increase in the polarity of C343⁻ in Pluronics on reline loading.

Grant et al. [46, 47] reported that C343 has a CLOGP (calculated value of the log of octanol‐water partition coefficient) of ‐1.09. This means C343 prefers to reside in the aqueous phase. C343 probes the hydrophilic regions of Pluronic, F88, which shows direct exposure of C343 to the bulk aqueous solution with some fraction near the hydrated PEO periphery of the micelles. In contrast, other reports by Singh et al. [48] and Anand et al. [49] reported the location of C343 in F88 or P105 with CTAB and in F127 with urea, respectively, stating that C343 exists more in the micellar phase than in the aqueous phase on the basis of several measurements.

To prove the location of C343⁻ in micellar systems over bulk phase, we have carried out controlled experiments in water with reline loading at x _reline = 0 and 0.14 and compared these steady‐state fluorescence emission results with the micellar microenvironment with similar reline content, as shown in Figure S4 (λ_ex = 420 nm) and Figure S5 (λ_ex = 405 nm) at 30 °C. Thus, a distinct difference in the spectral positions of C343⁻ is observed in micellar systems compared to the water system at reline loadings of x _reline = 0 and 0.14. This reveals that C343⁻ exists in the micellar system rather than the bulk aqueous phase.

Thus, the combination of all studies on C343⁻ in different Pluronics and through solvatochromic study, the already reported location of C343⁻ at the periphery of the PEO corona in Pluronics, F88, F127 and P105, with additives, and the controlled experiments in water versus micellar systems, collectively, reveals that C343⁻ is localized on the periphery of the polar PEO/water interface in Pluronics (5% w/v), P123 and F108, and even in the presence of reline at 30 °C.

Through steady‐state fluorescence emission studies (λ_ex = 420 nm and λ_ex = 405 nm), a decrease in the fluorescence intensity was observed with the gradual variation of reline from x _reline = 0 to 0.14, in Pluronics (P123 or F108) at 30 °C. Reline is viscous in nature and Pluronic‐reline systems (viscosities are tabulated in Table S2) may absorb a portion of incident excitation light (the primary inner filter effect (IFE)) or re‐absorb the emitted fluorescence before it reaches the detector (secondary IFE). The attenuation of both the excitation and emission radiation within a 1 cm path‐length of cuvette may result in apparent quenching or a reduction in intensity; to avoid such a situation, we used the correction factor [50], 10^{(Aex + Aem)/2}, and multiplied it by the observed fluorescence intensity spectrum (F_obs) to obtain the IFE corrected fluorescence spectra (F_c) of C343⁻ in Pluronics (5% w/v P123 and 5% w/v F108) upon reline loading of varying compositions (x _reline = 0, 0.04, 0.08, 0.11, 0.14) at both the excitation wavelengths, λ_ex = 420 nm (Figure S6) and λ_ex = 405 nm (Figure S7) at 30 °C. Here, A_ex is the absorbance value at the excitation wavelength of C343⁻ and A_em is the absorbance value at the emission peaks for the corresponding excitation wavelengths. Figures S6 and S7, showing IFE corrected fluorescence spectra in Pluronics, still exhibit the decrease in fluorescence intensity upon reline loading of varying compositions (x _reline = 0, 0.04, 0.08, 0.11, 0.14).

Additionally, due to the high refractive index (values tabulated in Table S3) of Pluronic systems upon the addition of reline, the fluorescence intensity may be affected; to avoid any artifacts, we applied the refractive index (RI) correction by multiplying the IFE corrected fluorescence emission spectra by the (n_s/n_r)² correction factor. Here, n_s and n_r are the refractive indices of sample and reference (water with a n_r = 1.3325 at 25 °C), respectively. Illustrative representations are shown in Figure S8 for P123 and Figure S9 for F108, where we found that RI corrected fluorescence spectra show an increase in the fluorescence intensity, that is a reversed trend at high reline loading (x _reline = 0.14) in Pluronics at both λ _ex = 420 nm and λ _ex = 405 nm at 30 °C to that observed for IFE corrected spectra. However, we observed no change upon refractive index correction to P123 system at λ _ex = 405 nm, and the same decrement in fluorescence intensity at a high reline loading of 0.14 mole fraction in the ternary system. This can be explained by the fact that the % intensity difference between the emission spectra in P123 and that in P123 with x _reline = 0.14 is ∼ 13%, while in other cases, it is in between ∼ 4.9 % and 11 %. Thus, the smaller the difference in the intensity more change upon refractive index correction would be observed provided that the solvent has a comparatively large refractive index. These intensity differences depend on the concentration of the dye, the slit used, and the integration time used to collect the fluorescence emission spectra. Thus, the refractive index correction would lead to erroneous and inconsistent results; therefore, it is not appropriate to use the RI correction factor, a concept originated from light scattering techniques, to understand the fluorescence intensity trends. This is also supported by the fluorescence experiment given for solvents of varying refractive indices from 1.33 to 1.63 by Raymond F. Chen [51]. Dielectric constants also affect the solvent properties in addition to the refractive index, thus, correcting the fluorescence spectra based only on one solvent property may lead to doubtful results.

Time‐resolved fluorescence emission studies of C343⁻ in P123 and F108, and reline loaded Pluronic systems are shown in Figure 8A,B at λ _ex = 405 nm. All decays were best fitted using a bi‐exponential function deduced through χ ² and residuals after deconvolution using the instrument response function (IRF). The fitted parameters are tabulated in Table 2. The biexponential fit to population decays of C343⁻ in 5% w/v P123 yielded twotime components. The short lifetime component, τ ₁, ranged from 0.08 to 1.94 ns, and corresponds to the dye localization in a less restricted microenvironment compared to the longer lifetime component, τ ₂, which ranged from 4.13 to 4.34 ns. This longer lifetime component of approximately 4 ns was also reported for C343 in literature (at 25 °C) for its localization in the PEO region of the 5% w/v P123 [52] micelle. Here, τ ₁ remained in the narrow range of 0.07–0.18 ns, while τ ₂ spanned 4.16–4.44 ns in the F108 system on varying compositions of reline loading. These results indicate that C343⁻ experiences distinct microenvironments in P123 and F108 upon reline loading, with stronger effects in P123 and partial stabilization of the probe in F108. From Figures S10A,B and 8C,D, the controlled time‐resolved fluorescence emission experiments of C343⁻ in water systems compared to that in Pluronics with reline loading (x _reline = 0 and x _reline = 0.14) at 30 °C proved that C343⁻ is localized in micellar systems at the PEO/bulk water interface, as also confirmed earlier through steady‐state experiments.

FIGURE 8.

Time‐resolved fluorescence emission decay profiles of C343⁻ in various aqueous micellar environments at 30 °C (λ_ex = 405 nm). Panels (A) and (B) show the decays in 5% w/v P123 and 5% w/v F108, respectively, with varying mole fractions of reline, x _reline = 0, 0.04, 0.08, 0.11, and 0.14. Panels (C) and (D) provide a comparison of the decay profiles in 5% w/v P123 and 5% w/v F108 at x _reline = 0.14 against the corresponding water–reline binary mixture.

TABLE 2.

Time‐resolved fluorescence emission decays parameters of C343⁻ in 5% w/v Pluronic P123 and F108 with the addition of various amounts of reline at 30 °C (λ_ex = 405 nm).

S. no.	Sample	a 1	𝝉1 (ns)	a 2	𝝉2 (ns)	Χ 2
01.	5% w/v P123 with x reline = 0	0.05	1.65	0.95	4.34	1.028
02.	5% w/v P123 with x reline = 0.04	0.02	1.89	0.98	4.31	1.041
03.	5% w/v P123 with x reline = 0.08	0.03	1.94	0.97	4.25	1.105
04.	5% w/v P123 with x reline = 0.11	0.07	0.18	0.93	4.16	1.061
05.	5% w/v P123 with x reline = 0.14	0.21	0.08	0.79	4.13	1.042
06.	5% w/v F108 with x reline = 0	0.20	0.07	0.80	4.44	1.077
07.	5% w/v F108 with x reline = 0.04	0.09	0.16	0.91	4.36	1.030
08.	5% w/v F108 with x reline = 0.08	0.09	0.18	0.91	4.29	1.029
09.	5% w/v F108 with x reline = 0.11	0.10	0.17	0.90	4.22	1.090
10.	5% w/v F108 with x reline = 0.14	0.10	0.17	0.90	4.16	1.083

These discussions reveal that the reline decreases the fluorescence intensity of C343⁻ in Pluronics. Furthermore, time‐resolved fluorescence emission decays of C343⁻ in Pluronics with reline loading led to a decrease in the lifetime values. To confirm whether this is due to the quenching effect of chloride ions from choline chloride, we carried out the quenching experiments using NaCl at varying concentrations (0–4 M) in aqueous systems of C343⁻, which led to an increase in the fluorescence intensity (Figure S11) and a small increment of fluorescence lifetime (Figure S12). This reveals that Cl⁻ ions do not act as a quencher to the C343⁻ dye due to the electrostatic repulsion between these negatively charged species. This indicates that the C343⁻ dye is interacting well in reline‐loaded Pluronic micellar media, due to the higher lifetime values of C343⁻ than in reline‐loaded water systems. In summary, the decrease in fluorescence intensity and lifetime values upon reline loading in Pluronics can be explained as follows: with increasing reline concentration, extensive hydrogen bonding between reline components (urea and choline chloride) and the PEO segments disrupts the hydration balance of the corona. This perturbation promotes partial relocation of C343⁻ toward more hydrated and polar regions, hence nonradiative relaxation pathways become more prominent, resulting in shorter lifetimes.

In P123, which possesses shorter PEO chains and a more compact corona, the addition of reline strongly perturbs the micellar interface. This effects dye stability within the corona and produces a change in lifetime. In contrast, the longer PEO blocks in F108 provide a more extensive hydration shell and multiple binding sites for reline.

3.4.3. Time‐Resolved Fluorescence Anisotropy Studies

Time‐resolved fluorescence anisotropy provides a quantitative measure of the rotational diffusion of a fluorophore upon excitation with polarized light [53]. The extent of polarization is inversely proportional to the rotational freedom of the fluorophore. The time‐resolved fluorescence anisotropy study investigated the microenvironmental effects on the rotational motion of C343⁻ dye within Pluronic micelles across varying reline compositions at 30 °C (λ_ex = 405 nm, Figure 9). All anisotropy data were best fitted to biexponential functions with good χ² values. The fitted parameters for C343⁻ in Pluronics and reline‐loaded systems are listed in Table 3. Typically, anisotropy decays in micellar systems originate from three independent motions: (i) the wobbling of dye within a restricted region, (ii) translational diffusion along the micellar interface, and (iii) the rotation of the micelle as a whole.

FIGURE 9.

Time‐resolved fluorescence anisotropy decays of C343⁻ in (A) 5% w/v P123, and (B) 5% w/v F108 and with the varying mole fraction of reline, x _reline = 0, 0.04, 0.08, 0.11, and 0.14 (λ_ex = 405 nm).

TABLE 3.

Time‐resolved fluorescence anisotropy decay parameters of C343⁻ in 5% w/v Pluronic P123 and F108 with the addition of various amounts of DES (reline) at 30 °C. (λ_ex = 405 nm).

S. no.	Sample	a1	𝝉1 (ns)	a2	𝝉2 (ns)	<𝝉rot> (ns)	χ2
01.	5% w/v P123 with x reline = 0	0.52	0.29	0.48	2.29	1.25	1.013
02.	5% w/v P123 with x reline = 0.04	0.71	0.22	0.29	2.24	0.81	0.980
03.	5% w/v P123 with x reline = 0.08	0.77	0.25	0.23	2.33	0.73	1.003
04.	5% w/v P123 with x reline = 0.11	0.78	0.27	0.22	2.46	0.75	1.060
05.	5% w/v P123 with x reline = 0.14	0.81	0.28	0.19	2.75	0.75	1.025
06.	5% w/v F108 with x reline = 0	0.89	0.20	0.11	1.13	0.30	1.143
07.	5% w/v F108 with x reline = 0.04	0.87	0.24	0.13	1.94	0.46	1.004
08.	5% w/v F108 with x reline = 0.08	0.88	0.27	0.12	2.11	0.49	0.909
09.	5% w/v F108 with x reline = 0.11	0.90	0.26	0.10	2.12	0.45	0.923
10.	5% w/v F108 with x reline = 0.14	0.92	0.31	0.08	2.64	0.50	1.050

In 5% w/v aqueous solutions of P123 and F108, the average rotational correlation times (<τ _rot>) of C343⁻ were found to be 1.25 ns and 0.30 ns, respectively. These results indicate that C343⁻ experiences a significantly more rigid and constrained microenvironment in P123 compared to F108 micellar systems, as also reflected by the higher fluorescence anisotropy in P123. Steady‐state and time‐resolved fluorescence measurements suggest that C343⁻ is predominantly localized at the interface between the PEO corona and bulk water. The observed differences in anisotropy arise from the intrinsic structural variations between the two Pluronics. P123, with a lower PEO‐to‐PPO ratio, forms a relatively dehydrated and compact corona that restricts probe rotation. In contrast, F108 contains a larger PEO fraction, resulting in a highly hydrated and flexible corona, thereby facilitating faster rotational diffusion and lower anisotropy.

The effect of reline incorporation on fluorescence anisotropy reveals distinct trends in the two systems. In P123, reline loading (x _reline = 0 to 0.14) leads to a significant decrease in <τ _rot> from 1.25 to ∼ 0.8 ns. This reduction is primarily driven by changes in the relative amplitudes of the fast and slow rotational components (a₁ and a₂). Notably, this behavior occurs despite a substantial increase in bulk viscosity (Table S2). In contrast, the F108 system exhibits an increase in <τ _rot> from 0.30 to ∼0.50 ns upon reline loading, along with an increase in both fast and slow rotational components. Although the bulk viscosity change in F108 is comparatively modest, the increase in <τ _rot> indicates a more restricted microenvironment.

A useful comparison can be drawn with the work of Heitz and co‐workers [54] on ethaline–methanol binary mixtures, where a transition from biexponential (x _methanol < 0.4) to single‐exponential decay (x _methanol ≥ 0.5) of C343 was observed with increasing methanol mole fraction and consequently, decreasing ethaline mole fraction. This transition reflects the evolution from a heterogeneous DES environment to a homogeneous solvent environment, where C343 experiences reduced restriction and faster rotational dynamics.

However, in the present study, even at very low reline mole fractions (x _reline = 0, 0.04, 0.08, 0.11, and 0.14), both P123 and F108 systems consistently exhibit biexponential decay behavior. This clearly indicates that, unlike simple DES–solvent binary mixtures, the micellar environment preserves microheterogeneity even at low DES content. In P123, the decrease in <τ_rot> (from ∼1.3 to ∼0.8 ns) without significant variation across small composition increments suggests partial disruption of reline into its components due to increased solvent exposure.

In the F108 system, reline loading leads to an increase in both individual rotational components and the average τ_rot (from ∼0.3 to ∼0.5 ns), indicating comparatively lower structural disruption of reline than in P123. Furthermore, the anionic nature of C343⁻ may promote electrostatic interactions with choline cations present in reline, particularly within the hydrated corona of F108.

3.5. Mechanistic Insight Into Reline‐Pluronic Interaction Using C343⁻

The concept of reline‐water mixtures with varying mole fractions of water is well established in the literature through several studies [2, 22, 23, 25, 55, 56, 57], including simulations, which is of great importance to understand the kind of mechanics that the present ternary system of aqueous Pluronic upon reline loading is experiencing. It has been found that in neat reline, the chloride ions play a big role by strongly interacting with the ammonium nitrogen in the urea group and the hydroxyl in the choline group. These further get stabilized due to the hydrogen bonding in urea‐urea and choline‐urea molecules. Also, this crowded nature of reline with these interactive groups, via hydrogen bonding, gives rise to a deep eutectic character to reline. In this section, we will discuss how the molecules of urea or choline chloride behave in reline on the addition of water of different weight percentages in three regimes—that is, for < 10 wt%, 20–40 wt% and > 40–50 wt% as per literature reports. Then we will compare the present ternary system with those binary systems in order to deduce the actual mechanics underway in these systems. For a water content of less than 10 wt%, the hydration of chloride ions begins, but still the bond of the hydrated chloride with urea and choline persists, which preserves the actual network of reline and its deep eutectic nature. Due to the increase in the water content from 10 wt% to between 20 and 40 wt%, the number of chloride ions surrounded by water molecules increases, leading to weak interactions between choline–chloride, urea–chloride, and choline–choline molecules. Contrary to this, urea‐urea hydrogen bonding becomes comparatively stronger due to the pull of the chloride ions by the increased number of water molecules. For the region of more than 40–50 wt% of water content in reline, the chloride ions become fully hydrated, which leads to a major disruption in the hydrogen bond network in reline. Due to the high water content, the association of choline—chloride diminishes, and the system becomes more hydrated and less ordered, in which the preferred interactions of urea‐urea and urea‐water exist. Pandey et al. [58] stated that in such a large water‐content system in reline, the system fuels towards polarity, and the capacity as a HBA decreases, and the properties of such binary systems tend towards a conventional aqueous electrolyte solution.

The present work lies under the third region, where the water content is > 50 wt%. This high water content is due to experimental constraints. Our aim of the work is to deduce the effect of reline in aqueous Pluronic systems using steady‐state and time‐resolved fluorescence spectroscopy. For this purpose, we chose a fluorophore, C343. The experiment requires the concentration of C343 to be constant over all the data acquisition for such spectroscopic measurements. Thus, by keeping the dye concentration of 5 µM in 2 mL of Pluronic solution, reline loading was done. This naturally increases the water content to be > 50 wt%. Importantly, it does not affect the validity of our results; instead, this kind of study provides unique insights into what happens in a system where reline is present in a more polar, more hydrated, and less structured state. Scientifically, the Pluronic micellar systems containing a PEO corona not only show the changes due to structured reline, but the PEO corona is also sensitive towards the reorganized components of reline, such as hydrated chloride, urea, and choline.

Upon reorganized reline loading in Pluronics, the PEO moiety becomes the new competitive hydrogen bonding site in addition to water molecules. In the P123 system, the compact PEO corona, due to the small PEO chains, limits the chances of hydration. The ether oxygens of the PEO unit form hydrogen bonding with the hydrated urea molecules easily, while the over‐hydrated chloride ions indirectly bother the first solvation shell. This displacement of hydrated water molecules increases the local polarity of dye sites, leading to red shifts in the absorption and emission spectra. However, the urea‐PEO and choline‐PEO dynamics destabilize the solvation environment, leading to non‐radiative decay pathways and hence a decrease in the lifetime. Moreover, the interruption in such structured hydrated networks upon reline loading decreases the microviscosity of the system, which leads to fast rotational diffusion, instead of the observed increase in the bulk viscosities of this medium upon reline loading.

Contrary to the above discussion related to the hydrophobic Pluronic P123, the hydrophilic Pluronic F108 contains an extended PEO corona, due to the long PEO chains, which provide a large number of interactive sites. In the F108 system, upon reline loading, the large PEO corona distributes the associations of urea and hydrated chloride with PEO sites more evenly. This balanced, distributive interaction with PEO sites prevents the system from full dehydration; upon reline loading in F108 system, the microenvironment around the C343⁻ dye will be less modulated. Consequently, spectral red shifts are smaller, lifetimes remain stable, and rotational diffusion slows down due to local stiffening of the corona as urea and choline form stabilizing hydrogen bonds with the extended PEO chains upon reline loading.

Thus, for more than 50 wt% of water content in reline, there is the existence of a weaker choline‐chloride interaction in addition to the predominant urea‐urea and chloride‐water interactions. The assimilation of these results provides an idea of how these reorganized species interact with the micellar PEO coronas. In the compact PEO corona, the P123 system experiences strong disruption upon reline loading. In contrast to P123, F108, containing a large PEO corona upon reline loading with various reorganized species, prevents the dye from much displacement and stabilizes it. Overall, the different photophysical results observed for C343 upon reline loading rely on the architectures of the Pluronics in which reline is being incorporated. This highlights the importance of the micellar framework to tune the properties of reline‐Pluronic systems. Therefore, the above probable mechanistic picture in the ternary system provides us with clarity on how the interactions are different from the water‐reline binary mixtures where the bulk hydrogen bond network restructures.

3.6. Small Angle Neutron Scattering (SANS) Studies

While DLS provides information on the hydrodynamic size of micelles in solution, it cannot unambiguously separate the contributions of the micellar core and the hydrated corona. In contrast, SANS enables the direct fitting of the scattering profile using appropriate models, which provides us the detailed structural parameters such as core radius, hard‐sphere radius, polydispersity, and volume fraction.

Therefore, SANS was employed in this work to go beyond the average hydrodynamic size obtained from DLS, and to resolve how the micellar core and overall hard‐sphere dimensions change upon addition of reline. This approach helps to establish not only the increase in micellar size (investigated through DLS data), but also why they grow—specifically, whether the increase arises from changes in the PPO core or from modifications in the PEO corona. As shown in Figure 10 and listed in Table 4, the core radius of 5% w/v P123 micelles increases from 6.08 to 6.77 nm upon varying the reline from x _reline = 0 to 0.14, accompanied by a concurrent increase in the hard‐sphere radius from 8.65 to 9.27 nm. A similar trend is observed for F108 micelles, where the core radius increases from 2.62 to 3.38 nm and the hard‐sphere radius from 10.5 to 11.1 nm, upon varying the reline from x _reline = 0 to 0.14. In Pluronic micelles, the core radius represents the hydrophobic PPO block, while the hard‐sphere radius encompasses the core, corona, and excluded volumes. The scattering data were fitted using the core shell hard sphere model (details are in Section S2).

FIGURE 10.

Coherent differential scattering cross‐section per unit volume (dΣ/dΩ) as a function of wave vector transfer (Q) plots for (A) 5% w/v P123 and with the addition of x _reline = 0.14, and (B) 5% w/v F108 and with the addition of x _reline = 0.14.

TABLE 4.

The fitted parameters obtained after applying the core‐shell hard sphere model for different reline‐Pluronic systems at 30 °C using the SANS technique.

S. no.	Sample	Core radius (nm)	Polydispersity	Hard sphere radius (nm)	Volume fraction
01.	5% w/v P123	6.08	0.24	8.65	0.058
02.	5% w/v P123 with x reline = 0.14	6.77	0.21	9.27	0.052
03.	5% w/v F108	2.62	0.30	10.5	0.040
04.	5% w/v F108 with x reline = 0.14	3.38	0.28	11.1	0.035

The fitted polydispersity values indicate that all Pluronic micellar systems remain moderately uniform in size distribution, with P123 showing slightly lower polydispersity (~0.21–0.24) compared to F108 (~0.28–0.30). This suggests that P123 micelles are relatively more homogeneous, whereas F108 micelles, owing to their longer PEO chains, exhibit broader size distributions. Upon reline loading, a marginal decrease in polydispersity is observed in both systems, implying that the presence of reline does not destabilize the micelles but rather promotes a more uniform organization.

The volume fraction values reveal a small but consistent decrease upon reline loading for both P123 (from 0.058 to 0.052) and F108 (from 0.040 to 0.035). This reduction is consistent with the observed increase in core and hard‐sphere radii, indicating that while individual micelles increase in size, their overall number density decreases, leading to a lower effective volume fraction. The volume fraction is governed by both the solute concentration (Pluronic + reline) and the degree of micellar hydration. The observed decrease in volume fraction can be attributed to a reduction in hydration, as the micelles swell to incorporate reline.

4. Conclusion

This study demonstrates how the incorporation of reline, a Type III DES composed of choline chloride and urea, reorganizes the microenvironment of Pluronic micelles in a manner that is both block‐selective and water‐fraction dependent. DSC revealed that reline lowers the critical micellization temperature of Pluronics by sequestering hydration water, thereby facilitating earlier dehydration of PPO blocks. FT‐IR spectroscopy further established that reline perturbs Pluronics selectively through hydrogen‐bonding interactions between choline chloride and PEO units, while urea indirectly reorganizes the network by weakening its native associations. DLS measurements revealed an increase in the hydrodynamic diameter of P123 micelles upon reline loading. In contrast, the F108 system exhibited a decrease in hydrodynamic diameter with appearance of only one peak confirming the existence of micelles with no unimer in the presence of reline, solely. Photophysical investigations using C343⁻ provided molecular‐level insight into how these structural reorganizations of reline affect the micellar systems. At > 50% water mole fraction, where reline primarily exists as a hydrated, less ordered medium dominated by urea–urea and chloride–water interactions, its components compete with PEO segments for hydrogen bonding. In compact P123 coronas, this competition strongly perturbs the PEO segments surrounded by water molecules, leading to red‐shifted spectra, significant lifetime decreases, and accelerated dye mobility upon reline loading. Conversely, in extended F108 coronas, the abundant hydration shells buffer against such displacement, yielding milder polarity changes, stabilized lifetimes, and locally stiffened dye environments, upon reline loading. Taken together, these results highlight a counterintuitive but crucial principle that although reline is inherently hydrophilic, it perturbs the hydrophobic P123 strongly than the highly hydrated F108. Even at very low reline mole fractions both P123 and F108 systems consistently exhibit biexponential anisotropy decays. This clearly indicates that, unlike simple DES–solvent binary mixtures, the micellar environment preserves microheterogeneity even at low DES content through time‐resolved anisotropy studies. This outcome underscores that DES–polymer interactions are dictated not only by bulk polarity but also by the availability of secondary and tertiary hydrogen‐bond donors/acceptors within the block copolymer architecture. Complementing these findings, SANS analysis confirmed that reline incorporation leads to an increase in both core and hard‐sphere radii with a concurrent decrease in micellar volume fraction, which can be understood in terms of decrease in the hydration amount as the micelles swell to accommodate reline. Thus, beyond advancing the mechanistic understanding of reline–Pluronic systems, our findings provide a conceptual framework for tuning micellar assemblies through reline additives. Such hybrid systems hold promise for the design of drug delivery vehicles and nanocarriers, where selective control of microenvironmental polarity, hydration, and dynamics can be strategically harnessed for biomedical and pharmaceutical applications.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: asia70840‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.