Design of fluorescent polymeric thermometers based on anthrapyrazolone functionalized oligo(ethylene glycol) methacrylates

Abstract

Temperature is one of the most important parameters for a wide range of applications, including biological and chemical systems, because it affects cellular activity by controlling metabolic processes within the cells as well as molecular diffusion and reaction kinetics. The anthrapyrazolone moiety has been used in a variety of applications due to its attractive photophysical characteristics. Nevertheless, its employment as a fluorescent probe for temperature sensing has been barely reported. In this contribution, we investigated the phase transition of poly(2-(2-methoxyethoxy)ethyl methacrylate) [P(MEO₂MA)] and poly[oligo(ethylene glycol) methyl ether methacrylates] [P(OEGMA₃₀₀ and OEGMA₅₀₀)], end-functionalized with a fluorescent anthrapyrazolone moiety [7-chloro-2-(2-hydroxyethyl) dibenzo[cd,g]indazol-6(2H)-one] (Cl-dye-OH) in aqueous solutions for the development of fluorescent polymeric thermometers. A fluorescence investigation revealed that P(MEO₂MA) displays nearly 100% fluorescence quenching above its T_CP, making it suitable as a sensor for a critical temperature, while the fluorescence intensity of the P(OEGMA) copolymers decreases gradually with increasing temperature, showing their promising potential for application as fluorescent thermometers. In vitro cytotoxicity tests on DC2.4 cells showed that none of the polymers were cytotoxic at the low concentrations that are used for sensing applications (i.e., 10 and 100 ng mL⁻¹). Unexpectedly, the P(OEGMA)-based fluorescence thermometers have a very broad sensing regime that spans the whole liquid water temperature range, which is significantly broader than the sharp LCST phase transition that usually spans only 10 °C. These polymers may be used to assess intracellular temperature since their fluorescence intensity decreases linearly with temperature. In contrast, P(MEO₂MA) can be used as a fluorescent probe to sense whether the temperature is below or above 25 °C. These polymeric temperature sensors are promising for future development of probes for intracellular temperature measurements.

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Introduction

Temperature is a fundamental physical parameter that governs all biological responses in living cells.^1,2 Biological actions that are critical for cellular activity occur either exothermically or endothermically at specific places inside a cell. According to medical research, the cellular pathogenesis of diseases is characterized by an excessive heat production.¹ As a result, measuring the internal temperature of live cells and tissue should lead to a better understanding of cellular activities and the development of innovative diagnostics and therapies.³ The smallest thermometers for this purpose are luminous molecular thermometers having temperature-dependent luminescence properties.⁴ Because of the small size of the molecules, luminous molecular thermometers work best in small spaces (<μm). Fluorescent dyes are generally considered for these applications for which they must have high photoluminescence quantum yield, a significant difference between excitation and emission strokes, biocompatibility, red to infrared emission for eliminating other biological environments in the background, and must be easy to synthesize.⁵

Pyrazolone and its derivatives are well-established molecules due to their broad spectrum of biological activity and wide-range application in designing bioactive molecules.⁶ Anthrapyrazolone molecules are fused systems of a pyrazole ring with different anthraquinone derivatives. These are very hydrophobic molecules and have limited solubility in polar solvents.⁷ In the last two decades, several anthrapyrazolone systems have been reported which are used as target-specific JNK inhibitors,^8–11 anti-cancer agents,^12–16 and DNA-intercalating agents.^17,18 Besides these applications, the acidic –NH protons in some anthrapyrazolone derivatives, like 1,9-pyrazoloanthrone, can be useful in the detection of fluoride and cyanide ions and thus extends their application as colorimetric and fluorometric turn-on chemosensors.¹⁹ Yang Hu et al. reported the synthesis of a fluorometric fluoride ion probe, based on anthra[1,9-cd]pyrazol-6(2H)-one.²⁰ Recently, Saravanan et al.²¹ developed copolymers functionalized with anthrapyrazolone derivatives that were successfully used for the detection of nitroaromatic compounds and other explosive analytes. Moreover, polymeric microparticles were reported for simultaneous adsorption and fluorescence detection of hazardous Cr(VI) ions.²² Although dyes with anthrapyrazolone moieties have been employed in various applications due to their appealing photophysical properties, their use as fluorescent probes in the biomedical field has been scarcely reported. Thus, the chemical incorporation of the anthrapyrazolone moiety within biocompatible polymers could lead to the synthesis of smart diagnostic materials.

Well-defined polymers are nowadays easy to access as controlled or living radical polymerization techniques showed a rapid development in the last few decades.²³ The reversible addition–fragmentation chain transfer (RAFT) polymerization is one of the most accessible controlled radical polymerization techniques due to its compatibility with various monomers and functionalities in the polymer chain, and easy access to polymers with advanced structures, pre-determined chain length, and molar mass.²⁴ The RAFT polymerization takes place in the presence of reversible chain transfer agents (CTAs), such as dithiobenzoates, xanthates, trithiocarbonates and dithiocarbamates.²⁵ The CTAs facilitate not only the synthesis of polymers with a narrow distribution and a high degree of functionality, but also the synthesis of advanced smart materials by incorporation of diverse stimuli responsive groups into their structure through chemical modification.^26,27

Thermoresponsive polymers having tunable lower critical solution temperature (LCST) with desired application can be synthesized via RAFT polymerization. Poly(oligo(ethylene glycol) methacrylate) (P(OEGMA)) based polymers have attracted great attention in the last couple of years due to their tunable LCST behavior.²⁸ Thermoresponsive LCST polymers show a solubility transition due to the change in entropy of solvation of the polymeric chain in aqueous solution as temperature changes.²⁹ Poly(N-isopropylacrylamide) (PNIPAM) is the most widely used thermoresponsive polymer having a sharp phase transition around 32 °C, which is between room and body temperature.³⁰ Thus, PNIPAM is a prominent candidate in biomedical applications.^31,32 However, PNIPAM possesses some inherent disadvantages, such as questionable biocompatibility and phase transition hysteresis. This hysteresis is because of dehydrated PNIPAM globules exhibiting intramolecular and intermolecular hydrogen bonding, leading to slow equilibration upon cooling. In contrast, ethylene glycol based macromonomers, the OEG methacrylate (OEGMA) based polymers, do not undergo intermolecular hydrogen bonding and thus exhibit limited hysteresis.^32–35 Poly(2-(2-methoxyethoxy) ethyl methacrylate) P(MEO₂MA) and P(OEGMA) are polymers principally composed of biocompatible oligo(ethylene glycol) units as poly(ethylene glycol) PEG showing interesting features such as higher biocompatibility and excellent blood circulation time which are suitable for biomedical applications.³⁵ To date, a great number of dyes have been used to synthesize polymeric thermometers either by incorporation into the side chain or as end groups by designing functional initiating or terminating agents.^36,37 Optical thermometers with naked eye detection were previously obtained by chemical modification of thermoresposive polymers such as PNIPAM or P(OEGMA) with merocyanine dye,³⁸ disperse red 1,³⁹ pyrene⁴⁰ or spiropyran.⁴¹ Although colorimetric thermometers have certain advantages, they are not suitable for intracellular temperature sensing and imaging where higher sensitivity is required. Thus, fluorometric thermometers have been thoroughly investigated, including linear polymers and cross-linked nanostructures such as nanogels and nanoparticles. Several soluble fluorometric thermometers which displayed a LCST behavior in the physiological temperature range were reported.^42–44 Furthermore, polymeric nanogel thermometers based on PNIPAM were successfully used for intracellular thermometry using microinjection technique.⁴⁵ Although the intracellular imaging results showed that the nanogel was distributed homogeneously in the cytoplasm, the techniques are only applicable to large cells. The macromolecular architecture of the thermoresponsive polymer was demonstrated to be crucial to the temperature-regulated fluorescence response. A comparison between PNIPAM and P(OEGMA) revealed that each polymer provides a different local micro-environment to the dye. For PNIPAM, an abrupt increase in fluorescence is found above the LCST due to a much more drastic change in chain packing density, whereas for P(OEGMA) polymers, minor changes can be observed.⁴⁶

Herein, we report our efforts to develop new polymeric fluorometric thermometers by harnessing the appealing photophysical properties of the anthrapyrazolone dye and the biocompatibility and well-defined characteristics of OEGMA-based polymers. In the first step, we synthesized a RAFT agent with an anthrapyrazolone moiety, 7-chloro-2-(2-hydroxyethyl)dibenzo[cd,g]indazol-6(2H)-one (Cl-dye-OH). Furthermore, we showed that the RAFT agent facilitates access to biocompatible and fluorescence labelled well-defined OEGMA homopolymers. Finally, the suitability of the polymers to act as fluorescent probes for temperature sensing is proven. As a result, these fluorescence labelled thermoresponsive homopolymers may be employed as thermometers for intracellular temperature monitoring in future work.

Experimental section

Materials

2-Hydrazinoethanol, dichloroanthraquinone, 4-cyano-4-(thiobenzoylthio)pentanoic acid, 4-dimethylamino pyridine (DMAP), triethylamine (TEA), 2-(2-methoxyethoxy)ethyl methacrylate (MEO₂MA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA₃₀₀ and OEGMA₅₀₀), ethylene dichloride (EDC) and 2,2′-azobis(isobutyronitrile) (AIBN) were purchased from Sigma Aldrich. AIBN was recrystallized twice from methanol before use. HPLC grade solvents N,N-dimethylacetamide (DMA), acetonitrile (ACN), diethyl ether (DEE), ethyl acetate (EtOAc), and dichloromethane were obtained from Sigma Aldrich, N,N′-dimethylformamide (DMF) was purchased from Biosolve and n-hexane was purchased from Fischer Scientific. All other solvents and chemicals used in this work were of analytical grade and used without further purification.

Instrumentation

¹H NMR spectra were recorded on a Bruker Avance 300 MHz at room temperature in chloroform-d (CDCl₃) purchased from Euriso-top. The chemical shifts are given in parts per million (δ), relative to the chemical shift of CDCl₃ at 7.24 ppm. Gas chromatography was performed on an Agilent 7890A system equipped with a VWR Carrier-160 hydrogen generator and an Agilent HP-5 column of 30 m length and 0.320 mm diameter. An FID detector was used and the inlet was set to 250 °C with a split injection ratio of 25 : 1. Hydrogen was used as carrier gas at a flow rate of 2 mL min⁻¹. The oven temperature was increased by 20 °C min⁻¹ from 50 °C to 120 °C, followed by a ramp of 50 °C min⁻¹ to 300 °C. Size-exclusion chromatography (SEC) was performed on an Agilent 1260-series HPLC system equipped with a 1260 online degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler (ALS), a thermostatted column compartment (TCC) at 50 °C equipped with two PLgel 5 μm mixed-D columns and a mixed-D guard column in series, a 1260 diode array detector (DAD) and a 1260 refractive index detector (RID). The eluent used was DMA containing 50 mM of LiCl at a flow rate of 0.500 mL min⁻¹. The spectra were analyzed using Agilent Chemstation software with the GPC add-on. The molar mass and dispersity values were calculated against PMMA standards from PSS. UV-Vis absorption spectra of the synthesized Cl-dye-OH molecule were recorded in methanol and ethylene glycol medium at different temperatures by using a UV-Vis spectrophotometer (Agilent, Cary 5000, double beam UV-Vis absorption spectrometer). UV-Vis spectra were also recorded on a Varian Cary 100 Bio UV-Vis spectrophotometer equipped with a Cary temperature and stir control. Samples were measured in quartz cuvettes with a path length of 1.0 cm in the wavelength range of 200–700 nm. Fluorescence measurements were performed using a fluorescence spectrometer Edinburgh FLS 1000. The emission spectra of the synthesized dye were measured at different excitation wavelengths, temperature, pH, and concentrations. The fluorescence measurements were also carried out on a Varian Cary Eclipse fluorescence spectrophotometer also equipped with a Cary temperature and stir control. The slit width of the excitation and emission were kept at 5 nm during the measurements. Deionized water was prepared with a resistivity less than 18.2 MΩ × cm using an Arium 611 system from Sartorius with a Sartopore 2150 (0.45 + 0.2 μm pore size) cartridge filter. Cloud point temperatures (T_CP) were measured on a Crystal16™ parallel crystallizer turbidimeter developed by Avantium Technologies connected to a recirculation chiller and dry compressed air. Aqueous polymer solutions (5 mg mL⁻¹) were heated from 20 to 95 °C with a heating rate of 0.5 °C min⁻¹ followed by cooling to 2 °C at a cooling rate of 0.5 °C min⁻¹. This cycle was repeated three times. The T_CP values are reported as the 50% transmittance temperature in the second heating run. Dynamic light scattering (DLS) was measured on a Zetasizer Nano-ZS Malvern apparatus (Malvern Instruments Ltd) using disposable cuvettes. The excitation light source was a He–Ne laser at 633 nm and the intensity of the scattered light was measured at an angle of 173°. This method measures the rate of intensity fluctuation and the size of the particles is determined through the Stokes–Einstein equation. The concentration of the polymer solution was 1.5 mg mL⁻¹ in all cases.

Cytotoxicity

MTT assay was performed at three concentrations, 1000, 100 and 10 ng mL⁻¹, for each sample in DC2.4 cells. In more detail, DC2.4 cells were seeded in 96-well plates at a density of 20 000 cells in 180 μL per well and incubated at 37 °C overnight. Each well was pulsed with 20 μL of the tested samples, and PBS or DMSO in equal volume were included as a negative control (100%) or a positive control (0%), respectively. After overnight incubation at 37 °C, the medium was removed and the cells were washed with 200 μL of PBS. To each well, 100 μL of MTT reagent (5 mg mL⁻¹ in PBS) was added and incubated for 2 h at 37 °C. The formed formazan crystals were dissolved by adding 100 μL of DMSO and incubated for 10 min. Quantification was carried out by measuring the absorbance at 590 nm using a microplate reader (n = 6, data shown as mean ± SD).

Methods

Synthesis of OH terminated fluorescent dye 7-chloro-2-(2-hydroxyethyl)dibenzo[cd,g]indazol-6(2H)-one (Cl-dye-OH)

7-Chloro-2-(2-hydroxyethyl)dibenzo[cd,g]indazol-6(2H)-one (Cl-Dye-OH) was synthesized as described by Hasinoff et al.⁴⁷ 2-Hydrazinoethanol (5.2 g, 68 mmol) was added to a suspension of dichloroanthraquinone (6.92 g, 25 mmol) in acetonitrile (120 mL) and DMF (10 mL). Triethylamine (4.0 mL) was then added and the mixture was refluxed for 60 hours (Scheme 1). The mixture was cooled to room temperature and placed in a freezer overnight. The resulting crystals were filtered, washed twice with cooled acetonitrile, and dried to afford orange crystals. After column chromatography separation using an EtOAc–hexane mixture, the product was obtained as pure yellow-orange color flakes (5.25 g; yield 70%). ¹H NMR and ¹³C NMR spectra of the compound are shown in (Fig. S1 and S2, ESI†) respectively.

Scheme 1 Reaction scheme for the synthesis of 7-chloro-2-(2-hydroxyethyl)dibenzo[cd,g]indazol-6(2H)-one.

Synthesis of fluorescent dye ester RAFT CTA

4-Cyano-4-(thiobenzoylthio)pentanoic acid (0.533 g, 1.9 mmol), Cl-dye-OH (0.57 g, 1.9 mmol), and DMAP (23.3 mg, 0.19 mmol) were introduced into a round-bottom flask and dissolved in anhydrous dichloromethane (DCM, 55 mL) (Scheme 2). The reaction mixture was cooled to 0 °C in an ice bath and a solution of EDC (403 mg, 2.09 mmol) in DCM (10 mL) was added drop-wise while stirring vigorously. The reaction mixture was stirred in an ice bath for 2 h and subsequently at room temperature overnight. The solvent was evaporated under vacuum. The crude product was purified by column chromatography on silica gel using dichloromethane (DCM) as the eluent. The first fraction was collected and the solvent was removed under reduced pressure to obtain the product as a red colored solid (0.82 g, yield 76%). ¹H NMR and LCMS spectra of the compound are shown in (Fig. S3 and S4, ESI†) respectively.

Scheme 2 Reaction scheme for the synthesis of RAFT CTA.

Synthesis of P(MEO₂MA)

MEO₂MA (2.0 g, 10.6 mmol), CTA (59.5 mg, 0.11 mmol), 2,2′-azobis(isobutyronitrile) (AIBN) (3.49 mg, 2.1 × 10⁻² mmol), and DMF (3.1 mL) were sealed in a 25 mL Schlenk tube ([MEO₂MA]/[CTA]/[AIBN] = 100/1/0.2). The polymerization mixture was first degassed by purging argon to remove residual O₂ for 50 minutes and then placed in a preheated oil bath at 70 °C. Samples were removed periodically using a syringe to determine the conversion using GC and the molar mass and dispersity (Đ) by size exclusion chromatography (SEC). The final polymer was isolated by precipitating into hexane/DEE (80/20) (50-fold excess). After removal of the solvents and residual monomers, the polymers were dried in a vacuum oven at 40 °C overnight prior to analysis.

A kinetic study of the RAFT polymerization of MEO₂MA with the CTA was performed by analyzing samples withdrawn from the polymerization mixture at different times with GC as described earlier. The RAFT polymerization of P(MEO₂MA) was well controlled, as demonstrated by the linear pseudo-first-order kinetics (Fig. 1). The systematic transition of SEC traces to lower retention times indicates the controlled growth of the polymer chains with time.

Fig. 1 (a) (left) (ln([M]₀/[M]_t)) plotted against time for the RAFT polymerization of MEO₂MA using CTA and (right) overlay of SEC traces that show the progress of the polymerization. (b) Corresponding number-average molecular weight (M_n) against conversion, including dispersities (Đ) for the homopolymerization of MEO₂MA.

Synthesis of P(OEGMA₃₀₀)

OEGMA₃₀₀ (2.0 g, 6.7 mmol), CTA (37.3 mg, 6.7 × 10⁻² mmol), 2,2′-azobis(isobutyronitrile) (AIBN) (2.19 mg, 1.3 × 10⁻² mmol), and DMF (2.4 mL) were sealed in a 25 mL Schlenk tube ([OEGMA₃₀₀]/[CTA]/[AIBN] = 100/1/0.2). The polymerization mixture is first degassed by purging argon to remove residual O₂ for 50 minutes and then placed in a preheated oil bath at 70 °C. Samples were removed periodically using a syringe to determine the conversion using ¹H NMR spectroscopy and the molecular weight and Đ by SEC. The final polymer was isolated by precipitating into hexane/DEE (80/20) (50-fold excess). After removal of the solvents and residual monomers, the polymers were dried in a vacuum oven at 40 °C overnight prior to analysis.

A kinetic study of the RAFT polymerization of OEGMA₃₀₀ with the CTA was performed by analyzing the samples withdrawn from the polymerization mixture at different times via¹H NMR spectroscopy using CDCl₃ solvent as described earlier (Fig. S5, ESI†). The RAFT polymerization of the poly(OEGMA₃₀₀) showed a long inhibition period and the polymer started forming after 3.5 h. SEC trace of the final sample is shown in (Fig. S6, ESI†).

Synthesis of P(OEGMA₅₀₀)

OEGMA₅₀₀ (2.0 g, 4.0 mmol), CTA (22.4 mg, 4.0 × 10⁻² mmol), 2,2′-azobis(isobutyronitrile) (AIBN) (1.31 mg, 8 × 10⁻³ mmol), and DMF (2.0 mL) were sealed in a 25 mL Schlenk tube ([OEGMA₅₀₀]/[CTA]/[AIBN] = 100/1/0.2). The polymerization mixture was first degassed by purging argon to remove residual O₂ for 50 minutes and then placed in a preheated oil bath at 70 °C. Samples were removed periodically using a syringe to determine the conversion using ¹H NMR spectroscopy and the molecular weight and Đ by SEC. The final polymer was isolated by precipitating into hexane/DEE (80/20) (50-fold excess). After removal of the solvents and residual monomers, the polymers were dried in a vacuum oven at 40 °C overnight prior to analysis.

A kinetic study of the RAFT polymerization of OEGMA₅₀₀ with the CTA was performed by analysing the samples withdrawn from the polymerization mixture at different times via¹H NMR spectroscopy using CDCl₃ solvent as described earlier (Fig. S7, ESI†). The RAFT polymerization of P(OEGMA₅₀₀) showed a slow increase in monomer conversion with time, which was supported by the SEC traces of the samples taken at different time intervals (Fig. S8, ESI†).

Results and discussion

The synthesis of well-defined dye-labeled P(MEO₂MA), P(OEGMA₃₀₀), and P(OEGMA₅₀₀) was performed by RAFT. These polymers were prepared in DMF, at 70 °C in the presence of AIBN and the Cl-dye-OH functionalized RAFT CTA. The successful synthesis of Cl-dye-OH was confirmed with ¹H-NMR and ¹³C-NMR spectroscopy (Fig. S1 and S2, ESI†). In the next step the dye modified RAFT-CTA was obtained by reacting Cl-dye-OH with 4-cyano-4-(thiobenzoylthio)pentanoic acid and its structure was confirmed by ¹H-NMR spectroscopy and mass spectrometry (Fig. S3 and S4, ESI†). The dye modified RAFT-CTA was used for the polymerization of MEO₂MA, OEGMA₃₀₀ and OEGMA₅₀₀ using previously optimized procedures.^48–50 The polymerization kinetics of the OEGMA monomers was investigated at 70 °C and the conversion was followed by GC for MEO₂MA and ¹H NMR for OEGMA₃₀₀ and OEGMA₅₀₀, respectively.

The first-order kinetic plots of monomer conversion with respect to the reaction time revealed a linear relationship (Fig. 1(a)) for MEO₂MA, thus demonstrating a constant amount of propagating species indicative of the absence of termination. A short initial inhibition period was commonly observed for dithiobenzoate mediated RAFT polymerization of methacrylate monomers (Fig. 1(a)).^51,52 SEC analysis revealed a linear increase in number average molecular weight (M_n) with conversion and relatively low dispersity (Đ < 1.25) for the polymer (Fig. 1(b) and (c)), demonstrating that the polymerization reactions proceeded in a controlled manner. The observed molecular weight data from SEC cannot however be compared with theoretical data as the PMMA standards used to perform the calibration are probably not suitable for the synthesized PMEO₂MA. After 6 h of polymerization, the MEO₂MA polymer with a maximum conversion of 42% and nearly 8.5 kg mol⁻¹ molecular mass was obtained. The final polymer SEC trace revealed a M_n of about 11 kg mol⁻¹ and a Đ of 1.23. The polymerization kinetics of other two monomers OEGMA₃₀₀ and OEGMA₅₀₀ were investigated using ¹H NMR spectroscopy. Interestingly, a very long inhibition period was observed during the polymerization of OEGMA₃₀₀. To determine the actual cause of this behavior for OEGMA₃₀₀, more research is required, albeit it may be speculated that the degassing was less efficient leading to more residual oxygen. After 8 hours of polymerization, a degree of polymerization (DP) of 28 was attained, resulting in a theoretical molar mass of around 9 kg mol⁻¹. The final polymer SEC trace revealed a M_n of about 12 kg mol⁻¹ and a Đ of 1.40. In contrast, kinetic investigation of OEGMA₅₀₀ by ¹H NMR spectroscopy and SEC revealed an increase in the conversion percentage and molecular weight with time. However, after 2 h of polymerization, no substantial increase in the molecular weight was observed. This might be due to an increase in viscosity, which reduces the probability of chain–chain contact, making the degenerative chain transfer process less likely.⁵³ After 6 h of polymerization, an OEGMA₅₀₀ polymer with a maximum conversion of 38% and nearly 19 kg mol⁻¹ molecular mass was obtained. The final polymer SEC trace revealed a M_n of about 12 kg mol⁻¹ and a Đ of 1.24.

Thermoresponsive behavior

The properties of the dye-functionalized polymers P(MEO₂MA), P(OEGMA₃₀₀), and P(OEGMA₅₀₀) were studied in an aqueous medium by turbidimetry and DLS. The cloud point temperatures (T_CP's) were measured in deionized water at a reference concentration of 5 mg mL⁻¹ (Fig. 2). Polymers possessing an LCST behavior will be insoluble in water above the T_CP and soluble below the T_CP. Clouding or precipitation of polymers can be observed when a clear polymer is heated across the T_CP if the concentration of the polymer is sufficiently high. This property enables us to accurately measure the T_CP's of polymers through turbidimetry, which usually is a fully reversible transition in a very small temperature range.⁵⁴ The turbidity curves for the as-prepared dye functionalized polymers from the second heating run are displayed in Fig. 2. At low temperatures, the polymer solution has close to 100% transmittance, indicative of a clear polymer solution. Upon increasing the temperature, the solution phase separates resulting in the formation of a high polymer concentration dispersed phase that scatters away light as indicated by 0% transmittance. The polymers showed sharp LCST transitions during heating with clouding of the solution in a very narrow temperature range of 1 °C to 3 °C. The T_CP values of P(MEO₂MA), P(OEGMA₃₀₀), and P(OEGMA₅₀₀) were found to be 21.8 °C, 61.8 °C, and 85.5 °C, respectively. The obtained values are within the range of reported values in the literature for the OEGMA polymers (e.g., 28, 65 and 90 °C for P(MEO₂MA), P(OEGMA₃₀₀), and P(OEGMA₅₀₀) respectively).^55–58 Nevertheless, the T_CP's of the herein synthesized polymers were slightly lower compared to previous reports due to the hydrophobic dye end-group that reduces the LCST.^51,59 Thus, the decrease in T_CP can be attributed to the presence of the bulky hydrophobic dye end groups possibly favoring the dehydration and phase separation of the polymer. The thermoresponsive phase transitions were found to be fully reversible although a very small hysteresis was observed in the cooling process.

Fig. 2 Transmittance versus temperature profiles for the second heating (solid lines) and cooling (dashed lines) cycles in deionized water (5 mg ml⁻¹; 0.5 °C min⁻¹).

It is obvious that the number of ethylene glycol units on each repeating unit has an effect on the T_CP of the polymer, whereby more ethylene glycol units lead to higher hydrophilicity and higher T_CP.^60,61 MEO₂MA seems to be the most promising candidate for in vivo biomedical applications, that is, fluorescence imaging or drug delivery, showing a response within the relevant biological window, ranging from room temperature to body temperature. Nevertheless, P(OEGMA₃₀₀) and P(OEGMA₅₀₀) can also be considered as viable candidates for in vitro applications such as cell culture and tissue engineering. Further on, DLS measurement of P(MEO₂MA) (Fig. 3) was conducted at different temperatures at 1.5 mg mL⁻¹ concentration to follow the changes in the particle size, size distribution and scattering intensity. Below T_CP, objects with a hydrodynamic diameter around 10 nm were detected, indicating the presence of individual polymer chains, but as the temperature increased to near T_CP, a rapid increase in diameter was observed to around 500–1000 nm. This result confirms the phase separation of the solution and the formation of a high polymer concentration dispersed phase.⁶⁰ The T_CP value determined from DLS shows a similar value with the one determined from turbidimetry measurements.

Fig. 3 Dynamic light scattering (DLS) data for a 1.5 mg mL⁻¹ aqueous solution of P(MEO₂MA) at various temperatures.

DLS temperature sweep experiments were also performed to study the influence of polymer solution concentration on the aggregation behavior during heating of the P(MEO₂MA) aqueous solutions (Fig. S9, ESI†). The phase separation into a high polymer concentration dispersed phase was observed when the solutions were heated above the T_CP, in all cases, while the T_CP seemed to be concentration independent at least in the studied polymer concentration interval (i.e, 0.5–3 mg mL⁻¹).

Photophysical properties

The photophysical properties of Cl-dye-OH, dye-functionalized P(MEO₂MA), P(OEGMA₃₀₀), and P(OEGMA₅₀₀) were investigated via UV-Vis and fluorescence spectroscopy. The absorption spectrum of Cl-dye-OH shows π → π* transitions at 280 nm and an intramolecular charge-transfer (ICT) band at 415 nm.²¹ Similar transitions were observed in the dye-functionalized polymers indicating that the conjugation to the polymer did not alter the electronic properties of the dye, which was expected as the coupling site was decoupled from the conjugated system of the dye (Fig. 4). For P(MEO₂MA), it is observed that the π → π* transitions are more broad as a result of the overlapped absorption from the dithiobenzoate ester of the RAFT end-group.⁶² The emission spectrum of Cl-dye-OH showed a strong fluorescence at 500 nm on excitation at 415 nm with a lifetime of 2.72 ns (Fig. S10, ESI†). A similar emission behavior is observed for the three polymers. Furthermore, the temperature sensing ability of the polymers in aqueous solution was investigated by temperature-controlled fluorescence spectroscopy at a polymer concentration of 1.5 mg mL⁻¹. The fluorescence emission spectra of the polymers were obtained upon excitation at 415 nm. To understand the temperature response behavior of the polymers, we have investigated the photophysical behavior of Cl-dye-OH as a function of temperature and excitation-dependent emission in methanol and highly viscous ethylene glycol media. The absorption spectra of Cl-dye-OH (0.99 μmol) in methanol and ethylene glycol media do not show any significant changes as a function of temperature in methanol and a minor decrease in ethylene glycol, which might be related to a decrease in viscosity (Fig. S11, ESI†).

Fig. 4 Absorption spectra of Cl-Dye-OH in methanol and P(MEO₂MA), P(OEGMA₃₀₀) and P(OEGMA₅₀₀) in deionized water.

The excitation dependent emission of Cl-dye-OH (0.99 μmol) in the ICT region (350–450 nm) shows that the obtained emission is independent of the excitation wavelength and results from the S₁ → S₀ state (Fig. S12, ESI†). The temperature-dependent emission in methanol reveals that an increase in the temperature from 20–55 °C leads to 22% quenching of emission intensity, while in ethylene glycol, 32% quenching was observed with an increase in the temperature from 20–80 °C (Fig. S13, ESI†). As these quenching results demonstrate only minor quenching upon heating the dye solutions, they further provoke to conjugate the dye to the thermoresponsive polymers P(MEO₂MA), P(OEGMA₃₀₀), and P(OEGMA₅₀₀) to enhance the temperature sensing ability.

The P(MEO₂MA) polymer exhibited near complete fluorescence quenching (∼96%) upon heating across the LCST phase transition (Fig. 5), which is similar to the previously reported systems in which the change in polarity around the dye suddenly changes upon dehydration of the polymer chains leading to aggregation of the fluorophores.^39,46,63 This behavior is in sharp contrast with the fluorescent molecular thermometers reported by Uchiyama, where an increase in emission was observed upon increasing the temperature. However, Uchiyama et al.,⁶⁴ used a fluorophore with a benzofuran structure that shows higher fluorescent yield with decreasing solvent polarity, whereas for anthrapyrazolone, the opposite behavior is expected.⁶⁵ The temperature at which the emission change occurs nicely corresponds with the T_CP of the polymers (Fig. 5 and Fig. S14, ESI†), thus demonstrating the applicability of polymers as fluorescent probes for temperature sensing. Although the P(MEO₂MA) polymer has the most attractive T_CP from a biomedical point of view, the high, sudden fluorescence quenching with increasing temperature makes it unsuitable for accurate temperature recording during cell imaging, besides determining whether certain compartments are beyond a critical temperature that induces the quenching. The polymer demonstrated a three times stronger quenching behavior than Cl-dye-OH. The effect of the polymer concentration on the fluorescence behavior was also examined (Fig. S14, ESI†) for P(MEO₂MA). The threshold of the emission increased with decreasing polymer concentration, reaching 30 °C at 0.5 mg mL⁻¹ concentration. The I/I₀ ratio versus temperature plot shows three distinct regimes. Below 20 °C, the I/I₀ ratio is constant suggesting that the polymer is fully soluble in an aqueous environment. From 20 to 30 °C, a strong decrease in the I/I₀ ratio can be noticed, while above 30 °C, the I/I₀ ratio shows a very minor decrease, suggesting that the polymer is found in the collapsed state. Therefore, P(MEO₂MA) can be used as a fluorescent thermometer to record the critical temperature with a temperature in between 20 to 30 °C, depending on the polymer concentration (Fig. S15, ESI†). This behavior is further confirmed by concentration dependent emission of Cl-dye-OH. With an increase in concentration, the emission intensity increased gradually and at higher concentrations, the emission intensity decreased presumably due to the formation of aggregated structures (Fig. S16, ESI†). Thus, we were able to tune the sensing regime of the fluorescent thermometers by simply varying the chemical structure of the monomer.

Fig. 5 Fluorescence spectra in DW as a function of temperature for: (a) P(MEO₂MA). (b) P(OEGMA₃₀₀), and (c) P(OEGMA₅₀₀). The polymer concentration was 1.5 mg mL⁻¹ and the excitation wavelength was 415 nm in all cases.

In contrast to the P(MEO₂MA), the anthrapyrazolone-functionalized POEGMA polymers revealed a continuous gradual decrease in the fluorescence intensity with increasing temperature over the entire liquid water temperature range (Fig. 5 and 6), which might be exploited for intracellular temperature detection. To quantify the sensing ability of the POEGMA polymers, the relative fluorescence intensity (I/I₀) versus temperature was plotted since this ratio is expected to be independent of fluctuations in polymer concentration, making the read-out of the sensor more robust. For both POEGMA polymers, the I/I₀ ratio shows a linear correlation with temperature with a very good coefficient of determination (Fig. 6). Moreover, the fluorescent thermometers show a very broad sensing regime spanning the entire liquid water temperature scale. This rather surprising observation can be ascribed to the much more hydrophilic nature of POEGMA compared to P(MEO₂MA) leading to stronger interactions with water. Hence, the degree of hydration more strongly depends on the temperature, and even above the T_CP the collapsed high polymer concentration phase retains a significant amount water which continues to gradually decrease with increasing temperatures.

Fig. 6 I/I₀ ratio of emission intensities as a function of temperature for POEGMA copolymers at 1.5 mg mL⁻¹ polymer concentration.

In vitro cytotoxicity

The in vitro cytotoxicity of three polymers, P(MEO₂MA), P(OEGMA₃₀₀), and P(OEGMA₅₀₀), at different concentrations was assessed in DC2.4 cells, an immortalized mouse dendritic cell line. As shown in Fig. 7, upon incubating for 24 h, at low concentration (i.e., 10 and 100 ng mL⁻¹), none of the polymers caused cytotoxicity to DC2.4 cells. At a higher concentration, i.e. 1000 ng m⁻¹, a slightly reduced cell viability was observed for all polymers, but still above the ISO norm of 70%.

Fig. 7 Viability of DC2.4 cells in the presence of polymers at different concentrations, as measured using MTT assay. (n = 6, mean ± SD).

Conclusions

In this study well-defined, thermoresponsive homopolymers of MEO₂MA, OEGMA₃₀₀, and OEGMA₅₀₀ end-functionalized with an anthrapyrazolone fluorescent dye were synthesized by RAFT polymerization. All three polymers showed LCST phase separation with a T_CP that increased with increasing polymer hydrophilicity, as expected. As the polarity around the anthrapyrazolone dye changes abruptly following dehydration of the polymer chains, fluorescence studies show a fluorescence quenching behavior above the T_CP of the polymer, covering the dye with the collapsed polymer. These mechanisms were supported by the turbidity and DLS studies. P(MEO₂MA) can be used as a sensor for critical temperatures, as it gives a sharp decrease in fluorescence when passing the T_CP and this critical temperature sensing range can be varied between 20 to 30 °C by varying the polymer concentration. In contrast, and rather surprisingly, the dye-functionalized P(OEGMA₃₀₀) and P(OEGMA₅₀₀) revealed a linear decrease in the fluorescence intensity, which could be used to detect the temperature of the solution in between 10 °C and 90 °C, which is an unusually broad sensing range spanning the entire liquid water temperature scale. This broad temperature sensing regime was correlated with the higher hydrophilicity of these POEGMA polymers, leading to more efficient hydration and enabling gradual dehydration upon heating, even in the high polymer concentration phase separated state. Thus, these fluorescence labelled thermoresponsive homopolymers are promising for future use as thermometers for intracellular temperature sensing.

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

S. S. and A. B. thank SRM IST for providing fellowship to support their PhD program. S. M. acknowledges SRMIST for providing SRMIST seed grant and the Science and Engineering Research Board (SERB), India, for a core research grant (CRG/2021/004203). R. H. thanks the research foundation Flanders (FWO) and Ghent University (BOF) for financial support. V. V. J. acknowledges the Romanian Ministry of Research, and Innovation and Digitization, CNCS/CCCDI – UEFISCDI for the financial support, project number PN-III-P1-1.1-TE-2019-1696 within PNCDI III.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: The structural characterization like NMR, LC-MS of the CTA, SEC results of P(OEGMA₃₀₀) and P(OEGMA₅₀₀), DLS data of P(MeO₂MA); photophysical properties of anthrapyrazolone dye and other additional data. See DOI: https://doi.org/10.1039/d3qm00019b

‡ These authors contributed equally to this work.

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