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The Temperature-Responsive Nanoassemblies of Amphiphilic Random Copolymers Carrying Poly(Siloxane) and Poly(Oxyethylene) Pendant Chains

Elisa Martinelli,a Luisa Annunziata,a Elisa Guazzelli,a Andrea Pucci,a,b Tarita Biver,a Giancarlo Gallia,b*

*Corresponding author: e-mail: giancarlo.galli@unipi.it

Novel amphiphilic random copolymers carrying poly(oxyethylene) and poly(siloxane) pendant chains were synthesized by atom transfer radical polymerization starting from either a fluorescent, julolidine-based initiator or a non-fluorescent initiator. For both copolymer systems, dynamic light scattering measurements carried out on aqueous solutions as a function of temperature revealed the occurrence of a sharp and fully reversible transition between two different self-associative states of individual, single-chain self-folded nanoassemblies, so-called unimer micelles, (Dh = 810 nm) and collapsed multi-chain aggregates (Dh = 7001400 nm) at a critical temperature Tc. Covalently linked julolidine terminal and added ethidium bromide were separately used as fluorescent probes and both proved the temperature-dependence of the different self-association of the copolymers in water.

1. Introduction

The selective self-folding, assembly, and sorting of amphiphilic random copolymers in aqueous solution is currently considered as one of the major tools to precisely build up globular soft nanoobjects with well-defined and functional compartments, capable of mimicking natural biomacromolecules.[1] In fact, amphiphilic random copolymers, in which the hydrophilic and hydrophobic co-units are statistically distributed along the macromolecular backbone, have been shown to self-fold in water into single-chain nanoassemblies, so-called unimer micelles, via the intramolecular interactions of the hydrophobic component.[2–6] Another current approach to the preparation of single-chain polymer nanoassemblies, so-called polymeric nanoparticles, exploits single-chain selective point folding and repeat unit folding of well-defined linear polymers.[7] This self-associative process basically contrasts that typical of the block copolymers, where the microphase separation of the blocks drives intermolecular self-assembly of multi-chain aggregates.[8–11]

Amphiphilic random copolymers consisting of hydrophilic poly(ethylene glycol) methacrylate (PEGMA) and various hydrophobic methacrylates carrying different pendant functionalities, including alkyl, fluoroalkyl and hydrogen-bonding amide groups were recently described as self-folding polymers in aqueous solutions.[12–17] In any case, the hydrophilic shell shielded the hydrophobic core and effectively stabilized the formation of single-chain nanoassemblies in water. On this basis, in this work we synthesized novel PEG-based amphiphilic random copolymers containing a poly(siloxane) methacrylate as the hydrophobic component, by copper-based atom transfer radical polymerization (ATRP) starting from either a new fluorescent julolidine-labeled initiator or a conventional non-fluorescent initiator. Polysiloxanes are water-repelling materials able to self-assemble in the bulk and at the surface as a result of their weak cohesion forces and low surface energy.[18–21] Fluorescence emission spectroscopy is a powerful technique to investigate self-folding and conformational changes of biomacromolecules and single-chain nanoparticles.[22,23] Fluorescent comonomers were also incorporated into amphiphilic self-folding polymers.[24] Here, we endowed the random amphiphilic copolymers with fluorescence properties by use of either an added water-soluble ethidium bromide or a covalently linked water-insoluble julolidine moiety to study the temperature-dependent association behavior of the copolymers in water. We demonstrate the occurrence of a reversible transition from an intramolecular, single-chain, self-association to an intermolecular multi-chain aggregation at a critical temperature Tc by combined dynamic light scattering and fluorescence emission spectroscopy measurements. This novel type of amphiphilic copolymers may constitute a versatile platform for application in diverse fields where precisely designed nanoobjects provide a soft, low Tg, functional compartment to express special chemical activities at a low and tunable Tc.

2. Results and Discussion

2.1. Synthesis of the copolymers

Novel amphiphilic random copolymers from hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 300 g mol–1, Đ = 1.2) and hydrophobic poly(dimethyl siloxane) methacrylate (SiMA, Mn = 680 g mol–1, Đ = 1.2) were synthesized via ATRP (Figure 1).

Figure 1. Reaction scheme for the synthesis of the initiator 9-[2-(bromo-2-methylpropanoyloxyethyloxycarbonyl)-2-cyanovinyl]julolidine (JBr); ATRP synthesis of the copolymers with different contents x of SiMA counits by JBr (x = 6 mol%) and EBPA (x = 5, 15 mol%) initiators; Chemical structure of the fluorophore ethidium bromide (EtBr).

The initial molar ratios monomer:initiator (100:1) and initiator:PMDETA:CuBr (1:1:1) were kept constant, while the SiMA content in the PEGMA/SiMA feed was varied in the range 1030 mol%, in order to yield water soluble copolymers with different amphiphilicity. The copolymers PEGMA-co-SiMAx (x = 5 and 15 mol% SiMA) were obtained by using a conventional non-fluorescent initiator, ethyl α-bromophenylacetate (EBPA), while the copolymer J(PEGMA-co-SiMAx) (x = 6 mol% SiMA) was synthesized by starting from a fluorescent initiator, 9-[2-(bromo-2-methylpropanoyloxyethyloxycarbonyl)-2-cyanovinyl]julolidine (JBr) (Figure 1), designed in such a way to impart to the isobutyryl terminal the viscosity-sensitive properties of a julolidine-based fluorescent molecular rotor (FMR).[25] Thus, the ATRP of the two comonomers by JBr- and EBPA-initiation enabled the synthesis of copolymers with identical composition of SiMA and PEGMA co-units. Relatively high molecular weight samples were obtained, eg Mn,SEC = 33200 g mol–1 for J(PEGMA-co-SiMA6), but narrower molecular weight distributions were detected for EBPA-initiated samples, eg Đ = 1.5 for PEGMA-co-SiMA5. These findings suggest that the julolidine moiety may interfere with the copper-mediated ATRP of SiMA/PEGMA.[26] Owing to the amphiphilic nature of the copolymers, extensive purification required numerous non-solvent precipitation cycles. The reactivity ratios evaluated were rPEGMA = 0.61 and rSiMA = 0.64 (Finemann-Ross method) and rPEGMA = 0.64 and rSiMA = 0.68 (Kelen-Tudos method) (Supporting Information), which shows that sequencing of the different co-units along the polymer backbone was random, with a slight tendency to alternation.

JBr was synthesized according to a four-step procedure (Figure 1); JBr is insoluble in water and its spectroscopic properties were evaluated in chloroform and mixtures of methanol/glycerol with different viscosities. Chloroform solutions showed an absorption maximum at 454 nm (ε = 52700 M–1 cm–1) and an emission maximum at 489 nm with a negligible fluorescence quantum yield (Φ = 1.4610–3), owing to the preferential formation of a non-emissive twisted intramolecular charge transfer (TICT) excited state[27,28] (Supporting Information, Figure S1). The resulting Stokes shift of 35 nm was similar to that typical of other cyanovinyl julolidine compounds.[25] JBr underwent a significant increase in fluorescence emission intensity and fluorescence quantum yield (Φ = 2.3510–3) when dissolved in viscous environments like methanol/glycerol mixtures at increasing contents of glycerol (Figure 2, Table S1). In agreement with the typical mechanism of julolidine-based FMRs,[29] the molecular internal rotation of JBr is hampered in viscous media, thus promoting the emission from the local excited (LE) state and, in turn, the increase in quantum yield. Fluorescence emission of JBr solutions followed a Förster-Hoffmann behavior, the viscosity sensitivity (x = 0.48) of JBr (for x, see Supporting Information) being consistent with its ability to act as an environmental microviscosity probe (Figure S2).[28] The spectroscopic properties of J(PEGMA-co-SiMA6) copolymer were comparable with those of the initiator JBr, showing absorption and emission peaks in chloroform solution at 454 nm and 489 nm (Φ = 6.3910–3), respectively (Figure S3). Therefore, the JBr chromophore did not experience significant variation in its optical properties when incorporated into the amphiphilic copolymer.

(a)(b)

Figure 2. (a) Fluorescence emission spectra of JBr in methanol/glycerol (v/v) solutions (exc = 410 nm, ~110–5 M); (b) Picture of JBr solutions (exc ~450 nm (Dark Reader 46B transilluminator Clare Chemical Research)). The corresponding UV-vis absorption spectra are reported in Figure S4.

2.2. Dynamic light scattering in water

Dynamic light scattering experiments of the copolymers were carried out to evaluate their ability to spontaneously single-chain fold in water. The DLS Dh distribution by volume for water solutions of copolymer J(PEGMA-co-SiMA6) is shown in Figure 3a (for other representative DLS distributions, see Figure S5a).

(a) (b)

Figure 3. DLS average Dh distributions for copolymer J(PEGMA-co-SiMA6) in water (5 mg mL–1); (a) At 25 °C (a); (b) At 60 °C.

The main signal centered at Dh ~10 nm was associated with the formation of nanoassemblies of single-chain folded copolymer, consistent with previous results on PEGMA-based amphiphilic random copolymers.[15,24] Formation of single-chain nanoassemblies was induced by the self-association of the hydrophobic poly(siloxane) SiMA chains in an inner core, which was shielded from the contact with water by a shell of hydrophilic poly(oxyethylene) PEGMA chains. A negligible population of larger particles (Dh ~200–500 nm) was also present in the volume Dh distribution (Figure 3a). All the copolymers presented a lower critical solution temperature- (LCST)-type phase separation in water at a critical temperature Tc, according to a thermoresponsive behavior of (co)polymers of PEGMA with ~2–9 oxyethylene units in the side chain.[15,24,30,31] At Tc the single-chain nanoassemblies collapsed into larger multi-chain aggregates (Dh ~700 nm) with a monomodal narrow distribution (Figure 3b and Figure S5b). Above Tc, these sub-micrometer aggregates were stable in size, but they reversed to single-chain nanoassemblies on cooling below Tc with no temperature hysteresis. The LCST transition occurred sharply and reversibly on repeated heating-cooling cycles. A cloud point (Cp) of the water solutions of copolymers was identified as the temperature of 50% transmittance by temperature-dependent measurements of the transmitted light (λ = 700 nm) (Figure 4 and Figure S6). Cp slightly decreased with increasing content of SiMA counits in the copolymer, passing from 52 °C for PEGMA-co-SiMA5 to 47 °C for PEGMA-co-SiMA15. A higher value of Cp of 58 °C was found for J(PEGMA-co-SiMA6). Thus, the covalent attachment of the fluorophore to the copolymer chain did not inhibit its self-assembling capacity but possibly introduced differences in the amphiphilicity of the two polymer systems. Differences in molar masses and molar mass distributions may also affect the value of Cp.

(a)(b)

Figure 4. Trend of light transmittance (λ = 700 nm) of PEGMA-co-SiMA5 (a) and J(PEGMA-co-SiMA6) (b) water solutions (5.0 mg mL–1) with temperature (heating-cooling rate and range: 5 °C min–1, 2570 °C).

2.3. Fluorescence emission in water

Fluorescence emission spectroscopy was employed to probe the single-chain folding and temperature-induced self-association of the copolymers in water by investigating the fluorescence response of two different fluorophores, i.e. the added ethidium bromide and the covalently linked julolidine terminal.

Ethidium bromide fluorophore: Ethidium bromide (EtBr) (Figure 1) is a water-soluble chromophore molecule capable of tightly intercalating into DNA with subsequent enhancement in fluorescence emission intensity.[32] By contrast, free EtBr molecules in water solution can dissipate excitation energy via non-radiative pathways, quenched by polar mobile solvent molecules. These interactions with the solvent are inhibited when EtBr is confined within DNA chains, so that relaxation is only possible through photon emission. The intensity of fluorescence emission of EtBr (λexc = 520 nm) was found to increase when it was added to a water solution containing PEGMA-co-SiMA5 (Figure 5a), manifesting a preferential hydrophobic interaction between the aromatic core of EtBr and the inner compartment of the single-chain self-folded copolymer.

(a)

(a)(b)

(c)(d)(e)

Figure 5. (a) Fluorescence emission spectra (λexc = 520 nm) in water of a PEGMA-co-SiMA5 solution (19.99 mg mL–1) containing EtBr (0.03 mg mL–1) at different temperatures from 25 to 65 °C; (b) Fluorescence emission intensity maxima at 606 nm and scattering intensity at 530 nm as a function of temperature for a PEGMA-co-SiMA5 solution (19.99 mg mL–1) containing EtBr (0.03 mg mL–1) (λexc = 520 nm); (c) Fluorescence emission spectra (λexc = 454 nm) of J(PEGMA-co-SiMA6) water solutions at different molar concentrations of julolidine terminal; (d) Fluorescence emission spectra (λexc = 454 nm) of J(PEGMA-co-SiMA6) water solutions (1.710–5 M julolidine terminal) from 25 to 65 °C; (e) Fluorescence intensity maximum of J(PEGMA-co-SiMA6) aqueous solutions (1.710–5 M julolidine terminal) as a function of temperature.

However, such an increase was markedly lower than that detected in the presence of nucleic acids,[32] showing that EtBr was not sheltered enough from the surrounding water molecules or blocked enough in its mobility, possibly because of the higher degree of flexibility of the self-folded inner compartments. Moreover, the fluorescence peak intensity of EtBr (at 606 nm) significantly decreased in going from 25 to 65 °C (Figure 5a). On the other hand, an increase in fluorescence scattering (at ~530 nm) due to formation of multi-chain aggregates at higher temperatures was evident (Figure 5a). These two opposite trends showed a discontinuity at 51 °C (20.0 mg mL–1 copolymer concentration) (Figure 5b). Above 51 °C, the interactions among the hydrophilic poly(oxyethylene) chains were broken, water was expelled from the shell and single-chain nanoassemblies collapsed into larger aggregates, which in turn caused the concomitant release of EtBr. Thus, a significant reduction in fluorescence emission intensity was produced. Solutions with different concentrations of PEGMA-co-SiMA5 exhibited similar trends at different values of LCST. In particular, for diluted copolymer solutions (0.13.1 mg mL–1), the release of EtBr was triggered at higher temperatures (59–53 °C) (Figure S7). Above 3.1 mg mL–1 copolymer concentration, and up to copolymer concentrations as high as 29.0 mg mL–1, the EtBr release was forced at lower temperatures (52–51 °C) (Figure S7). Such temperature values were consistent with those previously found by DLS and transmittance measurements (5.0 mg mL–1).

Julolidine fluorophore: By taking advantage of the covalent anchorage of the cyanovinyl-julolidine terminal of the J(PEGMA-co-SiMA6) chains, self-assembly behavior was studied at room temperature and as a function of temperature in heating-cooling cycles. Interestingly, the maximum fluorescence emission intensity of the copolymer solutions in water and the respective quantum yield (Φ = 5.2710–2) were almost one order of magnitude higher than those collected in chloroform (Φ = 6.3910–3) (cf. Figure 5c and Figure S8). Moreover, the quantum yield of the water solution of the julolidine-labeled copolymer was even higher than that of the fluorophore JBr in methanol/glycerol solution (1/9 v/v) (Φ = 2.3510–2) with a viscosity 630 times higher than that of water. The increase in fluorescence intensity was due to the preferential inclusion of the hydrophobic julolidine moiety into the inner compartment of the self-folded single-chain nanoassemblies, which caused a reduction in the FMR mobility and an associated emission from an LE state.

Fluorescence spectra of J(PEGMA94-co-SiMA6) solutions in water as a function of temperature on heating from 25 to 65 °C confirmed the existence of a critical aggregation temperature of the system (Tc = 54 °C) (Figure 5d,e), analogous to the transition detected by DLS and transmittance measurements. Fluorescence intensity of the copolymer solution decreased linearly with temperature, due to the reduction in solvent viscosity and higher mobility of the single-chain nanoassemblies, until Tc was reached (Figure 5e). Then, multi-chain sub-micron aggregates were formed and fluorescence emission intensity stabilized to an almost constant, much lower value (Figure 5e). The inclusion of the hydrophobic fluorescent probe in stable submicron-sized multi-chain aggregates led to a great decrease in its mobility, which sustained the fluorescence response with increasing temperature. The transition was reversible in temperature by cooling down from 65 to 25 °C (Figure S9).

3. Conclusions

Novel single-chain folding temperature-responsive PEGMA-based copolymers containing poly(siloxane) methacrylate as the hydrophobic component were synthesized by ATRP with new, fluorescent and conventional, non-fluorescent ATRP initiators. Both the covalently linked julolidine terminal and the added ethidium bromide fluorophores exhibited high values of fluorescence emission intensity and quantum yield when the copolymer was dissolved in water owing to hydrophobic compartmentalization. Thus, both fluorophores revealed the occurrence of a reversible transition from nanosized single-chain assemblies to submicron-sized multi-chain aggregates. The existence of such different temperature-triggered, interchangeable self-associative states covering the nano- to submicron- length scales may make these copolymers suitable for advanced applications, notably when soft, low Tg and hydrophobic core nanoobjects are created/disrupted at low Tc conditions. Further studies are needed to tailor the precision synthesis of polymers with composition, length and sequence distribution viable to effect controlled intramolecular–intermolecular temperature-responsive assembling.

4. Experimental section

4.1. Materials

N,N’-Dicyclohexylcarbodiimide (DCC), phosphorous oxychloride, 9-(2,2-dicyanovinyl)julolidine, ethylene glycol and piperidine (Pip) (Sigma Aldrich) were used as received. Anisole (Sigma Aldrich) was vacuum distilled over sodium. Tetrahydrofuran (Sigma Aldrich) was refluxed over Na/K alloy for 3 h and distilled under nitrogen. Triethylamine (Sigma Aldrich) was refluxed over KOH for 3 h and distilled under nitrogen. N,N-Dimethylformamide and dichloromethane (Sigma Aldrich) were refluxed over CaH2 for 2 h and distilled under nitrogen. α-Bromoisobutyryl bromide (Sigma Aldrich) was distilled under vacuum. CuBr (Sigma Aldrich) was extracted with glacial acetic acid then washed with diethyl ether, dried and stored under nitrogen. Cyanoacetic acid (Sigma Aldrich) was recrystallized from a toluene/acetone (2/3 v/v) mixture. N,N,N′,N′′,N′’′-Pentamethyldiethylenetriamine (PMDETA) and ethyl α-bromophenylacetate (EBPA, Sigma Aldrich) were freshly distilled before use. Monomethacryloyloxypropyl-terminated poly(dimethyl siloxane) (SiMA, Mn = 680 g mol–1, 69 cSt, Fluorochem) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 300 g mol–1, Sigma Aldrich) were filtered through basic alumina to remove inhibitors. Common laboratory solvents and other reagents (Sigma Aldrich) were used as received.

Ethidium bromide (EtBr, purity 99%, Sigma) solutions were prepared by dissolving weighed amounts in water and their molar concentration was verified spectrophotometrically ( = 5600 M–1·cm–1 at λ = 480 nm).

Synthesis of 9-formyljulolidine (1) and 2-hydroxyethyl α-bromoisobutyrate (2)

9-Formyljulolidine was synthesized according to a general Vilsmeier-Haack reaction starting from 9-(2,2-dicyanovinyl)julolidine, phosphorous oxychloride and N,N-dimethylformamide.[33]

1H-NMR (CDCl3, δ in ppm): 9.64 (1H, CHO); 7.34 (2H, aromatic); 3.32 (4H, NCH2); 2.81 (4H, NCH2CH2CH2); 2.04 (4H, NCH2CH2).

2-Hydroxyethyl α-bromoisobutyrate was synthesized according to a general esterification procedure from α-bromoisobutyryl bromide and ethylene glycol.[34]

1H-NMR (CDCl3, δ in ppm): 4.33 (2H, COOCH2); 3.89 (2H, CH2OH); 1.97 (6H, CH3).

Synthesis of (2-bromo-2-methylpropanoyloxyethyl) cyanoacetate (3)

A solution of DCC (0.883 g, 4.28 mmol) in of anhydrous dichloromethane (4.28 mL) was added dropwise to a solution of cyanoacetic acid (0.374 g, 4.34 mmol) and 2 (0.624 g, 4.30 mmol) in anhydrous dichloromethane (10.26 mL). The mixture was kept under stirring for 24 h at 25 °C. When the reaction was stopped the mixture was diluted with dichloromethane (20 mL) and the precipitate formed during the reaction was filtered off. The filtrate was dried under vacuum and the residue was purified by column chromatography on silica gel (230400 mesh) using n-hexane/ethyl acetate (50/50 v/v) as an eluent (36 % yield).

1H-NMR (CDCl3, δ in ppm): 4.49–4.46 (4H, COOCH2); 3.52 (2H, NCCH2); 1.96 (6H, CH3).

Synthesis of 9-[2-(bromo-2-methylpropanoyloxyethyloxycarbonyl)-2-cyanovinyl]julolidine (JBr)

Piperidine (0.311 mL, 3.15 mmol) was added to a solution of 1 (0.606 g, 3.01 mmol) and 3 (0.843 g, 3.03 mmol) in tetrahydrofuran (17 mL) and the mixture was stirred for 24 h at 25 °C. The solvent was then evaporated and the residue was purified by column chromatography on silica gel using chloroform/ethanol (99/1 v/v) as an eluent (63% yield).

1H-NMR (CDCl3, δ in ppm): 7.95 (1H, ArCH=C(CN)); 7.53 (2H, aromatic); 4.524.50 (4H, COOCH2); 3.35 (4H, NCH2); 2.77 (4H, NCH2CH2CH2); 1.98 (10H, CH3, NCH2CH2).

13C-NMR (CDCl3, δ in ppm): 171.55 (BrCCOO); 164.57 (CNCCOO); 154.77 (ArC=C); 147.93 (C=CN); 131.88 (C=CC=C); 120.85 (C=CN); 118.38 (C=CC=C); 117.80 (C=CCN); 90.75 (C=CCN); 63.4462.53 (COOCH2), 55.59 (C(CH3)2); 50.21 (N-CH2); 30.73 (C(CH3)2); 27.56 (NCH2CH2CH2); 21.08 (NCH2CH2).

Synthesis of copolymer J(PEGMA-co-SiMA6)

A solution of PEGMA (0.761 mL, 2.66 mmol), SiMA (0.209 mL, 0.29 mmol), PMDETA (5.85 μL, 0.028 mmol) and JBr (13 mg, 0.028 mmol) in anisole (2 mL) was degassed in a Schlenk tube with three freeze-pump-thaw cycles. Then, CuBr (4 mg, 0.028 mmol) was added and three more freeze-pump-thaw cycles were performed before the polymerization was carried out at 90 °C under nitrogen atmosphere for 24 h. The crude product was diluted with chloroform and precipitated into n-hexane. The copolymer was further purified by filtration through basic alumina to remove catalyst and by numerous, repeated precipitations from chloroform into n-hexane (yield 42%).

1H-NMR (CDCl3, δ in ppm): 4.0–4.4 (2.13H, COOCH2); 3.2–3.9 (18H, OCH2); 3.3 (3H, OCH3); 2.1–0.8 (3.25H, Si-CH2CH2CH2, CH2CH3, CH2CCH3); 0.5 (0.26H, SiCH2); 0.1 (2.73H, SiCH3).

The content of the julolidine terminal residue was determined by UV-vis absorption measurements of the copolymer solutions in chloroform, by using an absorbance calibration curve (A = ·conc, where = 52700 M–1 cm–1, R2 = 0.997) obtained for the respective JBr solutions (510–7–510–5 M) in chloroform. The variation in molar extinction coefficient variation from JBr to copolymer J(PEGMA-co-SiMA6) was assumed to be negligible.

The copolymers PEGMA-co-SiMAx were prepared according to the same experimental conditions as above using EBPA as initiator, instead of JBr.

The reactivity ratios of PEGMA/SiMA comonomers were determined by carrying out conventional free radical copolymerizations, using AIBN as an initiator (1 wt%) and anisole as solvent at 65 °C for 2 h. The initial feed PEGMA/SiMA ratio was varied from 90/10 to 10/90 mol/mol.

4.2. Characterization

1H-NMR solution spectra were recorded with a Bruker Avance DRX 400 spectrometer.

The number and weight average molecular weights of the polymers were determined by size exclusion chromatography (SEC) with a Jasco PU-2089Plus liquid chromatograph equipped with two PL gel 5 µm mixed-D columns, a Jasco RI-2031Plus refractive index detector and a Jasco UV-2077Plus UV/vis detector. Poly(methyl methacrylate) standards (1160–124300 g mol–1) were used for calibration.

Dynamic light scattering of polymer solutions were taken with a Beckman Coulter Delsa Nano C particle analyzer (detection angle = 166.22°). Intensity, volume and number distributions were obtained from the signal autocorrelation function through CONTIN analysis in the instrument software. Samples were prepared in previously filtered solvent (0.2 µm CA or PTFE filters) to reduce external contamination.

UV-vis absorption spectra were recorded at room temperature on a Perkin-Elmer Lambda 650 spectrometer. Fluorescence emission spectra were recorded with a Horiba Jobin-Yvon Fluorolog-3 spectrofluorometer equipped with a 450 W xenon arc lamp, double-grating excitation and single-grating emission monochromators. Fluorescence quantum yield was determined using fluorescein in 0.1 N NaOH solution as a standard,[1] by the equation:

where is the area under the emission curve; A is the absorbance at excitation wavelength; n is the refractive index; the st subscript refers to the fluorescein standard.

Fluorescence emission spectra of ethidium bromide solutions were recorded with a Perkin Elmer LS55 spectrofluorometer.

Acknowledgments

Work performed with partial financial support from the University of Pisa (fondi Progetti di Ricerca di Ateneo, PRA_2017_28).

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Conflicts of interest

There are no conflicts to declare.

Keywords: amphiphilic polymer, single-chain folding, ethidium bromide, fluorescent molecular rotor, self-folded nanoassembly.

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TOC

Amphiphilic random copolymers interchangeably self-assemble in single-chain nanoassemblies and collapsed multi-chain aggregates at a critical temperature in water.

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