Star-shaped and star-block polymers with a porphyrin core: from LCST–UCST thermoresponsive transition to tunable self-assembly behaviour and fluorescence performance

Abstract

Star-shaped copolymer poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with a porphyrin core (THPP(-PDMAEMA-Br)₄) was synthesized by atom transfer radical polymerization (ATRP). After reaction with excess NaN₃, THPP(-PDMAEMA-Br)₄ was transformed to THPP(-PDMAEMA-N₃)₄. Star-block copolymer THPP(-PDMAEMA-b-PEG)₄ was obtained by click reaction of THPP(-PDMAEMA-N₃)₄ and alkynyl poly(ethylene glycol) (alkynyl PEG). After reaction of the PDMAEMA segment with excess 1,3-propane sultone, the quaternized THPP(-PDMAEMA-Br)₄ (THPP(-PDMAPS-Br)₄) and quaternized THPP(-PDMAPS-b-PEG)₄ were obtained. These copolymers can self-assemble into spherical micelles by directly dissolving in water. The thermoresponsive micelle solutions showed transition from a lower critical solution temperature (LCST) of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAEMA-b-PEG)₄ to an upper critical solution temperature (UCST) of THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄, indicating that the LCST–UCST transition of the micellar solutions can be accomplished by the transition of PDMAEMA to PDMAPS. Furthermore, the presence of permanent hydrophilic PEG chains in star-block copolymers changed the values of LCST, UCST and hydrodynamic radius (R_h) of micelles. The micelle solutions presented obvious fluorescence performance. The fluorescence intensity decreased with the increase of temperature, indicating that the variation of temperature could alter the fluorescence intensity of these copolymer micelle solutions.

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Introduction

Thermo-responsive polymers have attracted considerable interest because the temperature can be easily manipulated and the corresponding polymers are extensively applied in catalysis, drug delivery, sensors, intelligent gels, and smart materials.^1–10 There are two types of thermoresponsive polymers based on the type of phase transition in water. The most widely investigated thermoresponsive polymers are of the type showing a lower critical solution temperature (LCST).^11–16 For example, poly(N-isopropylacrylamide) (PNIPAM) is one of the most common LCST-type polymers.^16–20 The LCST-type polymers are soluble in water at low temperature and precipitate from the solution upon heating above a critical temperature called the cloud point. The other type of thermo-responsive polymer shows an upper critical solution temperature (UCST). The UCST-type polymers are soluble in water only above the critical temperature.^21–28 However, only a few (co)polymers are known to exhibit UCST-type thermoresponsive property.^29–32 Sulfobetaine polymers, which carry permanently (pH independent) charged ammonium and sulfonate groups in every repeat unit, are promising candidates for aqueous UCST behaviour because their zwitterionic side groups can cause strong inter- and intra-polymer attractions through electrostatic interlocking at low temperatures resulting in insolubility.³³ Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA), a typical pH- and LCST-type temperature-responsive polymer, has attracted much attention due to its unique role during the transition of LCST–UCST, i.e. LCST-type PDMAEMA can be transferred to UCST-type polymer easily through reaction of PDMAEMA with 1,3-propane sultone.^34–37 Quaternized PDMAEMA (poly(3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate) (PDMAPS)) displays a UCST.³⁸ The LCST–UCST transition can be achieved by a zwitterionic transformation of PDMAEMA.

Porphyrin, as one of the important photosensitizers, is attractive due to not only its promising photoelectric functions for various applications containing light-harvesting materials, optoelectronic devices, but also its novel therapeutic capacities such as tumor targeting and photodynamic therapy for biomedical application.^39–45 In addition, the porphyrin can be used as initiator to initiate the polymerization of functional monomer, and the copolymers with porphyrin core will combine the properties of porphyrin and polymers, such as fluorescence property and copolymer self-assembly.^46–49 Dai and Pan et al. reported the synthesis, self-assembly, drug-release behavior and singlet oxygen research of star-shaped amphiphilic poly(L-lactide)-block-poly(ethylene glycol) with porphyrin core.⁵⁰ In our previous work, star-shaped amphiphilic poly(ε-caprolactone)-block-poly(oligo(ethylene glycol) methyl ether methacrylate) with porphyrin core was synthesized by combination of ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP), and the self-assembly and drug-release behaviour of the copolymer were investigated.⁵¹

Besides introduction of hydrophobic polymeric chains to porphyrin-cored copolymer to construct amphiphilic system, hydrophobic porphyrin core itself can aggregate to form polymeric micelle core. As a result, in this work, 5,10,15,20-tetrakis(4-(2-methyl-2-bromopropoxy)phenyl)-21H,23H-porphine (THPP(-Br)₄) initiator obtained from the reaction of 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine (THPP) with 2-methylpropionyl bromide was used to initiate ATRP reaction of DMAEMA (Scheme 1). Then star-shaped THPP(-PDMAEMA-Br)₄ was transformed to THPP(-PDMAPS-Br)₄ after quaternization reaction with 1,3-propane sultone (Scheme 2). The self-assembly behaviour, LCST–UCST thermoresponsive properties of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAPS-Br)₄ were investigated. Moreover, the fluorescence performance of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAPS-Br)₄ micelles was studied at different temperature. Because the temperature could induce the morphology transition of PDMAEMA and PDMAPS, the permanent hydrophilic PEG segment was introduced to these polymers and to form star-block copolymers, THPP(-PDMAEMA-b-PEG)₄ and THPP(-PDMAPS-b-PEG)₄. The effect of the presence of PEG chains on the self-assembly, thermoresponsive properties and fluorescence performance of copolymers were also investigated.

Scheme 1 Synthesis of star-shaped THPP(-PDMAEMA-Br)₄ and star-block THPP(-PDMAEMA-b-PEG)₄ by ATRP and click chemistry.

Scheme 2 Synthesis of star-shaped THPP(-PDMAPS-Br)₄ and star-block THPP(-PDMAPS-b-PEG)₄ by quaterization reaction.

Experimental

Materials

Propionic acid, acetic acid, nitrobenzene and N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA) were purchased from Aldrich and used as received. Pyrrole (Acros Organics) and 2-methylpropionyl bromide (Aldrich) were distilled under reduced pressure before use. Propargyl 3-(carbonylchloro) propanoate was synthesized by the reaction of succinic anhydride (Aldrich) and propargyl alcohol (Aldrich).⁵² Triethylamine, dichloromethane, tetrahydrofuran (THF) and dimethylformamide (DMF) was dried by CaH₂ and distilled under reduced pressure. N,N-Dimethylaminoethyl methacrylate (DMAEMA; Aldrich) was passed through a column of activated basic alumina to remove inhibitors. Copper bromide (CuBr) was treated by stirring in glacial acetic acid and washed with ethanol several times. 5,10,15,20-Tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin (THPP) was synthesized according to the literature.⁵³ Methoxy poly(ethylene oxide) with M_n = 1k denoted as mPEO-OH (Fluka) was dried by azeotropic distillation in the presence of toluene.

Characterization

Attenuated total internal reflectance Fourier transform infrared spectroscopy (ATR FT-IR). ATR FT-IR spectra of samples were recorded on an AVATAR 360 ESP FT-IR spectrometer (Thermo Nicolet, American).

Nuclear magnetic resonance (NMR). ¹H NMR spectra of samples were obtained from a Bruker DMX-500 NMR spectrometer with CDCl₃, DMSO-d₆ and D₂O as solvents. The chemical shifts were relative to tetramethylsilane.

Gel permeation chromatography (GPC). GPC analysis was carried out with a HLC-8320 (Tosoh, Japan) analysis system with two columns (TSK gel super AWM-H × 2, R0091 + R0093), using DMF with 10 mM LiBr as eluents at a flow rate of 0.6 mL min⁻¹ at 40 °C. PMMA calibration kit was used as the calibration standard.

Optical transmittances. The optical transmittances of copolymer micelles aqueous solution (30 mg mL⁻¹, deionized water was used as the solvent) at various temperatures were measured at a wavelength of 750 nm on a U-3310 UV-visible spectrophotometer (Hitachi, Japan) equipped with a temperature controller (0.1 °C accuracy). The temperature of the sample cell was thermostatically controlled using an external superconstant temperature bath. The solutions were equilibrated for 10 min at each measuring temperature. The LCST values of the copolymer micelles solutions were defined as the temperature producing a 50% decrease in optical transmittance. The UCST values of the copolymer micelles solutions were defined as the temperature producing a 50% increase in optical transmittance.

Dynamic light scattering spectrophotometer (DLS). The hydrodynamic radius (R_h) of the micelles of copolymer micelles was investigated using DLS techniques. The experiments were performed on a Malven Autosizer 4700 DLS spectrometer. The apparent R_h was obtained by a cumulant analysis. The variable temperature DLS test was measured by controlling the temperature of sample cell with programmed temperature device.

Transmission electron micrographs (TEM). The morphology of copolymer micelles was observed with a JEOL JEM-2010 TEM at an accelerating voltage of 120 kV. The samples for TEM observation were prepared by placing 10 μL of copolymer micelles solution on copper grids coated with thin films and carbon.

Fluorescence measurement. Fluorescence spectra were recorded using an F-2700 spectrometer (Hitachi, Japan) with a xenon lamp source. Fluorescence scans were performed at different temperature in the range of 390–700 nm, using a speed of 1500 nm min⁻¹, an excitation wavelength of 370 nm.

Synthesis of 5,10,15,20-tetrakis(4-(2-methyl-2-bromopropoxy) phenyl)-21H,23H-porphine (THPP(-Br)₄)

5,10,15,20-Tetrakis(4-hydroxyphenyl)-21H,23H-porphine (400 mg, 5.9 × 10⁻⁴ mol), triethylamine (1 mL) and dichloromethane (50 mL) were combined under Ar in an ice bath. 2-Bromo-2-methylpropionyl bromide (3.2 g, 1.4 × 10⁻² mol) in anhydrous dichloromethane (10 mL) was added dropwise over 15 min. The mixture was allowed to warm to room temperature and then stirred for 18 h. The reaction mixture was then washed with 1% aqueous NaOH solution, and the organic layer was reduced to a minimum under reduced pressure. The resulting solution was purified by silica flash chromatography (eluent: dichloromethane). After removal of the solvent the porphyrin was isolated as a purple powder.

¹H NMR (δ, DMSO-d₆, ppm): 8.89 (s, 8H, pyrrole-H), 8.25 (d, 8H, o-H, benzene), 7.55 (d, 8H, m-H, benzene), 2.2 (s, 24H, C(CH₃)₂Br).

Synthesis of star-shaped THPP(-PDMAEMA-Br)₄

5,10,15,20-Tetrakis(4-(2-methyl-2-bromopropoxy)phenyl)-21H,23H-porphine (40 mg, 3.12 × 10⁻⁵ mol), N,N-dimethylaminoethyl methacrylate (1.58 g, 0.01 mol), CuBr (54 mg, 3.78 × 10⁻⁴ mol) and PMDETA (65.25 mg, 3.78 × 10⁻⁴ mol) were combined in a Schlenk tube and subjected to three freeze–thaw cycles, then stirred at 60 °C under Ar for 3.5 h. After being cooled to room temperature, the reaction tube was opened to air, and the crude product was diluted with THF and passed through a neutral oxide alumina column to remove the copper catalysts. Then the filtered solution was purified by dialysis (molecular weight cut-off: 8000–14 000 Da) against water to remove unreacted DMAEMA. Water was removed by freeze-drying and the purified THPP(-PDMAEMA-Br)₄ was obtained (0.58 g, yield: 35.8%).

M_n,GPC = 26 100 g mol⁻¹, M_w/M_n = 1.29. ATR FT-IR (cm⁻¹): 3220–3660 (ν_N–H), 2692–3028 (ν_C–H), 1726 (ν_C=O). ¹H NMR (δ, CDCl₃, ppm): 4.07 (CH₂CH₂N), 2.59 (CH₂CH₂N), 1.72–2.06 (CH₂C(CH₃)), 0.81–1.14 (CH₂C(CH₃)).

Synthesis of star-shaped THPP(-PDMAEMA-N₃)₄

THPP(-PDMAEMA-Br)₄ (500 mg, containing 0.077 mmol of C–Br), NaN₃ (236 mg, 3.63 mmol), and DMF (30 mL) were added into a 100 mL round-bottom flask equipped with a magnetic stirrer, and the reaction was carried out at 50 °C for 24 h. After filtration of excess NaN₃, the solution was dialyzed against deionized water. After freeze drying, the resulting product was obtained (485 mg, yield: 88%).

ATR FT-IR (cm⁻¹): 3216–3660 (ν_N–H), 2692–3028 (ν_C–H), 2032 (ν_–N3), 1726 (ν_C=O).

Synthesis of alkynyl PEG

mPEG-OH (6 g, 6 mmol), propargyl 3-carboxylic-propanoate (4.68 g, 30 mmol), DCC (3.09 g, 15 mmol) and DMAP (1.83 g, 15 mmol) were dissolved in 40 mL of anhydrous dichloromethane, and the reaction was performed at room temperature for 40 h under argon atmosphere. The reaction byproduct dicyclohexyl carbodiurea was removed by filtration, and the solution was washed with aqueous NaHCO₃ solution and deionized water. Then the organic layer was dried over anhydrous MgSO₄ overnight. The purified product was obtained after removing of solvent and precipitating in ethyl ether (4.3 g, yield: 62.9%).

¹H NMR (δ, CDCl₃, ppm): 4.70 (COOCH₂C≡CH), 4.24 (CH₂OOC), 3.65 (OCH₂CH₂O), 3.38 (CH₃O), 2.69 (OOCCH₂CH₂COO), 2.50 (COOCH₂C≡CH).

Synthesis of star-block THPP(-PDMAEMA-b-PEG)₄

Star-block THPP(-PDMAEMA-b-PEG)₄ was prepared via click chemistry. A typical procedure was as follows. Alkynyl PEG (0.216 g, 0.2055 mmol) and THPP(-PDMAEMA-N₃)₄ (450 mg, containing 0.017 mmol of N₃) were dissolved in DMF (10 mL). Then CuBr (27 mg, 0.2055 mmol) and PMDETA (33 mg, 0.2055 mmol) were added into the above solution. After degassed via three freeze–evacuate–thaw cycles, the reaction was carried out at 55 °C for 48 h. The resulting copolymer was obtained by dialysis (dialysis membrane, MW cut: 8000–14 000 Da) with deionized water for 72 h to remove unreacted alkynyl PEG. The resulting product was obtained by precipitation from cold ethyl ether.

M_n,GPC = 29 200 g mol⁻¹, M_w/M_n = 1.25. ATR FT-IR (cm⁻¹): 3322–3646 (ν_N–H), 2686–3022 (ν_C–H), 1726 (ν_C=O), 1107 (ν_C–O–C). ¹H NMR (δ, CDCl₃, ppm): 5.22 (COOCH₂C(N)=C(N)), 4.06 (CH₂CH₂N), 3.66 (OCH₂CH₂O), 3.39 (CH₃O), 2.57 (CH₂CH₂N), 1.72–2.02 (CH₂C(CH₃)), 0.73–1.16 (CH₂C(CH₃)).

Synthesis of THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ by quaternization reaction

THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ were prepared by quaternization reaction of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAEMA-b-PEG)₄ with 1,3-propane sultone.

A typical procedure for preparation of THPP(-PDMAPS-Br)₄ was as follows. A 50 mL round-bottom flask was charged with 10 mL of dry THF, THPP(-PDMAEMA-Br)₄ (0.4 g), and a large excess of 1,3-propane sultone (3.0 g). The solution was stirred overnight at room temperature, and an insoluble residue was collected by filtration, washed with dry THF, and dried at 40 °C in vacuo.

A typical procedure for preparation of THPP(-PDMAPS-b-PEG)₄ was as follows. A 50 mL round-bottom flask was charged with 10 mL of dry THF, THPP(-PDMAEMA-b-PEG)₄ (0.4 g), and a large excess of 1,3-propane sultone (3.0 g). The solution was stirred overnight at room temperature, and an insoluble residue was collected by filtration, washed with dry THF, and dried at 40 °C in vacuo.

Micellization of copolymers

Amphiphilic THPP(-PDMAEMA-Br)₄, THPP(-PDMAEMA-b-PEG)₄, THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ were directly dissolved in deionized water and the micelles formed. The micelle concentration of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAEMA-b-PEG)₄ was 6 mg mL⁻¹. For THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄, the micelle concentration was 30 mg mL⁻¹.

Results and discussion

Syntheses of THPP(-PDMAEMA-Br)₄, THPP(-PDMAEMA-b-PEG)₄, THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄

The THPP(-Br)₄ initiator for ATRP reaction was synthesized by the reaction of THPP and 2-methylpropionyl bromide. Compared to the ¹H NMR spectrum of THPP (Fig. 1(a)), a new signal corresponding to methyl protons (a) appeared at 2.24 ppm in ¹H NMR spectrum of THPP(-Br)₄, as shown in Fig. 1(b), indicating that all the hydroxyl groups have been transferred to 2-methylpropionates. Star-shaped THPP(-PDMAEMA-Br)₄ copolymer was synthesized from THPP(-Br)₄ initiator and DMAEMA monomer via ATRP at 60 °C. The protons signals of PDMAEMA chains of THPP(-PDMAEMA-Br)₄ could be detected. However, due to the overlap of proton signals and low content of porphyrin core in copolymer, it is difficult to detect the proton signals of porphyrin from ¹H NMR spectrum of THPP(-PDMAEMA-Br)₄ (Fig. 1(c)). According to the ATR FT-IR spectrum of THPP(-PDMAEMA-Br)₄ shown in Fig. 2(a), the wide band at 3220–3660 cm⁻¹ was the absorption band of methylamino groups. In addition, the peak at 1726 cm⁻¹ assigned to the carbonyl absorption of PDMAEMA units could be observed. The number-average molecular weight measured by GPC (M_n,GPC) is 26 100 g mol⁻¹, as shown in Fig. 3. THPP(-PDMAEMA-N₃)₄ was obtained by the reaction of THPP(-PDMAEMA-Br)₄ with excess NaN₃. The azido groups can't be characterized by ¹H NMR spectrum, and therefore ATR FT-IR spectrum was used to confirm the presence of azide groups. As shown in Fig. 2(b), the characteristic absorption peak of the terminal azide group at about 2032 cm⁻¹ is observed. Alkynyl PEG was prepared by the DCC reaction of PEG-OH and excess propargyl 3-carboxylic-propanoate. Finally, using the click reaction, THPP(-PDMAEMA-N₃)₄ and alkynyl PEG were reacted to give the corresponding star-block copolymer THPP(-PDMAEMA-b-PEG)₄ in the presence of a CuBr/PMDETA catalyst system in DMF. From ¹H NMR spectrum of THPP(-PDMAEMA-b-PEG)₄ (Fig. 3(b)), the proton signals of PDMAEMA and PEG can be observed clearly. According to Fig. 2(c), the characteristic absorption peak of the terminal azide group disappeared, and the absorption peak of ether bond in PEG at 1107 cm⁻¹ was observed, which indicated that the successful preparation of THPP(-PDMAEMA-b-PEG)₄. Fig. 3 also shows the GPC curve of THPP(-PDMAEMA-b-PEG)₄ (M_n,GPC = 29 200 g mol⁻¹), and symmetrical unimodal peak is observed. The efficiency of click reaction of THPP(-PDMAEMA-N₃)₄ and alkynyl PEG was about 70.8%, according to the ¹H NMR spectrum of THPP(-PDMAEMA-b-PEG)₄ (Fig. 1(d)).

Fig. 1 ¹H NMR spectra of (a) THPP, (b) THPP(-Br)₄, (c) THPP(-PDMAEMA-Br)₄ and (d) THPP(-PDMAEMA-b-PEG)₄.

Fig. 2 ATR FT-IR spectra of (a) THPP(-PDMAEMA-Br)₄, (b) THPP(-PDMAEMA-N₃)₄, and (c) THPP(-PDMAEMA-b-PEG)₄.

Fig. 3 GPC traces of star-shaped THPP(-PDMAEMA-Br)₄ and star-block THPP(-PDMAEMA-b-PEG)₄ polymers.

LCST–UCST thermoresponsive transition of star-shaped and star-block copolymer micelles

As amphiphilic star-shaped and star-block copolymers, THPP(-PDMAEMA-Br)₄, THPP(-PDMAPS-Br)₄, THPP(-PDMAEMA-b-PEG)₄ and THPP(-PDMAPS-b-PEG)₄ can self-assemble into micelles by direct dissolution of the copolymer into water due to the high content of PDMAEMA or PDMAEMA-b-PEG hydrophilic chains in the copolymers. The hydrophilic PDMAEMA or PDMAEMA-b-PEG chains are mainly in the corona of the micelles, whereas the hydrophobic porphyrin segments are mainly in the core of the micelles. The LCST–UCST thermoresponsive properties, the hydrophilicity–hydrophobicity transition of PDMAEMA and PDMAPS, and the morphology transformation and aggregation of THPP(-PDMAEMA-Br)₄, THPP(-PDMAEMA-b-PEG)₄, THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ micelles/aggregates at different temperatures can be demonstrated from the schematic process as shown in Fig. 4.

Fig. 4 The schematic processes of LCST–UCST thermoresponsive properties of micelles/aggregates of THPP(-PDMAEMA-Br)₄, THPP(-PDMAPS-Br)₄, THPP(-PDMAEMA-b-PEG)₄ and THPP(-PDMAPS-b-PEG)₄.

Fig. 5(a) shows the transmittance curve of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAEMA-b-PEG)₄. It can be seen that the transmittance curve of THPP(-PDMAEMA-Br)₄ shows a sharp decreasing transition during heating process and the LCST value is 41.1 °C, indicating the hydrophilicity–hydrophobicity transition of PDMAEMA and the aggregation of micelles above LCST. However, the THPP(-PDMAEMA-b-PEG)₄ micelle solution remained high transmittance during heating process (Fig. S1† shows that the high transmittance was remained even the temperature increased to 90 °C). The main reason is that the presence of PEG segment ensured the stability of the micelles though PDMAEMA was transformed from hydrophilic state to hydrophobic state (PDMAEMA chains still possessed LCST-type transition). Fig. 5(b) shows the transmittance curves of THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄. It can be seen that the transmittance curves show sharp increasing transition during the heating process. But the UCST value of THPP(-PDMAPS-b-PEG)₄ (21.8 °C) is lower than that of THPP(-PDMAPS-Br)₄ (28.6 °C). The presence of hydrophilic PEG in the copolymer led to the decrease of UCST. In addition, due to the serious hydrophobicity of PDMAPS, the phase transition of THPP(-PDMAPS-b-PEG)₄ is obvious, which is different from THPP(-PDMAPS-Br)₄. The transmittance curves (cooling process, Fig. S2, ESI†) confirmed the reversibility of the thermal transition. Fig. 5(c) shows the plot of the hydrodynamic radius (R_h) of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAEMA-b-PEG)₄ in water as a function of temperature. In the lower temperature ranges (T < LCST), the R_h values of THPP(-PDMAEMA-Br)₄ micelles are relatively small and change slightly. When the temperature is higher than the LCST, the R_h values decrease to some extent. But in the higher temperature ranges, the R_h values increase rapidly. At lower temperatures, the PDMAEMA chains in the THPP(-PDMAEMA-Br)₄ copolymer chains exist in random coil conformation owing to the hydrogen-bonding interaction between the N,N-dimethylaminoethyl groups and water molecules. When the temperature increases to the LCST, the PDMAEMA chains would shrink into a globular structure because the hydrogen bonds between the N,N-dimethylaminoethyl groups of PDMAEMA chains and water molecules are obviously weakened or broken. For THPP(-PDMAEMA-b-PEG)₄, due to the presence of PEG, the micelles are still stable after the hydrophilicity–hydrophobicity transition of PDMAEMA segment. As a result, the size of micelles decreased when the temperature increased to LCST of PDMAEMA, but the aggregation of the micelles did not occur (Fig. S3† also confirmed the size change of micelles). Fig. 5(d) shows the plot of R_h of THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ in water as a function of temperature. At the lower temperature ranges (T < UCST), because of the ionic interactions between opposite charges of the quaternized groups, the quaternized N,N-dimethylaminoethyl groups interact with each other, which causes the quaternized units to collapse together to form aggregates with large sizes. When the temperature is higher than the UCST, the mutual intermolecular attraction of the quaternized groups is weakened due to the increase of the thermal energy of the system, and the quaternized PDMAEMA chains transform from a compact coil conformation to an expanding state. The quaternized PDMAEMA chains have excellent hydrophilicity at high temperature ranges. The R_h values of THPP(-PDMAPS-Br)₄ micelles decrease dramatically. The R_h decrease of THPP(-PDMAPS-b-PEG)₄ micelles occurred at lower temperature and the R_h values were smaller at low temperature region than those of THPP(-PDMAPS-Br)₄ micelles. This should be attributed to the presence of PEG which hindered the serious aggregation of micelles. Therefore, the micelles can accomplish the LCST–UCST transition by the transition of PDMAEMA to quaternized PDMAEMA (PDMAPS). The LCST, UCST or R_h values also could be adjusted though introduction of PEG to the copolymers.

Fig. 5 Transmittance curves of (a) THPP(-PDMAEMA-Br)₄ and THPP(-PDMAEMA-b-PEG)₄, (b) THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄, and temperature dependence of hydrodynamic radius (R_h) for (c) THPP(-PDMAEMA-Br)₄ and THPP(-PDMAEMA-b-PEG)₄, (d) THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄.

Fig. 6 shows the photographs of transparency–turbidity transition at different temperature of THPP(-PDMAEMA-Br)₄, THPP(-PDMAEMA-b-PEG)₄, THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄. Because the solutions presented colour, it is difficult to observe the transparency–turbidity transitions in photographs. Therefore the laser light beam was used to examine the transparency of solutions. The obvious scattering laser light in Fig. 6 indicated the turbidity of micelle solutions. The laser light scattering was obviously for THPP(-PDMAEMA-Br)₄ at high temperature, but for THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ at low temperature. For THPP(-PDMAEMA-b-PEG)₄, the laser light scattering didn't present obvious change. The transparency–turbidity transitions are in accordance with the results of Fig. 5.

Fig. 6 Photographs of transparency–turbidity transition at different temperature of (a) THPP(-PDMAEMA-Br)₄, (b) THPP(-PDMAPS-Br)₄, (c) THPP(-PDMAEMA-b-PEG)₄ and (d) THPP(-PDMAPS-b-PEG)₄.

Fig. 7(a) shows the TEM image of THPP(-PDMAEMA-Br)₄ micelles at 25 °C (T < LCST), and the spherical and separated micelles could be observed. However, when the temperature increased to 50 °C (T > LCST), the micelles tended to aggregate into aggregates with large size, as shown in Fig. 7(b), which should be ascribed to the transition of PDMAEMA chains from hydrophilicity to hydrophobicity. After linking PEG segments to PDMAEMA chains, the size of micelles (25 °C) increased to some degree, as shown in Fig. 7(c). When the micelle solution was heated to 50 °C, no obvious change occurred (Fig. 7(d)). The size of micelles at 50 °C was similar to that at 25 °C. The presence of PEG stabilized the micelles and prevented them from aggregating to large aggregates, which was consistent with the results of DLS. Fig. 7(e) and (f) revealed the morphologies of micelles of THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ at 40 °C. Because the UCST values of the two kinds of micelle solutions were lower than 40 °C, the PDMAPS segments were hydrophilic at 40 °C. Therefore, the spherical micelles were dispersed. The TEM results further indicated the LCST and UCST behaviours of these four copolymers micelles.

Fig. 7 TEM images of micelles of (a) THPP(-PDMAEMA-Br)₄ (25 °C), (b) THPP(-PDMAEMA-Br)₄ (50 °C), (c) THPP(-PDMAEMA-b-PEG)₄ (25 °C), (d) THPP(-PDMAEMA-b-PEG)₄ (50 °C), (e) THPP(-PDMAPS-Br)₄ (40 °C) and (f) THPP(-PDMAPS-b-PEG)₄ (40 °C).

Fig. 8 shows the ¹H NMR spectra of THPP(-PDMAEMA-Br)₄, THPP(-PDMAPS-Br)₄, THPP(-PDMAEMA-b-PEG)₄ and THPP(-PDMAPS-b-PEG)₄ in D₂O conducted at different temperatures. In D₂O, these star-shaped and star-block copolymers self-assembled to micelles and the hydrophobic porphyrin core couldn't be detected. The protons signals of PDMAEMA could be observed in Fig. 8(a), and the intensity of the proton signals at lower temperature ranges (T < LCST) are stronger than those at higher temperature ranges (T > LCST). By contrast, the intensity of the proton signals of quaternized PDMAEMA (PDMAPS) (Fig. 8(b)) at lower temperature ranges (T < UCST) are weaker than those at higher temperature ranges (T > UCST). The change tendency of proton intensity of PDMAEMA or PDMAPS in THPP(-PDMAEMA-b-PEG)₄ and THPP(-PDMAPS-b-PEG)₄ were similar to that of THPP(-PDMAEMA-Br)₄ and THPP(-PDMAPS-Br)₄. But the intensity of PEG protons didn't change. The behaviours of the copolymers in D₂O further confirm that the LCST–UCST transition can be achieved via the quaternization of PDMAEMA in the star-shaped and star-block copolymers.

Fig. 8 ¹H NMR spectra of (a) THPP(-PDMAEMA-Br)₄, (b) THPP(-PDMAPS-Br)₄, (c) THPP(-PDMAEMA-b-PEG)₄ and (d) THPP(-PDMAPS-b-PEG)₄ as a function of the temperature in D₂O.

Fluorescence of copolymer micelles

Due to the fluorescence of the porphyrin molecules, the THPP(-PDMAEMA-Br)₄, THPP(-PDMAPS-Br)₄, THPP(-PDMAEMA-b-PEG)₄ and THPP(-PDMAPS-b-PEG)₄ micelles may also present fluorescent properties. Because the micelle core was the aggregates of porphyrin group of copolymers, the fluorescence quenching behaviour would be serious. But during the ATRP reaction to prepare THPP(-PDMAEMA-Br)₄, part porphyrin molecules could form metal complex with copper ions of CuBr₂ (CuBr₂ was obtained from the oxidation of CuBr catalyst during ATRP reaction), which would lead to fluorescence property loss of part porphyrin groups and the amount of metal-free porphyrin decreased and corresponding fluorescence quenching behaviour was weakened. As a result, the micelles still presented relative high fluorescence intensity, as show in Fig. 9(a) and (b). Moreover, the fluorescence intensity of the micelles decreased with the increase of temperature, indicating that temperature has obvious effect on the fluorescence intensity of the micelles with porphyrin core. The main reason is that the increase of the probability of the interaction of porphyrin groups with the increase of temperature, which should cause the decrease of fluorescence intensity. After linking PEG chains to the copolymer, it can be found that the fluorescence intensity was much higher than that of star-shaped copolymer. There are two reasons. Firstly, the amount of free-metal porphyrin further decreased and corresponding fluorescence quenching behaviour was further weakened due to the click chemistry during the process of preparation of star-block copolymer. Cu²⁺ in CuBr₂ would further complex with metal-free porphyrin. Secondly, the presence of hydrophilic PEG caused the improvement of hydrophilicity of copolymer micelles, which would lead to the loose state of the micellar cores. Therefore, the space between porphyrin molecules increased and the fluorescence quenching decreased. Thus, the variation of temperature could be used to alter the fluorescence intensity of copolymer micelle solutions.

Fig. 9 Fluorescence spectra of (a) THPP(-PDMAEMA-Br)₄, (b) THPP(-PDMAPS-Br)₄, (c) THPP(-PDMAEMA-b-PEG)₄ and (d) THPP(-PDMAPS-b-PEG)₄ at different temperature.

Conclusions

Star-shaped copolymer THPP(-PDMAEMA-Br)₄ with porphyrin core was synthesized by ATRP. Then star-block copolymer THPP(-PDMAEMA-b-PEG)₄ was prepared by click reaction of THPP(-PDMAEMA-N₃)₄ and alkynyl PEG. These amphiphilic polymers can self-assemble into thermoresponsive micelles with LCST in water. After facile quaternization of the PDMAEMA segments, THPP(-PDMAPS-Br)₄ and THPP(-PDMAPS-b-PEG)₄ were obtained. The quaternized copolymers can also self-assemble into thermosensitive micelles but with the UCST in water. This indicates that the LCST–UCST transition of the micelles can be accomplished via a quaternization process of the PDMAEMA segments. The presence of PEG chains in star-block copolymers could change the LCST, UCST and R_h of the micelles. All micelle solutions presented fluorescence performance due to the porphyrin core. The fluorescence intensity decreased with the increase of temperature, indicating that the fluorescence intensity could be adjusted through altering the temperature of the micelle solutions. These copolymers and micelles have the potential applications in biomedical and smart materials fields.

Acknowledgements

The authors thank the financial supports of the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry and the National High Technology Research and Development Program (no. 2013AA032202).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra21647h

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