Supramolecular self-assembly of novel thermo-responsive double-hydrophilic and hydrophobic Y-shaped [MPEO-b-PEtOx-b-(PCL)2] terpolymers

Nonlinear amphiphilic block copolymer architectures with precisely controlled structures bring new challenges to biomedical materials research. The paper describes the straightforward synthesis of new “snake tongue“ Y-shaped terpolymers containing poly(ethylene oxide) (PEO), poly(2-ethyl-2-oxazoline) (PEtOx) and poly(3-caprolactone) (PCL) blocks into structure [AB(C)2] (herein referred to as [MPEO44-bPEtOx252-b-(PCL)2 44], [MPEO44-b-PEtOx252-b-(PCL)2 87], [MPEO44-b-PEtOx252-b-(PCL)2 131]). A series of well-defined Y-shaped terpolymers were successfully synthesised by a combination of living cationic and anionic ring-opening polymerization (ROP). The selected Y-shaped [MPEO44-b-PEtOx252-b(PCL)2 44] terpolymer self-assembly was characterised in detail by static and dynamic light scattering, nanoparticle tracking analysis and cryo-transmission electron microscopy. The physico-chemical properties as well as the molecular architecture effect on the self-assembled structures and on the LCST were compared with the Y-shaped [MPEO44-b-PEtOx252-b-(PCL)2 87] and the [MPEO44-b-PEtOx252-b(PCL)2 131] terpolymers. The results indicated a temperature-induced aggregation with an LCST between 60–63 C for the [MPEO44-b-PEtOx252-b-(PCL)2 44], at 60 C for the [MPEO44-b-PEtOx252-b-(PCL)2 87] and between 45–50 C for the [MPEO44-b-PEtOx252-b-(PCL)2 131] with significant differences in the supramolecular self-assembly behaviour compared with the analogous linear structure, clearly indicating the crucial effect of the molecular architecture. Furthermore, the increase of the molecular weight fractionof thehydrophobicblockon theY-shaped triblock terpolymers likely inducedadecreaseof theLCST.


Introduction
The key feature of successful and versatile polymer materials is the possibility to precisely control the polymer architecture and chemical functionality. Living polymerisation enables the preparation of such polymers with a broad variety of molecular architectures, compositions, side-and end-group functions, as well as the facile preparation of block copolymers with linear and nonlinear architectures. [1][2][3][4][5][6][7] Star-shaped copolymers are a unique and simple class of macromolecules with a complex architecture, and they constitute a topical area of research due to their intriguing properties, which can be tailored by varying their polymeric chains (arms).
Star-shaped block copolymers consisting of at least three linear polymeric arms with a radial arrangement around a central molecular fragment (core) 8,9 are usually prepared by the "arm-rst" or "core-rst" methods. The "arm-rst" approach involves the construction of polymer arms on a macroinitiator that contains a precise number of reactive sites. 10,11 The "core-rst" approach utilises multifunctional low-molecular-weight initiators, allowing for the synthesis of block copolymer chains. 12 A method based on difunctional monomers is mentioned in the literature as a third approach for the synthesis of star-shaped copolymers. 6 However, this method does not allow for strict control of the number of arms.
It is well known that amphiphilic star-shaped copolymers can easily self-assemble in aqueous media to form nanosized unimolecular micelles containing hydrophobic cores surrounded by hydrophilic shells. 13,14 These micellar systems are of great interest for medical uses such as the construction of micellar drug delivery systems. 9,[15][16][17][18][19] Signicant differences in the physicochemical properties of star-shaped copolymers compared with their linear analogues can be observed, such as smaller hydrodynamic volume and, radius of gyration and low melt and solution viscosities, which are benecial to drug loading and delivery. [20][21][22][23][24] It has been shown that linear amphiphilic copolymers have limited applications in drug delivery because they suffer from an initial burst release effect. Especially in systems with non-covalently incorporated drugs, the micellar stability and drug release are difficult to control. 25,26 Therefore, various starshaped copolymers with varying arm numbers and chemical compositions have received considerable attention because of the unique properties and advantages that they possess. [27][28][29] Y-shaped copolymers (typically referred to star copolymers) are another interesting vehicle for drug delivery because they exhibit distinct a micellization behaviour (special stability) compared with the amphiphilic copolymers with a linear architecture. [30][31][32] Furthermore, star copolymers bearing distinct polymeric arms have shown dynamic morphological changes (e.g., micelle-tounimer transition under certain conditions) because the constituting polymeric components could be designed to be individually responsive to external stimuli such as pH, temperature and solvent. 7,33-36 Moreover, a particular subject of even greater interest is the study of biocompatible thermo-responsive selfassembled polymer micelles of amphiphilic, double-hydrophilic and hydrophobic species of star copolymers. It should be noted that the number of such studies is quite limited, and more work is greatly needed to understand the particular characteristics involved in the micellar behaviour of such polymer systems.
In this paper, we describe the synthesis and the study of the self-assembly properties of new "snake tongue" Y-shaped terpolymers based on poly(ethylene oxide) (PEO), poly(2-ethyl-2oxazoline) (PEtOx) and poly(3-caprolactone) (PCL) with the general architecture [PEO-b-PEtOx-b-(PCL) 2 ] (Scheme 1). The newly synthesised Y-shaped terpolymers combine environmentally friendly blocks with possible applications in biomedicine. PEtOx was chosen because it exhibits similar chemical and biological properties to PEO; 37-39 both polymers are water-soluble and non-toxic. [40][41][42][43] PEO and PEtOx can be eliminated from the human body if they possess a low enough molar mass. Furthermore, PEtOx in an aqueous solution exhibits a lower critical solution temperature (LCST). [44][45][46][47] The LCST of PEtOx is $61-66.5 C and strongly depends on the polymer molecular weight (20-500 kDa) and polymer concentration. 48,49 PCL is a hydrophobic, nontoxic, biocompatible and fully biodegradable aliphatic polyester. 50 To the best of our knowledge, this is the rst time that these three blocks were combined in a nonlinear architecture using the "arm-rst" method and their supramolecular self-assembly behaviour was compared with analogous blocks of linear architecture and identical weight ratios and molecular weights.

Synthesis of [MPEO-b-PEtOx(OH) 2 ] diblock copolymer ((Stage 3) Scheme 1)
The polymerisation was carried out as follows; 0.45 g (0.20 mmol) of u-tosyl-MPEO (M n(NMR) $ 2200 g mol À1 ) was introduced into a 50 mL glass reactor with a magnetic bar. The macroinitiator was dissolved in dry toluene (15 mL), the solvent was evaporated and the azeotropic drying procedure was twice more repeated. Dry ACN (20 mL) was transferred to the glass reactor using a ame-dried and argon-ushed glass syringe equipped with a metallic cannula. The solution was heated to 80 C before to inject rapidly, through a septum, 5 mL (49.5 mmol) of 2-ethyl-2-oxazoline, and the polymerisation was continued for ve days. To terminate the polymerisation, diethanolamine (0.025 mL, 0.26 mmol, 1.3 eq.) was added and the reaction mixture was stirred for two more hours at 80 C. Then, the crude product was precipitated in cooled diethyl ether, ltered off, washed with diethyl ether and dried under vacuum overnight at 40 C. Yield: 4.85 g, (93%).

Synthesis of Y-shaped [MPEO-b-PEtOx-b-(PCL) 2 ] terpolymers ((Stage 4) Scheme 1)
Typically, 0.5 g (0.162 mmol) of [MPEO-b-PEtOx(OH) 2 ] diblock copolymer (M n(NMR) $ 25 750 g mol À1 ) was introduced into a 50 mL glass reactor with a magnetic bar. The macroinitiator was dissolved in dry toluene and dried three times by azeotropic distillations. A certain amount of freshly distilled 3-CL was then added. Aer heating to 110 C a 0.1 mL of 0.06 M Sn(Oct) 2 was rapidly injected through a septum and the polymerisation was carried out for 48 h at 110 C. The reactor was cooled to room temperature and the reaction mixture was dissolved in toluene. The Y-shaped terpolymer was collected by precipitation in cooled diethyl ether, ltrated, and dried overnight at 40 C in vacuum.

Preparation of the nanoparticles (NPs) solutions
To prepare the Y-shaped terpolymer NPs solutions, 5 mg of polymer was dissolved in 1.25 mL of acetone (40 C), and the polymer solution was added drop wise to 2.5 mL of pure water under magnetic stirring. The acetone was further removed by evaporation under reduced pressure and the solution was concentrated to 1.25 mL. For the scattering measurements the NPs samples were diluted with phosphate buffer saline pH 7.4 (PBS) to the nal concentrations of 2.0, 1.5, 1.0, and 0.5 mg mL À1 . All samples were ltered using a Millipore 0.45 mm lter (Milli-pore®, Czech Republic) before the scattering measurements.

characterisation techniques
The characterisation techniques such as proton nuclear magnetic resonance ( 1 H NMR), Fourier transform infrared spectroscopy (FT-IR), size exclusion chromatography (SEC), dynamic (DLS) and static (SLS) light scattering, nanoparticle tracking analysis (NTA), cryo-transmission electron microscopy (cryo-TEM) are described in detail in the ESI Section. †

Results and discussion
Synthesis of the a-methoxy-u-tosyl-poly(ethylene oxide) macroinitiator ((Stage 2) Scheme 1) MPEO end-capped with a tosyl group was prepared as a macroinitiator by esterication reaction. For this purpose, the u-hydroxyl end-group of commercially available MPEO (M n(NMR) $ 1800 g mol À1 ) was reacted with an excess of tosyl chloride using CH 2 Cl 2 as a solvent and TEA as a base (compound 2, in Scheme 1). The macroinitiator was fully characterised by 1 H NMR, FT-IR spectroscopy and SEC analysis, which are described in detail in our previous report. 51 Synthesis of the [MPEO-b-PEtOx(OH) 2 ] diblock copolymer ((Stage 3) Scheme 1) The living cationic ring-opening polymerisation (CROP) of 2-oxazolines (Ox)s (cyclic imino ether) was rst reported in 1966 by four independent research groups. [52][53][54][55] The CROP of 2-oxazolines can proceed in a "living" manner under appropriate conditions, meaning that neither undesired termination nor chain transfer occurs during the polymerisation. Depending on the nature of the monomer and initiator used, the CROP of (Ox)s can be ionic or covalent. 56 The living CROP of 2-oxazolines is a versatile method for the preparation of well-dened poly(2-oxazoline)s (POx)s, whereby both the initiation and termination steps provide the possibility of introducing a variety of functional groups. In the case of polymerisation of 2-ethyl-2-oxazoline (EtOx) using sulphonates as electrophilic initiators, the CROP proceeds via ionic species. [57][58][59] Termination of the CROP of (Ox)s can be achieved by nucleophilic attack on the 5-position of the oxazolinium species, which is the thermodynamically controlled and mostly favoured end-capping reaction. The most widely used nucleophilic terminating agents are aqueous or methanolic sodium hydroxide solutions 60,61 and carboxylic acid salts. Additionally, termination can occur by the in situ formation of carboxylic acids salts from the acid and 2,6-dimethylpyridine 62,63 and amines. 61 Here, a [MPEO-b-PEtOx(OH) 2 ] double-hydrophilic block copolymer was synthesised by CROP of EtOx using u-tosyl-MPEO as a macroinitiator and subsequent in situ end-capping by diethanolamine of the living oxazolinium species converted into u,u 0 -dihydroxyl groups, (compound 3, in Scheme 1). The doublehydrophilic block copolymer was obtained with a MPEO molecular weight of approximately 2200 g mol À1 and a PEtOx block molecular weight of approximately 23 550 g mol À1 . Aer purication, the structure of the double-hydrophilic block copolymer was conrmed by 1 H NMR and FT-IR spectroscopy. The M n of [MPEO-b-PEtOx(OH) 2 ] was determined by 1 H NMR spectroscopy and SEC. The 1 H NMR spectrum of the diblock copolymer ( Fig. 1) showed the characteristic signals of the protons belonging to the ethylene oxide (EO) and EtOx repeat units. The methylene protons of the EO repeat units marked with b were observed at d ¼ 3.64 ppm. The spectrum also showed a broad singlet signal observed at d ¼ 3.46 ppm and labelled as d that corresponded to the chemical shis of the protons in -N-CH 2 -CH 2 from the EtOx repeat units. Furthermore, the spectrum detected signals corresponding to the pendant group of the main polymer chain of PEtOx at d ¼ 2.40 ppm (e) that was attributed to the methylene protons of N-C(O)-CH 2 -CH 3 and another labelled as f at d ¼ 1.12 ppm that was assigned to the methyl group of N-C(O)-CH 2 -CH 3 . The 1 H NMR spectrum also showed a signal identied as c at d ¼ 3.70 from the last monomer unit of PEO that was connected to the PEtOx polymer chain. A triplet signal at d ¼ 2.84 ppm, which corresponded to the four protons from the N-CH 2 -CH 2 -HO end-capped group (g and g 0 ) that formed aer termination by diethanolamine, was observed. Nevertheless, the signals of the four -CH 2 -OH (h and h 0 ) hydrogen nuclei were not detectable because they were hidden by the signal of the CH 2 O units of the PEO repeat unit at d ¼ 3.64 ppm. Furthermore, the singlet signal at d ¼ 3.39 ppm attributed to CH 3 -Owas not detected in the spectrum because it was hidden by the peak of the -N-CH 2 -CH 2units of the PEtOx repeat unit at d ¼ 3.46 ppm.
Based on the molecular weight of the initiating u-tosyl-MPEO macroinitiator, the number-average molecular weight M n(NMR) of the diblock copolymer was calculated by eqn (1).
Â DP MPEO Â 99 + M n(NMR) (macroinitiator) (1) where I d and I b represent the integral values of the signals at d  Table 1). The structure of the obtained diblock copolymer was also conrmed by FT-IR spectroscopy (Fig. 2). The spectrum showed the absorption peaks characteristic of both components (MPEO and PEtOx): at 1625 cm À1 corresponding to the -amide bond (C]O stretching) of the 2-ethyl-2-oxazoline repeat units of the PEtOx block; at 1421 cm À1 (d CH from the CH 3 of POx); at 1197 cm À1 attributed to the -ether bond (C-O-C stretching) of the EO repeat units of the PEO backbone; at 1051 cm À1 (C-N stretching from POx); and at 2976 and 2885 cm À1 corresponding to the -C-H vibrations typical for both monomer units. The presence of hydroxyl groups at the u and u 0 positions in the double-hydrophilic block copolymer structure was proven by the appearance of a broad and intense absorption band with a maximum at 3442 cm À1 . Furthermore, a low-intensity absorption band at 3739 cm À1 indicating the existence of intramolecular hydrogen bonding between the hydroxyl groups, was observed.
The SEC analysis of the [MPEO-b-PEtOx(OH) 2 ] diblock copolymer (bold line, Fig. 3) (1, Table 1) showed a monomodal and narrow molecular weight distribution. The main molecular characteristics of the double-hydrophilic block copolymer are listed in Table 1. The obtained data reported in Table 1 conrmed that the polymerisation was controlled with a low polydispersity index and an experimental molecular weight dictated by the monomer-to-initiator ratio and monomer conversion.  Table 1).     Table 1. The good agreement between M n(theor.) and M n(NMR) conrmed that the expected Y-shaped [AB(C) 2 ] terpolymer was formed and that the polymerisation of the third block of the PCL from the symmetric u,u 0 -dihydroxyl groups of the double-hydrophilic block copolymer was controlled. The molecular weights and polydispersity indices of the synthesised Y-shaped [MPEO-b-PEtOx-b-(PCL) 2 ] terpolymers were determined by SEC. As evidenced the SEC curves were overlapped (see the dotted, dashed and dash dotted lines, Fig. 3). The SEC traces revealed relatively narrow molecular weight distributions commonly observed for "living" controlled polymerisation techniques. The SEC proles of the Y-shaped terpolymers were monomodal, and the elution times were shied towards lower values corresponding to higher molecular weights compared with those of the diblock copolymer macroinitiator (bold line, Fig. 3). A slight asymmetry was observed at longer elution time in the SEC curve (dashed line, Fig. 3), which strongly suggested that some unreacted double-hydrophilic block copolymer was present. The main characteristic molecular features of the Y-shaped [AB(C) 2 ] terpolymers are listed in Table 1.
(R H ) of the nanoparticles measured at a scattering angle of 90 . The diameter distribution (2R H ) and relative particle size intensity by NTA are displayed in Fig. 5b and c. The nanoparticles showed a mean particle diameter of 132 nm, and the values of d(0.1), d(0.5), and d(0.9) were 81, 134 and 176 nm, respectively.
The DLS data showed a single population of nanoparticles for each of the four solution concentrations. The size distributions of the particles were narrow, as indicated by NTA that displayed a span value of 0.7 (eqn (4) see ESI †). The R H values ( Table 2) were calculated using eqn (1) (see ESI †) through the diffusion coefficient values obtained from the slope of the linear ts of the relaxation rate dependence on q 2 (Fig. S1 †). The R G parameter was calculated from the linear t of the angular dependence of K c /R (q) based on the data in Fig. 6.
The diffusion coefficient (D) values and the R G /R H ratios were similar (Table 2) for the different concentrations (0.5 to 2.0 mg mL À1 ). At 25 C, the D values were constant regardless of the angle of observation, which was in agreement with the scattering contribution of spherical structures. 64 The R G /R H ratio, a sensitive structural parameter of the particles in solution, 65   The D values obtained from the slope of the linear ts of the relaxation rate dependence on q 2 (10 10 cm 2 s À1 , Fig. S1).     Fig. S3 and 4 †) showed only one population in all of the concentrations up to 55 C. The zeta potential values for the nanoparticle solutions (2 mg mL À1 ) varied from À2 to À6 mV (5-70 C) showing that the values did not change as a function of the temperature. The R G /R H ratio values (0.92-0.97) indicated the presence of structures corresponding to spherical nanoparticles at temperatures up to 50 C. In addition, nearly constant molecular weight values (1.53-1.85 Â 10 7 g mol À1 ) and aggregation numbers (349-462) suggested that no particle aggregation was observed in this temperature range (5 to 50 C). For the [MPEO 44 -b-PEtOx 252 -b-(PCL) 2Â87 ] ( Table 4) the DLS data showed only one scattering population for all temperatures up to 60 C (data not shown). The zeta potential values for the nanoparticle solutions varied from À4 to À8 mV (5-60 C) and as for the [MPEO 44 -b-PEtOx 252 -b-(PCL) 2Â44 ] did not change as a function of the temperature. According to the R G /R H ratio values (0.86-0.97) the presence of structures corresponding to spherical nanoparticles at temperatures up to 50 C are expected. In addition, nearly constant molecular weight values (0.60-0.74 Â 10 7 g mol À1 ) and aggregation numbers (118-132) suggested that no particle aggregation was observed in this temperature range (5 to 50 C). For the last synthesised [MPEO 44 -b-PEtOx 252 -b-(PCL) 2Â131 ] terpolymer (Table 5) the DLS data showed only one population in all of the concentrations up to 40 C. The zeta potential values for the nanoparticle solutions varied from À2 to À10 mV (5-50 C). For these terpolymers the R G /R H ratio values (0.97-1.06) indicated the presence of structures corresponding to spherical nanoparticles at temperatures up to 40 C. The molecular weight values (0.13-0.21 Â 10 7 g mol À1 ) and aggregation numbers (20-33) suggested no particle aggregation in the temperature range of 5 to 40 C.
At temperatures below the LCST, hydrogen bonds between the polymer carbonyl group and the water hydrogens were abundant, 72 and the NPs were swollen by water. The density values conrmed this swollen state, which was also previously veried for linear MPEO 44 -b-PEtOx 263 -b-PCL 87 NPs. 51 When  (Tables 3-5). The temperature dependence behaviour observed for the 3 terpolymer NPs was related to the thermodynamic effects of the LCST on the PEtOx block. 66 This result was observed because with the increase in temperature, the hydrogen bonds between the carbonyl (C]O) group of the PEtOx block and the water hydrogens were weakened. 73 The weakening of the hydrogen bonds favoured the expulsion of the water entrapped inside of the particles. Therefore, with an increase in the temperature, an increase in the particle interaction was expected due to the increase in its hydrophobicity. According the results, the onset of NP aggregation driven by PEtOx dehydration started to be observed at   aggregation, the R G /R H ratio values also provided valuable information related to this process. 64 Commonly, in the aggregated state, the increase in the R G of the NPs is more pronounced in comparison with R H ; thus, an increase in the R G / R H ratio is observed. According to previous observations, this behaviour is related to a shi in the position of the scattering centres between the non-aggregated and aggregated NPs. 51 In the non-aggregated NPs, the polymer density decreases from the centre to the shell, whereas in the aggregated NPs, the density is also relatively high in the periphery. The scattering centres of the aggregated particles are composed of a collection of hydrophobic collapsed particles, whereas they are water swollen in the non-aggregated particles. Therefore, higher R G /R H ratios would be expected as the aggregation proceeds with the temperature increase. At 60 C ([MPEO 44 -b-PEtOx 252 -b-(PCL) 2Â44 ]) and for nanoparticle concentrations of 0.5 and 1.0 mg mL À1 , two peaks were observed in the distribution of the hydrodynamic radii ( Fig. S4 c †). The scattering intensity related to the largest peak was approximately 450 nm for both concentrations, indicating a collapsed system at 60 C. At 61 C, for the nanoparticle concentration of 1.5 mg mL À1 , the presence of a scattering intensity peak corresponding to a size larger than 100 nm ($429 nm; aggregates) ( Fig. S4 d †) was observed. For the nanoparticle concentration of 2 mg mL À1 , the collapse was observed at 63 C (Fig. S5 †). Another evidence of the onset of aggregation and nanoparticle collapse was the increase in the diffusion coefficient with the increase on the temperature up to 60 C (1.95-7.48 Â 10 À8 cm 2 s À1 ; Fig. S2 †). However, at 62 C a decrease (6.67 Â 10 À8 cm 2 s À1 ) in the diffusion coefficient indicated the increase in the R H and nanoparticles aggregation.
All the SLS data were obtained from the data in Fig. S6 and 7. b NP density (r) was calculated by eqn (3) (Fig. 7). Cryo-TEM microscopy ( Fig. 8) was also performed on the NPs to verify the possible morphological changes induced by the temperature. For samples measured at 25 C, spherical NPs with a mean particle size of 70 nm were observed for [MPEO 44 -b-PEtOx 252 -b-(PCL) 2Â44 ] (Fig. 8a, top)  The experimental data suggest that the temperature increase caused size and morphological changes in the NPs. The mean diameter of the NPs determined from the cryo-TEM images was in agreement with that determined by DLS (Tables 3-5). It was possible to observe some morphologically ill-dened structures, which were related to inorganic salts (NaCl and KCl) presented in the saline buffer solution.

Conclusions
A series of nonlinear, amphiphilic, biocompatible, Y-shaped [MPEO-b-PEtOx-b-(PCL) 2 ] terpolymers with different molecular weights of the PCL block were successfully synthesised by a combination of living cationic and anionic ROP in a three-step synthetic procedure. The u-tosyl-MPEO macroinitiator was rst synthesised using an esterication reaction with tosyl chloride. In a second step, a [PEO-b-PEtOx(OH) 2 ] diblock copolymer was designed by cationic ROP of EtOx using the pre-synthesised u-tosyl-MPEO macroinitiator. The CROP was terminated in situ by diethanolamine in order to obtain two symmetrical hydroxyl groups (a primary alcohol), which were prone to initiate the anionic ROP of 3-CL catalysed by Sn(Oct) 2 , providing the nal Y-shaped [AB(C) 2 ] terpolymers. The terpolymers were synthesised with good control over the molecular characteristics of each product and with the capability of easily tuning the length of each block. The aqueous self-assembly of the "snake tongue" Y-shaped [MPEO 44 -b-PEtOx 252 -b-(PCL) 2Â44 ] terpolymer was characterised by SLS, DLS, NTA, and cryo-TEM and its physico-chemical properties as well as the molecular architecture effect on the self-assembly and on the LCST was compared with the Y-shaped [MPEO 44 2Â131 ] with signicant differences in the supramolecular self-assembly behaviour compared with the analogous linear structure, clearly indicating the crucial effect of the molecular architecture. Furthermore, in agreement with previous ndings a decrease in the LCST was observed with increase of the molecular weight fraction of the hydrophobic block (PCL) of the Y-shaped terpolymers.