Homo- and co-polymerisation of di(propylene glycol) methyl ether methacrylate – a new monomer

In this study, a new methacrylate monomer with two propylene glycol groups on the side chain, di(propylene glycol) methyl ether methacrylate (diPGMA), was synthesised via an esterification reaction. This new monomer was homo- and co-polymerised for the first time via group transfer polymerisation (GTP). Nine ABA triblock copolymers were synthesised via a “one-pot” GTP, with A and B blocks being based on the hydrophilic and the thermoresponsive oligo(ethylene glycol) methyl ether methacrylate, average M n 300 g mol –1 (OEGMA300), and the hydrophobic diPGMA, respectively. The molar mass (MM) and OEGMA300/diPGMA content was systematically varied and the effect of this on the self-assembly and thermoresponsive properties investigated. All copolymers were shown to self-assemble into aggregates and the size of these aggregates increased with both the MM of the polymer and the polymer content in diPGMA. A thermoresponse was observed in aqueous media, with the cloud point (CP) decreasing as the hydrophobic content increases, and the MM decreases. In concentrated aqueous solutions, the polymer with the highest MM and highest diPGMA content formed gels, whose storage modulus increases as a function of the concentration. This study reports a promising alternative hydrophobic monomer to be used in the fabrication of thermogelling materials.


Introduction
Thermo-responsive polymers (temperature-responsive polymers) are polymers that respond to temperature. [1][2][3][4][5][6][7][8][9][10][11] When aqueous solvents are present, the response is seen as a change in the hydrophilicity of the structure. When the hydrophilicity, and thus the water solubility, increases or decreases when the solution is heated, the polymers are characterised by either an upper critical or lower critical solution temperature, UCST or LCST, respectively. LSCT polymers have attracted much scientific interest for potential in biological applications, as they are soluble at low temperatures, at which they can be easily mixed with drugs and cells, without compromising their structural integrity. Under the appropriate external conditions, solvent, pH and temperature, depending on the molecular structure, the incompatibility with the solvent can be manifest by the formation of a 3D physical network. These networks are known as thermoresponsive gels (TRGs) and find numerous applications in biomedical engineering as injectable gels for tissue engineering and drug delivery, and 3D printing. 1-6, 12, 13 One of the most extensively studied families of thermoresponsive polymers are poloxamers, i.e. ABA triblock copolymers with A and B blocks being based on ethylene glycol (EG) and propylene glycol (PG), respectively. 14,15 Whilst poly(ethylene glycol) (PEG) is highly hydrophilic and thermoresponsive at high temperatures depending on its molar mass (MM), 16,17 poly(propylene glycol) (PPG) is thermoresponsive at lower temperatures. 18,19 More specifically, PPG is hydrophilic below room temperature, depending on the concentration, whilst it is hydrophobic at higher temperatures, thus promoting self-assembly. 18,19 Numerous registered tradenames are available for both the solid polymers and solutions, including Pluronic® (BASF), Lutrol® (BASF), Kolliphor® (BASF), Synperonic® (Croda) and Antarox® (Rhodia). 14,15,20 Various poloxamers are commercially available with differences in the EG/PG ratio and the total MM.
Poloxamers have been previously reported in studies concerning injectable gels, including poloxamer 407 (P407, Pluronic® F127), 21-23 poloxamer 188 (P188, Pluronic® F68), 24,25 poloxamer 124 (P124, Lutrol® L44), 22 and poloxamer 105 (P105, Pluronic® L35). 26 Amongst these, an extensively used material in thermogelling systems is Pluronic® F127. This polymer has a total MM of approximately 12600 g mol -1 , and a composition of EG-PG = 70-30 w/w%. 14 Its aqueous solutions form gels at a concentration of at least 15 w/w%, with gels being formed at room temperature at only 20 w/w%. Owing to its commercial availability and interesting thermogelling properties, many studies have been focused on applying it either as in vivo injectable gel [21][22][23] and as 3D printable material. 27,28 As PEG is highly hydrophilic and largely biocompatible, it has often been incorporated into biological systems to enhance biocompatibility. 29,30 (Meth)acrylate derivatives of PEG are commercially available and have gained significant scientific interest in the last two decades. [31][32][33][34][35][36][37][38][39][40][41][42] When polymerised, the Please do not adjust margins Please do not adjust margins backbone of the polymer consists of carbon-carbon bonds, with the ether bonds (originating from the EG units) being on the side chain and their thermoresponsive ability is affected by the number of EG repeating units. 34,40,41,43 Poloxamers contain both EG and PG repeating units in a linear polymer consisting of ether bonds along the backbone. Using (meth)acrylate PEG and PPG derivatives is advantageous as a wider variety of monomers can be used, including functional groups at which peptides or fluorescent groups might be attached post-polymerisation. In addition, these functional monomers could be polymerised at different points within the polymer chain with appropriate polymerisation methodology, unlike in poloxamers, in which the functionalisation can only take place at the ends of the polymer chain.
Inspired by poloxamers, this study focused on synthesising methacrylate ABA block copolymers with PEG and PPG side chains. For this, oligo(ethylene glycol) methyl ether methacrylate with average M n 300 g mol -1 (OEGMA300, OEG, giving average number of EG units = 4.5) was used to form the outer hydrophilic A blocks. For the central block, an in-house synthesised methacrylate monomer with two PG groups on the side chain, namely di(propylene glycol) methyl ether methacrylate (diPGMA, diPG), was used, Figure 1 Figure 1. This newly synthesised hydrophobic monomer was both homo-and co-polymerised via Group Transfer Polymerisation (GTP). Nine copolymers were synthesised in total which differ in the MM (5100, 10100, and 13100 g mol -1 ) and OEGMA300-diPGMA composition (80-20, 70-30, and 60-40 w/w%). The monomer and polymer synthesis, as well as the investigation of the aqueous solution properties of the polymers are presented and discussed below.

Chemical Structure of the Monomer
The hydrophobic diPGMA monomer was synthesised via the esterification reaction between methacryloyl chloride and di(propylene glycol) monomethyl ether (diPGOH), following a wellreported procedure for the synthesis of methacrylate monomers, [44][45][46] Figure 2. The successful synthesis of the monomer was confirmed via Fourier transform infrared (FT-IR), 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopies. The results of the analysis of diPGOH are presented for comparison. It should be noted that to the best of our knowledge, this is the first time the synthesis of diPGMA is reported.
The FT-IR analysis supports the successful esterification of the alcohol, diPGOH, to the corresponding methacrylate ester, diPGMA, Figure 3, Table S1, with the broad peaks of the PO-H stretching and bending at 3433.1 cm -1 and 1373.8 cm -1 , respectively, and PC-O stretching at 1202.5 cm -1 , disappearing when the esterification takes place. In addition to the disappearance of the peaks corresponding to the alcohol precursor, additional peaks corresponding to the ester group are present in the spectrum of diPGMA. Specifically, a strong peak corresponding to the PC=O stretching appears at 1715.2 cm -1 , whereas the PC-O stretching of the ester appears at 1293.7 cm -1 and 1165.4 cm -1 , and the C=C stretching appears at 1637.1 cm -1 .
The 1 H NMR analysis, Figure 4, confirms the successful esterification, as the peaks corresponding to the methacrylate vinyl bond appear between 5.5 to 6.0 ppm; the corresponding protons are denoted by a and b respectively. In addition, the peak which corresponds to the methyl group of the methacrylate group appears at 1.8 ppm. Concerning the protons derived from the alcohol precursor, their peaks appear at similar chemical shifts with the ones of the alcohol, as expected. Fig. 1 Chemical structures, names, and abbreviations of the monomers. OEGMA300 (OEG) and diPGMA (diPG) are the abbreviations of oligo(ethylene glycol) methyl ether methacrylate, average Mn 300 g mol R% , and di(propylene glycol) methyl ether methacrylate (mixture of isomers), respectively.

Fig. 2
Chemical reaction of the synthesis of diPGMA monomer (mixture of isomers, Figure 1), which is an esterification reaction between methacryloyl chloride and di(propylene glycol) monomethyl ether (diPGOH, mixture of isomers -from top to bottom: . The esterification was catalysed by triethylamine, in THF as solvent and the reaction was carried out in an ice-bath. Please do not adjust margins Similar observations are made by 13 C NMR analysis ( Figure 6). Three additional peaks are observed in the spectrum of the monomer, at S T 100ppm, which correspond to the carbons of the methacrylate part (double bond and ester/carbonyl bond).

Determination of Isomers
In scope of identifying the number of isomers of diPGOH and thus diPGMA, as well as their relative content, gas chromatography -(GC-FID) was used, Figure 6, Table S2.          1   56  57  58  59  60  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75  76  77  78  79  (ppm)   0  10  20  30  40  50  60  70  80  90  100  110  120  130  140  150  160  170  180 (ppm) 16      The measured M n values are slightly higher than the theoretical ones, similarly to previous GTP studies, due to the slight deactivation caused by humidity and acidic impurities. 43,[54][55][56][57][58][59] In addition, the Ð values are U 1.30, and they are satisfactorily low for the purpose of this study, while the experimental compositions agree with the targeted values. Generally, the data from GPC and 1 H NMR analysis support successful GTP formation of triblock copolymers with diPGMA as a central block. The 1 H NMR spectra of Polymer 2 and its precursors are shown in Figure S4, with a complete assignment of the peaks.

Glass Transition Temperature (T g )
diPGMA 7 (Polymer 1) and the triblock copolymers of the highest MM (Polymers 8, 9, and 10) were investigated via DSC and their glass transition temperatures (T g ) were determined, Table 2 and Figure S5. The diPGMA homopolymer, with M n 2200 g mol -1 , shows a transition from a glass to a rubbery state at -24 °C. When compared to an OEGMA300 homopolymer with similar MM (M n 3600 g mol -1 , synthesis reported elsewhere 43 ), the PG-based methacrylate homopolymer has higher T g (-24 °C for PG-based versus -59°C for EGbased). The ABA triblock copolymers based on OEGMA300 and Please do not adjust margins Please do not adjust margins ii) UV-Vis, as the temperature of 50% transmittance, and iii) DLS, as the temperature of 50% aggregation. The results are summarised in Table 4, while the effect of the MM and content in diPGMA on the CP is presented in Figure 10. The percentage in transmittance as a function of temperature is shown in Figure S8, while the size by intensity over temperature is shown in Figure S9, results summarised in Table S3, and Figures S10 and S11. Table 4 Theoretical structures, targeted molar mass (MM) and hydrophobic composition, and cloud points at 1 w/w% polymer solutions in deionised water, as determined by visual tests, UV-Vis spectroscopy, and dynamic light scattering (DLS). a The CP is determined visually as the temperature at which the solution turns cloudy. b The CP is determined via UV-Vis as the temperature at 50% transmittance. c The CP is determined via DLS as the temperature of 50% aggregation.
Generally, the CPs resulted by the three techniques are equal within experimental error, and the same trends are observed. When copolymers of the same MM are considered, it is observed that the CP decreases as the content in the hydrophobic diPGMA increases, as expected. 58 This trend has been previously observed and reported, 58, 60-62 and it is attributed to the enhancement of the "hydrophobic effect" when the hydrophobicity of the copolymers increases. Regarding the effect of MM, while the copolymers with comparable content in diPGMA and 10100 and 13100 g mol -1 do not show much difference in CP, as the MM difference is not significant enough, but the CPs of the copolymers with 5100 g mol -1 are much lower. This is in contrast with some previous studies, which observed the CP decreasing when increasing the polymer MM. 43,56,[63][64][65] These studies investigated: i) thermoresponsive homopolymers based on amine 56,64 and PEG-containing methacrylate units, 43 oxazoline 63 and N-vinylpiperidone, 65 ii) statistical bipolymers consisting of two different thermoresponsive oxazoline units, 63 and iii) triblock terpolymers consisting of a thermo-and pH-responsive block, one block which is hydrophilic and thermoresponsive at higher temperatures, and a hydrophobic block. 56 Thus, it is more accurate to compare the synthesised polymers with their Poloxamers counterparts, whose thermoresponse is attributed to both PPG and PEG blocks. As expected, the current trend of this study, i.e. decrease in CP with decrease in MM, is observed in Pluronics®, when the MM difference is profound. 14 For example, when Pluronics® of 30 w/w% EG are compared, the CP increases from 42 °C (1850 g mol -1 , Pluronic® L43), to 86 °C (4950 g mol -1 , Pluronic® P103), to 90 °C (5750 g mol -1 , Pluronic® P123). This is also the case when Pluronics® with 40 w/w% EG are compared; the CP increases from 58 °C (2900 g mol -1 , Pluronic® L64), to 74 °C (4200 g mol -1 , Pluronic® P84), to 81 °C (5900 g mol -1 , Pluronic® P104). 14 Interestingly, systems based on diblock copolymers of i) methyl tri(ethylene glycol) vinyl ether and isobutyl vinyl ether, 66 and ii) OEGMA300 and n-hexyl methacrylate, 67 in which only one block was thermoresponsive, also reported increase in the CP by increase in the MM. Patrickios et al. attributed this trend to the increased micelle size with increased MM, due to steric stabilisation of these colloidal systems. 66

Visual Gel Points
Solutions of the triblock copolymers in PBS were investigated for gelation by visual tests, and the phase diagrams are shown in Figure  11. The effect of the composition is shown from the left to the right, whereas the effect of MM is shown from top to bottom.
The diluted solutions at 1 w/w% in PBS present a CP, which (a) decreases as the diPGMA content increases (for polymers with similar MM), and (b) increases when the increase in MM is profound (for polymer with the similar composition). As expected, the CPs are lower in PBS than in DI water, which is caused by the presence of salt in solution. 59,68 Similarly, in more concentrated solutions, both a composition effect and a MM effect are observed. For the families of low and intermediate MM, all the polymers precipitate out of solution as the temperature increases, however, it is clearly observed that the temperature of phase separation decreases as the content in the hydrophobic diPGMA increases. This shows a clear effect of composition, as the increase in the hydrophilic OEGMA300 content shifts the thermoresponsiveness to higher temperatures and diminishes gelation if used at high content, in agreement with previous studies. 55,58 Interestingly, in the case of the polymers with high MM, gelation is observed at around 50 °C, shown in blue symbols, when the content of diPGMA is increased to 40 w/w%. This is in consistent with previous studies, which showed that increasing the MM promotes gelation. 56 Nevertheless, even though in most of the cases gelation was not observed, the results are promising, with diPGMA serving as an alternative hydrophobic monomer, to be used on promoting self-assembly and/or designing novel thermogelling systems. Cloud Point ( C) Experimental diPGMA Content (w/w%) 5100 g mol 1 10100 g mol 1 13100 g mol 1 Fig. 10 Effect of content in diPGMA on the cloud points, by UV-Vis, of solutions of the ABA triblock copolymers (1 w/w% in deionised water); where A and B are OEGMA300 and diPGMA, respectively. The effect of the molar mass is also shown in light blue squares (5100 g mol -1 ), dark blue triangles (10100 g mol -1 ), and red rhombi (13100 g mol -1 ).

Fig. 11
Phase diagrams of the ABA triblock copolymers (P2-P10) in phosphate buffered saline (PBS). The effect of diPGMA composition is shown from left to right, whereas the effect of the MM is shown from the top to bottom: (a) MM 5100 g mol -1 (top), (b) MM 10100 g mol -1 (middle), and (c) MM 13100 g mol -1 (bottom). Four phases are observed: i) runny solution coloured in white (square: clear, triangle: slightly cloudy) and black circles for cloudy, ii) viscous solution coloured in red (triangle and circle for transparent and cloudy, respectively), iii) the gel phase is shown in blue (triangle and circle for transparent and cloudy, respectively), the two-phase system is coloured in light green (rhombus and square for gel syneresis and precipitation, respectively).