Polymerization-Induced Self-Assembly via RAFT in Emulsion: Effect of Z-Group on the Nucleation Step

Polymerization-Induced Self-Assembly (PISA) has emerged as one of the most powerful and widely employed techniques for preparation of block copolymer and polymeric nanoparticles in dispersed systems. Its success relies on a rapid, easily scalable and straightforward process, associated with the ability to readily control nanoparticle morphology. In the present work, we have investigated effect of the Z-group on the nucleation step of aqueous RAFT PISA performed in environmentally friendly emulsion polymerization. Seven different poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) macroRAFTs were synthesized using RAFT agents containing Z-groups of different hydrophilicity. Slow polymerizations and incomplete chain extension reactions were observed for systems with a hydrophilic Z-group, while the more hydrophobic Z groups led to higher polymerization rates and very successful chain extensions. A mechanism based on Z-group induced RAFT exit is proposed to rationalize this surprisingly behaviour, providing important information on the mechanistic understanding and optimization of PISA in emulsion.


INTRODUCTION
2][3][4][5][6][7] The approach typically entails synthesis of a macromolecule that is soluble in a suitable solvent via reversible deactivation radical polymerization (RDRP), followed by chain extension with a second monomer forming amphiphilic chains which self-assemble into nano-objects.Note however that PISA can also be conducted based on non-living radical polymerization as exemplified by additionfragmentation chain transfer (AFCT) polymerization. 8The PISA process is attractive as it enables control of the particle morphology not only as conventional spherical particles but also sophisticated morphologies such as fibers, vesicles, jellyfish, 'yolk/shell', multi-shelled vesicles, etc.Furthermore, PISA processes also present the advantages of high polymerization rate with no intermediate purification steps, and the resulting dispersions can be obtained with high solids contents (30-50%).
Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization is the RDRP technique by far most commonly used for implementation of PISA due to the great versatility of RAFT polymerization towards a wide range of monomers, and also its

Synthesis of hydrophilic macroRAFT
In a typical experiment, 1.18 mmol of RAFT agent (RAFT3), 4.73 mmol of 1,3,5-trioxane, 56.9 mmol of AA and 0.0594 mmol of ACPA were introduced in a 25ml glass vial (Table SI1).The mixture was diluted with 15 mL of 1,4-dioxane.The [RAFT]/[I] and [M]/[RAFT] ratios were 20 and 44, respectively (unless otherwise stated), and solids content 24%.The flask was septum-sealed and purged with nitrogen for 30 min, and then immersed in an oil bath at 80°C with magnetic stirring at 300rpm.The reaction was conducted overnight.Conversion was calculated using NMR analysis and number average molecular weight (Mn) and dispersity (Ð) determined by SEC-THF.The theoretical molecular weight (Mn,th) was calculated according to the equation: (1)   where [] 0 and [] 0 are the initial monomer and RAFT concentrations, respectively,   is the molar mass of the monomer, X is the fractional conversion of monomer and   is the molar mass of the macroRAFT agent.The livingness was calculated based on the equation: (2)   where [CTA]0 and [I]0 are the initial concentrations of the RAFT agent and initiator, respectively, and fc is the coupling factor (termination by disproportionation was assumed for PMAA, fc = 0, while for PAA coupling was assumed, fc = 1).The term 2 ×  × [] 0 × (1 −  −   ) corresponds to the total number of radicals generated from the initiator over the polymerization time, where f is the initiator efficiency (assumed to be 0.6) and kd is the rate constant of decomposition for ACPA (kd = 1.26x10 16 x e -134/RT s -1 ). 32he macroRAFTs prepared in aqueous solution were used without purification.The macroRAFTs prepared in dioxane were purified by precipitating three times.The first precipitation was conducted directly from the reaction medium in cyclohexane.Two extra precipitations were performed using 5 ml of methanol as solvent and 50 ml diethyl ether as non-solvent.The polymer was recovered via centrifugation at 9000 rpm for 3 min.The purified macroRAFT (light-yellow fine powder) was obtained after drying in a high vacuum oven at 30 o C. Scheme 1 -Synthesis of hydrophilic macroRAFT agents via RAFT solution polymerization (Table S1).

PISA via RAFT emulsion polymerization
In a typical experiment (Latex 2, Table 1), 5 g of pre-synthesized macroRAFT solution (PMAA43-Ac, 1.2 10 -2 mol L -1 ), 4 mL of deionized water, 2.4 g of styrene and 1 mL of KPS stock solution (2.3 10 -3 mol L -1 ) were added into a 25 mL glass flask.The [M]/[RAFT] and [RAFT]/[I] ratios were kept at 200 and 5, respectively, and solids content = 22% (Table 1).The flask was septum-sealed with parafilm and wire, and purged with nitrogen for 30 min in an ice-water bath.After degassing, the flask was transferred to a thermostatically controlled oil bath pre-heated at 80°C under magnetic stirring of 500 rpm.The polymerization was conducted for 6h (unless otherwise stated).Samples were periodically withdrawn with a degassed needle to monitor conversion by gravimetry, particle size by DLS, Mn and Ð by SEC.

Dynamic light scattering (DLS)
Intensity mean average diameter (Zav) and polydispersity index (PdI) were measured using a Malvern Zetasizer Nanoseries (NanoZS).Measurements were conducted at 25°C using a 4 mW He-Ne laser with wavelength 633 nm, and a scattering angle of 173°.Samples for analysis were prepared by diluting 1 drop of the latex with deionized Milli-Q water.Zav and PdI were obtained using the fully automatic mode of the Zetasizer system and fitted with monomodal cumulant analysis.

RESULTS
The main focus of the present work has been to investigate to what extent the hydrophilicity of the Z-group of the RAFT agent affects the mechanism of PISA in emulsion polymerization.The one-pot PISA process adopted is based on the strategy reported by Chaduc et al. 23 Hydrophilic PMAA-and PAA-based macroRAFT agents with different Z-groups (Table S1 and Figure 1) were prepared via solution polymerization in water (except PMAA38-C12 and PAA46-C12 macroRAFTs, which were synthesized in dioxane due to poor water solubility of the RAFT agent).All polymerizations proceeded under RAFT control resulting in low dispersities (Ð = 1.1~1.2,Table S1) with monomodal and well-defined molecular weight distributions (MWDs; Figure 1).The degree of livingness was calculated according to equation 2, resulting in very high chain end-fidelity (L > 94%, Table S1).These macroRAFTs were subsequently chain extended with styrene, leading to self-assembly into polymer particles.M n = 2350 g mol -1 Đ = 1.14

PMAA-based system: Effect of Z-group hydrophobicity
Log M (g mol -1 ) Log M (g mol -1 ) Log M (g mol -1 ) Log M (g mol -1 ) Log M (g mol -1 ) Log M (g mol -1 ) Initially we focus our attention on PISA of styrene mediated by PMAA-based macroRAFT agents with different Z-groups.The experimental conditions and the results are summarized in Table 1.The experiments performed in the presence of the PMAA40-Ac macroRAFT (Latex 2) exhibited unexpected behaviour in terms of kinetics compared to PMAA43-C4 (Latex 3; Figure 2A).The emulsion polymerization in the presence of PMAA43-C4 proceeded very fast, reaching full conversion in 1h, resulting in small particle size (Zav = 71 nm), in agreement with previous work 25 performed with a similar macroRAFT agent (PMAA43-C3, Z-group: -S-CH2-CH2-CH3).On the contrary, PMAA40-Ac resulted in much lower polymerization rate with only 53% conversion in 6h and significantly larger particles (Zav = 190 nm).These two macroRAFTs have very similar structures (PMAA40-Ac and PMAA43-C4; Figure 1), composed of ca.40 units of methacrylic acid and the same R-group.The main difference is the additional carboxylic acid group of the Z-group for PMAA40-Ac (Figure 2).This seemingly very minor difference in structure drastically affects the kinetics of emulsion polymerization.Furthermore, the control of the MWD was also negatively impacted with incomplete consumption of PMAA40-Ac, while PMAA43-C4 resulted in successful chain extension (Figure 3B and 3C).How does this minor change in RAFT agent structure so dramatically influence the polymerization?Before discussing the polymerization mechanism, it is important to address the conformation of PMAA 25,30 in aqueous solution, and how this can affect the PISA process.Chaduc et al. 25 conducted fluorescence studies of short chain PMAA macroRAFT (< 5000 g mol -1 with Z-group S-(CH2)2-CH3) at different pH.A conformational transition from a PMAA hyper-coiled structure to a water-swollen state was observed between pH 4 and 6.Our group conducted further fluorescence studies to confirm if this change in conformation also applies to PMAA-based macroRAFT with other Z-groups. 30Interestingly, hyper-coiled PMAA chains were observed under acidic conditions regardless of Z-group hydrophobicity for the Z-groups investigated (-S-(CH2)11-CH3, -S-(CH2)3-CH3, -S-(CH2)3-COOH).Furthermore, DLS measurements indicated the formation of small aggregates comprising a few chains rather than single chain conformation.1).The number above each MWD indicates the conversion.Straight-lines represent RI signal and dashed-lines the UV-detection (λ = 325 nm).
Returning our attention to the PISA process using PMAA40-Ac and PMAA43-C4, we propose the mechanism illustrated in Figure 4 to explain the results.It is noteworthy that all polymerizations were performed at acidic conditions (pH < 3) above the critical aggregation concentrations (CAC, a.k.a.CMC; 5.4 x 10 -6 M (PMAA40-Ac) and 2.7 x 10 -6 M (PMAA43-C4)), so hyper-coiled structures are expected to lead to aggregate formation for both macroRAFTs.
Step 1 of the mechanism represents this hyper-coiled aggregated structure in aqueous solution.The presence of hydrophobic domains at low pH enhances the local monomer concentration in the vicinity of the PMAA chain end carrying the RAFT moiety leading to rapid chain growth.This behaviour was observed in the system using PMAA43-C4, i.e. a very fast nucleation step and high polymerization rate (Latex 3 in Figure 2).However, in case of PMAA40-Ac, despite the presence of aggregated hydrophobic domains (very similar fluorescence spectrum as PMAA43-C4 at pH 3, Figure SI1), 30 slow nucleation and low polymerization rate were observed.We propose that this can be explained by so-called "Zgroup induced RAFT exit" due to the more hydrophilic Z-group of PMAA43-Ac (Scheme 2).The Z-group RAFT species is the RAFT species generated by addition of a radical having entered the "precursor particle", followed by fragmentation to release a PMAA radical.Exit would occur during the RAFT pre-equilibrium, where the resultant RAFT agent Stn-Z (n = a few units, most likely 1) escapes the hydrophobic domain due to its relatively high hydrophilicity.Such exit results in the loss of RAFT agent from the locus of polymerization, negatively impacting the chain extension.This results in fewer amphiphilic chains being formed, which compromises the colloidal stability, and consequently larger particles form, resulting in a lower total number of particles (Table 1 and Fig. 2B).The reduced rate of chain extension leads to the particles swelling with less St monomer compared to if chain extension were more successful, given chain extension generates hydrophobic PSt domains, which would swell further with St.The polymerization rate is thus negatively impacted both by the lower number of particles (as per established emulsion polymerization kinetics) 33 and by the reduced extent of swelling.Exit of the expelled radical (R-group) has previously been invoked to explain results in both emulsion and miniemulsion polymerization.Importantly, however, such exit refers to the RAFT R-group as a radical species, not the Z-group as part of the RAFT agent Log M (g mol -1 ) PMAA 50 -C12 47% 99% (not a radical) as in the present workthis is a very important distinction.Z-group induced RAFT exit means that the RAFT moiety has exited, unlike R-exit which does not alter the location of the RAFT moiety (i.e. the trithiocarbonate moiety in this case).In regards to Rgroup exit as a radical, Huang et al. 34,35 reported on the emulsion polymerization of St mediated by a PAA-based macroRAFT agent.No polymerization 34 or a long inhibition 35 were observed for this system, which was attributed to exit of the PAA macroradical from the micelle-like structure during the pre-equilibrium step of RAFT process.In fact, the PAA-segment acts as the colloidal stabilizer for the pre-formed particle (or monomer swollen "micelle") and the loss of the macroradical would drastically affect the nucleation step.Macroradical exit has also been observed by other authors 36,37 in miniemulsion polymerization of St using PAA-based macroRAFT.The macroradical exit mechanism proposed by these authors [34][35][36] is consistent with our current observations.][40][41][42] The use of a hydrophilic macroRAFT with a lower number of MAA units was also explored, PMAA24-Ac (Latex 1 vs Latex 2 of PMAA40-Ac).Based on a traditional PISA mechanism one would expect that nucleation would be faster with a shorter hydrophilic block, given that a shorter hydrophobic block would then be sufficient for self-assembly to occur.However, no significant difference was observed when comparing the kinetics of the two systems using St as monomer (Figure 2).This can be rationalized by considering that hydrophobic domains and aggregates (Figure 4) would form also for PMAA24-Ac despite its lower molecular weight, 30 and the factor that limits the transformation of "precursor particles" to mature particles is Z-group induced RAFT exit, which restricts the extent of chain extension .1).Note that this is merely a schematic illustrating the principlesin reality the mature particles (in red) would comprise a significantly higher number of (blue) chains than displayed.Step 1: Polymerization within monomer-swollen hydrophobic domains after entry of radical from aqueous phase; Step 2: Chain extension within hydrophobic domains but also significant Z-group induced RAFT exit limiting the extent of chain extension in case of PMAA40-Ac; Step 3: The processes of Step 2 continue, accompanied by aggregation of precursor particles and further particle growth, while monomer droplets remain; Step 4: Same as Step 3 but monomer droplets are now depleted.Scheme 2 -Pre-equilibrium of the RAFT mechanism using PMAA-Ac as macroRAFT (Latex 1 and 2, Table 1)."I -" represents the sulfate radical anion originating from the initiator potassium persulfate (KPS).The species on the right is referred to as a "Z-group RAFT" species in the text.
It could be speculated that the different mechanisms of particle nucleation for the PMAA43-C4 and PMAA40-Ac systems may originate in different coil conformations given the different hydrophobicities of the Z-groups.However, in our previous work, 30 we demonstrated that these two macroRAFT species exhibit very similar behaviour in aqueous solution at acidic conditions (all latexes of the current work were prepared at pH < 3).The fluorescence spectra in the presence of Nile red were very similar for the two systems (Figure SI1), indicating that the dye is experiencing similar microenvironments, 30 i.e. the same hydrophobic character.Furthermore, the CAC values were also similar as mentioned above.The Zav obtained from DLS for both systems at pH 3 were also very similar, 2.2 and 2.7nm for PMAA43-Ac and PMAA41-C4, respectively. 30These results strongly indicate that these two macroRAFT species exhibit similar behaviour in aqueous solution at acidic conditions, although further investigations are necessary to confirm the exact coil conformations.
A PMAA-based macroRAFT containing a more hydrophobic Z-group (PMAA38-C12) was also tested (Latex 4, Table 1).Similar to the PMAA43-C4 system, the polymerization proceeded fast (Figure 1), reaching 95% in less than 2h.This is in agreement with our proposed mechanism (Figure 4), as the high hydrophobicity of the Z-group (-S-C12H25) would prevent Z-group induced RAFT exit.However, a larger particle size was obtained for the PMAA38-C12 system (Zav = 148 nm, Latex 4) compared to the PMAA43-C4 system (71 nm, Latex 3), although still significantly smaller than for PMAA20-Ac (Latex 1; 184 nm) and PMAA40-Ac (Latex 2; 190 nm).This difference may be associated with the initial size of the macroRAFT agent in aqueous solution -the PMAA38-C12 tends to form a larger aggregate than PMAA43-C4, i.e. a lower number of precursor particles. 30imodal MWDs were obtained for the PMAA-based macroRAFT containing the most hydrophilic Z-group, PMAA20-Ac and PMAA40-Ac (Latex 1 and Latex 2, Figure 3A and Figure 3B, respectively), resulting in very high dispersity (Ð > 2, Table 1).This observation further supports our mechanism, i.e. the extent of exit from hydrophobic domains would result in RAFT-end group loss from the polymerization locus (Scheme 2).Hence, the experimental Mn is higher than the Mn,th for both systems (Latex 1 and 2, Table 1), indicating unsuccessful RAFT polymerization.In contrast, due to the more hydrophobic character of the Z-groups in PMAA43-C4 and PMAA38-C12, the absence of such exit results in well-defined shifts toward high molecular weights (Figure 3C and D), Mn ≈ Mn,th and much lower dispersities (1.29 and 1.15, Table 1) in accordance with a controlled/living polymerization.Furthermore, UV detection (325 nm) resulted in good overlap between the RI and UV signals for Latex 3 and 4 (Figure 3C and D), indicating that the majority of the chains contain the trithiocarbonate RAFT endgroup consistent with successful chain extension for PMAA43-C4 and PMAA38-C12.

PAA-based system: Effect of Z-group hydrophobicity
We decided to further investigate how the hydrophobicity of the Z-group would affect PISA using a more hydrophilic macroRAFT based on acrylic acid.Emulsion polymerizations of styrene were conducted in the presence of PAA43-Ac, PAA40-C4 and PAA46-C12 (Latex 5-7, Table 1).Despite the different Z-groups, similar conversion-time profiles were observed (Figure 5).All polymerizations exhibited long inhibition periods (3-4 h) followed by very rapid polymerization after nucleation, reaching full conversion in less than 1 h after nucleation.One may anticipate that the longer inhibition period for PAA would be caused by its higher hydrophilicity compared to the PMAA systems, considering that a longer polystyrene block would be required before self-assembly.However, it was demonstrated above for the PMAA system that the length of the hydrophilic block does not have any significant impact on the kinetics, suggesting that the difference between PAA and PMAA systems would most likely be associated with presence of hydrophobic domains (hyper-coiled state of PMAA) at the early stages of the polymerization. 25haduc et al. 24 showed that PAA macroRAFT chains do not exhibit a hyper-coiled structure at low pH by use of fluorescence studies.Our recent work 30 confirms this behaviour, showing that regardless of the Z-group, significant hydrophobic domains were not observed at pH 3 for PAA-based macroRAFT species.Therefore, there are no aggregates present before polymerization and nucleation depends solely on chain extension and subsequent selfassembly.Since there are no significant hydrophobic domains, the local monomer concentration in the vicinity of the non-coiled PAA active chain is reduced dramatically due to the lower styrene concentration in water (schematically illustrated in Figure 6). 24This leads to a much slower growth of the PSt block and therefore a long inhibition period to reach the critical chain length required for self-assembly.An additional factor that may also affect the nucleation step is the fragmentation of the intermediate radical in favour of the polystyryl radical ("backwards fragmentation") rather than the PAA radical during the RAFT preequilibrium, which would also delay the nucleation step.However, once the growing macroRAFT reached the critical PSt length, a very high number of particles is generated (~10 18 L -1 , Table 1), which correlates directly with the small particle size for all PAA-based systems (<60 nm, Figure 5), leading to very rapid polymerization.Figure 5 -Conversion-time data (A) and intensity-mean average diameter (Zav, lines added as guide to the eye) and dispersity index (PdI) (B) for PISA of styrene using PMAA-and PAA-based macroRAFT with different Z-groups (Latex 1-5, Table 1).
The long inhibition period for PAA systems (Latex 5-7) also results in less effective chain extension compared to the PMAA-based systems.SEC-traces for both PAA systems (Figure 7) exhibit low MW tailing, which can be associated with dead chains generated during the long inhibition period.The more pronounced effect in the PAA43-Ac system (Latex 5) may be associated with the extent of Z-group induced RAFT exit due to the higher hydrophilicity of the Z-group.In the PAA-system, the particle from which exit occurs comprises PAA-b-PSt, hence the PAA-b-PSt macroRAFT is unable to exit, but the RAFT agent generated via additionfragmentation involving the entering radical is much more hydrophilic.4][45] We performed two polymerizations using PAA40-C4 with lower initiator concentration ([RAFT]/[I] = 10 and 20, Figure SI2).However, less than 10% conversion was observed in 23h, which originates in the low polymerization rate in the aqueous phase, thereby delaying nucleation.1).The number above each MWD indicates the monomer conversion.

CONCLUSIONS
The nucleation (particle formation) process in RAFT PISA implemented as an emulsion polymerization has been examined with respect to the effect of the Z-group of hydrophilic macroRAFT agents based on acrylic acid (AA) and methacrylic acid (MAA).Surprisingly, the polymerization of styrene mediated by PMAA-Ac (macroRAFT using the more hydrophilic Zgroup) led to low polymerization rates, poor chain extension and relatively large particles.On the other hand, when polymerizations were performed in the presence of PMAA-based macroRAFTs with more hydrophobic Z-groups, PMAA43-C4 and PMAA38-C12, high rates were observed resulting in full conversion, efficient chain extension and small particles.
A mechanism based on so-called Z-group induced RAFT exit is proposed to explain these different behaviours.Z-group induced RAFT exit refers to the RAFT agent generated by addition of a radical to the initial macroRAFT followed by "forward" fragmentation, resulting in a new RAFT agent where the initial PAA or PMAA segment has been replaced by an entering radical.The more hydrophilic character of the Z-group for PMAA-Ac (in combination with the R-group being a small moiety of relatively high hydrophilicity) leads to high probability of exit from the hydrophobic domains during the nucleation step, causing loss of the RAFT moiety.This ultimately leads to low polymerization rate, poor chain extension and, consequently, low degree of livingness. 46Z-group induced RAFT exit is also proposed to occur in PAA-based systems, leading to poor chain extension for PAA43-Ac while successful chain extension was observed for PAA40-C4 and PAA46-C12 (the latter two with more hydrophobic Z-groups).At low pH (all polymerizations in this study), PMAA chains form hyper-coiled structures as aggregates comprising a few chains which swell with hydrophobic monomer (the second block).Such behaviour is not exhibited by PAA.Consequently, the nucleation process (and thereby the time taken to reach high monomer conversion) is much slower for PAA-based systems because the local monomer concentration is not enhanced as it is for PMAA-based systems.
Overall, these results demonstrate that (i) PMAA-based macroRAFTs are preferable over PAA-based macroRAFTs, and (ii) the Z-group of the macroRAFT should be sufficiently hydrophobic for successful implementation of RAFT PISA as an aqueous emulsion polymerization.These findings have important implications for further development and optimization of PISA processes for synthesis of polymeric nanoparticles.Moreover, these systems are of a great interest for the preparation of multiblock copolymers 47,48 investigations are currently underway and will be reported in forthcoming publications.

Figure 1 -
Figure 1 -Structures of PMAA-and PAA-based macroRAFTs synthesized using RAFT agents with different Z-groups.

Figure 4 -
Figure4-Schematic illustration of proposed mechanism of particle formation/growth for PISA of St using PMAA40-Ac or PMAA43-C4 as hydrophilic macroRAFTs (Latex 2 and 3; Table1).Note that this is merely a schematic illustrating the principlesin reality the mature particles (in red) would comprise a significantly higher number of (blue) chains than displayed.Step 1: Polymerization within monomer-

Figure 6 -
Figure 6 -Schematic illustration of proposed mechanism of particle nucleation and growth for PISA polymerization of styrene using PAA43-Ac as hydrophilic macroRAFT (Latex 5, Table 1).Steps 1 and 2: Polymerization in the aqueous phase, generating some dead chains; Step 3: Nucleation accompanied by some Z-group induced RAFT exit; Step 4: Particle growth with monomer droplets being depleted.