Facile synthesis of thiol-functionalized amphiphilic polylactide–methacrylic diblock copolymers

Biodegradable amphiphilic diblock copolymers based on an aliphatic ester block and various hydrophilic methacrylic monomers were synthesized using a novel hydroxyl-functionalized trithiocarbonate-based chain transfer agent. One protocol involved the one-pot simultaneous ring-opening polymerization (ROP) of the biodegradable monomer (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione (L-lactide, LA) and reversible addition–fragmentation chain transfer (RAFT) polymerization of 2-(dimethylamino)ethyl methacrylate (DMA) or oligo(ethylene glycol) methacrylate (OEGMA) monomer, with 4-dimethylaminopyridine being used as the ROP catalyst and 2,2′-azobis(isobutyronitrile) as the initiator for the RAFT polymerization. Alternatively, a two-step protocol involving the initial polymerization of LA followed by the polymerization of DMA, glycerol monomethacrylate or 2-(methacryloyloxy)ethyl phosphorylcholine using 4,4′-azobis(4-cyanovaleric acid) as a RAFT initiator was also explored. Using a solvent switch processing step, these amphiphilic diblock copolymers self-assemble in dilute aqueous solution. Their self-assembly provides various copolymer morphologies depending on the block compositions, as judged by transmission electron microscopy and dynamic light scattering. Two novel disulfide-functionalized PLA-branched block copolymers were also synthesized using simultaneous ROP of LA and RAFT copolymerization of OEGMA or DMA with a disulfide-based dimethacrylate. The disulfide bonds were reductively cleaved using tributyl phosphine to generate reactive thiol groups. Thiol–ene chemistry was utilized for further derivatization with thiol-based biologically important molecules and heavy metals for tissue engineering or bioimaging applications, respectively.


Introduction
Block copolymers based on the same type of monomer (e.g. either vinyl or cyclic monomers) are traditionally prepared using a single "living" polymerization technique. 1 Recently, novel block copolymers with interesting properties have been prepared by combining two or more "living" polymerization chemistries to copolymerize dissimilar monomers.  Sequential polymerizations are most commonly used for such syntheses. [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] In principle, simultaneous polymerization can also lead to the synthesis of block copolymers. In practice, there are some examples in the literature for which incompatibility problems have been overcome to combine different polymerization techniques for the synthesis of well-dened block copolymers in a single step. [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] The combination of ring-opening polymerization (ROP) 34 for the controlled synthesis of biodegradable aliphatic polyesters 35,36 and reversible addition-fragmentation chain transfer (RAFT) polymerization, [37][38][39][40] allows the synthesis of dened block copolymer architectures (for a wide range of vinyl monomers). 9,11,15,[41][42][43][44] This approach bodes well for the synthesis of block copolymers for biomedical applications such as sutures, implants for bone xation, drug delivery vehicles and tissue engineering scaffolds. 45 A typical sequential polymerization strategy for the synthesis of polylactide (PLA)-based block copolymers is either to introduce a RAFT agent aer the ROP of lactide (LA) 2,10 or to initiate purication step is required. Clearly, simplifying the process to just one step where the two polymerizations proceed simultaneously is desirable. Exploring the simultaneous one-pot ROP-RAFT polymerization of LA with a vinyl monomer appears to be promising for the synthesis of new amphiphilic biodegradable PLA-based copolymers. Previous studies have focused on the simultaneous one-pot ROP of other monomers such as 3-caprolactone, [21][22][23]30,33 d-valerolactone, 22,33 trimethylene carbonate, 22 b-butyrolactone 28 and RAFT polymerization, and also the simultaneous ROP of LA and RAFT polymerization of acrylic monomers. 24,29 Simultaneous ROP of LA and RAFT polymerization of methacrylic monomers for the preparation of biodegradable PLA-containing block copolymers using a metal-free approach 46 has been previously described, but mainly in the context of the synthesis of hydrophobic diblock copolymers. 22 As far as we are aware, such an approach has not been explored for the preparation of amphiphilic PLA-based block copolymers, particularly when the water-soluble block is based on methacrylic repeat units.
Amphiphilic diblock copolymers have attracted the interest of many researchers because they undergo spontaneous selfassembly in aqueous solution. This results in the formation of various morphologies such as spherical micelles, worm-like micelles, vesicles or intermediate structures. 47,48 Oen the relatively high molecular weight (MW) and/or glass transition temperature can hinder the direct dissolution of amphiphilic diblock copolymers in water, since this makes the formation of micelles or vesicles extremely slow and inefficient. 49 These kinetic constrains can be overcome using either thin lm rehydration or by prior dissolution using water-miscible cosolvents. This latter approach, commonly known as the 'solvent switch' method, is widely used for block copolymer selfassembly. 50 Here, the block copolymer is initially dissolved in a water-miscible common organic solvent for both copolymer blocks. Then water is added gradually to the copolymer solution resulting in the formation of various nanostructures aer removal of the organic solvent. 47 One potential application for amphiphilic copolymers is in drug delivery. Here it is oen considered desirable to conjugate peptides, vitamins or sugars so as to confer cell/tissue specicity and targeting.
Amongst the various conjugation approaches, thiol-disul-de chemistry has been widely used for biomedical applications 51-54 because of its orthogonality, reversibility and redox activity. [55][56][57][58][59][60] The reduction of a disulde bond results in the formation of thiol functional groups. The presence of thiol functionalities during a radical-based vinyl polymerization such as RAFT is not desirable since they can act as efficient chain transfer agents. 61 We have previously reported post-polymerization formation of thiol-functional copolymers using a disul-de-based dimethacrylate (DSDMA) comonomer. [62][63][64][65][66] This disulde acts as an atom-efficient thiol-protecting group for the synthesis of branched methacrylic copolymers. In relatively dilute solution, DSDMA undergoes predominantly intramolecular cyclization on its statistical copolymerization with a methacrylic monomer. 63 Thus cleavage of the lightly branched copolymer results in the formation of near-monodisperse copolymer chains 62,64,65 bearing thiol functionality.

Experimental section
The MPC, OEGMA and GMA monomers were used without further purication. The DMA monomer was passed through a neutral alumina column before use to remove the inhibitor. LA was recrystallized 4-5 times from ethyl acetate prior to use. The solvents THF, DMF, chloroform and methanol were used as the mobile phase in GPC, whereas n-hexane and ethyl acetate were used in column chromatography. Regenerated cellulose dialysis membranes with molecular weight cut-off (MWCO) of 1000 Da were purchased from Spectra/Por. The disulde-based dimethacrylate (DSDMA) branching monomer was synthesized following a previously reported method. 64 Synthesis of hydroxyl-functional RAFT CTA CADB (3.00 g, 10.74 mmol) and 1,6-hexanediol (6.35 g, 53.70 mmol) were added in a round-bottom ask equipped with a magnetic stir bar. These reagents were dissolved in 75 mL CHCl 3 by stirring the contents of the ask for 15 min at 40 C. The reaction catalysts DCC (2.44 g, 11.81 mmol) and DMAP (131 mg, 1.07 mmol) were dissolved in 15 mL CHCl 3 at ambient temperature. The catalyst mixture was subsequently added with a syringe to the round-bottom ask. The reaction mixture was stirred under reux for 46 h and allowed to cool in the refrigerator overnight. Aer ltration, CHCl 3 was removed under reduced pressure using a rotary evaporator. The resulting oily residue was puried by column chromatography (silica gel/nhexane-ethyl acetate 50 : 50). Aer removal of the solvents using a rotary evaporator and a vacuum oven, an oily liquid was obtained (1.82 g, 45% yield). 1

Synthesis of PLA-based diblock copolymers
Simultaneous ROP-RAFT polymerization. For a typical simultaneous ROP-RAFT polymerization targeting PLA 30 -PDMA 30 block copolymer at 66.7% w/w solids, the protocol was as follows: LA (0.53 g, 3.65 mmol), DMA (0.62 mL, 0.57 g, 3.65 mmol), 1 (0.05 g, 0.12 mmol), AIBN (4.0 mg, 0.02 mmol), DMAP (0.06 g, 0.40 mmol) and 0.79 mL 1,2-dichloroethane were mixed together in a 5 mL round-bottom ask. The ask was equipped with a magnetic ea and sealed with a rubber septum. The reaction mixture was purged with nitrogen for 20 min before being placed in a preheated oil bath at 74 C for 24 h.
The syntheses of other PLA-PDMA and PLA-POEGMA block copolymers were conducted following the same one-step protocol. Representative 1 H NMR spectra of diblock copolymers prepared by simultaneous ROP-RAFT processes are given in Fig. 1(c-d).
Two-step ROP-RAFT polymerization. A protocol for the synthesis of a PLA 30 -PMPC 30 block copolymer is described. In a 10 mL round-bottom ask equipped with a magnetic stir bar and sealed with a rubber septum, LA (0.53 g, 3.65 mmol), 1 (0.05 g, 0.12 mmol), DMAP (0.06 g, 0.40 mmol) and 1.90 mL 1,2dichloroethane were added, the mixture was purged with nitrogen for 20 min, and then heated at 74 C for 24 h. The reaction mixture was cooled to 20 C and a sample was withdrawn for 1 H NMR and GPC analysis. Subsequently, MPC (1.08 g, 3.65 mmol), ACVA (8.5 mg, 0.03 mmol) and 8.71 mL anhydrous ethanol were added. The reaction solution was placed in a preheated oil bath at 78 C for 24 h.
A similar two-step protocol was followed for the synthesis of a PLA 200 -PMPC 30 block copolymer with a longer hydrophobic PLA block. The same method was applied for the preparation of PLA 30 -PGMA 30 block copolymer and PLA 30 -PDMA 30 block copolymer (for direct comparison with the one-step protocol). 1,2-Dichloroethane was used as a solvent for both polymerization steps. AIBN was the initiator for these two RAFT polymerizations.
All polymerizations (for both the simultaneous and the twostep protocols) were quenched by allowing the reaction solution to cool to 20 C. A sample was removed from each reaction solution for 1 H NMR and GPC analysis. The nal copolymers were puried by dialysis against acetone using membranes with a MWCO of 1000. For PLA-PGMA and PLA-PMPC block copolymers, this was followed by dialysis in methanol. The solvent was evaporated using a rotary evaporator. The copolymers were dried in a vacuum oven for 48 h. The resulting block copolymers were characterized by 1 H NMR spectroscopy (Table 1 and Fig. 1) and GPC (Table 1, Fig. 2 and 3).
Disulde cleavage of branched PLA 30 -P(OEGMA 30 -stat-DSDMA 1 ) diblock copolymer, functionalization of the resulting PLA 30 -P(OEGMA 30 -stat-TEMA 2 ) block copolymer with divinyl sulfone and its subsequent conjugation with Lglutathione 500 mg (275 mg solids content, 30.6 mmol disulde bonds) of the branched PLA 30 -P(OEGMA 30 -stat-DSDMA 1 ) polymer were added in a 50 mL round-bottom ask. Subsequently, 20 mL DMF (1.4% w/v) was added to the ask to dissolve the reagents. The ask was equipped with a magnetic stir bar and sealed with a rubber septum. The mixture was purged with nitrogen gas for 20 min and placed in an oil bath at 30 C. A solution of Bu 3 P (91.9 mmol, 18.6 mg, 3.0 eq. relative to disulde bonds) and Et 3 N (64.3 mmol, 6.5 mg, 2.1 eq. relative to disulde bonds) in 5 mL DMF was added under a nitrogen atmosphere via syringe to the roundbottom ask. This resulted in disulde cleavage, allowing the conversion of 1 eq. of DSDMA to 2 eq. of TEMA. The reaction was le in an oil bath for 2 h at 30 C to afford a thiol-functionalized linear PLA 30 -P(OEGMA 30 -stat-TEMA 2 ) block copolymer solution. A sample was extracted from the reaction ask for DMF GPC analysis (M n ¼ 38 800 g mol À1 , M w ¼ 53 100 g mol À1 , M w /M n ¼ 1.37). A degassed solution of divinyl sulfone (0.92 mmol, 108.6 mg, 15 eq. relative to the thiol -SH group) in 5 mL DMF was added to the block copolymer. The resulting mixture was stirred at 30 C for 15 h to convert the TEMA monomer units into VSTEMA monomer units. The nal solution was dialyzed (MWCO 1000 Da) against acetone (5 times/3 days). The solvent was removed by rotary evaporator, followed by vacuum oven drying. The dried PLA 30 -P(OEGMA 30 -stat-VSTEMA 2 ) was characterized by DMF GPC (M n ¼ 37 800 g mol À1 , M w ¼ 52 800 g mol À1 , M w /M n ¼ 1.40) and 1 H NMR (Fig. 4).
202 mg of the above dried vinyl sulfone-functionalized block copolymer (218.4 mmol vinyl sulfone, assuming 2 vinyl sulfones per polymer chain) were placed in a 10 mL glass vial equipped with a magnetic stir bar and sealed with a rubber septum. A mixture of 7.0 mg Glu (229.3 mmol Glu or free thiol group, 1.05 eq. relative to vinyl sulfone), 0.6 mg TCEP (0.1 eq. relative to vinyl sulfone) and 4 mL water was purged with nitrogen gas for 20 min. This solution was then added to the vial containing the copolymer. The resulting mixture was purged with nitrogen for another 10 min. The reaction was allowed to proceed a 20 C with stirring for 4 h. The nal copolymer solution was dialyzed (MWCO 1000 Da) against water (6 times/1 day). The puried PLA 30 -P(OEGMA 30 -stat-GluVSTEMA 2 ) was freeze-dried. The dried copolymer was dissolved in the appropriate solvent and characterized by DMF GPC (M n ¼ 23 600 g mol À1 , M w ¼ 34 100 g mol À1 , M w /M n ¼ 1.45) and 1 H NMR ( Disulde cleavage of the branched PLA 30 -P(DMA 30 -stat-DSDMA 1 ) diblock copolymer, functionalization of the resulting PLA 30 -P(DMA 30 -stat-TEMA 2 ) block copolymer with maleimido-monoamide-DOTA and subsequent In conjugation 182 mg (100 mg solids content, 10.3 mmol disulde bonds) of the PLA 30 -P(DMA 30 -stat-DSDMA 1 ) gel was placed in a 14 mL glass vial containing 5 mL DMF (2.0% w/v). The ask was equipped with a magnetic stir bar and sealed with a rubber septum. The mixture was placed in an oil bath at 30 C aer purging with nitrogen for 20 min. A solution of Bu 3 P (30.9 mmol, 6.3 mg, 3.0 eq. relative to disulde bonds) and Et 3 N (21.6 mmol, 2.2 mg, 2.1 eq. relative to disulde bonds) in 1 mL DMF was prepared and added under nitrogen atmosphere to the glass vial using a syringe. The reaction mixture was le at 30 C for 2 h, resulting in cleavage of the disulde bonds. With this reaction the gel network was converted to a thiol-functionalized linear PLA 30 -P(DMA 30 -stat-TEMA 2 ) block copolymer solution. Maleimido-monoamide-DOTA was reacted with the TEMA residues to produce DOTA-functionalized TEMA (DOTATEMA) residues. Specically, a degassed solution of maleimido-monoamide-DOTA (21.6 mmol, 17.0 mg, 2.1 eq. relative to disulde bonds) was added. The reaction was allowed to proceed at 30 C overnight. Then InCl 3 (4.6 mg, 20.6 mmol, 2.0 eq. relative to disulde bonds) were added and the mixture was stirred at 90 C for 1 h. The solution was dialyzed against acetone (MWCO 1000 Da/5 times/3 days) and the solvent was removed using rotary evaporator. The resulting In-conjugated PLA 30 -P(DMA 30 -stat-InDO-TATEMA 2 ) block copolymer was dried in a vacuum oven overnight and characterized by ICP-AES (1.16 In atoms per copolymer chain).

Self-assembly of amphiphilic block copolymers in dilute aqueous solution
Self-assembly of the block copolymers was carried out using the solvent switch method. Briey, PLA-PDMA and PLA-POEGMA block copolymers were dissolved in acetone to give 50 mg per mL of copolymer solutions. 4.8 mL deionized water was added drop by drop in 200 mL of the above solutions under stirring giving copolymer solutions with a nal concentration of 2.5 mg mL À1 . Similarly, an acetone-methanol mixture (3 : 1 v/v) was used to solubilize the PLA-PGMA block copolymer. These solutions were stirred vigorously at room temperature for 5 h, allowing acetone evaporation. For more efficient solvent evaporation the solution was le on a rotary evaporator for 1 min. Following a similar method, the PLA x -PMPC y block copolymers were dissolved in a 3 : 1 v/v chloroform-methanol mixture. This was followed by addition of water and stirring for 2 h to allow solvent evaporation. Dialysis was performed against deionized water for 4 h (5 times) to remove trace organic solvents. Finally, all copolymer solutions were stored at 4 C overnight to avoid copolymer degradation.

Polymer characterization
PLA-PDMA and PLA-POEGMA block copolymers were assessed using a GPC system purchased from Polymer Laboratories at 30 C. The system comprised two PL Gel 5 mm (7.5 Â 300 mm) Mixed-C columns in series with a guard column. These were connected with a WellChrom K-2301 refractive index (RI) detector operating at 950 AE 30 nm. THF containing 2% v/v triethylamine and 0.05 wt/v % of butylhydroxytoluene (BHT) (ow rate 1 mL min À1 ) was used as an eluent.
PLA-PMPC block copolymers were assessed using a Hewlett Packard HP1090 Liquid Chromatograph. Two PL Gel 5 mm Mixed-C columns in series with a guard column at 40 C and a Gilson Model 131 RI detector were used. The eluent was a 3 : 1 v/ v chloroform-methanol mixture with 2 mM lithium bromide (LiBr, ow rate of 1.0 mL min À1 ). Toluene (2 mL) was added to the sample as a ow rate marker.
Ten near-monodisperse linear poly(methyl methacrylate) (PMMA) standards (ranging from 1280 g mol À1 to 330 000 g mol À1 ) were purchased from Polymer Laboratories (UK). These were used to calibrate the above two RI detectors. For both GPC systems, data analysis was carried out using Cirrus™ GPC Soware supplied by the manufacturer.
The MWDs of the PLA-PGMA block copolymer, the branched PLA 30 -P(OEGMA 30 -stat-DSDMA 1 ) block copolymer, the disulde cleaved PLA 30 -P(OEGMA 30 -stat-TEMA 2 ) linear block copolymer and the functionalized linear block copolymers PLA 30 -P(OEGMA 30stat-VSTEMA 2 ) and PLA 30 -P(OEGMA 30 -stat-GluVSTEMA 2 ) were assessed by DMF GPC. This system comprised two Polymer Laboratories PL gel 5 mm mixed C columns and one PL polar gel 5 mm guard column. These were arranged in series and maintained at 60 C, followed by a Varian 390 LC RI detector. The DMF eluent containing 10 mM LiBr was kept at a ow rate of 1.0 mL min À1 . Ten near monodisperse PMMA standards with MWs ranging from 625 g mol À1 to 618 000 g mol À1 were used for calibration. 1 H (Fig. 1a) and 13 C NMR studies of the ROP-RAFT dual agent were performed in CDCl 3 . 1 H NMR spectra of the PLA macroCTAs and the copolymers were acquired in the appropriate solvent for each block copolymer ( Fig. 1b-f). A 250 or 400 MHz Bruker spectrometer was used for NMR analysis. The experimental DP values (DP NMR ) of the PLA polymer block were obtained by comparing the integrated signal intensities of the aromatic RAFT end-group at 7.10-7.80 ppm with those of the -CH-protons of PLA at 5.14-5.24 ppm in d 6 -DMSO. The experimental DP values (DP NMR ) of the RAFT-synthesized block were observed by comparing the integrated intensities of its characteristic protons with those of the methine protons from PLA. In more detail, for the calculations, the methyl protons of PDMA at 2.22-2.33 ppm in CDCl 3 (Fig. 1c), the -COOCH 2 protons of POEGMA block at 4.04 ppm in CDCl 3 (Fig. 1d), the -CH 2 OH protons of PGMA at 3.38 ppm in d 6 -DMSO (Fig. 1e), and the methylene protons of the PMPC polymer block (Fig. 1f, protons e, f and g) at 3.65-4.14 ppm were used for the RAFT-synthesized block. For the PLA block, the methine protons of PLA at 5.07-5.24 ppm in CDCl 3 , at 5.13-5.21 ppm in d 6 -DMSO or 4.84-5.01 ppm in 1 : 1 CDCl 3 : CD 3 OD were taken into account. All the results are presented in Table 1.
DLS experiments were performed using a Zetasizer Nano-ZS (Malvern Instruments, UK). Aqueous copolymer solutions (0.1% w/v) were analyzed using disposable cuvettes and data were averaged over three consecutive runs.
TEM imaging of copolymer samples was performed using a FEI Tecnai G2 Spirit TEM 120 kV instrument equipped with an Orius SC1000 camera. The TEM samples were prepared using in-house carbon-coated copper grids. Grids were plasma glowdischarged for 45 s to create a hydrophilic surface. Each aqueous copolymer solution (0.1% w/w) was placed onto a freshly glow-discharged grid for 30 s. The grid was then blotted with a lter paper to remove excess solution. For positive staining of the deposited nanoparticles, a uranyl formate solution (0.75 w/v %) was used. This was placed using a micropipette on the sample-loaded grid for 20 s. The grid was then blotted with lter paper and dried using a vacuum hose.

Results and discussion
In this work, amphiphilic diblock copolymers were synthesized consisting of a hydrolyzable aliphatic polyester block (PLA) and a methacrylic polymer block consisting of PDMA, POEGMA, PGMA or PMPC. The preparation of these copolymers is illustrated in Scheme 1. A combined ROP-RAFT agent 1 containing a Scheme 1 (a) Synthesis of hydroxyl-functional RAFT CTA for use as a ROP-RAFT dual agent in polymerizations. (b) Simultaneous ROP-RAFT copolymerization of (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5dione (L-lactide, LA) with 2-(dimethylamino)ethyl methacrylate (DMA) or oligo(ethylene glycol) methacrylate (OEGMA) at 74 C in 1,2dichloroethane to produce amphiphilic polylactide-methacrylic block copolymers 2. (c) Two-step ROP-RAFT polymerization; ROP of LA at 74 C in 1,2-dichloroethane to synthesize PLA macro-CTAs that are subsequently used in the RAFT polymerization of DMA and glycerol monomethacrylate (GMA) at 74 C in 1,2-dichloroethane or 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC) at 78 C in anhydrous ethanol for the preparation of amphiphilic polylactide-methacrylic block copolymers 3a and 3b, respectively. hydroxyl group as the initiating site for ROP and a dithiobenzoate functionality for the RAFT polymerization was utilized. This CTA is the monoester product of the reaction between CADB and 1,6-hexanediol (Scheme 1a). The esterication was conducted in chloroform under reux for 48 h using DCC (1.1 eq.) and DMAP (0.1 eq.) as catalyst. To ensure a monofunctional product, excess diol (5.0 eq.) was used. The ROP-RAFT dual agent 1 was obtained in 45% yield. Its chemical structure and high purity were conrmed by 1 H NMR spectroscopy (Fig. 1a), 13 C NMR and electrospray ionization mass spectroscopy (ESI-MS) analysis.
Subsequently, the ROP-RAFT dual agent 1 was used in the synthesis of block copolymers using both simultaneous onestep (Scheme 1b) and two-step (Scheme 1c) protocols. LA was used as the ROP monomer for the formation of all block copolymers (2, 3a and 3b). The resulting PLA homopolymer 3 was used as a macro-CTA in the second polymerization step (for the preparation of 3a or 3b). Various LA/1 molar ratios were used for the preparation of a PLA x -PDMA y block copolymer 2 via simultaneous polymerization (Scheme 1b). An LA/1 molar ratio of 30 was also utilized for the synthesis of a PLA-POEGMA block copolymer 2 by the same simultaneous polymerization protocol (Scheme 1b). The same molar ratio was also used for PLA-PDMA 3a, PLA-PGMA 3a and PLA-PMPC 3b block copolymer syntheses by two-step polymerization (Scheme 1c). In all cases, the molar ratio of the RAFT monomer (DMA, OEGMA, GMA or PMPC) to 1 was kept constant at 30.
More specically, for the synthesis of diblock copolymers by simultaneous ROP-RAFT polymerization (see Scheme 1b) either DMA or OEGMA was used as the RAFT monomer and 1,2dichloroethane (bp 84 C) as a solvent (55% w/w solids). A twostep process was required for the preparation of the diblock copolymer containing glycerol monomethacrylate (GMA) i.e. PLA-PGMA 3a. This was because the hydroxyl groups in the GMA monomer structure are capable of initiating unwanted ROP polymerization. Following the same two-step protocol (Scheme 1c), a PLA-PDMA block copolymer 3a was synthesized for comparison with its one-step synthetic process (Scheme 1b). Both steps were performed in 1,2-dichloroethane (36.0% and 55% w/w solids for rst and second step, respectively). A twostep protocol was also required for the synthesis of the PLA-PMPC block copolymers. Such copolymers cannot be synthesized by simultaneous ROP-RAFT polymerization due to the insolubility of the MPC monomer in any suitable aprotic solvent for ROP. First, ROP of LA produced a PLA homopolymer 3 ([LA] 0 : [1] 0 ¼ 30 or 200) in 1,2-dichloroethane (36.0% w/w solids). Subsequently, MPC was employed as the RAFT monomer in the second step. Anhydrous ethanol (b.p. 78 C, 20% w/w solids) was used for the synthesis of the PLA-PMPC block copolymers 3b (second step, Scheme 1c). Ethanol is a good solvent for both MPC monomer and PMPC. DMAP was selected as the catalyst for the ROP of LA instead of the more commonlyused catalyst stannous octoate (Sn(Oct) 2 ) for this polymerization technique. 6,7,16,17,46 DMAP can be used at lower temperatures, 46 making it compatible with the RAFT polymerization conditions. 24 2,2 0 -Azobis(isobutyronitrile) (AIBN) was used as the thermal initiator for RAFT polymerization. This initiator was utilized in both the one-step process 2 and the two-step process 3a ([1] 0 : [AIBN] 0 ¼ 1 : 0.20). ACVA was the initiator in the two-step process 3b ([1] 0 : [ACVA] 0 ¼ 1 : 0.25).
All reactions conducted in 1,2-dichloroethane (Scheme 1b and c polymers 3 and 3a) were performed at 74 C for 24 h. The PLA-PMPC block copolymer synthesis 3b (Scheme 1c) was conducted in ethanol at 78 C for 24 h. The monomer conversions were determined by 1 H NMR analysis. Conversions were calculated based on the resonance intensities of the remaining unreacted monomers: LA at 5.03-5.10 ppm in CDCl 3 (methine proton). DMA at 5.55 and 6.09 ppm, OEGMA at 5.52 ppm and 6.08 ppm in CDCl 3 , GMA at 5.66 and 6.05 ppm in d 6 -DMSO and MPC at 5.38 and 5.90 ppm in CDCl 3 /CD 3 OD (for four methacrylic protons for each monomer). As shown in Table 1, relatively high conversions were achieved for all monomers, ranging from 77% to 96% for the LA monomer and between 74% and 99% for the methacrylic monomers (DMA, OEGMA, GMA and MPC). The one-step synthesis of the PLA-POEGMA block copolymer gave the highest monomer conversions; 96% for LA and 99% for OEGMA (PLA 29 -POEGMA 29 ,  Table 1, entry 11) the resulting conversions were 93% for LA (rst step) and 85% for MPC (second step). In the onestep synthesis of PLA x -PDMA y block copolymers (Table 1, entries 1-6), the conversions of both LA and DMA monomers were not signicantly affected by changing the feed ratio of LA monomer to 1 ([LA] 0 : [1] 0 from 30 for entry 6 to 250 for entry 1). These values ranged from 77% to 93% for the LA monomer and between 79% and 97% for the DMA monomer.
All diblock copolymers were puried by dialysis against acetone. This was followed by methanol dialysis in the case of PLA-PGMA and PLA-PMPC block copolymers. This second dialysis step was essential for removal of any unreacted GMA or MPC monomer respectively, which are insoluble in acetone. Aer solvent evaporation, the dried block copolymers were characterized by 1 H NMR spectroscopy and gel permeation chromatography (GPC). The results are presented in Table 1, Fig. 1, 2 and 3.
The degrees of polymerization (DPs) calculated by 1 H NMR (DP NMR ) were found to be in agreement with the theoretical values (DP Calcd ) calculated from the monomer feed ratios. These results suggest the well-controlled formation of block copolymers both for the one-and two-step protocols, except in the case where MPC was used as a monomer due to the partial insolubility of the PLA macro-CTA in ethanol, which was the polymerization solvent used for this RAFT synthesis. The molecular weight distributions (MWDs) of these copolymers were relatively narrow (Fig. 2 and 3): polydispersities ranged between 1.30 and 1.43 (see Table 1). The two PLA 30 -PDMA 30 diblock copolymers had similar M n (NMR) values of 8.1 kDa and 7.6 kDa when prepared by either the one-step (Table 1, entry 6) or twostep (Table 1, entry 8) protocols, respectively.
The MWD for the PLA 30 -PDMA 30 diblock copolymer prepared by one-step polymerization was slightly narrower than that obtained via the two-step protocol (Fig. 3d). Polydispersities of 1.37 and 1.41 were obtained for the one-step and two-step protocols respectively (Table 1, entries 6 and 8). Thus using the more convenient protocol does not compromise the quality of the block copolymer. GPC analysis of PLA-based block copolymers using a refractive index detector is challenging because of the relatively low refractive index increment (dn/dc) of the aliphatic polyester component in common GPC eluents. For example, the dn/dc of PLA is 0.048 mL g À1 (ref. 70) in THF. This value is signicantly lower than that for PMMA calibration standards (0.087 mL g À1 (ref. 71) in THF), which leads to inaccuracies in the GPC characterization of PLA-based block copolymers, particularly in the low molecular weight range.
Simultaneous ROP-RAFT polymerization using 1 was also utilized for the synthesis of PLA-based branched block copolymers. Here, either OEGMA or DMA was used as the RAFT monomer and DSDMA was selected as the cleavable cross-linker. This synthetic route is presented in Scheme 2a. As shown in Fig. 4a, DMF GPC analysis (vs. PMMA calibration standards) of this disulde-containing block copolymer indicated a relatively broad MWD (M w /M n ¼ 2.36). This conrmed that the DSDMA comonomer had reacted not only intramolecularly but also intermolecularly, 63,65 resulting in branching. Cleavage of the disulde bonds (Scheme 2b) using excess Bu 3 P led to a much narrower MWD (Fig. 4a). The M w /M n was reduced from 2.36 to 1.37 for the resulting thiol-functionalized linear block copolymer PLA 30 -P(OEGMA 30 -stat-TEMA 2 ) (where TEMA denotes 2thioethyl methacrylate). Vinyl sulfone was reacted with the TEMA residues to afford 2-(2-(vinylsulfonyl)ethylthio)ethyl methacrylate (VSTEMA) units (Scheme 3a). For this thiol-ene reaction, a large excess of DVS (15 eq. relative to the thiol -SH group of TEMA) was used to ensure that only one of the DVS double bonds reacts with the thiol group. Thus possible interor intra-molecular cross-linking was avoided. The second double bond of the DVS thus remained unreacted and was utilized for further conjugation with thiol-containing molecules. 72 The success of the reaction for the synthesis of the vinyl sulfone-functionalized amphiphilic block copolymer, PLA 30 -P(OEGMA 30 -stat-VSTEMA 2 ), was conrmed by 1 H NMR studies (see Fig. 4b and Fig. S1, ESI †). Specically, in the 1 H NMR (CDCl 3 ) spectrum (Fig. S1 †) the signal at 6.0-7.0 ppm is due to the pendant vinylsulfone protons. The signal at 2.75 ppm is assigned to the -OCH 2 CH 2 SCH 2protons. DMF GPC analysis of this copolymer indicates an M w /M n of 1.40, compared to 1.37 for the PLA 30 -P(OEGMA 30 -stat-TEMA 2 ). This suggests that minimal branching occurred during RAFT copolymerization in this case. In principle, PLA 30 -P(OEGMA 30 -stat-VSTEMA 2 ) can be reacted with thiol-functional oligopeptides, which can be useful for biological applications. This concept was tested in a model reaction using Glu (Scheme 3b). The conjugation reaction was performed under mild conditions (20 C, 4 h, water). A Glu/ VSTEMA molar ratio of 1.05 and a small amount of TCEP catalyst ([TCEP] 0 : [thiol] 0 ¼ 0.10) were used. The resulting PLA 30 -P(OEGMA 30 -stat-GluVSTEMA 2 ) block copolymer had a similar M n /M w value (1.45 vs. 1.40) with the PLA 30 -P(OEGMA 30stat-VSTEMA 2 ) precursor. However, the former copolymer had a much lower M n (23 600 g mol À1 vs. 37 800 g mol À1 ) (DMF GPC, Fig. 4a) than the latter copolymer. This is attributed to partial hydrolysis of the PLA block in water during the conjugation reaction and purication by dialysis. Successful Glu conjugation was conrmed by 1 H NMR (CDCl 3 ) since the new signal at 3.51 ppm is assigned to the -CH 2 CH(NH 2 )COOH proton. Also, in Fig. S2b (ESI †) the same peak was obtained at 3.48 ppm when D 2 O was used as an NMR solvent.
A gel network was obtained when DMA was used as a comonomer for the synthesis of a disulde-containing branched amphiphilic block copolymer, PLA 30 -P(DMA 30 -stat-DSDMA 1 ) (Scheme 2a). Thus characterization of this precursor copolymer using GPC or 1 H NMR was not feasible. Gel formation indicates a signicantly higher degree of branching than for the analogous reaction using OEGMA instead of DMA. A viscous solution rather than a gel was obtained from the  synthesis of the PLA 30 -P(OEGMA 30 -stat-DSDMA 1 ) branched block copolymer. Presumably, the sterically congested nature of the OEGMA residues (vs. DMA residues) hinders intermolecular cross-linking. Subsequently, the disulde bonds of the PLA 30 -P(DMA 30 -stat-DSDMA 1 ) branched block copolymer were reduced using the same protocol as described above for the OEGMA-based branched block copolymer (Scheme 2b). This resulted in the formation of a thiol-functionalized PLA 30 -P(DMA 30 -stat-TEMA 2 ) block copolymer, which was functionalized with a DOTA ligand via thiol-ene chemistry. More specically, excess maleimido-monoamide-DOTA (2.1 eq. relative to disulde bonds) was used to convert the TEMA residues to DOTATEMA residues (Scheme 4a) to produce a PLA 30 -P(DMA 30stat-DOTATEMA 2 ) block copolymer. The DOTA is a well-studied macrocyclic that can be used for metal conjugation. It is known to form very stable metal complexes. [73][74][75] The high electron density of heavy metals enables high resolution imaging of the block polymer in transmission electron microscopy (TEM) studies, as discussed below. Indium was used to form a complex with the DOTA-functionalized polymer. This was achieved by using excess InCl 3 (2.0 eq. relative to disulde bonds, Scheme 4b). Successful metal complexation was conrmed by inductively-coupled plasma atomic emission spectroscopy (ICP-AES). It was calculated that there were approximately 1.16 In atoms per PLA 30 -P(DMA 30 -stat-InDOTATEMA 2 ) block copolymer chain. Self-assembly of the PLA 27 -PGMA 29 block copolymer during its synthesis in 1,2-dichloroethane was examined by TEM. Due to the insolubility of the PGMA block in this polymerization solvent the block copolymer self-assembled in situ to form various nanoparticles. Micelles and vesicles with diameters ranging from 50 to 500 nm were observed (see Fig. S4 †).  Self-assembly of all PLA-based amphiphilic block copolymers to form various nanostructures in water was obtained by the solvent switch method. Table 1 summarizes DLS hydrodynamic diameters observed for aqueous copolymer dispersions aer removal of the organic solvent. TEM images for representative block copolymer samples are displayed in Fig. 5. Addition of water to an acetone solution of copolymer was used for the self-assembly of PLA x -PDMA y and PLA 29 -POEGMA 29 block copolymers. A solvent switch from acetone/methanol to water was used for the PLA 27 -PGMA 29 and from chloroform/ methanol to water for the PLA x -PMPC y block copolymers. In the case of the PLA x -DMA y block copolymers, varying the PLA block DP led to different morphologies (Fig. 5a-f). Hydrodynamic diameters of these nanoparticles ranged between 113 nm and 685 nm ( Table 1, entries 1 to 6). In the case of the highly asymmetric PLA 173 -PDMA 24 , vesicles make up the majority of nanoparticles (214 nm, Table 1 entry 1), but a small number of micelles were also formed (Fig. 5a). The micelle fraction increased as the DP of the PLA was reduced (from Fig. 5a-f). For the PLA 26 -PDMA 25 block copolymer, micelles are the major population (Fig. 5f). Various nanostructures (vesicles, worm-like micelles and spherical micelles) were observed for PLA 29 -POEGMA 29 block copolymer (Fig. 5g). The TEM image of this sample also suggested partial crystallization of the PLA block, similar to previous studies on PLA-PEG block copolymers and blends. [76][77][78] The same sample had a hydrodynamic diameter of 550 nm (Table 1 entry 7) and a high polydispersity. The solvent switch protocol for the PLA 27 -PGMA 29 block copolymer resulted in a mixture of large vesicles (338 nm, Table 1 entry 9) and some micellar structures. The highly asymmetric PLA 184 -PMPC 23 block copolymer self-assembled to produce a mixture of vesicles (1430 nm, Table 1 entry 10), worm-like micelles and spherical micelles as judged by TEM (Fig. 5i).
Following the same solvent switch protocol, the selfassembly of the In-labelled PLA 30 -P(DMA 30 -stat-InDOTATEMA 2 ) was examined. For this sample, nanostructures with a mean DLS diameter of 265 nm were observed. These morphologies could be imaged by TEM (Fig. 5j and Fig. S4a †) without requiring a staining agent. This was possible because of the high electron density of the heavy metal conjugated to the copolymer chains.

Conclusions
A combination of two "living" polymerization techniques via either one or two steps was achieved using a novel hydroxylfunctionalized dithiocarbonate-based ROP-RAFT dual agent. Simultaneous polymerization of an aliphatic ester and a methacrylic monomer for the one-pot synthesis of amphiphilic block copolymers has been established using a facile metal-free formulation. This was achieved by ROP of LA (catalyzed using DMAP) and RAFT polymerization of DMA or OEGMA. In contrast, a two-step approach was essential for the synthesis of PLA-GMA and PLA-PMPC block copolymers. The simultaneous ROP-RAFT polymerization protocol was also utilized for the facile synthesis of new branched amphiphilic block copolymers, which serve as precursors for thiol-functionalized linear block copolymers. For this latter synthesis, the disulde cross-linking agent DSDMA was employed to incorporate latent thiol groups. Cleavage of the disulde bonds was performed under reducing conditions, resulting in thiol-functionalized linear block copolymers. These biodegradable block copolymers can be conjugated with molecules such as peptides or ligands for heavy metals using facile thiol-ene chemistry, which suggests potential biomedical applications.