ab diblock copolymer 
 self - assembly has attracted considerable attention 
 in recent decades as a convenient method for preparing organic nanoparticles 
 with spherical , wormlike , or vesicular morphologies . traditionally , block copolymer self - assembly is achieved using a 
 postpolymerization processing method such as a solvent switch . however , this approach typically requires relatively low copolymer 
 concentrations ( < 1% ) , which makes many potential commercial applications 
 economically unviable . over the past decade , polymerization - induced 
 self - assembly ( pisa ) 
 has been used to produce well - defined ab diblock copolymer nanoparticles 
 at high solids ( 1050% w / w ) . successful pisa requires a controlled / living polymerization technique 
 such as reversible addition  fragmentation chain transfer ( raft ) 
 polymerization which provides polymers with low dispersities and predictable 
 mean degrees of polymerization ( dp ) . in situ self - assembly occurs during polymerization when a soluble macromolecular chain transfer 
 agent ( macro - cta ) is extended with a second monomer that forms an 
 insoluble block . in principle , if appropriate monomers are selected , 
 then pisa can be conducted in any solvent . in practice , pisa syntheses in aqueous media are particularly attractive 
 from an environmental perspective , and 
 such diblock copolymer nano - objects can lead directly to potential 
 biomedical applications . successful pisa formulations based on raft aqueous dispersion 
 polymerization and raft aqueous emulsion polymerization have been reported . however , raft aqueous emulsion polymerization 
 typically results in kinetically trapped spherical nanoparticles , with rather few literature examples of worms or vesicles being accessed 
 using such formulations . on the other hand an essential prerequisite 
 for aqueous dispersion polymerization 
 is a water - miscible monomer that polymerizes to produce a water - insoluble 
 polymer . cia and co - workers reported successful pisa syntheses via 
 raft aqueous dispersion polymerization of a cationic core - forming 
 monomer , 2-aminoethylacrylamide hydrochloride , in the presence 
 of an anionic polyelectrolyte , which induces in situ polyion complexation . however , it is 
 much more common to use nonionic monomers such as 2-hydroxypropyl 
 methacrylate , n - isopropylacrylamide , n , n - diethylacrylamide , 
 2-methoxyethyl acrylate , or di(ethylene glycol ) methyl ether methacrylate . recently , a sixth nonionic monomer , diacetone acrylamide ( daam ) , has 
 been explored in the context of raft aqueous dispersion polymerization . daam is highly water soluble and forms a water - insoluble 
 homopolymer at a mean degree of polymerization ( dp ) as low as 50 . recently , daam has been utilized as the core - forming block in pisa 
 formulations . for example , jiang et al . prepared spherical nanoparticles 
 by chain - extending a poly(2-hydroxypropyl methacrylamide ) macro - cta 
 with daam . replacing small amounts of 
 daam with n-2-aminoethylacrylamide hydrochloride 
 produced primary amine - functionalized nanoparticles that could be 
 core - cross - linked using schiff base chemistry . an and co - workers reported the formation of well - defined 
 nano - objects 
 using a poly(n , n - dimethylacrylamide ) ( pdmac ) macro - cta . both pdmac  pdaam spheres and vesicles 
 could be fluorescently labeled by reacting fluorescein-5-thiosemicarbazide 
 with the ketone moiety in the daam residues . the same team prepared 
 vesicles via the raft aqueous dispersion copolymerization of daam 
 with allyl acrylamide using a pdmac macro - cta . comparable acrylamide 
 comonomer reactivities enabled vesicle formation via pisa , followed 
 by latent cross - linking within the vesicle membranes via the less 
 reactive pendent allyl groups . recently , gao et al . reported the formation of higher - order structures 
 such as pore - switchable nanotubes by chain extension 
 of a poly(2-hydroxypropyl methacrylamide ) macro - cta with 
 daam at high solids ( > 35% ) . these 
 workers 
 attribute the formation of these unusual higher - order nano - objects 
 to hydrogen bonding . however , as far as we are aware , there are as 
 yet no reports of pdaam - based block copolymer worms . this omission is perhaps not too surprising because numerous pisa 
 studies have shown that worms typically occupy a relatively narrow 
 phase space . given the literature precedent with other core - forming blocks such 
 as poly(2-hydroxypropyl methacrylate ) ( phpma ) , poly(n - isopropylacrylamide ) ( pnipam ) , and poly(2-methoxyethyl acrylate ) 
 ( pmea ) , if such pdaam - based worms could be obtained then stimulus - responsive 
 behavior might be anticipated as a result of variable hydration of 
 the core - forming chains and/or ionization of terminal carboxylic acid 
 groups on the stabilizer block . blanazs et al . 
 monitored the evolution of copolymer morphology 
 during the pisa synthesis of pgma47phpma200 diblock copolymer nano - objects using tem . the worm phase was shown to be one of several intermediate states 
 between spheres and vesicles . similar findings have been reported 
 for other pisa formulations , suggesting that this is generic behavior . pdaam pisa formulation , worms 
 should also be accessible if appropriate conditions can be identified . moreover , the ketone moiety within the daam residues has not yet 
 been exploited for covalent stabilization of diblock copolymer nano - objects . typically , cross - linking is achieved via the addition of a bifunctional 
 vinyl monomer such as ethylene glycol dimethacrylate ( egdma ) to form 
 a third hydrophobic block . this approach works well for spheres and vesicles 
 but can be problematic for the worm phase . this is because even minor perturbations to the copolymer composition 
 can lead to the formation of mixed phases ( e.g. , worms plus spheres 
 or worms plus vesicles ) . herein we utilize raft aqueous dispersion 
 polymerization to prepare 
 a series of pdmac  pdaam diblock copolymer nano - objects . the 
 mean dps of the pdmac stabilizer block and the pdaam core - forming 
 block have been systematically varied to produce well - defined spheres , 
 worms and vesicles at 20% w / w solids , and a phase diagram has been 
 constructed to facilitate reproducible syntheses of such pure phases . 
 moreover , we examine whether the worms exhibit either thermoresponsive 
 or ph - responsive behavior . finally , the cross - linking of such nano - objects 
 is explored via postpolymerization modification using a commercial 
 water - soluble adipic acid dihydrazide ( adh ) reagent at ambient temperature . 2-(dodecylthiocarbonothioylthio)-2-methylpropionic 
 acid ( ddmat ) , n , n - dimethylacrylamide ( dmac ) , and 2,2-azobis(2-methylpropionitrile ) 
 ( aibn ) were purchased from sigma - aldrich and used as received . diacetone 
 acrylamide ( daam ) , adipic acid dihydrazide ( adh ) , and 4,4-azobis(4-cyanovaleric 
 acid ) ( acva ) were purchased from alfa aesar and were used as received . dioxane was purchased from sigma - aldrich uk , and diethyl ether was 
 purchased from fisher scientific . all 
 nmr spectra were 
 recorded using a 400 mhz bruker avance iii hd 400 spectrometer in 
 deuterated methanol at 25 c ( 64 scans were required to ensure 
 high - quality spectra ) . vis 
 absorption spectra were recorded between 200 and 800 nm using a pc - controlled 
 uv-1800 spectrophotometer at 25 c using a 1 cm path length quartz 
 cell . a beer  lambert curve was constructed using a series of 
 ten ddmat solutions in methanol . the absorption maximum at 311 nm 
 assigned to the trithiocarbonate group was used for this calibration plot , and ddmat concentrations were 
 selected such that the absorbance always remained below unity . the 
 mean dp for each of the five macro - ctas was determined using the molar 
 extinction coefficient of 16  300  160 mol dm cm determined for the ddmat . the setup was comprised 
 of two agilent pl gel 5 m mixed - c columns and a guard column 
 connected in series to an agilent 1260 infinity gpc system equipped 
 with both refractive index and uv  vis detectors ( only the refractive 
 index detector used ) operating at 60 c . the gpc eluent was hplc - grade 
 dmf containing 10 mm libr at a flow rate of 1.0 ml min . calibration was achieved using 
 a series of ten near - monodisperse poly(methyl methacrylate ) standards 
 ( ranging in mp from 625 to 618  000 
 g mol ) . the 
 intensity - average 
 sphere - equivalent diameter of diblock copolymer nano - objects was determined 
 at 25 c by dls using a malvern zetasizer nanozs instrument via 
 the stokes all measurements were made on 0.1% w / w copolymer 
 dispersions in either acidic aqueous solution ( ph 2.5 ) or methanol 
 using disposable plastic cuvettes . for variable temperature dls studies , 0.1% w / w aqueous copolymer 
 dispersions were heated from 5 to 50 c , followed by cooling 
 to 25 c , at 5 c intervals allowing 15 min for thermal 
 equilibrium at each temperature . in this case , copolymer dispersions 
 were analyzed using a glass cuvette , and data were averaged over three 
 consecutive runs at each temperature . zeta potential measurements 
 were performed using a malvern zetasizer nano zs instrument on 0.1% 
 w / w aqueous copolymer dispersions at 25 c in the presence of 
 1 mm kcl . the initial copolymer dispersion was acidic ( ph 2.5 ) with 
 the solution ph being adjusted by addition of dilute naoh , with 5 
 min being allowed for equilibrium at each ph . copper / palladium 
 tem grids ( agar scientific , uk ) were coated in - house to yield a thin 
 film of amorphous carbon . the grids were then subjected to a glow 
 discharge for 30 s. individual 10.0 l droplets of 0.1% w / w 
 aqueous copolymer dispersions were placed on freshly treated grids 
 for 1 min and then carefully blotted with filter paper to remove excess 
 solution . to ensure sufficient electron contrast , uranyl formate ( 9.0 
 l of a 0.75% w / w solution ) was absorbed onto the sample - loaded 
 grid for 20 s and then carefully blotted to remove excess stain . imaging was performed using 
 a fei tecnai spirit 2 microscope fitted with an orius sc1000b camera 
 operating at 80 kv . an ar - g2 rheometer equipped 
 with a variable 
 temperature peltier plate and a 40 ml 2 aluminum cone was used 
 for all experiments . percentage strain sweeps were conducted at 25 
 c using a fixed angular frequency of 1.0 rad s. angular frequency sweeps were conducted at 25 c using a constant 
 percentage strain of 1.0% . ft - ir spectra 
 were recorded for 
 solid samples using a thermo scientific nicolet is10 ft - ir spectrometer 
 fitted with a golden gate diamond atr accessory . a typical 
 protocol for the synthesis of a pdmac68 macro - cta was conducted 
 as follows . 2-(dodecylthiocarbonothioylthio)-2-methylpropionic 
 acid ( ddmat ) ( 0.613 g , 1.68 mmol ) , aibn ( 27.0 mg 0.17 mmol , cta / aibn 
 molar ratio = 10.0 ) , and dmac ( 10.0 g , 0.101 mol ) were weighed into 
 a 100 ml round - bottomed flask . dioxane ( 24.8 ml ) was added to produce 
 a 30% w / w solution , which was purged with nitrogen for 30 min . the 
 sealed flask was immersed into an oil bath set at 70 c for 25 
 min ( final dmac conversion = 89% , as judged by h nmr spectroscopy ) , 
 and the polymerization was subsequently quenched by immersing the 
 flask in ice , followed by exposure to air . dioxane ( 50 ml ) was added 
 to the reaction solution , followed by precipitation into a 10-fold 
 excess of diethyl ether ( 1 l ) . the precipitate was redissolved in 
 dioxane and precipitated once more into excess diethyl ether . the 
 crude macro - cta was dissolved in deionized water , any residual diethyl 
 ether / dioxane was removed under reduced pressure , and the resulting 
 aqueous solution was freeze - dried for 48 h. the purified pdmac macro - cta 
 was obtained as a yellow solid . end - group analysis using uv spectroscopy 
 indicated a mean degree of polymerization of 68 , and the mn and mw / mn were 5700 g mol and 1.12 , respectively , 
 as judged by dmf gpc . the same protocol was used to synthesize a pdmac40 macro - cta , which had an mn and mw / mn of 3200 g mol and 1.12 , a pdmac46 macro - cta with an mn and mw / mn of 4600 g mol and 1.09 , 
 a pdmac58 macro - cta with an mn and mw / mn of 5100 g mol and 1.09 , and a pdmac77 macro - cta with an mn and mw / mn of 7100 g mol and 1.11 . the typical protocol for the synthesis of pdmac58pdaam230 vesicles at 20% w / w solids was as follows . pdmac58 macro - cta ( 0.136 g , 0.022 mmol ) , acva ( 0.6 mg , 
 0.002 mmol , cta / acva molar ratio = 10 ) , and daam monomer ( 0.864 g , 
 5.1 mmol ; target dp = 230 ) were weighed into a 14 ml vial . deionized 
 water adjusted to ph 2.5 with hcl ( 4.0 ml ) was then added to give 
 a 20% w / w aqueous solution , which was degassed for 15 min at 4 c 
 prior to immersion in an oil bath set at 70 c . this reaction 
 solution was stirred for 4 h and then quenched by exposure to air . the daam monomer conversion was greater than 98% as judged by h nmr spectroscopy , while the mn and mw / mn were 27  100 g mol and 1.54 , respectively , 
 as judged by dmf gpc . all other pisa syntheses were conducted at the 
 same initial volume ( 5.0 ml ) at 20% w / w solids . a 20% w / w aqueous dispersion of pdmac58pdaam230 vesicles ( 2.5 g ) prepared using 
 the previously stated protocol and adipic acid dihydrazide ( adh ; 0.045 
 g , 0.26 mmol , daam / adh molar ratio = 10.0 ) were added to a 14 ml vial . the raft 
 solution polymerization 
 of dmac in dioxane at 70 c using 2-(dodecylthiocarbonothioylthio)-2-methylpropionic 
 acid ( ddmat ) as a cta is outlined in scheme 1 . this water - soluble homopolymer precursor 
 was chain - extended with daam via raft aqueous dispersion polymerization 
 at 70 c and 20% w / w solids . pdmac100 showed that the dmac polymerization 
 proceeded to 98% conversion within 90 min ( see figure 1a ) . monomer conversions were 
 calculated from h nmr spectra by comparing the integrated 
 dmac vinyl signals between 5.5 and 7 ppm to the combined polymer / monomer 
 signals in the region between 2.3 and 3.25 ppm ( figure 2 ) . a linear semilogarithmic plot indicated 
 first - order kinetics with respect to dmac ( see figure 1a ) . the linear evolution of mn ( dmf gpc vs pmma standards ) with conversion was accompanied 
 by low dispersities throughout ( mw / mn  1.12 ) , which indicates a well - controlled 
 raft polymerization ( see figure 1b ) . subsequently , a range of pdmac macro - ctas were prepared 
 with mean degree of polymerizations of 40 , 46 58 , 68 , or 77 , as determined 
 by end - group analysis using uv spectroscopy ( see figure s1 for a typical beer  lambert plot obtained 
 for ddmat at its absorption maximum of 311 nm ) . gpc analysis indicated 
 low dispersities ( mw / mn = 1.091.12 ) for all five pdmac macro - ctas used 
 in this work . ( a ) dmac conversion vs time plot and corresponding semilogarithmic 
 plot and ( b ) evolution of number - average molecular weight ( mn ) and dispersity ( mw / mn ) vs dmac conversion for the raft 
 solution polymerization of dmac using a ddmat chain transfer agent 
 at 30% w / w in dioxane at 70 c conditions : ddmat / aibn molar ratio 
 = 10 when targeting a dmac / ddmat molar ratio of 100 . gpc analyses 
 were performed in dmf eluent using a series of near - monodisperse poly(methyl 
 methacrylate ) calibration standards . h nmr spectra recorded in cd3od for ( a ) 
 the ddmat raft cta used in this work , ( b ) a ddmat  pdmac40 macro - cta ( see entry 1 in table 1 ) , and ( c ) a ddmat  pdmac40pdaam85 diblock copolymer ( see entry 3 in table s1 ) . mn , th = ( ( [ dmac]0/[ddmat]0 )  dmac conv  
 mdmac ) + mddmat . determined by dmf gpc chain 
 extension of the pdmac macro - ctas was conducted via raft aqueous dispersion 
 polymerization of daam at 70 c and 20% w / w solids ( see scheme 1 ) . recently , lovett 
 and co - workers have shown that ionization of cta - derived carboxylic 
 acid end groups can influence the copolymer morphology of diblock 
 copolymer nano - objects prepared via pisa . thus , hcl was used to lower the solution ph to ph 2.5 so as to ensure 
 that the terminal carboxylic acid groups located on the pdmac stabilizer 
 chains remained in their neutral acid form during the pisa synthesis . a kinetic study of the chain extension of pdmac58 with 
 daam when targeting a dp of 120 for the core - forming block confirmed 
 that 99% conversion was obtained within 90 min ( see figure 3a ) . daam conversions 
 were determined by comparison of the residual vinyl signals at 5.46.4 
 ppm to the pdaam methyl signal labeled l  in figure 2 . the semilogarithmic 
 plot ( figure 3a ) indicated 
 more than a 5-fold increase in the rate of polymerization after approximately 
 25 min , which coincided with the reaction solution becoming distinctly 
 turbid . this indicates the onset of micellar nucleation , with the 
 immediate formation of monomer - swollen particles resulting in a relatively 
 high local daam concentration . a linear evolution 
 of mn with daam conversion was observed 
 ( see figure 3b ) , which 
 is consistent with a controlled radical polymerization . however , there 
 was also a modest increase in the copolymer dispersity with conversion , 
 resulting in a final mw / mn of 1.33 . ( a ) monomer conversion vs time curve and corresponding 
 ln[m0]/[m ] plot and ( b ) evolution of number - average molecular 
 weight 
 ( mn ) and dispersity ( mw / mn ) with conversion for 
 the raft aqueous dispersion polymerization of daam at 70 c and 
 ph 2.5 using a ddmat - pdmac58 macro - cta targeting ddmat  pdmac58pdaam120 . conditions : 20% w / w solids and 
 a macro - cta / aibn molar ratio of 10 . following this kinetic study , a series of pdmacx pdaamy diblock 
 copolymers 
 were prepared by systematically varying the target pdaam dp ( y ) , for each of the five pdmacx macro - ctas ( where x = 40 , 46 , 58 , 68 , or 77 ) . monomer 
 conversions exceeding 98% were achieved for all such pisa syntheses 
 within 4 h at 70 c ( table s1 ) . a 
 series of representative gpc chromatograms obtained for pdmac77pdaamy are provided in figure s2 . pdaam 
 diblock copolymer nano - objects were 
 characterized using transmission electron microscopy ( tem ) . the assigned 
 morphologies were used to construct a phase diagram at a fixed copolymer 
 concentration of 20% w / w solids . this is shown in figure 4 , along with representative 
 images of the pure spheres , worms , and vesicles . only a spherical 
 morphology could be accessed when using a relatively long pdmac stabilizer 
 block ( dp  68 ) because such formulations favor elastic collisions 
 between nascent spheres rather than the stochastic 1d sphere  sphere 
 fusion events that lead to the formation of worms . hence spheres represent 
 a kinetically trapped phase when targeting highly asymmetric diblock 
 compositions . for example , increasing 
 the pdaam dp from 78 to 620 when using a pdmac68 macro - cta 
 only resulted in a monotonic increase in mean sphere diameter from 
 40 to 150 nm , as determined by dls analysis . in contrast , worms and 
 vesicles could be accessed when using shorter pdmac macro - ctas ( dp 
  58 ) . for example , targeting pdmacx  pdaamy gave pure vesicles when x = 40 , 46 , and 58 and y  150 . the phase space for pure worms was extremely narrow and was bounded 
 by sphere / worm and worm / vesicle mixed phases . similar observations 
 have been reported by blanazs and co - workers for an all - methacrylic 
 raft aqueous dispersion polymerization formulation . this composition resulted in a free - standing 
 gel , most likely as a result of multiple inter - worm contacts . nevertheless , the phase diagram shown in figure 4 enables the elusive 
 pure worm phase to be reproducibly targeted . representative transmission 
 electron microscopy images showing 
 pure sphere , worm , and vesicle morphologies obtained for 0.1% w / w 
 aqueous dispersions of pdmacx  pdaamy diblock copolymer nano - objects at ph 2.5 : 
 ( a ) pdmac68pdaam207 ; ( b ) pdmac40pdaam99 ; ( c ) pdmac58pdaam201 . phase diagram constructed for a series of pdmacx  pdaamy diblock 
 copolymer nano - objects . s = spheres , s + w = mixed spheres and worms , 
 w = worms , w + v = mixed worms and vesicles and v = vesicles . reported that poly(glycerol 
 monomethacrylate)poly(2-hydroxypropyl 
 methacrylate ) ( pgma  phpma ) diblock copolymer nano - objects prepared 
 by raft aqueous dispersion polymerization using a carboxylic acid - functionalized 
 cta exhibit ph - responsive behavior . more specifically , 
 worm - to - sphere and vesicle - to - worm transitions were observed on increasing 
 the solution ph from ph 3.5 to ph 6 . such order order transitions 
 were attributed to ionization of the carboxylic acid end groups on 
 the pgma chains , which increases the effective volume fraction of 
 this hydrophilic stabilizer block . in the present study , the pdmac 
 stabilizer blocks also contain a terminal carboxylic acid group , so 
 similar ph - responsive behavior was anticipated . to examine this hypothesis , 
 dls and aqueous electrophoresis measurements were recorded for a series 
 of 0.1% w / w pdmac  pdaam aqueous dispersions as a function of 
 solution ph ( see figure s3 ) . in each case , 
 the zeta potential became more negative at higher ph as a result of 
 deprotonation of the carboxylic acid end - groups on the pdmac chains 
 originating from the ddmat raft agent . however , the sphere - equivalent 
 particle diameter remained essentially unchanged over the entire ph 
 range studied for pdma pdaam nano - objects synthesized using 
 a relatively long pdmac macro - cta ( dp  58 ) or containing a 
 pdaam block with a mean dp of at least 140 ( see figure s3a  d ) . clearly , end - group ionization is insufficient 
 to induce an order  order transition for such copolymers . in 
 contrast , pdmac40pdaam99 worms proved 
 to be weakly ph - responsive : their sphere - equivalent particle diameter 
 was reduced from 403 nm at ph 2.6 to 208 nm at ph 9.6 ( see figure s3e ) . tem studies indicated that this 
 is the result of a transition from pure worms to a mixed phase comprising 
 relatively short worms and spheres ( figure s3f ) . there are numerous literature examples of thermoresponsive 
 diblock 
 copolymer nano - objects prepared by raft aqueous dispersion polymerization . such behavior has been reported for relatively weakly hydrophobic 
 core - forming blocks such as phpma , pnipam , and pmea . given that 
 the daam monomer is fully miscible with water , the corresponding pdaam 
 block might be expected to be weakly hydrophobic and partially hydrated , 
 as previously reported for phpma . for 
 pdmac58pdaamy nano - objects , no change in either solution viscosity or turbidity was observed when 
 cooling 20% w / w aqueous dispersions of spheres , worms , or vesicles 
 to below 5 c or on heating up to 50 c . dls studies confirmed 
 that no discernible change in hydrodynamic diameter occurred on either 
 heating or cooling a 0.1% w / w aqueous dispersion of pdmac58pdaam170 vesicles at ph 2.5 ( figure s4a ) . [ one reviewer of this manuscript has suggested 
 that hydrogen bonding between the amide repeat units might be responsible 
 for this unexpected lack of thermosensitivity . ] in contrast , a modest 
 reduction in the sphere - equivalent particle diameter from approximately 
 360 nm to around 300 nm was observed for a 0.1% w / w aqueous dispersion 
 of pdmac40pdaam99 worms on heating from 
 20 to 50 c ( see figure s4b ) . tem 
 studies indicate that this is the result of a morphological transition 
 from worms to a mixture of short worms and vesicles ( see figure s4c ) . similar thermoresponsive behavior 
 has been previously observed for aqueous dispersions of diblock copolymer 
 nano - objects . this transition is believed to be related to the relatively narrow 
 phase space occupied by these pure worms ( see figure 4 ) . in summary , pdmacx  pdaamy diblock copolymer 
 nano - objects with x  58 or y  140 prepared 
 herein proved to be neither ph - responsive on raising the solution 
 ph to ph 10 nor thermoresponsive on lowering the solution temperature 
 to 5 c or heating to 50 c . in contrast , pdmac40pdaam99 worms proved to be weakly responsive with 
 respect to changes to either solution ph or temperature . however , 
 it is perhaps noteworthy that unlike the observations made by lovett 
 and co - workers , no additional change in copolymer morphology was observed when subjecting these 
 pdmac40pdaam99 worms to a dual stimulus - response ( i.e. , switching the solution ph to ph 9 while 
 simultaneously cooling to 5 c , or heating to 50 c ) . however , for the 20% w / w formulations reported 
 herein , the solution ph had risen in each case to approximately 4 
 after daam polymerization . fortuitously , this is the optimum ph for 
 subsequent cross - linking using adh , as reported by kessel et al . this reagent s hydrazide groups can react 
 with the pendent ketone groups on the pdaam chains via nucleophilic 
 substitution to form hydrazone linkages ( scheme 2 ) . if the two hyrazide groups on adh react 
 with different pdaam chains , then this should result in covalent stabilization 
 of these nano - objects . all such cross - linking reactions were conducted 
 at 25 c using various adh / daam molar ratios . if the pendent hydrazine group 
 then reacts with a ketone group on a second pdaam chain , this leads 
 to cross - linking . spectroscopic evidence for 
 the proposed cross - linking reaction 
 was obtained from ft - ir studies . first , a model reaction was conducted 
 whereby a stirred 20% w / w aqueous solution of daam monomer was reacted 
 with adh using an adh / daam molar ratio of 0.50 at 25 c . this 
 reaction mixture gradually became turbid , and after 6 h the crude 
 product was isolated by freeze - drying overnight . ft - ir spectra recorded 
 for adh alone , the daam monomer , and the freeze - dried crude product 
 are shown in figure 5 . ft - ir spectra recorded for ( a ) adipic acid dihydrazide ( adh ) cross - linker , 
 ( b ) daam monomer , and ( c ) the freeze - dried product obtained from the 
 reaction of adh with daam at 25 c for 6 h using an adh / daam 
 molar ratio of 0.50 . conditions : 20% w / w solution , ph 2.5 . this characteristic feature is absent 
 in the product , 
 indicating loss of the ketone moiety . complete attenuation of this 
 ketone band confirms efficient reaction of the adh with daam monomer 
 within 6 h at 25 c . following this successful model reaction , a ft - ir study of the 
 addition of adh to an aqueous dispersion of pdmac58pdaam230 vesicles was undertaken . figure 6 shows the ft - ir spectra recorded for ( a ) 
 adh alone , ( b ) the original linear freeze - dried pdmac58pdaam230 vesicles , and ( c ) a freeze - dried 20% 
 w / w pdmac58pdaam230 vesicle dispersion 
 after adh cross - linking using an adh / daam molar ratio of 0.50 for 
 6 h at 25 c . ft - ir spectra recorded for ( a ) the adipic acid dihydrazide 
 ( adh ) 
 cross - linker alone , ( b ) a freeze - dried 20% w / w aqueous dispersion 
 of pdmac58pdaam230 vesicles , and ( c ) 
 the freeze - dried product of the reaction of a 20% w / w aqueous dispersion 
 of pdmac58pdaam230 vesicles with adh . conditions : adh / daam molar ratio = 0.50 , 6 h , 25 c , ph 4 . the pendent ketone groups in the 
 pdaam chains exhibit a characteristic 
 band at 1707 cm , which is close to that observed 
 for daam monomer ( see above ) . after cross - linking with adh for 6 h 
 at 25 c , this spectral feature became substantially attenuated 
 relative to the other ir bands . pdaam vesicles suggests that cross - linking 
 remained incomplete after 6 h. it is also worth emphasizing that reaction 
 of the adh with the pendent ketone groups on the pdaam chains does 
 not necessarily guarantee that an intermolecular cross - link is obtained . it is likely that at least some of the adh is consumed in the formation 
 of intramolecular cycles via reaction with two ketones located on 
 the same pdaam chain . moreover , it is also possible that the adh might 
 only react once , with its second hydrazide group being simply unable 
 to react with another ketone group because of steric congestion . this 
 latter problem is more likely to occur at higher degrees of cross - linking 
 as the pdaam cores become more solidlike . ft - ir spectra recorded 
 when cross - linking pdmac58pdaam230 vesicles 
 using adh / daam molar ratios of 1.00 , 0.50 , 0.25 , 
 or 0.10 indicated that greater attenuation of the ketone band occurred 
 at higher adh concentrations ( see figure s5 ) . the effect of varying the adh concentration on the extent of cross - linking 
 ( and hence degree of covalent stabilization of the nano - objects ) was 
 studied using dls . accordingly , adh was added to a 20% w / w aqueous 
 dispersion of pdmac58pdaam230 vesicles 
 at adh / daam molar ratios of 0.010 , 0.025 , 0.050 , 0.075 , 0.100 , 0.150 , 
 or 0.200 and allowed to react at 25 c with continuous stirring 
 for 24 h. aliquots taken at various time intervals were diluted to 
 0.1% w / v in methanol , which is a good solvent for both pdmac and pdaam . thus , if no cross - linking had occurred , then molecular dissolution 
 would be expected in this solvent . all these dilute methanolic dispersions 
 were analyzed by dls to establish the minimum time required for sufficient 
 covalent stabilization to preserve the original nano - objects . as adh 
 cross - linking progressed , the vesicles became gradually more resistant 
 to methanol dissolution . for each adh concentration , the scattered 
 light intensity ( or derived count rate ) and the sphere - equivalent 
 particle diameter were monitored as a function of time ( see figure 7 ) . the former parameter 
 increased up to approximately 6 h , after which plateau values were 
 observed ( figure 7a ) . this suggests that the cross - linking was close to completion on this 
 time scale . moreover , maximum covalent stabilization was achieved 
 for adh / daam molar ratios 0.075 . time dependence for ( a ) 
 scattered light intensity count rate and 
 ( b ) dls diameter when cross - linking a 20% w / w aqueous dispersion of 
 pdmac58pdaam230 vesicles at ph 4 using 
 adh at adh / daam molar ratios of 0.200 , 0.150 , 0.100 , 0.075 , 0.050 , 
 0.025 , or 0.010 at 25 c . aliquots were extracted from the reaction 
 solution at regular time intervals prior to quenching via dilution 
 to 0.1% w / v solids using methanol ( which is a good solvent for both 
 blocks and hence causes molecular dissolution if the degree of vesicle 
 cross - linking is insufficient to ensure covalent stabilization ) . the dls diameter for a dilute 
 aqueous dispersion of pdmac58pdaam230 vesicles ( 0.1% w / w at ph 2.5 ) prior to 
 cross - linking was 402 nm . figure 7b indicates that larger particle diameters were observed 
 for all adh concentrations as a result of swelling of the cross - linked 
 vesicles when diluted in methanol . substantial swelling was observed 
 for the lightly cross - linked vesicles in the presence of methanol . in contrast , much less swelling occurred for adh / daam molar ratios 
 0.050 because more extensive cross - linking was obtained under 
 these conditions . tem images of the linear pdmac58pdaam230 vesicles and a series of vesicles cross - linked using various 
 adh / daam molar ratios are shown in figure s6 . cross - linking was 
 also conducted on aqueous dispersions of pdmac68pdaam207 spheres and pdmac40pdaam99 worms ( adh / daam molar ratio = 0.100 ; 6 
 h at 25 c ) . in both cases , the original copolymer morphology 
 was retained on exposure to methanol as determined by tem analysis 
 ( figure 8) . swelling 
 of the cross - linked pdmac68pdaam207 spheres 
 in methanol resulted in a larger dls diameter of 77 nm ( compared to 
 65 nm measured at ph 2.5 prior to cross - linking ) . conversely , the 
 sphere - equivalent diameter obtained for the cross - linked pdmac40-pdaam99 worms was lower than 
 that determined prior to cross - linking ( 317 nm vs 403 nm ) . given that 
 the tem images shown in figure 8 confirm retention of the worm morphology , one possible explanation 
 for these dls observations is that insufficient worm cross - linking 
 may result in partial worm fragmentation on exposure to methanol . tem images 
 and dls measurements recorded for 0.1% aqueous dispersions 
 of ( a ) linear pdmac68pdaam207 spheres 
 and ( b ) linear pdmac40pdaam99 worms 
 at ph 2.5 ; 0.1% methanolic dispersions of 
 ( c ) cross - linked pdmac68pdaam207 spheres 
 and ( d ) cross - linked pdmac40pdaam99 worms 
 after reacting with adh at an adh / daam molar ratio of 0.10 for 6 h 
 at 25 c . the storage modulus , g , of a 20% w / w pdmac40pdaam99 worm gel was determined by oscillatory 
 rheology before and after 
 adh cross - linking for 6 h at 25 c using a adh / daam molar ratio 
 of 0.10 . at a fixed angular frequency of 1.0 rad s and a constant strain of 1.0% , g increased 
 from 2 370 pa to 10 330 pa at 25 c ( see figure 9 ) . similar enhancements in gel strength on 
 cross - linking were also reported by both lovett et al . and this has been attributed to worm stiffening , which leads 
 to an increase in the worm mean persistence length . variation of gel moduli 
 ( g , red circles ; g , 
 blue squares ) with frequency at an applied strain 
 of 1.0% and variation of gel moduli ( g , red 
 circles ; g , blue squares ) with strain at 
 an applied frequency of 1 rad s for ( a ) linear 
 pdmac40pdaam99 diblock copolymer prepared 
 at 20% w / w solids in water at ph 2.5 and ( b ) cross - linked pdmac40pdaam99 diblock copolymer prepared at 
 20% w / w solids in water at ph 2.5 with subsequent cross - linking at 
 25 c for 6 h ( adh / daam in summary , a series of well - defined hydrophilic 
 pdmac macro - ctas 
 ( mean dps = 40 , 46 , 58 , 68 , or 77 ) were prepared using ddmat and subsequently 
 chain - extended with daam using a raft aqueous dispersion polymerization 
 formulation . the resulting amphiphilic diblock copolymers formed a 
 range of nano - objects via polymerization - induced self - assembly . a 
 phase diagram was constructed for various diblock copolymer compositions 
 at 20% w / w solids . the worm phase space was extremely narrow , which no 
 doubt explains why this copolymer morphology had not been previously 
 identified for this particular pisa formulation . pdaam nano - objects 
 proved 
 to be insensitive to changes in both solution temperature and ph . this behavior is atypical compared to other raft aqueous dispersion 
 polymerization formulations based on hpma , nipam , or mea , where such water - miscible monomers normally produce rather weakly 
 hydrophobic structure - directing blocks with significant degrees of 
 plasticization . however , the pdmac40pdaam99 worms did prove to be both weakly 
 ph - responsive and thermosensitive : this is attributed to the extremely 
 narrow phase space occupied by this copolymer morphology , and perhaps 
 also the relatively low mean dp for each block . concentrated 
 aqueous dispersions of covalently stabilized diblock 
 copolymer nano - objects could be prepared at ambient temperature using 
 adipic acid dihydrazide ( adh ) , which reacts selectively with the pendent 
 ketone groups on the hydrophobic pdaam chains to form hydrazone moieties . ft - ir studies provided direct spectroscopic evidence for this cross - linking 
 chemistry , while dls measurements performed in methanol ( a good solvent 
 for the pdmac and pdaam blocks ) confirmed that covalent stabilization 
 could be achieved within 6 h at 25 c using adh / daam molar ratios 
 as low as 0.075 . finally , rheological studies indicated a 4-fold increase 
 in worm gel strength when using a daam / adh molar ratio of 0.100 , presumably 
 because cross - linking leads to an increase in the worm persistence 
 length .
various carboxylic 
 acid - functionalized poly(n , n - dimethylacrylamide ) 
 ( pdmac ) macromolecular chain transfer agents ( macro - ctas ) were chain - extended 
 with diacetone acrylamide ( daam ) by reversible addition 
 fragmentation 
 chain transfer ( raft ) aqueous dispersion polymerization at 70 c 
 and 20% w / w solids to produce a series of pdmac  pdaam diblock 
 copolymer nano - objects via polymerization - induced self - assembly ( pisa ) . 
 tem studies indicate that a pdmac macro - cta with a mean degree of 
 polymerization ( dp ) of 68 or higher results in the formation of well - defined 
 spherical nanoparticles with mean diameters ranging from 40 to 150 
 nm . 
 in contrast , either highly anisotropic worms or polydisperse vesicles 
 are formed when relatively short macro - ctas ( dp = 4058 ) are 
 used . 
 a phase diagram was constructed to enable accurate targeting 
 of pure copolymer morphologies . 
 dynamic light scattering ( dls ) and 
 aqueous electrophoresis studies indicated that in most cases these 
 pdmac  pdaam nano - objects are surprisingly resistant to changes 
 in either solution ph or temperature . 
 however , pdmac40pdaam99 worms do undergo partial dissociation to form a mixture 
 of relatively short worms and spheres on adjusting the solution ph 
 from ph 23 to around ph 9 at 20 c . 
 moreover , a change 
 in copolymer morphology from worms to a mixture of short worms and 
 vesicles was observed by dls and tem on heating this worm dispersion 
 to 50 c . 
 postpolymerization cross - linking of concentrated aqueous 
 dispersions of pdmac  pdaam spheres , worms , or vesicles was 
 performed at ambient temperature using adipic acid dihydrazide ( adh ) , 
 which reacts with the hydrophobic ketone - functionalized pdaam chains . 
 the formation of hydrazone groups was monitored by ft - ir spectroscopy 
 and afforded covalently stabilized nano - objects that remained intact 
 on exposure to methanol , which is a good solvent for both blocks . 
 rheological studies indicated that the cross - linked worms formed a 
 stronger gel compared to linear precursor worms .