although ring - opening 
 polymerizations ( rops ) provide a method to 
 transform cyclic monomers into well - defined , functional , and degradable 
 polymers , it has been challenging , in general , with some exceptions , to overcome difficulties that are often experienced in the attempt 
 to introduce functional moieties within the cyclic monomer structures 
 and with incompatibilities of these desired functionalities during 
 the polymerizations .
chemistries 
 such as azide  alkyne huisgen cycloaddition , diels  alder reaction , and radical - mediated thiol 
ene / yne reactions have revolutionized polymer chemistry , allowing for efficient and 
 reliable routes toward postpolymerization modifications in the design 
 of complex and functional polymeric materials .
combined with the extraordinary 
 efficiency in the generation of 
 stable covalent linkages by 
click-type reactions in 
 biomaterials , acid - labile functionalities , 
 such as acetals , ortho esters , and hydrazones , are also of major interest in contemporary materials design , especially 
 toward biomedical applications . in spite 
 of the wide utilization of acetals / thio  acetals in synthetic 
 organic chemistry ( e.g. , as hydroxyl protecting groups ) ,
the most intriguing 
 characteristic of acetal / thio  acetal functionalities originates 
 from their unique property of behaving as a  dynamic covalent 
 bond  , i.e. , being cleavable upon exposure to acidic conditions 
 such as those found in the gastrointestinal tract , tissue at sites 
 of inflammation and in tumors , and in endosomal and lysosomal compartments , while being relatively stable at the normal physiological ph . in this context , 

acetals / thio  acetals are appealing for their incorporation 
 into biomacromolecules , where the dynamic covalent linkages can bridge 
 to biologically active molecules and , ultimately , release them at 
 a targeted site in a controlled manner . furthermore , introduction 
 of hydroxyl functionalities by controlled cleavage of acetals / thio  acetals 
 would improve the hydrophilicity of a given polymer , which has been 
 limited conventionally due to their incompatibility with the polymerization 
 processes .
the inherent acid - labile 
 property of acetals has been introduced 
 to the backbone or the side chains of functional polymers or cross - linkers 
 in the preparation of cross - linked nanoparticles .
for instance , among 
 several elegant works , reports on acetal - linked linear polymers or 
 dendrimers have elucidated the cleavability as well as the chemical 
 stability of acetal linkages in a broad spectrum of polymeric systems , 
 e.g. , degradable , biocompatible dextrans , by frechet et al .
; hydroxyl - group protection during 
 anionic polymerization and hyperbranched polyethers with acetal backbone 
 linkages by frey et al . ;
acetal - based degradable 
 shell cross - linked polymeric micelles by wooley et al . ; introduction of vinyl ether side chain moieties 
 along poly(ethylene glycol ) backbones by wurm et al . and
brocchini et al . ; and 
 acetal - linked backbone - cleavable aba - type triblock copolymers by ni 
 et al . here , our interest is the application 
 of acid - labile acetal / thio  acetal linkages to the side chains 
 in degradable polymeric systems for their potential use as drug delivery 
 carriers .
recently , acetal - linked prodrug micellar nanoparticles and 
 their ph - triggered controlled drug release from the side chains have 
 been highlighted by zhang et al . and 
 zhong et al
however , the polymeric nanoconstructs 
 used in these studies were hydrocarbon - based , nondegradable backbone 
 systems  polymethacrylates and poly(acrylic acid ) , respectively  which 
 limit their application as potential biomaterials because of the possible 
 long - term accumulation in the human body .
a major effort of 
 our group has been to develop polyphosphoesters 
 ( ppes ) tailored by manipulation of pendant functional groups on the 
 pentavalent phosphorus atoms and to integrate the ppe system into 
 new classes of degradable , biocompatible polymeric nanomaterials .
particularly , introduction of reactive vinyl ether moieties to degradable , 
 biocompatible ppes and their utilization 
 to conjugate a library of hydroxyl- or thiol - containing biologically 
 active molecules via multiple types of conjugation chemistries are 
 envisioned to expand the breadth of this chemical approach in the 
 development of functional biomaterials . herein , we report the 
 organocatalyzed rop of ethylene glycol vinyl 
 ether - containing cyclic phosphotriester monomer , as an initial fundamental 
 study toward unique degradable , functional polymer materials .
after 
 demonstrating the well - controlled homopolymerization of the monomer 
 with predetermined molecular weights and narrow molecular weight distributions , 
 the resulting vinyl ether - functionalized ppe scaffold was conjugated 
 with hydroxyl- or thiol - containing model small molecules via three 
 different types of conjugation chemistries  thiol  ene 
  click  reaction , acetalization , or thio  acetalization 
 reaction  revealing efficient routes for postpolymerization 
 modifications of functional polymers . furthermore , amphiphilic diblock 
 copolymers containing the ethylene glycol vinyl ether - functionalized 
 ppe as a hydrophobic block segment , also prepared by rop , afforded 
 well - defined micelles that showed a ph - dependent hydrolytic core degradability 
 of both backbone and side chains in aqueous solutions .
the degradation 
 products , as identified by mass spectrometry , were found to be nontoxic 
 toward two standard cell lines : raw 264.7 mouse macrophages and ovcar-3 
 human ovarian adenocarcinoma cells .
hence , this polymeric system can 
 have potential applications in the development of nanomedical devices 
 for a variety of biomedical applications .
a novel ethylene glycol 
 vinyl ether - functionalized cyclic phosphotriester monomer , 2-ethylene 
 glycol vinyl ether-1,3,2-dioxaphospholane 2-oxide ( evep ) , 1 , was synthesized by following a typical condensation method : coupling 
 of ethylene glycol vinyl ether ( eve ) to 2-chloro-3-oxo-1,3,2-dioxaphospholane 
 ( cop ) in the presence of triethylamine ( tea ) in dichloromethane ( dcm ) 
 at 4 c ( scheme 1 ) .
it was critical to 
 purify the monomer rigorously , by removing the residual starting materials 
 completely , because of the potential for undesired dual initiation 
 by residual eve and the possible formation of random / branched ( co)polymers 
 by remaining reactive cop during the polymerization process .
multiple 
 purification methods , including silica gel column chromatography , 
 ( vacuum ) distillation , extraction , and precipitation , were attempted , 
 but decomposition of the reactive cyclic monomer and/or incomplete 
 removal of the impurities were inevitable in all cases .
therefore , 
 use of an exact stoichiometric equivalence of reagents was attempted , 
 and then the purification process was applied for removal of the tea 
 salts by a series of recrystallization in dcm , precipitation in diethyl 
 ether , and filtration . even though tetrahydrofuran ( thf ) could be 
 used as a solvent ,
the quantitative conversion of eve and cop to 1 was supported by h , c , and p nmr spectroscopy ( figure s1 ) . 
 by c nmr spectroscopy , the methylene carbon of the hydroxymethyl 
 group of eve resonating as a singlet ( h - decoupled ) at 
 61.2 ppm disappeared upon coupling to cop to result in a doublet ( j(p , c ) ) having a coupling constant 
 of 10.2 hz at 66.1 ppm for the methylene carbon of the new phosphoester 
 linkage .
in addition , consumption of the cop was observed by p nmr spectroscopy as replacement of the cop phosphorus resonance 
 at 23.20 ppm with that for the monomer at 17.91 ppm .
although the 
 conversion to monomer appeared to be complete , h nmr analysis 
 indicated that there were residual solvents and tea salts that were 
 not removed ( 96% purity ) .
the rop of 1 was performed 
 in a glovebox at ambient temperature . for the kinetic study ,
1 and benzyl alcohol ( bnoh ) ( molar ratio of 100:1 ) were mixed 
 in dcm , and the polymerization began with the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene 
 ( dbu ) ( molar ratio to initiator of 3:1 ) .
after being stirred for a 
 predetermined period of time , an aliquot of the reaction mixture was 
 collected , immediately quenched by adding a solution of excess benzoic 
 acid in dcm , and then analyzed by p nmr spectroscopy . 

a portion of the collected samples was precipitated into diethyl ether 
 prior to injection into the gel permeation chromatography ( gpc ) instrument 
 ( figure s2 ) .
although routine decoupled p nmr spectroscopy has complications that may limit the quantitative 
 value , the distinct resonance frequencies of the phosphorus nuclei 
 of the monomer vs the polymer provided the best opportunity ( relative 
 to h or c nmr spectroscopy ) to monitor the 
 polymerization . therefore , the conversion was estimated from p nmr spectroscopy by comparing the integral ratio of two 
 distinct peaks of monomer 1 at 17.91 ppm and homopolymer , 
 pevep , at 0.68 ppm , on crude polymerization aliquots .
once 
 the polymerization was quenched and worked up , h nmr spectroscopy 
 end - group analysis of the degree of polymerization was found to be 
 in agreement with the monomer conversion calculated from the p nmr data .
the kinetic study displayed a rapid initial 
 polymerization rate , in which the monomer conversion reached at 23% 
 within the beginning 3 min .
however , the maintenance of linearity 
 of mn vs monomer conversion suggested 
 a living rop up to 79% conversion ( figure s2b ) .
the consistent low pdis ( 1.04 ) until the monomer conversion 
 reached at 79% indicated that there was minimal adverse transesterification 
 of the ppe backbone during the polymerization process . a kinetic plot 
 of ln([m]0/[m ] )
vs polymerization time illustrated pseudo - first - order 
 kinetics ( figure s2a ) , which is a typical 
 characteristic of rop .
the same molar ratio used for the kinetic 
 study was applied for a scaled - up production of 2 .
the 
 purification of 2 by precipitation in diethyl ether was 
 insufficient to remove the residual starting materials and benzoic 
 acid completely .
silica gel column chromatography was also attempted , 
 but instability of the ppe backbone was problematic .
dialysis ( mwco 
 68 kda ) of the reaction mixture against organic solvents , 
 switching from meoh to dcm , was the most assured purification method 
 among those tested .
dialysis in basic aqueous solutions ( e.g. , carbonate 
 buffer at ph 78 ) was also feasible , but the complete removal 
 of water was challenging , which is imperative for the following postpolymerization 
 modification reactions . being consistent with the kinetic study ,
quenching 
 the polymerization at a predetermined time ( 9 min ) , targeting at 50% 
 conversion , yielded the predicted molecular weight and pdi .
the degree 
 of polymerization ( dpn ) calculated based on p nmr spectroscopy - determined monomer conversion was in agreement 
 with that calculated from chain - end analysis by h nmr 
 spectroscopy , i.e. , by comparisons of the integrals of proton resonances 
 of the benzyl group ( 7.437.34 or 5.08 ppm , labeled as a or 
 b , respectively , in figure 1a ) of the initiated 
 chain end to those of the distinct double bonds ( 6.49 or 4.06 ppm , 
 labeled as f or g , respectively , in figure 1a ) or  protons on the substituents to the phosphorus 
 atom ( 3.943.86 ppm , labeled as e in figure 1a ) of 2 , which was indicative of retention of 
 the vinyl groups ( figure 1a ) .
in addition , 
 one distinct p resonance confirmed the stability of the 
 degradable ppe backbone during rop of 1 and the work - up 
 process of 2 .
h ( 300 mhz , cd2cl2 , ppm ) and p ( 121 mhz , cd2cl2 , ppm , inset ) nmr 
 spectra of ( a ) 2 , ( b ) 3 , ( c ) 4 , and ( d ) 5 .
ene 
  click  reaction with thiol - containing model small molecule , 
 2-(2-methoxyethoxy)ethanethiol .
ene 
  click  chemistry is a robust and versatile method that 
 tolerates a variety of functional groups in achieving a high degree 
 of functionalization on vinyl groups .
herein , this 
 efficient chemistry was applied to demonstrate the presence and chemical 
 availability of vinyl groups on 2 . to verify the integrity 
 of the ppe backbone in the presence of radicals during uv irradiation
, 
 a mixture of 2 and 2,2-dimethoxy-2-phenylacetophenone 
 ( dmpa ) in methanol - d4 ( meod4 ) was irradiated under uv light ( 365 nm , 6 w ) for several hours , 
 as a preliminary control reaction .
p nmr spectroscopy 
 confirmed that the polymer backbone was intact under these conditions . 

accordingly , an excess of 2-(2-methoxyethoxy)ethanethiol relative 
 to the vinyl ether bonds was employed with dmpa in meoh and uv irradiation 
 for 1 h to ensure a high coupling efficiency and to avoid undesired 
 cross - linking reactions between the double bonds along the backbone . 

the purified products were obtained by conducting precipitation in 
 diethyl ether followed by sequential dialysis ( mwco 68 kda ) 
 against meoh and dcm .
comparison of the h nmr spectra 
 of 1 and 2 before and after thiol 
ene 
  click  reaction , as shown in figures 1a and 1b , respectively , verified the 
 disappearance of the vinyl proton resonance ( 6.49 ppm , labeled as 
 f in figure 1a ) and the corresponding appearance 
 of two distinct proton resonances of methoxy ( 3.33 ppm , labeled as 
 f in figure 1b ) and -protons adjacent 
 to the sulfur atom ( 2.782.69 ppm , labeled as g and g in figure 1b ) in the thio  ether 
 functional group .
gpc analysis of 3 clearly showed a 
 peak shift to shorter elution time , relative to 2 , with 
 a monomodal peak having a pdi of 1.07 after thiol  ene  click  

reaction ( figure s3 ) . using 
 commercially available 4-methylbenzyl alcohol and a catalytic amount 
 of p - toluenesulfonic acid ( ptsa ) ,
the presence and 
 chemical availability of vinyl ether moieties of 2 were 
 demonstrated via acetalization . initially , to confirm the integrity 
 of the ppe backbone in the presence of alcohols and strong acidic 
 catalyst , ptsa ( pka  2.8 
 ( water ) ) , a mixture of 2 and 2 equiv of 4-methylbenzyl 
 alcohol ( relative to the absolute number of vinyl ether bonds ) or 
 that of 2 and 0.2 equiv of ptsa ( in relation to the absolute 
 number of vinyl ether bonds ) or that of 2 , 4-methylbenzyl 
 alcohol , and ptsa in n , n - dimethylformamide - d7 ( dmf - d7 ) was allowed to stir for a period of time , 
 as a preliminary control reaction .
an aliquot of each reaction solution 
 was collected at a predetermined time , quenched by the addition of 
 an excess of tea , and then evaluated by p nmr spectroscopy .
p nmr spectra confirmed the intact ppe backbone in the presence 
 of either 4-methylbenzyl alcohol or ptsa for several hours .
however , 
 the combination of 2 , 4-methylbenzyl alcohol , and ptsa 
 in the mixture solution was accompanied by undesired degradation / transesterification 
 along the ppe backbone structure after 6 min of reaction , as confirmed 
 by p nmr spectroscopy . accordingly , a scaled - up production 
 of 4 was conducted using the same molar ratio as used 
 for the preliminary control reaction , and the acetalization reaction 
 was quenched at 5 min by the addition of an excess of tea to ensure 
 the intact ppe backbone structure . with respect to the purification , 

employed an extraction method to purify their acetal - linked 
 linear poly(ethylene glycol ) by using water ; however , this extraction method was not compatible with the instability 
 of the ppe backbone in our system .
in addition , the complete removal 
 of ptsa and/or 4-methylbenzyl alcohol was not achieved by precipitation 
 of the desired polymer in organic solvent , such as diethyl ether . 

more importantly , this precipitation method was not desirable , especially 
 for acetal - bearing polymers as a result of the possible cross - linking 
 side reactions by trans - acetalization in the presence of a trace amount 
 of ptsa .
thus , sequential dialysis ( mwco 
 68 kda ) against two different organic solvents , dmf and dcm , 
 was conducted for purification .
finally , the desired product , 4 , was obtained after removal of the organic solvents , as 
 confirmed by h and p nmr spectroscopy ( figure 1c ) .
the conversion percentage of vinyl ethers 
 to acetals was calculated from chain - end group analysis by h nmr spectroscopy , i.e. , by comparison of the integrals of proton 
 resonances of the benzyl group ( 7.447.32 ppm , labeled as a 
 in figure 1c ) of the initiated chain end to 
 those of two distinct acetal linkages , methyl ( 1.33 ppm , labeled as 
 j in figure 1c ) and methylene ( 4.874.75 
 ppm , labeled as i in figure 1c ) , or 4-methyl 
 protons on the benzyl substituents ( 2.32 ppm , labeled as m in figure 1c ) . according to the chain - end analysis by h nmr spectroscopy
, approximately 18% of the initial vinyl 
 ether groups were converted into the acetal linkages , ca .
the hydrolysis side reaction was probably 
 attributed to a trace of water present in ptsa and/or highly viscous 
 ppe .
it is noteworthy that the complete disappearance of vinyl proton 
 resonances was observed with prolonged reaction time ( 12 h ) , 
 which could have enhanced the acetal conversion percentage .
however , 
 quenching the reaction at the optimal time , 5 min according to the 
 preliminary reaction , was essential for the integrity of the ppe backbone . 

this backbone stability was verified by the existence of one distinct p resonance peak at 0.68 ppm , observed by p nmr spectroscopy .
gpc analysis of 4 was not available 
 in thf because of its polar nature , conferred by the newly formed 
 hydroxyl groups on the side chains .
the same reaction protocol for the acetalization reaction was applied 
 to the thio  acetalization of 2 .
the h nmr spectrum of thio  acetal - bearing pevep50 , 5 , is shown in figure 1d . the conversion 
 percentage of vinyl ethers to thio 
acetals was calculated from 
 chain - end group analysis by h nmr spectroscopy , i.e. , 
 by comparisons of the integrals of proton resonances of the benzyl 
 group ( 7.447.32 ppm , labeled as a in figure 1d ) of the initiated chain end to those of the two distinct 
 thio  acetal linkages , methyl ( 1.51 ppm , labeled as j in figure 1d ) , methylene ( 4.71 ppm , labeled as i in figure 1d ) , or 4-methylbenzyl protons ( 2.31 ppm , labeled 
 as m in figure 1d ) . by h nmr chain - end 
 analysis , approximately 8% of the initial vinyl groups were found 
 to be converted into the thio 
36% of the repeat units underwent hydrolysis 
 of the side chains to present hydroxyl groups .
the intact ppe backbone 
 was affirmed by the presence of one distinct p resonance 
 peak at 0.67 ppm , as measured by p nmr spectroscopy . 

similar to the acetal linkage - bearing polymer , 4 , the 
 polar nature endowed by the newly formed hydroxyl groups on the side 
 chains did not allow for gpc analysis .
based on the kinetic study of 2 , amphiphilic diblock 
 copolymer , mpeg44-b - pevep33 , 6 , was prepared by rop using the molar ratio 100:1:3 
 of 1 , -methoxy--hydroxy poly(ethylene glycol ) 
 2000 da ( mpeg44-oh ) , and dbu , respectively ( scheme 2 ) .
the polymerization was quenched at 6 min by the 
 addition of a solution of excess of benzoic acid in dcm , and the desired 
 diblock copolymer product was obtained after sequential dialysis ( mwco 
 68 kda ) against organic solvents , meoh and dcm .
the dpn calculated based on p nmr spectroscopy - determined 
 monomer conversions was in agreement with that calculated from chain - end 
 analysis by h nmr spectroscopy , i.e. , by comparisons of 
 the integrals of the proton resonance of the methyl group of the initiated 
 chain end ( 3.33 ppm , labeled as a in figure 2a ) to that of the double bond ( 6.49 or 4.06 ppm , labeled as f or 
 g , respectively , in figure 2a ) 
 or -protons to the vinyl ether oxygen atom ( 3.943.86 
 ppm , labeled as e in figure 2a ) of the pevep 
 block segment .
additionally , one distinct p resonance 
 peak at 0.71 ppm confirmed the intact pevep block backbone 
 structure during the chain extension by rop of 1 and 
 the work - up process of 6 .
gpc analysis of 6 showed a distinct peak shift to lower elution time from that of 
 the macroinitiator , mpeg44-oh , after polymerization , with 
 a monomodal peak with a pdi of 1.09 ( figure 2b ) .
( a ) h ( 300 mhz , cd2cl2 , ppm ) 
 and p ( 121 mhz , cd2cl2 , ppm , inset ) 
 nmr spectra of 6 .
( b ) gpc traces of macroinitiator , mpeg44-oh , and diblock copolymer 6 , mpeg44-b - pevep33 , as a function of elution 
 time ( min ) .
the tg values of the prepared polymers 
 varied , as measured by differential 
 scanning calorimetry ( dsc ) , depending on the side chain substituents 
 ( table 1 ) .
the conjugation of the 2-(2-methoxyethoxy)ethanethioether 
 groups onto the side chains induced a decrease of the tg value from 39 c of 2 to 64 
 c of 3 after the thiol  ene  click  
 reaction .
the slight increase of tg value 
 of 4 and 5 , 27 and 31 c , 
 respectively , as compared with that of 2 , 39 
 c , was ascribed to the rigidity and  interactions 
 of the aromatic rings . meanwhile ,
the tg value of the diblock copolymer with the extended ethylene glycol 
 backbone units , 6 , was complicated to analyze but appeared 
 to give only a single tg value , similar 
 to that of 2 , 38 c .
in addition , no tm for the peg block segment was observed for 
 the diblock system ; therefore , an extensive investigation against 
 the homopolymers , mpeg44-oh and pevep50 , and 
 a mpeg44-oh / pevep50 blend was conducted .
these 
 studies confirmed that the pevep block fully suppresses peg crystallinity 
 in the diblock system , while the physical blend does little to suppress 
 peg crystallization ( full dsc traces for all systems upon heating 
 and cooling are presented with detailed discussions in the supporting information ( figure s9 ) ) .
the self - assembly behavior of the amphiphilic 
 diblock copolymer , 
 mpeg44-b - pevep336 , was studied by direct dissolution in nanopure water .
a high concentration 
 of 6 could be dispersed in nanopure water or buffer ( > 15 
 mg / ml ) without a significant turbidity or precipitation , which allowed 
 for analyses to be performed across a broad range of concentrations 
 and also serves as a promising criterion for its use as a drug delivery 
 carrier .
the morphology and surface charge of the resulting micellar 
 nanoparticles , 7 , were characterized by dynamic light 
 scattering ( dls ) , transmission electron microscopy ( tem ) , atomic force 
 microscopy ( afm ) , and -potential measurements ( figure 3 and figure s4 ) .
dls 
 and -potential analyses indicated narrow and monomodal size 
 distributions ( pdi = 0.114 ) with almost neutral charges and a negligible 
 difference at ph 5.0 and 7.4 , 4.52 and 7.64 mv , respectively . 

the tem images also revealed uniform nanoparticles 
 with an average diameter of 39  5 nm . although the hydrodynamic 
 and dry - state diameters measured by dls and tem , respectively , were 
 in agreement , afm indicated significant deformation of the micelles 
 upon deposition and drying on the mica substrate .
3 nm height 
 and 40  7 nm diameter indicate flattening of the micelles , which 
 is predicted to occur based upon the fluid shell and core components , 
 each being composed of a highly viscous polymer , peg ( tg = 17 c ) and pevep ( tg = 39 c , table 1 and figure s9 ) .
the afm data are useful qualitatively ; 
 however , further quantitative analysis is complicated by the presence 
 of substantial amounts of polymer debris and agglomerations of 7 across the substrate ( figure 3c , d ) , 
 which are also indicators of the fluidity of the micellar assemblies .
( a ) dls 
 results of 7 : dh(intensity ) = 49  7 nm , dh(volume ) = 46  
 7 nm , and dh(number ) = 44  6 nm 
 ( pdi = 0.114 ) .
( b ) tem image of 7 : dav = 39  5 nm , after counting more than 150 nanoparticles . 

afm height image ( c ) and three - dimensional image ( d ) of 7 : dav = 40  7 nm , after counting 
 more than 100 nanoparticles .
the phosphoester linkages of ppes 
 can be cleaved by spontaneous hydrolysis and/or enzymatic degradation ; 
 the hydrolysis rate of the phosphoester linkages is known to be highly 
 ph - dependent . in this study
, we postulated that the exposure of micelles , 7 , in acidic aqueous environment would induce a dual degradation 
 of the phosphoester backbone linkages and the vinyl ether side chain 
 moieties of the pevep block segment .
hence , the stability of the phosphoester 
 backbone linkage and the vinyl ether functionality of 7 were monitored in deuterated buffer solutions by p 
 and h nmr spectroscopies , respectively , in parallel to 
 measuring the micelle size and the intensity by dls .
furthermore , 
 the collected degradation products were identified using electrospray 
 ionization ( esi ) , gas chromatography ( gc ) , and matrix - assisted laser 
 desorption / ionization time - of - flight ( maldi - tof ) mass spectrometry .
the backbone 
 stability of the pevep segment in d2o at ph values of 5.0 
 and 7.4 and temperature of 37 c was monitored by p nmr spectroscopy ( figure 4 ) .
the integral 
 ratio of the p resonance of the intact pevep segment 
 at 0.71 ppm to that of the newly emerging peaks was analyzed . 

the ph - dependent degradation of the pevep backbone linkages became 
 apparent by the disappearance of p resonance corresponding 
 to the intact pevep backbone , coincident with the appearance of new p resonances as a result of hydrolytic degradation at ph 
 5.0 , whereas there was no significant change in the pevep p resonance when the samples were incubated at ph 7.4 .
( a ) rate of 
 the pevep backbone degradation of 7 at 
 ph 5.0 ( black line ) or ph 7.4 ( red line ) at 37 c as a function 
 of time , as measured by a comparison of the integrals of initial to 
 the newly appeared p nmr resonances .
changes in the p nmr resonance of pevep backbone of 7 at ph 
 5.0 ( b ) and 7.4 ( c ) at 37 c over a period of time . to compensate for potential complications in the detection of vinylic 
 proton resonances of the micelle core
, we evaluated the ph - dependent 
 hydrolytic reactivity of the vinyl ether functionalities by observing 
 both the polymer side chain functionalities and the small molecule 
 hydrolysis product using h nmr spectroscopy . to determine 
 the reactivity of the acid - labile vinyl ether functionality , the integral 
 ratio of a distinct vinyl proton resonance of the micelles at 6.49 
 ppm to the newly appeared proton resonance of acetaldehyde ,
one of 
 the hydrolysis products of the vinyl ether functionality , at 9.60 
 ppm was compared in d2o at ph values of 5.0 and 7.4 at 
 37 c over a period of time ( figure 5 ) . 
 for the micellar nanoparticles in ph 5.0 aqueous solution at 37 c , 

both the gradual disappearance of vinyl proton resonance intensity 
 and the appearance of an acetaldehyde proton resonance signal were 
 clearly observed within 1 day , and the equal ratio of these two different 
 proton resonances was reached after 14 days . by contrast , the vinyl 
 proton resonance of the micelles in ph 7.4 aqueous solution at 37 
 c remained consistent without any observable generation of acetaldehyde 
 over 39 days .
indeed , this observation bolstered our hypothesis that 
 the spontaneous cleavage of the vinyl ether moieties occurred in ph 
 5.0 aqueous solution simultaneously with the ppe backbone degradation , 
 but not at ph 7.4 .
rate of formation of acetaldehyde or disappearance of 
 vinyl proton 
 resonance at ph 5.0 ( a ) and ph 7.4 ( b ) and at 37 c as a function 
 of time , as measured by a comparison of the integrals of vinyl and 
 acetaldehyde proton resonance .
transition of proton resonances of 
 acetaldehyde and vinyl groups at ph 5.0 ( c ) or ph 7.4 ( d ) and at 37 
 c over a period of time .
it was hypothesized 
 that hydrolysis of the ppe backbone would decrease the proportion 
 of the hydrophobic : hydrophilic block segment ratio and that hydrolysis 
 of the side chain vinyl ether groups would increase the hydrophilicity 
 of the ppe backbone ; thereby , each would weaken the micelle assemblies . 
 in order to demonstrate the effects of ph - dependent hydrolytic degradation 
 of the polymer on the behavior and stability of the micelles , 7 ,
aqueous buffer solutions containing 6 at ph 
 5.0 and 7.4 were incubated at 25 and 37 c , and their degradation 
 profiles were assessed by measuring the changes in hydrodynamic diameter 
 and the intensity of light , scattered by micelles , when measured by 
 dls over a period of time ( figure 6 ) .
overall , 
 as predicted from the nmr degradation studies , the micelle assemblies 
 in ph 5.0 aqueous solutions , at both 25 and 37 c , became unstable 
 within 1 day , and the nanoparticles were undetectable within 7 and 
 2 days , respectively .
the swelling behavior of the micelles upon hydrolysis 
 of pevep segment was accounted for by the diffusion of water into 
 the core region ( figure 6a ) .
interestingly , 
 the unstable and dissociated micelles did not cause the formation 
 of visible precipitates , and thus , the micelle solutions remained 
 clear during monitoring . also , in agreement with the lack of backbone 
 or side chain hydrolysis observed by nmr spectroscopy , the nanoparticle 
 sizes in ph 7.4 aqueous solutions at 25 c remained consistent 
 over 39 days ( figure 6b ) .
surprisingly , when 
 the samples were incubated at ph 7.4 and 37 c , large aggregates 
 formed after 20 days , which persisted until day 43 when particles 
 were no longer detectable .
the lack of changes in the nmr data over 
 the same period of time and conditions suggests that transesterification 
 reactions may be a possible chemical change that produced subsequent 
 morphological changes . in accordance with these observations ,
the 
 intensity of light , scattered by the nanoparticles , as measured by 
 dls , was also dependent on the ph of water and temperature ( figure 6c ) . in the case of nanoparticles in ph 5.0 aqueous 
 solutions , both at 25 and 37 c ,
the signal intensities became 
 weaker rapidly , reaching below 20% as compared to the initial intensity , 
 and finally were undetectable within a week .
the signal intensity 
 of light scattered by micelles in ph 7.4 aqueous solution at 37 c 
 decreased gradually over a period of month .
meanwhile , there was no 
 significant change in the signal intensity for the micelles in ph 
 7.4 aqueous solution at 25 c throughout the monitoring period . 

taken together , the acidity of solutions was the primary determinant 
 of the micelle stability .
changes in the hydrodynamic 
 diameter of micelles at ph 5.0 ( a ) or ph 7.4 ( b ) and at a temperature 
 of 25 c ( black line ) or 37 c ( red line ) over a period 
 of time .
( c ) changes in the relative intensity of micelles in different 
 environments , ph and temperature , over a period of time .
the average 
 values and their standard deviations , from three measurements , are 
 shown .
there have been several reports where the 
 hydrolytic or enzymatic degradation behavior of ppe - containing micelles 
 were studied by using titration , nmr 
 spectroscopy , gpc , and/or dls methods ; however , to the best of our knowledge , the identification 
 of the actual degradation products of ppe has not been performed directly . 
 in this study
, we successfully identified the degradation products 
 of ppe qualitatively using electrospray ionization ( esi ) , gas chromatography 
 ( gc ) , and matrix - assisted laser desorption / ionization time - of - flight 
 ( maldi - tof ) mass spectrometry .
for this study , nanoparticles in aqueous 
 solution at ph 5.0 were incubated at 37 c until they were not 
 detectable by dls , and the complete disappearance of p resonance signal from the intact pevep backbone was confirmed by p nmr spectroscopy .
the mixture solution containing the degradation 
 products was analyzed by using esi , gc , and maldi - tof ms .
the 
 presence of oligomers and phosphoric acids was confirmed by esi ms 
 ( figure 7 ) , demonstrating the hydrolytic degradability 
 of phosphoester linkages of ppe .
tandem mass spectrometry was performed 
 on most of the precursor ions as a way of verifying the chemical structures 
 of these ions .
the ms / ms product ions are listed along with the structures 
 in figure 7 .
two series of oligomers 
 ( f and g series ) that differ by 44 da were 
 observed ( figure 7 ) . in order to verify that 
 the f series were not the fragments of the g series , i.e. , by losing 
 vinyl alcohol ( m / z 44 )
, ms / ms of 
 the lower mass precursor ions m / z 309 and 353 were performed ( figures s5 and s6 ) .
the predominant fragment ion for m / z 309 was 141 while that for m / z 353 was 185 , thus both losing a neutral repeating unit , 168 . in 
 addition , a loss of ethylene glycol was observed for both ions . however , 
 unlike the precursor ion m / z 353 , m / z 309 lost vinyl dihydrogen phosphate 
 to produce ion e. this major difference implied that the end group 
 of the f series oligomers is 2-hydroxyethyl dihydrogen phosphate , 
 while that of g series oligomers is bis(2-hydroxyethyl ) hydrogen phosphate . 
 therefore , the f series were not the fragment ions from the g series . because of its unionizable nature by electrospray ionization ,
the 
 presence of ethylene glycol , as one of the degradation products , was 
 confirmed by gc ms with electron ionization ( ei ) ( figure s7 ) .
neither vinyl- nor vinyl ether - containing compounds 
 were detected using gc ms , which agreed with our observation by h nmr spectroscopy , shown in figure 5 .
again , this absence of vinyl or vinyl ether functionalities within 
 the degradation products substantiated our hypothesis of a simultaneous 
 hydrolysis of the vinyl ether moieties during the degradation process 
 of the ppe backbone .
finally , maldi - tof ms analysis of the mixture 
 of the degradation products verified the presence of the intact peg 
 block segment with a single distribution having a spacing of 44 da , 
 corresponding to a peg repeat unit ( figure s8 ) .
mass spectra in negative ion mode ; m / z range of 1002000 ( a ) and 50180 ( b ) .
the cytotoxicities of the parent micelles , 7 , and their degradation products , 8 , were evaluated 
 toward two cell lines , raw 264.7 mouse macrophages and ovcar-3 human 
 ovarian adenocarcinoma cells , at a concentration range from 3 to 3000 
 g / ml for 24 h ( figure 8) . both 7 and 8 maintained high cell viability over the 
 range of the tested concentrations in both cell lines .
we have previously 
 observed low cytotoxicity and immunotoxicity of ppe - based micelles 
 with different surface charges , their shell cross - linked analogues , 
 and their degradation products even though we were not able to identify 
 the degradation products at that time .
these ppe - based nanoparticles are expected to have broad implications 
 in clinical nanomedicine as alternative vehicles to those involved 
 in several of the currently available medications , with precise control 
 over their molecular structures and overall architectures .
cytotoxicity 
 of the parent micelles of mpeg44-b - pevep337 ( black line ) and their 
 degradation products 8 ( red line ) at a concentration 
 range of 33000 g / ml for 24 h in raw 264.7 mouse macrophages 
 ( a ) and ovcar-3 human ovarian adenocarcinoma cells ( b ) .
in conclusion , a novel 
 polyphosphoester with ethylene glycol vinyl 
 ether side chain functionality was developed as a versatile template 
 for postpolymerization modifications , and its degradability and biocompatibility 
 were investigated .
a well - defined ( pdi 1.05 ) homopolymer with 
 vinyl ether side chain functionality was prepared by conducting rop 
 using an organocatalyst , dbu .
the kinetic study of this homopolymerization 
 revealed an excellent controllability during rop with predetermined 
 molecular weights and narrow molecular weight distributions .
subsequently , 
 the vinyl ether side chain moieties displayed chemical availability 
 and reactivity upon conjugation with hydroxyl- or thiol - containing 
 model small molecules via three different types of conjugation chemistries  thiol 
acetalization 
 reaction  resulting in modified polymers that contained either 
 stable thio  ether or hydrolytically labile acetal or thio 
acetal 
 linkages . despite the relatively low conversion percentages observed 
 during acetalization and thio  acetalization , ca . 18 and 8% , 

respectively , we anticipate that these degrees of conjugation efficiency 
 would be adequate to achieve a sufficient loading of diagnostic and/or 
 therapeutic molecules into this nanoparticle system
. meanwhile , amphiphilic 
 diblock copolymers , mpeg44-b - pevep33 , were also prepared by rop , and they afforded well - defined 
 micelles with a narrow and monomodal size distribution in water .
the 
 degradation study of the prepared micelles demonstrated a full acid - catalyzed 
 hydrolytic degradation behavior of both the side chain functionalities 
 and the backbone linkages .
finally , the parent micelles and their 
 degradation products , as identified qualitatively by mass spectrometry , 
 were found to be nontoxic toward raw 264.7 mouse macrophages and ovcar-3 
 human ovarian adenocarcinoma cells .
the fundamental understanding 
 of selective hydrolysis of the vinyl ether and/or acetal / thio  acetal 
 moieties for the introduction of hydroxyl groups to the ppe system , 
 which was conventionally limited to a cyclic ppe monomer , as a potential 
 protecting group strategy is currently under investigation .
moreover , 
 incorporation of biologically active molecules into these pevep - based 
 functional , degradable polymers via the presented conjugation chemistries 
 is underway .
 a novel 
 polyphosphoester ( ppe ) with vinyl ether side chain functionality 
 was developed as a versatile template for postpolymerization modifications , 
 and its degradability and biocompatibility were evaluated . 
 an organocatalyzed 
 ring - opening polymerization of ethylene glycol vinyl ether - pendant 
 cyclic phosphotriester monomer allowed for construction of poly(ethylene 
 glycol vinyl ether phosphotriester ) ( pevep ) . 
 this vinyl ether - functionalized 
 ppe scaffold was coupled with hydroxyl- or thiol - containing model 
 small molecules via three different types of conjugation chemistries  thiol  
 ene 
  click  reaction , acetalization , or thio - acetalization 
 reaction  to afford modified polymers that accommodated either 
 stable thio  ether or hydrolytically labile acetal or thio  
 acetal 
 linkages . 
 amphiphilic diblock copolymers of poly(ethylene glycol ) 
 and pevep formed well - defined micelles with a narrow and monomodal 
 size distribution in water , as confirmed by dynamic light scattering 
 ( dls ) , transmission electron microscopy , and atomic force microscopy . 
 
 the stability of the micelles and the hydrolytic degradability of 
 the backbone and side chains of the pevep block segment were assessed 
 by dls and nuclear magnetic resonance spectroscopy ( 1h 
 and 31p ) , respectively , in aqueous buffer solutions at 
 ph values of 5.0 and 7.4 and at temperatures of 25 and 37 c . 
 
 the hydrolytic degradation products of the pevep segments of the block 
 copolymers were then identified by electrospray ionization , gas chromatography , 
 and matrix - assisted laser desorption / ionization mass spectrometry . 
 
 the parent micelles and their degradation products were found to be 
 non - cytotoxic at concentrations up to 3 mg / ml , when evaluated with 
 raw 264.7 mouse macrophages and ovcar-3 human ovarian adenocarcinoma 
 cells . 