Synthesis and characterization of of a-cyclodextrins with thiolated 4-arm PEG using a

Synthesis and characterization of three-dimensional crosslinked networks based on self-assemly of a -cyclodextrins with thiolated 4-arm PEG using a three-step oxidationHuaiqing Yu,Zeng-guo Feng,*Ai-ying Zhang,Ling-gang Sun and Lijun QianReceived 5th December 2005,Accepted 2nd February 2006First published as an Advance Article on the web 16th February 2006DOI:10.1039/b517206cA kind of novel three-dimensional crosslinked networks based on the self-assemly of a -CDs with thiolated four-arm PEG was prepared by thiol-disulfide interchange reaction using a three-step oxidation.The supramolecular structure of the resulting hydrogels was characterized via FTIR,1H NMR,XRD,DSC and TGA measurements.The results demonstrated that the channel-type crystalline structure of inclusion complexes is still maintained after the multi-step oxidation.According to the swelling and degradation tests,the swelling behaviors and degradationproperties can be readily regulated by tuning the feed compositions of a -CDs and PEG-thiolated prepolymer.Furthermore,the threading a -CDs clearly protects disulfide bonds from an attack of a reduced form of glutathione as a result of prolonging the degradation time of the hydrogels in water.IntroductionSupramolecular architectures with topological characteristics have attracted considerable attention experimentally and theoretically.1Inclusion complex (IC)is a topological molecule composed of macrocycles threaded onto a linear polymer backbone via non-covalent interactions.2Because of the introduction of mechanical interlinking,it possesses different topologies and properties compared with a conventional small molecule or polymer.Cyclodextrins (CDs)are a kind of cyclic oligomers of a -D -glucose linked by a -1,4-glycoside bonds.3Their most familiar members are a -,b -,and c -CDs consisting of six,seven,and eight glucose units,respectively.Owing to the water-solubility and ability to selectively include a large range of guest molecules,cyclodextrins have been the most widely studied macrocyclic molecules.4Since Harada et al.first reported the inclusion complexes resulting from PEG and a -CDs in 1990s,5an enormous variety of ICs based on CDs have been extensively investigated.However,most of the inclusion complexes reported up to now are prepared by using linear polymer axles.6Making more complicated and well-ordered two or three-dimensional polyrotaxane systems is very interesting in terms of their novel topologies and properties.Jiao and coworkers reported ICs made from low molecular weight multi-arm PEG (three,four and six arms)with a -CDs.7Sabadini and Cosgrove described a synthesis of inclusion complexes with high molecular weight star-shaped PEG containing 13and 15arms.8Zhu et al.prepared inclusion complexes using hyperbranched polyether and a -CDs,9and found that these complexes show a novel lamellar morpho-logy and possess a juxtaposed structure.Furthermore,Okumura et al.disclosed a topological hydrogel by chemically cross-linking a -CDs threaded onto PEG molecular chain.10Unlike classical hydrogels,this polyrotaxane-based hydrogel can absorb water as high as 400times its dry weight and exhibits better flexible and tensile properties than the normal chemically crosslinked hydrogels.Recently,Sarvothaman and Ritter developed a mutually interlocked gel from host–guest complexes of a -CD and methylated b -cyclodextrin with diacrylates and dimethacrylates of small molecular diols in the presence of a redox initiator system in water.11Meanwhile,our group had developed a series of supra-molecular structured hydrogels by photopolymerization of physical hydrogels yielded from (meth)acryloyl terminated PCL–PEG–PCL 12and PLA–PEG–PLA 13with a -CDs.Owing to incorporating degradable polyester blocks into the pre-polymers,the resultant hydrogels are degradable in water.Recently,we have reported a kind of thermosensitive and supramolecular structured hydrogels via copolymerization of N -isopropylacrylamide with photocurable polypseudo-rotaxanes as cross-linkers under UV irradiation.14The results suggested that the original thermosensitivity of the prepoly-mers disappear upon threading a -CDs onto the prepolymer backbones and reappear after poly(N -isopropyl-acrylamide)is introduced.As is well known,thiol is a multi-functional reagent in the area of organic and polymeric chemistry.Descriptions about the use of thiols in chemical reactions can be traced back over 100years.Nowadays,the thiol-disulfide interchange reaction has resumed extensive interest,partially fuelled by its special role in DNA activation and repression,15as well as remarkable advantages in injectable tissue engineering.16On the one hand,disulfide bonds can be degraded into thiols in cytosol responding to the reduction potential mainly developed by high concentrations (5mM)of a reduced form of glutathione.On the other hand,disulfide bonds are stable in the oxidative environment of the cell exterior.17This dual character has beenBeijing Institute of Technnology,No.5South Street Zhongguancun,District Haidian,Beijing 100081,PR China.E-mail:sainfeng@;Fax:+861068912927;Tel:+861068912650PAPER /softmatter |Soft MatterD o w n l o a d e d o n 30 M a r c h 2011P u b l i s h e d o n 16 F e b r u a r y 2006 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 517206Capplied in some gene delivery materials.18In the present work,we intended to prepare a kind of novel three-dimensional crosslinked networks of polypseudorotaxanes made from the self-assembly of a -CDs with thiolated four-arm PEG by thiol-disulfide interchange reaction.It is expected that the resulting hydrogels have the potential to be used as drug and gene delivery carriers,biosensors as well as injectable tissue-engineering scaffolds.ExperimentalMaterialsFour-arm PEG (4A-PEG,M n =21100)was supplied by Liming Research Institute of Chemical Industries,Henan,China and a -CD was received from Sigma,USA.p -Toluenesulfonic acid,a reduced form of glutathione as well as thioglycolic acid (TGA)were supplied by VAS Chemical Reagents Company,Tianjin,China.TGA was distilled under reduced pressure before using.All other solvents and reagents were of analytical grade.Measurements1H NMR (400MHz)spectra were recorded as solutions on a Bruker ARX 400NMR instrument at room temperature and tetramethylsilane (TMS)as internal standard.FTIR spectra were measured using Shimadzu IR Prestige-21FTIR spectro-meter at room temperature in the range between 4000and 500cm 21,with a resolution of 2cm 21and 20scans.Powder samples were prepared by dispersing the samples in KBr and compressing the mixtures to form disks.Gel permeation chromatography (GPC)analysis was carried out with a chromatographic system equipped with a Waters 1515isocratic HPLC pump and a Waters 2414refractive index detector.Three columns were in series (Waters styragel HT3,HT4,and HT5,7.86300mm).THF was used as eluent at a flow rate of 1.0ml min 21.Monodispersed polystyrene standards were used to obtain a calibration curve.Wide-angle X-ray diffraction (WAXRD)measurements were performed on powder and film samples using Panaltic X 9pert PRO X-ray diffractometer.The radiation source used was Ni-filtered Cu K a radiation with a wavelength of 0.154nm.The voltage was set to be 40KV and the current 40mA.Samples were placed on a sample holder and scanned from 4.5to 60u in 2h at a speed of 0.0017u s 21.Thermogravimetric analysis (TGA)of samples was made using TA Instrument 2000thermogravimetric analyzer at a heating rate of 20u C min 21with nitrogen used as purge gas.Differential scanning calorimetry (DSC)measurements were carried out using a TA Instrument 2910differential scanning calorimeter.The DSC thermograms covered the temperature range of 2100to 150u C at a scanning rate of 10u C min 21.The swelling behaviors of dried gels were studied by a general gravimetric method.Dried films (a diameter of 10mm and a thickness of 2mm)were incubated in distilled water or DMSO at 25u C,and the swollen weight for each sample was recorded at regular time intervals after excess surface solvent was blotted carefully with moistened filter paper.Theprocedure was repeated until there was no further weight increase.The swelling ratio (SR)was calculated by the eqn (1):SR ~m 1{m 0m 0|100%(1)where m 0stands for the initial weight of dried gel and m 1the weight of the swelling gel at a prescribed time interval.Preparation of thiolated four-arm PEGFour-arm PEG was chain-end thiolated by esterification reaction with TGA using p -toluenesufonic acid as a catalyzer.Briefly,10mg p -toluenesufonic acid,10g of 4A-PEG,and 0.8g TGA were added to 100ml of toluene preheated to 120u C.The reaction proceeded for 10h under nitrogen atmosphere and was driven forward by the continuous removal of water produced.The thiolated prepolymer was purified by precipita-tion into anhydrous ether (200ml)at 5u C.This sequence was repeated three times using dichloromethane as a solvent.The product was then dried under vacuum at room temperature for 2days with yield of 90%.FTIR/cm 21:2866(CH 2,CH 3),1736(C L O),1109(C–O);1H NMR (CDCl 3)/ppm:d 4.298(s,8H,–CH 2–O–C(L O)–), 3.577–3.645(m,1980H,–OCH 2–CH 2–O–),3.287–3.307(d,8H,–O–C(L O)–CH 2–),2.145–2.150(t,4H,–SH).The GPC result showed a unimodal peak with a polydispersity index of 1.12.Preparation of polypseudorotaxaneA saturated aqueous solution containing a predetermined amount of a -CDs was added to a certain volume of 20wt%end-thiolated PEG solution in water at room temperature.The resulting mixture was sonicated for 10min and then stood for gelation.A gelation occurred rapidly to yield a physical hydrogel because of the supramolecular self-assembly of a -CDs with the prepolymer.The gel was washed with a small amount of distilled water and then directly freeze-dried to give rise to polypseudorotaxane.Preparation of gel film crosslinked by disulfide bond formation A physical gel was firstly prepared in PBS buffer (pH =7.4)containing a predetermined amount of the prepolymer and a -CDs.After being stirred vigorously,the gel was poured onto 1062mm poly(tetrafluoroethylene)disc and allowed to crosslink in air and dry spontaneously at room temperature for 3days.The sample was then dried under vacuum to constant weight and weighted as W i .Thereafter,air-crosslinked film disc was further oxidized by immersion in 0.1wt%H 2O 2for 1h.The film disc was then rinsed with distilled water and dried in air and at ambient temperature for 1day.The dried film disc was again immersed in 50wt%DMSO aqueous solution in order to attain thoroughly crosslinking.After 6h,the 50wt%DMSO aqueous solution was displaced with pure DMSO to leach out unthreaded a -CDs as well as uncrosslinked prepolymers presumably entrapped in the gel network.This course was carried out for 3days and the solvent was refreshed daily.Finally,after being rinsed with a large amount of distilled water,the film disc was dried at 40u C under vacuum to constant weight,and weighted as W t .D o w n l o a d e d o n 30 M a r c h 2011P u b l i s h e d o n 16 F e b r u a r y 2006 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 517206CFor the sake of expression,the supramolecular structured hydrogels studied in this work were designed as 4A-m ,in which m stands for the feeding molar ratio of a -CDs to the prepolymer.For comparison,the corresponding gel without adding a -CDs,denoted as 4A-0,was also prepared adopting the similar oxidation procedure.Results and discussionPreparationA physical gelation took place quickly at room temperature upon mixing a thiolated 4-arm PEG prepolymer solution with a saturated aqueous solution of a -CDs.The resulting physical hydrogel was freeze-dried to give IC or polypseudorotaxane.It is known that thiol can be transformed to disulfide bond using a number of oxidants,like dilute nitric acid,iodine,bromine,hypochlorous acid and so on.Furthermore,the disulfide bond may also be formed by oxygen in atmosphere under weak alkaline conditions.19In the present study,the physical gel formed from the self-assemly of a -CDs with thiolated 4-arm PEG in PBS buffer was firstly oxidized in air to form a film.The pure thiolated prepolymer without adding a -CDs was found to be crosslinked well under this condition.However,the threading a -CDs impaired the thiol-disulfide interchange reaction of the prepolymer to afford very weakly crosslinked hydrogels when only oxidized in air.For further crosslinking,the dried film sample was again immersed into 0.1wt%H 2O 2solution for 1h owing to longtime immersion could lead to the serious oxidation degradation of PEG molecular chains.As a result,this treatment greatly improved the crosslinked network integrity,but did not still provide the film with satisfactory mechanical properties.It was reported that DMSO oxidation had been often used in disulfide bond formation.20Thence,the film disc was finally immersed in 50wt%aqueous solution of DMSO.After 6h,the solution was displaced with pure DMSO to leach out unthreaded a -CDs as well as uncrosslinked prepolymers presumably entrapped in the hydrogel network.Consequently,the well crosslinked film disc was obtained by this three-step oxidation crosslinking process.The supramolecular self-assembly of a -CDs with thiolated 4-arm PEG and corresponding oxidation crosslinking reactions are shown in Scheme 1.A series of film discs consisting of 20,40and 80a -CDs per thiolated prepolymer were synthesized using the three-step oxidation procedure.The resultant film discs were opaque in water,but transparent in DMSO as depicted in Fig. 1.However,the thiol-disulfide interconversion was observed to hardly proceed any more when the feed molar ratio of a -CDs to the prepolymer exceeds 120.In order to determine the amount and percent coverage of the threading a -CDs,the film discs were degraded in 50mM of high concentration of a reduced form of glutathione,and then calculated via 1H NMR analysis.As presented in Table 1,the amount of the threading a -CDs per thiolated PEG prepolymer and percentage coverage of a -CDs are 26and 10.8%in sample 4A-20,61and 25.4%in 4A-40as well as 82and 34.1%in 4A-80,respectively.The amounts of the threading a -CDs are all higher than the theoretical feed values,possibly attributed to that most uncrosslinked prepolymers have been extracted out duringthe course of preparation of film disc samples.Moreover,the gel content (GC)was also calculated according to the eqn (2):GC ~W tW i|100%(2)As can be seen in Table 1,the gel contents of all supramolecular structured hydrogels are in the range from 76.2%to 81.4%after the three-stepoxidation.Scheme 1Schematic illustration of supramolecular self-assembly and oxidation crosslinkingprocesses.Fig.1The photograph of a supramolecular structured hydrogel (a)in H 2O and (b)in DMSO.Table 1Gel content with and without adding a -CDs,the threading amount of a -CDs in per thiolated prepolymer and percent coverage of a -CDs NameGel content a (%)CD/PEG bCoverage c (%)S4-096.300S4-2076.22610.8S4-4081.46125.4S4-8078.58234.1aGel content is calculated by (W t /W i )6100%.b The average number of a -CDs found per thiolated 4-arm PEG chain.cCoverage =(2CD/PEG)/(PEG repeat units),resuming 2PEG repeat units per a -CD.D o w n l o a d e d o n 30 M a r c h 2011P u b l i s h e d o n 16 F e b r u a r y 2006 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 517206CCharacterizationDMSO is a better solvent for disolving polypseudorotaxanes formed from PEG and a -CDs,but the dissolution will result in the dethreading of a -CDs substantially from PEG backbones.In general,the 1H NMR spectra of the polypseudorotaxanes in DMSO appear identical to that of a physical blend of the two components,indicating that these polypseudorotaxanes exist as a dynamical mixture of threaded and unthreaded species.In contrast,the formation of polyrotaxanes will broaden the resonance peaks of a -CDs attributing to the decrease in conformational flexibility caused by introducing bulky stop-pers to impede a -CDs sliding out of PEG axles.21Slight changes in the chemical shifts of a -CDs are generally observed because of the changes of chemical environments as a consequence of the host–guest interaction.221H NMR spectra of a pure a -CD(A)and a polypseudorotaxane made from the thiolated four-arm PEG prepolymer with a -CDs (theoretical feed molar ratio of a -CDs to prepolymer is 40)(B)are shown in Fig.2.However,the peaks of a -CD in the poly-pseudorotaxane were found to be slightly broadened as compared to pure a -CD,most likely resulted from the formation of disulfide bonds to yield polyrotaxane to some extent in DMSO-d 6owing to air oxidation during the course of measurement.The largest chemical shift of H(3)proton of a -CD was noticed to shift downfield in this case as compared with its free counterpart.Other resonance peaks in the polypseudorotaxane were assigned as follows:O(2)H at d 5.429–5.434,O(3)H at 5.504–5.521,H(1)at 4.792–4.799,and O(6)H at 4.462–4.488ppm,respectively.Multiple resonances appeared at d 3.506–3.791are the chemical shift of H(3),H(6)H(5)protons and PEG,and at 3.275–3.408ppm those of H 2O,H(2),and H(4).FTIR spectra of a -CD (A),4A-40(B)and thiolated four-arm PEG (C)are illustrated in Fig.3.The thiolated PEG spectrum exhibits vibrating bands at 1109and 1736cm 21,which are attributed to the C–O–C stretching mode and the characteristic stretching vibration of carbonyl group in ester,respectively.The pure a -CD spectrum displays extremely strong band around 3383cm 21,assigned to the symmetric and asymmetric O–H stretching mode.The broad hydroxyl band is shifted to higher frequency at 3419cm 21owing to the formation of ICs,which is most likely a consequence of the non-covalent interaction between O–H of a -CD and the prepolymer backbone.What is more,there are evident differences in the positions and intensities of the spectra between free a -CD and supramolecular structured hydrogel,suggesting a host–guest interaction indeed existed rather than a simple physical mixture of a -CD and thiolated PEG.TGA thermal analysis is a powerful technique from which the supramolecular interaction between host and guest molecules in ICs can be determined.The supramolecular structured hydrogels undergo a two-step thermal degradation process.The fist step is mainly attributed to the decomposition of a -CDs,while the second mainly to that of the guest prepolymers.Here,the temperature at which two tangents intersect is used as the decomposition temperature of a particular component (T d )to quantitatively evaluate the thermal stability of the samples.As outlined in Fig.4,the T d values for a -CD (A)and 4A-0(B)are 287and 392u C,separately.Upon threading a -CDs onto the prepolymer chains to form the supramolecular structured networks,the T d data for the component a -CD and prepolymer are 325and 398u C for 4A-20(C),321and 402u C for 4A-40(D),and 318and 397u C for 4A-80(E),respectively,and they are all higher than their free counterparts.This fact clearly implies that the thermal stability of individual component is sub-stantially improved as long as an inclusion complex is formed between them.Fig.5shows the DSC curves of pure a -CD (A),4A-0(B),4A-20(C),4A-40(D)and 4A-80(E).There is adistinctD o w n l o a d e d o n 30 M a r c h 2011P u b l i s h e d o n 16 F e b r u a r y 2006 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 517206Cendothermic peak at58.7u C corresponding to the melting point of PEG crystalline phase in the sample4A-0.Upon the formation of ICs,the corresponding endothermic peak is absent as evidenced in the supramolecular structured gels of 4A-40and4A-80,because the PEG molecular chain is included in the channel of the host a-CD lattice,which restricts its aggregating to form the crystalline phase.23 However,the endothermic peak in4A-20is still observable. This is possibly ascribed to that the threading amount of a-CDs in this sample is too low to thoroughly limit the aggregation and crystallization of PEG chain.Meanwhile,its fusion temperature moves to a lower range(at52.9u C),and the enthalpy change decreases dramatically with forming IC. Evidently,the decreasing in the fusion temperature and the enthalpy change are caused by the perturbation of crystal-lization resulted from the partial inclusion of PEG molecular chain into the channel of the host a-CDs.The XRD patterns of a-CD(A),thiolated four-arm PEG (B),a polypseudorotaxane consisting of the thiolated pre-polymer and a-CDs(theoretical feed molar ratio of a-CDs to prepolymer is40)(C),a pure gel of4A-0(D),and supra-molecular structured gels of4A-20(E),4A-40(F)and4A-80(G)are presented in Fig.6.The diffraction peaks of pure a-CD appear at12.36,22.09and26.70u,respectively.The thiolated prepolymer presents only two strong peaks at19.09and 23.24u.The diffraction pattern of the polypseudorotaxane is rather different from that of either a-CD or the thiolated PEG, showing a strong peak at2h=20.06u(d=4.42A˚).This is a characteristic peak to form a channel-type crystalline struc-ture.5While,the diffraction patterns in the supramolecular structured hydrogels resemble that of the polypseudorotaxane exhibiting a strong peak at about2h=20u.Thus,it can be deduced that there also exists the channel-type crystalline structure in these polyrotaxane-related hydrogel networks.But all the characteristic diffraction peaks slightly shift to lower range,such as the characteristic peak in4A-20appeared at 19.80u,4A-40at19.75u and4A-80at19.68u.Swelling behaviors of supramolecular structured hydrogels According to the feed compositions,a series of hydrogels with a-CDs and without a-CDs were prepared and their swelling ratios were measured in distilled water and DMSO at25u C, respectively.As illustrated in Fig.7,the hydrogel of4A-0(A) made from the pure thiolated prepolymer has the highest SR in water.The SR of the supramolecular structured hydrogels of 4A-20(B),4A-40(C)and4A-80(D)was found to decrease with increasing the content of a-CDs threaded onto the network chain.A rational explanation is that the channel-type crystalline structure formed from the self-assembly of a-CDs with the prepolymer impairs water to enter the hydrogel matrix.Furthermore,their SR in DMSO was also measured. As pictured in Fig.8,the swelling behaviors in DMSO evidently differ from that in distilled water.First,4A-0(A) hardly swells in DMSO,showing poor dissolution ability for PEG.Second,the SR of all supramolecular structured hydro-gels of4A-20(B),4A-40(C)and4A-80(D)is higher than that in distilled water,suggesting that DMSO is capable of unfolding crystalline congeries formed from theself-assembly Downloadedon3March211Publishedon16February26onhttp://pubs.rsc.org|doi:1.139/B51726Cof a-CDs.Third,the discipline of SR depended on the a-CDs to thiolated prepolymer feed molar ratio in DMSO is also different from that in water.The SR of the gel4A-40(C)is higher than that of other two samples of4A-20(B)and4A-80 (D).Although DMSO here is a better solvent able to com-pletely unfold the channel-type crystalline structure leading a-CDs to disperse along the whole molecular chain,it is not true for the pure thiolated PEG-based three-dimensional crosslinked hydrogel networks.Consequently,the resulting SR is the competing balance between DMSO to unfold the crystalline lattice of uncovered and covered PEG segments with a-CDs.In other words,the swelling behaviors of this kind of supramolecular structured hydrogels can be readily regulated via varying the feed compositions of a-CDs and thiolated4-arm PEG prepolymer.Degradation behaviors of supramolecular structured gelsFig.9shows a change in SR of the gels with a different amount of threading a-CDs and unthreading a-CDs in PBS buffer (pH=7.4)containing a high concentration(50mM)of a reduced form of glutathione.The SR of4A-0(A)quickly increases until about4h and then immediately decreases, indicating typical bulk erosion with water uptake.On the other hand,such a drastic increase of the SR is not observed in the supramolecular structured hydrogels,and their complete degradtaion times are prolonged with increasing the threading amount of a-CDs to20h for4A-20(B),36h for4A-40(C)and 84h for4A-80(D).This fact implies that a-CDs threaded onto the hydrogel network chains have protected disulfide bonds during the course of the reduction degradation.This is most likely owing to the threading a-CDs holding back the attacking of the reductive agent glutathione on disulfide bonds included in the cavities of a-CDs.Thiol-disulfide interchange reaction under mild conditions is very interesting.Controlling the formation and cleavage of the disulfide bonds in the supramolecular structured hydrogels through changing external conditions could help us to design and prepare a kind of novel smart polyrotaxane-related three-dimensional crosslinked hydrogels to achieve the drug and gene controlled release and tissue engineering scaffold injectable.In this study,a three-step oxidation crosslinking procedure was used,via O2in air,0.1wt%H2O2as well as DMSO,respectively,to fulfill the thiolated4-arm PEG crosslinked well,which is evidently too harsh to meet the demands for the drug and gene controlled delivery,and in situ crosslinking injectable tissue engineering.Therefore,looking for more biocompatible oxidation methods is our ongoing study.Furthermore,a great number of hydroxyl groups exit in each a-CD threaded onto the polymer backbones,which can be also used as active positions for further chemical modifications.ConclusionsIn this study,a kind of novel three-dimensional crosslinked hydrogel networks of polypseudorotaxanes based on the self-assemly of a-CDs with the thiolated4-arm PEG was synthesized via thiol-disulfide interchange reaction by means of a three-step oxidation.The channel-type crystalline structure of inclusion complexes was found to remain in the hydrogel network chains after the oxidation crosslinking process.The a-CDs threaded onto the network chains protected disulfide bonds from the reduction attack ofa Downloadedon3March211Publishedon16February26onhttp://pubs.rsc.org|doi:1.139/B51726Creduced form of glutathione.Their swelling behaviors and degradation properties can be tailored by changing the feed 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