science

Colloids and Surfaces B:Biointerfaces 81 (2010) 329–335Contents lists available at ScienceDirectColloids and Surfaces B:Biointerfacesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c o l s u r fbSelf-assembly behavior of peptide amphiphiles (PAs)with different length of hydrophobic alkyl tailsXiao-Ding Xu 1,Yue Jin 1,Yun Liu,Xian-Zheng Zhang ∗,Ren-Xi ZhuoKey Laboratory of Biomedical Polymers of Ministry of Education &Department of Chemistry,Wuhan University,Wuhan 430072,PR Chinaa r t i c l e i n f o Article history:Received 23April 2010Received in revised form 6July 2010Accepted 9July 2010Available online 16 July 2010Keywords:Peptide amphiphile Self-assembly Alkyl taila b s t r a c tAmphiphilic peptide and their derivatives,with distinguished advantages over conventional materials,have received extensively research interesting recently.In this work,four peptide amphiphiles (PAs1-4)with different length of hydrophobic alkyl tails (C9for PA1,C11for PA2,C13for PA3,and C15for PA4)were fabricated and their self-assembly behaviors in aqueous medium at different pHs were investigated systematically.It was found that all the peptide amphiphiles can self-assemble in water at a neutral pH of 7to form tightly packed nanofibers with a ␤-sheet conformation.When altering the solution environment to basic medium (pH 11),due to the strong hydrophobic interaction of long alkyl tails in PA3and PA4,the fibrous nanostructure self-assembled from PA3and PA4was not destroyed.However,the nanofibers self-assembled from PA1in which the length of alkyl tail was relatively short converted into loose spherical micelles with a ␤-sheet conformation.Due to the moderate length of alkyl tail in PA2,both nanofibers and micelles can be formed via the self-assembly of PA2when increasing the pH of the self-assembling system.© 2010 Elsevier B.V. All rights reserved.1.IntroductionThe self-assembly of peptides and their derivatives has attracted much attention as a powerful approach for construction of novel functional materials that promise broad applications in tissue engineering [1–3],3D cell culture [4,5],drug delivery [6,7],and bacteria inhibition [8,9].By exploiting the spontaneous or induced molecular arrangement upon external stimuli such as pH and/or temperature,peptides and their derivatives are able to grow from homogeneous solution into shape-specific nanostructures via non-covalent forces including ␲-stacking [10,11],hydrogen bonding [12,13],and hydrophobic interactions [14–16].Over the past few decades,a variety of structural motifs such as coiled-coils [17],␤-hairpins [12,13],␤-sheets [18,19],and peptide amphiphiles (PAs)[14–16],have been established for the self-assembly of peptides and their derivatives.Among them,peptide amphiphiles consisted of hydrophilic peptide headgroups and hydrophobic tails have been widely employed as building blocks for the self-assembly in water to form shape-specific nanostructures.One of the first designs of synthetic peptide amphiphiles was proposed by Kunitake [20]and consisted of a hydrophobic tail,a linker,a spacer,and a hydrophilic headgroup.By changing the structural segments of an amphiphilic molecule to tailor the fun-∗Corresponding author.Tel.:+862768754509;fax:+862768754509.E-mail address:xz-zhang@ (X.-Z.Zhang).1Authors contributed to this work equally.damental rules of the non-covalent forces,one can control the morphology,characteristics,surface chemistry,and function of the molecule.To date,a number of peptide amphiphiles have been reported,including peptide amphiphiles with different peptide headgroups [14–16]and one or more alkyl tails [20–22].These pep-tide amphiphiles have been shown to form various nanostructures such as micelles,vesicles,nanofibers,nanotapes,and ribbons.For examples,Gore et al.[21]investigated the influence of the number of tails and temperature on the self-assembly of collagen mimetic peptide amphiphiles.It was reported that the resulting morphol-ogy can transfer from spherical micelles to disc-like micelles which can stack up to form extended strand-like structures with the alternation of the length and number of tails.When changing the temperature of the self-assembling system,the triple-helical struc-ture of the peptide segment can be destroyed for the collagen mimetic peptide amphiphile with certain length of tail.We have also reported previously several peptide amphiphiles that exhibit different self-assembly behaviors upon the alter-nation of external pH and salt concentration [23].Moreover,it is interesting to know whether these peptide amphiphiles can self-assemble into different nanostructures including vesicles,micelles,and nanofibers when changing (from alkyl tail to aro-matic tail)or slightly increasing the length of the hydrophobic alkyl tails (from C9to C10)and solution pH.In this study,four peptide amphiphiles (PAs1-4)with different length of alkyl tails (C9for PA1,C11for PA2,C13for PA3,and C15for PA4)were designed and prepared via a standard FMOC chemistry (Fig.1).The self-assembly behaviors of these peptide amphiphiles in water0927-7765/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfb.2010.07.027330X.-D.Xu et al./Colloids and Surfaces B:Biointerfaces81 (2010) 329–335Fig.1.Molecular structures of the peptide amphiphiles (PAs1-4).were monitored by transmission electron microscopy (TEM),cir-cular dichroism (CD),and Raman spectroscopy.The data obtained indicate that the peptide amphiphiles having long alkyl tails (PA3and PA4)can self-assemble into tightly packed nanofibers with a ␤-sheet conformation at a pH range from 3to 11.However,the nanofibers self-assembled from PA1with short alkyl tail become loose micelles with a ␤-sheet conformation when altering the solution environment from acidic to basic medium.2.Materials and methods 2.1.MaterialsN -Fluorenyl-9-methoxycarbonyl (FMOC)protected l -amino acids (FMOC-Gly-OH,FMOC-Asp(OtBu)-OH,FMOC-Arg(Pbf)-OH,FMOC-Val-OH)and 2-chlorotrityl chloride resin (100–200mesh,loading 1.32mmol/g)were pur-chased from GL Biochem (Shanghai)Ltd.(China)and used as received.Piperdine,trifluoroacetic acid (TFA),o -benzotriazole-N,N,N ,N -tetramethyluroniumhexafluorophosphate (HBTU),and N -hydroxybenzotriazole (HOBt)were provided by Shanghai Reagent Chemical Co.(China)and used directly.10-Undecenoic acid,N,N -dimethylformamide (DMF),and diisopropylethylamine (DiEA)were obtained from Shanghai Reagent Chemical Co.(China)and distilled prior to uric acid,myristic acid,and palmitic acid were purchased from Shanghai Reagent Chemical Co.(China)and used after recrystallization from ethanol.Triisopropylsilane (TIS)was purchased from ACROS (USA)and used without further purification.All other reagents and solvents were of analytical grade and used directly2.2.Synthesis of peptide amphiphiles (PAs)All the peptide amphiphiles (PAs1-4)were synthesized man-ually in 1.98mmol scale on the 2-chlorotrityl chloride resin employing a standard FMOC chemistry.Before the synthesis,the resin was washed with CH 2Cl 2(three times)and DMF (three times)and then immersed in DMF for 30min.After draining off DMF solu-tion,a DMF solution of the mixture of FMOC protected amino acid (3equiv relative to resin loading)and DiEA (6equiv)was added to the resin and shaken for 2h at room temperature.After removing the reaction solution,the resin was washed with DMF (three times).Subsequently,20%piperdine/DMF (V/V)solution was introduced to the resin to remove the FMOC protected groups.After shaking for 30min at room temperature,the reaction solution was drained off and the resin was washed with DMF (three times).The presence of free amino groups was indicated by a blue color in the Kaiser test.Thereafter,a DMF solution of the mixture of FMOC protected amino acid (2equiv),HBTU (3equiv),HOBt (3equiv)and DiEA (4equiv)was added.After shaking for 1.5h at room temperature,the reaction solution was drained off and the resin was washed with DMF (three times).The absence of free amino groups was indicated by a yellow color in the Kaiser test.After the repetition of depro-tection and acylation reaction,the resin was finally washed with DMF (three times)and CH 2Cl 2(three times)and dried under vac-uum for 24h.Cleavage of the expected peptide and the removal of side chain protected groups from the dried resin were performed using a mixture of TFA,deionized water,and TIS in the ratio of 95:2.5:2.5.After shaking at room temperature for 2h,the cleav-age mixture and three subsequent TFA washing were collected.The combined solution was concentrated to a viscous solution byX.-D.Xu et al./Colloids and Surfaces B:Biointerfaces81 (2010) 329–335331rotary evaporation.Cold ether was added to precipitate the product. After washing with cold ether(five times)to remove TFA residual, the precipitate was dissolved in distilled water and then freeze-dried under vacuum for3days.The purities of the synthesized peptide amphiphiles were analyzed by high-pressure liquid chro-matography(HPLC)with a C18column and using a linear gradient of acetonitrile and DI water containing0.1%NH4OH.The masses of the peptide amphiphiles were examined by electro spray ionization mass spectrometry(ESI-MS,LCQ Advantage,Finigan,USA).PA1: purity,95.1%by HPLC;IR,∼3450cm−1,amide A band,∼1648cm−1, amide I band,∼1550cm−1,amide II band;MS,calculated698, [M−H]−found697.PA2:purity,93.8%by HPLC;IR,∼3455cm−1, amide A band,∼1652cm−1,amide I band,∼1556cm−1,amide II band;MS,calculated726,[M−H]−found725.PA3:purity,93.4%by HPLC;IR,∼3450cm−1,amide A band,∼1643cm−1,amide I band,∼1550cm−1,amide II band;MS,calculated754,[M−H]−found 753.PA4:purity,95.4%by HPLC;IR,∼3447cm−1,amide A band,∼1645cm−1,amide I band,∼1551cm−1,amide II band;MS,calcu-lated782,[M−H]−found781.2.3.Acid–base titrationThe solution of the peptide amphiphiles was prepared in dis-tilled water at a pH of4with a concentration of1.5mg/mL.Then 0.01M NaOH aqueous solution was added in5–10␮L increments. After each addition,the sample was constantly stirred for5min at room temperature and the pH value of the solution was measured using a PB-10Sartorius pH meter.Each sample was measured three times and the average value of the three measurements was col-lected.The p K a of the each peptide amphiphile was defined as the pH where half amount of carboxylic acid groups were ionized.2.4.Self-assembly of peptide amphiphiles(PAs)The solution of the peptide amphiphiles was prepared in dis-tilled water at a pH of11with a concentration of6mg/mL.Then concentrated HCl was added to in1–5␮L increments and the cor-responding pH value of the solution was measured on a PB-10 Sartorius pH meter after shaking the solutions for5min.The self-assembly behaviors of the peptide amphiphiles were investigated at various pH using the following transmission electron microscopy (TEM),circular dichroism(CD),and Raman spectroscopy.2.5.Transmission electron microscopy(TEM)The morphology of the self-assembled peptide amphiphiles was observed on a JEM-100CXa transmission electron micro-scope(TEM).Before the observation,the solution of the peptide amphiphiles was applied to a copper grid and stained by a0.2% (w/v)phosphotungstic acid solution.2.6.Circular dichroism(CD)The solution of the self-assembled peptide amphiphiles was fixed in a0.5mm quartz cell and analyzed on a Jasco J-810spec-tropolarimeter with4s accumulations every1nm and averaged over three acquisitions.2.7.Raman spectroscopyThe Raman spectra of the self-assembled peptide amphiphiles were recorded on a Jobin Yvon Raman microspectrometer(LABRAM HR800,helium-neon laser)with the citation wavelength at 632.8nm and long focal length.Before the analysis,the solutions of the self-assembled peptide amphiphiles were freeze-dried in a Freeze Drier(Labconco,CA)under vacuum.3.Results and discussion3.1.Self-assembly of the peptide amphiphiles(PAs)The molecular structures of the peptide amphiphiles(PAs1-4)are presented in Fig.1.All the peptide amphiphiles havea Fig.2.TEM images of the peptide amphiphiles in aqueous solution at pH7(a:PA1,b:PA2,c:PA3,d:PA4).332X.-D.Xu et al./Colloids and Surfaces B:Biointerfaces81 (2010) 329–335Fig.3.TEM images of the peptide amphiphiles in aqueous solution at pH 11(a:PA1,b:PA2,c:PA3,d:PA4).hydrophilic peptide headgroup of VRGDV sequence and hydropho-bic alkyl tail.And the hydrophobic alkyl chains were designed as C9,C11,C13,and C15,respectively in order to study the influence of the length of the tails on their self-assembly behaviors.These four PAs were dissolved in distilled water at a neural pH of 7with a concentration of 6mg/mL.PA1and PA2can dis-perse well in water to form clear solution.While for PA3and PA4,the solubility was comparatively decreased due to their rel-atively longer hydrophobic alkyl tails.Especially PA4,the solution was significantly viscous and some precipitates can be observed in the solution.When adding concentrated HCl (12M)to the solution of the peptide amphiphiles,the viscosity of the solution increased dramatically and three-dimensional hydrogels can be formed when the pH of the self-assembling system reached 3.Based on the experiments,the gelation can also occur even the con-centration of peptide amphiphiles deceased to 3mg/mL.However,by introducing concentrated NaOH solution to the self-assembling system,gel–sol transition took place and can be accomplished when increasing the pH to a neutral (pH 7)or basic value (pH 11).At this time,once adding concentrated HCl to the solution of the peptide amphiphiles at a neutral or basic pH,the viscosity of the solu-tion increased dramatically and three-dimensional hydrogels can be also formed.That is,this pH triggered phase transition of the peptide amphiphiles was reversible and can be repeatedly accom-plished.In order to examine the morphology of the self-assembled peptide amphiphiles,TEM measurements were performed.As pre-sented in Fig.2,all the peptide amphiphiles can self-assemble into tightly packed nanofibers with the width of around 25nm at a neutral pH of 7(Fig.2a–d).It is also found that there is no obvious change of the fibrous morphology for PA3(Fig.3c)and PA4(Fig.3d)with the increasing of the solution pH to a value of 11.However,besides nanofibers,micelles can be also observed in the solution of PA2at a pH of 11(Fig.3b).In case of PA1,after increasing the solution pH to 11,the original nanofibers are com-pletely replaced by spherical micelles with the width of ∼30nm (Fig.3a).The result of dynamic light scattering (DLS)measure-ment revealed in Fig.4indicates that the average diameter of the monodispersed micelles is around 33nm with a polydisper-sity index (PDI)of 0.55,which is in good accordance with the result of TEM measurement.Because PA1has been reported to have an ability to self-assemble into nanofibers at an acidic pH [23],as the derivatives,the TEM images of the self-assembled PAs2-4at an acidic pH are not shown.In fact,several recent reports have also demonstrated that the peptide amphiphiles with the similar molec-ular structures can self-assemble into nanofibers as the functional matrices of supramolecular hydrogels [14–16].As described above,we can see that PAs1-4with different length of alkyl tails present different self-assembly behaviors upon the alternation of external pH.It is known that the self-assembly of the peptide amphiphiles is related to the balance ofhydrophilicFig.4.Size distribution of the micelles self-assembled from PA1in aqueous solution at pH 11.X.-D.Xu et al./Colloids and Surfaces B:Biointerfaces81 (2010) 329–335333Fig.5.Acid–base titration curves of the peptide amphiphiles(a:PA1,b:PA2,c:PA3,d:PA4)at a concentration of1.5mg/mL. and hydrophobic interactions[14–16,20–22].The alternation ofthe solution pH can accomplish the protonation or deprotona-tion,resulting in the change of the hydrophilic interaction.Onthe other hand,changing in the length of the hydrophobic tailsalso has an influence on the hydrophobic interaction of the self-assembling systems.In this study,in order to determine whetherthe alternation of the solution pH induces the protonation or not,the classic acid–base titration was employed to measure the p K a sof the peptide amphiphiles.All the titrations started at a pHwhereFig.6.CD spectra of the peptide amphiphiles(PAs1-4)in aqueous solution at pH7 (a)and pH11(b).the molecules have already being the aggregated states to avoid kinetic effect of the self-assembly.From the data in Fig.5,the titra-tions of PA1(Fig.5a)and PA2(Fig.5b)present sharp transitions, corresponding to two apparent p K a s,mainly relating to the pro-tonation of the terminal carboxylic acid groups of PA1(Fig.5c) and PA2(Fig.5d).However,it is difficult to exactly determine the p K a of PA3and PA4,implying that their protonation/deprotonation processes occur slowly with variations of acidity due to thelocalFig.7.Raman spectra of the peptide amphiphiles(PAs1-4)in aqueous solution at pH7(a)and pH11(b).334X.-D.Xu et al./Colloids and Surfaces B:Biointerfaces81 (2010) 329–335Fig.8.Schematic illustration of the self-assembly of the peptide amphiphiles (PAs1-4)at acidic,neutral,and basic mediums.microenvironment within the nanofibers [15].According to the result of acid–base titration,the hydrophobic interaction of the alkyl tails at an acidic pH can make the unionized PAs1-4self-assemble into nanofibers.With the increasing pH of the self-assembling system,the peptide headgroups are ionized to provide electrostatic repulsion and the whole hydrophilicity of the system is improved,resulting in the morphology change for PA1from tightly packed nanofibers to loose micelles.However,due to the presence of relatively longer hydrophobic alkyl tails with strong hydrophobic interaction of PA3and PA4,there is no obvious morphology change in their self-assembled nanostructures.Because of the moderate length hydrophobic alkyl tail in PA2,both nanofibers and micelles can be formed via the self-assembly of PA2.From all these results,it is reasonable to conclude that the nanofibers self-assembled from peptide amphiphiles could be stabilized via the increasing of the length of the hydrophobic alkyl ly,the nanofibers become more tightly packed due to the strengthened hydropho-bic interaction,which is the driven force of the self-assembly of peptide amphiphiles,to resist the influence of pH changing.3.2.Secondary structures of the self-assembled peptide amphiphiles (PAs)In order to further understand the backbone orientation of the peptide amphiphiles in the self-assembled nanostructures,the sec-ondary structures were investigated using circular dichroism (CD).Because the CD analysis of peptide-based supramolecular materials is significantly affected by the status of the samples (tiny undis-solved pieces generally induce large light scattering effect),clear solution of the self-assembled PAs1-4was applied for CD analy-sis.As revealed in Fig.6,either at an acidic pH or basic pH,the CD spectra have a negative band at ∼219nm which shares a com-mon feature of ␤-sheet conformation of a polypeptide [14–16,24],suggesting that the self-assembly of PAs1-4leads to the formation of ␤-sheet conformation in the nanofibers or micelles.Besides CD analysis,the solution of the self-assembled PAs1-4was also freeze-dried for Raman analysis.As exhibited in Fig.7,the absorbance maxima of amide I frequency is at ∼1670cm −1,a typical band of ␤-sheet conformation formed via intermolecular hydrogen bond-ing interactions [25,26],which is consistent with the result of CDanalysis.It is noted that the Raman peak centered at ∼1450cm −1in Fig.7mainly corresponds to the variation of methylene groups of the peptide amphiphiles [25,26].Based on the information of CD and Raman analysis,the self-assembly mechanism of PAs1-4at various pH is proposed as that illustrated in Fig.8.At an acidic pH,the hydrogen bonding inter-action of the peptide headgroups and hydrophobic interaction of the alkyl tails are the driven forces to trigger the self-assembly of PAs1-4to form tightly packed nanofibers with a ␤-sheet conforma-tion.When increasing the pH of self-assembling system,leading to the deprotonation of the peptide headgroups,the generated elec-trostatic repulsion will disturb the self-assembly.However,the presence of hydrophobic interaction from the alkyl tails has an abil-ity to protect the self-assembled nanostructures.As a result,the nanofibers self-assembled from PA1in which the alkyl tail is too short to provide enough hydrophobic interaction can convert into loose spherical micelles,which is similar with self-assembly behav-iors of ionic surfactants [27–29].With the increasing length of the hydrophobic alkyl tail,the hydrophobic interaction is strengthened gradually,resulting in the co-existence of nanofibers and spherical micelles in the self-assembly of PA2.Further increasing the length of the hydrophobic alkyl tail,the hydrophobic interaction becomes stronger enough to tolerate the electrostatic repulsion and main-tains the fibrous nanostructures self-assembled from PA3and PA4regardless of pH changing.4.ConclusionsA series of peptide amphiphiles (PAs1-4)with different length of hydrophobic alkyl tails were designed.It is found that at an acidic pH,all these peptide amphiphiles can self-assemble into tightly packed nanofibers with a ␤-sheet conformation.Upon pH increasing,the nanofibers self-assembled from PA1having rela-tively short hydrophobic alkyl tail convert into spherical micelles.However,due to the strong hydrophobic interaction among the long hydrophobic alkyl tails in PA3and PA4,the fibrous nanostruc-tures remain unchanged regardless of the pH changing.Due to the moderate length of alkyl tail in PA2,both nanofibers and spherical micelles can be formed at a basic pH of 11.X.-D.Xu et al./Colloids and Surfaces B:Biointerfaces81 (2010) 329–335335AcknowledgementsWe acknowledge thefinancial support from the National Nat-ural Science 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