Spontaneous formation of narrowly-distributed self-assembly from polyamidoamine...

Spontaneous Formation of Narrowly-DistributedSelf-Assembly from Polyamidoamine Dendron-Poly(L-lysine)Block Copolymers Through Helix-Coil Transition ofPoly(L-lysine) Block

ATSUSHI HARADA, KENICHI NAKANISHI, SHINICHI ICHIMURA, CHIE KOJIMA, KENJI KONO

Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho,Naka-ku, Sakai, Osaka 599-8531, Japan

Received 5 November 2008; accepted 1 December 2008DOI: 10.1002/pola.23231Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: The self-assembly of head-tail type block copolymers composed of polya-midoamine dendron head block and poly(L-lysine) (PLL) tail block was studied usinga light scattering technique and transmission electron microscopy. A PLL tail blockin a head-tail type block copolymer exhibits a coil-to-helix transition as a result ofthe change in solvent quality from water to methanol. When the PLL tail block takesa helical conformation in high methanol content, the resulting head-tail type blockcopolymer has a defined three-dimensional structure like that of a protein molecule.Self-assemblies of such block copolymers having a totally fixed molecular shape spon-taneously form polymersome-like self-assemblies with an extremely narrow size dis-tribution through converging to a thermodynamically stable assembling state. VVC 2009

Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1217–1223, 2009

Keywords: block copolymer; dendron; helix-coil transition; nanoparticle; polymersome;polypeptide; self-assembly

INTRODUCTION

A variety of highly smart nano-materials systemsin nature are constructed by the association ofvarious kinds of biomacromolecules, includingproteins and DNA, through well controlled inter-molecular and/or intramolecular interactions.Protein molecules with well-defined conformationand amphiphilic character spontaneously formspatially-defined supramolecular complexes toachieve sophisticated biological functions: i.e.,capsid proteins in viruses and transmembrane

proteins in cells. A bioinspired approach toresearch on supramolecular association hasproved to be useful for the development of novelfunctional materials from engineered syntheticpolymers.1–5 The use of block copolymer self-assembly is one of the most intensively studiedapproaches. Most of amphiphilic block copolymerswhich formed self-assemblies in selective solventswere composed of flexible polymer strands. Thechain conformation of polymers plays an impor-tant role in controlling their phase behavior andassociation properties. Flexible polymer couldtake varying conformations; on the other hand,protein molecules have well-defined three-dimen-sional structure based on their amino acidsequences. This is significant difference bet-ween natural system and synthetic system.

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 47, 1217–1223 (2009)VVC 2009 Wiley Periodicals, Inc.

Correspondence to: A. Harada (E-mail: [email protected])

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Interestingly, it has been reported on the self-assemblies of block copolymers having rigid blockas one or both components,6–9 e.g., dendritic poly-mers and polypeptides, and they took variousunique morphologies including spherical micelles,rods, lamellas, and vesicles. Particularly, the stud-ies on block copolymers consisting of syntheticpolymers and polypeptides including the con-formational transition of polypeptide blockthrough the change in solvent quality have beenextensively carried out by many researchgroups.3,5,7,8,10–13 Also, the dendrimers and den-drons are highly branched synthetic moleculestypically based on AB2 monomers and are usuallyprepared by adding generations of monomers to acore molecule. These have well-defined structurewith no distribution as branched molecules, andthe self-assembly of the conjugates of dendriticmolecules with the other kinds of linear polymershave also been also investigated. However, therewas a few report on the self-assembly of blockcopolymers composed of dendritic block and poly-peptide block. We have recently reported the syn-thesis of head-tail type block copolymer composedof polyamidoamine (PAMAM) dendron head blockand poly(L-lysine) (PLL) tail block, which chemi-cal structure was shown in Scheme 1, as novelnonviral gene vector.14 This head-tail type blockcopolymer (PAMAM dendron-PLL) could alsoform self-assembly with unique morphology. Fromsuch motivation, we evaluate the self-assembly ofPAMAM dendron-PLL through the conforma-tional change of PLL tail block from random coilto helical conformation. PLL tail block couldchange from flexible coiled conformation to rigidhelical conformation even after the block copoly-merization with PAMAM dendron. Changing thecomposition of mixing solvent, methanol andwater (MeOH/H2O), PAMAM dendron-PLLformed monodisperse self-assemblies synchroniz-ing with coil-to-helix transition of PLL tail block.Totally holding of block copolymer conformationmight result in the formation of polymersome-likeself-assemblies with narrow size distributionthrough converging to a thermodynamically sta-ble assembling state.

EXPERIMENTAL

Synthesis of Block Copolymer

The block copolymer, PAMAM dendron-PLL, wassynthesized in two steps, the synthesis of thePAMAM dendron and the polymerization of the

PLL block with the PAMAM dendron, accordingto previous report.14 Tert-butyl N-(2-aminoethyl)-carbamate, which has one primary amino groupprotected by a Boc group, was used as the startingreagent. A PAMAM dendron with generation 3.5was synthesized following four Michael additionswith methylacrylate and three amidations withethylenediamine. The resulting compounds hadprimary amino groups protected by a Boc groupat the root. From the Boc-protected PAMAM den-dron, the Boc group was removed by trifluoroace-tic acid treatment. The polymerization of e-benzy-loxycarbonyl-L-lysine N-carboxy anhydride (NCA)was carried out in distilled DMF. The ring-open-ing polymerization of NCA was selectively initi-ated from the primary amino group at the root ofthe PAMAM dendron. The benzyloxycarbonylgroups at the side chain of the PLL block wereremoved by hydrogen bromide treatment, and themethyl esters at the periphery were also con-verted to carboxylate groups. The PAMAM den-dron-PLL thus obtained was confirmed to have aunimodal molecular weight distribution [the ratioof weight-averaged to number-averaged molecularweight (Mw/Mn) ¼ 1.3] by gel permeation chroma-tography. The final PAMAM dendron-PLL had aPAMAM dendron head block with generation 3.5and a PLL tail block with a number-averagedpolymerization degree of 70.

Scheme 1. Chemical structure of polyamidoaminedendron-poly(L-lysine) block copolymer.

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Journal of Polymer Science: Part A: Polymer ChemistryDOI 10.1002/pola

Characterization Techniques

Circular dichroism (CD) measurements were car-ried out using a 3 mm cell and a J-600 (JASCO)at 20 �C. All samples were prepared to have0.0025 wt % of polymer. Transmission electron mi-croscopy (TEM) observation was carried out usinga JEOL2000 with carbon-coated cupper grids, andthe sample was negatively stained with phospho-tungstic acid. Dynamic light scattering (DLS)measurements were carried out using an ELS-8000 (Otsuka Electronics, Japan) equipped with aHe/Ne laser (k ¼ 633 nm) at 20 �C. The detectionangle was fixed at 90�. In analysis of the DLSmeasurements, the general formula for the photo-electron count time correlation function had theform:

gð2ÞðsÞ ¼ 1þ bjgð1ÞðsÞj2 ¼ 1þ bexpð�2CsÞwhere g(2)(s) is the normalized second order corre-lation function, b is a parameter of the optical sys-tem (constant), g(1)(s) is the normalized first ordercorrelation function, s is the delay time, and C isthe average characteristic line width. g(1)(s) canbe expressed by the following equation:

gð1ÞðsÞ ¼Z

GðCÞexpð�CsÞdC

where G(C) is the distribution function of C. Theautocorrelation functions were analyzed using themethod of cumulants in which

gð1ÞðsÞ ¼ exp½�Csþ ðl2=2Þs2 � ðl3=3!Þs3 þ…�yielding an average line width C, and a variance(polydispersity index) l2/C

2. In the cumulantapproach, the z-averaged diffusion coefficient, D,was obtained from the average line width, C,based on the following equation:

C ¼ Dq2

q ¼4pnsinðh=2Þkwhere q is the magnitude of the scattering vector,and h is the detection angle. The correspondingmeans diameter, d, can then be calculated usingthe Stokes-Einstein equation:

d ¼ kBT=ð3pgDÞwhere kB is the Boltzmann constant, T is theabsolute temperature, and g is the solvent viscos-ity. Also, the size distribution was estimated fromthe correlation function profile using CONTINmethod.

RESULTS AND DISCUSSION

The conformational change of PLL tail block inPAMAM dendron-PLL through changing solventcomposition in MeOH/H2O mixture was evaluatedby CD and 1H NMR measurements. CD spectra ofPLL block of PAMAM dendron-PLL changed withvarying MeOH content [Fig. 1(a)]. Increasingwith MeOH content, it became to observe doubleminimum spectra at 208 and 222 nm demonstrat-ing a-helical conformation. The helicity of PLLtail block of PAMAM dendron-PLL was calculatedusing the mean residual ellipticity at 222 nm([h]222), characterizing a-helix structure, reportedby Padmanabhan et al.,15 in which the helicityis estimated using the following equation as�34,000 deg cm2/decimole for 100% helix and 640deg cm2/decimole for 0% helix.

Helicity ð%Þ ¼ ð640� ½h�222Þ=ð640þ 34;000Þ � 100

The helicity of PLL tail block steeplyincreased around MeOH content ¼ 50 vol %,reflecting cooperative transition, and PLL tailblock exhibit almost constant helicity at morethan 80 vol % of MeOH [Fig. 1(c)]. PLL tail blockof PAMAM dendron-PLL in MeOH/H2O mixturewith high MeOH content took a rigid helical con-formation, and PAMAM dendron-PLL had adefined three-dimensional structure as a wholemolecule. Helix-coil transition of PLL tail blockwas also confirmed from 1H NMR measure-ments. Figure 1(b) shows the 1H NMR spectrafor PAMAM dendron-PLL at various CD3ODcontent in CD3OD/D2O mixture. Obvious changewas observed for e-CH2 and a-CH peaks throughcoil-to-helix transition by increasing CD3OD con-tent. At random coiled conformation, the peaksof e-CH2 and a-CH were observed at 2.95 and 4.3ppm, respectively. Increasing CD3OD content,these peaks were separated and new peaks wereobserved at slightly upfield. This was due to coil-to-helix transition of PLL tail block with depro-tonation of amino groups. The helicity of PLLtail block was calculated from the peak area ra-tio of a-CH at 4.3 and 4.0 ppm, and the helicitydetermined from 1H NMR was also plotted inFigure 1(c). The helicity determined from CDand NMR showed identical profiles with thechange in MeOH content.

It has been reported that block copolymerscomposed of flexible synthetic polymer and poly-peptides including PLL were associated throughthe conformational transition,16,17 although the

NARROWLY-DISTRIBUTED SELF-ASSEMBLY 1219


degree of association was depended on variousfactors including kinds of polypeptide, molecularweight, and quality of solvent. When PAMAMdendron-PLL directly dissolved into MeOH/H2Omixtures with varying MeOH contents, the pre-cipitates were visibly observed for the mixing sol-vents over 60 vol % of MeOH. Because both ofMeOH and H2O are good solvent for PAMAM den-dron, low solubility of PAMAM dendron-PLL toMeOH/H2O with high MeOH content was attrib-uted to that of PLL tail block. However, PAMAMdendron-PLL was dissolved in MeOH/H2O mix-ture (50/50 vol %), in which PLL tail block hadlow helix content, and MeOH was dropwise addedto the solution with intensely stirring so as toincrease MeOH content to 80 vol %. The solutionexhibit bluish light scattering, indicating the for-mation of self-assemblies, and this solution wasno precipitation even over 1 month storage.

Figure 2(a) show the size distribution obtainedby DLS measurement for PAMAM dendron-PLLself-assemblies, which samples were stored for 1month. Unimodal peak was observed in DLS sizedistribution, and its mean diameter and polydis-persity index (l2/C

2) was detected to be 186 nmand 0.03, indicating that PAMAM dendron-PLLself-assemblies had narrow distribution. Lowerpolydispersity index means narrower size distri-bution, and zero of polydispersity index is com-pletely monodispersive size distribution. As well-known, block copolymers are typical examplesthat reveal a controlled balance of amphiphilicityand can assume various well-organized architec-tures including spherical, rod, vesicle, and lamel-lar structures. Also, the self-assemblies of dendri-tic-polypeptidyl block copolymers composed ofPLL dendron and poly(c-benzyl-L-glutamate)(PBLG), which might show similar self-assem-bling properties with PAMAM dendron-PLL stud-ied here, have been reported by Kim et al.9 Theyshowed that dendritic-polypeptidyl block copoly-mer could form various morphologies, althoughthey did not confirm the secondary structure ofpolypeptide block, i.e., PBLG block. PAMAM den-dron-PLL self-assemblies also had possibility totake various morphologies. PAMAM dendron-PLLin MeOH/H2O (80/20 vol %) mixture had totallydefined three-dimensional structure as describedearlier. The radius of gyration for PAMAM den-dron head block with generation 3.5 is 1.8 nm,18

and end-to-end distance of PLL tail block with po-lymerization degree of 70 is calculated to be 10.5nm, because the amino acid residue in a helicalpolypeptide expands over 1.5 A.19 On the

Figure 1. Change in CD spectra (a), 1H NMR spec-tra (b) and helicity of PLL tail block (c) with MeOHcontent for PAMAM dendron-PLL. Concentration ofpolymer for CD measurement was fixed to 0.0025 wt%, and CD measurements were carried out at 20 �Cusing quartz cell with length of 3 mm. In (c), closedsymbol, CD; open symbol, NMR. Helicity (CD) wascalculated using [h]222 as �34,000 deg cm2/decimolefor 100% helix and as 640 deg cm2/decimole for 0%helix. Helicity (NMR) was calculated from peak arearatio of a-CH at 4.3 and 4.0 ppm.

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calculation, the head-to-tail distance of PAMAMdendron-PLL used here is 12.3 nm. PAMAM den-don-PLL could not form core-shell type polymericmicelle, which is typical block copolymer self-as-sembly, with mean diameter of 190 nm, and theself-assembly of PAMAM dendron-PLL mighttake the other morphology. TEM observation wascarried out to confirm the morphology of PAMAMdendron-PLL self-assemblies, and the formationof polymersome-like self-assemblies with diame-ter of 200 nm was observed in TEM image [Fig.2(b)]. It should be noted that this polymer vesicleshad narrow size distribution in solution state asindicated from lower value of polydispersity index

in DLS maesurement. Discher et al. reported thatsonication followed by freeze-thaw plus extrusionwas required to obtain polymersomes with narrowsize distribution, which technique was common tothe preparation of liposome with narrow size dis-tribution.20 The results shown in Figure 2, spon-taneous formation of narrowly-distributed poly-mersome-like self-assemblies, was quite uniquefeature of PAMAM dendron-PLL self-assemblysystem.

Further, the interesting feature of PAMAMdendron-PLL self-assembly was observed in theobservation of time course on mean diameter andpolydispersity index in DLS measurements. Themean diameter of PAMAM dendron-PLL self-assemblies gradually increased in time course[Fig. 3(a)]. The rate of increasing diameter wasdependent on the polymer concentration. Higherpolymer concentration induced higher rate forincreasing mean diameter. After 3 weeks, meandiameter reached to 190 nm regardless of polymerconcentration, indicating PAMAM dendnron-PLLself-assembly system was a close association sys-tem, and there was no increase for further stor-age. This state was a thermodynamical equilib-rium state of PAMAM dendron-PLL self-assem-blies MeOH/H2O (80/20 vol %), and the resultsshown in Figure 2, size distribution and TEMimage, were obtained at this state. Also, thepolydispersity index decreased to less than 0.05accompanying with an increase in mean diameter[Fig. 3(b)], indicating that size distribution ofPAMAM dendron-PLL self-assemblies becamenarrow. Sendai virus and k-phage, which are nat-ural supramolecular assemblies, had polydisper-sity index of 0.08 and 0.06, respectively.21 In thecase of the comparison with these values, self-assemblies of PAMAM dendron-PLL for enoughstorage had extremely narrow size distributioncomparable with natural self-assemblies. Impor-tantly, the decrease in polydispersity index indi-cates that the association state of PAMAM den-dron-PLL spontaneously converges on one ther-modynamically stable state. Also, in this process,the size distribution kept unimodal [Fig. 3(c)].From these results, the self-assemblies ofPAMAM dendron-PLL shift to thermodynamicalequilibrium state from stable state underintensely stirring. Under stirring, there wasPAMAM dendron-PLL self-assemblies in the ther-modynamic equilibrium state with shearing force,but it is thought that the assembling state shift tothe thermodynamic equilibrium state withoutshearing force. This process was presumably

Figure 2. Size distribution (a) and TEM image (b)of the self-assemblies of the PAMAM dendron-PLL.The concentration was 2.0 mg/mL in final solution.The size distribution was obtained by DLS measure-ment, and the TEM image was negatively stainedwith phosphotungstic acid on a carbon grid.



through the fusion among the self-assemblies ofPAMAM dendron-PLL. Eisenberg et al. reportedthe thermodynamic size of the polymer vesiclesformed from poly(styrene)-poly(acrylic acid) block

copolymer (PS-PAA).22 In their study, PS-PAApolymer vesicles could show fusion and fissionbehaviors like liposomes. The fusion rate is de-pendent on the number of vesicles. As shown inFigure 3(a), the increasing rate of mean diameterdepended on the polymer concentration. Thistrend is reasonable for the fusion of PAMAM den-dron-PLL polymer vesicles.

CONCLUSIONS

The self-assembly of block copolymers theoreti-cally involves a thermodynamically stable con-verging state under equilibrium. In such funda-mental standpoint, the polymersome-like self-assemblies formed from the PAMAM dendron-PLL showed significantly important features,such as a narrow size distribution and a conver-gence of the self-assembling state. These featuresmight be based upon the defined three-dimen-sional structure of the PAMAM dendron-PLL as awhole molecule and the quality of mixing solvent.The detailed studies including the effect of molec-ular balance, i.e., molecular shape, of head-tailtype block copolymer and solvent quality will bereported elsewhere. Also, polymersomes havemany potential applications, especially the bio-medical field.23,24 The homogeneity of size is im-portant factor for nanoparticles including not onlypolymersomes, and affects the performance as bio-medical nano-devices.25 The control of the three-dimensional structure of the block copolymers haspotential importance not only from a fundamentalstandpoint, but also from the creation of novelnano-engineered materials.

This study was financially supported by a Grant-in-Aidfor Scientific Research form the Ministry of Education,Science, Sports, and Culture in Japan.

REFERENCES AND NOTES

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Figure 3. Change in mean diameter (a), polydisper-sity index (b) and size distribution (c) for the self-assemblies of the PAMAM dendron-PLL. The concen-trations were 4.0 (l), 2.0 (~) and 1.0 (n) mg/mL infinal solutions. The mean diameter and polydispersityindex were obtained from DLS measurements at20 �C. Size distribution was obtained by CONTINanalysis.

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