Eur. Phys. J. E 10, 25–35 (2003)DOI: 10.1140/epje/e2003-00006-1 THE EUROPEAN

PHYSICAL JOURNAL E

From supramolecular polymersomes to stimuli-responsivenano-capsules based on poly(diene-b-peptide) diblock copolymers

F. Checot1, S. Lecommandoux1,a, H.-A. Klok2, and Y. Gnanou1

1 Laboratoire de Chimie des Polymeres Organiques, Ecole Nationale Superieure de Chimie et Physique de Bordeaux,Universite Bordeaux 1, 16 Avenue Pey-Berland, 33607 Pessac, France

2 Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

Received 25 June 2002 and Received in final form 22 October 2002Published online: 11 March 2003 – c© EDP Sciences, Societa Italiana di Fisica, Springer-Verlag 2003

Abstract. This paper discusses the self-assembly of block copolymers into vesicular morphology. After abrief state of art of the field, a system based on an amphiphilic poly(butadiene)-b-poly(γ-L-glutamic acid)(PB-b-PGA) diblock copolymer in aqueous solution is discussed in detail. The aggregation behavior ofthis block copolymer has been investigated by means of fluorescence spectroscopy, dynamic (DLS) andstatic (SLS) light scattering as well as transmission electron microscopy (TEM). The diblock copolymerwas found to form well-defined vesicles in water. The size of these so-called polymersomes or peptosomescould be reversibly manipulated as a function of both pH and ion strength. Depending on the pH of theaqueous solution, the hydrodynamic radii of these vesicles were found to vary from 100 nm to 150 nm.By cross-linking the 1,2-vinyl double bonds present in the polybutadiene block, the ability to transforma transient supramolecular self-organized aggregate into a permanent “shape-persistent stimuli-responsivenanoparticle” has been demonstrated.

PACS. 87.16.Dg Membranes, bilayers, and vesicles – 83.80.Uv Block copolymers –05.65.+b Self-organized systems

1 Introduction and state of art

1.1 Block copolymer self-assembly

Most of today’s materials require additional modificationor processing in order to exhibit the properties that wouldmake them suitable for a particular application. As an al-ternative, routes that rely on the self-assembly of low mo-lar mass, oligomeric or polymeric building blocks attractincreasing attention [1–5]. By designing these buildingblocks in such a way that they contain all the informationnecessary to direct their self-assembly into functional ma-terials, additional processing or modification steps mightbecome superfluous. Whereas it is difficult to organize lowmolar mass organic molecules into periodic macroscopicassemblies, macromolecules can be assembled into a largevariety of ordered morphologies covering several length-scales. In this sense there is a great potential in the gen-eration and application of novel polymeric systems thatexhibit new and advanced morphological, chemical andphysical properties.

Amphiphilic diblock copolymers have been extensivelystudied, both in bulk as well as in solution. In most of

a e-mail: s.lecommandoux@enscpb.fr

these investigations, block copolymers were dissolved ei-ther in a non-selective or a selective solvent for one of theblocks [3–5]. In the first situation, the self-assembled struc-tures are formed during the solvent evaporation process,due to the incompatibility between the two blocks (Flory-Huggins χ-parameter). In the second case, supramolecularstructures occurring in solution are retained in the solidstate upon evaporation of the solvent. Depending on thevolume fraction of the blocks (which also depends on thedegree of swelling and thus the “quality of the solvent”),various morphologies can be observed (micelles, vesicles,rods...) [6,7].

Most of the block copolymers studied so far are coil-coil type diblock copolymers. Replacing one of the blocksof such a diblock copolymer by a stiff, rigid segment re-sults in a rod-coil type diblock copolymer. In this case,self-assembly is no longer solely determined by phase-separation, but is also affected by several other pro-cesses. One of the phenomena competing with phase-separation during the self-assembly of rod-coil diblockcopolymers is the aggregation of the rigid segments into(liquid-)crystalline domains. In addition, the introductionof a rigid segment results in a disparity of stiffness be-tween the constituent blocks. This stiffness asymmetryresults in an increase of the Flory-Huggins χ-parameter

26 The European Physical Journal E

in comparison with coil-coil diblock copolymers [8–10].As a consequence of the enhanced χ-parameter, phase-separation of rod-coil diblock copolymers can occur atlower molar mass than for coil-coil diblock copolymers.Rod-coil diblock oligomers, therefore, are of great inter-est since they might allow access to phase-separated mor-phologies with domain sizes that could not be attainedwith classical coil-coil diblock copolymers [8–11].

1.2 Polymeric vesicles

Besides conventional micellar core-shell structures, whichare generally obtained upon dissolving a block copolymerin a selective solvent, vesicular morphologies can also beproduced [12,13]. Vesicles have existed since the first celland play a critical role in compartmentalization functionssuch as drug transport and biological protection. Lipidsare natural amphiphiles that are well-known for their abil-ity to self-assemble into thin-walled vesicles (liposome, cellmembrane), but certain natural and synthetic polymerscan also generate vesicles in various media ranging fromorganic solvents to pure aqueous media. Such polymericarchitectures, or “polymersomes”, can be obtained fromdiblock copolymers in neat organic solvents, in pure wateror in solvent mixtures [14–17]. The Eisenberg group, forinstance, has prepared block copolymer vesicles frompolystyrene-b-poly(acrylic acid) (PS-b-PAA) in solutionsof dioxane/THF/H2O or DMF/THF/H2O [17]. Thevesicle sizes could be tuned reversibly by changing thesolvent composition, especially the water content. TEMstudies demonstrated that PS300-b-PAA44 vesicle sizesin THF/dioxane (44.4/55.6) solvent mixture vary from91 nm to 201 nm in response to increasing or decreas-ing the water content from 20% to 66.7%. Moreover,Eisenberg and co-workers have been able to correlatethe aggregation morphologies to molecular parameters(block ratios) and/or molecular environment (solventmixtures) in block copolymers. Another more recentexample is the work of the Bates group who investigatedthe formation of vesicles in water using poly(ethyleneoxide)(PEO)-based block copolymers [16]. The mainmethod used to study the morphological propertiesof poly(ethylene oxide)-b-polybutadiene (PEO-b-PB)as well as poly(ethylene oxide)-b-poly(ethylethylene)(PEO-b-PEE) copolymers was cryogenic transmissionelectron microscopy (cryo-TEM). All of the basic aggre-gation geometries were identified as a function of thePEO fraction and a transition from vesicles to micelles(low- to high-curvature structures; lamellar bilayers tospheres) was reported. To further elucidate the poly-mersome morphology, micromanipulation experimentswere performed on electroformed giant PEO40-b-PEE37

vesicles [15]. The results showed that polymersome mem-branes were almost an order of magnitude tougher andat least 10 times less permeable to water than commonphospholipid bilayers, due to the increased length andconformational freedom of polymer chains comparedto lipids. Furthermore they suggested that additionalcontrol over membrane properties can be achieved by

selective cross-linking of block copolymer hydrocar-bon chains (this particular aspect will be discussedlater). Vesicles were also obtained from triblock copoly-mers as reported by Jenekhe who used rod-coil-rodpolyphenylquinoline-b-polystyrene-b-polyphenylquinolinecopolymers [18]. Since block copolymers containingπ-conjugated polymer blocks exhibit electronic, opto-electronic, and photonic properties that can be variedwith the supramolecular morphology, one of the fun-damental objectives of his work was to understand therelationship between macromolecular architectures andthe self-assembly process on the one hand, and betweenthe morphology and the properties of the correspondingsupramolecular polymer assemblies on the other. Anotherinteresting feature of these rod-coil-rod copolymer vesiclesis their propensity to self-organize into large scale orderedmicrostructures. The authors suggested that orderedassemblies of such vesicles (highly ordered, close-packedhexagonal arrays) could be used to prepare photoniccrystals or serve as simple models of biological tissue.

Slightly different from original polymersomes, pepto-somes (polypeptide-based liposomes) systems were also re-ported with the biocompatibility function in mind [19–22].In fact polypeptide-based copolymers are known to formmicelles and are of great interest because polypeptidesare prone to hydrolysis via biodegradation, as alreadywidely exploited in the field of controlled drug release.Hence biodegradable vesicles for controlled release seemachievable using amphiphilic lipid-like blocks. Followingthis approach Cornelissen et al. reported one of thevery first examples of peptosomes [19]. Investigations onamphiphilic peptide-based diblock copolymers containinga polystyrene tail and a charged helical poly(isocyano-l-alanine-l-alanine) that self-assemble directly undersolvent-free aqueous conditions were carried out. Undermildly acidic conditions vesicular morphologies mixedwith rod-shaped and chiral architectures were observed.Very recently two groups independently reported the self-organization of diene-peptide based diblock copolymersin dilute aqueous solutions [21,22]. Polybutadiene-b-poly(γ-L-glutamic acid) (PB-b-PGA) amphiphilic diblock self-assemble directly in water giving rise to either micellar orvesicular morphologies, depending on the chemical com-position of the sample. The size and the shape of suchcopolymer aggregates were assessed by means of staticand dynamic light scattering techniques, freeze-fractureTEM and small angle neutron scattering with a view ofgathering the complete physico-chemical characteristics ofthese structures.

1.3 Stability enhancement

As already mentioned, low molar mass lipids havebeen known for more than 30 years for their ability toself-assemble into spherically closed bilayers (vesicles,liposomes). However, their overall stability is somehowlimited, even though they can be kinetically trapped afteraggregation upon polymerization of the reactive functionscarried by the lipid part or through surface grafting of

F. Checot et al.: From supramolecular polymersomes to stimuli-responsive nano-capsules 27

polymers. Indeed this insufficient stability induces, for in-stance, rapid clearance of vesicles after their intravascularadministration. In that sense polymersomes or pepto-somes are potentially very effective alternatives owing tothe enhanced stability of the self-assembled structure.However most aggregates are only stable within a certainrange of concentration, temperature or pH. A suitableapproach to improve the stability of self-organized struc-tures is to cross-link one of their blocks [23–28]. Applyingthis principle to polymersomes, only very few studieshave been reported. One approach has been developedby Meier et al. who used poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline)(PMOXA-b-PDMS-b-PMOXA) triblock copolymers car-rying polymerizable groups at both chain ends [29]. Afterformation of vesicular morphologies in dilute aqueoussolutions, the methacrylate end groups of the triblockcopolymer were polymerized within the aggregate usingan UV-induced free radical process. Cross-linking poly-merization did not lead to morphological modifications ofthese vesicles as evidenced by light scattering techniquesand TEM. Moreover authors reported that the resulting“shape-persistent nano-capsules” could be isolated fromthe aqueous solution and preserved their integrity. Morerecently Discher et al. reported water soluble cross-linkedpolymersome membranes based on PEO-b-PB diblockcopolymers [30]. Compared to common lipid vesiclesor the systems described by Meier et al., the use ofpolybutadiene as the hydrophobic cross-linkable blockoffers many more reactive groups and consequently makethis system suitable for the generation of extremely stablesupramolecular objects. Indeed cross-linked PEO-b-PBgiant vesicles were proved to be stable in organic solvent(as well as in water) and could also be dehydrated andrehydrated.

1.4 Toward stimuli-responsive nano-capsules

During the past few years increasing attention has beenpaid to water-soluble supramolecular organization in viewof potential applications such as coatings, drug deliverysystems, nanoparticles or nanoreactors. An important is-sue in order to make these self-assembled systems usefulfor such practical applications is their ability to respond toexternal stimuli such as temperature and/or pH [31–33].So far, in most of the systems that have been described, re-sponsiveness was solely based on electrostatic interactionsbetween the charged polymer blocks. Consequently, thisprocess is not completely reversible due to salt formationwith each pH variation cycle, which screens the electro-static interaction. Conversely, peptide-based systems ex-hibit this endless responsive property through order-orderconformation transitions [22].

To our knowledge the preparation of copolymeric self-assembled hollow spheres which exhibit at the same timeshape-persistent and stimuli-responsive capability is un-precedented. In this contribution, the way to obtainsuch “smart” materials using a “bottom-up” route is de-scribed. We report the formation and the characterization

of supramolecular structures that were obtained by self-organization of polybutadiene-b-poly(γ-L-glutamic acid)PB-b-PGA peptide-based rod-coil diblock copolymers inwater. The aggregation properties of this block copoly-mer as well as the size and shape of the supramolecu-lar structures were investigated by means of fluorescencespectroscopy, static and dynamic light scattering (SLSand DLS), UV-circular dichroism spectroscopy (CD), aswell as transmission electron microscopy (TEM). Ouraim was to exploit the pH-sensitivity of the peptide sec-ondary structure to induce order-order transitions in thesupramolecular organization of the diblock copolymers. Inaddition, the use of polybutadiene as a hydrophobic blockallowed us to “freeze” and capture upon UV curing thesupramolecular structure into “shape-persistent stimuli-responsive nanomaterials”.

2 Experimental section

2.1 Materials

1,3-Butadiene was successively stirred at −35 ◦C and cry-odistilled from sec-BuLi. Tetrahydrofuran (THF) was re-fluxed over CaH2, distilled, stored over Na/Benzophenone,cryodistilled over sodium mirror, and cryodistilled inhigh vacuum prior to use. 1-(3-Chloropropyl)-2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentane was prepared ac-cording to a literature procedure [34].

Ethylacetate (EtAc) was dried over molecular sieves(4 A). N,N’-Dimethylformamide (DMF) was distilled fromCaH2 under reduced pressure and subsequently storedover molecular sieves (4 A) under an argon atmosphere.γ-Benzyl-L-glutamate N-carboxyanhydride (Bn-GluNCA) was prepared according to a literature proce-dure [35]. All other solvents and reagents were purchasedfrom commercial suppliers and were used as received.

2.2 Physical and analytical methods

1H-NMR spectra were recorded at room-temperature on aBruker Avance 250 spectrometer using the residual protonresonance of the deuterated solvent as the internal stan-dard. GPC-analysis was performed with a set-up consist-ing of a Waters 510 pump and a series of three PSS-SDVcolumns (300 × 8 mm) with pore-sizes of 500 A, 105 Aand 106 A, respectively. THF for the polybutadiene pre-cursor or DMF for the polybutadiene-b-poly(γ-L-benzylglutamate) diblock copolymer were used as the mobilephase and the elution of the samples was monitored us-ing simultaneous UV- and RI-detection. The polybutadi-ene precursor that was investigated by GPC was a samplethat was withdrawn from the reaction mixture before end-functionalization. Elution times were converted to molarmasses using a calibration curve that was constructedwith narrowly disperse polystyrene standards. Fluores-cence spectra were recorded on a SAFAS Spectrofluorom-eter flx spectrometer. CD experiments were carried out ona JOBIN YVON CD6 Spex spectrometer (184–900 nm).

28 The European Physical Journal E

Scheme 1. Synthesis of the PB40-b-PGA100 diblock copolymer.

Static (SLS) and dynamic (DLS) light scattering measure-ments were performed on a ALV5000 goniometer equippedwith a ALV5000/E Multiple Tau digital Realtime corre-lator. The hydrodynamic radius (RH) and polydispersityindex (PDI) of the aggregates were obtained by a cumu-lant and CONTIN analysis of the experimental correla-tion functions. TEM pictures were recorded on a JEOLJEM100S microscope working at 100 KV. Samples wereprepared by spraying a 1 g l−1 solution of the block copoly-mer onto a TEM grid. UV-curing experiments were per-formed using a high pressure mercury lamp (450 W) andirradiating samples containing 9% (volume) (Darocure4265 from Ciba) photoinitiator for one hour.

2.3 Preparation of the polybutadiene-b-poly(glutamicacid) PB-b-PGA diblocks

All polymerizations were performed in flame-dried glass-ware employing standard techniques. Butadiene wasoligomerized at −78 ◦C in THF solution using sec-BuLi as the initiator. The oligomerization was quenchedby the addition of a five-fold molar excess of a THF-solution of 1-(3-chloropropyl)-2,2,5,5-tetramethyl-1-aza-2,5,-disilacyclopentane. After stirring at room tempera-ture overnight, the protecting group was removed by theaddition of a 0.1 M HCl (aq.) solution. The solution wasstirred at room-temperature and then evaporated to dry-ness. The residue was dissolved in CH2Cl2 and repeat-edly washed with a saturated NaHCO3-solution (aq.).The organic phase was separated, dried over MgSO4, fil-tered and evaporated to dryness. TLC-analysis (SiO2:CH2Cl2/MeOH 95/5 (v/v)) showed that in addition tothe desired compound, the crude product also contained

some unfunctionalized polybutadiene. These impuritieswere removed by means of flash-chromatography (SiO2).First the column was eluted with dichloromethane to sepa-rate the unfunctionalized oligobutadiene. Then the eluentwas changed to a mixture CH2Cl2/MeOH 95/5 (v/v) andthe desired ω-primary amine end-functionalized polybu-tadiene was collected. Evaporation of the solvent andvacuum-drying at room temperature finally afforded theprimary amine end-functionalized oligobutadiene in 80%yield. Then, a Schlenk-flask fitted with a stir-bar and adrying-tube was charged with the appropriate amount ofBn-Glu NCA in dry DMF (∼0.2 g ml−1). A THF-solutioncontaining the calculated amount of (PB)-NH2 was added,and the reaction-mixture (DMF/THF 1:1 v/v) stirred atroom temperature for 5 days. The PB-b-PBLG diblockoligomers were precipitated in diethylether and in water,filtered and vacuum-dried. Finally, a THF solution of PB-b-PBLG was prepared. A concentrated KOH solution wasadded (1 eq. per ester function) and the reaction mixturestirred at room temperature for 15 hours. THF was evap-orated from the resulting mixture. After neutralization ofthe solution and dialysis against ultra-pure water the am-phiphilic PB-b-PGA diblock copolymer was recovered byfreeze-drying process.

3 Results and discussion

3.1 Synthesis

The synthesis (Scheme 1) of the polybutadiene-b-poly(γ-l-glutamic acid) (PB-b-PGA) copolymer starts with the an-ionic polymerization of butadiene in THF at −78 ◦C usingsec-butyllithium (sec-BuLi) as initiator. Upon quenching

F. Checot et al.: From supramolecular polymersomes to stimuli-responsive nano-capsules 29

Fig. 1. 1H NMR spectrum of the PB40-b-PBLG100 diblockcopolymer in CDCl3.

oligobutadienyllithium with 1-(3-chloropropyl)-2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentane and acid aque-ous workup, a ω-amino oligobutadiene could be ob-tained. The number average degree of polymerization ofthis oligobutadiene was 40 as determined by gel per-meation chromatography (GPC) and vapor phase os-mometry. The primary amine end-functionalized oligomerwas then used to initiate the ring-opening polymeriza-tion of γ-benzyl-L-glutamate N-carboxyanhydride (Bn-GluNCA) in DMF/THF (1:1 v/v) mixture at room-temperature [36]. The length of the γ-benzyl-L-glutamatesegment could be controlled through the molar ratio ofBn-GluNCA to ω-amino oligobutadiene initiator leadingto the polybutadiene40-b-poly(γ-benzyl-L-glutamate)100block copolymer. Finally, after removal of the benzylestergroups by hydrolysis, the PB40-b-PGA100 amphiphilic di-block copolymer was obtained.

This block copolymer was analyzed by means of 1HNMR, 13C NMR and GPC. The 1H-NMR spectrum ofPB40-b-PBLG100 in CDCl3 is shown in Figure 1 and wasfound to be in accord with the expected chemical struc-ture of the copolymer. In order to determine the exactcomposition of the block copolymers, the respective in-tensity of signals due to the polybutadiene and the pep-tide blocks was compared: δ/ppm = 4.7−5.7 (6 + 9 + 10,= CH− += CH2) and 4.7−5.1 (17, −CH2−O).

3.2 Preparation and stability of the self-assembledaggregates

In a first series of experiments, the critical aggregationconcentration (c.a.c.), which is a measure of the stability

and of the physical properties of the aggregate, was deter-mined using pyrene as a fluorescent probe [37–40]. Parti-tioning of pyrene into the hydrophobic micro-domain, i.e.the polybutadiene one, causes changes in its photophysi-cal properties [41,42]. Among others, the change in probeenvironment is reflected in the absorption and emissionfluorescence spectra. By examination of a series of solu-tions with different copolymer concentrations but a con-stant probe concentration, the onset of aggregation (c.a.c.)of the copolymer in water could be determined.

To determine the c.a.c., solutions of PB40-b-PGA100

with concentrations ranging from 10−7 to 10−5 mol l−1

were prepared. The pyrene concentration in these solu-tions was kept constant at 6 × 10−7 mol l−1. Both ex-citation and emission spectra were recorded. The excita-tion spectra were obtained by illuminating with light from300 nm to 360 nm and recording the fluorescent light in-tensity at 371 nm. A red shift was observed in the excita-tion spectrum upon addition of the diblock copolymer.Plotting the position of the maximum excitation band(λmax) as a function of the PB40-b-PGA100 concentration,indicates a c.a.c. of 3 × 10−6 mol l−1.

In addition to the observed shift in the pyrene exci-tation spectrum, the vibrational structure of the pyrenemonomer emission also changes upon formation of a poly-meric micelle, due to changes in the local polarity (Hameffect [42]). Figure 2a, presenting the emission intensityat two diblock copolymer concentrations, shows this effect(intensity collected from 360 nm to 410 nm for an exci-tation wavelength of 334 nm). Plotting the II/IIII bandintensity ratio of the pyrene monomer emission versus thelogarithm of the PB40-b-PGA100 concentration indicatesa c.a.c. of 3×10−6 mol l−1 (Fig. 2b). This value is in goodagreement with the c.a.c. determined from the shift in theexcitation spectrum. This low c.a.c value corresponds to46 mg l−1 and indicates that the PB40-b-PGA100 diblockcopolymer forms stable aggregates in water.

3.3 Characterization of the supramolecularorganization

This PB40-b-PGA100 sample could be dissolved in waterupon addition of one eq. NaOH. According to the time de-pendence of the hydrodynamic radius (RH) as determinedby dynamic light scattering (DLS), perfect equilibrium isestablished after about 10 days of vigorous stirring. To fur-ther elucidate the supramolecular organization of PB40-b-PGA100, DLS experiments were performed on aque-ous solutions of different concentrations (from 0.31 g l−1

to 2 g l−1), at various angles (from 40◦ to 140◦), and withor without added salt (NaCl). The analysis of the normal-ized intensity autocorrelation functions C(q, t), where qis the scattering vector (q = [4πn0 sin(θ/2)]/γ0) and tthe time, was carried out following the method of cumu-lants [43], fitted with monoexponential decay functions.Once the first cumulant Γ is determined, the concentra-tion and angular dependence can be expressed as

Γ/q2 = DZ(1 + kDc + . . . )(1 + CR2

gq2 + . . .

)

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(a)

(b)

Fig. 2. (a) Fluorescence emission spectra of pyrene ([pyrene] =6 × 10−7 mol l−1) in the presence of PB40-b-PGA100 at twoconcentrations (10−5 M and 10−7 M). (b) II/IIII intensity ratiosfrom the pyrene emission spectra versus the logarithm of thePB40-b-PGA100 concentration.

where DZ is the z-average diffusion coefficient, C is aparameter that is characteristic of the molecular archi-tecture, kD is the effective interaction parameter. Fromthe diffusion coefficient, the z-average hydrodynamic ra-dius RH can be calculated from the Stokes-Einsteinequation

RH = kT/(6πηDZ)

(a)

(b)

Fig. 3. (a) Autocorrelation functions and CONTIN analysisof PB40-b-PGA100 vesicles in water at pH = 11 in “salt free”conditions (c = 1.8 g l−1) at two different scattering angles;open triangles: C(q, t) at 90◦; open squares: C(q, t) at 130◦; fullline: CONTIN at 90◦; dashed line: CONTIN at 130◦. (b) De-pendence of the first cumulant frequency (Γ ) with the squaremagnitude of the scattering vector (q2) in these conditions.

where η is the solvent viscosity. Dynamic light scatteringdata were also analyzed using the CONTIN method [44]to determine the distribution of hydrodynamic size.

As a representative example, the autocorrelation func-tions C(q, t) and the particle size distribution obtained byCONTIN analysis of DLS data are shown in Figure 3afor the PB40-b-PGA100 sample at pH = 11 without addedsalt and at two angles (90◦ and 130◦). This graph showsa unimodal and narrow distribution, independent of thescattering angle. The hydrodynamic radii of the aggre-gates were determined at ten different scattering anglesand analyzed using the cumulant method by extrapolationto 0◦ scattering angle. Figure 3b presents the evolution of

F. Checot et al.: From supramolecular polymersomes to stimuli-responsive nano-capsules 31

(a)

(b)

Fig. 4. (a) Autocorrelation functions and CONTIN analysis ofPB40-b-PGA100 vesicles in the presence of 1 M NaCl added saltin water at pH = 11 (c = 1.8 g l−1) at two different scatteringangles; open triangles: C(q, t) at 90◦; open squares: C(q, t) at130◦; full line: CONTIN at 90◦; dashed line: CONTIN at 130◦.(b) Dependence of the first cumulant frequency (Γ ) with thesquare magnitude of the scattering vector (q2) in these condi-tions.

Γ versus q2 for a PB40-b-PGA100 solution of 1.8 g l−1. Fit-ting these data gives a linear correlation with r2 = 0.98,indicating that only translational diffusion occurs. Fromthe Γ/q2 versus q2 plot, the intercept gives an apparentz-average diffusion coefficient DZ at this concentration.The consistent hydrodynamic radius of the correspondingspecies in water was found to be RH = 128 nm for so-lutions at pH = 11 without salt (by extrapolating RH(c)to c → 0).

Figure 4a presents the autocorrelation func-tions C(q, t) and the particle size distribution obtainedby CONTIN on PB40-b-PGA100 in the presence of 1 M

NaCl at pH = 11 and for two angles (90◦ and 130◦). Evenif the time scale is slightly different, only one populationis present in the solution with a very narrow size dis-tribution. The representation of Γ versus q2 also allowsthe determination of the hydrodynamic radius of theaggregate under these conditions (Fig. 4b). Extrapolationto zero concentration gives RH = 114 nm. One can clearlysee the so-called “screening effect” of the charges on thepolyelectrolyte copolymer PB40-b-PGA100 by adding salt(decrease in RH from 128 nm to 114 nm). This salt effecton polyelectrolytes is known to modify the hydrodynamicradius, but also the solution and aggregation propertiesof the block copolymers [45].

In addition to DLS, static light scattering experiments(SLS) were performed in water under the same conditions.The analysis of the scattering intensities using a Zimmplot yields a mass average molar mass, a z-average ra-dius of gyration and a second virial coefficient (of theosmotic pressure) [46]. However due to the well-knownproblem of static light scattering analysis of block copoly-mers these quantities have to be treated only as apparentvalues. The interpretation is further complicated by thecharged and amphiphilic character of the block copoly-mer. However, one may assume that with added salt, whencharges are screened, we actually measure the z-averageradius of gyration which should at least be close to thetrue radius of gyration Rg. Under these assumptions, SLSyields a Rg = 110 nm. Comparing this value to the hy-drodynamic radius RH also determined with added salt(RH = 114 nm), a ratio Rg/RH = 0.98 (the so-calledρ-parameter) is found, which is far different from the val-ues expected for a uniform sphere (0.774) and a polymercoil (1.50), but is very close to that predicted for a vesic-ular architecture (1.0) [47,48].

In order to confirm this self-organized aggregate mor-phology, we performed transmission electron microscopy(TEM). A sample of PB40-b-PGA100 (c = 1 g l−1) wassprayed on a TEM grid (Fig. 5) without any staining. Atlow magnification (Fig. 5a), spherical particles could beseen with a very low dispersion in size. Upon increasingthe magnification (Fig. 5b), we observed the characteristicresponse arising for a vesicle: a dark corona surroundinga light core. The apparent radius (RTEM) of ∼125 nm ofthe aggregates thus corresponds to the previous resultsobtained by DLS and SLS.

3.4 Stimuli-responsive effect

The effect of pH-induced conformational changes on thesize of the PB-b-PGA aggregates was investigated byUV-circular dichroism (CD) and DLS measurements. CDis a powerful tool to study peptide secondary struc-tures [49,50]. CD spectra for PB40-b-PGA100 in water atthree different pH values are presented in Figure 6. Sincethe CD spectra were recorded from solutions with blockcopolymer concentrations well above the c.a.c., changes inthe CD-spectra indicate changes in the secondary struc-ture of the peptide chains within the aggregates.

32 The European Physical Journal E

(a)

(b)

Fig. 5. TEM images of the PB40-b-PGA100 vesicles sprayedfrom a 1 g l−1 concentration solution in water (pH = 11);(a) low magnification (b) high magnification.

Fig. 6. CD spectra of PB40-b-PGA100 vesicles in aqueous so-lution of different pH.

Fig. 7. Hydrodynamic radius (RH) of PB40-b-PGA100 vesiclesin water measured by DLS (90◦) in “salt free” conditions (c =1.25 g l−1) as a function of pH.

The CD spectrum of PB40-b-PGA100 at pH = 4.5shows negative minima at 221 nm and 208 nm and a posi-tive maximum at 190 nm indicative of an α-helical confor-mation. In the ionized form, at pH = 11.5 PB40-b-PGA100

shows a doubly inflected CD curve with a small positivemaximum at 218 nm and a large minimum at 197 nm.This is characteristic of a random coil conformation. TheCD spectra of the PB-b-PGA diblock copolymers are thusperfectly similar to those obtained for a homopolypeptidePGA [49], showing the reversibility of the rod-coil to coil-coil transition in the polymersome made from our diblockcopolymer.

Figure 7 shows the pH-dependence of the hydrody-namic radius RH measured by DLS (90◦) on PB40-b-PGA100 (c = 1.25 g l−1) in the absence of any salt. Goingfrom basic to acidic pH values, first a plateau in the RH

values is observed up to about pH = 7 (RH ≈ 150 nm),followed by a strong decreases from pH = 7 to pH = 3(RH ≈ 100 nm). This critical pH value around pH = 7when the size of the polymersome is changing correspondsto the pH value where the peptide undergoes a transitionfrom an α-helix to a random coil conformation. In otherwords, this response in the aggregate size can be associ-ated to conformational changes in the peptide segment.

Other systems based on polyelectrolytes have alsoshown this ability to respond to pH variations [31–33].However, when adding salt, these systems do generallynot respond any more to this external stimulus due toa screening of the electrostatic character. Figure 8 bothshows the salt effect (ionic strength effect) in basic wa-ter and the pH effect. First, when looking at the uppercurve (empty squares), one can observe that the elec-trostatic interactions are screened when adding about0.5 mol l−1 NaCl at pH = 11.5. This results in a decreaseof the hydrodynamic radius RH from 152 nm to 130 nm.

F. Checot et al.: From supramolecular polymersomes to stimuli-responsive nano-capsules 33

Fig. 8. Hydrodynamic radius (RH) of PB40-b-PGA100 vesiclesin water measured by DLS (90◦) as a function of ionic strength(NaCl salt concentration) at pH = 11.5 (empty square) and pHwith 1 M NaCl (full circle).

The lower curve (full circle) shows the evolution of RH inPB40-b-PGA100 solutions with 1 mol l−1 NaCl (when theelectrostatic interactions are completely screened) as afunction of pH. We can still observe in that situation a de-crease from RH = 130 nm at basic pH value (11.5) to RH =102 nm at acidic pH value (3.5). Hence, one can clearlysee that when charges on PGA blocks are entirely screenedby added salt, the polymersome is still able to change insize due to pH variations. This response to pH is fullyreversible because it is related to the coil-to-helix transi-tion as evidenced by CD (Fig. 6). In conclusion, we cansay that our polybutadiene40-b-poly(γ-L-glutamic acid)100water soluble vesicles are “endless stimuli-responsiveaggregates”.

Figure 9 shows a schematic representation of the poly-mersomes formed by the PB40-b-PGA100 diblock copoly-mer; it can be viewed as a triple layered membrane consist-ing of an inner interdigitated polybutadiene layer betweentwo outer poly(γ-L-glutamic acid) ones.

3.5 Covalent capture

Finally, using the polybutadiene block as a cross-linkableblock, we tried to “freeze” the morphology adopted by thesystem and obtain “shape-persistent stimuli-responsivenano-capsules” from a supramolecular self-assembled ag-gregate. PB40-b-PGA100 vesicle solutions containing 9%initiator were UV-irradiated for one hour in a 2 ml, 10 ◦Ccooled, quartz cell. These first experiments were followed

Fig. 9. Schematic representation of the model proposed for thepolymersomes formed by self-organization of PB40-b-PGA100

in water.

Fig. 10. Hydrodynamic radius (RH) of PB40-b-PGA100

vesicles in water measured by DLS (90◦) as a function of thevolume fraction of THF added (%THF). Open circles: PB40-b-PGA100 vesicles without any cross-linking agent; open trian-gles: PB40-b-PGA100 vesicles before UV-curing; open squares:PB40-b-PGA100 vesicles after one hour UV-curing.

by DLS measurements which revealed a slight shrinkageof the vesicular size after UV curing, a phenomenon thattypically accompanies the cross-linking of the polybutadi-ene block.

These cross-linked vesicles could be swollen by addi-tion of organic solvents (THF, Toluene, DMF) as shownin Figure 10. Different information can be extracted fromthis plot. First, upon progressive addition of THF on na-tive vesicles (open circles), one can observe the swelling ofthe entire aggregate. Hydrodynamic radius RH vary fromaround 125 nm in pure water to 260 nm for 30% addedTHF, which corresponds to a swelling parameter approxi-mately equal to 2.1 (a swelling rate of 108%). The swellingparameter was calculated as the ratio of vesicle radii intheir swollen state, at a particular THF fraction, divided

34 The European Physical Journal E

by the radii in pure water. This first observation confirms(see Sect. 3.2) and points out the fact that we are ableto encapsulate – in the hydrophobic region of the vesicle– organic molecules such as simple solvents or fluorescentorganic molecules (pyrene for example). The second im-portant information that can be extracted from Figure 10is related to the cross-linking of the vesicles through reac-tions between the pendent vinyl double bonds of polybu-tadiene blocks. After addition of 9% in volume of UV-initiator, which is water insoluble, a subsequent swellingof the vesicle to a hydrodynamic radius RH

∼= 165 nm wasobserved as expected. Half of the sample was then UV-irradiated and the remaining part was removed from anylight source, until DLS experiments were carried out. Ineach sample (UV exposed or not) the THF fraction was in-creased up to 30% for comparison with native swollen vesi-cles. One can clearly observe that non-UV-exposed vesi-cles have a swelling behavior very similar to that of nativevesicles with a swelling parameter very close to 2.1. Incontrast, 1 hour UV-cured vesicles (open squares) exhib-ited a lower swelling parameter: for 30% added THF thisparameter is 1.7. As expected, cross-linking of 1,2-vinylbonds induced the formation of a covalent polybutadienenetwork within the hydrophobic compartment of the vesi-cles, thus reducing their swelling ability. Hence, this differ-ential swelling behavior whether vesicles were UV-exposedor not, confirmed that cross-linking indeed occurred.

4 Conclusions and perspectives

The peptide-based diblock copolymer presented in thiscontribution forms well-defined vesicular morphologies af-ter direct dissolution into water. Dissolution via dialy-sis and under different pH conditions, which may leadto other morphologies, is currently under investigation.The size of the aggregate can be manipulated reversiblyas a function of both pH and ionic strength. Even at highNaCl concentrations, where all the charges are effectivelyscreened, pH-induced changes in the polypeptide sec-ondary structure can be used to reversibly vary the aggre-gate dimensions. Compared to other polyelectrolyte-basedblock copolymers, this PB40-b-PGA100 diblock copoly-mer exhibits the unique feature that the polypeptideblock is capable of folding into a compact and well-defined secondary structure. Using the 1,2-vinyl doublebonds present in the polybutadiene block, it is possibleto covalently “capture” the morphology of the systemand transform a transient supramolecular self-organizedaggregate into a permanent “shape-persistent stimuli-responsive nanoparticle”. Such nanoparticles, nanocap-sules or polymersomes may be suitable for a number ofapplications including the encapsulation and/or the re-lease of hydrophilic as well as hydrophobic active speciesor their use as sensor nano-devices. As liposomes loadedwith anticancer agents have demonstrated clinical effec-tiveness [51], polymer vesicles may well be regarded asencapsulators of the future. Furthermore, most of thebiomimetic systems are based on bilayer-forming low mo-lar mass lipids. Hence polymeric systems are also very

useful in the understanding of natural processes (clear-ance mechanism from the blood circulation for instance)or for applications like vectorisation, though the liposomesformed are generally not stable enough [52]. In fact, com-pared to lipids, most polymer membranes are hyperthick,and can thereby achieve greater stability than any naturallipid membrane. Consequently, polymersomes and pepto-somes are reported to be much more robust and suitablefor this kind of applications, and it is already clear thatseveral biological membrane process can be actually repro-duced by polymer-based vesicles, as for instance biocom-patibility, encapsulation or protein integration [53,54].

Finally, significant efforts are currently directed to-wards the development of delivery methods compatiblewith noninvasive routes of administration such as pul-monary, nasal, oral, ocular or buccal formulations. Inaddition, if one can control the process of loading andrelease of the drug, which is a major issue for pharma-ceutical scientists, “ideal” drug delivery system capableof transporting in the body the required amount of ac-tive agent at the desired time and site of action would bereached. “Intelligent” or “smart” nanocapsules appear asa promising option in this regard, their smartness referringto their ability to receive, transmit a stimulus and respondthrough a useful effect. Our “shape-persistent stimuli-responsive nanoparticles” can be viewed smart or intelli-gent in the sense that they are able to perceive a stimulusand respond to it by exhibiting changes in their physicaland/or chemical behavior: this may be used to release en-trapped drug in a controlled manner. Moreover, because oftheir potential applications in therapeutics, product scale-up and cost considerations, internal stimuli-respondingsystems (responsive to pH, ionic strength, glucose) aremore interesting compared to those governed by externalstimuli (electric or magnetic fields, light radiations); andamong this category, pH-responsive systems attract hugeattention. Indeed, variations in pH are known to occurat several body sites, such as the gastro-intestinal tractor blood vessels, and these can provide a suitable basefor pH-responsive drug release. More precisely, one cananticipate that nanoparticles responding to pH changeswill function as ideal carriers for protein and drug de-livery considering the large change in pH from stomachto intestine. Hence, the introduction and exploitation ofsuch “shape-persistent polymeric stimuli-responsive nano-materials” may meet some of the therapeutic needs, butis also certainly very attractive in cosmetics, paints orlubricants...

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