α‑Acetal, ω‑Alkyne Poly(ethylene oxide) as a Versatile Building Blockfor the Synthesis of Glycoconjugated Graft-Copolymers Suited forTargeted Drug DeliveryHelene Freichels,†,# David Alaimo,† Rachel Auzely-Velty,‡ and Christine Jero me*,†

†Center for Education and Research on Macromolcules, University of Liege, B6a Sart-Tilman, 4000 Liege, Belgium‡Centre de Recherches sur les Macromolecules Vegetales (CERMAV-CNRS), BP53, 38041 Grenoble cedex 9, France (affiliated withUniversite Joseph Fourier, and member of the Institut de Chimie Moleculaire de Grenoble)

ABSTRACT: α-Acetal, ω-alkyne poly(ethylene oxide) wassynthesized as building block of glycoconjugated poly(ε-caprolactone)-graf t-poly(ethylene oxide) (PCL-g-PEO) co-polymers. The alkyne group is indeed instrumental for thePEGylation of a poly(α-azido-ε-caprolactone-co-ε-caprolac-tone) copolymer by the Huisgen’s 1,3 dipolar cycloaddition,i.e., a click reaction. Moreover, deprotection of the acetal end-group of the hydrophilic PEO grafts followed by reductiveamination of the accordingly formed aldehyde with anaminated sugar is a valuable strategy of glycoconjugation ofthe graft copolymer, whose micelles are then potential. A model molecule (fluoresceinamine) was first considered in order tooptimize the experimental conditions for the reductive amination. These conditions were then extended to the decoration of thegraft copolymer micelles with mannose, which is a targeting agent of dendritic cells and macrophages. The bioavailability of thesugar units at the surface of micelles was investigated by surface plasmon resonance (SPR). The same question was addressed tonanoparticles stabilized by the graft copolymer. Enzyme linked lectin assay (ELLA) confirmed the availability of mannose at thenanoparticle surface.

■ INTRODUCTION

Biocompatible amphiphilic copolymers with well-definedarchitectures are one of the challenging and rewarding areasin polymer science, because they are able to form micelles inwater with potential as drug delivery systems.1−4 Indeed, thehydrophobic core of core−shell micelles can serve as a reservoirfor hydrophobic drugs, which are accordingly protected fromcontact with the aqueous environment.5,6 In this field, aliphaticpolyesters, such as poly(ε-caprolactone) (PCL) and polylactide(PLA), are interesting hydrophobic candidates as a result oftheir degradability and biocompatibility. Moreover, poly-(ethylene oxide) (PEO) is one of the most desired hydrophiliccomponents because of nontoxicity, biocompatibility, lack ofimmunogenicity,7 and protein repellency that confers stealthi-ness to micelles.8−10

Last but not least, these amphiphilic copolymers can also beused as interfacial agents for the steric stabilization of polymericnanoparticles.11,12 In this context, previous works haveemphasized the higher efficiency of graft copolymers overdiblock copolymers of comparable hydrophilic−lipophilicbalance (HLB) in stabilizing PLA nanoparticles and in repellingproteins.13 Among the strategies reported in the scientificliterature about the synthesis of amphiphilic PCL(PLA)-g-PEOcopolymers, the grafting “onto” method has been widelystudied, i.e., the grafting of functional PEO onto reactivealiphatic polyesters. Huisgens 1,3-dipolar cycloaddition,8,14,15

atom transfer radical addition,16 Michael addition,17 and oxime

formation18,19 are the main grafting reactions used in thisrespect. Nevertheless, the lack of reactivity of the free α-end-group of the PEO grafts prevents any pilot molecule from beingattached to these copolymers, which are then useless in activetargeting. It is thus highly desirable to prepare nanocarriersdecorated with ligands able to bind to receptor-expressing cellsand to form ligand−particle complexes prone to internalizationinto the targeted cells by receptor-mediated endocytosis. Thetherapeutic activity of the entrapped drugs would thus beenhanced by the proper destination of the nanocarrier.20

In order to build up this type of intelligent nanocarrier with agraft architecture, α,ω-heterotelechelic PEO must be prepared.Tanigushi et al. reported a first strategy of grafting “onto” basedon PEO end-capped with an α-aminooxy for further graftingonto a ketone-containing PCL and with an ω-hydroxyl groupsuited to the attachment of targeting molecules.21,22 Thisheterotelechelic PEO was prepared in five steps, starting withan α,ω-hydroxyl PEO. One of the hydroxyl groups wasprotected by tetrahydropyranyl (THP), and the secondhydroxyl group was tosylated before being reacted with N-hydroxyphthalimide. Finally, the protecting end-group wasremoved at low pH and the hydroxyl group was converted intoan azide. However, the multistep synthesis of the difunctional

Received: December 19, 2011Revised: July 3, 2012

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PEO and the low yield are severe limitations for the preparationof the envisaged graft copolymers. There is thus a need for amuch more straightforward synthesis of PCL-g-PEO copoly-mers.The purpose of this work is to synthesize a PCL-graf t-α-

acetal PEO copolymer in three steps. The first two steps weredevoted to the synthesis of a heterotelechelic PEO: indeed, theanionic ring-opening polymerization of ethylene oxide by analkoxide flanked by an acetal group. The ω-hydroxyl end-groupof PEO was then converted into an alkyne (Scheme 1) reactivetoward the azide pendant groups of PCL by the Huisgens 1,3cycloaddition. This “click” reaction has the advantage of being

tolerant to many functional groups and being carried out undermild conditions that may prevent PCL from beingdegraded23−25 and was used by many research groups toprepare polymers, dendrimers, and hydrogels for drug deliveryapplications.26 Moreover, Suksiriworapong et al. show the lackof toxicity for NPs decorated with nicotinic acid and p-aminobenzoic acid by click reaction even if copper was usedduring the synthesis.27 Several copolymers were accordinglyprepared, in which the number and length of the PEO graftswere changed in such a manner that the HLB was maintainedclose to 5. This low HLB close to 5 was chosen, on purpose for

Scheme 1. Strategy for the Preparation of Poly(εCL)-graf t-(α-acetal PEO) Copolymers

Scheme 2. Decoration of Micelles by Reductive Amination of an Amino-Dye (when R = fluorescein) or Amino-Sugar (when R =mannose) with Reactive Poly[(εCL)-graf t-(α-aldehyde PEO)] Micelles

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the critical association concentration (cac) to be low, which is arequirement for biomedical application.28

The acetal end-group of the PEO grafts was hydrolyzed andthe released aldehyde was reacted with an amino derivative byreductive amination. This coupling reaction was selectedbecause it can occur in water, a good solvent for sugars, usedas pilot molecules in drug targeting, and it is tolerant towardchemical functions of sugars, which does not impose additionalprotection/deprotection reactions. As illustrated in Scheme 2, aspecial effort will be devoted to the direct decoration of themicelles of the amphiphilic copolymer with an amine ofinterest. Fluoresceinamine (Scheme 3a) will be first studied as a

model compound. This hydrosoluble fluorescent dye has theadditional advantage of being easily localized by fluorescence,which allows learning about the fate of labeled micelles in cellsor tissues.29 Finally, mannose will be used as a potentialtargeting agent (Scheme 3b) of dendritic cells30 and macro-phages.31

The bioavailability of mannose at the surface of micelles willbe studied by surface plasmon resonance (SPR). Indeed, thebinding of mannosylated micelles to lectin-modified sensorscan be directly probed by SPR. Two kinds of lectins(Concanavalin A (ConA) and Burkholderia cepacia complexlectin A (BclA)) will be immobilized on the sensorchips asmodels of mannose receptors at the surface of cells.The final copolymers will be used as steric stabilizer of PLA

nanoparticles. The targeting potential of these nanoparticleswill be estimated by enzyme linked lectin assay (ELLA).

■ EXPERIMENTAL SECTIONMaterials. Toluene (Chem-lab), THF (Chem-lab), diethyl

ether (VWR), 2-propanol (VWR), N,N-dimethylformamide(DMF) (Aldrich), heptane (VWR), CDCl3 (Aldrich), 3,3-diethoxy-1-propanol (Aldrich), copper iodide (Aldrich),triethylamine (Janssen Chimica), naphthalene (Aldrich), ethyl-ene oxide (Messer), potassium (Fluka), propargyl benzoate(Aldrich), propargyl bromide (Aldrich), Sephadex G25 andG50 (Sigma-Aldrich), sodium cyanoborohydride (1 M THFsolution, Aldrich), PLA (BioMerieux, Mn = 32 650 g/mol, Mw/Mn ∼ 1.5), dithranol (Aldrich, 97%), phenol (Sigma, 99%),sulfuric acid (JT Baker), bovine serum albumin (BSA, Sigma),glycine (Aldrich), tris(hydroxymethyl)aminomethane (Tris)(Acros), manganese chloride (Sigma), calcium chloride(Riedel-de Hae n), N-ethyl-N-[3-dimethylaminopropyl]-carbodi-imide (EDC, Fluka), N-hydroxysuccinimide (NHS,Acros), and Concanavalin A (ConA, Sigma-Aldrich) were usedas received. 2,2-Dibutyl-2-stanna-1,3-dioxepane (DSDOP) wasprepared as reported by Kricheldorf et al.32 Synthesis of α-chloro-ε-caprolactone (αClεCL)33 and of 2-aminoethyl-α-D-

mannopyroside (Man-NH2)34 was also reported elsewhere. ε-

Caprolactone (ε-CL) (Aldrich, 99%) was dried over calciumhydride under stirring at room temperature for 48 h andpurified by vacuum distillation just before use. Milli-Q waterwas used in all the experiments. The lectin BclA was preparedas previously reported.35

Characterization Techniques. 1H NMR spectra wererecorded in CDCl3 at 400 MHz in the FT mode with a BrukerAN 400 apparatus at 25 °C. Chemical shifts were given in ppmusing tetramethylsilane (TMS) as an internal reference.The number-average molecular weight (Mn) and polydisper-

sity (Mw/Mn) were determined by size exclusion chromatog-raphy (SEC) at 45 °C. The chromatograph was equipped witha UV−visible detector, a refractive index detector, and twopolystyrene gel columns (columns HP PL gel 5 μm, porosity:102, 103, 104, and 105 Å, Polymer Laboratories) that were elutedby THF at a flow rate of 1 mL/min. The columns werecalibrated with polystyrene and poly(ethylene oxide) standards,respectively (Polymer Laboratories).The hydrodynamic diameter and the particle size distribution

(PDI) of the nanoparticles were determined by quasi-elasticlight scattering measurements at 25 °C using the cumulantmethod and a Malvern Zetasizer NanoZS from Malverninstruments (U.K.). Each value was the average of at least fivemeasurements.The UV−visible spectra were recorded on spectrophotom-

eter Hitachi U3300.MALDI-TOF mass spectrometry was carried out with a

Bruker Reflex III equipped with a 337 nm N2 laser in thereflector mode at a 20 kV acceleration voltage. Dithranol wasused as the matrix. Sodium or potassium trifluoroacetate wasadded for ion formation. Samples were prepared by mixingmatrix (20 mg/mL), sample (10 mg/mL), and salt (10 mg/mL) in a 10:1:1 ratio. The number-average molecular weight(Mn) of polymers was determined in the linear mode.Surface plasmon resonance-based biosensors (SPR) experi-

ments were conducted with a Biacore X instrument at 25 °C.Synthesis. α-Acetal-ω-Hydroxy Poly(ethylene oxide) (α-

acetal-ω-hydroxy PEO). In a flame-dried and argon-purgedflask, 1.2 mL of 3,3-diethoxy-1-propanol (1.13 g; 7.6 mmol) in200 mL of anhydrous THF was titrated with potassiumnaphthalenide in a THF solution (0.72 M) under argon. Aftervigorous stirring at room temperature for 15 min, the mixturewas added into a 500 mL Parr reactor followed by 15 g ofethylene oxide (340 mmol). After 19 h of polymerization at 30°C, 2-propanol was added, and the polymer was precipitated inan excess of diethyl ether and vacuum-dried at 30 °C. Mn of therecovered α-acetal-ω-hydroxy PEO was 2000 g/mol asdetermined by SEC. The amounts of the reactants wereadapted to obtain polymers of other molar masses.1H NMR(CDCl3, 400 MHz) δ (ppm): 1.17 (t, 6H, CH3-CH2-O), 1.88(q, 2H, CH-CH2-CH2), 3.4 (q, 4H, CH3-CH2-O), 3.46 (t, 2H,CH2-CH2-OH), 3.6 (s, 4H × DPEO, O-CH2-CH2-O), 4.6 (t,1H, CH-CH2); recovered yield: 90%.

α-Acetal-ω-Alkyne Poly(ethylene oxide) (α-acetal-ω-al-kyne PEO). Three grams of α-acetal-ω-hydroxy poly(ethyleneoxide) (2000 g/mol) were dried by repeated (three times)azeotropic distillations of toluene before dissolution in dryTHF. Then, 0.75 equiv of potassium naphthalenide in THF(0.72 M) and 0.75 equiv of propargyl bromide were added tothe polymer solution, which was stirred at room temperatureovernight. The polymer was precipitated in ether at −20 °C,filtered, and dried at reduced pressure at room temperature. 1H

Scheme 3. Structure of the Amino-Derivatives Coupled toPoly(εCL)-graf t-(α-aldehyde-PEO) Micelles by ReductiveAmination

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NMR (CDCl3, 400 MHz) δ (ppm): 1.17 (t, 6H, CH3-CH2),1.88 (q, 2H, CH-CH2-CH2), 2.42 (t, 1H, CCH), 3.4 (q, 4H,CH3-CH2-O), 3.6 (s, 4H × DPEO, O-CH2-CH2-O), 4.18 (d,2H, O-CH2-C), 4.6 (t, 1H, O-CH-CH2); recovered yield:90%.Poly(α-azido-ε-caprolactone-co-ε-caprolactone) (Poly-

(αN3εCL-co-εCL)). This copolymer was prepared according toa reported procedure.33 Briefly, α-chloro-ε-caprolactone (0.996mg) was dried by repeated (three times) azeotropic distillationsof toluene just before polymerization. Then, 19 mL of εCL and80 mL of toluene were added to the reactor. When the solutionwas homogeneous, DSDOP (2.6 mL of a 0.52 M solution intoluene) was added and the polymerization allowed to proceedat 70 °C. After 140 min, 3.9 mL of pyridine and 3 mL of acetylchloride were added. After overnight reaction at roomtemperature, the copolymer was purified by precipitation inheptane. A copolymer with 5 units of αClεCL and 144 units ofεCL was recovered by precipitation in heptane and dried invacuo at room temperature.The chloride functions were converted into azide by reacting

8 g of poly(αClεCL-co-εCL) (dissolved in 80 mL of DMF)with 1.5 g of NaN3 (10 equiv in respect to the chloridefunction). The mixture was stirred overnight at roomtemperature. After elimination of DMF at reduced pressure,50 mL of toluene was added, and the insoluble salt wasremoved by centrifugation (5000 rpm at 25 °C for 15 min). Acopolymer with 5 units of αN3-ε-CL and 144 units of ε-CL wasrecovered by precipitation in heptane and dried in vacuo atroom temperature. The amounts of reactants were adapted forgetting copolymers of another azide content.

1H NMR (CDCl3, 400 MHz) δ (ppm): 1.38 (m, 2H ×(DPεCL + DPαN3αCL), O-((CH2)2-CH2-(CH2)2-CH(N3 or H)-CO), 1.61−1.65 (m, 4H × DPεCL, O-CH2-CH2-CH2-CH2-CH2-CO and 2H × DPαN3αCL, O-CHN3-CH2-CH2-CH2-CH2-CO), 1.95−2.00 (m, 2H × DPαN3αCL, CHN3-CH2-CH2), 2.05 (s, 6H, CH3C(O)O), 2.29 (t, 2H × DPεCL, O-(CH2)4-CH2-CO), 3.83 (t, 1H × DPαN3αCL, (CH2)3-CHN3-CO), 4.05 (t, 2H × DPεCL, CH2-CH2-O-C(O)-CH2), 4.15(t, 2H × DPαN3αCL, -CH2-O-C(O)-CHN3); recovered yield:90%.Poly(ε-caprolactone)-graf t-(α-acetal poly(ethyleneoxide)

(Poly[(εCL)x-graf t-(α-acetal PEOy)n]). One gram (0.17 mmolof azide function, 1 equiv) of Poly(αN3εCL3-co-PCL145) and449 mg (0.20 mmol, 1.2 equiv) of α-acetal, ω-alkyne PEO45were transferred into a glass reactor containing 10 mL of THF.The solution was stirred until complete dissolution of thepolymers. Then, 2 mg (0.02 mmol, 0.1 equiv) of NEt3 and 3.8mg (0.02 mmol, 0.1 equiv) of CuI were added into the reactor.The solution was stirred at 35 °C until the IR absorption of theazide at 2104 cm−1 disappeared completely, which occurredafter 10 h.Then, 30 μL of propargyl benzoate was added with a new

amount of CuI and NEt3. After 2 h of reaction, the graftcopolymer was recovered by precipitation in diethyl ether. Inorder to remove the nongrafted PEO chains whichcoprecipitated with the graft copolymer, the micelles of thepolymer mixture were eluted through a Sephadex column(mixture 1/1 of G25 and G50) and recovered by lyophilizationfor further characterization.From 1H NMR analysis, a composition poly[(εCL)148-graf t-

(α-acetal PEO45)3] was determined for the recovered graftcopolymer. These conditions were adapted in the preparationof other graft copolymers.

1H NMR (CDCl3, 400 MHz) δ (ppm): 1.17 (t, 6H ×DPαN3αCL, CH3-CH2), 1.38 (m, 2H × (DPεCL + DPαN3αCL), O-(CH2)2-CH2-(CH2)2-CO), 1.61−1.65 (m, 4H × DPεCL, O-CH2-CH2-CH2-CH2-CH2-CO and 2H × DPαN3αCL, O-CHN3-CH2-CH2-CH2-CH2-CO), 1.88 (q, 2H × DPαN3αCL,CH-CH2-CH2), 2.05 (s, 6H, CH3C(O)O), 2.29 (t, 2H ×DPεCL, O-(CH2)4-CH2-CO), 3.4 (q, 4H × DPαN3αCL, CH3-CH2-O), 3.62 (s, 4H × DPEO × DPαN3αCL, O-CH2-CH2-O),4.05 (t, 2H × (DPεCL + DPαN3αCL), CH2-CH2-O-CO), 4.6 (t,1H × DPαN3αCL, CH2-CH-(O)2), 4.7 (s, 2H × DPαN3αCL, C-CH2-O), 5.0 (t, 1H × DPαN3αCL (C(O)-CH-N), 7.80 (s, 1H ×DPαN3αCL, N-CH-C); recovered yield: 75%.

Fluorescein and Mannose Coupling to Poly(εCL)-graf t-(α-acetal PEO) Micelles. After complete dissolution of 100 mg of apoly(εCL)-graf t-(α-acetal PEO) copolymer in 1 mL THF, thesolution was added dropwise to 10 mL Milli-Q water. The pHwas then adjusted very slowly at 2 with diluted chlorohydricacid (0.1 M). After 2 h, the pH was slowly raised to 7.4 with aphosphate buffer (500 mM, pH 8), and then, 2 equiv of theamino derivative was added. After 1 h, a 10-fold excess ofNaBH3CN solution (43 μL, 0.043 mmol, 1 M solution inTHF) was added. After 96 h of reaction, the reaction mixturewas purified by dialysis (SpectraPor, cutoff of 6000−8000)against water for 3 days, by changing water 4 times/day. Thecopolymer was recovered by lyophilization for furthercharacterization. Recovered yield: 98%.

Quantification of Grafted Man-NH2 by ColorimetricAssay. To an aqueous solution of copolymer, 1 mL of a 5%aqueous phenol solution and 5 mL of concentrated sulfuric acidwere rapidly added. The tube was allowed to stand 10 min atroom temperature, before it was vigorously shaken and placedin a water bath at room temperature for 20 min before readingthe absorbance at 486 nm. The amount of sugar wasdetermined by reference to a standard curve of 2-aminoethyl-α-D-mannopyranoside.

Preparation of Micellar Solutions. Micelles wereprepared by a nanoprecipitation procedure as techniquedescribed.36 Briefly, 50 mg of copolymer were dissolved in2.5 mL of THF. The mixture was stirred until completedissolution of the copolymer. It was then added dropwise to1.775 mL of Tris buffer (0.1 M Tris, 0.03 mM CaCl2, 0.03 mMMnCl2, pH = 7.4) under moderate stirring. THF was removedby dialysis again (Tris buffer and the solutions stored at 6 °C).

Preparation of Nanoparticles (NPs). Nanoparticles ofPLA were prepared by a nanoprecipitation procedure aspreviously described.12,36 Briefly, 35.2 mg of copolymer and 50mg of PLA were dissolved in 2.5 mL of acetone. The mixturewas stirred until the complete dissolution of the polymers, thenadded dropwise to 1.775 mL of Tris buffer (0.1 M Tris, 0.03mM CaCl2, 0.03 mM MnCl2, pH = 7.4) under moderatestirring. Acetone was removed by evaporation under vacuum atroom temperature and the solutions stored at 6 °C.

SPR Experiments. Lectin Immobilization. CM5 sensorch-ip (carboxymethylated dextran covalently attached to a goldsurface) was used in each experiment. All the buffers werefiltered through a 0.45 μm PTFE filter (Millipore). The lectins,ConA and BclA, were immobilized by the followingprocedure35,37 at flow rate of 10 μL/min: the chip wasactivated with 70 μL EDC (75 mg/mL)/NHS (12 mg/mL)solution. Then, ConA in 0.1 M phosphate buffer (pH 7.4), orBclA in 10 mM acetate buffer (pH 4.5), was injected only intothe sample channel. The volume was adapted depending on therequired amount of bound proteins. Finally, the reactive groups

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of the sensor surface were blocked with 1 M ethanolamineadjusted at pH 8.5 with HCl (70 μL). The reference channelwas treated similarly, except for the lectin injection. 0.1 M Trisbuffer (pH 7.4) containing 0.03 mM CaCl2 and 0.03 mMMnCl2 was used in all the recognition experiments.SPR Measurements. After equilibration of the system by

Tris buffer, thus until a stable baseline was observed, thesolutions were injected in the reference and sample channels ata flow rate of 30 μL/min. The kinetic titration method38−40 wasused for each experiment, i.e., within a single binding cycle.Micellar solutions of increasing concentrations (90 μL) andTris buffer (4 min) were alternatively injected. The sensor-grams were recorded as a succession of association anddissociation phases without regeneration between eachinjection of the micellar solution. All the data were thenplotted after subtraction of the reference channel signal toremove any adverse contribution from refractive index noisedue to the bulk contribution of the sample injection.Biological Lectin Recognition Assay. This assay was

realized as previously reported.12,41

■ RESULTS AND DISCUSSIONSynthesis of α-Acetal, ω-Alkyne Poly(ethylene oxide).

The α-acetal, ω-alkyne poly(ethylene oxide) (α-acetal, ω-alkyne PEO) was synthesized in two steps (Scheme 1). Theanionic ring-opening polymerization of ethylene oxide was firstinitiated by potassium 3,3-diethoxy-1-propoxide, thus aninitiator that contains a protected aldehyde as previouslyreported by Nagasaki et al.42 In a second step, the α-hydroxylend-group of PEO was converted into an alkoxide by reactionwith potassium naphthalenide, followed by a substitutionreaction with propargyl bromide. Three α-acetal-ω-alkynePEO’s of different Mn were prepared by changing the monomerto initiator molar ratio (Table 1). 1H NMR spectroscopy

(Figure 1) showed the characteristic peaks of the acetal end-group at 1.18 ppm (methyl group), 4.62 ppm (acetal methineprotons), and 1.88 ppm (methylene groups) which confirmsthe effective initiation of ethylene oxide by the functionalinitiator. The Mn of PEO was determined by integration of thecharacteristic peaks of the end-groups, and the peak at 3.6corresponding to the methylene protons of PEO (Table 1).SEC analysis showed a monomodal elution peak (Figure 2a)and a low polydispersity (Table 1). Calibration of SEC wasperformed with PEO standards and allowed for the Mn to be

determined (Table 1). Values for Mn from SEC were lowerthan the values of Mn established by 1H NMR. Matrix-assistedlaser desorption ionization time-of-flight mass spectrometry(MALDI-TOF) elucidated the polymer composition. Asexemplified for the α-acetal-ω-alkyne PEO25, the MALDI-TOF spectrum (Figure 3) showed three distributions thatcorrespond to PEO chains centered around 1100 g/mol. Thetwo main distributions correspond to the targeted α-acetal, ω-alkyne PEO associated with a K+ or a Na+ cation, respectively.The third small distribution corresponds to α,ω-alkyne PEOassociated with Na+ (∼8.2%). Water trace during the anionicpolymerization of EO is a possible explanation for theformation of PEO end-capped with two hydroxyl groups andby two alkyne groups at the end of the second step. Waterwould actually contribute to the initiation of the EOpolymerization. Even in a low amount, these chains are ableto trigger cross-linking when used in the grafting “onto”reaction and thus to the gelation of the reaction medium. Inorder to avoid this side-reaction, α-acetal, ω-hydroxy PEO waspurposely reacted with less than the stoichiometric amount ofbromo-propyne. PEO was accordingly end-functionalized with66% (α-acetal, ω-alkyne PEO66), 72% (α-acetal, ω-alkynePEO45), and 71% (α-acetal, ω-alkyne PEO25) of alkyne end-group, respectively.

Synthesis of Poly(εCL)-graft-(α-acetal PEO). Synthesisof the graft copolymers by the grafting “onto” strategy relies onthe availability of azide pendent groups along the PCLbackbone reactive toward with the alkyne end-group of thePEO chains. For this purpose, poly(α-azido-ε-caprolactone-co-ε-caprolactone) poly(αN3εCL-co-εCL) random copolymerswere synthesized by ring-opening copolymerization of α-chloro-ε-caprolatone and ε-caprolactone in the presence ofDSDOP followed by substitution of the chloride by an azide, aspreviously reported.15,33 Nevertheless, it must be mentionedthat the protection of the hydroxyl end-groups of the PCLbackbone by reaction with acetyl chloride and pyridine wasrequired to prevent backbiting reactions and the copolyesterdegradation during further derivatization. In order to keep theHLB of the graft copolymers produced by the coupling of PEOchains of various lengths constant, three poly(α-azido-ε-caprolactone-co-ε-caprolactone) (poly(αN3εCL-co-εCL)) ofconstant polymerization degree (Mn ∼ 17 000 g/mol) butdifferent αN3εCL contents were synthesized. The αN3εCLcontents were kept at 1.3, 2.0, and 3.4 mol % of polymer,produced by addition of azido-comonomer. Again, 1H NMRallowed the composition of the azido-PCL backbones to bedetermined (Table 2).Poly(εCL)-graf t-(α-acetal PEO) copolymers were prepared

by the Huisgens alkyne−azide cycloaddition reaction, thus byreaction of α-acetal, ω-alkyne PEO with poly(αN3εCL-co-εCL)with CuI as a catalyst and triethylamine as a base. In each case,1.2 equiv of alkyne was used with respect to the azide pendantgroups. Although the reaction was complete with PEO grafts oflow molecular weight as assessed by the complete disappear-ance of the IR absorption of the azide groups at 2104 cm−1, noreaction was observed when the PEO66 (Mn = 2900 g/mol) wasreacted with the poly(αN3αCL2-co-εCL148), even for as longreaction time as 15 h, at higher catalyst content (10 equiv) andfor more concentrated polymer solutions. The coupling of PEOof the higher molecular weight and lower functionality was thuskinetically limited. In contrast, two graft copolymers 4 and 5were successfully prepared with PEO25 and PEO45, respectively,whose characteristics are summarized in Table 2.

Table 1. Characteristics of the Different α-Acetal-ω-alkynePEO

# sampleaMn

(SEC)bMw/Mn

bMn (

1HNMR)c

Mn (MALDI-TOF)

1 α-acetal-ω-alkynePEO25

1100g/mol

1.06 1200g/mol

1100 g/mol

2 α-acetal-ω-alkynePEO45

2000g/mol

1.05 2200g/mol

n. d.d

3 α-acetal-ω-alkynePEO66

2900g/mol

1.03 3200g/mol

2950 g/mol

aThe subscript numbers are the degree of polymerization (DP) ofPEO. bEstimated by SEC (THF) with poly(ethylene oxide)calibration. cDetermined by 1H NMR (CDCl3). The integrations (I)of the peak characteristic of PEO δ = 3.6 ppm and the acetal end-group at δ = 1.2, δ = 1.9, and δ = 4.6 ppm were used in the followingrelationship: Mn (PEO,

1H RMN) = 44 × (I3.6/4)/[(I1.2/6) + (I1.9/2)+ I4.6].

dNot determined.

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The excess of PEO used in the grafting reaction wasseparated from the amphiphilic copolymer. Purification of α-acetal-ω-alkyne PEO of low Mn (1100 g/mol) was straightfor-ward by the selective precipitation of the graft copolymer indiethyl ether. This technique was, however, not relevant for α-acetal-ω-alkyne PEO of higher Mn (2000 g/mol), because ofthe coprecipitation of PEO and the graft copolymer. Never-theless, the copolymer was purified in water, in which thecopolymer formed micelles in contrast to unreacted PEO thatremained highly soluble. This solution was eluted through acolumn of cross-linked dextran gel (Sephadex) used for gelfiltration chromatography. The collected fractions werecharacterized by SEC (Figure 2), which confirmed thesuccessful separation of PEO from the graft copolymer. 1HNMR analysis of the pure copolymers (Figure 4) confirmed thequantitative grafting of PEO onto the PCL backbone assupported by the integration of the peaks characteristic of PEO(methylene at 3.6 ppm) and the polyester backbone (4.2 ppm).This spectrum also confirmed that the α-acetal end-groupcapping of the PEO grafts was maintained during the grafting

“onto” reaction. As reported in Table 2, two copolymers with asimilar HLB but a different number of grafts were madeavailable.

Functionalization of the Graft-Copolymer Micelles inAqueous Media. The second purpose of this work was todecorate the micelles formed by the graft copolymers in waterby a sugar selected as a targeting unit. Because of the low HLBof the graft copolymers, the assistance of a water-misciblecosolvent (THF) was needed to prepare micellar solutions.THF is indeed a good solvent for both the PEO and PCLcomponents. It was easily eliminated when the copolymer iscompletely dissolved, which results in the copolymermicellization. 36 Micelles were collected with an averagediameter of about 80 nm for the copolymer 4 and 85 nm forthe copolymer 5 as determined by DLS.An amino-functional derivative of the targeting unit was then

reacted with the deprotected aldehyde end-group of the PEOgrafts, based on the hypothesis that these groups would beexposed at the surface of the micelles. The validity of thisassumption was corroborated by using an amino-dye, i.e.,

Figure 1. 1H NMR of the α-acetal-ω-alkyne PEO (Mn(SEC) 1100 g/mol) in CDCl3.

Figure 2. SEC traces in THF for (a) α-acetal, ω-alkyne PEO25 (regular line) and (b) poly(εCL)149-graf t-(α-acetal-PEO25)5 before (dotted line) andafter purification on a Sephadex column (bold line).

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aminofluorescein, easily detectable by UV, instead of amino-mannose (Scheme 3). The coupling reaction occurred in threesteps but in one pot as illustrated by Scheme 2. First, the acetalgroups on the copolymer micelles were converted into

aldehydes under mild acid conditions, i.e., at pH 2 for 2 h.43

Then, the pH was increased up to 7.4 with a phosphate bufferand 2 equiv of aminofluorescein was added in order to form theSchiff base. After one hour, the Schiff base was reduced by

Figure 3.MALDI-TOF spectrum in water for the α-acetal, ω-alkyne PEO (Mn(SEC) 1100 g/mol). Three populations are shown: (1) figures: α-acetal,ω-alkyne PEO ionized with K+; (2) circles: α-acetal, ω-alkyne PEO ionized with Na+; (3) stars: α,ω-alkyne PEO ionized with Na+.

Table 2. Synthesis of Poly[(εCL)x-graf t-(α-acetal-PEOy)n] Prepared by Click Reaction

copolymer (#)composition of the starting azido-PCL

backboneaDP of the starting α-acetal, ω-

alkyne PEObnumber of PEO segments per

copolymer chainbMw/Mn

c HLBd

poly[(εCL)149-g-(α-acetal-PEO25)5] (4)

poly(αN3εCL5-co-εCL144) 25 (1) 5 1.6 4.9

poly[(εCL)148-g-(α-acetal-PEO45)3] (5)

poly(αN3εCL3-co-εCL145) 45 (2) 3 1.6 5.2

poly[(εCL)150-g-(α-acetal-PEO66)2] (6)

poly(αN3εCL2-co-εCL148) 66 (3) n.d.e n.d.e 5.2

aDetermined by 1H NMR in CDCl3.bFrom Table 1. cDetermined by SEC (THF) with a poly(ethylene oxide) calibration. dDetermined with the

Griffin’s relationship: HLB = 20x{1 − [Mn(PCL)]/([Mn(PCL) + Mn(PEO)])}.55 eNot determined.

Figure 4. 1H NMR (CDCl3) of poly[(εCL)148-graf t-(α-acetal PEO45)3] (5) after purification.

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addition of a 10-fold excess of sodium cyanoborohydride(NaBH3CN) for 96 h. The control of the pH was critical inorder to avoid the hydrolytic degradation of the copolymer,particularly during the aldehyde deprotection. After reaction,the micelles were purified by dialysis against water andrecovered by lyophilization. Unexpectedly, the copolymerswere not soluble in THF anymore. A possible explanation wasthat a few azide groups left on the polyester backbone werereduced in amines reactive toward the aldehyde end-group ofthe PEO grafts, so leading to cross-linking. This reductionreaction was previously reported in methanol in the presence ofcopper(II) and NaBH4 at 5 °C.44 In this work, the copperadded during the click reaction could catalyze the azidereduction. Even though the content of these azide functionswas below the detection limit of 1H NMR, 5% might be enoughto induce the copolymer cross-linking. In order to avoid thisside reaction, a small molecule with an alkyne function(propargyl benzoate) was added to the reaction medium afterthe coupling of α-acetal-ω-alkyne PEO onto poly(αN3αCL-co-εCL) in order to consume any residual azide groups. As a resultof this additional step, the undesired cross-linking reaction wasno longer observed during reductive amination such that the

copolymers remained soluble in THF and could becharacterized further.The FA end-capped copolymers were analyzed by SEC in

THF by using a UV-detector at a wavelength of strongabsorption by fluorescein (274 nm). The chromatogram for thepoly[(εCL)149-graf t-(α-acetal PEO25)5] copolymer superposedquite well to the SEC traces recorded by a classical refractiveindex detector for the same copolymer before and afterreductive amination (Figure 5). Clearly, the copolymerabsorbed at 274 nm after reductive amination which confirmedthe successful coupling of FA. Moreover, the elution peakremained monomodal and symmetrical after reaction consistentwith the absence of copolymer degradation. Moreover, the yieldof the coupling reaction could be calculated from the areaunder the SECUV trace, which is actually proportional to thenumber of chromophores as exposed by eq 145

= ×A k nUV (1)

where AUV is the area under the SECUV trace, k is the specificUV response, which includes the instrumental constant and thespecific absorption for a given chromophore, and n is thenumber of chromophores. The need for an internal probe was

Figure 5. SEC chromatogram (RI detector in THF) of the graft copolymer 2 before (dotted line) and after (bold line) addition of fluoresceinamineonto the PEO chain ends by reductive amination. The dashed line is the chromatogram in THF with a UV detector at 274 nm.

Figure 6. SEC analysis (THF and UV detector at 274 nm) of a mixture of the graft copolymer 4 poly[(εCL)149-graf t-(α-FA PEO25)5] (ACopo) andstandard PEO (Mn = 2900 g/mol) (APEO). Mcopo = 27 mg and mPEO = 3 mg.

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met by adding the copolymer with a known amount of PEOchains end-capped by FA by the same recipe as the copolymer(Mn = 2900 g/mol; 44.6% of FA end-groups as determined by1H NMR). The relative area under the two elution peaks of thechromatogram was used to estimate the reaction yield providedthat the molecular weight of the copolymer and the PEOstandard was taken into account and their weight in the elutedsample. The average number of FA per standard PEO chainand PEO grafts per copolymer must also be considered (eq 2).Figure 6 is a typical chromatogram for the poly[(εCL)149-

graf t-(α-FA PEO25)5] copolymer. According to this method,20% of the PEO grafts were end-capped by FA (Table 3).

=m

m N

A

AYield

Mn 0.446

2900copocopo PEO

copo

copo

PEO (2)

where Acopo and APEO are the areas under the SEC curves forthe graft copolymer and the PEO, respectively, N is the numberof starting acetal groups per polymer chain, 0.446 is the averagenumber of FA end-groups coupled per PEO chain, and 2900 isthe Mn of the PEO standard.For sake of comparison, the yield of the reaction conducted

with micelles of the PCL148-b-PEO135 diblock copolymer wasalso 20%, which suggests that the copolymer architecture andthe number of PEO chain per copolymer do not affect thecoupling reaction that occurs at the surface of micelles inaqueous media. The advantage of the direct tagging of themicelles by a sugar in water is that additional steps ofprotection/deprotection of this sugar is not needed for makingit soluble in organic media.The experimental conditions used for the coupling of FA

were extended to the amino-mannose shown in Scheme 3b.Since mannose cannot be detected by UV−visible spectrosco-py, the coupling yield was determined by reaction of thecopolymer with phenol and sulfuric acid following theformation of a colored compound that absorbs at 486 nm.46

This colorimetric test specific to sugar concluded to a couplingyield close to 30% for both graft copolymers (Table 3), in linewith data reported for the diblock copolymer of same molecularweight and composition.47 The better coupling yield for theamino-mannose compared to FA more likely comes from the

higher nucleophilicity of the aliphatic primary amine ofmannose compared to the aromatic amine of FA.

Interaction of the Mannosylated Graft Copolymerswith Lectins. Even though the functionalization yield of thecopolymer was only 30%, the targeting ability of these micelleswas investigated. For this purpose, the bioavailability ofmannose at the surface of the poly(εCL)-graf t-(α-Man PEO)copolymer micelles was assessed by surface plasmon resonance(SPR). The micelles had a hydrodynamic diameter of 87 nmfor the poly[(εCL)149-graf t-(α-Man PEO25)5] 10 with PDI of0.2 and a hydrodynamic diameter of 90 nm for thepoly[(εCL)148-graf t-(α-Man PEO45)3] 11 with PDI of 0.25(determined by DLS). In order to monitor the real-timeinteraction between the mannosylated micelles and a lectin, theconcanavalin A (ConA), a protein able to form a complex withmannose, was immobilized onto the surface of a SPRsensorchip by a process described elsewhere.37 ConA is alegume lectin from the jack-bean Canavalia ensiformis and amember of the C-type lectins that requires calcium andmanganese cations for complexing mannose. Its monomericweight is of 25.5 kDa, and the lectin is a dimer at pH < 6 and atetramer at pH > 7. The binding site of ConA is specific tomannoside and glucoside residues.48,49 In this study, increasingconcentrations of mannosylated micelles were injected over thelectin modified SPR sensor, without regenerating the sensorbetween the injections. This method, which was previouslyreported for other types of complexes and named kinetictitration,38 does not require having the SPR sensorshipregenerated by a compound able to destroy the complexesbut not the bioactivity of the receptor.39,40 The recordedsensorgrams for the real-time interaction of the mannosylatedmicelles of the graft copolymers poly[(εCL)149-graf t-(α-ManPEO25)5] (10) and poly[(εCL)148-graf t-(α-Man PEO45)3] (11)with immobilized ConA are shown in Figure 7. 90 μL aliquotsof micellar solutions of increasing concentrations from 0.005 to2 mg/mL were injected over a period of 3 min at a flow rate of30 μL/min (association phase) followed by flowing pure buffer

Table 3. Coupling Yields for the Reductive Amination ofFluorescein Amine (FA) and 2-Aminoethyl-α-D-mannopyroside (Man-NH2), Respectively, with theAldehyde End-Group of the Graft or Block Copolymers

# copolymeramino-derivative

coupling yielda

(mol %)

7 poly[(εCL)149-g-(α-acetal-PEO25)5] (4)

FA 22b

8 poly[(εCL)148-g-(α-acetal-PEO45)3] (5)

FA 18b

9 α-acetal-PEO114-b-PCL149c FA 20b

10 poly[(εCL)149-g-(α-acetal-PEO25)5] (4)

Man-NH2 28d

11 poly[(εCL)148-g-(α-acetal-PEO45)3] (5)

Man-NH2 29d

12 α-acetal-PEO114-b-PCL149c Man-NH2 30d

amol % of amino-derivative coupled per acetal function. bDeterminedby SEC-UV (THF) at 274 nm with the FA conjugated PEO internalprobe. cCopolymer of linear architecture whose synthesis was reportedin ref 47. dDetermined by the colorimetric phenol/acid sulfuricmethod.

Figure 7. Real-time monitoring of the SPR signal for the interaction ofmicelles of poly[(εCL)149-graf t-(α-Man-PEO25)5] 10 (top curve) andpoly[(εCL)148-graf t-(α-Man-PEO45)3] 11 (down curve) copolymerswith ConA immobilized on the sensorchip (immobilized amount oflectin: 6300 RU). The kinetic titration method was used. Each step isindicated by an arrow that corresponds to the injection of 90 μL at aflow rate of 30 μL/min of micellar solution followed by 4 min flow ofbuffer. Values above the arrows indicate the sample concentrationexpressed in mg/mL. The inset is a representation of the interaction ofmicelles with the SPR surface modified lectins.

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for 4 min (dissociation phase) between each injection. Aconsiderable increase of the SPR signal was observed during theassociation phase for both the graft copolymers as result of thecomplexation of mannosylated micelles by the lectin ConAreceptors immobilized to the surface of the sensor. The lowestconcentration responsible for a detectable signal was 0.01 and0.02 mg/mL for copolymer 10 and 11, respectively. Thisdifference might be the result of the higher density of mannoseat the surface of the micelles formed by the copolymer 10.Indeed, the copolymer 10 contains more PEO grafts and thusmore mannose ligands than the copolymer 11 of similar HLBand sugar residue per PEO. Whatever the concentration, thecomplexation signal measured for the graft copolymer 10 wassystematically higher than for the copolymer 11. Thisobservation confirms the more extensive complexation of thecopolymer 10 micelles. A closer look at the SPR sensorgramsshows that the curve during the association phase is steeper forcopolymer 10 than for copolymer 11. Furthermore, theresponse curve during the association phases of copolymer11 approached equilibrium at the end of the injection. Thisobservation suggests that the complexation progresses morerapidly for copolymer 11 than for copolymer 10. Thisdifference in the complexation kinetics can be connected tothe difference in flexibility of the copolymers. Indeed, thelength of the PEO grafts in the two copolymers is not the same,and their mobility in the micellar shell must decrease as thelength is shorter.During the dissociation phase, the signal decreases in both

cases, which suggests a partial decomplexation of mannose fromthe lectin layer. These data usually give access to the associationand dissociation rate constants, and to the dissociation-bindingequilibrium constant by using specific softwares, e.g., CLAMP38

or MSK40 custom-made, for fitting the kinetic titration curves.Nevertheless, the experimental data could not be fitted by thesesoftware, which may suggest a multivalent interaction betweenthe mannosylated micelles and the lectin layer, which is beyondthe scope of these software as already reported.50

In order to confirm the binding of the micelles bycomplexation with a potential receptor, Burkholderia cepacialectin A (BclA) was immobilized onto a sensorchip. BclA is adimeric bacterial lectin know for stronger binding to α-D-mannopyranosyl residue than ConA (Ka = 3.6 × 105 M−1 35 forBclA and Ka = 8.2 × 103 M−1 for ConA 51). With this BclAlectin, attention was paid to the effect of the density of theimmobilized lectin on the SPR sensor surface, on theinteraction with the mannosylated micelles of copolymer 11.Sensors with different densities of BclA at the surface wereprepared by changing the volume of the lectin solution. Thequantity of immobilized lectins onto the sensor could be easilydetermined from the SPR signal variation before and after thelectin immobilization. Three sensors were thus modified bydifferent amounts of BclA that corresponded to a SPR signal of2560, 1140, and 740 response units (RU). Since 1 RUcorresponds to 1 pg/mm2 and the molecular weight of BclA is28 000 g/mol, the distance between two lectins can beestimated at 4.26, 6.39, and 7.93 nm, respectively (Table 4).These figures are only rough estimates for two main reasons.First, all the binding sites of the immobilized lectins may not beavailable to interact with the micelles. Second, the immobiliza-tion process could also impair the structure of some bindingsites. Therefore, the actual distance between near-neighboractive binding sites is most probably higher than the calculatedone.

Figure 8 shows the interaction of mannosylated micelles 11with the three different sensors of decreasing BclA density.Similarly to Con A, clear association curves are recorded, with aslope which is however steeper and thus a progress of BclA/micelles interaction which is slower compared to ConA. Duringthe buffer flow, no dissociation was observed with BclA whichreflects a more stable complex in good agreement with theassociation constant of the ConA and BclA to α-D-mannopyranose. For the lowest lectin density, partialdissociation of the complex was observed during the bufferflow, meaning that the complex was then weaker. This might beexplained by a decrease in the multivalency of the interactionsbetween micelles and BclA. Of course, in the case of 740 RU,the distance between near-neighbor lectins on the surface mightbe higher than the distance between two mannose ligands atthe surface of the micelles, so accounting for less multivalentinteractions and/or a lower number of multiple interac-tions,52−54 as schematized in Figure 9. Dissociation was alsoobserved with ConA for similar reasons.

Steric Stabilization of PLA Nanoparticles Stabilized byMannosylated Graft-Copolymers. Finally, the mannosy-lated graft copolymers were used to stabilize PLA nanoparticles(NPs) and to make them able to recognize lectins. The NPswere prepared by nanoprecipitation from an acetone solution(2.5 mL) containing 50 mg of PLA and 32.5 mg ofcopolymer.12 The hydrodynamic diameter of the PLA NPsstabilized with the mannosylated graft copolymers 10 and 11was around 365 nm with a PDI of 0.2. Recognition of BclA bythe PLA NPs was tested by the enzyme linked lectin assay(ELLA). For this purpose, biotin-labeled BclA was incubatedwith the PLA NPs.35 Biotinylated lectin on the surface of thePLA NPs was searched by using a streptavidine−peroxidaseconjugate followed by the addition of the enzyme substrate.This multistep procedure allowed the lectin to be detected byUV−visible spectroscopy (Scheme 4). Figure 10 compares theabsorbance of the PLA NPs stabilized by either each of the twomannosylated graft copolymers 10 and 11 or by a non-mannosylated copolymer 4 after interaction with biotin-labeledBclA. The absorbance was significantly higher for themannosylated nanoparticles, which assesses for the availabilityof mannose residues to interacting with lectin. The positiveresponse of the reference sample deprived of mannose might beexplained by nonspecific adsorption of the lectin or thestreptavidin−phosphatase conjugate.

■ CONCLUSIONWell-defined α-acetal, ω-alkyne poly(ethylene oxide)s weresynthesized and used as building blocks for the synthesis ofamphiphilic biodegradable PCL-graf t-PEO copolymers, whosePEO grafts were end-capped by an acetal. The micelles of thesecopolymers were successfully labeled by a fluorescent dye byreductive amination in water. This strategy has the advantage ofbeing implemented under mild aqueous conditions that

Table 4. Conversion of SPR Responses Unit (RU) intoDistances between Near-Neighbor Lectins onto the SensorSurface Based on 1 RU = 1 pg/mm2 and the Molar Mass ofthe BclA Lectin = 28 000 g/mol

amount of lectins (RU) distance between lectins (nm)

2560 4.261140 6.39740 7.93

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preserve the copolymer from degradation and allows thelabeling by aminated targeting agents. Indeed, mannoseresidues were successfully attached to the surface of micelleswithout the need of protection/deprotection steps of the sugar.The bioavailability of the sugar to complex appropriatereceptors was unambiguously shows by surface plasmonresonance. Finally, the mannosylated graft copolymers wereable to stabilize PLA nanoparticles for use in drug deliverysystems. The ELLA test also confirmed the availability of thesugar at the NPs surface to bind with a specific lectin. Themannosylated PCL-graf t-PEO copolymers are thus quitepromising for the preparation of nanocarriers for site-specificdrug delivery applications. The versatility of the strategydeveloped herein paves the way for preparation of labeled

micelles with other types of amino-functional and hydrosolubleligands, such as peptides.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: c.jerome@ulg.ac.be; Fax: +32 (0)43663497; Tel: +32(0)43663461.Present Address#Max Planck Institute for Polymer Research, Ackermannweg10, 55128 Mainz, Germany.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSH.F. and C.J. (CERM) are grateful to the “InteruniversityAttraction Poles Program (PAI 6/27) - Functional Supra-molecular Systems”, “Region Wallonne” in the framework of

Figure 8. Sensograms recorded by kinetic titration of BclA with mannosylated micelles of copolymer poly[(εCL)148-graf t-(α-Man-PEO45)3] (11)(2560, 1140, and 740 RU of immobilized lectin). The curve on the right-hand side is a zoom in the case of 740 RU. The figure above the arrowsindicates the micellar concentrations in mg/mL.

Figure 9. Possible cases for the divalent binding of a ligand to itsreceptor: (A) favorable case; (B) the linker molecule is shorter thanthe ideal length and the receptor conformation is deformed to allow asecond binding; (C) the linker is too short for a divalent interaction tooccur.

Scheme 4. Scheme of the Multistep ELLA Test That Probes the Availability of Mannose Residues at the Surface of PLANanoparticles Stabilized by a Mannosylated Graft Copolymer

Figure 10. Absorbance at 492 nm of PLA nanoparticles stabilized bythe mannosylated graft copolymer 10 (A) or 11 (B) and the non-mannosylated graft copolymer 4 (blank) after interaction with biotinlabeled BclA.

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the project VACCINOR and “Grant F.R.S.-FNRS-Televie” forfinancial support of this research. The authors are indebted tothe CGRI-FNRS-CNRS program for financial support of thisresearch in the framework of bilateral cooperation. We thankAnne Imberty for providing BclA lectin. The authors would liketo thank Veronique Schmitz (CERM), Gabriel Mazzuccheli(CART), Catherine Gautier (CERMAV), and ClaudineFraipont (CIP) for their technical support. SPR data wereobtained at the “Centre d’Ingenierie des Proteins” (CIP,University of Liege), which is supported by contract FRFCcredit no 9.4519.98. The authors express gratitude to R. Rivaand Ph. Lecomte for fruitful discussions.

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