Surface Plasmon Resonance Study on the Interaction between

DOI: 10.1039/b909656f. Dirk Grafahrend, Julia Lleixa Calvet, Kristina Klinkhammer, Jochen Salber, Paul D. Dalton, Martin Möller, Doris Klee. Control ...
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10334

Langmuir 2002, 18, 10334-10339

Surface Plasmon Resonance Study on the Interaction between Lactose-Installed Poly(ethylene glycol)-Poly(D,L-lactide) Block Copolymer Micelles and Lectins Immobilized on a Gold Surface Eduardo Jule,† Yukio Nagasaki,‡ and Kazunori Kataoka*,† Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and Department of Materials Science, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan Received April 4, 2002. In Final Form: October 7, 2002 Poly(ethylene glycol)-poly(D,L-lactide) (PEG-PLA) block copolymer micelles bearing lactose moieties on their surface were prepared, and their interaction with the lectin Ricinus Communis Agglutinin (RCA-I) was evaluated by surface plasmon resonance. The one-pot, ring-opening anionic polymerization used in the preparation of the R-acetoxy-PEG-PLA yielded one for which the molecular weight of the blocks (4900 and 4500 g/mol for the PEG and PLA blocks, respectively) was controlled by the initial monomer/initiator ratio. This process also conveyed a quite narrow molecular weight distribution (Mw/Mn ) 1.05). Micelles prepared from this copolymer had a size in the 30-nm range, also with low size distributions (polydispersity index < 0.1), as established by dynamic light scattering measurements. The coupling of p-aminophenylβ-D-lactopyranoside (Lac) moieties on the acetal-deprotected ends of the PEG block proceeded in a quantitative way, while the particle size remained unchanged, despite the chemical nature of the reaction. The lactose functionality, defined as the number of lactose molecules per 100 copolymer chains, was found to be as high as 80%. The interaction of Lac-installed micelles with surface-immobilized RCA-I lectins was found to be specific and to proceed in a cooperative manner. Further, the specificity in the complex formation provided a tool for more fundamental studies, including a review on the critical association concentration of these self-assembled structures, resulting in values in good accordance with the ones established using pyrene as a fluorescent probe (6-8 mg/L). Finally, the importance of multivalency was also stressed, as enhanced binding was observed for lactose functionalities higher than 40%.

1. Introduction Colloids are of an indisputable appeal, given the variety of physicochemical properties that can be formulated according to the purpose to be served. Further, many fundamental processes in nature occur at this scale, thus providing scientists with an invaluable source of inspiration to develop systems that should be of benefit to mankind.1 Among these colloids, polymeric micelles are macromolecular assemblies that find application in medical fields such as gene therapy or drug delivery.2 Certainly, the core-shell structure, in which a compact inner core formed by the hydrophobic block of an amphiphilic copolymer is surrounded by a palisade of hydrophilic chains, is a unique architecture allowing the entrapment and actual transport of drugs otherwise poorly soluble in water.3 Micelles based upon acetal-poly(ethylene glycol)poly(D,L-lactide) (Ac-PEG-PLA) block copolymers meet these requirements, given the exceptional nature of the hydrophilic block (PEG) constituting their outer corona which not only allows them to be water soluble but moreover lowers the protein adsorption that precedes * To whom correspondence should be addressed. Telephone: +81.3.5841.7145. Fax: +81.3.5841.7139. E-mail: kataoka@ bmw.t.u-tokyo.ac.jp. † The University of Tokyo. ‡ Science University of Tokyo. (1) Hemsley, A.; Griffiths, P. Philos. Trans. R. Soc. London 2000, 358, 547-564. (2) (a) Cammas, S.; Kataoka, K. Solvents and Self-Organization of Polymers; Kluwer: Dordrecht, 1996; pp 83-113. (b) Kataoka, K.; Kwon, G.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119-132. (3) Kwon, G.; Kataoka, K. Adv. Drug Delivery Rev. 1995, 16, 295309.

uptake by scavenger macrophages,4 resulting in particles that circulate in the bloodstream for prolonged periods of time.5-7 The PLA core, on the other hand, provides the hydrophobic interactions that ensure the cohesion of the macromolecular structure while creating a hydrophobic microenvironment capable of entrapping molecules otherwise poorly soluble in water (and thence in the bloodstream).8 PLA moreover degrades into a fully natural metabolite. The most innovative feature of Ac-PEG-PLA copolymers is the acetal end of the PEG block that yields, after deprotection, an aldehyde group9 by which further formulation of micelles can be achieved by the use of peptides10 or carbohydrates.11 Due to the extraordinary stereochemical encoding possibilities they offer, carbohydrates play a role of the uttermost importance in biological processes, from energy storage to cellular recognition.12 Furthermore, many of (4) Harris, J. M. Poly(ethylene glycol) Chemistry; Plenum Press: New York, 1992; pp 1-14. (5) Yamamoto, Y.; Nagasaki, Y.; Kato, Y.; Sugiyama, Y.; Kataoka, K. J. Controlled Release 2001, 77, 27-38. (6) Leroux, J.; Allemann, E.; De Jaeghere, F.; Doelker, E.; Gurny, R. J. Controlled Release 1996, 39, 339-350. (7) Furtado Mosqueira, V.; Legrand, P.; Gulik, A.; Bourdon, O.; Gref, R.; Labarre, D.; Barratt, G. Biomaterials 2001, 22, 2967-2979. (8) Govender, T.; Riley, T.; Ehtezazi, T.; Garnett, M.; Stolnik, S.; Illum, L.; Davis, S. Int. J. Pharm. 2000, 19, 95-110. (9) Scholz, C.; Iijima, M.; Nagasaki, Y.; Kataoka, K. Macromolecules 1995, 28, 7295-7297. (10) Yamamoto, Y.; Nagasaki, Y.; Kato, M.; Kataoka, K. Colloids Surf., B 1999, 16, 135-146. (11) Yasugi, K.; Nakamura, T.; Nagasaki, Y.; Kato, M.; Kataoka, K. Macromolecules 1999, 32, 8024-8032. (12) Dwek, R. A. Chem. Rev. 1996, 96, 683-720.

10.1021/la0258042 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/20/2002

Interaction between Copolymer Micelles and Lectin

these processes are to a certain extent regulated by carbohydrate-protein interactions, which consequently presents an immense therapeutic interest as specific recognition by the asialoglycoprotein receptor on the surface of hepatocytes could be sought in order to achieve enhanced retention at these sites. And although it is beyond the scope of this work to evaluate the therapeutic potential of carbohydrate-functionalized micelles from an in vitro standpoint, it is an objective of this work to insist upon the importance of the surface phenomena involved in active targeting. This shall be carried out by the assessment of the interaction between Ac-PEG-PLA micelles bearing a lactose homing device on their surface and the lectin Ricinus Communis Agglutinin (RCA-I) by surface plasmon resonance (SPR). Lectins are carbohydrate-binding proteins that agglutinate cells or precipitate glycoconjugates in a very specific manner and are involved in a wide array of biological processes.13 As shall be exposed, the use of SPR in the scope of simulating a cell surface has not only conveyed information about the high specificity and strength of such an interaction but has also given firm ground to discuss the structure of micelles in aqueous solution. Indeed, it has been possible to correlate the way in which unimers bearing lactose molecules on the distal end of their PEG block interact with a protein bed to their assembly state and thus to the critical association concentration (cac) of these block copolymers. Micelles are self-assembled systems for which the main driving force is a combination of intermolecular forces14 and thus respond to a cac under which they dissociate into unimers. A full acquaintance of this parameter is of deep relevance as, under the strain of injection in the bloodstream, these structures are prone to undergo a premature disruption accompanied by the release of the carried drug, whence exposing the patient to an unnecessary risk. A new approach to evaluating this parameter is introduced here, in which structure and binding ability are closely related, based on the direct interaction of carbohydrates and lectins. Moreover, the importance of multivalency is evidenced through separate studies on the influence of both the receptor and ligand densities. 2. Experimental Section 2.1. Materials and Methods. Commercial tetrahydrofuran (THF), 3,3′-diethoxypropanol (DEP), and ethylene oxide (EO) were purified by conventional distillations under an argon atmosphere. Commercial D,L-lactide (LA) was recrystallized from ethyl acetate twice under an argon atmosphere and sublimated under vacuum at 110 °C. Potassium naphthalene was used in a THF solution, the concentration of which was determined by an appropriate titration. Pyrene (Wako Pure Chemical Industries Co. Ltd., Japan) and RCA-I lectins (Funakoshi Corp., Japan) were used as received. Water was purified with a Milli-Q instrument (Millipore, Bedford, MA). Gel permeation chromatography (GPC) measurements of the prepared polymers were conducted using a JASCO liquid chromatograph equipped with TSK gel columns (G4000HHR and G3000HHR) and an internal refractive index (RI) detector (930RI, JASCO, Japan). Dimethyl formamide (DMF) containing 10 mM LiCl was used as the eluent at a flow rate of 0.8 mL/min. Molecular weight calibrations were done using a series of standard PEGs (Polymer Laboratories, U.K.). Samples dissolved in the eluent at a concentration of 2 mg/mL were injected into the GPC circuit through a 100 µL loop. The 1H NMR spectra of polymer samples were established at 80 °C in DMSO-d6 or at 25 °C in CDCl3-d1 with a JEOL GSX-270 spectrometer at 270 MHz. (13) Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637-674. (14) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113-131.

Langmuir, Vol. 18, No. 26, 2002 10335 2.2. Preparation and Characterization of Block Copolymers. The Ac-PEG-PLA block copolymer was synthesized via an anionic ring-opening polymerization at room temperature under an argon atmosphere. The typical procedure is described hereafter: 0.16 mL (1 mmol) of DEP and 3.2 mL (1 mmol) of a 0.31 M naphthalene potassium solution in THF were introduced in a flask containing 20 mL of dry THF and stirred for 10 min. EO (5.7 mL, 114 mmol) was then added, and its polymerization was carried out for 21/2 days. After a small volume of the solution was sampled for GPC analysis, 46 mL (40 mmol) of a LA solution in THF (0.88 mol/L) was introduced in the medium, and the polymerization was allowed to proceed for 4 h. The polymer was then precipitated into a 50-fold excess of ice-cooled isopropyl alcohol (IPA) and then centrifuged at 8000 rpm for 30 min. The polymer was then freeze-dried from benzene, conveying white flakes in a yield of 90%. The sample was stored at -20 °C until use. The molecular weight of the PEG segment was determined by GPC using the fraction sampled at the end of the first stage of the copolymerization. Given that interference of the block copolymers with the GPC separating columns, often yielding apparent lower molecular weights, has been observed, the size of the PLA block was calculated based on the molecular weight of the PEG block as obtained by GPC and the NMR spectrum of the block copolymer. Quite so, it is possible to correlate the number-average molecular weight (Mn) of the PEG block and the intensity ratio of methine protons on the PLA block (COCH(CH3)O: δ ) 5.2 ppm) and methylene protons on the PEG block (OCH2CH2: δ ) 3.6 ppm). 2.3. Micelle Preparation and Characterization. The AcPEG-PLA copolymer was dissolved in N,N-dimethylacetamide (DMAc), a good solvent of both the hydrophilic and hydrophobic blocks, in a 5 mg/mL concentration. The solution was then filtered through a 0.2 µm poly(tetrafluoroethylene) (PTFE) filter (Ekicrodisc, Japan) into a preswollen semipermeable membrane (Spectra/Por, Spectrum, Rancho Dominguez, CA; molecular weight cutoff, 3500) and dialyzed for 24 h against a 100-fold excess of distilled water. The dialysate was exchanged at times 2, 5, and 8 h. The micelles were eventually filtered through a 0.45 µm PTFE filter (Millipore). The hydrodynamic radii and the size distribution of micelles were determined by dynamic light scattering (DLS). All measurements were carried out at 25 °C on a light scattering spectrometer (DLS-7000, Photal, Otsuka Electronics, Japan), and the scattering was carried out with a vertically polarized incident beam at 488 nm supplied by an argon ion laser. A scattering angle of 90° was used for the DLS evaluations, and the size and distribution were estimated by the cumulant method.15 The critical association concentration of micelles was determined by either monitoring changes in the microenvironmentdependent fluorescence of pyrene or by evaluating the specific adsorption of lactose micelles on a carboxymethyl dextran (CM5) chip bearing RCA-I lectins by SPR measurements. In the fluorescence evaluation, micelles in a concentration range from 0.1 to 1000 mg/L were equilibrated with a pyrene-saturated aqueous solution overnight, shielded from light. Fluorescence measurements were carried out using a 770F fluorometer (Japan Spectroscopic Co., Ltd., Tokyo, Japan) at an excitation wavelength of 339 nm. As for the SPR evaluation, micelles in a similar concentration range were injected over a surface bearing RCA-I lectins and the absolute adsorption was established by internal reference subtraction on a BIACORE 3000 device (BIACORE Ac, Uppsala, Sweden). All experiments were carried out at 25 °C. 2.4. Lactose Installation on the Surface of Micelles. Deprotection of the acetal functions at the PEG block end was conducted by mild dropwise acidification of the corresponding Ac-PEG-PLA micellar solution (1.4 mg/mL) to pH 2, using a 0.1 M HCl solution, under moderate stirring. The solution was further allowed to stir for 2 h before being brought back to its initial pH (pH 5) with a 0.1 M NaOH solution, in a dropwise manner. At this point, a sample was taken to evaluate the (15) Gulari, E.; Gulari, E.; Tsunashima, Y.; Chu, B. J. Chem. Phys. 1979, 70, 3965-3972.

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aldehyde functionality. The system was then concentrated to 4 mg/mL and placed in phosphate-buffered saline (PBS; pH 7.4, I ) 0.1 M) before introducing a 10-fold molar excess of p-aminophenyl-β-D-lactopyranoside, the amine terminal group of which forms a Schiff base with the aldehyde previously obtained by deprotection of the acetal groups. After another hour of stirring, the Schiff base was reduced to a secondary amine using a 10-fold molar excess of NaH3BCN, and the system was allowed to stir for another 96 h. Unreacted species were removed by dialysis on a preswollen membrane (Spectra/Por, Spectrum; molecular weight cutoff, 1000). Micelles were eventually filtered through a 0.45 µm PTFE filter (Millipore), freeze-dried, and stored at -20 °C until use. 2.5. Quantitative Analysis of the Lactose Installation. The lactose functionality, defined as the number of lactose ends bound at the end of the PEG block per 100 polymer chains, was determined by NMR measurements of the freeze-dried polymer in DMSO-d6 at 80 °C. The peak intensity of the aromatic protons on the phenyl groups of the lactopyranoside at 6.4 and 6.8 ppm was related to the intensity of the methine peak on the PLA block (δ ) 5.2 ppm). 2.6. Surface Plasmon Resonance Measurements. 2.6.1. Principle. SPR evaluations were carried out on a BIACORE 3000 device. Proteins were bound to the carboxymethyl dextran gel on the surface of a CM5 chip by amine coupling. SPR measurements are based on the excitation of surface plasmons, free oscillating electrons propagating along an interface, through the total internal reflection phenomenon.16,17 2.6.2. Protein Coupling to the Surface of a Carboxymethyl Dextran Gel. Prior to SPR measurements comes the creation of a ligand bed on the surface of a carboxymethyl (CM) dextran gel. This process is carried out by chemical coupling of the amine terminal groups of proteins to the carboxylic ends of the carboxymethyl chains on the gel matrix. Both the ends of the gel matrix and the protein to be bound on the gel are electrostatically activated by flowing a buffer at a pH lower than the pI of the protein (acetate buffer, pH 5, I ) 0.01 M) as a preparation for the chemical binding of the proteins. After a stable baseline was reached, 70 µL of a freshly prepared 1:1 mixture of 0.2 M N-ethyl-N′-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (EDC) and 0.05 M N-hydroxysuccinimide (NHS) solutions was injected, chemically activating the surface of the gel. Following this was the injection of a 50 µg/mL RCA-I solution, the volume of which depends on the amount of bound proteins desired. Finally, the injection of 70 µL of ethanolamine/HCl pH 8.5 ensures the deactivation of nonreacted carboxyl ends on the surface of the gel. A similar procedure is carried out for a reference flow cell, for which the ligand injection is a blank injection of buffer. The buffer was subsequently changed to PBS (pH 7.4, 0.1 M) and flowed until reaching a stable baseline. All buffers were degassed and filtered through a 0.45 µm PTFE filter (Millipore). Once the protein installation was carried out, micelles were simultaneously injected over a flow cell containing lectins and an unmodified flow cell, the latter serving the purpose of an internal reference. All data presented in this report were then obtained by signal subtraction of the blank flow cell injection from the protein-bearing cell, to remove any adverse contribution from refractive index noise due to the bulk contribution of the sample injection.

3. Results and Discussion 3.1. Preparation of the Ac-PEG-PLA and of Micelles. When ring-opening, anionic polymerizations are used, initial monomer/initiator ratios can control the size of the blocks and confer low polydispersities on the so-obtained polymers and micelles.18 After polymerization of the first block (PEG), a small volume was sampled and analyzed by GPC. The number-average molecular weight (Mn) and the index of polydispersity Ip ) Mw/Mn were found (16) Green, R.; Frazier, R.; Shakesheff, K.; Davies, M.; Roberts, C.; Tendler, S. Biomaterials 2000, 21, 1823-1835. (17) Markey, F. BIA J. 1999, No. 1, 14-17. (18) Yasugi, K.; Nagasaki, Y.; Kato, M.; Kataoka, K. J. Controlled Release 1999, 62, 89-100.

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to be 4900 g/mol and 1.05, respectively. The actual Mn was in good accordance with the theoretically calculated value of 5016 g/mol. From the Mn found by GPC, it was possible to calculate the degree of polymerization (DP), that is, the number of monomers in the PEG block. And by correlation of this value to the ratio of the PLA methine and PEG methylene 1H NMR peaks on the final copolymer, the DP and the apparent molecular weight of the PLA block were readily determined. These results yielded a Mn of 4500 g/mol for the PLA block and a Mn of 9400 g/mol for the copolymer. The latter value differs from the one obtained by GPC (8100 g/mol, Ip ) 1.09), most probably due to interactions between the copolymer and the separating column. The molecular weight distribution remained low even after the copolymerization process. The z-averaged size of acetal-PEG-PLA micelles appeared to be 30 nm with a size distribution of 0.09, as determined by the cumulant method.15 3.2. Lactose-Installed Polymer and Micelle Characterization. The carbohydrate installation at the PEG ends exposed on the surface of micelles then proceeded, as thoroughly explained in section 2.4. Following which, both the aldehyde-poly(ethylene glycol)-poly(D,L-lactide) (Ald-PEG-PLA) and the lactose-poly(ethylene glycol)poly(D,L-lactide) (Lac-PEG-PLA) block copolymer samples were freeze-dried and analyzed in DMSO-d6 by NMR. It appeared, for the Ald-PEG-PLA, after relating the aldehyde peak intensity (HOC-CH2CH2O-: δ ) 9.8 ppm) to the intensity of the methine peak on the PLA block, that the aldehyde functionality varied between 65 and 80%, according to the analyzed sample. A second calculation, based on the correlation of the intensities of the R-methine proton peak (HOC-CH2CH2O-: δ ) 1.6 ppm) and the methine peak on the PLA block at 25 °C in CDCl3d1, was carried out and confirmed the previous results. The lactose functionality, that is, the number of copolymer chains containing a lactose end per 100 chains, was determined from the recorded 1H NMR spectra. The calculation based on the correlation of the intensities of the aromatic protons on the phenyl group of the p-aminophenyl-β-D-lactopyranoside (δ ) 6.4 and 6.8 ppm) and the methine peak on the PLA block was found to be again between 65 and 80%, denoting a quasi-quantitative reaction. The size of sugar-bearing micelles was measured by DLS, and their diameter was found to be 31.8 nm, with a size distribution of 0.12. 3.3. Interaction between Lactose-Installed Micelles and Lectins. 3.3.1. A Specific Interaction. RCA-I lectins were chosen to simulate cell surface glycoreceptors and provide the possibility of evaluating the most likely behavior of Lac-PEG-PLA micelles (Lacmicelles) when in the presence of cell lines bearing similar receptors. Indeed, these micelles are to be studied as candidates for the treatment of malignant hepatocytes. Lectins were hence immobilized on the surface of a carboxymethyldextran chip (CM) by amine coupling while micelles were injected in discrete pulses over the so-created protein bed.19 The injection of Lac-micelles at a concentration of 100 mg/L induced a considerable increase in the SPR signal on the protein-bearing flow cell (about 400 resonance units, RU), whereas only a very modest response (a mere 5 RU) was recorded for the reference cell. Acetal-PEG-PLA micelles (Ac-micelles) were used as a control and conveyed low responses on both flow cells (Figure 1). The increase in the SPR signal observed for Lac-micelles would be due (19) Karlsson, R.; Fa¨lt, A. J. Immunol. Methods 1997, 200, 121-123.

Interaction between Copolymer Micelles and Lectin

Figure 1. Typical sensorgram of the injection of Lac- and Ac-micelles (100 mg/L) on a CM surface bearing RCA-I lectins. Numbers next to plots represent the flow cell identification, where flow cell 2 is protein-functionalized and flow cell 1 is used as an internal reference. (a) Injection of Lac-micelles. (b) Dissociation of Lac-micelles. (c) Injection of an excess of free galactose. (d) Baseline stabilization. (e) Injection of Ac-micelles. (f) Dissociation of Ac-micelles. (g) Injection of Lac-micelles. (h) Dissociation of Lac-micelles. (i) Injection of an excess of free galactose.

to the specific association of lactose molecules on the surface of micelles with lactose binding sites on the immobilized proteins, whereas responses recorded for Ac-micelles would be due to the nonspecific adsorption of micelles on the dextran gel, as well as to the bulk contribution of injected samples. Regeneration of the surface was achieved by the injection of a small volume (10 µL) of an excess of free galactose, which as seen in Figure 1 yielded a baseline at the value observed before injection, so proving to be effective for this purpose. A subsequent injection of Lacmicelles produced a similar response, confirming the reproducibility in the evaluation (Figure 1). 3.3.2. The Critical Association Concentration of Micelles. Different polymer concentrations were injected over a protein bed containing about 7000 RU of bound proteins, and the complex formation levels were recorded, conveying interesting results. First of all, whereas the adsorption level of a polymer solution at 1 mg/L hardly reaches a mere 20 RU and remains at similar values for a wide range of polymer concentrations, it attains the enhanced value of 1400 RU for a polymer concentration of 200 mg/L. In the scope of establishing the relation between adsorption levels and polymer concentration, Lacmicelles in a concentration range between 0.1 and 500 mg/L were injected over the protein bed. As can be seen in Figure 2, the binding of Lac-micelles remains at discreet values for a concentration range up to 10 mg/L and then considerably increases with an increasing polymer concentration. Control micelles (Ac-micelles) yield only negligible responses due to nonspecific responses, for the tested concentration range. Second, at high polymer concentrations, the adsorption phases exhibit very steep slopes, denoting a fast-rate binding. But even more remarkable are the correspondent dissociation rates. The extremely gradual decrease in the recorded response denotes a notably slow dissociation from the protein bed. Figure 3 presents a plot of the derivative of the response against time (dR/dt) for both association and dissociation phases and thus provides information about the speed at which these two processes occur. And whereas dR/dt reaches high values for association phases, it remains at very low levels, close to 0, for dissociation phases. Hence, the binding of Lac-micelles to the lectin bed is extremely strong and stable. Such dissociation phases have been reported for systems in which multi-

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Figure 2. Sensorgrams for the binding of Lac-micelles on a CM surface bearing 7000 RU of RCA-I lectins. The intensity of complex formation or response (R), expressed in RU, is plotted against time, with t ) 0 being set as the start of the sample injection. Values above the sensorgrams represent sample concentrations, expressed in mg/L.

Figure 3. Derivatives of responses against time (dR/dt), as a function of time. Values next to curves represent sample concentrations, expressed in mg/L.

Figure 4. Binding of Lac-micelles on a CM surface bearing 7000 RU of RCA-I lectins as a function of polymer concentration as evaluated by SPR (left axis). On the right axis is the II/IIII ratio of pyrene, as evaluated by the fluorescence evaluation.

valent binding is involved20 and has been verified for the systems described here, as shall be discussed in detail in section 3.3.4 of this paper. Figure 4 presents the recorded response, as established at equilibrium, of all recorded sensorgrams, plotted as a function of the logarithm of the polymer concentration. The sigmoid shape sees its onset at a micelle concentration of 6.4 ( 0.5 mg/L. After this transition, the adsorption increases steeply in a linear manner. Interestingly enough, this onset concentration corresponds to the range of critical (20) Rheinnecker, M.; Hardt, C.; Ilag, L.; Kufer, P.; Gruber, R.; Hoess, A.; Lupas, A.; Rottenberger, C.; Plu¨ckthun, A.; Pack, P. J. Immunol. 1996, 157, 2989-2997.

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Figure 5. Binding of Lac-micelles and of the Lac-PEG homopolymer on a CM surface bearing 7000 RU of RCA-I lectins, as a function of polymer concentration.

association concentrations previously established for Lacmicelles based on the changes in the microenvironmentdependent fluorescence of pyrene.21 Since the pioneering works of Nakajima, it has been known that the intensities of the vibronic bands of pyrene exhibit a strong dependence on the solvent environment.22 Specifically, the intensity of the (0,0) band, II, undergoes a remarkable increase in polar environments, whereas of all the other bands, it is the intensity of the (0,2) band, IIII, which exhibits maximum variations when related to II. Thus, the II/IIII ratio has long been used to evaluate the change in the polarity of microenvironments surrounding pyrene monomers, especially in micellar solutions.23 In this evaluation, pyrene was excited at a wavelength of 339 nm, and its emission was recorded at both 374 nm (I) and 384 nm (III). Figure 4 also presents the plot of the II and IIII ratio (right axis) against the polymer concentration. The plot has also a sigmoidal shape for which the II/IIII ratio decreases with an increasing polymer concentration. Also, it exhibits values not incongruous with those of pyrene in water (1.87)24 at low polymer concentrations. As the latter increases, the ratio undergoes a gradual decrease, as pyrene molecules are transferred from water to environments of increased hydrophobicity. Stable values (ca. 1.52) are reached for concentrations above 20 mg/L, and the former values are not distant from that of pyrene in methyl acetate (1.48),24 an ester. Given the nature of PLA, this value is reasonable, and even though the change in the polarity of the environment surrounding pyrene molecules is not remarkable, the cac can be readily established as 7.7 ( 1.9 mg/L. Note that this value is in good accordance with the one otherwise found by SPR, for which the onset of the sigmoid occurs at 6.4 ( 0.5 mg/L. One further evaluation was carried out to confirm these observations, by injecting a lactose-PEG (Lac-PEG) homopolymer (Mn ) 5000 g/mol) over the RCA-functionalized surface. Such a polymer deprived of a hydrophobic block is not able to associate into micelles and should further convey proof of whether the observed transition is due to the association of unimers into micelles. The binding of the Lac-PEG is plotted in Figure 5, along with that of the Lac-PEG-PLA, as a function of polymer concentration. Binding levels to the protein bed attain only low levels, and aside from this, no transition or trend (21) Wilhelm, M.; Zhao, C.; Wang, Y.; Xu, R.; Winnik, M.; Mura, J.; Riess, G.; Croucher, M. Macromolecules 1991, 24, 1033-1040. (22) Nakajima, A. Bull. Chem. Soc. Jpn. 1971, 44, 3272-3277. (23) (a) Kalyanasundaram, K.; Thomas, J. J. Am. Chem. Soc. 1977, 99, 2039-2044. (b) Kwon, G.; Naito, M.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1993, 9, 945-949. (24) Dong, D.; Winnik, M. Can. J. Chem. 1984, 62, 2560.

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whatsoever is observed within this concentration range. It seems safe then to assume that the observed transition corresponds to the cac of Lac-micelles. The absence of a sharp inflection in the fluorescence evaluation could be attributed to a simple distribution of pyrene molecules among micelles, unimers, and water in solution. A more elaborated explanation would be that of a homogeneous distribution of pyrene molecules in environments of different association numbers and hence hydrophobicity, in turn due to the progressive association of unimers into micelles. As for the transition observed in the SPR evaluation, it can only be induced by an evolution in the structure of the polymer in solution leading to the formation of structures that are capable of displaying enhanced binding, brought about by multivalent responses, to the protein bed. Thus, the SPR evaluation constitutes a novel approach to determining the cac, as the macrostructure of unimers in solution can be related to their binding capacity, if one can put it this way, through the specific interaction between lactose molecules and lectins. 3.3.3. Protein Density on the Surface of the Chip. As a way of further examining the interaction between Lac-micelles and lectins, the influence of what will be defined here as the protein density was evaluated by installing different amounts of bound proteins on the surface of the chip. Furthermore, should quantitative analyses be undertaken in future works, a minimal amount of ligand installed on the gel bed is desirable, if one is to simulate an ideal 1:1 binding,25 while limiting mass transport phenomena.26 But more to the point for this work, special care is to be taken in studying the influence of the protein density on what has been concluded here to be the cac of Lac-micelles. Different amounts of proteins (900, 1900, 3300, and 5000 RU) were installed on a dextran gel as exposed in section 3.3.2, and polymer solutions were injected at a flow rate of 10 µL/min. No clear tendency in the binding of Lac-micelles could be deduced for the lowest evaluated protein density (900 RU, data not shown). Further, recorded responses were not reproducible, an observation that might be explained by the lack of a homogeneous enough protein bed. Quite so, should the interprotein distance become too important, regions containing higher (or lower) amounts of “available” proteins blur the evaluation of the absolute complex formation between micelles and lectins. For all lectin densities above that value, the plots of the absolute response against the polymer concentration exhibited a transition much in the way previously described in section 3.3.2. On the other hand, for any given concentration above the cac, the amount of complex formation increased with an increasing amount of proteins (Figure 6). This observation is not deprived of sense, as with an increasing protein density, the increasing level of binding sites conveys the possibility of forming complexes in higher levels. The slight decrease in responses recorded for a protein density of 6800 RU might be explained by an increasing steric hindrance between receptors that renders the latter less accessible for binding. Note as well how levels of complex formation are tremendously increased (around 3-fold) when the protein density increases from 3300 to 5000 RU, clearly stressing the importance of the receptor density at the target surface. Far more interestingly, the plot of the onset concentration for micellar binding was almost constant regardless (25) Biacore 3000 Instrument Handbook, Edition March 1999, Biacore AB: Uppsala, Sweden. (26) Rich, R.; Myszka. D. Curr. Opin. Biotechnol. 2000, 11, 54-61.

Interaction between Copolymer Micelles and Lectin

Figure 6. Binding of Lac-micelles on surfaces bearing different protein densities. Values next to symbols correspond to micelle concentrations, expressed in mg/mL.

of the protein density (data not shown), suggesting that the observed transition is an inherent property of the polymer in solution and not of the surface onto which the polymer binds. This observation seems to point in the direction that the observed transition corresponds indeed to the critical association phenomenon of Lac-micelles, occurring on average at a value of 6.9 ( 0.8 mg/L. 3.3.4. Lactose Density on the Surface of Micelles. Multivalency, in which binding between more than one ligand and one or many receptors occurs, has been widely reported throughout nature.27 Far more remarkable is the fact that multivalency usually triggers a far different response than the one that would be expected from single interactions. To evaluate this hypothesis, micelles bearing different lactose densities on their surfaces were prepared by mixing the Lac-PEG-PLA block copolymer with its Ac-PEG-PLA counterpart in different ratios, in DMAc. After the solution was properly homogenized, micelles were prepared by conventional dialysis. Quite predictably, the particle size was only affected in a negligible way (data not shown). In terms of kinetics, although the equilibrium dissociation rates of mono- or disaccharides, from lectins, are generally in the 10-3-10-4 M range, the same dissociation rates exhibit dramatically enhanced values when the affinity is measured with a neoglycoprotein or a cell.28 Multivalent interactions seem to bring about important changes in dissociation constants, not in association constants.29 As clearly observed in Figures 2 and 3, whereas association phases occur at extremely fast paces, the slopes in all recorded dissociation phases exhibit very low values, denoting an extremely slow dissociation from the protein bed, suggesting that Lac-micelles might form multivalent complexes with the lectin bed. And given the flexibility of a micelle’s structure, the multivalent binding of several lactose molecules to the lectin bed on the surface of the chip can indeed be expected. Micelles bearing different lactose functionalities were injected over a surface bearing about 2000 RU (set as the minimum protein density for reliable results) of lectins, at different concentrations (Figure 7). As previously observed, there is an enhancement in the binding for polymer concentrations above what has been concluded here to be the cac. Further, it dawns clearly that the binding of micelles is considerably enhanced for lactose (27) Mammen, M.; Choi, S.; Whitesides, G. Angew. Chem., Int. Ed. 1998, 37, 2754-2794. (28) Shinohara, Y.; Hasegawa, Y.; Kaku, H.; Shibuya, N. Glycobiology 1997, 7, 1201-1208. (29) MacKenzie, C.; Hirama, T.; Deng, S.; Bundle, D.; Narang, S.; Young, N. J. Biol. Chem. 1996, 271, 1527-1533.

Langmuir, Vol. 18, No. 26, 2002 10339

Figure 7. Binding of Lac-micelles containing different lactose functionalities on a CM surface bearing 2000 RU of RCA-I lectins, as a function of polymer concentration. Numbers next to symbols correspond to the lactose functionality of each batch.

functionalities above 40%. This evaluation provides evidence of the importance of multivalency for Lacmicelles, leading to stronger binding to a protein bed, a property that can only be a therapeutic asset for these drug delivery systems, as strong binding to cell surface receptors by its lactose targeting moieties is to be expected. These observations should furthermore yield an interesting ground for discussion if more quantitative evaluations such as, say, kinetic assays are to be carried out, as it would then be possible to discuss the influence of the ligand density on kinetic rates. In the case of Lac-micelles, for which the ligand density can be readily controlled, it would be possible to quantify what has been described here in a qualitative way and to gain insight on active targeting processes (to be published elsewhere). 4. Conclusion Lac-micelles appear indeed to be potential candidates for the active targeting of cellular glycoreceptors leading to an enhanced site-accumulation property. Indeed, as has been discussed in this paper, the binding between Lac-micelles and RCA-I lectins is one of a specific nature, Ac-micelles having yielded a negative control to this observation. Moreover, the intensity of complex formation between micelles bearing a carbohydrate moiety on their surface and RCA-I lectins is strong and stable, requiring the injection of an excess of free galactose. This property should lead to prolonged retention times at target sites and thence to enhanced uptake by cells expressing the asialoglycoprotein receptor. The intensity of complex formation is a function of both the protein density on the target surface and of the lactose density on the surface of micelles. In this regard, the role of multivalency has been proven to be considerable and at the origin of enhanced complex formation by a cooperative effect, an observation that it would be of interest to deepen on more quantitative analysis in yet another possibility offered by SPR analysis. Acknowledgment. This work was financially supported in part by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and by the Core Research Program for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST). E. Jule expresses his gratitude to Doctor Sandrine CammasMarion (CNRS Chaˆtenay-Malabry, France) and to the MEXT for the kind support of this work. LA0258042