Synthesis and Characterization of Poly (ethylene oxide)-block-poly

Peggy Studer,†,‡ David Limal,†,‡ Pascal Breton,† and Gérard Riess*,‡. VIRSOL, 46 Rue Boissie`re, 75116 Paris, France, and Laboratoire de ...
0 downloads 0 Views 109KB Size
Download PDF
Bioconjugate Chem. 2005, 16, 223−229

223

Synthesis and Characterization of Poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) Block Copolymers Bearing a Mannose Group at the PEO Chain End Peggy Studer,†,‡ David Limal,†,‡ Pascal Breton,† and Ge´rard Riess*,‡ VIRSOL, 46 Rue Boissie`re, 75116 Paris, France, and Laboratoire de Chimie Macromole´culaire, Ecole Nationale Supe´rieure de Chimie de Mulhouse/Institut de Chimie des Surfaces et Interfaces, 3 Rue Alfred Werner, 68200 Mulhouse, France. Received August 13, 2004; Revised Manuscript Received December 3, 2004

A poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) block copolymer (PEO-b-PMM 2.1.2) bearing a mannose moiety at the poly(ethylene oxide) chain end was synthesized by sequential anionic polymerization of ethylene oxide (EO) and methylidene malonate 2.1.2 (MM 2.1.2), followed by a coupling reaction between its poly(ethylene oxide) amino- or aldehyde-end group and a sugar derivative. Different coupling procedures, either in organic media or in aqueous micellar solutions, were examined in order to optimize the poly(ethylene oxide) end-glycosylation yield. The micellar size of the functionalized block copolymers was determined by dynamic light scattering.

INTRODUCTION

There is a growing interest in the development of novel drug delivery systems to reach an increased bioavailability of drugs with controlled release profiles to the targeted cells and to reduce their side effects, especially in the field of cancer chemotherapy (1, 2). To this aim, biocompatible amphiphilic block copolymers, which have the tendency to self-assemble into micelles in a selective solvent, can be utilized as potential carriers for hydrophobic drugs or other bioactive molecules (3-7). The good water solubility properties, the biocompatibility, and the lack of immunogenicity of poly(ethylene oxide) (PEO) provide these nanostructures with a suitable outer corona of PEO that prevents protein adsorption and cell adhesion, which are the first steps before their uptake by the mononuclear phagocyte system and their rapid blood clearance (8). The drug release kinetics being affected by the drug/polymer interactions as well as by the polymer microstructure and erosion/degradation properties, the chemical composition, and architecture of the polymers have to be tailored to accommodate drugs with varying hydrophobicity, molecular weight, etc. The polymer matrixes and particularly the hydrophobic block have to be chosen in order to achieve the desired biocompatibility, polymer degradation and/or erosion, and drug release kinetics. The synthetic degradable polymers used for biomedical applications are typically poly(amino acids), poly(esters), and poly(anhydrides) with hydrolyzable bonds along the backbone (9). Another category of biocompatible polymers bears enzymatically cleavable side chains leading to water-soluble polymers through a bioerosion mechanism. These bioerodible polymers can be poly(cyanoacrylates) (PACA) (10) or poly(methylidene malonate 2.1.2) (PMM 2.1.2). The latter is prepared by polymerization of 1-ethoxycarbonyl-1-ethoxycarbonylmethylenoxycarbonyl ethene (MM * To whom correspondence should be addressed. Tel +33-389-33-68-54; fax +33-3-89-33-68-54; e-mail: [email protected]. † VIRSOL. ‡ Ecole Nationale Supe ´ rieure de Chimie de Mulhouse/Institut de Chimie des Surfaces et Interfaces.

Scheme 1. Structure of Methylidene Malonate 2.1.2 (MM 2.1.2) and of the Mannose Derivatives a and b

2.1.2, Scheme 1) and has been shown to be less toxic than PACA (11, 12). More recently, it has been proposed to introduce reactive groups on the surface of this kind of polymeric micelles and to use them as a basic system for active targeting (4, 13). Thus, PEGylation chemistry is retaining more and more attention in the field of biomedical materials (14-16). To improve the potential utility of polymeric micelles for drug targeting, Nagasaki et al. (17) reported the functionalization of the outer surface of the micelle by an easy and quantitative method allowing the synthesis of heterobifunctional poly(ethylene oxide) and block copolymers such as poly(ethylene oxide)-b-poly(D,Llactide) (PEO-b-PDLLA), poly(ethylene oxide)-b-poly(dimethylaminoethyl methacrylate) (PEO-b-PDMAEMA) and many others. By using a sugar derivative as an initiator, the authors also developed a novel one-pot synthesis of PEO-b-PDLLA block copolymers bearing one sugar end-group, like glucosyl or galactosyl, at the PEO chain end (18, 19). Indeed, by an adequate protection of four out of five hydroxyl groups, a retained hydroxyl group can be used as an initiator for the monosaccharideinduced EO polymerization. As the synthesis of nonfunctionalized PEO-b-PMM 2.1.2 is already known using diphenylmethylpotassium as an initiator (20, 21), the aim of this study was to develop a novel route to prepare functionalized PEO-bPMM 2.1.2 block copolymers allowing the coupling of various ligands such as sugars (22), peptides (23), folic acid (24), fluorescent markers (25), etc. Our main purpose was to develop R-functionalized PEO-b-PMM 2.1.2 block copolymers and to demonstrate the reactivity of these

10.1021/bc0498065 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/24/2004

224 Bioconjugate Chem., Vol. 16, No. 1, 2005

Studer et al.

Scheme 2. Synthetic Route to r-Amino and r-Aldehyde-Functionalized PEO-b-PMM 2.1.2 Copolymers 1 and 3

PEO end functions, so that the latter are enabled to carry out the ligand-anchoring with targeting agents such as a mannosyl residue. In the present study, a mannose moiety was conjugated through two different coupling procedures, either in aqueous micellar solutions or in organic solvents, to the R-amino and R-aldehyde reactive PEO end groups of the PEO-b-PMM 2.1.2 block copolymers, which were introduced during the initiation step. EXPERIMENTAL PROCEDURES

Materials. All the reagents and chemicals used in this study were of the highest purity available. THF was purified using conventional methods (20). 2-Aminoethanol (Aldrich) was purified by distillation under CaH2. The sodium phosphate buffer solution pH 7.0 was prepared as described by Yamamoto et al. (13). A 100 mL sodium carbonate buffer solution pH 9.5 was prepared as follows: 4.8 mL of a 0.2 M sodium carbonate solution and 20.3 mL of a 0.2 M sodium bicarbonate solution were diluted to 100 mL with distilled water. Methylidene malonate 2.1.2, prepared according to the Bru-Magniez et al. procedure (26), was kept under sulfur dioxide at -20 °C. Potassium naphthalide was prepared by the method of Scherf et al. (27), and its concentration (0.3 M) was determined by titration. Preparation of the Sugar Derivatives. Sugar derivatives were prepared by combining different reaction steps described in the literature (28-31). (4-Aminophenyl)-R-D-mannopyranose a (Scheme 1) was obtained through a four-step synthesis (29). Briefly, mannose acylation according to Lerner et al. (28) consisted of the protection of the hydroxyl groups in positions 2, 3, 4, and 6 of R-D-mannopyranose, thus allowing the introduction on position 1 of the p-nitrophenyl group as described by Smits et al. (30). Deprotection of the hydroxyl groups was achieved on an Amberlyte resin, followed by a catalytic hydrogenolysis on Pd/C (31) that gave a with a global yield of 20%. As described by Monsigny et al. (31), (4isothiocyanatophenyl)-R-D-mannopyranose b (Scheme 1) was obtained with a 94% yield by the reaction of a with thiophosgene. Polymer Characterization. 1H and 13C NMR spectra were recorded in CDCl3 using a Bruker AC 250F spec-

trometer, respectively, at 250 and 62.9 MHz. Gel permeation chromatography measurements were performed by using a Waters 2690 Alliance system equipped with a sequence of Waters Styragel HR4, HR1 and HR0.5 columns and an internal RI Waters 410 detector (flow rate ) 1 mL/min). THF was used as an eluant, and standards were poly(ethylene oxide) and poly(styrene). Preparation and Characterization of the Polymeric Micelles. The procedure for micelle formation has been described previously (20). Briefly, 25 mg of copolymer was dissolved in 5 mL of a mixture THF/water 1/1 (v/v), and the polymer solution was transferred into a preswollen membrane SpectraPor 7 (molecular weight cut-off: 1000) and dialyzed against water for 24 h. The mean diameter of the polymeric micelles was determined by dynamic light scattering using a Coulter N4 Plus particle size analyzer (Coultronics). Synthesis of r-Amino PEO-b-PMM 2.1.2 Block Copolymers (1) (Scheme 2). Polymerizations were carried out under nitrogen atmosphere in reactors equipped with a magnetic stirring device. As described by Mosquet et al. (32) for PEO, a typical procedure for the synthesis of the amino-bearing PEO-b-PMM 2.1.2 block copolymers 1 was as follows: 3.3 mL of potassium naphthalide solution (1 mmol) was added to 150 mL of dry THF containing 1 mmol (60 µL) of 2-aminoethanol to prepare the insoluble metalated alcoholate initiator. Afterward, this heterogeneous system was stirred for 5 min, and then 114 mmol (5 g) of EO was added via a sealed ampule to the THF solution containing the initiator. The polymerization of EO was carried out at 35 °C for 2 days. The PEO solution was then divided into two equivalent parts. The first one was deactivated by a small amount of methanol and kept for the determination of the molecular weight, the polydispersity, and the end functionality of the PEO thus prepared. A degassed MM 2.1.2 in THF solution (1 g/15 mL) was quickly introduced under efficient stirring into the second reaction vessel, resulting in the discoloration of the solution, and the reaction was carried out for an additional 1 h at room temperature. After the polymerization was stopped by the addition of a few drops of methanol, the mixture was then concentrated under reduced vacuum and finally precipitated

Bioconjugate Chem., Vol. 16, No. 1, 2005 225

Synthesis of Mannosylated Amphiphilic Copolymers

into a large amount of diethyl ether. The precipitated polymer was collected by filtration and dried under vacuum at room temperature (yield ) 95%). HO-PEONH2 Homopolymer. GPC (THF, PEO standards): numberaverage molecular weight (Mn) ) 4470; weight-average molecular weight (Mw) ) 5810; polydispersity index (Ip) ) 1.30. 1H NMR (250 MHz, CDCl3, δ in ppm): 2.85 (t, CH2NH2, 2H), 3.60 (m, CH2 of PEO, 4H). 13C NMR (62.9 MHz, CDCl3, δ in ppm): 41.6 (CH2NH2), 60.1 (CH2OH), 70.0 (CH2 of PEO), 72,3 (CH2 in β position of OH), 73.3 (CH2 in β position of NH2). End functionality ) 98%. PMM 2.1.2-b-PEO-NH2 Copolymer. GPC (THF, PS standards): polydispersity index (Ip) ) 1.32. 1H NMR (250 MHz, CDCl3, δ in ppm): 1.30 (m, CH3 of PMM 2.1.2, 6H), 2.80 (m, CH2 of the main chain of PMM 2.1.2, 2H), 3.60 (m, CH2 of PEO, 4H), 4.10 (m, CH2 of CH2CH3 of PMM 2.1.2, 4H), 4.50 (m, CH2 of PMM 2.1.2, 2H). End functionality ) 98%. Synthesis of r-Acetal PEO-b-PMM 2.1.2 Block Copolymers (2) (Scheme 2). As reported by Yamamoto et al. (13) for PEO-b-PDLLA block copolymers, the synthesis of R-acetal-PEO-b-PMM 2.1.2 block copolymers 2 was as follows: 4.1 mL of potassium naphthalide (1.25 mmol) was added to 150 mL of dry THF containing 1.25 mmol (197 µL) of 3,3-diethoxypropan-1-ol to prepare the soluble metalated alcoholate initiator. After this mixture was stirred for 5 min, 114 mmol (5 g) of EO was then added via a sealed ampule to the THF solution containing the initiator. The polymerization of EO was carried out as described above. After dividing the solution into two equivalent parts, a degassed MM 2.1.2 in THF solution (4 g/15 mL) was quickly introduced under vigorous stirring into the second reaction vessel, and the reaction was carried out for an additional 1 h at room temperature. The polymerization was then stopped and the purification of the polymer was carried out as described before (yield ) 95%). HO-PEO-acetal Homopolymer. GPC (THF, PEO standards): number-average molecular weight (Mn) ) 4200; weight-average molecular weight (Mw) ) 4960; polydispersity index (Ip) ) 1.18. 1H NMR (250 MHz, CDCl3, δ in ppm): 1.18 (t, CH3, 6H), 1.88 (q, CH2, 2H), 3.60 (m, CH2 of PEO, 4H), 4.62 (t, CH, 1H). 13C NMR (62.9 MHz, CDCl3, δ in ppm): 15.3 (CH3), 34.0 (CH2), 60.1 (CH2OH), 61.4 (CH2 of CH2CH3), 61.5 (CH2O), 70 (CH2 of PEO), 72.3 (CH2 in β position of OH), 100.5 (CH). End functionality ) 98%. PMM 2.1.2-b-PEO-acetal Copolymer. GPC (THF, PS standards): polydispersity index (Ip) ) 1.32. 1H NMR (250 MHz, CDCl3, δ in ppm): 1.18 (t, CH3, 6H), 1.30 (m, CH3 of PMM 2.1.2, 6H), 1.88 (q, CH2, 2H), 2.80 (m, CH2 of the main chain of PMM 2.1.2, 2H), 3.60 (m, CH2 of PEO, 4H), 4.10 (m, CH2 of CH2CH3 of PMM 2.1.2, 4H), 4.50 (m, CH2 of PMM 2.1.2, 2H), 4.62 (t, CH, 1H). End functionality ) 95%. Synthesis of r-Aldehyde PEO-b-PMM 2.1.2 Block Copolymers (3) (Scheme 2). An aqueous micellar solution of the acetal-PEO-b-PMM 2.1.2 copolymer 2 was prepared as described above, and the acetal group was transformed into an aldehyde group by acid treatment as follows: the aqueous solution of R-acetal-PEO-b-PMM 2.1.2 micelles was acidified to pH 2.0 by 1 N HCl added dropwise. After being stirred for 2 h at room temperature, the solution was neutralized by 0.1 N NaOH. The micellar solution was then desalted by dialysis against water. After 24 h, the micelles were then freeze-dried. HO-PEO-CHO Homopolymer. GPC (THF, PEO standards): number-average molecular weight (Mn) ) 4290; weight-average molecular weight (Mw) ) 5060; polydispersity index (Ip) ) 1.18. 1H NMR (250 MHz, CDCl3, δ in ppm): 2.67 (td, CH2CHO, 2H), 3.60 (m, CH2 of PEO,

Table 1. Coupling Reactions Yields between Copolymer 1 and (4-Isothiocyanatophenyl)-r-D-mannopyranose b According to Experimental Conditions (Procedure A) run

reaction medium

1 2 3 4 5 6 7

micellar solutionb THF DMF

[b]/[(1)] (mol/mol)

R-end functionalization yield (%)a

5 10 25 10 25 10 25

18 18 21 40 59 23 40

a Coupling reaction with MM 2.1.2 -EO (7) (102)-NH2 block copolymer end functionalization yield ) 98%. b Carbonate buffer pH 9.5.

4H), 9.77 (t, CHO, 1H). 13C NMR (62.9 MHz, CDCl3, δ in ppm): 43.6 (CH2), 60.1 (CH2OH), 64.6 (CH2O), 70.0 (CH2 of PEO), 72.3 (CH2 in β position of OH), 201.0 (CHO). End functionality ) 87%. PMM 2.1.2-b-PEO-CHO Copolymer. GPC (THF, PS standards): polydispersity index (Ip) ) 1.30. 1H NMR (250 MHz, CDCl3, δ in ppm): 1.30 (m, CH3 of PMM 2.1.2, 6H), 2.67 (td, CH2-CHO, 2H), 2.80 (m, CH2 of the main chain of PMM 2.1.2, H), 3.60 (m, CH2 of PEO, 4H), 4.10 (m, CH2 of CH2-CH3 of PMM 2.1.2, 4H), 4.50 (m, CH2 of PMM 2.1.2, 2H), 9.77 (t, CHO, 1H). End functionality ) 95%. Coupling of Mannosyl Derivatives to r-Amino PEO-b-PMM 2.1.2 or r-Aldehyde PEO-b-PMM 2.1.2 Copolymers 1 and 3. Procedure A: Synthesis of Mannosylated PEO-b-PMM 2.1.2 Block Copolymers 4 by Conjugation between the Copolymer 1 and (4-Isothiocyanatophenyl)-R-D-mannopyranose b (Table 1). In a buffer solution pH 9.5: A typical procedure for coupling H2NPEO-b-PMM 2.1.2 copolymer 1 to an isothiocyanatebearing mannosyl derivative was as follows: a solution of 150 mg of copolymer 1 in 12 mL of N,N-dimethylacetamide was dialyzed against a carbonate buffer solution pH 9.5. Subsequently, 5, 10, and 25 mol equiv per polymer of b were added to the micellar solution, which was stirred for 24 h at room temperature. The micelle solutions were then dialyzed against water for 24 h and finally lyophilized. In organic solvent: 1 (150 mg) was dissolved in 20 mL of DMF or THF. Subsequently, 10 and 25 mol equiv per polymer of b and 50 mol equiv per polymer of N-methylmorpholine were added to the mixture. After a one-day reaction, unreacted mannosyl derivatives were removed from the solution by dialysis for 24 h. The micelle solutions were collected and lyophilized. 1H NMR (250 MHz, CDCl3, δ in ppm): 1.30 (m, CH3 of PMM 2.1.2, 6H), 2.80 (m, CH2 of the main chain of PMM 2.1.2, 2H), 3.60 (m, CH2 of PEO, 4H), 4.10 (m, CH2 of CH2CH3 of PMM 2.1.2, 4H), 4.50 (m, CH2 of PMM 2.1.2, 2H), 5.40 (d, Hano, 1H), 7.10 (d, aromatics, 2H), 7.22 (d, aromatics, 2H). Procedure B: Synthesis of Mannosylated PEO-b-PMM 2.1.2 Block Copolymers 5 by Reductive Amination between Copolymer 3 and (4-Aminophenyl)-R-D-mannopyranose a (Table 2). In buffer solution pH 7.0: A typical procedure for the coupling of OHC-PEO-b-PMM 2.1.2 block copolymers with an amino-bearing mannose was as follows: a solution of 150 mg of polymer 3 in 30 mL of N,Ndimethylacetamide was dialyzed against carbonate buffer solution pH 7.0. Subsequently, 5 and 10 mol equiv per polymer of a were added to the micelle solution. After 1 h stirring, 5 and 10 mol equiv per polymer of NaBH3CN were introduced into the reactor to reduce the Schiff’s base formed between the aldehyde groups of 3 and the primary amino groups of a. After a four-day reaction, the micelle solutions were then dialyzed against water for

226 Bioconjugate Chem., Vol. 16, No. 1, 2005

Studer et al.

Table 2. Coupling Reactions Yields between Copolymer 3 and (4-Aminophenyl)-r-D-mannopyranose a According to Experimental Conditions (Procedure B)

run

solvent

8 micellar solutionb 9 10 anhydrous DMF 11

ratio [a]/[(3)] (mol/mol) 5 10 5 10

reducing agent NaBH3CN NaBH(OAc)3

R-end functionalization yield (%)a 44 37 74 85

a Coupling reaction with MM 2.1.2 (30)-EO(96)-CHO block copolymer end functionalization yield ) 95%. b Phosphate buffer pH 7.

24 h in order to remove the unreacted mannosyl derivatives and the salts and finally lyophilized. In organic solvent: Anhydrous DMF (25 mL) was added to 100 mg of 3. Amounts of 5 and 10 mol equiv per polymer of a were then added to the mixture which was stirred for 1 h. Subsequently, 5 and 10 mol equiv per polymer NaBH(OAc)3 were introduced into the reactor, and the mixtures were stirred for 24 h. The purification procedure was the same as described before. 1H NMR (250 MHz, CDCl3, δ in ppm): 1.30 (m, CH3 of PMM 2.1.2, 6H), 1.85 (quin, CH2, 2H), 2.80 (m, CH2 of the main chain of PMM 2.1.2, 2H), 3.60 (m, CH2 of PEO, 4H), 4.10 (m, CH2 of CH2CH3 of PMM 2.1.2, 4H), 4.50 (m, CH2 of PMM 2.1.2, 2H), 5.38 (d, Hano, 1H), 6.53 (d, aromatics, 2H), 6.87 (d, aromatics, 2H) (Figure 1). As described by Monsigny et al. (33), the sugar contents in the polymer samples were determined by a microscale colorimetric assay, in which the mannose residue reacts with resorcinol in the presence of 75% sulfuric acid solution. This easy-to-handle microplate assay has been found to be suitable to quantitatively determine the sugar content of samples. Note that we checked by GPC that the dialysis-purified polymer samples do not contain free mannosyl derivative. RESULTS AND DISCUSSION

The synthesis of mannosylated PEO-b-PMM 2.1.2 block copolymers was carried out through the two following steps: (i) synthesis of R-amino or R-aldehyde PEOb-PMM 2.1.2; (ii) coupling of the mannosyl derivatives to these R-functionalized copolymers. The PEO precursor was analyzed by GPC using PEO standards for the determination of its molecular weight and polydispersity index. From the chemical composition of the purified copolymers determined by 1H NMR, molecular weights of copolymers were calculated on the assumption that the block copolymer samples contained neither unreacted PEO nor PMM 2.1.2 homopolymer. In fact, it was clearly established from GPC profiles that copolymer samples did not contain a detectable amount of PEO homopolymer. The elimination of eventual traces of PMM 2.1.2 homopolymer from block copolymers was achieved by precipitation of the copolymerization reaction products into a large amount of diethyl ether, a wellknown and good solvent of PMM 2.1.2. The R-end functionalization yield was determined as following. The number-average molecular weight of the R-functionalized PEO homopolymer, which is the precursor for the preparation of the functionalized PEO-b-PMM 2.1.2 copolymers, was determined by GPC and allowed the calculation of the number n of ethylene oxide units per chain and thus per end group, whatever its functionality. The relative intensity of the signals corresponding to the R-end group protons as compared to that of the methylene protons of the PEO segment were obtained

from the 1H NMR spectra and allowed the determination of the number of functionalized end groups per n units and thus the calculation of the R-end functionalization yield. Calculations of R-functionalized PEO-b-PMM 2.1.2 copolymers 2-5 made use of 1H NMR signals corresponding to the PMM 2.1.2 protons. Peaks corresponding to protons adjacent to or characteristic from amino, acetal, aldehyde, or mannosyl moieties were clearly observed in the spectra of the R-functionalized PEO homopolymers and/or of the PEO-b-PMM 2.1.2 block copolymers and enabled the easy determination of end functionalization yields. Furthermore, the chemical nature of the end group was confirmed by 13C NMR (29), and the values obtained were in good agreement with those given by Zalipsky (34). Synthesis and Characterization of r-Amino PEOb-PMM 2.1.2 Block Copolymers 1. Most of them involving amino-protected initiators, different methods have been previously reported in the literature for the synthesis of R-amino PEO (32, 35-38). We selected the easiest one that consists of initiating the polymerization of EO by aminoethanolate as described by Mosquet et al. (32). These authors showed that due to the high reactivity of the potassium alcoholate with respect to those of the amino group, the ring opening reaction of ethylene oxide by the amino group is avoided in a dry aprotic medium like THF. In our study, the numberaverage molecular weight and polydispersity index were 4470 g/mol and 1.30, respectively, for the R-amino PEO, i.e., a degree of polymerization of 102. The weightaverage molecular weight of the PMM 2.1.2 segment was estimated to be 1500 g/mol based on the molecular weight of the PEO segment and on the MM 2.1.2/EO ratio as determined by 1H NMR. Probably due to the low solubility of the potassium 2-aminoethanolate in THF, the polydispersity index of the R-amino PEO is slightly higher than those obtained with soluble initiators (32). This MM 2.1.2(7)-EO(102)-NH2 copolymer thus prepared has an excellent R-amino end-functionality of 98% which was confirmed by acido-basic titration with NaOH. Moreover, the elemental analysis of the nitrogen content was in good agreement with the NMR/GPC data (29). Synthesis and Characterization of r-Acetal PEOb-PMM 2.1.2 Block Copolymers 2. The synthesis of R-acetal and R-aldehyde PEO homopolymers or corresponding copolymers has already been extensively described by Kataoka et al. (39-42). For the R-acetal PEO synthesized in the present study, the number-average molecular weight and polydispersity index were 4200 g/mol and 1.18, respectively. As expected, when soluble initiators are used, the polydispersity index value is lowered. The weight-average molecular weight of the PMM 2.1.2 segment was estimated at 6760 g/mol based on the molecular weight of the PEO segment and on the MM 2.1.2/EO molar ratio obtained from 1H NMR data. This MM 2.1.2(30)-EO(96)-acetal copolymer showed a good R-acetal end-functionalization yield of 95%, which was in agreement with those given by Scholz et al. (41). Synthesis and Characterization of r-Aldehyde PEO-b-PMM 2.1.2 Block Copolymers 3. In water, core-shell-structured micelles were formed by the segregation of the hydrophobic PMM 2.1.2 blocks into the core, which is surrounded by a hydrophilic shell composed of PEO blocks. Thus, as confirmed by 1H NMR, this structure prevents possible acidic hydrolysis of the PMM 2.1.2 esters, the MM 2.1.2/EO ratio being the same before and after the 2-h hydrolysis. The MM 2.1.2(30)-EO(96)-CHO copolymer thus prepared showed an R-aldehyde end-

Synthesis of Mannosylated Amphiphilic Copolymers

Figure 1.

1H

Bioconjugate Chem., Vol. 16, No. 1, 2005 227

NMR spectrum of R-mannosyl-functionalized POE-b-PMM 2.1.2 5 prepared by reductive amination.

Scheme 3. Coupling Reaction between (4-Isothiocyanatophenyl)-r-D-mannopyranose b and the r-amino Ended Copolymer (1)

functionalization yield of 95%, similar to the previous one given above. Coupling of Mannosyl Derivatives to r-Amino PEO-b-PMM 2.1.2 and to r-Aldehyde PEO-b-PMM 2.1.2 Copolymers 1 and 3. Mannosyl derivatives were conjugated to the R-functionalized PEO end, either by reaction of the amine function of 1 with the phenylisothiocyanate group of b, or by the reaction of the aldehyde function of 3 with the amino group of a. The bound mannosyl derivative content was determined by the resorcinol colorimetric method after free unreacted mannosyl derivatives were completely removed by dialysis. As mentioned by Yamamoto et al. (13), there might be about 5% mol equiv ligand per polymer being noncovalently linked to the block copolymer; hence, the measured mannosyl derivative content could include a small amount of physically adsorbed sugar. In fact, a GPC calibration test showed that less than 0.08% (w/w) of free mannose derivatives (either a or b) per purified copolymer sample could be detected, that corresponded to a maximum of 3% mol equiv per polymer of unbound mannosyl derivative, in considering a polymer sample with a weight-average molecular weight of 10000 g/mol (29). Furthermore, the accuracy of the coupling yields were confirmed by 1H NMR analysis. The reaction conditions and the R-glycosylation yields are summarized in Table 1. No change in the GPC peak profile was observed by mannosylation of the precursor polymers. Our goal was to implement a strategy to compare the efficiency of two different coupling reactions: (i) one using R-amino copolymers 1; (ii) the other using R-aldehyde copolymers 3. H2N-PEO-b-PMM 2.1.2 Block Copolymer. The coupling reaction (Scheme 3) was performed either in a hetero-

geneous buffer solution at pH 9.5 (micellar solution) or in a homogeneous medium such as THF or DMF. The results given in Table 1 show that, probably because of the better solubility of copolymer 1 in these solvents, the highest coupling efficiency were achieved in organic solvents and especially in THF. OHC-PEO-b-PMM 2.1.2 Block Copolymer. The two most commonly used reductive amination methods differ in the nature of the reducing agent. In the first one, the catalytic hydrogenation involving platinum or palladium catalysts gives a mixture of products and has limited use especially in the presence of reducible functional groups. The second one utilizes hybride reducing agents such as borohydrides. The reductive amination between the R-aldehyde ended copolymer 3 and the amine-bearing mannosyl derivative a were carried out with two different reducing agents. The choice of the reducing agent was critical for the success of the reaction, since the latter must reduce imines selectively over aldehydes under the reaction conditions. In our procedure, the reductive amination was indirect, as the reaction involved the preformation of the imine intermediate followed by reduction in a subsequent separate step. Depending on the nature of the reducing agent, the coupling reaction (Scheme 4) was performed either in aqueous buffer solution pH ) 7.0 as described by Yamamoto et al. (13) or in anhydrous DMF as suggested by Abdel-Magid et al. (43). The results given in Table 2 show that the reductive amination was an efficient procedure. In the presence of NaBH3CN, which is a specific reducing agent in buffer solution, the coupling yields were similar to those of Yamamoto et al. (13) obtained for the coupling of amino acids with PDLLA-b-PEO-CHO. Note that excellent coupling yields could be reached with sodium

228 Bioconjugate Chem., Vol. 16, No. 1, 2005

Studer et al.

Scheme 4. Coupling Reaction between the (4-aminophenyl)-r-D-mannopyranose a and the r-Aldehyde Ended Copolymer (3)

Table 3. Size of Micelles Obtained with Various r-Substituted Copolymers copolymer

functionalization

average diameter in weight (nm)a

2 3 5

acetal CHO O-Man

56.2 ( 2.3 56.2 ( 2.0 56.0 ( 1.9

a Acetal-EO (96)-MM 2.1.2(30) block copolymer (end functionalization yield ) 95%) was used as a starting material.

triacetoxyborohydride NaBH(OAc)3 in DMF, this mild and selective reducing agent being suitable for reductive reactions to be carried out in organic solvents such as DMF as mentioned by Abdel-Magid et al. (43). Figure 1 shows the 1H NMR spectrum of R-mannosyl-functionalized PEO-b-PMM 2.1.2 as a typical example. From these results, it turned out that the synthesis of R-mannosyl-functionalized PEO-b-PMM 2.1.2 has preferably to be carried out in an organic medium such as THF or DMF which are common solvents of both sequences of the block copolymers. Furthermore, it appeared that the highest coupling yield (74-85%) is obtained in organic medium by reductive amination between R-aldehyde-ended copolymers 3 and amine-bearing mannosyl derivative a, this yield being quite comparable to those of 65% and 90% given by Nakamura et al. (18) and Yasugi et al. (19), respectively, which were obtained by initiating the polymerization with a protected monosaccharide residue. In addition, this procedure has a doubleadvantage: (i) compared to R-amino-functionalized copolymers 1, R-aldehyde-functionalized copolymers 3 have been obtained with a lower polydispersity index; (ii) the amine-bearing mannosyl derivative a was produced in a four-step synthesis, whereas the phenylisothiocyanatebearing mannosyl derivative b needed five steps. Micellization of the r-Acetal, r-Aldehyde and r-Mannosyl-Functionalized PEO-b-PMM 2.1.2 Block Copolymers 2, 3, and 5. As the primary application of these R-substituted PEO-b-PMM 2.1.2 block copolymers consists of preparing polymeric colloidal vectors for drug delivery, it was of interest to determine their micellar characteristics. In aqueous media, these block copolymers self-associate, leading to spherical micelles with a PMM 2.1.2-made hydrophobic core surrounded by a corona of hydrated PEO segments. A typical example of micellar characteristics of R-substituted block copolymers with various end groups is given in Table 3, the composition and molecular weight of the copolymer being kept constant. Dynamic light scattering studies indicated that, in our micellization procedure, the block copolymer leads to a monodisperse micelle population with an average diameter of 56 ( 2 nm, which is in agreement with the predicted value of 49 nm given by Larras et al. (20) for PEO-b-PMM 2.1.2 block copolymers. It was confirmed that the micelle diameter did not change, neither after the hydrolysis of the acetal group as pointed out by

Kataoka et al. (13) nor after the coupling reaction with the mannosyl derivatives a or b. CONCLUSION

Well-controlled molecular weight R-amino and R-aldehyde-functionalized PEO-b-PMM 2.1.2 block copolymers were synthesized with high mean yields. These block copolymers were then successfully conjugated to a mannosyl derivative for the preparation of bioerodible nanocarriers with the aim of developing active tissue targeting strategies. Reductive amination of the R-aldehyde endfunctionalized copolymer with amine-bearing mannosyl derivative in the presence of sodium triacetoxyborohydride in DMF appeared to be the most suitable functionalization procedure. A maximum of 85% of the PEO chain end was R-conjugated to mannosyl derivative without any change in the micellar size. Our studies result in a panel of novel macromolecular entities that can be used as starting materials on which to anchor various targeting ligands bearing various chemical functionalities. The latter can be adapted to the nature of the R-ending reactive function of the PEO-b-PMM 2.1.2 block copolymer, whereas the initiation process by a monosaccharide residue is limited to the sugar moiety only. ACKNOWLEDGMENT

The authors gratefully acknowledge the financial support by Dr. Bru. We thank Drs. Nathalie Colin and Sophie Ruiz for their assistance in the determination of the copolymer-bound mannosyl derivative. We are grateful to Drs. Virginie Larras and Serge Sagodira for their helpful discussions. LITERATURE CITED (1) Yang, L., and Alexandris, P. (2000) Physicochemical aspects of drug delivery and release from polymer-based colloids Curr. Opin. Colloid Interface Sci. 5, 132-143. (2) Nishiyama, N., Okazaki, S., Cabral, H., Miyamoto, M., Kato, Y., Sugiyama, Y., Nishio, K., Matsumura, Y., and Kataoka, K. (2003) Novel cisplatin-incorporated polymeric micelles can eradicate solid tumors in mice. Cancer Res. 63, 8977-8983. (3) Kwon, G. S. (1998) Diblock copolymer nanoparticles for drug delivery Crit. Rev. Ther. Drug Carrier Syst. 15, 481-512. (4) Kataoka, K., Harada, A., and Nagasaki, Y. (2001) Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Delivery Rev. 47, 113131. (5) Torchilin, V. P. (2001) Structure and design of polymeric surfactant-based drug delivery systems. J. Controlled Release 73, 137-172. (6) Harris, J. M., and Zalipsky, S. (1997) Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (Harris, J. M., and Zalipsky, S. Eds.) pp 99-116, Plenum Publishing Corporation, New York. (7) Allen, C., Maysinger, D., and Eisenberg, A. (1999) Nanoengineering block copolymer aggregates for drug delivery. Colloids Surf. B: Biointerfaces 16, 3-27.

Bioconjugate Chem., Vol. 16, No. 1, 2005 229

Synthesis of Mannosylated Amphiphilic Copolymers (8) Davis, S. S. (1997) Biomedical applications of nanotechnology - implications for drug targeting and gene therapy. Trends Biotechnol. 15, 217-224. (9) Riess, G. (2003) Micellization of block copolymers. Prog. Polym. Sci. 28, 1107-1170. (10) Couvreur, P. (1988) Polyalkylcyanocrylates as colloidal drug carriers. Crit. Rev. Ther. Drug Carrier Syst. 5, 1-20. (11) Lherm, C., Mu¨ller, R. H., Puisieux, F., and Couvreur, P. (1992) Alkylcyanoacrylate drug carriers: II. Cytotoxicity of cyanoacrylate nanoparticles with different alkyl chain length. Int. J. Pharm. 84, 13-22. (12) Lescure, F., Seguin, C., Breton, P., Bourrinet, P., Roy, D., and Couvreur, P. (1994) Preparation and characterization of novel poly(methylidene malonate 2.1.2)-made nanoparticles. Pharm. Res. 11, 1270-1277. (13) Yamamoto, Y., Nagasaki, Y., Kato, M., and Kataoka, K. (1999) Surface charge modulation of poly(ethyleneglycol)-poly(D,L-lactide) block copolymer micelles: conjugation of charged peptides. Colloids Surf. B: Biointerfaces 16, 135-146. (14) Harris, J. M. (1985) Laboratory synthesis of poly(ethyleneglycol) derivatives. Macromol. Chem. Phys. C25, 325-373. (15) Harris, J. M., Struck, A. C., Case, M. G., Paley, M. S., Van Alstine, and J. M., Brooks, D. E. (1984) Synthesis and characterization of poly(ethylene glycol) derivatives. J. Polym. Sci., Polym. Chem. 22, 341-352. (16) Zalipsky, S., Gilon, C., and Zilkha, A. (1983) Attachment of drugs to poly(ethyleneglycol)s. Eur. Polym. J. 19, 11771183. (17) Nagasaki, Y., and Kataoka, K. (1998) Heterotelechelic poly(ethylene glycol)s and their derivatives for active targeting drug delivery systems. ACS Polym. Prepr. 39, 190-191. (18) Nakamura, T., Nagasaki, Y., and Kataoka, K. (1998) Synthesis of heterobifunctional poly(ethylene glycol) with a reducing monosaccharide residue at one end. Bioconjugate Chem. 9, 300-303. (19) Yasugi, K., Nakamura, T., Nagasaki, Y., Kato, M., and Kataoka, K. (1999) Sugar-installed polymer micelles: synthesis and micellization of poly(ethylene glycol)-poly(D,Llactide) block copolymers having sugar groups at the PEG chain end. Macromolecules 32, 8024-8032. (20) Larras, V., Bru, N., Breton, P., and Riess, G. (2000) Synthesis and micellization of amphiphilic poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) diblock copolymers. Macromol. Rapid Commun. 21, 1089-1092. (21) Bru-Magniez, N., Larras, V., Riess G., Breton, P, Couvreur, P., and Roques Carmes, C. Novel surfactant copolymers based on methylidene malonate. WO 99/38898 (1999)/FR19980001001 (1998) (22) Seymour, L. W. (1994) Soluble polymers for lectin-mediated drug targeting. Adv. Drug Delivery Rev. 14, 89-111. (23) Gariepy, J. G., and Kawamura, K. (2001) Vectorial delivery of macromolecules into cells using peptide-based vehicles. Trends Biotechnol. 19, 19-26. (24) Sudimack, J., and Lee, R. J. (2000) Drug targeting via the folate receptor. Adv. Drug Delivery Rev. 41, 147-162. (25) Rihova, B., Ulbrich, K., Kopecek, J., and Mancal, P. (1983) Immunogenicity of N-(2-hydroxypropyl)-methacrylamide copolymers-potential hapten or drug carriers. Folia Microbiol. 28, 217-227. (26) Bru-Magniez, N., De Cock, C., Poupaert, J., De Keyser, J. L., and Dumont, P. (1988) Monoesters or diesters of endoethano-9,10-dihydro-9,10-anthracene dicarboxylic-11,11 acid, process for their preparation, and their use in the preparation of symmetric or asymmetric methylidene malonates. EP 0283364. (27) Scherf, G. W. H., and Brown, R. K. (1960) Potassium derivatives of fluorene as intermediates in the preparation of C-9-substituted fluorenes. II. Comparative reactivities of the alkali metals, lithium, sodium, and potassium, with fluorene in glycol dimethyl ether, glycol diethyl ether, tetrahydrofuran, and dioxane. Can. J. Chem. 38, 2450-2456.

(28) Lerner, L. M., and Mennitt, G. (1994) A new synthesis of L-talose and preparation of its adenine nucleosides. Carbohydr. Res. 259, 191-200. (29) Studer, P. (2001) Ph.D. Thesis, University of Haute-Alsace (France), Synthe`se et caracte´risation de polyme`res fonctionnalise´s et de copolyme`res a` base de me´thylide`ne malonate 2.1.2. Application a` la vectorisation de principes actifs. (30) Smits, E., Engberts, J. B. F. N., Kellogg, R. M., and Van Doren, H. A. (1996) Reliable method for the synthesis of aryl β-D-glucopyranosides, using boron trifluoride-diethyl ether as catalyst. J. Chem. Soc., Perkin Trans. 1 2873-2877. (31) Monsigny, M., Roche, A. C., and Midoux, P. (1984) Uptake of neoglycoproteins via membrane lectin(s) of L1210 cells evidenced by quantitative flow cytometry and drug targeting. Biol. Cell. 51, 187-196. (32) Mosquet, M., Chevalier, Y., Le Perchec, P., and Guicquero, J. P. (1997) Synthesis of poly(ethylene oxide) with a terminal amino group by anionic polymerization of ethylene oxide initiated by amino alcoholates. Macromol. Chem. Phys. 198, 2457-2474. (33) Monsigny, M., Petit, C., and Roche, A. C. (1988) Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod. Anal. Biochem. 175, 525-530. (34) Zalipsky, S. (1995) Functionalized poly(ethylene glycol) for preparation of biologicaly relevant conjugates. Bioconjugate Chem. 6, 150-165. (35) Kim, Y. J., Nagasaki, Y., Kataoka, K., Kato, M., Yokoyama, M., Okano, T., and Sakurai, Y. (1994) Heterobifunctional poly(ethylene oxide) - One pot synthesis of poly(ethylene oxide) with a primary amino group at one end and a hydroxyl group at the other end. Polym. Bull. 33, 1-6. (36) Yokoyama, M., Okano, T., Sakurai, Y., Kikuchi, A, Ohsako, N, Nagasaki, Y, and Kataoka, K. (1992) Synthesis of poly(ethylene oxide) with heterobifunctional reactive groups at its terminals by an anionic initiator. Bioconjugate Chem. 3, 275-276 (37) Nagasaki, Y., Iijima, M., Kato, and M., Kataoka, K. (1995) Primary amino-terminal heterobifunctional poly(ethylene oxide). Facile synthesis of poly(ethylene oxide) with a primary amino group at one end and a hydroxyl group at the other end. Bioconjugate Chem. 6, 702-704. (38) Cammas, S., Nagasaki, Y., and Kataoka, K. (1995) Heterobifunctional poly(ethylene oxide): synthesis of R-hydroxyω-amino PEOs with the same molecular weights. Bioconjugate Chem. 6, 226-230. (39) Otsuka, H., Nagasaki, Y., and Kataoka, K. (2000) Surface characterization of functionalized polylactide (PLA) through the coating with heterobifunctional poly(ethylene glycol)(PEG)/PLA block copolymers. Biomacromolecules 1, 39-48. (40) Scholz, C., Iijima, M., Nagasaki, Y., and Kataoka, K. (1998) Polymeric micelles as drug delivery systems: a reactive polymeric micelle carrying aldehyde groups. Polym. Adv. Technol. 9, 1-9. (41) Scholz, C., Iijima, M., Nagasaki, and Y., Kataoka, K. (1995) A novel reactive polymeric micelle - Polymeric micelle with aldehyde groups on its surface. Macromolecules 28, 72957297. (42) Nagasaki, Y., Kutsuna, T., Iijima, M., Kato, M., Kataoka, K., Kitano, S., and Kadoma, Y. (1995) Formyl-ended heterobifunctional poly(ethylene oxide): synthesis of poly(ethylene oxide) with a formyl group at one end and a hydroxyl group at the other end. Bioconjugate Chem. 6, 231-233. (43) Abdel-Magid, A. F., Carson, K. G., Harris, B. D., Maryanoff, C. A., and Shah, R. D. (1996) Reductive amination of aldehydes and ketones with sodium triacetoxyborohydride. Studies on direct and indirect reductive amination procedures. J. Org. Chem. 61, 3849-3862.

BC0498065