Heterostereocomplexes Prepared from d-Poly ... - ACS Publications

Biomacromolecules 2003, 4, 1308-1315

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Heterostereocomplexes Prepared from D-Poly(lactide) and Leuprolide. I. Characterization Joram Slager and Abraham J. Domb* Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University, Campus Ein Karem, Jerusalem 91120, Israel Received March 25, 2003; Revised Manuscript Received June 11, 2003

Heterostereocomplexes between D-PLA and L-peptides, obtained by spontaneous precipitation from acetonitrile solution, were characterized by thermal analysis and microscopic techniques. Differential scanning calorimetry showed two transition endotherms, one for the R form that melts at 178 °C and one for the β form of PLA that melts at 169 °C. A linear correlation was found between the enthalpy of both melt temperatures and the peptide concentration. The complexation was monitored by a change in morphology, which was imaged by AFM-tapping mode. The initial fibrous network of D-PLA changed to uniform disks of 100 nm in diameter and 2.5 nm in height of the heterostereocomplex. Rhodamine B labeled leuprolide was complexed selectively to D-PLA, which was chemically bound onto mica plates. Addition of L-PLA to the complex enabled displacement of the peptide, which was observed by fluorescent spectrometry and confocal microscopy. These results provide a method, which enables one to obtain an expression for the relative interaction strength between various stereoselective polymers and polypeptides with opposite enantiomeric configuration. Introduction The occurrence of complexes formed by two different small molecules with opposite enantiomeric configuration is a well-known phenomenon which can be used in the separation of a racemate into its enantiomers. Examples are the complexes of tartaric acid, strychnine, or brucine, with amino acids of complementary enantiomeric configuration. These complexes are commonly referred to as “diastereomers” or “diastereomeric salts”. In macromolecular chemistry, until recently, complexes have been reported consisting of pairs of polymeric enantiomers, such as D-PLA and L-PLA. These polymeric complexes are referred to as homostereoselective complexes.1 In the homostereocomplex formation, the polymer chains generally adopted helical conformations which intertwined or formed a crystal lattice with alternating D- and L-polymer chains2. The physicochemical properties of the stereocomplex were observed to differ from those of the enantiomeric parent compounds. Characteristic changes in properties of the complexed polymers were an increase in melt transition temperature and insolubility in common solvents.1,3 D- and L-poly(lactic acid) (PLA) were reported to form a stereocomplex which was characterized initially by Ikada et al.4 The complex was found to be insoluble in commonly used solvents such as chloroform, and the melt transition temperature had shifted from 178 to 230 °C.5,6 Also, block copolymers containing segments of D- and L-PLA with poly(ethyleneglycol),7,8 poly(glycolic acid),9 or poly(epsiloncaprolactone)10,11 or in graft-copolymers with dextrane12,13 * To whom correspondence should be addressed. Prof. Abraham J. Domb, Dept of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University, Campus Ein Karem, Jerusalem 91120, Israel, Phone: ++972-2-6757573. Fax: ++972-2-6758959. E-mail: [email protected].

or poly(2-hydroxyethyl methacrylate)7 were reported to form stereocomplexes. We recently discovered macromolecular diastereomer complexes, in which L-configured poly(amino acids) were observed to form a heterostereoselective complex with polyesters of D-configured lactic acid. No complexes were formed with the L-lactic or D,L-lactic acid polymers.14,15 These new type of macromolecular complexes are referred to as heterostereocomplexes. This discovery provides an alternative approach for controlled delivery of peptides and proteins. In our recent publications, controlled release of various peptide drugs from their heterostereocomplexes with D-PLA has been described.14-18 This work focuses on the characterization of the heterostereocomplex by thermal analysis and microscopic techniques. Leuprolide, a potent nonapeptide analogue of the luteinizing hormone releasing hormone (LHRH), has been used as a representative peptide in this study. Material and Methods Materials. Chemicals and solvents were purchased from Sigma-Aldrich Israel or from Mallinckrodt-J. T.Baker BV, Deventer, Holland. D- and L-lactides were purchased from Purac BV, Gorinchem, Holland. Stannous(II)bis-2-ethylhexanoate (Sn(Oct)2) was obtained from Sigma-Aldrich, Israel. Leuprolide acetate was purchased from Novetide Ltd., Israel; Lissamine-Rhodamine B L20 was obtained from Molecular Probes Inc., U.S.A. Thermal analysis was determined on a Mettler TA 4000DSC differential scanning calorimeter (DSC), calibrated with zinc and indium standards, at a heating rate of 10 °C/min. Scanning electron microscopy was performed on a SEM 505,

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Characterization of Heterostereocomplexes

Philips, Holland. Gold coating of samples prior to SEM analysis was performed using Polaron E 5100-2, (Holywell) Polaron Equipment Ltd., England. Molecular weights were estimated using a gel permeation chromatography (GPC) system consisting of an isocratic pump (Waters 1515), refractive index detector (Waters 2410), and Breeze 3.20 software (Waters Corp., Milford, U.S.A.). Samples were eluted with CHCl3 through a linear Styrogel column (Waters 7.8 × 300 mm, 10 µm pore size) at a flow rate of 1 mL/ min. The molecular weights were determined relative to polystyrene standards (Polyscience, Warrington, PA). NMR spectra were obtained on a Varian 300 MHz, using samples dissolved in deuterated chloroform. Atomic force microscopy (AFM) was performed on a Nanoscope III-Tapping Mode, Digital Instruments (Veeco, USA). Rectangular Si cantilevers (Nano-probes) were applied for the tapping mode experiments. Confocal microscopy was done on a Carl Zeiss 135M Confocal Microscope. Synthesis of PLA. Stereoselective and racemic PLA were synthesized by ring opening polymerization of D,D or L,L or a racemic mixture of lactides, using stannous 2-ethylhexanoate (Sn(Oct)2) as catalyst and different alcohols as cocatalysts3. In a typical polymerization reaction to form low molecular weight PLA (Mw 3 kDa), cocatalyst (benzyl alcohol, 3 g, 28 mmol) was added to a lactide solution in toluene (20 g, 140 mmol in 300 mL). The solution was dried by refluxing over a Dean-Stark apparatus for 3 h, and Sn(Oct)2 (280 mg, 0.7 mmol) was added. The mixture was stirred for 2 h, and the solvent was evaporated. The crude residue was dissolved in a small amount of chloroform and precipitated with 2-propanol. NMR: δ ) 5.15 (1H, q, CH); δ ) 1.60 (3H, d, CH3). Higher molecular weight polymers (>50 kDa) were prepared without alcoholic cocatalyst. After initial polymerization with Sn(Oct)2 for 3 h in toluene, the solvent was distilled off. The crude residue (Mw between 20 and 30 kDa) was further polymerized in bulk overnight at 135 °C to obtain the desired high molecular weight polymers. Heterostereocomplex Formation from Solution. Stereocomplexes of D-PLA with leuprolide (2 to 20% w/w in relation to polymer) were obtained by precipitation from an acetonitrile solution. In a typical experiment, D-PLA (18 mg) was mixed with leuprolide (2 mg, 10% w/w) in acetonitrile (1 mL) in glass ampules containing a micro stirrer. After sealing the ampule, the mixture was stirred at 60 °C for 3 days during which the clear solution turned turbid. A white precipitate was formed. It was isolated by either filtration or centrifugation. The precipitate was dried overnight in a vacuum over P2O5. The thermal behavior of the complexes was analyzed by DSC. SEM micrographs were taken from various complexes under high vacuum using 30 kV accelerating voltage. Particles were deposited on a carbon film followed by gold coating, performed in a vacuum for one minute at 20 mA. Binding Experiments on Mica Surface. Rhodamine-B Leuprolide. Leuprolide (2 mg) was dissolved in water (100 µL) and added to borate buffer pH 11 (1 mL). RhodamineB-sulfonyl chloride (lissamine) (2.89 mg, 5 µmol) in dimethyl formamide (DMF, 145 µL) was added, and the reaction

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mixture was shaken at room temperature for 30 min. The product was purified by separation on a G10 Sephadex column and monitored by HPLC (C18-column with a 70:30 v/v mixture of triethylammonium phosphate (TEAP) buffer pH 3 and acetonitrile at a flow rate of 1 mL/min; detection at 278 nm). Preparation of PLA Nitrophenyl Ester. PLA (3 kDa, 4 g, 1.6 mmol) was dissolved in dichloromethane (50 mL) together with p-nitrophenyl chloroformate (1.3 g, 6.4 mmol) and triethylamine (1.3 mL, 0.95 mg, 9.4 mmol). The reaction mixture was stirred overnight at room temperature. The polymer solution was concentrated by evaporation and precipitated with 2-propanol. 1 H NMR: 8.28 (d, 2H, p-nitrophenyl); 7.40 (d, 2H, p-nitrophenyl) and 5.15 (1H, q, CH); 1.60 (3H, d, CH3). (See Scheme 1 A.) Surface Modification of Mica. Freshly cleaved pieces of mica (0.5 × 0.5 cm) were treated with a solution of aminopropyl triethoxysilane (APTES) (5% v/v) in dry THF for 3 h in a tightly closed glass vial at room temperature. The mica surfaces were rinsed thoroughly with THF and ethanol and cured overnight at 120 °C. The modified mica plates were subsequently immersed in a dichloromethane solution (1 mL) containing D-PLA p-nitrophenyl ester (3 kDa, 10 mg, 1% w/v), in the presence of dimethylaminopyridine (DMAP, 10 mg), see Scheme 1 A. The reaction mixture was left at room temperature for 2 days. The colorless solution became yellow. The plates were thoroughly washed with dichloromethane and ethanol and dried under vacuum. Images at various stages of this process, measuring height and phase differences simultaneously, were recorded by AFM-tapping mode. Dansyl Labeled D- and L-PLA. Dansyl chloride (1 g, 3,7 mmol) was dissolved in acetone (15 mL) and added to an aqueous solution of 2-aminoethanol (270 mg, 4.4 mmol) and sodium carbonate (0.4 g, 3.8 mmol), see Scheme 1B. The reaction was followed by thin layer chromatography (TLC), 5% methanol in dichloromethane, while stirring at room temperature for 1 h. The product was isolated by extraction with dichloromethane. 1H NMR: 8.52 (d, 1H, naphthyl); 8.28 (m, 2H, naphthyl); 7.53 (m, 2H, naphthyl); 7.19 (d, 1H, naphthyl); 5.35-5.50 (br s, 1H, -SO2NH-); 3.6 (t, 2H, NH-CH2-CH2-OH); 3.04 (q, 2H, NH-CH2-CH2-OH); 2.89 (s, 6H, N(CH3)2). N-2-Hydroxyethyldansylsulfonamide (100 mg, 0.34 mmol, 0.01 eq.) was used as cocatalyst in the ring opening polymerization of D- or L-lactide (5 g, 34.7 mmol) in the presence of a catalytic amount of stannous 2-ethylhexanoate (141 mg, 1.4 mmol) in toluene (250 mL). The reaction was performed at 135 °C for 1 h. The crude polymer was dissolved in a small amount of chloroform and precipitated in 2-propanol. The properties of the polymers are given in Table 1. Leuprolide Binding Experiment. Freshly cleaved mica and mica modified with D- or L-PLA, were immersed in acetonitrile/water 9:1 v/v (1 mL), containing 0.05% w/v rhodamine B-labeled leuprolide, for different time periods. The plates were then thoroughly washed with acetonitrile/water solution and dried in a vacuum. The pieces were attached to a

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Scheme 1. A. Surface Modification of Mica with Aminopropyltriethoxy Silane (APTES) and p-nitrophenyl Activated Enantiomeric PLA and B. Inclusion of Dansyl-fluorophore in PLAa

a PLA was activated by coupling to p-nitrophenyl chloroformate in the presence of triethylamine in dichloromethane, while stirring overnight. Mica was treated with APTES solution in anhydrous THF for 3 hours, rinsed thoroughly, heated in oven overnight at 120 °C. It was then reacted by immersion in a dichloromethane solution of D-PLA p-nitrophenyl ester in the presence of dimethyl aminopyridine. B. Inclusion of dansyl-fluorophore in PLA. Dansyl sulfonyl chloride in acetone was added to an aqueous solution of 2-aminoethanol and sodium carbonate. The product was used as co-initiator together with stannous octoate in the ring opening polymerization of enantiomeric lactides in dry toluene.

Table 1. Properties of the Lactide Polymers Used in This Study polymer D-PLA D-PLAb D-PLAb L-PLA L-PLAb L-PLAb D,L-PLA

dansyl L-PLA

cocatalyst

mol. ratio LA:Cat

Mwa (Da)

polydispersity indexa

melt transition temp (°C)

benzyl alcohol octanol decanol benzyl alcohol octanol decanol octanol dansyl-2-hydroxy ethylamide

5:1 50:1 1000:1 5:1 250:1 1000:1 100:1 100:1

2800 10 000 122 000 2800 30 000 120 000 43 000 16 200

1.05 1.2 2.2 1.05 1.2 1.4 1.53 1.1

120 158 179 120 170 178 no transition 170

a Molecular weights were determined by GPC. b Polymers are soluble in acetonitrile at 50 °C. Other polymers are soluble in acetronitrile at room temperature. Polymers were purified by precipitation with a 1:1 v/v mixture of ether/petroleum ether or with isopropanol.

microscope slide and scanned face down under a confocal microscope, using He-Ne laser beam: excitation at 543 nm, emission at LP570 nm. Leuprolide Displacement Study. Freshly cleaved or surface modified mica pieces were immersed in acetonitrile/water (9:1 v/v) (1 mL), containing leuprolide (0.05% w/v) for 12 h, and subsequently washed thoroughly. The plates were then treated with a dansyl-labeled L-PLA solution (0.1% w/v) in acetonitrile for 45 min and 4 h. The mica plates were thoroughly washed with dichloromethane, dried in a vacuum, and scanned by confocal microscopy using Ar-Ne laser beam excitation at 488 nm, emission at LP515 nm. Sample Preparation on Mica for Atomic Force Microscopy (AFM). On a freshly cleaved piece of mica, a drop

of D-PLA 120 kDa solution in acetonitrile (0.1% w/v) was placed and left at 60 °C until the solvent had evaporated. Under identical conditions, a drop of a leuprolide solution (0.025% w/v) in 9:1 v/v acetonitrile/water was deposited on top of the D-PLA and left to dry at 60 °C followed by overnight vacuum. AFM was performed using the tapping mode. Results and Discussion Synthesis and Complex Formation. The synthesis of PLA was conducted according to known procedures by ring opening polymerization of chiral or racemic lactides. All



Figure 1. Scanning electron microscopy. Stereocomplexes of D-PLA and leuprolide or vaprotide were obtained by spontaneous precipitation from an acetonitrile solution at 60 °C. A. Complex of vaprotide 2% w/w with D- and L-PLA 120 kDa. B. Complex of leuprolide 5% w/w with D- and L-PLA 120 kDa. C. Hetero-stereocomplex of D-PLA (120 kDa) with vaprotide (2% w/w) D. Complex of vaprotide 2% w/w with D- and L-PLA 120 kDa.

polymers were characterized by DSC and GPC. The properties of the polymers used in this study are summarized in Table 1. When stirring a solution of D-PLA and leuprolide in acetonitrile at 60 °C, a precipitate was formed, consisting of uniform microparticles with a mean particle size of 1.7 microns (see Figure 1, parts A and B). Notable is the highly porous surface area of the particles (see Figure 1, parts C and D), as compared to common PLA microspheres having a compact and smooth surface.19 Using a particle size analyzer, the size of leuprolide/D-PLA hetero-stereocomplex particles was analyzed by suspending the precipitate in acetonitrile. The particle size was between 0.98 and 3.26 microns. When using L-PLA or racemic D,L-PLA instead of D-PLA, no turbidity or precipitation occurred when mixed with leuprolide or other peptides and the solution remained clear. Thermal Behavior. Unlike racemic PLA, which is amorphous and lacks any defined transition point, enantiomeric D- or L-PLA is crystalline and melts at around 178 °C, depending upon its molecular weight. The leuprolide/ D-PLA heterostereocomplex shows two transition endotherms: the normal melt transition at 178 °C and a new transition at around 169 °C. A change of the peptide concentration in the heterostereocomplex caused a change in the DSC thermogram, both in the temperature and in the enthalpy values (Figure 2).

Figure 2. DSC thermograms of heterostereocomplexes. The complexes were obtained by spontaneous precipitation from acetonitrile solution with different leuprolide concentrations (2-50% w/w) while stirring at 60 °C for 3 days. The precipitate was isolated and dried in a vacuum overnight prior to DSC analysis. DSC are from first run heating, at a rate of 10 °C/min.

The enthalpy of both transition states was observed to correlate with the peptide content in the heterostereocomplex. In heterostereocomplexes with a peptide content of 2% w/w, the mean enthalpy at the new transition was 13 J/g, whereas the mean enthalpy at 178 °C was 39 J/g. An increase of the peptide concentration to 5% w/w resulted in an inversion of the enthalpy values to 48.5 J/g for the new endotherm and 22.5 J/g for the 178 °C endotherm. These values were

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Figure 3. Influence of the peptide concentration of the endotherms of the heterostereocomplex. Complexes were prepared as described in Figure 2.

Figure 5. Section analysis, zoom of Figure 4B. Sample prepared as described in Figure 4.

Figure 4. Monitoring heterostereocomplex formation of D-PLA and leuprolide by AFM-Tapping mode. A. D-PLA 120 kDa solution in acetonitrile (0.1% w/v) was dropped on freshly cleaved mica and left at 60 °C. B. a drop of leuprolide solution in acetonitrile/water (0.025% w/v) was deposited on top of the D-PLA and left at 60 °C.

gradually restored to their former level by further increasing the peptide concentration in the complex to 50% w/w. The most significant effect was seen on the enthalpy of the new transition (Figure 3). PLA has been reported in the literature to adopt two distinct helical conformations: the most stable 103 R-helical conformation with 10 monomers every three helical turns and with a melt transition at 178 °C (R form) and a 31 helical conformation (β form), a slightly “stretched” R helix, with three monomers per turn, which is thermodynamically less

stable. Also the occurrence of the additional endotherm 10 °C below the melt endotherm was earlier observed in the thermal behavior of enantiomeric L-PLA, and was ascribed to the β conformation of the PLA helix.20,21 Hoogsteen reported that PLA was obtained in the β form of the R helix by spinning fibers of L-PLA from a hot chloroform/toluene solution (close to θ conditions). This β conformation was also observed to be present in the PLA stereocomplex.2,22 Sarasua found the occurrence of the additional transition state only in L-PLA “contaminated” with D-lactic acid monomers (80-60% optical purity) and, in addition, to be dependent on cooling and heating rates prior or during the calorimetry measurements.21 According to one of the explanations offered for the occurrence of the additional endotherm, initially crystals of low perfection in the sample would melt and undergo annealing simultaneously to the DSC experiment, then recrystallize, and melt again at a higher temperature. This would give rise to the second transition state, found for regular enantiomeric PLA.21 This observation, however, may indicate that the β form is mainly involved in the heterostereocomplex formation, losing some of its original crystal structure as it is engaged in complexation. The sharp inversion could originate from the actual change in helical conformation. Complex Formation Observed by Atomic Force Microscopy (AFM). To image crystals of the heterostereocomplex of D-PLA and leuprolide at ambient conditions AFM-tapping mode was applied, using complexes formed on freshly cleaved mica. AFM also enabled the visualization of morphological changes during the heterostereocomplexation by simultaneously monitoring phase differences. Phase



Figure 6. Height and phase images of mica surface modification by AFM-tapping mode. A. After treatment of mica surface (0.5 × 0.5 cm) with 5% v/v aminopropyltriethoxy silane (APTES) in anhydrous THF. B. After reaction with D-PLA p-nitrophenyl ester (3 kDa, 10 mg, 1% w/v) in the presence of dimethylaminopyridine (DMAP) in dichloromethane. See also Scheme 1.

Figure 7. Binding studies with leuprolide. Incubation of D-PLA surface modified mica with rhodamine B-labeled leuprolide for 5, 30, and 60 min, A, B and C, respectively. Scanned by confocal microscopy at 543 nm.

imaging is a technique that has exhibited the ability to provide qualitative information on material microstructure on the nanometer scale. Regions of a microstructure that exhibit incongruous mechanical properties like friction, elastic modulus, composition, and viscoelasticity are displayed, in the resulting image, as regions of differing contrast.23,24 Applying a solution of D-PLA on mica at 60 °C and subsequent evaporation led to the formation of a fibrous network (Figure 4A). Formation of disc-like structures was noticed when casting a leuprolide solution on top of D-PLA, deposited on freshly cleaved mica, whereas the previously observed fibrous network of D-PLA had almost completely disappeared (Figure 4B). The uniform disks were 100 nm in diameter and 2.5 nm in height (Figure 5). Although crystal structures depend very much on the way of sample preparation, disc-shape crystals are commonly found in stereocomplexes of both homo and block copoly-

mers of D- and L-PLA.17 Application of only leuprolide solution on mica did not lead to recognizable pattern formation. Binding Studies using Confocal Microscopy. Modification of mica with covalently bound enantiomeric PLA enabled the performance of initial binding and displacement studies in an attempt to evaluate the kinetics of the D-PLA/ leuprolide hetero-stereocomplex formation. The surface modification was performed according to known procedures using aminopropyltriethoxysilane (APTES) as linker between the mica and the PLA.25,26 The treatment of the mica with APTES and subsequently with a solution of PLA active ester was followed by AFM-Tapping mode, simultaneously monitoring height and phase differences in order to detect changes on the mica surface. The differences in surface properties is shown in the phase images in Figure 6. The dark and light colors originated from the mica surface and the bound

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Figure 8. Displacement of leuprolide from D-PLA surface modified mica with dansyl-labeled L-PLA. Mica surfaces chemically modified with D-PLA were immersed in a 9:1 v/v mixture of acetonitrile/water containing leuprolide (0.05% w/v) for 12 h. The plates were washed and treated with a dansyl-labeled L-PLA solution (0.1% w/v) in acetonitrile for 45 min and 4 h, washed, vacuum-dried, and scanned by confocal microscopy (λex is 488 nm, λem is 515 nm).

APTES, causing a difference in phase of the cantilever. From the height image, the thickness of the layer is measured to be 7 Å in average, with some roughness (maximal heights of 1 nm were observed). When PLA is covalently bound to the mica surface, a fibrous network is obtained, similar to Figure 4A, which was obtained by applying a drop of polymer solution on the mica surface. Using fluorescent labeled leuprolide, affinity to D-PLA was observed by scanning the mica plates with a confocal microscope. Mica, modified with D-PLA and incubated in a rhodamine B-labeled leuprolide solution for different time periods, showed increased levels of fluorescence with a maximum after incubation for 1 h (Figure 7). Confocal microscopy gave a clear indication of the stereoselectivity of the heterostereocomplex reaction, because only on mica surfaces modified with D-PLA fluorescence was observed. Mica modified with L-PLA did not bind any leuprolide as no fluorescence could be detected. In a preliminary experiment, mica plates chemically modified with D-PLA and further complexed with rhodamine B-labeled leuprolide (as verified by confocal microscopy) were immersed in a 9:1 v/v mixture of acetonitrile/water at 60 °C. A slow release of leuprolide to the acetonitrile/water solution was measured by fluorescent spectrometry. Up to 6% of the total complexed leuprolide was released in 14 h. Subsequent incubation of the same plates in a solution of low molecular weight L-PLA (3 kDa, 0.1% w/v) led to a 100% displacement and release of leuprolide from the mica plates within 12 h. Marking complementary L-PLA with the fluorescent dansyl probe, enabled the monitoring of complexation of L-PLA with D-PLA, replacing complexed leuprolide. In this displacement study, an indication about the strength of the leuprolide/D-PLA heterostereocomplex was attempted to be obtained by confocal microscopy. Incubation of the D-PLA modified mica surfaces in a solution of dansyl-labeled L-PLA led to a high level of fluorescence, indicating the formation of the D-PLA/L-PLA homostereocomplex. It was found that if the mica plates, modified with D-PLA, were first incubated in an aqueous leuprolide solution at 60 °C overnight, subsequent incubation in a labeled L-PLA solution led to lower levels of fluorescence (Figure 8). The D-PLA/leuprolide heterostereocomplexes hindered temporarily the formation of the D-PLA/L-PLA homostereocomplex. The level of the fluorescence was restored after incubation of the D-PLA/

leuprolide heterostereocomplex for 4 h in a dansyl-labeled L-PLA solution. This indicated that an almost total replacement of the leuprolide by L-PLA was obtained. The selectivity of these experiments was verified by incubating L-PLA modified mica plates in a dansyl labeledL-PLA solution or a rhodamine B labeled leuprolide solution, which did not lead to any detectable fluorescence. Untreated, freshly cleaved mica was also unable to bind either dansylPLA or rhodamine B labeled leuprolide. In summary, this study provides additional evidence for the formation of stereocomplexes between two complementary enantioselective polymers, demonstrated by D-PLA and the L-configured nonapeptide, leuprolide. Acknowledgment. We thank Dr. M. Tarshish for his help in Confocal Microscopy and Dr. E. Rachamim for supplying SEM micrographs. This work has been supported by a grant from the Israel Academy of Science. References and Notes (1) Slager, J.; Domb, A. J. AdV. Drug DeliVery ReV. 2003, 55 (4), 549583. (2) Brizzolara, D.; Cantow, H. J.; Diederichs, K.; Keller, E.; Domb, A. J. Macromolecules 1996, 29, 191-197. (3) Spinu, M.; Jackson, C.; Keating, M. Y.; Gardner, K. H. J. Macromol. Sci.-Pure Appl. Chem. 1996, A33, 1497-1530. (4) Ikada, Y.; Jamshidi, K.; Tsuji, H.; Hyon, S.-H. Macromolecules 1987, 20, 904-906. (5) Tsuji, H.; Hyon, S. H.; Ikada, Y. Macromolecules 1991, 24, 56575662. (6) Tsuji, H.; Horii, F.; Hyon, S. H.; Ikada, Y. Macromolecules 1991, 24, 2719-2724. (7) Lim, D. W.; Park, T. G. J. Appl. Polym. Sci. 2000, 75, 1615-1623. (8) Fujiwara, T.; Mukose, T.; Yamaoka, T.; Yamane, H.; Sakurai, S.; Kimura, Y. Macromol. Biosci. 2001, 1, 204-208. (9) Tsuji, H.; Ikada, Y. J. Appl. Polym. Sci. 1994, 53, 1061-1071. (10) Stevels, W. M.; Ankone, M. J. K.; Dijkstra, P. J.; Feijen, J. Macromol. Symp. 1996, 102, 107-113. (11) Pensec, S.; Leroy, M.; Akkouche, H.; Spassky, N. Polym. Bull. 2000, 45, 373-380. (12) de Jong, S. J.; De Smedt, S. C.; Wahls, M. W. C.; Demeester, J.; Kettenes-van den Bosch, J. J.; Hennink, W. E. Macromolecules 2000, 33, 3680-3686. (13) de Jong, S. J.; van Eerdenbrugh, B.; van Nostrum, C. F.; Kettenesvan de Bosch, J. J.; Hennink, W. E. J. Controlled Release 2001, 71, 261-275. (14) Slager, J.; Domb, A. J. Biomaterials 2002, 23, 4389-4396. (15) Slager, J.; Cohen, Y.; Khalfin, R.; Talmon, Y.; Domb, A. J. Macromolecules 2003, 36 (9), 2999-3000. (16) Slager, J.; Domb, A. J. Submitted for publication. (17) Slager, J.; Brizzolara, D.; Cantow, H. J.; Domb, A. J. Submitted for publication. (18) Slager, J.; Gladnikoff, M.; Domb, A. J. Macromol. Symp. 2001, 175, 105-115.

Characterization of Heterostereocomplexes (19) Okada, H. AdV. Drug DeliVery ReV. 1997, 28, 43-70. (20) Hoogsteen, W.; Postema, A. R.; Pennings, A. J.; Tenbrinke, G.; Zugenmaier, P. Macromolecules 1990, 23, 634-642. (21) Sarasua, J. R.; Prud’homme, R. E.; Wisniewski, M.; Le Borgne, A.; Spassky, N. Macromolecules 1998, 31, 3895-3905. (22) Okihara, T.; Tsuji, M.; Kawaguchi, A.; Katayama, K.; Tsuji, H.; Hyon, S. H.; Ikada, Y. J. Macromol. Sci.-Phys. 1991, B30, 119140.

Biomacromolecules, Vol. 4, No. 5, 2003 1315 (23) Tamayo, J.; Garcia, R. Langmuir 1996, 12, 4430-4435. (24) Fasolka, M. J.; Mayes, A. M.; Magonov, S. N. Ultramicroscopy 2001, 90, 21-31. (25) Umemura, K.; Ishikawa, M.; Kuroda, R. Anal. Biochem. 2001, 290, 232-237. (26) Weetall, H. H. Appl. Biochem. Biotechnol. 1993, 41, 157-188.

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