Synthesis, Characterization, and Bioavailability of Mannosylated Shell

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Synthesis, Characterization, and Bioavailability of Mannosylated Shell Cross-Linked Nanoparticles Maisie J. Joralemon, K. Shanmugananda Murthy,† Edward E. Remsen,‡ Matthew L. Becker,§ and Karen L. Wooley* Center for Materials Innovation and Department of Chemistry, Washington University, One Brookings Drive, Saint Louis, Missouri 63130-4899 Received November 14, 2003; Revised Manuscript Received January 20, 2004

Saccharide-functionalized shell cross-linked (SCK) polymer micelles designed as polyvalent nanoscaffolds for selective interactions with receptors on Gram negative bacteria were constructed from mixed micelles composed of poly(acrylic acid-b-methyl acrylate) and mannosylated poly(acrylic acid-b-methyl acrylate). The mannose unit was conjugated to the hydrophilic chain terminus of the amphiphilic diblock copolymer precursor, from which the SCK nanoparticles were derived, by the growth of the diblock copolymer from a mannoside functionalized atom transfer radical polymerization (ATRP) initiator. Mixed micelle formation between the amphiphilic diblock copolymer and mannosylated amphiphilic diblock copolymer was followed by condensation-based cross-linking between the acrylic acid residues present in the periphery of the polymer micelles to afford SCK nanoparticles. SCKs presenting variable numbers of mannose functionalities were prepared from mixed micelles of controlled stoichiometric ratios of mannosylated and nonmannosylated diblock copolymers. The polymer micelles and SCKs were characterized by dynamic light scattering (DLS), electrophoretic light scattering, atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). Surface availability and bioactivity of the mannose units were evaluated by interactions of the nanostructures with the model lectin Concanavalin A via DLS studies, with red blood cells (rabbit) via agglutination inhibition assays and with bacterial cells (E. coli) via TEM imaging. Introduction Among the various biological processes in which carbohydrate-protein binding events participate, pathogen attachment to host cells is of particular therapeutic interest.1-3 In a simplistic analysis, the surfaces of mammalian cells are decorated with carbohydrates, and those of pathogens with carbohydrate receptors, properties which have directed recent efforts toward new therapeutics based on the manipulation of pathogen attachment to host cells through agents that display carbohydrates.4-9 These agents have been designed to capitalize on the cluster glycoside effect,10,11 in which higher binding affinities are achieved through synergistic polyvalent interactions. Various substrates,12 for instance, linear polymers,13-15 calixarenes and cyclodextrins,16,17 dendrimers,18-26 micelles,27-31 glycoclusters,4,17,32,33 and glycoproteins,34 have served as scaffolds that present saccharides in a polyvalent array. The architecture of the multivalent scaffold from which the saccharide ligands are presented has been demonstrated to be an important determinant in the effectiveness of the ligand binding interactions with and clustering of their receptors.9 Moreover, the dynamic re* To whom correspondence should be addressed. Phone: (314) 9357136. Fax: (314) 935-9844. E-mail: [email protected]. † Present address: Rhodia, Inc., Building B, 259 Prospect Plains Road, Cranbury, NJ 08512-7500. ‡ Present address: Cabot Microelectronics Corporation, Core Technology Group, 870 North Commons Drive, Aurora, IL 60504-7963. § Present address: National Institute of Standards and Technology, Polymer Division, Biomaterials Group, Gaithersburg, MD 20899-8543.

organization of the multivalent presentation of supramolecularly assembled rotaxane-based saccharide ligands has been studied as a system for maximized receptor binding.16 Because of the significance of the scaffold size, shape, and flexibility for polyvalent ligand-receptor interactions, we chose to study shape-adaptable shell cross-linked (SCK) nanoparticles35,36 as a structure on which saccharide units can be clustered across a nanoscale surface area. The synthesis of mannose-functionalized SCKs is described herein, following a scheme that involves in situ cross-linking of self-assembled mixed polymer micelles comprised of amphiphilic block copolymers terminated at the hydrophilic chain end with a mannoside residue. Mannose was selected for the potential of the SCKs to target Gram negative bacterial cells. For instance, E. coli is a Gram negative bacterium, containing mannose receptors within the FimH region on type 1 pili, which allow for attachment to host endothelial cells.37,38 The surface accessibility and bioavailability of the mannose functionalities upon the surface of SCKs is demonstrated through interactions with mannosebinding proteins in solution and at the periphery of E. coli. Moreover, the amphiphilic interface between the hydrophobic core domain and the hydrogel shell material provide a potential reservoir39-42 for the sequestration of amphiphilic lipopolysaccharide endotoxins that are released from the Gram-negative bacterial cells upon cell death. This mode of action, which may provide for SCK utility in the prevention of endotoxic shock, is also demonstrated by DLS studies.

10.1021/bm0344710 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/27/2004

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Experimental Section Materials. The procedures were performed on a double manifold, vacuum (0.1 mmHg), 99.99% N2. Tetrahydrofuran (99%), tert-butyl acrylate (99%), and methyl acrylate (99%) were received from Sigma-Aldrich Company (St. Louis, MO) and distilled from sodium/benzophenone and calcium hydride, respectively, under N2 prior to use. Mannoside alcohol 3 was prepared according to a literature procedure.43 The synthesis and characterization data for the nonmannosylated diblock copolymer of acrylic acid and methyl acrylate was described previously.44 All other materials were used as received from Sigma-Aldrich Company. Prior to use, glassware, needles, syringes, and magnetic stir bars were dried in a 110 °C oven for a minimum of 1 h. Syringes were flushed with N2 prior to use. Spectra/Por membranes (Spectrum Medical Industries, Inc., Laguna Hills, CA) used for dialysis were obtained from Fisher Scientific (Pittsburgh, PA). Flash column chromatography was performed using 32-63 D 60 Å silica gel from ICN SiliTech (ICN Biomedicals GmbH, Eschwege, Germany). Measurements. Infrared spectra were obtained on a Perkin-Elmer Spectrum BX FT-IR system using diffuse reflectance sampling accessories. 1H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were recorded as solutions on a Varian Mercury 300 spectrometer with the solvent signal as reference. Size exclusion chromatography (SEC) was conducted on a Waters Chromatography, Inc. (Milford, MA) model 150CV, equipped with a model 410 differential refractometer, a Precision Detectors, Inc. (Bellingham, MA) model PD2040 dual-angle (15° and 90°) light scattering detector, and a threecolumn set of Polymer Laboratories, Inc. (Amherst, MA) gel mixed-bed styrene-divinylbenzene columns (PLgel 10 µm Mixed B, 300 × 7.5 mm columns). The system was equilibrated at 35 °C in anhydrous THF, which served as the polymer solvent and eluent (flow rate set to 1.00 mL/ min then determined gravimetrically). Data collection was performed with Precision Detectors, Inc. Precision Acquire software. Data analysis was performed with Precision Detectors, Inc. Discovery 32 software. Inter-detector delay volume and the light scattering detector calibration constant were determined from a nearly monodisperse polystyrene calibrant (Pressure Chemical Co., Mp ) 90 000 g/mol, Mw/ Mn < 1.04). The differential refractometer was calibrated with standard polystyrene reference material (SRM 706 NIST) of known specific refractive index increment dn/dc (0.184 mL/g). The dn/dc values of the analyzed polymers were then determined from the differential refractometer response. Glass-transition temperatures (Tg) were measured either on a Perkin-Elmer (Shelton, CT) DSC-4 differential scanning calorimeter that was upgraded with an Instrument Specialists, Inc. (Antioch, IL) temperature program interface-PE or on a Mettler Toledo (Columbus, OH) DSC822e differential scanning calorimeter. Data acquired on the Perkin-Elmer instrument were acquired and analyzed with Thermal Analysis PC software (version 2.11; Instrument Specialists). Data acquired on the Mettler were acquired and analyzed with STARe System software (version 7.01; Mettler Toledo).

Joralemon et al.

Heating rates were 10 °C/min, and the Tg was taken as the midpoint of the inflection tangent, upon the third heating scan. Hydrodynamic diameters (Dz, Dn) and size distributions for the SCKs in aqueous solutions were determined by dynamic light scattering (DLS). The DLS instrumentation consisted of a Brookhaven Instruments Limited (Worcestershire, U.K.) system, including a model BI-200SM goniometer, a model BI-9000AT digital correlator, a model EMI9865 photomultiplier, and a model 95-2 Ar ion laser (Lexel Corp.; Farmingdale, NY) operated at 514.5 nm. Measurements were made at 20 ( 1 °C. Prior to analysis, solutions were centrifuged in a model 5414 microfuge (Brinkman Instruments, Inc.; Westbury, NY) for 4 min to remove dust particles. Scattered light was collected at a fixed angle of 90°. The digital correlator was operated with 522 ratio spaced channels, an initial delay of 0.1 µs, a final delay of 5.0 µs, and a duration of 15 min. A photomultiplier aperture of 200 µm was used, and the incident laser intensity was adjusted to obtain a photon counting of 200 kcps. Only measurements in which the measured and calculated baselines of the intensity autocorrelation function agreed to within 0.1% were used to calculate the particle size. The calculations of the particle size distributions and distribution averages were performed with the ISDA software package (Brookhaven Instruments Company), which employed single-exponential fitting, cumulants analysis, and nonnegatively constrained least-squares particle size distribution analysis routines. The height measurements and distributions for the SCKs were determined by tapping-mode AFM under ambient conditions in air. The AFM instrumentation consisted of a Nanoscope III BioScope system (Digital Instruments, Veeco Metrology Group; Santa Barbara, CA) and standard silicon tips (type, OTESPA-70; L, 160 µm; normal spring constant, 50 N/m; resonance frequency, 246-282 kHz). The sample solutions were drop (2 µL) deposited onto freshly cleaved mica and allowed to dry freely in air. Transmission electron microscopy samples were diluted in water (9:1) and further diluted with a 1% phosphotungstic acid (PTA) stain (1:1). Carbon grids were prepared by a plasma treatment to increase the surface hydrophilicity. Micrographs were collected at 100 000× magnification and calibrated using a 41 nm polyacrylamide bead from NIST. Histograms of particle diameters were generated from the analysis of a minimum of 150 particles from at least three different micrographs. Sedimentation equilibrium experiments were conducted on a Beckman Instruments, Inc. (Fullerton, CA) model Optima XL-I analytical ultracentrifuge fitted with a model An60-Ti four-hole rotor, and Epon charcoal-filled, six-channel centerpiece sample cells with matched sapphire windows. All data were recorded using the instrument’s Rayleigh interferometric (refractive index) detection optics at 20 °C and 5000, 3000, and 2500 rpm, respectively, with a centrifugation time of 3-5 days to reach sedimentation equilibrium. The solution volume was 110 µL, and the optical path length was 12 mm. A Mettler-Parr model DMA 602 high-precision digital density meter was employed to determine the density at 20.0 °C for all solutions. All densities were an average of

Mannosylated Shell Cross-Linked Nanoparticles

5 runs, with measurements of one hundred periods per run. The weight-average degree of aggregation, Nagg, was computed as Nagg ) Mw/M0 where Mw is the nanoparticle peak average molecular weight obtained from sedimentation equilibrium measurements, M0 is the effective diblock molecular weight given by M0 ) fw,manMw,man + fw,polyMw,poly where Mw,man is the weight-average molecular weight of the mannosylated diblock copolymer, Mw,poly is the weightaverage molecular weight of the nonmannosylated diblock copolymer, fw,man is the weight fraction of mannosylated diblock copolymer used to prepare the mixed micelle, and fw,poly is the weight fraction of nonmannosylated diblock copolymer used to prepare the mixed micelle. Zeta potential (ζ) values for the SCKs were determined with a Brookhaven Instruments Limited, model ZetaPlus. Measurements were made following exhaustive dialysis of the SCK solutions into 1 mM KH2PO4, 1 mM KCl, pH 7.1 buffer. Data were acquired in the phase-analysis light scattering (PALS) mode45 following solution equilibration at 25 °C. Calculation of ζ from the measured nanoparticle electrophoretic mobility (µ) employed the Smoluchowski equation:46 µ ) ζ/η where and η are the dielectric constant and the absolute viscosity of the medium, respectively. Measurements of ζ were reproducible to within (2 mV of the mean value given by sixteen determinations of 10 data accumulations. Syntheses. 1-C-Propionyl bromide-2,3,4,6-tetra-O-triethylsilyl Mannopyranoside (2). A 50 mL two-neck roundbottom flask charged with a magnetic stir bar and fitted with a stopper and a sidearm adapter was flame dried under vacuum and allowed to cool to ambient temperature under N2. The flask was charged with a solution of alcohol 343 (1.8 g, 2.7 mmol) dissolved in THF (10 mL) and then triethylamine (0.75 mL, 5.4 mmol), via syringes. The mixture was then cooled to 0-5 °C and 2-bromopropionyl bromide (0.57 mL, 5.4 mmol) in THF (5 mL) was added via syringe. The reaction was allowed to stir under N2 at ambient temperature for 15 h. The reaction mixture was filtered and concentrated in vacuo, and the residue was taken up in a minimal amount of diethyl ether. The solution was then washed with dilute aqueous NaHCO3. The organic solution was dried over MgSO4, filtered, and concentrated by rotary evaporation. The residue was purified by flash column chromatography (4% ethyl acetate in hexane): Isolated yield 0.70 g, (32%). 1H NMR (300 MHz, CDCl3): δ 0.53-0.75 (m, 24H, SiCH2CH3), 0.89-1.05 (m, 36H, SiCH2CH3), 1.52-1.71 (m, 2H, CH2CH2O2C), 1.82 (d, J ) 6.9 Hz, 3H, CH(CH3)Br), 3.34-3.95 (m, 7H saccharide H-1, H-2, H-3, H-4, H-5, 2H-6), 4.18-4.48 (m, 3H, CH2CH2O2C and CH(CH3)Br) ppm. 13C NMR (CDCl3): δ 4.6-5.7, 7.0-7.5, 21.8, 31.1, 40.4, 61.5, 63.2, 66.1, 70.9, 71.7, 80.3, 82.1, 170.4

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ppm. LRMS (FAB) calcd for C35H75Br1O7Si4 (M + 2Li H): 811.4; observed: 811.0. 1-C-Poly(tert-butyl acrylate)-2,3,4,6-tetra-O-triethylsilyl Mannopyranoside (5). A 100 mL two-neck round-bottom flask charged with a magnetic stir bar and fitted with a sidearm gas inlet adapter, and the stopcock was flame dried under vacuum and allowed to cool to ambient temperature under N2. The flask was then charged with copper (I) bromide (0.12 g, 8.4 × 10-4 mol). A septum was placed over the stem of the stopcock and under positive pressure of N2, initiator 2 (0.42 g, 5.3 × 10-4 mol) dissolved in tertbutyl acrylate (14.97 mL, 0.102 mol) was added via syringe. Pentamethyldiethylenetriamine (PMDETA) (0.22 mL, 1.06 × 10-3 mol) was then added to the mixture under a positive pressure of N2 via syringe. After three freeze-pump-thaw degassing cycles, the reaction was allowed to stir for 15 h at 60 °C. The polymerization was quenched by submerging the flask in liquid nitrogen, and the mixture was then allowed to warm to ambient temperature. The reaction mixture was diluted with tetrahydrofuran and filtered through an alumina plug. The filtrate was precipitated twice from tetrahydrofuran into an 80% methanol, deionized water solution: Isolated Yield 7.98 g, (74%). Mn ) 20 500 g/mol, Mw/Mn ) 1.1 from SEC with dn/dc ) 0.054 mL/g. Tg ) 40 °C. IR: 30002800, 1740, 1690, 1450, 1280, 1180, 1045, 850, 750 cm-1. 1 H NMR (CDCl3): δ 0.50-0.76 (m, SiCH2CH3), 0.89-1.03 (m, SiCH2CH3), 1.2-1.7 (br, C(CH3)3), 1.7-2.0 and 2.02.4 (CH2 and CH of the polymer backbone), 3.3-4.4 (H’s of the chain termini) ppm. 13C NMR(CDCl3): δ 27.9-29.7, 35.4-38.0, 41.6-42.8, 80.6, 174.4 ppm. 1-C-Poly(tert-butyl acrylate-b-methyl acrylate)-2,3,4,6tetra-O-triethylsilyl Mannopyranoside (6). A 100 mL twoneck round-bottom flask fitted with a sidearm adapter, and the stopcock was charged with a magnetic stir bar, flame dried under vacuum, and allowed to cool to ambient temperature under N2. The reaction vessel was charged with copper (I) bromide (0.148 g, 1.03 × 10-3 mol). A septum was placed over the stem of the stopcock and macroinitiator 5 (1.660 g, 8.9 × 10-4 mol) in methyl acrylate (12.30 mL, 0.137 mol) was added via a syringe. PMDETA (0.215 mL, 1.03 × 10-3 mol) was added to the reaction vessel via a syringe. After three freeze-pump-thaw degassing cycles, the reaction was allowed to stir for 4 h at 50 °C under N2. The polymerization was quenched by immersion in liquid N2 and allowed to warm to ambient temperature. The reaction mixture was diluted with tetrahydrofuran and the copper catalyst was removed by filtration through an alumina plug. The filtrate was concentrated in vacuo and precipitated twice from tetrahydrofuran into a 70% methanol, deionized water solution. The polymer was dried under vacuum: Isolated yield 4.83 g (83%). Mn ) 46 000 g/mol, Mw/Mn ) 1.2 from SEC with dn/dc ) 0.061 mL/g. Tg (PMA) ) 19 °C, Tg (PtBA) ) 48 °C. IR: 3000-2800, 1750, 1670, 1450, 1280, 1180, 840, 760 cm-1. 1H NMR (CDCl3): δ 0.50-0.76 (m, SiCH2CH3), 0.89-1.03 (m, CH2CH3), 1.2-1.8 (m, CH2 of the polymer backbone), 1.3-1.5 (br, C(CH3)3), 1.4-2.0 and 2.1-2.5 (CH2 and CH of the polymer backbone), 3.6-3.7 (br, OCH3) ppm. 13C NMR (CDCl3): δ 27.9-28.7, 34.335.5, 41.3-41.6, 51.9, 80.2, 175.2 ppm.

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1-C-Poly(acrylic acid-b-methyl acrylate)mannopyranoside (1). A solution of trifluoroacetic acid (6.0 mL, 7.8 × 10-2 mol) in dichloromethane (20.0 mL) was added to 6 (2.70 g, 7.69 × 10-5mol). The reaction was allowed to stir at ambient temperature for 4 h. The solvent was removed under vacuum, and the resulting solid was taken up in tetrahydrofuran; the solution was transferred to a dialysis bag (MWCO 12-14 kDa); and the solution was dialyzed against a continuous flow of deionized water for 24 h. The final mannoside-terminated amphiphilic diblock copolymer was isolated by lyophilization: Isolated yield 1.70 g (75%). Tg (PMA) ) 16 °C; Tg (PAA) not observed. IR: 34402740, 2950, 1750, 1450, 1260, 1170, 830, 640, 510 cm-1. 1 H NMR (DMSO-d6): δ 1.3-1.9 and 2.1-2.4 (CH2 and CH of the polymer backbone), 3.3-3.4 (br, OCH3), 3.5-3.6 (br, OH) ppm. 13C NMR (DMSO-d6): δ 33.5-35.9, 40.8, 51.6, 174.4, 175.8 ppm. General Procedure for Micelle Formation. The relative amounts of nonmannosylated (7)44 and mannosylated (1) diblock copolymers were mixed as solutions in tetrahydrofuran, each at a concentration of 1.0 mg/mL. The mixture was allowed to stir at ambient temperature for 10 min. Nanopure water (18 MΩ-cm) was then added via a syringe pump at a rate of 15.0 mL/h, and the solution was allowed to stir at ambient temperature for 15 h. The respective solutions were transferred to presoaked and rinsed dialysis bags (MWCO 6-8 kDa) and dialyzed against a continuous flow of deionized water. The micelles were equilibrated in buffer (50 mM sodium phosphate, 50 mM NaCl, pH 7), and hydrodynamic diameters were determined by DLS. 0% Mannosylated Poly(acrylic acid-b-methyl acrylate) Micelle Formation. Nanopure water (56.30 mL) was added dropwise to a THF solution of 7 (1.0 mg/mL, 56.30 mL). The solution was dialyzed for 3 days against a continuous flow of deionized water. Dm z : 25 ( 2 nm. 1% Nominal Mannosylated Poly(acrylic acid-b-methyl acrylate) Micelle Formation. Nanopure water (50.50 mL) was added dropwise to a combined THF solution of 7 (1.0 mg/mL, 50.00 mL) and 1 (1.0 mg/mL, 0.50 mL). The solution was dialyzed for 36 h against a continuous flow of deionized water. Dm z : 26 ( 1 nm. 2% Nominal Mannosylated Poly(acrylic acid-b-methyl acrylate) Micelle Formation. Nanopure water (54.06 mL) was added dropwise to a combined THF solution of 7 (1.0 mg/mL, 53.10 mL) and 1 (1.0 mg/mL, 1.06 mL). The solution was dialyzed for 3 days against a continuous flow of deionized water. Dm z : 25 ( 2 nm. 10% Nominal Mannosylated Poly(acrylic acid-b-methyl acrylate) Micelle Formation. Nanopure water (55.00 mL) was added dropwise to a combined THF solution of 7 (1.0 mg/mL, 50.00 mL) and 1 (1.0 mg/mL, 5.00 mL). The solution was dialyzed for 36 h against a continuous flow of deionized water. Dm z : 31 ( 1 nm. 100% Mannosylated Poly(acrylic acid-b-methyl acrylate) Micelle Formation. Nanopure water (50.00 mL) was added dropwise to a solution of 1 (1.0 mg/mL, 50.00 mL). The solution was dialyzed for 3 days against a continuous flow of deionized water. Dm z : 57 ( 4 nm.

Joralemon et al.

General Procedure to Form SCK Nanoparticles. A solution of 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide in Nanopure water (18 MΩ-cm) was added to the respective micelle solutions. The solutions were allowed to stir at ambient temperature for 20 min. 2,2′-(Ethylenedioxy)bis(ethylamine) was added, and the reactions were allowed to stir at ambient temperature for 5 h. The respective SCK solutions were transferred to presoaked dialysis tubing (MWCO 6-8 kDa) and dialyzed against a continuous flow of deionized water for 4 days. The SCKs were equilibrated in buffer (50 mM sodium phosphate, 50 mM NaCl, pH 7) and characterized (see Experimental Sections below, Tables 2 and 3 and Figures 1 and 2). 0% Mannosylated Poly(acrylic acid-b-methyl acrylate) SCK Nanoparticle (8). An aqueous solution of 1-(3′dimethylaminopropyl)-3-ethylcarbodiimide methiodide (40.0 mg/mL, 1.34 mL, 1.80 × 10-4 mol) was added to a micelle solution of 7 in deionized water (194 mL, 0.28 mg/mL), followed by an aqueous solution of 2,2′-(ethylenedioxy)bis(ethylamine) (30.0 mg/mL, 0.89 mL, 1.8 × 10-4 mol). The SCK nanoparticle solution was dialyzed for 4 days against a continuous flow of deionized water. Tg ) 25 °C. IR: 3230-2660, 2950, 1750, 1650, 1560, 1450, 1270, 1170, 830 cm-1. 1% Nominal Mannosylated Poly(acrylic acid-b-methyl acrylate) SCK Nanoparticle (9). An aqueous solution of 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (100.0 mg/mL, 0.38 mL, 1.3 × 10-4 mol) was added to a micelle solution (1% 1, 99% 7) in deionized water (130 mL, 0.30 mg/mL), followed by 2,2′-(ethylenedioxy)bis(ethylamine) (0.019 mL, 1.3 × 10-4 mol). The SCK nanoparticle solution was dialyzed for 4 days against a continuous flow of deionized water. Tg ) 27 °C. IR: 3230-2660, 2950, 1750, 1650, 1650, 1560, 1450, 1270, 1170, 830 cm-1. 2% Nominal Mannosylated Poly(acrylic acid-b-methyl acrylate) SCK Nanoparticle (10). An aqueous solution of 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (40.0 mg/mL, 1.31 mL, 1.76 × 10-4 mol) was added to a micelle solution (2% 1, 98% 7) in deionized water (206 mL, 0.26 mg/mL), followed by an aqueous solution of 2,2′(ethylenedioxy)bis(ethylamine) (30.0 mg/mL, 0.87 mL, 1.8 × 10-4 mol). The SCK nanoparticle solution was dialyzed for 4 days against a continuous flow of deionized water. Tg ) 25 °C. IR: 3230-2660, 2950, 1750, 1650, 1560, 1450, 1270, 1170, 830 cm-1. 10% Nominal Mannosylated Poly(acrylic acid-b-methyl acrylate) SCK Nanoparticle (11). An aqueous solution of 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (100.0 mg/mL, 0.045 mL, 1.5 × 10-4 mol) was added to a micelle solution (10% 1, 90% 7) in deionized water (158 mL, 0.30 mg/mL), followed by 2,2′-(ethylenedioxy)bis(ethylamine) (0.022 mL, 1.5 × 10-4 mol). The SCK nanoparticle solution was dialyzed for 4 days against a continuous flow of deionized water. Tg ) 24 °C. IR: 32302660, 2950, 1750, 1650, 1560, 1450, 1270, 1170, 830 cm-1. 100% Mannosylated Poly(acrylic acid-b-methyl acrylate) SCK Nanoparticle (13). An aqueous solution of 1-(3′dimethylaminopropyl)-3-ethylcarbodiimide methiodide (40.0 mg/mL, 0.54 mL, 7.3 × 10-5 mol) was added to a micelle


solution of 1 in deionized water (178 mL, 0.27 mg/mL), followed by an aqueous solution of 2,2′-(ethylenedioxy)bis(ethylamine) (30.0 mg/mL, 0.36 mL, 7.4 × 10-5 mol). The SCK nanoparticle solution was dialyzed for 4 days against a continuous flow of deionized water. Tg ) 19 °C. IR: 3230-2660, 2950, 1750, 1650, 1560, 1450, 1270, 1170, 830 cm-1. Assays of Mannoside Bioactivity. Dynamic Light Scattering Experiments of Mixtures of ConA and Nanoparticles. The nanoparticles (0.4 mg/mL) and ConA (0.67 mg/ mL) were dialyzed into buffer (0.1 M Na2B2O4, 1 mM CaCl2, 1 mM MnCl2, pH 8.5), individually. Prior to analysis, the stock solutions were filtered (Acrodisc Syringe Filter, 0.1 µm Supor Membrane). The solutions were mixed together in the respective concentration ratios (0:1, 1:0, 20:1, 80:1, 320:1 [ConA]:36), and then centrifuged (15,000 rpm) for 4 min prior to data collection. Agglutination Inhibition Assay. Rabbit red blood cells (RBCs), obtained from Colorado Serum Company, were pelleted (1000 rpm) and resuspended (OD λ640 1.8-2.0) in phosphate buffered saline (PBS 25 mM sodium phosphate, 0.15 M NaCl, pH 7.0). The control inhibitor R-D-mannopyranoside was taken up in the PBS buffer, whereas the SCK inhibiting agents were dialyzed into the PBS buffer. The respective concentrations were determined by acoustical density measurements. Standard protocols were then used for the agglutination inhibition assay.15,47-49 ConA (CanaValia ensiformis, Sigma C-2010 Type IV) was taken up in HEPES buffer (20 mM HEPES, 100 mM CaCl2, pH 8.0), and dialyzed into PBS, and the concentration was determined at 298 nm. The minimum concentration of ConA required to agglutinate the RBCs was determined by incubating 2-fold serial dilutions of the stock solution with RBCs at room temperature for 4 h. Four times the minimum concentration of required ConA was then incubated at room temperature for 2 h with 2-fold serial dilutions of the respective inhibitor solutions. RBC suspension was then added and the plate was read after standing 4 h at room temperature. The experiment was repeated three times for each SCK. Transmission Electron Microscopy Images of E. coli and Nanoparticles. Escherichia coli (Stratagene XL-1 Blue) cells (10 µL ODλ600 ) 0.50 in LB media) were incubated for 14 h at 37 °C with the respective particle solution (225 µL, 134 µg 0% mannosylated SCK/L LB media, 67.5 µg 100% mannosylated SCK/L LB media). The samples were then diluted 9:1 in water and further diluted 1:1 with a 1% phosphotungstic acid (PTA) stain. Micrographs were collected at 100,000× magnification. SCK Sequestration of Amphiphilic LPS Endotoxins. The 0% mannosylated nanoparticle (0.4 mg/mL) and LPS from E. coli (serotype 0127:B8, 0.1 mg/mL) were respectively dialyzed from Nanopure water into PBS buffer (50 mM sodium phosphate, 50 mM NaCl, pH 7). Prior to data collection, the nanoparticle solution was filtered through a 0.4 µm filter (PTFE) and centrifuged (15 000 rpm) for 4 min, the LPS solution was centrifuged (15 000 rpm) for 4 min. The LPS solution (100 µL) and the filtered nanoparticle solution (100 µL) were combined and centrifuged (15 000 rpm) for 4 min prior to data collection.

Biomacromolecules, Vol. 5, No. 3, 2004 907 Scheme 1. Synthetic Route for the Preparation of the Mannose-terminated Amphiphilic Block Copolymer via Atom Transfer Radical Polymerization, Followed by Deprotectiona

a (i) Allyltrimethylsilane, BF ‚Et O, TMSOTf, CH CN, RT, 48 h, 74%; 3 2 3 (ii) CH3ONa, MeOH, RT, 52 h, 94%; (iii) TESOTf, pyridine, CH2Cl2, RT, 48 h, 90%; (iv) (a) O3, CH2Cl2, MeOH, -78 °C, 20 min. (b) NaBH4, -78 °C, 2 h, 52%.

Results and Discussion Synthesis of Mannosylated SCK Nanoparticles. The incorporation of mannose ligands into the shell of the SCKs began with the synthesis of the saccharide functionalized amphiphilic block copolymer precursor (1), which would serve as the building block for SCK nanoparticle preparation. To ensure chain end attachment, the protected mannoside moiety 2 (Scheme 1) was utilized as an initiator for atom transfer radical polymerization (ATRP).50-52 The mannoside-containing initiator 2 was synthesized via an intermediate mannoside alcohol, 3, originating from R-Dmannose pentaacetate, 4, according to methods described elsewhere.43 The mannosylated ATRP initiator 2 was afforded in 32% yield from the esterification of 3 by reaction with 2-bromopropionyl bromide in the presence of triethylamine and THF. This compound exhibited hydrolytic instabilities, which complicated its purification and storage. Therefore, the initiator was used for polymerization immediately after isolation. Poly(tert-butyl acrylate) homopolymer 5 was generated by the ATRP of tert-butyl acrylate neat at 60 °C for 15 h, initiated from mannosylated alkyl bromide 2, in the presence of CuBr and PMDETA. The chain was further extended by the ATRP of methyl acrylate neat at 50 °C for 4 h from the macroinitiator 5, to yield the mannosylated diblock copolymer composed of tert-butyl acrylate and methyl acrylate, 6. Selective removal of the tert-butyl ester and the triethylsilyl ether protecting groups, without cleavage of the methyl esters, was achieved through reaction of 6 with trifluoroacetic acid in dichloromethane,44 to yield the amphiphilic mannosylated poly(acrylic acid-b-methyl acrylate) block copolymer 1. The same procedures were followed for the preparation of an amphiphilic block copolymer of acrylic acid and methyl acrylate that lacked the mannoside chain end unit, 7. The number-average molecular weight value for each polymer was determined by both 1H NMR spectroscopy and size exclusion chromatography (SEC) analyses (Table 1). The

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Table 1. Molecular Weight and Molecular Weight Distribution Data, Determined by 1H NMR Spectroscopy and SEC polymer

na

mb

Mn (1H NMR), Da

Mn (SEC), Da

Mw/Mn

5 6 1 7

140 140 140 60

0 380 380 50

18 700 51 400 43 100 9300

20 500 46 000 NAc NAc

1.1 1.2 NAc NAc

a Number of tBA repeating units determined by 1H NMR end-group analysis. b Number of MA repeating units determined by comparison of unique MA protons (3.6-3.7 ppm) to overlapping MA and tBA protons (2.1 to 2.5 ppm) in the 1H NMR spectra. c Data not available due to the affinity of the PAA segment with the SEC column packing.

Scheme 2. Construction of Mannosylated SCK Nanoparticles, with Control over the Number of Mannose Residues Being Provided by the Formation of Mixed Micelles

molecular weight distribution values were also determined by SEC. The amphiphilic block copolymers were not characterized by SEC, due to the solubility differences of the two block segments and affinity to the SEC column packing material. The supramolecular assembly of the amphiphilic block copolymers in water afforded micelles, which were then cross-linked throughout the shell layer to produce robust SCK nanoparticles.53 The numbers of mannose units on the SCKs were controlled through the formation of mixed polymer micelles, using various stoichiometries of the chain-end mannosylated, 1, and nonmannosylated,44 7, block copolymers (Scheme 2). Mixtures of 1:7 at molar ratios of 0:100, 1:99, 2:98, 10:90, and 100:0 were prepared in THF solutions, followed by the slow addition of an equal volume of Nanopure water (18 MΩ-cm) and then extensive dialysis against deionized water (cellulose membrane with MWCO 6-8 kDa) to yield 0, 1, 2, 10, and 100 nominal % mannosylated polymer micelle solutions, respectively. Subsequent intramicellar cross-linking of nominally 50% of the acrylic acid sites, through reaction with 2, 2′-(ethylenedioxy)bis(ethylamine) in the presence of 1-[3′-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide, afforded SCK nano-

Table 2. Hydrodynamic Diameter Data for the SCKs Having Varying Degrees of Mannosylation SCK

nominal % mannosylation

Dz1 (nm)a

Dz2 (nm)b

Dz3 (nm)c

Dm z (nm)d

Dm n (nm)e

8 9 10 11 12

0 1 2 10 100

31 ( 3 24 ( 3 26 ( 3 17 ( 2 19 ( 4

80 ( 5 58 ( 5 60 ( 5 45 ( 4 49 ( 5

NA NA NA NA 117a

46 ( 3 41 ( 3 43 ( 3 39 ( 3 70 ( 5

30 ( 3 22 ( 2 26 ( 2 17 ( 2 19 ( 2

a D 1 ) intensity-weighted peak diameter of mode 1. b D 2 ) intensityz z weighted peak diameter of mode 2. c Dz3 ) intensity-weighted peak m diameter of mode 3. d Dz ) intensity-weighted mean diameter. e Dnm ) number-weighted mean diameter. f Weak mode, peak mode position not accurately determined.

particles 8-12 possessing 0, 1, 2, 10, and 100% mannosylation, respectively. Due to the composition of the mannosylated amphiphilic diblock copolymers, whereby the mannose unit was topologically located on the chain terminus of the hydrophilic block segment, it was anticipated that the mannose functionalities could reside on the surface of the SCKs. Moreover, the extended length of the hydrophilic chain segment for 1 vs 7 provided opportunity for the mannose groups to extend from the SCK surface. Of particular interest was the determination of the surface availability of the mannoside when presented as a ca. single unit from a nanostructure (e.g., 1% and 2%) in comparison to polyvalent presentation (10%) and complete coverage (100%). Characterization of the SCKs. Hydrodynamic diameter distributions for the mannosylated SCK nanoparticles in buffer solution were determined from DLS, and are summarized in Table 2. Two modes were found in the intensityweighted diameter distributions with the smaller-sized mode decreasing in peak diameter from 31 to 17 nm as the degree of mannosylation increased from 0 to 10%. In the case of 100% mannosylation, a larger, third mode was observed at 117 nm. For all levels of mannosylation, the smaller-size mode was the major component on a number-averaged basis and constituted at least 95% of the nanoparticle diameter distribution. The chemical nature of the larger modes was not revealed by DLS analysis. AFM and TEM imaging allowed for determination of the size and shape of the particles in the solid state. Average heights and diameters were calculated from measurements made on AFM images, and comparisons were made with the diameters measured from TEM images. Representative AFM images are shown in Figure 1 and TEM images in Figure 2, along with the histograms of the data. In each case, the diameters were found to be substantially larger than the heights, due to particle shape deformation upon adsorption onto the substrates resulting from the low-Tg core material.35,36 The percentage of mannosylation did not affect the average size of the nanoparticle. However, the 0% and 100% mannosylated SCKs exhibited larger Hav and Dav than the other nanoparticles. This corresponds to a greater number of polymer chains (Nagg, Table 3) forming the 0% and 100% mannosylated SCKs. The distribution of particle height was greatest for the 100% mannosylated SCK; however, the 0% mannosylated SCK had the largest distribution in diameter.



Figure 1. Average heights (Hav) and diameters (Dav) measured from AFM images are shown with the corresponding distribution, and a representative image. At least five different areas were imaged for each type of nanoparticle.

It is unknown whether the slight differences in particle sizes are due to scatter in the reproducibility of SCK formation, or if there is something unique between the homogeneous vs heterogeneous micellar assemblies. The diameters determined by DLS are smaller than those obtained from AFM measurements. As noted above, this difference is attributable to the fluid, low glass transition temperature (Tg) poly(methyl acrylate) core. Transmission Electron Microscopy (TEM). Average diameters and the distribution of diameters (Figure 2) were obtained from measuring particles visualized by TEM images. The diameters resulting from the TEM experiments were smaller than those obtained from AFM; however. the average diameters are within experimental error to the respective diameters resulting from DLS. This is likely due to the differential staining of the SCK core and shell by the phosphotungstic acid (PTA), which produces preferential

imaging of the core. In addition, the particles deform to a less extent on the carbon-coated copper grid used for TEM imaging, than they do upon the mica substrate for AFM imaging.35 The degree of mannosylation did not significantly affect the average diameter. Molecular Weight and Zeta-Potential Analysis. Sedimentation equilibrium (SE) analyses obtained with an analytical ultracentrifuge were used to determine the weightaverage molecular weight and aggregation number values for the SCKs. Representative normalized SE profiles for the SCKs, shown in Figure 3, display differing sedimentation equilibria as a function of the degree of mannosylation. The differences in the sedimentation profiles reflect both weight-average molecular weight (Mw) and partial specific volume (ν) variations resulting from differences in the degree of mannosylation. Prior to the calculation of Mw for the SCKs, accurate values of ν were determined using a

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Figure 2. Average diameters are shown with the corresponding distribution and a representative image. At least three different areas were imaged for each type of nanoparticle.

technique described previously.54 A dependence of ν on the degree of mannosylation was found between 0 and 10% mannosylation. The observed decrease in ν as the level of mannosylation increased is consistent with the change in ν that was observed with the addition of carbohydrates to proteins.55 However, the 100% mannosylated SCK yielded a ν value, as listed in Table 3, that was significantly larger than expected. Molecular weight analysis using the values of ν summarized in Table 3 was accomplished by calculation of apparent weight-average molecular weights (Mw,app) using single-component fitting to the sedimentation equilibrium profiles, followed by extrapolation of 1/Mw,app to limiting

Joralemon et al.

values at zero concentration, Mw. An increase in 1/Mw,app was noted as the concentration increased, suggesting nonideality due to intermolecular interactions between mannosylated SCKs.55 As summarized in Table 3, Nagg varied from approximately 83 to 352. Each of the SCKs derived from mixed micelles was of similar aggregation number, with no apparent effect of the mannoside unit. For the homogeneous assembly of the mannosylated block copolymer, the self-assembly process gave a substantially larger average aggregation number, which may be due to the substantially larger overall chain length and longer relative hydrophobic chain segment53 for the mannosylated vs nonmannosylated block copolymer. However, interpretation of the data for this sample is complicated by the presence of the large component (see DLS data of Table 2), which may be comprised of aggregated nanoparticles, and suggestive that this sample is prone to aggregation. Aggregation would be further promoted by the increased local concentrations that occur during the sedimentation experiments. The surface charge density of the mannosylated SCK nanoparticles was quantified by zeta-potential measurements. As expected, the zeta-potential values for mannosylated SCKs decreased relative to the value obtained for the parent SCK (see Table 3). The increase in zeta-potential at the 100% mannose level deviates from the expected trend, but direct comparison is not valid due to the differences in particle size and, therefore, electrophoretic mobility. Surface Characterization of the Mannosylated SCK Nanoparticles. The surface availability of the mannoside termini upon the SCKs in aqueous buffer solution was assessed through binding with the protein Concanavalin A (ConA), a known mannose receptor. DLS was utilized to observe aggregated species of ConA and nanoparticles. As shown in Figure 4, ConA gave a peak diameter of 7 nm, whereas the 2% mannosylated SCK had a peak diameter of 36 nm and a small fraction of aggregated particles at 130 nm. When the solutions were mixed together in a molar ratio of 1:80, SCK:ConA, some free ConA remained at 7 nm, whereas larger aggregated species appeared at peak diameters of 52 and 144 nm. No free SCK remained in solution. The ConA was in great excess to the SCK in order to probe the surface availability of the mannosides; therefore, the numberweighted averages reflected this excess and were not used for analysis. Similar experiments were performed with the 0% and 1% mannosylated SCKs, using increasing ratios of ConA to SCK. Figure 5 illustrates the dependence of the intensity-averaged size on the ratio of ConA to SCK. The 1% and 2% mannosylated SCKs behaved similarly and exhibited larger diameter species as the ratio of ConA to SCK increased. In contrast, the 0% mannosylated SCK displayed smaller diameter species as the ratio of ConA to SCK increased, demonstrating that there was no nonspecific association between the ConA and SCK. Therefore, the mannoside groups of the mannosylated particles are available to participate in binding interactions and are required for formation of the supramolecular aggregate formation between the synthetic constructs and proteins. This property is expected to be important for selective biological interactions



Table 3. Partial Specific Volumes, Weight-average Molecular Weights, Weight-average Aggregation Numbers, and Zeta-potentials for Mannosylated SCKs nominal % mannosylation

SCK

νav (mL/g)

MW106 (Da)

Naggb (chains/SCK)

z (mV)

0 1 2 10 100

8 9 10 11 12

0.752 ( 0.004 0.738 ( 0.002 0.713a 0.608 ( 0.18 0.816 ( 0.004

2.17 ( 0.02 1.80 ( 0.02 1.15 ( 0.04 1.56 ( 0.10 4.80 ( 0.05

159 ( 3 131 ( 2 83 ( 2 105 ( 7 352 ( 5

-34 ( 1 -23 ( 1 -25 ( 1 -3 ( 1 -29 ( 1

a Single determination. b Calculated based on the assumption that the mannosylated chains are homogeneously distributed among the respective nanoparticles (zeroth-order approximation).

Figure 5. Intensity-weighted diameter averages vs [ConA]/36 for 8 (filled circles), 9 (open circles), and 10 (filled squares). Figure 3. Overlay of sedimentation equilibrium profiles for SCK nanoparticles 8 (line), 11 (filled circles), and 12 (open circles).

Figure 4. Interaction between ConA and SCK 10 by comparison of hydrodynamic diameter distributions. (A) SCK 10 volume-weighted distribution, (B) ConA volume-weighted distribution, (C) SCK 10 + ConA intensity-weighted distribution.

and also for the design and preparation of well-defined hybrid nanostructured materials. Agglutination inhibition assays47 were also employed to study the interaction between ConA and the mannosylated nanoparticles. Rabbit red blood cells (RBCs) were incubated with 2-fold serial dilutions of ConA in PBS. Four times the minimum concentration of required ConA was then incubated with 2-fold serial dilutions of the respective inhibitors, followed by incubation with the RBCs. The experiment was repeated three times for each inhibitor. Table 4 shows the minimum concentration of inhibitor required to fully inhibit four times the minimum dose of

Table 4. Agglutination Inhibition Assay inhibitor

minimum inhibitor dose (M)

relative dose

R-methyl mannopyranoside 8 9 10 11 12

2.00 × 10-3 1.42 × 10-5 3.74 × 10-6 5.29 × 10-7 8.53 × 10-7 4.52 × 10-11

1 7.10 × 10-3 1.87 × 10-3 2.64 × 10-4 4.26 × 10-4 2.26 × 10-8

ConA from agglutinating the RBCs. Each SCK-based inhibitor required a lower minimum dose than the R-methyl mannopyranoside control, and these minimum dose values are lower than those reported for polyvalent linear polymers.15 Surprisingly, some nonspecific interaction was observed with the 0% mannosylated SCK. It is unclear from this assay if the nonmannosylated SCKs interact with the ConA protein or with the RBCs. In consideration of the DLS experiments with ConA, it is most likely that the 0% mannosylated SCKs (and perhaps each of the mannosylated SCKs) interact with the RBCs and therefore inhibit agglutination. The interaction between the mannosylated nanoparticles and E. coli was visualized through TEM. E. coli is a Gram negative bacterium possessing numerous type 1 pili protein structures which protrude from the cell wall. A mannose binding region is located at the tip of each pilus.38 E. coli cells were incubated with each of the respective particle solutions. The images in Figure 6 provide a qualitative view of the interaction between the nanoparticles and the bacterial cell. There is a greater population of particles located near the cell when the SCKs are functionalized with

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Figure 6. E. coli incubated with (A) SCK 12, (B) SCK 8, and (C) no SCKs, were imaged through TEM. A portion of an E. coli bacterium is shown in the lower left of (A) and (C), and in the upper right of (B).

Conclusions

Figure 7. Interaction between LPS and SCK 8 by comparison of volume-weighted hydrodynamic diameter distributions. (A) LPS, (B) SCK 8, and (C) SCK 8 + LPS.

mannose (Figure 6A) than when they are nonmannosylated (Figure 6B). Guest Sequestration in the Mannosylated SCK Nanoparticles. Lipopolysaccharide A (LPS) from E. coli was utilized to study the uptake of amphiphilic endotoxin guests into the nanoparticle. The amphiphilic character of the LPS molecule, which is constructed of hydrophobic aliphatic chains and a hydrophilic polysaccharide backbone, allows for favorable interactions with the respective hydrophilic shell and hydrophobic core of the SCK. DLS was employed to observe uptake of the LPS endotoxins into the 0% mannosylated SCKs. Figure 7 demonstrates the observed increase in average hydrodynamic diameter upon the addition of LPS to the nanoparticles. Due to the amphiphilicity of the LPS endotoxins, micelles form in aqueous solutions and are assumed to account for the populations with peak diameters of 7, 50, and 344 nm (Figure 7A). The 0% mannosylated SCKs were observed with a peak diameter of 32 nm (Figure 7B). Upon mixing of the LPS and the nanoparticle solutions in a 2 to 1 weight concentration ratio, a population with a peak diameter of 37 nm was observed, as well as a larger population around 182 nm (Figure 7C). The complete disappearance of the free LPS peak and the slight enlargement of the nanoparticle population demonstrate the ability of the SCK to sequester small molecule amphiphilic guests.

Mixed micelle methodologies were employed to construct nanoparticle scaffolds with a hydrophobic core, a hydrophilic cross-linked shell, and several different stoichiometries of surface-presented mannoside groups. Characterization of the fully assembled nanoparticles revealed variations in physical properties. The 100% mannosylated SCK, which was derived from the highest concentration of the high molecular weight amphiphilic block copolymer, exhibited the largest size and the broadest heterogeneity in particle diameter. Preliminary DLS studies of SCK diameter changes via interaction with ConA and TEM imaging of nanoparticles incubated with E. coli support surface availability of the saccharide chain-ends to bind with receptors. The agglutination inhibition assay results support semiquantitatively the surface availability of the mannose chain-ends, and indicate the possibility of polyvalent interactions to achieve a cluster glycoside effect. In addition to their design as a polyvalent56 nanoscaffold for use in enhanced receptor binding, SCKs have promise for use in targeted drug delivery applications.39-42,57 Sequestration interactions between the nanoparticle scaffold and LPS endotoxins were observed by DLS experiments. Ultimately, the SCKs are expected to serve as multifunctional nanoscale scaffolds to promote enhanced binding with bacterial pathogens, therapeutic treatment by release of antibiotics, and prevention of endotoxic shock through the sequestering of amphiphilic lipopolysaccharides released upon bacterial cell death. Further experiments to probe each of these features are in progress. Acknowledgment. Financial support for this work by the National Science Foundation (DMR-9974457 and 0210247) and the Department of Education, Graduate Assistance in Areas of National Need (P200A80221) is gratefully acknowledged. MJJ was supported in part by a Department of Energy Training Grant (DE F0101 NE23051). The authors thank Mr. G. Michael Veith (Washington University Electron Microscopy Laboratory) for expertise and assistance with the TEM measurements. References and Notes (1) Larsson, A.; Ohlsson, J.; Dodson, K. W.; Hultgren, S. J.; Nilsson, U.; Kihlberg, J. Bioorg. Med. Chem. 2003, 11, 2255-2261. (2) Liang, R.; Yan, L.; Loebach, J.; Ge, M.; Uozumi, Y.; Sekanina, K.; Horan, N.; Gildersleeve, J.; Thompson, C.; Smith, A.; Biswas, K.; Still, W. C.; Kahne, D. Science 1996, 274, 1520-1522.

Mannosylated Shell Cross-Linked Nanoparticles (3) Koeller, K. M.; Wong, C.-H. Nat. Biotechnol. 2000, 18, 835-841. (4) Burke, S. D.; Zhao, Q.; Schuster, M. C.; Kiessling, L. L. J. Am. Chem. Soc. 2000, 122, 4518-4519. (5) Sears, P.; Wong, C.-H. Angew. Chem., Int. Ed. Engl. 1999, 38, 23002324. (6) Ratner, D. M.; Plante, O. J.; Seeberger, P. H. Eur. J. Org. Chem. 2002, 826-833. (7) Lundquist, J. J.; Debenham, S. D.; Toone, E. J. J. Org. Chem. 2000, 65, 8245-8250. (8) Sofia, M. J.; Allanson, N.; Hatzenbuhler, N. T.; Jain, R.; Kakarla, R.; Kogan, N.; Liang, R.; Liu, D.; Silva, D. J.; Wang, H.; Gange, D.; Anderson, J.; Chen, A.; Chi, F.; Dulina, R.; Huang, B.; Kamau, M.; Wang, C.; Baizman, E.; Branstrom, A.; Bristol, N.; Goldman, R.; Han, K.; Longley, C.; Midha, S.; Axelrod, H. R. J. Med. Chem. 1999, 42, 3193-3198. (9) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124, 14922-14933. (10) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321-327. (11) Lee, R. T.; Lee, Y. C. Glycoconjugate J. 2000, 17, 543-551. (12) Houseman, B. T.; Mrkisich, M. Top. Curr. Chem. 2002, 218, 1-44. (13) Ohno, K.; Izu, Y.; Yamamoto, S.; Miyamoto, T.; Fukuda, T. Macromol. Chem. Phys. 1999, 200, 1619-1625. (14) Ohno, K.; Tsujii, Y.; Fukuda, T. J. Polym. Sci., Part A: Polym. Chem. 1998, 36, 2473-2481. (15) Mortell, K. H.; Weatherman, R. V.; Kiessling, L. L. J. Am. Chem. Soc. 1996, 118, 2297-2298. (16) Nelson, A.; Stoddart, J. F. Org. Lett. 2003, DOI: 10.1021/ol0353464. (17) Fulton, D. A.; Stoddart, J. F. Bioconjugate Chem. 2001, 12, 655672. (18) Turnbull, W. B.; Kalovidouris, S. A.; Stoddart, J. F. Chem. Eur. J. 2002, 8, 2988-3000. (19) Dubber, M.; Fre´chet, J. M. J. Bioconjugate Chem. 2003, 14, 239246. (20) Dubber, M.; Lindhorst, T. K. Chem. Commun. 1998, 1265-1266. (21) Ashton, P. R.; Hounsell, E. F.; Narayanaswamy, J.; Torill, N. M.; Spencer, N. J.; Stoddart, J. F.; Young, M. J. Org. Chem. 1998, 63, 3429-3437. (22) Bezouˆska, K. ReV. Mol. Biotechnol. 2002, 90, 269-290. (23) Boysen, M. M.; Lindhorst, T. K. Org. Lett. 1999, 1, 1925-1927. (24) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; Nepogodiev, S. A.; Meijer, E. W.; Peerlings, H. W. I.; Stoddart, J. F. Chem. Eur. J. 1997, 3, 974-984. (25) Gebhard, T.; Katopodia, A. G.; Voelcker, N.; Duthaler, R. O.; Streiff, M. B. Angew. Chem., Int. Ed. 2002, 41, 3195-3198. (26) Woller, E. K.; Cloninger, M. J. Biomacromolecules 2001, 3, 10521054. (27) Nagasaki, Y.; Yasugi, K.; Yamamoto, Y.; Harada, A.; Kataoka, K. Biomacromolecules 2001, 2, 1067-1070. (28) Narain, R.; Armes, S. P. Biomacromolecules 2003, 4, 1746-1758. (29) Serizawa, T.; Satoshi, Y.; Akashi, M. Biomacromolecules 2001, 2, 469-475. (30) Yasugi, K.; Nakamura, T.; Nagasaki, Y.; Kato, M.; Kataoka, K. Macromolecules 1999, 32, 8024-8032.

Biomacromolecules, Vol. 5, No. 3, 2004 913 (31) Kim, I.-S.; Kim, S.-H.; Cho, C.-S. Macrolmol. Rapid Commu. 2000, 21, 1272-1275. (32) Dubber, M.; Lindhorst, T. K. J. Org. Chem. 2000, 65, 5275-5281. (33) Dubber, M.; Lindhorst, T. K. Carboydr. Res. 1998, 310, 35-42. (34) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357-2364. (35) Huang, H.; Kowalewski, T.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 1659-1668. (36) Murthy, K. S.; Ma, Q.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. J. Mater. Chem. 2003, 13, 2785-2795. (37) Jones, C. H.; Pinker, J. S.; Roth, R.; Heuser, J.; Nicholes, A. V.; Abraham, S. N.; Hultgren, S. J. Proc. Natl. Acad. Sci., U.S.A. 1995, 92, 2081-2085. (38) Choudhury, D.; Thompson, A.; Stojanoff, V.; Langermann, S.; Pinker, J.; Hultgren, S. J.; Knight, S. D. Science 1999, 285, 1061-1066. (39) Baugher, A. H.; Goetz, J. M.; McDowell, L. M.; Huang, H.; Wooley, K. L.; Schaefer, J. Biophys. J. 1998, 75, 2574. (40) Huang, H.; Wooley, K. L.; Schaefer, J. Macromolecules 2001, 34, 547-551. (41) Kao, H.-M.; O’Connor, R. D.; Mehta, A. K.; Huang, H.; Poliks, B.; Wooley, K. L.; Schaefer, J. Macromolecules 2001, 34, 544-546. (42) Murthy, K. S.; Qinggao, M.; Clark, C. G., Jr.; Remsen, E. E.; Wooley, K. L. Chem. Commun. 2001, 773-774. (43) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053-12054. (44) Ma, Q.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 4805-4820. (45) Miller, J. F.; Schatzel, F.; Vincent, B. J. Colloid Interface Sci. 1991, 143, 537. (46) von Smoluchowski, M. Z. Phys. Chem. 1918, 92, 129. (47) Osawa, T.; Matsumoto, I. Methods Enzymol. 1972, 28, 323-327. (48) Choi, S. K.; Mammen, M.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 4103-4111. (49) Debenham, S. D.; Snyder, P. W.; Toone, E. J. J. Org. Chem. 2003, 68, 5805-5811. (50) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921-2990. (51) Matyjaszewski, K.; Wang, J.-S.; Grimaud, T.; Shipp, D. A. Macromolecules 1998, 31, 1527-1534. (52) Wang, J.-S.; Matyjaszewski, K. Macromolecules 1995, 28, 79017910. (53) Thurmond, K. B., II.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1997, 119, 6656-6665. (54) Remsen, E. E.; Thurmond, K. B., II.; Wooley, K. L. Macromolecules 1999, 32, 3685. (55) Laue, T. M.; Bhairavi, D. S.; Ridgeway, T. M.; Pelletier, S. In Analytical Ultracentrifugation in Biochemistry and Polymer Chemistry; Redwood Press Ltd.: Melsham, 1992; pp 90-125. (56) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754-2794. (57) Thurmond, K. B., II.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. Nucl. Acids Res. 1999, 27, 2966-2971.

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Synthesis, Characterization, and Bioavailability of Mannosylated Shell

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