Functional Disulfide-Stabilized Polymer−Protein Particles

The University of New South Wales (UNSW), Sydney, NSW 2052, Australia ... The micelles were subsequently decorated with (red fluorophore-labeled) ...
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Biomacromolecules 2009, 10, 3253–3258

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Functional Disulfide-Stabilized Polymer-Protein Particles Zhongfan Jia,†,‡ Jingquan Liu,‡ Cyrille Boyer,‡ Thomas P. Davis,*,‡ and Volga Bulmus*,§ Centre for Advanced Macromolecular Design (CAMD), School of Chemical Sciences and Engineering, and School of Biotechnology and Biomolecular Sciences (BABS), The University of New South Wales (UNSW), Sydney, NSW 2052, Australia Received July 19, 2009; Revised Manuscript Received September 8, 2009

Polymer-protein hybrid particles (PPHPs) have a significant potential in drug delivery, diagnosis, and biomedical imaging applications. Herein, we describe a simple route to disulfide cross-linked, poly(ethylene glycol)-streptavidin hybrid particles with tunable diameters. These particles have great versatility and potential for a number of reasons. First, they possess free biotin binding sites on their streptavidin (SAv) coated surface, enabling the conjugation of any biotinylated-molecule such as biotinylated antibodies. Second, core-stabilization can easily be controlled using reversible disulfide cross-links, and third, thiol- and ene-reactive functionalities in the core are available for the conjugation of drugs and labels. In detail, micelles having a biotinylated poly(ethylene glycol) corona and a disulfide cross-linked, reactive core were formed using R-biotin PEG-b-poly(pyridyldisulfide ethylmethacrylate) block copolymers synthesized via RAFT polymerization. Functionalization of the micelle core was performed in a one-pot reaction concurrent with the micellization and cross-linking processes by using a thiol-reactive model compound (a maleimide derivative of a green fluorophore). The resultant micelles displayed spherical morphology with a diameter of 54 ( 4 nm. Biotin functionality was largely exposed on the micelle corona (75 mol % availability), as determined by a streptavidin/HABA assay. The micelles were subsequently decorated with (red fluorophore-labeled) streptavidin (SAv) through the accessible biotins on the surface, yielding SAv-linked micelle aggregates with tunable dimensions (in the range between 350 nm and 2 µm), as determined by transmission electron microscopy. Fluorescent-labels on the particles were monitored using confocal microscopy, revealing that the SAv coats the periphery of the PPHPs.

Introduction Polymeric soft particles have great potential for drug delivery, biomedical diagnostics and imaging applications.1-3 To conform with the complex requirements of most biomedical applications, they often need to be endowed with multifunctionalities such as therapeutic drugs, specific biorecognition agents, transporter elements, detection, and imaging agents.1-4 Multifunctional particle designs inspired by biological systems are highly likely to address most of the criteria determined by specific application demands. The most recent development in bioinspired particle design is polymer-protein hybrid particles (PPHPs), described by a number of research groups.5-17 These earlier studies demonstrate that controlled/living radical polymerization techniques (CLRPs) such as atom-transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization provide efficient synthetic routes leading to well-defined building blocks and precursors to biohybrid materials, that is, polymer-protein conjugates,5,10,13,18,19 and also PPHPs. For example, the in situ ATRP of hydrophobic monomer units directly on proteins was shown to produce giant polymer-protein amphiphiles, subsequently yielding PPHPs.11 Conjugates of proteins with stimuli-responsive polymers have also been prepared via either in situ polymerization or by postpolymerization conjugation methods, producing PPHPs with assembly driven by environmental changes.5,10,13,15,20 Recently, polymer micelles with surface functional groups were prepared * To whom correspondence should be addressed. E-mail: vbulmus@ unsw.edu.au (V.B.); [email protected] (T.P.D.). † Current address: Australian Institute for Bioengineering and Nanotechnology (AIBN). ‡ Centre for Advanced Macromolecular Design (CAMD). § School of Biotechnology and Biomolecular Sciences (BABS).

from RAFT-synthesized block copolymers for conjugation to proteins, yielding PPHPs under optimized conditions.9,21 Biotinylated polymer micelles, built from both ATRP16 and RAFT21 polymers, have been described previously. Postpolymerization modification has also been employed to introduce biotin functionality on the surface of polymer micelles7,9 and also on polymer nanospheres synthesized via emulsion polymerization.7 Although interactions between biotinylated polymer micelles/nanospheres and streptavidin (SAv) have been investigated in a few publications,7,9,16 to the best of our knowledge, no attempt has been made to employ such interactions in the preparation of multifunctional PPHPs with tunable dimensions. In this study, we have used RAFT polymerization to generate biotinylated block copolymers comprised of a biocompatible block, poly(ethylene glycol) (PEG) and a functional block, poly(2-(2-pyridyldisulfide)ethylmethacrylate) (PPDSM). These block copolymers could then be assembled in water, forming polymer micelles with biotin functionalized coronas and thioland ene-reactive cores. Concurrent with the micellization, the cores were functionalized with a maleimide derivative of a green fluorophore. The micelle corona biotin functionality was then utilized for interactions with SAv, pretreated with free biotin at varying ratios, generating SAv-coated, disulfide stabilized, functional PPHPs with tunable dimensions. The synthetic route is summarized in Scheme 1. The PPHPs described herein can be regarded as generic particulate scaffolds for surfacemodification with any biotinylated agent using a simple, mild conjugation process. In addition, the PPHP cores can be functionalized with any thiol- or ene-reactive compounds, such as drugs and labels.

10.1021/bm900817a CCC: $40.75  2009 American Chemical Society Published on Web 09/30/2009

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Scheme 1. Procedure Followed To Prepare Multifunctional, Disulfide Stabilized PPHPsa

a The resultant PPHPs can potentially be functionalized with thiol- and ene-reactive compounds in the core, such as drugs and labels, and also biotinylated targeting agents, on the surface, via biotin binding sites of SAv coating the surface.

Scheme 2. Overall Synthetic Scheme of R-Biotin PEG-b-PPDSM Synthesis

Experimental Section Materials. Chemicals were purchased from Aldrich unless otherwise indicated. The initiator, 2,2′-azobisisobutyronitrile (AIBN) was recrystallized twice from methanol prior to use. High purity nitrogen (Linde Gases) was used for purging the reaction solutions before the polymerization. 2,2′-Dthiodipyridine (DTDP), 2-mercaptoethanol, and methacryloyl chloride (TCI) were used as received. Dichloromethane (Univar, reagent grade) were distilled prior to use. N,N-Dimethylacetamide, HPLC grade 99.9% (DMAc), used for polymerizations. O-[2(Boc-amino)ethyl]polyethylene glycol with Mn ) 5000 and tri(2carboxyethyl)phosphine (TCEP) hydrochloride were used as received. The precursor, hydroxethyl pyridyldisulfide (HPDS), and the monomer pyridyldisulfide ethylmethacrylate (PDSM) were synthesized according to the procedure reported elsewhere.22,23 4-(Cyanopentanoic acid)-4dithiobenzoate (CPADB) was synthesized according to the previously reported procedure.24 N,N′-Dicyclohexylcarbodiimide and biotin were used as received. Oregon Green 488 Maleimide (Molecular Probes) was used as received. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC · HCl), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), and 2-(4-hydroxyphenylazo) benzoic acid (HABA) were purchased from Pierce and used as received. Streptavidin from Streptomyces aVidinii (SAv) was received as lyophilized powder. Streptavidin DyLight 547 Conjugated was purchased from Pierce as a solution (1 mg/mL). Synthesis of r-Biotin PEG-b-PPDSM. The procedure for the synthesis of biotinylated PEG-b-PPDSM is shown in Scheme 2. (a) Deprotection of Boc-Amino PEG. A heterofunctional PEG O-[2(Boc-amino)ethyl]polyethylene glycol (Mn: 5000 g/mol) was used. First, the Boc-protected amine group of PEG (0.5 g) was deprotected in a

TFA/DCM mixture (1:1 v/v) for 2 h. After the removal of the solvent by evaporation, DCM (1 mL) was added to dissolve the product. The product was then precipitated in diethyl ether. After filtration, the product was dried in vacuo and then dissolved in water, dialyzed against water (pH ) 8.0) for 2 days by replacing water every 12 h. The final deprotected R-amino-, ω-hydroxyl PEG was recovered by freeze-drying. A white powder (0.44 g) was obtained (yield: 88%). 1H NMR spectra of the product showed the disappearance of Boc group at 1.42 ppm after hydrolysis. (b) Synthesis of Biotinylated PEG. Biotin (51.3 mg, 2.1 × 10-4 mol), EDC · HCl (80.5 mg, 4.2 × 10-4 mol) and Sulfo-NHS (45.6 mg, 2.1 × 10-4 mol) were dissolved in 3 mL of DMSO/MES buffer (pH ) 6.0; 1/1 V/V). The mixture was stirred for 4 h. The deprotected PEG (0.35 g, 7 × 10-5 mol) was then added to the biotin solution. The mixture was stirred overnight and then dialyzed against distilled water for 2 days, by replacing water every 12 h. The final product was recovered by freeze-drying. A white powder (0.28 g) was obtained (yield: 80%; 1 H NMR is shown in Figure S1). 1 H NMR (DMSO-d6, 298 K, 300 MHz), δ (ppm from TMS): 7.8 (1H, t, -CH2-NH-CO-), 6.32-6.39 (2H, d, -NH-CO-NH-), 4.28, 4.11, 3.17 (3H, m, methine protons of biotin), 2.80, 3.05 (2H, m, methylene protons of biotin), 2.05 (2H, t, -CH2-CH2-CO-), 1.25-1.6 (6H, m, methylene protons), 3.42-3.63 (methylene protons of PEG repeat units). (c) Synthesis of Biotinylated PEG Chain Transfer Agent. Hydroxyl group of biotinylated PEG was esterified using CPADB. Biotinylated PEG (0.25 g, 5.0 × 10-5 mol), CPADB (42 mg, 1.5 × 10-4 mol), DCC (41.2 mg, 2.0 × 10-4 mol), and DMAP (3.0 mg, 2.5 × 10-5 mol), in order, were dissolved in dry DCM (5 mL). The solution was stirred overnight. The white solid, dicyclohexyl urea (DHU), was

Polymer-Protein Particles removed by filtration. The filtrate was concentrated to 1 mL and then precipitated three times in diethyl ether. A pink powder (0.16 g) was obtained after drying in vacuo (yield: 64%; 1H NMR of the product is shown in Figure S1). 1 H NMR (DMSO-d6, 298 K, 300 MHz), δ (ppm from TMS): 7.9 (2H, d, aromatic protons), 7.8 (1H, t, -CH2-NH-CO-), 7.68 (1H, t, aromatic protons), 7.52 (2H, t, aromatic protons), 6.32-6.39 (2H, d, -NH-CO-NH-), 4.28, 4.11, 3.17 (3H, m, methine protons of biotin), 4.13 (2H, t, -CH2CH2-OCO-), 2.80, 3.05 (2H, m, methylene protons of biotin), 2.32-2.55 (4H, m, -CO-CH2-CH2-C(CN)(CH3)-), 2.05 (2H, t, -CH2-CH2-CO-), 1.9 (3H, s, methyl protons of CPADB), 1.25-1.6 (6H, m, methylene protons), 3.42-3.63 (methylene protons of PEG repeat units). (d) Polymerization of PDSM Monomer Using Biotinylated PEG Chain Transfer Agent. Typically, biotinylated PEG chain transfer agent (50 mg, 1 × 10-5 mol), PDSM monomer (0.26 g, 1 × 10-3 mol), and AIBN (0.55 mg, 3.4 × 10-6) were dissolved in DMAc (2.5 mL). The flasks were sealed with rubber septa and then purged with N2 for 40 min. The polymerization was carried out in a preheated oil bath at 70 °C. The flasks were taken out at predetermined time points and immersed in the liquid nitrogen to stop the polymerization. After thawing, the solutions were precipitated three times in diethyl ether and then dried in vacuo. Figure S2 shows the GPC traces of the block copolymers. 1 H NMR (DMSO-d6, 298 K, 300 MHz), δ (ppm from TMS): 8.42 (1H, bd, aromatic proton ortho-N), 7.62-7.81 (2H, m, aromatic proton meta-N and para-N), 7.19 (1H, m, aromatic proton, ortho-disulfide linkage), 6.32-6.38 (2H, d, amide protons of biotin), 4.11 (2H, bd, -OCH2CH2-SS-), 3.48 (4H, bd, methylene protons of PEG repeat units), 3.02 (2H, bd, -OCH2CH2-SS-), 0.8-2.0 (bd, methyl and methylene protons of backbone). One-Pot Preparation of Disulfide Cross-Linked Micelles. Typically, the block copolymer (10 mg) was dissolved in methanol (2 mL) and stirred overnight. TCEP (20 mg) was dissolved in 1 mL methanol. 0.2 mL from TCEP solution (the mole number of TCEP added: ∼1.4 × 10-5 mol) was added drop-by-drop to the polymer solution while the solution was stirred. The color of the solution changed to yellow gradually, which indicated the cleavage of the PDS groups and the formation of pyridine-2-thione. When conjugating Oregon Green 488 maleimide, the dye (1 mg) was added to the polymer solution and stirred for 30 min in dark before the addition of TCEP. After the addition of TCEP, the solution was stirred for 4 h. The solution was then dialyzed against DI water for three days by changing water every 12 h. The final micelle solution was stored for further characterizations and conjugations experiments. Preparation of Polymer-Protein Hybrid Particles. SAv or SAv DyLight 547 was pretreated with free biotin at varying ratios. In a typical experiment, biotin solution (0, 27, or 54 µL, 0.1 mM) was added to SAv solution (150 µL, 1 mg/mL) with or without SAv DyLight 547 (SAv: SAv DyLight 547 ) 4:1 mol ratio), followed by vortexing. According to the SAv-HABA assay, 0, 30, and 60% conjugation sites of SAv were preoccupied by biotins. Micelle solution (31 µL, 2 mg/ mL with 7.1 × 10-5 mol/mL biotins on the surface) was then added to the pretreated SAv solution and vortexed. The solution was then analyzed by DLS, TEM, and confocal microscope. Characterization. 1H NMR experiments were conducted using a Bruker Avance DMX300 spectrometer. Molecular weights and molecular weight distributions (PDI) were measured by gel permeation chromatography (GPC) using a Shimadzu modular LC system comprising a DGU-12A solvent degasser, a LC10AT pump, a SIL- 10AD autoinjector, a CTO-10A column oven, and a RID-10A refractive index detector. The system was equipped with a 50 × 7.8 mm guard column and four 300 × 7.8 mm linear columns (Phenomenex 500, 103, 104, and 105 Å pore size; 5 µm particle size). N,N′-Dimethylacetamide (DMAc; HPLC, 0.03% w/v LiBr, 0.05% BHT) was used as eluent at a flow rate of 1 mL min-1, while the columns temperature was maintained at 40 °C. Polymer solutions (3-5 mg

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mL-1) were injected in 50 µL volumes. Calibration was performed with narrow polydispersity polystyrene standards (Polymer Laboratories) in the range 0.5-1000 kDa. Particle size measurements were done using a Malvern Instruments Zetasizer NaNo ZS instrument equipped with a 4 mV He-Ne laser operating at λ ) 633 nm, an avalanche photodiode detector with high quantum efficiency, and an ALV/LSE-5003 multiple tau digital correlator electronics system with a measurement angle of 173°. Water was used as a solvent. JEOL 1400 transmission electron microscope (TEM) was used to investigate the morphology of the nanoparticles. It was operated at an acceleration voltage of 100 kV. UV-vis spectra were recorded using a VARIAN CARY 300 UV-vis spectrophotometer. Confocal microscopy images were taken using an Olympus FV1000 Laser Scanning Microscope (inverted). Two excitation wavelengths at 488 and 543 nm were used for Oregon green 488 and SAv-DyLight 547, respectively. A streptavidin/HABA (SAv/HABA) assay was used to quantify the biotinylation degree of both the PEG-chain transfer agent and the crosslinked micelles. The procedure and the calibration curves are given in the electronic Supporting Information (ESI).

Results and Discussion Synthesis of PEG-b-PPDSM. Successful living radical (co)polymerizations of a thiol reactive monomer, pyridyldisulfide ethylmethacrylate (PDSM), have been reported previously by Thayumanavan et al.25 and our group22,23 via ATRP and RAFT techniques, respectively. Herein, a biotinylated PEGmacroRAFT agent was first synthesized from a heterotelechelic PEG (Mn 5000 g/mol). 1H NMR analysis showed that approximately 100% of polymer chain ends were functionalized with biotin and dithiobenzoate groups (Figure S1, ESI). Subsequently, the PDSM monomer was used to chain-extend the R-biotin PEG chain transfer agent (PEG-CTA), yielding R-biotin PEG-b-PPDSM block copolymers (GPC chromatograms are shown in Figure S2, ESI). Block copolymers having number average molecular weights between 15000 and 29000 g/mol and polydispersity indices (PDI) between 1.24 and 1.39 were obtained with increasing monomer conversions. Relatively high PDI (1.39) at high conversion may be attributed to the transfer reactions due to PDSM‘s disulfide bond. In a previous study,23 the transfer of hydroxypyridyl disulfide (homologue of PDSM monomer without methacrylic bond) was investigated for MMA polymerization. The transfer onto hydroxypyridyl disulfide was found to be negligible at low to moderate monomer conversions. At high monomer conversions, low degree of chain transfer which broadened PDI was observed. The 1H NMR spectrum of the purified R-biotin PEG-b-PPDSM (Mn: 21000 g/mol, PDI: 1.25 by GPC; Mn: 20000 g/mol by 1H NMR) is shown in Figure 1. The spectrum shows a signal at 6.38 ppm indicating the presence of biotins at the chain ends. Preparation of Reversible Disulfide-Cross-linked Micelles with Surface-Accessible Biotins. The biotinylated block copolymer, PEG-b-PPDSM (Mn: 21000 g/mol, PDI: 1.25 by GPC; Mn: 20000 g/mol by 1H NMR) was used to form micelles in methanol. Micellization was performed simultaneously with core-cross-linking in one-pot, as described in a previous paper.22 Briefly, the micellization of the block copolymer in methanol was followed by reduction of the pyridyldisulfide (PDS) groups of the PPDSM block (in the micelle core) by addition of a reducing agent (tri(2-carboxyethyl)phosphine (TCEP), equivalent to 50 mol % of PDS groups, into the micellization solution, leading to the formation of intra- and interchain disulfide crosslinks in the core. Importantly, free thiols that are formed in the

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Figure 1. 1H NMR of the purified biotinylated PEG-b-PPDSM (Mn: 21000 g/mol, PDI: 1.25 by GPC; Mn: 20000 g/mol by 1H NMR) in DMSO-d6 (300 MHz).

Figure 2. Analysis of the particle hydrodynamic diameters in water by dynamic light scattering: (green triangle) SAv, (blue triangle) polymer micelles, (red circle) PPHPs with 60% preoccupied biotins, (black square) PPHPs without preoccupied biotins (the mol ratio of biotins on the micelles to SAv is 1:2).

core of the micelles during the reduction process can be used efficiently to conjugate “ene” bearing molecules. A maleimide derivative of a fluorophore (in this case, Oregon Green 488 maleimide) was used as an ene reagent. The concurrent micellization and cross-linking process was confirmed by GPC, DLS, and TEM. GPC results showed that the apparent molecular weight (Mn(app)) increased from 21000 to 550000 g/mol after the one-pot reaction (Figure S3, ESI). Such a significant increase can be attributed to the formation of a cross-linked structure. After the micelles were treated with a strong reducing agent, DTT, the Mn(app) decreased from 550000 to 16000 g/mol, in accord with our previous results.22 The decrease in molecular weight resulted from the cleavage of disulfide bonds generating unimers. The hydrodynamic diameter of the cross-linked micelles, measured by DLS, was 54 ( 4 nm (blue triangles in Figure 2) and the morphology of the micelles was spherical, as analyzed by TEM (Figure 3a). The size of the micelles after drying, as observed by TEM, was in accord with the hydrodynamic size determined by DLS. A streptavidin/HABA (SAv/HABA) assay was used to quantify the biotinylation degree of both the PEG-CTA and the cross-linked micelles (Figure 4). Biotinylated PEG-CTA and

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cross-linked micelles were tested separately. As expected, an inverse linear relationship was observed between the concentration of the samples and the absorbance of the SAv/HABA solution at 500 nm. The absorbance at 500 nm decreased gradually with increasing polymer concentration, consistent with HABA molecule displacement from the SAv/HABA complexes by biotin. Using a calibration curve built using a free biotin standard solution (Figure S4, ESI), the biotinylation degree of PEG-CTA was calculated to be 97 mol % of polymer chains, consistent with the NMR data. A SAv/HABA assay on the micelles showed that approximately 75 mol % of biotins presented on the micelle corona was accessible by SAv. The 25% loss in the accessibility of biotin can be attributed to both biotin embedment inside the polymeric corona and a steric hindrance effect resulting from the high density of biotins on the surface. Preparation of Polymer-Protein Hybrid Particles. A streptavidin (SAv) molecule can bind four biotin molecules with a high affinity (Ka ) 1015 M-1). The high binding affinity and the presence of four biotin binding sites have favored the widespread application of SAv as a universal biocrosslinker. In the case of biotinylated polymers, it has been reported that one SAv can interact with 2.5-3.5 biotinylated polymer chain depending on the molecular weight of polymer and binding kinetic.10,26-28 In the present study, the micelle surface was coated by SAv to create a generic particulate scaffold that can potentially be surface-modified with any biotinylated agent via a simple, mild conjugation process. It is unlikely that all biotin present on the micelle periphery binds with SAv as steric hindrance effects may play a significant role. As one SAv molecule can bind four biotin molecules, cross-linking between the micelles should occur. After incubation with SAv (biotin/ SAv mol ratio) 1:2) the micelles were analyzed by PAGE (Figure 4). SAv (lane 1), SAv with biotinylated PEG-CTA (lane 2), and micelles postincubation with SAv (lanes 3 and 4) can be seen in Figure 4. The protein bands, in lanes 3 and 4, that appear in the wells confirm that SAv molecules bind to the biotinylated micelles, yielding higher molecular weight (giant) structures (Scheme 1) that cannot migrate through the gel under the electric field applied. The hydrodynamic diameters of SAv-conjugated micelles (biotin/SAv mol ratio) 1:2), determined by DLS (black filled squares), are shown in Figure 2. Particles with diameters up to 1.8 ( 0.4 µm were observed. This can be attributed to the formation of giant particles composed of multimicelles crosslinked via SAv. The particle sizes could be controlled simply by using SAv pretreated with biotin (at different concentrations). For instance, when a SAv sample that had 2.4 out of 4 binding sites preoccupied with biotin was incubated with the biotinylated micelles, the hydrodynamic diameter of the multimicellar particles decreased to 380 ( 35 nm. This significant size decrease (from 1.8 µm to 380 nm) confirmed that the particles were formed by intermicelle cross-linking via affinity binding between the SAvs in solution and the biotins on the micelles, also concurred with TEM analyses. The micelles after incubation with SAv pretreated with free biotin at varying concentrations were investigated by TEM (Figure 3b-d). At all concentrations, particles with a spherical morphology were observed. The particles, formed from micelles incubated with SAv having 0 and 30% (1.2 out of 4) of binding sites preoccupied with free biotin, possessed an average diameter of 1.7 ( 0.3 µm and 720 ( 70 nm, respectively (Figure 3b,c). As the percentage of preoccupied biotin binding sites on SAvs was increased to 60% (2.4 out of 4), the particle size decreased to 350 ( 60 nm (Figure

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Figure 3. Transmission electron micrographs of (a) biotinylated micelles and (b-d) particles formed in the presence of SAv pretreated with free biotin at varying ratios: (b) 0%, (c) 30%, and (d) 60% of biotin binding sites of SAv (in each case, biotins on micelles/SAv mol ratio ) 1:2). The scale bar is 500 nm for the inset in b.

Figure 4. UV-vis spectra of SAv/HABA complex before and after the addition of biotinylated micelles and biotinylated PEG-CTA, and polyacrylamide gel electrophoresis of SAv (lane 1), SAv with biotinylated PEG-CTA (lane 2), and PPHPs (lanes 3 and 4).

3d). It is evident that higher prebinding would yield even smaller particle sizes. A higher resolution TEM micrograph of a single particle (Figure 3b, inset) confirmed that the giant particles were constituted by a number of subparticles (micelles). In summary, SAv proteins act as both surface functional and intermicellar cross-linking sites in the giant particles. The thiol-reactive PPDSM block in the micelle core can be employed as a versatile site for covalent conjugation of biomolecules or drugs.22,23,29 To exemplify the core functionality, a maleimide modified green fluorophore (Oregon Green 488 maleimide) was used as a model molecule. The fluorophore was conjugated in a one pot reaction concurrent with micellization and cross-linking processes. After incubation of the biotinylated and fluorescent-labeled micelles with SAv (60% of binding sites pretreated with biotin), spherical particles having green fluorescence were formed, observable by confocal microscopy (Figure 5A). When nonlabeled micelles were incubated with a mixture of red-fluorescent-labeled SAv (conjugated with Dy

Light 547) and nonlabeled SAv (biotin/SAv mol ratio ) 1:2), the formation of spherical, red fluorescing particles was observed (Figure 5B). The diameter of the green-fluorescent particles (264 ( 58 nm, measured from the confocal micrograph; in Figure 5A) was slightly smaller than that of the red particles (357 ( 79 nm measured from the confocal micrograph; in Figure 5B), suggesting different locations of the green and red fluorophores in the particle structure. The smaller green particles correspond to green fluorophores located in the particle cores, and the slightly larger red particles correspond to red fluorophores located at the particle surface. The micelle structures could also be probed by labeling both the micelle cores (with green fluorescence) and SAvs (with red fluorescence) and imaging at two different wavelengths (λex ) 488 and 540 nm). In Figure 5C, the first two images from the left show the same particle excited separately at 488 and 540 nm, respectively. Superimposition of these images shows the presence of a red fluorescent layer on the periphery of the particles. This observation is

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mental procedures for HABA assays. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes

Figure 5. Confocal fluorescence microscope images of PPHPs. (A) Oregon Green 488 conjugated micelles after incubation with nonlabeled SAv (having 60% of binding sites pretreated with biotin; λex ) 488 nm); (B) Nonlabeled polymer micelles after incubation with a mixture of SAv/SAv labeled with DyLight 547 (8/2 mol ratio; having 60% of binding sites pretreated with biotin; λex ) 540 nm); (C) Oregon Green 488 conjugated micelles after incubation with a mixture of SAv and SAv labeled with DyLight 547 (8/2 mol ratio; not pretreated with biotin): particle at the left; λex ) 488 nm, particle in the middle λex ) 540 nm, and the particle at the right was obtained by superimposition of the left and the middle images.

consistent with SAv molecules present not only at the surface of the micelles but also at the surface of the multimicellar particles.

Conclusion In summary, we have developed a method to make disulfide cross-linked PPHPs with tunable size and conjugation sites both in the core and on the capsid. The SAv- coating on the capsid potentially allows functionalization with any biotinylatedmolecule, such as biotinylated antibodies for recognition of surface receptors of specific cell types. Reversible disulfide cross-links in the particle structure creates the opportunity for intracellular disassociation (and potential release of actives). The presence of thiol- and ene-reactive functionalities in the particle cores provides a method for covalent conjugation of drugs and labels via cleavable (in the case of thiol-bearing molecules) or stable bonds (in the case of ene-bearing molecules). Overall, the simplicity and versatility of the presented synthetic approach and the resulting potential library of structures creates opportunities in both drug delivery and diagnostic technologies. Acknowledgment. V.B. and T.P.D. acknowledge the receipt of Discovery Grants from the Australian Research Council (ARC). T.P.D. is also thankful for a Federation Fellowship from the ARC. Supporting Information Available. GPC and 1H NMR characterizations for copolymers and their precursor; experi-

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