A Direct Probe of the Interplay between Bilayer Morphology and

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A Direct Probe of the Interplay between Bilayer Morphology and Surface Reactivity in Polymersomes Ya-Wen Chang, James A. Silas, and Victor M. Ugaz* Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843 Received April 23, 2010. Revised Manuscript Received June 10, 2010 Bilayer vesicles self-assembled from amphiphilic poly(ethylene oxide)-b-polybutadiene (PEO-b-PBd) copolymers are cell-like structures whose high stability and tunable membrane properties make them ideal for use as potential drug carriers and cell mimicry templates. Understanding how the surface interactions (reaction, binding, etc.) are governed by the bilayer structure is critical to enable construction of polymersomes with tailored colloidal behavior. Here, we adapt a previously established chemical labeling method by incorporating coumarin functionalized copolymer into the vesicular structure. This allows us to probe the effect of poly(ethylene glycol) (PEG) brush and surface architecture on the bimolecular quenching reaction occurring at the polymersome surface. Using these measurements, we have tracked quenching in free solution, on bare particles, and on two types of vesicle surfaces: one where the functionalized copolymer groups are longer than the surrounding unfunctionalized copolymer, and one where both functionalized and unfunctionalized groups are the same length. We find that quenching in the presence of the PEG brush proceeds at less than half the free solution rate in both vesicle architectures. However, the quenching rate is further reduced when the functionalized and unfunctionalized groups are the same length. The surface reaction appears to be dominated by quencher diffusion, a conclusion supported by conductivity measurements and ion partition studies indicating that these effects arise as a consequence of retarded ion mobility in the presence of the PEG brush rather than ion exclusion effects. These studies reveal the interplay between the vesicle bilayer architecture (copolymer composition, chain length, local concentration surrounding the active site) and the surface reaction rate, thereby providing useful insights that can help guide the design of polymersomes with desired functional properties.

Introduction Synthetic block copolymer amphiphiles, analogues to natural phospholipids, can self-assemble into enclosed bilayer structures similar to cell membranes.1-3 These polymeric vesicles (polymersomes) share many features in common with lipid vesicles such as a spherical enclosed structure capable of entrapping hydrophilic components in the interior aqueous region, but they also offer unique advantages including increased stability and resistance to thermal and mechanical stresses. This makes them superior to conventional phospholipid vesicles. Additionally, many of these properties (membrane bending modulus, rigidity, toughness, etc.) are widely tunable by varying block length or chemical structure.4,5 The ability to construct polymersomes with tailored properties critically depends on understanding how the kinetics of surface interactions (reaction, binding, etc.) are governed by the bilayer architecture. Previous efforts to probe these interactions have focused mainly on adhesion-based measurements. Surface force apparatus experiments, for example, have been employed to examine receptor-ligand interactions between a supported lipid *To whom correspondence should be addressed. Mailing address: Artie McFerrin Department of Chemical Engineering, Texas A&M University, Jack E. Brown Engineering Building 3122-TAMU, College Station, TX 77843-3122. Telephone: (979) 458-1002. Fax: (979) 845-6446. E-mail: [email protected]. (1) Discher, B. M.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Curr. Opin. Colloid Interface Sci. 2000, 5(1-2), 125–131. (2) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C. M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284(5417), 1143–1146. (3) Discher, D. E.; Eisenberg, A. Science 2002, 297(5583), 967–973. (4) Bermudez, H.; Hammer, D. A.; Discher, D. E. Langmuir 2004, 20(3), 540– 543. (5) Lin, J. J.; Silas, J. A.; Bermudez, H.; Milam, V. T.; Bates, F. S.; Hammer, D. A. Langmuir 2004, 20(13), 5493–5500.

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bilayer incorporating anchored biotinylated poly(ethylene glycol) (PEG) chains of varying length and a streptavidin functionalized surface.6,7 These studies provided precise measurement of specific and nonspecific forces between the PEG-anchored ligands and model receptor surfaces that were in good agreement with theoretical predictions and simulations.8,9 However, this method is restricted to planar systems, making it challenging to adapt for study of vesicular structures like polymersomes. Micropipet aspiration is another widely used technique that has been adapted to study adhesion in bilayer membranes such as red blood cells and liposome membranes.10-12 A further variation of the method, micropipet peeling tests, has been applied to assess adhesion strength between biotin-functionalized polymersomes and avidin-coated beads.5,13 These experiments were instrumental in identifying how surface adhesion depends not only on the bond strength but also on how the ligand is presented on the polymer brush surface. However, despite their simplicity, adhesion-based measurements primarily describe equilibrium interfacial interactions and provide little information about the reaction itself. Fluorescence-based spectroscopic methods enable interactions and dynamic events involving probe carrying molecules to be quantitatively measured with a high level of sensitivity. Fluorescence quenching is frequently used in biophysics to probe (6) Jeppesen, C.; Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S.; Marques, C. M. Science 2001, 293(5529), 465–468. (7) Moore, N. W.; Kuhl, T. L. Langmuir 2006, 22(20), 8485–8491. (8) Longo, G.; Szleifer, I. Langmuir 2005, 21(24), 11342–11351. (9) Longo, G. S.; Thompson, D. H.; Szleifer, I. Langmuir 2008, 24(18), 10324– 10333. (10) Evans, E. A. Biophys. J. 1985, 48(1), 175–183. (11) Evans, E.; Needham, D. Macromolecules 1988, 21(6), 1822–1831. (12) Evans, E. A. Biophys. J. 1985, 48(1), 185–192. (13) Lin, J. J.; Bates, F. S.; Hammer, D. A.; Silas, J. A. Phys. Rev. Lett. 2005, 95, 026101.

Published on Web 06/28/2010

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conformational changes, detect binding events, characterize membrane fluidity and permeability,14 and to elucidate the location of fluorescently tagged molecules inside the bilayer.15 Here, we adapt the fluorescence quenching technique to probe small molecule reaction kinetics on polymersome surfaces. We report the results of quenching studies performed to track reactions in the bulk solution, on bare particles, and on polymersome surfaces. Vesicles were constructed using poly(ethylene oxide)-b-polybutadiene (PEO-b-PBd) copolymer with two different lengths (PEO89-PBd120 and PEO20-PBd33). The long copolymer was end functionalized with coumarin and mixed with unfunctionalized copolymer to create labeled vesicles. We find that quenching is greatly reduced in the presence of PEG and that the polymersome surface in which the functionalized chains are the same length as the membrane brush displays a lower quenching rate then when these chains are surrounded by shorter brushes. Further experiments suggest that these observations can be explained by retarded ion mobility in the presence of the PEG polymer rather than ion exclusion effects. Reaction rates are quantified using a nonlinear Stern-Volmer analysis based on the finite sink approximation,16,17 and a modified Yasuda free volume model is used to describe ion diffusion.18 This analysis allows us to identify correlations between the quenching rate and ion diffusivity so that the local polymer concentration surrounding the fluorophore can be inferred. In this way, we are able to establish a relationship between the reaction rate and the bilayer surface structure, thereby providing a useful tool to understand how surface reactions can be manipulated by modifying the polymer vesicle formulation.

Materials and Methods Materials. Poly(ethylene oxide)-b-polybutadiene (PEO89PBd120, MW 10 400 g/mol; PEO20-PBd33, MW 1800 g/mol) block copolymers were purchased from Polymer Source Inc. (Canada). Sucrose (ACS reagent); sodium iodide (NaI); sodium chloride (NaCl); sodium thiosulfate (Na2S2O3); and phosphate buffered saline (PBS, 10
) were purchased from Fisher Scientific (Pittsburgh, PA). Chloroform (99.8þ% for analysis ACS, stabilized with ethanol) (CHCl3) and sodium chloride were purchased from Acros Organics (Morris Plains, NJ). Poly(ethylene glycol) (PEG) of molecular weight 1000, 2000, and 8000 was obtained from Clariant Corporation (Charlotte, NC). Cellulose ester dialysis tubing (MWCO 1000 Da) and the regenerated cellulose dialysis tubing kit (MWCO 8000 Da) (Spectra/Por Biotech grade) were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). N-Ethyl-N0 -(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Pierce (Rockford, IL). 4-Methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO); sodium hypochlorite (NaOCl); sodium bicarbonate (NaHCO3); sodium hydroxyde (NaOH); and 7-amino-4-(trifluoromethyl) coumarin (COU) were purchased from Sigma-Aldrich (St. Louis, MO). Carboxylate-modified FluoSpheres microspheres (polystyrene microspheres, red fluorescent) were purchased from Molecular Probes, Invitrogen (Carlsbad, CA). Functionalization and Formation of Polymersomes. Modification of the ethylene oxide block followed the protocol of Kinnibrugh et al.19 First, a two-phase oxidation reaction was carried out to from an end-carboxylated block copolymer. The block copolymers were dissolved in dichloromethane. An oxidizing (14) Paula, S.; Volkov, A. G.; Deamer, D. W. Biophys. J. 1998, 74(1), 319–327. (15) Lakowicz, J. R. Principles of fluorescence spectroscopy, 3rd ed.; Springer: New York, 2006; p 954. (16) Stevens, B. Chem. Phys. Lett. 1987, 134(6), 519–524. (17) Zeng, H.; Durocher, G. J. Lumin. 1995, 63(1-2), 75–84. (18) Yasuda, H.; Lamaze, C. E.; Ikenberry, L. D. I. Makromol. Chem. 1968, 118(1), 19–35. (19) Kinnibrugh, K. G.; Chang, Y.-W.; Casey, L. M.; Silas, J. A.; Cheng, Z. Functionalization of PEO Based Diblock Copolymer Vesicles. In preparation.

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agent TEMPO was also dissolved in dichloromethane, and a regenerating agent NaOCl buffered with NaHCO3 at pH 8.6 made up the aqueous phase. TEMPO was added in separate doses every 3-5 min. The pH was adjusted to g11 with 3 N NaOH to break down the formed emulsion once the reaction was completed. The organic phase was separated from the mixture and dried in a vacuum oven. Once the carboxylated block copolymers were made, a second labeling reaction could be done using a widely known EDC/NHS peptide linkage reaction. The oxidized PEO89-PBd120 acid block copolymers were dissolved in chloroform. EDC (dissolved in chloroform) and NHS (dissolved in THF) in 10-fold excess were then added and allowed to activate for 15 min at room temperature. An excess of COU (in chloroform) was then added to the reaction flask and allowed to react for 2 h. Extra doses of EDC and NHS were added every 2 h to increase linking efficiency. Finally, the unreacted products were separated using dialysis tubing (MWCO 8000 Da) against CHCl3, and the product was dried in a vacuum oven. Polymersomes for imaging and quenching experiments were formed using a thin film hydration method. First, a thin polymer film containing 2 mg of desired block copolymers is deposited on the bottom of a 20 mL glass vial by evaporation (8 h under vacuum). Then, the vesicles were formed by rehydration with 1.5-2 mL of 320 mOsm/kg sucrose solution (Osmometer model 3320, Advanced Instruments, Inc., Norwood, MA) for 24 h at 60
C. A stir bar was placed inside the vial after 18 h to gently agitate vesicles from the surface of the film. A small amount of the resulting polymersome was subsequently diluted with iso-osmotic PBS to provide contrast for imaging.

Coumarin Immobilization on Microsphere Surfaces. Covalent coupling of COU to carboxylate-modified microspheres followed a standard two-step EDC/NHS peptide linkage procedure at room temperature. First, 20 μL of carboxylated FluoSpheres microspheres (2 μm, surface charges range between 0.1 and 2.0 mequiv/g) were added to a mixture containing EDC and NHS (buffered to pH 6), each in 10-fold excess to maximize the number of carboxylate groups (and thus the surface charge) based on specifications provided by the manufacturer. The mixture was incubated for 15 min to form a semistable NHS-ester, after which COU prepared in 0.15 M PBS buffer (pH 7.13) was added and allowed to react for an additional 2 h. Dilute NaOH was then added dropwise to raise the pH to 7.3. Finally, a second dose of EDC/NHS was added and reacted for another 2 h for complete labeling. Centrifugation of the reaction mixture was carried out on an IEC Micromax microcentrifuge (model OM 3590, ThermoIEC., Milford, MA) to remove unreacted products, and the supernatant was replaced with DI water. Several additional washes were made to ensure purity of the particle sample. Sonication of the sample between washes and prior to imaging was performed to redisperse the particles and minimize agglomeration. Microscope Imaging and Fluorescence Microscopy. Microspheres and vesicles were imaged using a temporary sample chamber constructed with two Teflon strips sandwiched between a microscope slide and coverslip and sealed with vacuum grease. Bright field and phase contrast images were acquired with a 20
air or 63
oil immersion objective using a Carl Zeiss Axiovert 200 M inverted microscope equipped with a Zeiss AxioCam MRm camera. For fluorescence, sample illumination was provided by a mercury arc lamp and filtered using a DAPI 365 nm low-pass excitation filter and an emission filter centered at 445 nm (bandwidth, 50 nm). All images were processed with Image J. Fluorescence Quenching Measurements. Halide ions are known to quench coumarin fluorescence through a photoinduced electron transfer (ET) mechanism.20-22 Amino-coumarins are polarity sensitive dyes23 susceptible to quenching by halides and (20) (21) (22) (23)

Giri, R. Spectrochim. Acta, Part A 2004, 60(4), 757–763. Moriya, T. Bull. Chem. Soc. Jpn. 1984, 57(7), 1723–1730. Chris, D. G. Meas. Sci. Technol. 2001, No.9, R53. Rechthaler, K.; K€ohler, G. Chem. Phys. 1994, 189(1), 99–116.

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some aromatic amines.22,24,25 The lack of a twisted intramolecular charge transfer (TICT) state ensures a viscosity independent fluorescence lifetime23 so fluorescence intensity measurements are sufficient. To study fluorescence quenching, emission intensities of coumarin in aqueous solution containing graduated amounts of quencher were recorded. The stock solutions of quenchers used to vary the concentration of halide ions were around 0.16 M, and less than 3
10-4 M Na2S2O3 was added to prevent I3 formation. Since no noticeable quenching occurs when using chloride ions, NaCl was used to maintain constant ionic strength. Vesicle surface fluorescence quenching measurements were performed in the same fashion. After the vesicles were formed, 0.1 mL of the polymersome stock solution (1.8 mg/2 mL) was diluted into a series of 0.9 iso-osmotic NaCl solutions in which increasing amounts of NaCl had been replaced by iodide. Fluorophore concentrations in all samples were maintained below 0.5 μM to avoid inner-filter effects. It should be noted that these diblock copolymers are characterized by critical aggregation concentration (CAC) values in the 10-8 M range, nearly 3 orders of magnitude below the concentrations used in our fluorescence measurements. Although our experimental apparatus prevented us from acquiring fluorescence data below the CAC, the quenching behavior is expected to be similar to the polymer free case, with a possible slight decrease in quenching rate due to the fact that the probe is tethered onto the PEG terminus. Sample solutions were placed in a 1 cm path length plastic cuvette, and sample excitation was achieved by a xenon arc lamp over a wavelength range of 200-800 nm. The steady-state fluorescence spectrum was recorded using a Photon Technology International (PTI) (Birmingham, NJ) QuantaMaster UV VIS spectrofluorometer equipped with he FeliX32 software package. Fluorescence lifetime measurements were obtained using the same instrument with a 375 nm LED light source attachment and a PMT detector at 490 nm. Determination of Quenching Constants. Nonlinear SternVolmer plots were used to account for deviations from the ideal case where a linear Stern-Volmer relation would apply over all quencher concentrations.15 I0 ¼ 1 þ kq τ0 ½Q ¼ 1 þ KSV ½Q I

ð1Þ

Here I and I0 are the fluorescence intensities in the presence and absence of the quencher at a concentration [Q], respectively, τ0 is the lifetime of the fluorophore without a quencher, kq is a quenching rate constant, and KSV is the quenching constant. Estimation of the rate constant of the bimolecular quenching reaction was made by applying the sphere of action and static quenching models, along with the finite sink approximation.16,21,25 The latter model was used for the final data analysis to obtain the relative quenching rates and for comparison with normalized diffusion coefficients. The finite sink approximation incorporates a description of quenching rate constants based on Smoluchowski, Collins, and Kimball (SCK) solution flux equation.16 Integration of the timeindependent flux equation for biomolecular quenching yields 1 1 - ðR=r0 Þ 1 ¼ þ kq kd ka

ð2Þ

where ka is the activation energy controlled rate constant describing the reaction of encountered pairs at a reactive distance R and kd is the diffusion-limited reaction rate that is directly related to the diffusion coefficient D by kd = 4πNAVDR, where NAV is Avogadro’s number. Since an excited fluorophore is most susceptible to quenching by its nearest neighbor at some initial average (24) Nad, S.; Pal, H. J. Phys. Chem. A 2000, 104(3), 673–680. (25) Tablet, C.; Hillebrand, M. J. Photochem. Photobiol., A 2007, 189(1), 73–79.

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separation r0, only the first encounter is of interest in the case of efficient fluorescence quenching. The diffusive region of interest for a first encounter is R e r e r0, and contributions from all subsequent encounters in the range r0 e r e ¥ are eliminated. The upper limit of this boundary is defined as the sink radius r0, the most probable initial average separation (2πNAV[Q])-1/3, yielding the following. 0 -1 Þ KSV - 1 ¼ ðKSV

ð2πNAV Þ1=3 ½Q1=3 4πNDτ0

ð3Þ

The parameter KSV is calculated from experimental data using eq 1, yielding ((I/I0)-1)/[Q]. The value of K0SV(= τ0kd = τ04πNAVDR for a diffusion-limited reaction) is given by the intercept of plot KSV-1 against [Q]1/3, and the diffusion coefficient can be obtained from the slope. Relative magnitudes of diffusion coefficients with the same quencher-fluorophore pair in different solvent environments can also be determined by comparing K0SV values. By rearranging eq 3 and defining f = ka/(kd þ ka), a modified nonlinear Stern-Volmer plot can be constructed from I0 f τ0 ð4πNAV DRÞ½Q ¼ 1þ I 1 - f ð2πNAV Þ1=3 R½Q1=3

ð4Þ

For a diffusion limited reaction (ka . kd), the value of f goes to unity. The quenching rates determined by eq 4 will be denoted as Kq. Ion Partition Experiments. Ion partition experiments were conducted by first compartmenting PEG and salt solutions with a dialysis membrane that allows only small molecules to pass through. PEG solutions (MW 2000) of 15.5 and 50 wt % are placed inside a CE dialysis tubing (MWCO 1000 Da) and dialyzed against KCl salt solutions in an 8.2
16.5 cm Ziploc bag for at least 24 h. The volume ratios of PEG versus salt solution were 1:3 and 1:6, respectively. After the freely moving salt partitioned inside and outside of the tubing reached equilibrium, the concentration of the exterior solution was measured by solution conductivity calibrated by measurements of KCl solutions in the range of 0-10 mM. Conductivity and Viscosity Measurements. Conductivity measurements were performed at room temperature 25.0 ( 0.5
C, using an Oakton Acron series CON6 hand-held conductivity/
C meter. The cell was calibrated using three standard solutions with known conductivities. All PEGs and salt solutions were prepared in Milli-Q DI water. Solutions were magnetically stirred and equilibrated for 3 min prior to each measurement. A Parr-Physica MCR-300 cone-and-plate rheometer was used for bulk viscosity measurements (50 mm cone diameter, data are an average of 10 measurements over shear rates ranging from 20 to 300 s-1).

Results and Discussion Preparation of Surface Modified Particles and Formation of Labeled Vesicles. Immobilization of coumarin on carboxylated microsphere and vesicle surfaces was verified by optical microscopy. Coumarin was coupled onto bare particles to provide a control representing a surface condition where no PEG polymer brushes are present. Comparison of brightfield and fluorescence images from the microspheres indicates intense emission in the blue spectral region (Figure 1a, b). Vesicles incorporating 10 wt % of coumarin functionalized copolymer possess a well-defined shape with a fairly broad size range (Figure 1c). The labeled bilayer structure surrounding the polymersome is clearly visible in corresponding fluorescence images (Figure 1d). Although the bulk fluorescence intensity increased linearly with the amount of labeled copolymer incorporated up to 50 wt % (indicating that self-quenching effects do not play a significant role, data not shown), the corresponding vesicle sizes also became much smaller. Langmuir 2010, 26(14), 12132–12139

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Figure 1. (a) Brightfield and (b) fluorescence images of 2 μm FluoroSphere particles, surface modified with coumarin. (c) Phase contrast and (d) fluorescence images of PEO89-PBd120 vesicles functionalized with 10 wt % coumarin-labeled copolymer.

We therefore selected 10 wt % of labeled copolymer as an optimal concentration to ensure structural integrity of the vesicles while maintaining a strong fluorescence signal. The choice of coumarin dye also helped to minimize self-quenching effects as a consequence of its large Stokes shift (∼121 nm, versus