Design of Polypeptide-Functionalized Polystyrene Microspheres

Toluene, a good solvent for polystyrene, swells the cross-linked particle, giving the diblocks ..... Hayashi , S.; Seo , T.; Hata , H.; Hirai , T. J. ...
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Biomacromolecules 2008, 9, 1811–1817

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Design of Polypeptide-Functionalized Polystyrene Microspheres A. Bousquet,† R. Perrier-Cornet,† E. Ibarboure,† E. Papon,† C. Labruge`re,‡ V. He´roguez,† and J. Rodrı´guez-Herna´ndez*,† Laboratoire de Chimie des Polyme`res Organiques, LCPO-CNRS-Universite´ Bordeaux 1, ENSCPB-16, Avenue Pey Berland, 33607 Pessac-Cedex, France, and Institut de Chimie de la Matie`re Condense´e de Bordeaux, ICMCB-CNRS, 87, Avenue du Docteur Schweitzer 33608 Pessac-Cedex, France Received January 10, 2008; Revised Manuscript Received March 26, 2008

In this contribution, the principle of spontaneous surface segregation has been applied for the preparation of polypeptide-functionalized polystyrene microspheres. For that purpose, an amphiphilic diblock copolymer was introduced in the mixture styrene/divinylbenzene and polymerized using AIBN as initiator. During the polymerization, cross-linked particles were obtained in which the diblock copolymer was encapsulated. The amphiphilic diblock copolymers used throughout this study contain a hydrophilic polypeptide segment, either poly(L-lysine) or poly(L-glutamic acid) and a hydrophobic polystyrene block. After 4 h of polymerization, rather monodisperse particles with sizes of ∼3-4 µm were obtained. Upon annealing in hot water, the hydrophilic polypeptides migrate to the interface, hence, either positively charged or neutral particles were obtained when poly(L-lysine) is revealed at the surface and exposed to acidic or basic pH, respectively. On the opposite, negatively charged particles were achieved in basic pH water by using poly(L-glutamic acid) as additive. The surface chemical composition was modified by changing the environment of the particles. Thus, exposure in toluene provoked a surface rearrangement, and due to its affinity, the polystyrene block reorients toward the interface.

Introduction Polymer colloids, largely used in coatings, adhesives, or inks, have been more recently found of interest for other purposes such as medical and biological applications, for example, bioseparation,1 immunoassay and affinity diagnosis,2 or as a carrier for drug delivery purposes.3 Also, polymeric microspheres have been employed in optical and opto-electrical devices,4 in catalysis,5 or in micropatterning.6 The abovementioned applications require suitable particle sizes, nature, and density of the surface functional groups, and colloidal stability. Several methods are available for the preparation of polymer particles, including emulsion, dispersion, suspension polymerization, or precipitation polymerization.7 Each approach gives particles having different range of sizes below 100 nm (emulsion polymerization) to 100 µm (suspension polymerization), and some require the use of either surfactants or stabilizers. Precipitation polymerization, used throughout this study, leads to rather monodisperse microspheres and can be prepared with a large range of sizes (0.1-100 µm) free of surfactants or stabilizers. Equally, the use of divinylbenzene as comonomer affords highly cross-linked particles with a stable shape under harsh temperature and pressure conditions8 and exhibit thermal and solvent resistance.9 Microspheres have been typically functionalized in the past either by copolymerization of a functional comonomer or oligomer introduced in the feed or by postreaction on preformed latexes (functional group modification or surface grafting).10 In this contribution, we report an alternative approach to functionalize polystyrene/divinylbenzene (PS/DVB) particles prepared by precipitation polymerization with synthetic polypep* To whom correspondence should be addressed. Tel.: (33) 540003695. Fax: (33) 540008487. E-mail: [email protected]. † LCPO-CNRS-Universite´ Bordeaux 1. ‡ Institut de Chimie de la Matie`re Condense´e de Bordeaux.

tides. For that purpose, diblock copolymers, either polystyreneb-poly(L-lysine) or polystyrene-b-poly(L-glutamic acid), are introduced in the initial feed. The shape, the functionality, and the properties exhibited by the particles (reversibility and pH response) will be described as well. Transitions between the polypeptides secondary structure R-helical, random coil, and β-sheet can be finely tuned depending on the external conditions such as pH, ion strength or temperature. Hence, poly(L-lysine) or poly(L-glutamic acid) functional particles may find potential interest among others in medical and biological applications where both load and release of drug/active molecules may be modulated.11 In addition, poly(L-lysine) has been extensively used to complex with plasmid DNA and evaluated as gene transfection reagent.12

Experimental Section Chemicals. Styrene (Sty), divinylbenzene (DVB), and 1,4-diethylamine were purchased from Sigma-Aldrich. Styrene was dried under CaH2 and cryodistilled. Azobisisobutyronitrile (AIBN, 98%) as the initiator was recrystallized from methanol before use. Both γ-benzylester-L-glutamate N-carboxyanhydride and N-trifluoroacetyl-L-lysine N-carboxyanhydride were purchased from Isochem and recrystallized from ethylacetate/hexane before the polymerization. All other solvents were used as received unless otherwise specified. Characterization. 1H NMR spectra of the diblock copolymers were recorded at room temperature on a Bruker Avance 400 MHz spectrometer using the residual proton resonance of the deuterated solvent as an internal standard. Average molar masses and molar mass distributions were determined by size exclusion chromatography (SEC) using a Varian 9001 pump with both a refractive index detector (Varian RI-4) and a UV detector (spectrum studies UV 150). Calibration was obtained using narrowly distributed PS standards and THF as the mobile phase at a flow rate of 0.5 mL min-1. Synthesis. The synthesis of the diblock copolymers obtained combining both atom transfer radical polymerization (ATRP) and ringopening polymerization of R-amino acid N-carboxyanhydrides has been

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Figure 1. Methods reported for the preparation of functional microspheres by precipitation polymerization.

already described elsewhere.13 The preparation of the particles has been carried out by precipitation polymerization based on the following procedure (targeted cross-linking density of 40%): 0.79 mL of Sty, 0.79 mL of DVB, and 72 mg of AIBN were dissolved in 30 mL of an acetonitrile/toluene (v/v 95/5%) mixture. In a separate step, the diblock copolymer, either polystyrene-b-poly(L-glutamic acid) or polystyreneb-poly(L-lysine) (10% weight related to the total amount of Sty/DVB) was dissolved in the same solvent mixture and added to the previous solution. The polymerizations were carried out at a constant temperature of 85 °C during 4 h. After the polymerization was accomplished, the soluble polymer was separated from the insoluble fraction by vacuum filtration. The insoluble microspheres were extensively washed with tetrahydrofuran to remove surface adsorbed diblock copolymer. Annealing. The reversibility in terms of surface functionality has been evidenced by annealing the particles to different environments. Dispersed particles were annealed in hot water (90 °C, 2 days), inducing the hydrophilic block to migrate to the interface polymer/water. On the contrary, annealing in dry air (100 °C, 2 days) or in toluene (rt, 5-7 days), which is a good solvent of polystyrene, brought about a surface rearrangement where the hydrophobic polystyrene chains moved preferentially toward the interface. Before each annealing step, the particles were thoroughly dried under vacuum. Particle Size and Potential Zeta Analysis. Particle size, size distributions, and surface charge were measured using a Zetasizer 3000 (Malvern Instruments). All dynamic light scattering (DLS) measurements were carried out at wavelength of 633 nm, 25 °C, and detection angle of 90°. For the zeta potential measurements, the sample concentration was 1 mg/mL and the pH was varied between 3 and 10. Optical Microscopy. Optical images were obtained using an Axioskop 40 Zeiss microscope. The particle dispersion (1 mg/mL) was dropped in a glass plate to form a thin layer. Scanning Electron Microscopy (SEM). The morphological characterization of the functionalized microspheres was carried out with a scanning electron microscope (SEM, JEOL JSM-5200 scanning microscope). The particles were dropped onto a sample holder, placed under vacuum at room temperature, and gold-coated prior to examination. X-ray Photoelectron Spectroscopy (XPS). All XPS spectra were recorded with a 220i-XL ESCALAB from VG using the same analytical conditions. The particles, supported on indium, were put under UHV to reach the 10-8 Pa range. The nonmonochromatized Mg X-ray source was used at 100 W for all the samples, as well as a flood gun to compensate for the nonconductive samples. The detection occurs in a perpendicular plane to the sample surface. The spectra were calibrated in relation to the C1s binding energy (284.4 eV), which was applied as an internal standard. Fitting of the high-resolution spectra was provided through the AVANTAGE program from VG.

Table 1. Molecular Characteristics of the Block Copolymers Used Throughout this Study

1 2 3 4

compositiona

Mnb(g/mol)

PDc

PS34-b-PLys10 PS20-b-PLys7 PS39-b-PGA13 PS23-b-PGA22

4800 3000 6000 5200

1.25 1.3 1.23 1.2

a Composition calculated from the 1H NMR spectra (400 MHz) of the deprotected polymers carried out in a mixture THF-d6/D2O at room temperature. b Mn obtained from 1H NMR in THF at rt (400 MHz). c PD obtained from GPC measurements in DMF at rt carried out on the protected diblock copolymers.

FTIR (Transmission Mode). Spectra were taken in KBr pellets containing dispersed particles with concentration 1:100 at room temperature. The IR spectra were recorded at 20 ( 1 °C in the spectral range of 650-4000 cm-1 using a Perkin-Elmer Spectrum One spectrometer.

Results and Discussion The preparation of functional microspheres by precipitation polymerization can be carried out by following three main approaches (Figure 1). The first one (i) concerns the polymerization of a main monomer in the presence of one (or more) monomer14 or macromonomer15 containing the desired functions. Examples reported include the copolymerization of DVB with either water-soluble monomers such as acrylamide16 or hydrophobic monomers methacrylate17 and methyl methacrylate,18 chloromethylstyrene,19 or maleic anhydride.20 Highly cross-linked microspheres made by precipitation polymerization contain a large amount of residual vinyl groups. Hence, a second methodology (ii) consists of the modification of the final polymer latexes either by chemical treatments21 or by grafting polymers and block copolymers from/onto their surfaces.22 Both methods, that is, (i) and (ii), display several major drawbacks. The copolymerization of functional monomers is restricted to a limited number of monomers for two main reasons: the different reactivity of the comonomers and the presence of chain transfer reactions if the functional group present has, for instance, a labile proton.23 Equally, postmodification of latexes is difficult to perform in terms of reproducibility and generally needs additional steps to obtain the desired functionality.24

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Figure 2. Synthesis of polystyrene microspheres with either poly(L-lysine) or poly(L-glutamic acid) blocks at the interface.

Figure 3. Illustrative (a) SEM images obtained from 4 and (b) optical microscope images from the particles obtained using 2 as additive.

A novel strategy introduced by our group (iii) to prepare functionalized polymeric microparticles used an amphiphilic diblock copolymer incorporated within the monomer mixture during the precipitation polymerization.25 (Diblock copolymers have also been exploited for the stabilization of particles in emulsion polymerization.26) The diblock copolymers used throughout this study are designed to contain a block similar in nature to the monomers used during the reaction, that is, styrene (S) and divinylbenzene (DVB), that favors its incorporation during the microgel formation. The second block stands for the surface functionality. Once the reaction concluded and the particles formed, simple exposure to water will direct the hydrophilic segment of the block copolymer by surface segregation toward the interface.27 Equally, annealing either to air or toluene will induce surface reconstruction in which the lower surface-energy hydrophobic block will be located at the surface. The difference in surface energies of both blocks allows surface rearrangement to occur in response to a change in the environment, thus, the character of the particle can be reversibly changed from hydrophilic to hydrophobic. The preparation of functional particles by using this method improves several main aspects. Given that the diblock copolymer

used as additive is prepared in a separate step, chain lengths and polydispersity of the hydrophilic block that will form the particle shell can be easily modified.28 Hence, a homogeneous particle shell layer is formed by the hydrophilic chains having all similar lengths. More importantly, the density of functional groups, crucial for many applications and difficult to control following the postreaction approach, can be “a priori” controlled by the quantity of diblock added during the polymerization step. In this contribution, we explored the incorporation of novel amphiphilic diblock copolymers with either a positively or negatively charged hydrophilic block. The hydrophilic part is composed of a synthetic polypeptide: either poly(L-glutamic acid) negatively charged at basic pH values and neutral below 4.8 or poly(L-lysine) having positive amine groups at neutral to acidic pH and uncharged above 10. The additive, prepared in a separate step, has been obtained by the combination of two polymerization methods.12 The first block is synthesized by atom transfer radical polymerization (ATRP) having, thus, an endbromo functional group. The terminal bromo function can be modified by reaction with an R,ω-diamine into a primary amine group. Finally, the amine group was used as macroinitiator for the ring-opening polymerization of R-amino acid N-carboxy-

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Figure 4. FT-IR spectra of (a) particles having 1 as additive (red curve) and PS microspheres used as reference (black curve) (b) particles with 4 as additive (red curve) and PS microspheres used as reference (black curve).

Figure 5. XPS spectra of both particles having (i) PS-b-PGA (4) and (ii) PS-b-PLys (1) diblock copolymers within the structure. (Top) Spectra of particles annealed in hot water (85 °C) during 3 days. (Bottom) Spectra of the microspheres annealed in toluene (rt) during 3 days.

anhydrides (NCAs). The chemical characteristics of the diblocks used throughout this study are shown in Table 1. Functional particles were prepared directly by precipitation polymerization. as depicted in Figure 2. The copolymerization reaction takes place by mixing the initiator (AIBN), the monomers (styrene and divinylbenzene), and the diblock copolymer in an acetonitrile/toluene mixture. Toluene was used as cosolvent both to enhance the stability of growing particles and to facilitate the solubilization of the block copolymers. During the first minutes the cross-linking copolymerization leads to partially soluble microgels until the critical precipitation point is attained (depending both on the cross-linking ratio and chain length). Then, the microgels begin to aggregate, producing colloidally stable particles. Steric stabilization provided by the solvated polymer chains located at the slightly swollen surface of the particles prevented aggregation. The progressive incor-

poration of oligomer microgel conducts to the growth of particles as long as polymerization occurs. At the same time, the amphiphilic diblock copolymers incorporated within the microgels provided the affinity between the latter and the polystyrene block. The polymerization parameters, temperature, solvent mixture, amount of diblock copolymer (10%), and reaction time (4 h), were maintained invariable throughout all the experiments. The microspheres were first characterized by both SEM and optical microscopy. As an illustrative example, SEM micrographs of the particles obtained using PS23-b-PGA22 as additive and optical microscope images of the particles with PS20-bPLys7 are shown in Figure 3. In both cases, spherical morphologies were obtained with rather regular sizes 3-4 µm. Moreover, the sizes of the microspheres appeared to be independent of the chemical composition of the diblock copolymer intro-

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Figure 6. Evolution of the surface charge (ζ potential measured at pH 11) as a function of the annealing conditions for microspheres having block copolymer 4. Successive annealing of the microspheres lead to either to polystyrene enriched surfaces or hydrophilic surfaces having carboxylic functional groups.

Figure 7. Zeta potential measurements for water annealed particles having either 3 or 1. Whereas poly(L-lysine) modified particles are positively charged at acidic pH values and uncharged at basic pH values, poly(L-glutamic acid) modified particles exhibit a negative surface charge at basic to neutral pH values and are neutral in acidic pH solutions.

duced. Therefore, in principle, the use of a diblock copolymer with a particular composition and molar mass does not imply the modification of the polymerization conditions. After removal of the eventually absorbed diblock copolymer at the particle surface by washing with THF, the particles were characterized by FT-IR. The spectra obtained for the microspheres prepared with either PS34-b-PLys10 (1) or PS23-b-PGA22 (4) diblock copolymers are shown in Figure 4. An additional spectra (black curve) corresponding to pure PS microspheres without any additional additive has been included in both (a) and (b) and used as reference. The presence of the poly(L-lysine) block within the particles is evidenced by the presence of a large band at ∼3500 cm-1, which corresponds to amine functional groups. Similarly, for those particles in which PSb-PGA has been introduced in the initial monomer mixture, a band is observed at ∼1650 cm-1 due to the carbonyl groups present in the poly(L-glutamic acid) block. Both results confirmed the incorporation within the particles of the amphiphilic diblock copolymers during the precipitation polymerization.

Whereas FT-IR provided the average composition of the particle, XPS is more sensitive to the microsphere surface composition and permitted to establish differences between particles annealed either in hot water or in toluene. XPS spectra obtained after annealing in water and toluene are depicted in Figure 5: PS23-b-PGA22 (4; i) and PS34-b-PLys10 (1; ii). In those particles containing block copolymer 4, the oxygen signal (∼532.6 eV) has been compared before and after annealing. Upon exposure to hot water, important amounts of oxygen (∼7-10%) and nitrogen (∼2-2.5%) were measured, indicating the presence of a large quantity of PGA at the interface. On the contrary, annealing in toluene at room temperature reversed the situation and lead to polystyrene-enriched surfaces, as evidenced by the considerable decrease of the oxygen (∼1.4-1.6%) and nitrogen (∼1-1.1%) peaks. Similarly, the particles in which 1 has been used as additive in the initial mixture exhibit a larger quantity of nitrogen (∼2.5%) and oxygen (∼6-7%) due to the poly(L-lysine) block (signal at ∼398.6 eV) when exposed to water. In contact with toluene (O1s peak reduced to 1.5% and

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N1s to less than 1.0%), the polystyrene is preferred at the surface. These experiments clearly demonstrate that the particles can, depending on the environment in which the particles are dispersed, change the surface composition by surface migration/ rearrangement of the amphiphilic diblock copolymer. Hence, the modification of microspheres with amphiphilic diblock copolymers allows their dispersion both in organic nonpolar solvents and in polar/hydrophilic solvents. This unique behavior can enlarge the use of the particles by annealing in the appropriate conditions. The surface reorganization as a function of the environment described above has been demonstrated to be reversible as depicted in Figure 6 for particles prepared with 4. For that purpose, the variation of the surface charge after successive annealing to water and toluene has been determined by zetapotential measurements at basic pH values, where the poly(Lglutamic acid) is deprotonated. Whereas annealing in water was carried out at high temperature (90 °C) during 2 days, annealing by using toluene can be done at room temperature. Toluene, a good solvent for polystyrene, swells the cross-linked particle, giving the diblocks the mobility required for the surface rearrangement without the need of additional heat. Particles annealed in toluene were dried under vacuum and dispersed in water at room temperature. Because the particle structure is frozen at this temperature and the measurements were carried out in a short period of time, the surface rearrangement, if it occurs, can be neglected. To avoid possible desorption or solubilization of the diblock copolymer, these experiments have been carried out with particles containing 4, diblock copolymer with similar hydrophilic and hydrophobic block lengths. As depicted in Figure 6, the surface charge density increases upon water annealing, indicating the presence of a higher quantity of polar functional groups, that is, carboxylic acid groups. On the contrary, the density significantly decreases by annealing in toluene due to the preferential reorientation of the polystyrene block toward the surface. In addition, further successive annealing to water and toluene evidenced the reversibility of the surface rearrangement of one or the other block at the outmost surface. Equally, zeta-potential measurements have been carried out to evidence the response to pH of the microspheres. Water annealed particles having either PS34-b-PLys10 (1) or PS39-bPGA13 (3) have been dispersed in a solution at different pH values: acidic (pH ) 2) and basic (pH ) 11). As shown in Figure 7, modified particles with 1 have a positive surface charge when dispersed in acidic aqueous media (∼45-47 mV). In these pH conditions, the poly(L-lysine) primary amine groups are protonated. Changing the pH to basic values induces the deprotonation of the amine functional groups; hence, the surface charge is neutralized. The particles functionalized with poly(L-glutamic acid) show similar behavior but in a different range of pH. Whereas at low pH values the particles are uncharged, deprotonation of the carboxylic acid functional groups at pH values above 4.8 leads to negatively charged microspheres. Polypeptide tethered chains on inorganic surfaces exhibit similar pH transitions.29 These pH transitions have been associated with transitions between an R-helical secondary structure (where the side chain functional groups are neutralized) to random coil conformations (observed when the side chain functional groups are charged and impede by electrostatic repulsion the formation of a R-helical structure).

Bousquet et al.

Conclusions We described an alternative method for the design of polypeptide functionalized polymeric microspheres. In this approach, a preformed amphiphilic diblock copolymer is incorporated in the initial monomer mixture. The amphiphilic diblock copolymers used throughout this study consist of PSb-PLys and PS-b-PGA, in which the relative composition has been varied. Independent of the composition of the diblock copolymer used, spherical particles of 3-4 µm in diameter were obtained. The incorporation of diblock copolymer within the particles, evidenced both by FT-IR and XPS, allowed the reversible surface modification by exposure to water or to air/ toluene, where the hydrophilic block (either poly(L-lysine) or poly(L-glutamic acid)) is either revealed or hidden beneath the surface. Because polypeptides are sensitive to external conditions such as pH, ion strength, or temperature on the secondary structure, we evaluated, in addition, the response of the functionalized particles to pH. Depending on the environmental pH and the polypeptide used, we succeeded in the preparation of negatively or positively charged microspheres. Such peptide modified microspheres may be of potential use for such applications as bioseparation, immunoassay and affinity diagnosis, or as a drug delivery system in which the hydrophilic block could be used as a drug carrier.

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