Langmuir 2007, 23, 8003-8009
8003
Complex Coacervate Core Micelles with a Lysozyme-Modified Corona Maarten Danial,†,‡ Harm-Anton Klok,‡ Willem Norde,†,§ and Martien A. Cohen Stuart*,† Laboratory for Physical Chemistry and Colloid Science, Wageningen UniVersity, Dreijenplein 6, 6703 HB Wageningen, The Netherlands, Laboratoire des Polyme` res, Institut des Materiaux, Ecole Polytechnique Fe´ de´ rale de Lausanne, Baˆ timent MXD, Station 12, 1015 Lausanne, Switzerland, and Department of Biomedical Engineering, UniVersity Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands ReceiVed February 27, 2007. In Final Form: April 18, 2007 This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached. C3Ms were prepared by polyelectrolyte complex formation between PAA and mixtures containing different ratios of aldehyde and hydroxyl end-functionalized PQ2VP-PEO. This resulted in the formation of C3Ms containing 0-40% (w/w) of the aldehyde end-functionalized PQ2VP-PEO block copolymer (PQ2VP-PEO-CHO). Chemical conjugation of lysozyme was achieved via reductive amination of the aldehyde groups, which are exposed at the surface of the C3M, with the amine groups present in the side chains of the lysine residues of the protein. Dynamic and static light scattering indicated that the conjugation of lysozyme to C3Ms prepared using 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in the formation of unimicellar particles. Multimicellar aggregates, in contrast, were obtained when lysozyme was conjugated to C3Ms prepared using 30 or 40% (w/w) PQ2VP-PEO-CHO. The enzymatic activity of the unimicellar lysozyme-C3M conjugates toward the hydrolysis of the bacterial substrate Micrococcus lysodeikticus was comparable to that of free lysozyme. For the multimicellar particles, in contrast, significantly reduced enzymatic rates of hydrolysis, altered circular dichroism, and red-shifted tryptophan fluorescence spectra were measured. These results are attributed to the occlusion of lysozyme in the interior of the multimicellar conjugates.
Introduction There is an increasing interest in polymers for biomedical use, for example, as drug carriers and delivery systems1,2 or as components of antibacterial coatings.3 Interesting candidates for drug delivery systems are nanoparticles that carry an active ingredient. Polymeric micelles are one such type of nanoparticle. Polymeric micelles offer the advantage that they can solubilize functional ingredients into their cores, thereby protecting them against attack and degradation. The assembly of polymeric micelles can be achieved through several routes. One popular route involves the use of amphiphilic diblock copolymers consisting of a water-insoluble block and a water-soluble block.1,4,5 However, when this type of diblock copolymer is used, direct dissolution in water can be problematic (e.g., at very high weight fractions of the water-insoluble block) and may require an organic cosolvent4 that can later be removed by dialysis.5 An alternative method for preparing micelles is to use two different polymers, which form a complex in water.6 One way to achieve this, is to mix a diblock copolymer, consisting of a charged block and a neutral water-soluble block with either * Corresponding author. E-mail:
[email protected]. † Wageningen University. ‡ Ecole Polytechnique Fe ´ de´rale de Lausanne. § University Medical Center Groningen. (1) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113-131. (2) Ro¨sler, A.; Vandermeulen, G. W. M.; Klok, H. A. AdV. Drug DeliVery ReV. 2001, 53, 95-108. (3) Roosjen, A.; Kaper, H. J.; van der Mei, H. C.; Norde, W.; Busscher, H. J. Microbiology 2003, 149, 3239-3246. (4) Shen, H.; Zhang, L.; Eisenberg, A. J. Phys. Chem. B 1997, 101, 46974708. (5) Matejicek, P.; Podhajecka, K.; Humpolickova, J.; Uhlik, F.; Jelinek, K.; Limpouchova, Z.; Prochazka, K.; Spirkova, M. Macromolecules 2004, 37, 1014110154. (6) van der Burgh, S.; de Keizer, A.; Cohen Stuart, M. A. Langmuir 2004, 20, 1073-1084.
a homopolymer of opposite charge or a diblock copolymer with an opposite charged block and a neutral water-soluble block.6-9 As the charged blocks electrostatically attract each other, they tend to form a new phase, a process denoted as complex coacervation. Since the neutral water-soluble block remains in contact with the aqueous environment, unlimited growth and macroscopic phase separation cannot occur. Instead micelles are formed with a complex coacervate core. Such complex coacervate core micelles (C3Ms), also denoted as polyion complexes,10,11 block ionomer complexes,12 or interpolyelectrolyte complexes13 have been formed with various polymers. A broad variety of C3Ms has been prepared by mixing diblock copolymers with other oppositely charged diblock copolymers9,10 and homopolymers,13 as well as DNA,14 proteins,15 drugs,16 and ions.17 Since C3Ms are prepared by direct dissolution in aqueous solution and do not require the use of an organic cosolvent, C3Ms seem particularly attractive for applications involving sensitive biomolecules such as proteins and DNA. (7) Voets, I. K.; de Keizer, A.; Cohen Stuart, M. A.; de Waard, P. Macromolecules 2006, 39, 5952-5955. (8) Hofs, B.; Voets, I. K.; de Keizer, A.; Cohen Stuart, M. A. Phys. Chem. Chem. Phys. 2006, 8, 4242-4251. (9) Voets, I. K.; de Keizer, A.; de Waard, P.; Frederik, P. M.; Bomans, P. H. H.; Schmalz, H.; Walther, A.; King, S. M.; Leermakers, F. A. M.; Cohen Stuart, M. A. Angew. Chem., Int. Ed. 2006, 45, 6673-6676. (10) Harada, A.; Kataoka, K. Science 1999, 283, 65-67. (11) Vinogradov, S.; Batrakova, E.; Li, S.; Kabanov, A. Bioconjugate Chem. 1999, 10, 851-860. (12) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6797-6802. (13) Gohy, J. F.; Varshney, S. K.; Antoun, S.; Je´roˆme, R. Macromolecules 2000, 33, 9298-9305. (14) Miyata, K.; Kakizawa, Y.; Nishiyama, N.; Harada, A.; Yamasaki, Y.; Koyama, H.; Kataoka, K. J. Am. Chem. Soc. 2004, 126, 2355-2361. (15) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288-294. (16) Dufresne, M. H.; Leroux, J. C. Pharm. Res. 2004, 21, 160-169. (17) Yan, Y.; Besseling, N. A. M.; de Keizer, A.; Marcelis, A. T. M.; Drechsler, M.; Cohen Stuart, M. A. Angew. Chem., Int. Ed. 2007, 46, 1807-1809.
10.1021/la700573j CCC: $37.00 © 2007 American Chemical Society Published on Web 06/21/2007
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Harada and Kataoka, for example, prepared C3Ms with poly(aspartic acid)-b-poly(ethylene oxide) and hen egg-white lysozyme where the coacervate core consists of a positively charged protein lysozyme and a negatively charged poly(aspartic acid) block.15 Kataoka and co-workers have also prepared C3Ms from poly(L-lysine)-b-poly(ethylene oxide) and DNA and utilized these particles as nonviral vectors for gene delivery.14 In this report, we expand the range of presently available biological functional C3Ms. We describe the preparation and properties of protein-modified C3Ms, in which the protein is not entrapped in the core, but rather presented at the corona of the micelle. Such protein-functionalized particles could be of interest, for example, to catalyze (tandem) reactions in aqueous solutions. However, since C3Ms also readily adsorb onto surfaces such as polystyrene and silica,18 protein-functionalized C3Ms may also provide new opportunities to fabricate biologically active surfaces such as protein microarrays.19 The protein-functionalized nanoparticles reported here were obtained by chemical conjugation of the antibacterial enzyme hen egg-white lysozyme to C3Ms consisting of poly(N-methyl2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) and poly(acrylic acid) (PAA). The protein-modified C3Ms have been characterized by light scattering, and the structure of the conjugated lysozyme was studied with near-UV circular dichroism (CD) and steady-state tryptophan fluorescence. In addition, the enzymatic activity of the conjugated enzyme has been determined. Experimental Section Materials. Poly(2-vinyl pyridinine)-b-poly(ethylene oxide) (P2VP41-PEO204) and poly(acrylic acid) (PAA48) with polydispersities of 1.06 and 1.15, respectively, were used as received from Polymersource, Inc., Quebec, Canada. The subscripts indicate the number-average degree of polymerization of each block. Dess-Martin periodinane (97%), iodomethane (99%), sodium cyanoborohydride (95%), sodium periodate (99.8%), Purpald (99+%), acetaldehyde (99%), sodium nitrate (99%), hen egg-white lysozyme (95%), and Micrococcus lysodeikticus lyophilized bacteria were purchased from Sigma-Aldrich (Buchs, Switzerland). All solutions were prepared in Milli-Q water (>18 MΩ cm-1), and the pH was adjusted with 1 M NaOH or 1 M HNO3 if required. Dimethylformamide (DMF) and CH2Cl2 were dried over CaH2 and phosphorus pentoxide, respectively, and freshly distilled prior to use. Methods. Dynamic Light Scattering (DLS). DLS measurements were carried out on a Brookhaven Instruments Corp. system consisting of a BI-200SM goniometer and a BI-9000AT autocorrelator. A 100 mW Ar+ ion laser (Lexel Lasers) operating at 488 nm was used. All measurements were performed at a constant temperature of 25.0 ( 0.2 °C, which was maintained by a filtered decalin bath. All experiments were performed at a scattering angle of 90°. Borosilicate cuvettes were used with a minimum of 3 mL of the micellar solution. In DLS measurements, the measured normalized second-order autocorrelation function, g2(τ) can be expressed as the Siegert equation:20 g2(τ) ) 1 + β[g1(τ)]2
(1)
where β is an optical system constant depending on the geometry of the setup, g1(τ) is the normalized first-order correlation function, and τ is the delay time. g1(τ) can be expressed as follows: g1(τ) )
∫G(Γ) exp(-Γτ)dΓ
(2)
(18) Cohen Stuart, M. A.; Hofs, B.; Voets, I. K.; de Keizer, A. Curr. Opin. Colloid Interface Sci. 2005, 10, 30-36. (19) Tugulu, S.; Arnold, A.; Sielaff, I.; Johnsson, K.; Klok, H. A. Biomacromolecules 2005, 6, 1602-1607. (20) Berne, B. J.; Pecora, R. Dynamic Light Scattering with Applications to Chemistry, Biology and Physics; Dover Publications, Inc.: Mineola, NY, 2000.
Table 1. Refractive Index Increment Values and References for the Polymer Components of the Micelles Made (in mL g-1) PAA
PQ2VP+I-
PEO
PQ2VP-PEO
lysozyme
0.270a
0.210b
0.138c
0.173d
0.193c
a
b
c
d
Reference 35. Reference 36. Reference 15. Calculated value for PQ2VP-PEO.
where G(Γ) is a distribution function of the characteristic line width, Γ. The autocorrelation functions generated were analyzed according to the cumulant method,19 g1(τ) ) exp[-Γ h τ + (µ2/2!)τ2 - (µ3/3!)τ3 + ‚ ‚ ‚]
(3)
yielding µ2/Γ h 2 as a measure of the variance of the distribution (hence, the polydispersity) and an average characteristic line width Γ h , from which the diffusion coefficient D can be calculated according to Γ h ) Dq2
(4)
where q is the magnitude of the scattering vector defined as q ) 4πn sin(θ/2)/λ
(5)
θ is the detection angle, and λ is the wavelength of the incident laser beam. The hydrodynamic radius, RH was calculated according to the Stokes-Einstein equation: RH ) kBT/6πηD
(6)
where kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. Also, radius distributions were analyzed according to the CONTIN method.21 The radius distributions were analyzed for the 1-500 nm range. Static Light Scattering (SLS). SLS of dilute polymer solutions can be expressed as20 Kc/∆R(θ) ) 1/Mw(1 + q2Rg2/3) + 2A2c
(7)
where c is the total polymer concentration (in mg/mL), ∆R(θ) is the difference between the Rayleigh ratios for the solution and the solvent, Mw is the weight-average molar mass, Rg2 is the mean square radius of gyration, A2 is the second virial coefficient, and K is defined as follows: K ) 4π2n2(dn/dc)2/NAλ4
(8)
with n being the refractive index of the solvent, (dn/dc) being the refractive index increment of the sample (in g/mL), and NA being Avogadro’s number. The refractive index increments of the C3M solutions and of solutions of the lysozyme-modified C3Ms were calculated from the refractive index increments of the individual components, as described in the literature.6,15 The refractive index increments of the individual components are presented in Table 1. Micellar aggregation numbers were calculated at f + ) 0.5 using the method reported by Yuan and co-workers.22 Steady-State Fluorescence Spectroscopy. Steady-state fluorescence was measured with a Varian Cary fluorimeter at 25 °C using a 1-cm path length quartz cuvette. Fluorescence emission spectra were recorded in the 325-400 nm range using an excitation wavelength of 295 nm. All spectra were corrected for the background emission of 50 mM NaNO3. Near-UV CD. Near-UV CD spectra were recorded on a JASCO J-715 spectropolarimeter. Measurements were carried out at 25 °C using quartz cuvettes with a path length of 1 cm. For each measurement, an average of 16 spectra were recorded in the 280310 nm range. The resulting spectra were corrected for any contribution from the 50 mM NaNO3 solvent. The scan rate was 100 (21) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213-227. (22) Yuan, X.; Harada, A.; Yamasaki, Y.; Kataoka, K. Langmuir 2005, 21, 2668-2674.
C3Ms with a Lysozyme-Modified Corona
Langmuir, Vol. 23, No. 15, 2007 8005 Scheme 1
nm min-1 at a resolution of 0.2 nm. The observed ellipticities were converted into a mean residue ellipticity [θ] using the following expression: [θ]MRW ) θMr/10Ncl
(9)
where θ is the observed ellipticity (in deg), Mr is the molecular mass of the protein, N is the number of amide bonds present in the protein, c is the concentration of the protein (in g/mL), and l is the path length of the cuvette (in cm). Procedures. Synthesis of PQ2VP-PEO. The quarternization of P2VP-PEO was performed under nitrogen in DMF at 60 °C. Typically, 4.5 mL of iodomethane (72 mmol) was added dropwise to a stirred solution of 1 g of P2VP-PEO (3 mmol pyridine units) in 35 mL of dry DMF. After 48 h, an additional 1 mL (16 mmol) of iodomethane was added, and the reaction was allowed to proceed for an additional 12 h. The product was precipitated with an excess of cold diethyl ether and kept at 4 °C overnight, after which it was filtered, washed five times with cold diethyl ether to remove unreacted iodomethane, and dried in vacuo at 48 °C for 48 h. The degree of quarternization was determined by elemental analysis. Aldehyde End-Functionalization of PQ2VP-PEO. To a solution of 300 mg of PQ2VP-PEO in 20 mL of dry CH2Cl2, a 5 M excess of Dess-Martin periodinane (with respect to the number of block copolymer hydroxyl end groups) was added at 25 °C. The reaction mixture was stirred at this temperature for an additional 5 h. After that, the product was precipitated in cold diethyl ether and kept at 4 °C overnight before being filtered and washed with cold diethyl ether (three times). The product was dried in vacuo at room temperature for 24 h. As oxidation of aldehyde-modified PEO may take place,23 the dried polymers were stored in N2 at -20 °C prior to use. The degree of conversion of the PEO alcohol end-group into an aldehyde end-group was determined using the Purpald colorimetric assay. First, to convert measured UV-vis absorbances into aldehyde concentrations, a calibration curve was generated using acetaldehyde, as described in the literature.24 To estimate the degree of endfunctionalization of the PQ2VP-PEO block copolymer, a known amount of the polymer was first dissolved in 2 mL of Milli-Q water. Then, 200 µL of the block copolymer solution was added to 200 µL of a 30 mM solution of Purpald in 2 M NaOH. After equilibration for 30 min, 200 µL of a 30 mM solution of NaIO4 in 0.2 M NaOH was added, and the absorbance of the resulting purple solution was determined spectrophotometrically at 542 nm. Preparation of C3Ms. C3Ms were prepared by mixing polymer stock solutions at a molar fraction, f +, of 0.5. The molar fraction is defined as the ratio of positively charged polymers to the total amount of polymers present in the solution.7,8 Here, f + ) [PQ2VPPEO]/[PQ2VP-PEO+PAA]. First, aqueous solutions containing 1.4 mg/mL PAA and a 4 mg/mL mixture of PQ2VP-PEO and aldehyde end-functionalized PQ2VP-PEO (PQ2VP-PEO-CHO) were prepared in 50 mM NaNO3. The solutions were adjusted to pH 7.7 with 1 M (23) Bentley, M. D.; Roberts, M. J.; Harris, J. M. J. Pharm. Sci. 1998, 87, 1446-1449. (24) Quesenberry, M. S.; Lee, Y. C. Anal. Biochem. 1996, 234, 50-55.
NaOH or 1 M HNO3. The solutions were filtered three times through a 0.22 µm Acrodisc filter to remove dust. To 1.5 mL of PQ2VPPEO, 1.5 mL of PAA was added, and the pH was readjusted to pH 7.7 if required. Preparation of Lysozyme-Conjugated C3Ms. Lysozyme was dissolved in 50 mM NaNO3 at pH 7.7. Lysozyme solutions were filtered over a 0.22 µm Acrodisc filter prior to use. Lysozyme conjugation was carried out by adding 1.5 mL of a lysozyme solution to 2 mL of a 2 mg/mL C3M solution. The lysozyme concentrations were chosen such that there was a 10% molar excess of aldehyde groups with respect to protein in the final reaction mixture. The reaction mixture was stirred gently for 15 min, followed by the addition of 0.5 mL of a solution of 10 mM NaCNBH3 (in 50 mM NaNO3). To remove unreacted protein, the lysozyme-C3M mixture was placed in SpectraPor-6 dialysis membrane tubing (MWCO 25 kDa) and dialyzed against 50 mM NaNO3 at pH 7.7 for 60 h at 4 °C. The 50 mM NaNO3 medium surrounding the dialysis membrane was replaced after 12, 24, and 48 h. Lysozyme conjugation was confirmed with light scattering and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE. SDS-PAGE was conducted as described elsewhere.25 Briefly, a 12% (w/v) acrylamide separating gel and a 5% (w/v) stacking gel containing 1% SDS were prepared. Samples were treated with a Laemmli solution, as described elsewhere,26 containing 1% (v/v) β-mercaptoethanol and heated for 5 min at 95 °C. Electrophoresis was carried out at a constant current of 30 mA for 1.25 h. After electrophoresis, the gels were stained for protein with 0.025% (w/v) Coomassie Brilliant Blue R-250 solution. A 10-150 PLUS Roti Mark protein marker (Roth, Karlsruhe, Germany) was used to estimate the size of the protein-polymer conjugates. Lysozyme Enzymatic Assay. The enzymatic activity of lysozyme and lysozyme conjugated to C3Ms was measured by lysis of Micrococcus lysodeikticus using a method similar to the one described by Saint-Blancard and co-workers.27 A 0.015% (w/v) bacterial suspension (corresponding to an OD450 of ∼0.6) was prepared in 10 mM sodium phosphate buffer at pH 7.7 with 50 mM NaNO3. A solution containing lysozyme or lysozyme-C3M conjugates (0.2 mL of 0.1 mg/mL) was added to 2 mL of the cell suspension in 10 mM sodium phosphate buffer with 50 mM NaNO3 (pH 7.7). The initial decrease in the absorbance at 450 nm of the mixture caused by lysis of M. lysodeikticus cells was measured at 25 °C for 3 min with a Cary 100 Bio UV-vis spectrophotometer as described elsewhere.27,28 An example of an enzymatic curve is given in the inset in Figure 5. The initial slope of these curves was taken as a measurement of the enzymatic activity. The slopes were corrected for the activity of the unfunctionalized C3Ms by subtracting the corresponding rate measured for an unfunctionalized C3M and subsequently dividing by the lysozyme concentration of the respective solution. The resulting enzymatic rates are summarized in the bar chart in Figure 5. (25) Schagger, H.; Von Jagow, G. Anal. Biochem. 1987, 166, 368-379. (26) Laemmli, U. K. Nature 1970, 227, 680-685. (27) Saint-Blancard, J.; Chuzel, P.; Mathieu, Y.; Perrot, J.; Jolles, P. Biochim. Biophys. Acta 1970, 220, 300-306. (28) Nakamura, S.; Ban, M.; Kato, A. Bioconjugate Chem. 2006, 17, 11701177.
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Scheme 2
Results and Discussion C3M Preparation. The preparation of the lysozyme-functionalized C3Ms is outlined in Scheme 1. The first step involves the formation of aldehyde-functionalized C3Ms, which were obtained by combining mixtures of aldehyde- and hydroxylterminated PQ2VP-PEO and PAA. The quarternized block copolymer was synthesized by treating P2VP-PEO with iodomethane in DMF (Scheme 2). Elemental analysis indicated quantitative methylation of the 2-vinyl pyridine units. The hydroxyl group of PQ2VP-PEO was subsequently converted into an aldehyde end-group (PQ2VP-PEO-CHO) with the use of Dess-Martin periodinane following an improved version of a previously reported procedure.29 Using a 5-fold molar excess of Dess-Martin periodinane and a reaction time of 5 h, the block copolymer hydroxyl end-groups could be quantitatively converted into an aldehyde group. The original procedure, in contrast, which used an equivalent of Dess-Martin periodinane and a reaction time of 1 h, only resulted in a 37% conversion of alcohol to aldehyde end groups.29 1H NMR experiments indicated that quarternization and exposure of the block copolymer to DessMartin periodinane did not affect the relative block lengths. C3Ms were prepared by combining mixtures of PQ2VP-PEO and PQ2VP-PEO-CHO with PAA. The volumes and concentrations of the block copolymer and PAA solutions were chosen such as to obtain stoichiometric amounts of quarternized 2-vinyl pyridinium and acrylic acid repeat units in the final mixture. The amount of PQ2VP-PEO-CHO in the mixture of quarternized block copolymers was varied between 0 and 40% (w/w). The hydrodynamic radii of aldehyde-functionalized C3Ms, as determined with DLS, are summarized in Table 2. The CONTIN fits, which yield radial distributions, are shown in Figure 2. Conjugation of C3Ms with Lysozyme. Conjugation of lysozyme to the C3Ms was achieved via reductive amination29,30 of the aldehyde groups, which are presented at the surface of the micellar particles, with the amine groups present in the protein. Since lysozyme contains six -amine groups in the side chains of the lysine residues and one R-amine at its N terminus,31 there is a risk for the formation of higher order aggregates via the (29) McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 1349013491. (30) Roberts, M. J.; Bentley, M. D.; Harris, J. M. AdV. Drug DeliVery ReV. 2002, 54, 459-476. (31) van der Veen, M.; Norde, W.; Cohen Stuart, M. Colloids Surf., B 2004, 35, 33-40.
Figure 1. SDS-PAGE showing a marker (lane M), dialyzed lysozyme-modified C3Ms (lane 1), undialyzed lysozyme-modified C3Ms (lane 2), and lysozyme (lane 3) Table 2. Hydrodynamic Radii and Polydispersities Obtained from the Cumulant Fits PQ2VP-PEO-CHO content in C3M
C3M
lysozyme-modified C3M
% (w/w)
RH (nm)
µ2/Γ2
RH (nm)
µ2/Γ2
0 10 20 30 40
23.4 ( 3.2 23.9 ( 2.0 24.2 ( 2.1 23.7 ( 2.5 24.1 ( 1.5
0.07 0.06 0.06 0.07 0.07
23.6 ( 2.4 28.2 ( 1.6 34.2 ( 4.0 45.3 ( 9.0 56.9 ( 10.6
0.07 0.11 0.11 0.26 0.28
reaction of one lysozyme molecule with two or more C3Ms. To reduce this risk, the conjugation reactions were performed with a slight excess of aldehyde groups to lysozyme molecules. The progress of the conjugation could be monitored with SDS-PAGE. As an example, Figure 1 shows a gel of lysozyme (lane 3) and the product of the conjugation reaction between a C3M prepared with 10% (w/w) PQ2VP-PEO-CHO and lysozyme after a reaction time of 15 min before (lane 2) and after dialysis (lane 1). Since the 12% SDS-PAGE gel was run under denaturing conditions, the C3Ms dissociate into the individual components, that is, PAA, PQ2VP-PEO, lysozyme-conjugated PQ2VP-PEO-CHO, as well as unreacted PQ2VP-PEO-CHO. The gel was stained with Coomassie Brilliant Blue to visualize the protein bands. As a result, only PQ2VP-PEO-conjugated lysozyme as well as residual unreacted lysozyme were visualized in Figure 1. The gel of the crude reaction product of the reaction between a C3M consisting of 10% (w/w) PQ2VP-PEO-CHO and lysozyme (lane 2) shows a broad band corresponding to a molecular weight of 30-40 kDa. Since the average molecular weight of PQ2VPPEO is 19.2 kDa and lysozyme has a molecular weight of 14.3 kDa, this suggests that lysozyme has been conjugated with one block copolymer molecule, and multiple conjugation reactions did not occur. Lane 2 in Figure 1, however, also shows the presence of a significant amount of unconjugated lysozyme in the reaction mixture. The excess lysozyme was removed effectively by dialysis. Lane 1 in Figure 1 shows that this resulted in quantitative removal of the excess lysozyme. C3M Characterization. DLS experiments yielded an increase in the hydrodynamic radius of the lysozyme-conjugated C3M from 28 to 58 nm upon increasing the amount of PQ2VP-PEOCHO that was used for the preparation of the C3Ms from 10 to
C3Ms with a Lysozyme-Modified Corona
Langmuir, Vol. 23, No. 15, 2007 8007 Table 3. Results of SLS Experiments on the Unmodified and Lysozyme-Modified C3Ms PQ2VP-PEO-CHO content in C3M Mw,calca Mw,exptb lysozyme/ Rg (% (w/w)) (kg/mol) (kg/mol) C3Mc (nm) Rg/RH 0 10 20 30 40
n/a 1858 1970 2083 2196
1745 1923 2100 2618 4291
n/a 12.4 24.8 61.0 178.0
16.2 20.0 25.2 35.6 50.8
0.69 0.71 0.74 0.79 0.89
a Calculated cumulative molar mass of lysozyme-modified C3Ms assuming no multimicellar aggregation takes place. b Apparent molecular weight derived from Zimm or partial Zimm method. c Number of lysozyme molecules per C3M according to the experimental molar masses, Mw,expt, assuming no multimicellar aggregation takes place.
40% (w/w) (Figure 2, Table 2). Since the hydrodynamic radii of the unfunctionalized C3Ms and lysozyme were measured to be 24 and 2.2 nm, respectively, the hydrodynamic radius of the lysozyme-conjugated C3Ms would be expected to be about 28 nm. The radii in Table 2 show that the experimentally determined hydrodynamic radii of lysozyme-modified C3Ms that were prepared using block copolymer formulations containing 10 and 20% (w/w) PQ2VP-PEO-CHO are in good agreement with this predicted value. For lysozyme-conjugated C3Ms that were prepared with 30 and 40% (w/w) PQ2VP-PEO-CHO, however, considerably larger hydrodynamic radii and an increase in the size distribution were observed, which may indicate the formation of multimicellar aggregates. The unmodified and lysozyme-modified C3Ms were studied by SLS over an angular range from 30 to 140°. The data were evaluated using the (dn/dc) values presented in Table 1. Zimm and Berry analyses of unfunctionalized C3Ms at total micellar concentrations of 1.36, 2.71, 4.07, and 5.42 mg/mL revealed a molar mass Mw of 1745 kg/mol, a radius of gyration Rg of 16.2 nm, and a second virial coefficient of 4.5 × 10-5 mol mL g-2. From the molar mass we deduced an aggregation number of about 146. The second virial coefficient was sufficiently small to justify neglect of the (net) interaction between the micelles. The Rg/RH was calculated to be 0.69, a value consistent with results for other C3Ms and with the theoretical value for a homogeneous (“solid”) sphere (0.775).1,13,22,32 The deviation from a solid sphere is caused by a lower segmental density along the radial axis of the corona compared to the core. From the molar mass, the aggregation number of PAA and PQ2VP-PEO were calculated to be 67 and 79, respectively. This calculation is based on the assumption that there is complete charge neutralization and that there is no water in the core.15,22 Only partial Zimm and Guinier analyses could be carried out on the lysozyme-modified C3Ms because of the limited material available. In a partial Zimm analysis, only the angular dependence is measured, which is in contrast to a Zimm analysis where both the angular and concentration dependence of the scattering intensity is probed. Table 3 summarizes the results of the SLS experiments. The average number of lysozyme molecules per
C3M was obtained from the difference between the experimental molar mass Mw,expt of the respective lysozyme-modified C3Ms and the experimental molar mass of the unmodified C3M. For lysozyme-modified C3Ms from 10 and 20% (w/w) PQ2VPPEO-CHO, the Mw,expt and Mw,calc were in good agreement. For samples prepared from 30 and 40% (w/w) PQ2VP-PEO-CHO, the Mw,expt values were significantly larger than the Mw,calc values. The latter were calculated assuming the presence of unimicellar particles only. The relatively high average molar masses of lysozyme-modified C3Ms based on 30 and 40% (w/w) PQ2VPPEO-CHO, together with the increased hydrodynamic radii and polydispersities strongly suggest the formation of multimicellar aggregates. For the different lysozyme-modified C3Ms consisting of 10 and 20% (w/w) PQ2VP-PEO-CHO, we conclude that a structure close to a solid sphere is formed. The Rg/RH values deviate from values for a solid sphere because the shell (or corona) consisting of PEO-lysozyme, has a lower dn/dc value than the PQ2VP/PAA core, as discussed by Talingting and co-workers.33 For the lysozyme-modified C3Ms made from 30 and 40% (w/w) PQ2VP-PEO-CHO, the higher Rg/RH values imply that an intermediate structure between a core-shell particle and a homogeneous sphere are formed. However, we speculate that lysozyme is not only present on the corona of unimicellar particles, but also in the core of multimicellar aggregates. For such composite structures with large differences in dn/dc values between lysozyme, block copolymers and homopolymers (Table 1) may account for the exceptional Rg/RH values found. Effects of C3M Conjugation on Lysozyme Structure. NearUV CD and steady-state tryptophan fluorescence were employed to investigate whether structural changes occurred in lysozyme upon conjugation with the C3Ms. Figure 3 reveals significant differences between the CD spectra of lysozyme and those of the lysozyme-modified C3Ms. The CD spectra of the lysozymemodified C3Ms prepared from 10 and 20% (w/w) PQ2VP-PEOCHO are identical, but they show minor deviation from the spectrum of dissolved lysozyme. The CD spectra of the lysozymemodified C3Ms prepared from 30 and 40% (w/w) PQ2VP-PEOCHO are also nearly identical. The difference in the CD spectra of these two groups of lysozyme-modified C3Ms may be caused by the formation of multimicellar structures in the case of high lysozyme loading (30 and 40% (w/w) PQ2VP-PEO-CHO), as was already suggested above on the basis of DLS and SLS data. In addition to the CD spectra, the steady-state tryptophan fluorescence emission was measured by excitation of sample solutions at 295 nm (Figure 4). A comparison of the spectrum of lysozyme with those of the conjugate particles prepared from 10 and 20 (w/w) PQ2VP-PEO-CHO does not indicate any
(32) Douglas, J. F.; Roovers, J.; Freed, K. F. Macromolecules 1990, 23, 41684180.
(33) Talingting, M. R.; Munk, P.; Webber, S. E.; Tuzar, Z. Macromolecules 1999, 32, 1593-1601.
Figure 2. Radius distributions obtained from CONTIN analysis of lysozyme (a), aPQ2VP-PEO and PAA C3M (b), and lysozymemodified C3Ms containing (c) 10%, (d) 20%, (e) 30% and (f) 40% (w/w) PQ2VP-PEO-CHO.
8008 Langmuir, Vol. 23, No. 15, 2007
Figure 3. Near-UV CD spectra in 50 mM NaNO3. Lysozyme (a) and lysozyme-modified C3Ms containing (b) 10%, (c) 20%, (d) 30%, and (e) 40% (w/w) PQ2VP-PEO-CHO content.
Danial et al.
Figure 5. Relative enzymatic activity of lysozyme and lysozymemodified C3Ms in 10, 20, 30, and 40% (w/w) PQ2VP-PEO-CHO. The inset depicts a typical enzymatic rate profile for lysozyme. The arrow indicates the time at which the lysozyme is added to the cuvette, and the black line indicates the initial rate.
absorbance at 450 nm as a function of time. Although the CD spectra suggest minor structural changes (Figure 3), the data presented in Figure 5 demonstrate that the enzymatic activity of lysozyme was not negatively influenced upon conjugation to the C3Ms, at least not for the samples with 10 and 20% (w/w) PQ2VP-PEO-CHO. The relative lysis rates of these two samples are essentially identical to that of pure lysozyme. Conjugation of lysozyme to C3Ms prepared from 30 and 40% (w/w) PQ2VP-PEO-CHO, in contrast, resulted in a significant decrease in enzymatic activity. As was discussed before, these samples contained a considerable amount of multimicellar aggregates in which a significant fraction of lysozyme is not exposed at the particle surface but buried in its interior. This conclusion is consistent with the decrease in enzymatic activity (Figure 5).
Conclusions Figure 4. Steady-state tryptophan fluorescence spectra. From bottom to top, C3Ms conjugated to lysozyme with 10% (w/w) PQ2VPPEO-CHO (λmax ) 348 nm), 20% (w/w) PQ2VP-PEO-CHO (λmax ) 348 nm), 30% (w/w) PQ2VP-PEO-CHO (λmax ) 354 nm), 40% (w/w) PQ2VP-PEO-CHO (λmax ) 354 nm), and lysozyme (λmax ) 348 nm). The vertical bars indicate the position of the emission maximum in the different spectra. The spectra are vertically separated to facilitate comparison.
significant changes in the tryptophan emission intensity maximum. In contrast, the emission maxima of lysozyme-modified C3Ms consisting of 30 and 40% (w/w) PQ2VP-PEO-CHO are red-shifted and broadened. Such anomalous behavior of the conjugate particles prepared from 30 or 40% (w/w) PQ2VPPEO-CHO is again consistent with the formation of multimicellar aggregates. In these multimicellar structures, a fraction of the lysozyme molecules may not be exposed at the particle-water interface, but is rather buried in the core of these particles. This may lead to a change in the environment of the tryptophan residues and, hence, the observed changes in the fluorescence spectra. Enzymatic Activity of Lysozyme-Modified C3Ms. The activity of the conjugated lysozyme was determined and compared with that of the free lysozyme in solution using lyophilized Micrococcus lysodeikticus cells as the substrate. Lysozyme causes hydrolysis of the bacterial cell wall34 and leads to cell lysis and, hence, a decrease in the sample turbidity (Figure 5, inset). This process was followed spectrophotometrically by measuring the
In this paper we have reported the preparation of proteinmodified micelles by coupling the enzyme lysozyme to aldehydefunctionalized C3Ms composed of PQ2VP-PEO-CHO and PAA. Aldehyde-functionalized C3Ms were obtained by combining aqueous solutions consisting of the different relative amounts of hydroxyl and aldehyde end-functionalized block copolymers with aqueous solutions of PAA in a stoichiometric ratio, where complete charge neutralization occurs. The weight fraction of PQ2VP-PEO-CHO was used to control the degree of lysozyme modification. SLS and DLS experiments indicated that conjugation of lysozyme to the C3Ms prepared from 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in unimicellar particles comprising, on average, 12.4 and 24.8 lysozyme molecules per C3M, respectively. The enzymatic activity of these lysozyme-modified C3Ms was essentially the same as that of free lysozyme in solution, probably due to the high local enzyme concentration on the particle surface. Conjugation of lysozyme to C3Ms prepared from 30 and 40% (w/w) PQ2VP-PEO-CHO, in contrast, resulted in multimicellar aggregates, in which a significant fraction of the lysozyme molecules is not located at the periphery of the particle, (34) Nakimbugwe, D.; Masschalck, B.; Deckers, D.; Callewaert, L.; Aertsen, A.; Michiels, C. W. FEMS Microbiol. Lett. 2006, 259, 41-46. (35) Currie, E. P. K.; Sieval, A. B.; Avena, M.; Zuilhof, H.; Sudholter, E. J. R.; Cohen Stuart, M. A. Langmuir 1999, 15, 7116-7118. (36) Barten, D.; Kleijn, J. M.; Cohen Stuart, M. A. Phys. Chem. Chem. Phys. 2003, 5, 4258-4264.
C3Ms with a Lysozyme-Modified Corona
but probably in the particle interior. As a result, the enzymatic reaction rates measured for these particles showed a significant decrease compared to the unmodified enzyme. Acknowledgment. This work was partly supported by the European Science Foundation within the framework of the research networking programme STIPOMAT (short-term fel-
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lowship to M.D.). Ilja Voets of Wageningen University, The Netherlands, as well as Dr. Andrea Vaccaro and Prof. Michal Borkovec of the University of Geneva, Switzerland, are thanked for access to the light scattering equipment and for fruitful discussions. LA700573J
