Supporting Information Complex Formation Between Lysozyme and Stabilized Micelles with a Mixed Poly(ethylene oxide)/poly(acrylic acid) Shell Maria Karayianni 1,2, Valeria Gancheva 2, Stergios Pispas 1 and Petar Petrov 2 1 Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece 2 Institute of Polymers, Bulgarian Academy of Sciences, Akad. G. Bonchev Str., block 103-A, BG-1113 Sofia, Bulgaria S1
1. Synthesis of the Stabilized Polymeric Micelles Materials Poly(propylene glycol), PPG 34 (Fluka), was dried by azeotropic distillation of toluene. 2-Bromoisobutyryl bromide, triethylamine, CuBr, N,N,N,N,N - pentamethyldiethylenetriamine (PMDETA), trifluoroacetic acid, and pentaerythritol tetraacrylate were purchased from Aldrich and used as received. tert-butyl acrylate (tba, BASF) was stirred over calcium hydride (Merck) overnight and vacuum distilled just before use. All the solvents were purified by standard procedures. Silica- 60 gel (Merck) was used as supplied for removing residues of ATRP catalysts. Pluronic P85 was kindly donated by BASF. Block Copolymer Synthesis PPO Macroinitiator. The ATRP macroinitiators were synthesized by reacting PPG 34 with 2.5 mol equiv of 2-bromoisobutyryl bromide in dry toluene in the presence of triethylamine (2.5 mol equiv) at 20 C for 24 h. The reaction mixture was then filtered for removing the insoluble hydrobromide salt, added with activated carbon under stirring for 12 h, filtered again, and dried. The reaction product was added to basic water (NaOH, ph = 9), and the turbid solution was extracted several times with dichloromethane. The organic solution was dried over magnesium sulfate and filtered, and the solvent was finally removed under reduced pressure. The esterification yield (ca. 98%) was calculated from the 1 H-NMR spectrum. PtBA-PPO-PtBA Triblock Copolymers. In a typical procedure the Br-PPO 34 -Br macroinitiator (2.4 g; 4 mmol) was dissolved in acetone (0.6 ml). This solution was degassed by bubbling nitrogen under stirring for 45 min and then added with the PMDETA ligand (0.4342 ml; 2.08 mmol), the CuBr catalyst (0.2984 g; 2.08 mmol), and the freshly distilled and degassed tba monomer (12 ml; 0.083 mol). Polymerization was carried out at 50 C for 18 h. The copolymer was precipitated in hot water (60 C) and filtered out. It was dissolved in THF, and the solution was eluted through a silica gel column in order to remove the Cu(II) catalyst. Finally, THF was removed under reduced pressure and the copolymer was dried. PAA-PPO-PAA Triblock Copolymers. PtBA-PPO-PtBA copolymers were dissolved in freshly dried and distilled CH 2 Cl 2, followed by addition of a 5-fold molar excess of CF 3 COOH (with respect to the amount of the tert-butyl groups). The S2
reaction mixture was stirred at room temperature for 24 h, before being dialyzed against CHCl 3 for 3 days. The solvent was removed under reduced pressure and the copolymer was dried. A schematic diagram of the synthetic procedure is shown in Scheme S1. The copolymers were characterized by 1 H-NMR and Size Exclusion Chromatography (SEC) and the results are listed in Table S1. The number-average degree of polymerization (DP) of PtBA and the copolymer compositions were calculated from 1 H-NMR spectra (Figure S1, top). These determinations were based on the integrals of peaks assigned (i) to the PPO protons at δ = 3.55 ppm (2H, -O- CH 2 -CH-) and δ = 3.39 ppm (1H, -O-CH 2 -CH-), and (ii) to the PtBA protons at δ = 1.9-1.53 (2H, -CH 2 -C(C=O)H-) and δ = 1.44 (9H, O-C(CH 3 )3). SEC chromatograms of the two PtBA-PPO-PtBA copolymers (Figure S2) show a monomodal distribution and a low polydispersity, confirming the high efficiency of the macroinitiator. Based on the NMR and SEC data, one may conclude that the polymerization of the second block was well-controlled, resulting in well-defined triblock copolymers. The hydrolysis of PtBA with trifluoroacetic acid under mild conditions allowed for a quasi-quantitative conversion into PAA, as supported by the characteristic peak of the tert-butyl group that disappeared at δ = 1.44 ppm (Figure S1, bottom). This reaction did not alter the PPO blocks. Table S1. Molecular Characteristics of the Triblock Copolymers Synthesized by ATRP and Hydrolysis Copolymer composition ( 1 H-NMR) DP * of the outer blocks ( 1 H-NMR) M n (g mol -1 ) ( 1 H-NMR) M n (g mol -1 ) (SEC) M w /M n (SEC) PtBA 20 -PPO 34 -PtBA 20 20 7130 6035 1.14 PAA 20 -PPO 34 -PAA 20 20 4880 PtBA 40 -PPO 34 -PtBA 40 40 12250 11400 1.26 PAA 40 -PPO 34 -PAA 40 40 7760 * DP = degree of polymerization S3
Scheme S1: Synthetic procedure of the PAA-PPO-PAA triblock copolymers. S4
Figure S1. 1 H-NMR spectra of PtBA 40 -PPO 34 -PtBA 40 in CDCl 3 (top) and PAA 40 - PPO 34 -PAA 40 in DMSO (bottom). S5
Pt BA 40 -PPO 34 -Pt BA 40 Pt BA 20 -PPO 34 -Pt BA 20 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 Volume (ml) Figure S2. SEC eluograms of PtBA 20 -PPO 34 -PtBA 20 and PtBA 40 -PPO 34 -PtBA 40 triblock copolymer precursors with THF as the eluent. Micellization and Stabilization Polymeric micelles with a mixed shell were prepared as follows: 0.9 g of a binary mixture of triblock copolymers of a well-defined composition (Table S1) and 0.18 g of PETA (20 wt % to the copolymers amount) were dissolved in CH 2 Cl 2 under stirring. CH 2 Cl 2 was evaporated under reduced pressure, and 250 ml of basic water (NaOH, ph = 10) was added and temperature was increased to 40 C. Argon was bubbled through the solution for 45 min, followed by irradiation with a full spectrum UV light (Dymax 5000-ECUV curing equipment with a 400 W metal halide flood lamp) at 40 C for 45 min. The stabilized polymeric micelles were purified by dialysis against water using a cellulose membrane (Sigma, cutoff 12000 g mol -1 ) for 14 days. Stabilized micelles from the single PAA-PPO-PAA triblock copolymer were prepared by the same procedure. Characterization Nuclear Magnetic Resonance Spectrometry. The 1 H-NMR spectra were recorded in CDCl 3 and DMSO, with a 250 MHz Bruker AC-spectrometer. Size Exclusion Chromatography. PtBA-PPO-PtBA copolymers were analyzed by SEC at room temperature with PSS SDV-gel columns (5 µm, 60 cm, 1 linear (10 2 to 10 5 Å), 1 100 Å), with THF as an eluent (flow rate = ml/min) and a S6
refractometer for the detection. Molecular weights and polydispersity index (PDI) were determined with a polystyrene calibration. Pluronic P85 copolymer was analyzed by SEC at 70 C with a Waters chromatograph equipped with a refractive index detector and a Waters Styragel column eluted at 70 C with 0.5 wt % LiBr containing dimethylformamide (DMF) at a flow rate of 1 ml/min. PEO standards were used for calibration. S7
2. Analysis of the Light Scattering Measurements Dynamic light scattering (DLS) measurements of the normalized time autocorrelation function of the scattered light intensity g 2 (t) were performed. The obtained data were fitted with the aid of the CONTIN analysis and the distribution of relaxation times τ or the mean relaxation rate Γ = 1/τ were obtained. Assuming that the observed fluctuations of the scattered intensity are caused by diffusive motions, the apparent diffusion coefficient D app is related to the relaxation time τ as, D app = 1/τq 2, where q is the scattering vector defined as 4πn 0 sin( θ /2)/ λ0 with n 0, θ and λ 0 the solvent refractive index, the scattering angle and the wavelength of the laser in vacuum respectively. From the apparent diffusion coefficient D app, the hydrodynamic radius R h can be obtained, using the Stokes-Einstein relationship: R k T B h = (1) 6πη 0 Dapp where k B is the Boltzmann constant, T is the temperature and η 0 is the viscosity of the solvent. 1,2 Static light scattering (SLS) measurements were treated by the Zimm method using the equation: Kc 1 1 2 2 = 1 + Rg q + 2A2 c (2) R θ M 3 W where M W is the weight averaged molecular weight, is the average radius of gyration, A 2 is the second osmotic virial coefficient, c is the polymer concentration, R θ is the corrected Rayleigh ratio, which depends on the polymer concentration c and the magnitude of the scattering vector q, and the constant factor K is defined as: 4π n K = λ N 4 0 2 2 0 A n c where n 0, λ 0, N A are the refractive index of the solvent, the laser wavelength in vacuum, the Avogadro s number respectively and 2 (3) n / c is the refractive index increment of the sample solution with respect to the solvent. 1,2 For the solutions of the complexes the refractive index increment was calculated on the basis of the composition of the solution as the weighted average of ( n / c) HEWL and ( n / c) SPM, 1 Huglin, M. B. Light Scattering from Polymer Solutions, Academic Press: New York, 1972. 2 Chu, B. Laser Light Scattering: Basic Principles and Practice, Academic Press: New York, 1991. S8
which were measured using a differential refractometer (BI-DNDC, Brookhaven Instruments, Inc.) under the condition of osmotic equilibrium between the solution and the solvent. S9
3. Angular Dependence of the DLS Measurements for the SPMs The angular dependence of the relaxation rate, Γ, and the reduced translational diffusion coefficient, D, (D = Γ/q 2 ) of both peaks observed in the distributions obtained from the CONTIN analysis of the DLS measurements, for representative SPM-1, SPMMS-1, SPMMS-2, and SPMMS-3 solutions of 0.1 mg/ml concentration at ph 7 and 0.01 M ionic strength are shown in Figures S3 and S4, respectively. 9 8 7 a) SPM-1 9 8 7 b) SPMMS-1 6 6 Γ (ms -1 ) 5 4 3 Γ (ms -1 ) 5 4 3 2 2 1 1 0 0 9 8 7 c) SPMMS-2 9 8 7 d) SPMMS-3 6 6 Γ (ms -1 ) 5 4 3 Γ (ms -1 ) 5 4 3 2 2 1 1 0 0 Figure S3. Angular dependence of the relaxation rate, Γ, of both peaks observed in the distributions obtained from the CONTIN analysis of the DLS measurements for a) SPM-1, b) SPMMS-1, c) SPMMS-2, and d) SPMMS-3 solutions of 0.1 mg/ml concentration at ph 7 and 0.01 M ionic strength. S10
2.0 a) SPM-1 2.0 b) SPMMS-1 1.5 1.5 D (10-7 cm 2 s -1 ) 0.5 D (10-7 cm 2 s -1 ) 0.5 0.0 0.0 2.0 c) SPMMS-2 2.0 d) SPMMS-3 1.5 1.5 D (10-7 cm 2 s -1 ) 0.5 D (10-7 cm 2 s -1 ) 0.5 0.0 0.0 Figure S4. Angular dependence of the reduced translational diffusion coefficient, D, of both peaks observed in the distributions obtained from the CONTIN analysis of the DLS measurements for a) SPM-1, b) SPMMS-1, c) SPMMS-2, and d) SPMMS-3 solutions of 0.1 mg/ml concentration at ph 7 and 0.01 M ionic strength. S11
4. SLS Results Regarding, R h and ρ = /R h Values for the Solutions of the Complexes The combined SLS results regarding the, R h and ρ = /R h values for the solutions of the complexes of the SPM-1/HEWL, SPMMS-1/HEWL, SPMMS- 2/HEWL, and SPMMS-3/HEWL systems at ph 7 and 0.01 M ionic strength are shown in Figure S5. For all four different systems the observed increase in the values of and R h, as well as their characteristic ratio ρ is in accordance with the occurring secondary aggregation. S12
a) SPM-1/HEWL 200 b) SPMMS-1/HEWL, R h (nm) 80 70 60 R h Coacervation, R h (nm) 150 100 R h Coacervation 50 50 1.3 1.2 1.2 ρ = / R h 1.1 ρ = / R h 1.1 0.9 0.00 0.01 0.02 0.03 0.040.1 0.2 C HEWL (mg/ml) 0.9 0.00 0.02 0.04 0.06 0.1 0.2 C HEWL (mg/ml) c) SPMMS-2/HEWL d) SPMMS-3/HEWL, R h (nm) 150 100 R h Coacervation, R h (nm) 150 100 R h Coacervation 50 1.3 50 1.3 ρ = / R h 1.2 1.1 ρ = / R h 1.2 1.1 0.00 0.02 0.04 0.06 0.1 0.2 C HEWL (mg/ml) 0.9 0.00 0.02 0.04 0.06 0.08 0.10 0.150.20 C HEWL (mg/ml) Figure S5. SLS results regarding the, R h and ρ = /R h values for the solutions of the complexes of the a) SPM-1/HEWL, b) SPMMS-1/HEWL, c) SPMMS-2/HEWL, and d) SPMMS-3/HEWL systems at ph 7 and 0.01 M ionic strength. S13