Selective Enzymatic Degradation of Self-Assembled Particles from

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Selective Enzymatic Degradation of Self-Assembled Particles from Amphiphilic Block Copolymers Obtained by the Combination of N-Carboxyanhydride and Nitroxide-Mediated Polymerization Gijs J. M. Habraken,† Marloes Peeters,† Paul D. Thornton,‡ Cor E. Koning,† and Andreas Heise*,†,‡ †

Laboratory of Polymer Chemistry, Eindhoven University of Technology, Den Dolech 2, P.O. Box 513, 5600 MB Eindhoven, The Netherlands ‡ School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland S Supporting Information *

ABSTRACT: Combining controlled radical polymerizations and a controlled polypeptide synthetic technique, such as Ncarboxyanhydride (NCA) ring-opening polymerization, enables the generation of well-defined block copolymers to be easily accessible. Here we combine NCA polymerization with the nitroxidemediated radical polymerization of poly(n-butyl acrylate) (PBA) and polystyrene (PS), using a TIPNO and SG1-based bifunctional initiator to create a hybrid block copolymer. The polypeptide block consists of (block) copolymers of poly(L-glutamic acid) embedded with various quantities of L-alanine. The formed superstructures (vesicles and micelles) of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, resulting in controlled enzymatic degradation dictated by the polypeptide composition. The PBA containing block copolymers possessing 50% L-alanine in the polypeptide block showed a high degradation response compared to polymers containing lower L-alanine quantities. The particles stabilized by copolypeptides with L-alanine near the hydrophobic block showed full degradation within 4 days. Particles containing polystyrene blocks revealed no appreciable degradation under the same conditions, highlighting the specificity of the system and the importance of synthetic polymer selection. However, when the degradation temperature was increased to 70 °C, degradation could be achieved due to the higher block copolymer exchange between the particle and the solution. A number of novel biohybrid structures are disclosed that show promise as enzyme-responsive materials with potential use as payload release vehicles, following their controlled degradation by specific, target, enzymes.

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initiated by particular, target, enzymes, or combinations of enzymes, which are (over)expressed in affected regions in most disease states.8 Enzyme specificity in this process is dictated by the placement of the cleaved bond (endo- and exopeptidases) within the polypeptide and the combination of the adjacent amino acids residues that flank the cleavage site.9,10 Examples are copolypeptides of poly(L-glutamic acid) or poly(L-lysine) in combination with small hydrophobic amino acid monomers, such as L-alanine, L-leucine, and L-valine, which are degradable by endopeptidases such as trypsin and papain.11,12 For biohybrid micelles or vesicles, enzymatic degradation of parts of the polypeptide block should result in particle destabilization. Conceptually, this was shown for the enzymatic ester degradation of the hydrophobic block of poly(trimethylene carbonate-b-L-glutamic acid) vesicles.13 Lipases have also been used to degrade the core of poly(ethylene glycol) and oligo(caprolactone) micelles.14,15 This method

INTRODUCTION Biohybrid polymers combine natural polymers with synthetic polymers and are frequently proposed as natural mimics for biomedical applications. Superstructures, such as micelles and vesicles, are often the target structures for biohybrid polymers being considered for drug delivery applications.1−5 When a synthetic polypeptide block is used as the hydrophilic component, the core of the micelle, or the membrane of the vesicle, is formed by the hydrophobic synthetic polymer block. In many cases hydrophobic interactions are sufficient to stabilize the polymer assembly, although chemical cross-linking has been employed to further stabilize the structures. For example, our group prepared pH-responsive core cross-linked core−shell particles from block copolymers of polystyrene and poly(L-glutamate).6,7 Degradation of biohybrid block copolymers is a critical aspect for the lifetime of the superstructures in a biological environment and thus a key performance factor for their applicability. The main mode of degradation is enzymatic hydrolysis, which can either be specific or nonspecific. It has been shown that the enzyme-triggered release of therapeutic agents is of importance; release occurs exclusively when © 2011 American Chemical Society

Received: July 20, 2011 Revised: August 24, 2011 Published: September 9, 2011 3761

dx.doi.org/10.1021/bm2010033 | Biomacromolecules 2011, 12, 3761−3769

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prepared according to procedures mentioned in the literature. 20 NCAs of γ-benzyl-L-glutamate and L-alanine were prepared following literature procedures.21 The succinimide ester of 5(6)-carboxyfluorescein was prepared following literature procedures. 22 Methods. NMR was measured on a 400 MHz Varian Mercury 400 and 200 MHz Varian Mercury 200. Deuterated chloroform was used for the characterization of the NCA monomer and NMRP initiators. Deuterated TFA was used for measuring the polypeptide macroinitiators and the poly(n-butyl acrylate) block copolymers. The polystyrene block copolymers were measured in deuterated DMF. Size exclusion chromatography (SEC) was performed in N,N,dimethylacetamide at 60 °C on a system equipped with a Waters 2695 separation module, a Waters 2414 refractive index detector (50 °C), a Waters 486 UV detector, and a PSS GRAM guard column followed by two PSS GRAM columns in series of 100 (10 mm particles) and 3000 (10 mm particles), respectively. DMAc was used as eluent at a flow rate of 1 mL/min. The molecular weights were calculated using polystyrene standards. Before SEC analysis was performed, the samples were filtered through a 0.2 μm PTFE filter (13 mm, PP housing, Alltech). Matrix-assisted laser desorption/ionization−time of flight−mass spectroscopy (MALDI-ToF-MS) analysis was carried out on a Voyager DE-STR from Applied Biosystems (laser frequency 20 Hz, 337 nm, and a voltage of 25 kV). The matrix material used was trans-2(3-(4-tert-butylphenyl)-2-methyl-2-propenylidene)malononitrile (DCTB) (40 mg/mL). Potassium trifluoroacetic acid (KTFA) was added as cationic ionization agent (5 mg/mL). The polymer sample was dissolved in HFIP (1 mg/mL), to which the matrix material and the ionization agent were added (5:1:5), and the mixture was placed on the target plate. Fourier transform−infrared spectroscopy (FTIR) measurements were performed on a PerkinElmer SpectrumOne FT-IR spectrometer, using a universal ATR sampling accessory. Samples for the IR spectroscopy were taking from the reaction mixtures and measured without any further modification. High-performance liquid chromatography mass spectroscopy was carried out on an Agilent 1100 series setup with an Agilent MSD type SL (G1946D). The column used was a Zorbax RX-C18; 2.1 × 50 mm2; 5 μm. The eluent was 90% water and 10% methanol at 25 °C with a flow of 0.2 mL/min. Detection was done using UV and MS with atmospheric pressure electrospray interface. The conditions for the APESI were as follows: pressure, 30 psi; capillary voltage, 4000 V; and dry gas, 13 L/min, 350 °C. Gradient polymer elution chromatography (GPEC) was performed on an Agilent 1100 with a Alltech ELSD 2000 detector. The column used was a Zorbax SB-C18. The eluent was 100% toluene for the first 2 min and then was changed to 20%−15% DMF over a period of 28 or 30 min. Over a period of 2 min the eluent was changed to 100% toluene, and this was maintained for 3 min. The flow rate was kept constant at 1 mL/min at 25 °C. Injection volume was 2−10 μL of a 10 mg/mL solution. Dynamic light scattering was measured on a Coulter N4 plus in a 1 cm quartz cuvette at 90°. Static light scattering was measured with a Wyatt Dawn Heleos-II at 90°. Cryogenic transmission electron microscopy (cryo-TEM) measurements were performed on a FEI Tecnai 20, type Sphera TEM instrument (with a LaB6 filament, operating voltage = 200 kV). The sample vitrification procedure was performed using an automated vitrification robot (FEI Vitrobot Mark III). A 3 μL sample was applied to a Quantifoil grid (R 2/2, Quantifoil Micro Tools GmbH; freshly glow discharged for 40 s just prior to use) within the environmental chamber of the Vitrobot, and the excess liquid was blotted away. The thin film thus formed was shot into melting ethane. The grid containing the vitrified film was immediately transferred to a cryoholder (Gatan 626) and observed under low dose conditions at −170 °C. Synthesis. 2-Methyl-2-[N-tert-butyl-N-(1-diethoxyphosphoryl2,2-dimethylpropyl)aminoxy]-N-propionyloxysuccinimide. 23 The MAMA-SG1 (11.36 g, 29.80 mmol) and the N-hydroxysuccinimide

allowed for a full degradation of the hydrophobic core, resulting in fully dispersed polymer chains. The degradation of this block copolymer was suggested to occur in the bulk solution by unimer exchange from the micelles above the critical micelle concentration (cmc). Selective enzymatic degradation has been shown in other hybrid systems, such as hydrogels, where enzymatic hydrolysis was used as a release trigger. In oligopeptide (Asp-Ala-Ala-Arg) functionalized cross-linked PEG hydrogels, the Ala-Ala bonds were selectively cleaved, resulting in swelling of the nanoparticles due to the increased positive ionic repulsion of the remaining L-arginine-containing dipeptide.16,17 Swollen PEG networks, where a peptide sequence was used as a cross-linker, have been degraded by the enzyme trypsin, resulting in a fully degraded network.18 Recently, the same enzymatic trigger mechanism was used in a two-phase system with Asp-Ala-AlaArg oligopeptides coated porous silica particles to show the release of a fluorescent dextran.19 These examples illustrate the accessibility of the enzyme to the corona or peptide layer close to a hydrophobic surface, such as is the case in micelles and vesicles. However, to our knowledge, the effect of enzymatic polypeptide degradation in amphiphilic hybrid block copolymers on the stability of micelles or vesicles has not previously been investigated. Clearly, the effect of enzymatic degradation on amphiphilic block copolymers might be complex. For example, the nature of the hydrophobic, enzyme-neutral part of the block copolymers could influence polypeptide degradation. The unimer exchange process determines whether the majority of the degradation occurs in the particle corona or in the solution in the unimer state, influencing the speed of degradation and particle stability. Hydrophobic blocks with different properties need to be tested to determine these effects. By using noncrystalline hydrophobic polymer blocks with high and low glass transition temperatures, the difference between both processes can be studied. In this paper, the synthesis of block copolymers of polystyrene and poly(n-butyl acrylate) with homopolymers and copolymers of L-glutamic acid and L-alanine via a macroinitiation by nitroxide-mediated radical polymerization (NMRP) using bifunctional initiators is described. The deprotected polypeptide blocks and hydrophobic n-butyl acrylate or polystyrene block give the block copolymers amphiphilic properties. Micelles and vesicles were prepared from the deprotected hybrid block copolymers and the effect of peptidases tested on these particles. By altering the amino acid composition of the polypeptide block, the enzymatic degradation was tuned and a correlation made between materials with low and high Tg hydrophobic blocks. It is anticipated that this methodology is of relevance for the development of a novel enzyme-responsive release mechanism and adds to the understanding of the degradability of synthetic polypeptide hybrids.

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EXPERIMENTAL SECTION

Materials. THF, pentane, diethyl ether, DMF, NMP, DMSO, HFIP, and dichloromethane were purchased from Biosolve. DMAc was purchased from Aldrich. N-Hydroxysuccinimide dicyclohexylcarbodiimide, diaminoethane, MgSO4, TFA, HBr 33%/acetic acid solution, styrene, n-butyl acrylate, 5(6)-carboxyfluorescein, thermolysin, and lipase from Thermomyces lanuginosus were purchased from Sigma. Elastase was purchased from Worthington Biochemical Corp. Palladium (10%)/carbon was purchased from Merck. MAMA-SG1 and SG1 nitroxide were a gift from R. Hoogenboom (TU Eindhoven). All materials were used as received. Bifunctional initiator 2 was 3762


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(4.10 g, 35.6 mmol) were dissolved in 25 mL of THF. To this 25 mL of THF with dicyclohexylcarbodiimide (6.72 g, 32.6 mmol) was added. The reaction was left for 3 h, then filtered, concentrated, and precipitated in pentane. The solid was subsequently dried under vacuum at ambient temperature. Yield: 8.14 g, 17.0 mmol, 57.1% 1H NMR (400 MHz, CDCl3, δ, ppm): 1.13 (18H, s), 1.17 (18H, s), 1.26 (12H, q, J = 7.1 Hz, 9.0 Hz), 1.78 (6H, s), 1.85 (6H, s), 2.80 (8H, s), 3.24 (1H, s), 3.31 (1H, s), 3.9−4.3 (8H, m) (both stereoisomers) 13C NMR (400 MHz, CDCl3, δ, ppm): 16.17 (d, POCH2CH3, J(C,P) = 6.6 Hz), 16.57 (d, POCH2CH3, J(C,P) = 6.6 Hz), 21.91 (CHC(CH3)3), 25.63 ((CH3)2CON), 28.18 (s, (CH3)CN), 29.33 (s, CH2(CO)), 30.06 (d, (CH3)3CH, J = 5.6 Hz), 58.81 (d, POCH2CH3, J = 7.2 Hz), 61.90 (d, POCH2CH3, J = 6.2 Hz), 62.63 (s, NC(CH3)3), 69.21 (d, NCHP, J = 137.51 Hz), 83.67 (s, C(CH3)2O), 168.91 (s, CH2(CO)N), 170.25 (s, OC(O)C(CH3)). 31P NMR (200 MHz, CDCl3, δ, ppm): 25.8 (s). 2-Methyl-2-[N-tert-butyl-N-(1-diethoxyphosphoryl-2,2dimethylpropyl)aminoxy]-N-aminoethylpropionamide (Initiator 2). 2Methyl-2-[N-tert-butyl-N-(1-diethoxyphosphoryl-2,2-dimethylpropyl)aminoxy]-N-propionyloxysuccinimide (5.00 g, 10.5 mmol) was dissolved in 50 mL of dichloromethane. This was added slowly to a solution of diaminoethane (32.0 g, 532 mmol) in 100 mL of dichloromethane. After 5 h the solution was extracted with water 3 × 30 mL. The water fraction was extracted with dichloromethane. The organic fractions were collected and dried with MgSO4. The solution was subsequently concentrated at 20 °C, and under vacuum, the remaining solvent and diaminoethane were removed. The oil was stored in a freezer at −18 °C. Yield: 3.25 g, 7.67 mmol, 73.3%. MS: 424 Da (M + 1H)+, 446 Da (M + Na)+. 1H NMR (400 MHz, CDCl3, δ, ppm): 1.03 (18H, s), 1.15 (18H, s), 1.28 (12H, m), 1.50 (6H, s), 1.63 (6H, s), 2.74 (4H, t, J = 6.1), 3.24 (1H, s), 3.30 (1H, s), 3.36 (4H, q, J = 6.1 Hz) 3.9−4.2 (4H, m), 5.22 (2H, s), 8.32 (2H, s) (both stereoisomers) 13C NMR (400 MHz, CDCl3, δ, ppm): 16.10 (d, POCH2CH3, J(C,P) = 6.8 Hz), 16.57 (d, POCH2CH3, J(C,P) = 5,7 Hz), 24.34 (s, CHC(CH3)3), 27.34 (s, (CH3)2CON), 29.35 (s, (CH3)CN), 29.54 (d, (CH3)3CH, J = 5.4 Hz), 41.82 (s, CH2CH2NHC(O)), 42.89 (s, CH2NH2), 59.81 (d, POCH2CH3, J = 8.1 Hz), 61.56 (d, POCH2CH3, J = 3.7 Hz), 62.57 (s, NC(CH3)3), 69.55 (d, NCHP, J = 133.66 Hz), 85.82 (s, C(CH3)2O), 176.87 (s, OC(O)C(CH3)). 31 P NMR (200 MHz, CDCl3, δ, ppm): 28.0 (s). Polymerization of NCAs Initiated by Initiator 2. The following general procedure was applied for all combinations of BLG and Ala NCA. The bifunctional initiator 2 (134 mg, 0.316 mmol) was dissolved in 5 mL of DMF. The NCA of γ-benzyl-L-glutamate (5.02 g, 19.1 mmol) was dissolved in 20 mL of DMF in a Schlenk tube. The initiator solution was added quickly. The reaction was maintained under 0 °C for 4 days. The reaction mixture was precipitated in diethyl ether and filtered, and the sample was stored in the freezer. Yield: 3.10 g, 71.8 wt %. 1H NMR (400 MHz, D-TFA, δ, ppm): 1.4 (s, C(CH3)3, initiator) 1.5 (m, CH2, initiator), 1.6 (s, C(CH3)3, initiator), 1.8 (s, CH3, initiator), 1.9 (s, CH3, initiator), 2.0−2.4 (CH2), 2.6 (CH2), 4.8 (CH), 5.2 (CH2), 7.4 (C6H5). Block Copolymerization of Poly(γ-benzyl- L-glutamate15-co-Lalanine15)-b-poly(γ-benzyl-L-glutamate25-co-L-alanine5) Initiated by 2. The bifunctional initiator 2 (95.8 mg, 0.226 mmol) was dissolved in 1 mL of DMF and added to a solution of the NCAs of γ-benzyl-Lglutamate (1.50 g, 5.70 mmol) and L-alanine (0.133 g, 1.16 mmol) in 16 mL of DMF. The polymerization was performed at 0 °C under a nitrogen flow. After 3 days FTIR showed full conversion, and a solution of the NCAs of γ-benzyl-L-glutamate (0.9 g, 3.42 mmol) and L-alanine (0.393 g, 3.41 mmol) in 10 mL of DMF was added. The reaction mixture was precipitated in diethyl ether after reaching full conversion after 2 days. The polymer was dried under vacuum at room temperature. Yield: 2.29 g, 95.0 wt %. 1H NMR (400 MHz, D-TFA, δ, ppm): 1.6 (CH3, Ala), 1.9−2.2 (CH2, BLG), 2.6 (CH2, BLG), 4.7 (CH, Ala), 4.8 (CH, BLG), 5.2 (CH2, BLG), 7.4 (C6H5, BLG). NCA Random Copolymerization for MALDI-ToF-MS Analysis. The NCA monomers of γ-benzyl-L-glutamate (0.5 g, 1.90 mmol) and L-alanine (0.219 g, 1.90 mmol) were dissolved in 4 mL of DMF in a Schlenk tube. The solution was stirred at 0 °C until all monomer was

completely dissolved. To this a solution of benzylamine (0.51 mg, 0.272 mmol) in 1 mL of DMF was added. Samples were taken over time for IR and MALDI-ToF-MS measurements. For the MALDIToF-MS 100 μL was dissolved in 1 mL of HFIP with 1.5 M benzylamine and placed in the freezer at −18 °C. NMRP of n-Butyl Acrylate with Poly(γ-benzyl- L-glutamate) Macroinitiator (SG1). The macroinitiator (310 mg, 2.38 × 10−2 mmol), n -butyl acrylate (0.85 g, 6.6 mmol), and SG1 nitroxide (0.65 mg, 2.2 × 10−3 mmol) were dissolved in 2.0 mL of NMP. The solution was deoxygenized by three freeze−thaw cycles. The solution was subsequently placed in a oil bath of 60 °C. The temperature was increased to 90 °C for 1 h and then increased to 100 °C for 2.5 h. Monomer and solvent were removed by precipitation in methanol and filtration. Yield: 0.45 g. 1H NMR (400 MHz, D-TFA, δ, ppm): 1.0 (CH3, BA), 1.3−2.2 (C2H3, BA) 1.5 (CH2, BA), 1.8 (CH2, BA), 1.9− 2.2 (CH2, BLG), 2.6 (CH2, BLG), 4.3 (CH2, BA), 4.8 (CH, BLG), 5.2 (CH2, BLG), 7.4 (C6H5, BLG). Deprotection of Poly(γ-benzyl-L-glutamate)-b-poly(n-butyl acrylate). The hybrid block copolymer PBLG60-b-PBA (0.95 g) and 100 mg of palladium on carbon catalyst were dissolved in 20 mL of DMF and deoxygenized by a nitrogen flow. In a Parr setup the hydrogenation was performed under a 70 psi hydrogen gas atmosphere. After 6 h of reaction time the catalyst was removed by filtration. Most of the solvent was removed by high vacuum, and the polymer was precipitated in pentane. Yield: 110 mg, 50 wt %. 1H NMR (400 MHz, D-TFA, δ, ppm): 1.0 (CH3, BA), 1.3−2.2 (C2H3, BA) 1.5 (CH2, BA), 1.8 (CH2, BA), 1.9−2.2 (CH2, BLG), 2.6 (CH2, BLG), 4.3 (CH2, BA), 4.8 (CH, BLG). Deprotection of (γ-Benzyl-L-glutamate)-b-polystyrene. The hybrid block copolymer PBLG60-b -PS (1.07 g) was dissolved in 20 mL of dichloromethane. To this solution 6 mL of HBr 33%/acetic acid solution was added. The reaction was precipitated in diethyl ether after 24 h. The precipitated polymer was dried in a vacuum oven. Yield: 0.6 g, 95.9 wt %. The 1H NMR spectrum shown in Figure SI 7. Particle Formation of Deprotected Hybrid Block Copolymers. The block copolymer of PGlu60-b-PBA (44 mg) was dissolved in 10 mL of DMSO. To this 10 mL of phosphate buffer solution was added slowly over 3 h. The solution was dialyzed with a PBS buffer for 48 h. Synthesis of Fluorescein-Labeled Poly(γ-benzyl-L-glutamate). The NCA monomers of γ-benzyl-L-glutamate (1.50 g, 5.70 mmol) was dissolved in 14 mL of DMF in a Schlenk tube. To this a solution of benzylamine (15.3 mg, 0.141 mmol) in 1 mL of DMF was added. The reaction was left to stir in a cold water bath of 0 °C for 4 days under a dry nitrogen atmosphere. The succinimide ester of 5(6)carboxyfluorescein (0.200 g, 0.424 mmol) was added to the solution. The reaction was stirred for another 24 h and precipitated in diethyl ether twice. After this the polymer was dissolved in NMP and dialyzed (membrane cutoff of 3500 Da) for 16 h. After filtration the solution was precipitated in diethyl ether. The polymer was dried in vacuo under ambient temperature. Yield: 0.39 g, 29.3 wt %. 1H NMR (400 MHz, D-TFA, δ, ppm): 1.9−2.2 (CH2), 2.6 (CH2), 4.8 (CH), 5.2 (CH2), 7.4 (C6H5). Deprotection of Fluorescein-Labeled Poly(γ-benzyl- L-glutamate). The fluorescein-labeled PBLG (0.300 g, 6.35 × 10−2 mmol) was dissolved in 7 mL of TFA. To this 3 mL of HBr/acetic acid solution was added. The reaction was maintained overnight. After precipitation in diethyl ether the product was filtered and washed in diethyl ether for three times. Finally, the polymer was dried in a vacuum oven overnight at room temperature and stored in a freezer. Yield: 0.105 g, 51.3 wt %. 1H NMR (400 MHz, D-TFA, δ, ppm): 2.2− 2.4 (CH2), 2.7 (CH2), 5.0 (CH), 7.3−8.1 (fluorescein). Enzymatic Degradation of Fluorescent Functionalized (Co)polypeptides. The deprotected fluorescent labeled poly(L-glutamic acid) (6.1 mg) and elastase (2.2 mg, 4 units/mg) or thermolysin (2.2 mg, 42 units/mg) were dissolved in 1.5 mL of PBS buffer with 50 μL of concentrated NaHCO3 solution. The reaction was shaken at room temperature, and after 24 h the solution was measured by LC-MS. Enzymatic Degradation of the Selectively Deprotected Biohybrid Block Copolymers. 4.5 mL of the particle solution (1.25 mg/mL) of 3763


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Scheme 1. Synthesis of Block Copolymers by Polymerization of N-Carboxyanhydrides (NCA) and Styrene or n-Butyl Acrylate by Nitroxide-Mediated Polymerization Using Bifunctional Initiators 1 and 2

the biohybrid block copolymers was filtered through a 0.2 μm filter and added to a solution of 6.6 mg elastase (4 units/mg) or 1 mg of thermolysin (42 units/mg) in 1.5 mL of PBS buffer. The solution was divided over three reaction tubes. The solutions were shaken in a water bath at 37 °C and taken out for measurements by DLS.

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monomers in DMF. The monomer conversion was monitored by FT-IR following the characteristic peak of the NCA at 1786 cm−1. At full conversion the polypeptides were precipitated in diethyl ether and filtered. Homopolymers of PBLG and (block) copolymers with different chain compositions and Ala content were prepared (Table 1). All polypeptides were obtained with

RESULTS AND DISCUSSION

Synthesis of Polypeptide NMRP Macroinitiators. Polypeptide-containing block copolymers can be obtained by N-carboxyanhydride (NCA) ring-opening polymerization in combination with either convergent methods such as Huisgens cycloaddition or divergent methods, such as macroinitiation using controlled polymerization techniques.24−31 We and others have previously reported the use of bifunctional initiators combining NCA with controlled radical polymerizations.32−35 In particular, the synthesis of polystyrene-bpoly(γ-benzyl-L-glutamate) was successfully performed with an amine-functionalized nitroxide-mediated radical polymerization (NMRP) initiator.31 This TIPNO-based initiator was also applied in this study (initiator 1). For synthetic versatility, we decided to explore an alternative bifunctional NMRP initiator based on commercial MAMA-SG1 (initiator 2), which has been shown to be highly versatile in the polymerization of styrenes and acrylates.36−38 This initiator was synthesized with an overall yield of 42% in two steps based on a modified procedure reported by Vinas,22 reacting the N-succinimide activated MAMA-SG1 with an excess of diaminoethane. Both initiators were first employed in the initiation of γ-benzylglutamate (BLG) and/or L-alanine (Ala) NCA at 0 °C to obtain a homoor copolymer polypeptide block,20,39 followed by a NMRP of styrene or n-butyl acrylate, respectively (Scheme 1). The BLG of the polypeptide block was then deprotected by removal of the benzyl esters to obtain an amphiphilic hybrid block copolymer. For the NCA polymerization the bifunctional initiator 1 or 2 was dissolved in DMF and added to a solution of NCA

Table 1. Polypeptide Homo- and Copolymers Prepared by NCA Polymerization Using Initiators 1 (TIPNO) and 2 (SG1)a polymer

Mnb (g/mol)

PDIb

1 2

PBLG60-SG1 P(BLG50-co-Ala10) SG1 P(BLG30-co-Ala30)SG1 P(BLG15-co-Ala15)b-PBLG30-SG1 P(BLG15-co-Ala15)SG1 PBLG30-bP(BLG15-coAla15)-SG1 P(BLG25-co-Ala5)SG1 P(BLG15-co-Ala15)b-P(BLG25-coAla5)-SG1 PBLG20-TIPNO PBLG60-TIPNO P(BLG30-co-Ala30)TIPNO

13 000 14 500

1.1 1.1

5:1

5:1

11 200

1.1

1:1

1:1

13 300

1.1

2.5:1

3:1

7 600

1.2

1.1:1

1:1

13 600

1.1

2.6:1

3:1

7 100

1.1

5:1

5:1

11 600

1.1

2:1

2:1

5 900 13 500 12 100

1.1 1.1 1.1

1:1

1:1

3 4 5 6 7 8 9 10 11

BLG:Alac

BLG:Ala theor

entry

a

Superscript numbers refer to target degree of polymerization by monomer-to-initiator ratio. bMeasured in DMAc-SEC (polystyrene standards). cComposition determined by 1H NMR in d-TFA using peaks at 1.5 ppm (methyl protons of Ala) and 2.0 ppm (γ-benzyl-Lglutamate). 3764


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polydispersities of 1.2 or below and molecular weights (Mn) ranging from 5900 to 14 500 g/mol (polystyrene standards). Figure 1 shows the SEC traces of P(BLG15-co-Ala15) obtained

Figure 2. Contour plot of the copolymer P(BLG400-co-Ala400) obtained by MALDI-ToF-MS at 5% monomer conversion. The MALDI-ToFMS spectrum can be found in the Supporting Information. Figure 1. SEC plots of P(BLG15-co-Ala15) (entry 5, Table 1; solid line), PBLG30-b-P(BLG15-co-Ala15) macroinitiator (entry 6, Table 1; dashed line), and PBLG30-b-P(BLG15-co-Ala15)-b-PBA block copolymer (entry 6, Table 2; dotted line).

Table 2. Block Copolymers Obtained by Nitroxide-Mediated Polymerization (NMRP) from Polypeptide Macroinitiators

from initiator 2 (entry 5, Table 1). This polypeptide was then chain extended with 30 units of BLG (entry 6, Table 1). While the SEC trace of the first copolymers shows a small low molecular weight shoulder, a clear shift to higher molecular weight can be observed after the addition of BLG NCA. NMR analysis of the copolymers confirmed that the monomer composition of all polypeptides was in good agreement with the BLG/Ala NCA feed ratio. Of significant importance for the intended study is that the comonomers are randomly distributed along the polypeptide chain. First, when a selective enzyme is employed for the degradation of Ala-Ala bonds, a random distribution ensures that the extent of degradation is directly related to the copolymer composition (through the monomer feed ratio). Second, the random distribution of L-glutamic acid groups in the deprotected polypeptides throughout the polypeptide chain enhances the water solubility of the polymer compared to that of a block copolypeptide structure.40,41 In order to gain more information about the randomness of the copolymers, we employed MALDI-ToF-MS contour plot analysis.20 As a nonrandom distribution could only arise from different monomer reactivity ratios under the polymerization conditions, we invested the copolymerization of BLG and Ala NCA using a very high monomer-to-initiator ratio of 400:1 (per monomer). Following the conversion by IR, samples at low conversion were investigated by MALDI-ToF-MS. Figure 2 depicts the contour plot at 5% conversion showing that the obtained copolymer is close to the diagonal. This confirms that the composition of the copolymer is highly random throughout the polypeptide chains. NMRP Reactions and Polypeptide Deprotection. The library of polypeptide macroinitiators containing the SG1 and TIPNO-based NMRP groups was further used for the initiation of styrene (St) and n-butyl acrylate (BA) polymerization (Table 2). For the BA polymerizations, the macroinitiators were dissolved in N-methylpyrolidone (NMP) with BA and 10 mol % of the

entry

polymer

nitroxide

PDI

ratio BA/ S:amino acidb

1 2 3

PBLG20-b-PBA PBLG60-b-PBA P(BLG50-coAla10)-b-PBA P(BLG30-coAla30)-b-PBA P(BLG15-coAla15)-bPBLG30-b-PBA PBLG30-bP(BLG15-coAla15)-b-PBA P(BLG15-Ala15)b-P(BLG25-coAla5)-b-PBA PBLG60-b-PS P(BLG50-coAla10)-b-PS P(BLG30-coAla30)-b-PS

TIPNO SG1 SG1

7 500 19 000 20 400

1.21 1.47 1.17

1.08:1 2.86:1 2.64:1

SG1

19 500

1.38

3.70:1

SG1

20 300

1.33

2.97:1

SG1

18 000

1.14

0.55:1

SG1

14 400

1.08

0.41:1

TIPNO SG1

19 900 25 200

1.33 1.60

1.74:1 4.13:1

TIPNO

18 200

1.31

1.86:1

4 5 6 7 8 9 10

Mna

(g/mol)

a

Measured in DMAc-SEC (polystyrene standards). bComposition determined by 1H NMR: For PBA containing polymers using signals at 4.9 ppm (protons of the amino acid residues in the main chain of the polypeptide) and 1.1 ppm (n-butyl) measured in d-TFA. For PS containing polymers using signals at 5.3 ppm (methylene of benzylesters) and 6.6−7.8 ppm for PS measured in d7-DMF.

corresponding free nitroxide in a Schlenk tube. According to literature reports, better control over the polymerization reaction is achieved with additional free SG1 and at a lower reaction temperature.42,43 Following this procedure, the polymerization temperature was increased to 60 °C for several minutes and subsequently to 90 °C for 1 h.28 After an initial increase of the molecular weight at 90 °C the reaction seemed to stop, but by increasing the temperature above 100 °C, the molecular weight was found to increase again. However, at this temperatures high quantities of low molecular weight poly(n-butyl acrylate) (PBA) 3765


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Table 3. Deprotected Block Copolymers; Dynamic Light Scattering (DLS) Results of Particles Obtained from Amphiphilic Block Copolymers in Phosphate Buffer Solution (Zav: Size Average; PI: Polydispersity) and Critical Micelle Concentration (cmc) Measured by Static Light Scattering (SLS); Precipitation upon Treatment with Elastase entry 1 2 3 4 5 6 7 8

polymer

deprotectiona (%)

PGlu60-b-PBA P(Glu50-co-Ala10)-b-PBA P(Glu30-co-Ala30)-b-PBA PGlu30-b-P(Glu15-co-Ala15)-b-PBA P(Glu15-co-Ala15)-b-P(Glu25-co-Ala5)-b-PBA PGlu60-b-PS P(Glu30-co-Ala30)-b-PS P(Glu30-co-Ala30)-b-PS/P(Glu30-co-Ala30)-b-PBA (1:1)

91 90 93 94 90 100 100

Zav (nm) 129.6 168.8 149.7 63.2 37.7 48.5 129.5 151.3

PI 0.139 0.101 0.084 0.186 0.165 0.117 0.298 0.258

cmc (mg/mL) 8.0 9.0 7.0 1.5 1.5 2.5 2.0

× × × × × × ×

−3

10 10−3 10−3 10−1 10−1 10−1 10−3

precipb (days) none none 4 4 none none none 4

a

Conversion of the removal of benzyl esters was determined with 1H NMR using peaks at 7.4 ppm (aromatic protons of benzylester) and 4.9 ppm (protons of the amino acid residues in the main chain of the polypeptide) in d-TFA for PBA block copolymers. For PS block copolymers at 6.8−7.8 ppm (all aromatic protons) and 5.3 ppm (methylene of benzylesters) in d7-DMF were used. bPrecipitation determined visually during the enzymatic degradation with elastase.

Figure 3. pictures of (a) P(Glu60-b-BA) (2 mg/mL), (b) P(Glu30-co-Ala30)-b-PBA (10 mg/mL) and (c, d) P(Glu30-co-Ala30)-b-PS (2 mg/mL). Scale bars: 200 nm.

or NMP (for the polystyrene block copolymers). A 0.10 M phosphate buffer solution with a pH of 7.4 was added slowly over 3 h, and the solution was dialyzed against the phosphate buffer solution buffer for 2 days. The critical micelle concentration (cmc) of the deprotected block copolymers in buffer solution was determined using static light scattering (SLS) in the range from 2 × 10−3 to 1.5 × 10−1 mg/mL depending on the block copolymer composition (Table 3). Dynamic light scattering (DLS) was used to identify the particle size of the formed particles. Generally, the deprotected copolymers gave particles in the nanometer range. For some of the polymers with a polypeptide block with a higher Ala content larger overall particle sizes with a bimodal distributions were found, such as for P(Glu30-co-Ala30)-b-PS, suggesting the formation of larger aggregates (entry 7, Table 3). Cryo-TEM carried out on PGlu60b-PBA and P(Glu30-co-Ala30)-b-PBA block copolymers confirmed the formation of large spherical particles ranging from 20 to 80 nm in size (Figure 3a−c). The large particles are unlikely to be pure micelles but might also contain some PBA homopolymer (as was detected by GPEC).45 For the P(Glu30-co-Ala30)-b-PS solution cryo-TEM showed the presence of vesicles as well as micelles (Figure 3). The combination of these different sized particles in the solution explains the higher PDI obtained in DLS. Vesicles were also found for P(Glu30-co-Ala30)-b-PS, while for PGlu60-b-PS only micelles could be identified (Figure SI 8). Enzymatic Degradation. The ability of different proteolytic enzymes toward the polypeptide copolymers was first assessed in a model reaction using a fluorescently labeled P(BLG-co-Ala) random copolymer with a degree of polymerization of 40. Labeling was achieved by the addition of the succinimide ester of 5(6)-carboxyfluorescein following complete NCA conversion.46 The fluorescein-functionalized peptide was deprotected with HBr in acetic acid and TFA and after

were found. For conventional free radical polymerization of polyacrylates it was reported that radical backbiting occurs during the polymerization.44 The transferred radical on the polymer chain facilitates fragmentation and branching, resulting in free PBA. During nitroxide-mediated radical polymerization the same side reactions also occur, producing some free PBA. However, by selective precipitation in methanol most of the free PBA could be removed. Generally, both types of nitroxide radicals worked sufficiently well for the macroinitiation and resulted in block copolymers with polydispersities ranging from 1.1 to 1.6. As an example, Figure 1 shows the SEC trace of the block copolymer obtained from the macroinitiation of BA from PBLG30-b-P(BLG15-co-Ala15) (entry 6, Table 2), resulting in a clean shift of the trace to 18 000 g/mol. Further evidence for the block copolymer structure was obtained from gradient polymer elution chromatography (GPEC) (Figures SI 2−5) showing that the polymer product elutes at a time in between those of macroinitiator and PBA or PS, respectively. GPEC also showed the formation of traces of PBA and PS homopolymers in some reactions (entries 2, 3, 4, 6, and 8, Table 2). Self-Organization in Phosphate Buffer Solution. The benzyl esters of BLG monomer units in the PBA block copolymers were selectively deprotected by catalytic hydrogenation (Table 3) with the use of palladium catalyst to preserve the esters of the butyl acrylate. The conversion of the hydrogenation was monitored by the disappearance of the aromatic proton peaks at 7.4 ppm in the 1H NMR spectra (Figures SI 6 and SI 7). While full conversion was not achieved for the catalytic deprotections, it was above 90% for all reactions. A selective catalytic deprotection was not necessary for PS block copolymers due to the absence of labile ester bonds in PS. These block copolymers were therefore deprotected quantitatively by treatment with HBr. The amphiphilic block copolymers were then dissolved in DMSO 3766


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Figure 4. (a) DLS measurement of enzymatic degradation of P(Glu30-co-Ala30)-b-PBA (entry 3, Table 3) with elastase. (b) DLS measurement of enzymatic degradation of P(Glu30-co-Ala30)-b-PS (entry 7, Table 3) with elastase.

The particle size of the polystyrene block copolymer particles remained unaltered following exposure to elastase (Figure 4b). More surprisingly, no precipitation was observed for the micelles containing the Ala copolypeptide, which contrasts to the observed elastase-induced degradation of P(Glu30-co-Ala30)b-PBA. The difference in this degradation behavior must be attributed to differences between the synthetic hydrophobic blocks of PS and PBA. One obvious difference is the mobility of the chains due to the low Tg of PBA.48 First, this gives the polymer the opportunity to deform more easily due to the coalescence of particles during the degradation. Second, it influences the dynamic behavior of the block copolymers in the micelles, which are known to be in an equilibrium state with free chains in the solution.49 For PS block copolymers this process is observed only at elevated temperatures due to the high Tg of the material. This also implies that because of the low mobility the PS chains do not transfer into the water phase, while the PBA block copolymers are more likely to migrate.50−52 This could have a significant consequence for the enzymatic degradation, since most of the enzyme is dispersed in the bulk solution. Assuming that the majority of the degradation occurs in the solution, due to the sterical hindrance in the micelle (or vesicle) shell, a more dynamic system would degrade faster.13,53 In order to provide further evidence for this hypothesis, the degradation of the P(Glu30-coAla30)-b-PS systems was studied at higher temperatures using thermolysin, a thermostable enzyme with an optimum activity at 70 °C. Since the mobility of the polystyrene chains is increased above 60 °C, similar conditions as for the PBA containing copolymers with unimer exchange could be achieved. The sample incubated with thermolysin showed a significant increase in particle size compared to the blank sample maintained under the same conditions, indicating that enzymatic degradation indeed occurred (Figure SI 15). This result emphasizes the specific conditions required for polymeric degradation that may be further utilized for the highly controlled release of payload molecules, dependent on more than a single environmental factor. To show the selectivity of the enzymatic degradation with elastase both the P(Glu30-coAla30)-b-PS and P(Glu30-co-Ala30)-b -PBA solutions were mixed, and elastase was added to this solution. Precipitation was observed after 3 days, while DLS still showed the presence of

purification dissolved in a phosphate buffer solution. Two proteasesthermolysin and elastasewere selected to demonstrate their selectivity to cleave specific peptide bonds only. Elastase-induced release mechanisms are of particular interest due to the correlation between increased levels of elastase and specific biological events concerning disease or injury. 47 Elastase is known to selectively cleave peptide bonds flanked by small amino acids such as glycine and alanine in both P1 and P′1 positions, whereas thermolysin displays a preference to cleave when a hydrophobic residue is at the P′1 end of the cleaved peptide bond, while remaining unselective for the amino acid located in the P1 position. To identify if any peptide degradation was exclusively caused by protease activity, a lipase from Thermomyces lanuginosus was employed as a negative control. To the fluorescent polypeptide solutions the enzymes were added, and after 1 day the reaction solutions were analyzed by LC-MS (Figure SI 10). The LC-MS analysis confirmed the presence of fluorescently labeled L-alanine and L-glutamic acid fragments after the reaction with both peptidases as well as newly formed fragments corresponding to parts of the polypeptide that lack any original end groups. As negative controls, the homopolymer of poly(L-glutamic acid) gave no response to the peptidases as anticipated, and the lipase revealed no activity to degrade any of the polypeptides tested. These results confirm that both proteases are efficient enzymes for the hydrolysis of the polypeptide copolymers. Both enzymes were then added independently to the solution of the biohybrid polymer particles, and the particle size and polydispersity were monitored over time (Figure SI 16). Enzymatic degradation using elastase revealed large differences between the different polypeptide compositions. The PBA containing block copolymers with a high content of Ala demonstrated a quick increase in the particle size and polydispersity, observed after 3 days, and the polymer (partially) precipitated on the fourth day (entry 3, Table 3 and Figure 4a). A similar behavior was observed for PGlu30-b-P(Glu15-co-Ala15)-b-PBA (entry 4, Table 3). This again resulted in large clusters of particles with a size of a few micrometers and indicates that the destabilization of the particles is dependent on the enzymatic cleavage of the Ala-Ala amide bonds near the hydrophobic core. For the polymers with a lower Ala content in the main chain, less degradation was observed. 3767


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particles in the solution. The particle sizes did resemble that of the PS block copolymers. The precipitate was analyzed by 1H NMR and proved to be PBA (Figure 5), indicating that the

ASSOCIATED CONTENT S Supporting Information * MALDI-ToF-MS spectra, NMR spectra, HPLC-MS data, GPEC data, cryo-TEM, and degradation histograms. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ACKNOWLEDGMENTS This work formed part of the research program of the Dutch Polymer Institute (project #610). The authors thank Richard Hoogenboom for providing the SG1 and MAMA-SG1 for the NMRP experiments, Hector Tello Manon for the help with the SLS and DLS, Carin Diets and Hanneke Lambermont-Thijs for the LC-MS and GPEC measurements, and Rinske Knoop for the cryo-TEM measurements. A.H. is a SFI Stokes Senior Lecturer (07/SK/B1241).

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REFERENCES

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1

Figure 5. H NMR (CDCl3) of precipitated polymer after exposure of the mixed micelle solution P(Glu30-co-Ala30)-b-PS/P(Glu30-co-Ala30)-bPBA (1:1) to elastase.

degradation was very selective toward the P(Glu30-co-Ala30)-bPBA micelles.

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CONCLUSION Biohybrid block copolymers of PBA and (block co)polypeptides of PGlu and P(Glu-co-Ala) have been made using two different amine-functionalized bifunctional NMRP initiators based on the nitroxides TIPNO and SG1. Both bifunctional initiators successfully initiated the ROP of the amino acid NCAs selected before the macroinitiators were successfully employed for further chain extension with PS and PBA. The selective deprotection of the benzyl ester protecting groups from the block copolymers yielded amphiphilic polymers consisting of vesicles and micelles, the enzymatic stability of which was investigated. The extent of elastaseinduced degradation was reliant upon the amount of L-alanine present in the polypeptide block, offering the system tenability. At 50% L-alanine content the particles showed an increase in particle size after 3 days, due to degradation and consequent particle aggregation, under the applied conditions. By altering the peptide block copolymer composition, it was found that the composition of the polypeptide block near the hydrophobic block had the most profound effect on the stability of the particles. No degradation was seen for the particles containing a polystyrene block copolymer at 37 °C. This is attributed to the limited unimer exchange between the polymer micelle and the solution, confirmed by the successful degradation of polystyrene−peptide particles at 70 °C with thermolysin. The results suggest that polypeptide hybrid particles (micelles and vesicles) can be degraded selectively depending on the composition of the hydrophilic peptide block and the composition of the hydrophobic nondegradable block. These results offer guidelines for the specific design of particles possessing a desired composition for biological processes actuated by their interaction with target enzymes. 3768


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Selective Enzymatic Degradation of Self-Assembled Particles from

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