Biomacromolecules 2010, 11, 1891–1895
Copolymerization Behavior of N-(2-Hydroxypropyl)methacrylamide and a Methacrylated Coiled-Coil Peptide Derivative
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Scheme 1. Schematic Illustration of the Structure of Noncovalent, Coiled-Coil Based Polymer-Drug Conjugatesa
Bojana Apostolovic and Harm-Anton Klok* E´cole Polytechnique Fe´de´rale de Lausanne (EPFL), Institut des Mate´riaux and Institut des Sciences et Inge´nierie Chimiques, Laboratoire des Polyme`res, Baˆtiment MXD, Station 12, CH-1015 Lausanne, Switzerland Received May 16, 2010 Revised Manuscript Received June 12, 2010
Introduction Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) is a water-soluble, nontoxic, and nonimmunogenic polymer that is frequently used as a scaffold for the preparation of polymer-drug conjugates.1 N-(2-Hydroxypropyl)methacrylamide can be polymerized both using conventional free radical polymerization as well as via controlled radical polymerization,2 including reversible addition-fragmentation chain transfer polymerization (RAFT).3 The latter approach is particularly attractive as it allows precise control over polymer molecular weight and leads to polymers with narrow molecular weight distributions and well-defined end groups that can be further modified to introduce fluorescent labels or other functional groups. PHPMA-based polymer-drug conjugates have been prepared via two conceptually different approaches: (i) direct copolymerization of HPMA and a methacrylate functionalized derivative of the drug of interest4 or (ii) postpolymerization modification of HPMA copolymers containing appropriate side chain reactive (co)monomer units.5,6 In a recent study, Barz et al. compared the cell uptake behavior of HPMA-based homo, random, and block copolymers and found that, for a given molecular weight, random copolymers are more easily internalized than block copolymers or homopolymers.7 Because the relative reactivities, or reactivity ratios, of comonomers in a copolymerization reaction influence polymer architecture (microstructure) and as reactivity differences can lead to composition drifts and structural heterogeneities, these results underline the importance of a thorough understanding of copolymerization behavior to establish structure-activity relationships for polymer-drug conjugates. Surprisingly, only a very limited number of studies have appeared that report on the copolymerization behavior of HPMA. The examples that have been published include the copolymerization of HPMA with styrene and methyl methacrylate,8 with thiazolidine-2-thione functionalized, N-methacryloylated amino acids or short peptides (less than 5 amino acids),9 or with N-methacryloyloxysuccinimide.10 In this manuscript, we describe the copolymerization behavior of HPMA with a methacrylated coiled-coil peptide derivative (K3-MA). In a previous publication, we reported that copolymers of HPMA and K3-MA can be used as carriers to noncovalently bind cargo such as a drug or a fluorescent dye that is functionalized with a complementary coiled-coil peptide sequence (E3;11 Scheme 1). Binding of the guest to the polymer carrier proceeds in a highly specific manner via heterodimerization of the E3 and K3 peptides into a superhelical structure. * To whom correspondence
[email protected].
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addressed.
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a The polymer carrier is a PHPMA copolymer that is functionalized with multiple copies of one of the coiled-coil forming peptides, in this case, the K3 sequence. The cargo (R) is either a model anticancer drug (methotrexate, MTX) or a fluorescent dye (Oregon Green, OG), which is functionalized with a peptide sequence (in this case, the E3 peptide) that forms a heterodimeric coiled-coil with the sequence that is attached to the PHPMA carrier.
The position of the E3 and K3 peptides can be exchanged and noncovalent polymer drug-conjugates can also be constructed from E3 containing PHPMA carriers and K3 modified guests. Compared to the methacrylated peptide monomers used in earlier copolymerization studies, the peptide sequence of the comonomer in this contribution is much longer and, under appropriate conditions, can adopt stable secondary structures and form homo- or heterooligomeric assemblies. The objective of this contribution is to evaluate the possible influence of these factors on the copolymerization behavior of this monomer with HPMA, which may provide useful insights to establish and understand structure-activity relationships of these noncovalent polymer-drug conjugates.
Experimental Section Materials. Deuterated methanol (methanol-d4, 99.8 atom % D, CD3OD) was purchased from Sigma-Aldrich (Buchs, Switzerland). 2,2′Azobis(2-methylpropionitrile) (AIBN, purum g98% (GC)) and 4,4′azobis(4-cyanovaleric acid) (V-501, purum g98%) were used as received from Fluka Chemie GmbH (Buchs, Switzerland). 4-Cyano4-((thiobenzoyl)-sulfanyl)pentanoic acid (CTA, RAFT agent) was synthesized using a literature procedure.12 N-(2-Hydroxypropyl)methacrylamide (HPMA) and the coiled-coil peptide methacrylate (K3MA; MA-GYK (IAALKEK)2 IAALKEG-NH2; MW ) 2624 g/mol) were prepared as described previously.11 Methods. 1H NMR spectra were acquired on a Bruker (DRX-600) 600 MHz spectrometer at room temperature (27 °C) using deuterated methanol (methanol-d4, 99.8 atom % D, CD3OD) as solvent. Spectra were recorded with a relaxation time (d1) of 10 s by averaging 64 scans. Circular dichroism (CD) measurements were performed as described previously.13 Copolymerization Experiments. FRP copolymerizations were carried out in Schlenk tubes equipped with a magnetic stir bar, a nitrogen inlet, and a rubber septum. The comonomers were mixed in different molar ratios to give an initial monomer concentration ([HPMA + K3-MA]0) of 12.5 wt.%. The initial initiator (AIBN) concentration was 0.6 wt.%. Solid monomers were placed in a Schlenk tube and purged with nitrogen gas for 30 min at room temperature. In a separate flask AIBN was dissolved in degassed CD3OD, stirred and purged with nitrogen gas for 5 min. The reaction was initiated by introducing the desired volume of AIBN solution in CD3OD with a syringe into the
10.1021/bm100533g 2010 American Chemical Society Published on Web 06/24/2010
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Biomacromolecules, Vol. 11, No. 7, 2010
Notes
Table 1. Monomer Feed and Copolymer Composition Data for the Free Radical Copolymerization of HPMA and K3-MA in CD3OD at 50 °C feed composition (mol %)
1
experiment #
HPMA
K3-MA
HPMA
K3-MA
M1a (HPMA)
M2a (K3-MA)
m1a (HPMA)
m2a (K3-MA)
X) M1/M2
Y) m1/m2
G) X · (Y - 1)/Y
F ) X2/Y
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
95 95 95 95 95 93 90 90 85 85 80 80 80 75 75 70 70
5 5 5 5 5 7 10 10 15 15 20 20 20 25 25 30 30
1.86 2.88 2.53 4.31 3.14 2.57 1.96 1.02 2.06 2.91 2.41 2.38 2.63 3.32 3.23 2.47 4.31
9.67 9.99 8.84 17.08 13.57 10.51 15.29 8.47 11.93 14.53 14.64 8.47 8.88 9.34 12.28 6.50 11.27
0.95 0.95 0.95 0.95 0.95 0.93 0.9 0.9 0.85 0.85 0.8 0.8 0.8 0.75 0.75 0.7 0.7
0.05 0.05 0.05 0.05 0.05 0.07 0.1 0.1 0.15 0.15 0.2 0.2 0.2 0.25 0.25 0.3 0.3
0.79 0.85 0.84 0.83 0.81 0.76 0.54 0.52 0.49 0.53 0.40 0.53 0.54 0.52 0.44 0.47 0.47
0.21 0.15 0.16 0.17 0.19 0.24 0.46 0.48 0.51 0.47 0.60 0.47 0.46 0.48 0.56 0.53 0.53
19 19 19 19 19 13.29 9 9 5.67 5.67 4 4 4 3 3 2.33 2.33
3.67 5.48 5.45 4.79 4.39 3.24 1.15 1.09 0.98 1.14 0.66 1.12 1.18 1.07 0.79 0.89 0.89
13.82 15.53 15.51 15.03 14.67 9.19 1.20 0.72 -0.14 0.68 -2.08 0.44 0.62 0.19 -0.81 -0.30 -0.28
98.49 65.89 66.30 75.37 82.24 54.44 70.18 74.51 32.88 28.27 24.33 14.26 13.52 8.44 11.42 6.14 6.11
a
conversion by H NMR (mol %)
Composition of the initial mixture (M) and composition of the copolymer (m) are expressed in molar fractions.
Table 2. Monomer Feed and Copolymer Composition Data for the RAFT Copolymerization of HPMA and K3-MA in CD3OD at 70 °C
time experiment # (min)a 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 a
5 5 2 2 5 5 5 2 5 10 5 5 5 5 5 5
feed composition (mol %)
1
HPMA
K3-MA
HPMA
K3-MA
98 98 95 95 95 93 93 90 90 90 85 85 80 80 75 75
2 2 5 5 5 7 7 10 10 10 15 15 20 20 25 25
1.61 1.09 1.22 0.99 1.67 1.06 1.64 1.96 2.76 1.02 2.22 3.26 3.07 4.03 7.86 4.28
9.67 6.67 9.58 6.85 7.71 11.11 12.85 7.45 7.88 10.51 9.87 6.10 14.68 9.83 16.07 7.54
Polymerization time (min).
b
conversion by H NMR (mol %)
M1b M2b m1b m2b X) Y) G) (HPMA) (K3-MA) (HPMA) (K3-MA) M1/M2 m1/m2 X · (Y - 1)/Y F ) X2/Y 0.98 0.98 0.95 0.95 0.95 0.93 0.93 0.9 0.9 0.9 0.85 0.85 0.8 0.8 0.75 0.75
0.02 0.02 0.05 0.05 0.05 0.07 0.07 0.1 0.1 0.1 0.15 0.15 0.2 0.2 0.25 0.25
0.89 0.89 0.71 0.73 0.80 0.56 0.63 0.70 0.76 0.47 0.56 0.75 0.46 0.62 0.59 0.63
0.11 0.11 0.29 0.27 0.20 0.44 0.37 0.30 0.24 0.53 0.44 0.25 0.54 0.38 0.41 0.37
49 49 19 19 19 13.29 13.29 9 9 9 5.67 5.67 4 4 3 3
8.15 7.99 2.42 2.75 4.12 1.26 1.69 2.37 3.15 0.88 1.27 3.02 0.84 1.64 1.47 1.70
42.99 42.87 11.13 12.08 14.39 2.76 5.44 5.20 6.14 -1.28 1.21 3.79 -0.78 1.56 0.96 1.24
294.71 300.52 149.47 131.39 87.60 139.87 104.17 34.20 25.73 92.52 25.23 10.62 19.12 9.75 6.13 5.29
Composition of the initial mixture (M) and composition of the copolymer (m) are expressed in molar fractions.
Schlenk tube and immersing the Schlenk tube in the oil bath (50 °C) almost concurrently. After 2 min, the polymerization was stopped by exposing the reaction mixture to air and instantaneous quenching in liquid nitrogen. For each 1H NMR measurement, the quenched reaction mixture was diluted with CD3OD to a total volume of 1 mL. RAFT copolymerizations were carried out in Schlenk tubes equipped with a magnetic stir bar, a nitrogen inlet and a rubber septum. 4,4′Azobis(4-cyanopentanoic acid) (V-501) was used as primary radical source and 4-cyanopentanoic acid dithiobenzoate as the chain transfer agent (CTA). For all experiments, HPMA and K3-MA were mixed in different molar ratios at a total initial monomer concentration ([HPMA + K3-MA]0) of 0.5 M. The initial CTA to initiator ratio ([CTA]0/[I]0) was 5/1, and the targeted molecular weight was Mn ) 40000 g/mol. First, solid monomers were placed in the Schlenk tube, which was subsequently purged with nitrogen gas for 30 min at room temperature. In a separate flask, CTA and V-501 were dissolved in a small volume of degassed CD3OD, stirred and purged with nitrogen gas for 5 min. Copolymerizations were initiated by introducing the desired volume of V-501 and CTA solution in CD3OD with a syringe into the Schlenk tube, after which the Schlenk tube was immersed in an oil bath at 70 °C. After 2-10 min, the polymerization was stopped by exposing the reaction mixture to air and instantaneous quenching in liquid nitrogen. For each 1H NMR measurement, the quenched reaction mixture was diluted with CD3OD to a total volume of 1 mL. Determination of Copolymer Composition and Monomer Reactivity Ratios. The composition of the P(HPMA-co-K3-MA) copolymers (Tables 1 and 2) was determined by 1H NMR spectroscopy (Figure 1).
The HPMA conversion was determined by comparing the integrals of the HPMA CH3 protons labeled e (δ ) 1.10 ppm, d, 3H) with that of the dCH2 protons of the HPMA monomer (a, δ ) 5.64 ppm, s, 1H). The conversion of K3-MA was calculated by comparing the integrals of the peptide tyrosine protons (c′, δ ) 6.86 ppm, d, 2H) with those of the dCH2 protons (a′, δ ) 5.76 ppm, s, 1H) of the K3MA monomer. A 1H NMR spectrum of a 95/5 (mol/mol) reaction mixture of HPMA and K3-MA after 2 min of free radical copolymerization (experiment #1, Table 1) is shown in Figure 1C. The peaks assigned as a, e, a′, and c′, which are used to calculate monomer conversions, are well separated and do not overlap with other signals. The monomer reactivity ratios, r1 (monomer reactivity ratio of HPMA) and r2 (monomer reactivity ratio of K3-MA), were determined graphically using the Fineman-Ross14 method. The Fineman-Ross analysis plots the results of the copolymer composition analysis in form of a linear G versus F plot (eq 1), which provides r1 as the slope and r2 as the negative intercept. Values of G and F are given in Tables 1 and 2 for the different copolymerization experiments.
G ) r1F - r2
(1)
Results and Discussion To evaluate the copolymerization behavior of HPMA and K3MA, the reactivity ratios of these two monomers were determined. To this end, different series of copolymerization
Notes
Biomacromolecules, Vol. 11, No. 7, 2010
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Scheme 2. Synthesis of K3-Modified PHPMA Copolymers via (i) Reversible Addition-Fragmentation Chain Transfer Copolymerizationa
a Reagents and conditions: RAFT: 4-cyanopentanoic acid (dithiobenzoate; CTA), 4,4′-azobis(4-cyanopentanoic acid) (V-501), 70 °C, 2-10 min) and (ii) free radical copolymerization; FRP: AIBN, 50 °C, 2 min.
Figure 1. 1H NMR spectra of (A) N-(2-hydroxypropyl)methacrylamide (HPMA). The protons labeled a (5.64, s, 1H) and e (1.10, d, 3H) were used to calculate the monomer conversion; (B) K3-MA. The protons labeled a′ (5.76, s, 1H) and phenyl protons of tyrosine c′ (6.86 ppm, d, 2H) were used to calculate the K3-MA conversion; (C) Reaction mixture (experiment # 1, Table 1) in CD3OD after 2 min of FRP polymerization. Protons are assigned as in (A) and (B). Inset shows zoomed region from 5 to 7.5 ppm.
experiments were carried out (Scheme 2) in which the composition of the monomer feed was systematically varied. The copolymerizations were performed using both a conventional free radical initiator (AIBN) as well as using RAFT conditions. To avoid changes in the composition of the monomer feed, the copolymerizations were stopped at relatively low conversions, generally
