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Synthesis and Solution Properties of PCL‑b‑PHPMA Diblock Copolymers Containing Stable Nitroxyl Radicals Svetlana Petrova,*,† Damir Klepac,† Rafał Konefał,† Sami Kereïche,‡ Lubomír Kovácǐ k,‡ and Sergey K. Filippov*,† †

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic ‡ Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Albertov 4, 128 01 Prague 2, Czech Republic S Supporting Information *

ABSTRACT: This work focuses on the synthesis and the aqueous solution properties of novel amphiphilic PCL-b-PHPMA diblock copolymers possessing 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) stable radicals covalently conjugated to the hydrophobic poly(ε-caprolactone) (PCL) block. A new synthetic approach (a four-step pathway) combining ring-opening polymerization (ROP), carbodiimide chemistry (DCC method), a reversible addition−fragmentation chain transfer (RAFT) polymerization technique, and finally chemical oxidation was employed to successfully produce a series of TEMPO-containing PCL-b-PHPMA diblock copolymers for the first time. EPR spectroscopy was applied to verify successful oxidation of the synthesized diblock copolymers and to investigate the dynamics of the polymer chains before and after micellization. The diblock copolymers self-assembled in PBS solution into spherical radical-containing nanoparticles (RNPs), which were characterized by 1 H NMR spectroscopy, dynamic (DLS), static (SLS) light scattering, and cryo-transmission electron microscopy (cryo-TEM). These novel RNPs could find applications, e.g., as drug delivery systems and for the treatment of oxidative stress injuries. riers,10−13 and several anticancer drug−PHPMA conjugates have been clinically evaluated.14−16 Indeed, PHPMA is a watersoluble, nontoxic polymer possessing low immunogenicity and can reside well in blood circulation.17,18 Its physical properties and synthetic flexibility have been proven to be very useful when combined with biologically active agents.19,20 This polymer is a viable alternative to poly(ethylene oxide) (PEO) for many nanomedicine applications.19 In the past decade, a myriad of studies have been performed on the synthesis and supramolecular self-assembly of amphiphilic block copolymers based on PEO as the hydrophilic block and an aliphatic hydrophobic polyester, such as poly(ε-caprolactone) (PCL).21−26 On the other hand, it should be noted that PHPMA could be a good candidate as a comonomer to

1. INTRODUCTION Recently, there has been increased interest in the synthesis of amphiphilic block copolymers and in the use of their micellar structure for drug delivery applications.1−4 Generally, amphiphilic copolymers for which one block is hydrophilic and another is hydrophobic exhibit specific and unique behaviors in solution.5,6 In an aqueous medium, amphiphilic molecules orient themselves so that the hydrophobic blocks are removed from the aqueous environment to achieve a state of minimum free energy.7,8 Hence, the hydrophobic core is stabilized by the hydrophilic shell, which serves as an interface between the bulk aqueous phase and the hydrophobic domain.6,9 It is well-known that such nanosized micelles with a core−shell architecture can be successfully applied in medicine due to their great potential as drug delivery systems. Over the past 30 years, poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) and its copolymers have been among the most intensively studied polymeric drug car© XXXX American Chemical Society

Received: June 3, 2016 Revised: July 13, 2016

A

DOI: 10.1021/acs.macromol.6b01187 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

Scheme 1. Synthetic Route for the Preparation of the PCL-b-PHPMA Diblock Copolymer Containing the TEMPO Group

phobic core. Such RNPs composed of a self-assembling amphiphilic block copolymers show promising potential, and their study is rather limited. Yoshitomi et al. have recently designed nitroxyl NPs composed of a hydrophilic PEO and a hydrophobic poly(chloromethylstyrene) (PCMS).36 The RNPs were found to be effective for cerebral ischemia−reperfusion injury.37 Therefore, we sought to develop a novel synthetic approach for the synthesis of a well-defined biocompatible and biodegradable class of amphiphilic PCL-b-PHPMA diblock copolymers to bear nitroxyl radicals. We then evaluated the micelle formation of our novel diblock copolymers in solution. The developed RNPs could potentially be used for both drug delivery applications and the treatment of oxidative stress injuries. Furthermore, when used for drug delivery applications, these detectable nanoparticles allow for practical applications in monitoring drug routes in organisms. In this work, a series of amphiphilic PCL-b-PHPMA diblock copolymers carrying stable TEMPO radicals were successfully prepared. Various synthetic routes (i.e., ROP and the DCC method followed by “living” RAFT polymerization and oxidation) in four synthetic steps were employed, which resulted in new block copolymers (Scheme 1). In addition, the amphiphilic AB diblock copolymers self-assembled into regular spherical RNPs in buffer-simulated physiological conditions (phosphate buffered saline, PBS, pH 7.4). The dynamical processes during the self-assembly were studied via EPR spectroscopy. Moreover, the RNPs were characterized by 1H NMR spectroscopy, dynamic (DLS), static (SLS) light scattering techniques, and cryo-TEM.

produce biocompatible copolymers of PCL. Indeed, PCL exhibits a great potential as a biomaterial due to its unique combination of biodegradability and biocompatibility.25 However, few published studies regarding the synthesis and micellar characterization of block copolymers based on PCL and PHPMA have been reported. For example, Krimmer et al.27 reported the synthesis of an amphiphilic diblock copolymer by combining ring-opening polymerization (ROP) of ε-caprolactone (ε-CL) followed by “living” reversible addition− fragmentation chain transfer (RAFT) polymerization of HPMA. Furthermore, the authors investigated micelles based on PCL-b-PHPMA diblock copolymer as promising biocompatible, nonimmunogenic vehicles for drug delivery.27 The synthesis and micellar characterization of amphiphilic PCL-bPHPMA tri- and star-shaped copolymers using traditional freeradical polymerization to prepare the PHPMA blocks have been reported in studies by Lele and Leroux.28,29 However, to the best of our knowledge, there are no studies published on the synthesis and characterization of the radicalcontaining nanoparticles (RNPs) based on PCL and PHPMA. Nitroxyl radicals, such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), are organic molecules that have long been used as a spin labels or spin probes for electron paramagnetic resonance (EPR) spectroscopy studies of polymers and proteins, as they possess an unpaired electron that gives rise to the EPR signal.30,31 These nitroxyl radicals are known to catalytically scavenge reactive oxygen species (ROS), such as superoxide, hydroxyl radicals, and peroxy radicals.32 Numerous in vitro cell experiments have confirmed that nitroxyl radicals effectively scavenge ROS and regulate redox conditions, which improve cell viability and biofunctions.33 Under in vivo conditions, however, such low-molecularweight nitroxyl radicals pose several problems, such as nonspecific accumulation in normal tissues, preferential renal clearance, and their rapid reduction to the corresponding hydroxylamine form.34,35 One way to overcome these difficulties is to create the nanoparticles that carry the nitroxyl radicals in their hydro-

2. EXPERIMENTAL SECTION 2.1. Materials. Monomer N-(2-hydroxypropyl)methacrylamide (HPMA) was synthesized according to ref 46 using K2CO3 as a base (mp 70 °C; purity >99.8% (HPLC); elemental analysis: calcd C 58.72%, H 9.15%, N 9.78%; found C 58.98%, H 9.18%, N 9.82%) εCaprolactone (ε-CL, 99%) was received from Sigma-Aldrich. The εCL was dried over CaH2 under continuous stirring at room temperature for 48 h and distilled under reduced pressure before use. 2,2′-Azobis(2-methylpropionitrile) (AIBN, 98%, Sigma-Aldrich) B


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PHPMA copolymer (20 mg) was dissolved in dimethylformamide (DMF) (6 mL). The solution was then injected dropwise using a syringe (G = 26) into phosphate buffer saline (PBS) (14 mL) while stirring magnetically at room temperature. The organic solvent was removed via dialysis in PBS for 24 h using a 3−5 kDa molecular weight cutoff membrane. The final concentration of RNPs was 1.0 mg/mL. RNPs for NMR measurements were prepared using the aforementioned nanoprecipitation method but using deuterated water (D2O) instead of PBS. For the NMR measurements, NP samples were diluted with D2O to a final concentration of 1.0 mg/mL. The D2O solution samples were added to 5 mm NMR tubes, which were degassed and sealed under nitrogen. 2.7. Characterization Techniques. 1H NMR spectra (300 MHz) were obtained using a Bruker Avance DPX 300 NMR spectrometer with CDCl3 and DMSO-d6 as solvents at 25 °C. The chemical shifts were relative to TMS using hexamethyldisiloxane (HMDSO, δ = 0.05 ppm from TMS in 1H NMR spectra) as the internal standard. The 1H NMR spectrum of RNPs was acquired using a Bruker Avance III 600 spectrometer operating at 600.2 MHz with D2O as a solvent at 25 °C. The Fourier transform infrared (FTIR) spectra were recorded using a PerkinElmer Spectrum 100 equipped with a universal ATR (attenuated total reflectance) accessory with a diamond crystal. For all cases, the resolution was 4 cm−1, and the spectra were averaged over 16 scans. The number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (Mw/Mn) of the prepared α-TEMP-PCL prepolymer, the PCL MacroCTA RAFT Agent, and the PCL-b-PHPMA diblock copolymers before and after oxidation were determined based on size exclusion chromatography (SEC). The analyses were performed on using an SDS 150 pump (Watrex, USA), an autosampler Midas (Spark Holland) evaporative light scattering PL ELS 1000 (Polymer Laboratories), and UV (Watrex UVD 250) detectors. The separation system consisted of two PLgel MIXED-BLS columns (Polymer Laboratories) and was calibrated based on polystyrene (PSS, Germany). DMF was used as a mobile phase at a flow rate of 0.5 mL/min at 25 °C. Data collection and processing were performed using TriSEC (Viscotek Comp) software. Dynamic Light Scattering (DLS). The DLS measurements were performed using an ALV CGE laser goniometer. The scattered light of a 22 mW HeNe linear polarized laser (632.8 nm) was measured in a broad angle range of 40°−150° and was collected using an ALV 6010 correlator. The DLS experiments were conducted at body temperature, T = 37 °C. The data were collected using ALV Correlator Control software with various counting times from 100 to 300 s to accumulate an intensity correlation function g2(t) with a low signal-to noise ratio. The measured g2(t) was analyzed using the algorithm REPES (incorporated in the GENDIST program) resulting in a distribution of relaxation times, A(τ). The diffusion coefficient D was obtained according to the standard relation

was recrystallized twice from methanol prior to use. 4-Hydroxy-2,2,6,6tetramethylpiperidine (TEMPH, 98%), 4-cyano-4-(thiobenzoylthio)pentanoic acid (CTA, >97%), 4-(dimethylamino)pyridine (DMAP, 99%), N,N′-dicyclohexylcarbodiimide (DCC, 99%), m-chloroperbenzoic acid (mCPBA, ≤77%) and tin(II) bis(2-ethylhexanoate) (Sn(Oct)2, 95%, 0.06 M solution in toluene) were received from Sigma-Aldrich and used as received. N,N-Dimethylformamide (DMF, ≥99.5%, Sigma-Aldrich) was dried over CaH2 and distilled under reduced pressure. Dichloromethane (CH2Cl2, anhydrous, ≥99.8%, Sigma-Aldrich) was dried by refluxing over a benzophenone−sodium complex and distilled under an argon atmosphere. Toluene (99%, Labscan) was refluxed for 24 h over CaH2 under a dry argon atmosphere and then distilled. All other chemicals were used as received. 2.2. Synthesis of α-2,2,6,6-Tetramethylpiperidin-ω-HydroxyPCL (α-TEMP-PCL) ((1) Scheme 1). Approximately 0.1 g (6.36 × 10−4 mol) of 4-hydroxy-2,2,6,6-tetramethylpiperidine was added to a 50 mL glass reactor containing a magnetic stir bar and dried by three azeotropic distillations with anhydrous toluene. Freshly distilled ε-CL 3.77 g (33.0 × 10−3 mol) was then added. The reaction mixture was heated to 110 °C before rapid injection of 0.1 mL of 0.06 M Sn(Oct)2 solution through a septum. The polymerization was carried out for 24 h in bulk. Afterward, the reactor was cooled to room temperature, and the reaction mixture was dissolved in toluene. The solid was precipitated in cooled heptane, filtered, and precipitated again in cooled methanol to remove the nonreacted ε-CL. The α-TEMP-PCL was recovered via centrifugation and dried overnight under vacuum at 40 °C. Yield: 3.31 g (87.8 wt %). 2.3. Synthesis of PCL-CTA Macromolecular Chain Transfer Agent (PCL MacroCTA RAFT Agent) ((2) Scheme 1). In a 50 mL flame-dried and argon-purged two-neck round-bottom flask equipped with three-port valves, 1 g (1.89 × 10−4 mol) of α-TEMP-PCL (1) (Mn(NMR) ∼ 5300 g mol−1) was added. The prepolymer (1) was dissolved in dry toluene and dried three times via azeotropic distillation. Thereafter, 0.13 g of CTA (4.72 × 10−4 mol), 0.011 g of DMAP (9.43 × 10−5 mol), and 0.1 g of DCC (4.72 × 10−4 mol) were transferred to the flask under an inert gas blanket. A dry CH2Cl2 (15 mL) was then added to the flask using a flame-dried and argonflushed glass syringe equipped with a metallic cannula. The solution was stirred at room temperature for 72 h under argon. Next, the solvent was evaporated under vacuum, the solid residue was dissolved in THF, and the solution was filtered to remove the dicyclohexylurea byproduct. The pink solid was precipitated in cold methanol, filtered off, and dried under vacuum at room temperature. Yield: 0. 85 g (85 wt %). 2.4. Synthesis of Amphiphilic PCL-b-PHPMA Diblock Copolymer ((3) Scheme 1). The RAFT polymerization was carried out as follows: PCL macroCTA agent (2) 0.08 g (1.52 × 10−5 mol) (Mn(NMR) ∼ 5800 g mol−1), HPMA (the molar ratio of the monomer was varied to obtain PHPMA blocks with different molecular weights: [M]:[CTA]:[I] = 70:1:0.2, [M]:[CTA]:[I] = 105:1:0.2, and [M]: [CTA]:[I] = 140:1:0.2), and AIBN 0.00005 g (3.04 × 10−5 mol) were added to a 20 mL Schlenk tube with a magnetic stir bar. The reagents were dissolved in DMF (5 mL), purged with argon for 30 min, and then placed in an oil bath preheated to 70 °C. The polymerization was terminated by rapid cooling and freezing. The pink reaction product was precipitated into cooled diethyl ether, filtered off, washed three times with diethyl ether, and then dried overnight at 40 °C in a vacuum. 2.5. Oxidation of PCL-b-PHPMA Diblock Copolymer ((4) Scheme 1). The oxidation was realized as follows: The obtained diblock copolymer (3) (sample 2, in Table 1) (0.15 g) was dissolved in CH2Cl2 (10 mL), and mCPBA (0.018 g) was added to the solution. The reaction mixture was stirred at room temperature for 17 h and precipitated into cooled diethyl ether to yield the corresponding radical copolymer (4) as an orange elastomeric solid. Yield: 0.13 g (86.6 wt %). 2.6. Preparation of the Radical-Containing Nanoparticles (RNPs) from the PCL-b-PHPMA Diblock Copolymers. Nanoparticles were prepared by the nanoprecipitation method.47 PCL-b-

Γ = τ −1 = Dtrq2

(1)

where Γ is the relaxation rate, q = 4πn sin(θ/2)/λ is the scattering vector with λ corresponding to the laser wavelength, n is the refractive index of the solvent, and θ is the scattering angle. The apparent hydrodynamic radius (RH) of the nanoparticles was calculated from the Stokes−Einstein relation: RH =

kBT 6πηD

(2)

where kB is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the solvent, and D is the apparent diffusion coefficient of the nanoparticles. Static Light Scattering (SLS). The SLS measurements were carried out by varying the scattering angle from 30° to 150° with 10° stepwise increases. The molecular weight (Mw(NP)) and the gyration radius (RG) of the nanoparticles were estimated according to the Zimm approach as follows: R 2q2 ⎞ Kc 1 ⎛ ⎜1 + G ⎟ = Rθ M w(NP) ⎝ 3 ⎠ C

(3) DOI: 10.1021/acs.macromol.6b01187 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

Figure 1. 1H NMR spectrum of the α-TEMP-PCL prepolymer in CDCl3. where the K is the optical constant, Rθ is the excess normalized scattered intensity (toluene was applied as the standard solvent), and c is the polymer concentration given in mg mL−1. The refractive index increment was measured using a BI-DNDC differential refractometer. Cryo-Transmission Electron Microscopy (Cryo-TEM). Cryo-TEM measurements were carried out using a Tecnai G2 Sphera 20 electron microscope (FEI Company, Hillsboro, OR) equipped with a Gatan 626 cryo-specimen holder (Gatan, Pleasanton, CA) and a LaB6 gun. The samples for cryo-TEM were prepared by plunge-freezing.48 Briefly, 3 μL of the sample solution was applied to a copper electron microscopy grid covered with a perforated carbon film forming wovenmesh-like openings of different sizes and shapes (the lacey carbon grids #LC-200 Cu, Electron Microscopy Sciences, Hatfield, PA) and then glow discharged for 40 s with 5 mA current. Most of the sample was removed by blotting (Whatman no. 1 filter paper) for approximately 1 s, and the grid was immediately plunged into liquid ethane held at −183 °C. The grid was then transferred without rewarming to the microscope. Images were recorded at the accelerating voltage of 120 kV and with magnifications ranging from 11 500× to 50 000× using a Gatan UltraScan 1000 slow scan CCD camera in low-dose imaging mode, with the electron dose not exceeding 1500 electrons/nm2. The magnifications resulted in final pixel sizes ranging from 1 to 0.2 nm, and the typical value of the applied underfocus ranged from 0.5 to 2.5 μm. The applied blotting conditions resulted in the specimen thicknesses varying between 100 and ca. 300 nm. Brightness and contrast corrections of the acquired images were performed using ImageJ software. Electron Paramagnetic Resonance (EPR) Spectroscopy. EPR measurements were performed using a 20 μL capillary on a Bruker ELEXSYS E-540 X-band spectrometer equipped with a Bruker ER 049X microwave bridge and a Bruker ER4131VT variable temperature unit. Spectra were recorded at 37 °C with a sweep width of 100 G, a microwave power output of 6 mW, a modulation frequency of 100 kHz, and a sweep time of 22 min to improve the signal-to-noise ratio. The modulation amplitude was optimized to the line width of the spectrum (on the order of 1.0−2.0 G).

system in the presence of Sn(Oct)2 as a catalyst under anhydrous conditions, producing α-TEMP-PCL with a relatively narrow molecular weight distribution and a predictable molecular weight. This approach resulted in the formation of PCL with a hydroxyl group at one end and a 2,2,6,6-tetramethylpiperidine group at the other end. After purification, the obtained 1H NMR spectrum is shown (Figure 1), and the assignments of the different signals were realized. The 1H NMR spectrum of α-TEMP-PCL shows that the characteristic signals for the methylene protons of the ε-CL units were detected at δ = 4.05 ppm (h) −CH2−OC(O)−, δ = 2.29 ppm (d) −C(O)CH2−, δ = 1.61 ppm (e + g) −C(O)− CH2−CH2−CH2−CH2− and δ = 1.38 ppm (f) −C(O)−CH2− CH2−CH2−. Furthermore, a signal at δ = 3.61 ppm, marked as (h′), was also found originating from the methylene protons bonding to the hydroxy end-group, which is attached to another PCL terminal. The signal observed at δ = 5.15 ppm (a) is attributed to the methine proton of the TEMP end-group. Additionally, signal (b) from the methylene protons represents the TEMP end-group overlapped by the PCL signal labeled (f) at δ = 1.38 ppm. The spectrum clearly shows a signal attributed to protons from methyl groups in the composition of TEMP at δ = 1.35 ppm, which are all denoted (c). The degree of introduction of TEMP end-groups into the PCL was estimated as 83% from the relative intensity of the terminal hydroxymethylene (h′) and methine (a) of TEMP. The numberaverage molecular weight Mn(NMR) of the α-TEMP-PCL prepolymer (1, in Table 1) was estimated by eq 4. M n(NMR) = [(Ih /2)/(Ia)]114 + 157 + 17

(4)

where Ih and Ia are the integral values of the peaks at δ = 4.05 ppm (h, from PCL) and δ = 5.15 ppm (a, from TEMP), respectively. The value 114 is the molecular weight of the ε-CL monomer unit; 157 and 17 stand for the molecular weights of the two functional groups, end-chain TEMP and −OH, respectively. The effectiveness of ROP of ε-CL using the 4-hydroxyTEMP initiator system was confirmed via FTIR spectroscopy (Figure S1b). The FTIR spectra of 4-hydroxy-TEMP and the α-TEMP-PCL prepolymer are shown (Figure S1a,b). The IR

3. RESULTS AND DISCUSSION 3.1. Synthesis of α-2,2,6,6-Tetramethylpiperidin-ωhydroxy-PCL Prepolymer (α-TEMP-PCL) ((1) Scheme 1). Scheme 1 shows the strategy used to prepare the α-TEMP-PCL prepolymer (compound 1). In the first step, living ROP of εcaprolactone (ε-CL) was achieved using 4-hydroxy-2,2,6,6tetramethylpiperidine (4-hydroxy-TEMP) as an initiation D


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esterification in the presence of DCC and a catalytic amount of DMAP at room temperature (compound 2, in Scheme 1). 1 H NMR, FTIR, and SEC analyses were performed on compound 2 to ensure its purity. The 1H NMR spectrum of the PCL macroCTA RAFT agent was recorded, and the assignments of the different signals were made (Figure 3). 1H

spectrum of 4-hydroxy-TEMP (Figure S1a) shows characteristic vibrations; a broad absorption in the 3220−3065 cm−1 range due to the O−H stretch overlapped by a narrow absorption relative to the N−H stretch at 3249 cm−1. Secondary amines, as piperidines, correspond to a very narrow peak in the 3400−3100 cm−1 region, relative to the N−H stretch (Figure S1a).38 Additionally, the spectrum clearly shows the presence of C−H stretch vibrations for methyl and methylene groups between 2842 and 2985 cm−1. The FTIR spectrum of the α-TEMP-PCL prepolymer shows the appearance of a strong absorption band at 1720 cm−1 (−C O stretching mode), indicating the presence of ester groups in the polymeric backbone of PCL. Unfortunately, a characteristic absorption band attributed to N−H stretch at 3249 cm−1 due to the TEMP end-group in the composition of α-TEMP-PCL was not observed in the spectrum (Figure S1b), likely due to the high molecular weight of the prepolymer and because FTIR spectroscopy cannot detect characteristic N−H stretching vibration. The absorption bands for carbon−hydrogen groups were also observed, giving rise to C−H stretching absorption in the 2950−2866 cm−1 region of the IR spectrum. SEC analysis of the α-TMEP-PCL prepolymer in DMF revealed the monomodal curve, and no tailing at the lower and higher molecular weight sides was observed (bold line, in Figure 2), indicating a complete consumption of the ε-CL monomer units during ROP and successful synthesis of the target prepolymer (1, in Table 1).

Figure 3. 1H NMR spectrum of the PCL macroCTA RAFT agent in CDCl3.

NMR revealed complete disappearance of the signals at δ = 3.61 ppm (h′), attributed to the methylene protons neighboring the hydroxyl end-group of the α-TEMP-PCL prepolymer. A new set of signals at higher frequencies, δ = 7.90−7.39 (l + m + n), were assigned to aromatic protons of the phenyl group from CTA. Low-intensity signals in the spectrum characteristic of methylene and methyl protons at δ = 2.59 ppm from −C(O)CH2− (i), −C(O)−CH2−CH2− (j), and δ = 1.9 ppm −(CN)C(CH3)− (k) assignable to CTA were also detected. The signals at (h) 4.05, (d) 2.3, (e + g) 1.63, and (f) 1.39 ppm were all attributed to the methylene protons of the PCL main chain. The functionalization yield was quantitatively estimated from the relative intensity of the signals characteristic of both TEMP (a = 5.15 ppm) and CTA (l + m + n = 7.90−7.36 ppm) groups in the 1H NMR spectrum. The high yield (91%) obtained for the esterification reaction indicates the outcome of the synthetic strategy. The Mn of PCL macroCTA RAFT agent (2, in Table 1) was determined via 1H NMR spectroscopy and calculated according to eq 5.

Table 1. Macromolecular Characteristics of Functional PCL no.

PCL samples

conva (%)

Mnb (1H NMR)

Mnc (SEC)

Mw/Mnd (SEC)

1 2

α-TEMP-PCL PCL macroCTA

83 91

5300 5865

5950 6450

1.16 1.18

a

Conversion was calculated by 1H NMR spectroscopy (Figures 1 and 3). bMn was calculated via 1H NMR spectroscopy according to eqs 4 and 5. cMn was determined according to SEC calibrated with PS standards. dMw/Mn was determined according to SEC calibrated with PS standards.

M n(NMR) = [(Ih /2)/(Ia)] × 114 + 157 + 279

(5)

where Ih and Ia are the integral values of the peaks at δ = 4.05 ppm (h, methylene protons of PCL) and δ = 5.15 ppm (a, methine proton of TEMP), respectively. The value 114 is the molecular weight of the ε-CL monomer unit, and 157 and 279 represent the molecular weights of the two functional groups at the chain ends (TEMP and CTA, respectively). Furthermore, the structure of the obtained PCL macroCTA RAFT agent was determined by FTIR spectroscopy. Unfortunately, differences in the compositions and structures of the resulting PCL macroCTA RAFT agent after esterification via the DCC method were not observed. The characteristic absorption bands from the CTA end-group of the synthesized RAFT agent were not found in the IR spectrum. This result is again a consequence of its high molecular weight, and such

Figure 2. SEC chromatograms in DMF of the α-TEMP-PCL prepolymer (solid line) and the PCL macroCTA RAFT agent (dashed line).

3.2. Synthesis of PCL-CTA Macromolecular Chain Transfer RAFT Agent (PCL MacroCTA RAFT Agent) ((2) Scheme 1). The PCL-CTA macromolecular chain transfer RAFT agent (PCL macroCTA RAFT agent) was prepared as a macroinitiator via the DCC method. The resulting α-TEMPPCL (Mn(NMR) ∼ 5300 g mol−1) was end-capped with 4-cyano4-(thiobenzoylthio)pentanoic acid (CTA) via reaction of E


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corresponding to methylene protons from the pendant group (−C(O)−NH−CH2−) at δ = 2.88 ppm, marked as (s), is overlapped by the signal corresponding to residual DMF. The spectrum also shows a characteristic signal for methylene protons from the main polymer chain at δ = 1.70 ppm, labeled (p) ((CH3)C−CH2−), and signals in the range δ = 0.50−1.20 ppm (o and u) typical of methyl groups from the PHPMA backbone ((CH3)C−CH2−) and the pendant group (CH3− CH(OH)), respectively. Moreover, a broad singlet signal (r) was also detected at 7.18 ppm (−C(O)−NH−) for the amide group in the composition of PHPMA. Unfortunately, the spectrum of the AB diblock copolymer did not allow for clear identification of the signals labeled (b, c, i, j, and k), as they are hidden by signals from the main polymer backbone of PCL and PHPMA (a, l, m, and n) or cannot be detected, due to the high molecular weight of the diblock copolymer. The numberaverage molecular weight Mn(NMR) of the PCL-b-PHPMA diblock copolymers should correspond to eq 6.

small functional end-groups cannot be detected (FTIR spectrum is not shown). The SEC chromatogram from the corresponding PCL macroCTA RAFT agent obtained after the cycloaddition reaction shows a monomodal distribution, as indicated by the overlap of the SEC traces (dashed line, in Figure 2). Nevertheless, the SEC trace clearly demonstrates the absence of any duplicated products of higher Mn values. The analysis shows similar values for Mn with a shift toward higher Mn values for the PCL macroCTA RAFT agent and slight increases in polydispersity index compared to the starting α-TEMP-PCL prepolymer (2, in Table 1). 3.3. Synthesis of Amphiphilic PCL-b-PHPMA Diblock Copolymer ((3) Scheme 1). In recent years, RAFT polymerization has attracted much attention as a powerful tool for synthesizing polymers with well-defined structures. Indeed, RAFT polymerization is controlled/living free radical polymerization and is the most versatile and effective due to its compatibility with a very wide range of monomers under relatively simple conditions.39,40 PCL-b-PHPMA diblock copolymers were successfully synthesized via RAFT polymerization using a PCL macroCTA agent to mediate the RAFT polymerization of HPMA in the presence of AIBN as an initiator at 70 °C (compound 3, in Scheme 1). The polymerization of HPMA was performed with three different ratios of HPMA to PCL MacroCTA RAFT Agent to control the length of the PHPMA blocks, including 70:1, 105:1, and 140:1. The ratio of the PCL MacroCTA RAFT Agent to AIBN remained 1:0.2 for all cases. The structures, compositions, and molecular weights of the obtained PCL-b-PHPMA diblock copolymers, were characterized via 1H NMR, FTIR spectroscopy, and SEC analyses. The 1H NMR spectrum of the PCL-bPHPMA diblock copolymer (Figure 4) shows the characteristic

M n(NMR)(PCL‐b‐PHPMA) = [(Ih /2)/(It)] × DPPCL × 143 + M n(NMR)(PCL macroCTA RAFT agent)

(6)

where Ih and It are the integral values of the peaks at δ = 4.06 ppm (h, −CH2−OC(O)− of the PCL repeating unit) and at δ = 3.66 ppm (t, −CH−(OH) of the PHPMA repeating unit), respectively. The molecular weight of the HPMA unit is approximately 143, DPPCL is the degree of polymerization of the PCL macroCTA RAFT agent, and Mn(NMR) is the numberaverage molecular weight of the PCL macroCTA RAFT agent. The experimental degree of HPMA polymerization agreed well with the calculated theoretical values (Table 2). Table 2. Macromolecular Characteristics of the PCL-bPHPMA Diblock Copolymers no. 1 2 3

sample PCL61-bPHPMA70 PCL61-bPHPMA105 PCL61-bPHPMA140

Mna (theor)

Mnb (1H NMR)

Mnc (SEC)

Mw/Mnd (SEC)

17000

12250

13615

1.21

22000

28377

28200

1.25

27000

31860

32690

1.35

Mn = [M]0/[I]0 × 143 + Mn(PCL macroCTA RAFT agent). bMn was calculated via 1H NMR spectroscopy according to eq 6. cMn value relative to PS standards. dMw/Mn value relative to PS standards.

a

The structural analysis of the obtained PCL-b-PHPMA diblock copolymer was also confirmed via FTIR spectroscopy (Figure S1c). The following absorption bands characteristic of both the components (PCL and PHPMA) were observed: 1725 cm−1 for the ester bond (CO stretching) from the ε-CL repeated units in the PCL block; 1640 cm−1 for the amide bond (CO stretching) of the HPMA repeated units of the PHPMA backbone; 1530 cm−1 for N−H stretching from PHPMA; and 3950 and 2862 cm−1 for the C−H vibrations typical for both monomer units. Moreover, a broad peak was detected at 3350 cm−1 (CO−NH stretching mode), indicating the presence of the secondary amide group from the composition of PHPMA block. The molecular weight and polydispersity index of synthesized PCL-b-PHPMA diblock copolymers were determined via

Figure 4. 1H NMR spectrum of the PCL-b-PHPMA diblock copolymer (2, in Table 2) in DMSO-d6.

signals for the protons belonging to ε-CL and HPMA repeated units. The methylene protons of ε-CL repeating units were observed at 4.06 ppm (h) −CH2−OC(O)−, 2.29 ppm (d) −C(O)CH2−, 1.58 ppm (e + g) −C(O)−CH2−CH2−CH2− CH2−, and 1.34 ppm (f) −C(O)−CH2−CH2−CH2−. The spectrum also shows a broad singlet signal observed at 4.70 ppm (v) that corresponds to the chemical shifts of protons in the −OH group from the HPMA repeating units. The broad singlet signal at δ = 3.66 ppm (t) is attributed to the methine proton from the −CH−(OH) group. Furthermore, the signal F


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Macromolecules

spectra of PCL-b-PHPMA diblock copolymers measured at 37 °C are shown in Figure 6. A characteristic three-line spectrum of the nitroxyl radical was observed for all PCL-b-PHPMA copolymers in DMF and PBS solutions, confirming the presence of TEMPO radicals after oxidation. The signal arises due to anisotropic hyperfine interactions between the unpaired electron and nitrogen nucleus.31 The narrow EPR lines observed for the samples dissolved in DMF (Figure 6a) are characteristic for very fast motions of the nitroxyl radical. However, the EPR spectra observed after micellization of the copolymers (Figure 6b) were considerably broader. This broadening of the EPR signal reflects the slower dynamics of the nitroxyl radicals after micellization. The radical mobility depends on the flexibility of the chain that connects it to the backbone and on the tumbling of the entire macromolecule. This mobility can be quantified by the rotational correlation time, τR, which corresponds to the typical time during which a radical maintains its spatial orientation. To extract rotational correlation times, we simulated and compared the EPR spectra obtained at 37 °C. The spectra were simulated using the spectral fitting program NLSL, which is based on the stochastic Liouville equation and utilizes the modified Levenberg−Marquardt minimization algorithm to calculate the best fit with experimental spectra.42 The spin-label motion was assumed to follow the Brownian diffusion model with an axially symmetric rotational diffusion tensor. The components of the g and A tensors were determined by analyzing the rigid limit spectra. All spectra were simulated with a single spectral component. The fits were obtained by varying the parallel and perpendicular rotational diffusion coefficients (Rprp, Rpll), the diffusion tilt angle (βD) and the inhomogeneous line width tensor (W1). The quality of the fit was determined according to the correlation coefficient r, which varied from 0.995 to 0.999. The parameters used for the EPR spectral fitting and calculated rotational correlation times are given in Table 3. Rotational correlation times (τR) were calculated according to eq 742

SEC (Figure 5). The analysis clearly shows that the obtained curves are monomodal and symmetric with a relatively narrow

Figure 5. SEC chromatograms in DMF of the PCL-b-PHPMA diblock copolymers () (1, in Table 2), (- - -) (2, in Table 2), and (···) (3, in Table 2).

molecular weight distribution. The main molecular characteristics of the AB diblock copolymers are listed in Table 2. The data reported in the table suggest a well-controlled copolymerization. 3.4. Oxidation of the PCL-b-PHPMA Diblock Copolymer ((4) Scheme 1). Chemical oxidation of the 2,2,6,6tetramethylpiperidine end-group covalently conjugated to the PCL block in the resulting PCL-b-PHPMA diblock copolymers afforded the corresponding stable TEMPO radicals. The oxidation was performed using CH2Cl2 as a solvent in the presence of m-CPBA at room temperature. The molecular weight and polydispersity index of the obtained PCL-bPHPMA diblock copolymers containing stable nitroxyl radicals were evaluated via SEC. The analysis clearly showed that after chemical oxidation of the AB diblock copolymers the profiles of SEC curves remained the same. Monomodal distributions were observed in all cases with slight increases values for Mn and Mw/Mn (see Supporting Information, Figure S2). 3.5. EPR Spectroscopic Studies. Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for studying the motion of nitroxyl radicals covalently attached to a molecule of interest. It has been successfully applied to study the dynamics of various polymer systems, proteins and lipids.30,41 In this work, EPR spectroscopy was applied to verify successful oxidation of a series of synthesized PCL-bPHPMA diblock copolymers and to investigate the dynamics of the polymer chains before and after micellization. The EPR

1 6 3 R prp2R pll

(7)

The simulated EPR spectra of the PCL-b-PHPMA diblock copolymers before and after micellization are shown as dotted lines in Figure 6. The calculated rotational correlation times

Figure 6. EPR spectra of nitroxyl radical-containing PCL-b-PHPMA diblock copolymers (a) before micellization (in DMF) and (b) after micellization (in PBS solution) at 37 °C. Solid lines represent experimental spectra, and dotted lines represent simulated spectra. G


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Macromolecules Table 3. Parameters Used for the EPR Spectra Simulations and the Calculated Rotational Correlation Timesa before micellization

after micellization

parameter

PCL61-b-PHPMA70

PCL61-b-PHPMA105

PCL61-b-PHPMA140

PCL61-b-PHPMA70

PCL61-b-PHPMA105

PCL61-b-PHPMA140

gxx gyy gzz Axx Ayy Azz W1 βD Rprp/s−1 Rpll/s−1 τR/s

2.008784 2.005900 2.002330 7.72 3.95 35.34 1.510 10.29 3.40 × 109 2.21 × 109 5.7 × 10−11

2.008784 2.005900 2.002330 7.72 3.95 35.34 1.553 32.97 3.82 × 109 2.61 × 109 5.0 × 10−11

2.008784 2.005900 2.002330 7.72 3.95 35.34 1.563 86.89 6.88 × 109 2.58 × 109 3.4 × 10−11

2.008784 2.005900 2.002330 8.23 3.95 35.71 2.002 65.61 1.59 × 107 7.83 × 108 2.9 × 10−9

2.008784 2.005900 2.002330 8.23 3.95 35.71 1.453 65.23 1.72 × 107 6.31 × 108 2.9 × 10−9

2.008784 2.005900 2.002330 8.23 3.95 35.71 2.202 63.75 1.11 × 107 6.59 × 108 3.8 × 10−9

a

gxx, gyy, and gzz are the Cartesian components of the g tensor for the electronic Zeeman interaction, Axx, Ayy, and Azz are the Cartesian components of the electron/nuclear hyperfine tensor in gauss, W1 represents the isotropic spherical component of the inhomogeneous line-broadening tensor, Rprp and Rpll are the axial components of the rotational diffusion tensor in s−1, and βD is the diffusion tilt angle in degrees.

Figure 7. Distribution functions over RH values for different copolymers in PBS at T = 37 °C (a). Concentration dependence on the apparent diffusion coefficient for different copolymers in PBS at T = 37 °C (b).

that the RNPs and their aggregates were spherical (Figure 7a), and no distinct micellar structures were observed by DLS. By extrapolating to infinite dilutions, true diffusion coefficients were determined (Figure 7b). The calculated true RH values are presented in Table 4.

(Table 3) for the copolymers after self-assembly in PBS are approximately 2 orders of magnitude higher than the values obtained for the samples dissolved in DMF. The higher τR values clearly indicate slower dynamics of the nitroxyl radicals after micellization which confirms that the radicals are localized in the dense hydrophobic core where their motions are restricted compared to the almost free motion of the radicals at the polymer chain ends in an organic solvent. 3.6. DLS/SLS Studies of PCL-b-PHPMA RNPs. Radicalcontaining NPs were prepared from the PCL-b-PHPMA diblock copolymers, and their behaviors under PBS solution were evaluated via DLS and SLS techniques. Likewise, to obtain insight regarding possible nanoparticle shapes, a more detailed study of the nanoparticle solutions was performed. Based on multiangle DLS experiments, intensity-weighted distribution functions of apparent hydrodynamic radii (RH) and relaxation times (τ) were obtained. Figure 7a shows the distribution of RH for all the AB diblock copolymer micelles after the dialysis process and dilution with PBS, as measured via DLS. The distribution of RH for all diblock copolymers appeared as a single distribution of RH relative to the presence of the polymer NPs in PBS solution (Figure 7a). The dependence of the relaxation rate (Γ ) on the square of the wave vector (q2) was of diffusive nature, which allowed an apparent value of the diffusion coefficient (D) and an apparent value of the hydrodynamic radius RH to be calculated. The linear relationship observed between Γ and q2 for the particles demonstrated

Table 4. Light Scattering Data of Nanoparticles Composed of PCL-b-PHPMA Diblock Copolymers in PBS at T = 37 °C RG (nm)b A2b (10−7 mol cm3 g−2) (SLS)

no.

sample

Nagga

RH (nm) (DLS)

1

PCL61-bPHPMA70 PCL61-bPHPMA105 PCL61-bPHPMA140

25

21.9

19.5

−0.9

0.89

16

49.9

47.6

−8.0

0.95

70

63.6

73.3

−1.1

1.15

2 3

RG/RH

a

The aggregation number (Nagg) was calculated according to Nagg = Mw(NP)/Mw(unimer). bAll SLS data were obtained from the Zimm formalism (Figures S4−S6).

The sizes and aggregation numbers of the RNPs were directly dependent on the ratio between the PCL and PHPMA blocks (Table 4). For higher PCL/PHPMA ratios, smaller nanoparticles formed in solution. Furthermore, the second virial coefficient corresponded to negative values, which is a consequence of water being used as the thermodynamic solvent, which is not a good solvent for this type of copolymer. H


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Macromolecules

of ε-CL using 4-hydroxy-TEMP as an initiator in the presence of tin octoate. Next, a PCL macroCTA RAFT agent was synthesized via the DCC method using 4-cyano-4(thiobenzoylthio)pentanoic acid. The resulting PCL macroCTA RAFT agent was applied to the sequential RAFT polymerization of HPMA. Finally, oxidation of tetramethylpiperidine groups in the copolymers successfully afforded the corresponding TEMPO-containing block copolymers. This polymerization technique provided a facile way to synthesize PCL-b-PHPMA diblock copolymers with a narrow molecular weight distribution. Because all the blocks were prepared by living/controlled polymerization techniques, the length of each block can be easily tuned. Diblock copolymers with different molecular weights of the PHPMA block were synthesized, indicating the good control over the molecular characteristics of each product. The presence of TEMPO radical was confirmed via EPR spectroscopy. Furthermore, the EPR was used to study the dynamics of NPs under buffer-simulated physiological conditions. Applying various physical techniques, such as 1H NMR spectroscopy, DLS, SLS, and cryo-TEM, we found that the PCL-b-PHPMA diblock copolymers self-assembled into well-defined spherical RNPs and that the characteristics of the nanoparticles were closely related to the hydrophobic/hydrophilic balance resulting from different PHPMA block lengths. These materials open the possibility of introducing their selfaggregates to various biomedical applications such as in drug delivery and the treatment of oxidative stress injuries.

The calculated aggregation numbers agreed with the increases in gyration and hydrodynamic radii as a function of the PCL/ PHPMA ratio. Additional information can be obtained from the structure factor ρ = RG/RH, proposed by Burchard,43 which is used to assess the architecture of scattering objects based only on DLS/ SLS methods. A ρ value near 1 has been reported for glycogen44 and other compact nanoparticles with hyperbranched or similar structures. The structure factor value suggest that the nanoparticles can be described having a loose architecture. Further evidence that amphiphilic AB diblock copolymer self-assembled into RNPs was obtained from 1H NMR spectroscopy in D2O. Figure S3 shows the high-resolution 1H NMR spectrum of a D2O solution (c = 1.0 mg/mL) of the PCL61-b-PHPMA105 RNPs. Comparing the spectrum recorded in a solvent suitable for all blocks (DMSO-d6) and the spectrum recorded in D2O solution proves RNP formation. For example, the 1H NMR spectrum (see Figure 4) shows that the characteristic signals of the PCL and PHPMA segments are readily apparent, whereas the 1H NMR spectrum in D2O exhibits weak and broad signals of hydrophobic PCL blocks (d, e, f, g, h) and strong signals of hydrophilic PHPMA blocks (o, p, s, t, u), which indicates a core−shell formation in the D2O system (Figure S3). The small, broad signals in the NMR spectrum indicate restricted motions of these protons within the cores of the nanoparticles. These results demonstrate that the PCL protons are strongly restricted in mobility in the slightly hydrated solid-like core of NPs, whereas PHPMA creates a liquid-state shell.45 Cryo-TEM (Figure 8) was also performed to determine the morphologies and sizes of the RNPs obtained from the AB

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b01187. Figures S1−S6 (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*(S.P.) E-mail [email protected]. *(S.K.F.) E-mail fi[email protected]. Notes

The authors declare no competing financial interest.

■

Figure 8. Cryo-TEM micrographs of PCL61-b-PHPMA70 RNPs (A) and PCL61-b-PHPMA105 RNPs (B). Scale bars: 50 nm.

ACKNOWLEDGMENTS The authors thank Dr. P. Chytil (Biomedical Polymers Department at Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic) for providing the excellent quality material (N-(2-hydroxypropyl)methacrylamide). The authors also express their gratitude to Dr. A. Jäger (Supramolecular Polymer Systems Department at the Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic) for priceless advice on obtaining RNPs in PBS. We acknowledge financial support from the Ministry of Education, Youth and Sports of the Czech Republic, grant no. LH15213. L.K. and S.K. acknowledge the support of the Czech Science Foundation grant P302/12/G157 and the Prvouk/1LF/1 and UNCE204022 grants from Charles University in Prague.

diblock copolymers. Figure 8 shows TEM images of RNPs prepared from a PBS solution of PCL-b-PHPMA diblock copolymers. Well-defined spherical RNPs formed for all AB diblock copolymers. For the PCL61-b-PHPMA70 RNP sample a mean particle size of 20−45 nm was observed (Figure 8A), whereas the PCL61-b-PHPMA105 sample exhibited RNPs with an average size of approximately 30−40 nm (Figure 8B). In addition, the mean diameter of the RNPs determined based on the cryo-TEM images is in agreement with that determined by DLS.

4. CONCLUSIONS We developed micellar structures based on amphiphilic PCL-bPHPMA diblock copolymers containing stable nitroxyl radicals. A series of well-defined biodegradable and biocompatible diblock copolymers were successfully synthesized by combining ROP, the DCC method, RAFT polymerization, and chemical oxidation. A α-TEMP-PCL prepolymer was designed via ROP

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