Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
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Linear Amphiphilic Polyglycidol/Poly(ε-caprolactone) Block Copolymers Prepared via “Click” Chemistry-Based Concept Natalia Toncheva-Moncheva,† Pavel Bakardzhiev,† Stanislav Rangelov,† Barbara Trzebicka,‡ Aleksander Forys,‡ and Petar D. Petrov*,† †
Institute of Polymers, Bulgarian Academy of Sciences, Akad. G. Bonchev St. 103A, 1113 Sofia, Bulgaria Centre of Polymer and Carbon Materials, Polish Academy of Sciences, M. Curie-Sklodowskiej 34, 41-819 Zabrze, Poland
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‡
S Supporting Information *
ABSTRACT: We introduce a “click” chemistry-based concept for the preparation of amphiphilic block copolymers comprising linear polyglycidol (PG). Herein, a new class of linear polyglycidol/poly(ε-caprolactone) (PG/PCL) block copolymers of precisely designed macromolecular characteristics and composition was synthesized to demonstrate the convenience of this strategy. Monohydroxyl poly(ethoxyethylglycidyl ether) (PEEGE) precursors were synthesized by ring-opening anionic polymerization of ethoxyethylglycidyl ether (EEGE) and subsequently functionalized with a “clickable” alkyne end group. In parallel, a bifunctional PCL diol was modified to an azide-terminated macroreagent. PG/PCL block copolymers were obtained by “click” coupling reaction of the alkyn- and azide-functional macroreagents in the presence of a CuBr/PMDETA catalytic complex and subsequent cleavage of the protective ethoxyethyl groups of PEEGE. The amphiphilic block copolymers were not directly soluble in water, and defined nano-sized micelles were obtained via the solvent evaporation method. The synthesis pathway described here can be extended toward synthesis of various functional block copolymers comprising linear PG.
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INTRODUCTION elf-assembly of amphiphilic copolymers in solutions, at interfaces, and in bulk has been widely exploited for generating nanoscale structures of different shapes.1 The nanoscale features determine many characteristics of these systems relevant to their practical applications in cosmetics, the food industry, agrochemistry, uptake and pre-concentration of toxic organic compounds and heavy metal ions in water treatment, molecular templates for nanoelectronic devices, catalysis, and so on.2 Self-assembled structures of amphiphilic copolymers have also attracted increasing interest as carriers of biologically active substances (low-molecular-weight drugs, enzymes, DNA, and RNA), antifouling surfaces, biosensors, and so on. The requirements for biomedical applications favor nanostructures based on assembled amphiphilic macromolecules comprising functional blocks of biological origin and/or biocompatible synthetic polymers. In this respect, the most preferred systems are those composed of polymers approved by the Food and Drug Administration (FDA) for biomedical applications in humans.3
Recent advances in polymer chemistry have enabled the synthesis of amphiphilic macromolecules with controlled length, topology, and well-defined positions of functional groups along the chain. This offers many possibilities for fabricating desired nanoscale structures with tailored properties by design of novel copolymer molecules. For example, the pioneering works devoted to micellar carriers for nanomedicine have been focused mainly on the formation of core−shell micelles, loading with active substances, and in vitro release studies, while nowadays, more complex functional systems combining a variety of properties and allowing for the simultaneous performance of multiple functions in in vivo conditions are being investigated.4 It is not unexpected that the spectrum of micellar characteristics such as size, loading capacity, elimination time, cell penetration ability, and drug release profile has been remarkably improved or optimized.
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© XXXX American Chemical Society
Received: February 20, 2019 Revised: April 12, 2019
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DOI: 10.1021/acs.macromol.9b00366 Macromolecules XXXX, XXX, XXX−XXX
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ring opening polymerization of EEGE initiated by a polyester (e.g., PCL) macroinitiator in strong alkaline media. Besides several works describing preparation of non-linear architectures comprising PG and PCL segments,28−30 as far as we know, this is the first report on the synthesis of linear PG/PCL block copolymers by involving copper-catalyzed cycloaddition reaction of a monoalkyne-functionalized PEEGE and a bifunctional PCL bearing azide end groups. Further, the aqueous solution properties of these copolymers with a focus on the formation of nano-sized micellar aggregates are described in detail.
Among the variety of copolymer architectures studied for preparing micellar nanocarriers, the systems based on linear dior triblock copolymers dominate over others known from the scientific literature.5 With respect to polymer nature, three main polymer classes, poly(alkylene oxide)s, poly(L-amino acids), and poly(ester)s, can be emphasized as the most preferred building blocks.6 Polyesters such as poly(lactic acid), poly(lactic-coglycolic acid), and poly(ε-caprolactone) are hydrophobic, biodegradable, and bioresorbable and therefore are exploited as core-forming polymers.7−9 In addition, the crystallization of PCL for example leads to the formation of kinetically frozen micelles with increased stability upon dilution below the critical micelle concentration (CMC), which is beneficial for increased penetration in tumor cells.10 The size of such frozen structures strongly depends on the preparation protocol since the intermicellar copolymer exchange rates are extremely low.11,12 Poly(ethylene oxide) (PEO) due to its outstanding physicochemical and biological properties including hydrophilicity, low toxicity and immunogenicity, and high resistance to protein adsorption and cell adhesion stands out as the most frequently used corona-forming polymer.13 Actually, the majority of block copolymer micelles under different phases of clinical research comprise PEO segments.5 Other hydrophilic polymers used in micellar compositions are polyvinylpyrrolidone,14 poly(vinyl alcohol),15 chitosan,16 and poly(2-ethyl-2-oxazoline).17 Polyglycidol (also known as polyglycerol) is a hydrophilic biocompatible polyether that is structurally similar to PEO.18 Both polymers possess identical backbones; however, the presence of a pendant hydroxymethylene group at every repeating unit of PG implies unique physicochemical properties of the molecules themselves as well as of the nanoparticles on their basis. These functional groups offer the opportunity to attach covalently different small molecules (monoclonal antibodies, hydrophilic drugs, etc.) to PG chains, which enriches the therapeutic profile of the micellar carrier and expands the variety of possible biomedical and pharmaceutical applications.19 Generally, linear PG can be obtained via ring-opening anionic polymerization of glycidyl ethers (protected glycidols) and subsequent cleavage of the protective groups.20−23 The preparation of PG-based linear amphiphilic AB, ABA, and ABC block co(ter)polymers and other architectures usually involves polymerization of protected glycidol from a macroinitiator. Thus, polystyrene-b-polyglycidol diblock copolymers have been synthesized by Siebert et al. via sequential anionic polymerization of styrene initiated with sec-butyl lithium followed by polymerization of EEGE in the presence of a phosphazene base and deprotection of PEEGE blocks by acidic hydrolisis.24 Halacheva et al. synthesized various ABA polyglycidol-b-poly(propylene oxide)-b-polyglycidol triblock copolymers, using a CsOH deprotonated bishydroxy functionalized poly(propylene oxide) macroinitiator.25 Dimitrov et al. reported the synthesis of polyglycidol-b-poly(ethylene oxide)-bpoly(DL-lactide) terpolymers via multiple anionic polymerizations.26 In the last stage, the anionic polymerization of lactide was initiated with calcium amide instead of the alcoxide one due to the pronounced instability of polyesters in strong base media.27 The present study introduces a new strategy for the synthesis of linear amphiphilic triblock copolymers comprising PG segments by using the “click” chemistry method. The benefit of this strategy is the relatively easy protocol for preparation of copolymers with a pre-defined composition and macromolecular characteristics by a reaction pathway bypassing the
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EXPERIMENTAL SECTION
Materials. Glycidol (2,3-epoxypropanol, 96%, Aldrich), ethyl vinyl ether (99%, Aldrich), p-toluenesulfonic acid (ACS reagent, ≥98.5%, Sigma-Aldrich), tert-butanol (anhydrous ≥99.5%, Sigma-Aldrich), 2phenyl ethyl alcohol (≥99%, Aldrich), 4-pentynoic acid (95%, Acros Organics), 4-dimethylaminopyridine (DMAP, ReagentPlus, ≥99%, Sigma-Aldrich), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, commercial grade, powder, Sigma-Aldrich), triethylamine (TEA, ≥99.5%, Aldrich), methanesulfonyl chloride (≥99.7%, Aldrich), sodium azide (ReagentPlus, ≥99.5%, SigmaAldrich), sodium sulfate (≥99.99% trace metals basis, anhydrous, Sigma-Aldrich), AlCl3·6H2O (99%, Sigma-Aldrich), sodium hydrogen carbonate (anhydrous, ≥99.7%, Sigma-Aldrich), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%, Sigma-Aldrich), and copper(I) bromide (99.999% trace metals basis, Sigma-Aldrich) were used as received. Methylene chloride (>99.98%, Fisher Scientific), toluene (>99.8%, Fisher Scientific), and tetrahydrofuran (>99.5% Fisher Scientific) were dried with calcium hydride and freshly distilled before use. Dimethylformamide (>99.99%, Fisher Scientific) was dried using a molecular sieve. Deionized water was obtained using a Millipore MilliQ system and additionally filtered through a 220 nm PTFE filter and a 20 nm cellulose filter. Poly(ε-caprolactone) diol (OH-PCL35OH, CAPA 2402, molar mass of 4000 g mol−1, >99%, Cas. No 31831− 53-5, Perstorb) was dried by azeotropic distillation with toluene before use. Propylene oxide (PO, 99%, Sigma-Aldrich) was dried over CaH2, distilled, and stored over a 4 Å molecular sieve before use. Instrumentation. 1H-NMR measurements were conducted on a Bruker Avance II spectrometer operating at 600 MHz using CDCl3, DMSO-d6, or CD3OD at 25 °C. GPC analyses were performed on a Shimadzu Nexera HPLC chromatograph equipped with a degasser, a pump, an autosampler, an RI detector, and three columns: 10 μm PL gel mixed-B and 5 μm PL gel 500 Å and 50 Å. THF was used as the eluent at a flow rate of 1.0 mL min−1 and temperature of 40 °C. The sample concentration was 1 g L−1, and GPC was calibrated with polystyrene standards. FTIR spectra were measured with an attenuated total reflection (ATR) spectrophotometer (IRAffinity-1, Shimadzu, Japan) in the 450−4500 cm−1 range at a resolution of 1 cm−1. Spectrophotometric Determination of CMC. A micellar stock solution was diluted to 10 different concentrations (2 mL). Then, 20 μL of a 0.4 mM solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) in methanol was added to each sample. The samples were incubated in the dark for 24 h at 37 °C. UV−vis absorption spectra of DPH in the wavelength interval λ = 300−500 nm at 37 °C were recorded on a Beckman Coulter DU 800 UV−vis spectrometer. The intensities of the main absorption peak at 356 nm were plotted versus the polymer concentration. CMC values were determined as the inflection point in the absorbance intensity versus concentration curve. Static and Dynamic Light Scattering. Multiangle light scattering measurements were carried out on a Brookhaven BI-200 goniometer with vertically polarized incident light at a wavelength λ = 632.8 nm supplied by a He−Ne laser operating at 35 mW and equipped with a Brookhaven BI-9000 AT digital autocorrelator. The scattered light intensity was measured for dilute aqueous dispersions in the concentration range 0.15−1.0 mg mL−1 at 37 °C. Measurements were made at angles θ in the range of 50−130°. The autocorrelation functions were analyzed using the constrained regularized algorithm CONTIN to obtain the distributions of the relaxation rates (Γ).31 The B
DOI: 10.1021/acs.macromol.9b00366 Macromolecules XXXX, XXX, XXX−XXX
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portion of EEGE (2.0 g, 13.75 mmol) was added to the (CH3)3C− O−Cs+ initiator and the polymerization was carried out in bulk at 65 °C. Three additional portions of monomer (2.0 g) were added within time intervals of 48 h. After 8 days, the reaction was terminated with methanol, and the product was dialyzed against a methanol/water mixture (10:1 v/v; membrane, MWCO 1 kDa) for 24 h. Yield: 5.565 g (70%); 1H-NMR (CDCl3, δ ppm): 1.15−1.3 ppm (m, −CH(CH̲ 3)− O−CH2−CH̲ 3) side chain and (CH̲ 3)3−C−O− end group); 3.25− 4.05 ppm (m, −O−CH̲ 2−CH̲ (CH̲ 2−O)−O− main chain and −CH(CH 3 )−O−CH̲ 2 −CH 3 side chain); 4.68−4.76 ppm (t, = 6300 g mol−1, Mw/Mn = 1.2, CH̲ (CH3)−O−CH2−CH3); MSEC n NMR −1 Mn = 6600 g mol . Ph−PEEGE35-b-(PO)4−OH. The Cs-based initiator was prepared by the abovementioned procedure. In this case, CsOH·H2O (0.21 g, 1 mmol) was reacted with dry 2-phenyl ethyl alcohol (0.15 g, 1.2 mmol) dissolved in dry THF (1 mL). The polymerization of EEGE (10.0 g, 69 mmol) initiated by a Ph−O−Cs+ initiator was conducted at 65 °C for 12 days. EEGE was added at portions of 2 g within time intervals of 48 h. Then, the temperature was adjusted at 50 °C, and propylene oxide (0.346 g, 6 mmol) was added to the reaction medium via a Hamilton syringe. After additional 8 h, the reaction was terminated with methanol, and the product was dialyzed against a methanol/water mixture (10:1 v/v; membrane, MWCO 1 kDa) for 24 h. Yield: 9 g (0.75%); 1H-NMR (CDCl3, δ ppm), PEEGE: 1.15−1.3 ppm (m, −CH(CH̲ 3)−O−CH2−CH̲ 3) side chain); 3.25−4.05 ppm (m, −O− CH̲ 2−CH̲ (CH̲ 2−O)−O− main chain and −CH(CH3)−O−CH̲ 2− CH3 side chain); 4.68−4.76 ppm (t, CH(C ̲ H3)−O−CH2−CH3 side chain); 7.25 ppm (m, CH̲ 2 phenyl end group); PPO: 1.15−1.3 ppm (m, −CH2−CH(CH̲ 3)−O−); 3.25−4.05 ppm (m, −CH̲ 2−CH(C ̲ H3)− = 5300 g mol−1, MGPC = 3700 g mol−1; Mw/Mn = 1.15. O−); MNMR n n Monoalkyne-Terminated Poly(ethoxyethyl glycidyl ether) (t-Bu PEEGE45CCH). In a typical example, t-Bu−PEEGE−OH (1.9081 g, 0.3003 mmol) and 4-pentynoic acid (0.2356 g, 2.4026 mmol, 8 equiv) were dried by azeotropic distillation with toluene. The dry polymer, 4-dimethylaminopyridine (0.0727 g, 0.06006 mmol, 2 equiv), and (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.23027 g, 1.2012 mmol, 4 equiv) were dissolved in 5 mL of dry chloroform in a 50 mL round-bottom flask. The solution was purged with argon for 30 min, and 4-pentynoic acid dissolved in 1 mL of dry chloroform was added dropwise. The reaction was carried out under argon at 40 °C for 120 h in the dark. The reaction mixture was filtered (0.45 μm Teflon filter) and dialyzed against a methanol/water mixture (10:1 v/v; membrane, MWCO 1000 Da) for 24 h in the dark. Then, methanol was evaporated by a rotary vacuum evaporator, and then tBuPEEGECCH was collected by freeze-drying. Yield: 1.55 g (82%); degree of conversion: 86%. PhPEEGE35-b-(PO)4CCH. The PhPEEGE35-b-(PO)4 CCH macroreagent was obtained by reacting PhPEEGE35-b(PO)4OH with 4-pentynoic acid (8 equiv) at the reaction conditions described for t-BuPEEGE45CCH. Degree of conversion: 99%. Azide-Terminated Poly(ε-caprolactone) (N3−PCL35−N3). OH− PCL35−OH (5 g, 1.25 mmol, 1 equiv) was dissolved in dry CH2Cl2 (80 mL) in a 200 mL round-bottom flask under an inert atmosphere. Then, DMAP (0.152 g, 1.25 mmol, 0.5 equiv) and TEA (2.53 g, 25 mmol, 10 equiv) were added to the reaction mixture. After stirring for 5 min at 0 °C, methanesulfonyl chloride (2.8637 g, 25 mmol, 10 equiv) dissolved in 20 mL dry CH2Cl2 was added, and the reaction mixture was stirred overnight at 25 °C. The product was added to water, forming a turbid solution, and the polymer was extracted several times with CH2Cl2. The organic solution was dried using anhydrous Na2SO4 and filtered. The mesylated PCL was recovered after evaporation of CH2Cl2, precipitation in cold CH3OH (− 35 °C), and drying for 24 h in a vacuum oven at 25 °C. Yield = 4.6 g (92%); degree of conversion: 99%; 1H-NMR (CDCl3, δ ppm): 4.23 ppm (t, −CH2−CH̲ 2−O− SO2−), 4.05 (t, −CH̲ 2−O−(CO)-), 2.3 (t, O(CO)−CH̲ 2−), 1.7−1.55 (m, O(CO)−CH2−CH̲ 2−CH2−CH̲ 2−CH2−C(O)), 1.42−1.33 (m, O(CO)−CH2−CH2−CH̲ 2−CH2−CH2−C(O)). MnGPC = 7080 g mol−1, Mw/Mn = 1.32. The mesylated PCL (2.51 g, 0.6275 mmol, 1 equiv) was dissolved in dry N,N-dimethyl formamide (20 mL) under an inert atmosphere in a
latter provided distributions of the apparent diffusion coefficient (D = Γ/q2) where q is the magnitude of the scattering vector given by q = (4πn/λ)sin(θ/2) and n is the refractive index of the medium. The mean hydrodynamic radius was obtained by the Stokes−Einstein equation
R h = kT /(6πηD0)
(1)
where k is the Boltzmann constant, η is the solvent viscosity at temperature T in kelvin, and D0 is the diffusion coefficient at infinite dilution. The static light scattering (SLS) measurements were carried out in the interval of angles from 40 to 140° at 37 °C using the same light scattering set. The SLS data were analyzed using the Zimm or Debye plot software provided by Brookhaven Instruments. Information on the weight-average molar mass, Mw, the radius of gyration, Rg, and the second virial coefficient, A2, was obtained from the dependence of the quantity Kc/Rθ on the concentration (c) and scattering angle (θ). The scattering angle of the Debye plot method was 90°. Here, K is the optical constant given by K = 4π2n02(dn/dc)2/NAλ4 where n0 is the refractive index of the solvent (water), NA is Avogadro’s constant, λ is the laser wavelength, and Rθ is the Rayleigh ratio at θ. dn/dc is the refractive index increment measured in water in separate experiments on an Orange GPC19 DNDC refractometer. The dn/dc values of the investigated copolymers were in the range 0.120−0.122 mL g−1. Electrophoretic Light Scattering. The electrophoretic light scattering measurements were carried out on a 90Plus PALS instrument (Brookhaven Instruments Corporation) equipped with a 35 mW red diode laser (λ = 640 nm) at a scattering angle (θ) of 15°. ζ potentials were calculated from the obtained electrophoretic mobility at 25 °C by using the Smoluchowski equation ζ = 4πηυ/ε
(2)
where η is the solvent viscosity, υ is the electrophoretic mobility, and ε is the dielectric constant of the solvent. Cryogenic Transmission Electron Microscopy. Cryo-TEM micrographs were obtained using a Tecnai F20 X TWIN microscope (FEI Company, USA) equipped with a field emission gun operating at an acceleration voltage of 200 kV. Images were recorded on an Eagle 4K HS camera (FEI Company, USA) and processed with TIA software (FEI Company, USA). Specimen preparation was done by vitrification of the aqueous (HPLC-grade water) solutions on grids with a holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using a Femto plasma cleaner (Diener Electronic, Germany). Cryo-samples were prepared by applying a droplet (3 μL) of the solution to the grid, blotting with filter paper and immediate freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (FEI Company, USA). After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a cryo-TEM holder Gatan 626 (Gatan Inc., USA) and analyzed in the TEM at −178 °C. Pictures were processed using ImageJ software. Synthesis. Ethoxyethyl Glycidyl Ether. EEGE was synthesized following a procedure described elsewhere.32 p-Toluenesulfonic acid (1 g, 5.80 mmol) was added portion-wise to a magnetically stirred solution of 2,3-epoxypropanol (40.0 g, 0.54 mol) in ethyl vinyl ether (200 mL) maintaining the temperature below 40 °C. The reaction mixture was stirred for 3 h, and then saturated aq NaHCO3 (100 mL) was added. The organic layer was separated and dried over anhydrous Na2SO4, and ethyl vinyl ether was evaporated under reduced pressure. EEGE (bp of 152−154 °C) was collected by vacuum distillation at 44 °C and ∼1.103 Pa as a colorless liquid; a fraction with purity exceeding 98.1% (GC) was used. Monohydroxyl-Terminated Poly(ethoxyethyl glycidyl ether) (tBu−PEEGE45−OH). CsOH·H2O (0.167 g, 1 mmol) was weighed in a vial containing 2 mL of dry benzene, and the solution was transferred into a 100 mL two-neck round-bottom flask under an argon atmosphere. Benzene was evaporated under vacuum to obtain a white crystalline layer of CsOH. Next, dry (CH3)3COH (1 g, 13.5 mmol) was added, and the mixture was magnetically stirred at 90 °C for 2 h. The excess (CH3)3COH was removed under vacuum, and the crystalline solid was freeze-dried with 2 mL of dry benzene. Finally, a C
DOI: 10.1021/acs.macromol.9b00366 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. 1H-NMR spectra of t-Bu−PEEGE45−OH and t-BuPEEGE45CCH in CDCl3. 50 mL round-bottom flask, and NaN3 (2.4052 g, 37 mmol, 30 equiv) was added. The reaction was carried out at 25 °C overnight. The N3− PCL35−N3 macroreagent was recovered by the same procedure as described above. Yield = 2.3 g (95%); %. 1H-NMR (CDCl3, δ ppm): 4.05 (t, −CH̲ 2−O−C(O)-), 3.27 ppm (t, −CH2−CH̲ 2−N3), 2.3 (t, −O(CO)−CH̲ 2−), 1.7−1.55 (m, O(CO)−CH2−CH̲ 2−CH2−CH̲ 2− CH2−C(O)), 1.42−1.33 (m, O(CO)−CH2−CH2−CH̲ 2−CH2− = 7090 g mol−1, Mw/Mn = 1.29. CH2−C(O)); MGPC n Poly(ethoxyethyl glycidyl ether)-block-Poly(ε-caprolactone)block-Poly(ethoxyethyl glycidyl ether) (PEEGE 45 -b-PCL 35 -bPEEGE45). N3−PCL35−N3 (0.6031 g, 0.1507 mmol, 1 equiv) and CuBr (0.4303 g, 3 mmol, 10 equiv) were added to a 50 mL roundbottom flask under an argon atmosphere. Dry THF (3 mL) was added via a syringe, and the solution was purged with argon and stirred vigorously for 20 min. Monoalkyne-terminated PEEGE45 (1.9 g, 0.3015 mmol, 3 equiv) was dissolved in dry THF (2 mL) and added to the PCL solution along with PMDETA (0.7798 g, 4.5 mmol, 15 equiv). The “click” coupling reaction was carried out at 30 °C for 24 h. The reaction mixture was cooled to RT, diluted with THF (30 mL), and filtered through a column filled with neutral alumina to remove copper complexes. The excess THF was evaporated; the crude product was dissolved in methanol (10 mL) and dialyzed against a methanol/water mixture (10:1 v/v, membrane, MWCO 8 kDa) for 72 h. The methanol was removed using a rotary vacuum evaporator, and the copolymer was = 15,300 g recovered by freeze-drying. Yield: 1.42 g (78%); MGPC n mol−1, Mw/Mn = 1.8. PEEGE35-b-(PO)4-b-PCL35-b-(PO)4-b-PEEGE45. PEEGE35-b-(PO)4b-PCL35-b-(PO)4-b-PEEGE45 was obtained by “click” coupling reaction of N3−PCL35−N3 and PhPEEGE35-b-(PO)4CCH following = 16,300 g mol−1, Mw/Mn = 1.3. the procedure described above. MGPC n Cleavage of the Protective Ethoxyethyl Groups. PEEGE blocks were derivatized into PG ones by treatment with AlCl3·6H2O as described elsewhere.33,34 PEEGE45-b-PCL35-b-PEEGE45 (1.2 g, 100 equiv) was dissolved in methanol (8.9 mL, 3200 equiv) at 40 °C, and then AlCl3·6H2O (0.0152 g, 1 equiv) was added under stirring. The hydrolysis was conducted at the same temperature for 48 h until
complete disappearance of the methine proton signal at 4.75 ppm in the 1 H-NMR spectrum. The reaction mixture was filtered through Hyflo Super Cel diatomaceous earth, and then methanol was evaporated under reduced pressure. PEEGE35-b-(PO)4-b-PCL35-b-(PO)4-b-PEEGE45 (2 g, 100 equiv) was dissolved in methanol (15.1 mL, 4000 equiv) at 40 °C, and then AlCl3·6H2O (0.022 g, 1 equiv) was added under stirring. Then, the hydrolysis reaction was done following the procedure described above. Micelle Preparation Protocols. A: The copolymer (5 mg) was dissolved in methanol (5 mL). After that, the organic solution was added dropwise to purified water (5 mL) preheated to 37 °C under vigorous stirring (1000 rpm). The resulting mixture was stirred additionally for 30 min at the same temperature. Finally, the organic solvent was evaporated by a rotary vacuum evaporator at 37 °C, yielding a colorless aqueous micellar solution with a concentration of 1 g L−1. B: The copolymer (5 mg) was dissolved in methanol (2.5 mL). After that, the organic solution was added dropwise to purified water (5 mL) preheated to 60 °C under vigorous stirring (1000 rpm). The resulting mixture was stirred for 2 h at 60 °C. The organic solvent was evaporated by argon purging at 60 °C, yielding a colorless aqueous micellar solution with a concentration of 1 g L−1. C: The copolymer (5 mg) was dissolved in methanol (5 mL). After that, the organic solution was added dropwise to purified water (5 mL) preheated to 60 °C under vigorous stirring (1000 rpm.). The resulting mixture was stirred for 2 h at 60 °C. Finally, the organic solvent was evaporated by argon purging at 60 °C, yielding a colorless aqueous micellar solution with a concentration of 1 g L−1. D: The copolymer (5 mg) was dissolved in methanol (2.5 mL). After that, the organic solution was mixed with purified water (5 mL) at 25 °C. The resulting mixture was stirred for 30 min at the same temperature. Finally, the organic solvent was evaporated by a rotary vacuum evaporator at 25 °C, yielding a colorless aqueous micellar solution with a concentration of 1 g L−1. D
DOI: 10.1021/acs.macromol.9b00366 Macromolecules XXXX, XXX, XXX−XXX
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Figure 2. 1H-NMR spectra of Ph−PEEGE35-b-(PO)4−OH and PhPEEGE35-b-(PO)4CCH in CD3OH.
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pentynoic acid (Scheme S2 in the Supporting Information).36 Since the reaction of hydroxyl-terminated PEEGE with 4pentynoic acid has not been reported yet in the literature, our first experiment was conducted following the experimental conditions established for PEO and PCL.36−39 However, the reaction efficiency (degree of esterification) calculated by 1HNMR was only ∼14% (Figure S2; Table S1, run 1 in the Supporting Information). Therefore, a number of experiments aiming to improve the reaction efficiency by tuning the polymer/4-pentynoic acid ratio, reaction temperature, and/or reaction time were carried out (Table S1 runs 2−6 in the Supporting Information). As a result, a maximum degree of esterification ∼86% was achieved (Figure 1) by conducting the reaction at 40 °C for 120 h using an 8 equiv excess of 4pentynoic acid with respect to OH groups of PEEGE. The esterification reaction was also confirmed by FTIR spectroscopy (Figure S3). The alkyne functionalized PEEGE exhibits three characteristic peaks at around 3255, 1980, and 1738 cm−1 due to the stretching vibration between the terminal hydrogen and the carbon atom of the alkyne moiety, the stretching vibration between two carbon atoms of the triple bond, and the carbonyl stretch of ester groups, respectively. At first glance, the content of alkyne-functionalized PEEGE in the product is relatively high, and it can be reacted with the azide functional macroreagent, giving an appropriate excess. However, in order to answer the question as to why the reaction did not lead to a quantitative conversion, some additional experiments were done. We assumed that the bulky protecting side group in the PEEGE macromolecule may limit the access of acid molecules to the terminal hydroxyl end group. Therefore, at the end of EEGE polymerization, a given amount of propylene oxide was added to the reaction mixture. This allowed us to
RESULTS AND DISCUSSION Synthesis and Characterization of Block Copolymers. Amphiphilic copolymers comprising linear PG and PCL blocks were synthesized by a procedure involving copper-mediated “click” coupling reaction35 of monoalkyne functional PEEGE and azide end-capped bifunctional PCL and subsequent hydrolysis of the protective ethoxyethyl groups of PEEGE. This strategy based on the separate preparation of two “clickable” polymers and their coupling into a linear block copolymer architecture made it possible to achieve combinations of block copolymers inaccessible before. In addition, the coupling of the polymeric macroreagent of a defined degree of polymerization yielded polymers of predetermined molar mass and composition, which is not always possible by the classical approaches of sequential polymerization of different monomers. Indeed, our efforts to obtain well-defined triblock copolymers by ring-opening polymerization of EEGE using a deprotonated bifunctional PCL diol did not lead to a product with the desired macromolecular characteristics presumably due to instability of PCL in strong alkaline media at the stage of anionic polymerization of EEGE. Therefore, the separate preparation of two “clickable” building blocks was considered more appropriate for copolymer synthesis. First, a monofunctional PEEGE bearing a hydroxyl end group (t-Bu−PEEGE45−OH) was prepared by ring-opening anionic polymerization of EEGE in bulk using a (CH3)3C−O−Cs+ initiator (Scheme S1 in the Supporting Information), as described elsewhere.34 1H-NMR and GPC analysis confirmed the formation of well-defined PEEGE with a narrow molar mass distribution (Figure 1 and Figure S1 in the Supporting Information). Next, a monoalkyne functional macroreagent (t-BuPEEGE45CCH) was obtained via esterification of t-BuO−PEEGE45−OH with 4E
DOI: 10.1021/acs.macromol.9b00366 Macromolecules XXXX, XXX, XXX−XXX
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having in mind the limits of the two analytical methods, the reaction efficiency at the reported experimental conditions was significantly improved. The superior reaction efficiency should be attributed to the incorporation of short PO spacers between the reactive OH group and the PEEGE chain. Thus, by extending the PEEGE molecule with a few PO units, the negative effect (steric hindrance) of the bulky protective groups was suspended. The bifunctional N3-PCL-N3 macroreagent was obtained by a two-step procedure involving mesylation of the primary hydroxyl end groups of PCL35-diol and reaction with NaN3 (Scheme S4 in the Supporting Information). The successful functionalization of PCL with mesylate end groups was confirmed by 1H-NMR analysis of the purified reaction product (Figure 4). The signals at 3.64 ppm assigned to the methylene protons next to the hydroxyl end group (−CH2OH) entirely disappeared, and a new signal at 4.23 ppm corresponding to methylene protons adjacent to the mesylate end group appeared. In the second step, the mesylated precursor was derivatized into azide end-functional PCL as evidenced by the 1H-NMR spectra. Here, a new signal at 3.27 ppm assigned to the methylene protons adjacent to the azide end group was detected, while the signal at 4.23 ppm completely disappeared. In addition, the integral ratio of the proton signals from CH2N3 (3.98 H) and the methylene groups of PCL backbone (70 H at 4.69 ppm) was almost equal to the theoretical value. GPC analysis of the modified PCL revealed a monomodal symmetric peak, and the calculated dispersity and number-average molar mass (Mn) were 1.29 and 7090 g mol−1, respectively (Figure 3 and Figure S7). The successful functionalization of PCL with azide end groups was additionally confirmed by FT-IR spectroscopy (Figure S8). As seen from the figure, the typical azide stretching absorption peak at 2100 cm−1 is present in the spectrum of the modified polymer unlike the spectrum of PCL-diol.31 The general scheme of the preparation of block copolymers via copper-catalyzed “click” coupling of azide end-capped
extend the PEEGE block (Scheme S3 in the Supporting Information) with a very short poly(propylene oxide) spacer (degree of polymerization = 4) as confirmed by 1H-NMR analysis (Figure 2 and Figure S4). In addition, a phenyl end group was substituted for the tert-butyl one to facilitate the calculation of homo-PEEGE molar mass from the 1H-NMR spectrum. In fact, a well-defined Ph−PEEGE35-b-(PO)4−OH diblock copolymer of a narrow molar mass distribution (MGPC = n 3700 g mol−1, Mw/Mn = 1.15; see Figure 3 and Figure S5) was
Figure 3. GPC traces of Ph−PEEGE35-b-(PO)4−OH, N3−PCL35−N3, and the resulting PEEGE35-b-(PO)4-b-PCL35-b-(PO)4-b-PEEGE35 copolymer.
prepared and used for further modification. The monoalkyne functional macroreagent (PhPEEGE35-b-(PO)4CCH) was obtained by reacting Ph−PEEGE35-b-(PO)4−OH with 4pentynoic acid (8 equiv) at 40 °C for 120 h. In this case, the 1HNMR and FTIR analysis of the purified copolymer revealed a very high conversion of OH-end groups into alkyne ones (Figure 2 and Figure S6). Based on the peak integral ratio of four methylene (i and l) and one methine (m) protons and five Ph protons (h), the calculated conversion was 99%. In other words,
Figure 4. 1H NMR spectra of the PCL35-diol precursor, mesylate end-capped PCL35, and azide end-capped PCL35 macroreagent in CDCl3. F
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Scheme 1. Schematic Representation of the Synthesis of PEEGE35-b-(PO)4-b-PCL35-b-(PO)4-b-PEEGE35 Copolymer by CopperCatalyzed “Click” Coupling Reaction and Subsequent Hydrolysis Leading to Amphiphilic PG35-b-(PO)4-b-PCL35-b-(PO)4-bPG35 Bock Copolymer
conditions leading to well-defined linear PG/PCL block copolymers (Scheme 1 and Table 1). The conversion of PEEGE blocks into PG ones at the reported experimental conditions was evident form the complete disappearance of the signals assigned to the protective ethoxyethyl group (Figure 5 and Figure S10 in the Supporting Information). In addition, a new signal assigned to OH groups of PG was detected at 4.46 ppm. One should note that this reaction did not alter the PCL block. In summary, the reaction pathway described in the present study allowed the preparation of a new class of amphiphilic copolymers based on linear polyglycidol and poly(ε-caprolactone) segments. This strategy is not limited only to the examples given in the paper and can be exploited for the synthesis of many different copolymers comprising linear polyglycidol blocks. Aqueous Solution Properties. Determination of the Critical Micelle Concentration. The synthesized amphiphilic PG/PCL block copolymers are not directly soluble in water, and therefore the solvent evaporation method was employed to form defined nano-sized aggregates. First, the triblock copolymer was dissolved in methanol (common solvent for PCL and PG), and then the organic solution was added dropwise to purified water at 37 °C under vigorous stirring. Finally, methanol was evaporated at 37 °C, and a colorless aqueous micellar solution with a concentration of 1 g L−1 was obtained. Based on polymer characteristics, the formation of core−shell micelles comprising a biodegradable hydrophobic PCL core and a functional hydrated PG shell is assumed. The CMC was determined by a standard procedure using the hydrophobic dye 1,6-diphenyl1,3,5-hexatriene as a probe.40 It is known that the DPH absorbance in water is minimal, whereas in hydrophobic environments, it increases substantially, and a spectrum with a
bifunctional PCL and monoalkyne functional PEEGE macroreagents is shown in Scheme 1. A proper excess of PhPEEGE-b-(PO)4CCH (or tBuPEEGECCH) was used to ensure high coupling efficiency of the two macroreagents. PEEGE blocks were derivatized into PG ones by treatment with the Lewis acid AlCl3· 6H2O, following the procedure developed by Namboodiri and Varma33 and modified by Dimitrov et al.34 The non-reacted polymer was removed afterward by ultrafiltration. The reaction efficiency at each step was monitored by 1H-NMR and GPC analyses. The first proof for coupling of PEEGE and PCL was provided by the 1H-NMR spectra of the purified product. A signal characteristic for the CH proton of the triazole ring at 7.93 ppm was registered along with the characteristic peaks of PCL and PEEGE (Figure 5). At the same time, the signals assigned to the alkyne protons at 2.01 ppm and the two types of methylene protons adjacent to the alkyne end functionality at 2.51 and 2.59 ppm completely disappeared. The integral ratio of two PEEGE methyne protons (68.7 H at 4.7 ppm) to two PCL methylene protons (70 H at 4.05 ppm) was approximately equal to the theoretical value (70:70), indicating effective coupling reaction. This statement was also confirmed by GPC analysis. Indeed, the GPC trace of the block copolymer was shifted to a lower retention time as compared to the traces of N3−PCL35−N3 and Ph−PEEGE35-b-(PO)4−OH precursors (Figure3; see also Figure S9 in the Supporting Information for PEEGE45-bPCL35-b-PEEGE45). Therefore, taking into account the symmetric monomodal GPC curves and the low dispersity values, one may conclude that well-defined linear block copolymers comprising PEEGE and PCL blocks were synthesized by “click” coupling reaction (Table 1). Finally, the protective ethoxyethyl groups of PEEGE were cleaved at mild G
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Figure 5. 1H NMR spectra of (a) PEEGE35-b-(PO4)-b-PCL35-b-(PO4)-b-PEEGE35 in CDCl3 and (b) PG35-b-(PO4)-b-PCL35-b-(PO4)-b-PG35 in DMSO-d6, respectively.
Light Scattering Experiments. Static and dynamic light scattering experiments were carried out at 37 °C in the concentration range 0.1−1.0 g L−1, which was above the CMCs of the two copolymers. As expected, the preparation protocols (see Experimental Section) strongly affected the light scattering parameters of the aggregates. PG45-b-PCL35-b-PG45 exhibited
characteristic maximum at 356 nm can be recorded. The CMC values were determined from the break of the absorbance intensity plotted against copolymer concentration, as shown in Figure 6. The CMC values of PG45-b-PCL35-b-PG45 and PG35-b(PO)4-b-PCL35-b-(PO)4-b-PG35 were 0.1 and 0.08 g L−1, respectively. H
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Macromolecules Table 1. Composition and Molar Mass Characteristics of the Synthesized Copolymers copolymer composition1H NMR
NMR M1H (g mol−1) n
PEEGE45-b-PCL35-b-PEEGE45 PG45-b-PCL35-b-PG45 PEEGE35-b-(PO)4-b-PCL35-b-(PO)4-b-PEEGE35 PG35-b-(PO)4-b-PCL35-b-(PO)4-b-PG35
a
17,400 11,000b 15,000a 10,000c
d MGPC (g mol−1) n
Mw/Mn
15,300
1.8
16,300
1.3
a
CDCl3. bCD3OD. cDMSO-d6. dUsing THF as eluent, polystyrene standards.
essentially a monomodal relaxation time distribution (proportional to the hydrodynamic radius) from DLS (Figure 7a,b). Additional slow or fast modes of low amplitudes were only occasionally observed at certain angles and practically did not influence the linearity of the angular dependences of the relaxation rates from the slope of which the diffusion coefficients were determined (Figure 7c). The latter were extrapolated to zero concentration to obtain the diffusion coefficient at infinite dilution, D0 (Figure 7d), which was used to calculate the hydrodynamic radius, Rh, by the equation of Stokes−Einstein. The values of Rh of the aggregates obtained by the different preparation protocols together with the zeta potential values are summarized in Table 2. As seen, the aggregates varied considerably in size. Therefore, to determine the static light scattering parametersmolar mass of the aggregates (Magg W ), the second viral coefficient (A2), and radius of gyration (Rg) different methods of data treatment were applied: the smaller ones were evaluated by the Debye plot method at a single angle of 90° (Figure 8a) since angular dependence of the quantity Kc/ Rθ was not observed, whereas the Zimm plot method was used to
Figure 6. Determination of CMC of the PG45-b-PCL35-b-PG45 copolymer using the DPH absorbance at 356 nm in aqueous media at 37 °C.
Figure 7. Relaxation time distributions measured at 90° for aggregates of PG45-b-PCL35-b-PG45 at a concentration of 1.0 g L−1 prepared via (a) protocol A and (b) protocol B. (c) Relaxation rate, Γ, as a function of sin2(θ/2) for PG45-b-PCL35-b-PG45 dispersion at a concentration of 0.5 g L−1 prepared via protocol A. (d) Concentration dependence of diffusion coefficients, D, for PG45-b-PCL35-b-PG45 aggregates prepared via protocol A. All measurements were performed at 37 °C. I
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Table 2. Static, Dynamic, and Electrophoretic Light Scattering Characterization Data for the Aggregates of the Copolymer PG45b-PCL35-b-PG45 Formed by Different Preparation Protocolsa preparation protocol
Rh (nm)
ζ (mV)
−1 10−3 × Magg W (g mol )
Nagg
103 × A2 (mL mol g−2)
Rg (nm)
Rg/Rh
A B
27.3 58.1
5.63 ± 1.28 8.10 ± 1.98
300.0 1,350.0
28 127
9.500 −0.237
40.9
0.70
Measurements were performed in pure water at 37 °C.
a
Figure 8. (a) Debye plot and (b) Zimm plot for determination of the static light scattering parameters of the aggregates of PG45-b-PCL35-b-PG45 prepared via protocols A and B in (a) and (b), respectively. Measurements were performed at 37 °C.
PG45, prepared via protocol A. A2 was invariably positive, indicating favorable particle−solvent interactions. In accordance with the non-ionic nature of the copolymer, ζ potential was small and slightly positive. In all cases, however, the number of particles was dominated by the smaller ones as evidenced by recalculating the size distributions from DLS by the number (Figure 9d−f). Considering the contour length of the copolymer chain, one may suggest that the aggregates responsible for the fast modes (that is, the smaller ones) are closest in structure to the classical core−shell micelles. It is tempting to assume that the π−π stacking interactions of the phenyl residues might be involved in bridging several micelles into larger structures, to which the presence of slow modes can be attributed. Such objects were repeatedly observed by cryo-TEM. Cryo-TEM Measurements. Direct evidence of the morphology, size, and size distribution of the micelles was provided by cryo-TEM analysis. Representative micrographs of PG45-bPCL35-b-PG45 and PG35-b-(PO)4-b-PCL35-b-(PO)4-b-PG35 aggregates in water at a concentration of 1 g L−1 are shown in Figure 10. In the case of PG45-b-PCL35-b-PG45 dispersion, only uniform spherical micelles were observed (Figure 10a), while in the PG35-b-(PO)4-b-PCL35-b-(PO)4-b-PG35 sample, uniform micelles coexist with some larger aggregates, obviously formed by two or more bridged micelles (Figure 10b). The average diameter of particles calculated for 100 objects is 51.0 ± 2.3 nm for PG45-b-PCL35-b-PG45 (protocol A) and 28.8 ± 1.5 nm for PG35-b-(PO)4-b-PCL35-b-(PO)4-b-PG35 (protocol D). The cryo-TEM results are in good agreement with light scattering data and confirmed that the amphiphilic PG/PCL triblock copolymers are able to form nano-sized micelles in aqueous media. The small size in combination with the hydrophobic nature of the PCL core, which can gradually degrade under physiological conditions, makes the micelles obtained a candidate for drug delivery application. In addition, the hydrophilic polyglycidol moiety, which is similar to
treat the data for the larger aggregates (Figure 8b). All static light scattering parameters are collected in Table 2. Data in Table 2 revealed that the aggregates prepared at 60 °C (protocol B) were ∼2 times larger in size than those prepared at 37 °C (protocol A), whereas the molar mass (and hence, Nagg) was more than 4.5 times higher. This implied that the particles prepared by protocol B were much denser. The enhanced density was further reflected by the Rg/Rh ratio, which was found to be slightly lower than the value of 0.775, which is consistent for hard spheres.41,42 It is noteworthy that the ζ potential was not affected by the preparation protocol, whereas A2 turned from positive to slightly negative indicating worsening of the solvent conditions. The most distinctive feature of the copolymer PG35-b-(PO)4b-PCL35-b-(PO)4-b-PG35 is the presence of phenyl residues at the two distant ends of the copolymer chain. These residues might be involved in attractive non-covalent interactions referred to as π−π stacking. In addition, compared to PG45-bPCL35-b-PG45, this copolymer is characterized with slightly shorter PG chains that are connected to the PCL middle block via short PPO spacers. Accordingly, the aqueous solution behavior of this copolymer was different from that of PG45-bPCL35-b-PG45. The results indicate that the dominating feature that governs the behavior of PG35-b-(PO)4-b-PCL35-b-(PO)4-bPG35 in aqueous solution is the presence of phenyl residues. Several preparation protocols were used to obtain aggregates from this copolymer; however, monomodal particle size distributions were not observed: the distributions were invariably bimodal with two well-separated modes (Figure 9a−c), indicating formation of two populations of particles. The preparation protocols affected the magnitude and position of the two modes. The values of the hydrodynamic radius accounted at 90° (Rapp h ) for the fast and slow modes are shown in Table 3 together with the apparent molar masses, A2, and ζ potential. The apparent molar masses varied between 121.2 and 181.0 × 103 g mol−1 corresponding to an aggregation number between 16 and 24, which was comparable to that of PG45-b-PCL35-bJ
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Figure 9. Size distributions from DLS of aggregates obtained from PG35-b-(PO)4-b-PCL35-b-(PO4)-b-PG35 via preparation protocols (a, d) B, (b, e) C, and (c, f) D. Panels (a)−(c) and (d)−(f) represent size distributions by intensity and by number, respectively. Measurements were performed at 37 °C, concentration of 1.0 g L−1, and θ = 90°.
Table 3. Static, Dynamic, and Electrophoretic Light Scattering Characterization Data for the Aggregates of the Copolymer PG35b-(PO)4-b-PCL35-b-(PO)4-b-PG35 Formed by Different Preparation Protocolsa Rapp h (nm) preparation protocol
fast
slow
−1 10−3 × Mapp W (g mol )
103 × A2 (mL mol g−2)
ζ (mV)
B C D
25.1 23.3 13.9
36.15 63.15 40.55
121.2 174.0 181.0
1.08 5.71 1.31
2.27 ± 0.87 4.75 ± 3.60 4.43 ± 1.37
Measurements were performed in pure water at 37 °C. The apparent values of molar mass, Mapp W , and A2 were obtained by Debye plots at θ = 90°.
a
K
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Figure 10. Cryo-TEM micrograph of (a) the PG45-b-PCL35-b-PG45 triblock copolymer (protocol A) and (b) PG35-b-(PO)4-b-PCL35-b-(PO)4-b-PG35 block copolymer (protocol D) dispersion in water.
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poly(ethylene oxide) that is widely used for such purpose, has one −OH group in each monomer unit and can offer more functionalities of the nanocarriers developed.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.9b00366. Additional reaction schemes, 1H-NMR spectra, GPC chromatograms, and FTIR spectra (PDF)
CONCLUSIONS
The “click” chemistry method was successfully applied for the synthesis of linear block copolymers comprising PEEGE (protected PG) and PCL. This strategy allowed preparation of copolymers having predefined composition and macromolecular characteristics via copper-catalyzed cycloaddition reaction of a monoalkyne-functionalized PEEGE and a bifunctional PCL bearing azide end groups. The reaction pathway described in this work was considered to be more convenient as compared to the classical approaches of sequential ROP for obtaining a linear PG block chain architecture. It allowed preparation of block copolymers bypassing the stage of polymerization of EEGE initiated by a deprotonated bifunctional PCL diol in strong alkaline media. The incorporation of short PO spacers (4 units) between the reactive −OH end group and the PEEGE chain eliminated the negative steric hindrance of the bulky ethoxyethyl groups and thus contributed to achieving a quantitative functionalization of PEEGE with alkyne groups. The conversion of PEEGE into PG blocks by hydrolysis with the Lewis acid AlCl3 was selective and did not alter the PCL block. Amphiphilic PG/PCL block copolymers formed in aqueous media rather uniform spherical nano-sized micelles with slightly positive ζ potential with the aid of the solvent evaporation method. We anticipate a core−shell structure comprising a biodegradable hydrophobic PCL core and a functional hydrated PG shell. The small size in combination with the favorable physicochemical properties of the building blocks makes the micelles a promising candidate for multifunctional nanocarrier development. One should take into account that the preparation protocol affects to some extent the size of micelles; while the phenyl residues at the two distant ends of the copolymer chains trigger some undesired attractive π−π stacking interactions between the micelles. The present synthesis strategy is not restricted to the copolymers described in this work and can be extended toward developing a variety of novel copolymers comprising linear PG.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Petar D. Petrov: 0000-0002-5777-2066 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Bulgarian National Science Fund (Project DN 09/1 − 2016). A scientific cooperation agreement between the Bulgarian Academy of Sciences and the Polish Academy of Sciences made cryo-TEM analysis possible.
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REFERENCES
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