Article pubs.acs.org/Macromolecules
Organocatalytic Copolymerization of a Cyclic Carbonate Bearing Protected 2,2-bis(hydroxymethyl) Groups and D,L‑lactide. Effect of Hydrophobic Block Chemistry on Nanoparticle Properties Yanet Elised Aguirre-Chagala,†,‡ José Luis Santos,† Rafael Herrera-Nájera,‡ and Margarita Herrera-Alonso†,* †
Department of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, United States Departamento de Ingeniería Química, Facultad de Química, Universidad Nacional Autónoma de México, Distrito Federal 04510, México
‡
S Supporting Information *
ABSTRACT: The organocatalytic copolymerization of a derivative of trimethylene carbonate bearing protected 1,3diols and D,L-lactide was studied. Homopolymerization of the cyclic carbonate from a PEG macroinitiator exhibited a controlled character, obeying pseudo first-order kinetics up to a conversion of ∼60%. Longer reactions resulted in more polydisperse materials. Kinetics of copolymerization with DLLA showed a considerable accelerative effect of the ester on the polymerizability of the carbonate, attributed to relief of steric limitations that characterize the polymerization of this and other bulky cyclic carbonates. Postpolymerization hydrogenolysis, conveniently adjusted though catalyst concentration, yielded functional poly(ester carbonate)s (PECs) with enhanced chain mobility and hydrophilicity compared to protected analogues. PEG-b-PECs were further used as stabilizers for the formation of fluorophore nanoparticles via flash nanoprecipitation. Nanoparticle size and core properties were discussed in terms of hydrophobic block chemistry. The regulation offered by organocatalysts for the synthesis of diol-functionalized PECs with precise molecular characteristics provides access to biodegradable materials with readily tunable physicochemical properties through controlled installation of reactive handles. In the context of hydrophobic solute encapsulation, regulated functionalization allows fine-tuning of nanoparticle core properties, ultimately impacting solute loading, release, and stability.
■
INTRODUCTION Aliphatic polycarbonates and polyesters are two important classes of biocompatible and biodegradable materials widely used for drug delivery and tissue engineering applications. Polycarbonates are mostly known for their good mechanical properties and for their minimally toxic and nonacidic degradation products.1−4 Certain polyesters are interesting as biodegradable and bioassimilable materials.2,3 Nevertheless, the growing need for increasingly versatile biomaterials has spurred the development of functional versions of traditionally used polymers, including polycarbonates and polyesters, to enable facile tuning of their physicochemical and biological properties by installation of sites for covalent conjugation.5−10 Aliphatic polycarbonates and polyesters are generally synthesized by ring-opening polymerization (ROP) of cyclic carbonates and esters through anionic, cationic, coordination/ insertion and nucleophilic routes.11−13 Metal-catalyzed ROP proceeds through a coordination/insertion mechanism involving electrophilic monomer activation by Lewis acids and insertion of the monomer to the metal−oxygen bond.14 The more commonly used catalysts are based on transition metals © XXXX American Chemical Society
(e.g., Sn(Oct)2) and polymerization is generally carried out in the bulk at elevated temperatures. Recently, more attention has been devoted to the use of organocatalysts to replace metalbased Lewis acids particularly for biomedical applications where trace metals may have cytotoxic and inflammatory effects.15 Equally important is the fact that organocatalysis provides better control over polymerization, yielding near monodisperse polymers.9,16−18 The mechanistic pathways for organocatalyzed ROP rely on electrophilic or nucleophilic activation of the monomer and/or the initiator to achieve chain growth; the exact mechanism depends on the choice of initiator and catalyst.16,17,19 Organic catalysts from neutral basessuch as amidines, guanidines, and phosphazinesshow high activities for ROP under mild conditions in nonpolar solvents and provide control over the polymerization of functional and nonfunctional cyclic carbonates and esters, yielding near monodisperse linear polymers. Received: April 30, 2013 Revised: June 24, 2013
A
dx.doi.org/10.1021/ma400894t | Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
■
The “superbases” more commonly used to mediate ROP of cyclic carbonates and esters include the amidine 1,8diazabicycloundec-7-ene (DBU) and the guanidine 1,4,7triazabicyclodecene (TBD). Bifunctional activation is also an alternative for the lesser reactive systems.20 In this case, the catalytic effect is achieved by combination of a thiourea and an amine to simultaneously effect electrophilic activation of the monomer and nucleophilic activation of the initiator, respectively.21 These systems exhibit particularly high selectivity toward ring-opening (vs chain transesterification) and are widely tolerant of functional substrates. Strategies for imparting functionality to polycarbonates depend on the reactive groups of interest; functional groups that interfere with ROP conditions (e.g., amines, carboxylic acids, and hydroxyls) are generally introduced through the use of monomers containing an appropriate protecting group which is removed postpolymerization to yield the desired functionality.6,7,11,22,23 Another strategy is by modification of the monomer with functional groups orthogonal to ROP conditions (e.g., allyl, alkyne, azide), which eliminates the need for protection/deprotection reactions but may also require postpolymerization derivatization.9,24,25 Recent examples illustrate the use of the appended functional groups for the synthesis of polyelectrolyte block copolymers used in gene transfection,26−28 of substrates for polymeric prodrugs,6,29 and to mediate cell adhesion.5,6 A vast amount of work regarding the synthesis of functional polycarbonates focuses on their use as drug delivery agents as films or in the form micelles or nanoparticles of well-controlled size and core properties.11,30,31 Micelles from block copolymers comprised of carbonate monomers are known to exhibit good thermodynamic stability, a characteristic crucial for drug delivery applications wherein carrier stability affects the success of drug encapsulation and transport to the target site.11 On several of these instances, functional cyclic carbonates have been copolymerized with cyclic esters to modulate core degradation kinetics and mechanism, particularly when the polycarbonate has hydrophilic moieties.32,33 The chemical characteristics of the core-forming hydrophobic block can also be tuned to enhance polymer-drug compatibility resulting in better sustained release profiles.34 We are interested in the synthesis of amphiphilic copolymers of aliphatic polycarbonates containing pendant hydroxyl groups and the properties of their self-assembled structures. Specifically, we focus our attention on methods for the installation of 1,3-diols onto polycarbonate backbones as they can serve as sites for covalent attachment of biologically relevant molecules, mediate the formation of dynamic networks, and allow modulation of the hydrophobic character of the micellar core. To this end, we studied the polymerization of a benzylprotected dihydroxylated cyclic carbonate, 9-phenyl-2,4,8,10tetraoxaspiro[5,5]undecan-3-one (PTO)5,33,35−38 by an organocatalyzed ring-opening reaction. In this communication we discuss, for the first time, details regarding the copolymerization of PTO and D,L-lactide (DLLA) from a a monomethoxypoly(ethylene glycol) macroinitiator, DBU and a thiourea, to generate polymeric amphiphiles. This polymerization route yields poly(ester carbonate) scaffolds with controlled molecular characteristics which, aside from their use as stabilizers for hydrophobic solute nanoparticles as discussed here, can serve as a platform for biodegradable materials mediated by the chemical versatility of hydroxyl groups.
Article
EXPERIMENTAL SECTION
Materials. All reagents were commercially available and used as received unless otherwise specified. Solvents were dried by passage through activated alumina columns. Dichloromethane was distilled from calcium hydride immediately before use. PTO (9-phenyl2,4,8,10-tetraoxaspiro[5,5]undecan-3-one) (1) and 1-(3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2-thiourea (3) were synthesized according to previously published methods and stored in a desiccator under an inert atmosphere.33,39 D,L-Lactide was recrystallized from anhydrous ethyl acetate and stored in a desiccator. 1,8Diazabicyclo[5.4.0]undec-7-ene (DBU) was kept over molecular sieves (3 Å). Deionized water was purified in a Barnstead Nanopure system to a final resistance of 18.2 mΩ; it will be referred to as Nanopure water. Instruments. Gel Permeation Chromatography (GPC) was performed on a Waters 1515 Isocratic HPLC equipped with two Styragel columns (HR4 and HR3, 300 mm ×7.8 mm) connected in series, a differential refractive index detector (Waters 2414) and a UV−visible detector (Waters 2489). HPLC grade THF was used as the eluent, at a flow rate of 1 mL/min. Molecular weights are reported referenced to polystyrene standards (Shodex SL-105). 1H and 13C NMR spectra were recorded on a Bruker AV 400 MHz spectrometer in either CDCl3 or DMSO-d6. Spectra were referenced to CHCl3 (7.26 ppm) or DMSO-d6 (2.50 ppm). Fluorescence spectroscopy was carried out on a Fluorolog-3 system (HORIBA Jobin Yvon Inc., NJ). Dynamic light scattering experiments were conducted on a Malvern Instruments Nano-ZS ZetaSizer equipped with a 4 mW He−Ne laser operating at 633 nm. All measurements were performed at 25 °C at a scattering angle of 173°. Autocorrelation functions of backscattered light were analyzed using a DTS 6.12 software. Cumulants method was used to obtain hydrodynamic radius and polydispersity. Measurements were carried out five times with a duration of 150 s each. Differential scanning calorimetry (DSC) was carried out on a Perkin-Elmer DSC 8000. Each sample was subject to two scanning cycles, with a heating rate of 10 °C/min from −40 to +150 °C, equilibration at 150 °C for 1 min, and cooling from +150 to −40 °C at 30 °C/min. Reported thermograms correspond to the second cycle. Nanoparticle Formation. Block copolymer-stabilized solute nanoparticles were produced by flash nanoprecipitation in a fourstream vortex mixer. A detailed description and characterization of the mixer are provided elsewhere.40 The block copolymer (5 mg/mL) and solute of interest (pyrene or Nile red) were allowed to dissolve in tetrahydrofuran for a minimum of 12 h at room temperature. Solutions were filtered through 0.22 μm PVDF syringe filters (Millipore). The THF:water volumetric ratio used was 1:9, with mixing speeds of 12 mL/min and 108 mL/min (36 mL/min per stream) for the organic and aqueous phases, respectively, resulting in suspensions with a concentration of 0.05% (wp/w). Nanoparticle suspensions were then dialyzed (6−8 kDa MWCO, Spectrapor) against Nanopure water for 8 h. Water was replenished five times throughout the dialysis process. Suspensions were stored in clean scintillation vials for further use. Methods. Synthesis of PTO Homopolymers. PTO (303 mg, 0.06 mmol) and monomethoxypoly(ethylene glycol) mPEG−OH (195 mg, 0.782 mmol) were dried by azeotropic distillation under vacuum to prevent thermally induced polymerization of the cyclic carbonate. The flask was placed under vacuum (60 mTorr) for 4 h to completely remove the solvent and backfilled with nitrogen. Dry dichloromethane (5 mL) was then added via syringe and the reagents were allowed to dissolve completely prior to the addition of TU 3 (14.46 mg, 0.39 mmol) and DBU (5.8 μL, 0.039 mmol). Polymerizations proceeded at room temperature under an inert atmosphere with continuous stirring. Aliquots (∼0.2 mL) were withdrawn at regular intervals and polymerization was quenched by addition to scintillation vials containing benzoic acid (1.1 mg, 9 μmol). The final mPEG-b-PPTO sample was also quenched with benzoic acid (15.9 mg, 0.13 mmol), precipitated in diethyl ether, filtered and dried under vacuum at 50 °C for 12 h. Synthesis of PTO and D,L-Lactide Triblock Copolymers from mPEG Macroinitiator [mPEG-b-PPTO-b-PDLLA]. mPEG-b-PPTO was B
dx.doi.org/10.1021/ma400894t | Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Article
Figure 1. (A) Ring-opening polymerization of PTO (1) initiated by mPEG−OH (5 kDa) and catalyzed by a bifunctional catalytic system consisting of DBU (2) and a thiourea (3). Polymerization was carried out at room temperature in dichloromethane with molar ratios of [PTO]:[OH]:[DBU]: [TU] = 20:1.5:1:1. (B) Chromatograms of the reaction mixture showing the increase of copolymer molecular weight and the development of oligomeric PPTO (∼18.5 min). (C) Polymerization kinetics monitored by 1H NMR to 24 h; polymerization proceeded with a controlled character within the first 7 h exhibiting a linear dependence of conversion on reaction time (inset). (D) Dependence of molecular weight and polydispersity on conversion. Fluorescence Measurements. Critical micelle concentrations (CCMC) of block copolymer amphiphiles were determined by fluorescence spectroscopy using pyrene as probe.30,41,42 Samples were equilibrated at 25 °C for 10 min prior to measurements. Pyrene (6.0 × 10−5 M in acetone, 10 μL) was added to a 5 mL scintillation vial. The solvent was allowed to evaporate completely prior to the addition of 50 μL of a block copolymer solution (in acetone) of known concentration. Water (1 mL) was then added, and samples were stirred with a vortex mixer for 1 min. The final concentration of block copolymer would range from 0.05 μg/mL to 1 mg/mL. The solvent was allowed to evaporate at room temperature and samples were equilibrated for 12 h prior to measurements. The final pyrene concentration in each sample was 6.0 × 10−7 M. Pyrene excitation was scanned from 300 to 360 nm at an emission wavelength of 390 nm. Excitation and emission bandwidths were set at 2 nm. The intensity ratio from signals at 336 and 334 nm (I336/I334) was analyzed as a function of polymer concentration. CCMC values were read from the intersection between curve tangents at low and high concentrations. Nanoparticle core hydrophobicity was determined by Nile red encapsulation. Emission from loaded nanoparticles was measured from 560 to 800 nm at an excitation wavelength of 550 nm. Excitation and emission bandwidths were set at 2.5 nm. Transmission Electron Microscopy. Bright-field TEM imaging was performed on an FEI Tecnai 12 TWIN Transmission Electron Microscope operated at an acceleration voltage of 100 kV. All TEM images were recorded by a SIS Megaview III wide-angle CCD camera. TEM grids were treated under plasma to render carbon films hydrophilic. Sample grids were prepared by placing a carbon-coated copper grid (Electron Microscopy, Hatfield, PA) onto a droplet of
synthesized as specified above. The block copolymer (500 mg, 0.0769 mmol) was dried by azeotropic distillation.D,L-lactide (213 mg, 1.48 mmol) was then added, and the flask was placed under high vacuum for 4 h. Both reagents were dissolved in dichloromethane (2.1 mL) prior to the addition of DBU (2.22 μL, 0.0149 mmol). Polymerization proceeded for 1 h at room temperature under an inert atmosphere with continuous stirring. Reaction aliquots and the final sample were quenched with benzoic acid and purified as specified above. Synthesis of PTO and D,L-Lactide Copolymers from mPEG−OH [mPEG-b-P(PTO-co-DLLA)]. PTO (586 mg, 2.35 mmol) and mPEG− OH (909 mg, 0.18 mmol) were dried by azeotropic distillation under vacuum. D,L-lactide (505 mg, 3.51 mmol) was then added and the flask was placed under high vacuum for 4 h. Dichloromethane (11 mL) was introduced via syringe and the reagents were allowed to dissolve completely prior to the introduction of TU (43.3 mg, 0.12 mmol) and DBU (17.5 μL, 0.12 mmol). Polymerizations proceeded at room temperature under an inert atmosphere with continuous stirring. Aliquots and the final sample were quenched with benzoic acid. Polymers were precipitated in diethyl ether, filtered, and dried under vacuum at 50 °C for 12 h. PPTO Deprotection. mPEG-b-P(PTO-co-DLLA) samples (700 mg) were dissolved in methanol or THF:MeOH mixtures (12 mL of 1:3 vol), and to these solutions was added Pd(OH)2/C. Mixtures were refluxed for 24 h, allowed to return to room temperature, and filtered through a short bed of Celite which was later rinsed with THF. The solution was concentrated under vacuum and precipitated into diethyl ether, and the resulting samples were dried under vacuum at 50 °C for 12 h. C
dx.doi.org/10.1021/ma400894t | Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Article
Figure 2. (A) Copolymerization of D,L-lactide (LA) and PTO, initiated by mPEG−OH (5 kDa) and catalyzed by DBU−thiourea (2 + 3). Polymerization was carried out at room temperature in dichloromethane with molar ratios of monomers:[OH]:[DBU]:[TU] = 50:1.5:1:1. Molar ratio of LA:PTO was 3:1. (B) Chromatograms of the reaction mixture showing the increase in molecular weight of the copolymer with monomer consumption. (C) Polymerization kinetics monitored by 1H NMR and GPC to 7 h, showing differences in polymerizability between LA and PTO, and copolymer polydispersity. nanoparticle suspension (30 μL). After 5 min, the grid was washed under 8 drops of Nanopure water and placed onto a drop of a 2 wt % aqueous uranyl acetate solution for 20 s. Grids were then blotted with filter paper and allowed to dry at room temperature prior to imaging.
characteristics is relevant for the synthesis of biodegradable polymer scaffolds mediated by the diol functionality. PTO Homopolymerization. The homopolymerization of PTO (1) (Figure 1A) was studied at room temperature using monomethoxypoly(ethylene glycol) (mPEG114−OH, subscripts represent repeat units) as a macroinitiator and DBU(2)− TU(3) as catalysts (PTO:DBU:OH = 20.1:1.0:1.5 molar). The reaction was monitored by GPC and 1H NMR to 24 h. GPC traces acquired during polymerization show evolution of the copolymer with monomer consumption and no observable residues of unreacted macroinitiator (Figure 1B). Under the reaction conditions used in this study, PTO homopolymerization reached a limiting conversion of 75% after ∼15 h (Figure 1C). Similar reaction profiles have been reported for polymerizations of other sterically hindered cyclic carbonates.24,44 After ca. 30 min of reaction, the appearance of a small broad peak at an elution time intermediate to that of the copolymer and the monomer (∼18.5 min) was observed, with increasing intensity and variable peak maximum and width over time, which we discuss below. DBU plays a complex role in the ring-opening polymerization of cyclic carbonates and esters. For example, it can act as an initiator for the anionic homopolymerization of trimethylene carbonate derivatives, affording high molecular weight polymers.45 Additionally, it was recently shown that in the absence of alcohol initiators, organocatalysts derived from amidines including DBUmediate the nucleophilic zwitterionic polymerization of cyclic and linear poly(lactide).46 These results suggest that DBU-catalyzed reactions can operate through a combination of mechanisms, and reaction conditions or choice of reagents will strongly influence selectivity and polymerizability. Transesterification should also be included in the list
■
RESULTS AND DISCUSSION The ease of synthesis of functional derivatives of trimethylene carbonate has broadened the applicability of their homo- and copolymers particularly in the biomedical field.7,13 Equally evolving are their polymerization methods, and in this respect, we examined the organocatalytic ring-opening polymerization (ROP) of a cyclic carbonate monomer bearing protected 2,2bis(hydroxymethyl) groups. Organic catalysts from neutral bases show high activity toward ROP under mild reaction conditions and provide good control over the polymerization of functional cyclic carbonates, yielding near monodisperse linear polymers. Exact choice of catalyst and polymerization conditions largely depend on monomer structure and catalytic activity and selectivity, which are chosen to minimize undesirable side-reactions such as transesterification or, in the case of polycarbonates decarboxylation. We chose to study the ROP of PTO (9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3one) and its copolymerization with D,L-lactide (DLLA), catalyzed by 1,8-diazabicycloundec-7-ene (DBU) and 1-(3,5bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2-thiourea (TU). We used monomethoxy poly(ethylene glycol) as a macroinitiator since we were interested in the synthesis of amphiphilic block copolymers with a biodegradable core. In this respect, the resulting 1,3-diol can be used to modulate core hydrophobicity and degradability, act as a reactive site for the conjugation of biologically relevant molecules, or mediate core cross-linking.5,6,43 In a more broad sense, optimization of the conditions to achieve PTO-based copolymers with precise molecular D
dx.doi.org/10.1021/ma400894t | Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Article
Figure 3. 13C NMR spectra for mPEG-b-PPTO (1), mPEG114-b-P(PTO-co-DLLA) (2−4), and mPEG-b-PDLLA (5) showing triad peak assignments. Spectra 2, 3, and 4 correspond to copolymerization times of 7, 0.5, and 0.05 h, respectively.
distribution of the triblock copolymer can be attributed to the occurrence of side reactions during polymerization of the cyclic carbonate, as mentioned before. The low polydispersities observed for mPEG-b-PPTO copolymers are strongly influenced by that of the macroinitiator, in this case ∼1.02 for mPEG114. To eliminate the contribution of PEG, a small molecule initiator (1-dodecanol) was used for the ROP of PTO. Polymerization kinetics were followed as before (Supporting Information, Figure S2). The reaction again showed excellent agreement with pseudofirst order kinetics and an apparent reaction rate coefficient of kapp = 0.12 h−1 was determined. This value, which is very similar to that of the mPEG-initiated system (kapp = 0.16 h−1), indicates no appreciable effect of the initiator on polymerization kinetics. PPTO polydispersity was higher than that for the mPEG-based system (1.2 vs 1.03 for similar conversion) but was still relatively low. PTO−DLLA Copolymerization. We examined the ringopening random copolymerization of DLLA and PTO, initiated by mPEG114−OH and catalyzed by DBU(2)-TU(3) over 7 h (Figure 2A). The ratio of PTO to catalyst used was approximately the same as that for PTO homopolymerization (PTO:DBU:OH = 20.1:1.0:1.5) and the PTO:DLLA feed ratio was 3, such that the molar ratio of reagents added was monomers:DBU:OH = 50.0:1.0:1.5. Chromatograms of the copolymers showed an increase in molecular weight over time and the absence of unreacted macroinitiator (Figure 2, B). As for PTO homopolymerization, chromatograms of its copolymer with DLLA showed a small presence of oligomeric species, again suggesting the occurrence of side reactions in the comonomer system. Copolymer ĐM remained relatively low throughout the reaction (
