A Combined Experimental and Computational Study of the Substituent Effect on Micellar Behavior of γ‐Substituted Thermoresponsive Amphiphilic Poly(ε-caprolactone)s

The effect of the core substituent structure on the micellar behavior of thermoresponsive amphiphilic poly(εcaprolactone) diblock copolymer micelles was investigated through a combination of experimental and computational methods. The polycaprolactone (PCL) amphiphilic block copolymers used in this study consisted of a hydrophilic poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone} block, which also endowed the polymer with thermoresponsiveness, and various hydrophobic poly(γ-alkoxy-ε-caprolactone) blocks. Five different substituents have been attached to the γ-position of the ε-caprolactone of the hydrophobic block, namely octyloxy, ethylhexyloxy, ethoxy, benzyloxy, and cyclohexylmethoxy, which self-assembled in aqueous media to generate the core of the micelles. All five synthesized diblock copolymers formed micelles in water and displayed thermoresponsive behavior with lower critical solution temperature (LCST) in the range of 36−39 °C. The impact of different substituents on the micelle properties such as size, stability, and phase transition behavior was investigated. Drug loading and release properties were also studied by employing doxorubicin (DOX) as payload. Molecular dynamics modeling was used to predict the variation of particle size, free volume, and drug loading capacity. The drug loading capacity predicted from molecular dynamics simulation was found to be comparable with the experimental data, which suggests that molecular dynamic simulations may be a useful tool to provide valuable selection criteria for the engineering of polymeric micelles with tunable size and drug loading capacity. ■ INTRODUCTION Polymeric micelles are core−shell structures formed by selfassembly of amphiphilic block copolymers. The hydrophobic core acts as a microreservoir for the encapsulation of drugs, while the hydrophilic shell acts as a corona which protects the micelles from protein adsorption and cellular adhesion. Among the polymeric micelle systems under clinical study, micelles formed from amphiphilic aliphatic polyesters have shown great promise due to their superior biocompatibility and biodegradability. Moreover, due to the flexibility and feasibility of chemical modification of polyesters, the properties of these micelles can be tailored by introducing functional groups to the core or shell to optimize delivery efficacy and maximize the therapeutic effect. For instance, micellar cores have been conjugated with functional groups, drug molecules, or cross-linked to enhance drug loading capacity, micelle stability, and controlled release properties. Moreover, micellar shells have been engineered to achieve active targeting, enhanced cellular uptake, and stimuliresponsive drug release properties. Imparting thermoresponsive properties to the micelles allows the micelle to release encapsulated molecules in a controlled manner upon temperature change. Oligo(ethylene glycol) (OEG) polymers have emerged as a new class of thermoresponsive polymers. The phase transition behavior of the OEG-functionalized polymers have been shown to be insensitive to ionic strength, concentration, etc., and thus are superior to N-substituted acrylamide polymers, like PNIPAM. In addition, polymeric micelles from OEG grafted polymers have shown controllable drug release and improved cellular uptake in response to temperature increase. In our previous studies, we have reported the synthesis of tri(ethylene glycol)substituted amphiphilic polycaprolactone diblock copolymer (PMEEECL-b-POCTCL) and its self-assembled micelle. The drug loading, cyctotoxicity, and thermo-induced drug release behavior of this polymeric micelle system have also been studied. The obtained experimental results indicated that this polyester-based amphiphilic diblock copolymer is an ideal polymeric micelle system for controlled drug release, thus deserving further investigation. Micelle core engineering represents a promising methodology to optimize various properties of a micelle as a drug Received: April 25, 2013 Revised: May 30, 2013 Published: June 5, 2013 Article pubs.acs.org/Macromolecules © 2013 American Chemical Society 4829 dx.doi.org/10.1021/ma400855z | Macromolecules 2013, 46, 4829−4838 vector. Rationally designed functionalities of the micelle core are expected to enhance the overall performance of micelle nanocarriers by tuning the drug loading capacity, stability, and “smartness”. Previously, we employed an octyloxy substituent attached to the γ-position of the ε-caprolactone to form the micelle core. However, only moderate drug loading and stability were achieved. To further optimize this class of amphiphilic thermoresponsive polycaprolactone block copolymers as drug carriers and systematically study the substituent effect on the micellar assembly, we synthesized five polycaprolactone amphiphilic block copolymers with different hydrophobic substituents. Octyloxy, ethylhexyloxy, ethoxy, benzyloxy, and cyclohexylmethoxy were used as substituents on the core-forming block. The substituents were chosen to compare linear vs branched aliphatic substituents, long vs short aliphatic substituents, and aromatic vs nonaromatic ring substituents. These functionalities on the core segment were expected to interact intraand intermolecularly with the encapsulated drug molecules by noncovalent interactions, such as hydrophobic, π−π stacking, and hydrogen bonding. Doxorubicin (DOX) was employed as a model drug as its interactions with polymer chains govern the encapsulation behavior by polymeric micelles. In summary, the substituent effect on thermal-induced phase transition, thermodynamic and kinetic stability, drug loading, and thermo-induced drug release of DOX were investigated for this library of polycaprolactone amphiphilic block copolymers. In addition to exploring the substituent effect experimentally, we were also interested in whether the micellar behavior can be predicted by molecular dynamics (MD) simulation. While molecular dynamics has been applied to study micellation behavior, this is the first report of using MD methodology to study the substituent effect of polymeric micelles. MD simulations were performed for micelles with the same polymer backbone and similar functional hydrophobic substituents as the experimentally synthesized polymers. The drug loading behavior was predicted by using phenol as a DOX alternative. The free volume of the micellar core was calculated for both drug loaded and unloaded micelles. The results indicated that the interaction between the drug and polymer is more important than the void volume of the micelle cores in determining the drug loading capacity (DLC). ■ MATERIALS AND METHODS Materials and Characterization. All commercial chemicals were purchased from Aldrich Chemical Co., Inc., and were used without further purification unless otherwise noted. Stannous(II) 2-ethylhexanoate was purified by vacuum distillation prior to use. γ-Octyloxyε-caprolactone and γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone monomers were synthesized according to the previously reported procedure. H NMR spectra of the synthesized monomers and polymers were recorded on a Bruker 500 MHz spectrometer at 30 °C in CDCl3. H NMR data are reported in parts per million as chemical shift relative to tetramethylsilane (TMS) as the internal standard. GC/MS was performed on an Agilent 6890-5973 GC-MS workstation. The following conditions were used for all GC/MS analyses: injector and detector temperature, 250 °C; initial temperature, 70 °C; temperature ramp, 10 °C/min; final temperature, 280 °C. Molecular weights of the synthesized polymers were measured by size exclusion chromatography (SEC) analysis on a Viscotek VE 3580 system equipped with ViscoGEL columns (GMHHR-M), connected to a refractive index (RI) detector. GPC solvent/sample module (GPCmax) was used with HPLC grade THF as the eluent, and calibration was based on polystyrene standards. Running conditions for SEC analysis were flow rate = 1.0 mL/min, injector volume = 100 μL, detector temperature = 30 °C, and column temperature = 35 °C. All the polymers samples were dissolved in THF, and the solutions were filtered through PTFE filters (0.45 μm) prior to injection. General Procedure for the Synthesis of Amphiphilic Diblock Copolymers P1−P5. All the monomers were dried by azeotropic distillation from toluene before the reaction. Dried γ-2-[2-(2methoxyethoxy)ethoxy]ethoxy-ε-caprolactone (0.5 g, 1.8 × 10−3 mol) was transferred into a flame-dried 10 mL Schlenk flask under a nitrogen atmosphere. Stock solutions of Sn(Oct)2 (0.016 g, 3.6 × 10 −5 mol) in hexane and benzyl alcohol (0.004g, 3.6 × 10−5 mol) in hexane were added to the Schlenk flask under a nitrogen atmosphere. The reaction mixture was deoxygenated by three consecutive freeze− pump−thaw cycles, and the vacuum of the last cycle was canceled with nitrogen. The reaction flask was heated in a thermostated oil bath at 110 °C for 4 h. At this time a sample was collected to determine the monomer conversion and molecular weight by H NMR analysis. Deoxygenated monomers (M1−M5) (1.8 × 10−3 mol) were added to the reaction flask under a nitrogen atmosphere, and the reaction was left overnight at 110 °C. Poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}-bpoly(γ-octyloxy-ε-caprolactone) (P1). H NMR (500 MHz, CDCl3): δH 0.89 (t, 3H), 1.27 (m, 10H), 1.54 (m, 2H), 1.80 (m, 8H), 2.38 (m, 4H), 3.38 (m, 6H), 3.60 (m, 13H), 4.15 (m, 4H) (the polymer contains 53.5% PMEEE). Poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}-bpoly[γ-(2-ethylhexyloxy)-ε-caprolactone] (P2). H NMR (500 MHz, CDCl3): δH 0.87 (m, 6H), 1.27 (m, 9H), 1.80 (m, 8H), 2.37 (m, 4H), 3.38 (m, 3H), 3.60 (m, 16H), 4.16 (b, 4H) (the polymer contains 54.1% PMEEE). Poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}-bpoly(γ-ethoxy-ε-caprolactone) (P3). H NMR (500 MHz, CDCl3): δH 1.17 (t, 3H), 1.8 (m, 8H), 2.39 (m, 4H), 3.38 (s, 3H), 3.47 (m, 3H), 3.59 (m, 13H), 4.17 (t, 4H) (the polymer contains 62.8% PMEEE). Poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}-bpoly(γ-benzyloxy-ε-caprolactone) (P4). H NMR (500 MHz, CDCl3): δH 1.80 (m, 8H), 2.37 (m, 4H), 3.38 (s, 3H), 3.59 (m, 14H), 4.15 (b, 4H), 4.47 (m, 2H), 7.30 (b, 5H) (the polymer contains 54.4% PMEEE). Poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}-bpoly(γ-cyclohexylmethoxy-ε-caprolactone) (P5). H NMR (500 MHz, CDCl3): δH 0.93 (m, 2H), 1.24 (m, 3H), 1.81 (m, 14H), 2.40 (m, 4H), 3.22 (m, 2H), 3.40 (s, 3H), 3.62 (m, 14H), 4.18 (m, 4H) (the polymer contains 53.4% PMEEE). Preparation of Polymeric Micelles and Drug Encapsulation. Each polymer (20 mg) was dissolved in 1 mL of THF, and 40 μL of the polymer THF solution was added dropwise to 4 mL of deionized water under vigorous agitation. THF was removed by dialyzing the mixture against deionized water for 1 day (MWCO = 3 kDa). The micelle suspension was filtered through a 0.45 μm filter before characterization. Doxorubicin (DOX) was encapsulated in polymeric micelles as a model hydrophobic guest molecule. DOX was first treated with 3 equiv of triethylamine in DMSO. The neutralized DOX solution was mixed with each polymer THF solution at a mass ratio of 1:10. The polymer/DOX mixture (40 μL) was added dropwise to 4 mL of deionized water under vigorous agitation. The DOX-loaded micelle suspension was dialyzed against deionized water for 1 day and then filtered with a 0.45 μm filter before characterization. Equivalent DOX concentration in the micelles was determined by fitting readout absorbance of DOX loaded micelles at 485 nm to a pre-established standard curve of DOX. Loading efficiency (LE) and loading capacity (LC) of all polymeric micelles, assuming there was no loss of polymer during sample preparation, were calculated according to the following equations: = × LE (wt %) weight of encapsulated DOX weight of total DOX 100% = × LC (wt %) weight of encapsulated DOX weight of polymer 100% Macromolecules Article dx.doi.org/10.1021/ma400855z | Macromolecules 2013, 46, 4829−4838 4830