Interplay between Molecular Packing, Drug Loading, and Core Cross

Feb 8, 2017 - Amphiphilic copolymers with bottlebrush architecture provide opportunities for the refinement of materials properties that may not be at...
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Interplay between Molecular Packing, Drug Loading, and Core CrossLinking in Bottlebrush Copolymer Micelles Hande Unsal,†,‡ Sebla Onbulak,† Filiz Calik,§ Meriem Er-Rafik,∥ Marc Schmutz,∥ Amitav Sanyal,§ and Javid Rzayev*,† †

Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, United States Department of Chemical Engineering, Hacettepe University, Ankara, Turkey § Department of Chemistry, Bogazici University, Istanbul, Turkey ∥ Institut Charles Sadron, CNRS-Strasbourg University, Strasbourg 67034, France ‡

S Supporting Information *

ABSTRACT: Amphiphilic copolymers with bottlebrush architecture provide opportunities for the refinement of materials properties that may not be attainable from their linear analogues. In this study, we investigated the effect of polymer architecture on an interplay between molecular packing inside micelle cores, cargo loading, and core crosslinking. Four families of polylactide-b-poly(ethylene oxide) (PLA−PEO) bottlebrush block copolymers with different sidechain arrangements were synthesized by a combination of grafting-through and grafting-from methods. Copolymers with double-graft PLA side chains produced smaller and more uniform micelles than those with single-graft PLA branches. Photoactive coumarin groups, installed at PLA side chain ends, improved paclitaxel loading efficiencies of the copolymer micelles and allowed for the preparation of uniform, core-cross-linked PLA nanoparticles. The highest paclitaxel uptake (up to 30 wt % of the micelle core) was observed for micelles prepared from bottlebrush copolymers with branched PEO side chains, with paclitaxel uptake increasing with the size of PEO side chains. On the other hand, micelle photo-cross-linking efficiency was the highest (up to ∼90%) for copolymers with linear PEO side chains and decreased with increasing size of the hydrophilic headgroup. These trends were attributed to the decrease in molecular packing efficiency inside micelle cores for copolymers with larger and more rigid hydrophilic headgroups. For poorly packed micelles, paclitaxel loading improved core photo-cross-linking efficiencies, suggesting structural rearrangements inside micelles with cargo uptake. Preliminary results also showed that paclitaxel release from bottlebrush micelles was slowed down with increasing degree of core cross-linking.

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range of sizes and physical properties.2,16 Compared to small surfactants, polymer amphiphiles afford better structural control of material properties and enhanced thermodynamic stability of the formed aggregates. Drug delivery has been a particularly challenging arena where amphiphilic block copolymers are poised to make an impact.17−21 One of the most widely studied systems in this field are linear block copolymers of poly(lactic acid) (PLA) and poly(ethylene oxide) (PEO),22−24 which are in the late stage clinical trials for the delivery of anticancer drug paclitaxel.25,26 While polymeric micelles prepared from linear amphiphilic block copolymers carry a tremendous potential in biomedical applications, numerous challenges remain pertaining to improving their in vivo stability, drug loading, and controlled release without compromising biocompatibility and biodegradability.27−29 In an attempt to alleviate some of the

INTRODUCTION Amphiphilic block copolymers, composed of covalently connected hydrophilic and hydrophobic polymer chains, have been utilized in a variety of applications as compatibilizers, emulsifiers, rheological modifiers, and drug delivery vehicles thanks to their ability to self-organize at interfaces and form micellar aggregates in solution.1−3 Linear block copolymer amphiphiles have been a workhorse in the majority of these applications. On the other hand, polymeric surfactants with highly branched architectures offer some unique structural advantages for the refinement of materials properties.4−15 Herein, we report a new class of bottlebrush polymer amphiphiles with the emphasis on how subtle variations in molecular architecture can be used to manipulate cargo loading and core cross-linking in the polymeric micelles produced on their basis. Self-assembly of linear amphiphilic block copolymers in a selective solvent has been exploited for the fabrication of spherical, cylindrical, and bilayer vesicle aggregates with a wide © XXXX American Chemical Society

Received: October 6, 2016 Revised: January 19, 2017

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large number of end groups, for both PLA and PEO side chains, that can be potentially utilized for the attachment of various functional units to improve the overall effectiveness and utility of the micellar aggregates. In this report, we describe the synthesis of coumarinfunctionalized PLA−PEO bottlebrush block copolymers with poly(oxanorbornene) backbones and four different side-chain architectures and explore the effect of molecular packing on their capacities for drug loading and core cross-linking. Coumarin groups attached to the ends of hydrophobic PLA side chains provide an effective means of fabricating highly stable, core-cross-linked spherical nanoparticles without compromising their biodegradability. We show that side-chain architecture plays a critical role in controlling molecular packing within the micellar core, which affects the efficacy of both cargo loading and cross-linking.

inherent limitations of linear amphiphilic block copolymers, we recently reported30 a new class of amphiphilic PLA−PEO block copolymers with a bottlebrush architecture31−33 (Figure 1).

Figure 1. Self-assembly of amphiphilic bottlebrush block copolymers in water.

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These giant, comb-shaped surfactants are composed of PLA and PEO side chains densely grafted along a vinyl polymeric backbone in a blocky arrangement. Steric repulsion between the side chains forces the backbone to stretch out and results in a persistent cylindrical macromolecular shape, which can be easily manipulated by the appropriate choice of side chains.34−36 We have shown that judicious sculpting of bottlebrush amphiphiles can be used to produce well-defined nanoparticles by single-molecule templating as well as aqueous self-assembly methods.37−39 Thus, PLA−PEO bottlebrush block copolymers with carefully chosen side chain asymmetry assemble into highly uniform spherical micelles with nanomolar critical micelle concentrations.30 There are several unique attributes of bottlebrush copolymer amphiphiles that merit further investigation. Unlike linear block copolymer surfactants, where chain stretching of the hydrophobic block plays an important role in accommodating for different interfacial curvatures, bottlebrush copolymers are characterized by a persistent molecular shape with limited backbone flexibility, which can manifest itself in packing frustrations within the micellar core. This concept is illustrated in Figure 2, where chain stretching and molecular packing in

EXPERIMENTAL SECTION

Materials and Methods. All solvents and reagents were used as received unless noted otherwise. Poly(ethylene oxide) methyl ether methacrylate (PEOMA, Mn = 500 g/mol) was purified by passing over basic alumina. Dichloromethane (DCM) was dried using a commercial solvent purification system (Innovative Inc.). DL-Lactide (LA) was recrystallized from ethyl acetate. Oxanorbornene-functionalized diol (1) was synthesized according to the literature procedure.40 7(Carboxymethoxy)-4-methylcoumarin,41 poly(ethylene oxide) (PEO) macromonomer (4) (Supporting Information),42 and alkyl bromide initiator (5) (Supporting Information)43 were also synthesized with slight modifications of the previously published procedures. All 1H NMR spectra were recorded on a Varian INOVA-500 (500 MHz) spectrometer by using CDCl3 as a solvent. GPC analysis was performed by using Viscotek GPC Max and TDA 302 Tetradetector Array system equipped with two PLgel MIXED-C columns (Agilent). The detector unit contained refractive index, UV, viscosity, low (7°), and right angle light scattering modules. Measurements were carried out in THF with 1 vol % triethylamine as a mobile phase at 30 °C. Further GPC data were obtained from Viscotek GPC system equipped with a VE-3580 refractive index (RI) detector, a VE 1122 pump, and two mixed bed organic columns (PAS-103 M and PAS-105M). DMF (HPLC grade) with 0.1 M LiBr was used as a mobile phase with a flow rate of 0.5 mL/min at 55 °C. Both GPC systems were calibrated with 10 polystyrene (PS) standards from 1.2 × 106 to 500 g/mol. Photocross-linking was performed by using a 15 W high-pressure mercury UV bench lamp (Ultraviolet Products XX-15L). UV−vis spectra were recorded on an Agilent UV−vis 8453 spectrophotometer. Dynamic light scattering (DLS) analysis was conducted on Zetasizer Nano ZS90 (Malvern) at room temperature. Cryo-TEM analysis was performed by applying 5 μL of the sample onto a 300 Cu grid covered with a lacey carbon film that was freshly glow discharged to render it hydrophilic (Elmo, Cordouan Technologies). The grid was rapidly plunged into liquid ethane slush by using a homemade freezing machine with a controlled temperature chamber. The grids were then mounted onto a Gatan 626 cryoholder and observed under low dose conditions on a Tecnai G2 microscope (FEI) operating at 200 kV. The images were recorded with a slow scan CCD camera (Eagle 2k2k FEI). Synthesis of Double-Graft Polylactide (PLA) Macromonomer (2). To a round-bottom flask, diol 1 (0.50 g, 1.48 mmol) and DL-lactide (2.88 g, 20.0 mmol) were added in a glovebox. Dried DCM (50 mL) was then added, and the mixture was stirred until all polymer dissolved. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 30.0 μL, 0.20 mmol) was then injected into the flask. After stirring at room temperature for 1 h, the reaction was quenched by adding benzoic acid (250 mg, 2.05 mmol). The resulting polymer was precipitated from DCM into hexanes (2×) and into methanol/water (1:1) (2×). The polymer was dried under vacuum at room temperature for 24 h. Yield = 1.9 g (66%). SEC (PS standards): Mn = 3.1 kg/mol, Đ = 1.12 1H NMR: n(LA) = 15.

Figure 2. Self-assembly of (top) flexible amphiphilic block copolymers and (bottom) hypothetical rod-like amphiphiles with progressively increasing hydrophilic blocks.

flexible polymer amphiphiles are contrasted with those of hypothetical rigid amphiphiles with increasing hydrophilic headgroup. In the latter case, an increase in the rigid head size leads to inefficient packing inside the micelle core. The behavior of bottlebrush surfactants is expected to fall somewhere between these two extremes. Another unique aspect of such a branched architecture is the availability of a B

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Scheme 1. Four Families of Amphiphilic Bottlebrush Copolymers with Different Side-Chain Architectures Studied in This Work

μmol) were mixed, evacuated, and refilled with nitrogen three times. Deoxygenated PEOMA (2 mL) was added to the flask containing the catalyst mixture, and the contents were stirred under nitrogen until a homogeneous brown-red catalyst was obtained (∼2 h). The catalyst solution (0.1 mL) was transferred to the reaction tube via a nitrogenflushed syringe under heavy nitrogen flow, and the polymerization was carried out at 45 °C for 18 h. The reaction mixture was then cooled down and precipitated in hexanes (4×). Obtained viscous brush copolymer was dried under vacuum. Aqueous Self-Assembly. Block copolymers were dissolved in dichloromethane in a round-bottom flask. After the solvent was completely evaporated, ultrapure water (18.2 MΩ·cm) was added to the flask to obtain the polymer concentration of 1 mg/mL, and the solution was stirred at 60 °C overnight. Core Cross-Linking of Bottlebrush Block Copolymer Micelles. Aqueous bottlebrush block copolymer micellar solutions (1 mg/mL) in a glass vial were exposed to UV irradiation at 365 nm (600 mW/cm2) for 2 h. For the determination of the intermolecular cross-linking efficiency, the cross-linked samples were dried and then redissolved in DMF (0.1 M LiBr) for SEC analysis. The intermolecular cross-linking efficiency is calculated from the area ratios of the peaks belonging to molecular copolymers and the core-cross-linked micelles. Paclitaxel (PTX) Loading Capacity. Paclitaxel (0.2 mg) was transferred to a round-bottom flask via acetonitrile stock solution, and the solvent was evaporated to dryness. Block copolymer dissolved in DCM was added to the same flask to form a homogeneous PTX− copolymer film after complete solvent evaporation. Ultrapure water was added, and the mixture was stirred at 60 °C overnight. The resultant drug-loaded micelle solution was filtered through a 0.45 μm filter. The samples were dried and redissolved in DMF (0.1 M LiBr) prior to measurements. PTX loading capacity was measured by SEC (DMF) analysis. Release Studies. The drug-loaded polymer micelle solution was enclosed in a regenerated cellulose dialysis membrane with a MWCO of 3500 g/mol to allow selective drug passage. The membrane containing the micelle solution was suspended in 100 mL of PBS at 37 °C. Micelle solution aliquots (0.5 mL) were taken periodically to measure the remaining PTX concentration by using SEC (DMF) analysis.

Synthesis of Coumarin-Functionalized Double-Graft PLA Macromonomer (3). PLA macromonomer 2 (0.1 g, 0.04 mmol), 7(carboxymethoxy)-4-methylcoumarin (0.05 g, 0.2 mmol), and 4(dimethylamino)pyridine (DMAP, 0.003 g, 0.07 mmol) were mixed in 2.5 mL of anhydrous 4/1 (v/v) DCM/DMF for 30 min at 0 °C under a N2 atmosphere. Then, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) (0.042 g, 0.2 mmol) in 3 mL of DCM was added dropwise at 0 °C, and the reaction mixture was allowed to stir for 20 h at room temperature. The resultant reaction mixture was extracted several times with saturated NaHCO3 solution, 1 M HCl solution, and water. The final product was obtained by precipitation from hexanes. SEC (PS standards): Mn = 3.2 kg/mol, Đ = 1.14 1H NMR: functionalization = 80+%. Synthesis of Bottlebrush Copolymers with Double-Graft PLA and Linear PEO Side Chains (L2cO-x). In a glovebox, two separate vials were prepared containing solutions of macromonomers in dry DCM. Vial 1: PEO macromonomer 4 (30 mg, 12.3 μmol) dissolved in 0.1 mL of DCM. Vial 2: double-graft PLA macromonomer 3 (23 mg, 7.85 μmol) dissolved in 0.1 mL of DCM. In a glass vial, third-generation Grubbs’ catalyst (3.8 mg, 4.3 μmol) was dissolved in 1 mL of DCM. Once dissolved, 0.1 mL of the catalyst solution (0.38 mg) was injected to vial 1 and allowed to stir for 30 min. Then, a small aliquot (50 μL) was extracted and quenched with ethyl vinyl ether, and PLA solution in vial 2 was quickly added to vial 1. After 4 h, vial 1 was quenched with ethyl vinyl ether. The resulting brush polymers were precipitated in hexanes and dried under vacuum for 24 h. Synthesis of Double-Graft PLA Macroinitiator Block Copolymers (6). In a glovebox, two separate vials were prepared containing monomer solutions in dry DCM. Vial 1: alkyl bromide initiator 5 (50 mg, 0.125 mmol) dissolved in 2 mL of DCM. Vial 2: double-graft PLA macromonomer 3 (40 mg, 13.7 μmol) dissolved in 0.3 mL of DCM. In a glass vial, third-generation Grubbs’ catalyst (5.5 mg, 6.2 μmol) was dissolved in 1 mL of DCM. The whole catalyst solution (5.5 mg) was injected to vial 1 and allowed to stir for 15 min. Then, an aliquot of 0.2 mL was extracted and quickly added to vial 2. The leftover solution in vial 1 (2.8 mL) was immediately quenched with ethyl vinyl ether. After 4 h, vial 2 was also quenched with ethyl vinyl ether. The resulting polymers were precipitated in hexanes and dried under vacuum for 24 h. Synthesis of Bottlebrush Copolymers with Double-Graft PLA and Branched PEO Side Chains (L2cOb-x). PEOMA was bubbled with nitrogen for 30 min in a septum-capped vial to remove oxygen. Double-graft PLA macroinitiator 6 (40 mg, 9.9 μmol of bromide groups), toluene (1.0 mL), and deoxygenated PEOMA (0.85 mL) were placed in a reaction tube and subjected to three freeze− pump−thaw cycles. In a separate flask, CuCl (19.6 mg, 198 μmol), CuCl2 (2.6 mg, 7.4 μmol), and 4,4-dinonyl-2,2′-dipyridyl (82 mg, 200

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RESULTS AND DISCUSSION Molecular Design. We prepared amphiphilic PLA−PEO bottlebrush block copolymers with four different side-chain arrangements to explore the effect of polymer architecture on molecular packing, cargo loading, and cross-linking. The utilized bottlebrush copolymers were based on polyC

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the primary factor governing the molecular shape and the interfacial curvature during polymer assembly.30 Polymer Synthesis. Scheme 2 summarizes the synthesis of bottlebrush copolymers with double-graft PLA side chains. PLA macromonomers 2 were synthesized by DBU catalyzed ringopening polymerization48 of DL-lactide from oxanorbornene initiator 1 with two hydroxy groups and were subsequently endfunctionalized by reaction with 7-(carboxymethoxy)-4-methylcoumarin to produce macromonomer 3. Successful coumarin functionalization was confirmed by 1H NMR analysis by the appearance of characteristic coumarin peaks in the aromatic region (Figure 3). By comparing integral areas of the coumarin signal at 6.18 ppm relative to the PLA signal at 5.20 ppm, we calculated the functionalization efficiency to be 85 ± 5%. The SEC trace of the functionalized polymer 3 shifted to slightly lower elution volume compared to the precursor polymer 2 likely due to a slight increase in molecular weight as a result of coumarin addition (Figure 3). Amphiphilic bottlebrush copolymers with linear PEO and coumarin-functionalized, double-graft PLA side chains (L2cO) were synthesized by sequential ROMP of the corresponding macromonomers 4 and 3. An aliquot taken upon completion of PEO macromonomer polymerization and before the addition of the PLA macromonomer allowed for the determination of the number of repeat units in the first block by SEC-LS analysis using PEO dn/dc = 0.057 (THF, 30 °C). SEC characterization of the final bottlebrush copolymer revealed effective reinitiation from the first block and complete consumption of both macromonomers (Figure 4). 1H NMR analysis of the PLA−PEO bottlebrush copolymer was used to determine the length of PLA brush

(oxanorbornene) backbones, synthesized by a modular grafting-through process based on ring-opening metathesis polymerization (ROMP) of the corresponding macromonomers.44−47 Four bottlebrush block copolymer systems explored in this study are shown in Scheme 1 and are composed of (1) single-graft PLA and linear PEO side chains (L1cO-x), (2) single-graft PLA and branched PEO side chains (L1cOb-x), (3) double-graft PLA and linear PEO side chains (L2cO-x), and (4) double-graft PLA and branched PEO side chains (L2cOb-x). In the polymer coding system used throughout this text, subscripts “1” or “2” indicate the number of PLA branches per repeat unit, subscript “c” indicates the presence of coumarin groups at the end of PLA side chains, and subscript “b” indicates the branched nature of PEO side chains. The number immediately following the polymer sample code is the weight fraction of PEO in a given copolymer. For the copolymer system with single-graft PLA and branched PEO side chains, we also prepared coumarin-free analogues (L1Ob-x) as a control. For all polymers used in this study, PLA molecular weight per brush backbone repeat unit was equal to 2.2−2.4 kg/mol. Thus, double-graft PLA side chains were half as long as single-graft PLA ones (and had double number of coumarin end groups). Branched PEO side chains had a “brush-like” architecture themselves and were expected to form a much more compact and rigid hydrophilic block than linear PEO side chains. Additionally, PLA and PEO brush backbone lengths are roughly symmetric in all copolymers with branched PEO side chains, and polymer asymmetry was controlled by the length of PEO side chains. In our previous work on bottlebrush amphiphiles, we demonstrated that side chain asymmetry is D

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Figure 5. 1H NMR (CDCl3) spectrum of the bottlebrush copolymer L2cO-57.

chains by ATRP grafting50,51 of PEOMA (Mn = 500 g/mol) initiated from pendant bromide groups of 6. The average number of PEOMA units per bottlebrush backbone repeat unit was calculated from 1H NMR spectrum of the final copolymer, where integrated areas of the PLA signal at 5.20 ppm were compared to that of PEO end group signal at 3.40 ppm (Figure 6). The formation of well-defined copolymers was confirmed by SEC analysis (Figure 7). Amphiphilic bottlebrush copolymers with single-graft PLA side chains in the hydrophobic block and linear (L1cO) and branched PEO side chains (L1cOb) in the hydrophilic block as well as coumarin-free bottlebrush copolymers (L1Ob) were synthesized by similar methods using single chain PLA

1

Figure 3. H NMR and SEC characterization of double-graft PLA macromonomer (2) and coumarin end-capped PLA macromonomer (3).

Figure 4. SEC traces of double-graft PLA macromonomer (3), PEO macromonomer (4) and its homopolymer aliquot poly(4), and final bottlebrush block copolymer L2cO-57.

backbone by comparing integral areas of the PLA signal at 5.20 pm and that of the PEO signal at 3.65 ppm (Figure 5). For the synthesis of bottlebrush copolymers with doublegraft PLA and branched PEO side chains (L2cOb), we utilized a combination of grafting-through and grafting-from methods because direct ROMP of branched PEO macromonomers proved challenging and did not yield complete macromonomer conversions (Figure S9). First, we prepared a block copolymer of PLA brush and macroinitiator for atom transfer radical polymerization (ATRP)49 by sequential ROMP polymerization of bromo-functionalized oxanorbornene derivative 5 and PLA macromonomer 3. An aliquot taken after the first block allowed us to confirm complete monomer consumption of 5 and to measure the degree of its polymerization by SEC-LS (dn/dc = 0.13 in THF, 30 °C). The length of double-graft PLA brush was then calculated by 1H NMR analysis where the integral area of the PLA signal at 5.20 ppm was compared to that of signal at 1.95 ppm, corresponding to methyl groups of poly(5) block (Figure S10). In the next step, we installed branched PEO side

Figure 6. 1H NMR (CDCl3) spectrum of the bottlebrush copolymer L2cOb-68. E

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Figure 7. SEC traces of double-graft PLA macromonomer (3), macroinitiator homopolymer aliquot poly(5), PLA brush macroinitiator diblock copolymer (6), and final bottlebrush block copolymer L2cOb-68.

macromonomers, as described in the Supporting Information. Structural characteristics of all synthesized PLA−PEO bottlebrush block copolymers are summarized in Table 1. Aqueous Assembly. Micellar aggregates from amphiphilic bottlebrush block copolymers were prepared by a direct dissolution method, where a polymer film at the bottom of a vial was hydrated with enough water to reach 1 mg/mL concentration and stirred at 60 °C (above PLA Tg) for a day. Dynamic light scattering (DLS) analysis confirmed the formation of micellar aggregates with hydrodynamic diameters varying from 52 to 150 nm (Table 1). On the basis of the PEO content (43−93 wt %) in the chosen copolymers and our previous study on bottlebrush amphiphiles,30 we expected the formation of spherical micelles for all copolymers utilized in this work. For samples L2cOb-68 and L2cO-57, the formation of uniform spherical micelles was corroborated by cryogenic transmission electron microscopy (cryo-TEM, Figure 8). Core diameters of the spherical aggregates were measured directly from cryo-TEM images to be 25 ± 5 nm for L2cOb-68 and 31 ± 5 nm for L2cO-57. These dimensions are close to the double of the end-to-end distance of a hypothetical fully stretched PLA

Figure 8. Cryo-TEM analysis of polymer micelles prepared from copolymers (A) L2cOb-68 and (B) L2cO-57.

brush backbone (∼0.6 nm per oxanorbornene repeat unit), suggesting highly stretched conformations of the bottlebrush backbone inside the micellar cores. For L2cO-57, we could also distinguish a hydrophilic shell composed of rod-like brushes

Table 1. Structural Parameters of the Synthesized PLA−PEO Bottlebrush Block Copolymers and Their Micellar Aggregates in Aqueous Solutions backbonea sample

nPLA

nPEO

L1Ob-64 L1Ob-80 L1cOb-62 L1cOb-69 L1cOb-87 L1cOb-93 L2cOb-59 L2cOb-60 L2cOb-68 L1cO-45 L1cO-51 L2cO-43 L2cO-47 L2cO-57

33 28 25 26 26 29 31 31 24 41 35 38 40 29

38 35 27 38 38 36 28 35 24 33 37 27 33 38

side chainb Mn,PLA (kg/mol) 2.3 2.3 2.3 2.3 2.3 2.3 2× 2× 2× 2.3 2.3 2× 2× 2×

1.1 1.2 1.1

1.1 1.1 1.2

Mn,PEO (kg/mol)

Mnc (kg/mol)

Đd

wPEOe

Dhf (nm)

PDIf

PTXg (%)

3.5 7.5 3.5 3.5 11 26 3.5 3.0 4.5 2.3 2.3 2.3 2.3 2.3

231 347 168 213 498 1023 184 197 175 178 173 153 172 160

1.15 1.16 1.13 1.25 1.26 1.28 1.32 1.35 1.19 1.26 1.25 1.27 1.29 1.21

0.64 0.80 0.62 0.69 0.87 0.93 0.59 0.60 0.68 0.45 0.51 0.43 0.47 0.57

58 52 150 123 106 88 112 129 77 149 148 92 102 117

0.26 0.22 0.29 0.41 0.32 0.50 0.19 0.10 0.15 0.29 0.28 0.10 0.18 0.23

2.7 2.3 5.2 6.5 3.7 0.6 6.0 6.5 5.4 4.3 4.4 2.2 4.8 2.3

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.5 0.2 1.0 0.1 0.1 1.0 1.0 1.0 0.7 0.9 0.3 1.0 0.1

PTXcoreh (%)

f x‑linki (%)

± ± ± ± ± ± ± ± ± ± ± ± ± ±

n/a n/a −j 71 15 (32) −j 65 (71) 60 51 (73) 72 (76) 80 (86) 86 (85) 88 (88) 85

7 12 14 21 30 9 15 16 17 8 9 4 9 5

0.3 2.6 0.5 3.2 0.8 1.5 2.5 2.5 3.1 1.3 1.8 0.5 1.8 0.2

a Number of repeat units in the bottlebrush backbone for each block. bAverage molecular weight of the side chain for each block. cTotal molecular weight of the final bottlebrush block copolymer obtained by a combination of SEC-LS and 1H NMR. dDispersity of the final bottlebrush block copolymer from SEC with polystyrene calibration. eWeight fraction of PEO in the bottlebrush block copolymers. fHydrodynamic diameter and polydispersity values of the bottlebrush copolymers aggregates in water obtained by DLS. gWeight percentage of paclitaxel loaded into the bottlebrush copolymer micelles. hWeight percentage of loaded paclitaxel relative to the PLA core. iCross-linking efficiency of empty bottlebrush copolymer micelles. Numbers in parentheses indicate cross-linking efficiencies of micelles loaded with paclitaxel. jNot measured.

F

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The comparison between PTX loading capacities of copolymers L1Ob vs L1cOb suggests that coumarin derivatization of PLA chain ends leads to a significant increase in paclitaxel uptake. This result is consistent with the increase in hydrophobicity of the PLA block upon conversion of PLA hydroxy end groups to coumarins, which creates a more favorable environment for paclitaxel encapsulation. On the other hand, for copolymers with linear PEO side chains, double-graft PLA amphiphiles (L2cO) show lower paclitaxel loading capacities than single-graft PLA amphiphiles (L1cO) despite having a higher number of coumarin groups per copolymer. Another trend that can be observed from data in Table 1 is that copolymers with branched PEO side chains seem to have a higher paclitaxel uptake capacity than those with linear PEO branches. This effect is most vividly illustrated by comparing copolymers L2cOb-60 and L2cO-57, which possess similar structural characteristics, such as the molecular weight and PEO weight fraction, but differ only in the architecture of PEO side chains. Among these two copolymers, the one with branched PEO side chains (L2cOb-60) seems to have a markedly higher uptake capacity for paclitaxel. We hypothesize that hydrophilic brushes with branched (brush-like) PEO side chains produce a more conformationally restricted headgroup than those with linear PEO side chains. Combined with the inflexible nature of PLA brushes in the hydrophobic core, this creates a scenario where packing of hydrophobic brushes within the micellar core cannot be optimized due to the size and rigidity of the hydrophilic headgroup (see Figure 2), which allows for an increased uptake of the hydrophobic cargo. This hypothesis is reinforced by the observation that increasing the size of PEO block (via installment of longer PEO side chains) leads to an increase in the amount of paclitaxel inside the micelle cores, as measured by PTXcore parameter. For example, PTXcore loading capacity of copolymers L1cOb-x increases with increasing PEO block size, reaching a maximum of 30% for L1cOb-87. The latter number is noteworthy, as it implies that for this copolymer a third of the hydrophobic micelle core is composed of paclitaxel. Such extremely high local concentrations of paclitaxel are rarely achieved with linear PLA−PEO copolymer micelles, even though their overall PTX loading capacities (per mass polymer) might be higher.52−58 This unusual behavior is consistent with the presence of a large hydrophilic headgroup and inflexible hydrophobic brushes creating a loosely packed micellar cores capable of higher cargo uptake. There is a limit to how big of a headgroup can be tolerated. For example, for copolymer L1cOb93, the PEO block becomes so big that micelle formation is disrupted, thus lowering the amount of uploaded PTX. For this copolymer, we had to use concentrations of upward of 4 g/L to observe any aggregation by DLS. The dependence of paclitaxel loading within the micelle core on the size of the hydrophilic block is global (Figure 9) and is observed for copolymer systems with branched PEO side chains (filled circles) and linear PEO side chains (filled squares). In Figure 9, we plot PTXcore loading capacities versus PEO weight fraction of the copolymer, which we take to be a good measure of the size of the hydrophilic group since all copolymers have a similar size PLA block. Open circle markers in Figure 9 represent loading capacities of coumarin-free copolymers, and they are consistently lower than those of their coumarin-functionalized analogues (filled markers). Given that filled markers represent copolymers with different architectures in Table 1, the scatter in the data is understandable, but the

protruding from the central core (Figure 8B). The thickness of the hydrophilic shell was measured to be ∼15 nm, shorter than the end-to-end distance of a fully stretched PEO brush backbone (22 nm). Under the assumption that the density of micellar cores is equal to melt density of PLA, we calculated the aggregation numbers for L2cOb-68 and L2cO-57 to be 110 and 170, respectively. The analysis of data in Table 1 reveals that coumarinfunctionalized amphiphilic bottlebrushes with single-graft PLA side chains (L1cOb) produce much larger micelles than their coumarin-free analogues (L1Ob) with similar compositions (for example, L1Ob-64 vs L1cOb-69). We hypothesize that replacing hydroxy end groups of PLA side chains in L1Ob with coumarin functionalities in L1cOb increases the hydrophobicity of the PLA block and leads to increased chain stretching of the PLA brush backbone away from the hydrated PEO interface, thus swelling the dimensions of the micelles. This suggests that there is a certain degree of conformational freedom available to bottlebrush amphiphiles with single-graft PLA side chains, and they do not behave like the hypothetical rod-like surfactants depicted in Figure 2. We also note that bottlebrush amphiphiles with double-graft PLA side chains produce more uniform micelles (as measured by DLS PDI values) than those with single-graft PLA side chains with otherwise similar compositions. This trend holds for both amphiphiles with linear PEO side chains (L1cO vs L2cO) and branched PEO side chains (L1cOb vs L2cOb). In fact, the most uniform micelles with PDI values of 0.10 were produced from double-graft PLA copolymers L2cOb-60 and L2cO-43 (Table 1). Additionally, double-graft PLA amphiphiles appeared to form slightly smaller aggregates than their single grafted PLA analogues, but this trend could be related to the aforementioned differences in polydispersity, since the z-averaged hydrodynamic diameters will be heavily affected by the changes in the micelle size distribution. Drug Loading. Hydrophobic anticancer drug paclitaxel (PTX) was effectively solubilized in water using amphiphilic bottlebrush copolymers. Paclitaxel was chosen both due to its clinical relevance and as a model cargo compound to elucidate molecular packing inside the micelle cores. After PTX was codissolved in water with amphiphilic bottlebrushes at 60 °C, the solution was filtered with 0.45 μm Teflon filters to remove any large PTX aggregates that were not incorporated into micelles. Paclitaxel loading capacities were measured by SEC analysis in DMF, where refractive index detector signals from a copolymer and PTX can be easily resolved and the amount of PTX is quantified against a calibration curve constructed using standard PTX solutions. For amphiphilic bottlebrush copolymers used in this work, paclitaxel loading capacities of up to 6.5 wt % (Table 1) were obtained, which is in general agreement with the values reported for PLA−PEO block copolymers.52−58 However, it must be noted that there is a large variation in the reported numbers for PTX loading capacities of PEO−PLA copolymers, which we believe stems from different sample preparation protocols. Failure to remove large PTX aggregates that form in water upon sonication (even in the absence of the copolymer) will lead to overestimation of loading capacities. In this study, we were mostly concerned with paclitaxel entrapped inside the micellar cores, and so large aggregates were filtered out. As shown in Table 1, we also calculate weight percentage of paclitaxel relative to PLA block only (PTXcore), which is a better estimate of the loading capacity of the hydrophobic micelle core. G

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Figure 9. Dependence of micelle core paclitaxel loading on the size of hydrophilic headgroup. Open circles: coumarin-free copolymers L1Obx; filled circles: branched PEO copolymers L1cOb-x and L2cOb-x; filled squares: linear PEO copolymers L1cO-x and L2cO-x. Blue markers indicate double-graft PLA amphiphiles.

Figure 10. SEC (DMF) traces of bottlebrush copolymer L2cO-57 before (A) and after (B) photo-cross-linking in aqueous solution.

bottlebrush solutions are those corresponding to core-crosslinked micelles and intramolecularly cross-linked individual bottlebrushes with nothing in between could suggest that the first intermolecular reaction between two bottlebrushes within the core is the most difficult, and once it takes place, further cross-linking is facilitated. We calculated cross-linking efficiency ( f x‑link) by comparing RI detector signal areas of peaks corresponding to cross-linked micelles and intramolecularly cross-linked copolymers. Thus, f x‑link represents the percent of bottlebrush amphiphiles that were incorporated into the crosslinked micelle upon UV exposure. Concentrations of coumarin groups inside the polymer micelle cores were calculated to be around 1 M for L2cO-57 and L2cOb-68 based on core diameters and aggregation numbers obtained from cryo-TEM images. For all doublegraft PLA copolymers, the weight fraction of coumarin relative to the PLA block was ∼10%, while for single-graft PLA copolymer it was half of that. Despite such high and similar coumarin concentrations inside the micelle cores, cross-linking efficiencies obtained for different bottlebrush amphiphiles varied from 15% to 88% (Table 1). Copolymers with linear PEO side chains generally exhibited better cross-linking efficiencies than their counterparts with branched PEO side chains. For example, copolymer L2cOb-60 with branched PEO side chains was found to have f x‑link of 60%, while its analogue with linear PEO side chains (L2cO-57) exhibited a much higher cross-linking efficiency of 85%. Since the reaction between two coumarin groups requires them to be in a close proximity with each other, we hypothesize that micelle cores with better packed PLA brushes will show higher cross-linking efficiencies. This behavior is consistent with better molecular packing of copolymers with linear PEO side chains, similar to what we observed for paclitaxel loading experiments. When all measured f x‑link values are plotted against the weight fraction of PEO in the copolymer (Figure 11), a clear trend emerges: increasing the size of the hydrophilic headgroup in the copolymer leads to diminished cross-linking within its micelle core. Core-cross-linking reactions were conducted at room temperature (below PLA Tg) in what would be considered a frozen state for micelle cores. Carrying out photo-cross-linking at 60 °C (above PLA Tg) did not lead to increase in crosslinking efficiency (for example, 72% cross-linking efficiency was obtained for L1cOb-69 at 60 °C compared to 71% at room temperature), indicating that increased segmental mobility does

overall trend is unmistakable: paclitaxel amount within the hydrophobic core increases with the size of the hydrophilic headgroup (as measured by PEO weight fraction in the copolymer). The trend breaks down for very asymmetric copolymers (wPEO > 0.9 for copolymers with branched PEO side chains), where micelle formation is disrupted by the presence of an extremely large hydrophilic headgroup. Core Cross-Linking. The presence of coumarin end-group functionalities at PLA side chains not only increases paclitaxel loading capacities of the PLA−PEO bottlebrush amphiphiles but also provides a handle for covalent fixation of the aggregates via photoactivated 2π + 2π cycloaddition.59 Since core crosslinking requires a reaction between different bottlebrush molecules, it can also shed light on molecular packing within the micelle cores. Empty and paclitaxel-loaded polymeric micelles were cross-linked in water upon exposure to UV light (365 nm) for 2 h at a concentration of 1 g/L, which is 2−3 orders of magnitude higher than expected critical micelle concentrations for these copolymers.30 Complete consumption of coumarin groups was confirmed by UV−vis analysis, where the characteristic absorption band at 320 nm completely disappeared after UV exposure (Figure S11).60,61 However, not all coumarin dimerization necessarily contributes to micelle cross-linking. Reactions between two coumarin groups within the same PLA brush molecule would not lead to micelle crosslinking, while producing the same UV signal. Therefore, we quantified micelle core cross-linking by SEC analysis in DMF, which is a good solvent for both PLA and PEO and can easily distinguish between covalently cross-linked micelles and the precursor copolymer. As shown in Figure 10, upon micelle photo-cross-linking, the original copolymer peak completely disappeared, and a new peak appeared at a much lower retention time, which we attributed to core-cross-linked micelles. This signal appeared to be monomodal with no shoulders at either side, suggesting rapid micelle core fixation without micelle−micelle reactions. Additionally, a new signal was observed at a higher elution volume (smaller size) than the original copolymer, which we attributed to the intramolecularly cross-linked copolymer that did not get incorporated into the cross-linked micelle. Intramolecular cross-linking led to the expected decrease in molecular dimensions. The fact that the only signals observed after UV irradiation of aqueous H

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tional changes. Rather, we hypothesize, paclitaxel loading leads to a conformational reorganization within PLA brushes that creates higher local concentrations of coumarin groups between brushes facilitating the cross-linking process. One possible scenario is where π−π interactions between coumarin groups and paclitaxel promote partial unfolding of PLA side chains and create a higher local concentration of coumarin groups in the vicinity of paclitaxel molecules segregated between the brushes. Another important observation from Figure 13 is that there is a correlation between the amount of paclitaxel loaded into micelle cores (PTXcore) and the empty micelle core-crosslinking efficiency (f x‑link(empty)). Since these two parameters were determined completely independently, the results suggest that the underlying forces governing both of these properties are the same, which we hypothesize to be the molecular packing of PLA brushes within the micelle core. The latter can be controlled by the proper choice of bottlebrush amphiphile molecular architecture. Core cross-linking of paclitaxel-loaded bottlebrush micelles produces stable spherical nanoparticles that maintain their biodegradability because coumarin groups are connected to the main polymer backbone via degradable PLA chains. Additionally, our preliminary studies on the release of paclitaxel from these nanoparticles suggests that the rate of drug release might be controlled by the cross-linking density within the micelle cores. Thus, for copolymers L1cOb-87, L2cOb-68, and L1cO-45 with progressively increasing cross-linking efficiencies, 6 h paclitaxel release in a phosphate buffer was measured to be 74%, 42%, and 13%, respectively. These results suggest a promising strategy for creating PLA−PEO nanoparticles with controlled and sustained release of hydrophobic drugs by architectural manipulation of the copolymers.

Figure 11. Dependence of empty micelle core cross-linking on the weight fraction of PEO in the parent copolymer (size of hydrophilic headgroup) for branched PEO copolymers L1cOb-x and L2cOb-x (circles) and linear PEO copolymers L1cO-x and L2cO-x (squares). Blue markers indicate double-graft PLA amphiphiles.

not facilitate brush−brush coupling reactions inside the micelle core. Core-cross-linking experiments were also conducted for PTX-loaded micelles (Figure 12) to create cargo-loaded PLA

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Figure 12. Synthesis of core-cross-linked paclitaxel-loaded bottlebrush copolymer micelles.

CONCLUSIONS In summary, we have synthesized amphiphilic PLA−PEO bottlebrush copolymers with four different side-chain architectures by a combination of grafting-through and grafting-from protocols. The four families of studied copolymers were characterized by the presence of single-graft and double-graft PLA side chains and linear and branched PEO side chains in different combinations. PLA side chains were end-functionalized with photoactive coumarin groups to provide a capacity for core cross-linking. All copolymers formed uniform aggregates in aqueous solutions with hydrodynamic diameters ranging from 50 to 150 nm, as obtained by DLS and corroborated by cryo-TEM analysis of selected samples. Paclitaxel loading capacities of bottlebrush amphiphiles varied from 0.6 to 6.5%, while concentration of paclitaxel inside micellar cores reached 30 wt %. Coumarin functionalization of PLA chain ends improved paclitaxel loading efficiencies. Additionally, photodimerization of coumarin groups attached to PLA side chains was used to prepare highly uniform, corecross-linked bottlebrush micelles with high degrees of efficiency. Increasing the hydrophilic block size and rigidity led to improved paclitaxel uptake by copolymer micelles while decreasing the photo-cross-linking efficiency of empty micelles. Both trends, which were obtained from independent measurements and appeared to be correlated, were attributed to decreasing packing efficiency of PLA brushes inside the micelle cores. Paclitaxel loading aided micelle core-cross-linking efficiency, indicating structural rearrangements inside the micelle core upon cargo uptake. Our preliminary results also

nanoparticles. Surprisingly, the presence of paclitaxel inside the micelle cores did not diminish cross-linking efficiencies and in some cases even led to an increase in f x‑link, as shown in Figure 13. Since the concentration of coumarin inside micelle cores actually decreases upon addition of paclitaxel, the observed results cannot be explained on the basis of average composi-

Figure 13. Increase in micelle cross-linking efficiency ( f x‑link) after paclitaxel loading (filled circles, left axis) and correlation between empty micelle cross-linking efficiency and paclitaxel loading in micelle cores (open squares, right axis). I

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(11) Luo, H.; Raciti, D.; Wang, C.; Herrera-Alonso, M. Macromolecular Brushes as Stabilizers of Hydrophobic Solute Nanoparticles. Mol. Pharmaceutics 2016, 13, 1855−1865. (12) Luo, H.; Santos, J. L.; Herrera-Alonso, M. Toroidal structures from brush amphiphiles. Chem. Commun. 2014, 50, 536−538. (13) Garofalo, C.; Capuano, G.; Sottile, R.; Tallerico, R.; Adami, R.; Reverchon, E.; Carbone, E.; Izzo, L.; Pappalardo, D. Different Insight into Amphiphilic PEG-PLA Copolymers: Influence of Macromolecular Architecture on the Micelle Formation and Cellular Uptake. Biomacromolecules 2014, 15, 403−415. (14) Aguilar-Castillo, B. A.; Santos, J. L.; Luo, H.; Aguirre-Chagala, Y. E.; Palacios-Hernandez, T.; Herrera-Alonso, M. Nanoparticle stability in biologically relevant media: influence of polymer architecture. Soft Matter 2015, 11, 7296−7307. (15) Sant, S.; Poulin, S.; Hildgen, P. Effect of polymer architecture on surface properties, plasma protein adsorption, and cellular interactions of pegylated nanoparticles. J. Biomed. Mater. Res., Part A 2008, 87A, 885−895. (16) Blanazs, A.; Armes, S. P.; Ryan, A. J. Self-Assembled Block Copolymer Aggregates: From Micelles to Vesicles and their Biological Applications. Macromol. Rapid Commun. 2009, 30, 267−277. (17) Adams, M. L.; Lavasanifar, A.; Kwon, G. S. Amphiphilic block copolymers for drug delivery. J. Pharm. Sci. 2003, 92, 1343−1355. (18) Duncan, R.; Ringsdorf, H.; Satchi-Fainaro, R. Polymer therapeutics: Polymers as drugs, drug and protein conjugates and gene delivery systems: Past, present and future opportunities. In Polymer Therapeutics I: Polymers as Drugs, Conjugates and Gene Delivery Systems, 2006; Vol. 192, pp 1−8. (19) Torchilin, V. P. Micellar nanocarriers: Pharmaceutical perspectives. Pharm. Res. 2006, 24, 1−16. (20) Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem. Soc. Rev. 2013, 42, 1147−1235. (21) Gaucher, G.; Dufresne, M. H.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J. C. Block copolymer micelles: preparation, characterization and application in drug delivery. J. Controlled Release 2005, 109, 169−188. (22) Xiao, R. Z.; Zeng, Z. W.; Zhou, G. L.; Wang, J. J.; Li, F. Z.; Wang, A. M. Recent advances in PEG-PLA block copolymer nanoparticles. Int. J. Nanomed. 2010, 5, 1057−1065. (23) Saffer, E. M.; Tew, G. N.; Bhatia, S. R. Poly(lactic acid)poly(ethylene oxide) Block Copolymers: New Directions in SelfAssembly and Biomedical Applications. Curr. Med. Chem. 2011, 18, 5676−5686. (24) Cohn, D.; Younes, H. Biodegradable PEO/PLA block copolymers. J. Biomed. Mater. Res. 1988, 22, 993−1009. (25) Liggins, R. T.; Burt, H. M. Polyether-polyester diblock copolymers for the preparation of paclitaxel loaded polymeric micelle formulations. Adv. Drug Delivery Rev. 2002, 54, 191−202. (26) Ma, P.; Mumper, R. J. Paclitaxel Nano-Delivery Systems: A Comprehensive Review. J. Nanomed. Nanotechnol. 2013, 4, 1000164. (27) Owen, S. C.; Chan, D. P. Y.; Shoichet, M. S. Polymeric micelle stability. Nano Today 2012, 7, 53−65. (28) Eetezadi, S.; Ekdawi, S. N.; Allen, C. The challenges facing block copolymer micelles for cancer therapy: In vivo barriers and clinical translation. Adv. Drug Delivery Rev. 2015, 91, 7−22. (29) Kim, S.; Shi, Y.; Kim, J. Y.; Park, K.; Cheng, J.-X. Overcoming the barriers in micellar drug delivery: loading efficiency, in vivo stability, and micelle-cell interaction. Expert Opin. Drug Delivery 2010, 7, 49−62. (30) Fenyves, R.; Schmutz, M.; Horner, I. J.; Bright, F. V.; Rzayev, J. Aqueous Self-Assembly of Giant Bottlebrush Block Copolymer Surfactants as Shape-Tunable Building Blocks. J. Am. Chem. Soc. 2014, 136, 7762−7770. (31) Verduzco, R.; Li, X.; Pesek, S. L.; Stein, G. E. Structure, function, self-assembly, and applications of bottlebrush copolymers. Chem. Soc. Rev. 2015, 44, 2405−2420.

suggest that the degree of core cross-linking slows down paclitaxel release from micelles and thus opens avenues for the rational design and preparation of biodegradable PLA nanoparticles with controlled and sustained release characteristics.

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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.6b02182. Experimental details for the synthesis of single-graft PLA copolymers with linear (L1cO) and branched PEO (L1cOb) side chains and coumarin-free analogues (L1Ob); SEC, NMR, and UV characterization (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail jrzayev@buffalo.edu (J.R.). ORCID

Amitav Sanyal: 0000-0001-5122-8329 Javid Rzayev: 0000-0002-9280-1811 Author Contributions

H.U. and S.O. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Science Foundation (DMR-1409467) and Kapoor Award from the University at Buffalo. M.E.-R. and M.S. acknowledge the EM facility of the ICS for the use of the instruments.

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REFERENCES

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