Flow-Directed Loading of Block Copolymer Micelles with Hydrophobic

Article pubs.acs.org/Langmuir

Flow-Directed Loading of Block Copolymer Micelles with Hydrophobic Probes in a Gas−Liquid Microreactor Chih-Wei Wang,† Aman Bains,† David Sinton,‡ and Matthew G. Moffitt*,† †

Department of Chemistry, University of Victoria, P.O. Box 3065, Victoria, BC, Canada V8W 3V6 Department of Mechanical and Industrial Engineering, University of Toronto, 5 King’s College Rd., Toronto, ON, Canada, M5S 3G8

‡

S Supporting Information *

ABSTRACT: We investigate the loading efficiencies of two chemically distinct hydrophobic fluorescent probes, pyrene and naphthalene, for self-assembly and loading of polystyreneblock-poly(acrylic acid) (PS-b-PAA) micelles in gas−liquid segmented microfluidic reactors under different chemical and flow conditions. On-chip loading efficiencies are compared to values obtained via off-chip dropwise water addition to a solution of copolymer and probe. On-chip, probe loading efficiencies depend strongly on the chemical probe, initial solvent, water content, and flow rate. For pyrene and naphthalene probes, maximum on-chip loading efficiencies of 73 ± 6% and 11 ± 3%, respectively, are obtained, in both cases using the more polar solvent (DMF), an intermediate water content (2 wt % above critical), and a low flow rate (∼5 μL/min); these values are compared to 81 ± 6% and 48 ± 2%, respectively, for off-chip loading. On-chip loading shows a significant improvement over the off-chip process where shear-induced formation of smaller micelles enables increased encapsulation of probe. As well, we show that on-chip loading allows off-chip release kinetics to be controlled via flow rate: compared to vehicles produced at ∼5 μL/min, pyrene release kinetics from vehicles produced at ∼50 μL/min showed a longer initial period of burst release, followed by slow release over a longer total period. These results demonstrate the necessity to match probes, solvents, and running conditions to achieve effective loading, which is essential information for further developing these on-chip platforms for manufacturing drug delivery formulations.

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INTRODUCTION

Microfluidic approaches to loading polymeric colloidal nanostructures with drugs or small probe molecules have only recently been pursued,31−37 spurred by the potential for improved control over current nanocarriers, fast and efficient screening of process parameters and formulations, and portable platforms for patient-specific point-of-care drug preparation.31,32 Although compared to bulk preparation methods, the output volume of microfluidic systems is extremely low, the low throughput should be well matched to applications such as drug delivery, where small, patient-specific quantities would be the desired output. Gas−liquid segmented microfluidic reactors are a particular class of reactor in which compartmentalized liquid plugs are segmented by a regular stream of gas bubbles;1,5 compared to their single-phase counterparts, these reactors are characterized by increased mixing rates associated with chaotic advection within the liquid phase.1,5 In addition, it has been shown that flow-variable high shear regions in the corners of the liquid plugs can strongly influence the sizes and morphologies of polymeric colloids,38,39 including block copolymer micelles,40,41 which should provide a convenient top-down handle with which to tune the structures for drug

Microfluidic reactors have provided unique environments for the synthesis of nanomaterials, with advantages derived from nanoscale volumes and laminar flow that enable fast mixing and fine control of reagents.1−5 For example, the synthesis of semiconducting nanoparticles (CdS, CdSe, and CdSe/ ZnS),6−14 metal nanoparticles (Au, Ag, Cu and Co),15−17 titanium oxide nanorods,18 and Janus and ternary polymeric particles,19−21 have been recently demonstrated on various types of microfluidic chips. In recent years, there has been considerable interest in using block copolymer micelles as nanoscale drug delivery vehicles.22−26 Block copolymer micelle morphologies include spherical, cylindrical, and vesicle forms,27,28 allowing critical drug delivery parameters such as transport, biodistribution, and circulation times to be tuned to specific drugs and patient needs.23,26 Furthermore, in comparison with their smallmolecule amphiphilic counterparts, block copolymer micelles generally exhibit higher thermodynamic and kinetic stabilities, with much lower critical micelle concentrations (cmc, 10−8− 10−9 M)29 than surfactants (∼10−3 M),29 and lower rates of dissociation, which enable slower release, greater therapeutic potential, and ultimately higher drug accumulation at a target site.30 © XXXX American Chemical Society

Received: January 2, 2013 Revised: June 3, 2013

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delivery applications.26 As well, strong and localized shear forces could also have a considerable influence on the loading efficiency of hydrophobic molecules into polymer micelles during on-chip assembly and on the subsequent release kinetics. To our knowledge, no previous study has investigated the onchip self-assembly and loading of polymeric nanocarriers in gas−liquid segmented reactors. In this paper, we study the on-chip self-assembly and loading of polystyrene-block-poly(acrylic acid) (PS-b-PAA) copolymer micelles with each of two fluorescent hydrophobic probes, pyrene and naphthalene. The self-assembly of this copolymer has been previously studied in an identical reactor in terms of the influence of shear on micelle morphologies,40,41 and so it provides a useful and well-characterized model system for loading and release studies of hydrophobic probes. These two hydrophobic compounds were selected as established and chemically distinct probes for studies of loading into copolymer micellar systems;42,43 since one probe has a higher affinity for the PS-core (pyrene) than the other (naphthalene),42,43 comparing the loading efficiencies of these probes under the same conditions should allow relationships between molecule solubility and on-chip shear effects to be characterized. To this end, the on-chip loading efficiency of each probe is evaluated for different chemical and flow conditions, and compared to values obtained using a conventional off-chip bulk nanoprecipitation method,26,44 involving dropwise water addition to a solution of block copolymer and dye molecules. For optimized chemical conditions for on-chip loading, we also evaluate the effect of flow rate on the subsequent release kinetics of the hydrophobic probe. These results show gas− liquid segmented microreactors to be promising platforms for flow-tunable loading and release of hydrophobic cargo molecules into polymer nanocarriers, with implications for various applications, including medical imaging and drug delivery.

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pure deionized water to remove organic solvent and unincorporated dyes. During dialysis, water was changed every hour for the first 8 h, and then micelle/dye solutions were allowed to dialyze overnight. The dialyzed dispersions of probe-loaded micelles were weighed on an analytical balance to determine the final copolymer concentrations. Microfluidic Chip Fabrication. High-quality silicon wafers (Silicon Quest International, Santa Clara, CA) and negative photoresist, SU-8 100 (Microchem Inc., Newton, MA), were used to fabricate negative masters. Prior to use, new silicon wafers were heated on a hot plate to at 200 °C for 20 min to remove all moisture. SU-8 films of 150 μm thickness were spin-coated onto the silicon wafers, followed by heating at 95 °C for 60 min to remove residual SU-8 solvent. A photomask was placed over the film for exposure under UV light for 180 s. The device was heated for an additional 20 min at 95 °C. After that, the device was submerged in SU-8 developer (Microchem, Newton, MA) until all unexposed photoresist was removed. The reactor has a set channel depth of 150 μm and consists of a sinusoidal mixing channel 100 μm wide and 100 mm in length, and a sinusoidal processing channel 200 μm wide and 740 mm in length. To further stabilize the process of bubble generation, we employed external resistor chips, which were connected in series between the Ar gas tank and the microfluidic chip. The PDMS resistors were fabricated in an identical fashion to the reactor chips using an appropriate photomask. The design and dimensions of the resistor chips, containing channels 1000 mm long, 400 μm wide, and 150 μm deep, are given in the Supporting Information (Figure S3). In operation, these high pressure drop resistors serve to effectively dampen the pressure fluctuations caused by the Ar gas tank and the bubble generation process itself. The resistors were designed in a way that the total pressure drop in the resistors was at least 1 order of magnitude higher than the pressure drop in the reaction channel. Microfluidic chips were fabricated from poly(dimethylsiloxane) (PDMS) using a SYLGARD 184 silicon elastomer kit (Dow Corning, Auburn, MI) with an elastomer base-to-curing agent ratio of 10:1. The elastomer and curing agent were mixed together and degassed in a vacuum chamber. The degassed PDMS was poured onto the negative master in a Petri dish and then degassed again until all remaining air bubbles in the PDMS were removed. The PDMS was then heated at 85 °C until cured (∼50 min). The microfluidic chips were cut and peeled off the master, and holes were punched through its reservoirs to allow for the insertion of tubing. A thin PDMS film was formed on a glass slide by spin-coating and was permanently bonded to the base of the microfluidic reactor after both components were exposed to oxygen plasma for 30 s. Flow Delivery and Control. Pressure-driven flow of liquids to the inlets of the reactors was provided using 250 μL and 1 mL gastight syringes (Hamilton, Reno, NV) mounted on syringe pumps (Harvard Apparatus, Holliston, MA). The microchip was connected to the syringes with 1/16 inch (OD) Teflon tubing (Scientific Products and Equipment, ON). Gas pressure was adjusted by via an Ar tank regulator as well as a downstream regulator for fine adjustments (Johnson Controls Inc.). For gas flow, connections were joined using Teflon tubing of 1/16 in. (OD) and 100 μm (ID) (Upchurch Scientific, Oak Harbor, WA). The liquid flow rate (Qliq) was programmed via the syringe pumps, and the gas flow rate (Qgas) was varied by tuning the pressure regulator. Due to the compressible nature of the gas and the high gas/liquid interfacial tension, discrepancies arise between the nominal (programmed) and actual values of Qgas, Qgas/Qliq, and the total flow rate (Qtotal).39−41 Therefore, actual gas flow rates were calculated from the frequency of bubble formation and the average volume of gas bubbles, determined from image analysis of the mean lengths of liquid and gas plugs, Lliq and Lgas, respectively, under a given flow set of conditions (Supporting Information Table S1). This method of flow determination has been previously employed in the context of gas−liquid multiphase flow in microfluidic devices.39,47 For all experiments in this paper, the relative gas-to-liquid flow ratio (Qgas/Qliq) was ∼1, and single nominal total flow rate was investigated: Qtotal = ∼5 μL/min (low flow rate case).

EXPERIMENTAL SECTION

Materials. The polystyrene-block-poly(acrylic acid) block copolymer used in this study, PS(665)-b-PAA(68), was synthesized by anionic polymerization of the corresponding polystyrene-block-poly(tert-butyl acrylate) block copolymer, followed by hydrolysis of the ester block; numbers in parentheses indicate number-average degrees of polymerization.45,46 Dimethylformamide (DMF) (Aldrich, 99.9+%, HPLC grade, H2O < 0.03%) and 1,4-dioxane (dioxane) (Aldrich, 99.0%, reagent grade, H2O < 0.05%) were used as received without further purification. The dyes, pyrene (Aldrich, ≥99.0%, fluorescence grade) and naphthalene (Aldrich, ≥99.7%, analytical standard), were used as received without further purification. Microdialysis tubes (Dtube, Dialyzer Midi, MWCO 6−8 kDa) were purchased from Novagen. Off-Chip Probe Loading. The copolymer PS(665)-b-PAA(68) was dissolved in either DMF or dioxane to a concentration of 1.0 wt % and allowed to stir overnight to form the copolymer stock solution. Pyrene or naphthalene were also dissolved in DMF or dioxane to form appropriate dye stock solutions. The dye and copolymer stock solutions were blended to give a ratio of 1 × 10−2 g dye/g polymer in either DMF or dioxane; for this work, the ratio of dye to polymer was held constant and was selected based on the results of a previous study.44 The blend dye/copolymer solutions were diluted with the appropriate pure organic solvent to a copolymer concentration of 0.33 wt % and allowed to stir for 4 h in the dark. To induce micellization and probe loading, deionized water (DI H2O) was added dropwise to the dye/copolymer solutions at a constant rate of 20 μL every 10 s with moderate magnetic stirring (600 rpm) until a target water concentration of 80 wt % was reached. The micelle/dye solutions were allowed to stir overnight in the dark. They were then dialyzed against B

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On-Chip Probe Loading. The copolymer was dissolved in DMF or dioxane to a concentration of 5.0 wt % and allowed to stir overnight to form the copolymer stock solution. Pyrene or naphthalene was also dissolved in DMF or dioxane to form appropriate dye stock solutions. The dye and copolymer stock solutions were blended to give a ratio of 1.0 × 10−2 g dye/g polymer (identical ratio to that employed in offchip loading). To form solutions for the copolymer/dye stream, blend solutions were diluted with pure DMF or dioxane to yield copolymer concentrations of 1.0 wt %. The blend solutions were stirred for 4 h in the dark. As well, to form solutions for the water-containing stream, suitable amounts of water were added to pure DMF or dioxane, taking into account the on-chip dilution factors. For on-chip micellization/probe loading experiments (Figure 1), three streams were combined at equal flow rates to form gas-separated

ments. The dialyzed dispersions of probe-loaded micelles were weighed on an analytical balance to determine the final copolymer concentrations. Determination of Probe Loading Efficiency. To determine the probe loading efficiency, probe-loaded micelles were broken down into singles chains and free probes by adding a small weighed aliquot of micelle dispersion (1−2 drops) to a known quantity of DMF (selected so that maximum absorbance values were ≤0.1) and stirring for 4 h in the dark. Fluorescence intensity−concentration calibration curves for pyrene at the emission maximum (λex = 337 nm; λem = 373 nm, Supporting Information Figure S1) and naphthalene (λex = 278 nm; λem = 322 nm, Supporting Information Figure S2) in DMF solutions were determined in the linear range; fluorescence intensities of dissolved micelle solutions were measured at the same wavelengths and under identical conditions to those of the calibration standards, and within 5 min of obtaining the calibration curves. Before calculating integrated photoluminescence intensities, a solvent background was subtracted and a correction for the detector response was applied to each sample and standard measurement. The dye concentrations were then determined from the calibration curves. After correcting for dilution factors, the loading efficiency was determined using the following expression:48

loading efficiency (%) =

mass of probe in micelles (g) × 100% total mass of probe used (g)

The various steps in the loading and characterization of loading efficiency are depicted in Figure 2. Errors on loading efficiencies were determined from standard deviations of values for three repeat loading experiments under the same conditions.

Figure 1. PS-b-PAA self-assembly process and multiphase microfluidic reactor approach. (A) Cartoon representation of the self-assembly of PS-b-PAA into micelles and concomitant probe loading in the gas− liquid microfluidic reactor. (B) Schematic of the microfluidic reactor, showing liquid and gas inlets at the injector, followed by the mixing channel (represented in A). The inset of (B) shows a select optical microscopy image of the reactor under stable operation. The white scale bar indicates 500 μm. Figure 2. Cartoon representation of probe loading, along with the measurement process for determining loading efficiencies. (A) The probes and single chains are codissolved in a common solvent (e.g., DMF). (B) Water addition causes micellization/probe entrapment when water content is above the critical water content (cwc). (C) The micelles/loaded probes are dialyzed to remove organic solvent as well as unincorporated dyes. (D) The micelles are broken up to release the incorporated dyes for quantitative fluorescence measurements of the amount of loaded dye.

liquid plugs within the reactor: (1) 1.0 wt % PS(665)-b-PAA(68) with dye concentration of 1.0 × 10−2 g dye/g polymer in DMF or dioxane; (2) DMF or dioxane only (separator stream); and (2) DMF or dioxane containing different amounts of deionized water. Combining the three liquid streams yielded a steady-state on-chip concentration of 0.33 wt % polymer with 1.0 × 10−2 g dye/g polymer ratio, identical to the off-chip loading experiments, but with various final water contents. When a steady-state on-chip water content of 80 wt % was targeted in some cases, the water/solvent stream was replaced by a pure water stream and the flow rates of the three liquid streams were adjusted accordingly; also, the concentration of the copolymer in the copolymer stream was increased 3-fold to 3.0 wt % in these cases so that the steady state copolymer concentration was 0.33 wt %. For all on-chip experiments, 50 μL of solution produced on-chip was collected into 200 μL DI of H2O such that the same final polymer concentration was obtained as in the off-chip experiment. For all experiments, the relative gas-to-liquid flow ratio (Qgas/Qliq) was ∼1, and two different nominal total flow rates were investigated: Qtotal = ∼5 μL/min (low flow rate case) and ∼50 μL/min (high flow rate case). All on-chip samples were dialyzed using microdialysis tubes following the same dialysis procedure described for off-chip experi-

Determination of Pyrene Probe Release Kinetics. To determine probe release kinetics under near-“perfect sink” conditions,44 the following experiments were carried out. A dialysis bag (MWCO: 50 000) containing 1.5 mL suspension of pyrene-loaded micelles was placed in a 250 mL beaker filled with tap water. The beaker was then placed in a crystallization dish (190 × 100) equipped with a valve for out-flowing water. Tap water was allowed to flow into the beaker (flow rate = ∼480/min) via a Tygon tube. The beaker was allowed to overflow into the crystallization dish and subsequently flow out into the sink. At various times, t, aliquots of the probe-loaded micelles were taken out of the dialysis bag and put into a quartz C

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microcuvette and subsequently analyzed using fluorescence spectroscopy (λex = 337 nm; λem = 373 nm) to determine I(t). After analysis, the aliquot of probe-loaded micelles were returned to the dialysis bag and the near-“perfect sink” conditions. For each measurement time, the percentage of dye released was calculated using % dye released = {1 − [I(t)/I(t = 0)]} × 100%. Due to the long time scale of the experiment (several days), intensity measurements were corrected for time-dependent instrumental fluctuations (i.e., lamp intensity and detector response) by periodically measuring a standard pyrene solution in DMF, at the same excitation and emission wavelengths as the sample, and normalizing measured intensity data for the sample relative to changes in intensity for the standard. Variations in measured intensities due to instrument fluctuations over the course of the experiment were small,