Scalable, Semicontinuous Production of Micelles Encapsulating

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Scalable, Semicontinuous Production of Micelles Encapsulating Nanoparticles via Electrospray Anthony D. Duong,† Gang Ruan,†,⊥ Kalpesh Mahajan,† Jessica O. Winter,†,‡ and Barbara E. Wyslouzil*,†,§ †

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 W. 19th Ave., Columbus, Ohio 43210, United States ‡ Department of Biomedical Engineering, The Ohio State University, 1080 Carmack Rd., Columbus, Ohio 43210, United States § Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Ave., Columbus, Ohio 43210, United States ⊥ Department of Biomedical Engineering, College of Modern Engineering and Applied Sciences, Nanjing University, Nanjing 210008, Jiangsu, China S Supporting Information *

ABSTRACT: Nanoparticle encapsulation within micelles has been demonstrated as a versatile platform for creating water-soluble nanocomposites. However, in contrast to typical micelle encapsulants, such as small molecule drugs and proteins, nanoparticles are substantially larger, which creates significant challenges in micelle synthesis, especially at large scale. Here, we describe a new nanocomposite synthesis method that combines electrospray, a top-down, continuous manufacturing technology currently used for polymer microparticle fabrication, with bottom-up micellar selfassembly to yield a scalable, semicontinuous micelle synthesis method: i.e., micellar electrospray. Empty micelles and micellar nanocomposites containing quantum dots (QDs), superparamagnetic iron oxide nanoparticles (SPIONs), and their combination were produced using micellar electrospray with a 30-fold increase in yield by weight over batch methods. Particles were characterized using dynamic light scattering, transmission electron microscopy, and scanning mobility particle sizing, with remarkable agreement between methods, which indicated size distributions with variations of as little as ∼5%. In addition, new methodologies for qualitatively evaluating nanoparticle loading in the resultant micelles are presented. Micellar electrospray is a broad, scalable nanomanufacturing approach that should be easily adapted to virtually any hydrophobic molecule or nanoparticle with a diameter smaller than the micelle core, potentially enabling synthesis of a vast array of nanocomposites and self-assembled nanostructures.

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INTRODUCTION Self-assembling micelles composed of amphiphilic materials have been used to encapsulate hydrophobic drugs, dramatically improving effective drug concentration and delivery via passive and/or active targeting.1,2 More recently, micellar encapsulation has also proved to be a powerful route for creating watersoluble nanostructures from hydrophobic nanomaterial precursors.3−7 Of particular interest are micellar composite nanoparticles that combine features of two or more constituent particles into a single construct.8,9 For example, particles that combine the fluorescence of quantum dots (QD) and the magnetism of superparamagnetic iron oxide nanoparticles (SPIONS) in a single nanocomposite have attracted significant interest for medical applications.6,10−12 Nanocomposite fluorescence enables imaging, whereas the magnetic property permits both imaging (e.g., magnetic resonance imaging, MRI) and particle manipulation via external magnetic fields. Thus, these particles can be used for simultaneous isolation and molecular profiling of cancer cells,13 multimodal in vivo fluorescence/MR imaging,6 and magnetic drug targeting.14 © 2014 American Chemical Society

Micellar encapsulation offers several advantages over other approaches to composite nanoparticle synthesis since constituent particles can be individually optimized during synthesis and then combined in a matrix that affords protection against the external environment. The approach has been demonstrated to be a versatile way to create both individual and composite aqueous nanostructures, incorporating a wide array of hydrophobic nanomaterials, including nanorods and spheres, and resulting in both spherical and wormlike micelle nanostructures.4−7 However, established methods to synthesize micelles encapsulating hydrophobic drugs,15 such as direct dissolution,16 dialysis,17 or solvent evaporation−film rehydration,6 display several limitations when translated to nanoparticle encapsulants. Nanoparticle encapsulants are substantially larger than small molecule drugs or proteins, and therefore, steric interactions between the particle and the amphiphile can be Received: December 11, 2013 Revised: February 20, 2014 Published: March 17, 2014 3939

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Figure 1. (a) A coaxial electrospray configuration. In this example (i.e., magnetic quantum dot (MagDot) synthesis), the organic phase, including chloroform, flows through the inner needle and the aqueous phase, 5% w/v poly(vinyl alcohol) (PVA) in water, flows through the outer needle. (b) Image of the cone-jet. (Left) In the absence of an electric field, concentric droplets with a spheroidal meniscus are formed. (Center) As voltage is applied (∼2 kV), the outer aqueous meniscus deforms. (Right) As voltage is increased (∼3.5 kV), a convergent electrospray jet is emitted.

significant and may hinder the self-assembly process. We4,7 and others5,6 have observed that the size of the micelle formed can be influenced by the encapsulant. Furthermore, large nanoparticle encapsulants can hinder process scale-up. Methods that easily produce particles in 1 mL batches cannot be adapted to 15 mL batches (e.g., Supporting Information Figure S.1) in a straightforward manner. This failure may stem from the large energy input needed to form the dispersed phase for emulsion,18 interfacial instability,7,19 and thin film hydration methods3 used to create micelles encapsulating nanoparticles. At larger processing scales, the lack of control over droplet size in the conventional emulsification method can lead to large droplets that require evaporation times that are incompatible with the interfacial instability process, resulting in kinetically trapped polymer aggregates, a phenomenon that has been documented for high molecular weight block copolymers.20 Finally, most conventional methods of micelle synthesis are not suitable for adaptation to a commercial process. For example, direct dissolution cannot be used for block copolymers with large hydrophobic blocks, dialysis methods are time intensive, and solvent evaporation−film rehydration requires large surface areas and heat transfer. Newer preparation methods such as flash nanoprecipitation21 are emerging; however, these are still in their infancy. Given these difficulties, it is clear that micelles encapsulating nanoparticles represent a new class of nanomaterials that are substantially different from traditional micelles and, as such, will require novel methods for their synthesis at commercial scale. Electrohydrodynamic spraying (electrospray) is one promising alternative fabrication approach. Colloidal synthesis via electrospray has been demonstrated for the dispersion of hydrophobic material in aqueous medium,22 and electrospray is well established for the production of polymer particles.23 Particles produced via electrospray are monodisperse and can encapsulate proteins and drugs without degrading the payload.24,25 Most importantly, electrospray can be operated as a continuous process, and scalability has been demonstrated through parallel operation of a planar array of electrospray nozzles.26 Despite these advantages, however, most polymer particles produced by electrospray are in the micrometer and submicrometer size range with particle size limited by the initial

aerosol droplet size and polymer concentration as well as the solvent removal rate.23 Almeria and Gomez27 have recently fine-tuned process parameters of standard electrospray to produce PLGA particles with diameters of 59 nm. However, considerable effort and diminutive flow rates were required to satisfy the multiple constraints to achieve stable electrospray of nanometer sized particles. Thus, there is still a need for innovations that compliment standard electrospray in a way that diversifies the options for polymer nanoparticle production. Here, we demonstrate a novel synthesis method, i.e., micellar electrospray, which combines the benefits of bottom-up selfassembly with the high-throughput, top-down production capabilities of electrospray. In this approach, particle size is controlled by the thermodynamic stability of the self-assembled structure and the kinetics of solvent removal in addition to primary droplet size, thus allowing nanometer (∼30 nm) sized polymeric particles to be produced from much larger primary droplets when compared to standard electrospray. The current work expands upon our previous efforts to create selfassembled structures, including lipid28,29 and polymer-based30 particles via electrospray, and here, we demonstrate a semicontinuous, scalable method for generating empty micelles, micelle-protected nanoparticles, and micellar composite nanoparticles. In particular, we synthesized empty micelles and micelles encapsulating quantum dots (QDs) (i.e., MultiDots4), superparamagnetic iron oxide nanoparticles (SPIONs) (i.e., SuperMags), and their combination (i.e., magnetic QDs) (i.e., MagDots 7 ). Using MagDots as a model system, we demonstrate the potential of micellar electrospray for continuous production of micelles encapsulating nanoparticle cargoes and provide one of the first applications of bottom-up self-assembly synthesis strategies in electrospray. Additionally, resultant particle size, size distribution, and constituent encapsulation within the micelle are evaluated.

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EXPERIMENTAL SECTION

Materials. Hydrophobic SPIONs were synthesized according to the literature31 and suspended in chloroform. Hydrophobic QDs (λem = 615 nm) were purchased from Invitrogen (Carlsbad, CA). Quantum dots were received in decane, precipitated in isopropanol/methanol, dried, and then reconstituted in chloroform. Poly(styrene-b-ethylene 3940

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oxide) (PS 9.5 kDa:PEO 18.0 kDa) was purchased from Polymer Source (Montreal, Canada). Poly(vinyl alcohol) (PVA) (5 mg/mL, 100 μL, 13−23 kDa, 87−89% hydrolyzed) was purchased from SigmaAldrich. Ammonium acetate buffer was prepared by dissolving 0.77 g of ammonium acetate (crystalline/HPLC; Fisher Chemical) in 500 mL of double distilled water (dd-H2O) and then adding 0.75 mL of ammonium hydroxide, 1.0 N (LabChem Inc.). The pH of the resulting buffer was measured to be 8.0 whereas the conductivity was 0.2 S/m. Micellar Electrospray Synthesis. Micelles were synthesized using a coaxial electrospray configuration as shown in Figure 1a. The inner capillary needle was a 27 gauge (410 μm o.d.; 210 μm i.d.) stainless steel capillary, and the outer needle was a 20 gauge (910 μm o.d.; 600 μm i.d.) stainless steel three-way connector. The nozzle tip was positioned 0.5 cm above a grounded copper ring and 10 cm above a grounded aluminum collection dish. The composition of the organic phase employed depended on the type of micelle generated: empty micelles, quantum dot-loaded micelles (MultiDots), SPION-loaded micelles (SuperMags), or micelles with both SPIONs and quantum dots (MagDots). In all cases, each of the components (SPIONs, QDs and PS−PEO) was first individually dissolved in chloroform at the specified concentrations and then mixed together in the specified volumes. For empty micelles, PS−PEO (10 mg/mL, 90 μL) in chloroform was added to 675 μL of pure chloroform. For QD-loaded micelles, the organic phase consisted of red quantum dots (0.1 μM, 150 μL), green quantum dots (0.1 μM, 150 μL), and PS−PEO amphiphiles (10 mg/mL, 600 μL) in chloroform. SPION-loaded micelles were prepared by adding SPIONs (1 mg/mL Fe, 225 μL) and PS−PEO amphiphiles (10 mg/mL, 90 μL) in chloroform to 450 μL of pure chloroform. Finally, for MagDots, the organic phase was prepared by mixing SPIONs (1 mg/mL Fe, 225 μL), QDs (0.1 μM, 450 μL), and PS−PEO amphiphiles (10 mg/mL, 90 μL) in chloroform, based on scaling up the previously developed batch method.7 The organic phase was delivered to the inner stainless steel capillary at a flow rate of 0.48 mL/h using a syringe pump. An aqueous phase was prepared by dissolving PVA in dd-H2O at 50 mg/mL. The aqueous solution was delivered to the outer annulus of the coaxial needle at a flow rate of 2.8 mL/h using a second syringe pump. After allowing both flow rates to stabilize, compound droplets with an organic core and aqueous shell could be observed dripping from the coaxial nozzle using a high-resolution camera and monitor. When both flow rates stabilized, a power supply was used to apply a positive high voltage to the coaxial nozzle with respect to a grounded copper ring positioned 0.5 cm below the nozzle tip. At a voltage in the range of 3− 4 kV, a concave cone-jet (i.e., a convergent jet32) was observed at the tip of the coaxial nozzle. An aluminum dish containing 14 mL of an aqueous phase (either dd-H2O or ammonium acetate buffer) was placed 10 cm below the nozzle tip to collect the aerosol droplets. After 1 h of collection, an emulsion was visible in the bottom of the collection dish. This was transferred to a 15 mL centrifuge tube. After 2 h, the emulsion vanished, leaving a transparent suspension as illustrated in Figure 2. Dynamic Light Scattering. Hydrodynamic particle size was characterized using dynamic light scattering (Brookhaven Instruments Corperation, BI 200SM). Samples were taken from the micelle suspension described above and diluted with distilled water, if necessary, to reduce the intensity of scattered light to the acceptable range of the instrument (between 10 and 200 kCPS). Mean particle diameter weighted by number was determined using a laser wavelength of 633 nm, a pinhole of 200 μm, and a detection angle of 90° over 2 min. The CONTIN algorithm, in the Brookhaven Software, was used to analyze the data. Transmission Electron Microscopy. Images of micelles from the transparent suspension were obtained using an FEI Tecnai G2 Bio Twin TEM. First, 10 μL droplets of samples were pipetted onto a clean silicone pad. Micelles were loaded onto Formvar/carbon-coated nickel grids by placing the grid over the sample droplet with the support film facing down. Micelles were allowed to collect on the support film for 2 min, after which time the excess liquid was wicked away using filter paper. Next, the grid was placed over a 10 μL droplet of phosphotungstic acid (PTA, 1%). Negative staining with 1% PTA

Figure 2. Example of solution collected during MagDot synthesis. (a) The initial emulsion in water. (b) The emulsion is easily dispersed by tipping the tube. (c) The final clear solution (∼2 h postspraying) indicating the formation of micelles. Grid size = 6 mm × 6 mm.

was allowed for 2 min, and the excess liquid was wicked away. The grid was then imaged. TEM images were analyzed for particle size using ImageJ software.33 The TEM micrographs were divided into three regions, and 100 particle samples from each region were measured to yield three, number-weighted particle size distributions each with a mean and a standard deviation. The average of three mean particle diameters was calculated, and the error is represented by the standard deviation of the three samples. Scanning Mobility Particle Size (SMPS) Distribution. Empty and loaded micelles synthesized via micellar electrospray were dispersed in ammonium acetate buffer (to increase the conductivity of the solution) and aerosolized in a second, postsynthesis electrospray step using a TSI 3480 electrospray aerosol generator. The aerodynamic size distribution of the resulting aerosol was measured using a TSI 3936 scanning mobility particle sizer (SMPS). According to the criterion established by Lenggoro and Okuyama,34 the concentration of micelles in ammonium acetate buffer was well below the level required for droplets created by electrospray to contain at most one micelle. The aerosol was classified by electrical mobility using an electrostatic classifier (TSI 3080) with a differential mobility analyzer (DMA, TSI 3081). The particles were then quantified using an ultrafine water condensation particle counter (UWCPC, TSI 3786). The equipment was programmed to scan through a range of electrical mobility particle sizes from 9 to 400 nm, counting the particles at each size. Three measurements were taken for each sample. The average of three mean particle diameters was calculated, and the error is represented by the standard deviation. Particle Characterization via Tracking with Fluorescence Microscopy and Magnetic Attraction. The fluorescent and magnetic functions of the particles generated by electrospray were tested by particle tracking with fluorescence microscopy (Olympus IX71) alone and in the presence of a neodymium magnetic needle.35 For red quantum dots, a TRITC filter was used with the following wavelengths: λex = 545 ± 25 nm and λem = 605 ± 70 nm. For green quantum dots, a FITC filter was used with λex = 470 ± 20 nm and λem = 520 ± 25 nm. To calibrate fluorescence intensity, unencapsulated QDs were evaporated from chloroform on a microscope slide.4 The fluorescence above background of 50 spots was averaged to yield the mean fluorescence of a single QD. For all particle tracking experiments, a 10 μL sample of particles was observed for 500 frames at an exposure time of 150 s per frame for a total observation time of 75 s. Videos were then compiled from still frames and image analysis performed using ImageJ software33 to yield a graph of mean-square displacement (MSD) vs time. For fluorescent observations in the presence of a magnet, the sample of MagDots was placed in a glass dish containing a small cylindrical segment (1/16 in. in diameter and 1 in. in length) of a neodymium magnetic needle (K&J Magnetics, D1X0). Particle motion was observed using the same exposure and observation time as above. The resultant data were analyzed as described in the Results section and in the Supporting Information to 3941

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yield the average magnetic velocity and a graph of the Brownian motion component of velocity (MSD) as a function of time.

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RESULTS Electrospray Synthesis of Micelles and Comparison to the Batch Emulsion Process. The basic physical principles that govern the electrospray process are quite well understood. When a charged liquid is pumped through a capillary at high voltage relative to the ground, electrodynamic forces can overcome surface tension and inertial stresses in the fluid, yielding ejection of a thin liquid jet from the tip of a coneshaped meniscus (i.e., cone-jet electrospray). This jet then breaks up into droplets because of varicose or kink instabilities.32,36−41 Multiphase emulsion droplets can be created by employing a coaxial configuration (Figure 1a), in which concentric needles are used to produce droplets containing both an organic and an aqueous phase. In this case one of the fluids, designated as the driving liquid, accumulates charge and responds to the electrostatic force. This force is transferred to the driven liquid by shear stress, as exemplified by the photos in Figure 1b.39 Micelles were synthesized using the coaxial electrospray approach illustrated in Figure 1a, with chloroform solvent, poly(styrene-block-ethylene oxide) (PS−PEO) amphiphiles, and nanoparticles (if any) flowing through the central needle, while a mixture of poly(vinyl alcohol) (PVA) surfactant and water flowed through the outer needle. Here, the conductive PVA aqueous solution responded to the electric field and acted as the driving liquid, transferring force to the inner, chloroform solution via shear stresses. A coaxial configuration is required because direct spraying of the polymer−particle solution into the water-filled collection dish yields micrometer-size, solid polymer aggregates that result from rapid chloroform solvent evaporation. Thus, the role of the outer water phase of the droplet is to prevent significant evaporation of the organic phase in the 30−50 ms transit time from the exit of the jet to the collection plate. The transit time estimate is based on an electrospray droplet velocity of 2−3 m/s42 and the distance between the tip of the capillary and the collection dish. Flow rates and voltages were adjusted to achieve a stable cone-jet as shown in Figure 1b. For all experiments in this work, the electrospray jet had a concave cone shape, generally referred to as a convergent jet.32,43 Aerosol droplets generated via electrospray were collected for 1 h in an aluminum dish containing distilled water, yielding an emulsion that segregated to the bottom of the collection dish (e.g., red-brown for MagDots, Figure 2a,b). With time the solution became transparent, indicating micelle formation (Figure 2c). The configuration in Figure 1 was used to synthesize empty micelles, micelles containing multiple QDs with different emission wavelengths (i.e., MultiDots4), micelles containing SPIONs (SuperMags), and fluorescent−magnetic composite nanoparticles (i.e., MagDots7). Figure 3 illustrates typical TEM images of each micelle type, qualitatively showing the ability of electrospray to synthesize monodisperse nanoparticles of each class. Figure 3 demonstrates that TEM is not always able to reveal nanostructures encapsulated within a micelle, highlighting the need for additional characterization methods such as tracking of fluorescent particles. Comparing micellar electrospray to the conventional batch interfacial instability method,7 the weight of micelles produced by the micellar electrospray procedure (see Experimental Section) was ∼30 times higher than that yielded by batch

Figure 3. Transmission electron micrograph (TEM) images of (a) empty micelles, (b) micelles encapsulating SPIONs (SuperMags), (c) micelles encapsulating QDs (MultiDots), and (d) MagDot composite nanoparticles encapsulating both QDs and SPIONs. All particles were produced using micellar electrospray. Scale bar = 100 nm in all images.

interfacial instability over the same time period. The scale of this method could be increased further by increasing the concentration of polymer and constituent nanostructures used and also by using a multiplexed array of nozzles operating in parallel. Although multiplexed coaxial electrospray is a more complex problem than single needle electrospray, Deng et al.44 present a multiplexing system with potential to be adapted to a coaxial electrospray. To validate the micellar electrospray process, MagDot particles were studied in more detail and compared to their counterparts synthesized via the interfacial instability batch method.7 Figure 4a−c shows dynamic light scattering (DLS) and transmission electron microscopy (TEM) data for MagDots prepared by the micellar electrospray method. The MagDot particle size distribution from DLS (Figure 4a) clearly shows unimodality, with a number-weighted mean particle diameter of 36 ± 6 nm, based on five measurements. Analysis of 100 particles across three TEM images (e.g., Figure 4b) yielded a number mean particle diameter of 35.3 ± 0.2 nm (Figure 4c), a value that is in good agreement with the particle size obtained from DLS. Both particle characterization methods show that the micellar electrospray process yields particles with uniform size and a very narrow size distribution. Furthermore, the observed particle size and size distribution are nearly identical to those of PS−PEO micelles produced by the batch interfacial instability approach (e.g., 38 ± 5 nm) reported earlier19 and comparable to the distributions of MagDots (Figure 4d−f) produced using the conventional interfacial instability approach (e.g., average diameter of 46 nm via DLS and 36 nm via TEM). These results demonstrate that the electrospray micelle synthesis method is highly comparable to the previously established interfacial instability approach, while demonstrating a 30-fold increase in throughput by weight. Characterizing Particle Loading. Unfortunately, few methods are available to confirm particle loading, yet alone 3942

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Figure 4. (a) Dynamic light scattering size distribution (geometric mean particle diameter Dpg = 34 and geometric standard deviation σpg = 1 nm) for MagDots prepared using the micellar electrospray approach. (b) Transmission electron micrograph of electrosprayed MagDots. (c) Size distribution of electrosprayed MagDots measured from TEM using ImageJ (Dpg = 43 and σpg = 1 nm). (d) Dynamic light scattering size distribution for MagDots prepared using the conventional interfacial instability approach (Dpg = 46 and σpg = 1 nm). (e) Transmission electron micrograph of MagDots produced using the interfacial instability approach. (f) Size distribution of MagDots produced using interfacial instability as measured from TEM using ImageJ (Dpg = 37 and σpg = 1 nm). All particle size distributions are normalized.

Figure 5. (a) Particle trajectories observed for three particle aggregates under a fluorescence microscope. (b) Mean-square displacement plotted versus time for particles A, B, and C. The lines are linear fits to the data with intercept set to zero and fit parameters and R2 values noted.

⟨x 2⟩ = 4Dt

provide a quantitative measure of particles encapsulated. For example, evaluating encapsulation efficiency via TEM is challenging. Nanoparticles are not always visible in EM images; they may be confounded by the negative stain often used to visualize the micelle, and images also provide only a twodimensional projection of the three-dimensional nanostructure. QD encapsulation can be confirmed by verifying a fluorescence signal, and fluorescence intensity can be used to determine the number of QDs in each particle. However, it is difficult to distinguish small aggregates from single micelles, as both display sizes below the diffraction limit. To address these concerns, we combined Brownian motion particle tracking methods with fluorescence intensity measurements and magnetic mobility tracking to provide qualitative evidence of QD/SPION encapsulation and an order of magnitude estimate of nanoparticle loading. Since MagDots contain QDs, the random motion of particles in solution as observed under a fluorescent microscope, i.e., mean-square displacement (MSD), can be used to estimate particle size via the Stokes−Einstein equation. The MSD of a particle in two-dimensions (i.e., the x−y plane), ⟨x2⟩, is related to the diffusivity of the particle (D) and time (t) by eq 1.

(1)

Assuming a spherical particle shape and a Newtonian fluid, the value of D is in turn related to the particle diameter (dp) through the Stokes−Einstein equation:

D=

kBT 3πμd p

(2)

where kB is the Boltzmann constant, T is temperature, and μ is the viscosity of the surrounding medium (i.e., in this case water with a small amount of PVA). Observing the trajectories of three particles from a batch of electrosprayed MagDots (Figure 5a,b), this method was used to estimate particle diameters of 61 ± 4 and 64 ± 1 nm for two smaller particles and 314 nm for a significantly larger aggregate. If we assume that each micelle in the aggregate has an average dp of 36 nm (from DLS data) and that the micelle packing in the aggregate is that of close-packed hard spheres, a volume balance suggests that these aggregates contain approximately 4, 4, and 500 micelles, respectively. In a separate experiment, the number of QDs per aggregate was determined by comparing the fluorescence intensity of single, QD nanoparticles as evaluated using fluorescence 3943

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microscopy4 to that of a tracked aggregate. For aggregates of 150 and 225 nm, the estimated QD loading was in the range of 0.1−1 QDs/micelle. This loading is consistent with the goal of one QD per MagDot, which maximizes magnetic content and therefore potential for force application. In contrast, our prior work,4 which encapsulated QDs only, was designed for a much higher QD loading (i.e., 4−5 QDs/micelle vs 1 QD/micelle in the MagDot). Finally, for MultiDots, which encapsulate QDs with multiple emission wavelengths, coencapsulation was confirmed using fluorescence microscopy with the appropriate filters to verify the presence of signals in both emission channels (Figure S.2). Fluorescence particle tracking was then combined with magnetic mobility measurements to confirm coencapsulation of QDs and SPIONs and to estimate the number of SPIONs encapsulated. Here, particle mobility was tracked under a fluorescence microscope in the presence of a neodymium magnetic needle (Figure 6a−f). Migration of fluorescent particles toward the needle indicated colocalization of SPIONs and QDs, as particles containing only SPIONs are too small to be visualized with traditional optical microscopy and particles containing only QDs would not respond to the magnet. The additional observation that fluorescence (white) was highly concentrated near the neodymium magnet surface further supports that SPIONs and QDs are coencapsulated in the same micelles. To estimate particle size of the particle observed in Figure 6a−f, the MSD in 1 dimension (i.e., the dimension in which the magnetic field gradient was known to be uniform) was evaluated (Figure 6g,h). Particle motion in Figure 6g reflects both the directed motion resulting from the magnetic field and random motion occurring because of Brownian fluctuations. The velocity, v, of particle motion in a magnetic field is governed by v=

M(B0 )dp 2 18μ

∇B

Figure 6. (a−f) Still images following the movement of a fluorescing particle toward the magnetic needle demonstrate that micelles exhibit both fluorescent and magnetic functionalities. The neodymium magnet is to the right of the red line shown in frame (a). The boundary of this needle appears fuzzy because of surface roughness of the magnet. (g) Particle trajectory as a function of time in the X−Y plane. The magnet boundary is shown by the dotted line, and the direction that is perpendicular to the magnet boundary is shown by the dashed-dotted line. (h) Particle one-dimensional mean-square displacement after subtraction of the average directed motion (i.e., magnetic) velocity component.

(3)

where M(B0) is the magnetization of the particle and B is the magnetic field. Near the magnet, in the direction normal to the magnet surface, the gradient of the magnetic field, ∇B, is reported by the manufacturer of the magnet to be constant. Further, it is assumed that B is high enough to ensure M(B0) is constant (i.e., magnetic saturation). Based on these assumptions, the average velocity of the particle should be constant in the direction along the gradient, i.e., normal to the magnet surface. The average velocity attributed to directed (i.e., magnetic) motion can then be calculated by evaluating the distance traveled normal to the needle surface over the observation time. As this velocity is assumed constant, it is then possible to deconvolve the motion of the particle into its directed (i.e., magnetic) and fluctuating (i.e., Brownian) components in frame by frame analysis. (Further details are presented in the Supporting Information.) As illustrated in Figure 6h, the 1D MSD resulting from the Brownian motion component of particle velocity is a linear function of time as expected, validating the reasonability of the assumptions made in regards to the directed velocity component. From the slope of the line and eqs 1 and 2, we can determine the size of the micelle aggregate tracked. However, since only one dimension is evaluated instead of two, the slope is equal to 2D instead of 4D, and in this case, dp = ∼140 ± 20 nm, and a volume balance suggests that this aggregate contains ∼40 micelles. Equation 3

can then be used to determine M(B0) and, finally the number of SPIONs in this aggregate, NS, can be estimated using M(B0 ) = NSMSVS

(4)

where MS and VS are the magnetic susceptibility (obtained from superconducting quantum interference device, SQUID, measurements) and volume of a single SPION (obtained from TEM), respectively. In this case, NS ∼ 400, suggesting that each micelle in this aggregate contains on average ∼10 SPIONs. Both particle tracking methods rely on the presence of a fluorescence signal. However, for SuperMags, and other particle types that are not fluorescent, loading can be qualitatively evaluated using scanning mobility particle sizing (SMPS), a well-established method that determines the size distribution of the particles based on their electrical mobility. For example, SMPS was used to evaluate the size distributions of two micelle samples (N = 3): empty micelles (Figure 7a,c) and SPION3944

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Figure 7. TEM images of (a) empty micelles and (b) SPION-loaded micelles. Typical particle size distributions for (c) empty micelles and (d) SPION-loaded micelles were measured from TEM images. (e) Typical SMPS distribution measurements of empty micelles and micelles loaded with SPIONs.

loaded micelles (Figure 7b,d). The SMPS results (Table 1) show that the number-average electrical mobility diameters for

both empty and loaded micelles are comparable to the values determined by TEM and DLS (Figure 4), albeit systematically smaller. Investigations of micelles in the gas phase45,46 have demonstrated that amphiphiles have the same block orientation in the gas phase as in solution (e.g., in this case, the hydrophilic block comprises the outer surface layer, the hydrophobic block comprises the core). The fact that the diameters in the aerosol phase are smaller than the hydrodynamic diameters suggests that the micelles become more compact upon evaporation of water, possibly as a result of corona collapse. There was a statistically significant (p = 4 × 10−6) increase in the geometric mean diameter of SPION-loaded micelles compared to empty micelles, as indicated by SMPS. There are at least two reasons why this diameter would increase with iron loading in the micelles. The first reason is an increase in

Table 1. Mean Diameters Measured by TEM Image Analysis and SMPS for Empty and Loaded Micellesa TEM empty loaded

SMPS

Dpg/nm

σg/nm

Dpg/nm

σg/nm

25.1 ± 1.4 24.1 ± 1.0

1.225 ± 0.006 1.216 ± 0.004

17.3 ± 0.2 21.1 ± 0.1

1.41 ± 0.03 1.50 ± 0.01

a

Uncertainties are determined using the standard deviation of three repeated measurements (N = 3). The geometric mean and geometric standard deviation (Dpg and σg, respectively) are reported since the particle sizes appear to be log-normally distributed (see Figure 7e).

Figure 8. Schematic diagram of the key steps in the micellar electrospray synthesis process. (a) The coaxial electrospray generates a compound aerosol droplet containing PS−PEO and hydrophobic species dissolved in chloroform and PVA dissolved in water. The aerosol droplets are collected in a larger water bath to yield (b), the organic emulsion droplets. PS−PEO will adsorb to the emulsion droplet interface resulting in (c), an emulsion droplet with surface wave instabilities that spontaneously eject smaller emulsion droplets. The smaller emulsion droplets undergo further interfacial instabilities until they form (d), micelles encapsulating the hydrophobic species. Complete removal of the organic solvent through instabilities and evaporation results in (e), a suspension of micelles containing hydrophobic species. 3945

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is the generation of the emulsion in a continuous, automated fashion. As such, micellar electrospray offers the promise of semicontinuous or even continuous operation with additional process improvements. Finally, this work presents an innovative fluorescence tracking analysis that provides a quantitative estimate of the payload in the micelles, a quantity that DLS does not provide and where TEM using negative stain can yield ambiguous results. The assumption is that the micelles in an aggregate are a representative sample of the entire population in terms of SPION and QD loading. This assumption will be tested in the future by tracking a large number of aggregates simultaneously to elucidate the relationship between aggregate size and particle loading.

the physical size because of the inclusion of iron nanoparticles. This is unlikely because the mean physical particle size of micelles, as calculated from TEM images (Table 1) and evaluated via the student t test, does not increase significantly with the inclusion of SPIONs (p = 0.36). The second possibility is that the presence of solid SPIONs may make the micelles more resistant to corona collapse as they dry, suggesting that SMPS may be useful in qualitatively confirming SPION loading.

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DISCUSSION Based on the experimental observations, the following mechanism for particle formation via micellar electrospray is proposed (Figure 8). Block copolymer is dissolved in a water immiscible, common solvent (i.e., chloroform), and a PVA− water solution comprises the aqueous phase. Similar to traditional coaxial electrospray, a thin compound jet of these two fluids is emitted from the cone-shaped meniscus formed by the dual capillary, coaxial needles (Figure 1a). This jet breaks up into a monodisperse population of compound aerosol droplets that travel toward the grounded collection dish. Based on this configuration, the chloroform solution is initially coated by an aqueous droplet that serves to prevent rapid chloroform evaporation. Although Figure 8 illustrates these droplets as core−shell structures with PVA−water solution in the outer phase, the true structure of the droplet is most likely more complex47,48 and will be influenced by the surface tensions of the chloroform (27 dyn/cm) and the PVA−water solution (50 dyn/cm),49 the interfacial tension between the two phases, and the ability of one phase to wet the other. Nevertheless, as evidenced by the brown emulsion observed immediately after collection (Figure 2a), the spray emitted from the cone-jet clearly produces discrete droplets rich in the organic phase. Micelle formation then occurs at the surface of the collected emulsion droplets through the interfacial instability effect. This is supported by the observation that, with time and gentle agitation, this emulsion disappears (Figure 2c), indicating micelle formation. The current picture of micelle formation via interfacial instability is based on the work of Granek et al.50 and Zhu and Hayward.19 To summarize the mechanism proposed by this previous work, the amphiphile first adsorbs to the interface of the emulsion droplet. As the organic solvent diffuses into the continuous phase and evaporates, the block copolymer becomes concentrated at the interface, causing a decrease in the interfacial surface tension. This change in surface tension, coupled with the flux of amphiphile to the droplet interface, results in wavelike interfacial instabilities and the ejection of small, amphiphile-rich droplets from the parent droplet surface. As the solvent continues to evaporate, subsequent interfacial instabilities occur until the amphiphile reaches a high enough concentration for micelles to selfassemble. In the conventional interfacial instability process, the organic−water phase emulsion consisting of block copolymer in a common organic solvent (e.g., chloroform) and a continuous aqueous phase sometimes containing emulsifying polymers (e.g., PVA−water) is formed using conventional techniques, including vortexing, homogenization, or sonication, all of which require substantial energy input to both phases, even though only the organic phase needs to be dispersed. This energy input is the primary limitation that hinders potential scale-up of the interfacial instability approach. Thus, the major role played by electrospray in the micellar electrospray process

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CONCLUSION Electrospray is an established technique32,36−40 that has been used to synthesize solid polymer particles for encapsulation of pharmaceutical agents.24,51,52 In our earlier work we showed that electrospray could also be used as a one-step method to generate self-assembled liposomes for nucleic acid delivery.28 Here, for the first time, the electrospray approach was extended to the synthesis of micelles from block copolymers. Electrospray has previously been used to generate particles from a liquid emulsion;53−55 but to our knowledge, it has never been used to generate an emulsion for micelle self-assembly through the interfacial instability method. This combination of topdown electrospray with self-assembly thus yielded much smaller polymeric nanoparticles than generally possible using electrospray. Further, the method has remarkable versatility. In theory, any hydrophobic nanomaterial smaller than the micelle core could be encapsulated, and here we demonstrate encapsulation of QDs, SPIONs, and their combination. Many other methods have been studied to produce micelles, including dialysis,17 flash precipitation,21,56,57 inkjet printing,58 microfluidic flow focusing,59 and dewetting/hydration;60 however, scale-up for commercial production remains a challenge. Micellar electrospray offers a promising new method to scalable nanomanufacturing of micelles and micellar nanocarriers with an initial 30-fold increase in production yield by weight. Addition of stirring and flow through the collection region could make the micelle formation process continuous, with further scaling enabled by a multiple nozzle configuration. Thus, the micellar electrospray process provides a semicontinuous route to the synthesis of micelles and micelles encapsulating nanoparticles at increased scale.

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ASSOCIATED CONTENT

S Supporting Information *

Fluorescence microscopy images of electrosprayed MultiDots, demonstrating colocalization of red and green quantum dots; a detailed description of the analysis methods used to determine particle size and SPION loading from the particle trajectories obtained using fluorescence microscopy in the presence of a neodymium magnet. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (B.E.W.). Notes

The authors declare no competing financial interest. 3946

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation Grants EEC-0914790, CMMI-0900377, and DMR-1206745; National Institutes of Health Grant 1RC2AG036559-01, and the William G. Lowrie family (H. C. “Slip” Slider Professorship to J.O.W.).

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