Microfluidic Platform for the Continuous Production and

Dec 7, 2016 - A microfluidic platform combined with synchrotron small-angle X-ray scattering (SAXS) was used for monitoring the continuous production ...
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Microfluidic Platform for the Continuous Production and Characterization of Multilamellar Vesicles: A Synchrotron SmallAngle X‑ray Scattering (SAXS) Study Aghiad Ghazal,†,‡,# Mark Gontsarik,‡,# Jörg P. Kutter,‡ Josiane P. Lafleur,‡ Davoud Ahmadvand,§ Ana Labrador,∥ Stefan Salentinig,⊥ and Anan Yaghmur*,‡ †

Niels Bohr Institute, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark § Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, Iran ∥ MAX IV Laboratory, Lund University, 223 62 Lund, Sweden ⊥ Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland ‡

S Supporting Information *

ABSTRACT: A microfluidic platform combined with synchrotron small-angle X-ray scattering (SAXS) was used for monitoring the continuous production of multilamellar vesicles (MLVs). Their production was fast and started to evolve within less than 0.43 s of contact between the lipids and the aqueous phase. To obtain nanoparticles with a narrow size distribution, it was important to use a modified hydrodynamic flow focusing (HFF) microfluidic device with narrower microchannels than those normally used for SAXS experiments. Monodispersed MLVs as small as 160 nm in size, with a polydispersity index (PDI) of approximately 0.15 were achieved. The nanoparticles produced were smaller and had a narrower size distribution than those obtained via conventional bulk mixing methods. This microfluidic platform therefore has a great potential for the continuous production of monodispersed NPs.

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sheath streams.15,18 Unilamellar (ULVs)12,15 and multilamellar (MLVs)25 vesicles can be formed in microfluidic systems. Their formation is affected by the lipid composition. MLVs are primarily used for encapsulating substrates to be released upon disruption of the membrane at a later time, which is relevant for drug delivery applications.13 The transition from MLVs to ULVs can be achieved by applying postprocessing steps, for instance using membrane extrusion26 or sonication.27 In this report, we combined microfluidics with synchrotron small-angle X-ray scattering (SAXS) to monitor the early dynamic structural features occurring during the continuous production of multilamellar vesicles (MLVs) based on the binary phytantriol (PHYT)/glyceryl dioleate-PEG12 (DOPEG12) lipid mixture on exposure to phosphate saline buffer (PBS) containing the triblock polymeric stabilizer Pluronic F127. Moreover, we investigated the dependence of the vesicle size and size distribution on the microfluidic device geometry, the flow rate ratio (FRR) which is the ratio of sheath to center stream, and the total volumetric flow rate (TFR).

icrofluidic devices have gained popularity in the last two decades for a wide range of technological applications including tissue engineering,1−3 stem cell growth and differentiation,4,5 cell sorting,6−8 and organ-on-a-chip that mimics the functionalities of human guts, lungs, and heart.9−11 Microfluidic devices are also attractive and promising platforms for pharmaceutical applications. They have been used to form lipidic and polymeric nanoparticulate formulations such as liposomes with controllable size by mixing miscible liquids and controlling the diffusion process by implementing hydrodynamic flow focusing (HFF) mixing.12−18 In addition to HFF mixing, different microfluidic techniques can be employed for the production of liposomes such as electroformation and hydration,19 extrusion,20 pulsed jetting,21 and droplet emulsion transfer,22 among others. However, HFF is considered the most common technique for the production of liposomes and other soft lipidic and polymeric nanoparticulate formulations due to its high efficiency in controlling the size and size distribution of the formed nanoparticles (NPs).23,24 Typically in HFF mixing, a stream of lipid (or lipid mixture) dissolved in an organic solvent flows through the center channel of the microfluidic system and then meets with streams of an aqueous medium, the latter being referred to as © XXXX American Chemical Society

Received: October 23, 2016 Accepted: December 7, 2016 Published: December 7, 2016 73

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and drug delivery applications. We found therefore that the hydrophilic lipid DO-PEG12 can be used to facilitate the formation of such PEGylated PHYT-based NPs with a relatively narrow size distribution. DO-PEG12 alone formed MLVs when dispersed in excess buffer, which is in agreement with previous studies.31 However, its inclusion in the PHYTbased dispersion, as presented below, was associated with a colloidal transition from cubosomes to vesicles. In this context, it is worth noting that the structural characteristics of these NPs are affected by the lipid composition. At ambient temperatures, mixing PHYT (a lipid with nonlamellar propensity)30,32−35 with DO-PEG12 (a lamellar-forming lipid)31 in excess water triggers in a concentration dependent manner a structural transition from bicontinuous cubic phase of the symmetry Pn3m to a lamellar (Lα) phase. It was reported in the literature that the lipid composition and the applied experimental procedure play an important role in the formation of uni-, oligo- or multilamellar vesicles.36,37 For instance, a transition from multi- (MLVs) and oligo-lamellar (OLVs) vesicles to unilamellar vesicles (ULVs) can be achieved by means of extrusion or when applying a high energy-input (e.g., emulsification by using ultrasonic processors).27 In this respect, the formation of multilamellar vesicles in the present work is most likely attributed to the investigated lipid composition and the used microfluidic device. Regarding the lipid composition, PHYT tends to form nonlamellar phases at ambient temperature, whereas the used PEGylated lipid DO-PEG12 forms multilamellar vesicles with a d-spacing value of about 6.45 nm when dispersed in excess buffer. In this study, the investigated NPs can also be formed using a low energy emulsification method, in which an ethanol (EtOH) solution of the binary PHYT/DO-PEG12 mixture is mixed with PBS buffer containing the polymeric stabilizer F127 by

Figure 1. (a) A schematic representation of the thiol−ene chip with mixing-microchannel dimensions of 320 × 225 μm2 and length of 1 cm used for the SAXS investigations of the continuously produced MLVs. The inset illustrates the production of polydispersed MLVs. (b) Polyimide chip with mixing-microchannel dimensions of 90 × 125 μm2 and length of 9 cm. The inset illustrates that this microfluidic device can be used to form MLVs with a narrower size distribution.

In recent studies, PHYT was suggested as an alternative to the most commonly used lipid monoolein (MO) as it displays similar nonlamellar phases in excess water at ambient temperatures.28 PHYT does not contain an ester bond in its molecular structure and is therefore less vulnerable to hydrolysis than MO.29 Therefore, it has most likely a better chemical stability on exposure to a biological environment than MO, which broadens its practical application in the development of drug delivery systems.29 In the presence of an efficient stabilizer such as Pluronic F127, PHYT can be dispersed to form cubosomes.28−30 In the present work, it was our interest to introduce a microfluidic platform for the continuous production of monodispersed PEGylated MLVs that can be used in further studies as injectable nanocarriers for bioimaging

Figure 2. (a) A schematic representation of the thiol−ene HFF chip used to form PHYT/DO-PEG12 MLVs. (b) Formation pathway of the vesicles in microfluidics. (c) SAXS patterns with corresponding reaction times along the microchannel. SAXS data were acquired at different positions along the exit channel. The black profile represents a SAXS measurement for the dispersion prepared using a low-energy emulsification method by vortexing an ethanol solution of the binary PHYT/DO-PEG12 mixture in buffer containing the polymeric stabilizer F127 for a few minutes. The investigated dispersion under equilibrium conditions has the following composition: PHYT 1.25 wt %, DO-PEG12 1.25 wt %, ethanol 1.5 wt %, 95.5 wt % PBS buffer containing 1 wt % F127 (sample 1). (d) A representative cryo-TEM image of MLVs. (e) An isotropic 2D detector image of undistorted MLVs at TFR of 5 μL/min. (f) Anisotropic 2D image of slightly aligned MLVs. (g) A depiction of aligned MLVs. 74

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microfluidic channel walls by maintaining the X-ray beam confined between the walls of the microfluidic channel. Otherwise, scattering from the channel walls occurs and may significantly decrease the quality of the obtained scattering signal. Therefore, to use the polyimide-based chip (90 μm wide channels) for SAXS characterization, an X-ray beam that is smaller than 90 μm in diameter is needed. A reduction in the size of the X-ray beam to such small dimensions would significantly reduce the photon flux and makes the data acquisition difficult due to the aforementioned limitations. Therefore, we had to use the thiol−ene chips with channel width of 320 μm for SAXS experiments, and limit the use of the polyimide chips to the formation of MLVs with a lower polydispersity (Figure 1). To gain insight into the structural characterization of continuously produced NPs using hydrodynamic focusing in the thiol−ene chip, SAXS data were acquired at different positions with a distance of 1 mm between each position along the exit channel (Figure 2a). The possible pathway for the formation of MLVs that involves transformation from single molecules to lipid bilayers that eventually induces the formation of MLVs is depicted in Figure 2b. Referring to the mechanism of vesicle formation, Lasic40 proposed that lipid molecules first assemble into intermediate flat and disk-shaped nanostructures (known as bilayered phospholipid fragments (BPFs)), which are characterized by their hydrophobic edges. Once exposed to water, these edges would be markedly destabilized so they close on each other to form vesicles, where no edges are exposed any longer.41 Jahn et al.15 suggested also that the vesicle formation occurs as the organic solvent around the lipids dissipates inducing a close contact of the lipid molecules with more water molecules through a mutual diffusion (water molecules diffuse into the focused stream; whereas the molecules of the organic solvent diffuse out). The associated self-assembly of the amphiphilic lipids with changing the continuous environment due to an exposure to excess water and a mutual lipid and water diffusion leads to the formation of small vesicles. It is most likely that the pathway for the formation of MLVs in this work (as depicted in Figure 2b) is similar to that proposed by Lasic40 and Jahn et al.15 In addition, we do not exclude that an uneven distribution of the two embedded lipid molecules at the interfacial lipid−water area could promote the formation of an asymmetric curved state bilayer with nonzero spontaneous curvature (C0 ≠ 0) that eventually induces self-closure of the vesicles.42−44 The formation of the PHYT/DO-PEG12 MLVs by applying a low-energy emulsification method is consistent with previous reports42,45,46 and it is most likely due to the important role of PEGylated surfactants such as DO-PEG12 in enhancing the formation of asymmetric curved state bilayers as suggested by Lasic.43 Our results suggest also that the final size of these vesicles is critically dependent on the actual time provided for these asymmetric bilayers to grow. This could explain why relatively bigger vesicles were obtained under slower mixing conditions. The corresponding SAXS patterns for the different positions along the mixing channel are presented in Figure 2c. In addition, a SAXS pattern of an already prepared dispersion is shown in Figure 2c (black line, top). This was done to compare the structural characteristics of NPs continuously produced using the HFF microfluidic device with the final nanoparticulate product. The components had more time to diffuse and to take part in the self-assembly process when moving further down from the initial contact point. The formation of the MLVs is so

vortexing the lipid mixture for a few minutes. This dispersion (sample 1) was colloidally stable for more than one month, as observed by visual inspection, and composed of 1.25 wt % PHYT, 1.25 wt % DO-PEG12, 3.0 wt % EtOH, 1 wt % F127, and 93.5 wt % phosphate saline buffer (PBS) with pH 7.4. Decreasing the ethanol concentration from 3.0 wt % (sample 1) to 1.5 wt % (sample 2) has no significant effect on the structure, most likely due to the preferential redistribution of ethanol molecules in excess buffer during the formation of these nanoparticles (black and red SAXS profiles from these two samples are presented in Figure S1 in the Supporting Information). Dispersions with different lipid ratios were also investigated; however, they proved to be colloidally unstable (Table S1 in the Supporting Information). For the continuous production of these lipidic NPs using HFF microfluidics, PBS streams acting as a sheath fluid confined and hydrodynamically focused a lipid-containing central fluid into a jet like stream having rectangular crosssection (Figure 2a). The central stream consisted of the binary PHYT/DO-PEG12 mixture dissolved in ethanol (EtOH), and its width was adjusted by varying the FRR between the sheath and the central streams. This means that mixing the sheath and center fluids, which eventually induces the formation of the vesicles, is governed by the diffusion of water and lipid molecules at the liquid interface between the lipid-containing center and the two PBS streams containing 1 wt% F127. Mixing the center and sheath streams enhances a decrease in EtOH concentration below the solubility limit of lipids. This triggers the self-assembly in excess buffer and eventually initiates the vesicular formation process.14,15 To prepare the lipidic NPs in the microfluidic device, the applied volume mixing ratio of the buffer to the injected EtOH solution of the lipid mixture was kept constant at 17.2. It should be noted that the lipid concentration is affected by varying FRR. Therefore, caution should be taken when making direct size and size distribution comparisons between NPs formed conventionally using a highenergy batch emulsification method such as ultrasonication with those continuously produced using microfluidic devices.38,39 Two different HFF microfluidic chips based on thiol−ene and polyimide (Kapton) with different designs were used (Table S2). The thiol−ene-based HFF chip featuring a 320 × 225 μm2 mixing channel was described in details in a recent report32 and used to monitor the in situ formation of the MLVs by synchrotron SAXS, where a fixed FRR of 17.2 was used to achieve a desired final concentration of the binary lipid mixture of 2.5 wt %. The calculations of the mixing time and distance are given in the Supporting Information. TFR of 5 and 10 μL/ min were applied with corresponding linear velocities of 1.15 and 2.31 mm/s, respectively. This means that a diffusive mixing was achieved at two positions (37 and 74 μm down the microfluidic channel from the initial contact point). This corresponds to a time for diffusive mixing of 32 ms for both TFRs. The second polyimide-based (Kapton) microfluidic chip, which features a narrower mixing microchannel (90 μm wide) than the thiol−ene based microchannel, is deemed necessary for improving control over NP size and size distribution of the continuously produced lipid nano-self-assemblies. The width of the center stream ranges from 8 to 1.7 μm for FRR of 10 and 50, respectively. The width of the channel, through which the X-ray radiation travels, has to be bigger than the width of the Xray beam (250 μm). This is important to ensure successful SAXS experiments and to avoid parasitic scattering from the 75

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Figure 3. DLS results for the MLVs produced using batch (a low-energy emulsification based on vortexing for few minutes) and continuous (an employed polyimide-based microfluidic chip) methods. Effects of FRR and TFR on the size (a) and size distribution (b) of the produced MLVs using a polyimide-based microfluidic chip. It was possible to achieve an average size of MLVs as small as 160 nm (a), and PDI as low as 0.15 (b). The continuous production of these MLVs was performed at different FRR and TFR in the ranges of 10−50 and 50−200 μL/min, respectively.

fast that the structure is almost identical to that of the final product after about 0.86 s of mixing (yellow, Figure 2c). At about 0.43s, the SAXS pattern at a position near the intersection area (light orange) shows a weak reflection at a q value of about 1.25 nm−1, while the bottom green signal corresponds to data acquired near the outlet after 3.46 s. A second reflection started to appear at a q value of about 2.50 nm−1 after an estimated time of 0.86 s (yellow). These two reflections are characteristic for a lamellar (Lα) phase and indicate the formation of MLVs. For these MLVs, the d-spacing was calculated and there is only a slight increase from 5.01 to 5.26 nm when probing reaction times of 0.43 and 3.46 s, respectively. This is most likely due to a decrease in EtOH concentration in the newly formed lipidic NPs and a slight increase in the solubilized water content in the nano-selfassemblies with increasing reaction time. This is attributed to a simultaneous mixing of the center and sheath streams, which is associated with the diffusion of a considerable amount of EtOH to the surrounding excess buffer. The dispersion formed using the polyimide microfluidic system was also investigated using cryo-transmission electron microscopy (cryo-TEM) to complement the SAXS results (Figure 2d). The representative cryoTEM image given in Figure 2d supports the SAXS findings showing multilamellar vesicular structure. To gain further insight into the structural features of the continuously produced vesicles, we further analyzed the SAXS patterns with a model-dependent fitting method using the multilamellar vesicle model presented in the software Sasview.47 The experimental SAXS profile fitted well with this model (Figure S2) and the fitting parameters are summarized in Table S3. The total number of bilayers in the MLVs was ∼10. It is most likely that we have an existence of closely packed MLVs in the microfluidic channel. The occurrence of such closely packed vesicles was also observed by Cryo-TEM (the red circle in the cryo-TEM image in Figure 2d). Such closely packed vesicles in the present study could also affect the number of lamella layers estimated by SAXS analysis. However, caution must be taken when comparing the number of lamella layers obtained from SAXS with the number obtained from cryo-TEM images since

SAXS provides statistical information on the nanostructural self-assembled features, while a few cryo-TEM images provide local information about a few representative vesicles. It should be noted that the azimuthal profile of the SAXS peak intensity is isotropic for all positions at the applied TFR of 5 and 10 μL/min. This indicates that the formation of these MLVs is not affected by the flow’s strain and shear stresses. A representative example for the isotropic azimuthal profile at a position in the microchannel with a corresponding to a reaction time of 1.73 s is presented in Figure 2e. However, a slight anisotropy was observed when applying a slightly higher TFR (>15 μL/min) and can be seen in the two-dimensional (2D) SAXS data (Figure 2f). This detected anisotropy indicates the presence of slightly aligned MLVs in the flow direction (Figure 2g). A similar effect was reported in recent studies, where a slight shear-induced deformation and alignment of MLVs were observed.48,49 This could pave the way for investigating the alignment of similar lipid assemblies during the continuous formation process under shear stresses rather than just studying the alignment and deformation of ready prepared lipid nanoself-assemblies as recently discussed in the reports of Poulos et al.48 and Gentile et al.49 DLS was used to gain important information on the size characteristics of the produced MLVs. In these measurements, MLVs prepared using batch (a low-energy emulsification) and continuous (a polyimide-based microfluidic chip) methods were investigated (Figure 3). It was important to evaluate the polydispersity of these nano-self-assemblies by determining the PDI, which is a measure of size distribution and takes into account not only the variance in size but also the size itself (see Supporting Information). A significant effect on the size characteristics was obtained when using the polyimide-based microfluidic chip with a smaller channel width and a greater length than that of the thiol−ene chip. A relatively long serpentine channel (90 mm) was used to form NPs with narrower size distribution. It is suggested that the formation of vesicles (or at least BPFs) might occur when the fast diffusive mixing of EtOH and buffer molecules is completed.12 Therefore, using an appropriate lipid concentration and long 76

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HFF microfluidic devices can be modified to allow combination of two or more processes in series. Integrating typically postproduction processes as well as analytical techniques into one microfluidic platform provides even more control over the production process. This method is suitable for a scale-up as flow rates can be increased and several microfluidic systems can be run in parallel.52 This paves the way for developing controlled and efficient methods for forming monodispersed drug nanocarriers.

exit channel may facilitate the formation of vesicles with controllable size and eliminate the formation of any lipid aggregates.12 We found that the FRR plays a key role in modulating the size and size distribution of the continuously produced nanoself-assemblies. The FRR between the sheath fluids (PBS) and center fluid (lipids dissolved in ethanol) was varied between 10 and 50 with an increment of 10 for each experiment. Under these conditions, the TFR ranged from 50 to 200 μL/min. Altering the TFR had a less pronounced effect on the size of the lipid NPs than altering the FRR. Changing the FRR from 10 to 50 for a given TFR (100 μL/min) for example, led to a significant reduction of about 23.5 and 27% in both the mean size (a decrease from 224.0 to 171.5 nm) and the PDI (a decrease from 0.26 to 0.19), respectively. It was even possible to obtain NPs with smaller sizes (a mean size of 160 nm and a PDI as low as 0.15) when applying FRR and TFR at 50 and 200 μL/min, respectively. These results are in agreement with previous studies on the role of FRR on controlling the size and size distribution of lipid liposome and niosome NPs using microfluidic systems.14−18 MLVs prepared by using a lowenergy emulsification (vortexing for few minutes) had an average size of 220 nm and a PDI of almost 0.3. The thiol−ene chip was also tested for the formation of vesicles; however, longer diffusion lengths are obtained due to the presence of larger channel dimensions (320 × 225 μm2) as compared to the polyimide-based chip. This explains the formation of MLVs that were comparable in size and PDI to the samples prepared using a low-energy emulsification method based on vortexing the binary lipid mixture in PBS containing the polymeric stabilizer F127. It is worth noting that vesicles larger than 200 nm in size tend to have multilamellar structure50 and a decrease in their size below 200 nm could facilitate a transition from MLVs and OLVs to ULVs.37,36 Since the optimized polyimide microfluidic chip with narrower mixing channel has the ability to produce vesicles that are 160 nm in size, it would be interesting for future work to perform the in situ characterization of these vesicles by using a microfocus SAXS setup. The HFF microfluidic thiol−ene system coupled with synchrotron SAXS was successfully used to monitor the continuous production of MLVs based on the binary PHYT/ DO-PEG12 mixture in order of fractions of a second to few seconds. MLVs with a more controllable size were obtained when using another HFF microfluidic system featuring narrower microfluidic channels. Depending on different flow parameters including TFR and FRR, MLVs with a mean size ranging from 161 to 224 nm and PDI in the range of 0.15−0.26 were produced in a continuous process. In addition to synchrotron SAXS, the production of the MLVs was confirmed by cryo-TEM. The application of slightly higher TFR induces a slight alignment of MLVs as indicated by the anisotropic azimuthal profile observed in the obtained 2D SAXS images. The combination of HFF microfluidic system with SAXS shows great prospects in basic and applied research, particularly in the development of nanocarriers for delivering drugs, as it allows for the formation of monodispersed NPs without the need for further processing such as extrusion and ultrasonication, which can be costly and time-consuming. In another important aspect, the need for monodispersed NPs is crucial when assessing the efficiency of lipid and polymer NPs as drug carriers in vivo as polydispersed NPs could behave differently in terms of drug release and circulation time in the body.51 Moreover, in one continuous production line, the presented



EXPERIMENTAL METHODS Phytantriol (PHYT, 3,7,11,15,-tetramethyl-1,2,3-hexadecanetriol) with purity of >96.4% (from product specifications by gas chromatography) was purchased from DSM Nutritional Products Ltd. (Basel, Switzerland). DO-PEG12 (DioleinPEG12 or glyceryl dioleate-PEG12) was obtained from Hydrior AG (Wettingen, Switzerland). Pluronic F127 was a gift from BASF SE (Ludwigshafen, Germany). The 67 mM PBS (pH 7.40) was made using Dulbecco’s phosphate buffer saline purchased from Sigma-Aldrich (St. Louis, Missouri, USA). SAXS measurements were performed at the beamline I911SAXS at the MAX IV synchrotron facility (MAX II storage ring, Lund University, Sweden) at an operating electron energy of 1.5 GeV and a wavelength of 0.91 Å.53 Bli9114 software (developed by Christer Svensson, Prekat AB, Kåseberga, Sweden) was used for data reduction. The detector covered the q-range of interest from 0.1 to 4.0 nm−1 (with q = 4π sin θ/ λ, where q is the magnitude of the scattering vector, λ the wavelength and 2θ is the scattering angle). Silver behenate (CH3-(CH2)20-COOAg) with a d spacing value of 58.4 Å was used to calibrate the q-range. The scattering patterns were recorded with 2D pixel detector (Pilatus 1M, Dectris Ltd., Baden, Switzerland) using collection times of 60 s per frame. Background measurements of the solvent (the PBS buffer) in the microchannels were recorded at each position and subtracted from the presented SAXS profiles. All measurements were performed at room temperature by positioning the microfluidic device in the beamline that the X-ray beam would be irradiated through the center channel, where the solutions are mixed. For the characterization of the MLVs, the d-spacing of the lamellar phase was calculated as follows: d = 2πh/qh

(1)

where d is the d-spacing, h is the order of the Bragg peak, and qh is the q-value of the hth-order Bragg peak.54 Phytantriol, DOPEG12 and EtOH were weighed into a vial at a weight ratio of 1.25:1.25:3, followed by vortexing until the solution became isotropic. PBS containing 1% Pluronic F127 was used to prepare the dispersion. The polyimide-based (Kapton) chip was made using adhesive Kapton films (Kapton FN), and a micromachining laser was used to pattern the channels. Polyimide Kapton HN and FN with thicknesses of 127 and 28 μm, respectively, were purchased from American Durafilm Co. Inc. (Holliston, MA, USA). MicroSTRUCT Vario from 3DMicromac laser micromachining system was used for polyimide cutting. The laser power used was 3.7 W with a laser beam diameter of 15 μm, an iteration rate of 40, and a linear speed of 1000 mm/s. The chips were designed using computer-aided-design software (Autodesk Inventor Professional 2014, San Rafael, CA, USA). The chip consists of three layers, with Kapton HN layer sandwiched and bonded between two adhesive Kapton FN layers. The 77

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designed microchannel width is 90 μm and 9 cm long. Kapton FN is comprised of Kapton HN films, which are coated on one or both sides with adhesive Teflon FEP fluoropolymer. Once exposed to a high temperature (above 300 C°) and pressure, the FEP presents adhesive properties. All Kapton films were cleaned using isopropanol and dried with nitrogen gas before bonding. Temperature was first set at 330 C° with 2 kg cylindrical metal weight placed on top of it. The metal weight exerted an approximate pressure of 5 kPa on the 2 × 2 cm2 chip. Once the metal weight reached the same temperature as the hot plate, the glass slides with the Kapton chip were placed between the weight and the hot plate. DLS measurements were performed in triplicate at 25 °C using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.) equipped with a 633 nm laser and 173° detection optics. Malvern DTS v.5.10 software (Malvern Instruments) was used for data acquisition and analysis. The samples were diluted 100 times and PBS was used for dilution. For Cryo-TEM investigations, 3−4 μL of the dispersion was applied to a lacey carbon 300 mesh copper grid (Ted Pella Inc., California, USA). The sample was then plunged into liquidnitrogen-cooled ethane (−180 °C) after quick blotting with filter paper (Fei, Vitrobot, Holland). A Gatan 626 cryoholder (Gatan, Abingdon, U.K.) was used to observe the samples in a Tecnai G2 20 transmission electron microscope (FEI, Eindhoven, The Netherlands) at a high voltage of 200 kV at a low-dose rate (∼5 e/Å2 s). The images were recorded using Fei Eagle 4 × 4k camera (Fei, Holland) at a nominal magnification of 69 000× resulting in a final image sampling of 0.22 nm/pixel.



REFERENCES

(1) Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Microscale Technologies for Tissue Engineering and Biology. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 2480−2487. (2) Kimura, H.; Yamamoto, T.; Sakai, H.; Sakai, Y.; Fujii, T. An Integrated Microfluidic System for Long-Term Perfusion Culture and on-Line Monitoring of Intestinal Tissue Models. Lab Chip 2008, 8, 741−746. (3) Choi, N. W.; Cabodi, M.; Held, B.; Gleghorn, J. P.; Bonassar, L. J.; Stroock, A. D. Microfluidic Scaffolds for Tissue Engineering. Nat. Mater. 2007, 6, 908−915. (4) Zhang, Q.; Austin, R. H. Applications of Microfluidics in Stem Cell Biology. BioNanoScience 2012, 2, 277−286. (5) Wu, H.-W.; Lin, C.-C.; Lee, G.-B. Stem Cells in Microfluidics. Biomicrofluidics 2011, 5, 013401. (6) Voldman, J. Electrical Forces for Microscale Cell Manipulation. Annu. Rev. Biomed. Eng. 2006, 8, 425−454. (7) Gossett, D. R.; Weaver, W. M.; Mach, A. J.; Hur, S. C.; Tse, H. T. K.; Lee, W.; Amini, H.; Di Carlo, D. Label-Free Cell Separation and Sorting in Microfluidic Systems. Anal. Bioanal. Chem. 2010, 397, 3249−3267. (8) Piyasena, M. E.; Graves, S. W. The Intersection of Flow Cytometry with Microfluidics and Microfabrication. Lab Chip 2014, 14, 1044−1059. (9) Kim, H. J.; Huh, D.; Hamilton, G.; Ingber, D. E. Human Gut-ona-Chip Inhabited by Microbial Flora That Experiences Intestinal Peristalsis-like Motions and Flow. Lab Chip 2012, 12, 2165−2174. (10) Huh, D.; Matthews, B. D.; Mammoto, A.; Montoya-Zavala, M.; Hsin, H. Y.; Ingber, D. E. Reconstituting Organ-Level Lung Functions on a Chip. Science 2010, 328, 1662−1668. (11) Agarwal, A.; Goss, J. A.; Cho, A.; McCain, M. L.; Parker, K. K. Microfluidic Heart on a Chip for Higher Throughput Pharmacological Studies. Lab Chip 2013, 13, 3599−3608. (12) Balbino, T. A.; Aoki, N. T.; Gasperini, A. A. M.; Oliveira, C. L. P.; Azzoni, A. R.; Cavalcanti, L. P.; de la Torre, L. G. Continuous Flow Production of Cationic Liposomes at High Lipid Concentration in Microfluidic Devices for Gene Delivery Applications. Chem. Eng. J. 2013, 226, 423−433. (13) van Swaay, D.; DeMello, A. Microfluidic Methods for Forming Liposomes. Lab Chip 2013, 13, 752−767. (14) Jahn, A.; Stavis, S. M.; Hong, J. S.; Vreeland, W. N.; DeVoe, D. L.; Gaitan, M. Microfluidic Mixing and the Formation of Nanoscale Lipid Vesicles. ACS Nano 2010, 4, 2077−2087. (15) Jahn, A.; Vreeland, W. N.; DeVoe, D. L.; Locascio, L. E.; Gaitan, M. Microfluidic Directed Formation of Liposomes of Controlled Size. Langmuir 2007, 23, 6289−6293. (16) Hong, J. S.; Stavis, S. M.; DePaoli Lacerda, S. H.; Locascio, L. E.; Raghavan, S. R.; Gaitan, M. Microfluidic Directed Self-Assembly of Liposome−Hydrogel Hybrid Nanoparticles. Langmuir 2010, 26, 11581−11588. (17) Mazzitelli, S.; Capretto, L.; Quinci, F.; Piva, R.; Nastruzzi, C. Preparation of Cell-Encapsulation Devices in Confined Microenvironment. Adv. Drug Delivery Rev. 2013, 65, 1533−1555. (18) Lo, C. T.; Jahn, A.; Locascio, L. E.; Vreeland, W. N. Controlled Self-Assembly of Monodisperse Niosomes by Microfluidic Hydrodynamic Focusing. Langmuir 2010, 26, 8559−8566. (19) Aimon, S.; Manzi, J.; Schmidt, D.; Poveda Larrosa, J. A.; Bassereau, P.; Toombes, G. E. S. Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles. PLoS One 2011, 6, e25529. (20) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Vesicles of Variable Sizes Produced by a Rapid Extrusion Procedure. Biochim. Biophys. Acta, Biomembr. 1986, 858, 161−168. (21) Stachowiak, J. C.; Richmond, D. L.; Li, T. H.; Liu, A. P.; Parekh, S. H.; Fletcher, D. A. Unilamellar Vesicle Formation and Encapsulation by Microfluidic Jetting. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 4697−4702.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b02468. Detailed information on the colloidal stability, mixing in the microfluidic chip, dynamic light scattering, and SAXS data analysis (PDF)



Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]. ORCID

Anan Yaghmur: 0000-0003-1608-773X Author Contributions #

Contributed equally

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS A.G. is a recipient of a Ph.D. Scholarship from the Neutron and X-ray Techniques (CoNeXT) initiative (Copenhagen University, Copenhagen, Denmark). We acknowledge financial support from the Danish Natural Sciences Research Council (DanScatt) for SAXS experiments. The authors thank Kell Mortensen, Camilla Foged, and Susan Weng Larsen for fruitful discussions. The authors would also like to thank the staff at MAX IV laboratory for their support. This work benefitted from SasView software, originally developed by the DANSE Project under NSF Award DMR-0520547. 78

DOI: 10.1021/acs.jpclett.6b02468 J. Phys. Chem. Lett. 2017, 8, 73−79

Letter

The Journal of Physical Chemistry Letters

(40) Lasic, D. D. The Mechanism of Vesicle Formation. Biochem. J. 1988, 256, 1−11. (41) Dopico, A. M. Lipid Phases. In Methods in Membrane Lipids; Methods in Molecular Biology; Humana Press: New York, 2007; Book 400, pp 15−26. (42) Azmi, I. D. M.; Wibroe, P. P.; Wu, L.-P.; Kazem, A. I.; Amenitsch, H.; Moghimi, S. M.; Yaghmur, A. A Structurally Diverse Library of Safe-by-Design Citrem-Phospholipid Lamellar and NonLamellar Liquid Crystalline Nano-Assemblies. J. Controlled Release 2016, 239, 1−9. (43) Lasic, D. D. Spontaneous Vesiculation and Spontaneous Liposomes. J. Liposome Res. 1999, 9, 43−52. (44) Shearman, G. C.; Ces, O.; Templer, R. H.; Seddon, J. M. Inverse Lyotropic Phases of Lipids and Membrane Curvature. J. Phys.: Condens. Matter 2006, 18, S1105−S1124. (45) Martiel, I.; Sagalowicz, L.; Mezzenga, R. Phospholipid-Based Nonlamellar Mesophases for Delivery Systems: Bridging the Gap between Empirical and Rational Design. Adv. Colloid Interface Sci. 2014, 209, 127−143. (46) Kamo, T.; Nakano, M.; Leesajakul, W.; Sugita, A.; Matsuoka, H.; Handa, T. Nonlamellar Liquid Crystalline Phases and Their Particle Formation in the Egg Yolk Phosphatidylcholine/Diolein System. Langmuir 2003, 19, 9191−9195. (47) SasView V.4.0.1, a Small Angle Scattering Analysis Software Package, originally developed as part of the NSF DANSE project. http://www.sasview.org/ (accessed Nov. 22 2016). (48) Poulos, A. S.; Nania, M.; Lapham, P.; Miller, R. M.; Smith, A. J.; Tantawy, H.; Caragay, J.; Gummel, J.; Ces, O.; Robles, E. S. J.; et al. Microfluidic SAXS Study of Lamellar and Multilamellar Vesicle Phases of Linear Sodium Alkylbenzenesulfonate Surfactant with Intrinsic Isomeric Distribution. Langmuir 2016, 32, 5852−5861. (49) Gentile, L.; Behrens, M. A.; Porcar, L.; Butler, P.; Wagner, N. J.; Olsson, U. Multilamellar Vesicle Formation from a Planar Lamellar Phase under Shear Flow. Langmuir 2014, 30, 8316−8325. (50) Lasic, D. D.; Barenholz, Y. A Handbook of Nonmedical Applications of Liposomes: Models for Biological Phenomena; CRC Press: Boca Raton, FL, 1996. (51) Simon, B. H.; Ando, H. Y.; Gupta, P. K. Circulation Time and Body Distribution of 14C-Labeled Amino-Modified Polystyrene Nanoparticles in Mice. J. Pharm. Sci. 1995, 84, 1249−1253. (52) Capretto, L.; Carugo, D.; Mazzitelli, S.; Nastruzzi, C.; Zhang, X. Microfluidic and Lab-on-a-Chip Preparation Routes for Organic Nanoparticles and Vesicular Systems for Nanomedicine Applications. Adv. Drug Delivery Rev. 2013, 65, 1496−1532. (53) Labrador, A.; Cerenius, Y.; Svensson, C.; Theodor, K.; Plivelic, T. The Yellow Mini-Hutch for SAXS Experiments at MAX IV Laboratory. J. Phys.: Conf. Ser. 2013, 425, 072019. (54) Rappolt, M. The Biologically Relevant Lipid Mesophases as “Seen” by X-Rays. In Advances in Planar Lipid Bilayers and Liposomes; Elsevier: Waltham, MA, 2006; Vol. 5, Chapter 9, pp 253−283.

(22) Ota, S.; Yoshizawa, S.; Takeuchi, S. Microfluidic Formation of Monodisperse, Cell-Sized, and Unilamellar Vesicles. Angew. Chem., Int. Ed. 2009, 48, 6533−6537. (23) Jahn, A.; Vreeland, W. N.; Gaitan, M.; Locascio, L. E. Controlled Vesicle Self-Assembly in Microfluidic Channels with Hydrodynamic Focusing. J. Am. Chem. Soc. 2004, 126, 2674−2675. (24) An, S. Y.; Bui, M.-P. N.; Nam, Y. J.; Han, K. N.; Li, C. A.; Choo, J.; Lee, E. K.; Katoh, S.; Kumada, Y.; Seong, G. H. Preparation of Monodisperse and Size-Controlled Poly(ethylene Glycol) Hydrogel Nanoparticles Using Liposome Templates. J. Colloid Interface Sci. 2009, 331, 98−103. (25) Mizuno, M.; Toyota, T.; Konishi, M.; Kageyama, Y.; Yamada, M.; Seki, M. Formation of Monodisperse Hierarchical Lipid Particles Utilizing Microfluidic Droplets in a Nonequilibrium State. Langmuir 2015, 31, 2334−2341. (26) Batzri, S.; Korn, E. D. Single Bilayer Liposomes Prepared without Sonication. Biochim. Biophys. Acta, Biomembr. 1973, 298, 1015−1019. (27) Maulucci, G.; De Spirito, M.; Arcovito, G.; Boffi, F.; Castellano, A. C.; Briganti, G. Particle Size Distribution in DMPC Vesicles Solutions Undergoing Different Sonication Times. Biophys. J. 2005, 88, 3545−3550. (28) Dong, Y.-D.; Larson, I.; Hanley, T.; Boyd, B. J. Bulk and Dispersed Aqueous Phase Behavior of Phytantriol: Effect of Vitamin E Acetate and F127 Polymer on Liquid Crystal Nanostructure. Langmuir 2006, 22, 9512−9518. (29) Mat Azmi, I. D.; Wu, L.; Wibroe, P. P.; Nilsson, C.; Østergaard, J.; Stürup, S.; Gammelgaard, B.; Urtti, A.; Moghimi, S. M.; Yaghmur, A. Modulatory Effect of Human Plasma on the Internal Nanostructure and Size Characteristics of Liquid-Crystalline Nanocarriers. Langmuir 2015, 31, 5042−5049. (30) Rizwan, S. B.; Dong, Y.-D.; Boyd, B. J.; Rades, T.; Hook, S. Characterisation of Bicontinuous Cubic Liquid Crystalline Systems of Phytantriol and Water Using Cryo Field Emission Scanning Electron Microscopy (Cryo FESEM). Micron 2007, 38, 478−485. (31) Koynova, R.; Tihova, M. Nanosized Self-Emulsifying Lipid Vesicles of Diacylglycerol-PEG Lipid Conjugates: Biophysical Characterization and Inclusion of Lipophilic Dietary Supplements. Biochim. Biophys. Acta, Biomembr. 2010, 1798, 646−653. (32) Ghazal, A.; Gontsarik, M.; Kutter, J.; Lafleur, J. P.; Labrador, A.; Mortensen, K.; Yaghmur, A. Direct Monitoring of Calcium-Triggered Phase Transitions in Cubosomes Using SAXS Combined with Microfluidics. J. Appl. Crystallogr. 2016, 49, 2005−2014. (33) Azmi, I. D. M.; Moghimi, S. M.; Yaghmur, A. Cubosomes and Hexosomes as Versatile Platforms for Drug Delivery. Ther. Delivery 2015, 6, 1347−1364. (34) Nilsson, C.; Østergaard, J.; Larsen, S. W.; Larsen, C.; Urtti, A.; Yaghmur, A. PEGylation of Phytantriol-Based Lyotropic Liquid Crystalline ParticlesThe Effect of Lipid Composition, PEG Chain Length, and Temperature on the Internal Nanostructure. Langmuir 2014, 30, 6398−6407. (35) Yaghmur, A.; Glatter, O. Characterization and Potential Applications of Nanostructured Aqueous Dispersions. Adv. Colloid Interface Sci. 2009, 147−148, 333−342. (36) Hope, M. J.; Bally, M. B.; Mayer, L. D.; Janoff, A. S.; Cullis, P. R. Generation of Multilamellar and Unilamellar Phospholipid Vesicles. Chem. Phys. Lipids 1986, 40, 89−107. (37) Maherani, B.; Arab-tehrany, E.; Kheirolomoom, A.; Reshetov, V.; Stebe, M. J.; Linder, M. Optimization and Characterization of Liposome Formulation by Mixture Design. Analyst 2012, 137, 773− 786. (38) Laouini, A.; Jaafar-Maalej, C.; Limayem-Blouza, I.; Sfar, S.; Charcosset, C.; Fessi, H. Preparation, Characterization and Applications of Liposomes: State of the Art. J. Colloid Sci. Biotechnol. 2012, 1, 147−168. (39) Carugo, D.; Bottaro, E.; Owen, J.; Stride, E.; Nastruzzi, C. Liposome Production by Microfluidics: Potential and Limiting Factors. Sci. Rep. 2016, 6, 25876. 79

DOI: 10.1021/acs.jpclett.6b02468 J. Phys. Chem. Lett. 2017, 8, 73−79