Microwell Array Guided Assembly of Lipoplex Nanoparticles

6-Mercaptohexanoic acid (MHA) was purchased from Sigma-Aldrich Inc. (674974, St. Louis, MO). SiGLO red fluorescent siRNA was purchased from Thermo ...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/Langmuir

Microwell Array Guided Assembly of Lipoplex Nanoparticles Containing siRNA Megan C. Terp,†,‡ Yun Wu,† Bo Yu,† Kwang Joo Kwak,† and L. James Lee*,†,‡ †

Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, 174 W 18th Ave, Room 1012, Columbus, Ohio 43210, United States ‡ William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 W 19th Ave, Room 125A, Columbus, Ohio 43210, United States ABSTRACT: Nucleic acid based therapeutics has been widely explored to treat genetic and acquired diseases. However, the clinical translation of nucleic acid based therapies has been challenged by low delivery efficiency, off-target effects, poor cellular uptake, and limited serum stability. Lipopoplex nanoparticles, as one of the major nanocarrier systems, have shown great potential in overcoming these challenges. Current techniques for lipoplex nanoparticle preparation rely on selfassembly at macroscale, which suffers from limited control over particle structure and composition due to local fluctuations in the concentration of the constituent materials. We have developed a discontinuous dewetting/imprinting method that guided the assembly of lipoplex nanoparticles containing siRNA in a microwell array, which achieved much better control on particle size and composition. The lipoplex nanoparticles prepared by the discontinuous dewetting/imprinting method showed unilamellar core−shell-like structure in contrast to the multilamellar onion-like structure generally observed in lipoplex nanoparticles prepared by the conventional bulk mixing method.

■

INTRODUCTION Nucleic acid based therapeutics, such as siRNA, antisense oligodeoxynucleotides (ODN), and microRNAs, offers treatments for many diseases. While its potential is high, realization of clinical nucleic acid therapies is facing many challenges such as low delivery efficiency, off-target effects, limited cellular uptake, and poor serum stability.1,2 Many nanocarrier systems have been developed to overcome these challenges.3 Among them, lipoplex nanoparticles are especially promising due to good biocompatibility and their ability to incorporate targeting ligands and both hydrophilic and hydrophobic cargo with very high encapsulation efficiency.3,4 Several liposomal drug delivery systems have already been approved by the FDA, including DOXIL and AmBisome. Lipoplex nanoparticles are spherical particles composed of amphiphilic lipid molecules arranged in a bilayer around an aqueous core. Most techniques for lipoplex nanoparticle preparation, such as dry film hydration, ethanol injection, or solvent/surfactant dialysis, rely on the self-assembly process. Since these methods are conducted on the macro-scale or in “bulk”, which is much larger than the nanoparticles themselves, they provide limited control over particle structure and composition due to local fluctuations in the concentration of the constituent materials. To overcome these challenges, various microfluidic devices have been developed to produce lipoplexes nanoparticles in a better controlled manner.5−10 © 2014 American Chemical Society

Microfluidic devices have been developed to rapidly prepare nanoparticles from a large number of materials to quickly screen various formulations and facilitate clinical translation.11,12 However, the dimensions of these microfluidic devices, usually hundreds of micrometers, are still several orders of magnitude larger than the sizes of nanoparticles. The ideal production method is to prepare particles on the same scale as the particles themselves. To achieve this goal, discontinuous dewetting and imprinting techniques have been developed to guide the assembly of micro/nanoparticles. Wu et al. used the discontinuous dewetting technique to prepare lipoplex nanoparticles in the microwell array.13 The microwell array mediated delivery of lipoplex nanoparticles containing ODN and microRNAs showed much higher delivery efficiency than conventional bulk mixing. Guan et al. successfully used discontinuous dewetting and imprinting techniques to make polymer microcapsules of poly(lactic-co-glycolic acid) (PLGA) in 30 μm PDMS microwells.14 In their process, an initial PLGA layer that served as the bottom layer of the particles was applied to a PDMS microwell array by dip-coating. Each well was then filled with a drug solution, and finally a second PLGA layer that served as the top layer of the particles was added using dipReceived: November 12, 2013 Revised: January 12, 2014 Published: February 24, 2014 2873

dx.doi.org/10.1021/la404366p | Langmuir 2014, 30, 2873−2878

Langmuir

Article

Figure 1. Schematic diagram of discontinuous dewetting and imprinting technique to fill microwells with siRNA and lipids before particle hydration and assembly. (a) In step 1, the wells are filled with a predefined mass of siRNA determined by the initial solution concentration. (b) In step 2, a layer of lipids is deposited on top of the siRNA. (c) In step 3, the lipoplex nanoparticles containing siRNA are self-assembled in the microwells upon water vapor hydration and flushed out of the wells.

discontinuous dewetting and imprinting process. Using discontinuous dewetting, the microwells (1 μm in diameter) were first uniformly filled with the siRNA solution, then a layer of lipids was deposited on top of the dried siRNA, and the lipoplex nanoparticles containing siRNA were self-assembled in the microwells upon water vapor hydration and flushed out of the wells by the imprinting process. The discontinuous dewetting/imprinting method was able to fill each microwell with a predefined quantity of siRNA and lipids, thereby systematically controlling the nanoparticle size and composition. Although 1 μm microwells were used as the reactors to produce nanoparticles, the formed particles were much smaller because of electrostatic conjugations of the negatively charged siRNA molecules and the positively charged lipids.

coating. The excess polymer on the ridges of the wells was removed by hot microcontact printing before the microcapsules containing the drug cargo were released into water where they swelled up due to osmotic forces, indicating the polymer sealing was adequate. Similarly, the PRINT technology (Particle Replication In Nonwetting Templates, Liquidia Technologies Inc.) can produce versatile and monodisperse polymer-based nanoparticles, from 20 nm to 100 μm. This process uses a printing technique to fill wells composed of a low surface energy substrate with a polymer/cargo mixture and then photocures the solution in place to form the particles without a “scum” layer.15,16 Nanoparticles composed of the biocompatible polymers such as PEG and PLGA and even quantum dots and proteins were successfully prepared in a wide variety of shapes and sizes.17−19 Essentially, the PRINT is a bulk mixing process carried out in discrete micro- or nanosized wells to form uniform shape-specific micro- or nanoparticles. Within formed particles, the composition may still vary because of the “bulk mixing” nature. Furthermore, the UV radiation required for curing could be damaging to nucleic acids and has been known to break bonds between base pairs. Glangchai et al. and Buyukserin et al. also used nanoimprint lithography to form polymeric nanoparticles for biological applications.20,21 The discontinuous dewetting/imprinting method has been investigated intensively for polymer based particles but not yet for the formation of lipoplex nanoparticles. In this work, we use a microwell array to guide the assembly of lipoplex nanoparticles containing siRNA with precise control over the component ratios and particle size. As shown in Figure 1, this guided assembly process was accomplished using a modified

■

MATERIALS AND METHODS

Materials. 1,2-Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) was purchased from Avanti Polar Lipids Inc. (890890P, Alabaster, AL). Cholesterol was purchased from SigmaAldrich Inc. (C3045, St. Louis, MO). Methoxypoly(ethylene glycol) (MW ≈ 2000 Da)−distearoylphosphatidylethanolamine (PEG2K− DSPE) was obtained from Lipoid (Newark, NJ). 1,2-Dioleoyl-snglycero-3-phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (Fluorescein-DOPE) was purchased from Avanti Polar Lipids Inc. (810332C, Alabaster, AL). λ-DNA was purchased from Thermo Scientific Inc. (SD0011, Pittsburgh, PA). YOYO-1 iodide (491/509) was purchased from Life Technologies (Y3601, Carlsbad, CA). Thiolmethoxyl PEG1k (mPEG-SH) was purchased from Nanocs Inc. (PG1TH-1k, New York, NY). 6-Mercaptohexanoic acid (MHA) was purchased from Sigma-Aldrich Inc. (674974, St. Louis, MO). SiGLO red fluorescent siRNA was purchased from Thermo Scientific Inc. (D001620-02, Pittsburgh, PA). 2874

dx.doi.org/10.1021/la404366p | Langmuir 2014, 30, 2873−2878

Langmuir

Article

Fabrication of Microwell Array. First, polydimethlysiloxane prepolymer (PDMS) and curing agent (Sylgard 184, Dow Corning, Midland, MI) were mixed at a 10:1 weight ratio and degassed before pouring onto a silicon wafer patterned with wells that had 1 μm diameter and 1 μm depth. After 2 days of curing at room temperature, the pattern was demolded from the silicon wafer, and the resulting features on the PDMS were 1 μm diameter pillars with 1 μm height. These pillars were then coated with gold for 8 min and 10 mA in an Emitech K550X sputter coater (Quorem Technologies, Kent, United Kingdom) and then covered with a self-assembled monolayer (SAM) of 1-hexadecanethiol (Sigma-Aldrich) as a demolding agent. Next, a second round of PDMS was cast upon the gold- and hexadecanethiolcoated PDMS pillars and allowed to cure for 2 more days at room temperature. When the two PDMS pieces were separated, the resulting pattern was a replica of the original silicon mold, that is, wells with 1 μm diameter and 1 μm depth, which were then used for the following experiments. The gold/hexadecanethiol layer remained on the pillar features after the demolding, leaving clean 1 μm wells. Surface Modification to 1 μm PDMS Wells. Surface modifications to PDMS were investigated to facilitate particle formation and release from the microwell array. Since redissolution of nucleic acids after the first dewetting step was problematic with unmodified PDMS, gold−thiol chemistry was used to create SAMs of compounds chosen to encourage nucleic acids release from the PDMS surface, with either a hydrophilic group mPEG1K-SH or a hydrophilic and negatively charged group, 6-mercaptohexanoic acid (MHA). To accomplish this, the PDMS microwell array was coated with gold in the sputter coater for 45 s, and the gold on the ridges was carefully removed with Scotch tape so that only gold remained in the wells. Then the stamps were soaked overnight in a 1 mM mPEG1K-SH solution, 1 mM mixture of 90% mPEG1K-SH and 10% MHA, or 1 mM MHA. The stamps were removed, rinsed with ethanol and water, and then air-dried. Release of Nucleic Acid from Modified PDMS Surfaces. The release of nucleic acids from modified PDMS surfaces was studied using λ-DNA since it is a large molecule that can be easily identified with fluorescence microscopy. A droplet of 10 μL of 25 μg/mL YOYO-1-labeled λ-DNA was placed on surface and removed by pipet after about 10 s. The resulting surface was imaged on a Nikon TE Eclipse 2000S inverted microscope and captured with a Photometrics Coolsnap HQ camera to determine the initial amount of DNA adhesion. Metamorph software was used for image acquisition. Next the stamps were rinsed with distilled water and then imaged again to evaluate the DNA release efficiency. Guided Assembly of Lipoplex Nanoparticles Containing siRNA Using Microwell Array. PDMS microwell stamps were cut into 1 cm by 1 cm squares. To prepare lipoplex nanoparticles containing siRNA, the microwell array was first filled with siRNA solution and then filled with a layer of lipids as shown in Figure 1. In step 1, 10 μL of 0.2 mg/mL siGLO red fluorescent siRNA solution was pipetted onto a clean glass slide, and the PDMS microwell stamp was placed on top of the droplet. Care was taken to ensure that no air bubbles were present between the stamp and the droplet by applying gentle but firm pressure. When it was visually apparent that all wells were filled with the reagent, the stamp was peeled away from the droplet on the glass slide. From the discontinuous dewetting, siRNA solution was uniformly distributed in each well and quickly dried, leaving behind a predetermined amount of siRNA (mass is dependent on the initial solution concentration). If a small droplet of solution remained on the edge of the stamp after the dewetting process, it was gently blotted onto a Kimwipe to remove it. In step 2, the PDMS microwell stamp was placed on 10 μL of 2 mg/mL lipid solution (DOTAP/Cholesterol/PEG2K-DSPE/Fluorescein-DOPE in ethanol at a molar ratio of 48.5/48.5/3/0.5) and peeled away, leaving a thin film of lipids on top of the siRNA and on the ridges between microwells. Excess lipid layer on the ridges between microwells was removed by a simple stamping process, similar to microcontact printing. Finally, in step 3, water vapor was applied using a 100% humidity chamber of water so that each lipoplex nanoparticle containing siRNA could self-assemble in the microwell, and then the

stamp was exposed to bulk water to rinse the nanoparticles out of the wells. The lipid/siRNA mass ratio was 10. Preparation of Lipoplex Nanoparticles Containing siRNA Using the Bulk Mixing Method. The conventional bulk mixing method used to prepare lipoplex nanoparticles containing siRNA was the ethanol injection method. Briefly, 100 μL of 10 mg/mL lipid solution was drawn into a syringe and injected into 900 μL of PBS buffer while vortexing, forming 1 mg/mL empty liposomes that were then bath sonicated for 5 min. Lipoplexes were formed by adding an equal volume of siRNA to the empty liposomes to achieve final lipid/ siRNA mass ratio of 10. Cryo-Transmission Electron Microscopy (Cryo-TEM). Lipoplex nanoparticles containing siRNA were prepared for cryo-TEM imaging in a controlled environment vitrification system (CEVS) at 25 °C and 100% relative humidity and then plunged into liquid ethane at its freezing point, as described previously.22,23 Imaging of the vitrified samples was performed at 120 kV on an FEI Tecnai T12 G2 TEM with a Gatan 626 cryo-holder system that was maintained at less than −178 °C. Images were captured with a Gatan US1000 high-resolution camera and Digital Micrograph software in the low dose imaging mode to reduce the amount of radiation damage. Atomic Force Microscopy (AFM). One drop of lipoplex nanoparticles containing siRNA was put on the mica, air-dried, and observed using AFM. AFM was performed using a MFP-3D-Bio-AFM (Asylum Research, Santa Barbara, CA) in air environment. The image was acquired using a normal intermittent contact mode (ac mode) with a scan rate of 0.5 Hz, a resonant frequency of 315 kHz, and a set point of 0.75 V (25% reduced from free amplitude of the AFM cantilever). Without foreside coated and with backside Al coated, a Si pyramidal cantilever with a nominal spring constant of 40 N/m (NSC15, Micromash) was mounted in a standard cantilever holder. The Asylum software was used to analyze the surface profile and characterize the size and shape of dried lipoplex nanoparticles.

■

RESULTS AND DISCUSSION Fabrication of PDMS Microwells and Discontinuous Dewetting. The 1 μm microwell array was prepared using the “replica molding” (REM) soft lithography process, which is a multistep molding process that generates a PDMS duplicate of the patterned silicon master.24,25 As shown in Figure 2, large arrays of 1 μm PDMS wells were successfully created using the replica molding soft lithography process. To prove the discontinuous dewetting prcess, 10% sodium chloride solution

Figure 2. SEM images of (a) PDMS 1 μm wells created using the replica molding (REM) soft lithography process and (b) sodium chloride crystals in 1 μm PDMS microwells after discontinuous dewetting procedure in 10% solution. 2875

dx.doi.org/10.1021/la404366p | Langmuir 2014, 30, 2873−2878

Langmuir

Article

Figure 3. SEM images of gold stamped onto carbon tape from 1 μm PDMS wells coated with 25 nm of gold with light pressure (a) and high pressure (b). The gold from the ridges was removed with light pressure and the gold cups from the wells were stamped out with high pressure. Light micrographs of gold stamped onto Scotch tape (c) and the resulting uniform PDMS wells (d) with no gold on the ridges.

Figure 4. YOYO-1-labeled lambda DNA stretched by pipet on unmodified PDMS or gold-coated PDMS surfaces soaked overnight in 1 mM solutions of 100% mPEG1K, 9:1 mPEG1K:MHA, or 1 mM MHA in EtOH. DNA adhesion was minimized on the surfaces modified with 9:1 PEG:MHA.

was used to fill the wells, and due to the high surface-to-volume ratio of the microwells, the water quickly evaporated. SEM image in Figure 2c showed that salt crystals were present uniformly in the wells, indicating successful discontinuous dewetting. Surface Treatments to PDMS. In order to accomplish the selective surface modifications of the two different features (wells and ridges) that helped control the wettability and particle release, a layer of gold in the wells, but not on the ridges, was necessary to attach the SAM molecule using the gold−thiol bond. After the PDMS microwell stamp was coated with a 25 nm layer of gold, the stamp was then placed on a SEM carbon tape and slight pressure was applied, so that the gold layer on the ridges came off. This simple stamping process was sufficient to remove the gold from the ridges, but not from the wells, as the black holes where the wells were present were clearly visible, as shown in Figure 3a. To confirm that the gold

left behind after the first printing process was left in the wells, the same stamp was then placed on another piece of carbon tape and high pressure was applied, which pushed the gold “cups” out of the wells as shown in Figure 3b. Uniform removal of gold from the ridges was also accomplished with Scotch tape as shown in Figures 3c,d. Selective gold deposition to the wells but not the ridges was accomplished using this process, and therefore we were able to coat SAM only in the wells using molecules with a thiol end group. Since both siRNA and lipids in the dewetting process were in a dried state after the water or solvent evaporated, it was essential that both components were able to redissolve when exposed to water to allow the nanoparticles to self-assemble in the well. Lipids redissolve easily in water and were not found to have a very strong interaction with PDMS (data not shown). However, nucleic acids were more challenging to redissolve from the PDMS surface as they seemed to stick strongly on the 2876

dx.doi.org/10.1021/la404366p | Langmuir 2014, 30, 2873−2878

Langmuir

Article

Figure 5. Cryo-TEM (a, b) and AFM (c, d) images of lipoplex nanoparticles containing siRNA prepared by stepwise discontinuous dewetting process (a, c) and bulk mixing method (b, d).

microwells, leaving a thin lipid layer only inside microwells as shown in Figure 1b. Finally, the stamp was exposed to water vapor to allow the siRNA and lipids self-assemble into lipoplex nanoparticles in the well. Then the PDMS microwell array was exposed to bulk water to flush the lipoplex nanoparticles containing siRNA out of the wells (Figure 1c). Characterization of Lipoplex Nanoparticles by CryoTEM and AFM. Cryo-TEM and AFM were used to characterize the lipoplex nanoparticles containing siRNA prepared using the stepwise discontinuous dewetting process and compared with those prepared by the conventional bulk mixing method. Cryo-TEM images (Figures 5a,b) showed that lipoplex nanoparticles formed in the microwell array had the unilamellar structure, while those produced by the bulk mixing method had the multilamellar structure. Imprinted lipolplex nanoparticles therefore had the “core−shell” type structure, as opposed to the onion-like structure of particles formed using bulk mixing methods. Lipoplex nanoparticles containing siRNA were also visualized using AFM. The lipoplex nanoparticles prepared by the stepwise discontinuous dewetting process appeared to have a “flying saucer” type structure (Figure 5c), indicating that the siRNA payload was in the center of the nanoparticles, especially compared to AFM images of lipoplex particles formed by the bulk mixing method (Figure 5d). The structure difference was mainly caused by the preparation method. In the conventional bulk mixing method, empty liposomes were mixed with siRNA to prepare lipoplex nanoparticles. In this process, free siRNA served as a “glue” between two empty liposomes, and then one liposome opened up and wrapped on the other liposome to form a bilamellar structure. This process repeated several times and multilamellar structured lipoplex nanoparticles were thus formed. This is the so-called “zipper” effect.26 However, in the stepwise discontinuous dewetting process, siRNA and lipids were dried in the microwells, and no empty liposomes existed. When exposed to water vapor in step 3, lipids and siRNA quickly formed lipoplex nanoparticles by self-assembly. In addition, both cryo-TEM and AFM images indicated that lipoplex nanoparticles assembled in

PDMS surface. SiRNA was particularly challenging as it has two base overhangs on each strand, leaving the hydrophobic bases exposed and possibly leading to hydrophobic interactions with the PDMS. Therefore, a surface modification to the PDMS that allowed the components to redissolve easier was desired. The more hydrophilic the surface, the less interaction nucleic acids have with it, making the dried nucleic acid molecule easier to be released back in water. The gold-coated flat PDMS surface was treated overnight in a 1 mM mPEG1K-SH solution, the 1 mM mixture of 90% mPEG1K-SH and 10% MHA, and 1 mM MHA. SAMs of the compounds were formed on flat PDMS surfaces. YOYO-1-labeled λ-DNA molecules were used to investigate the nucleic acid release efficiency from the treated PDMS surface. As shown in Figure 4, DNA had a strong interaction with the untreated PDMS, as the DNA was stretched along the surface and remained after the surface was rinsed with water. Adding the PEG surface modification decreased the hydrophobic interaction, as the DNA was coiled in particles on the surface instead of stretched but the DNA still remained after the surface was rinsed with water. A SAM of 100% MHA worked well, but the mixture of 90% PEG and 10% MHA had the best results, as the interaction with the DNA was minimized since no stretching was seen and virtually no DNA remained after the surface was rinsed. Therefore, the 9:1 PEG/MHA combination was used for surface modifications to the PDMS microwell stamp for the guided assembly experiments. Formation of Lipoplex Nanoparticles Containing siRNA in the Microwell Arrays. To prepare the lipoplex nanoparticles containing siRNA in the microwell arrays, the PDMS microwell stamp was first filled with the red fluorescent siRNA through discontinuous dewetting (“step 1” in Figure 1a). The fluorescence microscopy image confirmed the uniform distribution of siRNA molecules in all microwells. Next, the siRNA in the microwells were covered with the green fluorescent lipid solution. Nucleic acids are not soluble in organic solvents like ethanol, so siRNA was not disturbed by the second step. A simple stamping process was used to carefully remove excess lipid layers on the ridges between 2877

dx.doi.org/10.1021/la404366p | Langmuir 2014, 30, 2873−2878

Langmuir

Article

(10) Kim, Y.; Fay, F.; Cormode, D. P.; Sanchez-Gaytan, B. L.; Tang, J.; Hennessy, E. J.; Ma, M.; Moore, K.; Farokhzad, O. C.; Fisher, E. A.; Mulder, W. J. M.; Langer, R.; Fayad, Z. A. Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics. ACS Nano 2013, 7, 9975−9983. (11) Chen, D.; Love, K. T.; Chen, Y.; Eltoukhy, A. A.; Kastrup, C.; Sahay, G.; Jeon, A.; Dong, Y.; Whitehead, K. A.; Anderson, D. G. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J. Am. Chem. Soc. 2012, 134, 6948−6951. (12) Valencia, P. M.; Pridgen, E. M.; Rhee, M.; Langer, R.; Farokhzad, O. C.; Karnik, R. Microfluidic platform for combinatorial synthesis and optimization of targeted nanoparticles for cancer therapy. ACS Nano 2013, 7, 10671−10680. (13) Wu, Y.; Terp, M. C.; Kwak, K. J.; Gallego-Perez, D.; NanaSinkam, S. P.; Lee, L. J. Surface-mediated nucleic acid delivery by lipoplexes prepared in microwell arrays. Small 2013, 9, 2358−2367. (14) Guan, J.; He, H.; Lee, L. J.; Hansford, D. Fabrication of particulate reservoir-containing, capsulelike, and self-folding polymer microstructures for drug delivery. Small 2007, 3, 412−418. (15) Rolland, J. P.; Maynor, B. W.; Euliss, L. E.; Exner, A. E.; Denison, G. M.; DeSimone, J. M. Direct fabrication and harvesting of monodisperse, shape-specific nanobiomaterials. J. Am. Chem. Soc. 2005, 127, 10096−10100. (16) Gratton, S. E. A.; Pohlhaus, P. D.; Lee, J.; Guo, J.; Cho, M. J.; DeSimone, J. M. Nanofabricated particles for engineered drug therapies: A preliminary biodistribution study of PRINT nanoparticles. J. Controlled Release 2007, 121, 10−18. (17) Euliss, L. E.; DuPont, J. A.; Gratton, S.; DeSimone, J. M. 2006. Imparting size, shape, and composition control of materials for nanomedicine. Chem. Soc. Rev. 2006, 35, 1095−1104. (18) Wang, J.; Tian, S.; Petros, R. A.; Napier, M. E.; DeSimone, J. M. The complex role of multivalency in nanoparticles targetingthe transferrin receptor for cancer therapies. J. Am. Chem. Soc. 2010, 132, 11306−11313. (19) Merkel, T. J.; Herlihy, K. P.; Nunes, J.; Orgel, R. M.; Rolland, J. P.; DeSimone, J. M. Scalable, shape-specific, top-down fabrication methods for the synthesis of engineered colloidal particles. Langmuir 2010, 26, 13086−13096. (20) Glangchai, L. C.; Caldorera-Moore, M.; Shi, L.; Roy, K. Nanoimprint lithography based fabrication of shape-specific, enzymatically- triggered smart nanoparticles. J. Controlled Release 2008, 125, 263−272. (21) Buyukserin, F.; Aryal, M.; Gao, J.; Hu, W. Fabrication of polymeric nanorods using bilayer nanoimprint lithography. Small 2009, 5, 1632−1636. (22) Xu, Y.; Szoka, F. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 1996, 35, 5616−5623. (23) Lu, J. J.; Langer, R.; Chen, J. A novel mechanism is involved in cationic lipid-mediated functional siRNA delivery. Mol. Pharmaceutics 2009, 6, 763−771. (24) Xia, Y.; Whitesides, G. M. Soft lithography. Annu. Rev. Mater. Sci. 1998, 28, 153−184. (25) Gates, B. D.; Xu, Q.; Gates, B. D.; Xu, Q.; Love, J. C.; Wolfe, D. B.; Whitesides, G. M. Unconventional nanofabrication. Annu. Rev. Mater. Res. 2004, 34, 339−372. (26) Weisman, S.; Hirsch-Lerner, D.; Barenholz, Y.; Talmon, Y. Nanostructure of cationic lipid-oligonucleotide complexes. Biophys. J. 2004, 87, 609−614.

microwell array showed smaller and more uniform size compared with their counterpart prepared by the bulk mixing method.

■

CONCLUSIONS In this work, a microwell array based guided assembly method was developed to produce lipoplex nanoparticles in a wellcontrolled manner. The siRNA and lipid solutions were uniformly distributed in the PDMS microwell array by stepwise discontinuous dewetting, thereby exactly controlling the nanoparticle composition and guiding their assembly into nanoparticlesessentially eliminating the “bulk mixing” nature. Comparing with the conventional bulk mixing method, lipoplex nanoparticles containing siRNA formed in the microwell array showed the unilamellar core−shell-like structure instead of the multilamellar onion-like structure. The stepwise discontinuous dewetting can be scaled up to produce large quantity of lipoplex nanoparticles containing siRNA or other nucleic acid therapeutics for their therapeutic efficacy evaluation.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], Tel 614-292-2408, Fax 614-292-8685 (L.J.L.). Present Address

Y.W.: Department of Biomedical Engineering, University at Buffalo, State University of New York, 332 Bonner Hall, Buffalo, NY 14260. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by the National Science Foundation under Grant EEC-0425625. REFERENCES

(1) Pecot, C. V.; Calin, G. A.; Coleman, R. L.; Lopez-Berestein, G.; Sood, A. K. RNA interference in the clinic: challenges and future directions. Nat. Rev. Cancer 2010, 11, 59−67. (2) Davidson, B. L.; McCray, P. B. Current prospects for RNA interference-based therapies. Nat. Rev. Genet. 2011, 12, 329−40. (3) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2, 751−760. (4) Tseng, Y. C.; Mozumdar, S.; Huang, L. Lipid-based systemic delivery of siRNA. Adv. Drug Delivery Rev. 2009, 61, 721−731. (5) 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. (6) 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. (7) Koh, C. G.; Zhang, X.; Liu, S.; Golan, S.; Yu, B.; Yang, X.; Guan, J.; Jin, Y.; Talmon, Y.; Muthusamy, N.; et al. Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing. J. Controlled Release 2010, 141, 62−69. (8) Yu, B.; Zhu, J.; Xue, W.; Wu, Y.; Huang, X.; Lee, L. J.; Lee, R. J. Microfluidic assembly of lipid-based oligonucleotide nanoparticles. Anticancer Res. 2011, 31, 771−776. (9) Valencia, P. M.; Basto, P. A.; Zhang, L.; Rhee, M.; Langer, R.; Farokhzad, O. C.; Karnik, R. Single-step assembly of homogenous lipid-polymeric and lipid-quantum dot nanoparticles enabled by microfluidic rapid mixing. ACS Nano 2010, 4, 1671−1679. 2878

dx.doi.org/10.1021/la404366p | Langmuir 2014, 30, 2873−2878