Communication pubs.acs.org/cm
Microfluidics-Driven Strategy for Size-Controlled DNA Compaction by Slow Diffusion through Water Stream Ciprian Iliescu*,† and Guillaume Tresset*,§ †
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669, Singapore Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France
§
B
distributions not suitable for practical delivery applications as mentioned earlier. The convective-diffusive mixing of miscible liquids can be conveniently controlled at nanometer length scales by microfluidic hydrodynamic flow focusing.33−35 We have recently employed this technique for tuning the size of nanoparticles made of flexible anionic polyelectrolytes condensed with cationic surfactant.36 Hydrodynamic flow focusing allowed us to control the mixing time τmix between a stream of polyelectrolytes sandwiched by two other streams containing surfactants. When τmix was much smaller than the adsorption time τad for surfactants to bind to polyelectrolytes, the nanoparticle size was found to be minimal and we could achieve hydrodynamic diameters as small as 50 nm. However, substituting flexible polyelectrolytes with DNA led to large nanoparticle sizes (>100 nm in hydrodynamic diameter) and broad distributions due to the higher linear charge density and rigidity of DNA. As a consequence, we adopted an alternative strategy that consisted of changing rapidly the solvent quality of soluble surfactant-bound DNA molecules dissolved in 35% ethanol.37 In that latter case, τmix was about 1 order of magnitude shorter (∼4 ms) than that obtained by direct mixing. Resultantly, a hydrodynamic diameter of 70 nm could be achieved for surfactant−DNA nanoparticles. A major drawback in the above-mentioned method was the presence of organic solvent, i.e., ethanol, to solubilize surfactant molecules. Even after solvent exchange, traces of ethanol still remained in the nanoparticle solution, which might cause deleterious effects for delivery applications. Accordingly, we devised an organic solvent-free method in which the adsorption time τad was considerably increased by slow diffusion through a water stream so as to satisfy τmix < τad. For the first time, we could achieve hydrodynamic diameters as small as 30 nm with a fair monodispersity, which suggested close-to-monomolecular complexes synthesized in a repeatable manner. The microfluidics approach circumvents the use of harsh formulation processes. The device could be integrated into more elaborate systems in which complex assembly schemes would finely control the synthesis of multicomponent soft nanoparticles with tailored physicochemical properties. The working principle is depicted in Figure 1a. A stream of pure water was sandwiched between a stream of DNA and another one containing surfactants. DNA and surfactants slowly diffused across the water stream while flowing along the microchannel. The slow diffusion had for effect to bring very few surfactants at a time in contact with DNA chains. As a
ottom-up nanotechnologies and synthesis of innovative nanomaterials mostly relies on molecular self-assembly, a process by which individual components spontaneously form elaborate ordered structures with emerging functions.1,2 The driving forces pushing molecular subunits toward each other are noncovalent long-range interactions, either weak (H bond, hydrophobicity, entropic effects) or strong (electrostatics, van der Waals forces).3−5 Polyelectrolyte-based complexes constitute an important class of soft nanoparticles that rely on electrostatics for their formation. In their simplest form, they are made of a polyelectrolyte condensed via a coil−globule transition occurring in the presence of oppositely charged agents.6−9 When the polyelectrolyte is made of therapeutic nucleic acids, the resulting complexes hold a great potential for gene therapy. The development of gene therapy requires a proper design and development of safe and effective delivery vectors.10 Viral vectors11 exhibit safety restrictions related to carcinogenesis12 and immunogenicity13 but also to limited DNA packaging efficiency.14 Physical methods for injection of naked DNA, such as gene gun, sonoporation, electroporation, or magnetofection,15−18 expose the nucleic acids to degradation. As an example, the half-life of plasmid DNA intravenously injected in mice is estimated to be around 10 min.19 Synthetic vectors, i.e., nanocarriers that entrap the therapeutic gene, elude the safety issues that characterize viral vectors. The therapeutic nucleic acids can be “adsorbed” on biodegradable or inorganic nanoparticles,20 or condensed using cationic agents such as lipids,21−24 surfactants,25,26 and polymers.10,27−29 The size and the polydispersity of these complexes are critical aspects for transfection. Large nanoparticles yield good transfection efficiencies in vitro thanks to sedimentation but are irrelevant in vivo due to diffusion limitation and to their inability to penetrate deeply into tissues. In addition, the hydrodynamic and shear forces exerted on large nanoparticles work against their attachment to target cells.30 As a result, achieving a reduced hydrodynamic diameter of the nonviral vectors is desirable. For short segments of nucleic acids such as mRNA, small interfering RNA (siRNA), and microRNA (miRNA), controlling the size and the distribution is not a challenge. By contrast, the condensation of long DNA chains (>2 kbp) becomes an important aspect that requires an adapted strategy. In this context, microfluidics has emerged as a state-of-the-art technique that enables the synthesis of a large variety of soft micro- and nanoparticles with excellent control on composition, morphology, and size distribution.31,32 The assembly of DNA-based complexes is driven by kinetics due to strong electrostatic interactions, and bulk nanoparticles are often trapped in metastable states that translate into large size © XXXX American Chemical Society
Received: October 26, 2015 Revised: December 4, 2015
A
DOI: 10.1021/acs.chemmater.5b04129 Chem. Mater. XXXX, XXX, XXX−XXX
Communication
Chemistry of Materials
Figure 1. (a) Working principle of the surfactant−DNA association using hydrodynamic focusing through a diffusion stream. (b) Layout of the microfluidic device. (c) Optical image of the device. (d) Microscope image of the microchannels at the confluence of the three streams containing DNA, water, and surfactant.
streams of interest, we obtain the corresponding flow rates. In the case of the DNA stream for example, we arrive at a normalized flow rate RDNA ≈ 3 (wDNA/wt)2, where wDNA is the width of the DNA stream supposed to be small with respect to wt. Likewise, we have Rsurf ≈ 3(wsurf/wt)2 with wsurf the width of the surfactant stream. After rearranging wt = wc + wDNA + wsurf, we can readily establish that
result, the surfactant−DNA coil−globule transition36,37 occurred nearly in dilute regime, which prevented the aggregation of several DNA chains into a same nanoparticle. The microfluidic device (see Figure 1b−d) was made of a 300μm-thick polished silicon wafer patterned into microfluidic channels by lithography and deep reactive ion etching process. The wafer was subsequently anodically bonded onto a glass wafer, then diced prior to mounting fluidic connections. The mixing channel had a cross section of 80 μm × 40 μm (width × depth) for a length of 15 mm. The device could be easily cleaned after each experiment by flowing N-methylpyrrolidone and rinsed with deionized water. The three fluids were injected with a MFCS-FLEX pumping system equipped with mass flow controllers for each channel (Fluigent, France). The flow rates were feedback-controlled by automatically tuning the applied pressure. Let wt be the width of the channel and wc be the width of the central stream containing water. We denote RDNA and Rsurf the flow rates of the DNA and surfactant streams, respectively, normalized to the total flow rate of the fluids in the channel. Assuming that the fluids are incompressible, the flows are laminar, and in the limit of large aspect ratio, that is, when the channel depth is considered much larger than the channel width, the longitudinal fluid velocity v(x) obeys to the Poisson equation with a no-slip condition on the channel wall:
wc/wt ≈ 1 −
3 ( RDNA + 3
R surf )
We can see that the width of the central stream weakly varies with the flow rate ratios allowing a fine control. The diffusion coefficient of a 2-kbp DNA molecule is about 4.0 × 10−12 m2/s whereas that of surfactants below the critical micelle concentration is Dsurf = 6.0 × 10−10 m2/s, i.e., 2 orders of magnitude higher. As a consequence, surfactants diffuse faster than DNA and the assembly is therefore driven by surfactants. A crude estimate for the mixing time can be τmix ≈ (wc + wDNA)2/Dsurf where wDNA is the width of the DNA stream and can be expressed by wt (RDNA/3)1/2. For RDNA = 0.075, Rsurf = 0.3 and wt = 60 μm, it comes τmix ≈ 2.8 s. It corresponds to the typical time to homogenize the surfactants in the channel and shows that the assembly occurs in a slow diffusion regime. Nanoparticles were assembled in deionized water by combining a stream of calf thymus DNA (CT-DNA; Invitrogen), a stream of deionized water, and a third stream of dodecyl trimethylammonium bromide (DTAB; SigmaAldrich). Figure 2 gives the hydrodynamic diameter of surfactant−DNA nanoparticles versus the water flow rate. Hydrodynamic diameters and polydispersity indexes (PDIs) were measured by dynamic light scattering using a Zetasizer
⎛ x2 ⎞ v(x) = vmax ⎜1 − 4 2 ⎟ wt ⎠ ⎝
where x is the transverse coordinate and νmax the maximal fluid velocity at the channel center. By integrating v(x) over the B
DOI: 10.1021/acs.chemmater.5b04129 Chem. Mater. XXXX, XXX, XXX−XXX
Communication
Chemistry of Materials
Figure 2. Influence of the water flow rate on the hydrodynamic diameter and PDI of surfactant−DNA nanoparticles. The flow rate of the CT-DNA solution (0.5 g/L) was set to 6 μL/min and that of the DTAB solution (5 mM) was set to 24 μL/min. The inset shows the precipitation of DNA in the channel when the water flow is turned off. The values in red are PDIs.
Figure 4. Effect of the surfactant stream on the size of surfactant− DNA nanoparticles. (a) Influence of the surfactant flow rate. The DTAB concentration was fixed to 5 mM. (b) Influence of the surfactant concentration, the flow rate being set to 24 μL/min. In all cases, the water flow rate was 50 μL/min and the 0.5 g/L DNA solution was flowing at 6 μL/min. The values in red are PDIs.
model ZS-90 (Malvern Instruments, Ltd., UK). In the absence of water stream, surfactant and DNA streams encountered brutally and produced large aggregates in an uncontrolled manner. As the water flow rate was increased, the nanoparticle size steadily decreased and saturated above 35 μL/min. The saturation was due to the fact that the width of the water stream no longer increased when the water flow rate ratio approached 1, and subsequently the assembly kinetics remained unchanged. Figure 3 illustrates the effects of the DNA stream. The hydrodynamic diameter was decreasing with the DNA flow rate
remained around 71 nm, it steeply increased up to the micrometer scale for flow rate greater than 36 μL/min. The best results were obtained at low surfactant concentration (3 mM) for a mild flow rate (24 μL/min): in that case, we achieved nanoparticle sizes of 33 nm with a PDI lower than 0.1 that indicated fairly monodisperse nanoparticles. These were the smallest surfactant−DNA nanoparticles that we managed to form including with previous microfluidics-based strategies.36,37 As the surfactant concentration was increased, the nanoparticle size increased as well as the polydispersity through PDI. In our previous works, we also noticed that the surfactant concentration had a much higher impact on the nanoparticle size distribution than the DNA concentration, which makes important the careful selection of the proper surfactant concentration to optimize the size of the nanoparticles. At last, we carried out observations by transmission electron microscopy of nanoparticles obtained in the best conditions mentioned above, i.e., for which the hydrodynamic diameter was estimated to be 33 nm. The measured PDI of 0.09 suggested a relative standard deviation of 30%, which means that 68% of the nanoparticles had a hydrodynamic diameter comprised between 23 and 43 nm. Figure 5 shows a large field view of a dry sample imaged by negative staining (1% v/w ammonium molybdate) at 200 kV with a field emission gun JEOL 2010F transmission electron microscope. The nanoparticles exhibited a fair monodispersity with actual diameters ranging between 30 nm up to 40 nm for most of them, in good agreement with the values inferred from dynamic light scattering. The insets give evidence of a globular morphology, which was verified for the vast majority of them. In conclusion, we devised an effective microfluidics-based strategy for the directed self-assembly of surfactant−DNA nanoparticles. The method relied on the controlled diffusive mixing of surfactant and DNA solutions through a water stream of tunable width. Unlike our previous study,37 the assembly process did not involve any organic solvent and the final nanoparticle solution was therefore suitable for downstream applications requiring biocompatible and nontoxic materials such as those in gene delivery. The smallest nanoparticles achieved were about 30 nm in hydrodynamic diameter, meaning that most of them contained a single DNA molecule (
