Fabrication of Functionalized Double-Lamellar Multifunctional Envelope-Type Nanodevices Using a Microfluidic Chip with a Chaotic Mixer Array

Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>105) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency

Structure of the microfluidic chip with a chaotic mixer array.

<p>(A) Two-dimensional design of the chip: i₁, an inlet for f-NA and STR-R8; i₂, an inlet for SUV and D-MEND; o₁, an outlet. (B) Schematic diagram of the staggered chaotic mixer: h₁=75 µm, w=200 µm, h₂=25 µm, c=50 µm, s=50 µm. (C) SEM images of the parts of the staggered chaotic mixers and herringbone ridges in the reaction channel: 150× (left) and 450× (right). The SEM images show the fabricated staggered chaotic mixer array and herringbone ridges.</p

Fluorescence images tracing the progress of the MEND precursor mixing in the microchannel.

<p>The rhodamine-stained SUV solution and the f-NA solution were mixed in the microchannel and fluorescence images (<i>xy</i>-plane) were recorded using a confocal microscope in (A) 0, (B) 1, (C) 5, (D) 10, and (E) 15 cycle regions of the chaotic mixer array. (F) Accumulated scanned fluorescence images along the <i>z</i>-axis direction in the 15 cycle region. (G) Image (<i>xy</i>-plane) of the 15 cycle region before precursor introduction. Flow rate=10.0 µl min⁻¹. Scale bars=300 µm. The fluorescence images indicate homogeneous mixing of the two precursor solutions in the early cycle region of the chaotic mixer array.</p

Schematic representation of a D-MEND.

<p>It consists of a condensed DNA/polycation core and a lipid bilayer membrane envelope. Its surface is further modified with the membrane penetrating peptide stearylated octaarginine to improve cellular uptake and intracellular trafficking.</p

Precursor flow rate-dependent changes in the particle size and zeta potential of the R8-MENDs.

<p>As the precursor flow rate increased, their average particle size (blue circles) increased whereas their predominant particle size (wine red starts did not change. Their zeta potential values (red squares) were highly positive due to the presence of the positively charged STR-R8 on their surface.</p