High Throughput Production of Nanoscale Liposomes Using Extreme Aspect Ratio Hydrodynamic Flow Focusing

HIGH THROUGHPUT PRODUCTION OF NANOSCALE LIPOSOMES
USING EXTREME ASPECT RATIO HYDRODYNAMIC FLOW

FOCUSING
R.R. Hood, E.L. Kendall, and D.L. DeVoe*
University of Maryland College Park, USA
ABSTRACT
A robust microfluidic platform which enables high throughput production of low-polydispersity liposomal nanoparticles is described. High aspect ratio microfluidic hydrodynamic focusing (HAR-MHF)
overcomes the limitations of traditional microfluidic nanoparticle synthesis, which is typically performed
in microfluidic devices with limited channel aspect ratios and reduced throughput, by providing extreme
aspect ratio structures for highly uniform flow and improved production rates. Here the HAR-MHF technique is utilized to generate populations of small, nearly-monodisperse liposomal nanoparticles with exceptionally high production rates and excellent sample homogeneity in comparison to established microfluidic techniques.
KEYWORDS: liposomes; nanoparticles; flow focusing
INTRODUCTION
Microfluidic hydrodynamic focusing (MHF) is a powerful technique for nanoparticle synthesis,
offering multiple advantages over bulk-scale methods including excellent size control and sample
uniformity. However, the utility of MHF-directed nanoparticle production is limited by low throughput,
particularly for the preparation of nanomedicine where large quantities of nanoparticles at high
concentrations are necessary for clinical utility [1]. Here we describe a thermoplastic device enabling
robust high throughput synthesis of nanoscale liposomes using high aspect ratio microfluidic
hydrodynamic focusing (HAR-MHF). The HAR-MHF technology employs a bonded multilayer
thermoplastic microfluidic system containing a vertical stack of extreme aspect ratio microchannels. In
conventional MHF for liposome synthesis [2], fabrication constraints typically limit channel aspect ratios
to ~5:1 or lower, resulting in low overall nanoparticle synthesis rates and adverse effects on nanoparticle
homogeneity due to non-uniform flow profiles within the focusing zone. While devices with parallel
focusing zones for increased production rates have been explored [3], parallel synthesis requires careful
tuning of flow rates within these individual zones to maintain low polydispersity. Furthermore, parabolic
flow within low aspect ratio channels imposes significant variations in flow velocity along the channel
cross-section which negatively impact size variance
of the resulting nanoparticles. The HAR-MHF
technique differs from typical MHF in that shallow
and wide microchannels separated by thin polymer
films form the desired flow focusing zone, where
focusing occurs in the vertical direction (Fig. 1).
Because each HAR-MHF device contains only a
single focusing element, the technique is not subject
to flow rate variations between multiple parallel Figure 1. Photograph of a COC HAR-MHF defocusing elements that can degrade homogeneity of vice which possesses a channel height of 50 m
the resulting nanoparticles. Additionally, HAR-MHF and width of 5 mm, yielding an aspect ratio of
is performed within shallow channels which are 100:1 along the focusing axis. The multilayer fabrealized using simple fabrication techniques. Here rication process is highly scalable, enabling miwe exploit the HAR-MHF technique to execute crochannels with extremely high aspect ratios.
rapid, continuous-flow production of high quality
978-0-9798064-7-6/TAS 2014/$2014CBMS-0001
686
18th International Conference on Miniaturized

Systems for Chemistry and Life Sciences
October 26-30, 2014, San Antonio, Texas, USA
nanoscale liposomes with tunable diameters,

and demonstrate the method for realizing
significantly higher nanoparticle throughput
than conventional microfluidic flow focusing
systems.
EXPERIMENTAL
For
demonstration
of
HAR-MHF,
microfluidic devices were fabricated using a
combination of cyclic olefin copolymer (COC)
plaques and thin COC films. Buffer routing
channels were produced in COC plaques
through a programmable CNC milling machine.
Thin (50 m) COC films were used to provide a
uniform channel height throughout the entire
Figure 2. Comparison between MHF and HAR-MHF mixing channel. The COC films were patterned
at FRR 30 and identical flow conditions. (a) HAR- using an automated craft cutter to be 5 mm in
MHF (100:1) produces lipid nanoparticles at a rate width, resulting in a device with an aspect ratio
nearly 2 orders of magnitude faster than its MHF of 100:1 in the focusing zone. For comparison,
(5:1) and MHF (0.5:1) counterparts (~100 mg/h lipid traditional MHF was performed within hybrid
versus ~4 mg/h lipid and 0.5 mg/h mg/h lipid, respec- PDMS-glass devices, which were produced
tively). (b) HAR-MHF produces comparably sized lip- using standard soft lithographic techniques from
osomes (45~50 nm) as MHF under identical flow con- SU-8 molds with raised features, creating
ditions, with a decrease in polydispersity devices with aspect ratios of either 5:1 or 0.5:1
corresponding to an increase in aspect ratio of the fo- in the focusing zone.
cusing channel. (c) PDI decreased with increasing asThe HAR-MHF devices were used to
pect ratio across a variety of FRRs, along with in- produce liposomes under identical flow
creasing production rates.
conditions to MHF devices for comparison. A
mixture of DMPC, cholesterol, and PEG2000-PE at a molar ratio of 6:3:1 and starting concentration of 20
mM in anhydrous ethanol was used for the lipid phase. Liposomes were prepared as described previously
[2]. The ratio of the buffer flow rate to the ethanol-lipid phase flow rate (FRR) was set to 30, with a
linear flow velocity of 0.1 m/s. COMSOL
Multiphysics software was used for 3D
numerical simulations to depict flow
conditions within the various devices.
RESULTS AND DISCUSSION
The HAR-MHF technique incorporates
shallow but wide microchannels formed
through thin polymer films to precisely define the desired flow focusing zone. Owing
to this feature, the characteristic diffusion
lengths are comparable to conventional
MHF along the focusing axis, while the
width of the focusing zone is least an order
of magnitude larger in the perpendicular axis (Fig. 2), providing highly uniform flow
profiles within the microchannel (Fig. 3).
In this work, the HAR-MHF technique
Figure 3. Fluid velocity at the midpoint of the channel

width from a numerical simulation of the HAR-MHF
system (COMSOL Multiphysics). As the aspect ratio
increases, flow velocity become more uniform across
the width of the channel (all profiles normalized to effective channel height and maximum flow velocity).
687
was used to enable liposome production

at rates up to 2 orders of magnitude higher than traditional MHF (Fig. 4a) while
maintaining identical average flow velocities and flow rate ratios. Liposome production rates as high as ~100 mg/h were
achieved for HAR-MHF (aspect ratio
100:1) versus ~4 mg/h and ~0.5 mg/h for
MHF devices with aspect ratios of 5:1
and 0.5:1, respectively. Additionally,
HAR-MHF produces comparably sized
liposomes as MHF (45~50 nm) under
identical flow conditions, with a decrease
in polydispersity index (PDI) corresponding to an increase in aspect ratio of the
focusing channel (Fig. 4b). A decrease in
PDI, along with increasing production
rates, with increasing aspect ratio was observed across a variety of flow conditions
(Fig. 4c).
Figure 4. Comparison between MHF and HAR-MHF at FRR

30 and identical flow conditions. (a) HAR-MHF (100:1)
produces lipid nanoparticles at a rate nearly 2 orders of
magnitude faster than its MHF (5:1) and MHF (0.5:1) counterparts (~ 100 mg/h lipid versus ~4 mg/h lipid and 0.5 mg/h
mg/h lipid, respectively). (b) HAR-MHF produces comparably sized liposomes (45~50 nm) as MHF under identical flow
conditions, with a decrease in polydispersity corresponding
to an increase in aspect ratio of the focusing channel. (c)
PDI decreased with increasing aspect ratio across a variety
of FRRs, along with increasing production rates.
CONCLUSION
The HAR-MHF technique for nanoparticle synthesis provides an approach
for generating small and highly uniform
populations of liposomes at exceptionally
high production rates. Optimization of
device design, including the use of higher
aspect ratio channels, is expected to enable further increases in production rate
beyond this initial demonstration. Finally,
HAR-MHF technology is amenable to the
synthesis of a wide range of other nanoparticles beyond liposomes, providing a promising route to high
throughput generation of microfluidic-enabled nanoparticles for clinical applications and beyond.
REFERENCES
[1]
P. M. Valencia, O. C. Farokhzad, R. Karnik, and R. Langer, Microfluidic technologies for
accelerating the clinical translation of nanoparticles., Nat. Nanotechnol., vol. 7, no. 10, pp. 623
9, Oct. 2012.
[2]
A. Jahn, S. M. Stavis, J. S. Hong, W. N. Vreeland, D. L. DeVoe, and M. Gaitan, Microfluidic
Mixing and the Formation of Nanoscale Lipid Vesicles, ACS Nano, vol. 4, no. 4, pp. 20772087,
Apr. 2010.
[3]
J.-M. Lim, N. Bertrand, P. M. Valencia, M. Rhee, R. Langer, S. Jon, O. C. Farokhzad, and R.
Karnik, Parallel microfluidic synthesis of size-tunable polymeric nanoparticles using 3D flow
focusing towards in vivo study., Nanomedicine, vol. 10, no. 2, pp. 4019, Feb. 2014.
CONTACT
* D.L. DeVoe; phone: +1-301-405-8125; ddev@umd.edu
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HIGH THROUGHPUT PRODUCTION OF NANOSCALE LIPOSOMES

USING EXTREME ASPECT RATIO HYDRODYNAMIC FLOW

18th International Conference on Miniaturized

nanoscale liposomes with tunable diameters,

Figure 3. Fluid velocity at the midpoint of the channel

was used to enable liposome production

Figure 4. Comparison between MHF and HAR-MHF at FRR

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