1LIPOSOME NANOPARTICLESSYNTHESIS USING 3D MICROFLUIDICHYDRODYNAMIC FOCUSINGINTRODUCTION
Despite considerable progress in recent years, various diseases’ diagnosis and treatment continue
to present constraints, such as low sensitivity or specificity, drug toxicity, and severe side effects1.
Cancer represents one of the best examples of a disease where localized delivery of therapeutics
is of high importance, as the potent yet toxic mechanisms of action of such compounds canlead to an effective response or side effects. Today, most drug formulations are not capable oftargeting specific sites of interest. Nanoparticle-based drug delivery platforms have emerged
as suitable vehicles for overcoming these limitations [2]. Nanoparticles, such as liposomes, have
proven advantageous at preserving therapeutic material and allowing for extended half-lives ofdrugs within the body[3].
Liposomes were discovered in the 1960s. These hollow nanoparticles are phospholipid vesicles
consisting of at least one lipid bilayer (figure 1). This bilayer is usually composed of amphiphilic
phospholipids that have a hydrophilic phosphate head and a hydrophobic tail consisting of two
fatty acid chains. This structural feature has facilitated liposomes’ applications, including theiruse as artificial cell membranes, carriers for drug delivery systems, encapsulating agents forfood ingredients, and analytical tools [4–8].
During the COVID-19 pandemic, the first vaccines to reach clinical trials were based on viral
vector and nucleic acid technologies. One of the most promising vaccine candidates was basedon nucleoside-modified mRNA and encapsulated within liposome nanoparticles [9]. This only
confirms the need for liposome nanoparticles for present and future drug delivery applications.

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TYPE DE DOC
APPLICATION NOTEINTRODUCTION
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dui faucibus.
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Hac habitasse platea dictumst vestibulum rhoncus. Vulputate ut pharetra sit amet aliquam
id diam maecenas. Sit amet mauris commodo quis. Pellentesque adipiscing commodo elit at
imperdiet dui accumsan. Semper eget duis at tellus.
Figure 1: An example of a lipid nanoparticle (LNP) composed of phospholipids, homing peptide, drugs, and nucleic
acids.

The size of these nearly spherical lipid vesicles can range from a few nanometers to several
micrometers. However, liposomes applied to medical use typically range between 50 and 450 nm[1].

i.

Microfluidic approach for highly-controlled liposome production

Size and size distribution are essential properties that determine the clinical successes of thenanocarriers. Researchers have observed that size has an impact on liposome accumulation andpenetration efficiency at a targeted tumoral site. [1] In the past, different methods have been
proposed for the production of liposomes. Macroscale batch methods are widely employed forliposome production (e.g. sonication technique, hydration method10), or extrusion method [11].
These techniques often show broad size distribution, and inconsistent encapsulation efficiency10,
and are inadequate if one requires controllability and reproducibility.
Recently,
improvements
have been made with the development of microfluidic production
Pellentesquenec nammethods,inwhichliposome
aliquam sem et. Donec formation occurs within a confined microenvironment. Using
micrometerchannelsof varying diameters (typically up to 500 µm), fluid streams result in laminarenim diam
vulputate
flow.
In
a
typicalmicrofluidic
ut pharetra sit. Odio ut liposome production system, an organic phase (ethanol, isopropyl
alcohol,
containing
phospholipids flows in parallel with an aqueous phase (distilled water,
enim etc.)
blanditvolutpatPBS,etc.).
This
resultsindiffusivemixing and local dilution of the organic phase. The organic phasemaecenas
volutpatconcentration
decreases
blandit aliquam
etiam.until it reaches solubility limit of the lipids. This triggers self-assembly ofphospholipids into liposomes, that are subsequently collected. Under microfluidic conditions, it ispossible to precisely tune parameters, such as aqueous and organic flow rates, to achieve different
liposome sizes and encapsulation efficiencies. These methods have demonstrated higher control
over the physical properties of the end product, particularly in terms of liposome size and sizeForadditional information,
contact us by email : contact@fluigent.com or consult our website : www.fluigent.com
distribution.
[12]

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TYPE DE DOC
APPLICATION NOTEINTRODUCTION
Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut
labore et dolore magna aliqua. Vitae purus faucibus ornare suspendisse. Dapibus ultrices in
iaculis nunc sed augue lacus viverra. Dignissim diam quis enim lobortis scelerisque fermentum
duiTable
faucibus.
1: Comparison of liposome synthesis specification between standard batch method, and Fluigent microfluidic
methodMagnis dis parturient montes nascetur ridiculus mus mauris. Duis convallis convallis tellus id
ii.
A glass
capillarya erat
devicefor
improved
particle
sizetellus
distribution
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nam atlectusurna. Rutrum
quisque non
orci ac auctor
augue mauris. Lereo dui is ut diam quam nulla. Nibh cras pulvinar mattis nunc. Fames ac turpis
egestas sed tempus urna et pharetra. Odio aenean sed adipiscing diam donec adipiscing.
Manymicrofluidic
makeuse of the ultricies
microfluidichydrodynamic
focusing(MHF)
technique
Tristiquesenectussystems
et netus
et. Maecenas
mi egetmauris. Egetmaurispharetraettosynthetize
In standard
designs(two-dimensional(2D) MHF), an organic phase withultricesnequeliposomes.
ornare aenean.
Suscipit
tellus maurisa diam maecenas.
lipids flows in the center of a rectangular channel, and an aqueous buffer flows on both sides ofit(figure2A). However,as 2D-MHF
chips relyrhoncus.
on a planar
flow-focusing
configuration,
they show
Hac
habitasse
platea dictumst
vestibulum
Vulputate
ut pharetra
sit amet aliquamapoor
surfacetovolumeratio,
limiting
liposome
synthesis
at
the
two
lipid/aqueousinterfaces.
id diam maecenas. Sit amet mauris commodo quis. Pellentesque adipiscing commodo
elit atSide
effectsarealso encounteredat theupper
and lower parts of the chip. These designs usuallyimperdietdui
accumsan.
Semper egetduis
at tellus.
require specific surface coatings depending on the fluids used. To overcome these limitations, glass
capillary devices have been developed to generate a hydrodynamically focused 3D stream, where
the aqueous phase fully surrounds the lipid phase (figure 2B). As a result, radially symmetric mixingof the fluidic inputs is realized, facilitating the production of highly uniform nanoscale liposomes.
This design has shown narrower size distribution compared to 2D-MHF13. These devices usually do
not require any surface coating as there is no contact between the lipidic phase and the wall of thecapillary in which the liposomes are formed.

Figure 2: Two microfluidic designs for liposome synthesis. A) two-dimensional microfluidic hydrodynamic focusing(2D MHF) showing mixing at the two lipid/water interfaces and B) three-dimensional microfluidic hydrodynamic

In this application note, we show highly monodisperse liposome synthesis using theRaydrop™(Raydrop™ is a registered trademark of Secoya Technologies): a commercially-available
glass capillary-based microfluidic device, with 3D axisymmetric geometry.
For additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com

4

TYPE DE DOC
APPLICATION NOTE
MATERIALS AND METHODSINTRODUCTION

Figure 3: Setup for liposome synthesis

I. Materialsi.

Microfluidic system

Pressure controller
Two Fluigent Flow EZ with 2 bar are used in the setup presented here.
Flow sensor
The Flow Unit is a flow sensor that allows real time flow rate measurement.
By combining a Flow Unit with the Flow EZ, it is possible to switch frompressure control to flow rate control, allowing for the generation of highly
monodispersed droplets over a long period of time. Two Flow Unit M are used
to monitor and control the flow rates of the dispersed and continuous phaseduring the run.
2-Switch
A 3-port/2-way microfluidic valve is used to switch between PBS and lipid+ EtOH solution. This allows for better reproducibility by priming the system
and adds a cleaning step during the experiment.

For additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com

5

APPLICATION NOTE
Microfluidic device
The Raydrop™ is a versatile microfluidic device that can be used for highlyprecise fluid mixing. It is based on the alignment of two glass capillaries
immersed in a pressurized chamber containing the outer phase. The inner
phase exits the capillary through a 3D-printed nozzle, placed in front of asecond capillary, where it is surrounded by the outer phase. This nonembedded implementation of axisymmetric flow-focusing is referred to coflow-focusing, and allows one to generate a hydrodynamically focused 3D
stream. The advantage lies in his geometry that prevents wettability issues
that could appear in other microfluidic chips. This allows for the generationof highly monodispersed nanoparticles with any type of fluid.

ii.

Reagents

Inner phase
Absolute ethanol (LiChrosolv Supelco, Merck, Germany), Lipids 10 mg/mL (Phospholipon 90
G, Lipoid, Switzerland), and Dimethyldioctadecylammonium (DDAB) 2.5 mg/mL (from Merck,
Germany) filtered with 0,2 µm syringe filter.
Outer phase
Phospate-buffered saline (PBS) (pH 7.2) filtered with 0,2 µm syringe filter.
Inner phase for priming and cleaning
Absolute ethanol (LiChrosolv Supelco, Merck, Germany) filtered with 0,2 µm syringe filter.

II. Liposome synthesis and size analysis
As mentioned previously, a stream of lipid in alcohol solution is surrounded by an aqueous phasewithin a glass capillary. The alcohol solution containing lipids diffuses into the aqueous solution(and reciprocally the water diffuses into the alcohol), until the alcohol concentration decreases
to the solubility limit of the lipids. As a consequence, this diffusion triggers the formation ofliposomes by a mechanism described as “self-assembly”, where lipids assemble into a moreenergetically favorable structure. In microfluidic liposome synthesis, two parameters are usually
utilized for tuning the size of the focused stream: the flow rate ratio (FRR) between the aqueous
and the alcohol phases, and the total flow rate (TFR). Jahn et al. first demonstrated that themean size of the liposome produced is inversely proportional to FRR. Conversely, a small effecton liposome size is observed when changing the TFR. [14,15] These results were confirmed inadditional studies. These parameters are utilized in this application note.

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APPLICATION NOTE

Figure 4: Scheme for liposome synthesis

The liposome production system is illustrated in figure 4. Two reservoirs of 15 mL containing
absolute ethanol, and lipids in ethanol are connected to a 2-SWITCH, which is connected to themicrofluidic device via 1/16 in. PEEK tubing (inner phase). One reservoir of 50 mL containing PBS
is connected to the second inlet of the device (outer phase). The tubing passes through flow
units allowing flow rate measurement and control. The outlet tubing can be connected to asecond 2-SWITCH to switch between waste and particle recovery.

i.

Liposome synthesis

1.
The 15 mL reservoir containing absolute ethanol is selected by the 2-SWITCH. Ethanol is
injected as inner phase. At the same time, PBS (surrounding outer phase) flow is started to prime
the system and generate a co-flow of ethanol and PBS. Identical pressure should be applied on
both reservoirs.
2.
As soon as a steady co-flow is reached, the solution of lipids in ethanol can be easily
injected by switching the 2-SWITCH to the second position (lipids/ethanol).
3.
Liposomes should be produced in less than 1 min (time for the liquid to switch)
4.
Pressure and/or flow rate can be then adjusted to target the stream diameter (figure 5),
desired TFR and FRR, and the related liposome size.

Figure 5: A liquid stream of ethanol with lipid, surrounded by PBS

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APPLICATION NOTE
5.
To stop the experiment, identical pressure is applied on both reservoirs. Absolute ethanol
is injected by switching the 2-SWITCH to first position (at 10 µL/min the liquid replacement takes
approx. 70 s, depending on the tubing used). The dispersed phase is then stopped, and thetubing at the injection entry is removed to allow for the injection capillary to be filled with theaqueous phase. Pure water should be used instead of PBS for cleaning.
For more information about assembly, filling and cleaning using the Raydrop™, please refer tothe Good practice guide on droplet generation process.
In this application note, we determine the effects of the FRR and the TFR on liposome size. Table
2 summarizes the flow rates used.

Table 2: Summary of the stream generated using different flow ratesii.

Liposome size analysis

Analysis using a Dynamic light scattering (DLS) system (Nanoflex 180, Microtrac, USA), assuming
n=1.45 for liposomes, n =1.33 for the water phase (PBS) and a spherical shape for the liposomes.
For each sample, 3 measurements of 60 s each are performed and the mean value is calculated.

RESULTS & DISCUSSION
Table 2 shows the used pressures, flow rates, TFR, FFR and the corresponding generated innerstream diameters of the lipids/ethanol solution. We observe from the images a well-centeredand steady inner flow, confirming enhanced flow stability using the Raydrop™. We also notefrom the same table that the stream diameter increases when the FRR increases. Lipid solubility
limit is reached, and liposomes are synthesized.

Figure 5: A liquid stream of ethanol with lipid, surrounded by PBS

APPLICATION NOTE

Once synthetized, liposome size analysis is performed using DLS. For each sample, 3measurements were performed, and the median diameter is calculated, associated with its
polydispersity index (measure of the distribution). Figure 6 shows the liposome median diameterand its corresponding polydispersity index as a function of the FRR. TFR was kept constantaround 100 µL/min. We observe that liposomes with a diameter ranging from ~30 nm to ~155 nm
were generated under these conditions. In addition, we observe that when the FRR increases,
the liposome diameter decreases. In fact, a diameter of 87 nm is obtained using a FRR of 4,
while a diameter of 29 nm is obtained using a FRR of 9. This result is in good agreement with theliterature using microfluidic methods [14,15], and is likewise attributed to the increasing degree

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APPLICATION NOTE
of focusing experienced by the center stream of solubilized lipid [13]. This level of control on theliposome diameter is not possible to reach using standard batch methods. Depending on theparameters used, a wide range of liposome sizes is demonstrated. This size range achieved is
wider than typical alternative microfluidic systems [13]. From the same figure, we observe that
the PDI ranges from 0,07 to 0,15.

Figure 6: Liposome mean diameter and polydispersity index (PDI) as a function of the flow rate ration (FRR)

Finally, the impact of the TFR on liposome size was investigated. The
FRR was kept constant using around 6,33, while varying the TFR. Table
4
shows
the
liposome
diameter
and
PDI
as
a
function
of
the
TFR.

Table 4: Liposome diameter and related PDI as a function of the TFR. The FRR is kept constant around 6,33

We observe that by doubling the TFR from 110 µL/min to 220 µL/min the liposome diameter
varies from 50 to 59 nm. This result is also in good agreement with the previous studies. [14,15]
The generation of highly monodisperse liposomes at sizes ranging from approximately 30 nm to
150 nm were generated, confirming the great potential of the Raydrop™ for liposome synthesis.
It is worth noting that it is possible to reach a broader liposome size range by using other valuesof FRR.

APPLICATION NOTECONCLUSION
Liposome nanoparticles prove advantageous at solubilizing therapeutic substances.
Macroscale batch methods widely employed for liposome production lack control
on liposome morphology, size, and distribution. Microfluidic systems allow for the
production of highly monodisperse liposome nanoparticles. We have demonstrated
the production of liposomes using a microfluidic system consisting of pressure-based
flow controllers and the Raydrop™ microfluidic device with standard configuration.
Liposomes ranging from 30 to 150 nm were generated. Sizes can be adjusted bycontrolling the device flow input parameters, particularly the flow rate ratio (FRR).
The polydispersity index (PDI) ranges from 0,07 to 0,15. This system enables thesynthesis of liposomes for drug delivery applications, as encapsulating agentsfor food ingredients, or for other applications requiring nano-sized and sphericalliposomes.
A complete, cost-effective, and commercially-available platform for the on-demand
production of monodisperse liposome nanoparticles is now available. This allows
for control of liposome size and frequency by adjusting flow parameters.

REFERENCES
1.

Bozzuto, G. & Molinari, A. Liposomes as nanomedical devices. Int. J. Nanomedicine 10, 975–999 (2015).

2.

Blanco, E., Shen, H. & Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to
drug delivery. Nat. Biotechnol. 33, 941–951 (2015).

3.

Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 4,
145–160 (2005).

4. Bally, M. et al. Liposome and lipid bilayer arrays towards biosensing applications. Small 6, 2481–2497(2010).
5.

Fathi, M., Mozafari, M. R. & Mohebbi, M. Nanoencapsulation of food ingredients using lipid based
delivery systems. Trends Food Sci. Technol. 23, 13–27 (2012).

6. Grimaldi, N. et al. Lipid-based nanovesicles for nanomedicine. Chem. Soc. Rev. 45, 6520–6545 (2016).
7.

Andrew Pohorille & David Deamer. Artificial cells: prospects for biotechnology. Trends Biotechnol.
Biotechnol. 31- (2002).

8. Rongen, H. A. H., Bult, A. & Van Bennekom, W. P. Liposomes and immunoassays. J. Immunol. Methods
204, 105–133 (1997).

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APPLICATION NOTE
9. Vogel, A. B. et al. A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lunginfection in non-human primates. bioRxiv 2020.09.08.280818 (2020).
10. Pattni, B. S., Chupin, V. V. & Torchilin, V. P. New Developments in Liposomal Drug Delivery. Chem. Rev.
115, 10938–10966 (2015).
11. Mui, B., Chow, L. & Hope, M. J. Extrusion Technique to Generate Liposomes of Defined Size. Methods
Enzymol. 367, 3–14 (2003).
12. Carugo, D., Bottaro, E., Owen, J., Stride, E. & Nastruzzi, C. Liposome production by microfluidics: Potential
and limiting factors. Sci. Rep. 6, 1–15 (2016).
13. Hood, R. R., Devoe, D. L., Atencia, J., Vreeland, W. N. & Omiatek, D. M. A facile route to the synthesisof monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric
capillary array. Lab Chip 14, 2403–2409 (2014).
14. Jahn, A., Vreeland, W. N., Devoe, D. L., Locascio, L. E. & Gaitan, M. Microfluidic directed formation ofliposomes of controlled size. Langmuir 23, 6289–6293 (2007).
15. Jahn, A. et al. Microfluidic mixing and the formation of nanoscale lipid vesicles. ACS Nano 4, 2077–2087(2010).

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