PLGAMICROPARTICLESYNTHESISABSTRACT
When PLGA is used as an active pharmaceutical ingredient carrier it is important to produce highly monodispersed particles for drug release reproducibility. The most common
production process of PLGA particles is solvent based and can involve hazardous solutions. Ethyl acetate is preferred as it shows better biocompatibility than other conventional
solvent such as dichloromethane. We present here a novel PLGA microparticle production station based on a new droplet production technology (RayDrop system designed by
Secoya) and high precision pressure pumps (Flow EZTM, Fluigent). The station is adapted
for working with ethyl acetate solvent and shows outstanding monodisperse particle production. We present an overall characterization of the device including phase diagramsfor several PLGA concentrations. This station is particularly suitable for researchers
who want to test different API encapsulation conditions with highly reproducible results.

This application note was made in collaboration with Adrien Dewandre at
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APPLICATION NOTEINTRODUCTION
In recent years, biodegradable microspheres/microparticles have gained widespread importance in the delivery of bioactive agents [1]. The copolymers of Poly
(D, L-lactic-co-glycolic acid) (PLGA)/poly (lactic acid) (PLA) microparticles are one of
the most successful new drug delivery systems (DDS) in labs and clinics. Becauseof good biocompatibility and biodegradability, they can be used in various areas,such as long-term release systems, vaccine adjuvant, and tissue engineering [2].
Using PLGA/ PLA has demonstrated longer and better efficiency, reduction of administration frequency [3–5] by many administration routes, including subcutaneous injection,
intramuscular injection, oral administration, pulmonary administration [6,7], and ocularadministration [8].
PLGA/PLA microparticles can be used to encapsulate hydrophobic as hydrophilic therapeutics drugs and allowing for reduced dosing frequency and less potential toxicity. [9–11]
In PLGA microparticle production for drug release and delivery, microparticle size is a paramount parameter. Indeed, the microparticle size is directly related to the microparticles degradation rate as well as the drug release rate [12].
Although PLGA microparticle synthesis appears to be a successful drug delivery system,
the current processes and tools to produce PLGA microparticles have many limitations
such as wide microparticle size distribution, poor repeatability, and agressive preparation
conditions [2].
To solve these problems, microfluidics, and more exactly droplet-based microfluidics, offers an efficient method for improvement. Droplet based microfluidics is a powerful tool
which enables of micrometric monodispersed droplet generation and manipulation.

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APPLICATION NOTE
Microfluidic droplet size control and generation rate allows highly monodispersed droplet
productions opposed to other batch emulsion methods and provides an “In-line” continuous droplet production process. There are many different PLGA particle production methods such as solvent extraction, solvent evaporation or solvent exchange. In all cases,
the nature of the solvent is an important parameter that must be taken into account.
Indeed, it affects the PLGA precipitation time and must be biocompatible with theactive agent.
Currently in every microfluidic system for PLGA microparticles production highlighted
by researchers, dichloromethane is the most used and widespread solvent. Nevertheless, some researchers have highlighted some limitations of this solvent
due to its potential toxicity [13]. Ethyl acetate has been considered as an alternative dispersed solvent useful in preparing PLGA microspheres because of its properties less hazardous. It is thus more suitable for biological applications [14]. Moreover, due to high solubility, ethyl acetate allows a reduced PLGA precipitation time.
According to the U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER) classification, dichloromethane is recognized as a class
2 solvent whereas ethyl acetate is class 3 which makes it more suitable for biological applications. Even though, some end-user production devices using ethyl acetate
are available on the market but they all present limitations for PLGA microparticles,
which are released with the new microfluidic device presented in this application note.

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APPLICATION NOTEDESCRIPTION
Fluigent presents a new microfluidic device and system for PLGA microparticle synthesis

Figure 1: PLGA microparticle production station

All the products currently available in the market have critical drawbacks. Fluigent in collaboration with Secoya, a spin-off of the Université libre de Bruxelles,has developed a new
breakthrough technology leading to outstanding particle size monodispersity and production flexibility.
In this application note we will describe a novel platform and protocol to use ethyl
acetate at different concentrations for PLGA polymerization and continuous synthesis. This application note characterizes the Fluigent solution for generating
PLGA microparticles. The monodispersity and solvent extraction time are measured and discussed. Wide and detailed phase diagrams for various PLGA concentration are provided showing parameters for manipulating droplet size and frequency.
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APPLICATION NOTE
MATERIALS AND METHODS
Reagents• Priming fluid: Ethyl acetate filtered with 0.2 μm pore filter and used without further
modification (Sigma Aldrich, CAS Number: 141-78-6)• Droplet fluid: Resomer® RG 753 S, Poly(D,L-lactide-co-glycolide) ester terminated,
Lactide: Glycolide 75:25 is dissolved in ethyl acetate at room temperature by stirring
over the course of an hour (Sigma aldrich,CAS Number: 26780-50-7).
• Continuous phase: 2 % (w/v) Poly (vinyl alcohol) (PVA) surfactant in water is filteredwith a 0.2 μm pore filter (Sigma Aldrich,CAS Number: 9002-89-5).

Droplet generation experimental set-up
PLGA microparticle production station, full packFigure 2: Schematic of PLGA microparticle production station set-up, full pack

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APPLICATION NOTE
The droplet generation set-up is illustrated in the previous picture.Two different pressure
controllers (Flow EZTM, Fluigent) are used to handle fluids. A 3/2 valve (2-SWITCHTM, Fluigent) valve is used to switch between pure ethyl acetate and PLGA dissolved in ethyl acetate
and a second valve is also used in particle production output to switch between a waste
and recover sample (see the detailed protocol in the attachment down page). Two FLOWUNIT sensors are used to monitor and control the internal and external phases flow rates
during he runall. The new, innovative RayDrop system is used to generate PLGA droplets.

Droplet monitoring and Analysis
To observe droplet generation an Inverted microscope (Nikon, Eclipse Ti 2)
coupled with High speed camera (Nikon, PhotronFastcam) have been used.
For droplet sizes analysis the software PhotronFastcam Viewer has been used.
To estimate

dropletfrequency

thefollowing

formula

has

been

used.

Droplet rate (Hz) = Droplet phase flow rate (µL/s) / Droplet volume (µL).

Microparticle Precipitation
Once droplets are generated, the solvent inside the microparticles in the presence of the continuous phase will continuously diffuse out of the droplet.
With solvent diffusion outside of the droplet the PLGA concentration will increase and precipitate to form a solid microparticle smaller than the droplet.
A

B

400 µm

400 µmFigure 3: Particle shrinking during solvent evaporation: A) Particle size just after
droplet formation B) Particle size after few seconds
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APPLICATION NOTE
Microparticle Recovery
Microparticles are recovered in a vial containing the continuous phase: water with 2%
PVA. The microparticle size and monodispersity are determined under a microscope. To
do so, collect a small sample by placing the outlet tubing directly on a glass cover slip.

RESULTS
Dispersed
phase /
Dropletphase

Dispersed phase: 2% (w/v) PLGA in ethyl acetate
Continuous phase: 2% (w/v) PVA in wateContinuous
phase

Microparticles

Droplets

Quantity

Picture
Q
Q
P
P
(µL/
(µL/
(mbar)(mbar)min)
min)

Diameter
(µm)

Rate
(Hz)

Diame- Quantertity(µm) (mg/h)

492

3

1348

100

64

364

19

4.7

592

5

1348

100

68

506

20

7.65

893

10

1374

100

76

725

26

24.1

1052

12.5

1400

100

80

778

27

29.1

1222

15

1454

100

80

933

27

35

Figure 3: Table showing how flow-rate changes affect PLGA particle size and production rate
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APPLICATION NOTE
After generation, a microparticle size population has been analyzed to determine microparticles monodispersity at 1%. The monodispersity has been analyzed on a microparticle population (around 100 microparticles). The flow rates
have been adapted to generate 20 µm diameter particle size (cf phase diagram).
B

40 µm

Count

Particle size (µm)

Figure 5: Particle size distribution: A) Microscope observation of PLGA microparticles
just after the generation B) Particle monodispersity graph

Figure 6: Droplet size diagram with 2% (w/v) PLGA concentration

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APPLICATION NOTE

Figure 7: Particle size diagram with 2% (w/v) PLGA concentration

Results with Other Concentration
Dispersed phase: 5% (w/v) PLGA in ethyl acetate
Continuous phase: 2% (w/v) PVA in water

Figure 8: Droplet size diagram with 5% (w/v) PLGA concentration
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Dispersed phase: 10% (w/v) PLGA in ethyl acetate
Continuous phase: 2% (w/v) PVA in water

Figure 10: Droplet size diagram with 10% (w/v) PLGA concentration
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APPLICATION NOTE

Figure 11: Particle size diagram with 10% (w/v) PLGA concentration

Figure 12: Particle shrinking for different PLGA concentration

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APPLICATION NOTECONCLUSIONPLGA microparticles ranging from 15 to 50 µm in diameter have been successfully genesignificantly improved monodispersity (CV < 2%) as compared to other methods on thepid startup with a short learning curve. Fluigent has developed a PLGA synthesis package
droplet microfluidic and pressure-based solution advantages to focus on the experiment

Traditional methods(Batch methods)

Fluigent PLGA microparticle
production station

Particle size distribution

~20%

~2%

Reproducibility

Low

High

API mixing

UnevenUniform

Live particle size control

No

PreciseContinuous/in line production

No

Yes

Microfluidic methods available Fluigent PLGA microparticle
on the market
production station
Particle size distribution

~5%

~2%

Semi automated production

No

Yes

Ethyl acetate dedicated protocol

No

Yes

No (glass chip changed when
clogged)Non standard, user dependantquality (leakage, blockage)

Yes (the RayDrop can be maintained)
Standard fittings for better
sealing

Device regeneration
Connectors

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APPLICATION NOTEDROPLET GENERATION PROTOCOL
1. Set-up installation

Recoveryoutletphase inlet

1.1 Preparation
Install the PLGA microparticle production station as shown in the scheme below (the useof filtered solutions is highly recommended). If you own a different pack, please refer tothe good practise guide explaining in details each PLGA microparticle production station
pack installation.

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APPLICATION NOTE
1.2 Filling the Raydrop
State
The dispersed phase inlet is not yet connected to union before the device.
The continuous phase channel is connected to the RayDrop.
The output tubing is connected to the device outlet.
The RayDrop cavity is empty.
The first 2-SWITCHTM valve is in the ethyl acetate position.
The second 2-SWITCHTM is in the waste position.

Steps to reproduce
Before the first use of the RayDrop, the cavity has to be filled with the continuous phase.
This operation has to be done once only. (Find a step by step detailed protocole on thegood practice guide)

1 Connect the continuous phase

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APPLICATION NOTE
2 Hold the RayDrop vertically, with the continuous phase channel on the bottom.

3 Set the flow rate to a high value (high pressure) on the continuous phase. As you canobserve through the windows, the cavity is progressively filled with the continuous phase.
If air bubbles are remaining in the chamber you can agitate the RayDrop to easily remove
bubbles.
4 When continuous phase starts to flow from the top tubing capillary, close the purge outlet with the plug. The continuous phase is now filling both capillaries, flushing out the air
trapped inside. (You can use a plug on the outlet to help flushing every channel but makesure that all tubing is flushed with continuous phase)

5 When there is no more air trapped into capillaries and tubings, decrease the flow rate toa low value to avoid wasting continuous phase.
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APPLICATION NOTE
2. Priming the system and generating microparticles
This part explains how to generate microparticles with the RayDrop and the station.
State
The dispersed phase inlet is not yet connected to union before the device.
The continuous phase channel is connected to the RayDrop.
The output tubing is connected to the device outlet.
The Raydrop cavity has been filled with the continuous phase.
The first 2-SWITCHTM valve is in the ethyl acetate position. Make sure that you are in
the right 2-SWITCHTM configuration to inject the ethyl acetate and not the PLGA yet.
The second 2-SWITCHTM is in the waste position.

Steps to reproduce
1 Set the flow-rate of the continuous phase to a high value (1200mbar) until the continuous phase starts to flow from both the input and output channels.

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APPLICATION NOTE
2Settheflow-rateonthedropletphasechannel
to a high value until you observe the ethyl acetate flowing at the tubing outlet and no more
air bubbles. (100 mbar should be enough)3 Connect the dispersed inlet to the RayDrop.

At this step, the ethyl acetate flow mightchange drastically and back flow could beobserved
4 Increase the Ethyl acetate pressure to reach a positive flow rate (around 10 µl/min).
Make sure to always have, at the beginning, at least 10 times more continuous phase thanthe dispersed phase.
At this step:
Adjust as needed to reach the state:- Water flow rate= 100 µl/min- Ethyl acetate flow rate= 10 µl/min

Or use the MAT protocol if you have the Automated pack and skip all the next step.

After approximatively one minute, a jetting state appears.

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APPLICATION NOTE
5 Decrease sequentially the two pressures until
you observe droplet formation. Always decrease
the ethyl acetate first and after decrease the continuous phase.

6 As soon as a suitable ethyl acetate droplet formation regime is reached, switch the first
2-SWITCHTM valve to allow now a flow of PLGA solution (at this step you might see the dispersed phase flow rate decreasing a bit).

7 Then wait for the PLGA solution to come into the input channel.
8 As soon as all the ethyl acetate solution is pushed out from the input channel you should
be in the PLGA droplet generation state.
9 Switch the second 2-SWITCHTM to recover the microparticles generated.
10 If you need to achieve a specific size and rate of droplets, adjust the pressures to reacha certain continuous/droplet phases flow rate combination.
ATTENTION: to avoid the dispersed phase to enter in the cavity, note to always work with acontinuous flow-rate at least twice the droplet flow-rate.
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APPLICATION NOTE
3. Stop generation and shutting down the experimentThis part explains how to properly stop droplet generation

and

avoid

any

channel lockage for further experiments (for a complete review of cleaning methods and current issues, please refer to the good practise guide)State
The dispersed phase inlet is connected to union before the device.
The continuous phase channel is connected to the RayDrop.
The output tubing is connected to the device outlet.
The RayDrop cavity has been filled with the continuous phase.
The first 2-SWITCHTM valve is in the PLGA position.
The second 2-SWITCHTM is in the recover position.
Steps to reproduce
1 Switch the first 2-SWITCHTM valve to the ethyl acetate position, allowing a flow of ethyl
acetate (at this step you might see the dispersed phase flow rate change a bit)
2 Switch the second 2-SWITCHTM valve to the waste position

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APPLICATION NOTE
3 Wait for the ethyl acetate solution to come in the input channel.
4 As soon as all the PLGA solution is pushed out of the input channel wait a little moment
to completly flush the output channel.
5 Then you can start decreasing the solvent solution flow rate and disconnect the dispersed phase input, without changing the flow rate of the continuous phase. Stop the flowof ethyl acetate once disconnected.

6 The continuous phase should now flow from the outlet and the inlet.To allow for flushingand cleaning, wait a little time to make sure that all PLGA solution is totally removed fromall channel and avoid plugging for next experiment. (you can use a plug on the outlet tohelp flushing every channel)
7 Then you can lock everything with the plugs and set the pressures to zero.

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APPLICATION NOTEREFERENCES[1] Soppimath, K. S., &Aminabhavi, T. M. (2002). Ethyl acetate as a dispersing solvent in
the production of poly ( DL -lactide-co-glycolide ) microspheres : e ect of process, 19(3),281–292. https://doi.org/10.1080/02652040110105319[2] Qi, F., Wu, J., Li, H., & Ma, G. (2018). Recent research and development of PLGA / PLA
microspheres / nanoparticles: A review in scientific and industrial aspects.
Kwak H H, Shim W S, Choi M K, Son M K, Kim Y J, Yang H C, Kim T H, Lee G I, Kim
B M, Kang S H, et al. Development of a sustained-release recombinant human growthhor- mone formulation. Journal of Controlled Release, 2009, 137(2): 160–165[3][4]. Kim B S, Oh J M, Hyun H, Kim K S, Lee S H, Kim Y H, Park K, Lee H B, Kim M S. Insulin-loaded microcapsules for in vivo delivery. Molecular Pharmaceutics, 2009, 6(2):353–365[5]. Kwak H H, Shim W S, Hwang S, Son M K, Kim Y J, Kim T H, YoonZH,YounHJ,LeeGI,KangSH,etal.Pharmacokineticsand efficacy of a biweekly dosage formulation of exenatide in Zucker diabetic fatty (ZDF) rats. Pharmaceutical Research, 2009, 26(11): 2504–
2512[6]. Gaspar M C, Gregoire N, Sousa J J S, Pais A A C C, Lamarche I, Gobin P, Olivier J C,
Marchand S, Couet W. European Journal of Pharmaceutical Sciences, 2016, 93: 184–
191[7]. Feng T S, Tian H Y, Xu C N, Lin L, Xie Z G, Lam M H W, Liang H J, Chen X S. Synergistic co-delivery of doxorubicin and paclitaxel by porous PLGA microspheres for pulmonary
inhalation treatment. EuropeanJournalofPharmaceuticsandBiopharmaceutics,2014,88(3): 1086–1093[8]. Salama A H, Mahmoud A A, Kamel R. A novel method for preparing surface-modified
fluocinolone acetonide loaded PLGA nanoparticles for ocular use: In vitro and in vivo evaluations. AAPS PharmSciTech, 2016, 17(5): 1159–1172

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APPLICATION NOTE

[9]. Qi F, Wu J, Fan Q Z, He F, Tian G F, Yang T Y, Ma G H, Su Z G. Preparation of uniform-sized exenatide-loaded PLGA microspheres as long-effective release systemwith high encapsulation efficiency andbio-stability. Colloids and Surfaces. B, Biointerfaces,2013,112: 492–498[10]. Parumasivam T, Leung S S Y, Quan D H, Triccas J A, Britton W J, Chan H K. Rifapentine-loaded PLGA microparticles for tuberculosis inhaled therapy: Preparation and in vitro
aerosol characterization. European Journal of Pharmaceutical Sciences, 2016, 88: 1–11
[11]. Nath S D, Son S, Sadiasa A, Min Y K, Lee B T. Preparation and characterization of
PLGA microspheres by the electrospraying method for delivering simvastatin for boneregeneration. International Journal of Pharmaceutics, 2013, 443(1-2): 87–94[12] Anderson, J. M., & Shive, M. S. (1997). Biodegradation and biocompatibility of PLA
and PLGA microspheres, 28, 5–24.
[13] Sani, S. N., Das, N. G., &Das, S. K. (2009). Effect of microfluidization parameters on
the physical properties of PEG-PLGA nanoparticles prepared using high pressure microfluidization, 26(6), 556–561. https://doi.org/10.1080/02652040802500655[14].Cho, M., &Sah, H. (2005). Formulation and process parameters affecting protein
encapsulation into PLGA microspheres during ethyl acetate-based microencapsulation
process, 22(February), 1–12. https://doi.org/10.1080/02652040400026269

CONTACT⽇本代理店: 株式会社ASICON
Local distributor in Japan: ASICON Tokyo Ltd.
asicon-tokyo@asicon-tokyo.com
www.asicon-tokyo.com

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