CHITOSANMICROCAPSULESSYNTHESISAPPLICATION NOTE

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APPLICATION NOTE CHITOSAN MICROCAPSULES SYNTHESISINTRODUCTION
Over the past few decades, core-shell microcapsules have been extensively used for the delivery
and release of materials in the pharmaceutical, cosmetic, and food industries Microencapsulation of lipophilic drugs has attracted special attention because highly effective, but poorly
water-soluble drug candidates are common outcomes of drug discovery programs. In thiscontext, microcapsules consisting of a chitosan shell and an oily core have been extensively
studied as chitosan, a cationic polysaccharide, exhibits numerous benefits.
These include: excellent biological activity, good biocompatibility and biodegradability andpH sensitivity for acid-triggered oral delivery. Traditional microencapsulation methods suchas coacervation, spray drying, and solvent evaporation, require complex processes and equipment and are difficult to control the size and load of the microcapsules. Microfluidics allows
for the production of monodisperse double emulsions with a high level of control over boththe size and the structure.
In this Application Note, chitosan-shell/oily-core microcapsules are generated using the Raydrop double emulsion chips, a capillary based microfluidic device and Fluigent pressure-based
flow controllers. This allows for the generation of double emulsions in a user friendly manner.
The influence of the fluidic parameters on the size and the release from the oil across the shell
are studied and presented.

MATERIALS
1. Setup
The production of droplets has been performed with the Raydrop Platform, a lab system integrating all the components needed to produce simple and double emulsions. This platform isdivided into three parts: mechanical, fluidic, and optical.

Figure 2: Raydrop Platform

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APPLICATION NOTE
• Mechanics: The mechanical assembly includes x-y-z displacement plates that allow to adjust
the focus and the observation window in the Raydrop.
• Fluidics: The fluidic section includes the Fluigent Flow EZ controllers and the required tubing and valves which permit automated fluidic injection. A pressure is set on each reservoir,and fluids are delivered to the microfluidic chip. It also includes Falcon reservoirs and theRaydrop, in which double emulsions are generated.
• Optics: The optical assembly contains an LED light source and a color USB 3.0 camera. Thiscamera is connected to a computer to observe the droplet formation in real time, to control
the stability of the emulsion and measure the size of interest (core, shell).

Figure 3: Experimental set-up to produce double emulsion

Fluid reservoirs
Falcon identification

F1F2F3F4Volume (mL)

50505015Phase*ContinuousCoreShell (primingand cleaning)

ShellComposition

1-octanol + 2% Soybean oil Water + 2%
Span80+ Sudan IV acetic acid

Water + 2% chitosan 30100 cps + 1% PLURONIC®
F-127 + 2% acetic acid

*Each phase must be filtered to avoid clogging the tubing or the nozzle of the Raydrop. Therefore, there is a filter after each Falcon. In this case, the Falcons’ filters F1 and F4 have a 10 µmfilter pore size and the Falcons’ filters F2 and F3 have a 2 µm filter pore size.
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APPLICATION NOTE CHITOSAN MICROCAPSULES SYNTHESIS
2. System components
Microfluidic system:•Pressure controller: Fluigent LineUpTM Flow EZ 7 bar1*•
Flow rate sensor: Fluigent Flow Unit M (x2) and L (x1)
•Microfluidic droplet generator: Raydrop double emulsion**Raydrop™ is a registered trademark of Secoya Technologies

Picture of the Raydrop

Scheme of the Raydrop

Figure 4: Microfluidic droplet generator

Nozzle information
PartCore nozzle

Size-shell nozzle

Size-extraction capillary

Inside diameter (µm)

7090450

Software information
Fluigent OxyGEN software (formerly AiO software)1

The pressure controllers used are 7 bar full scale. The maximum pressure used for the generation of double emulsions withlarge shells is 2440 mbar (corresponding to a shell phase flow rate of 24.3 µL/min). Except from this scenario, maximum working
pressure is 1650 mbar. Priming and cleaning steps can require a pressure higher
than 2 bar.

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APPLICATION NOTE
3. Reagents
Core phase:
Soybean oil (8001-22-7, Sigma-Aldrich) containing red dye Sudan IV (Sigma-Aldrich)

Shell phase:
Water containing 2% chitosan (viscosity 30-100 mPa·s, Glentham Life Sciences UK), 2% acetic
acid (Sigma-Aldrich), 1% Pluronic® F-127 (Sigma-Aldrich).

Note that the chitosan is very viscous.
Therefore, the preparation of this solution
should be done in the following order:
add Pluronic® F-127 to the water whilestirring with a bar magnet and, once thePLURONIC® F-127 is dissolved, gradually

Continuous phase:
1-octanol (Glentham Life Sciences UK) containing 2% Span 80 (Sigma-Aldrich)Collection phase:
Heptane (VWR) containing 2% Span 80 (Sigma-Aldrich) and 0,3 wt% glutaraldehyde (50% in
H2O, Glentham Life Sciences UK)

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Monodisperse chitosan particle synthesis is performed in 2 main steps:•
Generation of monodisperse double emulsion in the Raydrop•
Capsule formation by reticulation of the chitosan shell in the collection bath

1. Double emulsion generation
To generate droplets, the system must first be primed with pure solvent in the shell phase(here water + 2% acetic acid). Once droplet formation is stable, the shell phase is switched tothe chitosan-based solution. This avoids clogging issues during the transient phase. The user
should follow the steps below, using the typical flow rates shown in the table below:
1.
Set the valve on the Falcon F3 (priming solution)2.
Fill the Raydrop with the continuous phase (refer to the user guide for more information)
3.
Set the continuous phase (F1) to the desired flow rate4.
Set the shell phase (F3) to the desired flow rate. At this point, a co-flow of 1-octanol andwater is generated.
5.
Set the core phase (F2) to the desired flow rate to generate double emulsions
6.
When the double emulsion is stabilized, switch the valve to the chitosan-based shellsolution (F4)7.
Wait** until the chitosan solution crosses the tubing and reaches the Raydrop to form adouble emulsion with a chitosan solution shell and a soybean oil in the 1-octanol continuous
phase

Continuous phase

Shell

CoreComposition

1-octanol + 2%
Span 80

Water + 2% chitosan 30-100cps+ 1% PF127 + 2%
AcOH

Soybean oil

Flow rate (µL/min)

1301512For any additionnal information, please contact us by email: contact@fluigent.com or consult our website: www.fluigent.com
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2

Figure 5: Generation of double emulsion droplets in the Raydrop. Red dye Sudan IV
is added in the core phase to increase the contrast.

8.
If necessary, stabilize the double emulsion by varying the flow rates
9.
Adjust the flow rates to obtain the desired droplet diameter and shell thickness
10.
Collect the droplets at the outlet of the Raydrop in a plastic container e.g. polypropylene(droplets will tend to wet a glass container).

Figure 6: chitosan-shell/oil-core double emulsion collected in the 1-octanol continuous phase

Before stopping the experiment, flush the shell tubing (T3) and the nozzle of the Raydrop withthe priming solution (F3). This priming/cleaning solution only contains water and acetic acid,
which allows the evacuation of the chitosan. This cleans the tubing clogging is avoided.
11.
To flush the chitosan out of tubing and Raydrop, switch the valve on the priming solution(F3)12.
Wait** until the cleaning solution crosses the tubing and reaches the Raydrop to forma double emulsion with a water solution shell and a soybean oil in the 1-octanol continuous
phase
13.
Stop the flow of the core phase
14.
Then, stop the flow of the shell phase
15.
Finally, stop the flow of the continuous phase** This can take 10 to 15 minutes, depending on the flow rate of the chitosan phase and thediameters and length of the tubing.

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APPLICATION NOTE CHITOSAN MICROCAPSULES SYNTHESIS
2. Capsule formation
After generation, the droplets are collected in a cross-linking solution of 0.3% glutaraldehyde inhexane. The chitosan reacts with glutaraldehyde by solvent extraction and chemical cross-linking based on the Schiff base reaction. The droplets are solidified and become glutaraldehyde
cross-linked chitosan microcapsules.

Figure 7: Glutaraldehyde cross-linked chitosan microcapsules on the cross-linking bath3. On the left,
after 4 minutes in the cross-linking bath. On the right, after 1h in the cross-linking bath. The shell
thickness decreases and becomes progressively yellow, as a part of its water content diffuses in thecontinuous phase. Expelled water is clearly visible wetting the capsules.

RESULTSIn this Application Note, different parameters were studied. First, the evolution of the droplets
over time was observed. Then, the influence of the middle and the outer phase flow rates werestudied.

1. Evolution of the droplet diameter during the cross-linking process
After generation, the double emulsion droplets are collected into the collection solution. An
analysis of the size of the capsules is performed using a microscope and measurement software (ToupView). For a given sample, several measurements of the capsule diameter are doneat different times. The evolution of the diameter is highlighted in Figure 8 and the operating
conditions are shown in Table 1.

2DCA-MAM-019-video1-frame-rate-4.75

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APPLICATION NOTE
Table 1: Operating conditions for the evaluation of the diameter of capsulesContinuous phase

ShellCoreComposition

1-octanol + 2%
Span 80

Water + 2% chitosan 30-100cps+ 1% PF127 + 2%
AcOH

Soybean oil + Sudan IV

Pressure (mbar)

164

1650

1384

Flow rate (µL/min)

162

16,413,6

Figure 8: Size of chitosan capsules as a function of time

The capsules have a diameter ranging from 342 µm (7 min) to 202 µm (>1 000 min, not displayed in the graph). We observe that during the cross-linking, the diameter of the capsules
decreases. In fact, a diameter of 332 µm is obtained 15 minutes after droplet formation, while adiameter of 263 µm is obtained 60 minutes after droplet formation. This result agrees with thepublication of Du et al. [3] and can be explained by the fact that during cross-linking, water inthe shell phase is extracted.

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2. Influence of the middle phase flow rate
After analyzing the size of the capsules over time, the influence of the middle phase flow rate isobserved. We varied the shell flow rate at fixed continuous and core phase flow rates. For eachshell phase flow rate, a video was recorded and the diameter of the shell and the core weremeasured using software. The evolution of these two diameters is underlined in Figure 9 andthe operating conditions are shown in Table 2. Figure 10 shows the evolution of the thicknessof the droplet with the evolution of the shell flow rate.

Table 2: Operating conditions for the evaluation of the size of capsules for a middle
flow rate variation

Continuous phase

ShellCoreComposition

1-octanol + 2%
Span 80

Water + 2% chitosan 30-100cps+ 1% PF127 + 2%
AcOH

Soybean oil + Sudan IV

Pressure (mbar)

174

variable1378

Flow rate (µL/min)180

2 – 27 µL/min

13,6Figure 9: Core size and shell size as a function of the shell liquid flow rate

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We observe that the shell diameter remains relatively constant when varying the shell liquidflow rate. A shell diameter of 308 µm is obtained using a shell flow rate of 3 µL/min, while asimilar shell diameter of 305 µm is obtained using a shell flow rate of 24 µL/min. However, weobserve that when the flow rate of the shell phase increases, the diameter of the core decreases.
Indeed, a core diameter of 257 µm is obtained using a shell flow rate of 3 µL/min, while a core
diameter of 183 µm is obtained using a shell flow rate of 24 µL/min. This phenomenon can be
explained by the fact that the size of the shell is constrained in a limited range by the geometryof the system when the continuous phase flow is constant.

Figure 10: Thickness of the shell as a function of the shell liquid flow rate

Figure 10 underscores the increase of the thickness of the shell when the shell liquid flow rateincreases. The shell thickness increases with the shell liquid flow rate. A shell thickness of 25µm is obtained when the shell liquid flow rate is 3 µL/min, while a shell thickness of 61 µm is
obtained when the shell liquid flow rate is 24 µL/min. As the diameter of the droplet remains
almost constant (the diameter being fixed by the continuous phase flow rate and by thegeometry of the system) but the flow rate of the shell phase increases, the droplet generation
frequency increases and the core size decreases.

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APPLICATION NOTE CHITOSAN MICROCAPSULES SYNTHESIS
3. Influence of the outer phase flow rate
Previously, the continuous phase flow rate and core flow rate were fixed and the shell flow rate
was varying. In this part, the shell flow rate and core flow rate are fixed but the continuous phaseflow rate is varying. Once again, for each continuous phase flow rate, a video was recorded andthe diameter of the shell and the core were measured. The change in diameter as a functionof flow rate is shown in Figure 11 and the operating conditions are shown in Table 3.
Table 3: Operating conditions for the evaluation of the size of capsules for a continuous flow rate variation

Continuous phase

ShellCoreComposition

1-octanol + 2%
Span 80

Water + 2% chitosan 30-100cps+ 1% PF127 + 2%
AcOH

Soybean oil + Sudan IV

Pressure (mbar)

variable

1553

1378

Flow rate (µL/min)variable

16,4

13,6

Figure 11: Core size and shell size as a function of the outer liquid flow rate

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APPLICATION NOTE

We observe that the shell diameter remains relatively constant when varying the shell liquidflow rate. A shell diameter of 308 µm is obtained using a shell flow rate of 3 µL/min, while asimilar shell diameter of 305 µm is obtained using a shell flow rate of 24 µL/min. However, weobserve that when the flow rate of the shell phase increases, the diameter of the core decreases.
Indeed, a core diameter of 257 µm is obtained using a shell flow rate of 3 µL/min, while a core
diameter of 183 µm is obtained using a shell flow rate of 24 µL/min. This phenomenon can be
explained by the fact that the size of the shell is constrained in a limited range by the geometryof the system when the continuous phase flow is constant.

4. Capsule release analysis
Collected droplets are separated in two samples with different cross-linking times. The firstsample contains droplets that have cross-linked for 17 minutes and the second contains droplets that have cross-linked for 45 minutes. Each sample is then washed with a buffer solution
at pH 7.3 and then stored in the buffer.
Analysis of the release of the dye contained by the capsules is performed using a spectrophotometer. This allows one to determine the absorbance of the Sudan IV dye of the solution
containing the capsules according to a specific wavelength.
For each sample, 15 measurements were made over a period of 24 hours. The results are presented in Table 4. No significative difference was observed between the two samples. In bothcases about 95% of the encapsulated oil remained after 24h when stored in a water solution,
demonstrating that a compact shell structure preventing oil leakage is formed in less than 20
minutes of cross-linking.
Table 4: Release rate of capsules

Release rate of the red dye in the buffer
solution containing the capsules

Capsules cross-linked for 17 minutes

Capsules cross-linked for 45 minutes

< 5%

< 5%

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APPLICATION NOTE CHITOSAN MICROCAPSULES SYNTHESIS
5. Conclusion
The production of stable, monodispersed microcapsules with a solid chitosan shell and a liquidoil, non-polar core using a microfluidic system has been successfully achieved. The Fluigent
microfluidic platform also allows one to tune the core diameter and the shell thickness byadjusting the flow rates of the different fluids. Due to excellent oil encapsulation properties anda very limited leakage over time, these microcapsules can be used in a wide range of applications, including the encapsulation of volatile products like mint oil [3] as well as specific drugs,
which will be delivered according to the pH acidity [2].

6. References[1] KILDEEVA, N. R., PERMINOV, P. A., VLADIMIROV, L. V., NOVIKOV, V. V. and MIKHAILOV, S.
N., 2009. About mechanism of chitosan cross-linking with glutaraldehyde. Russian Journalof Bioorganic Chemistry. 1 May 2009. Vol. 35, no. 3, p. 360–369. DOI 10.1134/S106816200903011X.
[2] LIU, Li, YANG, Jian-Ping, JU, Xiao-Jie, XIE, Rui, LIU, Ying-Mei, WANG, Wei, ZHANG, Jin-Jin,NIU, Catherine Hui and CHU, Liang-Yin, 2011. Monodisperse core-shell chitosan microcapsulesfor pH-responsive burst release of hydrophobic drugs. Soft Matter. 3 May 2011. Vol. 7, no. 10, p.
4821–4827. DOI 10.1039/C0SM01393E.
[3] DU, Yuhan, MO, Liangji, WANG, Xiaoda, WANG, Hongxing, GE, Xue-hui and QIU, Ting, 2020.
Preparation of mint oil microcapsules by microfluidics with high efficiency and controllability
in release properties. Microfluidics and Nanofluidics. June 2020. Vol. 24, no. 6, p. 42. DOI 10.1007/
s10404-020-02346-2.

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