IOP Publishing Journal Title

Journal XX (XXXX) XXXXXX https://doi.org/XXXX/XXXX

Alteration of microcapsules permeability by addition of alginate

Author Name1, Author Name2 and Author Name1,2

1 Department One, Institution One, City One, Country One
2 Department Two, Institution Two, City Two, Country Two

E-mail: xxx@xxx.xx

Received xxxxxx
Accepted for publication xxxxxx
Published xxxxxx

Abstract
Cellulose microcapsule containing alginate was prepared by inoculating Acetobacter sp. in a single emulsion solution. The
single emulsion of water in oil (W/O) is produced using customized syringe system, where the water phase containing the
medium was introduced into the oil phase of wax under controlled flow rate. The gelation in aqueous phase of W/O is induced
by crosslinking of alginate with cellulose. The structural characteristics of the microcapsules were studied by observing the
flow pattern of vitamin B under optical microscope. It was determined that the pore size and alginate concentration in the
medium affects the outward permeation of dye from the capsule. Optical microscopic images were analysed using Image J to
find the distance traced by the vitamin B1 dye from the capsule. On correlating the distance of dye displacement to the respective
concentration of alginate in the microcapsule, it was found that increase in alginate percentage decreases the permeability of
microcapsule.

Keywords: term, term, term

1. INTRODUCTION comprises of unbranched beta (1.4) D-glucopyranosyl units.

Alginates, the anionic polysaccharide composed of beta-D-
mannuronic acid and a-L-guluronic acid, is a highly
biocompatible and biodegradable polymer. Alginate
microcapsules can be generated either by droplet generation
or ionic crosslinking. Ionic crosslinking is advantageous over
droplet generation technique due to its potential of adjusting
the pore size. The external gelation for preparation of
microcapsules is initiated by introducing a crosslinker. The
cross linker can be dissolved either in the oil phase or the
aqueous phase. Alginates are polysaccharides naturally
derived from bacterial strains such as Azotobacter,
Acetobater, Pseudomonas and lot more diverse species.
Alginate monomers can appear in homo polymeric blocks of
consecutive G-residues, consecutive M-residues or alternating
M and G-residues. Alginates possess a high degree of
physiochemical heterogeneity and differ in the M and G
residue in its back chain depending on source. On gelation,
alginate crosslinks the chain through “egg-box” model. Slow
crosslinking of alginate with cellulose creates uniform
structures with mechanical integrity. Cellulose nanocrystals
have gained the attention owing to its good mechanical
properties. (Podsiadlo et al., 2005). Cellulose polymer
These chains have high tendency to form hydrogen bonds with
alginate thereby cross linking into a highly ordered structural
entities.
Acetobacter species has gained attention due to its cellulose’s
mechanical strength. The bacterial medium comprises of
sugar and tea bags, which serves as the source of cellulose
synthesis. The synthesis of cellulose is as follows: formation
of uridine diphosphoglucose (UDPGlc), followed by glucose
polymerization into chains of the β-1→4 glucan chain. The
mechanical strength and Young’s modulus are attributed to the
microfibril’s biological origin and hydrogen bonds. The
structural heterogeneity of fiber is because of the partial
hydration of water.
In single emulsion technique, the natural polymer (cellulose)
is excreted in the aqueous medium by Acetobacter sp.
Followed by cross linking of cellulose with alginate present in
the aqueous phase. The microcapsules are formed after
incubation period which can be filtered out from the emulsion.
2. MATERIALS AND METHODS
Bacterial culture: The bacteria from freezer stock was

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retrieved again in the nutrient medium Emulsion incubation:

by incubating at 27°C for a week. All the experiments were The prepared emulsion was taken in 100mL jars for
carried out with the acetobacter mother culture. The inoculation of the bacterial culture. Bacterial culture was
retrieved mother culture was stored for future experiments. incorporated into emulsion with the help of a syringe system
2% Alginate: The 2% alginate solution was prepared by at a constant rate of 5mL/min. The samples are incubated
adding weighed quantity of alginate (Grindsted FD 156) in under constant shaking at a temperature of 27°C for 7 days.
respective volume of MilliQ water. The sample was placed Different concentrations of culture were inoculated as per the
under vigorous stirring for achieving complete dispersion of following table to characterization of alginates effect on the
the alginate in the solution. The prepared solution was stored microcapsules.
in room temperature for further use.
Emulsion composition: The single emulsion comprising of
Content Control 0.05 0.2 0.5 1% 1.5
outer oil phase and inner aqueous phase, consists of canola
(mL) % % % %
oil and a suspension of Acetobacter sample respectively. Alginate (mL)
Aqueous phase: The aqueous phase of emulsion consists of
the acetobacter culture and alginate. The experiments were Mother 10 10 10 10 10 10
carried out with different concentration of alginate (0.05%, culture
0.2%, 0.5%, 1% and 1.5%). A control sample was prepared New 10 9.5 8 5 2.5 0
culture
comprising only of mother culture and new culture. The
Alginate 0 0.5 2 5 7.5 10
cultures were adjusted to pH 4 by addition of concentrated
Table 1: Composition of medium
Acetic acid.
Bacterial staining: Microcapsules retrieval: The inoculated emulsion was
The bacterial culture was stained for better visualization of heated at 95 °C for a while until the emulsion melts out. Few
the jelly fishes using naked eye. Two drops of Congo red dye milliliters of milliQ water is added into the jar containing the
(Fluoresbrite Carboxylate 10 µM Microsphere) were added emulsion while it is on the heating plate. The jar is under the
into 10 mL bacterial culture. treatment of heat until two different layers of water phase and
Oil phase: The oil phase consists of 2% castor wax (add oil phase separates out visibly. Now the jellyfish present in
company name) dissolved in canola oil. the water phase, is retrieved using a plastic dropper into a
The drops were developed using a syringe system and a clean jar. The retrieval of jellyfish is repeated until a clear
microfluidic device to control the sample flow through the water phase is obtained. The jellyfish is stored in the water
syringe. phase under refrigeration until further studies.

Visualization of cell structure and HCO gel matrix:

The cell structure was visualized under optical microscope.

Few drops of bacterial culture were spread on to the glass
slide and covered with a glass rod. The cells were then
analyzed using optical microscope (Leica Microsystems,
Heidelberg, Germany) under the objective 5X.
The gel matrix of HCO gel was also imaged using optical
microscope (Leica Microsystems, Heidelberg, Germany)
under 5X objective.

Figure 1: Single emulsion technique for

microcapsule formation

Figure 2: Microcapsule structure

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Characterization of microbial cell structure and HCO gel

Permeability determination of microcapsules:
The permeability of microcapsules is determined by the
rate of release of dye from the capsule. 2µg/mL of Vitamin
B is added into the solution containing microcapsules. The
solution is let to stay steady for 120 seconds. Then the
microcapsules are sucked out followed by repetitive
washing in pure milliQ water. The washed microcapsule is
imaged in optical microscope
(Leica Microsystems, Heidelberg, Germany) under
Fluorescence. Pore size of the capsule is related to the
permeability of the capsule and diffusivity of the dye from
the capsule. The distance of dye displacement from the
capsule is measured using Image J (NIH).
3. RESULTS AND DISCUSSION
Determination of microcapsule permeability
The permeability of microcapsules is correlated to its ability
in diffusing out its content. The analysis of microcapsules in
fluorescence shows the rate at which vitamin B diffuses from
various microcapsules with different alginate concentration.
The snapshots of dye diffusion for every 5 seconds are
analyzed using Image J software to find the distance of
displacement from the capsule. (Fig. ) Evident bright red color
path depicting vitamin B is found under fluorescence in an
optical microscope in the filter number 2. Significant
difference in diffusion could not be seen visually due to
variations in the range of microns.
A B C
wax The Acetobacter sp. was found to be rod shaped under
10X objective in optical microscope. The bacterial culture
analyzed was cultured at 27˚C for a week. There were evident
colonies found in the microscopic analysis, which may have
been due to the overgrowth of the culture. On further
magnification few cell ruptures were found. The overgrowth
of culture and handling might have caused cell ruptures (Fig.
). Hydrogenated castor oil exhibits ice flake like structure
when characterized using optical microscope. The ice flake
structures depict the wax’s crystallinity (Fig. ). The cell count
was found using optical microscope, where few drops of
culture was spread on to a slide and the image was captured in
a digital camera. The analysis of Acetobacter cells in Image J
shows that there are 73 cells/ mL, with average size of 1.70
microns.
A B
Figure : Acetobacter structure in (A) 5X and (B) 10X
objective visualized in optical microscope under yellow
and red filters respectively
A B

Figure : The snapshots of vitamin B diffusing from the

microcapsules (1% alginate) onto the slide observed in
optical microscope under 10X objective
A B C Figure : Characterization of HCO wax in optical microscope
under 5X and 10X objective

Figure : The snapshots of vitamin B diffusion from

microcapsules (control) onto the slide observed in optical
microscope under 10X objective Figure : Snapshot of Image J process calculating the
number of cells

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Characterization of the microcapsules of the method. The results proved diffusion of dyes to be more
significant than the reproducibility error.
The spherical microcapsules were of irregular sizes, varying
due to the inconsistency in microfluidic device control. The
cells were contained deep into the microcapsule as shown in
Fig. (). The thick outer layered is the formed due to the 5.967
crosslinking of alginate with cellulose nanofibrils. This 6
crosslinking incorporates high elasticity and mechanical 5.8126
strength better than that in the native microcapsules. The 5.8 5.706 5.7035
capsule matrix breaks off under intolerable stress causing

DISTANCE (µm)
5.6133
exposure of cells as in Fig. (). 5.5304
5.6
A B
5.4

5.2

ALGINATE PERCENTAGE

Figure : Distance transverse by dye from different

Figure : Microscopic image of (A) containg bacterial cell
entrapped in the microcapsule matrix and (B) a broken
capsule excretion of entrapped cells into the environment
The cellulose nanofibrils microcapsule where characterized to
be spherical in shape. The size of the capsule was found using
the Image J software form the Threshold image. The capsule
diameters were found in the range of 10 to 750µm. From the
8-bit image of microcapsules (control), the diameter was
found to be 15.34 µm (Fig. ).
microcapsules
Diffusion of dye from 1.5% alginate microcapsule increases
linearly with time, as per the equation y = 0.3838x (Fig. ).
Similar linearity is found in microcapsules with various
alginate concentrations. It clearly indicates that the presence
of alginate in the medium, induces cross linking with cellulose
in the microcapsule shell. There is a significance decrease in
the pore size of microcapsules containing 1.5% alginate when
compared to control. The movement of dye out of the capsule
was analyzed for every 5 seconds. Significant variation was
found when comparing control to microcapsules containing
1.5% alginate. Fig. () shows the variation in dye displacement
for every 10 seconds in the control and 1.55 alginate
microcapsules.

2.5
DISTANCE (µm)

2
y = 0.3838x
1.5 R² = 0.9371
Figure : Measurement of capsule size in image
1
Determination of permeability of microcapsules
0.5
The diffusion distance of dye from microcapsule into the
environment was measured using Image J. The diffusion 0
distance from microcapsules containing different percent of 0 20 40 60 80 100 120
alginate was compared against control. Vitamin B1 was found TIME (S)
to diffuse to the greatest distance from control microcapsules.
This could be due to the absence of cross linking between Fig : Linearity in the distance traced by dye from the
alginate and cellulose, thereby the control microcapsules had (1.5% alginate) microcapsules with respect to time
greater pore size than the ones containing alginate. The
diffusion distance changed linearly with respect to alginate
concentration, but the significance in the value was minimal.
The experiment was repeated twice to test the reproducibility

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A A B
3.5
3 control
Distance (µm)

2.5
0.05%
2
0.20%
1.5
1 0.50%

0.5 1.00%
0 1.50%
0 20 40 60 80 100 120
Time (s) Fig : The image shows the coiled structure of cellulose
microfibril and alginate network
B 5. Conclusion

3 The alginates have gained interest for its application in

1.382 2.7652 controlled drug delivery system and decreasing the pore size
2.5 2.553
of microcapsules by crosslinking, owing to its swelling
capacity, ability of sol/gel transition, crosslinking and
Distance (µm)

2 0.974 1.993
biodegradability. Incorporation of alginate into the medium
1.5 has helped in decreasing the pore size of cellulose
1.362 1.50%
1 0.53
microcapsules produced in the emulsion. These microcapsules
0.933 control
have a greater scope of application in the field of drug delivery
0.5 0.42 due to its potential in improving safety and effectiveness of
0 0 drug. Usage of a customized syringe system assists in
0 20 40 60 80 100 120 achieving uniform sized microcapsules.
Time (s)

Figure : Comparison of distance traced by the dye with respect

to time in (A) different microcapsules and (B) specific
comparison of control and (1.5% alginate) microcapsule.
4. Structural analysis using confocal microscopy
From the confocal microscopy analysis of microcapsules, the
fibril network was found to be 10 µm in microcapsules
containing 0.5% alginate. The bundled structures of cellulose
nanofibril and alginate networks were found in the bottom
view of the capsules (Fig. ). A clear thick network was
observed on the capsule outer layer as in Fig. . Some coiled
structure were also observed apart from bundled structures.
The hydroxyl groups of cellulose nanofibrils binding
covalently to alginate is evident from the network mesh
visualized.
A B

Fig : The confocal microscopic image of (A) 0.5%

alginate microcapsules and (B) image in red filter light