Novel biodegradable laminarin microparticles for biomedical applications

Article abstract of DOI:10.1246/bcsj.20200034

Fabrication of biocompatible polymeric carriers for sustained/ controlled drug-delivery have been extensively explored over the years. Furthermore, systems based on polymers from natural origins exceed conventional polymers in biocompatibility, biodegradability and cost efficiency. Polysaccharides are one of the most common biopolymers found in nature and they can achieve a high degree of complexity and fine biological properties. Herein, is proposed a biodegradable and biocompatible microcarrier synthesized from laminarin, a low Mw marine polysaccharide based on glucose units with great biological activity, such as immune modulation and antimicrobial properties. Within this work, controlled size microparticles were obtained from novel modifications of laminarin. Microparticles showed 40% release of fluorescein isothiocyanatedextran (70 kDa) after 24 h and full degradability after 11 days, when in physiological conditions. When incubated with human adipose stem and L929 cell lines (up to a microparticle concentration of 100 μg/mL) no cytotoxicity was perceived, and neither membrane or nucleus disturbance. Thus, microparticles synthesized from laminarin, proved to be a cost efficient, biocompatible and biodegradable system.

Full text of DOI:10.1246/bcsj.20200034

Advance Publication Cover Page
Novel biodegradable laminarin microparticles for biomedical applications
Edgar J. Castanheira, Tiago R. Correia, João M. M. Rodrigues,* and João F. Mano*
Advance Publication on the web March 18, 2020
doi:10.1246/bcsj.20200034
© 2020 The Chemical Society of Japan
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Novel biodegradable laminarin microparticles for biomedical applications
Edgar J. Castanheira, Tiago R. Correia, João M. M. Rodrigues,* and João F. Mano*
CICECO – Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
E-mail: jrodrigues@ua.pt, jmano@ua.pt
João M. M. Rodrigues
João Rodrigues received his Ph.D. in Chemistry from University of Aveiro, Portugal, in 2015 under the supervision of
Professors João Tomé and José Cavaleiro. Then he moved to UK for a Postdoctoral Research Assistant position at University
of Hull under the supervision of Professor Ross Boyle. He joined the COMPASS Research Group from CICECO – Aveiro
Institute of Materials at University of Aveiro, Portugal, in 2017 as a Junior Research Fellow.
João F. Mano
João F. Mano received his PhD degree from Technical University of Lisbon, Portugal, in 1996. Currently he is the leader of
the COMPASS Research Group in CICECO – Aveiro Institute of Materials, University of Aveiro, Portugal, where holds the
position of Full Professor in the Chemistry Department. His research interests include the use of biomaterials and cells
toward the progress of transdisciplinary concepts to be employed in regenerative and personalized medicine.
Abstract
Fabrication of biocompatible polymeric carriers for
are commonly used as emulsifying agents16, 17. Furthermore, a
system based on natural polysaccharide and immiscible polymer
blends has proven to be a good approach for the production
homogeneous and degradable polysaccharides microparticles
using mild conditions18. Accordingly, proteins, photosensitive
and thermosensitive drugs can be encapsulated in this type of
carriers, due to unnecessary use of organic solvents, temperature
or irradiation.
a
sustained/controlled drug-delivery have been extensively
explored over the years. Furthermore, systems based on
polymers from natural origins exceed conventional polymers in
biocompatibility, biodegradability and cost efficiency.
Polysaccharides are one of the most common biopolymers found
in nature and they can achieve a high degree of complexity and
fine biological properties. Herein, is proposed a biodegradable
and biocompatible microcarrier synthesized from laminarin, a
low Mw marine polysaccharide based on glucose units with a
great biological activity, such as, immune modulation and
antimicrobial properties. Within this work, controlled size
microparticles were obtained from novel modifications of
laminarin. Microparticles shown 40% release of fluorescein
isothiocyanate-dextran (70 kDa) after 24 h and full degradability
after 11 days, when in physiological conditions. When incubated
with human adipose stem and L929 cell lines (up to a
microparticle concentration of 100 µg/mL) no cytotoxicity was
perceived, and neither membrane or nucleus disturbance. Thus,
microparticles synthesized from laminarin, proved to be a cost
efficient, biocompatible and biodegradable system.
Laminarin (LAM), also called laminaran, is a neutral small
polysaccharide (Mw <10 kDa; 20 to 50 units of glucose) and is
the main energy storage polysaccharide found in brown algae19,
a marine specie rich in different elements important for
biomedical applications20-24
biological activity aspects such as, immune modulation25, 26
.
Mainly due the important
,
antioxidation27, 28 and antimicrobial properties27. Additionally,
some authors reported the capability of LAM to trigger apoptosis
on different human colon cancer cell lines29-31. LAM has a linear
structure with a degree of side branch through the β-(1,3; 1,6)
bonds between glucose units. Further, the ratio between both
bonds may differ between algae specie and form a triple helical
conformation32, 33, which may difficult their modification. There
are only few examples in the literature surrounding the
modification of LAM, either by sulfonation or by adding a
methacrylic or propargyl moieties. LAM sulfonation has shown
to significantly enhance the antitumor activity of LAM 34, while
methacrylation of LAM has been used to obtain microparticles
and hydrogels for biomedical applications35-38. Furthermore, the
modification with propargyl was used in a Cu(I) catalyzed
Huisgen cycloaddition in a way to synthesize nanoparticles for
drug delivery through the interaction between the
polysaccharides and proteins24. Besides particles and hydrogels,
LAM has also been used to produce regular size and shape
organized layered nanostructures similar to the natural process
of biomineralized structures found in nature33.
Keywords: Marine polysaccharides, laminarin,
microparticles, biocompatible, biodegradable, L929 &
hASCs.
1. Introduction
Polysaccharides particles are excellent biocompatible and
biodegradable carriers with an array of possible bioactivities
according to their structure, thus augmenting certain drugs
effects 1-6. Different techniques can be used to produce several
size particles and accordingly, the modification of the
biopolymer and its charge hold important factors. For instance,
modifications with hydrophobic moieties allow a self-assembly
into micelles, which may be excellent carriers for poor water-
soluble drugs7, 8. Different type of processes can be applied after
the emulsification process to produce polysaccharides carriers
with different sizes, namely, solvent diffusion9, solvent
In this study is reported the modification of LAM with alkyne
and azide moieties as a way of polymerization trough the
formation of a triazole linker using click-chemistry. The
obtained modified polymers were characterized by infrared and
1H-NMR spectroscopy. The combination of Cu(I) catalyzed
Huisgen cycloaddition and micro-emulsification of immiscible
polymers, are combined to synthesize novel LAM biodegradable
microparticles. Accordingly, when a stable aqueous biphasic
system is disturbed, a microemulsion is created. Then, the Cu (I)
11
13
evaporation10,
,
photo-reticulation12,
and the use of
superhydrophobic surfaces14,
.
Biopolymers, such as
15
polysaccharides are used as stabilizers in the development of
food emulsions, avoiding the aggregation of micelles. Hence, it
is possible to replace surfactants, solid particles or proteins that
1

catalyzed Huisgen cycloaddition enables the formation of solid
microparticles. This type of procedure has been used before for
the synthesis of degradable microparticles of dextran18. LAM
microparticles morphology and size dispersity was assessed by
scanning electron microscopy (SEM) and fluorescent
microscopy. Microparticles’ degradability and release profile of
macromolecule chains attached with a fluorophore were
performed in physiological conditions and used to prove the
degradability and sustain release of such microcarriers.
Moreover, microparticles biocompatibility were tested through
live-dead assays with two different cell lines (human adipose
stem cells and fibroblasts mouse cells). As far as the authors
know it is the first time that is reported the modification of LAM
with azides, their assembly into biodegradable LAM
microparticles and their consequent characterization.
against distilled water, while the dialysis solvent was changed
twice a day. Purified LAM-N3 was obtained by lyophilization as
a white fluffy powder with a degree of substitution (DS; i.e. the
number of azide moieties per 100 glucose units) equal to 11%
and stored at a dry place protected from light. 1H NMR (300.13
MHz, DMSO): δ (ppm) 1.79-1.95 (m, 2H, CH2-N3), 2.90-3.88
(m, LAM backbone), 4.08-4.28 (m, 2H, CH2-O), 4.30-5.62 (m,
LAM backbone).
Synthesis of propargyl carbonylimidazole (PA-CI). A
dry round bottomed flask was charged with 14.60 g (90 mmol)
of CDI and 100 mL dichloromethane yielding a turbid
suspension. 2.90 mL (50 mmol) of propargyl alcohol (PA) was
added under vigorous stirring yielding a clear solution upon
dissolution of the PA. After 1 h reaction at room temperature the
mixture was extracted three times with 100 mL water. The
dichloromethane was removed by rotary evaporation and PA-CI
was obtained as a dry powder (5.6 g, η = 75%). 1H NMR (300.13
MHz, CDCl3): δ (ppm) 2.62 (t, 1H, J = 2.5 Hz, HC≡C), 4.99 (d,
2H, J = 2.5 Hz, CH2-O), 7.03-7.1 (m, 1H, C=CH-N), 7.44 (t, 1H,
J = 1.4 Hz, N-CH=C), 8.15 (s, 1H, N-CH=N).
2. Experimental
Materials: Used reagents and materials in this work were
purchased from Sigma-Aldrich, Fluka, Merck, Fisher Scientific,
Sarstedt and Spectrum Labs at analytical grade and used without
any further purification.
Synthesis of laminarin-propargylcarbonate (LAM-
C≡C). In a dry round bottomed flask 1 g of LAM (corresponding
to 5.56 mmol of glucose repeating units) was dissolved in 20 ml
of anhydrous DMSO. To this mixture 169 mg (1.1 mmol) of PA-
CI was added and the reaction was stirred overnight at 50 °C
under N2 atmosphere. The reaction solution was transferred to
dialysis bags (MWCO 3.5 kDa) and dialyzed for 5 days under
stirring against distilled water, while the dialysis solvent was
changed twice a day. Purified LAM-C≡C was obtained by
lyophilization as a white fluffy powder (DS = 14%) and stored
1
Instrumentation: H spectra was recorded on a Bruker
Avance-300 spectrometer at 300.13 MHz. Tetramethylsilane was
used as internal reference. Fourier-transform infrared
spectroscopy (FTIR) spectra were recorded using an IR-FT
Mattsson 7000 galaxy series with a Golden Gate ATR.
Microscopy images were obtained using a Zeiss Axio Imager 2
fluorescence microscope. Confocal laser scanning microscopic
(CLSM) images were acquired using a Zeiss laser scanning
microscope 880 Airyscan. Morphological images were obtained
using Hitachi SU-70 SEM. Particle size distribution analysis was
taken by numerical assessment of SEM and microscopy
fluorescence images using ImageJ software.
Synthesis of 3-azidopropanol (AP). 3-Bromopropanol
(10.0 g; 72 mmol) and sodium azide (7.66 g; 118 mmol) were
dissolved in a mixture of acetone (120 mL) and water (20 mL)
and stirred at 65 °C overnight. Acetone was removed under
reduced pressure and 100 mL of water was added to the mixture.
The mixture solution was three times extracted with 100 mL
diethyl ether and dried over sodium sulfate. The diethyl ether
was removed by rotary evaporation and AP was obtained as a
colorless oil (5.2 g, η = 71%). 1H NMR (300.13 MHz, CDCl3):
δ (ppm) 1.73 – 1.82 (m, 2H, C-CH2-C), 3.39 (t, 2H, J = 6.6 Hz,
CH2-N3), 3.67 (t, 2H, J = 6.0 Hz, CH2-OH).
Synthesis of 3-azidopropyl carbonylimidazole (AP-CI).
A dry round bottomed flask was charged with 18.5 g (114 mmol)
of 1,1'-carbonyldiimidazole (CDI) and 200 mL ethyl acetate
yielding a turbid suspension. AP (7.1 mL, 76 mmol) was added
dropwise under vigorous stirring while the reaction mixture
turned into a clear solution. After 2 h reaction at room
temperature, the solution was extracted three times with 200 mL
of water. The organic layer was dried over sodium sulfate,
evaporated by rotary evaporation obtaining AP-CI as a pale-
yellow oil (10.1 g, η = 68%) 1H NMR (300.13 MHz, CDCl3): δ
(ppm) 1.92–2.06 (m, 2H, CH2-CH2-CH2), 3.42 (t, 2H, J = 6.5 Hz,
CH2-N3), 4.44 (t, 2H, J = 6.2 Hz, CH2-O), 6.90–7.03 (m, 1H,
C=CH-N), 7.35 (t, 1H, J = 1.4 Hz, N-CH=C), 8.06 (s, 1H, N-
CH=N).
Synthesis of laminarin-azidopropylcarbonate (LAM-
N3). In a dry round bottomed flask 1 g of LAM (corresponding
to 5.56 mmol glucose repeating units) was dissolved in 20 mL
anhydrous dimethyl sulfoxide (DMSO). To this mixture 214 mg
(1.1 mmol) of AP-CI was added and the reaction was stirred
overnight at 50 °C under a N2 atmosphere. The reaction solution
was transferred to dialysis bags with a molecular weight cut-off
(MWCO) of 3.5 kDa and dialyzed for 5 days under stirring
1
at a dry place protected from light. H NMR (300.13 MHz,
DMSO): δ (ppm) 2.28 (m, 1H, HC≡C), 2.92-4.67 (m, LAM
backbone), 4.72-4.86 (m, 2H, O-CH2-C≡), 4.89-5.70 (LAM
backbone).
Fabrication of LAM microparticles. Microparticles
were fabricated using a microemulsion method based on the
immiscibility between an aqueous LAM phase and polyethylene
glycol phase18. 12.5 mg of LAM-N3 and 9.8 mg of LAM-C≡C
were dissolved in 210 µL of MilliQ water, as a way to obtain 1:1
ratio of the modified LAM moieties. This solution was added to
268 μL of a 50% (w/w in pure water) polyethylene glycol (8
kDa). A solution of CuSO4.5H2O (50 mg/mL) in water was
added to an ice-cold solution of sodium ascorbate (50 mg/mL)
in water. To the reaction mixture, 34 µL from the resulting Cu(I)
solution was added, and vortexed for 60 s. The reaction was
allowed to proceed for 30 min, with no further stirring or vortex.
After that, 2 mL of water was added to quench the reaction and
the resultant was centrifuged (5000g/10 min) 3 times with
washing steps using 2 mL of water. Finally, the obtained LAM
microparticles were freeze-dried and stored protected from light.
In order to incorporate a macromolecule as an imaging agent, 2.5
mg of fluorescein isothiocyanate-dextran (FITC-dextran, 70
kDa) was added onto the 210 µL of water prior to the
microemulsion step. Microparticles were achieved with an
average of 71% yield and 85% of FITC-dextran incorporation.
Microparticles mass loss assay. Degradability essays
were conducted by creating a suspension of 0.6 mg / mL in
phosphate-buffered saline (PBS) at pH 7.4 and placing them in a
heating bath (37 ºC at 80 rpm) to mimic physiological conditions.
The samples after each time point (1, 3, 7 and 11 days) were
freeze-dried for 2 days and weighted. Microparticles NMR
solution were performed by dissolving 10 mg of the
microparticles into a 500 µL solution of D2O/NaOD (95/5) at pH
12.
Microparticles
degradability.
Microparticles
degradation was carried out in PBS solution at 37 ºC under
2

physiological pH (7.4). To understand the influence of culture
medium on particles degradation the same assay was performed
using Dulbecco’s modified Eagle’s medium low glucose
(DMEM-LG) and alpha modification of Eagle's minimum
essential medium (α-MEM), respectively. Briefly, particle
suspensions at a concentration of 2 µg/mL were incubated in a
μ-slide 8-well plate (ibidi GmbH) for 72 h. Different conditions
were tested, i.e., α-MEM medium with and without fetal bovine
serum (FBS) and DMEM-LG with and without FBS, in PBS and
in MilliQ water with exact concentration of carbonate ions
present in DMEM-LG (26 mM) or in α-MEM (44 mM).
Microparticles release studies. Encapsulation of FITC-
dextran (70 kDa) into the microparticles was quantified by
centrifuging the particles (5000g/10 min) after synthesis and
reading the absorbance at 490 nm of the supernatant. Release
profile was drawn by creating a suspension of 25 mg of the
microparticles loaded with 2.5 mg of FITC-dextran in 5 mL of
PBS (at pH 7.4). The experiment was conducted for 11 days, in
a heating bath at 37 ºC and at a stirring velocity of 80 rpm, till
achieve microparticles full degradability. For each time point,
the sample was centrifuged (5000g/10 min) and then 0.5 mL of
the supernatant was taken and replaced with 0.5 mL of fresh PBS.
The samples were frozen at -20 ºC and protected from the light.
To analyze, the samples were defrosted, and the absorbance at
490 nm was measured on a microplate reader.
SEM. This technique was utilized as a mean to evaluate
the size and morphology of the biodegradable LAM
microparticles. After freeze-drying, the particles were deposit on
a metal stub using an ethanol dispersion that was dried for 1 day
at room temperature. The sample was then sputtered in gold and
analysed in a Hitachi SU-70. Unmodified LAM morphology was
assessed by dissolving LAM in deionised water and by applying
1 µL on a polished silicon surface, letting it dry and repeat, after
a couple of cycles the sample was cover with carbon and
analysed in a Hitachi SU-70.
Cell Seeding. L929 mouse fibroblasts (ATCC ® CRL-
6364™) and human adipose stem cells (ATCC® PCS-500-
011™) (hASCs) were seeded in a tissue culture flasks T-175
(Sarstedt) using DMEM-LG and α -MEM (supplemented with
10% FBS and 1% antibiotic/antimycotic) under controlled
atmosphere of 5% CO2 and 37 °C, respectively. Upon reach
confluence, cells were trypsinized and seeded in contact with
different microparticles concentrations (100, 10, 1 µg/mL) at a
density of 1x104 cells mL-1 on 96-well plates for 72 h. Besides
the cell incubation with the micros, controls were made using the
copper concentration utilized in the synthesis, unmodified LAM
(100 µg / mL) and a control without particles.
3. Results and Discussion
Synthesis of AP, AP-CI and PA-CI. AP was synthesized
according literature and has been successfully synthesized by
SN2 type reaction from the azide anion as an excellent
nucleophile and the alkyl halide in 77% yield40 (Scheme 1). The
chemical structure of AP was confirmed by 1H NMR
spectroscopy where the aliphatic protons appeared at 1.77, 3.37
and 3.69 ppm (Figure S1). 3-azidopropyl carbonylimidazole
(AP-CI) and propargyl carbonylimidazole (PA-CI) were
obtaining according procedure in literature, by activating PA and
AP, respectively with CDI18 (Scheme 1). The final chemical
structures of AP-CI and PA-CI were also confirmed by 1H NMR
spectroscopy (Figure S2 and S3). The alkyne proton of PA-CI
appeared at 2.62 ppm, while the aliphatic protons of AP-CI
appear at 2.00, 3.42, and 4.44 ppm. The aromatic protons of the
diazole ring for AP-CI and PA-CI appear in the form of multiple
signals around 7.0, 7.4 and 8.1 ppm confirming the expected
structure.
Synthesis of LAM-N3 and LAM-C≡C.
LAM-N3 and LAM-C≡C were synthesized with a DS of 11 and
14, respectively, as determined by 1H-NMR spectroscopy. LAM-
N3 and LAM-C≡C were obtained by reacting LAM with
activated carbonylimidazoles (AP-CI and PA-CI) in DMSO at
50 ºC under nitrogen atmosphere conditions (Scheme 1),
resulting in the formation of a carbonate ester between the LAM
backbone and the carrying azidopropyl (LAM-N3) and propargyl
(LAM-C≡C) moieties, respectively. Also, by 1H NMR
spectroscopy is possible to see the changes in the LAM
backbone. The conjugation of PA-CI with the LAM backbone
was confirmed by the appearance of the alkyne proton signal at
2.28 ppm for LAM-C≡C (Figure 1). Concerning the insertion
of the AP-CI for the formation of LAM-N3, a multiplet signal
corresponding to CH2-N3 was found at 1.79-1.95 ppm. By
comparison (Figure 2A), in the FTIR spectrum of LAM and the
respective derivatives, is possible to confirm the vibration of the
azide group at 2170 cm-1. At 1270 cm-1 (C-O) and 1750 cm-1
(C=O) is also possible to confirm the vibrations of the esters
groups peaks and regarding the propargyl modification the
C≡CH, and C≡C vibration may be masked by the other
vibrations, but the esters peaks are still visible at 1270 (C-O) cm-
1 and 1750 (C=O), respectively.
Fabricating LAM Microparticles. LAM has proven to
have attractive properties for biomedical applications38 and so,
carriers based on such polysaccharide may provide new ways of
drug-delivery with low side-effects. LAM was modified with
azide and alkyne groups in order to obtain microparticles
through microemulsion combined with a Huisgen cycloaddition
Live-dead staining. Cell viability in contact with the
microparticles was evaluated by incubating the different
concentrations with Calcein-AM/propidium iodide (PI) (live-
dead Kit, ThermoFischer Scientific) for 20 min, according to the
manufacturer’s protocol. This assay was performed for the 24
and 72 h timepoints. Fluorescence CLSM was used to image the
stained cells. Acquired data was processed in Zeiss ZEN v2.3
blue edition software. Live/dead results quantification was
performed by ImageJ software analysis.
Cell Morphology Analysis. Cell morphology was
assessed by imaging cells in contact with the microparticles
using fluorescence CLSM. To do so, cells were fixed by
incubating them in a 4% solution of paraformaldehyde for 20
min, as previously reported39. Then, cell structures, namely,
membrane and nucleus were labelled using wheat germ agglutin
(WGA) (1:100 in Dulbecco's phosphate-buffered saline (DPBS))
solution for 20 min, and 4´,6-diamidino-2-phenylindole (DAPI)
(1:1000 in DPBS) solution during 15 min, respectively. The
samples were then analyzed at 24 and 72h timepoints.
Figure 1 – Characterization of the microparticles precursors by
1H-NMR in DMSO. LAM (unmodified); LAM-N3 (modified
with azide); LAM-C≡C (modified with propargyl).
3

Scheme 1 - Synthesis of carbonylimidazole (AP, AP-CI, PA-CI) and laminarin (LAM-N3, LAM-C≡C, LAM microparticles) derivatives and their
reaction conditions. Highlighted at green the cleavable bonds at pH above 7.
reaction catalyzed by copper (I). Particle size was evaluated
through both fluorescence microscopy and SEM. By
fluorescence microscopy analysis was, shown that the particles
seemed to emit a low auto-florescence with an average size of
15 ± 4 µm (Figure S4). Similarly, SEM allowed to understand
that the particles were spherical, monodispersed, with a smooth
surface (Figure 2B and 2C) and an average size of 7 ± 4 µm
(Figure S5). The difference in the results may be explained by an
auto-of-focus fluorescence effect present in the fluorescence
microscope, thus indication a higher size than expected. This
procedure had previously been developed and used in higher
molecular weight polymers, like dextran18. As the synthesis were
performed through the usage of esters, the microparticles have
an enhanced biodegradability, since this groups are known to
suffer hydrolysis above pH 7. Thus, the particles will be
degraded into the LAM polymer in different rates according to
the different pH’s. To prove this point, microparticles were
dispersed in D2O/NaOD (at pH 12), where their dissolution
occurred in less than 5 minutes. The result was then analyzed by
1H-NMR (Figure S6), where the signal from the peak of the
triazole formed during the Huisgen cycloaddition, was clear to
see at 8.39 ppm. This result is a complementary information to
the formation of the microparticles through the Cu (I) catalyzed
reaction. Furthermore, to understand the cumulative release
profile of the microparticles in comparison to their degradability,
FITC-dextran (70 kDa) was encapsulated inside the
microparticles. The release profile and particle degradability
were evaluated in physiological conditions, through the
incubation of the particles in PBS at pH 7.4 and at 37 ºC.
Additionally, the microparticle cytocompatibility was assessed
by live-dead assay and morphology analysis using fluorescence
CLSM.
Release Studies and Particle Biodegradability. In order
to evaluate the potential of the microparticles as drug-delivery
agents, the release profile was determined. For that, the particles
were loaded with FITC-dextran (70 kDa), a high molecular
weight polymer coupled to a fluorophore, as a way to prove the
high uptake potential of macrosystems, such as proteins. Thus,
with an incorporation of 85% of FITC-dextran (70 kDa), the
microparticles were left on a heating bath during 11 days in
physiological-like conditions (Figure 3A). A fast release during
the first day occurred, reaching 41 ± 6% release of the overall
cargo loaded, and then a slower release occurs releasing around
10% per day till the fourth day. From there, a slower stabilization
occurs, and was followed till the eleventh day, reaching a total
of 80 ± 5% of release. This result when compared to the previous
published dextran microparticles, that have a similar degree of
Figure 2 – (A) FTIR analysis of the microparticles precursors. LAM
(unmodified); LAM-N3 (modified with azide); LAM-C≡C (modified
with propargyl) and LAM click microparticles, where no vibration
corresponding to the azide or propargyl group could be found. (B and C)
SEM morphology analysis of the LAM microparticles at two different
magnifications. (D) SEM morphology analysis of unmodified LAM.
4

hASCs were used to evaluate the cytotoxicity profile of LAM
microparticles through live-dead assays. After 24 h (Figure 4) of
incubation at 37 ºC, cell viability was analyzed by image
acquisition. In order to evaluate the different aspects that could
affect the toxicity of the microparticles, cells were also seeded
with copper (II) chloride and LAM (Figure S8). Microparticles
cytotoxicity, when incubated with L929 or hASCs was similar to
that of the control, indicating that cell viability was not affected.
Also, microparticle at different concentrations (1, 10 and 100
µg/mL) seemed to have no effect on cell viability (Figure 4).
Additionally, the cells incubated in the presence of the LAM
polymer did not affect either cell proliferation or viability. In the
case of cells incubated with copper, there was a decrease on the
viability of both cell types viability when compared with the
control. This result led us to two conclusions: first, the copper
previously detected onto the microparticles may be coordinated
with the polysaccharides41, and secondly, even after full
degradability, the copper that may be released is not sufficient to
damage the cells42. Therefore, LAM microparticles, from the
first results, and up to 72h, which in DMEM-LG medium
corresponded to full degradability, seemed to be compatible in
both type of cells. As a complementary assay, the morphology
A
B
Figure 3 – (A) Cumulative release profile of FITC-dextran from LAM
microparticles in PBS, 37 ºCat pH 7.4. (B) Microparticle degradation
assessed by mass percentage loss of the microparticles.
substitution, and presented by the same method, seemed to have
a release two times faster18. Microparticles biodegradability was
also performed in physiological-like conditions and followed by
11 days, leading to what may be considered a full degradability
of the particles (Figure 3B). The release of the macromolecules
seemed to occur prior to the full degradability of the
microparticles, due the FITC-dextran diffusion through the pores
formed on the first days of degradation. Furthermore, to evaluate
the basicity of the different ions presented in the distinct
mediums used for cell culture, more assays were conducted in α-
MEM and DMEM-LG, in which DMEM has a difference of
almost 2-fold of the carbonate ion concentration. All the
experiments were conducted during 72 h, at pH 7.4 and in a
humidified incubator at 37 ºC, to mimic the physiological
conditions (Figure S7). As expected, the higher concentration of
carbonate ions (equal to DMEM-LG), triggered the particles
degradation, and after 72 h particles were fully degraded. On the
other hand, in α-MEM, particles degradation was also clear, but
full degradability was still far from accomplished. Moreover,
similar results were obtained using the same mediums but
without the supplementation with FBS, confirming that the
proteins in the serum have no impact on their degradation.
Figure 5 – Fluorescence morphology images of L929 fibroblasts and
hASCs after 24h of incubation with different microparticles
concentrations. Laminarin microparticles – green channel (FTIC-
dextran); Cell membrane – red channel (WGA); Cell nucleus – blue
channel (DAPI).
analysis of L929 and hASCs was performed for 24h (Figure 5)
and 72 h (Figure S9), where cells’ adhesion, spreading and
proliferation could be observed. Additionally, microparticles did
not suffer uptake (Figure 5), making this type of microcarriers
good to reduce the cytotoxicity or enhance hydrophilicity of
target specific macromolecules (e.g. tumor specific
dendrimers43) Furthermore, after 72 h of incubation with L929
fibroblasts, no particles were visible (Figure S9), due to the full
degradability in DMEM-LG, and faster dispersion and
photobleaching of the FITC-dextran molecules. Lastly, no live-
dead assay was performed at 72 h due to proliferation behavior
observed in morphology images after 72 h upon incubation
(Figure S9).
Figure 4 – Live-dead images of L929 fibroblasts and hASCs incubated
with different microparticles concentrations and without particles
(control) at 24 h. Live cells – green channel (Calcein-AM); dead cells
– red channel (PI).
Furthermore, degradation was evaluated in equimolar ratios (to
DMEM-LG or α-MEM) of a carbonated aqueous solution.
Likewise, to the higher concentration of carbonate ions (equal
molarity to DMEM-LG), microparticles full degradability was
achieved, while for the lower concentration (equal molarity to α-
MEM), the degradation was clear but not complete (Figure S7).
This assay was performed to verify the impact of the ionic
strength on microparticles degradability since, at the same pH,
the two mediums used, differ on the rate of the microparticle
degradability. Each medium was chosen due to compatibility to
cell line. Mainly, sodium carbonate is the only ionic compound
that may have a direct impact on the microparticles degradability
and still diverge quite substantially, in quantity, from each
medium. Thus, it was shown, that not only the increment of the
pH affect the degradability but also the ionic strength of
carbonate ions, a basic specie, can interfere.
4. Conclusion
In this work, we reported the synthesis of LAM microparticles
stabilized by click chemistry, a bioactive polymer that exhibits
relevant properties for biomedical applications. Microparticles
were synthesized in mild conditions and evaluated as micro
carriers through the potential biodegradability and sustained
release in physiological conditions. Moreover, the encapsulation
of FITC-dextran (70 kDa), proved the possibility to include
macromolecules, such proteins, inside the particles. Furthermore,
microparticles were incubated with human adipose stem cells
and mouse fibroblasts, where no cytotoxicity was found by
evaluation of cell proliferation and viability. Additionally, cancer
cells’ overexpression of glucose transporters44, 45 and LAM’s
potential to induce apoptosis of the colon cancer cells29-31 may
Cell Viability and Morphology. L929 mouse fibroblasts and
5

be an indicative as a new way of treatment. This is expected due
to a higher selectivity of the colon cancer cells towards the sub
product (LAM polymer–glucose terminals) of the microparticles
degradation. Degradability into LAM (Mw < 10 kDa) is likely,
as most small linear polymers46, to be cleared from the organism
by the kidneys, thus reducing in-blood circulation time and
consequent accumulation issues. Concluding, this type of drug-
delivery system may be applied in different fields due to the
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Acknowledgement
This work was developed within the scope of the project
CICECO-Aveiro Institute of Materials, UIDB/50011/2020 &
UIDP/50011/2020, and project COP2P (PTDC/QUI-QOR/
30771/2017), financed by national funds through the FCT/MEC
and when appropriate co-financed by FEDER under the PT2020
Partnership Agreement. E.J. Castanheira also acknowledges
FCT for his PhD grant (SFRH/BD/144880/2019).
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