Carbohydrate Research 534 (2023) 108973

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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres

An influence of molecular weight, deacetylation degree of chitosan xerogels

on their antimicrobial activity and cytotoxicity. Comparison of chitosan
materials obtained using lactic acid and CO2 saturation
Szymon Mania a, *, Adrianna Banach-Kopeć a, Karol Staszczyk a, Jolanta Kulesza b,
Ewa Augustin b, Robert Tylingo a
a
Department of Chemistry, Technology and Biotechnology of Food, Faculty of Chemistry, Gdansk University of Technology, 11/12 G. Narutowicza Street, 80-233,
Gdansk, Poland
b
Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, 11/12 G. Narutowicza Street, 80-233, Gdansk,
Poland

A R T I C L E I N F O A B S T R A C T

Keywords: This paper presents a comparison of the antimicrobial activity and cytotoxicity against L929 cells of chitosan
Chitosan xerogels prepared by dissolving the polymer in a solution of lactic acid (LA) or carbonic acid (CO2) and then
Antimicrobial properties freeze-drying. There was no simple relationship between the antimicrobial activity and cytotoxicity of the
Cytotoxicity
samples obtained using both techniques (LA and CO2). Chitosan materials obtained by the LA method in a 1:1
Molecular weight
Deacetylation degree
dilution were characterized by the highest cytotoxicity against L929 cells (~20%). For the same diluted samples
prepared using the CO2 saturation method, the viability of L929 cells was approximately 2.5 times greater. Some
of the tested chitosan materials obtained by the innovative method were characterized by significantly lower
antimicrobial activity, for example, reduction of E. coli bacteria for MMW-LA and MMW-CO2 samples by 6.00 and
0.75 logarithmic order, respectively. This clearly indicates that in many applications, the presence of the acid
necessary to dissolve chitosan is responsible for the antimicrobial activity of the polymer solution and its
products.

1. Introduction biodegradability, non-toxicity, antimicrobial activity, hemostatic prop­

erties, and ability to accelerate wound healing, make it an object of
Chitosan is a natural polysaccharide with a positive charge, and is a research and application in designing dressing materials [5], scaffolds
derivative of chitin. It consists of the monomers N-acetyl-D-glucosamine [6], and carriers for the controlled release of drugs and other biologi­
and D-glucosamine linked by β-1,4-glycosidic bonds [1]. Chitosan occurs cally active pathways [7]. Chitosan can also be used as a component of
sporadic in the biosphere, therefore this polymer is obtained from chitin, food packaging or as a food additive, inhibiting the development of
which is one of the main components of the exoskeletons of arthropods microorganisms and thus prolonging the freshness of the product u [8].
such as shrimp, crabs, lobsters and arachnids [2]. The most common One of the most significant features of the potential action of chito­
method for obtaining chitosan is the deacetylation of chitin, which is a san is the antimicrobial activity of the polymer, which is the result of
two-stage nucleophilic substitution reaction that takes place in an many different factors, including DD and MW [9]. Zheng and Zhu [9]
alkaline solution. Depending on the origin of chitin and parameters of and Tavaria et al. [10] reported an increase in the antimicrobial activity
the deacetylation process, chitosan can be obtained with different mo­ of chitosan against Staph. aureus bacteria with increasing MW [9,10].
lecular weights (MW) and degrees of deacetylation (DD). These pa­ Despite the relatively well-determined effect of the MW of chitosan on
rameters affect the physicochemical and biological properties of its antimicrobial activity against Gram-positive bacteria, many incon­
biopolymer-based materials based on this biopolymer [1–4]. sistent results of its activity against Gram-negative bacteria can be found
The properties of chitosan, such as biocompatibility, [11]. Some studies on E. coli and Salmonella have indicated an increase

* Corresponding author.
E-mail addresses: [email protected] (S. Mania), [email protected] (A. Banach-Kopeć), [email protected] (K. Staszczyk), jolanta.
[email protected] (J. Kulesza), [email protected] (E. Augustin), [email protected] (R. Tylingo).

https://doi.org/10.1016/j.carres.2023.108973
Received 6 May 2023; Received in revised form 3 October 2023; Accepted 10 October 2023
Available online 14 October 2023
0008-6215/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
 S. Mania et al. Carbohydrate Research 534 (2023) 108973

in antimicrobial activity with decreasing MW [9,12,13]. Other results lyophilization.

for the same class of bacteria showed an increase in performance as the Standard methods of producing chitosan xerogels involve pre-
MW increased to the determined value and then decreased with dissolving the polymer in an acid solution, freezing the solution and
increasing MW, or different performances for different MW and different freeze-drying it. During this operation, water is removed from the ma­
types of bacteria of the same class ([14,15], n.d.). The justification for terial by sublimation, and acid anhydrides are formed, which remain in
these differences is the method of processing the chitosan material, the the material in an amount depending on its volatility and boiling point.
pH of the solution, or the DD [16]. In addition, the source of chitin from After contact with water, acid is formed again in the chitosan xerogel, i.
which chitosan was produced, as well as the method of processing, play e. hydrogen cations and anions of the acid residues used to dissolve the
an important role in the antimicrobial activity of chitosan materials polymer are present. In the CO2 saturation method, chitosan is initially
[17–19]. dissolved in a solution of hydroxyacetic acid (hydrogen cations,
Several models have suggested that the antimicrobial activity of hydroxyacetic anions), then precipitated with sodium hydroxide, as a
chitosan is due to its cationic nature. The electrostatic interaction be­ result of which chitosan is precipitated in a microxystalline form to
tween positively charged polymers and negatively charged microbial produce sodium hydroxyacetate and water as a result of reaction. The
cell membranes is predicted to be responsible for cellular lysis, and is precipitate is washed thoroughly until the pH is neutral (removal of
assumed to be the main antimicrobial mechanism [20]. sodium cations and hydroxyacetic anions). In the last stage, the chitosan
The second mechanism is based on the binding of chitosan to bac­ precipitate is suspended in distilled water and saturated with carbon
terial DNA, which leads to the inhibition of protein synthesis as a result dioxide gas, which acidifies the environment (production of weak car­
of chitosan penetration inside the cells of microorganisms. This model bonic acid), which dissolves the polymer. Carbonic acid is a weak acid
assumes that chitosan can pass through the walls and/or membranes of that dissociates only slightly and is unstable, decomposing fast into
bacterial cells, as confirmed by scanning techniques [2]. carbon dioxide and water, which means that the chitosan hydrogel
The third mechanism of the antimicrobial activity of chitosan is ready for freeze drying does not contain any additional compounds that
related to its ability to chelate metal ions. It consists of binding the would affect the biological properties of the chitosan xerogels created.
polymer to nutrients necessary for the growth of microorganisms [20].
This mechanism applies in particular to the alkaline environment 2. Experimental section
because the amino groups are in the unprotonated state, and the electron
pair present on the nitrogen of these groups can interact with cations 2.1. Materials
[21,22].
Chitosan is generally considered safe, which has been confirmed by Chitosan polymers with low (20–300 cps, DD ≥ 75%, Cat.
numerous studies presenting in vitro tests carried out on cell lines No.:102473649, LOT: BCCG5629), medium (200–800 cps, DD ≥ 75%,
[23–25], and in vivo tests carried out on model organisms living ([26, Cat. No.:102466463, LOT: BCCG9377), high (800–2000 cps, DD ≥ 75%,
27], 1997b). Despite the proven harmlessness of chitosan, some studies Cat. No.:102515456, LOT: BCCH4876) MW, chitosan isolated from crab
have indicated the possible cytotoxicity of materials produced through (Cat. No.:101167160, LOT: BCBH3811V) and shrimp shells (DD ≥ 75%,
its participation [28,29]. It is assumed that the cytotoxicity may depend Cat. No.:1003507957, LOT: SLCP5257),phosphate buffer saline (PBS,
indirectly on the method of processing and the origin of chitin because Cat. No.:1002795530, LOT: SLBZ3711), Tryptic Soy Agar (TSA, Cat.
the influence of the DD and the MW of chitosan on the cytotoxicity No.:1.05458.0500, LOT: VM1009758 216), Tryptic Soy Broth (TSB, Cat.
phenomenon was observed. The lower molecular weight (LMW) chito­ No.:1.05459.0500, LOT: VM899959 942), Lactic acid (LA; Cat.
san showed less cell-damaging effects than its higher molecular weight No.:1003429706, LOT:SHBP4889), hydroxyacetic acid (Cat.
(HMW) counterpart. This phenomenon is probably correlated with the No.:102527954, LOT: STBK8247) were purchased from Merck. Chitosan
surface charge density distribution and amount of free amino groups. with MW of 150 kDa (Cat. No.: 22741, LOT: 407568/1) was purchased
Their greater amount in the case of chitosan with a higher MW results in form Fluka.Peptone K (Cat. No.:S-0011, LOT: S011130306) was pur­
Downloaded from mostwiedzy.pl

stronger electrostatic interactions between the chitosan chains, and thus chased from BTL Sp. Z o.o.(Poland). Acetic acid (Cat. No.:568760114,
the formation of a more developed polycation molecule that attaches LOT:1024/04/19), hydrochloric acid (Cat. No.: 115752837
more easily to the cell [30,31]. LOT:210305322), and sodium hydroxide (Cat. No.: 115752837
There is a lot of information in the scientific literature on the bio­ LOT:210305322) were purchased from Avantor Performance Materials
logical properties of chitosan, but finding an answer to the relationship Poland S.A. (Gliwice, Poland). Viscosity standards in the form of mineral
between the cytotoxic effect and antimicrobial activity of chitosan ma­ oils for viscosimeter calibration were purchased from IKA (10 mPas Cat.
terials is practically impossible. The biological properties of these ma­ No.: 25000398, LOT: 280038/1, 100 mPas (Cat. No.: 25000434, LOT:
terials are subject to high variability resulting from the source of the 07040, 1000 mPas (Cat. No.: 25000436, LOT: 07119) (Warsaw, Poland)
polymer, method of extraction and processing, and different conditions Viscosimeter standards – poly(ethylene glycol) with MW of 200 kDa
of freezing and freeze-drying to obtain porous materials [32–34]. This (Cat. No.: 102511497 LOT:BCCG7893, 400 kDa Cat. No.: 102550289
paper presents the change in antimicrobial activity and cytotoxicity of LOT:BCCH5871, 1000 kDa (Cat. No.: 8.07488.1000 LOT:S8283588 247.
xerogel chitosan materials resulting from the difference in the DD and The CO2 used to saturate the chitosan precipitate was obtained from
MW of the polymer, and the technique of its dissolution. The influence of Linde Gaz Polska Sp. Z o. o. (Gdansk, Poland). For microbiological tests,
the use of an innovative procedure for obtaining chitosan xerogel ma­ the following bacterial species were used: Gram-negative Escherichia coli
terials consisting of dissolving the polymer by saturation of its suspen­ (ATCC 25922) and Gram-positive Staphylococcus aureus (ATCC 29213)
sion with carbon dioxide (CO2) [35] on the above parameters is from the Polish Collection of Microorganisms, Ludwik Hirszfeld Institute
presented. To date, no attempt has been made in the scientific literature of Immunology and Experimental Therapy of the Polish Academy of
to assess these relationships, taking into account the above variables. Sciences (Wrocław, Poland). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe­
According to the research hypothesis, the lack of acid residues in nyltetrazolium bromide (MTT; Cat. No.:1003404689, LOT: MKCR748),
xerogel chitosan materials obtained by the innovative method of satu­ medium, antibiotics, and supplements necessary for cell culture were
ration with gaseous CO2 will allow the actual assessment of the anti­ obtained from Merck (Germany). MilliQ water was used to prepare all
microbial activity of chitosans resulting from the structures of the the aqueous solutions (Milli-Q® IQ 7005 Water Purification System,
polymer itself and differences in MW and DD. In addition to maintaining Millipore, USA). All other reagents were of analytical grade or higher.
this activity, the prepared materials will be characterized by lower
cytotoxicity in relation to the L929 model cell line in comparison to
xerogels created by classical chitosan dissolution in acid, freezing and

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 S. Mania et al. Carbohydrate Research 534 (2023) 108973

2.2. Chitosan characterization Condenser temperature: − 80 ◦ C, Sample shelf temperature: 50 ◦ C).

Before use in the tests, the samples were stored in a dry, tight package
2.2.1. Degree of deacetylation under cool conditions.
The DD of chitosan was determined using the potentiometric titra­
tion method [36]. The titration solution was prepared by dissolving 2.4. Solubility test with visualization
0.25 g of chitosan in 10 mL of 0.3 M hydrochloric acid and filled up to
200 mL). It was titrated with a 1 M solution of sodium hydroxide, which To compare the behavior of chitosans xerogel immersed in water,
allowed the pH vs. amount of added NaOH titration curve to be plotted. 0.5 g was suspended in 50 mL of distilled water for 24h at 37 ◦ C. Sub­
Then, using MatLab software (R2022a, The MathWorks, Inc.), the in­ sequently, the samples were imaged using the TOP Show 3D automatic
flection points of the titration curve were determined. The mass of used rotation system (Wroclaw, Poland).
chitosan (M), value of measured pH for the first inflection point (x), and
value for the second inflection point (y) were applied in equation (1) to 2.5. Antimicrobial activity
calculate the percentage of chitosan-free amino groups (%NH2).
% NH2 = 16.1 (y − x)/M (1) The antimicrobial properties of the xerogel materials were evaluated
according to the quantitative ASTM E2149 method with slight modifi­
cations using E. coli and S. aureus species [38]. Colonies of bacteria were
2.2.2. Molecular weight
first subcultured in TSB for 24 h at 37 ◦ C. Then, from cultured bacteria
The MW of the chitosan polymers was determined by an indirect
test medium in TSB were prepared by adjusting the number of bacterial
method using dynamic viscosity measurements with a Brookfield Digital
cells in the range 1.0–5.0 × 107 CFU/mL with a spectrophotometer,
Model DVIII Ultra viscometer equipped with a temperature-controlled
measuring the absorbance at 600 nm (optical density 0.1). The change in
bath (Middleboro, MA, USA). The viscometer was calibrated using vis­
absorbance of a bacterial culture is proportional to the number of bac­
cosity standards in the form of mineral oils with viscosities of 10 mPas,
teria present in the sample. This is a relationship that describes how
100 mPas, and 12500 mPas (IKA, Warsaw, Poland) to measure spindles
absorbance (A) changes as a function of the number of bacterial cells in
SC4-18, SC4-27, and SC4-25, respectively, whose measuring range
the sample. This relationship can be described by the general equation:
corresponds to the viscosity of the measured chitosan solutions. Then,
A (Absorbance) = ε absorption coefficient) *b (length of the light path
the viscosity dependence of the standard glycol solutions and chitosan
through the sample) *c (concentration of absorbing substances in the
solution with a defined MW (150 kDa; 1% m/v solutions in 1% v/v
sample). The more bacterial cells there are, the more light they absorb,
acetic acid) as a function of shear rate at 25 ◦ C was determined. In order
leading to a higher absorbance value. The value of the absorption co­
to determine the MW of chitosans, a 1% solution (m/v) in 1% acetic acid
efficient (ε) is specific to a given type of bacteria and the wavelength of
(v/v) was prepared from each of them, and their viscosities were
light used for measurement.The materials for the study were prepared
measured at 25 ◦ C using an appropriate measuring spindle adjusted
by cutting squares with a side of 4 cm from the chitosan xerogels.
based on the generated strain as a result of shear stresses between the
Polyethylene foil of the same dimensions was used as the control sample,
liquid layers during the measurement. For each of the measuring spin­
which showed no antimicrobial activity. The cut materials were steril­
dles, the relationship between MW and dynamic viscosity was used to
ized with UV radiation for 30 min on each side. Then, 0.4 mL of diluted
determine the MW of chitosans with unknown MW. All rheological
bacterial inoculum was applied to the surface of the samples. Each
measurements were performed in triplicate.
square of the inoculated surface was covered with a sterile polyethylene
film with the same dimensions to ensure contact of the cell suspension
2.3. Chitosan xerogels preparation with the material on the surface of 16 cm2. After 24 h incubation at
37 ◦ C, samples were placed separately in 10 mL of PBS solution and
Xerogel materials based on chitosan were prepared using two vortexed intensively for 25 s. Next ten-fold serial dilutions were pre­
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dissolution methods: the classic method using a 0.1 M LA solution [37] pared, then seeded on TSA plates, and incubated for 48 h at 37 ◦ C. After
and the method of CO2 saturation, an aqueous suspension of chitosan in incubation, only plates containing 30 CFU–300 CFU were counted.
the microcrystalline form [35]. The chitosan concentration in the solu­ When no colonies were recovered in non dilluted sample, the number of
tion was 1% v/v. The solutions in LA were prepared by systematically bacteria was recorded as “10.” The viable count of bacteria (CFU/mL)
pouring chitosan powder into a LA solution during mechanical mixing at was recorded using the following formula:
a speed of 300 RPM (RA 2020, Heidolph Instruments GmbH & Co. KG,
Kelheim, Germany) and stirred until the polymer was completely dis­ VC = N • D (2)
solved (approximately 1 h). The chitosan solution was prepared by CO2
where Vc is the bacterial concentration in colony forming units per mL
saturation, as follows: In the first step, a 1,5% chitosan solution in 0.1 M
(CFU/mL), N is the average value in colony forming units (CFU) from
hydroxyacetic acid was obtained by indirect dissolution of the polymer
Petri dishes, and D is the dilution factor from the counted plates. The
in a proper acid solution during mechanical stirring at a speed of 300
antimicrobial activity on a logarithmic scale was calculated using the
RPM (RA 2020, Heidolph Instruments GmbH & Co. KG, Kelheim, Ger­
following formula:
many). Then, during mixing 0.5 M solution of sodium hydroxide solu­
tion was added until a pH value in the range of 9–10 was reached. This R = log(B / A) (6)
was equivalent to the complete precipitation of chitosan in the micro­
crystalline form. The precipitated chitosan was filtered using a seepage where A is the average of the number of viable cells on the test sample
kit under reduced pressure and washed five times with distilled water. after 24 h incubation at 37 ◦ C (CFU/mL) and B is the average of the
Finally, the precipitated chitosan was weighed and suspended in number of viable cells in the control sample after 24 h incubation at
distilled water to obtain a solution of 1% relative to the dry matter of the 37 ◦ C (CFU/mL). A percentage reduction of bacteria on logarithmic (R)
polymer. The suspension was homogenized at 10000 RPM for 3 min scale equal to 1, 2, and 3 corresponded to a reduction of 90%, 99%, and
(Silent Crusher M, Heidolph Instruments GmbH & Co. KG, Kelheim, 99.9%, respectively.
Germany), and then saturated with CO2 with simultaneous mechanical
mixing using a hollow shaft stirrer for gas saturation (BIOMIX BMX-10, 2.6. Cytotoxicity
Gdansk, Poland) until completely dissolved. The obtained solutions
were poured into flat forms of 10 × 20 cm size to a height of 5 mm, 2.6.1. Cell culture
frozen at − 80 ◦ C, and then freeze-dryed (Pressure: 0.94 mbar, Adult mouse fibroblast L929 cells were purchased from the American

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 S. Mania et al. Carbohydrate Research 534 (2023) 108973

Type Culture Collection (ATCC, Manassas, VA, USA) and tested negative molecular weight (LMW), medium molecular weight (MMW), and high
for mycoplasma using a Universal Mycoplasma Detection Kit (ATCC, molecular weight chitosan (HMW) are consistent with literature data,
Manassas, VA, USA). The L929 cell line was cultured in low-glucose for which the MW range are 50–150 kDa, 150–500 kDa and 500–2000
Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. kDa, respectively [37,39,40]. Data from the specification of chitosan
Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine from shrimp shells and crab shells indicate that these are HMW poly­
serum (FBS; Biowest, Nuaille, France), 100 μg/mL streptomycin, and mers, which is also confirmed by our results. In addition, the manu­
100 U/mL penicillin. Cells were incubated at 37 ◦ C in a 5% CO2 atmo­ facturer described chitosan from crab shells as highly viscous. This is
sphere. All experiments were performed using cells in the exponential consistent with the results, as the mass of chitosan was as high as 4000
phase of growth. kDa. The highest measured MW of chitosan reported in literature is
approximately 10 million dalton [41]. However, the MW of chitosan can
2.6.2. Cell viability and morphology assessment vary depending on its source, method of preparation, and DD. The LMW,
To estimate the in vitro cytotoxicity of the extracts, the 3-(4,5- MMW and HMW chitosans do not differ significantly in the DD. Signif­
dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay icant differences were observed only for chitosans with HMW (Table 1).
was used, according to ISO 10993–5:2009(E). Briefly, L929 cells were Therefore, the results were used to determine the variability of the
seeded in 96-well plates at a density of 1 × 104 cells/well and 100 μL of biological properties of chitosan materials (antimicrobial activity and
culture medium (blank) was dispensed into the peripheral wells. After cytotoxicity) depending on the MW with the same DD (LMW, MMW,
24h of incubation at 37 ◦ C in a 5% CO2 atmosphere, the culture medium HMW) and the DD with the same HMW (H66, H79, H83). Chitosan
was removed and replaced with either fresh medium (positive control materials were obtained by the classical method using LA dissolved in
and blank) or fresh medium containing samples (1:1 v/v, 1:5 v/v, and water (Fig. 1. B). This indicates that their preparation method, which
1:9 v/v of chitosan extract). Samples for cytotoxicity studies were pre­ includes a lyophilization step, retains the acid in the final product. The
pared by suspending 0.5 g of the chitosan xerogel in 50 mL of distilled acid trapped in the dried material causes it to re-dissolve when
water for 24h at 37 ◦ C. After this time, the liquid part of the sample immersed in water. Photographs in Fig. 1 indicate that the chitosan
(extract) was passed through a 0.22 μm filter. Following 24h incubation, materials obtained by CO2 saturation did not dissolve in water because
images of cells were taken using a 20× objective in an OLYMPUS I×83 they did not contain acid. Only swelling of the materials can be
inverted microscope with an XC 50 camera and cellSens Dimension observed, which is a well-known characteristic of this polymer. Ac­
software. The culture medium was then removed, and 50 μL of the MTT cording to literature, chitosan materials can absorb liquids 30 times
solution (1 mg/mL in medium without supplements and phenol red) was their own weight or more. ([42]; Zhang et al., 2019).
added to each well and incubated for 2h at 37 ◦ C in a 5% CO2 atmo­
sphere. Next, the MTT solution was removed, and the formazan crystals 3.2. Antimicrobial activity
were dissolved in 100 μL isopropanol and shaken for 10 min. Absor­
bance was measured at 540 nm using a microplate reader (iMarkTM, The antimicrobial activity of the obtained chitosan xerogels was
Bio-Rad, Hercules, CA, USA). The results were obtained from four in­ determined using the modified ASTM E2149 method because of its high
dependent experiments (n = 4). accuracy in relation to the given form of material [38]. Fig. 2 shows the
antimicrobial properties of chitosan xerogels as a function of MW and
DD against E. coli and S. aureus. The antimicrobial activity of all chito­
2.7. Statistical analysis
sans dissolved in lactic acid, for both Gram-positive and Gram-negative
bacteria, was greater than 99.99%. The reduction of bacteria by more
STATISTICA software (StatSoft, Inc., Tulsa, OK, USA) was used for all
than five logarithmic orders proved the bactericidal activity of the ma­
the analyses. Statistical significance was set at P < 0.05. All data re­
terials. It can be seen that such high activity is caused by one factor, i.e.
ported are based on the means of four replicates (n = 4). Experimental
the presence of LA in the samples. The mechanism of action of this acid is
results are expressed as mean ± standard deviation (SD). Student’s t-test
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based on the degradation of the cell membrane, causing leakage of

and one-way analysis of variance (ANOVA) were applied. The Mann-
proteins from the inside of the cell and, thus, cell death [43].
Whitney U test was used to analyze the differences between the results
Stanojević-Nikolić and co-workers conducted a study to investigate the
of the cytotoxicity assay for LA and CO2—obtained materials (*p <
antimicrobial effect of lactic acid against different pathogen and
0.05).
spoilage microorganisms [powinno by]. They the determined minimal
inhibitory concentration (MIC) and minimal bactericidal concentration
3. Results and discussion (MBC) against E. coli and S. aureus. For both species, the MIC and MBC
were 0.25% and 0.50%, respectively [44,45]. The methodology of the
3.1. Chitosan characteristic parameters antimicrobial test (number of samples, volume of applied inoculum)
shows that the concentration of lactic acid in our test system was
Five different chitosan polymers were used to evaluate the rela­ approximately 0.36%, which results in a registered killing effect (at least
tionship between the antimicrobial properties and cytotoxicity of the 3 logarithmic orders of reduction), which could also be intensified by the
materials prepared using two different methods. Table 1 shows the re­ synergistic effect of the lactic acid and chitosan. Chitosan materials
sults of the DD measurements and their MW. produced by the CO2 saturation method were characterized by lower
The indirect measurement of the MW by viscometry confirmed the antimicrobial activity in relation to the same samples prepared using the
information provided by the chitosan manufacturer. The values for low classical method (Fig. 2), and were more effective in inhibiting the
growth of the S. aureus than E. coli. Considering the MW, the antimi­
Table 1 crobial activity against both bacterial species was similar to that ob­
Description of the tested chitosan samples, their DD and MW (n = 3, p < 0.05). tained for materials produced using LA. MMW was characterized by the
Seller chitosan name DD [%] MW [kDa] Given symbol lowest antimicrobial activity, amounting to 1.35 and 0.75 logarithmic
Low molecular weight 75.7 ± 5.7a a
89 ± 4 LMW
for S. aureus and E. coli, respectively, which concern materials obtained
Medium molecular weight 81.7 ± 4.8a 280 ± 10b MMW by CO2 saturation.
High molecular weight 78.8 ± 1.5a 591 ± 26c HMW/H79 Chung and Chen investigated the antibacterial activity of LMW chi­
From shrimp shellsa 66.2 ± 3.1c 545 ± 22c HMW/H66 tosan (30 kDa) by assessing the mortality rates of E. coli and S. aureus,
From crab shellsa 83.2 ± 4.6b 4000 ± 142d HMW/H83
and demonstrated that chitosan can destroy the cell structure of both
a
High molecular weight chitosan. bacterial cells, resulting in the leakage of enzymes and nucleotides from

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 S. Mania et al. Carbohydrate Research 534 (2023) 108973

Fig. 1. Results of water extraction of xerogel materials prepared A) by CO2 saturation, B) by dissolving with LA.
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Fig. 2. Antimicrobial properties of chitosan xerogels of a) different MW against E. coli, b) different DD against E. coli, c) different MW against S. aureus, d) different
DD against S. aureus. The results were analyzed by one-way ANOVA with comparisons vs. control: ns (not statistically significant, p > 0.05), *p < 0.05.

different cell locations [44]. Jeon et al. used fluorescence-labeled chi­ penetration of chitosan into the cell [9]. If so, the antimicrobial effect
tosans and monitored changes in zeta potential values of bacteria with must be the result of several mechanisms (Fig. 3). Moreover, differences
chitosan coating, confirming the flocculation and adsorption behavior of in antimicrobial activity were also observed between chitosans H66 and
this polymer (Jeon et al., 2001). Zheng et al. reported that the antimi­ H83. A bactericidal effect was achieved for both bacterial species and
crobial activity of chitosan against Gram-positive S. aureus increased materials obtained using lactic acid. H83 produced by CO2 saturation
with an increase in the MW. In addition, for Gram-negative E. coli, the showed the lowest activity compared to H66 and H79, but it was also the
antimicrobial activity of chitosan increased with a decrease in MW. The chitosan with the highest viscosity and molecular weight. The H66
authors suggested the following two mechanisms for the antimicrobial sample produced by the CO2 saturation method was characterized by
activity. In the case of S.aureus, chitosan on the cell surface can form a lower activity against S.aureus than H79 and higher than H83, which in
polymer membrane, which inhibits nutrients from entering the cell. For turn could be the result of weak protonation and, at the same time, the
E. coli, activity, especially in the case of low MW, is related to the amount of amino groups in chitosan, which determine the strength of

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 S. Mania et al. Carbohydrate Research 534 (2023) 108973

Fig. 3. Schematic showing the proposed mechanism of antimicrobial activity of chitosan depending on the method on polymer dissolution method (LA and CO2),
MW and DD against Gram-positive and Gram-negative bacteria.
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the antimicrobial effect. innovative method of CO2 saturation. A greater DD means a greater
There is a significant difference in the structure of the cell walls of amount of free amino groups capable of carrying a charge, even at pH
Gram-positive and Gram-negative bacteria, which leads to differences in values lower than pKa (pH < 6.3).
their charge. Gram-positive bacteria have a thicker layer of peptido­ In the case of materials prepared with LA, the change in the DD does
glycan in their cell walls, which is surrounded by a polysaccharide and not affect the antimicrobial activity of chitosan, which is a very high -
lipid layer. Peptidoglycan contains a large amount of positively charged reduction of bacteria by five and seven logarithmic orders for E. coli and
amino acids such as lysine, which makes the cell wall of Gram-positive S. aureus, respectively (Fig. 2b and d). There was no strict relationship
bacteria highly positively charged. Gram-negative bacteria are sur­ between antimicrobial activity and the DD of chitosans obtained by CO2
rounded by a cell membrane composed of two lipid layers, the outer saturation. This may mean that DD has a lower priority in imparting
layer of which contains lipopolysaccharides (LPS). LPS is a complex activity to the materials than the MW because samples H66, H79, and
molecule that consists of lipids, polysaccharides, and proteins. LPS also H83 are high-molecular chitosans. However, the MW of H83 was
includes many carboxyl groups, which are responsible for the negative significantly higher than those of the other two chitosans (H66 and
charge on the outer membrane of Gram-positive bacteria [46]. The H79). This may mean that molecules that are too large lose their anti­
positive charge of the amino group (NH3+) at pH values lower than pKa microbial activity due to the formation of aggregates (intramolecular
(pH < 6.3), at which this functional group carries 50% of its total hydrogen interactions).
electrical charge, allows interactions with the negatively charged mi­ For acid-free chitosans, it is highly likely that a third antimicrobial
crobial cell of the membrane, which is prone to the leakage of intra­ mechanism may occur, involving chelation of metal ions found on the
cellular components [47]. In the case of chitosan prepared using CO2 surface of the bacteria or in its nutrients. According to the literature, this
saturation (above pKa), the positive charge transfer of chitosan was mechanism occurs more often at pH > 6.5, owing to the ability of the
negligible. This results in very limited electrostatic interactions between deprotonated amino group to donate the nitrogen lone pair. At pH
the polymer and cell. values of 7–9, metal ion chelation occurs through both the amine groups
Most studies have shown that an increase in DD and a decrease in pH and the two deprotonated hydroxyl groups, forming a more stable
improves the antimicrobial properties of chitosan (Jeon et al., 2001). complex than the two –NH2 receptor groups at lower pH [20].
The dependence of the increase in antimicrobial activity on the increase Fig. 3 shows a diagram of the proposed mechanism of antimicrobial
in DD should also be maintained for materials prepared using the activity of chitosans, differentiated by the method of dissolution, MW,

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DD, and type of bacteria. In the case of Gram-negative bacteria and the 3.3. Cytotoxicity
LA environment, the cell membrane is lysed under the influence of this
acid and LMW chitosan. This causes migration of intracellular compo­ The MTT assay was used to investigate the cytotoxicity of the studied
nents from the cell. For Gram-positive bacteria in the same environment, chitosan xerogels against adult mouse fibroblast L929 cells at different
there is a hybrid mechanism consisting of coating the cell with HMW, dilutions (1:1 v/v, 1:5 v/v, and 1:9 v/v).
thus preventing the uptake of nutrients and damage to the cell mem­ The obtained results were expressed as the percentage of viable cells
brane. In the case of chitosans dissolved by CO2 saturation and Gram- compared to the control without the materials. The data presented in
positive bacteria, the cell membrane was not lysed, and only the sur­ Fig. 4 clearly show that chitosan materials obtained by the LA method in
face of the cells was coated with HMW chitosan. For LMW chitosan and a 1:1 dilution are characterized by the highest cytotoxicity against L929
Gram-negative bacteria, membrane lysis should be limited. In addition, cells (~20%). For the same dilution and samples prepared using the CO2
for all chitosans obtained by CO2 saturation, the mechanism of chelation saturation method, the viability of L929 cells was approximately 2.5
of ions constituting the components of the cell membranes is activated. times higher (~50%). aStatistical analysis showed lower cytotoxicity of
This difference may also be due to the influence of the environment on tests performed using the CO2 method for all chitosan extracts with the
the activity of chitosans differing in the DD, where the presence of acid lowest dilution of the extract (1:1 v/v) administered to cells. For samples
enhances the interaction of polymers with the cell membrane in a series with the highest dilution of chitosan extract (1:9 vv/v), no statistically
of increasing activities: H83(CO2) < H66(CO2) < H79(CO2) < H66(LA); significant differences were found (Fig. 4).
H79 (LA); H83(LA). Hismiogullari and colleagues conducted a study to investigate the
effects of organic acids, including lactic acid, on murine fibroblast cells
from the NIH 3T3 cell line. Their research showed a similar relationship
despite the use of different cell types. For samples administered to cells
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Fig. 4. The cytotoxicity of chitosan materials following 24 h incubation at different dilution factors (1:1 v/v, 1:5 v/v, and 1:9 v/v) against L929 cells. Data are
expressed as the mean ± standard deviation of four independent experiments. The results were analyzed by one-way ANOVA with comparisons vs. control: ns (not
statistically significant, p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001. The Mann-Whitney U test was used to analyze the differences between the results of the
cytotoxicity assay for LA and CO2—obtained materials (*p < 0.05).

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 S. Mania et al. Carbohydrate Research 534 (2023) 108973

with lactic acid concentrations of 0.5%, 0.25% and 0.1%, cell survival other hand, cytotoxicity studies of chitosan with different MW (17 kDa,
was ~20%, ~70% and 90–100%, respectively [48]. Comparable results 45 kDa, and 240 kDa) by Zhang et al. showed an increase in cytotoxicity
were achieved for sample extracts from chitosan materials produced with a decrease in MW of <40%, ~60%, and >90%, respectively [49].
using lactic acid, in which the concentration of this acid was: 0.45%, Results reported by Jeon et al. showed that chitosan oligosaccharides
0.15%, 0.09%. Therefore, for both lines of mouse fibroblasts, the cyto­ with a higher DD exhibited higher cytotoxicity against L929 cells [50].
toxic effect of drug acid was very similar. The exact mechanism of chitosan-induced cytotoxicity in L929 cells is
Cell viability improved with increasing dilutions of extracts prepared not fully understood and may involve multiple factors, such as cell
from chitosan materials. The same trend was observed for all materials, membrane disruption through the interaction of positively charged
regardless of the MW and DD of chitosan. Huang et al. also confirmed chitosan particles with negatively charged cell membranes, induction of
that the MW does not affect the cytotoxicity of chitosan [31]. On the reactive oxygen species (ROS) generation by chitosan, activation of
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Fig. 5. Representative images at 200 × magnification of L929 cells following 24 h treatment with chitosan materials. The scale bar is 50 μm (LA-samples prepared
with lactic acid, CO2-samples prepared with CO2 technology).

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 S. Mania et al. Carbohydrate Research 534 (2023) 108973

pro-apoptotic pathways or inhibition of anti-apoptotic pathways, and to 120 h [57]. The biocompatibility of the manufactured titanium alloy
induction of cell cycle arrest at different stages, depending on the cell materials was also evaluated using the MTT test with L929 fibroblast
type and chitosan concentration or mitochondrial dysfunction leading to cells and MG-63 osteoblasts. Analysis of the MTT test data showed that
the release of pro-apoptotic factors and subsequent cell death [51,52]. the survival rate of L929 cells after 120 h of incubation was significantly
The cytotoxicity data obtained here were confirmed by a morphological higher than that of MG-63 osteoblasts [58]. In contrast, bioassessment of
study of L929 cells treated with the tested chitosan extracts for 24 h the Ti17Mg composite material showed no difference in the abundance
(Fig. 5). Owing to the very similar cytotoxicity of all tested chitosans, the of viable cells of both L929 and hDPSC dental pulp stem cells, which was
imaging results are presented in a comparison of tested dilutions only for comparable in both cases [59]. However, in the case of cancer cells, their
the selected sample, H66. Fig. 5 clearly shows that as the dilution of the sensitivity is different. For example, Elsayed et al. examined the effects
tested extracts increases, there is a marked increase in the number of of Moringa oleifera seed essential oil on HeLa, human cervical cancer;
cells visible in the microscopic images. Moreover, L929 cells showed HepG2, human hepatocellular carcinoma; MCF-7, human breast cancer;
unchanged morphology (retained their fibroblast shape) after treatment CACO-2, Caucasian colon adenocarcinoma and L929, mouse fibroblast
with extracts at 1:5 and 1:9 dilutions, relative to the control sample. cell lines. The researchers showed that cytotoxicity depended not only
Significant differences were observed in the morphology of cells treated on the concentration of the test compound. but also on the cell line. HeLa
with chitosan extracts at 1:1 v/v dilution. In the case of cells treated with proved to be the most sensitive cells, followed by HepG2, MCF-7, L929
the chitosan extract obtained by the innovative method of CO2 satura­ and CACO-2 [60]. Similar conclusions were reached by Samarghandian,
tion, the morphology of the majority of the L929 cells was preserved. who observed that the cytotoxic effect of ethanolic saffron extract on the
The cells were oval and elongated in shape (H66–CO2 1:1). For the human non-small lung cancer cells (A549) was significantly greater than
H66-LA 1:1 sample, two observations were made. First, the microscopic on L929 cells [61].
image is blurred. Chitosan LA-extracts diluted 1:1 were characterized by In conclusion, the use of the L929 line in the MTT assay to assess the
the highest viscosity, resulting from the increased solubility of the biosafety of a given material is a standardized method that allows a
polymer under such conditions, making it difficult to visualize the cell preliminary assessment of the cytotoxic effect. One of the main reasons
morphology. Second, the number of cells in the H66-LA 1:1 sample in for using fibroblasts as a model line is that they are cells present in every
the same measuring field of the microscope was lower than that in the tissue except blood. Therefore, if a particular compound has a detri­
other images, indicating substantial inhibition of cell proliferation and mental effect on a fibroblast line then its use will adversely affect the
induction of cell death of a significant number of cells. Contours of cells entire body. In addition, these are the most commonly used cell lines in
with only spherical shapes are visible, revealing a change in L929 cell experiments which is due to their simple culture [62]. This approach
morphology. Undoubtedly, the cytotoxic effect of lactic acid was makes it possible to compare specific materials, composites or com­
evident. This acid affects the function and viability of the cells, which pounds with each other. However, when conducting further research to
has been confirmed in many studies. The cytotoxic effect of lactic acid is evaluate the applicability of a given composite, it is necessary to conduct
attributed to mechanisms such as the generation of free radicals, which extended studies on their effects on specific cell lines.
affect the activity of intracellular enzymes, such as dehydrogenases
(causing disturbances in energy metabolism), and gene expression 4. Conclusions
(disruption of DNA replication and transcription) [52–54].
In vitro methods, which are crucial when evaluating medical devices Chitosan is a well-known and widely studied compound. Several of
such as implants, cannot capture all the complexities of the human body. its advantages are often pointed out, such as biocompatibility, biode­
Therefore, standardized tests for biosafety evaluation have been devel­ gradability, bioadhesiveness, coating ability, and antimicrobial activity.
oped, as described in ISO 109933 “Biological Evaluation of Medical Due to the many mechanisms determining the resultant antimicrobial
Devices.” In the section that provides a set of recommendations, pa­ activity of chitosan and its cytotoxicity, it remains a challenge to pre­
rameters and conditions for conducting the test ISO 10993–5:2009 cisely identify and link those properties.
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recommends testing on a cell line derived from mouse fibroblasts L929 However, it can certainly be unequivocally stated that the use of
[55]. Accordingly, many authors in their studies focus precisely on using innovative technology for CO2 saturation in the production of chitosan
the L929 line as a model, a benchmark. However, in cases where authors materials significantly expands the application possibilities of this
focus their studies on a specific tissue, organ, or site of action of a polymer owing to the reduction of its cytotoxic effect, regardless of the
compound in the body, they conduct additional studies confirming the MW and DD, compared to the classical method of dissolution in LA and
biosafety of a given implant, on target cell lines, in order to confirm this certainly in other acid solutions. The results of the antimicrobial activity
cytotoxic effect or lack thereof. measurements indicate that the acid present in the chitosan materials,
As an example, Dodero et al. compared the effect of the method used which is necessary for the dissolution and processing of this polymer, is
to crosslink electrospun chitosan-based membranes on their biosafety. partly responsible for its high antibacterial activity. In the next work, we
The toxicity of the methods was tested on three cell lines, i.e. L929, plan to perform an additional test confirming the absence of the sodium
HaCaT and human Saos-2 osteoblasts. For both chemical and physical salt of hydroxyacetic acid in the rinsed chitosan suspension to
crosslinking of the test material, the survival rate of L929 cells after 24 h completely exclude the potential impact of this compound on the ac­
of contact was the highest and significantly higher than for the other tivity of the polymer.The results showed that the same materials pro­
cells. Moreover, only the L929 cells showed no cytotoxic effect of the duced by the CO2 saturation technique may show much lower
samples tested. In addition, comparing the other two lines with each antimicrobial activity against microorganisms, which results from the
other, HaCaT cells had a higher survival rate after exposure to the test resultant mechanisms related to the MW of the polymer and the DD.
materials than Saos-2 cells [56]. In another study conducted by Cas­ Nevertheless, the antimicrobial effect of materials produced by CO2
tellano et al. the biological safety of electrospun chitosan-collagen saturation is still sufficient to design bacteriostatic or even bactericidal
nanofibers was also examined by MTT for two cell lines, i.e. L929 and materials.
human HaCaT keratinocytes. The study showed differential behavior of The results indicate that chitosan is a safe raw material and can be
these two cell lines in interaction with different substrates over time. In used in many industries, even in the food industry (registered as a food
the case of L929 cells, for all tested materials, a decrease in cell counts additive in some countries), as well as in the packaging industry.
was observed after 48h, followed by an increase after 72h. Moreover, the However, it is an organic compound of natural origin, which is often
survival rate of this cell line, compared to HaCaT cells, after 24h contact difficult to process technologically and ensure appropriate mechanical
with the tested material was higher in most samples. In the case of properties of the final products. This creates the need to use it as an
HaCaT cells, cell abundance decreased with increasing exposure time up additive and not the main raw material, which still requires strict

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control, including potential cytotoxic effects. fabric, J. Appl. Microbiol. 112 (5) (2012) 1034–1041, https://doi.org/10.1111/
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B–K., K.S., J.K., E.A., and R.T All authors have read and agreed to the com/locate/ijfoodmicro.
[16] P. Chhabra, Y.-W. Huang, J.F. Frank, R. Chmielewski, K. Gates, Fate of
published version of the manuscript.
Staphylococcus aureus, Salmonella enterica serovar typhimurium, and Vibrio
vulnificus in raw oysters treated with chitosan, J. Food Protect. 69 (Issue 7) (2006).
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[17] M. Kong, X.G. Chen, K. Xing, H.J. Park, Antimicrobial properties of chitosan and
mode of action: a state of the art review, Int. J. Food Microbiol. 144 (1) (2010)
The authors declare that they have no known competing financial 51–63, https://doi.org/10.1016/j.ijfoodmicro.2010.09.012.
interests or personal relationships that could have appeared to influence [18] S. Mania, R. Tylingo, E. Augustin, K. Gucwa, J. Szwacki, H. Staroszczyk,
Investigation of an elutable N-propylphosphonic acid chitosan derivative
the work reported in this paper.
composition with a chitosan matrix prepared from carbonic acid solution,
Carbohydr. Polym. 179 (2018) 196–206, https://doi.org/10.1016/j.
Data availability carbpol.2017.09.082.
[19] R. Tylingo, P. Kempa, A. Banach-Kopeć, S. Mania, A novel method of creating
thermoplastic chitosan blends to produce cell scaffolds by FDM additive
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Acknowledgement Polímeros - Ciência Tecnol. 19 (3) (2009) 241–247.
[21] M. Hosseinnejad, S.M. Jafari, Evaluation of different factors affecting antimicrobial
This study was supported by NCN Miniatura 6 grant (2022/06/X/ properties of chitosan, Int. J. Biol. Macromol. 85 (2016) 467–475, https://doi.org/
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