ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 10, pp. 1727-1743 © Pleiades Publishing, Ltd., 2024.
1727
Bacterial Cellulose-Chitosan Composite
for Prolonged-Action L-Asparaginase
in Treatment of Melanoma Cells
Anastasia N. Shishparenok
1
, Egor R. Petryaev
2
, Svetlana A. Koroleva
3
,
Natalya V. Dobryakova
1
, Igor D. Zlotnikov
4
, Elena N. Komedchikova
5
,
Olga A. Kolesnikova
5
, Elena V. Kudryashova
4
, and Dmitry D. Zhdanov
1,a
*
1
Institute of Biomedical Chemistry, 119121 Moscow, Russia
2
Moscow Polytechnic University, 107023 Moscow, Russia
3
Patrice Lumumba Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia
4
Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia
5
Moscow Institute of Physics and Technology (National Research University),
141701 Dolgoprudny, Moscow Region, Russia
a
e-mail: zhdanovdd@mail.ru
Received May 30, 2024
Revised July 12, 2024
Accepted July 22, 2024
Abstract— A significant challenge associated with the therapeutic use of L-ASP for treatment of tumors is its
rapid clearance from plasma. Effectiveness of L-ASP is limited by the dose-dependent toxicity. Therefore, new
approaches are being developed for L-ASP to improve its therapeutic properties. One of the approaches to
improve properties of the enzymes, including L-ASP, is immobilization on various types of biocompatible poly-
mers. Immobilization of enzymes on a carrier could improve stability of the enzyme and change duration of its
enzymatic activity. Bacterial cellulose(BC) is a promising carrier for various drugs due to its biocompatibility,
non-toxicity, high porosity, and high drug loading capacity. Therefore, this material has high potential for ap-
plication in biomedicine. Native BC is known to have a number of disadvantages related to structural stability,
which has led to consideration of the modified BC as a potential carrier for immobilization of various proteins,
including L-ASP. In our study, a BC–chitosan composite in which chitosan is cross-linked with glutaraldehyde was
proposed for immobilization of L-ASP. Physicochemical characteristics of the BC–chitosan films were found to
be superior to those of native BC films, resulting in increase in the release time of L-ASP invitro from 8 to24h.
These films exhibited prolonged toxicity (up to 10h) against the melanoma cell line. The suggested strategy for
A-ASP immobilization on the BC–chitosan films could be potentially used for developing therapeutics for treat-
ment of surface types of cancers including melanomas.
DOI: 10.1134/S0006297924100067
Keywords: L-asparaginase, bacterial cellulose, chitosan, kinetic models, cytotoxicity, melanoma
Abbreviations: BC,bacterial cellulose; EcA, ErA, and EwA, therapeutic forms of L-ASP; L-ASP,L-asparaginase; SEM,scanning
electron microscopy; PEG,polyethylene glycol.
* To whom correspondence should be addressed.
INTRODUCTION
L-asparaginase (L-ASP, EC3.5.1.1) is an enzyme that
catalyzes hydrolysis of asparagine with formation of
ammonia and L-aspartic acid. Various forms of this en-
zyme are used in pharmaceutical and food industries
[1-6]. Native L-ASP of Escherichia coli (EcAII), aspara-
ginase from E.coli conjugated with polyethylene glycol
(PEG–asparaginase), and L-ASP of Erwinia chrysanthe-
mi (ErA) are successfully used for treatment of acute
SHISHPARENOK et al.1728
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
lymphoblastic leukaemia [7-9]. It has been shown in
recent studies that L-ASP used in treatment of acute
leukaemia could potentially be used for treatment
of several aggressive types of solid tumors including
breast cancer[10], glioblastoma, pancreatic cancer, and
hepatocellular carcinoma [11]. Use of L-ASP, however,
is associated with multiple side effects, short half-life
time of the drug, and toxicity [12]. Period of inactiva-
tion of L-ASP reduces sharply from 18-24 h to 2.5 h
due to generation of neutralizing antibodies and pro-
teolytic degradation [13]. Therapeutic efficiency of the
most often used form of L-ASP, EcAII[9], is decreased
due to the relatively high glutaminase activity [14].
Glutaminase activity is associated with the develop-
ment of such side effects as thrombosis, pancreatitis,
hyperglycemia, and toxicity [9, 15]. In comparison with
the native EcAII, the PEG-from of this enzyme exhib-
its longer half-life time and lower frequency of devel-
opment of toxic side effects [9], nevertheless, 30% of
the patients develop hypersensitivity reaction [16, 17].
Preparations based on the ErA enzyme and its homo-
log from Erwinia carotovora(EwA) were developed as
the second-line therapy in the case of development of
hypersensitivity to EcA [17]. ErA and EwA have low-
er toxicity due to the lower specificity to glutamine
and, as a consequence, lower number of side effects
[18, 19]. However, EwA is less active and less stable in
comparison with EcA[19, 20]. Hence, development of
the methods to prolong the action of L-ASP [21, 22], in
particular of EwA, and decrease the number of side
effects is an urgent task. One of the main approached
to solve these problems is search for new sources of
L-ASP, as well as development of less immunogenic
and more stable variants of L-ASP with the help of
genetic engineering [6, 23].
Immobilization of L-ASP on various supports com-
prises another approach to increase stability, half-life
time, and to decrease toxicity [24, 25]. Immobilization
allows optimizing catalytic activity and minimizing
side effects [26, 27]. Various supports have been used
to immobilize L-ASP such as synthetic supports (PAG,
polyimide, polyacrylamide), hybrid supports (PEG–al-
bumin, PEG–chitosan, polymethyl methacrylate–starch,
PEG–polyethylenimine), natural supports (carbohy-
drates– cellulose, dextran, starch, chitosan, chitin, and
proteins – albumin, gelatin, collagen, silk fibers, and
silk fibroin) [12, 28, 29]. One of the immobilization
strategies involves modification of the L-ASP enzyme
structure, such as covalent binding (conjugation) with
PEG[24, 25] or chitosan[30, 31]. Another approach in-
volves embedding enzyme into a protective structure
or encapsulation[32]; this allows to decrease toxicity,
prolong its half-life in vivo, increase stability, ensure
targeted delivery and controlled release of the enzyme
[12]. Erythrocytes, solid lipid nanoparticles, liposomes,
polymers (poly lactic-co-glycolic acid, polyacrylamide,
polyaniline, and others) have been used for L-ASP en-
capsulation [12]. Benefits of both these strategies in-
volve protection of the enzymes against deactivation
and degradation with the help of immobilization[32].
At present the most popular approach for L-ASP im-
mobilization is chemical modification of the enzyme
via covalent binding with PEG [33]. This modification
allowed increase half-life of the preparation, but, un-
fortunately, did not decrease side effects [21, 34].
Drug delivery systems based on bacterial cellu-
lose (BC) attract attention of the researchers [35] due
to its unique characteristics. BC displays high purity
(absence of lignin typical for plant-derived cellulose),
mechanical strength [36], high porosity, biocompati-
bility, high specific surface area and high density of
the fibril network, i.e., characteristics affecting adsorp-
tion and release of a drug from the matrix [37]. BCis
nontoxic [38].
Drawbacks of the non-modified BC involve low
water holding capacity, and compounds adsorbed on
this type of BC prone to leaching. That is why in order
to achieve prolonged release of a drug, BC is subject-
ed to various chemical modifications [39-42]. One of
the promising compounds used for BC modification
is chitosan [43]. High reactivity of amino groups in
chitosan facilitates crosslinking via such linkers as
glutaraldehyde. It has been shown previously [44-48]
that crosslinking of cellulose with chitosan increases
sorption capacity due to surface availability of the re-
active hydroxyl groups of cellulose and amino groups
of chitosan[49]. Introduction of crosslinks into the bio-
polymer network changes its texture, hydration and
mechanical properties, as well chemical resistance
to biodegradation [50]. Applicability of this approach
has been confirmed by the studies on creation of
chitosan-BC composites for immobilization of lipase
[51, 52], albumin and fibronectin [53], controlled re-
lease of quercetin [54], quetiapine fumarate [55], and
ibuprofen [43].
One of the main advantages of using BC is the
possibility of targeted delivery of drugs to tumor tis-
sues and controlled release; as a result, systemic side
effects are minimized [40, 56]. In this study we devel-
oped a BC–chitosan composite for immobilization of
Erw. carotovora L-ASP in order to prolong time of ef-
ficient cytotoxic activity against melanoma tumor cells.
MATERIALS AND METHODS
Preparation of bacterial cellulose. A Koma-
gataeibacter hansenii strain (VKMP no. B-11239) was
used for producing biofilms. The strain producer was
cultivated in a Hestrin-Schramm medium of the follow-
ing composition (per one liter): 10 g glucose (Sigma,
USA); 5 g peptone (Diaem, Russia); 5 g yeast extract
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1729
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
(PanReac Applichem, Spain); 1.15 g citric acid monohy-
drate (Merck Millipore, Germany); 2.7 g Na
2
HPO
4
·2H
2
O
(PanReac Applichem). The medium was prepared with
deionized water, ph was adjusted to 4.5 and autoclaved
in flasks. Next, 1 ml of 90% ethanol was added to
200 ml of the medium as well as 15 ml of the medi-
um with K. hansenii B-11239; 2-ml aliquots of the mix-
ture were placed into the wells of 24-well plates (Wuxi
NEST Biotechnology, China) and cultivated under static
conditions at 27°C. Bacteria were grown for 48, 72,
and 96 h to prepare BC films with different thickness.
The obtained BC films were treated with a mixture
of 0.1 M NaCl (Diaem) and 1% sodium dodecyl sulfate
(Merck Millipore) for 72 h for lysing and removal of
bacteria, which was followed by washing films with
deionized water to remove cell residues and adjust
ph to neutral.
Preparation of BC–chitosan composites. BC–chi-
tosan composite was prepared using ex situ strategy
[37, 57,58]. Solutions of chitosan–HCl with mean mo-
lecular mass 7 kDa (degree of deacetylation – 80%;
provided by the Federal Research Center “Fundamen-
tals of Biotechnology”, Russian Academy of Sciences,
Russia) were prepared with concentrations 0.05, 0.1,
0.5, and 1%. For this purpose, chitosan was dissolved
in a 1% solution of CH
3
COOH and incubated for 1 h
in a water bath with temperature 50°C until complete
dissolution. Wet BC membranes were dried for 20min
at 60°C to remove excess of moisture, immersed into
1 ml of chitosan solution, and incubated for 1, 2,
3, or 6 h, with constant shaking (100 rpm) at 37°C.
Next, 0.5 ml of aqueous solution of 1% glutaralde-
hyde (pH 7.0) (neoFroxx, Germany) was added to BC
membranes and incubated at 23°C for 1, 2, 3, or 4 h.
BC films were lyophilized to determine their physical
characteristics. For this purpose, films were frozen in
a liquid nitrogen and lyophilized for 24 h at –50°C and
pressure 0.07 mbar in a freeze dryer Alpha 2-4 LD,
type 101042 Lab (Christ, Germany).
Determination of water content in films, their
porosity and adsorption capacity. Water contents in
the films was evaluated from the change in weight
after lyophilization and expressed in percent(%) using
equation (1):
Water content (%) =
A − B
A
· 100%, (1)
where A– weight of a wet sample; B– weight of a dry
sample.
Porosity was calculated using equation suggested
by Kitaoka et al. [59]. First, BC samples with water
excess removed with filter paper were weighted. Next,
samples were lyophilized, and films were incubated in
a deionized water for 12 h at room temperature and
weighted again. Porosity was calculated according to
equation (2):
P (%) =
Q
0
− Q
1
Q
0
− Q
2
· 100%, (2)
where P – membrane porosity; Q
0
– weight of a wet
sample (g); Q
1
– weight of a dry sample (g); Q
2
–
weight (g) after 12-h incubation of the dried sample
in water.
Adsorption capacity of the films was measured
according the technique suggested by Wu et al. [60]
by incubation of a lyophilized film sample in a HEPES
buffer (50 mM (pH 8.0); Serva, Germany) for 24 h
at 37°C. The following equation (3) was used for cal-
culations:
Adsorption capacity (%) =
W
t
− W
0
A
· 100%, (3)
where W
0
and W
t
– sample weight before and after
incubation in a buffer, respectively.
Determination of chemical composition of the
films with infrared (IR) spectroscopy. IR-spectra of
lyophilized samples were recorded with a Fourier
transform IR-microscope MIKRAN-3 (SIMEX, Russia).
Spectra were recorded in an absorption mode; 36
scans were carried out with resolution 4cm
–1
at 22°C
in a spectral range 4000-900 cm
–1
.
Morphology. Morphology of lyophilized BC sam-
ples and of BC–chitosan composites was examined us-
ing scanning electron microscopy (SEM). Film samples
were coated with gold and examined with the help of
a MAJA3 scanning electron microscope (Tescan, Czech
Republic)[61]. Sample images were acquired at accel-
eration voltage 7kV and 20,000× magnification.
Immobilization of L-ASP on BC. L-ASP EwA from
the Erw. carotovora producer (595 IU/mg; molecular
mass– 37 kDa; 349amino acid residues; purity– 98.1%;
isoelectric point – 8.1; GenBank ID: AAP92666.3) was
provided by the laboratory of medical biotechnology
of the Institute of Biomedical Chemistry [62]. L-ASP
was immobilized on BC via physical adsorption in a
50-mM HEPES buffer (pH 8.0). For this purpose, BC
films were immersed in 0.5 ml of the enzyme solution
with concentration 0.05 mg/ml (28 IU/ml) and incu-
bated at 4°C for 12 h. Amount of immobilized L-ASP
was determined with the help of UV-VIS-spectroscopy
(Aquarius CE 7400 Spectrophotometer; Cecil Instru-
ments Ltd., Great Britain) using protocol suggested
by Dawsonetal.[63]. Percent of adsorbed L-ASP was
determined from difference between the initial protein
content and protein content in the supernatant after
incubation.
Evaluation of L-ASP release. Release of L-ASP
from BC–chitosan and BC films was determined by in-
cubation in 25 ml of 50 mM HEPES (pH 8.0) without
substrate for 24 h. Every hour 300-µl aliquots were
sampled and enzyme activity was determined. For this
purpose, 100 µl of L-asparagine solution were added
SHISHPARENOK et al.1730
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Fig. 1. Scheme of experiment for determination of L-ASP
release from BC and BC–chitosan films. BC and BC–chitosan
films with immobilized enzyme are incubated in 25 ml of
50 mM HEPES buffer. Immobilized L-ASP is released from
films and accumulated in the buffer. Every one hour 300-µl
samples are taken (simultaneously 300 µl of fresh buffer
solution is added to maintain reaction volume) and enzyme
activity is assayed by determination amount of ammonia
using method of direct nesslerization after incubation with
thesubstrate.
to the samples (40 µM; Diaem) and the reaction mix-
ture was incubated for 5 min at 37°C followed by
determination of ammonia concentration using the
method of direct Nesslerization with a Nessler reagent
(PanReac Applichem, Spain) [64, 65]. Amount of en-
zyme that catalyzes release of 1µmol of ammonia per
1 min at 37°C was defined as one unit. After each sam-
pling 300 µl of fresh buffer was added to the reaction
mixture to maintain its volume. Scheme of the exper-
iment is presented in Fig. 1.
The data on the rate of enzyme release were used
to construct kinetic models for the release: zero-order
model, first-order model, Higuchi’s model, Korsmeyer–
Peppas model, and Hixson–Crowell model [66]. Tode-
scribe the mechanism of release Korsmeyer–Peppas
model was used and diffusion coefficient(n) was deter-
mined characterizing differences in the L-ASP release
from BC matrices.
Evaluation of cytotoxicity. Human melanoma
cell lines A375, A875, and MelJuso, as well mouse
melanoma cells B16F10 (obtained from the collection
of the Blokhin Russian Cancer Research Center) were
cultivated in a RPMI-1640 medium (PanEco, Russia).
Human fibroblast cell line WI-38 (ATCC, USA) culti-
vated in a DMEM medium (PanEco) was used as a
conditionally normal cell line. Cultivation medium
contained 5% fetal bovine serum (Capricorn Scientific,
Germany) and 1% sodium pyruvate (PanEco). Cells
were grown in an atmosphere of 5% CO
2
in a CO
2
-in-
cubator at 37°C. Prior to experiment cells were tested
for the presence of mycoplasma with the help of a
PlasmoTest™ reagent kit (InvivoGen, USA).
To determine the IC
50
level (enzyme concentration
decreasing cell viability by 50%) cell were cultivated
for 72 h in a 96-well plate (Wuxi NEST Biotechnology)
in the presence of free EwA in concentration range
0.01-10 U/ml. Cytotoxicity was determined using MTT
test by monitoring transformation of the tetrazolium
salt, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenylterazolium
bromide (Serva), into formazan [67]. Formazan absorp-
tion at wavelength 540 nm was determined with the
help of a SuPerMax3000 plate reader (Flash Spectrum,
China). IC
50
values were calculated from the depen-
dencies of cell viability on enzyme concentration [68].
To identify the cell line most sensitive to EwA im-
mobilized on the BC–chitosan composite, cells were
incubated in the presence of the composite in a 24-well
plate (Wuxi NEST Biotechnology) for 24 h. Cytotoxicity
was determined using MTT test after 48-h incubation
with films.
To determine duration of action of the immobi-
lized enzyme, cells of the human uveal melanoma cell
line A875 and conditionally normal fibroblasts of the
WI-38 cell line were seeded into a 24-well plate with
density 5 × 10
4
cells per well and cultivated for 24 h.
Next BC and BC–chitosan films with immobilized L-ASP
were placed into wells and incubated for 3h followed
by transfer into new wells with cells. Cytotoxicity was
evaluated 48h after end of incubation with the films
using MTT test. Optical images of cell morphology
72h after incubation were obtained with an inverted
microscope Biomed 3I (Biomed, Russia) in the bright
field mode.
Statistical analysis. Quantitative data are pre-
sented as a mean ± standard deviation of the mean
calculated based on the data from three indepen-
dent experiments. All data were analyzed using the
MS Office Excel 2016 software (Microsoft Inc., USA).
Values p<0.05 were considered statistically significant.
RESULTS
Optimization of conditions of BC–chitosan com-
posite preparation. To identify optimal condition for
preparation of BC–chitosan composite the following
parameters were varied: time of BC-producing bacte-
ria cultivation, time of crosslinking of chitosan with
glutaraldehyde, chitosan concentration, and time of
incubation of chitosan with BC. Conditions which did
not results in the loss of enzyme activity, i.e., activity
of the enzyme on the BC–chitosan film was the same
as on the BC film after 24 h.
Kinetics of L-ASP release from the BC and BC–chi-
tosan films is presented in Fig. 2. The rate of L-ASP
release from the BC–chitosan film (chitosan concentra-
tion 0.05%) depended on time of cultivation of BC pro-
ducer, i.e., thickness and porosity of the films (Fig. 2a).
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1731
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Fig. 2. Effect of condition of BC–chitosan composite preparation on the 24-h enzyme release. a)Effect of time of film growth;
b)effect of chitosan concentration. Activity of enzyme immobilized on the BC films is shown in the panel for comparison.
c)Effect of time of incubation with chitosan; d)effect of time of incubation with glutaraldehyde.
Maximum rate of release was observed from the films
that grew for 72h. Films grown for 48 and 96 h demon-
strated 2-fold lower rate of release. Such difference
could be explained by the different inner structure of
the films. Addition of chitosan to BC leads to formation
of three-dimensional structure, and, as a result, size
of pores and adsorption capacity of the films changes.
It is known that insertion of an exogenous molecule,
as a rule macromolecule, changes the structure of BC
fibrils, which affects size and volume of pores [69].
Hence, films grown for 72h displayed optimal porosity
characteristics for immobilization and release of L-ASP.
Chitosan concentration in the composite did
not affect proportionally the rate of enzyme release
(Fig. 2b). The composites with chitosan concentration
0.1, 0.5, and 1% demonstrated lower rate of the en-
zyme release in comparison with the composite with
chitosan concentration 0.05%. Optimal time of incu-
bation of BC with chitosan (with concentration0.05%)
was 2 h (Fig. 2c), as in this case maximum activity of
the immobilized L-ASP was observed. These differenc-
es could be explained by the short time of incuba-
tion of BC in chitosan solution (2 h). In other studies
[70-72] longer incubation time of BC with chitosan or
another component was used, and it was shown that
penetration of chitosan into BC increased significantly
during the incubation period from 5 to 20 h, and after
that it practically did not change[73]. Hence, chitosan
concentration in the case of 1-2-h incubation of BC
with chitosan does not affect the rate of L-ASP release.
It was revealed in the course of optimization of
the incubation time of BC–chitosan composite with glu-
taraldehyde that the highest L-ASP activity after 24 h
was observed for the films with chitosan incubated
with glutaraldehyde for 1 h (Fig. 2d). Increase of this
incubation time to 4 h caused significant decrease of
the enzyme activity, and after 24-h incubation activity
of the enzyme decreased almost to zero.
Optimization of conditions for preparation of BC–
chitosan composite demonstrated that optimal time of
cultivation of the BC-producers for film production was
72 h, optimal concentration of chitosan – 0.05%, time
of incubation with chitosan – 2 h, time of incubation
of BC–chitosan with glutaraldehyde – 1 h. The films
produced under these optimal conditions were used
in the following experiments.
Kinetic models of drug release. The data on dy-
namics of L-ASP release obtained by measuring the
enzyme activity were used for modeling kinetic pa-
rameters of L-ASP release from BC and BC–chitosan
composites. Release from the BC–chitosan composite is
best described by the Higuchi’s model(Fig.3c; Table1),
Table 1. Regression coefficients (R
2
) for kinetic models
of L-ASP release for 8h from BC and BC–chitosan films
Kinetic model BC BC–chitosan
Zero order model 0.960 0.928
First order model 0.959 0.948
Higuchi’s model 0.937 0.971
Hixson–Crowell model 0.962 0.915
Korsmeyer–Peppas model 0.985 0.961
SHISHPARENOK et al.1732
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Fig. 3. Graphic representation of kinetic models for release of L-ASP immobilized on BC–chitosan composite and BC. a)Zero
order model; b)First order model; c)Higuchi’s model; d)Korsmeyer–Peppas model; e)Hixson–Crowell model.
in which release of a drug is controlled by diffusion
exhibiting first-order dependence on concentration
gradient[74]. Release from the non-modified BC corre-
sponds to the Korsmeyer–Peppas kinetic model (Fig. 3d;
Table 1), in which release of a drug is controlled by
the processes of matrix relaxation and rearrange-
ment [75].
Within first 8 h 52% of L-ASP was released from
BC films, and from the BC–chitosan films – only 29%
(Fig. 2b; Fig. 3a). After 24 h 65% of the enzyme was re-
leased from the BC films, and 63%– from the BC–chi-
tosan films. These data indicate that a relatively fast
release is observed from the non-modified film within
first 8 h, which slows down later. The films modified
with chitosan demonstrate slower, prolonged release
of the enzyme without sharp release within first 8 h.
Table 2. Characteristics of BC–chitosan and BC films
Parameter BC–chitosan BC
Weight
of a wet film, mg
401.8 ± 0.7 402.0 ± 0.9
Weight of
a lyophilized film, mg
5.3 ± 0.6 3.7 ± 0.6
Weight of a film
after incubation
in water, mg
154.3 ± 18.2 63.7 ± 23.1
Water content, % 98.67 ± 0.14 99.09 ± 0.14
Porosity, % 160.77 ± 11.47 119.52 ± 7.95
Adsorption capacity, % 2794 ± 341 1745 ± 630
Moreover, the BC and BC-films differ in the
mode of diffusion. Diffusion coefficient (n) calculated
for the BC–chitosan composite for the first 8 h was
0.246 ± 0.029, and for the BC– 0.743 ± 0.142 (p < 0.05).
In the first case the L-ASP diffusion is described by
the Fick’s law (n < 0.45), in which the rate of diffu-
sion is much slower than the rate of polymer matrix
relaxation [76]. In the second case release of the en-
zyme is characterized by the non-Fickian diffusion
(0.45 < n < 0.89). Fick’s diffusion is related to the pro-
cess of solute transport within the polymer with relax-
ation time significantly longer than the time of solute
diffusion. When the time of polymer relaxation is com-
parable with the solute diffusion time, macroscopic
release of the drug becomes abnormal or non-Fickian
[77], which was observed for the case of L-ASP immo-
bilized on BC.
Effect of BC modification on characteristics of
the films. Analysis of water holding capacity of BC
films before and after modification demonstrated that
modification with chitosan practically does not affect
water holding capacity of the films (Table2). However,
adsorption capacity for water in the BC–chitosan films
after lyophilization was 1.6-fold higher than in the case
of BC films. Increase of porosity of the inner structure
of the BC–chitosan composite (1.34-fold) in comparison
with the BC film was also revealed.
Improvement of adsorption capacity and porosity
of the modified BC after lyophilization could be asso-
ciated with higher melting temperature of the bound
water due to lower accessibility and better water hold-
ing capacity in comparison with the control. Formation
of pores depends on the number of ice crystals formed
by the water adsorbed by the material. Ice crystals
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1733
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Fig. 4. IR spectra of BC–chitosan and BC films. a)Range 1000-4000cm
–1
; b)range 1000-2000cm
–1
.
are completely sublimated in the process of drying,
and the volume occupied by ice becomes a pore [78].
Higher ratios of the surface area to volume in thinner
non-modified BC films leads to higher rates of forma-
tion of unbound water and its evaporation [79], which
results in the lower water adsorption capacity.
IR spectra of the films. In order to determine
chemical composition of the BC–chitosan and BC films
used for L-ASP immobilization their infrared spectra
have been recorded (Fig. 4).
The spectra of BC-based films have common
peaks corresponding to cellulose: broad peak at
3335 cm
–1
(Fig. 4a) typical for valent vibrations of –OH
group; peak at 2876 cm
–1
typical for valent vibrations
of aliphatic CH
2
-groups, and peak at 1435 cm
–1
as-
signed to deformation vibration of –CH groups [80].
The peaks observed at wavenumbers 1618, 1435, 1323,
1155, and 1066 cm
–1
correspond to various cellulose
groups: deformation vibrations of –CH
2
groups, defor-
mation vibrations of –CH groups, asymmetric stretch-
ing of glycosidic bonds C–O–C, and stretching of C–O
bonds, respectively (Fig. 4b).
IR spectrum of BC–chitosan films was similar to
the spectrum of BC, because majority of the functional
groups are common for both cellulose and chitosan
[81, 82]. Peaks of valent vibrations of –OH groups at
3359 cm
–1
, –CH groups at 2837 cm
–1
, and peak of defor-
mation vibrations at 1435 cm
–1
typical for α-CH
2
groups
were observed (Fig. 4a) [83]. Unlike in the spectrum
of BC, the spectrum of BC–chitosan has a peak of the
chitosan N–H groups (deacetylated amino groups,
primary amino groups) at 1589 cm
–1
(Fig. 4b). Addi-
tionally, the spectrum of the BC–chitosan composite
has a peak typical from the –CH=N– group observed
at~1682cm
–1
, which indicated formation of the Schiff
base [84]. The spectrum of the BC–chitosan film with
immobilized L-ASP (Fig. 4b) also have peaks typical for
amide I (1500-1600 cm
–1
), amide II (1600-1700 cm
–1
),
and amide III (1200-1350 cm
–1
). It indicated that the
reaction of glutaraldehyde with primary amino groups
of chitosan leading to formation of covalent crosslinks
occurred in the BC–chitosan composite after incuba-
tion with glutaraldehyde [85].
Morphology of BC-based films. Cultivation of the
stain producer K. hanseii in a 24-well place allows pro-
ducing biofilms with diameter 16 mm (Fig. 5a). Mor-
phological changes of the BC structure before and
modification were analyzed based on SEM images.
Lyophilization of BC films results in generation of void
spaces in the network organization, which determine
final porous structure [71]. Microstructures of BC- and
BC–chitosan films are shown in Fig. 5. The BC–chitosan
films (Fig. 5b) form more ordered porous layered
structure with large number of pores of smaller size in
comparison with BC films (Fig. 5c). The BC nanofibers
in BC–chitosan films are intertwined and folded form-
ing a denser structure in comparison with the loosely
arranged fibers in BC films. Therefore, different degree
of density of the three-dimensional network in BC and
BC–chitosan could affect swelling, adsorption capacity,
and rate of L-ASP release.
Prolonged cytotoxic action of L-ASP immobi-
lized on BC–chitosan films. To select cells most sensi-
tive to the action of L-ASP the IC
50
values for the native
EwA enzyme were determined. Among the melanoma
cell lines, the cells of A875 cell line were shown to be
most sensitive, and the cells of A375 cells line were
shown to be the least sensitive (Table 3). As expected,
SHISHPARENOK et al.1734
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Fig. 5. Film morphology. a)Appearance of BC film grown in a well of 24-well plate. b)SEM image of cross section of BC–chi-
tosan film. c)SEM image of cross section of BC film.
the control fibroblasts of the WI-38 cell line were the
most resistant to the enzyme action.
Toxicity of L-ASP released from the film is de-
termined by the rate of enzyme release and rate of
enzyme degradation, i.e., by the amount of active
enzyme in the medium. In order to identify cell line
most sensitive to EwA immobilized on the BC–chitosan
composite, survival of cells after 24-h incubation with
the films was determined. The survival data were in
good agreement with the IC
50
values. The most sensi-
tive melanoma cells were the cells of A875 cell line,
and the least sensitive – cells of the A375 cell line
(Table 3). The conditionally normal fibroblasts WI-38
were the most resistant and demonstrated the highest
viability.
To evaluate duration of the action of L-ASP immo-
bilized on BC or BC–chitosan on the A875 melanoma
cells and on the conditionally normal fibroblasts the
study was conducted involving transfer of the cells
every 3 h into the new wells with the cells (Figs. 6
and7). The enzyme immobilized on the BC films mod-
ified with chitosan preserved its cytotoxicity activity
even after four sequential incubations with the cells
(Fig.6). After 6 h of incubation viability of tumor cells
was ~50%, and after 12 h– 80%. At the same time en-
zyme in the BC film did not exhibit cytotoxic activity
after the first incubation with the cells, and already
after 3 h of incubation viability of the cells was at the
level of control cells not treated with L-ASP. Hence,
immobilization of L-ASP on the BC–chitosan compos-
ite facilitates its prolonged action on the melanoma
tumor cells.
As expected, the EwA enzyme immobilized on the
films, practically did not exhibit any cytotoxic effect
on the conditionally normal fibroblasts WI-38(Fig. 7).
Slight decrease in the cell viability was observed only
during the first 3-h incubation to the level of 66.5-
85.4% for the EwA, immobilized on BC, and to the
level of 71.7-93.0% for the enzyme immobilized on the
BC–chitosan film.
Hence, immobilization of L-ASP on the BC–chi-
tosan composite facilitates prolonged action of the
enzyme on the tumor melanoma cells. No prolonged
action on the conditionally normal cell was observed.
DISCUSSION
Despite the unique properties of native BC, its ap-
plication as a support of therapeutic agents is limited
by the profiles of adsorption and release of a thera-
peutic agent. Very often high rate of these processes is
limited to the few first hours [37, 70, 86,87]. Insome
cases, this could result in the initial local high accu-
mulation of the drug and cause toxic effects in the
cells from normal tissues [88].
Up to now several composites (semi-penetrable
hydrogels) based on BC and chitosan with improved
mechanical and antibacterial properties have been
developed [43, 51, 55, 89]. Hydrogels are usually pre-
pared by mixing BC with chitosan solution followed
Table 3. IC
50
values for free EwA and survival of cells
after incubation with EwA immobilized on BC–chitosan
films
Cell line IC
50
, U/ml Survival, % of control
A875 0.04 11.49 ± 6.93
MelJuso 0.12 22.89 ± 2.66
B16F10 0.24 34.26 ± 6.92
A375 0.38 50.81 ± 10.51
WI-38 8.22 93.61 ± 17.24
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1735
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Fig. 6. Duration of cytotoxic action of L-ASP immobilized on BC and BC–chitosan films on A875 melanoma cells. a)Viability
of cells evaluated with MTT-test after incubation with films and sequential transfer every 3 h to new wells. Bright-field mi-
crophotographs of the cells synthesizing formazan crystals after incubation with BC–chitosan films(b) and BC films(c) with
immobilized L-ASP. *p≤0.05 in comparison with the control cells incubated with BC or BC–chitosan films not containing L-ASP.
Fig. 7. Duration of cytotoxic action of L-ASP immobilized on the BC and BC–chitosan films on the conditionally normal fibro-
blasts of the WI-38 cell line. a)Viability of the cells measured using MTT-test after incubation with the films and sequential
transfer of the films to new well after each 3 h. Bright-field microphotographs pf the cells synthesizing formazan crystals
after incubation with BC–chitosan(a) and BC(b) films with immobilized L-ASP. *p≤0.05 in comparison with the control cell
incubated with BC or BC–chitosan films not containing L-ASP.
by crosslinking with glutaraldehyde. In this system one
polymer is crosslinked and another polymer remains
linear. However, the linear polymer remains physically
bound with the crosslinked polymer via Van-der-Waals
forces, electrostatic interactions, hydrogen bonds, or
combination of these interactions [89]. It is known
that crosslinking of hydrogels could prevent prema-
ture proteolytic degradation of immobilized highly la-
bile macromolecules (such as antibodies), which could
facilitate increase of half-life of a drug in blood and
improve stability of hydrogels via increasing density
of crosslinks [90].
It was observed in our study that the BC films
grown for 96 h released L-ASP slower than the films
grown for 72 h, which was comparable with the re-
lease from the films grown for 48 h (Fig. 2a). This
result is in agreement with the results reported by
Pavaloiuetal.[43], in which the polyvinyl alcohol–chi-
tosan–BC composites were used to achieve controlled
release of ibuprofen. Ibuprofen diffusion decreases
with increase of BC concentration in the film compo-
sition. In our case it indicates that the thickness of BC
films (which depends on the time of cultivation of the
producer) affects significantly the 24-h L-ASP release:
activity of the films grown for 72 h was 2-fold higher
than of the films grown for 48 or 96 h.
Analysis of the L-ASP release from the BC and
BC–chitosan films revealed that chitosan concentration
did not affect L-ASP activity (Fig.2b; Fig. 3). This could
be explained by the fact that the chitosan particles
could aggregate, and chitosan was not able to com-
pletely penetrate the BC film. This hypothesis received
an indirect confirmation in the study by Lietal.[72],
where it was shown that adsorption capacity of the
BC–hyaluronic acid composite did not depend on con-
centration of the latter in the first hours of incubation.
Previously, the BC–chitosan composites were main-
ly developed as wound dressing materials [91, 92]; and
BC with chitosan were incubated for 6-12 h. We sug-
gested to optimize time of chitosan incubation with
BC for creation of L-ASP delivery agent to melanoma
tumor cell. Prolonged incubation time of BC with chi-
tosan (6 h) (Fig. 2c) resulted in the decrease of released
L-ASP activity practically to zero, while incubation
SHISHPARENOK et al.1736
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
of BC with chitosan for 1-2 h did not affect activity
of L-ASP released after 24 h. This indicates that for
immobilization of the investigated enzyme the time of
incubation of BC with chitosan should be significantly
reduced.
Moreover, the released enzyme activity was af-
fected by the time of incubation of BC–chitosan with
glutaraldehyde (Fig. 2d). Conditions of the reaction of
glutaraldehyde with chitosan have been optimised in
the earlier study by Monteiroet al.[85]. The reaction
was carried out in neutral medium, and time of in-
cubation was 1 h at room temperature. Hence, these
conditions are suitable for preparation of the BC–chi-
tosan composite for immobilization of L-ASP.
Evaluation of the L-ASP release from the BC–chi-
tosan composite (chitosan concentration 0.05%) re-
vealed that the cumulative release for 24 h in this
case was more prolonged than in the case of native
BC films. These data are in agreement with the results
of application of BC–chitosan composite hydrogel beads
for immobilization of lipase from Candida rugosa[51].
As a result of immobilization half-life of lipase in-
creased more than 3-fold. Crosslinking of chitosan
with glutaraldehyde resulted in formation of addition-
al three-dimensional structure of the BC fibers, which
caused increase of the fiber thickness (confirmed by
the SEM images in Fig.5) and more effective retention
of L-ASP in the composite.
The data on the pattern of L-ASP release from the
BC films or from the composite, as well application of
the Higuchi’s kinetic model and diffusion coefficient
of the value n < 0.45 indicate that the enzyme release
is controlled by diffusion [93]. The Higuchi’s kinetic
model suggests that the release of a drug obeys the
Fick’s law of diffusion, where the main result is de-
pendence of the drug transport on the square root of
time [94, 95]. At the same time, the release of L-ASP
from the non-modified BC films was better described
by the Korsmeyer–Peppas model for which abnormal
diffusion (n > 0.45) with characteristics of subdiffusion
[96] is typical, as well as faster release of L-ASP within
first hours.
Diffusion of a compound is closely associated with
the structure of a material through which the diffusion
occurs [95], hence, modification of BC with chitosan
allowed achieving prolonged release of L-ASP. Adsorp-
tion capacity of a protein on BC is affected by such
characteristics as its porosity and density of fibers,
which, in turn, depend on time of cultivation of the
producer and composition of the culture medium[97].
In our study porosity of the BC–chitosan composite
was 1.6-fold higher than in the case of non-modified
BC, and following crosslinking with glutaraldehyde
facilitated pore size decrease. This created steric hin-
drance for fast release of the enzyme from the matrix
and prolonged action on the cells in vitro. The most
important feature of the films is the size of pores
formed by the BC fibrils. The size affects steric in-
teraction between BC and a therapeutic agent, which
eventually determines how the drug is released from
the matrix [90]. The effect of steric hindrance blocks
the drug inside the network until the moment of the
network disruption or the pore size increase due to
swelling or deformation [98].
The data obtained for the cells (Fig.6) correspond-
ed to the general trend of L-ASP release from BC and
BC–chitosan composite in solution. The immobilized
enzyme exerted cytotoxic effect on the tumor cells
of uveal melanoma A875, and the use of the BC–chi-
tosan composite prolonged cytotoxic action to four
sequential incubations. This indicates that even in
the case of several transfers of the composite films,
small amount of the released L-ASP was sufficient for
cytotoxic effect, while practically 100% of L-ASP was
released from the BC films during the first incuba-
tion. No prolonged cytotoxic effect was observed in
the case of conditionally normal WI38 cells. Cytotoxic
activity of free L-ASP was demonstrated in the recent
studies for several solid tumor cell cultures [99], and
the PEG-L-ASP preparation exhibited good antitumor
activity against the malignant melanoma in the phase I
clinical trials[100]. The EwA conjugated with PEG[22],
albumin[101], glycol–chitosan[102], and PEG–chitosan
[103] have been developed previously. The EwA conju-
gates with PEG–chitosan demonstrated 3-5-fold higher
specific activity against the K562 and Jurkat cells, and
composition and structure of the conjugate essentially
affected the degree of cytotoxic effect [103]. At pres-
ent the randomized clinical trial of the efficiency of
L-ASP preparation encapsulated into erythrocytes for
treatment of triple-negative breast cancer is under-
way [104]. Furthermore, it is known that switching
to alternative types of L-ASP is recommended for the
patients with hypersensitivity, immune or proteolytic
inactivation of the enzyme [105]. Melanomas represent
surface type of cancer, which are treated efficiently
with surgical treatments [106]. However, there are
clinically and genetically different subgroups of mel-
anomas for which surgical interventions are inefficient
[106,107], which makes development of new strategies
for treatment of certain types melanomas very import-
ant. That is why melanoma cell lines were selected
for this study.
Some traditional methods for melanoma treat-
ment were proved to be inefficient, which promoted
investigation of combination approaches for treatment
of this disease [106]. Therapy based on depletion of
amino acids with the help of enzyme cleaving amino
acids could be potentially used for treatment of vari-
ous types of tumors [108], including melanomas. This
effect on melanoma has been demonstrated for such
enzymes as arginine deiminase and arginase [109].
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1737
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Several systems for drug delivery to the cells with
minimal side effects have been developed. One of the
types of supports for these drug delivery systems are
hydrogels based on natural and synthetic polymers
[110]. Transdermal drug delivery has several advan-
tages in comparison with injection of proteins into
systemic blood flow– these include non-invasiveness,
prevention of first-pass metabolism, prolonged and
controlled action, decrease of the frequency of drug
administration, improved bioavailability, simplicity
of administration by the patient [111, 112]. Delivery
of EwA with the help of BC could allow increasing
EwA concentration at the tumor site, and also could
be an alternative treatment for the patients, who can-
not be treated surgically or have contraindication for
systemic chemotherapy. However, this approach has
a number of limitations. Epidermis acts as a barrier
for large molecules, including proteins [113]. Another
limitation is the fact that L-ASP immobilized on BC
films could be applied only for treatment of surface
tumors. Such tumor cells could become resistant to
L-ASP due to the high expression asparagine synthe-
tase, which would require therapy with higher doses
of L-ASP [114]. Determination of the level of asparag-
ine synthetase expression [109] in the cells prior to
therapy could help to determine potential sensitivity
of the cells to this enzyme.
Hence, localized continuous release of EwA L-ASP
could be potentially beneficial for treatment of solid
tumors providing prolonged local cytotoxic effect on
melanoma tumor cells with decreased frequency of
the drug administration.
CONCLUSIONS
Commonly known mechanism of L-ASP cytotox-
icity is asparagine hydrolysis. However, alternative
mechanisms such as suppression of telomerase [115-
117], release of the 2-HS glycoprotein fetuin [118], and
degradation of concovaline receptors [119] could also
be involved in the development of cytotoxic effects.
Wide spectrum of the known producers of various
forms of L-ASP with different individual character-
istics, primarily thermophilic organisms [120-122], as
well approaches to generation of recombinant ana-
logues of this enzyme make in possible to produce
biofilms with pre-set characteristics of their biological
actions [6]. New BC–chitosan composites were success-
fully produced by mixing chitosan suspension with BC
membranes followed by crosslinking with the help of
glutaraldehyde. In comparison with the non-modified
films, the BC–chitosan composites have higher poros-
ity, water holding capacity, and smaller average pore
size. L-ASP from Erw. carotovora immobilized on the
BC–chitosan composites exhibited 3-fold longer release
and maintained its cytotoxic properties against mel-
anoma cells, but did not affect conditionally normal
fibroblasts (even after three consecutive transfers),
while toxicity of the films with L-ASP immobilized on
BC was observed only in the course of first incubation
with the cells.
Acknowledgments. The authors are grateful to
Viktoriya Olegovna Shipunova (Moscow Institute of
Physics and Technology) for help in acquiring SEM
images and to Andrei Fedorovich Kozlov (Orekhovich
Research Institute of Biomedical Chemistry) for help
in lyophilization of films. Equipment purchased with
the support of the Program of Lomonosov Moscow Uni-
versity Development (Fourier transform IR-microscope
MIKRAN-3) was used in the study.
Contributions. A.N.S. concept of the study, de-
sign and conducting experiments on modification of
the films and analysis of cytotoxicity, writing draft of
the paper; E.R.P. and S.A.K. conducting experiments
on investigation of the enzyme release; E.N.K. and
O.A.K. obtaining SEM images; N.V.D., I.D.Z., and E.V.K.
experiments with IR spectroscopy; D.D.Z. concept of
the study, general supervision of the work, attracting
funding, editing of the final version of the paper.
Funding. This work was financially supported by
the Program of Fundamental Scientific Studies in the
Russian Federation for the prolonged period (2021-
2030) (project no.122022800499-5).
Ethics declarations. This work does not contain
any studies involving human and animal subjects.
Theauthors of this work declare that they have nocon-
flicts of interest.
REFERENCES
1. Pokrovskaya, M. V., Pokrovsky, V. S., Aleksandrova,
S.S., Sokolov, N.N., and Zhdanov, D.D. (2022) Molecu-
lar analysis of L-asparaginases for clarification of the
mechanism of action and optimization of pharmaco-
logical functions, Pharmaceutics, 14, 599, https://doi.
org/10.3390/pharmaceutics14030599.
2. Castro,D., Marques, A.S.C., Almeida, M.R., de Paiva,
G.B., Bento, H.B.S., Pedrolli, D.B., Freire, M.G., Ta-
vares, A.P.M., and Santos-Ebinuma, V.C. (2021) L-as-
paraginase production review: bioprocess design and
biochemical characteristics, Appl. Microbiol. Biotech-
nol., 105, 4515-4534, https://doi.org/10.1007/s00253-
021-11359-y.
3. Chand, S., Mahajan, R. V., Prasad, J. P., Sahoo, D.K.,
Mihooliya, K.N., Dhar, M.S., and Sharma,G. (2020)
A comprehensive review on microbial L-asparagi-
nase: bioprocessing, characterization, and industrial
applications, Biotechnol. Appl. Biochem., 67, 619-647,
https://doi.org/10.1002/bab.1888.
SHISHPARENOK et al.1738
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
4. Suresh, S. A., Ethiraj, S., and Rajnish, K. N. (2022)
Asystematic review of recent trends in research on
therapeutically significant l-asparaginase and acute
lymphoblastic leukemia, Mol. Biol. Rep., 49, 11281-
11287, https://doi.org/10.1007/s11033-022-07688-4.
5. Jia,R., Wan,X., Geng,X., Xue,D., Xie,Z., and Chen,C.
(2021) Microbial L-asparaginase for application in
acrylamide mitigation from food: current research
status and future perspectives, Microorganisms, 9,
1659, https://doi.org/10.3390/microorganisms9081659.
6. Shishparenok, A. N., Gladilina, Y. A., and Zhdanov,
D. D. (2023) Engineering and expression strategies
for optimization of L-asparaginase development and
production, Int. J. Mol. Sci., 24, 15220, https://doi.
org/10.3390/ijms242015220.
7. Chan, W.K., Horvath, T.D., Tan,L., Link,T., Harutyu-
nyan, K.G., Pontikos, M.A., Anishkin,A., Du,D., Mar-
tin, L. A., Yin, E., Rempe, S. B., Sukharev, S., Kono-
pleva, M., Weinstein, J. N., and Lorenzi, P.L. (2019)
Glutaminase activity of L-Asparaginase contributes to
durable preclinical activity against acute lymphoblas-
tic leukemia, Mol. Cancer Ther., 18, 1587-1592, https://
doi.org/10.1158/1535-7163.MCT-18-1329.
8. Tsegaye,K., Tsehai, B. A., and Getie, B. (2024) Desir-
able L-asparaginases for treating cancer and current
research trends, Front. Microbiol., 15, 1269282, https://
doi.org/10.3389/fmicb.2024.1269282.
9. Liu,W., Wang,H., Wang,W., Zhu,M., Liu,C., Wang,J.,
and Lu, Y. (2016) Use of PEG-asparaginase in newly
diagnosed adults with standard-risk acute lympho-
blastic leukemia compared with E.coli-asparaginase:
a retrospective single-center study, Sci. Rep., 6, 39463,
https://doi.org/10.1038/srep39463.
10. Mazloum-Ravasan, S., Madadi, E., Mohammadi, A.,
Mansoori, B., Amini, M., Mokhtarzadeh, A., Barada-
ran, B., and Darvishi, F. (2021) Yarrowia lipolytica
L-asparaginase inhibits the growth and migration
of lung (A549) and breast (MCF7) cancer cells, Int. J.
Biol. Macromol., 170, 406-414, https://doi.org/10.1016/
j.ijbiomac.2020.12.141.
11. Van Trimpont, M., Peeters, E., De Visser, Y., Schalk,
A. M., Mondelaers, V., De Moerloose, B., Lavie, A.,
Lammens, T., Goossens, S., and Van Vlierberghe, P.
(2022) Novel insights on the use of L-asparaginase as
an efficient and safe anti-cancer therapy, Cancers (Ba-
sel), 14, 902, https://doi.org/10.3390/cancers14040902.
12. Talluri, V.P., Mutaliyeva,B., Sharipova,A., Ulagana-
than,V., Lanka, S.S., Aidarova, S., Suigenbayeva,A.,
and Tleuova, A. (2023) L-Asparaginase delivery sys-
tems targeted to minimize its side-effects, Adv. Col-
loid Interface Sci., 316, 102915, https://doi.org/10.1016/
j.cis.2023.102915.
13. Bahreini, E., Aghaiypour, K., Abbasalipourkabir, R.,
Mokarram, A. R., Goodarzi, M. T., and Saidijam, M.
(2014) Preparation and nanoencapsulation of l-aspar-
aginaseII in chitosan-tripolyphosphate nanoparticles
and invitro release study, Nanoscale Res. Lett., 9, 340,
https://doi.org/10.1186/1556-276X-9-340.
14. Aghaeepoor, M., Akbarzadeh, A., Mirzaie, S., Had-
ian,A., Jamshidi Aval,S., and Dehnavi,E. (2018) Se-
lective reduction in glutaminase activity of l-Aspara-
ginase by asparagine248 to serine mutation: a com-
bined computational and experimental effort in blood
cancer treatment, Int. J. Biol. Macromol., 120, 2448-
2457, https://doi.org/10.1016/j.ijbiomac.2018.09.015.
15. Chan, W. K., Lorenzi, P.L., Anishkin,A., Purwaha,P.,
Rogers, D.M., Sukharev,S., Rempe, S. B., and Wein-
stein, J.N. (2014) Theglutaminase activity of l-aspar-
aginase is not required for anticancer activity against
ASNS-negative cells, Blood, 123, 3596-3606, https://
doi.org/10.1182/blood-2013-10-535112.
16. Rizzari,C., Möricke,A., Valsecchi, M.G., Conter,V., Zim-
mermann,M., Silvestri,D., Attarbaschi,A., Niggli, F.,
Barbaric, D., Stary, J., Elitzur, S., Cario, G., Vinti, L.,
Boos, J., Zucchetti, M., Lanvers-Kaminsky, C., von
Stackelberg, A., Biondi, A., and Schrappe, M. (2023)
Incidence and characteristics of hypersensitivity reac-
tions to PEG-asparaginase observed in 6136children
with acute lymphoblastic leukemia enrolled in the
AIEOP-BFM ALL 2009 study protocol, HemaSphere, 7,
e893, https://doi.org/10.1097/HS9.0000000000000893.
17. Figueiredo, L., Cole, P. D., and Drachtman, R. A.
(2016) Asparaginase Erwinia chrysanthemi as a com-
ponent of a multi-agent chemotherapeutic regimen
for the treatment of patients with acute lympho-
blastic leukemia who have developed hypersensi-
tivity to E. coli-derived asparaginase, Expert Rev. He-
matol., 9, 227-234, https://doi.org/10.1586/17474086.
2016.1142370.
18. Ko, R.H., Jones, T.L., Radvinsky,D., Robison,N., Gay-
non, P. S., Panosyan, E. H., Avramis, I. A., Avramis,
V.I., Rubin,J., Ettinger, L.J., Seibel, N.L., and Dhall,G.
(2015) Allergic reactions and antiasparaginase anti-
bodies in children with high‐risk acute lymphoblastic
leukemia: a children’s oncology group report, Cancer,
121, 4205-4211, https://doi.org/10.1002/cncr.29641.
19. Papageorgiou, A. C., Posypanova, G. A., Andersson,
C.S., Sokolov, N.N., and Krasotkina,J. (2008) Struc-
tural and functional insights into Erwinia carotovora
L-asparaginase, FEBSJ., 275, 4306-4316, https://doi.org/
10.1111/j.1742-4658.2008.06574.x.
20. Faret, M., de Morais, S. B., Zanchin, N. I. T., and de
Souza,T. (2019) l-Asparaginase from Erwinia carotov-
ora: insights about its stability and activity, Mol. Biol.
Rep., 46, 1313-1316, https://doi.org/10.1007/s11033-018-
4459-2.
21. Singh, M., Hassan, N., Verma, D., Thakur, P., Panda,
B.P., Panda, A.K., Sharma, R.K., Mirza,A., Mansoor,S.,
Alrokayan, S.H., Khan, H.A., Ahmad,P., and Iqbal,Z.
(2020) Design of expert guided investigation of native
L-asparaginase encapsulated long-acting cross-linker-
free poly (lactic-co-glycolic) acid nanoformulation in
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1739
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
an Ehrlich ascites tumor model, Saudi Pharm. J., 28,
719-728, https://doi.org/10.1016/j.jsps.2020.04.014.
22. Melik-Nubarov, N.S., Grozdova, I.D., Lomakina, G.Y.,
Pokrovskaya, M. V., Pokrovski, V. S., Aleksandrova,
S.S., Abakumova, O.Y., Podobed, O.V., Grishin, D.V.,
and Sokolov, N. N. (2017) PEGylated recombinant
L-asparaginase from Erwinia carotovora: produc-
tion, properties, and potential applications, Appl. Bio-
chem. Microbiol., 53, 165-172, https://doi.org/10.1134/
S0003683817020119.
23. Fonseca, M.H.G., da Silva Fiúza,T., de Morais, S.B.,
Souza,T., and de Trevizani,R. (2021) Circumventing
the side effects of L-asparaginase, Biomed. Pharma-
cother., 139, 111616, https://doi.org/10.1016/j.biopha.
2021.111616.
24. Meneguetti, G.P., Santos, J.H.P.M., Obreque, K.M.T.,
Barbosa, C.M.V., Monteiro,G., Farsky, S.H.P., Marim
de Oliveira,A., Angeli, C. B., Palmisano, G., Ventura,
S. P. M., Pessoa-Junior, A., and de Oliveira Rangel-
Yagui,C. (2019) Novel site-specific PEGylated L-aspara-
ginase, PLoS One, 14, e0211951, https://doi.org/10.1371/
journal.pone.0211951.
25. Feenstra, L. R., Gehring, R., van Geijlswijk, I. M.,
König,T., Prinsen, H.C.M.T., Vandemeulebroecke,K.,
Lammens, T., Krupa, A., and Teske, E. (2022) Eval-
uation of PEG-L-asparaginase in asparagine sup-
pression and anti-drug antibody development in
healthy Beagle dogs: a multi-phase preclinical study,
Vet. J., 286, 105854, https://doi.org/10.1016/j.tvjl.
2022.105854.
26. Mohamad, N. R., Marzuki, N. H. C., Buang, N. A.,
Huyop, F., and Wahab, R. A. (2015) An overview of
technologies for immobilization of enzymes and
surface analysis techniques for immobilized en-
zymes, Biotechnol. Biotechnol. Equip., 29, 205-220,
https://doi.org/10.1080/13102818.2015.1008192.
27. Zhang, D.-H., Yuwen, L.-X., and Peng, L.-J. (2013)
Parameters affecting the performance of immobi-
lized enzyme, J.Chem., 2013, 946248, https://doi.org/
10.1155/2013/946248.
28. Ulu, A., and Ates, B. (2017) Immobilization of L-as-
paraginase on carrier materials: a comprehensive
review, Bioconjug. Chem., 28, 1598-1610, https://
doi.org/10.1021/acs.bioconjchem.7b00217.
29. Dobryakova, N.V., Zhdanov, D.D., Sokolov, N.N., Alek-
sandrova, S.S., Pokrovskaya, M.V., and Kudryashova,
E. V. (2023) Rhodospirillum rubrum L-asparaginase
conjugates with polyamines of improved biocatalyt-
ic properties as a new promising drug for the treat-
ment of leukemia, Appl. Sci., 13, 3373, https://doi.org/
10.3390/app13053373.
30. Qian,G., Zhou,J., Ma,J., He, B., and Wang,D. (1997)
Chemical modification of L-asparaginase with N,
O-carboxymethyl chitosan and its effects on plasma
half-life and other properties, Sci. China Ser. B Chem.,
40, 337-341, https://doi.org/10.1007/BF02877748.
31. Dobryakova, N.V., Zhdanov, D.D., Sokolov, N.N., Alek-
sandrova, S.S., Pokrovskaya, M.V., and Kudryashova,
E. V. (2022) Improvement of biocatalytic properties
and cytotoxic activity of L-asparaginase from Rhodo-
spirillum rubrum by conjugation with chitosan-based
cationic polyelectrolytes, Pharmaceuticals, 15, 406,
https://doi.org/10.3390/ph15040406.
32. Imam, H.T., Marr, P.C., and Marr, A.C. (2021) Enzyme
entrapment, biocatalyst immobilization without cova-
lent attachment, Green Chem., 23, 4980-5005, https://
doi.org/10.1039/D1GC01852C.
33. Zalewska-Szewczyk,B., Gach,A., Wyka,K., Bodalski,J.,
and Młynarski,W. (2009) Thecross-reactivity of an-
ti-asparaginase antibodies against different l-asparagi-
nase preparations, Clin. Exp. Med., 9, 113-116, https://
doi.org/10.1007/s10238-008-0026-9.
34. Wang,N., Ji,W., Wang,L., Wu,W., Zhang,W., Wu,Q.,
Du,W., Bai,H., Peng,B., Ma,B., and Li,L. (2022) Over-
view of the structure, side effects, and activity assays
of L-asparaginase as a therapy drug of acute lympho-
blastic leukemia, RSC Med. Chem., 13, 117-128, https://
doi.org/10.1039/D1MD00344E.
35. Liang, S. (2023) Advances in drug delivery applica-
tions of modified bacterial cellulose-based materials,
Front. Bioeng. Biotechnol., 11, 1252706, https://doi.org/
10.3389/fbioe.2023.1252706.
36. Shah,N., Ul-Islam,M., Khattak, W. A., and Park, J. K.
(2013) Overview of bacterial cellulose composites: a
multipurpose advanced material, Carbohydr. Polym.,
98, 1585-1598, https://doi.org/10.1016/j.carbpol.
2013.08.018.
37. Adepu, S., and Khandelwal, M. (2020) Ex-situ mod-
ification of bacterial cellulose for immediate and
sustained drug release with insights into release
mechanism, Carbohydr. Polym., 249, 116816, https://
doi.org/10.1016/j.carbpol.2020.116816.
38. Gregory, D.A., Tripathi,L., Fricker, A.T.R., Asare,E.,
Orlando,I., Raghavendran,V., and Roy,I. (2021) Bacte-
rial cellulose: a smart biomaterial with diverse appli-
cations, Mater. Sci. Eng. R Reports, 145, 100623, https://
doi.org/10.1016/j.mser.2021.100623.
39. Stumpf, T.R., Yang, X., Zhang,J., and Cao, X. (2018)
In situ and ex situ modifications of bacterial cellu-
lose for applications in tissue engineering, Mater.
Sci. Eng. C, 82, 372-383, https://doi.org/10.1016/j.msec.
2016.11.121.
40. Cacicedo, M.L., Islan, G.A., León, I.E., Álvarez, V.A.,
Chourpa, I., Allard-Vannier, E., García-Aranda, N.,
Díaz-Riascos, Z. V., Fernández, Y., Schwartz, S., Jr.,
Abasolo, I., and Castro, G. R. (2018) Bacterial cel-
lulose hydrogel loaded with lipid nanoparticles
for localized cancer treatment, Colloids Surfaces B
Biointerfaces, 170, 596-608, https://doi.org/10.1016/
j.colsurfb.2018.06.056.
41. Autier, L., Clavreul, A., Cacicedo, M. L., Franconi, F.,
Sindji,L., Rousseau,A., Perrot,R., Montero-Menei,C.N.,
SHISHPARENOK et al.1740
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
Castro, G.R., and Menei, P. (2019) Anew glioblasto-
ma cell trap for implantation after surgical resection,
Acta Biomater., 84, 268-279, https://doi.org/10.1016/
j.actbio.2018.11.027.
42. Pandey,A., Singh, M.K., and Singh,A. (2024) Bacterial
cellulose: a smart biomaterial for biomedical applica-
tions, J. Mater. Res., 39, 2-18, https://doi.org/10.1557/
s43578-023-01116-4.
43. Pavaloiu, R.-D., Stoica-Guzun,A., Stroescu,M., Jinga,
S.I., and Dobre,T. (2014) Composite films of poly(vinyl
alcohol)-chitosan-bacterial cellulose for drug con-
trolled release, Int. J. Biol. Macromol., 68, 117-124,
https://doi.org/10.1016/j.ijbiomac.2014.04.040.
44. Mohamed, M. H., Udoetok, I. A., Wilson, L. D., and
Headley, J. V. (2015) Fractionation of carboxylate
anions from aqueous solution using chitosan cross-
linked sorbent materials, RSC Adv., 5, 82065-82077,
https://doi.org/10.1039/C5RA13981C.
45. Udoetok, I. A., Dimmick, R. M., Wilson, L. D., and
Headley, J. V. (2016) Adsorption properties of cross-
linked cellulose-epichlorohydrin polymers in aque-
ous solution, Carbohydr. Polym., 136, 329-340, https://
doi.org/10.1016/j.carbpol.2015.09.032.
46. Mohamed, M. H., and Wilson, L. D. (2016) Seques-
tration of agrochemicals from aqueous media us-
ing cross-linked chitosan-based sorbents, Adsorp-
tion, 22, 1025-1034, https://doi.org/10.1007/s10450-
016-9796-7.
47. Wilson, L.D., and Xue,C. (2013) Macromolecular sor-
bent materials for urea capture, J.Appl. Polym. Sci.,
128, 667-675, https://doi.org/10.1002/app.38247.
48. Poon, L., Wilson, L. D., and Headley, J.V. (2014) Chi-
tosan-glutaraldehyde copolymers and their sorption
properties, Carbohydr. Polym., 109, 92-101, https://
doi.org/10.1016/j.carbpol.2014.02.086.
49. Udoetok, I.A., Wilson, L.D., and Headley, J.V. (2016)
Self-assembled and cross-linked animal and plant-
based polysaccharides: chitosan-cellulose composites
and their anion uptake properties, ACS Appl. Mater.
Interfaces, 8, 33197-33209, https://doi.org/10.1021/
acsami.6b11504.
50. Udoetok, I.A., Wilson, L.D., and Headley, J.V. (2018)
“Pillaring Effects” in cross-linked cellulose biopoly-
mers: a study of structure and properties, Int. J.
Polym. Sci., 2018, 6358254, https://doi.org/10.1155/
2018/6358254.
51. Kim, H.J., Jin, J.N., Kan,E., Kim, K.J., and Lee, S.H.
(2017) Bacterial cellulose-chitosan composite hydrogel
beads for enzyme immobilization, Biotechnol. Biopro-
cess Eng., 22, 89-94, https://doi.org/10.1007/s12257-
016-0381-4.
52. Ribeiro-Viana, R. M., Faria-Tischer, P. C. S., and
Tischer, C.A. (2016) Preparation of succinylated cel-
lulose membranes for functionalization purposes,
Carbohydr. Polym., 148, 21-28, https://doi.org/10.1016/
j.carbpol.2016.04.033.
53. Wacker, M., Riedel, J., Walles, H., Scherner, M.,
Awad, G., Varghese, S., Schürlein, S., Garke, B., Ve-
luswamy,P., Wippermann,J., and Hülsmann,J. (2021)
Comparative evaluation on impacts of fibronectin,
heparin-chitosan, and albumin coating of bacterial
nanocellulose small-diameter vascular grafts on endo-
thelialization invitro, Nanomaterials, 11, 1952, https://
doi.org/10.3390/nano11081952.
54. Jantarat, C., Attakitmongkol, K., Nichsapa, S., Sir-
athanarun,P., and Srivaro, S. (2020) Molecularly im-
printed bacterial cellulose for sustained-release deliv-
ery of quercetin, J.Biomater. Sci. Polym. Ed., 31, 1961-
1976, https://doi.org/10.1080/09205063.2020.1787602.
55. Arikibe, J. E., Lata, R., Kuboyama, K., Ougizawa, T.,
and Rohindra, D. (2019) pH-responsive studies of
bacterial cellulose/chitosan hydrogels crosslinked
with genipin: swelling and drug release behaviour,
ChemistrySelect, 4, 9915-9926, https://doi.org/10.1002/
slct.201902290.
56. Mohammadi,S., Jabbari,F., and Babaeipour,V. (2023)
Bacterial cellulose-based composites as vehicles for
dermal and transdermal drug delivery: a review, Int.J.
Biol. Macromol., 242, 124955, https://doi.org/10.1016/
j.ijbiomac.2023.124955.
57. Pasaribu, K. M., Ilyas, S., Tamrin, T., Radecka, I.,
Swingler,S., Gupta,A., Stamboulis, A.G., and Gea,S.
(2023) Bioactive bacterial cellulose wound dressings
for burns with collagen in-situ and chitosan ex-situ
impregnation, Int. J. Biol. Macromol., 230, 123118,
https://doi.org/10.1016/j.ijbiomac.2022.123118.
58. Abu Hasan, N. S., Mohamad, S., Sy Mohamad, S. F.,
Arzmi, M.H., and Supian, N. N.I. (2023) Ex-situ de-
velopment and characterization of composite film
based on bacterial cellulose derived from oil palm
frond juice and chitosan as food packaging, Pertanika
J. Sci. Technol., 31, 1173-1187, https://doi.org/10.47836/
pjst.31.3.03.
59. Kitaoka, K., Yamamoto, H., Tani, T., Hoshijima, K.,
and Nakauchi, M. (1997) Mechanical strength and
bone bonding of a titanium fiber mesh block for in-
tervertebral fusion, J. Orthop. Sci., 2, 106-113, https://
doi.org/10.1007/BF02489521.
60. Wu,S., Wu,S., and Su,F. (2017) Novel process for im-
mobilizing an enzyme on a bacterial cellulose mem-
brane through repeated absorption, J. Chem. Tech-
nol. Biotechnol., 92, 109-114, https://doi.org/10.1002/
jctb.4994.
61. Isobe, N., Lee, D.-S., Kwon, Y.-J., Kimura,S., Kuga,S.,
Wada,M., and Kim, U.-J. (2011) Immobilization of pro-
tein on cellulose hydrogel, Cellulose, 18, 1251-1256,
https://doi.org/10.1007/s10570-011-9561-8.
62. Krasotkina, J., Borisova, A. A., Gervaziev, Y. V., and
Sokolov, N.N. (2004) One‐step purification and kinet-
ic properties of the recombinant l‐asparaginase from
Erwinia carotovora, Biotechnol. Appl. Biochem., 39,
215-221, https://doi.org/10.1042/BA20030138.
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1741
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
63. Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones,
K. M. (1989) Data for Biochemical Research, 3rd ed.
Oxford, Clarendon Press.
64. Meister, A. (1955) Glutaminase, asparaginase, and
α-keto acid-ω-amidase, Methods Enzym., 2, 380-385,
https://doi.org/10.1016/S0076-6879(55)02216-7.
65. Cooney, D. A., Capizzi, R. L., and Handschumacher,
R.E. (1970) Evaluation of L-asparagine metabolism in
animals and man, Cancer Res., 30, 925-935.
66. Dave, P. N., Macwan, P. M., and Kamaliya, B. (2023)
Biodegradable Gg-cl-poly(NIPAm-co-AA)/-o-MWCNT
based hydrogel for combined drug delivery system
of metformin and sodium diclofenac: in vitro stud-
ies, RSC Adv., 13, 22875-22885, https://doi.org/10.1039/
D3RA04728H.
67. Looi, C. Y., Moharram, B., Paydar, M., Wong, Y. L.,
Leong, K.H., Mohamad,K., Arya,A., Wong, W.F., and
Mustafa, M.R. (2013) Induction of apoptosis in mela-
noma A375 cells by a chloroform fraction of Centrath-
erum anthelminticum(L.) seeds involves NF-kappaB,
p53 and Bcl-2-controlled mitochondrial signaling
pathways, BMC Complement. Altern. Med., 13, 166,
https://doi.org/10.1186/1472-6882-13-166.
68. Denizot, F., and Lang, R. (1986) Rapid colorimet-
ric assay for cell growth and survival, J. Immunol.
Methods, 89, 271-277, https://doi.org/10.1016/0022-
1759(86)90368-6.
69. Horue, M., Silva, J. M., Berti, I.R., Brandão, L.R., da
Silva Barud,H., and Castro, G.R. (2023) Bacterial cel-
lulose-based materials as dressings for wound heal-
ing, Pharmaceutics, 15, 424, https://doi.org/10.3390/
pharmaceutics15020424.
70. Ul-Islam, M., Khan, T., and Park, J. K. (2012) Water
holding and release properties of bacterial cellulose
obtained by in situ and ex situ modification, Car-
bohydr. Polym., 88, 596-603, https://doi.org/10.1016/
j.carbpol.2012.01.006.
71. Li,G., Nandgaonkar, A. G., Habibi, Y., Krause, W. E.,
Wei,Q., and Lucia, L.A. (2017) An environmentally
benign approach to achieving vectorial alignment
and high microporosity in bacterial cellulose/chi-
tosan scaffolds, RSC Adv., 7, 13678-13688, https://
doi.org/10.1039/C6RA26049G.
72. Li,Y., Jiang,H., Zheng,W., Gong,N., Chen,L., Jiang,X.,
and Yang, G. (2015) Bacterial cellulose-hyaluronan
nanocomposite biomaterials as wound dressings for
severe skin injury repair, J. Mater. Chem. B, 3, 3498-
3507, https://doi.org/10.1039/C4TB01819B.
73. Ul-Islam,M., Shah,N., Ha, J.H., and Park, J.K. (2011)
Effect of chitosan penetration on physico-chemical
and mechanical properties of bacterial cellulose, Kore-
an J. Chem. Eng., 28, 1736-1743, https://doi.org/10.1007/
s11814-011-0042-4.
74. Ojagh, S.M.A., Vahabzadeh,F., and Karimi,A. (2021)
Synthesis and characterization of bacterial cellu-
lose-based composites for drug delivery, Carbohydr.
Polym., 273, 118587, https://doi.org/10.1016/j.carbpol.
2021.118587.
75. Bayer, I.S. (2023) Controlled drug release from nano-
engineered polysaccharides, Pharmaceutics, 15, 1364,
https://doi.org/10.3390/pharmaceutics15051364.
76. Zhu,W., Long,J., and Shi,M. (2023) Release kinetics
model fitting of drugs with different structures from
viscose fabric, Materials (Basel), 16, 3282, https://
doi.org/10.3390/ma16083282.
77. Fu, Y., and Kao, W. J. (2010) Drug release kinet-
ics and transport mechanisms of non-degradable
and degradable polymeric delivery systems, Ex-
pert Opin. Drug Deliv., 7, 429-444, https://doi.org/
10.1517/17425241003602259.
78. Hu,Y., Catchmark, J. M., Zhu,Y., Abidi, N., Zhou, X.,
Wang, J., and Liang, N. (2014) Engineering of porous
bacterial cellulose toward human fibroblasts in-
growth for tissue engineering, J.Mater. Res., 29, 2682-
2693, https://doi.org/10.1557/jmr.2014.315.
79. Rebelo, A.R., Archer, A. J., Chen,X., Liu, C., Yang,G.,
and Liu,Y. (2018) Dehydration of bacterial cellulose
and the water content effects on its viscoelastic and
electrochemical properties, Sci. Technol. Adv. Mater.,
19, 203-211, https://doi.org/10.1080/14686996.2018.
1430981.
80. Numata,Y., Kono,H., Mori,A., Kishimoto,R., and Ta-
jima,K. (2019) Structural and rheological characteri-
zation of bacterial cellulose gels obtained from Glu-
conacetobacter genus, Food Hydrocoll., 92, 233-239,
https://doi.org/10.1016/j.foodhyd.2019.01.060.
81. Munim, S.A., Saddique, M.T., Raza, Z.A., and Majeed,
M.I. (2020) Fabrication of cellulose-mediated chitosan
adsorbent beads and their surface chemical charac-
terization, Polym. Bull., 77, 183-196, https://doi.org/
10.1007/s00289-019-02711-4.
82. Urbina, L., Guaresti, O., Requies, J., Gabilondo, N.,
Eceiza,A., Corcuera, M.A., and Retegi, A. (2018) De-
sign of reusable novel membranes based on bacte-
rial cellulose and chitosan for the filtration of cop-
per in wastewaters, Carbohydr. Polym., 193, 362-372,
https://doi.org/10.1016/j.carbpol.2018.04.007.
83. Kalyani, P., and Khandelwal, M. (2021) Modulation
of morphology, water uptake/retention, and rheolog-
ical properties by in situ modification of bacterial
cellulose with the addition of biopolymers, Cellulose,
28, 11025-11036, https://doi.org/10.1007/s10570-021-
04256-0.
84. Maity,M., Pramanik,U., Hathwar, V.R., Brandao,P.,
Mukherjee,S., Maity,S., Maity,R., Maity,T., and Chan-
dra Samanta,B. (2022) Biophysical insights into the
binding capability of Cu(II) schiff base complex with
BSA protein and cytotoxicity studies against SiHa,
Heliyon, 8, e11345, https://doi.org/10.1016/j.heliyon.
2022.e11345.
85. Monteiro, O. A., and Airoldi, C. (1999) Some studies
of crosslinking chitosan-glutaraldehyde interaction in
SHISHPARENOK et al.1742
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
ahomogeneous system, Int.J. Biol. Macromol., 26, 119-
128, https://doi.org/10.1016/S0141-8130(99)00068-9.
86. Vasconcellos, V., and Farinas, C. (2018) The effect
of the drying process on the properties of bacteri-
al cellulose films from Gluconacetobacter hansenii,
Chem. Eng. Trans., 64, 145-150, https://doi.org/10.3303/
CET1864025.
87. Ciecholewska-Juśko, D., Żywicka, A., Junka, A.,
Drozd, R., Sobolewski, P., Migdał, P., Kowalska, U.,
Toporkiewicz,M., and Fijałkowski,K. (2021) Superab-
sorbent crosslinked bacterial cellulose biomaterials
for chronic wound dressings, Carbohydr. Polym., 253,
117247, https://doi.org/10.1016/j.carbpol.2020.117247.
88. Ciecholewska-Juśko,D., Junka,A., and Fijałkowski,K.
(2022) Thecross-linked bacterial cellulose impregnat-
ed with octenidine dihydrochloride-based antiseptic
as an antibacterial dressing material for highly-ex-
uding, infected wounds, Microbiol. Res., 263, 127125,
https://doi.org/10.1016/j.micres.2022.127125.
89. Wahid, F., Hu, X.-H., Chu, L.-Q., Jia, S.-R., Xie, Y.-Y.,
and Zhong,C. (2019) Development of bacterial cellu-
lose/chitosan based semi-interpenetrating hydrogels
with improved mechanical and antibacterial prop-
erties, Int. J. Biol. Macromol., 122, 380-387, https://
doi.org/10.1016/j.ijbiomac.2018.10.105.
90. Li,J., and Mooney, D.J. (2016) Designing hydrogels for
controlled drug delivery, Nat. Rev. Mater., 1, 16071,
https://doi.org/10.1038/natrevmats.2016.71.
91. Kim, J., Cai, Z., Lee, H. S., Choi, G.S., Lee, D.H., and
Jo, C. (2011) Preparation and characterization of a
Bacterial cellulose/Chitosan composite for potential
biomedical application, J. Polym. Res., 18, 739-744,
https://doi.org/10.1007/s10965-010-9470-9.
92. Lin, W.-C., Lien, C.-C., Yeh, H.-J., Yu, C.-M., and Hsu,S.
(2013) Bacterial cellulose and bacterial cellulose-chi-
tosan membranes for wound dressing applica-
tions, Carbohydr. Polym., 94, 603-611, https://doi.org/
10.1016/j.carbpol.2013.01.076.
93. Shishparenok, A. N., Koroleva, S. A., Dobryakova,
N.V., Gladilina, Y.A., Gromovykh, T.I., Solopov, A.B.,
Kudryashova, E.V., and Zhdanov, D.D. (2024) Bacteri-
al cellulose films for L-asparaginase delivery to mela-
noma cells, Int.J. Biol. Macromol., 276, 133932, https://
doi.org/10.1016/j.ijbiomac.2024.133932.
94. Paul, D.R. (2011) Elaborations on the Higuchi mod-
el for drug delivery, Int.J. Pharm., 418, 13-17, https://
doi.org/10.1016/j.ijpharm.2010.10.037.
95. Siepmann, J., and Peppas, N. A. (2011) Higuchi
equation: derivation, applications, use and mis-
use, Int. J. Pharm., 418, 6-12, https://doi.org/10.1016/
j.ijpharm.2011.03.051.
96. Danyuo, Y., Ani, C. J., Salifu, A. A., Obayemi, J. D.,
Dozie-Nwachukwu,S., Obanawu, V.O., Akpan, U.M.,
Odusanya, O. S., Abade-Abugre,M., McBagonluri, F.,
and Soboyejo, W.O. (2019) Anomalous release kinet-
ics of prodigiosin from poly-N-isopropyl-acrylamid
based hydrogels for the treatment of triple negative
breast cancer, Sci. Rep., 9, 3862, https://doi.org/10.1038/
s41598-019-39578-4.
97. Drozd,R., Szymańska,M., Przygrodzka, K., Hoppe, J.,
Leniec,G., and Kowalska,U. (2021) Thesimple method
of preparation of highly carboxylated bacterial cellu-
lose with Ni- and Mg-ferrite-based versatile magnetic
carrier for enzyme immobilization, Int.J. Mol. Sci., 22,
8563, https://doi.org/10.3390/ijms22168563.
98. Ciolacu, D. E., Nicu, R., and Ciolacu, F. (2020) Cel-
lulose-based hydrogels as sustained drug-delivery
systems, Materials (Basel)., 13, 5270, https://doi.org/
10.3390/ma13225270.
99. Kislyak, I. A., Pokrovskaya, M. V., Zhanturina, D.Y.,
and Pokrovsky, V.S. (2023) Theuse of L-asparaginase
for the treatment of solid tumors: data from experi-
mental studies and clinical trials, Russ. J. Oncol., 28,
79-94, https://doi.org/10.17816/onco562802.
100. Taylor, C. W., Dorr, R.T., Fanta, P., Hersh, E.M., and
Salmon, S.E. (2001) AphaseI and pharmacodynam-
ic evaluation of polyethylene glycol-conjugated L-as-
paraginase in patients with advanced solid tumors,
Cancer Chemother. Pharmacol., 47, 83-88, https://
doi.org/10.1007/s002800000207.
101. Nerkar, D.P., and Gangadharan,M. (1989) Modifica-
tion of L-asparaginase from Erwinia carotovora using
human serum albumin, Mol. Biother., 1, 152-154.
102. Sukhoverkov, K. V., and Kudryashova, E. V. (2015)
PEG-chitosan and glycol-chitosan for improvement
of biopharmaceutical properties of recombinant
L-asparaginase from Erwinia carotovora, Biochem-
istry (Moscow), 80, 113-119, https://doi.org/10.1134/
S0006297915010137.
103. Sukhoverkov, K.V., Sokolov, N.N., Abakumova, O.Y.,
Podobed, O.V., and Kudryashova, E.V. (2016) Thefor-
mation of conjugates with PEG-chitosan improves
the biocatalytic efficiency and antitumor activity of
L-asparaginase from Erwinia carotovora, Moscow
Univ. Chem. Bull., 71, 122-126, https://doi.org/10.3103/
S0027131416020073.
104. Rossi, L., Pierigè, F., Aliano, M. P., and Magnani, M.
(2020) Ongoing developments and clinical progress in
drug-loaded red blood cell technologies, BioDrugs, 34,
265-272, https://doi.org/10.1007/s40259-020-00415-0.
105. Burke, M.J., and Zalewska-Szewczyk,B. (2022) Hyper-
sensitivity reactions to asparaginase therapy in acute
lymphoblastic leukemia: immunology and Clinical
consequences, Futur. Oncol., 18, 1285-1299, https://
doi.org/10.2217/fon-2021-1288.
106. Villani, A., Potestio, L., Fabbrocini, G., Troncone, G.,
Malapelle,U., and Scalvenzi,M. (2022) Thetreatment
of advanced melanoma: therapeutic update, Int.J. Mol.
Sci., 23, 6388, https://doi.org/10.3390/ijms23126388.
107. Klinac, D., Gray, E. S., Millward, M., and Ziman, M.
(2013) Advances in personalized targeted treatment
of metastatic melanoma and non-invasive tumor
BC-BASED COMPOSITE FOR PROLONGED-ACTION CYTOTOXIC L-ASP 1743
BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024
monitoring, Front. Oncol., 3, 54, https://doi.org/10.3389/
fonc.2013.00054.
108. Pokrovsky, V. S., Chepikova, O. E., Davydov, D. Z.,
Zamyatnin, A.A.,Jr., Lukashev, A.N., and Lukasheva,
E.V. (2019) Amino acid degrading enzymes and their
application in cancer therapy, Curr. Med. Chem., 26,
446-464, https://doi.org/10.2174/0929867324666171006
132729.
109. Pokrovsky, V. S., Abo Qoura, L., Morozova, E., and
Bunik, V. I. (2022) Predictive markers for efficien-
cy of the amino-acid deprivation therapies in can-
cer, Front. Med., 9, 1035356, https://doi.org/10.3389/
fmed.2022.1035356.
110. Vishnubhakthula, S., Elupula, R., and Durán-Lara,
E.F. (2017) Recent advances in hydrogel-based drug
delivery for melanoma cancer therapy: a mini re-
view, J. Drug Deliv., 2017, 7275985, https://doi.org/
10.1155/2017/7275985.
111. Rastogi, V., and Yadav, P. (2012) Transdermal drug
delivery system: an overview, AsianJ. Pharm., 6, 161,
https://doi.org/10.4103/0973-8398.104828.
112. Ramadon, D., McCrudden, M. T. C., Courtenay, A. J.,
and Donnelly, R.F. (2022) Enhancement strategies for
transdermal drug delivery systems: current trends
and applications, Drug Deliv. Transl. Res., 12, 758-791,
https://doi.org/10.1007/s13346-021-00909-6.
113. Kalluri,H., and Banga, A.K. (2011) Transdermal de-
livery of proteins, AAPS PharmSciTech, 12, 431-441,
https://doi.org/10.1208/s12249-011-9601-6.
114. Shakambari, G., Sameer Kumar, R., Ashokkumar, B.,
Ganesh,V., Vasantha, V.S., and Varalakshmi,P. (2018)
Cloning and expression of L-asparaginase from Bacil-
lus tequilensis PV9W and therapeutic efficacy of Solid
Lipid Particle formulations against cancer, Sci. Rep., 8,
18013, https://doi.org/10.1038/s41598-018-36161-1.
115. Zhdanov, D.D., Pokrovsky, V.S., Pokrovskaya, M.V.,
Alexandrova, S. S., Eldarov, M. A., Grishin, D. V.,
Basharov, M.M., Gladilina, Y.A., Podobed, O.V., and
Sokolov, N.N. (2017) Rhodospirillum rubrum L-aspar-
aginase targets tumor growth by a dual mechanism
involving telomerase inhibition, Biochem. Biophys.
Res. Commun., 492, 282-288, https://doi.org/10.1016/
j.bbrc.2017.08.078.
116. Zhdanov, D.D., Pokrovsky, V.S., Pokrovskaya, M.V.,
Alexandrova, S. S., Eldarov, M. A., Grishin, D. V.,
Basharov, M.M., Gladilina, Y.A., Podobed, O.V., and
Sokolov, N.N. (2017) Inhibition of telomerase activity
and induction of apoptosis by Rhodospirillum ru-
brum L-asparaginase in cancer Jurkat cell line and
normal human CD4
+
Tlymphocytes, Cancer Med., 6,
2697-2712, https://doi.org/10.1002/cam4.1218.
117. Plyasova, A.A., Pokrovskaya, M.V., Lisitsyna, O.M.,
Pokrovsky, V.S., Alexandrova, S.S., Hilal,A., Sokolov,
N.N., and Zhdanov, D.D. (2020) Penetration into can-
cer cells via clathrin-dependent mechanism allows l‐
asparaginase from Rhodospirillum rubrum to inhibit
telomerase, Pharmaceuticals, 13, 286, https://doi.org/
10.3390/ph13100286.
118. Bosmann, H. B., and Kessel, D. (1970) Inhibition of
glycoprotein synthesis in L5178Y mouse lukaemic
cells by L-asparaginase invitro, Nature, 226, 850-851,
https://doi.org/10.1038/226850a0.
119. Ankel, E.G., Zirneski, J., Ring, B. J., and Holcenberg,
J. S. (1984) Effect of asparaginase on cell mem-
branes of sensitive and resistants mouse lymphoma
cells, In Vitro, 20, 376-384, https://doi.org/10.1007/
BF02619582.
120. Dumina, M.V., Zhgun, A. A., Pokrovskay, M.V., Alek-
sandrova, S. S., Zhdanov, D. D., Sokolov, N. N., and
El’darov, M.A. (2021) Comparison of enzymatic activ-
ity of novel recombinant L-asparaginases of extremo-
philes, Appl. Biochem. Microbiol., 57, 594-602, https://
doi.org/10.1134/S0003683821050057.
121. Dumina,M., Zhgun,A., Pokrovskaya,M., Aleksandro-
va,S., Zhdanov,D., Sokolov,N., and El’darov,M. (2021)
A novel L-asparaginase from hyperthermophilic ar-
chaeon Thermococcus sibiricus: heterologous expres-
sion and characterization for biotechnology applica-
tion, Int. J. Mol. Sci., 22, 9894, https://doi.org/10.3390/
ijms22189894.
122. Dumina,M., Zhgun,A., Pokrovskaya,M., Aleksandro-
va,S., Zhdanov,D., Sokolov,N., and El’darov,M. (2021)
Highly active thermophilic L-asparaginase from Me-
lioribacter roseus represents a novel large group
of type II bacterial L-asparaginases from chlorobi-
ignavibacteriae-bacteroidetes clade, Int. J. Mol. Sci.,
22, 13632, https://doi.org/10.3390/ijms222413632.
Publisher’s Note. Pleiades Publishing remains
neutral with regard to jurisdictional claims in published
maps and institutional affiliations. AI tools may have
been used in the translation or editing of this article.
