International Journal of Biological Macromolecules 148 (2020) 218–225

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Chitosan-modified hyaluronic acid-based nanosized drug carriers

Árpád Turcsányi a, Norbert Varga a, Edit Csapó a,b,⁎
a
Interdisciplinary Excellence Centre, Department of Physical Chemistry and Materials Science, University of Szeged, H-6720, Rerrich Béla Square 1, Szeged, Hungary
b
MTA-SZTE Biomimetic Systems Research Group, Department of Medical Chemistry, Faculty of Medicine, University of Szeged, H-6720, Dóm Square 8, Szeged, Hungary

a r t i c l e i n f o a b s t r a c t

Article history: Fabrication possibilities, detailed size and structural characterization of biodegradable chitosan (Chit)
Received 9 December 2019 polysaccharide-modified hyaluronic acid (HyA)-based colloidal carriers are demonstrated. The negatively
Received in revised form 11 January 2020 charged and highly hydrophilic HyA polymer chains have been ionically modified by positively charged pure
Accepted 12 January 2020
Chit and crosslinked Chit macromolecules at various Chit/HyA weight ratios, which resulted in the formation
Available online 15 January 2020
of carrier nanoparticles (NPs) having three different nanostructures depending on the polymer concentrations.
Keywords:
Electrostatically-compensated Chit/HyA polymer coils with loose colloidal structure, tripolyphosphate (TPP)-
Hyaluronic acid crosslinked Chit-TPP/HyA NPs having interpenetrating polymer network and well-defined Chit-TPPcore-HyAshell
Chitosan NPs with diameters of 100–300 nm were also prepared and were loaded with tocopherol (TCP) and cholecalcif-
Nanocarrier erol (D3) having Vitamin E and D activity, respectively. By using rheological, particle charge titration and conduc-
tivity studies we first confirmed that the expected 1:1 Chit/HyA monomer molar ratio is strongly influenced by
the pH of the polymer solutions as well as the deacetylation degree of Chit which are crucial factors for the sol-
ubility, purity and the quality of the commercially available biocompatible Chit in aqueous medium. Encapsula-
tion studies revealed that D3 could be better incorporated in every system, especially in Chit-TPP/HyA NPs, while
for TCP the simple Chit/HyA polymer coils were the most promising carriers.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction acetyl-D-glucosamine monomers with β-(1 → 4) linkages, and these

Natural polysaccharides are promising materials in medical and
pharmaceutical applications thanks to their hydrophilic, non-toxic and
biodegradable properties [1–3]. Some of these biomaterials possess
negatively or positively charged side chains, like hyaluronan (HyA)
and chitosan (Chit), respectively. HyA is a negatively charged polyelec-
trolyte composed of repeating disaccharide units of β-(1 → 4) linked D-
glucuronic acid and β-(1 → 3) linked N-acetyl-D-glucosamine. It has nu-
merous biological functions and it is widely used as a component of dif-
ferent drug carrier systems [4]. HyA has high hydrophilicity and great
swelling properties, which contribute to its huge biological importance,
like maintaining tissue hydration, lubrication in joints, and it also has a
role in inflammation and wound healing [5]. The functional groups of
HyA monomer can be modified to obtain crosslinked, self-assembled
nanosized drug carriers [6,7], or hydrogels for tissue engineering [8].
Thanks to its negatively charged carboxyl groups, HyA can form com-
plexes with positively charged surfactants, proteins and polymers and
these can be efficient in drug loading [9–11]. Chit is a cationic polysac-
charide, where the linear polymer consists of D-glucosamine and N-
⁎ Corresponding author at: MTA-SZTE Biomimetic Systems Research Group,
Department of Medical Chemistry, Faculty of Medicine, University of Szeged, H-6720,
Dóm Square 8, Szeged, Hungary.
E-mail address: juhaszne.csapo.edit@med.u-szeged.hu (E. Csapó).
basic units are randomly distributed in the material. Due to the presence
of amine group(s) the crosslinking [12], chemical modifications [13]
and formation of electrostatical interactions with negatively charged
surfactants, peptides or polyelectrolites is feasible [14]. Chit and its de-
rivatives can also be effectively applied as drug carriers [15] and in
gene delivery because of their biocompatibility and biodegradability,
and their mucosal adhesiveness can be utilized for targeting certain tis-
sues [2]. For these applications, Chit is present in hydrogel, film, coating
and nanoparticle form, and several types of drugs can be encapsulated,
e.g. non-steroidal anti-inflammatory drugs (NSAIDs), proteins, nucleic
acids [16] and cancer therapeutics [17,18].
The complexes of Chit and HyA are extremely important materials,
and they are mainly used in the medical field as gels, scaffolds or
sponges for tissue growth and wound healing [19–21]. These 3D struc-
tures can be fabricated by simple mixing of the polymer solutions. Coat-
ing and film making is also possible with these polysaccharides, where
the layer-by-layer (LbL) approach is mostly used [22,23]. Chit/HyA com-
plex NPs can also be prepared by homogenizing the diluted solutions of
these materials [24–27]. Aside from polymer NPs, the common drug
carriers for medical and pharmaceutical applications include liposomes,
microspheres, polymeric micelles and metal (usually silver or gold) NPs.
Due to the applied preparation methods and reagents, these carriers can
contain several hazardous materials, e.g. organic and halogenated sol-
vents, residues of heavy metal ions etc. In case of Chit/HyA-based

https://doi.org/10.1016/j.ijbiomac.2020.01.118
0141-8130/© 2020 Elsevier B.V. All rights reserved.
 Á. Turcsányi et al. / International Journal of Biological Macromolecules 148 (2020) 218–225
carriers, these additives and contaminations are not present, because
the preparation process can be easily executed in aqueous medium as
well as only mild reagents are needed; furthermore, their biodegrad-
ability is also a desirable property compared to inorganic particles. So
far, the interaction between Chit and HyA polysaccharides has been
partly investigated, but only the effect of pH and ionic strength on the
structure of these biopolimers has been examined [28,29]. In our actual
work, the negatively charged HyA macromolecules have been neutral-
ized with positively charged Chit at various ratios. On one hand, the
main motivation of this work was to determine quantitatively the
charge compensation point and the structural changes due to the inter-
action of the polymers at different ratios and pH values by using several
physico-chemical techniques. Moreover, based on these quantitative
and structural data the formation possibilities of three different
nanosized drug carriers were also investigated. Furthermore, the drug
loading of these carriers was also tested using tocopherol (TCP) and
cholecalcipherol (D3), as model hydrophobic drugs. The blank and
loaded NPs were characterized and the relationship between the struc-
ture and the phobicity of the carriers and the studied model vitamins
and the drug loading has been interpreted.
2. Materials and methods
2.1. Materials
Medium (MMW, 190–310 kDa, degree of deacetylation 75–85%) and
high (HMW, 310–375 kDa, N75% deacetylated) molecular weight Chito-
san (Chit) were purchased from Sigma-Aldrich, Hungary. Hyaluronic
acid sodium salt (HyA, 1.5–1.8 · 106 Da, ≤1% protein), ±-α-tocopherol
(TCP, ≥96%) and cholecalciferol (D3, ≥98%) were purchased from
Sigma-Aldrich, Hungary. Tripolyphosphate (TPP, for BOD determina-
tion) was obtained from Merck KGaA, Germany. Sodium dodecyl sul-
phate (SDS, ≥95%) and sodium hydroxide (NaOH, ≥99.0%) were
purchased from Molar Chemicals, Hungary. Acetic acid (AcOH, ≥99.5%)
was obtained from Erdőkémia Co, Hungary. Highly purified water was
obtained by deionization and filtration with a Millipore purification ap-
paratus (18.2 MΩ·cm at 25 °C). All the applied solvents and reagents
were of analytical grade and no further purifications were carried out.
2.2. Preparation of Chit-modified HyA carrier NPs
2.2.1. Electrostatically compensated polymer coils (Chit/HyA NPs: I)
Type I carriers were prepared by physical mixing of the polymer so-
lutions. The possible formation of colloidal NPs with compositions from
Chit- (80:1) to HyA-excess (1:8) weight ratios was investigated in this
section. Namely, MMW Chit and HyA were dissolved in 2% (v/v) AcOH
solution and in deionized water, respectively, to obtain stock solutions
with c = 2 mg/mL. On one hand, the Chit solution was further diluted
to 0.023–0.364 mg/mL (depending on the mass ratios between
1:8–2:1 Chit:HyA), with final cHyA being 0.042 mg/mL. For the other
part, cChit was kept at 0.364 mg/mL, while cHyA varied from 6.7 μg/mL
to 0.134 mg/mL (4:1–80:1 Chit:HyA mass ratios). The appropriate solu-
tions were mixed under vigorous magnetic stirring (2–3 min). The pH
= 4.40–4.70 was set using NaOH and the final colloidal dispersions
were stored at 4 °C.
2.2.2. TPP-crosslinked Chit/HyA NPs (Chit-TPP/HyA NPs: II)
The Chit-TPP/HyA NPs were fabricated by ionic gelation method.
Briefly, stock solutions were made first (cChit = 2 mg/mL in AcOH
using HMW Chit; cHyA = 0.615 mg/mL). The Chit solution was then di-
luted to 0.727 mg/mL, and HyA stock solution was diluted to 7.7 μg/mL-
0.062 mg/mL. Then 2.365 mL of the HyA solutions was mixed with 365
μL of 2 mg/mL TPP solution (Chit:TPP mass ratio was 2:1). To obtain NPs,
the full amount of HyA-TPP mixture was added in one step to 2 mL of
diluted Chit solution under strong magnetic stirring. The final Chit:
HyA mass ratios varied from 80:1 to 10:1.
219
2.2.3. Chit-TPPcore – HyAshell NPs (III)
First, the core NPs (Chit-TPPcore) were prepared by adding 1.25 mL of
TPP solution (c = 2 mg/mL) to heavily stirred 6.875 mL of HMW Chit so-
lution (cChit = 0.727 mg/mL) in 50 μL portions, waiting 5 s for mixing
before the following addition of TPP. The formed suspension was stirred
for a few minutes, and then stored in 4 °C for 24 h before the next step.
To make the HyA coating, 2 mL Chit-TPPcore suspension was mixed to
2 mL vigorously stirred HyA solution (c = 7.7 μg/mL − 0.062 mg/mL),
where the Chit:HyA mass ratios were varied in the range of 80:1–10:1.
2.3. Preparation of Chit-modified HyA carrier NPs loaded with TCP and D3
The vitamins were dissolved in absolute ethanol (c = 1 mg/mL). For
type I, the polymer stock solutions were diluted before the synthesis of
drug loaded carriers (cHyA = 0.057 mg/mL, cChit = 0.010–0.333 mg/mL).
The TCP or D3 solutions (250–250 μL) were added to the Chit solution
before the mixing. The Chit:HyA mass ratios varied between 1:8–4:1.
For type II NPs, 145–145 μL of the TCP and D3 solutions were added
to the HyA-TPP mixture before preparating the carriers. The Chit:HyA
mass ratios used here were the same as the blank carriers.
For type III NPs, the vitamins were added to the TPP solution before
formulating the core NPs. For TCP-containing particles, the Chit stock
solution was diluted with 875 μL deionized water, and 125 μL TCP solu-
tion was added to the TPP solution. The TPP-TCP mixture was added to
the Chit solution in a single step under heavy stirring. Due to the dilu-
tion of Chit, 2 mL of core NP dispersion was added to 1.780 mL of HyA
solution. For D3 loaded carriers, the Chit stock solution was 5.875 mL
(c = 0.851 mg/mL). The TPP solution was modified with 875 μL water
and 125 μL D3. The TPP-D3 mixture was added in a single step to the
Chit solution. The coating was executed the same way, and the Chit:
HyA mass ratios were the same as for blank NPs.
2.4. Methods for characterization
2.4.1. Charge titration
The charge titration of Chit-HyA system was performed in a Mütek
Particle Charge Detector PCD-04 model. In short, 10 mL of HyA solution
(0.045 mg/mL at different cAcOH) was placed in the PCD, and 5 mL of
MMW Chit solution (c = 0.091 mg/mL at the same cAcOH) was added
in 125–125 μL portions. The streaming potential values were recorded
20 s after every addition of Chit. The acquired results were analyzed
and fitted taking into consideration the fact that the regular sigmoidal
character of charge titration curves is strongly distorted in our case.
For this reason, a modified version of the sigmoidal Boltzman equation
(Eq. (1)) [30] has been used to fit the experimental data and to deter-
mine the charge neutralization point (in our case, the composition
ratio which corresponds to the point of zero charge).
a1 x þ a2
U str ¼ þ a5 x þ a6 ð1Þ
1 þ exp½ðx−a4 Þ=a3 
In this form of the Boltzman equation x represents the molar ratio of
Chit to HyA, which is the independent variable of the experimental titra-
tion curves (measured streaming potential of the colloid system (Ustr/
mV) plotted versus the molar ratio of opposite charged species (nChit/
nHyA)), while the ai coefficients are fitting parameters. The a1 and a2
are asymptotic values for lower and upper values of the streaming po-
tential curve, while the nominator and the second term in the right-
hand side of the expression account for the drift of the baseline and
the plateau of the sigmoid. The a3 is the width of the surface charge tran-
sition, a4 is the x value at the inflection point, and the (a1a4) + a2 term
measures the height at the inflection point. Knowing the value of the
fitting parameters it becomes possible to calculate the x coordinate of
the model function at Ustr = 0 mV, which corresponds to the neutraliza-
tion point of the colloidal system.
220 Á. Turcsányi et al. / International Journal of Biological Macromolecules 148 (2020) 218–225
2.4.2. Rheology
Changes in the rheological behavior of Chit-HyA system were de-
tected with an Anton Paar Physica MCR 301 Rheometer (Anton Paar,
GmbH, Germany) at 25.0 ± 0.1 °C, and the system was equipped with
concentric cylinder geometry (CC27-SN12793). Here MMW Chit and
HyA were dissolved in 1% (v/v) AcOH to reach 4 mg/mL and 1 mg/mL
concentrations, respectively. 19 mL of HyA solution was loaded into
the rheometer, and altogether 1.0 mL of Chit solution was added in con-
sequential steps with 3 min intervals. The viscosity of HyA solution and
Chit-HyA systems was recorded between the additions of 40–40 μL Chit.
The effect of dilution was also investigated for HyA using 1% (v/v) AcOH.
2.4.3. Conductometry
The conductivity of different SDS:Chit and SDS-HyA:Chit systems
was examined with a Radelkis OK-114 conductometer equipped with
an electrode with sheet plates. First, different amounts of SDS (0.360,
0.721 or 2.89 mg/mL in 0.727% (v/v) AcOH) and HyA (0.727 mg/mL in
0.727% (v/v) AcOH) solutions were mixed together, producing
11.0 mL SDS-HyA mixture (for the simple SDS:Chit measurement,
10.0 mL of 0.360 mg/mL stock solution was mixed with 1 mL 0.727%
(v/v) AcOH). To these surfactant-polymer mixtures, 5–7 mL MMW
Chit stock solution (0.727 mg/mL in 0.727% (v/v) AcOH) was added in
125–125 μL portions. Between each step the conductivity was recorded
after ~1 min stirring (after the value was stable). The amount of HyA so-
lution changed from 0.5 to 8 mL in the initial solutions.
2.4.4. Infrared spectroscopy
The Fourier transformed infrared spectroscopy (FT-IR) measure-
ments were conducted with a Jasco FT/IR-4700 spectrometer using an
ATR PRO ONE Single-reflection accessory (ABL&JASCO, Hungary). The
spectra for lyophilized Chit-HyA polyelectrolyte complexes (PECs,
[28]), MMW Chit and HyA were registered between 4000 and
500 cm−1 by averaging 128 interferograms, using 2 cm−1 optical reso-
lution. The samples were prepared by mixing 2 mg/mL HyA and
MMW Chit solutions under heavy stirring (cAcOH = 1% (v/v)). Lyophili-
zation of all samples was carried out in a CHRIST Alpha 1–2 LD Plus
freeze dryer.
2.4.5. Thermoanalytical studies
Differential scanning calorimetry (DSC) studies were carried out
using a Mettler-Toledo 822e calorimeter. The materials were heated
from 25 to 500 °C with 5 °C/min heating rate. Thermogravimetric (TG)
measurements were performed using a Mettler-Toledo TGA/SDTA
851e Instrument. The observed temperature range was 25–1000 °C,
using 5 °C/min heating rate.
2.4.6. Determination of particle size and size distribution
Dynamic light scattering (DLS) and zeta potential measurements
were performed on HORIBA SZ-100 NanoParticle Analyzer (Retsch
Fig. 1. (A) Change of the streaming potential of HyA titrated with medium molecular weight Chit at different AcOH concentrations. cHyA: 0.045 mg/mL, cChit: 0.091 mg/mL.
(B) Concentration distribution curves of HyA (black) and Chit (green) (cHyA: 0.040 mg/mL, cChit: 9.0 μg/mL); and correspond to individual pH in Chit-HyA systems at charge
neutralization point (from left to right: pH = 2.53; 2.70; 2.77; 2.91), [HChit]+ is equivalent to Chit in the text.
Technology GmbH, Germany) at 25 °C holder temperature, using semi-
conductor laser excitation solid laser (λ = 532 nm) as light source, with
90° scattering angle detection. For the measurements, the viscosity (η,
mPa·s or T (K) dependent equation) of the solvent and the refractive
index of the measured particles must be given, then the photon correla-
tion method is used to acquire the auto correlation function G2(τ) vs.
scattering light intensity I(t). To obtain the particle size distribution
(dZ-average, nm), the software uses the cumulant method for calculation,
then the histogram method is applied to get the mean and the standard
deviation of the distribution (d ± S.D. in nm). Transmission
electronmicroscope (TEM) images were taken using a Tecnai G2 20 X-
Twin type instrument operating with 200 kV accelerating voltage. The
recorded images were analyzed with ImageJ software.
3. Results and discussion
3.1. Quantification of the interaction of Chit with HyA
3.1.1. Charge titration
The interaction between HyA and Chit was first investigated by
charge titration (Figs. 1A and S1), where the charge neutralization
point was obtained at 0.210–0.312 mChit/mHyA mass ratios (Fig. S1)
and 0.496–0.738 nChit/nHyA monomer molar ratios (Fig. 1A) under dif-
ferent acidic conditions. The negative charge of HyA continually de-
creased with increasing Chit amount and then shifted to positive
values following neutralization and aggregation of the polymers. The
monomer ratio was lower than the anticipated 1:1, because in the pH
= 2.53–2.91 range the carboxyl groups of HyA are partially protonated
(67–45%) (pKa: 2.83 Fig. 1B, symbols, [31]), and due to the deacetylation
degree of Chit the polymer only possessed ca. 80% positively charged
groups (pKa: 6.41 [32]).The titration was also carried out at pH =
4.50, where [HyA]− and [HChit]+ are almost 100%. In this case, 0.537
mChit/mHyA mass ratio and 1.271 nChit:nHyA monomer molar ratio was
determined which can be easily interpreted solely by the deacetylation
degree of Chit. The titrations cannot be performed at higher pH because
of the extremely decreasing solubility of Chit.
3.1.2. Rheology
The charge neutralization point was also determined by parallel rhe-
ology experiments (Figs. 2A, S2). The viscosity of HyA gradually de-
creased with each addition of Chit due to the loss of charge, which
probably decreased the hydrophilicity. Near the charge neutralization
point of 0.261 mChit/mHyA (0.617 nChit/nHyA), which was similar to the
appropriate PCD measurement, the viscosity barely changed, and after
the charge shift it started to increase due to the presence of free Chit.
3.1.3. Conductometry
Conductometric measurements were also carried out to confirm our
results. However, the interaction between the polysaccharides could
 Á. Turcsányi et al. / International Journal of Biological Macromolecules 148 (2020) 218–225
Fig. 2. (A) Viscosity (black) and streaming potential (blue) values of HyA-Chit system at different nChit/nHyA monomer ratios (1% (v/v) AcOH, pH = 2.70). (B) Conductometric
measurements of SDS-Chit and SDS + HyA-Chit systems (0.727% (v/v) AcOH, pH = 2.77).
not be followed directly under the applied conditions, because their
contribution to conductivity is zero due to low concentrations (1/100
of cAcOH). To solve this problem, SDS was introduced to the system.
This surfactant is completely charged in this medium (pKa~0), and the
interaction with Chit results in decreasing conductivity. During the ex-
periment, SDS and different amounts of HyA were simultaneously ti-
trated with Chit (Fig. 2B), thus the reaction could be successfully
tracked through the whole range of interest. Until the negatively
charged reactants were completely neutralized, the conductivity gradu-
ally decreased, reaching a minimum value, and this process was also sig-
naled by the aggregation of the polymers. Following neutralization, only
small changes were observed, because the Chit surplus had zero effect,
as mentioned previously. After titrating every HyA-containing solution,
the acquired results (mSDS/mChit in completely neutralized systems)
were plotted against mHyA/mChit. These values showed linear decrease
with increasing cHyA. Extrapolating to y = 0, we could calculate the neu-
tralization ratio of HyA with Chit (0.277 ± 0.023 mChit/mHyA, 0.656 ±
0.053 nChit/nHyA), which is in good agreement with the charge titration
result (Table 1).
3.1.4. FT-IR and thermoanalytic studies
The FT-IR studies of Chit, HyA and Chit-modified HyA systems were
performed to confirm the formation of electrostatic interactions be-
tween the polysaccharides. The spectra of the complexes in Fig. 3A
showed that they were slightly different from the basic polymers. No
new peaks appeared in any sample (Fig. 3A) showing that no new cova-
lent bonds appeared. The peak intensity ratios were almost identical
with varying intensities, so we could assume that the acquired PECs
were nearly the same. There was no perceptible Chit excess in any sam-
ple, which is a clear sign of no interaction between the neutralized PEC
and Chit. The single remarkable difference was observable in the
1600–1580 cm−1 region, where the stretching vibrations of C\\O of car-
boxylates (COO− form, 1610–1540 cm−1), and N\\H deformation of
−1
amine salts (NH+ 3 , ~1585 cm ) are present [33]. For the basic poly-
mers, only the peak for carboxylates (1602 cm−1) for HyA, and the
peak of amine salts (1588 cm−1) for Chit were distinguishable. For
Table 1
Charge neutralization points (nChit/nHyA monomer molar ratios) of Chit/HyA system determined by PCD, rheology and conductometry studies at different AcOH conditions.
cACOH Fraction of protonated HyA (%)
(v/v%)
0.500 45.4
0.727 53.4
1.000 57.4
2.000 66.6
a
For calculations, 80% average Chit deacetylation degree is used.
b
For rheological studies the measuring error was 2.5%.
221
PECs, these bands appeared as a wide, common peak at 1595 cm−1
confirming the formation of only secondary bonds.
The DSC curves of PECs in Fig. 3B did not contain the peaks charac-
teristic to either polysaccharide, these only appeared as steps in the
exotermic region. The first endothermic peak was attributed to water
evaporation, and the minimum values were present between the HyA
and Chit peaks. The lack of tendential changes based on composition
shows that the difference between the samples could not originate
from the rising Chit content. A new endothermic peak appeared near
200 °C, which was the starting temperature for degradation during TG.
This value can be connected to the electrostatic interaction between
the polyelectrolites. The heat flow for Chit:HyA samples did not de-
crease during the last part of the experiment, and this also supports
the successful preparation of the PECs. In all samples the amount of
Chit was larger than required for charge compensation, thus we can hy-
pothesize that after complete neutralization of HyA the unreacted Chit
surplus remained in the supernatant, and was washed away during
the cleaning process. This Chit excess could also be detected during rhe-
ology, where it increased the viscosity of the system.
The results from TG experiments (Fig. S3) were also in accordance
with the beforementioned analyses. The Chit/HyA complexes behaved
differently compared to the simple polymers. The curves of different
compositions were almost identical, which proves that the actual
mass ratio of Chit:HyA in the lyophilized PECs was very similar. In the
first part of the curves, the polymers remained stable below 180 °C,
only the water content of the materials evaporated. The 1:0.5 HyA:
Chit PEC had the largest amount of water, and the endothermic peak at-
tributed to water evaporation was the smallest among the complexes.
Regarding the weight losses, it can be stated that by increasing the ini-
tial Chit content the lyophilized composite contains more water (9.7,
11.6, 14.0 and 15.5% weight loss at 180 °C). After the loss of water, the
degradation of the polymers and complexes occurs. Based on the
onset temperature of degradation, Chit:HyA PECs are less resistant to
heating compared to the simple polymers (~180 °C for PECs, 200 °C
for HyA and 220–225 °C for Chit). This difference clearly shows that
there was electrostatical interaction between them. Another important
nChit/nHyA monomer molar ratiosa
PCD Rheology Conductometry
0.738 ± 0.015 – –
0.696 ± 0.022 – 0.656 ± 0.053
0.622 ± 0.018 0.617b –
0.496 ± 0.033 – –
222 Á. Turcsányi et al. / International Journal of Biological Macromolecules 148 (2020) 218–225

Fig. 3. FT-IR spectra (A) and DSC curves (B) of Chit, HyA and their PECs at several ratios.

conclusion was that there was no excess Chit in any composites, the ad-
ditional polysaccharide in the system did not react with the neutralized
Chit-HyA PEC and it could be easily washed out.
Based on these findings, we can clearly state that the mixing of these
polysaccharides using appropriate mass or molar ratios and mostly
polymer concentrations results in the formation of possible colloidal
drug carriers and the presence of only physical polymer mixture can
be excluded.
3.2. Structural characterization of Chit-modified HyA-based nanosized drug
carrier particles
3.2.1. Effect of the formulation types and polymer weight ratios on the size
of unloaded carriers
Based on the main quantitative results of Section 3.1, we proposed
three different Chit-modified HyA-based drug carrier systems, as Fig. 4
presents. For syntheses of nanocarriers, we chose the range of pH =

Fig. 4. Schematic representation of the preparation and possible structure of the three different Chit/HyA carriers.
 Á. Turcsányi et al. / International Journal of Biological Macromolecules 148 (2020) 218–225
4.40–4.70, where both HyA and Chit are practically in their fully charged
forms (Fig. 1B). The size and size distribution of prepared carriers have
been meticulously analyzed by DLS. The size distribution curves in
Fig. 5A show major differences between the formulations. The average
size for type I was d = 208 ± 132 nm. These particles had broad distri-
bution and moderate size, even though cChit (0.364 mg/mL) and cHyA
(0.027 mg/mL) were lower than the concentrations used for type II
and III. For type II, the average diameter (d = 182 ± 63 nm) decreased,
and the particles gained in here were more uniform. The largest size (d
= 243 ± 94 nm) was determined for type III, but the size distribution
was narrower compared to type I NPs, indicating better size control.
The size of type III was higher than the core NPs (d = 147 ± 45 nm),
so the HyA-coating was effective. We can conclude that the fabrication
of the three different nanostructures was successful. Type I NPs have a
loose colloidal structure based on size, distribution and initial polymer
concentrations, where the electrostatic interaction between the macro-
molecules is dominant. Thanks to the introduction of TPP having size-
controlling effect, the structure of type II NPs is more compact, produc-
ing smaller monodispersed particles possessing interpenetrating poly-
mer network. It was also observed that the type III NPs possess larger
average size compared to the type II. This can be explained with the dif-
ferent molecule size of HyA and Chit (the latter is 6.6 times smaller). The
Chit macromolecules formed a compact core after crosslinking with
TPP, and the large HyA polymer chains could not penetrate this net-
work, providing a core-shell structure. Analysing the effect of Chit:
HyA mass ratio on the different carrier types in the wide range of
80:1–1:8, we concluded that simple type I system formed particles
throughout most of the investigated region (Fig. 5B). For particles con-
taining more Chit than required for charge compensation (from 40:1
to 1:1 mChit:mHyA), the values exponentially increased from d = 168
to 864 nm. In the 80:1–45:1 mChit:mHyA range, no particles were per-
ceivable visually or with DLS, because cHyA was so large compared to
cChit that the formation of NPs was inhibited. Between 40:1–25:1, the
presence of small particles (dZ-average = 168 nm) was detectable, but
no size distribution curves were available due to the lack of appropriate
number of NPs. From 20:1 to 1:1 mChit:mHyA mass ratio, the size in-
creased from d = 195 to 864 nm, but the compositions close to neutral-
ization gave completely aggregated systems, because the loss of surface
charge destabilized the forming NPs. For HyA-excess systems (1:2–1:8
mChit:mHyA), the size was relatively low (d = 199–103 nm). On both
ends of the investigated range, the acquired data suggested a minimum
Chit:HyA mass ratio required for carrier formation using these concen-
trations. For type II and III (Fig. S4), the presence of crosslinking agent
yielded more particles with better controlled size in the 80:1–10:1
mChit:mHyA range. Type II gave smaller particles with every HyA content,
suggesting a more compact internal structure. Due to the core-shell
structure, the free positions for electrostatic interactions are reduced
Fig. 5. (A) Representative DLS curves of the three different formulations of Chit-HyA before drug loading, cHyA = 5% (w/w). (B) Change of the average particle size in type I carriers at
various Chit/HyA mass ratios.
223
and that could explain why the type III with 10% (w/w) HyA
aggregated.
3.2.2. Size and structural characterization of vitamin-loaded NPs
After loading the vitamins (Figs. S5–S6) as model hydrophobic
drugs, the size increased for type II and III, but the beforementioned ef-
fects of Chit:HyA ratio remained the same for all systems. The TCP in
type I carriers slightly increased the size for HyA-excess NPs (from d
= 103–199 nm to 144–211 nm), while drastically decreased it for com-
positions with more Chit (from d = 461–864 nm to 342–375 nm). D3
increased the size to d = 133–209 nm for carriers with more HyA. We
concluded that TCP had a size controlling effect for 80:1–1:1 mChit:
mHyA, while both vitamins increased the size of 1:2–1:8 mChit:mHyA for-
mulations. The size of the blank and vitamin-loaded NPs was also
checked by DLS during storage. The size of every TPP-containing
nanocarrier generally increased, and the effect was more significant
for larger HyA contents (type III with 5% (w/w) HyA even aggregated).
The particles also grew in type I with more HyA, while the size de-
creased for systems with more Chit. The presence of TPP in drug con-
taining formulations drastically decreased the zeta potential from ζ =
+39.2 ± 1.9 mV (type I) to ζ = +9.8 ± 2.1 mV (type II and III) through
electrostatical binding of Chit.
Evaluating the TEM images of drug-containing nanocarriers taken
under the TEM conditions, it was evident that the structure of loaded
carriers was heavily dependent on the synthesis method and the used
vitamin. Type I NPs were efficient in encapsulation, however while
TCP was homogeneously distributed in the polymeric carriers as small,
d = 14 ± 4 nm sized particles (Fig. 6A), D3 was only present on the sur-
face as a thin layer (d = 4–13 ± 2 nm) (Fig. 6B). Although Fig. 6A shows
spherical particles with TCP loading (d = 142 ± 73 nm), several 0.5–2
μm sized composites were also detected on TEM images, which was
also confirmed by DLS. In case of D3 (Fig. 6B) all particles were spherical
with d = 59 ± 19 nm, which is far lower than the size measured with
DLS. In case of type III NPs, the drug loading was checked first for the
Chit-TPPcore NPs. The TCP was poorly encapsulated, and the carrier
NPs were hardly visible. Most of TCP was present as roughly shaped, d
= 48 ± 10 nm sized particles, and the polymer NPs (d = 103 ±
41 nm) were amorphous. Comparing the images with the DLS results,
we could surmise that the TCP-loaded NPs were successfully created,
but the dialysis damaged the structure and TCP was released as parti-
cles. The formation of D3 loaded carriers was more stable, giving
round shaped particles with d = 119 ± 28 nm diameter. The polymer
encapsulated most of D3, and the drug was present on the surface of
all NPs, and several also contained it as a core. This formulation survived
the cleaning process without losing the molecules, but after a longer
storing period, the D3 came off from the surface in the form of small par-
ticles (Fig. 6B).
224 Á. Turcsányi et al. / International Journal of Biological Macromolecules 148 (2020) 218–225
Fig. 6. Representative TEM images of TCP (A) and D3 (B) encapsulated in type I NPs.
Coating the vitamin-loaded Chit NPs with HyA gave mixed results.
For TCP, the carriers remained amorphous. The surface HyA layer col-
lected some drug particles, yielding d = 202 ± 59 nm sized NPs with
79 nm core. The outer layer was not washed off during dialysis, and it
somewhat enhanced the encapsulation efficiency. For the complexes
with D3, HyA coating resulted in a few not well-defined, d = 115 ±
14 nm sized NPs, where the formation of core-shell structure could
not be clearly verified. However, most of the visible drug carriers were
identical to core NPs, with diameter of 75 ± 21 nm (Fig. 7B). In these
samples, the HyA layer was probably washed off, so we can conclude
that HyA was weakly connected to the Chit core, maybe due to the D3
layer present on all NPs. Taking the DLS results into account, we could
assume that HyA formed a layer on all particles regardless of the loaded
vitamin, and these carriers remained stable in the initial buffer solution.
Aside from the effectiveness of coating, comparing the cores without the
HyA layer to the Chit-TPPcore NPs, we could see that the presence of HyA
stopped the growth of core NPs, resulting in smaller particles with both
vitamins.
The encapsulation of vitamins into type II systems also ended in very
different formulations. For TCP, we obtained non-spherical, amorphous
structures with d = 122 ± 48 nm. The drug was better encapsulated
than in type III, but it was clearly worse than type I. For D3 loading, d
= 74 ± 23 nm sized, well-defined round particles were observed
(Fig. 7A), with D3 evenly distributed in the polymer matrix. The size
was smaller for both carriers than type III, further supporting our hy-
pothesis presented in Fig. 4, that a more compact nanocarrier can be
achieved with this fabrication method.
Based on the theoretical composition and TEM images of type 1 car-
riers, we could calculate an ideal and estimated drug encapsulation effi-
ciency for TCP and D3. For the preparation of TCP-loaded type I NPs, the
expected maximal weight of the TCP in the composite NPs is 35%, while
12% for composites having D3 content (55% and 14% calculating for
loaded drug(s) per only polymer weight). After preparation and purifi-
cation steps, the actual calculated values were 13 ± 5% and 37 ± 12%
loaded drug per total volume (15 ± 8 and 66 ± 34% drug per polymer)
Fig. 7. Representative TEM images of D3 encapsulated in type II (A) and type III (B).
for TCP and D3, respectively. The difference in the case of TCP (from 35%
to 13 ± 5%) may be the result of drug loss during purification steps and
quite inefficient encapsulation, while for D3 the higher calculated value
(37 ± 12%) instead of the expected encapsulated D3 content (12%) orig-
inates from the non-uniform encapsulation efficiency of the carrier NPs.
4. Conclusion
In this work we investigated quantitatively the charge compensation
between the positive Chit and negative HyA. Rheological, particle
charge titration and conductivity studies clearly confirmed that the
1:1 Chit/HyA monomer molar ratio is strongly influenced by the pH as
well as the deacetylation degree of Chit which are crucial factors for
the solubility, purity and the quality of the commercially available
Chit. Based on the quantitative results, we suggested three different
Chit-modified HyA-based nanosized (d = 100–300 nm) drug carriers,
introducing TPP as a cross-linking and size-controlling reagent. The
DLS results proved the successful synthesis of NPs in most cases, and
the effect of Chit:HyA molar ratio on the formulations was assessed.
The encapsulation of vitamins TCP and D3 was effective based on the
change of NP size, therefore TEM was used to verify the structure and
drug loading efficiency of the carriers.
Summarizing the results, we confirmed that D3 could be better en-
capsulated with Chit:HyA systems, with more successful drug loading
attempts compared to TCP. For TCP encapsulation, type I was the best
with the drug incorporated into the polymer matrix, however the
shape and size was not well controlled. In case of D3, type II gave opti-
mal results with high encapsulation, well defined spherical NPs and ho-
mogenous drug distribution. Beside this nanocarrier, type I and the
Chit-TPPcore NPs were also promising. Based on the images of type 1 car-
riers, we could calculate an estimated drug encapsulation efficiency for
TCP (small particles in polymer) and D3 (coating on polymer). The
values were 13 ± 5% and 37 ± 12% loaded drug per total volume (15
± 8 and 66 ± 34% drug per polymer), respectively.
 Á. Turcsányi et al. / International Journal of Biological Macromolecules 148 (2020) 218–225
CRediT authorship contribution statement
Árpád Turcsányi: Methodology, Investigation, Resources, Writing -
original draft, Visualization. Norbert Varga: Methodology, Investiga-
tion, Resources, Visualization. Edit Csapó: Conceptualization, Writing -
review & editing, Visualization, Supervision.
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgement
The research was supported by the National Research, Development
and Innovation Office-NKFIH through GINOP-2.3.2-15-2016-00060 and
FK 131446. This work was supported by the János Bolyai Research
Fellowship of the Hungarian Academy of Sciences (E. Csapó) and the
UNKP-19-3-SZTE-321 New National Excellence Program of the Ministry
of Human Capacities (N. Varga). The authors thank Viktória Hornok
(University of Szeged, Department of Physical Chemistry and Materials
Science) for the registration of several TEM images. The Ministry of Hu-
man Capacities, Hungary grant TUDFO/47138-1/2019-ITM is
acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2020.01.118.
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