Serum protein-hyaluronic acid complex nanocarriers: Structural characterisation and encapsulation possibilities

Carbohydrate Polymers 251 (2021) 117047

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Serum protein-hyaluronic acid complex nanocarriers: Structural
characterisation and encapsulation possibilities
´ ´
´cs a, Norbert Varga a, Ad
´sz a, b, Edit Csapo
´ a, b, *
Alexandra N. Kova
am Juha
a
b

Department of Physical Chemistry and Materials Science, University of Szeged, H-6720, Rerrich B. Square 1, Szeged, Hungary
MTA-SZTE Biomimetic Systems Research Group, Department of Medical Chemistry, Faculty of Medicine, University of Szeged, H-6720 D´
om Square 8, Szeged, Hungary

A R T I C L E I N F O

A B S T R A C T

Keywords:
Hyaluronic acid
Bovine serum albumin
Protein-polymer nanoconjugates
Charge neutralisation

Encapsulation capacity
Drug release

A protein-polysaccharide-based potential nanocarrier system have been developed via a simple, one-step prep­
aration protocol without the use of long-term heating and the utilization of hardly removable crosslinking agents,
surfactants, and toxic organic solvents. To the best of our knowledge, this article is the first which summarizes in
detail the pH-dependent quantitative relationship between the bovine serum albumin (BSA) and hyaluronic acid
(HyA) confirmed by several physico-chemical techniques. The formation of colloidal complex nanoconjugates
with average diameter of ca. 210–240 nm is strongly depend on the pH and the applied BSA:HyA mass ratio.
Particle charge titrations studies strongly support the core-shell type structure, where the BSA core is covered by
a thick HyA shell. Besides the optimization of these conditions, the drug encapsulation capacity and the disso­
lution profiles have been also studied for ibuprofen (IBU) and 2-picolinic acid (2-PA) as model drugs.

1. Introduction
Due to the outstanding biocompatible and biodegradable nature, the
utilization of natural polysaccharides as sustained-release carriers is
beneficial for pharmaceutical fields (Mohamed, El-Sakhawy, &
´, 2020). The targeted drug
El-Sakhawy, 2020; Turcs´
anyi, Varga, & Csapo
delivery is possible by using HyA, which is a negatively charged
glycosaminoglycan, but the chemical modification of its hydroxyland/or carboxyl-group is required (Yamanlar, Sant, Boudou, Picart, &
Khademhosseini, 2011). The HyA has a prominent role at biomedical
´
applications as well (Huerta-Angeles
et al., 2020). The HyA-based
nanohydrogels/nanoparticles (NPs) and films are implied as a prom­
ising area of the cancer treatment, tissue engineering, gene delivery etc.
(Graỗa, Miguel, Cabral, & Correia, 2020). BSA is a water soluble glob­
ular protein consist of 583 amino acid residues (Ghosh & Dey, 2015);

wide ranges of active compounds are able to bind at the appropriate
ă mo
ăto
ăr et al., 2018). Depending on the pH,
binding sites of the protein (Do
the charge of the BSA is shifted from the positive to the negative value
reaching the isoelectric point (pI⁓5.1), that regulates the interaction
between the BSA-polymers and BSA-drugs via electrostatic interactions
(Varga, Hornok, Sebok, & D´ek´
any, 2016).
Polysaccharide-protein conjugates may represent a new dimension
in the design of drug delivery systems. The utilization of these complex

conjugates enhances the colloid stability, targeted efficiency, biocom­
patibility or the reduced drug toxicity (Gaber et al., 2018), which is
published previously for e.g. BSA/Chitosan (Karimi, Avci, Mobasseri,
Hamblin, & Naderi-Manesh, 2013), Ovalbumin/Chitosan (Yu, Hu, Pan,
Yao, & Jiang, 2006), Protamine/HyA (Mok, Ji, & Tae, 2007) or Lyso­
zyme/Alginate (Fuenzalida et al., 2016) nanocarriers. The
polysaccharide-protein nanoconjugates are generally prepared by elec­
trostatic complexation (Antonov et al., 2019), chemical conjugation
´n, & Blanco, 2011) and electrospinning
(Martínez, Iglesias, Lozano, Teijo
techniques (Torres-Giner, Ocio, & Lagaron, 2009). In some cases, the
Maillard reaction is also implied, but the reaction requires long term
heating (60 ◦ C; for at least 3 h) (Edelman, Assaraf, Levitzky, Shahar, &
Livney, 2017) which is specifically unfavourable for heat-sensitive
drugs. The fabrication of carrier NPs is usually carried out by chemical
coupling in the presence of crosslinking agents, like glutaraldehyde
´nyi et al., 2020), which

(Chen et al., 2013), or tripolyphosphate (Turcsa
components can be difficult to remove during the purification process.
Based on these facts, the demand for the development of an effective
polysaccharide-protein nanoconjugates with tuneable-size is strongly
required, where the long-term heating and the utilization of hardly
removable crosslinkers and surfactants is excluded.
In this work we first demonstrate a preparation possibility of BSA/
HyA conjugates by a simple, controllable charge neutralization

* Corresponding author at: Department of Physical Chemistry and Materials Science, University of Szeged, H-6720, Rerrich B. Square 1, Szeged, Hungary.
E-mail address: (E. Csap´
o).
/>Received 15 July 2020; Received in revised form 1 September 2020; Accepted 1 September 2020
Available online 7 September 2020
0144-8617/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( />

A.N. Kov´
acs et al.

Carbohydrate Polymers 251 (2021) 117047

technique without protein degradation and in the absence of cross­
linking agents and organic solvents. Moreover, the pH-dependent
quantitative characterisation of the interaction between BSA and HyA
was also investigated by multiple techniques, which is not published
previously in detail. All the previous studies present only the charac­
terization of the drug-containing composites by using only dynamic light
scattering (DLS) and transmission electron microscopy (TEM) images,
however the pharmacokinetics of these complex nanocarriers are
demonstrated (Huang, Chen, & Rupenthal, 2017; Martins et al., 2014;

˘u et al., 2020; Pigman, Gramling, & Holley, 1961; Shen & Li,
Pas¸cala
2018). Only one article was found (Chen et al., 2013) which contains
some information on the interaction between BSA and the encapsulated
drug in the presence of HyA by differential scanning calorimetry (DSC)
and infrared spectroscopy studies, but the interaction between serum
protein and HyA was not investigated. Two articles focus on the inter­
pretation of the HyA- serum protein interaction by NMR (Filippov,
Artamonova, Rudakova, Gimatdinov, & Skirda, 2012) and integrated
computer modeling (Grymonpr´
e, Staggemeier, Dubin, & Mattison,
2001), but only the presence and strength of the electrostatic interaction
was mentioned. Moreover, we also considered to demonstrate the suc­
cessful encapsulation of two drugs with slightly different hydrophilicity
(2-PA: logP = -0.1; IBU: logP = 1.74) at the optimized weight ratio. Both
molecules have one aromatic ring and one carboxylic group, but the
charge of the molecules at pH = 4.5 is different. The IBU was selected for
our experiments as a model drug because this molecule is often studied
non-steroidal anti-inflammatory compound and its binding mechanism
to serum albumin binding sites is well-known. 2-PA is a neuroprotective,
immunological and antiproliferative compound, it has not been encap­
sulated as active component in any drug delivery system. Drug loading
(DL%) and the release mechanism of the encapsulated molecules are
examined, and the results are compared with other nanocapsule-based
carrier systems containing same drugs.

Alpha 1–2 LD plus) and the solid samples were stored at -70 ◦ C.
2.2.2. Synthesis of the 2-PA- and IBU-loaded BSA/HyA conjugates
For the drug-loaded derivatives, similar preparation and purification
protocol was used than for fabrication of unloaded NPs, but 1− 1 mg of

drugs was also dissolved in the BSA solution (2 mL, cBSA = 2 mg mL− 1).
The synthesis was carried out at mBSA/mHyA = 2.00 mass ratio based on
the experimental results presented in Chapter 3.
2.2.3. Surface plasmon resonance (SPR)
The SPR studies were performed in a two-channel device improved at
the Institute of Photonics and Electronics (Prague, Czech Republic). The
light source was an Ocean Optics HL-2000 type tungsten halogen light
source with 6.8 mW output power, while the reflected light intensity is
monitored in the 574–1000 nm spectrum range using an IPE AS CR
S2010 spectrometer. The sensorgrams were registered by SPR UP
1.1.11.3 (2014 IPE AS CR) control software. Firstly, the protein solution
(cBSA = 10 μM) was flowed under a constant flow rate (50 μL min− 1)
above the gold-coated SPR chip in order to immobilize the protein to the
´ et al., 2016). On the
surface of the sensor via Au-S covalent bond (Csapo
next step, the HyA solution was flowed across the protein-functionalized
surface with 50 μL min-1 flow rate. During studies, the following con­
ditions were used: T = 15− 30 ◦ C, cHyA = 2.5–10.0 μg mL− 1 and the pH
range of 3.6− 5.5.
2.2.4. Particle charge detector (PCD)
The PCD measurements were performed by a PCD-04 Particle Charge
Detector (Mütek Analytic GmbH, Germany) with manual titration ac­
´nyi et al., 2020).
cording to our previously detailed technique (Turcsa
Firstly, the HyA was dissolved in acetate buffer at four different pH
values (pH = 3.6; 4.0; 4.5; 5.0), while the concentration kept constant
(20 mL of cHyA=0.36 mg mL− 1). The BSA solution (10 mg mL− 1) was
added dropwise in 100− 100 μL portions to the HyA solution at 25 ◦ C and
the streaming potential values (mV) were registered. The acquired re­
sults were analyzed and fitted with the modified version of the sigmoidal

Boltzman equation.

2. Experimental
2.1. Materials
BSA (~66,000 Da), HyA sodium salt (1.5–1.8⋅106 Da), IBU sodium
salt (≥98 %), and 2-PA (≥99 %) were purchased from Sigma-Aldrich.
The disodium hydrogen phosphate (Na2HPO4; ≥99 %), the sodium
dihydrogen phosphate monohydrate (NaH2PO4⋅H2O; ≥99 %), the so­
dium acetate 3-hydrate (CH3COONa⋅3H2O; ≥99 %), and sodium hy­
droxide (NaOH; ≥96 %) pastilles and the hydrochloric acid (HCl, ≥99
%) were bought from Molar Chemicals. Acetic acid (AcOH, ≥99 %) was
˝k´
purchased from Erdo
emia Ltd. Company. Highly purified water was
obtained by deionisation and filtration with a Millipore purification
apparatus (18.2 MΩ cm at 25 ◦ C). All reagents and solvents used were of
analytical grade without further purification.

2.2.5. Rheology
Anton Paar Physica MCR 301 Rheometer (Anton Paar, GmbH, Ger­
many) equipped with cylinder geometry (CC27-SN12793) was used; the
changing of the viscosity was followed at 25 ± 0.1 ◦ C and at 37 ± 0.1 ◦ C
at different pH values using acetate buffer (pH = 3.6; 4.0; 4.5). 10 mg
mL− 1 BSA solution was added drop by drop in 19 mL of 0.1 mg mL− 1
HyA solution at 40 μL/3 min dosing speed. The effect of solvent dilution
´ et al.,
was also considered as described in our previous article (Csapo
2018).
2.2.6. Thermal behaviour
The thermal behaviour of the BSA, HyA, and the lyophilized powders

of BSA/HyA conjugates at 6 different mass ratios were studied with
thermogravimetric (TG) and DSC. The TG studies were performed with
the use of a Mettler-Toledo TGA/SDTA851e instrument with 5 ◦ C min− 1
between the range of 25− 1000 ◦ C, under constant air flow (50 mL
min− 1). The DSC studies were performed with the use of a MettlerToledo DSC822e calorimeter with 5 ◦ C min− 1 in the range of 25− 500
◦
C under nitrogen stream (50 mL min− 1).

2.2. Methods
2.2.1. Synthesis of the unloaded BSA/HyA conjugates
The BSA/HyA conjugates were prepared by charge neutralization
method. The 1.6 mg mL− 1 HyA and the 2 mg mL− 1 BSA stock solutions
were prepared in acetate buffer (0.010 M acetic acid/0.0057 M sodiumacetate; pH = 4.5) and in MilliQ water, respectively. In the first step, the
HyA stock solution was stirred at 350 rpm for 1 h and stored overnight at
4 ◦ C. During the synthesis, several BSA/HyA conjugates were prepared
within the range of mBSA/mHyA = 0.25–5.00 mass ratios. Namely, 2 mL
of 2 mg mL− 1 BSA was added dropwise into the 10 mL of 0.08–1.6 mg
mL− 1 HyA solutions under 1000 rpm magnetic stirring at 25 ◦ C. After
mixing the appropriate amounts of BSA to HyA, the samples were
further stirred for 2 h under 500 rpm. Finally, the samples were cen­
trifugated at 5000 rpm for 5 min; the supernatant was removed, and the
samples were redispersed in acetate buffer. The cleaning method was
repeated three times. The cleaned products were freeze-dried (Christ

2.2.7. Fourier transformed infrared spectroscopy
The FT-IR spectra were registered with a Jasco FT/IR-4700 spec­
trometer with the use of an ATR PRO ONE Single-reflection accessory
(ABL&E-JASCO, Hungary). The spectra were recorded at a resolution of
2 cm− 1 between 4000 and 500 cm− 1 by accumulating 128 interfero­
gram. The samples prepared in the same method discussed in the pre­

vious section.

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Carbohydrate Polymers 251 (2021) 117047

Fig. 1. (A) Representative SPR reflectance curves before (black) and after (blue) addition of HyA solution at pH = 3.6 and (B) the registered sensorgrams at different
pH values (cHyA = 2.5 μg mL− 1, 50 μL min− 1 flow rate, t = 25 ± 0.1 ◦ C). (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

2.2.8. Circular dichroism spectroscopy
CD spectra were recorded by using Jasco J-1100 CD spectrometer
(ABL&E-JASCO, Hungary) at 25 ± 1 ◦ C using 1 cm optical pathlength
quartz cuvette. All spectra were recorded at 100 nm min− 1 scanning
speed in the middle UV-region (200− 300 nm) under N2 flow (3 L min− 1)
and represents the average of three scans. The light source was a watercooled, high-energy xenon lamp (450 W). The raw data was converted
into mean residue ellipticity (MRE) using the Eq. (1), and the ratio of the
α-helix content was calculated from the Eq. (2).
MRE208 =

observed CD(mdeg)
10Cp nl

α − helix (%) =

− MRE208 − 4000

× 100
33000 − 4000

spectrophotometer in a 1 cm quartz cuvette. The measurements were
carried out at room temperature in 200− 500 nm wavelength range. The
exact concentration of the non-encapsulated free IBU and 2-PA was
calculated from the calibration curves, where the characteristic absor­
bance band of the IBU and 2-PA were appeared at λ = 222 nm and λ =
264 nm in acetate buffer (pH = 4.5) medium, respectively (Fig. S1). The
DL% and EE% values were defined by Eqs. (3) and (4).

(1)
(2)

DL% =

encapsulatedmassofdrug
× 100
totalmassofthenanoparticles

(3)

EE% =

encapsulatedmassofdrug
× 100
totalmassofdruginsynthesis

(4)

2.2.10. In vitro release study
The in vitro dissolution profiles of the IBU- and 2-PA-containing BSA/
HyA conjugates were measured by UV–vis. The release measurements
were performed in phosphate buffer solution (pH = 7.4 ± 0.1) at 37 ±
0.5 ◦ C and a semipermeable cellulose membrane (avg. flat width = 25
mm; Mw cut-off = 14,000; Sigma-Aldrich) was used. The data points
were registered for 250 min. The concentrations of the IBU and 2-PA in
the release medium were determined by calibration curves (Fig. S2). The
possible release kinetics and the proposed mechanism can be defined
´nyi, Hor­
from the fitting of the Weibull kinetic models (Varga, Turcsa
nok, & Csap´
o, 2019; Veres et al., 2017).

where the Cp is the molar concentration of the protein, n is the number of
amino acid residues, and l is the pathlength of the cuvette.
2.2.9. Characterisation of the BSA/HyA NPs
The average size, size distribution, morphology, polydispersity index
and the Zeta-potential values were measured by DLS using a HORIBA SZ100 NanoParticle Analyzer (Retsch Technology GmbH, Germany). The
light source was a semiconductor laser (λ = 532 nm, 10 mW) and
photomultiplier tubes (PMT) were used as detector at 90◦ scattering
angle. For registration of TEM images a Jeol JEM-1400plus equipment
(Japan) at 120 keV accelerating voltage was applied. To determine the
encapsulation efficiency (EE%) and DL%, the absorbance spectra of the
supernatants of the centrifuged drug-loaded BSA/HyA conjugates were
registered
by
Shimadzu
UV-1800
UV–vis

double
beam

Fig. 2. (A) Change of the streaming potential of pure BSA and HyA as a function of pH (B) Change of the streaming potential of HyA titrated with BSA at different pH
values (starting concentrations: cHyA =0.36 mg mL− 1, cBSA =10.0 mg mL-1, VBSA = 100-100 μL).
3


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Carbohydrate Polymers 251 (2021) 117047

Fig. 3. Apparent viscosity values of BSA/HyA conjugates (marked with (⸰)) and the calculated streaming potential curves (grey continuous lines) as a function of
mBSA/mHyA at pH = 3.6 (A); pH = 4.0 (B) and pH = 4.5 (C) at 25 ◦ C. (starting concentrations: cHyA =0.10 mg mL− 1, cBSA =10.0 mg mL-1, VBSA = 40-40 μL).

3. Results

opposite charges. Fig. 2B shows that the initial negative charge of the
HyA is shifted to higher values by the addition of BSA. At pH = 3.6 and
4.0 the course of the curves is steeper, which is much longer at pH = 4.5.
In accordance with SPR results, we found that there is no measurable
change in the streaming potential values at pH = 5.0. The following
neutralization points (where the streaming potential is 0 mV) are ob­
tained by fitting the measured points by the modified Boltzmann
equation: mBSA/mHyA = 2.04 ± 0.01 (pH = 3.6), 2.69 ± 0.01 (pH = 4.0)
and 5.05 ± 0.01 (pH = 4.5). It is also observed that the inflection points
of the titration curves (1.97, 2.51 and 4.46, respectively) appear before
the neutralization points, which suggests structural changes between the
macromolecules and the possible formation of BSA/HyA colloidal NPs

before charge neutralization. To confirm this observation rheological
studies were also performed.

3.1. Surface plasmon resonance spectroscopy
The pH-dependence SPR studies have been performed at five
different pH using acetate buffers (pH = 3.6; 4.0; 4.5; 5.0; 5.5) at 25 ±
0.1 ◦ C. The concentration of HyA solution was fixed at 2.5 μg mL− 1 in
every cases. The registered sensorgrams are presented in Fig. 1.
The decrease in the pH of the HyA solutions causes a greater shift of
the signal of the sensor response which suggests a pH-dependent inter­
action between the HyA and BSA. If the pH of the HyA solution exceeds
the pH = 5.0, no significant interaction can be observed (pH = 5.5). The
concentration- and the temperature-dependence of the interaction of the
two studied macromolecules was also investigated, while the pH of the
HyA solution was remain the same (pH = 4.5; acetate buffer). At this pH
= 4.5, the HyA are in fully deprotonated form and it is well-known that
no measurable structural change occurs in the secondary structure of
´ et al., 2016). For both series
BSA at this slightly acidic conditions (Csapo
of measurements, merely a slight shift can be observed in the signal of
the SPR gold-coated biosensor (Fig. S3). Based on these results, it can be
concluded that the interaction between the polysaccharide and the
protein is strongly depends on pH, and the effect of the temperature and
the concentration is negligible under the studied conditions.

3.3. Rheological studies
By the addition of the BSA stock solution to HyA the viscosity values
are continuously decreased to a given point and then a slightly constant
values are measured. The intersection point of the fitted lines gives a
breaking point. This trend is observed at all the studied pH values

(Fig. 3).
The breaking points can be given at the following mBSA/mHyA ratios
at 25 ◦ C: 1.43 (pH = 3.6), 2.26 (pH = 4.0) and 4.14 (pH = 4.5). The
determined breaking points can be obtained at nearly similar mBSA/
mHyA ratios than the inflection points of the PCD curves (1.97 (pH =
3.6), 2.51 (pH = 4.0) and 4.46 (pH = 4.5)). The rheological studies have
been carried out at 37 ◦ C and similar trend was observed that at 25 ◦ C,
but the breaking points shifted towards the smaller values because of the
different solvatated states of the macromolecules (breaking points at 37
◦
C: 0.91 (pH = 3.6), 1.79 (pH = 4.0) and 3.63 (pH = 4.5). It can be
concluded that, before neutralization, a structural change is occurred,
which strongly indicates the possible formation of colloidal NPs via
electrostatic interaction of BSA and HyA.

3.2. PCD measurements
The interaction between HyA and BSA was also confirmed in detail
by PCD titrations, where the neutralization points were determined at
different pH values. However, the acid-base property of HyA is well´nyi et al., 2020)), but for quantitative
known (pKa = 2.83 (Turcsa
interpretation of the results the isoelelectric points of BSA and HyA were
determined by PCD (Fig. 2A).
The titration clearly proved that the BSA has positive charge below
pH = 5.0; the neutralization point is obtained at pH = 5.10 which value
is in good correspondence with the pI of BSA (Varga et al., 2016). For
HyA, the negative surface charge is dominant in wide pH range (pH =
2–11). In case of BSA/HyA system the titrations were carried out in the
pH range of pH = 3.5–5.0, where the macromolecules have well-defined
4

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Carbohydrate Polymers 251 (2021) 117047

Fig. 4. (A) The hydrodynamic diameter (●, left y-axis) and the turbidity values ( , right y-axis) of the BSA/HyA system as a function of increasing mBSA/mHyA (cBSA
= 2 mg mL− 1) with the representative photos of the samples at mBSA/mHyA = 2 and mBSA/mHyA = 4. (B) DLS curve of BSA/HyA NPs using mBSA/mHyA = 2 with the
TEM images of the sample.

Fig. 5. CD curves of BSA (continuous red line) and BSA/HyA conjugates (dotted grey lines) in MilliQ water (A) and in acetate buffer (pH = 4.5) (B) t = 25 ◦ C, cBSA =
2.78 μg mL− 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. Characterisation of the drug-free BSA/HyA nanocarrier systems

negatively charged HyA. To confirm these theory, further PCD studies
have been performed. The pure HyA solution and the probably HyAcoated BSA-based particles-containing dispersion were titrated with
the same CTABr solution under similar conditions. We hypothesized
that, if HyA functions as a thick shell, nearly the same amount of CTAB
will compensate the negative surface charge as would be expected on the
free macromolecule as well. For pure HyA solution and for composites
the charge compensation points are obtained at nCTABr /nHyA(monomer) =
0.88:1.0 and at 0.93:1.0, respectively (Fig. S5). This means that ca. one
CTABr compensates one HyA monomer unit. This observation is in good
´ et al., 2018). The presence of
agreement with our previous result (Csapo
core-shell-type NPs instead of “alloy-like” structure is more preferred.
The adsorption of the BSA on the surface of HyA can be ruled out.

3.4.1. DLS and TEM investigations

To prove the formation of BSA/HyA colloidal NPs, DLS and turbidity
studies have been also performed. The measured hydrodynamic di­
ameters (Z-average) and the turbidity values of the BSA/HyA
conjugates-containing aqueous dispersion at pH = 4.5 are presented in
Fig. 4. The parallel registered Zeta-potential values are seen in Fig. S4.
Fig. 4A clearly indicates the formation of colloidal NPs according to
the increasing turbidity values within the range of mBSA/mHyA =
0.25–3.75 mass ratios. If the mass ratio exceeds the mBSA/mHyA = 4.0
value, the aggregation of NPs can be observed, as the inserted photo also
represents. This observation is confirmed by DLS. At the above
mentioned mBSA/mHyA = 0.75–3.50 mass ratios the average diameter of
240 – 210 nm is obtained depending on the mass ratios. For small BSA
content (mBSA/mHyA <0.50), larger diameters (300–400 nm) can be
measured because of the still large excess of HyA, while at high BSA
content (mBSA/mHyA >3.75) both the adhesion and the aggregation of
NPs is feasible. The results of DLS and turbidity studies are in good
agreement with the main conclusions of both PCD and rheological
measurements. The change of the Zeta-potential values as a function of
mBSA/mHyA also shows similar trend. The values continually decrease
with increasing mBSA/mHyA (ζ = − 50.2 ± 1.2 mV (mBSA/mHyA = 2); ζ =
− 37.4 ± 1.5 mV (mBSA/mHyA = 4)). Fig. 4B. represents the DLS curve of
BSA/HyA NPs at mBSA/mHyA = 2.0, where the average diameter is the
smallest. The representative TEM image of this system also supports the
formation of NPs with nearly 200 nm average diameter and a welldefined core-shell structure. The negative surface charge may indicate
the formation of core-shell structure, where the BSA is covered by

3.4.2. CD, FT-IR and thermal behaviour
Detailed structural studies of the BSA/HyA conjugates at several
mBSA/mHyA ratios have been also carried out by CD, FT-IR as well as TG/
DSC. The main text contains the results of BSA/HyA NPs prepared at

mBSA/mHyA = 2.0 ratio; other data are summarized in (Figs. S6–S7).
Fig. 5A, B represents the CD curves registered for the pure BSA and BSA/
HyA conjugates.
In both cases the characteristic negative bands of the pure BSA are
occurred at 208 and 220 nm (Zhou, Wu, Zhang, & Wang, 2017). In
MilliQ water (Fig. 5A), by the presence of HyA, only a slight shift is
observed in the intensity of the negative band at 208 nm, which confirms
that, there is no significant change in the secondary structure of BSA.
According to Eqs. (1) and (2), the calculated α-helix content: 54.49 % for
pure BSA and 57.76 % for BSA/HyA nanoconjugates. This is in good
correspondence with previous values (Zhou et al., 2017). At nearly
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Carbohydrate Polymers 251 (2021) 117047

Fig. 6. FT-IR spectra (A), DSC (B) and TG (C) curves of BSA, HyA and the lyophilized powder of BSA/HyA nanoconjugates.

neutral pH, both macromolecules have negative charge and thus the
potential for electrostatic interactions is low. In contrast, the α-helix
content of the BSA in the BSA/HyA conjugates is drastically decreased in
the presence of HyA at acidic conditions (Fig. 5B). The proportion of the
α-helix content in acidic conditions is: 41.52 % for pure BSA and 13.56
% for BSA/HyA nanoconjugates. Parallel with the decreasing α-helix
content the ratio of the β-sheets is increased to ca. 75 % supported by the
fitting of the measured CD curve by Reed model (Reed & Reed, 1997). At
pH = 4.5 the macromolecules have well-defined opposite charge and

most probably the serum protein chains are charge compensated with
the approx. 20-fold larger HyA and the protein chains are partially
unfolded and arranged to form a core(BSA)-shell(HyA) structure sup­
ported by TEM, Zeta-potential and PCD studies (Fig. S5). The FT-IR
measurements clearly indicate that the presence of both HyA and the
protein in the composite; the determinative bands of amide I and II as
well as the vibrations of COO− and C-O(H) are presented in Fig. 6A.
The data are in good agreement with previously published values for
same macromolecules (Zhou et al., 2017). The FT-IR results did not
confirm obviously the observations of CD studies, which can be
explained by the fact that the FT-IR spectra were recorded in solid
powder form, while the CD studies were measured in aqueous solution.

The degradation temperature (Tg) and the composition of the BSA/HyA
conjugates were determined by TG and DSC (Fig. 6B, C). Based on
Fig. 6B, it can be seen that the intensive exotherm peak of the HyA (230
◦
C) and the endotherm peak of the BSA (222 ◦ C) does not appear in the
conjugates which indicates the effective washing procedure and the
composite does not contain macromolecules in free form. By fitting of
the DSC curves, the Tg values of the BSA and HyA are 202 ◦ C and 222 ◦ C,
while for conjugates is 181 ◦ C. The decrease is presumably due to the
formation of electrostatic interaction between the macromolecules. The
Tg value was also determined by TG, where similar data can be obtained:
204 ◦ C (BSA), 224 ◦ C (HyA) and 185 ◦ C (BSA/HyA conjugates).
Considering the weight changes and the shape of the curves, the com­
posite contains both BSA and HyA, but the BSA content is more domi­
nant and the presence of only physical mixture can be excluded (Figs. S8,
S9)
3.5. Characterisation of the drug-loaded BSA/HyA nanocarrier systems

After the comprehensive study of the drug-free BSA/HyA NPs, the
encapsulation of two model drugs is performed using mBSA/mHyA = 2.0
mass ratio. The average size, the size distributions and the morphology

Fig. 7. Size distribution curves of IBU- and 2-PA-loaded BSA/HyA colloidal particles by DLS with the representative TEM images of these particles.
6


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acs et al.

Carbohydrate Polymers 251 (2021) 117047

Fig. 8. Dissolution profiles of the 2-PA (A) and IBU (B) molecules before (⸰) and after loading () at pH = 7.4 ± 0.1 (in phosphate buffer solution) at 37 ◦ C. (the dotted
lines represents the fitting of the primer data via Second-order (free molecules) and Weibull kinetic models (drug-loaded particles).
•

of the drug-loaded BSA/HyA conjugates are presented in Fig. 7.
It can be stated that the capsulation was successful; the size of the
drug-loaded NPs is greater than the size of the drug-free NPs (dDLS = 210
± 56 nm) and spherical morphology is observed. The results of DLS
(dDLS, IBU-loaded = 250 ± 80 nm, ζ = -38.9 ± 1.4 mV; dDLS, 2-PA-loaded
= 276 ± 74 nm; ζ = -42.0 ± 1.1 mV) and TEM (dTEM, IBU-loaded = 247
± 92 nm; dTEM, 2-PA-loaded = 264 ± 80 nm) are in good agreement.
However, the TEM images do not present core-shell structure but based
on strongly supported structure of unloaded NPs and the preparation
conditions, most probably the BSA-drug conjugates form the inner core
and the outer shell contains dominantly HyA. The measured Zetapotential values (presented above) also indicate this supposition. The
EE% and the drug loading also calculated by the Eqs. (3)− (4). For IBUloaded BSA/HyA NPs the EE % is 40 % and the DL % is 6 %. In case of the
2-PA-loaded BSA/HyA NPs the EE % is 14 % and the DL% is 2 %.

Although no previously published data on the encapsulation of 2-PA
were found, but it can be clearly stated for IBU that the presence of
´
HyA slightly increases the drug content from DL% = 4–4.5 % (Csapo
et al., 2016) to 6 % compared the DL% of our previously published pure
BSA-based systems. After encapsulating the active substance, the
dissolution profiles of the drugs are also investigated at pH = 7.4 (in
phosphate buffer solution) at 37 ◦ C. The registered curves are presented
in Fig. 8.
For the 2-PA-loaded BSA/HyA conjugates almost the 28 % of the
encapsulated drug is released in the examined period (t = 240 min),
while for IBU-loaded BSA/HyA conjugates ca. 52 % of the encapsulated
IBU is liberated. The primer data points are fitted by different kinetic
models (First-Order, Second-Order, Weibull, Korsmeyer–Peppas, Higu­
chi), but for IBU- and 2-PA-loaded particles the measured data fit well to
the Weibull expression. To compare the rate of dissolutions, the disso­
lution data of several IBU-containing nanocomposites synthesized in our
lab were considered and the corresponding half-time (t1/2) data were
compared. We can compare the t1/2 values because similar technique
was used for the registration of the dissolution profiles and same kinetic
models were applied for fitting. In case of mesoporous SiO2 (Varga et al.,
´ et al., 2016), the change of t1/2 of the IBU is
2015) and pure BSA (Csapo
not determinative in the presence of these carriers (t1/2(IBU) =0.11 h, t1/2
(SiO2/IBU) =0.08 h, t1/2(BSA/IBU) = 0.13 h). In case of our BSA/HyA
complex NPs the following t1/2 values are calculated under the applied
conditions: t1/2(IBU) =0.6 h, t1/2(BSA/HyA/IBU) = 2.2 h. These data clearly
confirm that the combination of HyA with BSA strongly facilitates the
prolonged release of IBU at pH = 7.4 (in phosphate buffer solution),
where nearly fourfold drug retention is achieved.

rheology, turbidity, DLS, TEM and CD proved that the optimized fabri­
cation as well purification protocols resulted in the formation of
colloidal drug carriers with average diameter of ca. 200− 210 nm via the
partially charge compensation of these macromolecules. The commonly
used long-term heating as well as the application of crosslinking agents,
surfactants and toxic organic solvents were eliminated. It was confirmed
that the pH as well as the applied BSA/HyA mass ratios strongly influ­
ence the size, the size distribution, and the proposed core-shell structure
of the potential drug carrier particles. This work clearly highlights the
importance of detailed physico-chemical characterization of the pure
carriers and drug-containing carriers to design effective nanosystem for
encapsulation. Our results may successfully contribute to the develop­
ment of promising drug delivery and controlled drug release colloidal
systems in the future.
CRediT authorship contribution statement
´cs: Methodology, Investigation, Writing - orig­
Alexandra N. Kova
inal draft. Norbert Varga: Methodology, Investigation, Visualization.
´ a
´m Juha
´sz: Validation, Formal analysis, Visualization. Edit Csapo
´:
Ad
Conceptualization, Resources, Writing - review & editing, Supervision.
Acknowledgements
This research was supported by the National Research, Development
and Innovation Office -NKDIH through GINOP-2.3.2-15-2016-0034,
GINOP-2.3.2-15-2016-0060 and FK131446. The research is supported
´nos Bolyai Research Fellowship of the Hungarian Academy of

by the Ja
Sciences (E. Csap´
o). The authors thank the registration of TEM images
´ria Hornok (University of Szeged, Department of Physical
for Vikto
Chemistry and Materials Science).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi: />References
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