BioNanoScience

https://doi.org/10.1007/s12668-024-01323-1

RESEARCH

Doxorubicin Folic Acid‑Chitosan Conjugate‑Based Super Paramagnetic

Erythrocyte‑Loaded Nano Delivery System as Onco‑theranostic
Platform
Niloofar Dezham1 · Abdolhossein Zarrin2 · Elahehnaz Parhizkar1 · Parisa Ghasemiyeh3 ·
Soliman Mohammadi Samani1,3

Accepted: 1 February 2024

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024

Abstract
Doxorubicin (DOX) is a broad-spectrum antineoplastic agent that is used in hepatocellular carcinoma (HCC) treatment
regimens. In this study, DOX was loaded in folic acid (FA)-chitosan (Cs) conjugate-coated superparamagnetic iron oxide
nanoparticles (SPIONs) (DOX@FA-Cs/SPIONs) and then further encapsulated within erythrocytes to act as a liver-
specific onco-theranostic system for HCC management. In this regard, SPIONs were prepared through the co-precipitation
technique. The particle size and morphology of the prepared SPIONs were assessed through transmission electron microscopy
(TEM). Then, FA and Cs were conjugated through a chemical bond and were characterized through the FTIR and 1H
NMR spectrum. After that, FA-Cs conjugate was used to coat SPIONs, and finally, DOX was loaded and DOX@FA-Cs/
SPIONs were stabilized by the ionic gelation technique using sodium tripolyphosphate (TPP) as cross-linker. Particle size
and morphology, zeta potential, drug loading, drug release, and paramagnetic behavior of the particles were assessed in
the optimized formulation. Finally, the prepared DOX nanoparticles were further encapsulated in erythrocytes, and they
were optimized and characterized. FTIR and 1H NMR spectrum confirmed the FA-Cs conjugate formation. TEM results
revealed that DOX@FA-Cs/SPIONs were spherical in shape with an average diameter of ~ 20 nm, and their zeta potential
was + 15.3 mV. The results revealed that the entrapment efficiency and loading capacity of DOX within FA-Cs/SPIONs
were 36.0 ± 1.5% and 11.8 ± 0.5%, respectively. The results of the drug release revealed a much higher and faster release
rate in acidic pH. Vibrating sample magnetometry results revealed the preserved superparamagnetic properties of FA-Cs/
SPIONs. Encapsulation of DOX@FA-Cs/SPIONs within erythrocytes was accompanied by a high entrapment efficiency of
57 ± 2% and could preserve their magnetic properties. This novel DOX delivery system would be a promising liver-specific
onco-theranostic system for HCC management.

Keywords Doxorubicin · Folic acid-chitosan conjugate-coated superparamagnetic iron oxide nanoparticles (SPIONs) (FA-
Cs/SPIONs) · Erythrocytes · Hepatocellular carcinoma

1 Introduction

Primary liver cancer is the sixth most prevalent cancer and the
third leading cause of death among cancers worldwide [1].
* Soliman Mohammadi Samani Hepatocellular carcinoma (HCC) is the most common type
smsamani@sums.ac.ir (80–90%) of primary liver cancer [2]. Due to the enhanced
1 rate of alcohol consumption and also increased prevalence of
Department of Pharmaceutics, School of Pharmacy, Shiraz
University of Medical Sciences, Shiraz-Marvdasht Hwy, obesity, the incidence of liver cancer has increased in some
Karafarin St, Shiraz 71468 64685, Fars Province, Iran nations including the USA [3]. Liver cancer is considered
2
Medicinal and Natural Products Chemistry, Shiraz University a poor prognosis cancer. Various therapeutic interventions
of Medical Sciences, Shiraz, Iran including surgical approach, trans-arterial chemoemboliza-
3
Pharmaceutical Sciences Research Center, Shiraz University tion (TACE), and chemotherapy have been considered based
of Medical Sciences, Shiraz, Iran on the stages of liver cancer [4]. Doxorubicin (DOX) is an

Vol.:(0123456789)

anthracycline chemotherapeutic agent with broad-spectrum
anti-tumor effects and can be used in HCC management. The
main challenge of DOX therapy is the potential cardiotoxic-
ity occurrence, especially at higher cumulative doses [2]. In
order to prevent or at least reduce the risk of DOX-induced
cardiotoxicity, the recruitment of nanotechnology and drug
encapsulation within the nanocarriers would be promising
[5]. Furthermore, encapsulation of DOX within nanoparticles
with desired characteristics can lead to specific targeted drug
delivery to the cancer cells [6].
Superparamagnetic iron oxide nanoparticles (SPIONs) are
made of iron oxide cores with internal superparamagnetic
potential that can be specifically directed to the desired
target areas through the application of external magnets. In
this regard, spatial drug targeting in cancer treatment can
be achieved through the encapsulation of chemotherapeutic
agents within the SPIONs. Therefore, the general systemic
distribution of the administered chemotherapeutic agents and
further unwanted adverse drug reactions could be avoided.
Furthermore, the spatial targeting potential of SPIONs might
reduce the required dose of the chemotherapeutic agents
which in turn can lead to a substantial reduction in adverse
drug reactions [7]. Cs, as a poly-cationic, biocompatible, and
biodegradable polymer, has been used widely in the coating
of SPIONs. SPIONs coating with hydrophilic polymers can
prevent nanoparticle aggregation and also can reduce SPIONs
toxicity,therefore, Cs-coated SPIONs would be more desired
for cancer diagnosis and therapy purposes [8]. In addition, due
to the over-expression of folic acid (FA) receptors at the surface
of cancer cells [9], FA-Cs conjugate-coated SPIONs would be
promising delivery carriers as a nano-theranostic system in
cancer management with active cellular targeting potential [10].
Red blood cells (RBCs) or erythrocytes are considered safe
drug delivery systems in cancer management due to their high
drug-loading capacity with low toxicity potential [11]. In addition,
specific liver organ targeting can be achieved through the recruit-
ment of erythrocytes as nanocarriers in HCC management [12].
The main purpose of this study was to design and charac-
terize a novel onco-theranostic system for liver cancer man-
agement. In this regard, first of all, FA-Cs conjugate-coated
SPIONs (FA-Cs/SPIONs) were prepared, and then DOX was
encapsulated (DOX@FA-Cs/SPIONs). After that, the pre-
pared DOX nanocarriers were further encapsulated within the
erythrocytes for targeted drug delivery to the liver organ and
hepatocytes with over-expressed folate receptors.
2 Results
2.1 DOX Assay Validation
Reversed-phase high-performance liquid chromatography
(RP-HPLC) method for DOX assay was validated according
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to the ICH guidelines. The results revealed that the validated
RP-HPLC method is specific for DOX analysis in pharma-
ceutical matrices. This validated method had sufficient linear-
ity (R2 > 0.999) in concentration ranges of 1–50 µg/mL, high
sensitivity (with a limit of detection (LOD) and limit of quan-
tification (LOQ) of 0.3 µg/mL and 1 µg/mL, respectively),
and adequate intra- and inter-day precision (%CV < 10%) and
accuracy (89–99%) values. As shown in Fig. 1, DOX had a
retention time of 5.3 min at λmax of 233 nm, and no interaction
was observed between DOX and solvent peaks.
2.2 Synthesis and Characterization of FA‑Cs
Conjugate
2.2.1 Fourier Transform Infrared Spectroscopy (FTIR)
As shown in Fig. 2, the FTIR results revealed that in the area
of 3200 ­cm−1, the peaks related to the hydroxyl (–OH) and
amine (–NH2) group of Cs were observed in the blue spectrum,
respectively, while this peak was softened in the FA-Cs conju-
gate (green spectrum). In addition, results revealed that in the
area of 1020–1230 ­cm−1, the peaks related to the C–O bond
were observed for Cs, while this peak was shifted in the FA-Cs
conjugate spectrum due to the amide bond formation. Further-
more, in the area of 1650–1693 ­cm−1, the peaks related to the
amide and carboxylic acid functional groups were observed
in the FA (blue) spectrum. A sharp peak in the area of 1600
­cm−1 was observed for the FA-Cs conjugate spectrum which
is correspondence to the newly formed amide bond between
the –NH2 group of Cs and –COOH group of FA.
2.2.2 Proton Nuclear Magnetic Resonance (.1H NMR)
As shown in Fig. 3, the signals at 0.930–1.002, 2.083,
2.424–2.799, and 3.519–3.872 ppm were assigned to the
resonance of the monosaccharide residue protons, –CH3,
–CH–NH–, –CH2–OH–, and –CH2, respectively. In addi-
tion, the signals observed at 6.403–8.174 ppm were
attributed to the resonance of the FA aromatic protons.
Therefore, 1H NMR results revealed that the FA has been
successfully conjugated to Cs through an amide bond.
2.2.3 Conjugation Efficiency
Based on the results, a conjugation efficiency of 75 ± 2%
was obtained that confirmed sufficient conjugation of Cs
with FA molecules has occurred.
2.3 Optimization of DOX@FA‑Cs/SPIONs
DOX@FA-Cs/SPIONs, as an onco-theranostic system, were
prepared and optimized in terms of iron to Cs ratio, sodium
BioNanoScience

Fig. 1  A representative chromatogram of doxorubicin (DOX) (2.5 µg/mL) assay using the RP-HPLC method

tripolyphosphate (TPP) to Cs ratio, and drug to Cs ratio in stability and no precipitation during the storage stability
order to achieve an optimum formulation with the highest assessments.

Fig. 2  FTIR spectra of folic acid (red), chitosan (blue), and folic acid-chitosan conjugate (green)
 BioNanoScience

Fig. 3  1H NMR spectrum of folic acid-chitosan conjugate

2.3.1 Optimization of Iron to Cs Ratio 2.3.2 Optimization of TPP to Cs Ratio

Among the various ratios of iron to Cs in DOX Among the various ratios of TPP to Cs in DOX nano-
nanoparticle formulations, the ratio of 1:3 with the most particle formation, the ratio of 0.33:1 with the higher
stability and no precipitation during 5 days of stability stability and no precipitation during 5 days of stability
assessment was selected and used for further assessments. assessment was selected. The sample without TPP had the
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Table 1  Entrapment efficiency (EE%) and loading capacity (LC%) of 2.4.2 Particle Size
doxorubicin (DOX) nanoparticles with various drug to chitosan ratios
Drug to chitosan ratio EE% LC% As shown in Fig. 5, the results of particle size analyzer
(PSA) confirmed that the size of the FA-Cs/SPIONs was
1:1 36.0 ± 1.5% 11.8 ± 0.5%
increased up to 92 nm after Cs coating.
0.5:1 16.8 ± 0.8% 3.3 ± 0.2%
0.25:1 23.0 ± 1.5% 2.5 ± 0.2%
2.4.3 Zeta Potential

Fig. 4  TEM of SPIONs (A) and folic acid-chitosan conjugate-coated SPIONs (B)

lowest storage stability and showed early precipitation.

The zeta potential of the prepared DOX@FA-Cs/SPIONs
The electrostatic reaction between the positive charge of
was + 15.3. The positive zeta potential confirmed the locali-
the amine group ­(NH3+) of Cs, and the negative charge of
zation of Cs with a net positive charge on the surface of the
the phosphate group of TPP can lead to the aggregation of
nanoparticles. Furthermore, this positive zeta potential can
Cs particles and TPP can act as a cross-linker among them.
lead to a better interaction with cancer cells.
2.3.3 Optimization of the Drug to Cs Ratio
2.4.4 Vibrating Sample Magnetometry (VSM)
Among the various ratios of the drug to Cs in DOX
Vibrating sample magnetometry was performed for both
nanoparticles, the ratio of 1:1 of DOX to Cs with the highest
SPIONs and FA-Cs conjugate-coated SPIONs. As it is obvi-
EE% and LC% values was selected according to Table 1.
ous in Fig. 6, the results revealed that although coating of
SPIONs with FA-Cs conjugate reduced their superparamag-
2.4 Characterization of DOX@FA‑Cs/SPIONs
netic intensity, however, these nanoparticles still are super-
paramagnetic and respond to the magnetic field accordingly.
2.4.1 Transmission Electron Microscopy (TEM)
Therefore, it seems that the superparamagnetic capability of
SPIONs still remained after coating with FA-Cs conjugate,
TEM was performed to characterize and compare the mor-
and they could be affected by the external magnetic field to
phology and particle size of the DOX@SPIONs and DOX@
help target the cancer tissues at specified sites of the body.
FA-Cs/SPIONs. As shown in Fig. 4, the results revealed that
the prepared SPIONs had an average particle size of ~ 20
2.4.5 Drug Loading Assessment
nm and were spherical in shape. The DOX@FA-Cs/SPIONs
showed the same particle size ranges and also were spherical
Drug loading assessment through direct and indirect
in shape. In addition, the FA-Cs conjugate coating on the
techniques led to the same results. As shown in Table 1,
surface of the prepared SPIONs was confirmed and is com-
nanoparticles with a drug to Cs ratio of 1:1 had the highest
pletely obvious in Fig. 4. As it is obvious from this figure,
entrapment efficiency (EE%) of 36.0 ± 1.5% and loading
the density of black color was reduced profoundly due to the
capacity (LC%) of 11.8 ± 0.5%.
coating of SPIONs by FA-Cs conjugate.

Fig. 5  PSA of folic acid-chitosan conjugate-coated SPIONs
Fig. 6  VSM diagrams of SPIONs and folic acid-chitosan conjugate-
coated SPIONs
2.4.6 Drug Release Assessment
As shown in Fig. 7, the results revealed that drug release
from DOX@FA-Cs/SPIONs during 72 h is higher in an
acidic pH of 5.4 in contrast to the physiologic pH of
7.4. Therefore, it seems that the prepared nanoparticles
can release more drugs within the acidic environment
of cancer cells in comparison to the normal tissues of
the body. Moreover, since after the entrance of DOX
nanoparticles into the cells, they will internalize to the
lysozymes with much more acidic pH; therefore, faster
release rate with a higher extent would be predictable.
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Fig. 7  Doxorubicin (DOX) release pattern from folic acid-chitosan con-
jugate-coated SPIONs during 72 h at two various pHs of 5.4 and 7.4
2.5 Encapsulation of DOX Nanoparticles Within
Erythrocytes
2.5.1 Assessment of the DOX@FA‑Cs/SPIONs Loading
Within Erythrocytes
In this regard, first of all, a calibration curve of Fe(NO3)3 was
designed using the atomic absorption spectrometry method.
The calibration curve resulted in an R2 value of > 0.999 with
the linear equation of y = 0.2085x + 0.0626 at the concentration
ranges from 1 to 20 µg/mL. At first, the amount of internal
iron within erythrocytes was calculated as 573 µg/mL through
the obtained equation. Since the average amount of iron within
erythrocytes of the normal population is about 600–800 mg/L,
therefore the obtained value was in acceptable ranges.
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Table 2  Iron concentrations Formulation Osmolarity of the buffer
of the prepared doxorubicin (Osm)
(DOX) nanoparticles
encapsulated in erythrocytes F1 0.50 Osm
F2 0.50 Osm
F3 0.67 Osm
Table 3  A comparison of the iron concentrations, iron amounts,
and entrapment efficiency (EE%) of iron within doxorubicin (DOX)
nanoparticles encapsulated in erythrocytes, sham samples, and whole
blood
Iron concentration (µg/mL) EE%
Whole blood 572.5 ± 22.5 µg/mL - 2.6 Erythrocyte Indices Assessments
Sham sample 547.5 ± 27.5 µg/mL − 4.3
DOX nanoparticles 690.0 ± 32.5 µg/mL 57.0 ± 2.0
encapsulated in
erythrocytes
As shown in Table 2, among the prepared samples with
various osmolarities and DOX nanoparticle amounts, F3
with the highest iron concentration was selected as the opti-
mum formulation for further characterizations.
After that, the amount of iron encapsulated within
the erythrocyte nanocarriers was assessed for optimum
formulation (F3), and the obtained results were compared
with that of the sham sample and the whole blood as
summarized in Table 3. According to the results, EE% of
iron within erythrocyte nanocarriers was 57.0 ± 2.0%. Based
on the results, the sham sample revealed a reduced iron
concentration in comparison to the whole blood and also a
negative EE% value due to the hemoglobin depletion from
the erythrocytes membrane. However, DOX nanoparticles
that were encapsulated within erythrocytes showed a higher
iron concentration due to the high EE% of the DOX@FA-Cs/
SPIONs within the erythrocytes.
2.5.2 Magnetometry
Results of the magnetometry assessment via holding of a
strong external magnet revealed that the sham sample and
whole blood had no super para magnetic behavior; however,
DOX@FA-Cs/SPIONs that were encapsulated within
erythrocytes showed an intense magnetic movement.
2.5.3 Scanning Electron Microscopy (SEM)
As it is obvious in Fig. 8, SEM results revealed that although the
structure of erythrocytes in the sham sample had no significant
difference with whole blood; however, SEM of DOX@FA-Cs/
SPIONs that encapsulated within erythrocytes encountered
Amount of DOX Iron concentration (µg/mL)
nanoparticles (mL)
1 mL 75.0 ± 12.5 µg/mL
2 mL 532.5 ± 30.0 µg/mL
1 mL 690.0 ± 32.5 µg/mL
significant changes in erythrocytes structure. Therefore, these
changes in erythrocytes as nanocarriers can result in accumula-
tion within the reticuloendothelial system and liver, and hence
targeted delivery to the liver can be achieved more efficiently.
As summarized in Table 4, all erythrocyte indices including
mean corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH), and mean corpuscular hemoglobin
concentration (MCHC) were reduced in the sham sample and
also in DOX@FA-Cs/SPIONs nanoparticles encapsulated
within erythrocytes in comparison with the whole blood
sample. This reduction was much higher for sham samples in
comparison to the prepared erythrocyte-loaded nanocarriers.
3 Discussion
3.1 Synthesis and Characterization of FA‑Cs
Conjugate
The conjugation of Cs to FA was confirmed through the FTIR
and 1H NMR methods. The obtained results were in agree-
ment with the results of the previously published study [10].
In our previous study, the formation of the conjugation was
also confirmed through thermal gravimetric assay (TGA)
in which all FA molecules were conjugated to the Cs, and
the remaining unconjugated ones were removed completely
through the dialysis membrane [10]. In addition, the conjuga-
tion efficiency of 75 ± 2% was compatible with the results of
the previous study on FA-Cs conjugated nanoparticles with a
conjugation yield range of 53.2 to 81.6% [13]. Another study
on FA-modified carboxymethyl Cs nanoparticles revealed a
conjugation efficiency of 75% which was compatible with
the results of the current study [14]. Moreover, the results of
another study on SN-38-loaded FA-conjugated Cs nanopar-
ticles revealed a conjugation efficiency of 73.3% [15].
FA modification can induce active tumor-targeting
potential, especially in liver cancer and hepatocellular car-
cinoma, through the enhanced intracellular uptake of the
prepared nanoparticles. Therefore, it can inhibit the tumor
cell growth process, induce apoptosis, enhance reactive
oxygen species (ROSs), and, as a result, reduce the tumor
cell recovery and migration [16].
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Fig. 8  SEM of whole blood (A), sham sample (B), and doxorubicin (DOX) nanoparticles (DOX@FA-Cs/SPIONs) encapsulated within erythro-
cytes (C)

was compatible with the results of a previous study on metroni-

Table 4  Comparison of erythrocyte indices of whole blood, sham dazole-loaded Cs-TPP nanoparticles with an optimized TPP to
sample, and doxorubicin (DOX) nanoparticles encapsulated within
Cs ratio of 0.25:1 [17]. Another study on Cs-dextran-SPIONs
erythrocytes
reported an optimum TPP to Cs ratio of 0.16:1 [18]. In addi-
Erythrocyte Whole blood Sham sample DOX nanoparticles tion, the optimum molar ratio of iron to Cs in the current onco-
indices encapsulated in
erythrocytes theranostic system was 1:3, while this ratio was 1:1 in a previous
study on magnetic ­Fe3O4-Cs nanoparticles [19]. Furthermore,
MCV1 (fL) 91.3 ± 0.7 80.8 ± 1.0 82.1 ± 1.5 the optimum drug to Cs ratio in the current study was 1:1 which
MCH2 (pg) 30.9 ± 1.8 20.5 ± 1.3 25.4 ± 0.9 was much higher than that of a previous study on aceclofenac-
MCHC3 (g/dL) 34.7 ± 1.2 21.7 ± 0.8 27.5 ± 2.1 encapsulated Cs microspheres with an optimum drug to Cs ratio
1 of 1:3 [20]. These differences could be attributed to the different
Mean corpuscular volume
2 nature of the loaded compounds and also probably the different
Mean corpuscular hemoglobin
3
molecular weights of the Cs that are used in different studies.
Mean corpuscular hemoglobin concentration

3.2 Optimization of DOX@FA‑Cs/SPIONs
DOX@FA-Cs/SPIONs, which could act as an onco-theranostic
system, were prepared and optimized in this study. In this regard,
the optimum formulation had a TPP to Cs ratio of 0.33:1 which
3.3 Characterization of DOX@FA‑Cs/SPIONs
3.3.1 Transmission Electron Microscopy (TEM)
TEM results revealed that the prepared DOX@FA-Cs/SPIONs
were spherical in shape with an average diameter of ~ 20 nm.
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These results were compatible with the results of the previous
study [10]. In Addition, TEM results of a previous study on Cs-
coated SPIONs showed that the prepared nanoparticles were
spherical in shape and had an average diameter of < 35 nm that
were also in accordance with that of the current study [21].
3.3.2 Zeta Potential
The prepared DOX@FA-Cs/SPIONs had a zeta potential
of + 15.3. This positive zeta potential can induce the desired
stability in prepared nanoparticles. The SPIONs have negative
zeta potential [22, 23], however, due to the predominance of the
amino groups in FA-Cs/SPIONs, the net charge was switched to
positive [10, 24]. Furthermore, this positive zeta potential can
lead to better interaction with cancer cells [25, 26].
3.3.3 Vibrating Sample Magnetometry (VSM)
VSM results revealed that although the coating of
SPIONs with FA-Cs conjugate was accompanied by a
reduction in superparamagnetic properties, however, their
superparamagnetic properties were still preserved after
coating with polymers including Cs. This reduction in
superparamagnetic potential in FA-Cs conjugate-coated
SPIONs can be attributed to the protective effect of polymer
coat on the surface of the SPIONs [10]. In addition, the lack
of hysteresis loop in the VSM profile of both nude SPIONs
and FA-Cs/SPIONs confirmed their superparamagnetic
properties. The preserved amount of superparamagnetic
behavior and magnetization of the prepared FA-Cs/SPIONs
is sufficient enough to target the desired cancer cells through
the application of an external magnetic field [27].
3.3.4 Drug Loading
The results revealed the EE% and LC% of 36.0 ± 1.5%
and 11.8 ± 0.5%, respectively. These obtained values
were somewhat lower than those of the previous studies
on SPIONs [28, 29, 27]. These differences also could be
attributed to the nature of the loaded compound and different
loading techniques.
3.3.5 Drug Release
Results of the drug release from DOX@FA-Cs/SPIONs
revealed that the amount of released drug at an acidic
pH of 5.4 was much more than that of the physiologic
pH of 7.4. Therefore, it seems that DOX release from the
prepared nanocarriers would be higher within the acidic
tumor microenvironment in comparison to the normal
cells with physiologic pH [30]. In this regard, this delivery
system would be more capable of active targeting of the
cytotoxic drug to the tumor site. The obtained results
were also compatible with the results of a previous study
on multifunctional DOX-loaded SPIONs which showed a
higher release rate along with a higher extent at the acidic
pH of 5.1 in comparison to the pH of 7.4 [31]. Another study
on DOX-loaded SPIONs revealed 58% burst release in the
first hour at the acidic pH, while this amount was 38.6% at
the neutral pH [32].
3.4 Encapsulation of DOX Nanoparticles Within
Erythrocytes
The prepared DOX@FA-Cs/SPIONs can obtain spatial tar-
geting through the recruitment of an external magnet [33,
34]. In order to achieve special organ targeting for the pur-
pose of liver cancer diagnosis and treatment, the prepared
DOX nanoparticles were further encapsulated within eryth-
rocytes to specifically target the liver organ. This goal can
be achieved through the hypotonic pre-swelling of eryth-
rocytes after the encapsulation of DOX@FA-Cs/SPIONs
within their structure. Therefore, erythrocyte morphologi-
cal changes during the loading process in turn can lead
to erythrocyte accumulation within the reticuloendothelial
system of the liver, and specific drug delivery to the liver
cancer cells would be achieved. In addition, the presence of
FA on the surface of SPIONs can lead to specific cellular
targeting due to the over-expression of the FA receptors at
the surface of the cancer cells [35].
3.5 Characterization of DOX Nanoparticles
Encapsulated in Erythrocytes
According to the results, EE% of DOX@FA-Cs/
SPIONs that encapsulated within erythrocytes was
57 ± 2% which was relatively superior to the results
of the previous study on valproate-loaded hydrogel
nanoparticles that encapsulated within erythrocytes with
EE% of 42.07 ± 3.6% [36]. The results of a recent study
on SPIONs-loaded erythrocytes revealed an iron oxides
entrapment wide range of 1.6 to 16.4 mM Fe based on the
amount of SPIONs added to 1 mL of erythrocytes [37].
The results revealed that the prepared DOX@FA-Cs/
SPIONs encapsulated within erythrocytes had magnetic
properties, while sham cells and the whole blood failed to
show magnetic properties in the presence of a strong external
magnet. These results were the same as the results of a pre-
vious study on SPIONs-loaded erythrocytes which showed
sufficient magnetic properties for magnetic resonance imag-
ing (MRI) purposes [38]. In addition, another study on albu-
min and magnetic nanoparticle-loaded erythrocytes reported
the same results [38]. Moreover, the results of a previous
study on SPIONs encapsulated in erythrocytes revealed the
high superparamagnetic potential of the prepared carriers in

comparison to naïve erythrocytes [39] which was compatible
with the results obtained in the current study.
4 Conclusion
In conclusion, FA-Cs/SPIONs were prepared and then DOX
was encapsulated. After that, the prepared DOX@FA-Cs/
SPIONs were further loaded within the erythrocytes for
targeted drug delivery to the liver organ and hepatocytes
with over-expressed folate receptors. The prepared DOX@
FA-Cs/SPIONs showed a higher and faster release rate at
acidic environment; therefore, this delivery system would be
more capable of both active and passive targeting of DOX to
the tumor site. In addition, VSM results also confirmed the
superparamagnetic properties of FA-Cs/SPIONs which in turn
can lead to spatial targeted delivery through the application
of an external magnet. DOX@FA-Cs/SPIONs encapsulation
within the erythrocytes was accompanied by a high entrapment
efficiency, and their magnetic properties were still retained.
Consequently, this novel DOX delivery system can act as a
liver-specific onco-theranostic system for HCC management.
5 Materials and Methods
5.1 Materials
Sodium tripolyphosphate (TPP), glacial acetic acid, ammonia
­(NH3), iron(III) chloride hexahydrate ­(FeCl3.6H2O), iron(II)
chloride tetrahydrate (­ FeCl2.4H2O), hydrochloric acid (HCl),
sodium acetate, dimethyl sulfoxide (DMSO), sodium chlo-
ride, potassium chloride, sodium acetate, disodium hydrogen
phosphate, potassium dihydrogen phosphate, nitric acid, and
phosphoric acid were purchased from Merck, Germany. Cs
(MW of 600 kDa), 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide (EDC), and N-hydroxysuccinimide (NHS) were
obtained from Sigma-Aldrich, USA. Acetonitrile and metha-
nol were HPLC grade and purchased from Samchun Chemi-
cals, Korea. FA was kindly gifted by Jalinous Pharmaceutical
Company, Iran.
5.2 DOX Assay
DOX was assessed through a validated RP-HPLC method
equipped with a UV detector (Cecil, Milton Technical
Centre, England). For this purpose, a C18 HPLC column
(C18-5 µm, 250 × 4.6 mm, Hichrome, UK) was utilized as
the static phase. The loop volume was 20 µL. The mobile
phase consisted of acetonitrile to phosphate buffer (pH = 3)
with a ratio of 30:70%v/v. The flow rate was set to 1 mL/
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min, the column temperature was fixed at 25 °C, and the
λmax was set at 233 nm.
DOX hydrochloride was dissolved in distilled water,
and the prepared standard solutions with a concentration
range of 1–50 µg/mL were assessed in triplicate on three
different days to assess intra- and inter-day precision and
accuracy of the validated HPLC method.
5.3 Preparation of Superparamagnetic Iron Oxide
Nanoparticles (SPIONs)
SPIONs were prepared through a modified co-precipitation
technique [40, 10]. In this regard, 1.62 g ­F eCl 3.6H 2O
(10 mmol F ­ e3+) and 0.6 g F
­ eCl2.4H2O (4.7 mmol F ­ e2+)
were dissolved in 30 mL and 60 mL deionized water,
respectively. After preparation of these two separate
solutions, the solutions were mixed together and then
6 mL ammonia (25%w/v) was added dropwise to the
aqueous solution under continuous stirring. During
ammonia addition, the solution’s color switched to black
which was a confirmation of F ­ e3O4 super para magnetic
nanoparticles formation. After that, the ­N2 gas was purged
to the prepared solution under vigorous stirring for 15
min. Finally, the iron oxide ­(Fe3O4) nanoparticles were
separated by an external magnet.
5.4 Characterization of Superparamagnetic Iron
Oxide Nanoparticles (SPIONs)
5.4.1 Particle Size and Morphology Assessment
The particle size and morphology of the prepared SPIONs
were assessed using particle size analyzer (PSA; Shimadzu,
SALD-2101, Japan) and also transmission electron micros-
copy (TEM) (EM10C, Zeiss, Germany). In this regard, a drop
of the prepared sample was fixed on a carbon-coated copper
grid, and the grid was assessed using the TEM technique.
5.5 Synthesis of FA‑Cs Conjugate
In order to induce chemical binding between Cs (MW = 600
kDa) and FA, first of all, the carboxylic group of the FA should
be activated [10]. In this regard, 100 mg FA (0.2 mmol), 62 mg
EDC (0.4 mmol), and 46 mg NHS (0.4 mmol) were dissolved
in 4 mL DMSO and mixed using a magnetic stirrer for 1 h (in
a dark room). Then, 70 mg Cs (0.05 mmol) was dissolved in
25 mL acetate buffer (pH = 4.7) and continuously mixed for 1
h. After that, the Cs solution was added to the FA mixture and
mixed again for 16 h in order to form a chemical bond between
the carboxyl group of FA and the amine group of Cs (with a
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stoichiometric ratio of 4:1). After that, in order to precipitate The prepared onco-theranostic system, DOX@FA-Cs/SPIONs,
the fabricated FA-Cs conjugate, NaOH 1 N was added drop- was separated through centrifugation (16,000 rpm for 20 min).
wise to change the pH of the final solution to 9. Afterward, the
sample was poured into a dialysis membrane (Dialysis tub- 5.8 Optimization of DOX@FA‑Cs/SPIONs
ing cellulose membrane, Sigma-Aldrich, cut off 12 kDa) and
floated in phosphate-buffered saline (PBS, pH = 7.4) for 3 days 5.8.1 Optimization of Iron/Cs Ratio
in order to separate the unconjugated FA molecules. Finally,
the sample within the dialysis membrane was freeze-dried, and In this regard, various molar ratios of iron to Cs including
the FA-Cs conjugate was collected. 1:1, 1:2, 1:3, and 1:4 were prepared. The SPIONs were
added to the FA-Cs conjugate solution and mixed for 18
h using a magnetic stirrer. The optimum molar ratio of
5.6 Characterization of FA‑Cs Conjugate iron/Cs was selected through the stability assessment. The
formulation with the highest stability and no precipitation
5.6.1 Fourier Transform Infrared Spectroscopy (FTIR) after 5 days of preparation was chosen as the optimum one.
Assessment
5.8.2 Optimization of TPP/Cs Ratio
FA-Cs conjugate was confirmed using the FTIR technique
(Vertex 70, Bruker, Germany). In this regard, the FTIR spec-
To do so, various weight ratios of TPP to Cs including 0.16:1,
trums of FA, Cs, and FA-Cs conjugate were assessed in a
0.33:1, 1:1, and the sample without TPP (0:1) were assessed.
range of 400–4000 c­ m−1 in potassium bromide disks, and the
TPP was added dropwise to the FA-Cs conjugate and mixed
results were compared to confirm the conjugate formation.
for half an hour. The optimum Cs to TPP ratio was selected
through the stability assessment.
5.6.2 1H NMR Assessment
5.8.3 Optimization of Drug/Cs Ratio
FA-Cs conjugation was also confirmed using the 1H NMR
technique (Ascend 600, Bruker, Germany) at 400 MHz.
In this regard, various weight ratios of DOX to Cs including
0.25:1, 0.5:1, and 1:1 were assessed. The optimum ratio that
5.6.3 Conjugation Efficiency Assessment could induce the highest drug loading amount within the
nanoparticles was selected among the assessed ratios.
The conjugation efficiency of FA-Cs conjugate was assessed
through a validated UV spectrophotometric method (UV1650PC, 5.9 Characterization of DOX@FA‑Cs/SPIONs
Shimadzu, Germany). In this regard, the unconjugated (free)
FA was analyzed at λmax of 368 nm. Therefore, the conjugation 5.9.1 Particle Size and Morphology Assessment
efficiency was assessed indirectly according to Eq. 1.
Total folic acid − Unconjugated folic acid The particle size and morphology of the prepared DOX@FA-Cs/
%Conjugation ef f iciency =
Total folic acid
× 100 (1) SPIONs were assessed using TEM (EM10C, Zeiss, Germany).
In this regard, a drop of the prepared sample was fixed on a
carbon-coated copper grid, and the grid was assessed using the
5.7 Preparation of DOX@FA‑Cs/SPIONs TEM technique. The obtained results were compared to SPION
morphology.
DOX was loaded after FA-Cs conjugate coating of SPIONs and
FA-Cs/SPIONs were stabilized through the ionic gelation tech-
5.9.2 Zeta Potential
nique using sodium tripolyphosphate (TPP) as a cross-linker
[10]. In this regard, 10 mg of the previously prepared SPIONs
The zeta potential of the prepared DOX@FA-Cs/SPIONs was
were dissolved in FA-Cs conjugate solution 1%w/v (pH = 4.7)
assessed using Zetasizer (Microtrac, USA).
and underwent stirring for 18 h. Then, the prepared FA-Cs/
SPIONs were separated through centrifugation (16,000 rpm
5.9.3 Vibrating Sample Magnetometry (VSM)
for 20 min). After that, DOX was added to the prepared sample
and mixed through a magnetic stirrer for 24 h. Finally, the ionic
The magnetic properties of the prepared samples were assessed
gelation process was induced through the dropwise addition of
through the vibrating sample magnetometer (Lake Shire 7400,
TPP to the prepared solution under the magnetic stirrer (Hei-
USA) at room temperature from − 18,500 to + 18,500 Oe.
dolph, Germany), and stirring was continued for half an hour.

5.9.4 Drug Loading Assessment
Loading capacity (LC%) and entrapment efficiency (EE%) of
DOX were assessed using both direct and indirect methods. In
the direct method, the separated nanoparticles through the cen-
trifugation technique (the precipitant which contained DOX@
FA-Cs/SPIONs) were dispersed in acetate buffer (pH = 4.7).
Then, the prepared sample was diluted, and the DOX amount
was analyzed through the validated HPLC method. In the indi-
rect method, the supernatant, which contained the free DOX,
was diluted and analyzed with HPLC. LC% and EE% were
assessed according to Eqs. 2 and 3 for both methods.
Weight of loaded drug
%LC =
Weight of loaded drug + Weight of nanoparticles
× 100
Loaded drug
EE% = × 100
Total drug
5.9.5 Drug Release Assessment
In order to assess the drug release profile from DOX@
FA-Cs/SPIONs, 2 mg of the prepared nanoparticles were
dispersed in 2 mL of phosphate buffer at 2 different pHs of
5.4 and 7.4. Drug release assessment was performed at 37
°C and a stirring rate of 150 rpm. Sampling was done at 0,
1, 2, 3, 6, 12, 20, 24, 48, and 72 h and replaced with aliquot
amounts of the fresh phosphate buffer. The amount of DOX
in the prepared samples was analyzed using the HPLC
method, and the DOX release profile from the designed
onco-theranostic system was compared at pH of 5.4 and 7.4.
5.10 Encapsulation of DOX Nanoparticles Within
Erythrocytes
In order to encapsulate DOX nanoparticles within
erythrocytes, whole blood, which was unknown, was
obtained from Blood Transfusion Organization, Shiraz,
Iran, and stored in heparinized tubes. Erythrocytes were
separated through the centrifugation of whole blood at 5000
rpm for 10 min. In this regard, the plasma and buffy coat
were separated, and the remaining packed red blood cells
(RBCs) were washed with isotonic phosphate buffered saline
(PBS, pH = 7.4) in triplicate. Then, 1 mL of the isolated
erythrocytes were treated with 4 mL of hypotonic phosphate
buffer to deplete hemoglobin from erythrocytes and form
the erythrocyte ghosts. In order to assess the effect of buffer
osmolarity on erythrocyte lysis, the isolated erythrocytes
were separated into 3 tubes (each containing 1 mL of packed
RBCs) and treated with hypotonic buffer with osmolarity
of 0.5, 0.5, and 0.67 Osm, respectively. After that, the
samples were centrifuged at 2000 rpm for 10 min and the
BioNanoScience
precipitated swollen cells were separated. Afterward, 200
µL of hemolysate (erythrocyte: distilled water with 1:1 v/v
ratio) was added to the swelled cells. Subsequently, 1, 1,
and 2 mL of DOX nanoparticles were added to the samples
with the osmolarity of 0.5, 0.67, and 0.5 Osm, respectively,
and centrifuged at 2000 rpm for 5 min. Then, 400 µL of
hypertonic phosphate buffer 10 × was added to each sample
and incubated at 37 °C. Finally, the erythrocytes were
washed with 10 mL of isotonic PBS in triplicate to remove
the free drug and hemoglobin from the prepared samples.
5.10.1 Assessment of Iron Loading Within Erythrocytes
(2) In order to assess the amount of external iron encapsulate
levels (SPIONs) within erythrocytes, at first, the amount of
internal iron levels stored in erythrocytes was calculated. In
(3) this regard, 1 mL of whole blood sample was diluted with 4 mL
of nitric acid 65%w/w. After that, 1 mL of this diluted sample
was further diluted with 49 mL of nitric acid 1 Nz. Finally, the
prepared sample was analyzed using the atomic absorption
spectrometer (Varian, SpectrAA-plus, USA) at the wavelength
of 372 nm. In this regard, iron was dissolved in nitric acid 1
N, and after appropriate dilution at the concentration range
of 1–20 µg/mL, absorbance of the standard solutions was
assessed through atomic absorption spectrometry.
The loading capacity of SPIONs within erythrocytes was
calculated through the comparison of iron amount in free
erythrocytes and erythrocytes encapsulated with DOX@
FA-Cs/SPIONs. In addition, in order to assess the effect of
processes’ impact on erythrocytes and their structure and pos-
sible destruction occurrence, a sham sample was processed in
the same way; however, DOX nanoparticles were not included.
5.10.2 Formulation Optimization
Among the prepared formulations with different phosphate
buffer osmolarities and DOX nanoparticle amounts, the
optimum formulation with the highest iron concentration
was selected.
5.10.3 Magnetic Behavior Assessment
The magnetic properties of DOX@FA-Cs/SPIONs encapsu-
lated in erythrocytes, sham samples, and whole blood were
assessed and compared using a strong external magnet.
5.10.4 Scanning Electron Microscopy (SEM)
The morphology of the prepared samples was assessed using
the SEM technique (Hitachi, F4160, Japan). In this regard,
glutaraldehyde 4% buffered solution, as a fixing agent, was
added to the samples, and the samples were stored in the
BioNanoScience
refrigerator for 2 h. Then, glutaraldehyde was removed
and sodium cacodylate buffer 0.1 M was added. After that,
the samples were fixed at osmium tetroxide for 2 h in the
refrigerator. Thereafter, all samples were washed using the
PBS buffer solution, and then samples were dehydrated by
pure ethanol and were exposed to hexamethyldisilazane.
After 40 min, hexamethyldisilazane was removed, and the
samples were dried at room temperature for 2 h. Finally,
samples were covered with gold and became ready for SEM
assessment.
5.10.5 Erythrocyte Indices Assessments
In order to assess the possible changes in erythrocytes,
various indices including mean corpuscular volume
(MCV), mean corpuscular hemoglobin (MCH), and
mean corpuscular hemoglobin concentration (MCHC)
were analyzed for DOX@FA-Cs/SPIONs encapsulated in
erythrocytes, sham sample, and whole blood.
Abbreviations HCC: Hepatocellular carcinoma; TACE: Trans-arterial
chemoembolization; DOX: Doxorubicin; SPIONs: Superparamagnetic
iron oxide nanoparticles; FA: Folic acid; Cs: Chitosan; FA-Cs/
SPIONs: Folic acid-chitosan conjugate coated SPIONs; DOX@
FA-Cs/SPIONs: Doxorubicin-loaded folic acid-chitosan conjugate-
coated SPIONs; TPP: Sodium tripolyphosphate; NH3: Ammonia;
FeCl3.6H2O: Iron(III) chloride hexahydrate; FeCl 2.4H2O: Iron(II)
chloride tetrahydrate; HCL: Hydrochloric acid; DMSO: Dimethyl
sulfoxide; EDC: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide;
NHS: N-hydroxysuccinimide; RP-HPLC: Reversed-phase
high-performance liquid chromatography; Fe 3 O 4 : Iron oxide;
TEM: Transmission electron microscopy; PSA: Particle size
analyzer; NaOH: Sodium hydroxide; PBS: Phosphate-buffered saline;
FTIR: Fourier transform infrared spectroscopy; VSM: Vibrating sample
magnetometry; RBCs: Red blood cells; LC%: Loading capacity;
EE%: Entrapment efficiency; SEM: Scanning electron microscopy;
MCV: Mean corpuscular volume; MCH: Mean corpuscular
hemoglobin; MCHC: Mean corpuscular hemoglobin concentration;
LOD: Limit of detection; LOQ: Limit of quantification; AUC​: Area
under the curve; TGA​: Thermal gravimetric assay; ROSs: Reactive
oxygen species; MRI: Magnetic resonance imaging
Acknowledgements This study was a part of the Pharm. D. thesis of
Niloofar Dezham. We would like to sincerely appreciate the finan-
cial support of the Vice-Chancellor for Research of Shiraz University
of Medical Sciences [Grant No. 9069] and also Iran’s National Elites
Foundation. The authors would like to appreciate the valuable com-
ments of Dr. Kobra Rostamizadeh and Prof. Mehrdad Hamidi during
this project.
Author Contribution ND contributed to data curation and formal
analysis. AZ contributed to methodology, formal analysis, and
investigation. EP contributed to investigation and formal analysis. PG
contributed to formal analysis, investigation, writing-original draft,
and writing-review & editing. SM-S contributed to conceptualization,
supervision, project administration, resources, methodology, formal
analysis, writing-original draft, writing-review & editing. all authors
approved the final version of the manuscript.
Funding This study was supported by the Vice-Chancellor for
Research of Shiraz University of Medical Sciences [Grant No. 9069].
Data Availability The datasets used and/or analysed during the current
study are available from the corresponding author on reasonable request.
Declarations
Ethical Approval This study was performed in line with the principles
of the Declaration of Helsinki. Approval was granted by the Ethics
Committee of Shiraz University of Medical Sciences (Approval date
07/26/2016, Approval No. IR.SUMS.REC.1395.S655).
Competing Interests The authors declare no competing interests.
Research Involving Humans and Animals Statement None.
Informed Consent None.
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