Alimohammadvand et al.

BMC Biotechnology (2024) 24:108 BMC Biotechnology

https://doi.org/10.1186/s12896-024-00939-1

RESEARCH Open Access

Aripiprazole-loaded niosome/chitosan-gold
nanoparticles for breast cancer chemo-photo
therapy
Sajjad Alimohammadvand1,2, Masoumeh Kaveh Zenjanab1, Parvin Samadi Pakchin1, Elaheh Dalir Abdolahinia1,3,
Jaleh Barar4, Yadollah Omidi4, Mohammad M. Pourseif1,5,6, Marziyeh Fathi1* and Jalal Shayegh7*

Abstract
Introduction Breast cancer, a formidable global health challenge for women, necessitates innovative therapeutic
strategies with enhanced efficacy and minimal side effects. Aripiprazole (ARI), a widely used schizophrenia
medication, exhibits promising potential in the treatment of breast cancer. As cancer therapy evolves towards a
combination approach, multimodal nano-based delivery systems, such as ARI-loaded niosomes (NIOs) combined with
Chitosan-Au nanoparticles for chemo-photothermal therapy, show promise over traditional chemotherapy alone by
enhancing targeted efficacy and minimizing side effects.
Methods In this study, a niosomal formulation was designed, incorporating ARI and chitosan-coated AuNPs (i.e.
NIOs/AuNPs-CS/ARI), to study the synergistic effect of photothermal/chemotherapy in breast cancer cells.
Results The nanosystems were characterized using UV-Vis spectroscopy and Fourier-transform infrared spectroscopy
(FT-IR), confirming the successful synthesis steps. The hydrodynamic diameter of NIOs/AuNPs-CS was determined
to be 44.62 nm with a zeta potential of -0.836. Also, Transmission Electron Microscopy (TEM) and Field-Emission
Scanning Electron Microscopical (FE-SEM) analysis were performed to assess the size and morphology of NPs. The
loading efficiency of ARI in NIOs and NIOs/AuNPs–CS was 75% and 88%, respectively. Furthermore, the release rate of
the drug from NIOs/AuNPs–CS is higher than blank NIOs at two pH values (5.8 and 7.4). The cellular uptake of AuNPs-
CS-encapsulated NIOs was considerably higher than that of blank NIOs. The Annexin V/PI staining assay showed that
the apoptosis/necrosis rate was high in NIOs/AuNPs-CS/ARI (46%) and NIOs/ARI (36%) in 48 h. The results of MTT
assessments demonstrated higher cytotoxicity by ARI-loaded NPs. The viability of MCF-7 cells treated with NIOs/
AuNPs-CS/ARI was reduced from 60% and 50% to 40% and 20%, respectively, after 24 and 48 h upon laser irradiation.
Conclusion The results of this experiment demonstrated the remarkable effectiveness of NIOs/AuNPs-CS/ARI in
cancer treatment, owing to their unique properties, including the PTT capability and pH sensitivity.
Keywords Niosomes, Aripiprazole, Photothermal therapy, Combination therapy, Breast cancer, Chitosan, Gold
nanoparticles

*Correspondence:
Marziyeh Fathi
fathi.marziyeh@yahoo.com; fathim@tbzmed.ac.ir
Jalal Shayegh
jalalshayegh@gmail.com
Full list of author information is available at the end of the article

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Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 2 of 13

Introduction in the biological milieu [19]. One approach for stabiliz-

Breast cancer is the most common and life-threatening ing AuNPs is surface modification, and encapsulation
cancer in females worldwide [1]. Despite having distinct within chitosan (CS)-based NPs [20]. CS is a natural
subtypes, breast cancer cells differ significantly from polysaccharide with abundant amine functional groups
healthy cells. For example, some receptors are highly that offers unprecedented potential for the fabrication
expressed in cancerous cells that control fundamental of multifunctional targeted/smart delivery systems [21].
cellular functions such as proliferation, adhesion, dif- In addition, it has been indicated that CS-stabilized
ferentiation, and survival [2, 3]. Additionally, their dis- AuNPs demonstrate improved biocompatibility and sta-
tinct metabolic mechanisms result in an altered tumor bility [20]. Due to the associated serious side effects, and
microenvironment (e.g. hypoxia and acidic extracellular resistance development with the customary chemother-
pH) [4]. Although surgery and chemotherapy hold great apeutics, there has been a great interest in finding new
promise in breast cancer therapy, the efficacy is subopti- alternatives, such as drug repurposing [22]. This strategy
mal due to side effects -including severe nausea, hair loss, is gaining popularity among scientists as it offers a more
and fatigue [5]- as well as the development of resistance viable and effective alternative to traditional drug discov-
by cancer cells. Cancer cells can acquire genetic muta- ery approaches [23]. In this regard, aripiprazole (ARI), a
tions that modify the drug target, reducing drug binding, high-affinity partial agonist of the dopamine D2 receptor
and efficacy. These mutations can alter the target struc- that is commonly used as an antipsychotic drug to treat
ture, making it difficult for the drug to exert its intended schizophrenia [24], has attracted much attention. How-
effect [6]. The new treatment modalities have attracted ever, more recent studies have shown that ARI also has
much attention and offer attractive and more effective antiproliferative effects on different cancer cells, such
alternatives to combat this disease. Several fields, includ- as colon, gastric, and breast cancer cells in increased
ing biomedicine, have witnessed an increase in nano- apoptosis [23]. ARI demonstrates a sensitization effect,
technology research, applying nanoparticles (NPs) for especially in drug-resistant cancer cells and enhances
cancer treatment [7]. Nanoparticle-based drug delivery radiosensitizing effects in various cancer cells [25, 26].
has emerged as a promising cancer therapy approach [8]. In this study, we aim to develop a multimodal nanosys-
This technology offers distinct advantages, such as tar- tem for the combination therapy of breast cancer. To this
geted delivery, improved drug solubility [5], the ability to end, a niosomal formulation was proposed that encapsu-
overcome drug resistance, and controlled release [9]. lates ARI as a chemotherapeutic agent, and AuNPs-CS
Among various NP-based delivery systems, niosomes for PTT therapy of breast cancer cells. This drug delivery
(NIOs) are considered suitable platforms due to their system was engineered and characterized, utilizing physi-
biocompatibility, low toxicity, and facile synthesis [10]. cochemical properties, encapsulation efficiency, and drug
They are vesicular nanocarriers and suitable for carrying release properties in different conditions. The bioimpacts
both hydrophilic and hydrophobic drugs, being regarded of NPs on breast cancer cells were studied by evaluating
as viable alternatives to liposomal [11, 12]. the cell uptake, cytotoxicity, and apoptosis/necrosis regu-
Moreover, a hybrid system of combination therapy, lation. Further, the effectiveness of the chemo/PTT ther-
using photothermal therapy (PTT) and chemotherapy, apy combination of the synthesized NIOs/AuNPs-CS/
is reported to be more effective due to the synergistic or ARI was investigated on MCF-7 cancer cells under laser
complementary impacts of such an emerging combined irradiation.
treatment strategy [13].
PTT offers a localized, minimally invasive cancer Methods
therapy option, that uses a photothermal agent to con- Materials
vert light into heat to raise the temperature of the tumor Chloroauric acid (HAuCl4. 3H2O), chitosan (CS)
site [14]. Gold nanoparticles (AuNPs) are one of the best (medium molecular weight), cholesterol, polysorbate 80
photothermal agents that have photoacoustic and photo- (Tween 80), and surfactant sorbitan monostearate (Span
thermal properties at specific wavelengths [15], leading 60) were purchased from Sigma-Aldrich Corp (St. Louis,
to properties that make them attractive for hyperther- USA). Breast cancer cells (MCF-7) were purchased from
mal therapy of cancer cells and for medical imaging [16, the National Cell Bank of Iran, Pasteur Institute (Tehran,
17]. Near-infrared (NIR) laser is commonly employed in Iran). RPMI 1640 medium and FBS were prepared from
AuNPs-induced PTT as the optimal biological optical Gibco, Invitrogen (Paisley, UK). The Annexin V-FITC kit
window, where the absorption by hemoglobin, melanin, for the detection of apoptosis/necrosis was provided by
and water is reduced, allowing deeper light penetration eBiosciences (MA, USA ). Cell culture flasks and plates
into fluids and tissues [18]. However, AuNPs must be were purchased from SPL Life Science (South Korea).
stabilized for clinical applications to prevent aggregation
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 3 of 13

Preparation of AuNPs-CS excluding the addition of ARI and AuNPs-CS during the
The AuNPs-CS were synthesized according to our previ- synthesis procedure, respectively. The final solution was
ously published method with slight modifications [27]. centrifuged at 12,000 rpm for 10 min at room tempera-
In this study, AuNPs-CS were prepared in lower con- ture to remove the unreacted reagents.
centrations of CS and HAuCl4 solutions as well as lower
temperature to achieve the smaller size of NPs. In brief, Characterization of NIOs and NIOs/AuNPs-CS
50 mg of CS was dissolved in 10 mL acetic acid solu- The dynamic light scattering (DLS) Zetasizer Nano ZS
tion (1% v/v). Then 200 µL of HAuCl4. 3H2O (12.5 mM) (Malvern Instruments Ltd., Malvern, UK) was used to
was added dropwise to the CS solution under stirring determine the zeta potential and size distribution of
conditions at 45 °C. After 3 h, the color of the solution NIOs and NIOs/AuNPs-CS. The morphology of NIO
was slowly changed from yellow to wine-red, which was also analyzed using transmission electron micros-
indicated the formation of AuNPs-CS. For purification, copy (TEM). The sample was dispersed on copper grids
the obtained AuNPs-CS solution was centrifuged at at 1 mg/mL concentration without staining. LEO 906E
12,000 rpm for 10 min. TEM (Carl Zeiss, Oberkochen, Germany) with an accel-
erating voltage of 80 kV was used to image the TEM
Synthesis of NIOs/AuNPs-CS, ARI-loaded NIOs, and ARI- micrographs. Additionally, the morphology and size
loaded NIOs/AuNPs-CS of the NPs were also determined using field-emission
NIOs were synthesized using the thin-film hydration scanning electron microscopy (FE-SEM, S4160, Hitachi,
method [28]. Briefly, Span 60 (8 mg), Tween 80 (20 mg), Japan). The samples were spread on aluminum sheets,
cholesterol (4 mg), and ARI (4 mg) were dissolved in dried at 60 °C for 10 min, and then the sample was
methanol (3 mL) and chloroform (3 mL). Then this solu- applied on the SEM stage under vacuum at 25 kv. The
tion was put in a rotary evaporator at 120 rpm and 60 °C chemical structure of the synthesized nanosystems was
for 1 h and the solvent was removed. The proniosomes investigated through Fourier-transform infrared spec-
were hydrated with 5 mL of PBS containing 500 µL of troscopy (FT-IR) to verify the presence of specific chemi-
AuNPs-CS and the mixture was then subjected to probe cal bonds and functional groups on the NPs. The FT-IR
sonication for 5 min in a pulsatile manner (50 s sonica- spectra were acquired in the 4000 –400 cm− 1 range using
tion with 10 s pause) with 30% amplitude to form ARI- KBr discs on an FT-IR Tensor 27 spectrometer (Bruker
loaded NIOs/AuNPs-CS (Fig. 1). The NIOs/AuNPs-CS, Optik GmbH, Ettlingen, Germany).
and NIOs/ARI were prepared using the same strategy,

Fig. 1 Schematic representation of NIOs/AuNPs-CS/ARI synthesis. First, chitosan-coated AuNPs were prepared (a). NIOs were produced using solvent
evaporation technique, loaded with AuNPs-CS and ARI to obtain final nanosystems, i.e. NIOs/AuNPs-CS/ARI (b)
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 4 of 13

Drug loading and in vitro releases by MTT assay. Cells at a density of 5 × 103/well were cul-
UV-Vis spectroscopy was used to determine the entrap- tured in a 96-well plate. Following overnight culture, the
ment efficacy of ARI in the NIOs/AuNPs-CS. ARI-NIOs/ cells were treated with 12, 24, 48, and 96 µM concentra-
AuNPs-CS was placed into the dialysis bag (12,000 Da) tions of ARI, NIOs, NIOs/ARI, NIOs/AuNPs-CS, and
immersed in 100 mL PBS (pH = 7.4) and stirred (4 °C, NIOs/AuNPs-CS/ARI. After 24 and 48 h of treatment,
30 min). The concentration of encapsulated ARI was then the media was removed, and MTT solution was added
measured using UV-Vis spectroscopy at 250 nm. The fol- and then incubated for a further 4 h. Subsequently, the
lowing formula was used to calculate the efficiency of MTT solution was replaced by DMSO, and an ELISA
drug encapsulation (DE). reader (Elx808, BioTek Instruments, Winooski, VT, USA)
at a wavelength of 570 nm was used to determine the
initial drug concentration − unload drug concentration
DE% = × 100 absorbance.
initial drug concentration

Apoptosis/necrosis assessment
The release of ARI from NIOs and NIOs/AuNPs-CS was An Annexin V/PI apoptosis detection kit was utilized
evaluated at pH = 7.4 and 5.8. For this purpose, NPs were to examine the impact of ARI, NIOs, ARI/NIOs, NIOs/
placed in the dialysis bag immersed in 100 mL PBS and AuNPs-CS, and NIOs/AuNPs-CS/ARI with concentra-
agitated on a shaker (90 rpm and 37 °C). At predeter- tion of 48 μm on the apoptosis/necrosis of MCF-7 cells.
mined time intervals, 2 mL of PBS was withdrawn and To this end, 5 × 105 MCF-7 cells per well were seeded into
replaced with an equal volume of fresh PBS. The amount 6-well plates. After 24 h, cells were treated with samples
of ARI released was measured at 250 nm and calculated and then incubated for 48 h. Subsequently, the cells were
using the following equation: washed, trypsinized, and centrifuged at 160 g for 5 min-
∑n utes. After washing with PBS, cells were resuspended in
Ci Vt + (Ci−1 Vs )
The amount of drug release (% ) = i=1
× 100 binding buffer (100 µL), and then 10 µL of FITC-Annexin
mt
V and PI were added for staining. Samples were incu-
bated at 4 °C for 10 min, then the cells were analyzed
Where Ci represents the concentration of the drug using FACS flow cytometry (BD FACSCalibur, BD biosci-
release at time t, Vt represents the volume of the envi- ence, CA, USA).
ronment in which the drug is released, Vs represents the
volume extracted from the release medium, and mt rep- The effect of hyperthermia on cytotoxicity
resents the mass of the loaded ARI. A variety of mathe- To determine the effects of ARI in combination with
matical models were used to determine the drug kinetics hyperthermia on cancer cells, MCF-7 cells were seeded
of ARI-NIOs/AuNPs-CS and NIOs/ARI at normal and in a 96-well plate with a density of 5 × 103 per well. After
acidic pH, including Zero-order, First order, Higuchi, 24 h, cells were treated with ARI, NIOs, NIOs/ARI,
Power law, Square root of mass, Hixson, Crowell, Three NIOs/AuNPs-CS, and NIOs/AuNPs-CS/ARI at a con-
seconds’ root of mass, Weibull, and Reciprocal. centration of 48 µM. After 4 h of treatment, the cells were
exposed to the laser radiation (525 nm) (Mustang@ 2000,
Cellular uptake Russia) for 1 min. The cells were further cultured for 24,
The cellular uptake of NIOs and NIOs/AuNPs-CS by and 48 h, and then the MTT assay was performed.
MCF-7 cells was investigated. First, NIOs and NIOs/
AuNPs-CS were labeled by FITC, to this end, they were Statistical analysis
incubated with FITC (4 mg/mL methanol), overnight In this study, each experiment was repeated at least three
with continuous shaking at 4 °C. MCF-7 cells were seeded times, and the results were expressed as the mean ± stan-
in six-well plates with a density of 5 × 105 cells/well. After dard deviation (SD). ANOVA, a multiple comparison test
24 h, the cells were treated with FITC-labeled NPs (48 µM). involving three or more groups, was used to analyze the
An untreated group was included as a control, where cells data and statistical significance was determined with a
were treated with PBS. After 4 h, the cells were washed p-value less than 0.05. Prism, version 9.3, was used for all
with PBS, trypsinized, and centrifuged at 160 g for 5 min. statistical analysis.
Finally, NIOs and NIOs/AuNPs-CS uptake by MCF-7 cells
was determined using FACS Calibur flow cytometry (BD Results and discussion
FACSCalibur, BD bioscience, CA, USA ). NIOs possess distinctive properties that make them
favorable drug delivery systems (DDSs). These include
Cytotoxicity assessment their ability to simultaneously load both hydrophilic and
Cytotoxicity of ARI, NIOs, NIOs/ARI, NIOs/AuNPs-CS, hydrophobic drugs, ease of synthesis, low toxicity, and
and NIOs/AuNPs-CS/ARI on MCF-7 cells was evaluated biocompatibility [29]. In this study, our objective was to
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 5 of 13

engineer a suitable niosomal nanosystem for the com- morphology assessment, which showed the prepared
bined chemo/photothermal therapy of breast cancer. NIOs and NIOs/AuNPs-CS have a spherical shape with a
Chitosan-coated AuNPs, as pH-responsive PTT agents size of around 50 nm for NIOs (Fig. 3a and c) and almost
were synthesized. Subsequently, ARI and AuNPs-CS 38 nm for NIOs/AuNPs-CS (Fig. 3b). Morphological
were loaded in the hydrophobic domain and hydrophilic characterization of NIOs acquired by TEM and SEM
core of NIOs, respectively (Fig. 1). Then their physico- analysis indicated a smaller size than the DLS measure-
chemical characteristics and biological impacts were ment since the zeta sizer indicates the hydrated diameter
studied in MCF-7 breast cancer cells. form of NPs, which is consistently larger than NPs’ accu-
rate diameters [35]. Further, the zeta potential of NIOs/
NIOs/AuNPs-CS characterization AuNPs-CS (-0.836) was found to be more neutral com-
The particle size is a crucial factor in the development pared to NIOs (-14.8) (Fig. 2b, d), which can be attrib-
of nanotechnology-based DDSs. Recent advancements uted to the presence of CS. The positively charged amino
indicate that NPs with a size of about 50–100 nm have groups of CS influence the zeta potential of the fabricated
significant potential for cancer therapy [30]. Owing to a NPs [36]. NPs with a zeta potential of -10 to + 10 mV and
high rate of angiogenesis, solid tumors display abnormal a size of less than 100 nm promote their function and
leaky vasculature with an irregular shape and a lack of a enhance their qualities for DDSs [37, 38]. Therefore, the
smooth muscle layer. These characteristics can be har- NIOs/AuNPs-CS have suitable size and zeta potential for
nessed to increase the NPs penetration from the blood biomedical applications.
circulation to the tumor tissues through enhanced per- FT-IR spectra were used to determine the chemi-
meability and retention (EPR) [31]. These characteristics cal structure of NIOs, and NIOs/AuNPs-CS (Fig. 4a).
make them highly advantageous for biomedical applica- The peaks identified were at 1738 cm− 1 attributed to a
tions. The size of the prepared NIOs and NIOs/AuNPs- 5-membered ring [39]. In the spectrum of NIOs/AuNPs-
CS were 54.77, and 44.62 nm, respectively (Fig. 2a, c), as CS, the bands at 2900 cm− 1 and 3400 cm− 1 are related
evaluated by dynamic light scattering. Previous research to the stretching vibrations of CH2 and hydroxyl groups,
has demonstrated the high sensitivity of NIOs to aggre- respectively [40]. The peaks at 1382 and 1646 cm− 1 are
gation. Following sonication, NIOs can exhibit signifi- for C-N stretching and C = O amide stretching of CS,
cant aggregation [32, 33]. In our study, NIOs are slightly while 3417 cm− 1, and 1106 cm− 1 could be related to
larger than NIOs/AuNPs-CS, which could be due to the the asymmetric stretch of C–O–C groups of CS and
aggregation of NIOs. In our previous study, it was also NIOs [41, 42]. The peak at 1580–1650 cm− 1 is related
observed that the size of bare NIOs were larger than to AuNPs-CS for the amines group of CS [43], which
NIOs/AuNPs and NIOs/AuNPs-Polyamidoamine [34]. does not exist in bare NIOs. The characteristic peaks of
TEM and SEM analysis were performed for size and 1457 cm− 1 which indicate alkanes and the aromatic ring

Fig. 2 The size (a, c) and zeta potential (b, d) distribution of NIOs and NIOs/AuNPs-CS respectively. Zeta potential of (b) NIOs, (d) NIOs/AuNPs-CS
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 6 of 13

Fig. 3 The SEM images (scale bar = 2.54 μm) of (a) NIOs, (b) NIOs/AuNPs-CS, and (c) the TEM image of NIOs

Fig. 4 (a) The FT-IR spectra of NIOs and NIOs/AuNPs-CS. (b) The UV-Vis spectrum of NIOs/AuNPs-CS

stretch functional group were found in Tween 80, cho- AuNPs-CS nanosystems were used for delivering ARI as
lesterol, and Span 60 [44]. The lipophilic region is chains a hydrophobic drug. The loading efficiency of ARI in sim-
made up of alkanes [45], which are similar in both NIOs ple NIOs and NIOs/AuNPs–CS was 75 and 84%, respec-
and NIOs/AuNPs-CS indicating that AuNPs-CS did not tively, indicating excellent loading efficiency. The high
interact in lipophilic regions. As shown in Fig. 4b, a clear drug loading in nanostructures can be attributed to span
and single peak at 525 nm was observed in the UV-Vis 60, characterized by its low hydrophobic moiety values,
spectrum indicating that AuNPs-CS was successfully promoting the formation of robust and stable NIOs with
synthesized and can be used in PTT. exceptional entrapment efficiency [48]. Similarly, choles-
terol is known to positively influence the permeability,
Drug entrapment efficiency drug release rigidity, leakage, and entrapment efficiency of NIOs [49].
As amphiphilic NPs, NIOs could entrap the hydropho- The synthesis of AuNPs using chitosan would improve its
bic and hydrophilic drugs simultaneously [46]. As previ- surface properties for binding of biomolecules which can
ously demonstrated, NIOs exhibit a significant capability increase drug loading efficiency [50].
for carrying hydrophobic drugs such as paclitaxel, with Developing a smart nanocarrier with a high encapsula-
remarkable %EE values of 98.5% [47]. In this study, NIOs/ tion efficiency, and regulated release property is a critical
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 7 of 13

Fig. 5 The cumulative in vitro release of ARI from (a) NIOs and (b) NIOs/AuNPs-CS at pH = 7.4 and 5.8 at 37 °C, ∗, ∗∗ represented p < 0.05 and p < 0.01,
respectively (n = 3)

Table 1 The kinetics models of ARI release from NIOs/ARI

Kinetics model Equation Coefficient of determination (R2)
pH = 5.8 pH = 7.4
Zero-order F = k0 t 0.8576 0.7367
First order ln(1 − F ) = −kf t 0.9322 0.7844
√
Higuchi F = kH t 0.969 0.8811
Power lawa ln F = ln kP + P ln t 0.9903 0.9516
√
Square root of mass 1 − 1 − F = k1/2 t 0.8977 0.7609
√
Hixson-Crowell 1 − 3 1 − F = k1/3 t 0.9099 0.7688
Three seconds’ root of mass
√ 0.8849 0.7529
3 2
1− (1 − F ) = k2/3 t
Weibull b
ln(− ln(1 − F )) = −β ln td + β ln t 0.9937 0.9618
(1 )
Reciprocal powered timec −1 = m 0.9885 0.9701
F tb
a
pH = 5.8: kP=0.1645 and P = 0.2315; pH = 7.4: kP=0.0955 and P = 0.2708
b
pH = 5.8: β = 0.2661 and td=615.2458; pH = 7.4: β = 0.2948 and td=2391.1066
c
pH = 5.8: m = 4.9973 and b = 0.3046; pH = 7.4: m = 9.3633 and b = 0.3204

challenge in cancer therapy [51]. On the other hand, the possess unique pH-responsive drug release properties,
acidic tumor microenvironment (TME), primarily due to making them a suitable smart DDS. Various kinetic mod-
anaerobic glucose metabolism, presents an opportunity els were employed to fit the release of ARI from NIOs/
for developing intelligent, pH-driven, controlled-release ARI and NIOs/AuNPs-CS/ARI as presented in Tables 1
DDSs [51]. Previous research has demonstrated that the and 2. The analysis indicated that at the pHs of 7.4 and
hydrophobicity of tween 80 may favor the retention of 5.8, the release curves best fit with the Power law, Weibull,
hydrophobic drugs, such as ARI on the vesicle’s surface and Reciprocal powered time kinetic models (as shown
rather than encapsulation within the system [48]. The in Tables 1 and 2) . The results of this study are consis-
decoration of AuNPs-CS on the surface of NIOs influ- tent with our prior work, which demonstrated the release
ences their pH sensitivity. This is due to the protonation of silibinin from NIOs with similar behavior [53]. The
of amino groups of CS under acidic conditions, thereby Weibull and Power law release kinetics indicated the drug
increasing their solubility and drug release [51]. More- release profile is largely based on diffusion while swelling
over, in previous research, the NIO formulation allowed and erusion mechanisms might be somewhat involved
for a more gradual release of ARI in physiological pH due since the biopolymer is subjected to degradation [54].
to its lipophilicity, demonstrating the NIO’s effectiveness The Reciprocal powered time model also indicated a con-
in controlled drug delivery applications [52]. The cumula- trolled release behavior [55]. Based on the release kinetics
tive in vitro release profile of ARI from formulated NIOs data, Power Law model seems to be a better fit for the drug
and NIOs/AuNPs-CS composite at pH = 7.4 and 5.8 at release, in which the release exponent is smaller than 0.5,
37 °C is shown in Fig. 5. The rates of ARI released from indicating the drug release is primarily governed by Fick-
NIOs/AuNPs-CS are higher than NIOs in both pH condi- ian diffusion through the polymer matrix at both pHs.
tions. These findings demonstrated that NIOs/AuNPs-CS However, we speculate that upon the initial release of the
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 8 of 13

Table 2 The kinetics models of ARI release from NIOs/AuNPs-CS/ARI

Kinetics model Equation Coefficient of determination (R2)
pH = 5.8 pH = 7.4
Zero-order F = k0 t 0.7917 0.7627
First order ln(1 − F ) = −kf t 0.832 0.7845
√
Higuchi F = kH t 0.7211 0.892
Power lawa ln F = ln kP + P ln t 0.9866 0.922
√
Square root of mass 1 − 1 − F = k1/2 t 0.8123 0.7736
√
Hixson-Crowell 1 − 3 1 − F = k1/3 t 0.819 0.7773
Three seconds’ root of mass
√ 0.8055 0.77
3 2
1− (1 − F ) = k2/3 t
Weibull b
ln(− ln(1 − F )) = −β ln td + β ln t 0.99 0.9217
(1 )
Reciprocal powered timec −1 = m 0.9913 0.9211
F tb
a
pH = 5.8: kP=0.1809 and P = 0.3186; pH = 7.4: kP=0.1316 and P = 0.2623
b
pH = 5.8: β = 0.3916 and td=58.5851; pH = 7.4: β = 0.2917 and td=798.3528
c
pH = 5.8: m = 4.3540 and b = 0.4792; pH = 7.4: m = 6.4734 and b = 0.3237

Fig. 6 Cellular uptake (a) untreated group, (b) NIOs, and (c) NIOs/AuNPs-CS

drugs, the polymer swelling and possible degradation pro- the positively charged NPs are taken up faster than nega-
cess might be involved in drug release. Furthermore, based tively charged NPs [59, 60]. Figure 6 presents the uptake
on Weibull kinetics model (with a shape parameter, b < 1), of NIOs and NIOs/AuNPs-CS by MCF-7 cancer cells. It
the rate of drug release decreases over time, which further was shown that NIOs can be transported into the cells
indicates that drug release profile is largely dependent because of their small size and sphere shape. However,
upon the diffusion phenomenon. cell uptake of NIOs/AuNPs-CS was significantly higher
due to the positive charge of NIOs/AuNPs-CS compared
Cellular uptake to NIOs and enhanced cell membrane interaction with
Cellular uptake of NPs plays a key role in assessing the the particles.
efficacy of DDSs. Particle size, shape, and surface charge
can influence the mechanism of cellular uptake and the Cell cytotoxicity evaluation by MTT assay
efficiency of the therapeutic agent [56]. Previous stud- The MCF-7 cells were treated with free ARI, NIOs, ARI/
ies have demonstrated that the uptake of different NPs NIOs, NIOs/AuNPs-CS, and NIOs/AuNPs-CS/ARI at
by nonphagocytic cells is highly correlated to the size of concentrations of 12, 24, 48, and 96 µM for 24 and 48 h,
NPs [57]. Uptake reaches an optimum level at around and then the MTT assay was performed. As shown in
50 nm, then declines for particles of higher sizes [58]. Fig. 7, NIOs and NIOs/AuNP-CS formulations have neg-
In addition, the shape of NPs directly influences cellular ligible cytotoxicity against the MCF-7 cells. Such cyto-
uptake, and spherical NPs show the highest uptake after compatibility is mainly attributed to the low cytotoxicity
rod-NPs [59]. Owing to the presence of phospholipids, of the ester surfactants (span and tween) used to prepare
the cell membrane has a slight negative charge, and cell NIOs, indicating the great potential of NIOs formula-
uptake is driven by electrostatic attractions. Therefore, tions for drug delivery applications [61]. Previous studies
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 9 of 13

Fig. 7 The cytotoxicity of ARI, NIOs, NIOs/ARI, NIOs/AuNPs-CS, and NIOs/AuNPs-CS/ARI on the MCF-7 cells after (a) 24 h and (b) 48 h, p* < 0.05, (n = 4)

have shown that ARI inhibits the proliferation of MCF-7 and c-myc [23]. In various studies, loading drugs into
cells in a concentration-dependent manner [23]. How- almost the same nanoplatforms has shown significantly
ever, some studies have indicated that free ARI exhibits higher apoptosis rates in cancer cells compared to
relatively weaker effects in inhibiting cell proliferation administering free drugs. For instance, a formulation of
when compared to its efficacy in combination with other doxorubicin and vincristine loaded into CS-coated NIOs
agents, such as Cisplatin [26] and in some cancer cell demonstrated a notable increase in apoptosis in cancer
lines, achieving the half-maximal inhibitory concentra- cells compared to the effects of free drugs [67]. Our latest
tion (IC50) may require the use of very high concentra- study reported that loading paclitaxel onto NIOs/AuNPs-
tions of ARI (> 100 µM) [62]. Our study has demonstrated Polyamidoamine platform produced a higher apoptotic
that ARI-loaded NIOs exhibit higher cytotoxicity as com- rate in cancer cells than free paclitaxel [34]. Our find-
pared to free ARI, due to increased solubility and sub- ings reveaedl that ARI (48 µM) loaded in a composite
sequently enhanced intracellular levels in MCF-7 cells nanosystem of NIOs/AuNPs-CS significantly increases
[63]. Moreover, targeted NPs can enter the target cells, the apoptosis and necrosis rate, specifically in the cells
through receptor-mediated endocytosis, increasing the treated with NIOs/AuNPs-CS/ARI reaching %46 after
concentration of drug molecules within the cell. During 48 h. This surpasses the corresponding rates observed
this process, P-gp does not recognize drug molecules and with NIOs/ARI and free ARI, which are %36 and %26,
fails to pump out the free drug molecules from the cell respectively (Fig. 8). This observation may be attributed
[64]. Besides, in our latest studies, niosomal formulations to enhanced cellular uptake facilitated by improved inter-
were highly biocompatible, as there was no significant action with the cell membrane.
cytotoxicity on the HHF-2 and HEK-293 (normal cell
lines) [35, 53]. NIOs/AuNPs-CS/ARI have more inhibi- Combination therapy
tory effects on MCF-7 compared to NIOs/ARI (50% VS The combined chemo/PTT therapeutic efficacy of
53%, at the concentration of 48 µM after 48 h), which is drug-loaded NIOs and NIOs/AuNPs-CS on MCF-7
not statistically significant (p > 0.05) between two groups. cancer cells was investigated after 24 and 48 h. These
Since sample size and measurement variability can easily findings have shown that NIOs did not have cytotox-
influence the statistical results, a nonsignificant outcome icity, while NIOs/AuNPs-CS have shown significant
does not imply that the new therapy or treatment proto- cytotoxicity. Previous studies have demonstrated that
col is not clinically useful [65]. The reason for this slight small gold nanospheres have negligible damage to the
increase may be due to the high uptake of NIOs/AuNPs- mitosis and DNA synthesis at normal growth tem-
CS/ARI by MCF-7 cells [66]. The IC50 of ARI loaded in perature, while this defect was exacerbated by mild
NPs was about 48 µM after 48 h. This concentration was hyperthermia conditions [68]. Laser irradiation itself
used for the subsequent experiments and combination has also been shown to have no cytotoxic effects on
therapy step. the untreated cells (control +), because PTT works by
exerting local cytotoxic hyperthermia on the treated
Flow cytometry assay cancer cells through a photothermal contrast agent.
Studies have demonstrated that ARI increases the apop- In this approach, a photothermal contrast agent is
tosis rate in the various cancer cells by regulating the used to deliver radiation energy to the tumor tissue
expression of key proapoptotic genes, such as BCL10 and [69]. Local hyperthermia mediated by AuNPs induced
caspases 3, along with anti-apoptotic genes like BCL2L1, by a mild PTT can result in the apoptosis/necrosis of
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 10 of 13

Fig. 8 Flow cytometry analysis of MCF-7 cells. (a–f) represents the cell population for the untreated control group and treated with NIOs, NIOs/AuNPs-CS,
ARI, NIOs/ARI, and NIOs/AuNPs-CS/ARI after 48 h. (g) The viable, early apoptotic, late apoptotic, and necrotic cell population upon 48 h treatment. ∗, ∗∗
represented p < 0.05 and p < 0.01, respectively (n = 2)

cancer cells by denaturing proteins and enzymes, dis- 24 h. In contrast, at the same concentration, IC50 was
rupting metabolic signals, and swelling the endothe- observed at 48 h without PTT. Notably, the cytotox-
lium, among other effects [70]. Moreover, combination icity of NIOs/AuNPs-CS/ARI with PTT was found to
therapy decreases the IC50 in MCF-7 cells treated be substantial, resulting in a cell death rate of approxi-
by NIOs/AuNPs-CS/ARI which IC50 has shown in mately 60% in 24 h and around 80% in 48 h (Fig. 9).
Alimohammadvand et al. BMC Biotechnology (2024) 24:108 Page 11 of 13

Fig. 9 The cytotoxicity of ARI, NIOs, NIOs/ARI, NIOs/AuNPs-CS, and NIOs/AuNPs-CS/ARI on the MCF-7 cells, with and without laser irradiation after (a) 24
and (b) 48 h, ∗, ∗∗ represented p < 0.05 and p < 0.01, respectively, (n = 4)

Data availability
Conclusion All data that support the findings of this study are included in the article.
Transitioning from monotherapy to combination ther-
apy for cancer presents numerous advantages, including Declarations
reduced side effects with efficient eradication of can-
Ethical approval
cer cells. In this research, we successfully synthesized This study was approved by the Research Ethics Committee of Tabriz
and characterized NIOs/AuNPs-CS/ARI for breast can- University of Medical Sciences (IR.TBZMED.VCR.REC.1400.539).
cer treatment. Considering the amphiphilic proper-
Competing interests
ties of NIOs, we loaded ARI into the hydrophobic core The authors declare no competing interests.
while decorating the surface with AuNPs-CS. The use of
AuNPs for PTT serves to convert light into heat, thereby Author details
1
Research Center for Pharmaceutical Nanotechnology, Biomedicine
enhancing the effect of ARI against breast cancer cells. Institute, Tabriz University of Medical Sciences, Tabriz, Iran
Furthermore, incorporating AuNPs-CS into the fabri- 2
Department of Basic Sciences, Faculty of Veterinary Medicine, Shabestar
cation of DDS results in a positively charged surface for Branch, Islamic Azad University, Shabestar, Iran
3
Department of Oral Science and Translation Research, College of Dental
NIOs/AuNPs-CS, promoting uptake by cancer cells com- Medicine, Nova Southeastern University, Fort Lauderdale, FL 33313, USA
pared to NIOs. This research underscores the potential of 4
Department of Pharmaceutical Sciences, College of Pharmacy, Nova
NIOs/AuNPs-CS/ARI, with their unique properties, as Southeastern University, Fort Lauderdale, FL, USA
5
Department of Reproductive Biology, Faculty of Advanced Medical
a promising strategy for targeting breast cancer. Future Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
investigations, including in vivo studies or the use of dif- 6
Engineered Biomaterial Research Center, Khazar University, Baku,
ferent ligands, could provide essential insights needed to Azerbaijan
7
Department of Microbiology, Faculty of Veterinary and Agriculture,
advance this approach toward clinical trials. This com- Islamic Azad University, Shabestar Branch, Shabestar, Iran
bination therapy has the potential to integrate smoothly
into existing breast cancer treatment protocols, improv- Received: 7 October 2024 / Accepted: 16 December 2024
ing patient survival by reducing side effects and address-
ing drug resistance.
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