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Expert Opinion on Drug Delivery
ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/iedd20
Enzyme-responsive mannose-grafted magnetic
nanoparticles for breast and liver cancer
therapy and tumor-associated macrophage
immunomodulation
Gholam Hossein Darya, Omid Zare, Hamid Reza Karbalaei-Heidari, Sedighe
Zeinali, Heather Sheardown & Banafsheh Rastegari
To cite this article: Gholam Hossein Darya, Omid Zare, Hamid Reza Karbalaei-Heidari,
Sedighe Zeinali, Heather Sheardown & Banafsheh Rastegari (06 May 2024): Enzyme-
responsive mannose-grafted magnetic nanoparticles for breast and liver cancer therapy and
tumor-associated macrophage immunomodulation, Expert Opinion on Drug Delivery, DOI:
10.1080/17425247.2024.2347300
To link to this article: https://doi.org/10.1080/17425247.2024.2347300
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ORIGINAL RESEARCH
Enzyme-responsive mannose-grafted magnetic nanoparticles for breast and liver
cancer therapy and tumor-associated macrophage immunomodulation
Gholam Hossein Darya
a,b
, Omid Zare
c
, Hamid Reza Karbalaei-Heidari
d,e
, Sedighe Zeinali
f
, Heather Sheardown
g
and Banafsheh Rastegari
a
a
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz,
Iran;
b
Department of Comparative Biomedical Sciences, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical
Science, Shiraz, Iran;
c
Department of Biology, Islamic Azad University, Tehran, Iran;
d
Molecular Biotechnology Lab, Department of Biology, Faculty
of Science, Shiraz University, Shiraz, Iran;
e
Department of Chemistry, Faculty of Science, University of Manitoba, Winnipeg, MB, Canada;
f
Department of Nanochemical Engineering, School of Advanced Technologies, Nanotechnology Research Institute, Shiraz University, Shiraz, Iran;
g
Department of Chemical Engineering, McMaster University, Hamilton, ON, Canada
ABSTRACT
Background: Chemo-immunotherapy modifies the tumor microenvironment to enhance the immune
response and improve chemotherapy. This study introduces a dual-armed chemo-immunotherapy
strategy combating breast tumor progression while re-polarizing Tumor-Associated Macrophage
(TAM) using prodigiosin-loaded mannan-coated magnetic nanoparticles (PG@M-MNPs).
Methods: The physicochemical properties of one-step synthetized M-MNPs were analyzed, including
X-ray diffraction, FTIR, DLS, VSM, TEM, zeta potential analysis, and drug loading content were carried
out. Biocompatibility, cancer specificity, cellular uptake, and distribution of PG@M-MNPs were investi-
gated using fluorescence and confocal laser scanning microscopy, and flow cytometry. Furthermore, the
expression levels of IL-6 and ARG-1 after treatment with PG and PG@M-MNPs on M1 and M2 macro-
phage subsets were studied.
Results: The M-MNPs were successfully synthesized and characterized, demonstrating a size below 100
nm. The release kinetics of PG from M-MNPs showed sustained and controlled patterns, with enzyme-
triggered release. Cytotoxicity assessments revealed an enhanced selectivity of PG@M-MNPs against
cancer cells and minimal effects on normal cells. Additionally, immuno-modulatory activity demon-
strates the potential of PG@M-MNPs to change the polarization dynamics of macrophages.
Conclusion: These findings highlight the potential of a targeted approach to breast cancer treatment,
offering new avenues for improved therapeutic outcomes and patient survival.
ARTICLE HISTORY
Received 4 December 2023
Accepted 4 March 2024
KEYWORDS
Mannan; breast cancer;
magnetic nanoparticles;
macrophage; drug delivery
1. Introduction
Among the newly diagnosed cases in women worldwide,
breast, lung, and colorectal cancer make up 51% of the total,
with breast cancer alone accounting for almost one-third [1].
In the advanced stages of breast tumors, chemotherapy is
a primary treatment strategy for patients. Nonetheless, the
recommended therapeutic dose of chemotherapy medications
can lead to various complications due to their poor cell selec-
tivity, side effects, and bioavailability. Nanotechnology offers
numerous benefits in treating cancer through site-specific, and
target-oriented delivery of precise medicines [2].
Chemo-immunotherapy is a promising approach in can-
cer treatment that aims to modify the tumor microenviron-
ment (TME) to enhance the immune response of immune
cells and improve the effectiveness of chemotherapy with
fewer side effects [3]. Various immune cells, such as tumor-
associated macrophages (TAMs), natural killer (NK) cells,
dendritic cells (DCs), and lymphocytes, play a critical role
in tumor progression. However, TAMs, prevalent in the
tumor microenvironment, are associated with poor prog-
nosis and resistance to chemotherapy [4]. The significance
of TAMs in triggering tumor initiation and development is
becoming increasingly recognized. Primary cancer cells can
evade local innate immune control, transform into cancer
cell clones, effectively recruit monocytes from the circula-
tion, and reprogram resident TAMs. TAMs are believed to
contribute to the innate tumor immune system of the
microenvironment and can constitute up to 50% of the
tumor mass [5]. TAMs originate from two main sources,
tissue-resident macrophages and circulating monocytes,
which are recruited to the tumor by growth factors and
chemokines such as M-CSF, CCL2, and CCL5 [6]. Tissue-
resident macrophages can identify cancer cells and possess
the inherent capability to eliminate cancer cells in the early
stages of development. Earlier studies have demonstrated
that tissue-resident macrophages in mouse lungs contribute
to the reservoir of TAMs and promote tumor growth in vivo,
whereas monocyte-derived TAMs contribute to tumor
CONTACT Banafsheh Rastegari brastegari@sums.ac.ir Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences,
Shiraz University of Medical Sciences, Shiraz 7143918596, Iran
Supplemental data for this article can be accessed online at https://doi.org/10.1080/17425247.2024.2347300.
EXPERT OPINION ON DRUG DELIVERY
https://doi.org/10.1080/17425247.2024.2347300
© 2024 Informa UK Limited, trading as Taylor & Francis Group
progression through metastasis [7]. As the relationship
between TAMs and malignant tumors has become more
evident, they have emerged as a promising target for the
development of novel cancer therapies. The high conver-
sion plasticity of both M1 and M2 macrophages led to
changes in the tumor microenvironment, resulting in micro-
environment changes and thereby resulted in effective ther-
apeutic interventions [8].
Recently, carbohydrates such as mannose and mannose-
6-phosphate have been gaining increasing attention as poten-
tially effective targeting agents in the treatment of cancer. With
the remarkable gelling capacity and biocompatibility of
Ceratonia siliqua mannan, there has been increasing interest in
the use of this polymer for topical, ocular, buccal, colonic, and
particularly, oral drug delivery in controlled-release tablets [9,10].
The asialoglycoprotein receptor (ASGPR) is a specific recep-
tor used for smart targeting, primarily found on breast and
hepatic cancer cells, and minimally presents in normal cells
[11–13]. Another receptor found on breast and hepatic cells,
with poorly understood significance in cancer pathology, is
the mannose receptor (MR) or CD206. This C-type lectin is
expressed on the surface of macrophages and some immature
dendritic cell subsets [14–17].
Regarding breast cancer cells, the mannose-6-phosphate
receptor plays a crucial role in ensuring the accurate localization
of enzymes to lysosomes. A recent study using immunoblotting
techniques has indicated that the expression of mannose recep-
tors is significantly elevated in MCF-7 cells compared to other
breast cancer cell lines and non-tumorigenic cells [18]. Moreover,
previous studies have demonstrated the presence of mannose
receptors on the cellular membrane of HepG2 cells, indicating
a potential approach for directing drug delivery through mannose
receptor-mediated endocytosis in HepG2 and MCF 7 cells [19].
In this study, a dual-armed chemo-immunotherapy strategy
was introduced for the detection and overcoming breast
tumor progression and TAM re-polarization.
2. Materials and methods
2.1. Nanoparticle synthesis and characterization
Mannose-coated magnetic nanoparticles (M-MNPs) were synthe-
sized in a one-step manner according to a previously described
procedure with some modifications [20]. In the first step, 4.0 g of
FeCl
2
•4 H
2
O (Merck Millipore, Germany) and 10.4 g of FeCl
3
•6 H
2
O (Merck Millipore, Germany) were dissolved independently in
100 ml distilled water. The ferrous and ferric solutions were
mixed, and the mixture was incubated for 30 min at 90°C under
N
2
protection. In the next step, 0.4 g of mannan solution at a final
concentration of 2% (W/V) (Locust bean gum from Ceratonia
silique, Sigma-Aldrich, St. Louis, MO, U.S.A.) is added slowly to
the mixture, followed by adding 8.5 ml of NH
4
OH (30% V/V,
Merck Millipore, Germany) drop-wised within 20 min. The black
solution was then kept at 90°C for 45 min, followed by cooling
with double distilled water. Finally, the M-MNPs were harvested
from the ammonium solution using a 0.5 T permanent magnet,
rinsed twice, and dried in a vacuum oven at 70°C for further
analysis.
2.2. Nanoparticle physicochemical characterization
The magnetite crystalline structure and phase identification of
the M-MNPs were investigated using a Cu-Kα radiation X-ray
diffractometer within the range of 10°<2θ°<90° (Bruker,
D8ADVANCE, Germany). The presence of the mannan polymer
coating was confirmed using a Fourier transform infrared
(FTIR) spectrometer (Thermo Fisher Scientific Inc., Ma, U.S.A.)
in the frequency range 4000–400 cm
−1
. The magnetic proper-
ties of M-MNPs were analyzed at room temperature using
a vibrating sample magnetometer (VSM, Dexing, Model: 250).
The magnetic field was applied between −10 and +10 kOe,
with a sensitivity of 10
−3
emu. The dried size and morphology
of the M-MNPs were observed using Transmission Electron
Microscopy (JEOL-JEM 1200EX) at 80 kV. In this proposal, the
M-MNPs TEM imaging was fixed onto carbon support copper
grids (200 mesh) and the grid surface was air-dried at room
temperature. Dynamic light scattering (DLS) analysis was done
to determine the hydrodynamic size and polydispersity index
with the help of HORIBA SZ-100 (HORIBA LTD, Japan) in ethy-
lene glycol. The net charge of the M-MNPs was investigated in
PBS buffer using zeta potential analyzers (HORIBA SZ-100,
HORIBA Ltd. Japan).
The red pigment prodigiosin (PG) was isolated from the
gram-negative bacterium Serratia sp. S2B and purified in three
steps, acidified methanol, chloroform, and silica gel column as
described previously [21]. The 10 mM prodigiosin stock solu-
tion was prepared in methanol using an extinction coefficient
of ε=112500 M
−1
cm
−1
at λ535 nm [22].
The adsorption process was used to load the prodigiosin
onto mannan magnetic nanoparticles (PG@M-MNPs). M-MNPs
(5 mg) were initially dispersed for 10 min (UP100H, Hielscher,
Germany) in 5 ml PBS solution, pH 7.2–7.4. A total volume of
500 µl of 10 mM prodigiosin was added dropwise to the nano-
particles, followed by rotation for 1 h at room temperature.
The PG@M-MNPs were washed three times with PBS using
a 0.56 T external permanent magnet. Unbounded prodigiosin
was calculated using a standard curve at a wavelength of 535
nm. Drug loading was estimated as follows:
The encapsulation efficiency (EE) of PG on the PG@M-MNPs
was calculated according to the following equation. The drug
content was determined with the addition of 1 volume of
acidified ethanol (ethanol: HCl 1 N, 24:1) to PBS and measuring
the PG content at λ
535
PG. The drug encapsulation efficiency
was estimated as follows:
2.3. Prodigiosin release
The in vitro PG release kinetics of PG@M-MNPs were studied
in a shaker incubator at 100 RPM in the dark at 37°C.
Initially, 5 mg of PG@M-MNPs containing 1.9 mg of PG
were dispersed in 10 mL PBS (pH ~8.0) and dialyzed in the
2G. H. DARYA ET AL.
presence of 1 mg/mL BSA to simulate the bloodstream
condition. Also, 5 mg of PG@M-MNPs/BSA mixture was pre-
pared in PBS solution, pH ~5.0, and 1 U of mannanase
(endo-1, 4- β-mannanase from Aspergillus niger, Megazyme,
Ireland) was added to the cocktail. Then, 500 µL of each
sample was collected after 15, 30, 45, 60, and 90 min and
mixed with an equal volume of acidified ethanol to deter-
mine the PG content.
2.4. Cell culture and toxicity assessments
Cell toxicity and cancer specificity were evaluated using
MTT assay against two cancer cell lines MCF-7/GFP (breast
cancer), HepG2 (liver cancer), and NIH/3T3 normal fibroblast
cell lines. Cells were seeded into 96-well plates at an initial
density of 1.0 × 10
4
for MCF-7/GFP, 1.5�10
4
for HepG2, and
1.5�10
4
cells/well for 3T3 in Dulbecco’s Modified Eagle’s
Medium (DMEM, Gibco Gaithersburg, U.S.A.) for overnight
in complete medium containing 10% heat-inactivated fetal
bovine serum (Gibco, Gaithersburg, U.S.A.) and 1% penicil-
lin/streptomycin at 37.0°C under a humidified atmosphere
of 5% (V/V) CO
2
.
The cytotoxicity of M-MNPs and PG@M-MNPs against 3T3
cells was investigated by incubation with increasing
amounts of MNPs ranging from 0 to 600 µg/mL (0, 50,
100, 200, 300, 400, 500, and 600 µg/mL) for 24 h. To inves-
tigate cancer cell specificity, after initial incubation of the
cell lines, the medium was replaced with 100 μL fresh cul-
ture medium containing varying concentrations of free pro-
digiosin ranging from 0 to 18.5 µM (0, 0.58, 1.16, 2.31, 4.62,
9.25, 13.87, and 18.50 μM), and/or PG@M-MNPs equal to
free prodigiosin (0, 6.25, 12.5, 25, 50, 100, 150, and 200
µg/mL of PG@M-MNPs). After the appropriate time (4, 8, 12,
and 24 h), floating nanoparticles were discarded by washing
the wells with PBS. The cells were then incubated in
a medium supplemented with 10 μL of the 5.0 mg/mL MTT
solution (Sigma-Aldrich, St. Louis, MO, U.S.A.). Then, the
plates were incubated for an additional 4 h in the dark
under the optimum cell culture conditions mentioned
above. Subsequently, the culture medium was replaced
with a solubilization solution (40.0% (v/v) DMF, 16.0% (w/
v) SDS, pH ~4.7) and incubated at room temperature for 2 h.
Finally, the formazan maximum absorbance and back-
ground were measured at 570 and 630 nm (Tecan infinite-
200 M Pro, Tecan Co, Switzerland), respectively. The survival
percentage was estimated according to Formulation 1:
2.5. Fluorescence microscopy, confocal laser scanning
microscopy (CLSM), and flow cytometry analysis
Since prodigiosin is an auto-red fluorescent pigment, it can be
used for cellular uptake and imaging studies. Cellular uptake
and distribution of prodigiosin were investigated using fluor-
escence microscopy and flow cytometry, respectively. Initially,
the total population of 2.5�10
4
of MCF-7/GFP, HepG2, and
NIH/3T3cells were pre-cultured in 24 well plates for 16 h. Then,
the culture medium containing 9.25 µM PG and 100 µg/mL
PG@M-MNPs was incubated for 30 min, 4 h, and 8 h). The
cells were then carefully washed twice with PBS to remove
free particles and PG. For DAPI staining, cells were fixed with
3.7% formaldehyde solution for 10 min. After removing the
supernatant, the cells were washed twice with PBS and stained
with 0.3 µM 4´, 6-diamidino-2-phenylindole (DAPI, Sigma-
Aldrich, U.S.A.) in PBS for 5 min. After washing the cells three
times with PBS, they were imaged under red, blue, and green
filters using an inverted fluorescence microscope (Olympus;
IX51, Japan) at 200 × magnification.
For flow cytometry analysis, MCF-7/GFP, HepG2, and NIH/
3T3 cells were seeded in six-well plates at a density of 5 × 10
5
for 16 h. The next day, the culture medium was replaced with
a fresh culture medium containing 50 µg/mL of PG@M-MNPs
and 4.6 μM PG, followed by incubation for 24 h under opti-
mum cell culture conditions. The cell supernatant containing
detached cells was collected, and adherent cells were added
to the mixture using the TrypLE digestion procedure. The cells
were centrifuged at 700�g for 5 min, and the supernatant was
discarded. After washing once with PBS, the cells were re-
suspended in a FACS buffer containing 0.5 mg/mL of BSA.
Finally, fluorescence emission histograms were obtained
using a flow cytometer (BD LSR-II; Becton & Dickinson and
Co., U.S.A.). The gating was set to red fluorescence to analyze
20,000 gate events per histogram.
Two-dimensional image reconstructions of the endosomal
vesicles with fluorescein-dextran (average MW of 10,000,
Sigma-Aldrich, U.S.A.) and PG@M-MNPs or PG were obtained
using MP TCS-SP5 confocal laser scanning microscope (Leica
Ltd., Germany). The total population of 2�10
5
cells was pre-
cultured in 6 well plates under optimum culture conditions on
microscope slides for 24 h. The following day, fresh medium
containing 1 mg/mL FITC-Dextran and either 100 µg/mL
PG@M-MNPs or 9.25 µM PG was replaced and incubated for
an additional 6 h. Finally, the culture medium was removed,
and the cells were washed three times with PBS, fixed with
3.7% paraformaldehyde for 20 min, and fixed with a refractive
glue solution for 16 h. Slides were photographed using
a confocal microscope (Leica MP TCS-SP5, Leica Co. Germany).
2.6. M1 and M2 subset stimulation, cell toxicity, RNA
extraction, and real-time PCR
The murine macrophage, J774A was initially stimulated with 100
ng/mL LPS (Sigma-Aldrich, St. Louis, Missouri, U.S.A.), and 50 µg/
mL IFN-γ (Biolegend, CA, U.S.A.), and 75 µg/mL IL-4 (Biolegend
LTD., CA, U.S.A.) to generate M1 and M2 subset, respectively. For
this purpose, the total population of 2 × 10
6
cells were seeded in
6 well plates for 16 h under optimum cell culture conditions. The
cells were then stimulated with LPS+IFN-γ or IL-4 for 24 h. The
next day, the cells were treated with different concentrations of
PG, M-MNPs, and PG@M-MNPs for 24 and 48 h. Total RNA was
extracted using RNA extraction kit (Parstus Co., MA, Iran), accord-
ing to the manufacturer’s protocol. Reverse transcription of
mRNA was performed using RT synthesis kit with oligo-dT pri-
mers according to the manufacturer’s protocol (SMOBio Inc.,
Taiwan). The expression levels of M1 and M2 biomarkers, IL-6
EXPERT OPINION ON DRUG DELIVERY 3
and ARG-1, were determined with the help of real-time PCR using
ARG1 primers (Forward primer: 5′-AAGACAGGGCTCCTTTCAGG
-3′ and Reverse primer: 5′-AGCAAGCCAAGGTTAAAGCC-′); IL-6
(Forward primer: 5′-TCTGCAAGAGACTTCCATCCA-3′ and
Reverse primer: AGACAGGTCTGTTGGGAGTG-3′), and β-actin as
reference gene (Forward primer: 5′-
CCAGGGTGTGATGGTGGGAATG-3′, and Reverse primer: 5′-
TGTAGAAGGTGTGGTGCCAGATC-3′). The mixture of 2.0 µL of
cDNA, 5 pmol of each primer, 12.5 µL of SYBR Premix (without
Rox, Amplicon, Inc., Denmark), and 9.5 µL of RNase-free H
2
O was
prepared by thermal cycling using Rotor-Gene Q (Qiagen,
Germantown, MD, U.S.A.). The cycling program was as follows:
initial denaturation for 5 min at 95°C, 40 cycles of denaturation
for 30 s at 95°C; annealing for 30 s at 60°C, and extension for 45
s at 72°C. Melting curve analysis was performed from 55 to 95°C
with 0.3°C increments at 5 s/step. The 2
ΔΔCt
method was used for
the relative expression levels of IL-6 and ARG-1 in comparison
with β-actin as a reference gene. Also, the cell toxicity of the
macrophages was evaluated in 3 × 10
4
cells using the MTT assay
with the above-mentioned procedure.
2.7. Flow cytometry
The cell death mechanism of the PG and PG@M-MNPs (apop-
tosis/necrosis) was investigated using Annexin-V-PE/7-AAD
according to the manufacturer’s protocol (Biolegend LTD.,
CA, U.S.A.), and analyzed on a BD FACS Calibur Flow
Cytometer (BD Bioscience LTD., CA, U.S.A.) set at 10,000
events. Finally, FlowJo software (Tree Star Inc., U.S.A.) was
used for the analysis.
2.8. Statistical analysis
The results from triplicate MTT and real-time PCR experiments
were statistically analyzed using the Student’s t-test and the
non-parametric one-way ANOVA (Kruskal–Wallis). The fitness
curves of the release kinetics were calculated using GraphPad
Prism 8.0.2. Statistical significance was set at p < 0.05.
3. Results
3.1. Nanoparticle characteristics
Secondary metabolites have gained special attention due to
their unique lifespan properties, including antimicrobial, anti-
parasite, anti-cancer, immunosuppressive, cholesterol-
lowering, and anti-HIV effects. Since many secondary meta-
bolites have minimal side effects in disease treatment, there
is increasing hope for the treatment of many untreatable
diseases, such as Alzheimer’s, multiple sclerosis, cancer, cys-
tic fibrosis, and parasitic and fungal diseases [23,24].
Prodiginin is a family of microbial secondary metabolites
that has garnered significant attention from researchers in
recent years due to its selective immunomodulatory and
anticancer properties [25]. The formation of the Cu(II)-PG
pyrrole ring results in oxidative stress as a result of reduction
of Cu(II) to Cu(I). Reactive oxygen species, particularly hydro-
xyl radicals, subsequently cause single- or double-strand
DNA breaks [26,27].
With exclusive advantages such as improved bioavailability,
controlled release, bypassing drug resistance, and cost-
effectiveness, many scientists have been motivated to utilize
modern drug delivery systems. To enhance the therapeutic
properties of PG, a new delivery system has been introduced
using mannan as a dual targeting vehicle. Mannan which is
easily decomposed by lysosomal enzymes, and its in vitro
anticancer and immunomodulatory effects were studied. For
this purpose, a one-step synthesis of M-MNPs was performed
by co-precipitation of ferrous/ferric ions with ammonium solu-
tions in the presence of mannan as a capping polymer. The
main backbone structure of the mannan polymer consists
mainly of a β-(1–4)-linked D-mannose backbone with α(1–6)-
linked D-galactose side chains, which belongs to the galacto-
mannan groups with the 4:1 ratio of mannose:galactose units,
respectively [8,28,29]. The M-MNPs are built with several mag-
netite nanoparticles which are entrapped with the mannan
polymer (Figure 1(a)).
The presence of the functional groups of the M-MNPs was
examined by FT-IR spectroscopy at 400–4000 cm-
−1
wavenumber (See Figure 1(b)). The coupled υ (C–C/C–O)
stretch vibration is detectable in wave numbers 1059, 1110,
1492, and 1712 cm
−1
in both the mannan and M-MNP spectra.
Also, the peak at 945 cm
−1
in M-MNP was related to the
antisymmetric glycoside υa (C–O–C), which may improve the
presence of the mannan polymer as a capping agent [30].
Moreover, the C-H groups at 2922 and 2861 cm
−1
in the
mannan polymer were detected in the M-MNPs. The –OH
stretching vibration was also detectable with a broadband
peak at 3200–3400 cm
−1
in both the mannan M-MNPs. The
specific peaks at 586 cm
−1
showed Fe–O tetrahedral sites in
both MNPs and M-MNPs spectra. The wide peak at 3200–3600
cm
−1
represented the O-H group at the surface of the MNPs,
mannan sugar polymer, and mannan-grafted MNPs. From an
overall point of view, the FTIR spectrum shows the successful
synthesis of the mannan-grafted magnetic nanoparticles. As
illustrated in Figure 1(c), the six distinct peaks at 2θ values of
30.15°, 35.60°, 43.05°, 54.55°, 57.50°, and 63.45° M-MNPs XRD
correspond to the diffractions of the 220°, 311°, 400°, 422°,
511°, and 440° crystal faces of the Fe
3
O
4
spinel structure.
The TEM images showed a well-crystalized spherical mag-
netite with a core size of 10 to 20 nm (see Figure 1(d)). The
M-MNPs showed a size of 86.96 ± 10.23 nm with a thin bright
coating of mannan as a shell. The hydrodynamic size was also
investigated with the help of a particle size analyzer.
According to Figure 1(e), the relative mean size was estimated
to be 86.40 ± 23.50 nm with a PDI of 0.28 which was more
dispersed and smaller than previously reported mannan-
coated magnetite nanoparticles with a hydrodynamic size of
124.37 nm and a PDI of 0.36 [31]. The sigmoid shape of the
magnetic diagram and reaching zero magnetic fields of VSM
analysis revealed the superparamagnetic behavior of M-MNPs
(see Figure 1(f)). It was also determined that the magnetism of
bare nanoparticles is 60.01 emu cm−3 and the mannan coat-
ing decreased the saturation level to 53.96 emu cm
−3
. The net
charge of the nanoparticles was also determined using zeta
potential analysis. According to the zeta potential analysis
results, the net charge of the MNPs was shifted from −26.67
± 3.46 to −9.68 ± 2.57 mV which showed the presence of
4G. H. DARYA ET AL.
Figure 1. (a) The schematic illustration of the one-step synthesis of M-MNPs. (b) The IR spectrum of MNPs, mannan, and M-MNPs. (c) The crystal structure of the
M-MNPs with the help of XRD analysis of MNPs and M-MNPs. (d) The TEM images and (e) the hydrodynamic size of the M-MNPs using dynamic light scattering. (f)
The magnetic behavior of the MNPs and M-MNPs. (g) The net charge of the MNPs, mannan, and M-MNPs.
Figure 2. (a) The release kinetics of the PG after treatment with 0.02 U of mannanase. The baseline release of the M-MNPs at neutral pH. The release study was done
in three independent experiments and represented as mean ± STDEV. (b) The fitting model of PG release behavior of PG loaded mannan containing magnetite
nanoparticles (M-MNPs), after treatment with 0.02 U of mannanase compared to control.
EXPERT OPINION ON DRUG DELIVERY 5
closely neutral mannan polymer (+2.24 ± 0.98 mV) as
a capping polymer on M-MNPs (See Figure 1(g)).
Due to the hydrophobic nature of the PG, the adsorption was
used for the drug’s loading process in an aqueous solution. The
PG loading efficiency and loading capacity of PG on M-MNPs
were calculated to be 90.21 ± 8.38 and 58.97 ± 0.78, respectively.
3.2. PG release
The enzyme-sensitive PG release kinetics were also investigated
using a commercially available mannanase enzyme substitute for
lysosomal β-mannosidase. The timing of the release closely
aligns with the endosome/lysosome maturation process within
the phagosome/lysosome, which typically ranges from 30 min to
several hours, varying based on the cell type and the character-
istics of the particle surface [32]. According to Figure 2, the
enzyme-triggered percentage of PG in PBS medium was up to
38.03 ± 5.53% within 60 min post-treatment. The unwanted leak-
age was estimated to reach up to 4.08 ± 0.88% after 90 min
without using the mannan degrading enzyme.
The current study analyzed the release kinetics of PG-loaded
M-MNPs using various kinetic models including zero-order, first-
order, Higuchi, Korsmeyer–Peppas, and Hixon-Crowell kinetics.
The results, as depicted in Figure 2(b), indicated that the release
kinetics of PG were best described by the first-order kinetic
model, exhibiting a high regression coefficient of 0.987 (R
2
).
This suggests that the release of PG from PG@M-MNPs followed
a sustained but controlled-release pattern. Conversely, the
release kinetics of the M-MNPs’ unwanted leakage displayed
a different behavior, with the Korsmeyer–Peppas model being
identified as the most suitable model for the mannan polymer,
with an R2 value of 0.989. This model delineates the process of
drug release as a two-phase mechanism. Initially, the release of
the drug is regulated by the diffusion of PG through the mannan
polymer matrix of the nanoparticles. Subsequently, the drug
release is governed by the desorption of the PG from the surface
of the nanoparticles [33].
3.3. Cytotoxicity and drug targeting
To investigate the cytotoxicity of the delivery system on breast
and hepatocellular carcinoma (MCF-7 and HepG2), and also
the bio-safe threshold dosage of PG, M-MNPs, and
PG@M-MNPs on normal fibroblast and macrophage cells
(NIH/3T3, and J774A), MTT assay was performed. As illustrated
in Figure 3(a), the cell toxicity threshold of bare Fe
3
O
4
was
estimated to be up to 200 µg/mL of nanoparticles, while
M-MNPs on two normal cells, NIH/3T3 and J774A showed
the safe threshold concentration of up to 500, and 200 µg/
mL even after 72 h of incubation, respectively.
The neoplastic cell toxicity and targeted delivery potential
of PG and PG@M-MNPs were done using breast (MCF-7) and
liver (HepG2) cancer cell lines, respectively. Also, the
unwanted side effects and the immune toxicity of the delivery
systems were investigated against fibroblast (NIH/3T3) and
Macrophage (J774A) cell lines, respectively. As illustrated in
Figure 3(b), free administrations of the PG showed significant
toxicity effects on MCF-7 from 16 h post-treatment with IC50
values of 3.75 ± 0.45 µg/mL and finally reached 1.22 ± 0.20 µg/
mL. In the case of HepG2, the toxicity was initiated even after
4 h post-treatment with IC50 values of 3.31 ± 0.15 µg/mL and
last up to 0.57 ± 0.18 µg/mL at 48 h. This trend revealed the
relative resistance of MCF-7 cell lines against PG compared to
HepG2. Treatment of PG against two normal cells, NIH/3T3 and
J774A initiated the cellular toxicity at 4 and 24 h post incuba-
tion with IC50 values of 3.96 ± 0.43, and 4.29 ± 0.22 µg/mL. The
main difference between cancer and normal treatments is that
the cancer cell treatment showed a time-dependent manner,
while the normal cells showed relatively equal toxicity at each
time frame. This trend might relate to the constant release
kinetics of PG@M-MNPs.
The Anticancer Selectivity Index (ASI) value helps estimate
the cancer selectivity and targeted strategies of the PG and
PG@M-MNPs which are calculated as IC50 values of normal
cells/and IC50 values of cancerous cells, respectively [34].
According to Table 1, PG naturally represented a selective
anticancer ability against both MCF-7 and HepG2 with final
ASI values of 2.41 and 5.17, respectively. The PG delivery to
MCF-7 improved the anticancer selectivity about 2.23-fold
(5.38/2.41) which relatively represented the successful target-
ing ability of the PG@M-MNPs to tumor and minimal
unwanted side effects on normal breast cells. In the second
place, PG@M-MNPs to HepG2 decreased the SI values to 0.62-
fold (3.16/5.17) which might be due to the low content of
Figure 3. (a) The cellular toxicity analysis of magnetite nanoparticles (MNPs), and Mannan coated magnetite nanoparticles (M-MNPs) on NIH/3T3 and J774A cell lines at 72
h post incubation. (b) The IC50 values of PG against MCF-7, HepG2, NIH/3T3, and J774A cell lines. (c) the IC50 values of PG@M-MNPs against MCF-7, HepG2, NIH/3T3, and
J774A cell lines. The IC50 values greater than 6 µg/mL was considered to be 6 µg/mL. The MTT analysis were done in three independent replicates and the statistical
significance was reported using the one-way ANOVA (Kruskal-Wallis) method is defined as follows: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), and **** (p ≤ 0.0001).
6G. H. DARYA ET AL.
galactose on mannan polymer (1 unit of galactose to 4 units of
mannose). The Immune Selective Index (ISI) also indicated the
immune-targeting and immune safety of PG and PG@M-MNPs.
As shown in Table 1, the ISI values of the PG also show the
natural selective immunomodulatory effects of PG against
immune cells like macrophages. Accordingly, the ISI values of
the PG are higher than the ASI values on both breast and liver
cell lines, with 3.52 and 7.53, respectively. Similar to the ASI
values, the immune selective index of the breast cells is
improved about 2.11-fold (7.41/3.52), while in the case of
liver cells, the ISI is decreased to 0.58-fold (4.35/7.53).
3.4. Cellular uptake study
Due to the auto-red fluorescence nature of PG, the specific
affinity and endosome/lysosome uptake of PG and
PG@M-MNPs were also investigated using fluorescence and
confocal laser microscopy, as well as flow cytometry on MCF-
7/GFP, HepG2, and NIH/3T3 cells. As illustrated in Figures 4(a–c),
the cellular uptake of PG in the first 30 min in MCF-7/GFP,
HepG2, and NIH/3T3 cells showed the high potential ability of
PG to pass through the cell membrane. Also, the nearly equal
red emission of the PG at 4, 8, and 24 h post-treatment showed
that the cellular uptake reached equilibrium after 30 min of
incubation. The cellular uptake of the PG@M-MNPs was also
monitored after 30 min, 4 h, and 8 h on MCF-7/GFP, and HepG2
compared with 24 h post-treatment in NIH/3T3 cells (see
Figure 4(d,e)). A few scattered red dots were detected in MCF-
7/GFP and HepG2 cell lines after 30 min, while there was
a significant difference between the cytoplasmic fluorescence
emission of MCF-7/GFP and HepG2 cells. The emission differ-
ence also showed the relative receptor-mediated cellular
uptake rate of PG@M-MNPs. Moreover, fluorescence emission
was only detected 24 h post-treatment in NIH/3T3 normal cells.
Overall, the cellular uptake of the PG@M-MNPs was greater in
MCF-7/GFP, and in second place HepG2 cancer cells compared
to the normal cell, NIH/3T3. The time-dependent manner of PG
uptake in PG@M-MNPs also suggests a continuously specific
mannose, and in second place, galactose receptor-mediated
endosome/lysosome root of the delivery system in the breast
and liver cancer cell lines, respectively. Additionally, the limited
PG uptake in normal cell after 24 h approves the strong specific
targeting of the delivery system and minimal side effects on
normal cells.
Cellular uptake was also investigated using flow cytometry
in MCF-7/GFP, HepG2, and NIH/3T3 cells. As illustrated in
Figure 5(a), the fluorescence intensity difference within the
MCF-7/GFP cell population indicated the significant efficacy
of cellular absorption of free (p < 0.0001) and PG@M-MNPs
(p = 0.0026) compared to the unstained control, which might
suggest improved drug incorporation into these cells. In addi-
tion, the fluorescence intensity of HepG2 (compared with
MCF-7/GFP) showed a relatively lower but still significant
amount of PG (p = 0.046) compared with free PG administra-
tion (p = 0.002), confirming the moderate ability of
PG@M-MNPs to target HepG2 liver cells (Figure 5(b)). Both
results have shown the targeting ability of M-MNPs against
breast, and in second place, liver tumors. Moreover, the non-
significant cellular uptake of PG@M-MNPs compared to the
unstained control may decrease the adverse effects of PG on
normal cells (See Figure 5(c)).
Receptor-mediated cellular uptake with the help of over-
expressed lectin-type receptors can be used as a dual-function
vehicle for diagnosis, selective targeting, and drug delivery to
cancer cells through clathrin-mediated endocytosis, offering
major ability and minor toxicity in healthy tissues [35].
Moreover, enzyme-mediated degradation of the mannan poly-
mer with lysosomal hydrolase enhanced the cytotoxic effect of
the released prodigiosin. To take a brief look at the cellular
uptake mechanism, a phagocytosis/pinocytosis natural poly-
mer, FITC-labeled low molecular weight dextran (FITC-LMWD)
was used to investigate the probable cellular uptake mechan-
ism of PG@M-MNPs (similar to GLUT1 mediated uptake of
FITC-LMWD to cancerous cells). As mentioned in Figure 6, PG
mainly entered through the cell membrane rather than endo-
some/lysosome root in MCF-7/GFP, HePG2, and NIH/3T3, while
co-localization of FITC-LMWD with PG@M-MNPs revealed that
the majority of the PG@M-MNPs population was localized in
the early endosome, which facilitated enzyme-mediated PG
release, endosomal escape, and finally localization to the cyto-
plasm of the cells.
3.5. Immuno-modulatory activity
The overexpression of mannose receptor (MR, CD206) on the
surface of the macrophages would allow their immune-targeting
ability of the PG@M-MNPs and subsequent modulation to
improve the chemotherapy treatments. The results of J774A treat-
ments showed a significant shift of the M0 subset of macrophages
Table 1. The anticancer selectivity Index (ASI) and immune selectivity Index (ISI) values of PG and
PG@M-MNPs on MCF-7 and HepG2 cell lines.
Drugs
Time (h) MCF-7 HepG2
ASI ISI ASI ISI
PG 4 0.66 1.00 1.19 1.81
8 0.68 1.13 1.58 2.63
12 0.79 1.60 2.18 4.38
24 1.54 2.67 4.42 7.66
48 2.41 3.52 5.17 7.53
PG@M-MNPs 4 1.00 1.00 1.00 1.00
8 1.65 2.05 0.85 1.00
12 2.68 3.68 1.35 1.85
24 4.52 6.52 2.12 3.06
48 5.38 7.41 3.16 4.35
EXPERT OPINION ON DRUG DELIVERY 7
to M1 when using LPS (100 ng/mL)/IFN-γ (50 µg/mL) on expres-
sion levels of IL-6 for 24 h. The M2 subsets were also assessed for
ARG1 expression using IL-4 (75 µg/mL) after 24 h of incubation.
The expression levels of M1 (IL-6) and M2 (ARG1) markers were
assessed after treating the cells with different concentrations of PG
and PG@M-MNPs for 24 and 48 h (see Figure 7a–d).
As shown in Figure 7(a), treatment of M1 cells with different
concentrations of PG at low concentrations (<1 µM) had
a significant stimulating effect (P-value = 0.039, 0.034, 0.028,
0.0078, and 0.041) on the M1 biomarker. This illustrates the
pro-inflammatory effects of PG on the active M1 macrophages.
However, at higher concentrations (>1 µM), this process sti-
mulated a population of M1 cells to differentiate toward the
M2 subset, indicating the induction of an anti-inflammatory
environment in cells exposed to this concentration of PG. In
other words, the effect of PG at higher concentrations (2.5, 5.0,
and 5.7 µM) diminished the expression of IL-6 and, in some
cases, a mixture of M1 and M2 subsets. As illustrated in
Figure 7(b), after 48 h of treatment, the cell’s response was
similar to the 24-h treatment, with the difference that the
expression of IL-6 was much higher after 48 h. This indicates
that the treatment of cells with free PG displays time-
dependent behavior. Additionally, it can be said that similar
to the 24-h treatment, low concentrations of PG were more
successful in inducing cell stimulation (p = 0.041, 0.023, 0.0084,
0.0052, 0.0012, and 0.017).
Figure 4. The fluorescence images of (a) MCF-7/GFP, (b) HepG2, and (c) NIH/3T3 after 30 min, 4 and 8 h post treatment with PG. The fluorescence images of (d) MCF-
7/GFP, and (e) HepG2 cells after 30 min, 4 and 8 h incubation of PG@M-MNPs compared to 24-h incubation with NIH/3T3 cell line. The fluorescence images were
captured under magnification is 200×.
8G. H. DARYA ET AL.
The effect of PG@M-MNPs is also presented in Figures 7(c,d)
after 24 and 48 h of incubation. As shown in Figure 7(c),
PG@M-MNPs did not show any significant effect on the
expression levels of IL-6 in the M1 subset after 24 h of treat-
ment compared with M1-stimulated cells as the control.
Loading an equal amount of PG onto M-MNPs resulted in
a relative decrease in IL-6 gene expression compared with
free PG treatment. Additionally, at lower concentrations of
PG, no significant changes were observed in M1 marker gene
expression (P-value = 0.029, 0.035, and 0.043) which indicates
that using concentrations less than 0.25 µM does not affect
changing the M1 subset population in the short term. At
higher concentrations (>0.5 µM), administering PG@M-MNPs
resulted in a decreased expression of the IL-6 gene, leading
to a reduction in the stimulated M1 subset population. This
treatment showed a concentration-dependent behavior simi-
lar to the free PG treatment, but the expression of both marker
genes changed in a way that indicates macrophage cells
approached their baseline state (M0) before stimulation.
The effect of PG on the M2 subset was presented in
Figures 8(a,b), in 24 and 48 h of incubation with different
concentrations of PG. Accordingly, short-term exposure of PG
at low concentrations (<0.1 µM), had an inhibitory effect on
the expression levels of the M2 biomarker, ARG1, in a way
Figure 5. After a 24-h incubation period with a PG dosage of 2.0 μg/mL and PG@M-MNPs dosage of 50.0 μg/mL, flow cytometry analyses were conducted on MCF-7/
GFP, HepG2, and NIH/3T3 cells. Negative controls were tested using a PBS solution, whereas positive controls were tested using free PG. The mean fluorescence
intensity (MFI) of PG was analyzed in three independent replicates and the statistical significance was reported using the one-way ANOVA (Kruskal-Wallis) method is
defined as follows: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), and **** (p ≤ 0.0001).
EXPERT OPINION ON DRUG DELIVERY 9
Figure 6. CLSM images of MCF-7/GFP, HepG2, and NIH/3T3 cells after 6 h of incubation with 2.0 μg/mL of PG, and 50 μg/mL of PG@M-MNPs. The green and red dots
represent the FITC-LMWD and PG, respectively.
Figure 7. The expression levels of IL-6 and ARG1 genes in LPS/IFN-γ stimulated J774A macrophages after treatment with PG for a) 24 and b) 48 h of incubation and
PG@M-MNPs for c) 24 and d) 48 h of incubation. The presented results in the figure are represented with Mean ± STDEV obtained from three independent
repetitions. The significant level of changes was calculated using the Mann–Whitney method, where * (P-value ≤ 0.05), ** (P-value ≤ 0.01), *** (P-value ≤ 0.001), and
**** (P-value ≤ 0.0001) are defined.
10 G. H. DARYA ET AL.
that J774A cells were not statistically M2-stimulated (p =
0.089, 0.197, 0.389, 0.870, 0.76). Also, a decreasing trend in
IL-6 expression levels at concentrations of 0.5 and 1.0 µM was
observed. However, high concentrations of PG (5.0 and 7.5
µM) showed a significant elevation in expression levels of the
IL-6 gene (p = 0.028, 0.017), indicating an induction toward
the pro-inflammatory subset, M1. As shown in Figure 8(b),
treatment of the M2 subsets with different concentrations of
free PG for 48 h was also investigated. Low concentrations of
PG (<0.25 µM), had an inhibitory effect on the expression of
the M2 biomarker, ARG1 gene so that the population was
returned to the M0 unstimulated subsets (p = 0.103, 0.276,
0.572). The expression of the IL-6 showed a decreasing
trend at concentrations of 0.125 and 0.062 µM, however, at
concentrations of 0.25 µM and above, the expression levels of
the IL-6 elevated in the way that the population of the M2
subset was significantly shifted to the M1 subset at
a concentration of 2.500 µM (P-value = 0.022, 0.015, 0.0084).
Figure 8(c,d) illustrate the investigation of the expression
levels of both M1 and M2 macrophage marker genes by
loading an equal amount of PG@M-MNPs to investigate
their effects. According to the results, non-significant changes
in the expression levels of both markers at concentrations of
0.067, 0.125, and 0.250 µM (p = 0.13, 0.281, 0.309) suggest
that using low concentrations of PG results in decreased
expression levels of anti-inflammatory factors such as ARG1,
and this trend continues after 48 h. At higher concentrations
of 0.5 µM and above, the administration of PG@M-NMPs
showed a significant increase in the expression levels of the
IL-6 after 24 h compared to free PG treatment, indicating
specific uptake and gradual drug release from the nanostruc-
ture, leading to a reduction in macrophage polarization
toward the inflammatory subset. Therefore, at concentrations
greater than 0.5 µM, a significant shift was observed from M2
to the M1 subset of macrophages (p = 0.035, 0.018, 0.005,
0.0014).
As shown in Figure 8(d), the treatment of PG@M-MNPs
showed a time-dependent trend in IL-6 expression. Similar to
the 24-h treatment, treatment at low concentrations (≥0.5 µM)
exhibited a significant trend in inducing IL-6 expression and
inhibiting ARG1 expression at the final concentration of 5.0 µM
(p = 0.041, 0.008, 0.0013, 0.0019). Overall, PG@M-MNPs treat-
ment creates a mixture of both active subsets at higher
concentrations.
4. Discussion
To enhance cell type-specific targeting and minimize the
negative side effects of selective anticancer drug, prodigiosin,
a dual drug delivery system was developed. The ultimate goal
is to simultaneously destroy the tumor cells and strengthen
the immune system by changing the phenotype of macro-
phages, an all-out fight against solid tumor tissue. The delivery
system consists of a magnetic nanoparticle core to address the
Figure 8. The expression levels of IL-6 and ARG1 genes in IL-4 stimulated J774A macrophages after treatment with PG for a) 24 and b) 48 h of incubation and
PG@M-MNPs for c) 24 and d) 48 h of incubation. The presented results in the figure are represents with Mean ± STDEV obtained from three independent repetitions.
The significant level of changes was calculated using the Mann–Whitney method, where * (P-value ≤ 0.05), ** (P-value ≤ 0.01), *** (P-value ≤ 0.001), and ****
(P-value ≤ 0.0001) are defined.
EXPERT OPINION ON DRUG DELIVERY 11
drug toward tumor sites using an external magnet with clea-
vable mannose polymer with the ability to penetrate more
efficiently into breast (and in second place, liver) cancer cells
through endocytosis. For this purpose, mannan-grafted MNPs
(M-MNPs) were synthesized using one spot alkaline co-
precipitation method and were subsequently investigated for
breast cancer cell-specific internalization. The mannose and
galactose are cleavable with the help of hydrolytic lysosomal
β-D-mannosidase and α-D-galactosidase A [36,37]. On the
other hand, due to the presence of mannose receptors on
the surface of tumor-associated macrophages (TAMs), the
delivery system is also capable of uptake via M2 macrophages
to overcome immunosuppressive tumor microenvironment,
helping the delivery system to enhance the therapy with two-
armed chemo- and immune-mediated cancer therapy.
Our study investigates the cytotoxicity of a delivery system
on breast and hepatocellular carcinoma, as well as the bio-safe
threshold dosage of certain components on normal fibroblast
and macrophage cells. Bare magnetic nanoparticles had a cell
toxicity threshold of up to 200 µg/mL of nanoparticles, while
M-MNPs showed safe threshold concentrations of up to 500
µg/mL on normal fibroblast cells and 200 µg/mL on macro-
phage cells. Free administrations of PG showed significant
toxicity effects on breast cancer cells, in a time-dependent
manner, while normal cells showed slight adverse effects.
The Anticancer Selectivity Index of PG@M-MNPs shows
improved anticancer selectivity for breast cells but decreased
selectivity for liver cells. The Immune Selective Index values
demonstrated the immunomodulatory effects of PG against
immune cells, with higher ISI values for breast and liver cell
lines, indicating selective immunomodulatory effects.
The expression levels of IL-6 and ARG1 genes showed that
a low dose of prodigiosin treatment in the early stages of
cancer (M1 subset) stimulates the inflammatory cytokines. In
controversy, high concentrations of prodigiosin (>1 µM) lead
to generating a neutral, but active population of M1/M2 sub-
sets. Therefore, it can be said that a low dose of free PG
administration in the early stages of the disease can be effec-
tive in messing up the chronic early inflammation to late stage
(M2 subset) and altering the tumor-supporting interleukins, IL-
6, IL-1β, etc., to terminate the malignant cell supporting TME
[38]. Administration of equal PG@M-MNPs acts similar to that
of free PG; however, the inflammatory stimulation is relatively
lower. Treatment of low concentrations of PG (<1 µM) at the
late stage of cancer, with a dominant population of M2 sub-
sets, has led to suppressing the anti-inflammatory activity in
the way that it generates into the basic state of macrophages,
i.e. M0. In contrast, higher concentrations of prodigiosin
(>1 µM) significantly transformed the M2 population into M1
subsets both in the short and long terms. Treatment of low
concentrations of PG@M-MNPs (<0.5 µM) macrophage cells at
the late phase of cancer causes a significant shift of cells from
M2 to unstimulated, M0 subsets. Even at higher concentra-
tions (>0.5 µM), a strong conversion of the M2 to M1 subset is
formed, even much higher than in PG administration, and this
process continues in the long term.
One of the interesting hallmarks of cancer treatment is
related to the targeting of narrow differences of cancer cells
out of normal ones. Recent studies have suggested that slight
pH decreases and Cu ion level elevation were detectable in
several tumor tissues [39]. The multifaceted roles of copper in
cancer: a trace metal element with dysregulated metabolism,
but also a target or a bullet for therapy [39]. Previous research
has shown that the time-dependent toxicity of PG may be
influenced by the higher concentration of Cu
2+
and Zn
2+
ions,
as well as the low pH of cancer cells, leading to accelerated
DNA breakage [40,41]. Additionally, PG may inhibit the over-
expressed repairing enzymes called topoisomerase, leading to
a loss of control in the entire process [42]. As same as other
stimuli-responsive nanoformulations, controlled drug release
of PG@MNPs provides a higher retention time compared to
free administration [43]. The potential mechanisms underlying
this phenomenon might be related to the highly hydrophobic
nature of PG. According to the fitting curve models of release,
after enzyme-related mannan decomposition and evacuation
of a huge part of the cargo, the portion of the drug that
accumulated into the condition might be reabsorbed into
the nanostructures, which result in an increase in the release
time.
Tumor-associated macrophages (TAMs) are one of the main
types of infiltrating immune cells in the tumor environment,
and are generally transformed into one of two functionally
opposing subsets, including classically activated M1 and M2
subsets of macrophages. At the early stages of tumor forma-
tion, activated M1 macrophages usually exert antitumor func-
tions, including direct cytotoxicity and antibody-dependent
cytotoxicity (ADCC), while M2 subsets enhance the progres-
sion and metastasis of tumor cells through interfering with
T cell’s anti-tumor immune responses and enhancing tumor
angiogenesis [8,44]. Earlier studies have shown that hypoxia in
the tumor triggers the M1 population into the M2 subset [44].
Interestingly, chemotherapy led to the depletion of quiescent
and monocyte-derived macrophages. Monocyte-derived
macrophages were able to recover and provide tumor clear-
ance by phagocytosis [7]. Tumor-associated macrophages
(TAM) are involved in the growth and metastasis of cancer
cells through the secretion of cytokines, chemokines, and
growth factors like TGF-β1, EGF, PDGF, HGF, and BFGF
[45,46]. Both M1 and M2 macrophages have a high degree
of plasticity and therefore can be converted to each other by
changing the tumor microenvironment or therapeutic inter-
ventions. M1 subsets have fast (a few minutes) and slow (1–3
days) antitumor effects through directly mediated cytotoxicity
against tumor cells, like antibody-dependent cytotoxicity
(ADCC) induction and ROS and NO release [8,45,47]. M2
macrophages specifically contributed to cancer metastasis,
angiogenesis, and proliferation through various anti-
inflammatory mechanisms and induced immunosuppression
by secreting immunosuppressive molecules such as IL-10, TGF-
β, and human leukocyte antigen G (HLA-G) into the TME
[46,48,49]. Additionally, M2 macrophages directly interact
with myeloid-derived suppressor cells (MDSC) and actively
suppress T cell-mediated antitumor responses [50]. M2 macro-
phages also mediated a key role in the recruitment of regula-
tory T cells to the TME through the chemokine receptors CCR4
and M2-derived CCL17/CCL22 [51]. The M2 subset also shows
enhanced expression of PD-L1 and cytotoxic T lymphocyte
antigen ligand 4 (CTLA4) both of which have specific targets
12 G. H. DARYA ET AL.
for cytotoxic T cells (CTLs) to block their cancer cell-killing
ability [52].
Improving cancer therapies remains one of the biggest
obstacles to overcome in medicine today. Chemotherapy
resistance is often attributed to elevated levels of IL-6 and
prostaglandin E2 (PGE2), which are both inflammatory media-
tors that cause differentiation of tumor-promoting M2 macro-
phages which are more resistant to chemo- and radio-therapy
than M1 macrophage subtypes and pose a challenge to cancer
treatment [53,54]. Several nanostructures have been utilized
to target macrophages like liposomal clodronate (Clo-Lipo-
DOTAP), which is a typical strategy for reducing TAMs in
B16/F10 melanoma tumor models. Even though some cancer
treatments, such as immunotherapy, cannot benefit from
TAMs reduction [55]. Nevertheless, not all cancer treatments
can benefit from TAMs reduction, particularly complementary
therapies like immunotherapy, in which the promptness of
innate immune stimulation is elevated. In such cases, trans-
forming M2 TAMs into pro-inflammatory M1 macrophages
may prove to be a potential new anti-tumor immunotherapy.
This can be achieved via facilitation of receptor-dependent cell
phagocytosis (e.g. with antibodies) or by inhibiting CD47-
SIRPα signaling. For instance, some monoclonal antibodies,
such as rituximab, which is used to treat non-Hodgkin’s lym-
phoma, and trastuzumab which is used to treat HER2-
overexpressing breast cancer, have been shown to increase
macrophage phagocytosis and induce macrophage-mediated
phagocytic killing in vitro and in vivo [56,57].
The application of nanotechnology in cancer treatment
has gained increasing attention today. These nanostructures
infiltrate tumor sites via the bloodstream and move into the
interstitial fluid through the vascular wall by passive diffusion
before being absorbed by the tumor cells [58]. Ligand-
receptor-mediated active targeting strategies, stimulus-
sensitive systems, and cell-mediated systems are currently
among the most significant techniques employed in deliver-
ing NPs to tumor tissues and enhancing tissue distribution.
Noteworthy tumor biomarkers include prostate-specific
membrane antigen (PSMA) in prostate cancer [59], epidermal
growth factor receptor (EGFR) in lung cancer cells [60,61],
and human epidermal growth factor receptor 2 (HER2) in
gastric and breast cancers [62,63]. Mannose is a well-known
ligand for targeting macrophages because of the high
expression of mannose receptors on macrophages.
Numerous new methods that utilize mannose as the target
ligand have been developed in recent years. Among the
strategies designed to target macrophages, TAMs depletion
and reprogramming are recognized as the most effective
[64,65]. For instance, inhibition of receptor signaling (CSF1-
CSF1R), using anti-CSF1R monoclonal antibody, or the CCL2-
CCR2 signaling pathway monocyte recruitment inhibitor, PF-
04136309, led to apoptosis induction in TAMs and blockage
of circulating inflammatory populations toward in vitro and
preclinical studies, respectively [66–71]. Also, Toll-like recep-
tor (TLR) agonists, cytokines, antibodies, and microRNAs like
R848-loaded β-cyclodextrin NPs (CDNP-R848), poly(β-amino
ester)s copolymer including histamine, mPEG-NH2, and
HDDA with IL-12 cargo, and miR-125b@hyaluronic acid–poly-
ethyleneimine (HA-PEI) have led to a change in the
phenotype of TAMs and finally induced the transformation
of macrophages from M2 to M1 phenotype, respectively [72–
75]. Combined with anti-PD-1 therapy, TLR agonists act as
protective agents against the challenge of tumor recurrence
and improve immunotherapeutic responses by inducing
tumor regression and increasing efficacy in animal models
[76,77].
Receptors on immune system cells were preferred, mak-
ing mannose a popular ligand for targeting macrophages
[78]. Albumin nanoparticles modified with dual ligands
including transferrin receptor (TfR) containing T12 peptide
and mannose effectively prevent the proliferation of glioma
cells and by reprogramming M2- to M1-TAMs subsets by
reconstructing the TME and enhancing the anti-cancer
potential of the delivery systems through the interaction
of TAM, Treg, and CD
8+
cells as well as effective cytokines
[79]. In another study, twin-like core-shell mannose
anchored-IMD-0354 nanoparticles were introduced for
smart TAM targeting of Sorafenib [80].
5. Conclusion
In the current study, a new method for dual cancer therapy
was investigated using magnetic nanoparticles for chemo-
immunotherapy against breast tumors using the mannan fab-
ricating magnetic nanoparticles harboring the anticancer drug,
prodigiosin. The designed delivery system aimed to address
the tumor while also modifying the tumor microenvironment
to enhance the power of CTL (cytotoxic T lymphocytes) in
combating the remaining cells. Further research is necessary
to fully understand the effectiveness of the mannan delivery
systems loaded with prodigiosin.
Funding
The research was partially supported by the collaboration between Shiraz
University and Shiraz University of Medical Sciences [no. 1170-1399].
Declaration of interest
The authors have no relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript. This
includes employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or pending, or
royalties.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
ORCID
Banafsheh Rastegari http://orcid.org/0000-0001-5620-2670
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EXPERT OPINION ON DRUG DELIVERY 13
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EXPERT OPINION ON DRUG DELIVERY 15
