Journal of Inorganic Biochemistry 217 (2021) 111383

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Antitumor potential of cisplatin loaded into SBA-15 mesoporous silica

nanoparticles against B16F1 melanoma cells: in vitro and in vivo studies
Dijana Drača a, David Edeler b, Mohamad Saoud b, Biljana Dojčinović c, Duško Dunđerović d,
Goran Đmura e, Danijela Maksimović-Ivanić a, Sanja Mijatović a, Goran N. Kaluđerović b, f, *
a
Department of Immunology, Institute for Biological Research”Siniša Stanković” National Institute of Republic of Serbia, University of Belgrade, Bulevar despota Stefana
142, 11060 Belgrade, Serbia
b
Department of Bioorganic Chemistry, Leibniz-Institute of Plant Biochemistry, Weinberg 3, D 06120 Halle (Saale), Germany
c
University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Njegoševa 12, 11000 Belgrade, Serbia
d
Institute of Pathology, School of Medicine, University of Belgrade, dr Subotića 1, 11000 Belgrade, Serbia
e
Animal Facility, Institute for Biological Research”Siniša Stanković” National Institute of Republic of Serbia, University of Belgrade, Bulevar despota Stefana 142, 11060
Belgrade, Serbia
f
Department of Engineering and Natural Sciences, University of Applied Sciences Merseburg, Eberhard-Leibnitz-Straße 2, DE-06217 Merseburg, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: CP (cisplatin) and mesoporous silica SBA-15 (Santa Barbara amorphous 15) loaded with CP (→SBA-15|CP) were
Drug carrier tested in vitro and in vivo against low metastatic mouse melanoma B16F1 cell line. SBA-15 only, as drug carrier, is
SBA-15 found to be not active, while CP and SBA-15|CP revealed high cytotoxicity in lower μM range. The activity of
Cisplatin
SBA-15|CP was found similar to the activity of CP alone. Both CP and SBA-15|CP induced inhibition of cell
Cytotoxicity
Apoptosis
proliferation (carboxyfluorescein succinimidyl ester - CFSE assay) along with G2/M arrest (4′ ,6-diamidino-2-
Autophagy phenylindole - DAPI assay). Apoptosis (Annexin V/ propidium iodide - PI assay), through caspase activation
(apostat assay) and nitric oxide (NO) production (diacetate(4-amino-5-methylamino-2′ ,7′ -difluorofluorescein-
diacetat) - DAF FM assay), was identified as main mode of cell death. However, slight elevated autophagy
(acridine orange - AO assay) was detected in treated B16F1 cells. CP and SBA-15|CP did not affect production of
ROS (reactive oxygen species) in B16F1 cells. Both SBA-15|CP and CP induced in B16F1 G2 arrest and subse­
quent senescence. SBA-15|CP, but not CP, blocked the growth of melanoma in C57BL/6 mice. Moreover, hepato-
and nephrotoxicity in SBA-15|CP treated animals were diminished in comparison to CP confirming multiply
improved antitumor potential of immobilized CP. Outstandingly, SBA-15 boosted in vivo activity and diminished
side effects of CP.

1. Introduction ototoxicity and nephrotoxicity, as well as acquired tumor resistance

[3,4]. Various mechanisms of CP resistance have been proposed based
Cisplatin (CP) is used in clinic as a first-line agent against various on resistant cell models, such as cellular CP accumulation, detoxifica­
type of tumors (for instance ovarian, testicular, lung cancers), alone or in tion, apoptosis inhibition as well as DNA repair processes [5,6]. More­
combined therapy [1]. Despite, CP causes severe side effects [2]. The over, enhancement of CP therapeutic efficacy should be improved too
application of CP is restricted due to dose-dependent toxicities, i.e. [7]. This issue is a result from the prompt CP inactivation through non-

Abbreviations: AO, acridine orange; B16F1, low metastatic mouse melanoma cell line; CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′ ,6-diamidino-2-
phenylindole; DAF FM, diacetate(4-amino-5-methylamino-2′ ,7′ -difluorofluorescein-diacetat); DHR, dihydrorhodamine; EDTA, ethylenediaminetetraacetic acid; FBS,
fetal bovine serum; CP, cisplatin; MCM-41, (Mobil Composition of Matter No. 41) mesoporous silica nanoparticles; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe­
nyltetrazolium bromide; NO, nitric oxide; PBS, phosphate-buffered saline; PI, propidium iodide; ROS, reactive oxygen species; RPMI-1640, Roswell Park Memorial
Institute-1640 medium; SA-β-Gal, senescence-associated beta-galactosidase; SBA-15, (Santa Barbara amorphous 15) mesoporous silica nanoparticles; SBA-15p,
functionalized SBA-15 with 3-chloropropyltriethoxysilane; SBA-15|CP, mesoporous silica nanoparticles loaded with CP; CV, crystal violet.
* Corresponding author at: Department of Engineering and Natural Sciences, University of Applied Sciences Merseburg, Eberhard-Leibnitz-Straße 2, DE-06217
Merseburg, Germany
E-mail address: [email protected] (G.N. Kaluđerović).

https://doi.org/10.1016/j.jinorgbio.2021.111383
Received 21 June 2020; Received in revised form 24 January 2021; Accepted 25 January 2021
Available online 3 February 2021
0162-0134/© 2021 Elsevier Inc. All rights reserved.
D. Drača et al. Journal of Inorganic Biochemistry 217 (2021) 111383

specific interaction in the body, on the first line with the blood proteins were prepared in DMF and culture medium at concentration 10 mM
[8]. All these obstacles could not properly be resolved by chemical and 2 mg/mL, respectively. Working concentration were prepared using
manipulations of the platinum ligand sphere (e.g. carboplatin, neda­ appropriate stock solution and diluting it with completed nutrient me­
platin, oxaliplatin, satraplatin) [9,10], neither by using CP-polymer dium. BD FACS Aria III was used for flow cytometry experiments and
conjugates [11,12]. results were analyzed using BD FACSDiva software (BD Biosciences,
However, using nanoconstructs, acting as drug vehicles through Heidelberg, Germany) and Cyflogic (V 1.2.1, CyFlo Ltd.). For viability
physical adsorption and trapping, has become an interesting field over assays 5000 cells/well in 96 well plate was used, while for flow
the last years [13,14]. Justification for drug loading in such nano­ cytometry measurements 2.5 × 105 cells/well in 6 well plate was used.
materials is in drug hydrolysis hindrance, prevention of non-specific
protein-drug interaction, impact on drug accumulation as well as 2.2. Viability assessment by MTT and CV assays
customized drug release [15,16]. Various nanoparticles are shown to be
physiologically harmless, without toxicity, however this depends on The viability of the cells was measured by CV (crystal violet) and
their size [17,18]. These nontoxic materials [19], particularly meso­ MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
porous silica materials SBA-15 (Santa Barbara Amorphous 15) [20], are assays as described previously [48]. Absorbance was measured using a
found to be very promising as transporters for different anti- 96-well plate reader (Tecan Spectra, Crailsheim, Germany). IC50 [μM]
inflammatory and anticancer drugs [13,14,21–23]. and MC50 [μg/mL] values, defined as the concentration of the com­
Two nonplatinum-metal based anticancer active compounds, tita­ pounds at which 50% of cell inhibition occur were calculated using four-
nium(IV) and tin(IV), were loaded at first in mesoporous silica, SBA-15 parameter logistic function and presented as mean from three inde­
and MCM-41 (Mobil Composition of Matter No. 41) [24–28]. From all of pendent experiments. The results of cell viability are presented as means
them the most active in vitro and the most promising in vivo were found ± SD. The significance of the differences between various treatments
SBA-15 particles loaded with Ph3Sn(CH2)6OH (Sn6) [24]. The most was assessed by analysis of variance (ANOVA), followed by a Student-t-
interesting fact for this system is that SBA-15p|Sn6 (SBA-15p = func­ test and p value less than 0.05 was considered significant. MC50 for SBA-
tionalized SBA-15 with 3-chloropropyltriethoxysilane) transform sur­ 15|CP: 1.23 ± 0.13 μg/mL, CV; 0.94 ± 0.18 μg/mL, MTT. MC50 for SBA-
vived malignant melanoma B16 cells into nonproliferative melanocyte- 15: > 100 μg/mL, CV and MTT.
like phenotype presenting nonaggressive suppression of tumor growth.
Besides, different mesoporous silica nanoparticle types or sizes, as well, 2.3. Uptake study
as functionalized ones were used for delivery of CP. Moreover, code­
livery with other drugs and or proteins with CP has been reported too B16F1 cells were seeded in nine flasks (8 × 105 cells/flask). After 24
[29–42]. Recently we reported on drug delivery system SBA-15 loaded h incubation, the cells were washed with PBS and treated with CP (IC50
with CP (→ SBA-15|CP; loaded amount 12.1 wt% Pt) and its anticancer dose; four flasks) or SBA-15|CP doses (MC50 dose; four flasks), in addi­
potential [43]. CP from SBA-15|CP in a lower concentration expressed tion, one control flask was kept without treatment, however the medium
higher anticancer potential against mouse high metastatic melanoma was changed at the same day. The treatments were sampled after 2, 6, 24
B16F10 cell line [44]. This reflected through minimal apoptosis induc­ and 48 h treatment, afterwards, cells were washed with PBS and de­
tion, accompanied by enhanced autophagy and ROS (reactive oxygen tached using trypsin. Cell count was evaluated using cell counter
species) production. Furthermore, SBA-15|CP abrogated proliferation of Countess II FL. The control sample was processed at 48 h time point. The
B16F10 cells. Importantly, survived clones differentiated in the senes­ pellets were stored at − 20 ◦ C until the time of measurement. The acid
cent cells. extracts of the samples for ICP-MS studies was performed with 2 mL
As continuation of our work, herein investigation of SBA-15|CP, hydrochloric acid (HCl, 37 wt%, Suprapur®, Merck KGaA, Darmstadt,
along with CP as control and carrier SBA-15, against low metastatic Germany). The Thermo Scientific iCAP Qc ICP-MS (inductively coupled
variant B16F1 cell line was conducted. This cell line showed lower plasma mass spectrometry, Thermo Scientific, Bremen, Germany) in­
sensitivity to CP in comparison to other tumor cell lines [45–47]. strument was optimized for optimum performance gas H2/He mixture in
In this pioneer work, similarities and differences between free and Collision Cell Technology (CCT) mode using the supplied autotune
immobilized CP were studied in vitro against melanoma B16F1 model protocols and operational software Qtegra. The ICP-MS instrument was
cells and in vivo syngeneic melanoma model in C57BL/6 mice to prove if tuned using a solution TUNE B iCAP Q (1 μg/L of each: Ba, Bi, Ce, Co, In,
it is justified the use of SBA-15 as suitable vehicle for conventional drug, Li, U) provided by the manufacturer Thermo Scientific, Germany. The
CP. Subcutaneous syngeneic model of melanoma induced by imple­ external calibration solutions were made from certified plasma standard
mentation of B16F1 cells, enabled us to precisely follow the develop­ solutions Specpure®, Pt 1000 μg/mL and Specpure®, Si 1000 μg/mL
ment of tumors in time and in the context of physiologically active (Alfa Aesar GmbH & Co KG, Germany). The ICP-MS measurement for
immune and stromal environment. These circumstances allowed better each sample was carried out in triplicate (n = 3). The reliability of
definition of the overall impact of applied drugs as well as differences measurements was approved by relative standard deviation, RSD <
between free and nano-formulated cisplatin. 0.5%. For water extracts, the limit of quantitation (LOQ) for platinum
and silicon were determined to be 46 and 94 ng/L, respectively. The
2. Experimental measurement were performed on isotope 194Pt and 28Si.

2.1. Reagents and cells 2.4. Cell cycle analysis

Cisplatin (Sigma) was purchased and used as received from distrib­ In 6 well plates, cells were incubated with CP (IC50, 2 × IC50 doses) or
utor. SBA-15|CP was prepared as previously described [43]. RPMI-1640 SBA-15|CP (MC50, 2 × MC50 doses) for 48 h and cell cycle analysis was
(PAA Laboratories GmbH), phosphate-buffered saline (PBS, Dulbecco’s), performed using DAPI stain [49].
fetal bovine serum (FBS, Biochrom AG), penicillin/streptomycin (PAA
Laboratories GmbH), trypsin-EDTA (ethylenediaminetetraacetic acid) 2.5. Proliferation potential (CFSE assay)
0.05%/0.02% (PAA Laboratories GmbH) were used. Tumor cell line
mouse melanoma B16F1 was cultivated in completed nutrient medium: Investigated cells were pre-stained with CFSE (carboxyfluorescein
RPMI-1640 (Roswell Park Memorial Institute-1640 medium) supple­ succinimidyl ester) as described previously [50]. Intensity of fluores­
mented with 10% FBS and 1% penicillin/streptomycin; at 37 ◦ C, 5% CO2 cence decreases after each division, enabling the quantification of cell
in a humidified atmosphere. The stock solutions of CP and SBA-15|CP proliferation. Afterwards, B16F1 cells were seeded in 6 well plate and

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D. Drača et al. Journal of Inorganic Biochemistry 217 (2021) 111383

Fig. 1. Dose-dependent survival of B16F1 cells treated with CP [μM], as well as SBA-15 and SBA-15|CP [μg/mL] (48 h; * p < 0.05 refers to untreated cultures).

treated with CP (IC50, 2 × IC50 doses) or SBA-15|CP doses (MC50, 2 × overnight staining with a SA-β-Gal reagent at 37 ◦ C with 5% CO2.
MC50 doses). Samples were investigated using flow cytometry after 48 h Samples were analyzed using flow cytometry (FACSAria III, BDsciences,
of treatment. USA) to investigate the presence of cellular senescence.

2.6. Apoptosis detection (Annexin V/PI and apostat assays) 2.11. Induction of tumors and in vivo treatment

In 6 well plate, cells were incubated with CP (IC50, 2 × IC50 doses) or Tumors were induced by application of 2.5 × 105 B16F1 cells sub­
SBA-15|CP (MC50, 2 × MC50 doses) for 48 h. Afterwards, Annexin V/PI cutaneously into the right dorsal lumbosacral region of syngeneic
or apostat procedure was applied as described in the literature [51]. C57BL/6 mice. Treatment started on the 7th day after cell inoculation
when tumors became palpable. CP was administrated as 2 mg/kg in 2%
2.7. Autophagy detection (AO assay) DMF in PBS 3 times a week. SBA-15|CP was applied as 10 mg/kg in PBS
the same treatment regime as CP, while control received 2% DMF in PBS
In 6 well plate, cells were incubated with CP (IC50, 2 × IC50 doses) or as vehicle. Tumor growth and behavior of animals were monitored
SBA-15|CP doses (MC50, 2 × MC50 doses) for 48 h. Afterwards, AO assay daily, and animals were sacrificed on the 21st day of experiment. Tu­
was performed as previously described [52]. mors were isolated, measured in three dimensions and volume was
calculated as 0.52 × a × b2, where “a” is the longest and “b” is the
2.8. ROS (DHR) and NO detection (DAF FM assay) shortest diameter. Animals used in this study were female inbred
C57BL/6 mice from our own facility at the Institute for Biological
Investigated cells were pre-stained with DHR (dihydrorhodamine) as Research “Siniša Stanković”. Animals were kept under the standard
described previously [53]. Afterwards, B16F1 cells were seeded in 6 well laboratory conditions (non-specific pathogen free) with ad libitum
plate and treated with CP (IC50, 2 × IC50 doses) or SBA-15|CP doses regime of food and water intake. The handling of animals and the study
(MC50, 2 × MC50 doses). DAF FM staining in cells exposed to treatment protocols were in agreement with the rules of the European Union and
for 48 h was done exactly as previously reported [54]. Samples were were approved by the Institutional Animal Care and Use Committee at
investigated using flow cytometry after 48 h of treatment. the Institute for Biological Research “Siniša Stanković”, University of
Belgrade (1–02/14).
2.9. Morphological evaluation
2.12. Histopathological examination
B16F1 cells were seeded overnight in 6 well plate (50,000 cells/
well). After 24 h, IC50 and MC50 doses of CP and SBA-15|CP were applied At the end of the treatment animals were euthanized using carbon
and further incubated for 48 h. For evaluation of the morphological dioxide and tumor tissue, livers and kidney were collected from all
changes the medium was discharged from the wells and cells were groups, fixed in 10% formalin and processed exactly as described pre­
washed with PBS, afterwards fixed. The cells were observed under the viously [55]. Slides were mounted and analyzed under Olympus BX50
EVOS® FL Auto Imaging System using 40× magnification phase- microscope (Olympus, Tokyo, Japan) with Olympus DP70 camera at
contrast imaging. 200× or 400× magnification (n = 3 in each group).

2.10. Senescence assay (SA-β-Gal) 2.13. Statistical analysis

B16F1 cells were seeded overnight on 6 well plate (50,000 cells/ The results of cell viability are presented as means ± standard de­
well). After 24 h, IC50 and MC50 doses of CP and SBA-15|CP were applied viations of triplicate cultures from one representative of three repeated
and further incubated for 48 h. For evaluation of the cellular senescence experiments. The significance of the differences between various treat­
using flow cytometry, the medium was discharged from the wells and ments was judged by analysis of variance (ANOVA), followed by a
the cells were washed with PBS. Subsequently the cells were detached Student’s t-test. For in vivo experiment, statistical significance between
using trypsin and transferred to falcon tubes for further fixation and the treated groups was estimated by the Mann-Whitney test. For eval­
uation of differences in histopathological changes between treatment
groups and control group was used Oneway ANOVA test. Statistical
Table 1
IC50 [μM] values of CP and SBA-15|CP determined with CV and MTT assays (48
significance was considered if p value was less or equal 0.05. Statistics
h) are calculated as mean ± SD from 3 repeated experiments. was done in program IBM Statistics SPSS 20.

Compound/Material CV MTT

IC50 [μM]

CP 0.73 ± 0.11 0.72 ± 0.17

SBA-15|CPa 0.76 ± 0.08 0.58 ± 0.11
a
recalculated from platinum content (EDX) [43].

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D. Drača et al. Journal of Inorganic Biochemistry 217 (2021) 111383

assay) were evaluated using ICP-MS analysis (Fig. 2). Thus, Pt and Si
content in the B16F1 cells were determined on different time points
upon treatment (0, 2, 6, 24 and 48 h). After only 2 h of action naked and
immobilized CP was intercellularly found at concentrations which do
not differ significantly. Recently, our groups reported that another de­
rivative of SBA-15 containing organotin(IV) compound started to
internalize even after 15 min in melanoma A375 cells [56], while after 2
h by passive fluid-phase uptake and macropinocytosis particles were
allocated in cells. Accordingly, also here a high accumulation of Pt (and
Si) was observed after only 2 h of action. Therefore, it might be assumed
that the release of CP occurs in the cells, however the leakage of the CP
in medium should not be excluded. Though, both free and immobilized
CP exhibited similar accumulation at examined time points in the B16F1
Fig. 2. Metal uptake (Pt and Si) in B16F1 cells treated with CP and SBA-15|CP.
cell line. Consequently, efficiency and uptake of free and immobilized
CP in B16F1 cells showed similar high cytotoxic potential.
3. Results and discussion

3.1. Viability of B16F1 cells treated with CP and SBA-15|CP correlates 3.2. CP and SBA-15|CP cause G2/M arrest and inhibit B16F1 cell
with metal uptake division

Viability of B16F1 cells upon treatment with SBA-15, CP and SBA-15| To investigate mode of cell death caused by SBA-15|CP, at first cell
CP was determined using CV and MTT assays (Fig. 1). As observed cycle distribution using flow cytometry in treated B16F1 cells was
previously on different cell lines [24,43], herein SBA-15 was found analyzed (Fig. 3.A). Both CP and SBA-15|CP induced cell cycle arrest in
inactive against B16F1 cells (up to 100 μg/mL). On the other hand, CP G2/M phase, with the similar extent (IC50 doses). This arrest was more
was found highly efficient on B16F1 cells. Taking low amount of loaded pronounced with 2 × IC50 concentrations. A slight increase of the cells in
CP into SBA-15 (12.1% Pt) in consideration [43], SBA-15|CP exhibited sub-G1 population was observed upon the treatment indicating that
slightly higher activity than CP according to MTT test (Table 1). On the apoptosis may succeed G2/M arrest. Furthermore, the influence on
other hand SBA-15|CP (IC50 = 0.43 ± 0.03 μM, CV; 0.72 ± 0.20 μM, cellular proliferation of CP and SBA-15|CP on B16F1 cells was investi­
MTT) was 3.5 times more efficient than CP (IC50 = 1.49 ± 0.14 μM, CV; gated using CFSE assay (Fig. 3.B). It can be observed, consistent with cell
1.29 ± 0.16 μM, MTT) against high metastatic melanoma cell line cycle results, that CP (IC50: 69.2%, divided; 30.8% undivided) and SBA-
B16F10 [43]. However, no significant difference exits in between CP 15|CP (IC50: 78.2%, divided; 21.8% undivided) remarkable inhibited
and SBA-15|CP treated B16F1 cells. proliferation of B16F1 cells (control: 96.7%, divided; 3.3% undivided).
Uptake studies of CP and SBA-15|CP in B16F1 cells (IC50 values; CV This effect was more pronounced with higher applied concentration of
CP (2 × IC50: 43.4%, divided; 56.6% undivided) as well as SBA-15|CP (2

Fig. 3. Cell cycle distribution (DAPI assay) (A) and proliferation potential (CFSE assay) (B) of B16F1 cells treated with CP and SBA-15|CP (48 h).

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Fig. 4. Apoptosis (Annexin V/PI assay) (A) and caspase induction (apostat assay) (B) of B16F1 cells treated with CP and SBA-15|CP (48 h).

Fig. 5. Autophagosomes presence (AO assay) in the B16F1 cells treated with CP and SBA-15|CP (48 h).

× IC50: 52.1%, divided; 47.9% undivided). Both free and immobilized cells apostat assay was conducted. Results showed massive caspase
CP significantly inhibited proliferation of the B16F1 cells upon activation in B16F1 cells cultivated in the presence of both CP and SBA-
treatment. 15|CP (Fig. 4.B). Obviously, it is found that caspase activation was
concentration dependent and more pronounced with higher doses.

3.3. CP and SBA-15|CP induce caspase triggered apoptosis in B16F1 cells

3.4. Enhanced NO production can be responsible for drugs action
As apoptosis may be responsible for decreased viability of B16F1, its
presence was investigated using Annexin V/PI double staining assay Having in mind that autophagic process may contribute to cell death
(Fig. 4.A). Clear evidence both early and late apoptotic cells was seen and even to antagonize apoptosis [57–59] it was of interest to evaluate
upon treatment of B16F1 cells with CP and SBA-15|CP. Also here, this its eventual contribution to drugs antitumor activity. Because of that in
effect was more pronounced with 2 × IC50 doses. On the other hand, B16F1 cells treated with CP and SBA-15|CP the autophagosomes pres­
naked CP induced enhanced apoptosis in B16F1 cells in comparison to ence, as the hallmarks of autophagy, were analyzed (Fig. 5). Moderate
immobilized CP. To verify the involvement of caspases in B16F1 treated elevation of autophagosomes (less than 9%) was detected in B16F1 cells

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D. Drača et al. Journal of Inorganic Biochemistry 217 (2021) 111383

Fig. 6. Effect of CP and SBA-15|CP on NO (DAF FM assay) (A) and ROS production (DHR assay) (B) in B16F1 cells (48 h).

Fig. 7. Morphology evaluation (A) and senescence induction (B) of B16F1 cells treated with of CP and SBA-15|CP (48 h).

upon CP and SBA-15|CP treatment. Contrarily to data in this study, a production of ROS in treated B16F1 cells (Fig. 6.B). Contrary to that,
significant autophagy presence (31%) was detected only when the same drugs applied against metastatic model B16F10 induced elevated
B16F10 metastatic cells were treated with 2 × IC50 dose of SBA-15|CP production of these toxic metabolites [43].
[43]. Since the number of apoptotic cells multiply overcome detected
autophagy and the quantity of autophagic cells was not affected by the
application of 2 × IC50 doses, it can be speculated that in this settings 3.5. Senescence is triggered through irreversible cell cycle arrest in G2
autophagy is not of pivotal significance for drugs action. phase
Having in mind the significance of NO and ROS as mediators of cell
death, their production upon the treatment with experimental com­ As CP provokes senescence, beside in other cell lines (e.g. ovarian:
pounds was investigated. Boosted NO production was determined in the A2780, SKOV3 and TOV-21G; nasopharyngeal: CNE1; lung: A549 and
treated B16F1 cells with both CP as well as CP in nano form (Fig. 6.A). It H292; hepatocellular: HepG2 and SMMC-7721) [64], also in A375 and
is well known that NO can regulate diverse processes, thus can also B16F10 melanoma [65], herein necessity to investigated potential of
affect apoptosis and autophagy in a cell-specific manner [60,61]. Pre­ immobilized and free CP against B16F1 cell line on this specific
viously, it was shown that release of NO in B16F1 cells support apoptosis phenotype change has arisen. Treated B16F1 cells with CP or SBA-15|CP
[62]. Accordingly, a massive apoptosis, both early and late, may be (48 h) were examined by light microscopy and flow cytometry analysis
connected to NO production in cells treated with free as well as loaded (SA-β-Gal). Enlargement of cellular morphology (Fig. 7.A) could be
CP. This potent cellular messenger may inhibit autophagosome syn­ easily observed in cells treated with CP or SBA-15|CP which might point
thesis [63]. Therefore, remarkable NO production can be associated to senescence transition. Using senescence-associated β-galactosidase
with a slight autophagy in B16F1 cells. Precisely it may be involved in (SA-β-Gal) as a biomarker, a canonical marker of cell senescence [65],
modulation of mentioned processes. On the other hand, independently confirmed morphological changes (Fig. 7.B). Senescence is defined as an
from applied doses (IC50 or 2 × IC50) of these two drugs there is no irreversible cell cycle arrest in G1 (G1 exit) or in G2 phase (G2 exit) [66].
In concordance with cell cycle outcome of treated B16F1 cells (G2/M

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hunching or unusual interactions (see Supporting Information Table S1).

All these signs were not observed in animals receiving SBA-15|CP
indicating that novel drug form is less toxic to animals in comparison
to CP alone. Histopathological review of tumor tissue revealed massive
necrosis and inflammatory infiltration in SBA-15|CP treated groups that
multiply overcome one observed in CP receiving group, confirming once
more increased antitumor potential CP when immobilized into SBA-15
(Fig. 9). Necrosis, evaluated as percentage of necrosis on cross section
in CP, SBA-15|CP and control groups were 32, 56 and 22%, respectively
(p > 0.05).
On the other hand, nephrotoxicity is more obvious in CP treated
group. In concordance with literature data showing injury of tubules and
kidney vasculature [71], in CP group necrotic changes, vascular mal­
formation, inflammation and atrophy of cortex were detected (Fig. 10).
Therefore, in control group width of kidney cortex was 2396.6 ± 132.9
μm, in SBA-15|CP 2122.4 ± 179.8 μm and in CP treated group it was
2055.6 ± 219.5 μm (p < 0.01 between control group and treatment
groups). Moreover, in kidney of SBA-15|CP treated animals lymphocyte
infiltration and vascular degeneration was also noticed but the level of
injury was lower than in CP group. However, there was no statistical
significance in effects between treated groups (p > 0.05). Similarly,
Fig. 8. Effect of CP and SBA-15|CP on tumor growth in vivo. CP was applied at
2 mg/kg, SBA-15|CP 10 mg/kg 3 times a week. Animals (n = 7 per group) were
following the microscopic evaluation, in liver of both groups focal
sacrificed at day 21. Tumor volume was calculated as indicated in subsection inflammation, oligo-cellular necrosis and portal vein dilatation were
2.11. *p = 0.048 in SBA-15|CP in comparison to control group. observed indicating the hepatotoxicity but it was more pronounced in
CP group in comparison to SBA-15|CP treated animals. Those changes in
phase arrest), both naked and immobilized CP induced G2 exit, which is both experimental groups are quite discrete, and to evaluate full impact
usually related with permanent DNA damage-inducing agents such as CP (and also differences between groups) larger sample is needed.
[67].
4. Conclusions
3.6. SBA-15 boosted activity of CP in vivo
This study confirmed high potential of SBA-15|CP against less
invasive mouse melanoma B16F1 cell model. Drug carrier, SBA-15, was
Finally, the efficacy of SBA-15|CP was investigated in vivo in syn­
found to be inactive, as expected, against these melanoma cells. A strong
geneic model of mouse melanoma induced in C57BL/6 mice. As SBA-15
anticancer potential of SBA-15|CP, similar to CP, against B16F1 cells
particles did not show any in vitro inhibition potential against B16F1
was observed. SBA-15|CP inhibited cell division along with G2/M phase
cells and previously was demonstrated that SBA-15 has no influence on
arrest in B16F1 cells. Caspase dependent apoptosis, supported by NO
tumor growth in vivo [24], drug vehicle was excluded from in vivo
production, was identified as main mode of cell death. Autophagy is
experiment. The treatment with CP (2 mg/kg) or SBA-15|CP in equi­
slightly present indicating that it doesn’t have a pivotal role for drugs
molar concentration (10 mg/kg, recalculated from EDX) started at day
activity. ROS do not play any role in B16F1 cells treated with CP or SBA-
7., when the first palpable tumor was detected in animals. Animals
15|CP.
received 6 doses of treatment (3 times a week) and were sacrificed on
As DNA damage mediator, naked and immobilized CP, triggered the
day 21. While application of CP did not decrease the growth of tumor,
G2 exit program leading to senescence. Moreover, SBA-15|CP expressed
SBA-15|CP significantly diminished tumor volume (Fig. 8.).
similar anticancer potential against low B16F1 and high B16F10 [43]
In vivo observed improvement in efficacy of nanoformulated CP in
metastatic melanoma cell models, nevertheless mechanism of action
comparison to naked one, could be ascribed to unique solid tumor tissue
against these two cell lines of SBA-15|CP is cell-type specific. SBA-15|CP
architecture, abnormal blood vessel structure, specific characteristics of
significantly diminished tumor volume in syngeneic model of mouse
endothelial cells and finally, blockage of drug efflux by the transporters.
melanoma induced in C57BL/6 mice, while application of CP showed no
In addition to enhanced permeability and retention (EPR) effect as a
effect. Even though that certain degree of nephro- and hepatotoxicity
basic mechanism of nanoparticle accumulation in tumor tissue, trans­
was detected in vivo after application of immobilized CP, results reported
cytosis was found as leading alternative for the transfer of mesoporous
herein indicated that SBA-15|CP possesses improved antitumor poten­
silica nanoparticles from endothelial cells directly to tumor stroma and
tial in comparison to its free form – clinically used drug CP – deserving
cancer cells, independently from tumor blood vessel fenestration [68]. It
high attention and pointing out necessity for further studies to prove it.
is assumed that the same process enables efficient and safe transfer of
mesoporous silica nanoparticles through the blood brain barrier, indi­
cating the importance of their usage as a driver for the drugs necessary Synopsis
to reach brain tissue as a target for the treatment [69]. Finally, having in
mind that mesoporous silica affects the activity of drug transporters and Naked cisplatin (CP) and immobilized onto SBA-15 (Santa Barbara
therefore drug efflux from the cell, this drug delivery system empowers amorphous mesoporous silica), similar in the in vitro activity, in B16F1
the intracellular accumulation of the compound carried by nanoparticles cells induced cell proliferation obstruction, caspase activated apoptosis,
[70]. nitric oxide production, G2 arrest and subsequent senescence.
Outstandingly, SBA-15 boosted in vivo activity in melanoma C57BL/6
mice model and diminished side effects of CP.
3.7. SBA-15 diminished side effects of CP in vivo

Cisplatin treated animals significantly lost their body weight during Declaration of Competing Interest
treatment (10–15%) and revealed obvious signs of intoxication such as
piloerection, vocalizations, heavy breathing, aggravated moving, The authors declare no competing financial interest.

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D. Drača et al. Journal of Inorganic Biochemistry 217 (2021) 111383

Fig. 9. Histopathological examination of tumor tissues. First row (A,B) control tumor tissue (H/E stain, 40×, 400× respectively); Second row (C, D) tumor tissue
treated with cisplatin (H/E stain, 40×, 400× respectively); Third row (E, F) tumor tissue treated with cisplatin bound to nanoparticles (H/E stain, 40×, 400×
respectively). White arrowheads point to tumor necrosis (A, C, D). Not obvious at first glance are intratumoral confluent cellular necrosis (black stars).

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D. Drača et al. Journal of Inorganic Biochemistry 217 (2021) 111383

Fig. 10. Histopathological examination of liver and kidney. First row (A, D, G) control kidney (2×) and liver tissue (H/E stain, 40×, 200×, 400× respectively);
Second row (B, E, H), kidney (2×) and liver tissue of animals treated with cisplatin (H/E stain, 200×, 400× respectively); Third row (C, F, I), kidney (2×) and liver
tissue of animals treated with cisplatin bound to nanoparticles (H/E stain, 200×, 400× respectively). In the first column is noticeable atrophy of the kidney cortex (B
and C) in comparison to control tissue (A). More cellular kidney interstitium (E; white arrows), and presence inflammatory cells (white arrows) with some destruction
and microcystic change (white stars) (F), compared to control tissue in D. Presence of confluent necrosis with inflammatory cells and apoptotic bodies (black stars;
H, I).

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