The antitumor activity of bismuth lipophilic nanoparticles (BisBAL NPs) on human glioblastoma is higher than temozolomide

Abstract

Background:

Glioblastoma multiforme (GBM) is the most common type of brain tumor and it is considered as one of the most aggressive malignancies (1.5 years of survival rate).

Objective:

Determine the antitumor potential of bismuth lipophilic nanoparticles (BisBAL NP) on a human glioblastoma cell-line.

Methods:

BisBAL NP were characterized by scanning electron microscopy (SEM). BisBAL NP entry and intracellular distribution on U-87 MG cells were observed by transmission electron microscopy (TEM). The effect of BisBAL NP on tumor cells was evaluated by MTT assay (IC₅₀ value), Calcein AM staining, Live/dead assay, apoptosis quantification, and comet assay.

Results:

BisBAL NP-induced cytotoxicity more efficient than temozolomide (TMZ). The IC₅₀ value of BisBAL NP was 12.7 µM. For the first time, direct interaction between BisBAL NP and plasmatic U-87 MG cell membrane was obtained by TEM. Calcein AM assay revealed loss of permeability of tumor cells after 24 h-exposure to 25 µM of BisBAL NP. 25 µM of BisBAL induced 48.5% of apoptosis, while 50 µM of BisBAL induced a higher rate of apoptosis 77.2%.

Conclusion:

BisBAL NP inhibited U-87 MG cell growth through membrane attack and loss membrane permeability, apoptosis induction and later promoting genotoxicity among tumor cells.

Keywords

antitumor activity bismuth lipophilic nanoparticles chemotherapy genotoxicity glioblastoma nanomedicine temozolomide

Introduction

Glioblastoma multiforme (GBM) constitutes the most frequent malignant primary tumor of the brain and central nervous system in adults and the elderly.¹ It is considered as one of the most aggressive malignancies with a very poor prognostic (1.5 years of survival rate). GBM originates from astrocytes and is localized more frequently in the supratentorial compartment specifically in the frontal lobe.² Based on histological criteria, four types of GBM are distinguished: 1) the isocitrate dehydrogenase (IDH) type, 2) the ID-mutant, 3) GBM not otherwise specified (NOS), and 4) not-elsewhere-classified (NEC) GBM. GBM has an incidence of about 3–4 per 100,000 people-years.³ Most incident cases of elderly patients (90%) belong to the IDH type.⁴ GBM is cataloged as a grade IV cancer. It is one of the most aggressive malignancies and has a very poor prognostic (1.5-year survival rate).

Treatment options for GBM depend on the stage of the disease, size and localization of metastases, and the general health condition of the patient. In most cases, treatment start with surgery, followed by treatment with temozolomide (TMZ).⁵ However, GBM patients are highly heterogeneous (GBM-type, genetics, tumor characteristics, patient-related characteristics), which complicates the selection of specific therapeutic targets. Medications directed to the brain have to pass the blood-brain barrier, which reduces the treatment options. Innovative nanomedicine options could help get across the blood-brain barrier⁶ to deliver the antitumor agent at the tumor site.⁷ Several types of nanocarriers and nanoparticles (gold,⁸ silver,⁹ gold-platinum¹⁰ and iron oxide¹¹) with GBM growth reduction potential have been reported. Our group has reported the antitumor activity of bismuth lipophilic nanoparticles (BisBAL NPs) against 9 different types of human cancer including breast, cervicouterine, prostate and colon.^12,13 However, it is unknown if BisBAL NPs inhibit the GBM growth. Bismuth-based nanomaterials have been attracted the attention in biomedical field by their low cytotoxicity, long-term biodistribution and renal excretion in animal models, suggesting a great potential to be applied in treatment of several diseases including cancer.¹⁴ There are not previous reports about the use of BiNPs on human glioblastoma cells but has been reported the maximum safe doses of BiNPs on several cell lines like HeLa, MCF-7, HePG2 is rated between 100–3000 μg/mL. The objective of this study was to evaluate the in vitro antitumor activity of BisBAL NPs on glioblastoma cells and analyze their entry, subcellular localization and effect on cell metabolism and possible genotoxicity.

Material and methods

Synthesis and characterization of BisBAL NPs

BisBAL NPs were developed by a colloidal method employing bismuth nitrate Bi(NO₃)₃ as starting salt and reduced by sodium borohydride (NaBH₄) like previously reported.¹⁵ The BisBAL NPs characterization was made with scanning electron microscopy (SEM; Carl Zeiss Auriga FIB-SEM & TEM, Oberkochen, Germany) obtaining data about their shape, average size, and distribution. BisBAL NP batch was kept at room temperature until further studies.

Cell culture and drug exposure

The human U-87 MG cell line (ATCC HTB 14, U-87 MG) was grown in DMEM/F12 culture media supplemented with 10% fetal bovine serum and antibiotics as previously described.¹⁶ Cell cultures were incubated at 37°C and 5% CO₂ until a confluent monolayer was obtained. For drug exposure, 1 × 10⁵ cells were seeded in wells of a 96-well plate and exposed to the specified concentrations of BisBAL NP (0.1–100 µM) or positive control drugs: 100 µM temozolomide (TMZ; Sigma-Aldrich, St Louis, MO) for the MTT assay, 20 mM hydroxyurea (HU) for the histone H2AX assay (BD Bioscience, NJ, USA), 100 µM etoposide (Sigma-Aldrich Corporation, St Louis, MO, USA) for the comet assay, and 1 mM H₂O₂ for the apoptosis assay, for different incubation periods as specified in results. Drug-free cultures serve as growth controls.

MTT cell viability assay and IC₅₀ value

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay¹³ was used to analyze the antitumor effect of BisBAL NP on U-87 MG cells and to determine the IC₅₀ value. After drug exposure (as specified in results), cells were washed three times with cold 0.1 M phosphate-buffered saline, pH 7.2 (PBS) and incubated with 10 µL MTT (2.5 mg/mL) for 2 h at 37°C and 5% CO_2. Next, formazan crystals were dissolved in 100 µL DMSO, and absorbance at 570 nm was read with a plate spectrophotometer. The assays were performed in triplicate.

Live/dead assays

The Live/Dead kit for mammalian cells (Molecular Probes Inc, OR, USA) was used to confirm viability/cytotoxicity of U-87 MG cells exposed to BisBAL NP. After treatments, cells were washed three times with PBS and stained with 2 µM calcein AM/4 µM Ethidium homodimer-1 in 100 µL. Cells were observed with an EVOS Cell Imaging System (Thermo Fisher Scientific, CA, USA) equipped with FITC and rhodamine filters.

TEM assays

To follow the fate of BisBAL NP in U-87 MG cells, cultures were exposed to 5 µM BisBAL NP for 2 h. Next, cells were fixed with 2.5% glutaraldehyde-formaldehyde in PBS pH 7.4 for 1 h and, after three washes with cold PBS, with 2% OsO₄ in PBS for 1 h. After another triple wash with cold PBS pH 7.4, cells were dehydrated with ascendant concentrations of alcohol and propylene oxide and embedded in Epon 812 resin. Ultrathin sections were stained impregnated with alcoholic uranyl (Luff) and Pb and Lufflead. The samples were observed in a JOEL 10/10 TEM.

Calcein AM assay

To evaluate the integrity and functionality of U-87 MG cell cultures after drug exposure (1, 25 or 50 µM BisBAL NP; 24 h), a Calcein AM assay (Biotium, Hayward, CA, USA) was performed according to the manufacturer's instructions. Cells that had been washed three times with cold PBS were observed with an EVOS Cell.

Apoptosis assay

To evaluate whether apoptosis occurs, the Annexin V/Propidium iodide kit (Annexin V-Fluos staining kit; Manufacturer data) was employed according to the manufacturer's instructions.¹⁷ Briefly, after a 2-h exposure to 0, 5 and 10 µM BisBAL NP or 1 mM H₂O₂ (positive control), cells were collected in 4 mL PBS-EDTA by centrifugation (300g, 5 min) and washed with 1 mL PBS (300g, 5 min). For labeling, cells were resuspended in 100 µL incubation buffer (2 µL Annexin V; 2 µL Propidium iodide) and incubated for 15 min in the dark. Next, 400 µL of incubation buffer was added and samples were analyzed by FACSCanto II flow cytometry (BD, Biosciences, and software FACS-Diva 8.0.2). Results are product from at least three independent experiments showed as means ± standard deviation. Statistical differences were tested by one way analysis of variance and Dunnett's post hoc test, a p value ≤ 0.05 was considered statistically significant. The analysis was performed using GraphPad version 6 software.

Comet assay

To evaluate the genotoxicity of BisBAL NP on U-87 MG cell cultures, the comet assay (Cell Biolabs, INC, San Diego, CA, USA) was employed according to the manufacturer's instructions.¹³ Briefly, after drug exposure, U-87 MG cells were collected, washed with sterile bidistilled water and incubated with 70% cold acetone for 5 min. Genomic DNA was stained with 4′,6-diaminidino-2-phenylindole (DAPI; 1 µL/mL; 100 µL/well). DNA damage was evaluated by microscopy (DAPI filter; EVOS microscope; Thermo Fisher Scientific, CA, USA).

Statistical analysis

Linear regression was used to evaluate the effect of concentration gradients or exposure times. One-way ANOVA with Tukey correction was used to compare among groups. A significance level of α = 0.05 was considered for all tests.

Results

BisBAL NPs characterization

In this study the antitumor effect of BisBAL NP on GBM cells was evaluated. Typical BisBAL nanostructures were formed as previously was reported.¹⁵ The colloidal synthesis throws circular bismuth nanoparticles with an average size of 24 nm and spider web arrangement (Figure 1A and B) after their characterization by scanning electron microscopy (SEM). Figure 1C and D show a close up of BisBAL NP illustrating their morphology and size.

Figure 1.

Characterization of bismuth lipophilic nanoparticles by scanning electron microscopy (SEM). An aliquot of BisBAL NP batch was observed by using a Carl Zeiss Auriga FIB-SEM & TEM to get data about their shape, average size and distribution. (A) and (B) show bismuth NPs shape, size and conglomeration. (C) and (D) represent BisBAL NPs with a distribution more isolated.

Antitumor activity of BisBAL NP on U-87 MG cells

A 24-h exposure to 25 µM BisBAL NP decreased U87 MG cell survival by 86% and was 28 times more efficient than the first-line drug TMZ (Figure 2). In contrast, the positive control of temozolomide only reduced 3% of the U-87 MG cell growth (100 µM; 24 h-exposition: Figure 2). The same experiment but measured after 48h-exposition, showed a slight increase in the inhibition percentage after BisBAL NP treatment (92%: Figure 2). TMZ after 48 h-exposition decreased 51% of the tumor cell growth (p < 0.0001) (Figure 2). Altogether these results indicate that BisBAL NP is a better agent than TMZ to interfere with tumor U-87 MG cell growth. The IC₅₀ value of BisBAL NP was measured after 24 h-exposition and it was estimated to be 12.7 µM (Figure 2Bii) and correlates with our findings with live/dead assays at 12 µM BisBAL NP (Figure 2Bi).

Figure 2.

Effect of BisBAL NPs on glioblastoma cell growth. (A) The antitumor activity of 25–100 µM of BisBAL NPs was measured by MTT cell viability assay on glioblastoma cells after 24–48 h post-incubation times. 100 µM of Temozolomide (TMZ) was employed as positive inhibition control. Cell viability (% respect to untreated cells considered 100%) was evaluated with an MTT assay. (B) i) Live/dead assay and ii) IC₅₀ value were analyzed by MTT assay employing a range of final concentrations (1–25 µM) of BisBAL NP following the method described above. Bars and error bars indicate means ± SD respectively; (n = 4); *, statistically significant (p < 0.0001; α = 0.05).

BisBAL NP interaction with plasmatic cell membrane of U-87 MG cells

With the aim of getting evidence about the entry process of BisBAL NP inside the tumor cells, TEM assays were developed. After 2 h-exposition to 5 µM of BisBAL NP, U-87 MG cells were fixed and observed by using a JOEL 10/10 transmission electron microscopy. TEM micrographs showed how BisBAL nanostructures bind directly to the plasmatic cell membrane of U-87 MG cells (Figure 3B). Tumor cells free-drug were employed as growing control to illustrate U-87 MG cell morphology (Figure 3A). Figures 3C and D show how the spider web arrangement of BisBAL NP approach to the U-87 MG cell plasmatic membrane to bind and initiate the entry process to the cell cytoplasm.

Figure 3.

Entry of BisBAL NPs into glioblastoma cells through plasmatic cell membrane. After 2 h of exposure to 5 µM of BisBAL NP, U-87 MG cells were observed by using a JOEL 10-10 microscope and an AMT Camera System. (A) Cells drug-free were used as control. (B), (C) and (D) show TEM micrographs of tumor cells exposed to BisBAL NP at direct magnification of 7200X-80, 000X.

Cell membrane permeability evaluation after BisBAL NP-exposition

Calcein AM assay was used to evaluate the integrity of U-87 MG cell after interaction with BisBAL nanoparticles. After 24 h-exposition to increasing concentrations (1, 25 and 50 µM) of BisBAL nanostructures the internalized fluorescent calcein was delivered (Figure 4), suggesting that during BisBAL NP crossing plasmatic cell membrane promote damage on plasmatic U-87 MG cell membrane and maybe due cellular lysis.

Figure 4.

Cell permeability loss of U87 cells after exposition to increasing concentration of BisBAL NP. 1 × 10⁵ cells were incubated with 1, 25 or 50 µM BisBAL NP (24 h; 37°C and 5% of CO₂), U87 cells with only culture media were employed as growing control. 2 µM Calcein AM were used to stain the cytoplasm of tumor cells (30 min; 37°C and 5% of CO₂). After washing the cells with PBS, fluorescent Calcein AM retained in the inside of tumor cells was analyzed with an EVOS Cell Imaging System (Thermo Fisher Scientific, CA, USA) using FITC filter.

BisBAL NP storing at U-87 MG cell cytoplasm

Once BisBAL NP gets inside of tumor cells, they are stored at the cytoplasm of U-87 MG cell after only 2 h of exposition like was observed by TEM assays (Figure 5B). U-87 MG cells free-drug were used as growing control (Figure 5A). Figures C and D show accumulations of BisBAL NP at the cytoplasm, suggesting that bismuth nanostructures internalization is a fast process.

Figure 5.

Intracellular localization of BisBAL NPs on glioblastoma cells. After 2 h of exposure to 5 µM of BisBAL NP, U-87 MG cells were observed by using a JOEL 10-10 microscope and an AMT Camera System. A) Cells drug-free were used as control. (B), (C) and (D) show TEM micrographs of tumor cells exposed to BisBAL NP at direct magnification of 1000X-100000X.

When the metabolism of tumor cells was analyzed after short exposition times to 25 µM BisBAL NP (30–240 min) to get insights about a possible alteration by bismuth nanostructures presence, the obtained results showed a decreasing rate of cellular growth while BisBAL NP concentration increased (Figure 6(i)). Interestingly, after 240 min-exposition to 25 µM BisBAL NP, 97% of U-87 MG cell growth was inhibited (p < 0.0001) (Figure 6ii).

Figure 6.

Live/dead and MTT cell viability assays were employed to analyze the metabolism of U-87- MG cells after 30–240 min. of exposition (37°C and 5% of CO₂) to 25 µM of BisBAL nanostructures. i) Images obtained after stain with Live and Dead assay. ii) Quantification of Cell viability assay by MTT. % Cell viability of BisBAL NPs treatment compared to all groups (*p < 0.0001). Error bars indicate mean ± SD (n = 3), asterisk indicate statistical differences (α = 0.05).

Apoptosis induction after BisBAL-exposition by U-87 MG cells

In our experiments, BisBAL NP induces apoptosis (early and late apoptosis) as mechanism of death cell on U-87 MG cells. U87 cells were exposed to 25 and 50 µM of BisBAL for 2 h and cell death was determined by double stained with Annexin-V and IP and quantified by flow cytometry, whereas H₂O₂ was used as a positive cell death. The results indicate that BisBAL were able to significantly induce apoptosis (sum of early and late apoptosis; Figure 7). 25 µM of BisBAL induced 48.5% of apoptosis, while 50 µM of BisBAL induced a higher rate of apoptosis 77.2% (compared to 16.6% of apoptosis in CT cells, p < 0.05; Figure 7A and B). In the case of H₂O₂ an apoptosis index of 44.45% was determined (p < 0.05 vs CT). For necrosis, cells treated with 25 µM of BisBAL present an index of 24.8%, while 50 µM of BisBAL induced 20.4% of necrosis and H₂O₂ present 26.6% of necrotic cells (compared to 4.4% of necrosis in CT cells, p < 0.05; Figure 7C).

Figure 7.

Apoptosis/necrosis among U87 cells after exposition to BisBAL NP. Apoptosis/necrosis was determined by Annexin-V and IP double staining assay. (A) Representative histograms of apoptosis cell index derived from flow cytometric analysis after two hours of BisBAL exposure. (B) Quantitative result of apoptosis expressed as percentage (Show the sum of early and late apoptosis). (C) Necrotic cells after 2 h-exposure to 25–50 µM BisBAL NP or 1 mM of H2O2 as positive control of necrosis. Values represent results from minimum three experiments with the mean ratio ± standard error. *Treatments significantly different from the control at p < 0.05.

Nuclear localization of BisBAL NP and their genotoxicity

TEM revealed that BisBAL NP also reached the nuclei of tumor cells. The bismuth lipophilic nanoparticles were traced along their way inside tumor cells and finally they were localized in U-87 MG cell nucleus (Figure 8B). BisBAL NP showed a heterogeneous distribution inside of the nucleus cells (Figure 8C and D) and U-87 MG cells free-drug did not observe nanostructures in their nucleus (Figure 8A).

Figure 8.

Nuclear internalization of BisBAL NPs into glioblastoma cells. After 2 h of exposure to 5 µM of BisBAL NP, U-87 MG cells were observed by using a JOEL 10-10 microscope and an AMT Camera System. (A) Cells drug-free were used as control. (B), (C) and (D) show TEM micrographs of tumor cells exposed to BisBAL NP at direct magnification of 5800X-40000X.

The possible genotoxic effect of BisBAL NP on genomic DNA of U-87 MG cells was analyzed by comet assay and fluorescence microscopy. 10 and 25 µM BisBAL NP induced the formation of classic comet steel on U-87 MG cell nucleus after 24 h post-exposition (Figure 9). 100 µM etoposide promote same kind of comet stela (Figure 9). Altogether these data indicate that once bismuth nanoparticles ingress to the cell nucleus and damage the genomic DNA of U-87 MG cells as a consequence of their interaction.

Figure 9.

Genotoxicity of BisBAL NP on U-87 MG cells. Comet assay to determine a possible genotoxic effect of 10–25 µM BisBAL NP on U-87 MG cells (24h-exposure; 37°C and 5% of CO₂). Nucleuses were observed by fluorescence microscopy (DAPI filter; EVOS microscope; Thermo Fisher Scientific, CA, USA). Bar indicates 50 µm.

Discussion

GBM is the most frequent and aggressive brain malignancy.¹⁸ GBM has a bad prognosis and patients require surgery as first treatment option, followed by daily radiations for several weeks.¹⁹ The median survival after this treatment is about 1.5 years.²⁰ One of the main challenges in GBM chemotherapy is to develop new molecules able to penetrate the blood-brain barrier and gain access to the tumor to halt growth and stop metastasis without adverse side effects. Innovative nanotechnology-based drug delivery options seem promising.^21,22 In this study the antitumor effect of BisBAL NP on GBM cells was evaluated. Typical BisBAL NP spheres (average size, 24 nm) formed web-like conglomerates as previously reported.¹⁵ This property of form conglomerates is common among metal nanoparticles like silver and gold NPs. Interestingly, these conglomerates are not covalent linked because we detect individual BisBAL NP spheres in the interior of GBM cells. A 24-h exposure to 25 μM BisBAL NP decreased U-87 MG cell survival by 86% and was 28 times more efficient than the first-line drug TMZ. Recently, a TMZ hexadecyl ester targeted plga nanoparticle has been reported to inhibit glioblastoma cell growth.²³ Interestingly, TMZ hexadecyl ester and BisBAL NP share properties such as hydrophobicity, affinity to cell membranes, and antitumor capacity. Our hypothesis is that BisBAL NP antitumor potential is related to their lyophilic and cationic characteristics.^13,15 The nanomaterials have the advantage (versus traditional drugs) to pass the blood-brain barrier and get access to fight against neurodegenerative diseases, brain cancer, and/or hemorrhages.²⁴ Biomimetic keratin-coated gold nanoparticles (Ker-Au NP) in combination with photo-thermal therapy is another innovative and promising proposal against GBM tumor cells.²⁵ In our experiments, after a 48-h exposure, BisBAL NP performed 1.8 times better than TMZ. Ultramicrographs of U-87 MG cells after a 2-h exposure to BisBAL NP conglomerates revealed adherence of the web-like conglomerates to the cell membrane and entry of bismuth particles into the cytoplasm. This explains why BisBAL NP had a faster antitumor effect on U-87 MG cells than temozolomide. Interestingly, Tsymbal et al., 2021 have reported that copper-containing NPs and organics complexes induced irreversible membrane damage leading to fast cell death.²⁶ Unfortunately, evidence of a direct interaction between copper-containing NPs with membrane of tumor cell was not achieved. Our experiments with calcein AM and fluorescence microscopy showed that after increasing amounts of BisBAL NP, the fluorescent calcein molecule was released, suggesting damage in the membrane of U-87 MG cells, product of the BisBAL NP entry process. Membrane damage has also been reported due to anionic magnetite NP.²⁷ In our study, it seemed that cell membrane alterations observed upon BisBAL NP entry were reversible as fluorescent calcein was retained within the cells even after a 24-h exposure. However, the cell morphology was lost and substituted by erratic forms. Live/dead and MTT assays suggest that cytoplasmic BisBAL NP interferes with metabolism and cell replication.^12,13 It has been reported that cisplatin induces cell dead by apoptosis among U-87 MG cells.²⁸ But, according to our Annexin V results, the induction of apoptosis is not the main mechanism of action of BisBAL PN. TEM revealed that BisBAL NP also reached the nuclei of tumor cells. The comet assay in our study suggests that BisBAL NP caused DNA damage in the U-87 MG cells, as has been previously reported for human breast cancer cells.¹³ Several strategies to ensure nuclear delivery of NPs or antitumor agents have been reported.^29–31 Wu et al., 2022 synthesized pH/enzyme dual-sensitive nuclear-targeting dendrimer NPs that delivered doxorubicin into of the nuclei of tumor cells and achieved a 88.4% tumor reduction.³² Early, Jian et al., 2022 developed a nanocomposite, containing AgNPs and peptide-functionalized doxorubicin, capable of penetrating the nucleus and effectively inhibiting breast cancer cell growth.³³ Our hypothesis based on TEM and Calcein AM assays is that BisBAL NP use mainly permeability alteration as the main action mechanism, instead of apoptosis.

In summary, we present evidence that the in vitro antitumor potential of BisBAL NP on GBM cells is superior to TMZ, the current first-line drug against GBM. Also, for the first time, ultramicrographs evidence the interaction between tumor cells and BisBAL NP, their entry into the cells and their nuclear localization. Calcein AM and live/dead assays support the hypothesis of an interference with the metabolism required for cell survival. Furthermore, the comet assay was confirmed that genomic DNA damage is not repaired.

Footnotes

Acknowledgments

The authors want to thank the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCyT) for the grant provided. Instituto Nacional de Cancerología - INCan Mexico, RAI, UNAM - Advanced Microscopy Applications Unit (ADMiRA), RRID:SCR_022788.

ORCID iDs

Gustavo Israel Martinez-Gonzalez

Rene Hernández-Delgadillo

Yesennia Sánchez-Pérez

Claudia María García-Cuellar

Irene Meester

Claudio Cabral-Romero

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Koshy

Villano

Dolecek

, et al. Improved survival time trends for glioblastoma using the SEER 17 population-based registries. J Neuro-Oncol 2012; 107: 207–212.

Grochans

Cybulska

Simińska

, et al. Epidemiology of glioblastoma multiforme-literature review. Cancers (Basel) 2022; 14: 2412.

Fabbro-Peray

Zouaoui

Darlix

, et al. Association of patterns of care, prognostic factors, and use of radiotherapy-temozolomide therapy with survival in patients with newly diagnosed glioblastoma: a French national population-based study. J Neuro-Oncol 2019; 142: 91–101.

Louis

Perry

Reifenberger

, et al. The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 2016; 131: 803–820.

Cruz

JVR

Batista

Afonso

, et al. Obstacles to glioblastoma treatment two decades after temozolomide. Cancers 2022; 14: 3203.

Czerniawska

. Experimental investigations on the penetration of ¹⁹⁸Au from nasal mucous membrane into cerebrospinal fluid. Acta Otolaryngol 1970; 70: 58–61.

Neganova

Aleksandrova

Sukocheva

, et al. Benefits and limitations of nanomedicine treatment of brain cancers and age-dependent neurodegenerative disorders. Semin Cancer Biol 2022; 86: 805–833.

Hsing

Hsu

Chang

, et al. Improved delivery performance of n-butylidenephthalide-polyethylene glycol-gold nanoparticles efficient for enhanced anti-cancer activity in brain tumor. Cells 2022; 11: 2172.

Kabir

Islam

Asaduzzaman

AKM

. Biogenic silver/silver chloride nanoparticles inhibit human cancer cells proliferation in vitro and Ehrlich ascites carcinoma cells growth in vivo. Sci Rep 2022; 12: 8909.

10.

Stavropoulou

Theodosiou

Sakellis

, et al. Bimetallic gold-platinum nanoparticles as a drug delivery system coated with a new drug to target glioblastoma. Colloids Surf B: Biointerfaces 2022; 214: 112463.

11.

Chen

Wen

. Iron oxide nanoparticles loaded with paclitaxel inhibits glioblastoma by enhancing autophagy-dependent ferroptosis pathway. Eur J Pharmacol 2022; 921: 174860.

12.

Cabral-Romero

Solís-Soto

Sánchez-Pérez

, et al. Antitumor activity of a hydrogel loaded with lipophilic bismuth nanoparticles on cervical, prostate, and colon human cancer cells. Anti-cancer Drugs 2020; 31: 251–259.

13.

Hernandez-Delgadillo

García-Cuéllar

Sánchez-Pérez

, et al. In vitro evaluation of the antitumor effect of bismuth lipophilic nanoparticles (BisBAL NPs) on breast cancer cells. Int J Nanomed 2018; 13: 6089–6097.

14.

Badrigilan

Heydarpanahi

Choupani

, et al. A review on the biodistribution, pharmacokinetics and toxicity of bismuth-based nanomaterials. Int J Nanomed 2020; 15: 7079–7096.

15.

Badireddy

Hernandez-Delgadillo

Sánchez-Nájera

, et al. Synthesis and characterization of lipophilic bismuth dimercaptopropanol nanoparticles and their effects on oral microorganisms growth and biofilm formation. J Nanopart Res 2014; 16: 2456.

16.

Maszczyk

Banach

Karkoszka

, et al. Chemosensitization of U-87 MG glioblastoma cells by neobavaisoflavone towards doxorubicin and etoposide. Int J Mol Sci 2022; 23: 5621.

17.

Wang

Liu

Chen

, et al. Paradol induces cell cycle arrest and apoptosis in glioblastoma cells. Nutr Cancer 2022; 74: 3007–3014.

18.

Wang

Zhang

. Neoantigen discovery and applications in glioblastoma: an immunotherapy perspective. Cancer Lett 2022; 550: 215945.

19.

Cruz Da Silva

Mercier

Etienne-Selloum

, et al. A systematic review of glioblastoma-targeted therapies in phases II, III, IV clinical trials. Cancers 2021; 13: 1795.

20.

El-Khayat

Arafat

. Therapeutic strategies of recurrent glioblastoma and its molecular pathways ‘Lock up the beast’. Ecancermedicalscience 2021; 15: 1176.

21.

Sandbhor

Goda

Mohanty

, et al. Targeted nano-delivery of chemotherapy via intranasal route suppresses in vivo glioblastoma growth and prolongs survival in the intracranial mouse model. Drug Deliv Transl Res 2022; 13: 608–626.

22.

Mao

Calero-Pérez

Montpeyó

, et al. Intranasal administration of catechol-based Pt(IV) coordination polymer nanoparticles for glioblastoma therapy. Nanomaterials 2022; 12: 1221.

23.

Wang

, et al. Temozolomide hexadecyl ester targeted plga nanoparticles for drug-resistant glioblastoma therapy via intranasal administration. Front Pharmacol 2022; 13: 965789.

24.

Rhaman

Islam

Akash

, et al. Exploring the role of nanomedicines for the therapeutic approach of central nervous system dysfunction: at a glance. Front Cell Dev Biol 2022; 10: 989471.

25.

Chirivì

Bearzi

Rosa

, et al. Biomimetic keratin-coated gold nanoparticles for photo-thermal therapy in a 3D bioprinted glioblastoma tumor model. Int J Mol Sci 2022; 23: 9528.

26.

Tsymbal

Moiseeva

Agadzhanian

, et al. Copper-containing nanoparticles and organic complexes: metal reduction triggers rapid cell death via oxidative burst. Int J Mol Sci 2021; 22: 11065.

27.

Hasan

Karal

MAS

Akter

, et al. Influence of sugar concentration on the vesicle compactness, deformation and membrane poration induced by anionic nanoparticles. PLoS One 2022; 17: e0275478.

28.

Contreras-Ochoa

López-Arellano

Roblero-Bartolon

, et al. Molecular mechanisms of cell death induced in glioblastoma by experimental and antineoplastic drugs: new and old drugs induce apoptosis in glioblastoma. Hum Exp Toxicol 2020; 39: 464–476.

29.

Sukocheva

Liu

Neganova

, et al. Perspectives of using microRNA-loaded nanocarriers for epigenetic reprogramming of drug resistant colorectal cancers. Semin Cancer Biol 2022; 86: 358–375.

30.

Mikušová

Mikuš

. Advances in chitosan-based nanoparticles for drug delivery. Int J Mol Sci 2021; 22: 9652.

31.

Nikezić

AVV

Bondžić

Vasić

. Drug delivery systems based on nanoparticles and related nanostructures. Eur J Pharm Sci 2020; 151: 105412.

32.

Shen

Lang

, et al. Ph/enzyme dual sensitive and nucleus-targeting dendrimer nanoparticles to enhance the antitumour activity of doxorubicin. Pharm Dev Technol 2022; 27: 357–371.

33.

Jiang

Chen

Guo

, et al. Combating multidrug resistance and metastasis of breast cancer by endoplasmic reticulum stress and cell-nucleus penetration enhanced immunochemotherapy. Theranostics 2022; 12: 2987–3006.

The antitumor activity of bismuth lipophilic nanoparticles (BisBAL NPs) on human glioblastoma is higher than temozolomide

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

Introduction

Material and methods

Synthesis and characterization of BisBAL NPs

Cell culture and drug exposure

MTT cell viability assay and IC50 value

Live/dead assays

TEM assays

Calcein AM assay

Apoptosis assay

Comet assay

Statistical analysis

Results

BisBAL NPs characterization

Antitumor activity of BisBAL NP on U-87 MG cells

BisBAL NP interaction with plasmatic cell membrane of U-87 MG cells

Cell membrane permeability evaluation after BisBAL NP-exposition

BisBAL NP storing at U-87 MG cell cytoplasm

Apoptosis induction after BisBAL-exposition by U-87 MG cells

Nuclear localization of BisBAL NP and their genotoxicity

Discussion

Footnotes

Acknowledgments

ORCID iDs

Funding

Declaration of conflicting interests

References

MTT cell viability assay and IC₅₀ value