Efficacy of Antineoplastic Nanocarriers on 3D Oral Cancer Spheroids

Abstract

Background/Aim: Cancer is a leading cause of death worldwide. Conventional treatments as surgery, chemotherapy, radiotherapy, and combined therapies are commonly used. However, these therapies have several limitations and side effects. To address these issues, innovative research is being conducted on nanocarriers (NCs) functionalized with antineoplastic agents. These NCs aim to overcome limitations and improve patients’ lives. However, before they can be used clinically, these NCs are primarily assessed on a lab scale to determine their efficacy.

Materials and Methods: A primary cell culture was established from a lymphoblastic neoplasm in the maxilla. After characterization, the cells were cultured in 2D to evaluate the dose-effect of nanoparticles (NPs), such as Zinc oxide (ZnO) and Magnesium oxide (MgO), as well as those of free drugs of 5-fluorouracil (5-FU) and cisplatin (Cis). Based on the results, a 3D spheroid culture was used for further study. Finally, the spheroids were histologically processed for immuno-morphological observation.

Results: To evaluate spheroid cell viability, we conducted an MTT assay. Treatment of cell spheroids with ZnONPs, 5-FU, and NPs conjugated with antitumor agents such as 5-FU-ZnO and Cis-ZnO decreased cell viability by >25%, >60% and >10% and <20% at a concentration of 0.06, 0.015 and 0.015 & 0.03 mg/ml, respectively.

Conclusion: Nanoparticles conjugated with antitumor agents showed promising antineoplastic effects on both 2D and 3D cell cultures. However, the efficacy of the nanoparticles varied between the different models. This highlights the importance of selecting appropriate in vitro culture models for the evaluation of biomedical agents.

Cancer remains a significant public health problem and is one of the leading causes of mortality and morbidity worldwide. In the United States, it is estimated that there will be an increase of 1,958,310 new cases and 609,820 cancer deaths by 2023 (1). Hematological neoplasms are the third most common type of cancer after lung and breast neoplasms. In Mexico, cancer is the ninth leading cause of death, with lymphomas, leukemias, and myelomas being the most frequent types, accounting for 10% of all cancer diagnoses (2-5).

In recent years, cancer nanotechnology has become increasingly important for the diagnosis and treatment of cancer, with the goal of improving human well-being. The field has seen substantial development, with the approval of over 50 nano-based drugs by the US Food and Drug Administration (FDA). These drugs have shown promise in improving the effectiveness of cancer treatment while minimizing side effects (6-8). Malignant neoplasms of the head and neck are traditionally treated with a combination of surgery, radiation therapy, or both, depending on the size, type, and location (9-11). In recent years, nanoparticles (NPs) have received increasing attention due to their unique and promising characteristics. Among the different types of nanoparticles, nanocarriers (NCs) have shown potential in inhibiting the proliferative activity of cancer cells by inducing the formation of reactive oxygen species (ROS) and activating apoptotic pathways. These properties make NCs an attractive option for cancer treatment and have led to further research and developments in this area (12-14).

To ensure the safety and effectiveness of designed NCs for cancer treatment, it is crucial to evaluate their properties and mechanisms under standard laboratory conditions before proceeding to clinical use. In vitro cellular models are a common tool used for preliminary investigation, but there are concerns about their ability to accurately reflect the complexity of tumor biology. As a result, there is a growing interest in transitioning from traditional 2D cell cultures to 3D cultures, which offer a more realistic and comprehensive representation of the tumor microenvironment. By utilizing 3D cultures, researchers can obtain more reliable and relevant data regarding the development and optimization of NCs for cancer treatment, ultimately improving patient outcomes and minimizing side effects (15-19).

These therapies are evolving along with nanomedicine, a medical field that applies nanotechnology to take advantage of the qualities of nanometric materials for the development of antineoplastic medicines. Among the most used NPs are inorganic ones (20), including ZnONPs and MgONPs; they are used for their cytotoxic selectivity and ROS induction. Thus, many investigations suggest that NCs can have a significant antineoplastic effect by decreasing the number of viable cancer cells, causing changes to their morphology and the expression of tumoral proteins (21-25).

Therefore, this study aimed to determine the antitumor effect of the NCs of commercial ZnONPs, MgONPs, biofunctionalized with cisplatin (Cis) and 5-fluorouracil (5-FU) and compare the efficacy of naked NPs through a cytotoxicity assay primarily in a 2D monolayer model and contrast it with a 3D spheroid model of lymphoid origin. Finally, it aimed to characterize the spheroids with histological studies to determine the effect of NCs on cell immuno-morphology.

Materials and Methods

Chemicals and reagents. All the reagents were procured from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise mentioned.

Tissue biopsy sample. A tumor biopsy from the right maxillary region of a 53-year-old female patient was obtained at the Maxillofacial Surgery Department of the General Hospital of Leon, Mexico. The sample was divided into two halves; one half was sent for histopathological examination (Oral and maxillofacial pathology area) and the other half for primary cell culture (Nanostructures and biomaterials area). Both areas are part of the Interdisciplinary Research Laboratory, National School of Higher Studies (ENES) Leon unit, National Autonomous University of Mexico (UNAM). The isolation of cultures was approved by the bioethical committee of the ENES Leon Unit, UNAM, with the registration code CE_16 004_SN.

Histopathological findings. The tissue biopsy was immersed in 4% buffered formalin for 24 h, treated with a histology tissue processor (HistoKinette, Leica, Wetzlar, Germany), passing through a graded series of alcohol (60-100%), and then immersed in xylol, l & ll, paraffin l & ll; this process lasted 12 h, 1 h per reagent. Later, they were embedded in paraffin and cut into 3-micron sections using a microtome (Leica, Germany, Wetzlar). The sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope.

Primary cell culture. The process was performed inside the horizontal laminar flow hood (Biobase, Shandong, PR China). The tissue was sectioned into 1×1 mm explants and incubated with minimum essential medium eagle (MEM, Sigma-Aldrich) medium supplemented with 20% fetal bovine serum (FBS, Sigma-Aldrich), 1% glutamine (Sigma-Aldrich), and 1% antibiotic (PenStrep, Sigma-Aldrich) in 10-cm petri dishes at 37˚C with 5% CO₂ and 95% humidity for 21 days.

Cell characterization. The primary cancer cells exhibited a fibroblast-like shape and were detached with trypsin-Ethylenediaminetetraacetic acid (0.025%-EDTA-2Na, Sigma-Aldrich) in Phosphate-buffered saline (PBS). The cells were grown in subcultures of supplemented MEM medium. The cells grew to a 90% confluence, indicating a high proliferation rate over the electrocharged cover slide. Cells at their four population doubling levels (PDL) were analyzed through immunocytochemistry using cyclin D1 and CD3 antibodies (Sigma-Aldrich).

Preparation of stock solution and NCs. The following stock solutions were prepared: for 5-FU (Sigma-Aldrich) and Cis (Sigma-Aldrich), a solution of 1 mol was prepared; for NPs, a solution of 1 mg/ml was prepared. For the nanocarriers, a 1:1 proportion (5 ml) was prepared, and the composite was used for further studies. All stock solutions were prepared using distilled water.

Cell-culture and dose-response study.

2D monolayer. The cancer cells were subcultured (5 passages) in a 96-well microplate overnight. Then, the cells were washed with PBS and detached using trypsin, and the number of cells was counted using a hemocytometer under the inverted light microscope (Leica, Germany). Cells were subcultured at a density of 1×10⁵ cells/ml for 48 h to allow complete attachment. Then, the cells were incubated with the different free drugs (5-FU or Cis) and NPs loaded with various concentrations (0-1 mg/ml) of the compounds. The treatment groups were as follows: 1) MgONPs (≤50 nm, Sigma-Aldrich), 2) ZnONPs (≤50 nm, Sigma-Aldrich), 3) 5-FU, 4) Cis, 5) 5-FU+MgONPs, 6) 5-FU+ZnONPs, 7) Cis+MgONPs, 8) Cis+ZnONPs. Treatment was performed for 24 h in fresh culture medium. The cell viability was assessed using 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich), which was incubated with the cells for 7 h. After incubation, the formazan produced was dissolved with dimethyl sulfoxide (DMSO, Karal, Leon, Guanajuato, Mexico), and the absorbance at 570 nm was measured using a microplate spectrophotometer reader (Multiskan Go, Thermo-scientific, Helsinki, Finland). The cytotoxicity was evaluated according to ISO 10993-5:2009, and the 50% cytotoxic concentration (CC₅₀) was calculated for each group.

3D spheroids. The spheroids were formed using a scaffold-based model with the liquid overlay method with 1.5% agarose (Sigma-Aldrich). The agarose solution was prepared by autoclaving the solution for 15 min, and 50 μl was added to a round-bottomed 96-well plate, which was allowed to solidify for 20 minutes under sterile conditions. Then, the lymphoid cells were subcultured (6 passages) at a concentration of 2.5×10² cells/ml/well and incubated for 72 h. The growth of the spheroids was recorded using a phase contrast microscope (Leica), and the medium was replaced every two days. The spheroids were treated with the compounds mentioned above at their CC₅₀ dose (n=9). Later the spheroids were evaluated for cell viability using MTT added directly to the plates as above mentioned. Furthermore, the spheroids were collected by centrifugation (5-10 min, 4˚C, 2,000 rpm), fixed with 4% buffered formalin and then processed with H&E and immunohistochemical staining.

H&E staining and immunohistochemistry process. All the spheroids were incubated with 4% buffered formalin for 24 h at histology tissue processor (HistoKinette, Leica), embedded in paraffin, refrigerated at 4ºC for 30 min, and then sliced into 3-micron sections using a microtome (Leica). The sections were suspended in a water bathtub (Leica), fixed onto a glass slide, and left on a hot plate for 24 h. Subsequently, they were subjected to: 100% xylol for 20 min, 100% and 96% alcohol for 30 s, hematoxylin for 10 min, lithium carbonate for 15 s, eosin for 10 min, 96% and 100% alcohol for 10 s and 100% xylol for 20 min. Finally, they were mounted with resin (El Crisol, Queretaro, Mexico) and covered with coverslips for observation under light microscopy at 40× (Leica).

In the case of immunohistochemistry, the paraffin cube was sectioned with a microtome at 5 microns and allowed to float in a water bathtub (Leica) with distilled water at a temperature of 40˚C. Then, it was fished with a slide with enzymatic glue, left to dry at 60˚C for 30 min and stained for cyclin D and CD3.

The dataset includes the mean, percentage, and standard deviation for a sample size of n=9, which was tested for normality using the Shapiro-Wilk test and subjected to ANOVA posthoc-Tukey test in the Statistical Package for Social Science software V16 (SPSS, Chicago, IL, USA). A p<0.05 was considered significant and a 95% confidence level was used.

Results

Histopathological findings and primary cell culture. The biopsy’s histopathological findings, displayed in Figure 1 at various magnifications, indicate the presence of a malignant neoplasm comprised of pleomorphic plasma cells with large central nuclei and scant clear cytoplasm. The neoplastic cells infiltrated and destroyed the underlying tissue, and many blood vessels exhibited lymphoblastic neoplasia (A and B). Additionally, isolated germinal centers resembling lymphoblastic cells were present. Subsequent immunocytochemistry examinations of primary cell cultures confirmed the presence of specific markers, such as strong positive expression of cyclin D (C and D), and negativity for the CD3 marker (E and F), indicating lymphoid cancer cells.

Figure 1. Histopathological findings and lymphoid cancer cell characterization. Microphotograph A) viewed at 4× magnification, and A*) viewed at 40×, show the H&E staining of the incisional biopsy with several characteristics of lymphoid neoplasm. Primary cell culture characterization. Near confluent (90%, 4 PDL) cells were subcultivated for 48 h. Microphotograph B) viewed at 20×, and B*) viewed at 40×, show strong positive staining for cyclin D. Microphotograph C, viewed at 20×, and C*) viewed at 40×, show negative staining for CD3. The results of the immunohistochemistry examination confirm the presence of lymphoid cancer cells. H&E: Hematoxylin and eosin; PDL: population doubling level.

Dose-response study with 2D monolayer. The dose-response evaluation of NPs, drugs and NCs using 2D monolayer cell cultures is shown in Figure 2 and Table I. Treatment of cells grown in 2D cultures with different concentrations of the various compounds reduced the number of viable cells in the following order, beginning from the most cytotoxic: 5-Fu+ZnONPs<5-Fu+MgONPs<5-Fu<Cis+ZnONPs< ZnONPs<Cis+MgONPs<Cis<MgONPs. Based on the obtained results, the 50% cytotoxic concentration (CC₅₀) was determined and presented in Table II.

Figure 2. Compound and nanocarrier dose-effects. Near confluent lymphoid cancer cells (90% confluence, 5 PDL) were incubated for 24 h. The cells were treated with different concentrations from 0-1 mg/ml of antineoplastics and nanocarriers for 24 h. After incubation, the relative viable cell number was determined using the MTT assay. Each value represents the mean±SD of triplicate assays (n=9). PDL: Population doubling level; MTT: 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; SD: standard deviation; ZnONPs: zinc oxide nanoparticles; Cis: cisplatin; 5-Fu: 5-fluoracil; MgONPs: magnesium oxide nanoparticles.

Table I. Viable cell number (%) of control, treated with antineoplastics, and functionalized nanocarriers.

Significant intragroup difference at p<0.05* p<0.01**. Significant differences between groups at p<0.05+ p<0.01++; ANOVA post-hoc Tukey test.

Table II. 2D cytotoxic CC₅₀ concentration and anticancer effect (%) in 3D spheroids.

3D spheroids formation. The formation of spheroids using the liquid overlay method was closely monitored. After four days, spheroids with a mean size of 150±25 μm and a spherical shape were chosen, as shown in Figure 3A. H&E staining revealed the presence of homogeneous and compact structures that exhibit concentrically organized cells with well-defined cellular structures. Notably, the nuclei were prominent, and the cytoplasm and membranes were intact, indicating a high degree of structural integrity as demonstrated in Figure 3B.

Figure 3. 3D spheroid formation. The spheroids were generated using a liquid overlay method with 1.5% agarose cultivated with lymphoid cancer cells (2.5×10² cells/ml, population doubling level 6) for 4 days to achieve a spherical shape. A) A spheroid with compact round-shape and mean size of 150±25 μm at four days. B) Hematoxylin and eosin staining of spheroids. Compact, concentrically organized cells with well-defined structures suitable for protein identification are observed. Prominent nuclei, intact cytoplasm, and membranes indicate high structural integrity packed in the inner region and somewhat flattened at the periphery. Over time, the cells in all spheroids aligned themselves to reduce configurational energy, as anticipated and reported before (26). Magnification 40×.

Cytotoxic assay in 3D spheroids. The pre-determined CC₅₀ from the 2D cell culture study was used to examine the antineoplastic effect of the various compounds on spheroids. Following treatment for 24 h, the MTT assay was carried out. The results are presented in Figure 4. A decrement in the percentage of viable cells was observed in the following order: ZnONPs=47.4±0.8% <Cis+MgONPs=53.4±1.51% <5-FU+ZnONPs= 57±3.1% <5-Fu+MgONPs=69.4±1.2% <MgONPs= 71.9±4.7% <Cis=72.3±4.1 <Cis+ZnONPs= 72.4±4.1% <5-Fu=84.5±3.8% <Control=100±3.9%. To study the effect of the above-mentioned compounds on the morphology of the spheroids, they were imaged using a microscope and distorted spheroids were observed for ZnONPs, Cis, and 5-FU (Figure 5A, D, and G). When stained with H&E, the reduction in nuclei was realized as expected, as well as a loss of the spherical shape (Figure 5B, E, and H). The spheroids were subsequently stained with the immunomarker Cyclin D, and scarce labeling of cell nuclei was observed. This indicated that there is little tumor activity in the lymphoid cell spheroids (Figure 5C, F, and I).

Figure 4. The cytotoxic effect of CC₅₀ doses of the carious compounds and their combinations on cell spheroids. Near confluent lymphoid cancer cells (90% confluence, population doubling level 6) were incubated for 24 h with CC₅₀ doses of the carious compounds and their combinations. After incubation, the relative viable cell number was determined using the MTT assay. A significant decrease in viable cell count (p<0.01), indicating a high level of sensitivity to the treatments was observed in the following order: ZnONPs=47.4±0.8% <Cis+MgONPs=53.4±1.51% <5-Fu=57±3.1%<MgONPs=71.9±4.7% <Cis=72.3±4.1%<Cis+ZnONPs=72.4±4.1%<5-Fu=84.5±3.8% <Control=100±3.9%. Each value represents the mean±SD of triplicate assays (n=9), *p<0.05, **p<0.01 ANOVA Post-hoc Tukey test. MTT: 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; ZnONPs: zinc oxide nanoparticles; Cis: cisplatin; 5-Fu: 5-fluorouracil; MgONPs: magnesium oxide nanoparticles; SD: standard deviation.

Figure 5. Cytotoxicity of compounds in 3D lymphoblastic cell spheroids. Near confluent lymphoid cancer cells (90% confluence, population doubling level 6) were incubated for 24 h. Cell spheroids were treated with CC₅₀ doses of the different compounds and their combinations. A, B and C) Spheroids treated with ZnONPs; D, E and F show spheroids treated with Cis, finally, G, H, and I show spheroids treated with 5-Fu. Spheroids are viewed at 40× magnification. A, D, and G viewed using phase contrast microscope at 40×; B, E, and H spheroids stained with H&E were viewed using a light microscopy at 40× and C 40×, F 100×, and I 100× views of spheroids immunolabeled for cyclin D1 using light microscopy. The spheroids were subsequently stained with the immunomarker Cyclin D, and scarce labeling of cell nuclei was observed. MTT: 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; ZnONPs: zinc oxide nanoparticles; Cis: cisplatin; 5-Fu: 5-fluorouracil; MgONPs: magnesium oxide nanoparticles; SD: standard deviation.

Discussion

Spheroid cultures present certain advantages over monolayer culture, such as a more faithful reproduction of intercellular interactions and the tumor microenvironment. Several studies based on biomarker expression have reiterated the importance of three-dimensional cultures and described criteria such as co-aggregates and spheroid length that should be considered for biomarker identification. Some researchers have suggested that spheroids formed by cellular aggregation tend to have more unstable connections. Therefore, the optimal conditions for spheroid formation result from clonal growth through cellular proliferation rather than cellular aggregation (19).

In the present study, a 3D spheroid model was generated from primary lymphoid cancer cells. Saraiva et al. also generated spheroids using the liquid superimposition technique, but interestingly they reported that modifying the protocol with a scaffold-based model resulted in better spheroid formation (27). This finding is consistent with our own results using a scaffold-based method.

Atashi et al. (28) compared the efficacy of 5-FU and Cis and found that 5-FU alone was associated with a higher frequency of resistance compared to Cis alone. This finding is inconsistent with our investigation, where lymphoid neoplasms treated with Cis alone were less responsive to treatment. Furthermore, Atashi et al. also agreed that combining therapies with other drugs would be more effective, which is consistent with the results of our study, where NPs conjugated with Cis were more effective. Similarly, Ghosh conducted a study on the use of cisplatin conjugated to nanomaterials, including inorganic nanoparticles such as carbon, gold, silica, and iron oxide. Their main objective was to enhance the uptake of the nano-drug delivery system through endocytosis in cells (29).

Abhishek et al. (30) explored the potential of ZnO NPs in treating various types of cancer, including cervical, breast, lung, and head and neck cancers, and found promising results. However, their review highlighted a lack of studies on using ZnO NPs as a nanocarrier for antineoplastic drugs and evaluating their efficacy. Similarly, Deng et al. (31) conducted a study on tumor spheroids treated with doxorubicin conjugated with Zn and Cu NPs and found that the conjugates of antineoplastics with Zn NPs were significantly more cytotoxic at a concentration of 0.01 mg/ml (10 μg/ml). In contrast, Chia et al. (32) compared cell cytotoxicity in a 2D versus 3D model of two different cancer cell lines, one of them of hematological origin, and found that the 2D model was more susceptible to ZnO cytotoxicity at any dose compared to the 3D model. It was because the cells of the outermost layers in the 3D model exhibited a protective effect against ZnO cytotoxicity at concentrations lower than 0.5 mg/ml, in contrast to the internal cells of the spheroid. This finding is consistent with our study, where we used a lower concentration (0.04 mg/ml) to obtain cytotoxicity.

While Al-Fahdawi et al. (21) observed a decrease in cell viability at doses of 6.12 and 12.5 μg/ml in a 2D culture using platinum-impregnated nanoparticles, our study showed significant cytotoxicity starting at a higher dose of 0.25 mg/ml (250 μg/ml), indicating differences in the effectiveness of the two nanoparticle formulations. In another study by Franke et al., (33) administering Cis through nanoparticles resulted in a significant induction in DNA damage at low doses (0.015 mg/ml), which is in contrast to our findings that showed a cytotoxic effect at a concentration greater than 0.03 mg/ml, highlighting the importance of understanding the nuances of different nanoparticle-drug formulations in achieving the desired therapeutic outcomes. Manisekaran et al. (34) conducted a comprehensive review of the literature on the use of MXene nanoparticles as antineoplastic agents in a 2D model. MXenes are a new class of 2D materials that have shown promise in various biomedical applications due to their unique physicochemical properties, including their high surface area, excellent biocompatibility, and tunable surface chemistry. The authors highlighted several studies that demonstrated the efficacy of MXene nanoparticles in cancer treatment when used alone or in combination with other nanostructures. The MXene-based nanostructures showed improved cancer cell inhibition and apoptosis compared to conventional chemotherapy drugs.

From a broader perspective, the findings of this study could potentially pave the way for the development of more effective and targeted cancer therapies that can selectively target cancer cells while minimizing damage to healthy tissues. The use of 3D spheroid models in cancer research is a significant advancement as it better mimics the complex tumor microenvironment and provides more accurate results. This study could contribute to the development of new treatment strategies that could ultimately improve patient outcomes and quality of life.

Conclusion

In conclusion, a 3D scaffolded model of lymphoblastic cell culture from a head and neck lesion was generated to compare the cytotoxic effects of antineoplastics and NPs with those in a 2D model in order to emphasize the significant differences in cell viability. It is worth noting that the impact of ZnO NPs alone and as a Cis nanocarrier on cancer cells is promising because it is the antineoplastic used as the first line of chemotherapy in most malignant neoplasms.

One of the essential points regarding nanocarriers is their endocytosis-mediated uptake by cancer cells. Hence, several reports on the capacity of inorganic NPs as nanocarriers of anticancer agents drive research for the future of nanomedicine.

Conflicts of Interest

The Authors declare no conflicts of interest in relation to this study.

Authors’ Contributions

NLM-M, ADC-G, RG-C: experimentation, writing – original draft, KMA-J, FGV-S, NP-C, JDR-B, EAC-M & UV-Z: conceptualization, methodology, review, and editing: NLM-M, ADC-G, RG-C:, project administration: RG-C, writing – review and editing, formal analysis: KMA-J, FGV-S, NP-C, JDR-B, EAC-M & UV-Z.

Acknowledgements

The Authors acknowledge the financial support from the UNAM-DGAPA-PAPIME and PAPIIT grants PE201622 and IT200922.