Nanocomposite fibers based on cellulose acetate loaded with fullerene for cancer therapy: preparation, characterization and in-vitro evaluation

The current prevalence of cancerous diseases necessitates the exploration of materials that can effectively treat these conditions while minimizing the occurrence of adverse side effects. This study aims to identify materials with the potential to inhibit the metastasis of cancerous diseases within the human body while concurrently serving as therapeutic agents for their treatment. A novel approach was employed to enhance the anti-cancer properties of electrospun cellulose fibers by incorporating fullerene nanoparticles (NPs) into cellulose acetate (CA) fibers, resulting in a composite material called Fullerene@CA. This development aimed at utilizing the anti-cancer properties of fullerenes for potential therapeutic applications. This process has been demonstrated in vitro against various types of cancer, and it was found that Fullerene@CA nanocomposite fibers displayed robust anticancer activity. Cancer cells (Caco-2, MDA-MB 231, and HepG-2 cells) were inhibited by 0.3 and 0.5 mg.g−1 fullerene doses by 58.62–62.87%, 47.86–56.43%, and 48.60–57.73%, respectively. The tested cancer cells shrink and lose their spindle shape due to morphological changes. The investigation of the prepared nanocomposite reveals its impact on various genes, such as BCL2, NF-KB, p53, Bax, and p21, highlighting the therapeutic compounds' effectiveness. The experimental results demonstrated that the incorporation of NPs into CA fibers resulted in a significant improvement in their anti-cancer efficacy. Therefore, it is suggested that these modified fibers could be utilized as a novel therapeutic approach for the treatment and prevention of cancer metastasis.


Preparation of spinning solutions
Many experiments with dichloromethane and acetic acid were carried out to determine the optimal concentration of acetic acid to produce bead-free fibers.Finally, CA (10%) in acetic acid: dichloromethane (1:1) might produce smooth and bead-free fibers.Subsequently, Fullerene@CA fiber was made using different concentrations of fullerene (0.1, 0.3, and 0.5) mg produced by thermal catalytic cracking of mineral water waste bottles 43,44 to one gram of polymer.

Fabrication of fullerene@CA nanocomposite fibers
The nanocomposite fibers were prepared using an electrospinning apparatus consisting of a flow rate machine, a metal plate (used as a collector), and a high voltage source.Fibers were gathered (coated with aluminum foil) using a collector.The experimental conditions encompassed a spinning flow rate of 3 ml/h, a spinning voltage of 17 kV, a receiving collector distance of 20 cm, and the experiments were conducted at room temperature.Following the completion of the electrospinning process, the electrospun Fullerene@CA nanocomposite fiber was subjected to a vacuum oven at ambient temperature for a duration of 24 h in order to remove any residual solvent.

Characterization of fullerene@CA nanocomposite fibers
The surface morphology of all the electrospun Fullerene@CA nanocomposite fibers was observed using Transmission Electron Microscope (TEM, JEOLJEM 1230, Japan) and Scanning Electron Microscopy (SEM, JEOL JSM, Japan) after coating with gold.The softwarImage-Pro 6.0 image analysis software was used to measure the diameter of each sample, and the average value was calculated from randomly selected about 60 measurements.FTIR spectra were captured using the Shimadzu FTIR-8400 S (Japan) instrument from 4000 to 400 cm −1 wavelength with a 4 cm −1 resolution for all spectra.

Determination of fullerene release profile
The optimal excitation and emission wavelengths of free fullerene were determined through spectrofluorometry, employing a broad range of measurements, using spectrofluorometry (BMG Labtech, Germany).Then, the release rate of fullerene from the fullerene@CA nanofiber scaffold was assessed after 6, 24, 48, and 72 h incubations in phenol red free-culture medium via measuring it at excitation of 355 nm and emission of 590 nm.

Determination of cytotoxicity of fullerene@CA nanocomposite fibers to normal human cell line
Normal human lung fibroblast Wi-38 cell line (passage#30) was used to detect cytotoxicity of the prepared Fullerene@CA nanocomposite fibers, supplied from the American type culture collection (ATCC, USA).Wi-38 cell line was cultured in DMEM medium 10% fetal bovine serum (FBS), seeded as 5 × 10 3 cells per well in a 96-well cell culture plate, and incubated at 37 °C in a 5% CO 2 incubator.After 24 h for cell attachment, 0.1, 0.3 and 0.5 mg of free-fullerene and Fullerene@CA nanocomposite fibers were incubated with Wi-38 cells for 72 h.Cell viability was assayed by the MTT method.The wells were filled with 20 μl with 5 mg/ml MTT (Sigma, USA), and the plate was then incubated at 37 °C for 3 h.100 μl of DMSO was added after the MTT solution was eliminated, and a microplate reader was used to measure each well's absorbance at 570 nm (BMG LabTech, Germany).The Graphpad Instat program calculated the investigated substances' effective, safe concentration (EC100) value (at 100% cell viability).

Investigation of the anticancer activity
The anticancer effect of Fullerene@CA nanocomposite fibers was assayed using three human cancer cell lines that were obtained from (ATCC, USA).Colon cancer cell line (Caco-2, passage#32), triple-negative breast cancer cell line (MDA-MB 231, passage#35), and liver cancer cell line (HepG-2, passage#40) were cultured in DMEM (Lonza, USA) contained with 10% FBS (GEBCO, USA) supplemented with 10% FBS.All cancer cells (4 × 10 3 cells/well) were seeded in sterile 96-well plates.After 24 h, 0.1, 0.3, and 0.5 mg.g −1 Fullerene@CA and CA freefullerene were incubated with three cancer cell lines for 72 h at 37 °C in a 5% CO 2 incubator.The MTT method was done as described above.The percentage of growth inhibition of three tested cancer cell lines was calculated at each corresponding dose, relative to the untreated cells.Furthermore, cellular morphological changes before and after treatment with the most effective and safest anticancer compounds were investigated using a phase contrast inverted microscope with a digital camera (Olympus, Japan).

Statistical analysis
The data are expressed as mean ± standard error of the mean (SEM) and the significant values were considered at p < 0.05.One-way analysis of variance (ANOVA) by Tukey's test was used to evaluate the difference between the mean values of the studied treatments.The analysis was done for three measurements using SPSS software version 16.

Results and discussion
Characterization of fullerene@CA nanocomposite fibers SEM images for CA and Fullerene@CA nanocomposite fiber are shown in Fig. 1(a,b,c and d).Evidently, smooth, bead-free nanofibers are obtained and no fullerene aggregates are observed on the surface of these nanofibers, implying that the as-loaded fullerene is perfectly incorporated well within the nanofibers.The diameter of the neat CA nanofibers are 486 nm.These results align with the previous work of Suwantong et al. when CA was used for biomedical applications and the average diameter was 300 nm 45 .However, the diameter in our work is higher than that due to the solvent effect we used.Solvent has a critical role in determining the shape and diameter of the nanofiber 26 .In contrast, the diameters of the Fullerene@CA nanofiber ranged between 441 and 506 nm with no particular dependency on the initial amount of the as-loaded fullerene.TEM analysis was conducted to observe fluorine particles that were loaded into the CA nanofibers (Fig. 1e and  f).The pristine CA nanofiber is homogenous and does not possess any particles derived from fluorine (Fig. 1e).The image of Fullerene@CA (Fig. 1f) reveals the presence of fluorine-associated particles with varying sizes.The aforementioned findings are consistent with earlier TEM analysis of CA when incorporated with TiO 2 and AgNPs and also indicate a similar outcome 46 .
FTIR spectra for CA and Fullerene@CA nanocomposite fibers with different loading ratios are presented in Fig. 2. Prepared fullerene has a band around 1650 cm −1 corresponding to stretching carbonyl group (C = O) and C = C vibration, in addition to the band at 1095 cm −1 which was ascribed to the intrinsic characteristic of sp2 graphitic materials and oxygen functionalities of skeletal stretching vibration of C = O group and the broad peak at 3441 cm −1 representing O-H stretching vibration of hydroxyl functional groups (Fig. 2a) 43,44 .CA has strong peaks at 3455 cm −1 and 1044 cm −1 which are assigned to the hydroxyl group and ring of cellulose ether (Fig. 2b) 47 .Furthermore, it has characteristic peaks of C = O, C-O, and C-H of the acetyl group (CHCOO) at 1755 cm −1 , 1384 cm −1 , and 1234 cm −1 , respectively 48 .These results agree with earlier FTIR analyses for CA 49 .Fullerene@CA nanocomposite fibers show the same peaks for CA and fullerene.Nevertheless, the peaks have more intensity and become sharper (Fig. 2 c,d,e).FTIR data indicate that the prepared fullerene has been successfully incorporated into CA fibers.
As demonstrated in Fig. 3, the release rate of fullerene increased in a time-dependent manner from 7.43, 34.96, and 67.69% to 99.46% at 6 h, 24 h, and 48 h, respectively, to 72 h.

Cytotoxicity of fullerene@CA nanocomposite fibers to normal human cell line
An assessment of their safety profile was carried out using cytotoxicity screening on normal fibroblasts (Wi-38), followed by a comparison of their anticancer potential with 5-fluorouracil (5-FU) against three selected cancer cell lines (Caco-2, MDA-MB 231 and HepG-2).Fullerene@CA nanocomposite fibers, loaded with fullerene concentrations (0.1, 0.3, and 0.5 mg.g −1 ), are initially used as prescription carriers for anticancer treatments because fullerene has anticancer properties-according to previous studies [50][51][52] .The effect of Fullerene@CA nanocomposite fiber as an anti-cancer material was revealed, and it began to be evaluated as a standalone drug for anti-tumor growth where Bacakova et.al.investigated nanocellulose/nanocarbon composites in biomedical applications 53 .In contrast, Hamouda et al. use Au/cellulose nanocomposite as an anticancer 54 .However, numerous markers are used in cell viability studies to identify metabolically active (living) cells 55 .
The Fullerene@CA nanocomposite fibers loaded with 0.1 mg fullerene-maintained Wi-38 cell viability above 87% relative to < 60% in the case of fullerene which stands alone as free-fullerene.Conversely, when considering higher concentrations (0.3 and 0.5 mg), there are no significant differences in the viability of regular cells (< 51%) when comparing the free-fullerene and Fullerene@CA forms (Fig. 4).

Investigation of the anticancer activity
Included: cytotoxicity of the tested compounds on human cancers cell lines, flow cytometric analysis of the apoptotic effect of the tested compounds, and relative change in the expression of critical genes in the treated cancer cells.CA fibers and Fullerene@CA nanocomposite fibers exhibited potent anticancer activity, in a dosedependent manner, against Caco-2, MDA-MB 231, and HepG-2 cells.Figure 5  www.nature.com/scientificreports/nanocomposite fibers loaded with 0.1, 0.3, and 0.5 mg.g −1 of fullerene inhibited the growth of these cancer cells by 58.62-62.87,47.86-56.43%,and 48.60-57.73%,respectively.However, the lowest dose of the loaded fullerene (0.1 mg.g −1 ) significantly suppressed the proliferation of the studied cancer cell lines by 60.56 ± 0.56, 49.00 ± 3.29 and 51.53 ± 2.47%, for Caco-2, MDA-MB 231 and HepG-2 cells respectively, compared with free-fullerene that stands only form 51.94 ± 0.06, 41.71 ± 1.29 and 43.07 ± 0.40%, respectively.In addition, it has been observed that at this particular concentration, the safest dosage tested on Wi-38, there are morphological changes in human cancer cells, such as cell shrinkage and the loss of spindle shape.The CA fibers and Fullerene@CA nanocomposite fibers exhibit distinct variations in their efficient experiences with respect to size reduction.This finding is primarily attributed to the confinement of electrons to smaller surfaces and the corresponding increase in the edge-plane ratio.The increased density of edges facilitates a greater degree of electrochemical activity.Due to the higher edge density, this enables more electrochemical activity.Despite being less electrochemically active, the oxygenated equivalents exhibit superior stability and dispersibility, rendering them highly effective in cancer treatment 56 .Conversely, fullerenes have received much attention due to their potential use in human medicine 57 .
Several studies have employed cellulose nanofibers in the treatment of cancer cells.For instance, the combination of cellulose nanofibers with a bilayer of poly(diallyldimethylammonium chloride) (PDADMAC) and polyacrylic acid (PAA) through electrostatic interactions has demonstrated promising outcomes.This can be Figure 1.SEM images and diameter distribution histograms of CA and Fullerene@CA nanocomposite fibers loaded with different concentrations of fullerene: (a) without adding fullerene, and with (b) 0.1 mg.g −1 , (c) 0.3 mg.g −1 , and (d) 0.5 mg.g −1 ; and their diameter distribution; TEM images of (e) CA and (f) Fullerene@CA.attributed to the negative surface charge exhibited by cellulose fibers 58 .In addition, gold-silver NPs, electrospun silk fibroin, and CA (CA/SF/Au-Ag) composite nanofiber were used as anticancer where the fabricated CA/SF/Au-Ag nanofiber has an efficient IC50 value and substantially induced the cytotoxic effects against the human breast cancer cells MCF-7 and MDA-MB-231 59 .A critical step in drug screening and cancer research is the development of nanofibrous scaffolds for in vitro cancer cells using graphene oxide (GO) and (CA) 60 .The mechanical properties of GO/CA scaffolds exhibited a clear superiority over bare CA scaffolds, with an observed enhancement as the concentration of GO increased.In vitro cell experiments revealed that cancer cells exhibited significantly enhanced vitality, improved cell adherence, and accelerated growth when cultured on GO/CA scaffolds compared to those cultured on bare CA scaffolds.To the best of our understanding, there has been no prior investigation into the utilization of nanocomposite electrospun fibers comprising fullerene NPs with cellulose acetate (CA) for the specific targeting and treatment of various cancer cell types, including colon cancer cell line (Caco-2), breast cancer cell line (MDA-MB 231), and liver cancer cell line (HepG-2).Therefore, this study contributes a novel discovery to the existing body of knowledge in this particular domain.

Flow cytometric analysis of the apoptotic effect of the tested anticancer compounds
Quantification of annexin-stained apoptotic cells was done and the apoptotic powerful effect on Caco-2 was confirmed by a high percentage of annexin-stained apoptotic population after 72 h treatment relative to the untreated cells (Fig. 6 a,b).The Fullerene@CA nanocomposite fibers loaded with 0.1 mg.g −1 have more substantial  apoptotic potential than free-fullerene, where the loaded fiber can induce apoptosis by 49.79 ± 1.12% compared to 42.88 ± 1.86% in fullerene-treated Caco-2.The production of ROS in cells clarified the molecular pathways behind fullerene-induced apoptosis 61 .Fullerene improved the ROS-independent MEK-ERK pathway stimulation composed of the extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) 62 .

Quantitative detection for the change in the expression of treated (Caco-2, MDA-MB 231, and HepG-2 cells)
qRT-PCR analysis of genes expression RT-PCR analysis of cells after treatment with fullerene and Fullerene@ CA nanocomposite fibers was studied to determine the differences after treatment of cancer cells as presented in other studies.Both materials are nearly equipotent in the treated Caco-2 cells, critical genes of proliferation and apoptosis are measured at the mRNA level.Both forms can suppress the expression of BCl2 and cyclin D by ~ 5 and 2 folds, respectively.Furthermore, in its loaded and free forms, fullerene can induce the expression of p53 by 4 and 3 folds, respectively.The downstream (Bax and p21 genes) are also upregulated by 2 and 3 folds, respectively, in the treated cells, with no significant difference between loaded and free (Fig. 7).Multiple genes play a crucial role in the regulation of cellular division and developmental processes.The cell cycle refers to the systematic and sequential mechanism through which a cell undergoes replication to produce an identical copy of itself.The aforementioned process is subject to stringent regulation in order to ensure the accurate duplication of DNA in dividing cells, the rectification of any DNA errors, and the equitable distribution of a complete set of chromosomes to each progeny cell.The observed fold change in gene expression levels of the P53 gene, a known tumor suppressor gene, is higher in Caco-2 cells than other genes utilized to demonstrate the genetic impact on treated Caco-2 cells.In contrast, utilizing Fullerene@CA nanocomposite fibers with a loading concentration of 0.1 mg.g-1 demonstrates greater efficacy than pure fullerene.Specifically, the relative fold change in gene expression levels of p53 increased by 0.72 in the presence of Fullerene@CA nanocomposite fibers.Following the occurrence of double-stranded DNA strand breaks, the levels of p53 increase due to the stabilization process.Additionally, kinases promptly phosphorylate histone H2AX at or in proximity to the sites of DNA damage, resulting in the formation of C-H2AX 63,64 .The p53 protein in the cell binds DNA, activating a different gene to make the p21 protein, which interacts with a protein that promotes cell division (cdk2).The relative fold change in gene expression levels of the BAX gene for Caco-2 cell increases than NF-KB and less than (P53 & p21) where, the first pro-apoptotic member of the Bcl-2 protein family to be discovered is the BAX gene 65 .A moderating variable's value affects how the mediating variable (lincRNA-p21) behaves 66 .Based on the current data, lincRNA-p21 is a promising indicator of hematotoxicity therapy response.While, BCL-2, an anti-apoptotic protein, is found to have significantly decreased levels; this links the research materials to the apoptotic process in Caco-2 cells.The interaction between BCL-2 and VEGFA (vascular endothelial growth factor A) has been investigated in several cancer cells.BCL-2 has been shown to increase the expression of the VEGFA gene 67,68 .

Conclusion
This study presents a comprehensive methodology for the fabrication of biocompatible electrospun cellulose acetate (CA) materials specifically designed for use in biomedical applications.The incorporation of fullerene, a carbonaceous nanomaterial, was carried out in CA nanofibers at varying weight ratios (0.1, 0.3 and 0.5 mg.g −1 ).The addition of fullerene to CA nanofiber showed an insignificant effect on the morphology of neat CA.FTIR data confirmed the presence of fullerene in CA nanofiber (Fullerene @CA).The cytotoxicity of CA free-fullerene and Fullerene@CA nanocomposite fiber was assessed against Wi-38 cells.It was observed that the fullerene encapsulation (0.1-Fullerene@CA) demonstrated the ability to protect normal cells while still exhibiting its anticancer properties against Caco-2, MDA-MB 231, and HepG-2 cells.The findings indicate that Fullerene@ CA nanocomposite fiber prepared by electrospinning could be a promising candidate for biomedical applications as a cancer-fighting material.Based on the current study data, further research is required to validate the findings obtained in this area.

Figure 4 .Figure 5 .
Figure 4.The cytotoxicity effect of Fullerene@CA nanocomposite fibers with different fullerene-loaded ratios on Wi-38 cell viability compared to free-fullerene.(Value are presented as mean ± SEM and considered statistically significant at * P ≤ 0.05.NS refers to non-significant variation).

Figure 6 .
Figure 6.Flow cytometric analysis of Fullerene@CA nanocomposite fibers loaded with 0.1 mg.g −1 treated Caco-2 cells, after 72 h incubation, comparing with the untreated control cells and treated cell with fullerene only, (a) Dot charts of Annexin-propidium iodide-stained cells (FL1/FL2) with (b) the apoptosis percentages.(Value are presented as mean ± SEM and considered statistically significant at * P ≤ 0.05.NS refers to nonsignificant variation).

Figure 7 .
Figure 7. Relative fold change in the expression of oncogenes and apoptotic genes in loaded CA fibers and Fullerene@CA nanocomposite fibers loaded with 0.1 mg.g −1 -treated (a) Caco-2, (b) MDA-MB 231 and (c) HepG-2 cells.(Value are presented as mean ± SEM).