Introduction

Cancers are the second globally threatening/fetal diseases that endanger millions of peoples yearly1. The incidence and mortality due to cancers diseases were not declined in the last 30 years, despite substantial progresses in their therapy and prevention protocols. Regretfully, the incidences/mortalities of colorectal cancers (CRC) have been increased from the year 2000 in young adults below the 50th age2. CRC is currently the 3rd frequently reported cancer type among American citizens3.

Numerous common and advanced strategies were suggested for targeting specific cancerous cells, to constrain tumors’ development, evolution, and metastasis with less potential consequences4. Alongside the chemically fabricated anticancerous agents (chemotherapy), numerous potential anticancer formulations with diverse action modes were introduced based on plant extracts and phytocompounds5. From these precious phytocompounds, curcumin (Cur) that is the highest valuable constituent of Curcuma longa L. rhizomes (turmeric), proved its bioactivity and bio-functionality in various fields, including the anticancer, anti-inflammatory and antioxidant propereties6,7.

The key reported mechanisms of Cur anticancer action included the induction of apoptosis, the inhibition of tumors invasion and proliferation through restricting vital signaling pathways of cancers’ cells8. Various Cur delivery systems were introduced, which are mainly depending on nanotechnological approaches, to enhance Cur biofunctions and targeting; the formulations of Cur nanoparticles (i.e. NCur) was confirmed to augment their bioactivities and reactivity9, whereas the usage of biopolymers’ nanoforms as carriers of Cur could significantly improve its bioactivity, biodegradability, functionality, and water-solubility, with minimal biotoxicity risks10,11.

Fucoidan (Fu), the sulfated, fucose-rich polysaccharide, was principally extracted from marine brown macroalgae (e.g. Phaeophyta; Fucaceae, Laminariaceae, Chordariaceae and Alariaceae), in addition to further marine sources with minor quantities (e.g. invertebrates, sea cucumber, urchins eggs and seagrass)12. Fu is an exceptionally active biopolymer, with remarkable potentialities and bioactivities; including the anti-inflammatory, antioxidant, anti-coagulant, antithrombotic, antifungal, antibacterial and antiviral activities, in addition to the potent anticancer and anti-proliferative properties toward numerous cancer cell types13,14,15. The ionic nature of Fu (negative charges from sulfate residues in its C2, C4 and C3 positions) is the key factor for its potential applications in pharmaceutical and biomedical technologies, due to their interactions with positively-charged molecules16,17. Fu also possesses outstanding biocompatible, biodegradable, and non-toxic nature, which is approved from FDA “Food and Drug Administration” as GRAS “Generally Recognized As Safe” food ingredient. The functionalities/bioactivities of Fu were significantly augmented by its transformation to smaller sizes (e.g. nanoparticles, microparticles, liposomes . etc.), or with its conjugation with other biopolymers and nanomaterials16,17,18.

The nanomaterials and nanoparticles (NPs) have the sizes in “Nano” scale (e.g. ≤ 100 nm for nanometals and ≤ 1000 nm for nanopolymers), which provides them with the greatest surface areas, reactivity and functionality. The synthesis methods of NPs can be environmentally-harmful or expensive, but the biological approaches (i.e. biosynthesis) could offer highly efficient, eco-friendly, cost-effective, stable, biocompatible and biosafe NPs using natural-based precursors19. The plant extracts and phyto-constituents include divers bioactive molecules and secondary metabolites (e.g. phenolics, flavonoids, alkaloids, terpenoids, proteins and enzymes) that can be involved in reduction/synthesis of metals NPs, in facile and eco-friendly processes19,20. Likewise, the phyto-biosynthesis of metal NPs could mostly generate more uniformed shape and size particles, with minimized needs for further stabilizing or capping agents, which provide better bioavailability and efficiency to such biosynthesized NPs21. Selenium (Se); the essential nonmetal trace element, could have numerous vital and human-health benefiting purposes (e.g. such as enzymes-activation, immune-stimulation, anti-inflammatory antioxidation and anticancer potentialities)22. Selenium nanoparticles (SeNPs) was particularly the ideal candidate for implementing Se into clinical and biomedical practices, as the SeNPs have many unique physicochemical physiognomies, including the elevated biocompatibility, bioavailability and stability, with the minimal toxicity21,22. Besides the numerous biological and health-benefiting consequences of SeNPs, their antiproliferative and anticancerous potentialities were stated and recommended to develop novel attitudes for cancer prevention, managing and treatment23,24. The SeNPs can selectively trigger apoptosis in diverse cancer types, with protection of normal somatic cells from impairment21,25. The employment of capping agents to hold/encapsulate SeNPs can positively enhance their characteristics, biosafety attributes and preventing their over-growth/aggregation to be applied in biomedical uses26.

Nanocomposites (NCs) term refer to combined materials that have at least one “nano-scale” dimension (e.g. within 1–100 nm range). The NCs frequently exhibit higher performance, activities and uniqueness of design potentials than their original constituents do; they can comprise ≥ two different molecules with dissimilar physical/chemical properties and interface phases27,28. The biopolymers-based NCs showed enhanced ability for regulating chemicals and drugs releases, with more biocompatibility and safety manners29.

The applications of nanomaterials/NCs for suppressing and inhibiting diverse cancer cells were encouragingly investigated, using miscellaneous anticancerous nanomaterials’ types including protein-based nanoformulations, nanometals, biopolymers nanoparticles, biomimetic nanoparticles and their conjugations30,31,32,33,34,35. The main functions of such nanoformulations, toward diverse cancer cells’ types, were reported to include the apoptotic/anti-proliferative actions, increasing pro-apoptotic mechanisms, synergistic stimulation of apoptosis and anti-proliferative genes’ expressions33,34,35,36,37.

As the biosynthesis of NCur/SeNPs nanocomposite and their conjugation with Fu to generate potential anticancerous candidates was not formerly investigated or accomplished by earlier studies, thus the innovative biosynthesis of SeNPs using NCur and their conjugation with Fu were planned in this study, aiming to provide effectual anticancer NCs for suppressing colorectal cancer lines.

Materials and methods

Materials

The entire used materials, reagents, dyes and media were attained from Sigma-Aldrich Co., Saint Louis, MO (unless other sources are stated). The used materials included fucoidan (Fu) from Undaria pinnatifida brown algae (CAS No: 9072-19-9), curcumin (Cur) from Curcuma longa (CAS No: 458-37-7), gum Arabic (GA), Na2SeO3 (≥ 98%), HCl, KOH, NaOH, methanol (99%), chloroform, ethanol (95%), DMEM “Dulbecco’s modified eagle’s medium”, fetal bovine’s serum, streptomycin, L-glutamine, penicillin, MTT “3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide”, ethidium bromide (EB), toluidine blue, osmium tetroxide, lead citrate, uranyl acetate, dimethylsulfoxide (DMSO), and PBS “phosphate buffer solution”. The colorectal cancerous cell lines, i.e. CaCo-2 (ATCC- HTB-37) and HT-29 (ATCC-HTB-38), were purchased from ATCC “American Type Culture Collection, Manassas, VA”.

Preparation of nanomaterials and nanocomposites

The fabrication of Fu/NCur/SeNPs nanocomposites involved the following steps of preparation: to prepare NCur, GA solution (1.0% concentration, w/v) was prepared using deionized water (DIW), and Cur solution (0.1% concentration, w/v) was prepared in ethanol (70%), then both solutions were individually homogenized (IKA T25, ULTRA-TURRAX, Staufen, Germany), then thoroughly mixed via dropping Cur into GA solution (using 10:22 ratio, respectively)9,38. The homogenization (7200 x g) was continued for 50 min, then the formed NCur composite was cleansed by ethanol and centrifuged (10500 x g) to exclude unbounded molecules.

A solution of Na2SeO3 (sodium selenite) in DIW was made with 0.173% (w/v) concentration, then 10 mL of it was thoroughly dropped into 12 mL of NCur solution (0.1%, w/v), and vigorously stirred (680 x g) for 35 min. The emergence of deep brownish-orange color was the indicator of NCur/SeNPs synthesis. The formed NPs were harvested via centrifugation (11400 x g, 32 min), rinsed with ethanol and DIW, re-centrifuged, and lyophilized9. To attain plain SeNPs, the NCur/SeNPs nanocomposite pellet was comprehensively rinsed with ethanol and DIW (5 cycles each) and harvested after each cycle, and the NCur-free SeNPs were finally oven-dried.

Fu, dissolved in DDW with 0.1% (w/v/) concentration, was thoroughly mixed with equal volume of NCur/SeNPs solution (1 mg/mL) and stirred for 150 min at 7500 x g. The formed Fu/NCur/SeNPs nanocomposites were harvested and washed through successive centrifugation cycles, and then the resulted pellet was freeze-dried18.

Characterization of interacted molecules/composites

UV-vis (UV-visible) analysis

The UV-vis spectroscopic analysis (UV-2450, Shimadzu, Japan) was conducted for NCur/SeNPs color assessment, at wavelengths spectra range of 200–800 nm. The uppermost absorbance wavenumber (λmax) was recorded for interacted molecules’ solutions21.

FTIR “Fourier transform infrared” spectroscopic analysis

The distinctive biochemical bonding and interactions between molecules (Cur, Cur/GA, NCur/SeNPs, Fu and Fu/NCur/SeNPs) were inspected using infrared spectroscopy (JASCO; FT-IR-360, Tokyo, Japan). Samples were prepared for FTIR analysis via blending of their dried powders with KBr, and then their transmission patterns were perceived within 4000 –450 cm− 1 wavenumbers’ range18.

Particles’ size (ps) and zeta (ζ) potential appraisal

The Ps distribution and surface charges (ζ potential) of synthesized NPs/NCs were appraised using DLS “dynamic light scattering”, photon correlation spectroscopy approaches, employing Malvern™ Zetasizer (Malvern, UK)9,21.

TEM “Transmission electron microscopy” imaging

The TEM pictures (JEM-1010, JEOL Ltd., Tokyo, Japan) of NCur/SeNPs were captured at 200 kV accelerated voltage, to inspect the structure, morphology and Ps of the NCs particles. The dissolved NCs in DIW were drop-casted onto TEM carbon grids, air-dried and screened9,21.

Further characterization of NCur-mediated SeNPs, e.g. via TEM-EDX “energy dispersive X-ray” (Supplementary materials; Fig. 1-S) and XRD “X-Ray diffraction analysis” (Supplementary materials; Fig. 2-S), were performed to specify the elemental composition and crystallinity nature of synthesized materials.

Anticancer assaying

The HT-29 cell and CaCo-2 cell lines were operated for assessing the NPs/NCs anticancer potentialities. Cancerous cells were multiplied at 37 °C in 5% CO2 atmosphere T-flasks; the used DMEM medium was supplemented with L-glutamine (8 mM), glucose (4.5 g/L), bovine serum (10%), and antibiotic mixture (streptomycin/penicillin, 1%).

Viability assessment via MTT protocol

Active grown cancerous cells in (DMEM) were implanted into microtiter 96-well plates (with ∼20 k cells/well); the negative (untreated) control cells were planted in plain medium without adjustment. The incubation was sustained for 24 h, then the wells were supplied with gradual concentrations (1–250 µg/mL) from each NPs/NCs. After incubation for further 24 h, media are detached then 20 µL of MTT solution (5 mg/mL) were imparted to each wells and cancerous cells were incubated for additional 4 h. DMSO (~ 150 µL) was carefully added to each well, mildly agitated, and the absorbance (optical density) of these mixtures were measured with microplates reader (at 570 nm)39.

DNA damage assay using Comet “single cell gel electrophoresis”

Comet assay technique was accompanied within alkaline environment, as slightly modified from standard procedure40. Slides were handled after treating cells (as exceeding protocol), neutralized using cold buffer (Tris, 0.4 M, pH 7.5), and investigated under fluorescent microscope using imaging software “Komet 5.5, Olympus, Japan”. The lengths of damaged DNA tail (µm) indicate for severity of DNA destruction after cells’ treatments with anticancerous NCs.

Ultrastructure imaging of treated CaCo-2 cancer cells

Within their logarithmic phase, grown CaCo-2 cells were treated with 2 x IC50 form the Fu/NCur/SeNPs nanocomposite, whereas the DMSO-treated cells were the control group. For TEM analysis, after 24 h of incubated treatment, cells were rinsed by PBS, harvested via centrifugation (1200 x g) and fixed with 2.5% icecold glutaraldehyde. The samples were successively rinsed with PBS (0.1 M), post-fixed with osmium tetroxide (1.0%), dehydrated through 30–90% graded ethanol solutions series, and administered to Epon™ embedding “Momentive Specialty Inc., Columbus, OH”. Semithin (600–800 nm) sections were stained by toluidine blue, then representative parts were subjected to ultrathin sectioning (50–70 nm), stained with lead citrate and uranyl acetate, and examined under TEM device41.

For the SEM “scanning electron microscopy, IT-100, JEOL Ltd., Japan”, ~ 2 × 105 of CaCo-2 cells were planted onto 0.17-mm coverslips in 6-well plates. After incubated for 24 h, cells were exposed to 2 × IC50 from the NCs, and incubated for further period then rinsed, fixed and dehydrated as above. The cells were then frozen overnight, then samples were sputtered with palladium/gold coating and subjected to SEM imagining42.

Statistical analysis

ANOVA and Student’s t-test were employed to analyze the comparisons between treatments/groups, using statistical (SPSS 18.0) software (Chicago, IL). The triplicates means of experiments ± SD “standard deviation” were compared with p values of < 0.05.

Results and discussion

The current investigation targeted the biosynthesis of NCur/SeNPs and their conjugation with Fu to achieve more effectual and natural anticancer alternatives. The biosynthesis and characterization of nanoparticles (NPs) and nanocomposites (NCs) were achieved.

Visual/structural characterization nanomaterials

The construction of NCur /SeNPs composite involved 3 steps: first, NCur fabrication using GA, then (2) preparation of the selenite “Na2SeO3” solution, and (3) homogenizing both solutions together. This procedure resulted in emergence of deep brownish-orange color, indicated the successful synthesis NCur /SeNPs composite30,31,32,33,34. The color change (to brownish orange) was instantly observed after mixing of the two solutions (within 30 min) [Fig. 1(1)]. The biosynthesis of SeNPs using Cur was verified with UV-vis spectroscopic assessment [Fig. 1(2)]. Where the λmax of Cur solution was recorded at 426 nm, the synthesis solution that contained SeNPs had λmax value of 267 nm. The distinctive SPR “surface plasmon resonance” of SeNPs solution with λmax near 270 nm was reported as indicative for NPs biosynthesis43. The color change of biosynthesized SeNPs solution is frequently associated with the size, dispersion and shapes of NPs in addition to the employed resources for their reductions9,44. The structural attributes (size/shape) and dispersion of both NCur and SeNPs were proved via TEM imaging [Fig. 1(3)]; the NCur particles had mean diameter size of 34.83 ± 4.81 nm, whereas SeNPs mean diameter was ~ 5.21 ± 1.08 nm. Numerous SeNPs were embedded/encapsulated within NCur particles, where some other particles appeared in Free State out of NCur particles [Fig. 1(3)]. This evidently appoints the competence of NCur to reduce, stabilize and hold Se in the nanoform9,45.

Fig. 1
figure 1

Optical observations of biosynthesized selenium nanoparticles (SeNPs) with curcumin (Cur) nanoparticles, including their visual appearance (V), their UV-vis spectra (U), and their transmission microscopy imaging (T).

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Infrared analysis of materials/nanocomposites

The FTIR analysis can provide accurate information about the constructions, structural components and interactions of the screened compounds. The IR spectra of Cur and NCur (Cur and Cur/GA in Fig. 2) exhibited typical characteristic groups, with altered intensities of their peaks; Cur spectrum signified distinctive bands at 3502 and 1622 cm− 1, attributed to vibrated stretching of (O–H) and (C = O), respectively. The representative band at 1496 cm− 1 appointed the (C = C) vibrated stretching, while the aromatic (C–O) vibration is signified at 1254 cm− 1, and the bending (= C–H) at 908 cm− 1, respectively38,46. The IR peaks of Cur at 733, 812, and 954 cm− 1 designated vibrated bending of –CH in alkenes groups39. In NCur spectrum, the (C = O) vibrated stretching in RCOOR’ ester appeared at 1723 cm− 1. Additionally, the distinctive (O–H) band became much wider, mainly due to more combinations of these groups from Cur and GA. The changed peaks in NCur from their relevant in Cur are principally attributed to involvement of GA groups in the structure of NCur9,38,46.

Fig. 2
figure 2

Infrared spectral analysis (FTIR) of composited materials, including curcumin (Cur), gum Arabic (GA), selenium nanoparticles (SeNPs) and fucoidan (Fu).

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In the spectrum of NCur\SeNPs composite (Cur/GA/SeNPs in Fig. 2), the interactions between Se and NCur/GA were evidenced from the disappearance or lower intensities of numerous bonds in NCur spectrum (e.g. within 508–720 cm− 1 and 1220–1270 cm− 1 ranges, and at 1462, 1496, 1243, and 2902 cm− 1), which strongly appoint the occupation of functional groups in NCur/GA with SeNPs during their reduction and interaction. The emergence of new peaks in NCur/SeNPs spectrum at 641, 1303, 1688, 3741 cm− 1, comparing with NCur spectrum, indicated the formation of novel bonds/groups between Se and NCur through the synthesis of SeNPs45,47.

The IR spectrum of Fu (Fig. 2-Fu) illustrated the existence of key distinctive peaks that designate native Fu; the wide band within 3420 cm− 1 indicated the –OH occurrence (hydroxyl group); at 2942 cm− 1 appoints the aliphatic C–H, at 810 cm− 1 indicated the equatorial/axial sulfate position; and at 1638 cm− 1 indicates the (O–C–O) functional carbonyl groups associated to uronic acid17,48. The Fu spectrum also provided evidences for the sulfated polysaccharides groups; where the level of sulfation correlated with peaks’ intensities. At 1071 cm− 1 (this band indicates the symmetric vibrated stretching of sulfate esters O = S = O); the 834 cm− 1 band indicated the C–S–O, whereas the 697 cm− 1 band is distinctive for fucose dexoysugars49. Moreover, the 538 cm− 1 strong band indicates the C‒O‒S secondary axial sulfate existence at C-4 in fucopyranose17,48,50.

The spectrum of the entire compounds conjugates (Fig. 2-Fu/Cur/GA/SeNPs) showed several designative peaks from both Fu and Cur/Ag/SeNPs spectra (marked with blue lines for Fu and red lines for Cur/Ag/SeNPs derived peaks), which indicates the electrostatic interactions between the compounds. The appearance of Fu-derived peaks in the conjugates’ spectrum was prevailing than derived peaks from Cur/Ag/SeNPs spectrum, which may propose the encapsulation of Cur/Ag/SeNPs within Fu molecules, where the surface groups are mainly from Fu molecules.

Particles’ sizes and zeta (ζ) potential of fabricated nanomaterials

Table 1 Particles’ sizes and zeta (ζ) potential of fabricated nanomaterials.
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The diameters’ sizes (Ds) of nanomaterials and their charging (ζ potential) were appraised by DLS technique (Table 1), which emphasized that each of NCur, SeNPs, and NCur/SeNPs composite was negatively charged (e.g. with − 24.3, − 28.6, and − 36.5 mV, respectively). Both DLS and TEM results were matched for the screened nanoparticles [Fig. 1(3)], and their ζ potentials could provide high stabilities for synthesized nanocomposites, as the higher ζ values (e.g. >30 mV), provided higher NPs stability and dispersion9,51. The well dispersion and stability of synthesized nanoparticles could be evidenced from their TEM imaging [Fig. 1(3)]. The mean Ds of SeNPs was 5.17 nm, which indicates the powerful reduction activities of NCur to generate that minute sizes. The mean Ds of NCur/SeNPs composites (38.39 nm) was slightly greater than the mean Ds of individual NCur and SeNPs, which suggests the encapsulation of SeNPs within NCur particles, as earlier appointed in Fig. 1(3).

MTT cytotoxicity assay

Table 2 Anticancer potentialities of fucoidan (Fu), nanocurcumin (NCur), NCur-mediated SeNPs (NCur/SeNPs) and their composites against colorectal adenocarcinoma cells.
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The anticancer potentialities of Fu, NCur, NCur/SeNPs and their composite (Fu/NCur/SeNPs), compared to cisplatin (standard anticancer drug) are appointed, using MTT assay (Table 2). The IC50 values of screened agents indicated that NCur, NCur/SeNPs and Fu/NCur/SeNPs had remarkable anticancerous activities toward colorectal adenocarcinoma cells (CaCo-2 and HT-29); the nanocomposite (Fu/NCur/SeNPs) was significantly the most powerful, with IC50 of 10.35 and 19.44 mg/L, respectively. Interestingly, the cytotoxicity assay (MTT) results for NCur/SeNPs were comparable to cisplatin, whereas the activity of Fu/NCur/SeNPs significantly exceeded cisplatin, toward both cell lines (Table 2). The CaCo-2 cells were generally more susceptible to the entire agents than HT-29 cells.

Cisplatin, the frequently employed chemotherapy, could intermediate its anticancerous activity via diverse cytotoxicity mechanisms, e.g. DNA damaging, apoptotic pathways’ activation and cellular damage inflicting through oxidative stress and inflammation induction52. Harmonized results of adenocarcinoma cells’ treatment with cisplatin were reported53; they appointed that HT-29 (colon cancer line) and Sk-OV-3 (ovarian cancerous cells) were more resistant to cisplatin than CaCo-2 cells, after 24 and 48 h of exposure.

The combination between Cur-loaded SeNPs with other anticancer agents led to synergistic actions toward cancerous cells45, which was verified here with the conjugation of Fu with NCur/SeNPs composites.

The main anticancerous functions of Cur/SeNPs composite were supposed to associate with greater cellular uptake of conjugated anticancer agents, increment of ROS “reactive oxygen species” levels, induced apoptosis/cell cycle arresting, reduce the potentials of mitochondrial membrane and metastatic ability in cancerous cells9,45. The surplus generation of ROS triggers oxidative damages in several cells’ components and affects numerous signaling pathways, which leads to cellular apoptosis, whereas the metastasis (the most fatal feature of cancers) involves the capability of tumor cells to spread from the primary location to further distant locations/organs within the body54,55.

Additionally, the fabricated nanocomposites are assumed to trigger cells ‘autophagy, inflammation, and EMT “epithelial-mesenchymal transition”-metastasis. Both Se56, and Cur7, were capable of down-regulating the EMT process, where the epithelial cells mislay their cell–cell polarity and adhesion capabilities.

DNA damage using comet assay

The DNA damage assessment using Comet assay indicated the severity of NCs to affect CaCo-2 cancerous cells (Fig. 3). The control cells did not exhibit apparent DNA damaging (C in Fig. 3); the tail DNA intensity was only 4.26 ± 0.92%. On contrary, the treatment with Fu/NCur/SeNPs composite led to vigorous DNA damage with tail DNA intensity of 43.32 ± 3.57%. Additionally, the DNA damages after CaCo-2 cells’ treatment with NCur and NCur/SeNPs were obvious, with tail DNA intensity of 18.64 ± 3.11and 27.06 ± 2.26%, respectively (Fig. 3). The greater percentage of tail length (e.g. distance between DNA head and the DNA tail end) in comet assay indicates direct evidence for DNA damages, which provide sensitivity to the assay for evaluating genoprotective/genotoxic properties57. The attained results imply that NCs could induce severe DNA damages after cellular apoptosis58.

Fig. 3
figure 3

The DNA damage in the CaCo-2 cancerous cells after treatment with nanocurcumin (NCur), synthesized SeNPs with NCur (NCur/SeNPs) and their conjugation with fucoidan (Fu/NCur/SeNPs), comparing to control cells (C), using the Comet assay. Cells were treated with 2 X IC50 of each examined agent and incubated for 24 h at 37 °C.

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The application of Comet assay to assess DNA damages in CaCo-2 cells after treatments with different nanometals’ oxides (e.g. TiO2, SiO2, MgO and ZnO) was reported59; they concluded the direct DNA damage after ZnO and TiO2 NPs treatment without relation to particles’ size or shape. Additionally, the consequences of many plant extracts (e.g. rosemary, oregano, sage and Echinacea) on CaCo-2 viability, antioxidant status, membranes and DNA integrities, were reported60, which support the attained results here regarding the Cur and NCur/SeNPs actions as potential phytocompounds with documented anticancer powers7,39,61.

Fig. 4
figure 4

Electron microscopy imaging, including scanning (S) and transmission (T) images of control (1) and treated (2)* CaCo-2 cancerous cells with fucoidan/ nanocurcumin/selenium nanoparticles. *The nanomaterials around and inside cancer cell are appointed with blue zones, whereas the emerged autophagosomes are indicated by red arrows and zones in T-2 image.

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The anticancerous potentialities of NC (Fu/NCur/SeNPs) were further elucidated via electron microscopy approaches (i.e. SEM and TEM imaging) after treatment of CaCo-2 cells with 2 X IC50 form the NC (Fig. 4).

Using SEM imaging, the control cells [Fig. 4(1)] appeared with intact structures and have normal size/shapes; no abnormal features or distortions were observed in such control cells. In treated cells with Fu/NCur/SeNPs, [Fig. 4(2)], cells were obviously collapsed and numerous membranes’ perforation and vacuolization are observed. Harmonized deformation and distortion signs were formerly observed42, with the SEM imaging of treated cells using anticancer candidates, which indicates the efficacy of current nanocomposite in deforming CaCo-2 cells. Diverse abnormal ultra-structural protrusions and self-assembled construction types can exist on tumor cells’ surface, which could not be detected via regular light microscopy and need SEM for their elucidation. These surfaces’ structures have crucial functions in the cells’ migration and motility, and they are exceedingly important in cancer biology understanding, to emphasize cells communications and interactions with outer environment62.

The TEM images of treated CaCo-2 cells with Fu/NCur/SeNPs nanocomposite also provided more evidences of the cells’ autophagy and apoptosis [Fig. 4(4)]. The control cell image [Fig. 4(3)] indicated the normal structures of cells’ cytoplasm/membranes and the absence of membranous, autolysosomes or autophagosomes vacuoles. On contrarily, Numerous autophagosomes, autolysosomes and membranous vacuoles, comprising both digested residues and nanoparticles, were appeared in cytoplasm of treated cells with nanocomposite [Fig. 4(4)]. Several particles of SeNPs could be evidently observed inside and outside treated CaCo-2 cells, which further indicated that NPs could interact with the surface of tumor cells and with their interior organelles after penetration inside cells63,64. The autophagy processes frequently commence with doublemembrane vacuoles formation (i.e. autophagosomes), which embrace cytoplasmic components. Autophagosomes could subsequently fuse with progressed endosomes/lysosomes to develop into autolysosomes, where both inner membranes and interior contents are mostly degraded41,64. The ultrastructures of treated CaCo-2 cells with Fu/NCur/SeNPs nanocomposite could appoint the strong potentialities of fabricated NCs to suppress/kill the screened colorectal cancerous cells.

Conclusion

The biosynthesis of NCur/SeNPs nanocomposite and their conjugation with Fu was innovatively achieved to produce more effectual and natural anticancer alternatives for suppressing colorectal cancerous cells. The NCur particles had mean diameter size of 34.83 nm, whereas SeNPs mean diameter was ~ 5.21 nm. The Fu/NCur/SeNPs nanocomposites had vigorous activities against colorectal adenocarcinoma (CaCo-2 and HT-29), which exceeded the action of cisplatin. The cytotoxicity, DNA damage and ultrastructure’s deformation effects were verified after treatment of colorectal adenocarcinoma with fabricated natural nanocomposite. The bio-based constituents of the fabricated Fu/NCur/SeNPs nanocomposites and their powerful anticancerous actions could advocate their prospective practical applications for preventing/managing colorectal adenocarcinoma. Further investigations of the drug release kinetics and in vivo biotoxicity are recommended for prospective works.