Introduction

Cancer is one of the major reasons for mortality worldwide and is a great apprehension among researchers for therapeutic findings1. The World Health Organization (WHO) has stated that by 2040, it is expected to reach 27.5 million new cancer cases and 16.3 million mortalities2. It is instigated by uninhibited uncharacteristic cell development1. At present, chemotherapy and radiation therapy are leading in the treatment of cancer which has substantial side effects like hair loss, nausea, vomiting, reduced red blood cells, deficiency of leukocytes, and low platelet count which affects the patient’s well-being and quality of life during treatment3. Hence, the necessity to find an efficient treatment to overcome the disadvantages of the above treatment must be found4. Herein, nanomedicine is a significant root that finds a solution to cure cancer and kill cancerous cells using different nanoparticles (NPs) without many side effects5. Nanotechnology deals with materials at the molecular level and has enormously reformed the arena of science6. NPs with size 1–100 nm have eminent properties compared to bulk materials7.

Several methods are available for the synthesis of NPs including physical and chemical methods with the use of toxic chemicals that are exclusive and injurious to human beings8. Hence, evolved the greener approach for the nontoxic synthesis of NPs using aqueous extracts of different organisms like plants9,10, algae11,12, bacteria13,14, and fungi15,16,17. Amongst these, algae-assisted production of NPs is comparatively less discovered. This method is not only cost-effective but causes insignificant menace to ecological health18. Although, algae are abundantly present in bulk extents at coastal areas19,20. However, less research has been carried out globally.

Seaweed or macroalgae is a significant renewable source in the coastal area. Red, brown, and green seaweeds play crucial roles in developing several pharmaceutical products21. In the field of medicine, seaweeds are widely used to cure stones in gall bladder, stomach disorders, eczema, cancer, kidney failure, scabies, asthma, atherosclerosis, cardiovascular disorders, lung ailments, ulcers, etc.22. The presence of biomolecules such as terpenoids, sterols, lectins, pigments, phlorotannins, ketones, alkanes, fatty acids, polysaccharides, carotenoid, glycoproteins, haloforms, hydroquinones, and phenolic compounds aids the use of seaweeds for drug discovery23. In the past decades, derivatives of brown seaweed Rosenvingea intricata (R. intricata) have shown antifungal, anticancer, antiallergic, antidiabetic, antiaging, anti-inflammatory, and antioxidant activities24. Seaweed-mediated NPs have been demonstrated as efficient nanomaterials for the treatment of cancer25.

Metal oxide NPs are assumed to be stable and benign for human beings and among iron oxide NPs especially magnetite (Fe3O4) being a semiconductor material finds applications in wastewater decontamination, sensors, purging of organic dyes, carbon capture, energy devices, magnetic storage devices, magnetic hyperthermia treatment, targeted drug delivery, and crop development26,27. Realizing the medical importance of R. intricata and Fe3O4 NPs, the objectives of this work are as follows: (1) Synthesis of Fe3O4 NPs using aqueous extract of R. intricata, (2) characterization of Fe3O4 NPs using various spectroscopic techniques and (3) testing its efficacy against human hepatocarcinoma (Hep3B) and pancreatic (PANC1) cancer cells. Hence, Fe3O4 NPs were synthesized using an aqueous extract of R. intricata collected from the tropical coastal region of Collinpur, South Andaman, India. Synthesized Fe3O4 NPs were characterized using various spectroscopic methods. 3-(4,5-dimethylethiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, morphological analysis and dual staining assay were used to determine the anticancer efficacy of R. intricata mediated Fe3O4 NPs.

Materials and methods

Study area description

Mature and well-grown thallus of R. intricata were collected from the coastal area of the Collinpur region, which was sandy coast along with various types of rocky substratum spread over the shoreline that helps in the sustenance of the luxuriant development of seaweed flora. Latitude (11° 41′ 33.9" N) and Longitude (92° 35′ 55.1"E) of R. intricata collection site was noted from handheld GPS (Garmin etrex Vista H), and the study area map (Fig. 1) was generated using ArcGIS software (ArcGIS, Version: 10.8.2).

Figure 1
figure 1

Location of the seaweed collection site map (ArcGIS 10.8.2, inset: R. intricata).

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Collection and identification of seaweed

The collected seaweed sample was preserved and species-level identification was done based on the external and internal morphology of specimens and sourcing the original protocol and standard flora28. Rosenvingea intricata (= Rosenvingea endiviifolia) is one of the lesser-known wild species that dwell in the east (Tamil Nadu and Andhra Pradesh) and west coast (Gujarat) of India and Andaman Nicobar Islands21,29,30. For the present study, the biomass of this species was collected from the Collinpur region of South Andaman. The species level of identification is done by acquiring the morphological and anatomical character sourcing the original protologue of the species31 and referring to the standard Indian seaweed flora29,32,33. The collected specimens have been preserved in the form of herbarium (Specimen No: ACOSTI-NIOT-027) and deposited at the Atal Centre for Ocean Science and Technology, National Institute of Ocean Technology, Port Blair.

Processing of seaweed

The collected seaweed sample was well rinsed with seawater followed by freshwater and Milli Q water to eliminate all debris such as epiphytes, sand particles including other adhering detritus materials. The water content in the seaweed sample was removed using blotting sheets. Cleaned seaweed was shade-dried for 5–7 days and destined for pulverization to get a coarse powder34,35.

Preparation of aqueous extract of R. intricata

10 g dry powder of R. intricata was mixed with 100 mL milli Q water and boiled at 90 °C for 15 min. It was vacuum-filtered to yield the aqueous extract of R. intricata. Stored at -4 °C for further usage.

Phycosynthesis of Fe3O4 NPs using aqueous extract of R. intricata

To 50 mL aqueous extract of R. intricata, 0.6 molar (M) ferric chloride hexahydrate (FeCl3.6H2O) was added and stirred for 1 h at 80 °C at 600 rpm. Further, 0.5 M sodium hydroxide (NaOH) was added dropwise to this for the precipitation of NPs. pH is maintained to be 11 (Fig. 2). It was further stirred for 30 min, centrifuged, and dried in a hot air oven (Model: ASI-HO-121212) at 60 °C. Powdered using an agate mortar and pestle11.

Figure 2
figure 2

Schematic illustration of R. intricata-mediated phycosynthesis of Fe3O4 NPs as a promising agent against pancreatic and liver cancer cells.

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Characterization of Fe3O4 NPs

Various methods for the description of synthesized Fe3O4 NPs in the present work were used for NPs characterization such as:

Ultra violet-visible diffused reflectance spectrophotometer (UV–Vis DRS)

The optical behavior of Fe3O4 NPs obtained using an aqueous extract of R. intricata was analyzed using UV–Vis DRS (Model: Jasco V-750). The sample was prepared by compacting it into a wafer and fitting the wafer to the reflectance sample holder. Optical band gap energy is estimated using Eq. (1).

$$\alpha h\upsilon = (h\upsilon - E_{g} )^{n}$$
(1)

where α = absorption coefficient, hν = photon energy, Eg = optical band gap energy, and n = ½ for a direct transition36.

Fluorescence analysis

Fluorescence property was studied using a fluorometer (Model: PerkinElmer LS 45)32. R. intricata-mediated Fe3O4 NPs were diluted with Milli Q water and the fluorescence spectra were attained by exciting the sample using LED lamps with a resolution of 0.5 nm.

Fourier transform infra-red spectrometer (FTIR)

Biomolecules adhered to the sample were premeditated using FTIR (Model: PerkinElmer Spectrum Two)36. Briefly, 3 mg R. intricata-mediated Fe3O4 NPs was mixed and ground in an agate mortar with 250 mg of pure spectroscopic grade potassium bromide (KBr) earlier dried at 300 °C in a muffle furnace (Model: Gefran 400) for 4 days. The ultrapure transparent pellet of 13 mm diameter was attained by smearing a force weight of 10 tons for nearly 10 min. IR absorbance was scanned in the range 450–4000 cm−1.

X-Ray diffractometer (XRD)

Crystalline phases of Fe3O4 NPs were analyzed using XRD (Model: Bruker D8 Advance) with a Cu radiation source. Briefly, the sample was ground into a fine powder using an agate mortar and pestle. It was filled in the empty sample holder and gently pressed using a glass slide. The excess sample was removed from the sample holder edges. The sample holder was carefully placed in the XRD slot. Scanned in the range of 20°–70°. The crystallite size was determined using Scherrer’s Eq. (2):

$${\text{D}} = {\text{K}}\lambda /\beta {\text{Cos}}\theta$$
(2)

where D = crystallite size (nm), K = shape factor, λ = wavelength of X-ray radiation, β = full with at half maximum (FWHM) and θ = Bragg angle36.

Dynamic light scattering (DLS)

Measurement of size is a main stage in the design of NPs production and its thriving usage. DLS utilizes scattered light to measure the extent of diffusion in nanomaterials29,30. Particle size measurement was done using DLS (Model: Micromeritics Nano Plus). Fe3O4 NPs are distributed in Milli Q water and sonicated for 30 min. The measurement state is maintained with a refractive index of 1.3328 and viscosity of the dispersion as 0.8878 (cP) at temperature 25º C36,37.

High-resolution scanning electron microscope (HRSEM) coupled with energy dispersive X-ray spectrometer (EDX)

HRSEM analysis is apt for the visualization of the morphology and size of the nanomaterials with their exterior texture35. The sample was mounted on a stub of metal with adhesive coated with gold and then visualized in the scanning electron microscope HRSEM (Model: F E I Quanta FEG 200). HRSEM coupled with EDX was used for the elemental profiling36.

High-resolution transmission electron microscope (HRTEM) coupled with selected area electron diffraction (SAED)

Surface texture and particle size were observed using HRTEM (Model: Jeol/JEM 2100). The sample was prepared through drop-coating on carbon-coated TEM grids36. HRTEM coupled with SAED was used for the identification of crystalline phases of Fe3O4 NPs36. The d spacing was calculated using the electron diffraction Eq. (3)

$${\text{L }}\lambda \, = {\text{ d R}}$$
(3)

where L is the distance between the sample and electron micrograph, λ is the X-ray wavelength, and R is the radius of diffraction rings26,36.

Vibrating sample magnetometer (VSM)

Magnetic properties of R. intricata-mediated Fe3O4 NPs are determined using VSM (Model: Lakeshore, USA, 7407) at moderate conditions and the magnetization value is analytically noted with the applied magnetic field to an extent of 15,000 Gauss. The squareness ratio is estimated using relation (4).

$${\text{Squareness}}\;{\text{ratio}} = \frac{{M_{r} }}{{M_{s} }}$$
(4)

where Mr = remanent magnetization and Ms = saturation magnetization36,37.

Anticancer studies

Cell culture conditions

The Human liver cancer cells Hep3B and pancreatic cancer cells PANC1 were purchased from the National Centre for Cell Science (NCCS, Pune, India). Cells were cultured in Dulbecco’s modified Eagle's medium with high glucose (DMEM-HG) media ((Make: Himedia; Cat No: AT151-5L)) containing 10% FBS (Make: Himedia; at No: RM9955-500 ml), Antibiotic Antimycotic solution 100X (Make: Himedia; Cat No: A002) and 37 °C temperature provided with 5% CO2. During subculture, once every 2–3 days, the medium was replaced with a fresh one. Cell-based experiments were performed after reaching 80% to 90% confluency.

In vitro cytotoxic studies using MTT assay

The human liver cancer cells (Hep3B) and human pancreatic cancer cells (PANC1) cells (Table 1) were independently seeded (1 X 104 cells/well) in 96 well plates with DMEM medium augmented with 10% FBS, 1X Antibiotic Antimycotic solution in CO2 incubator at 37 °C with 5% CO2. Cells were washed with 200 μL of 1 × phosphate-buffered saline (PBS), afterwards the cells were treated with different doses of R. intricata-mediated Fe3O4 NPs (25, 50, 100, 250, and 500 µg/mL) and with half-maximal inhibitory concentration (IC50) of Doxorubicin (3.87 μg/mL for Hep3B) and (8 μg/mL for PANC1)) as a positive control in serum-free DMEM medium and incubated for 24 h. The medium was aspirated from cells towards the end of the treatment period. Subsequently, the cells were washed with sterile PBS and imaged using an inverted phase contrast microscope (Make: Optika, Germany). MTT (0.5 mg/mL) prepared in 1 × PBS was added to 100 µl which was added to each well and incubated for a period of 4 h at 37 °C in a CO2 incubator. Once the incubation time was accomplished, the MTT was cast off from the cells and washed using 200 μL of PBS. The washing phase is for the exclusion of residual non-reduced MTT in the supernatant. Formed formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO) and carefully mixed. Progress of color intensity was estimated at 570 nm. The dissolution of formazan crystals yielded a purple-blue color. Absorbance was measured at 570 nm using a microplate reader (Model: Lark LIPR-9608). Blank well contained only medium served as a negative control. Experiments were repeated thrice and the IC50 values were attained from the mean of triplicate measurements36,37,38. The amount of cytotoxicity was evaluated from the MTT protocol. From produced purple formazan crystals, the metabolic functioning of live cells is measured37.

Table 1 Details of cell lines used.
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The Percentage of cell viability is calculated using Eq. (5).

$${\text{Percentage}}\;{\text{of}}\;{\text{cell}}\;{\text{viability}} = \frac{{{\text{OD}}\;{\text{Control}} - {\text{OD}}\;{\text{sample}}}}{{{\text{OD}}\;{\text{Control}}}} \times 100\%$$
(5)

where OD control = absorbance value of untreated cells, OD sample = absorbance value of treated cells37.

Morphological analysis

Hep3B and PANC1 cancer cells were cultured on a coverslip in DMEM medium overnight and treated with R. intricata-mediated Fe3O4 NPs for 24 h. Then, the medium was discarded and washed with 1 × PBS, later the coverslip was fixed with ethanol and acetic acid solution (3:1 v/v). Coverslips were slightly placed on glass slides and the change in shape of cells was detected using a phase contrast microscope38.

Apoptosis analysis using acridine orange/ethidium iodide assay

Apoptosis is a biotic progression usually categorized by dissimilar structural phenomena by actuating the precise signaling pathways counting the caspase death receptor pathway39. Hep3B and PANC1 cancer cells were developed individually in a 6-well plate for 24 h before attachment. Cells were treated with IC50 concentrations of R. intricata-mediated Fe3O4 NPs and incubated for 24 h in a CO2 incubator. The cells were trypsinized, centrifuged, washed twice with 1 × PBS, and resuspended in PBS solution. The cell suspension was withdrawn on the slide trailed by adding 1 mL of acridine orange (1 mg/mL) and 1 mL of ethidium bromide (1 mg/mL) and placed with a coverslip. Additional fluorescent dye was washed with PBS and the apoptotic features were captured under a fluorescence microscope38.

Statistical analysis

All tests were conducted a minimum of thrice in triplicate. T-test was used for the approximation of intergroup variations using GraphPad Prism 5 software. The data was expressed as mean and standard error mean (SEM). When a p value was fewer than 0.05, it was assumed statistically substantial.

Results and discussion

Optical properties of Rosenvingea intricata-mediated Fe3O4 NPs

UV-DRS analysis

The absorbance peak at 260 nm (Fig. 3a) corresponds to the transition of ligand to metal charge transfer (LMCT) in the tetra-coordinated Fe3+ (t1—t2 and t1—e) and the broadness in the peak around 400–550 nm was accredited to the existence of Fe3+ and Fe2+ ions at the octahedral sites. Also, the enlargement of the absorbance band denotes NPs synthesis. Peak amplification owes to the oxygen to metal charge transfer (O2- –Fe3+)40. The direct optical band gap was calculated to be 2.30 eV by inferring the tangent curve41 (Fig. 3b).

Figure 3
figure 3

(a) Ultraviolet–visible spectrum and (b) Tauc plot for direct optical band gap of R. intricata aqueous extract-mediated Fe3O4 NPs.

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Band gap arises due to the electrically conductive nature of a material. Semiconductors are materials with a narrow band gap. The versatility of semiconductors is that this optical phenomenon makes these materials appropriate for photocatalytic and antiaging applications. The band gap is calculated to be 2.30 eV which is quite narrow proving the possibility of using R. intricata-mediated Fe3O4 NPs in sunscreen lotions and wastewater treatment plants as catalysts37.

Fluorescence analysis

The fluorescence spectrum (Fig. 4) of R. intricata-mediated Fe3O4 NPs exhibited sturdy emission with an intense peak at 660 nm which is credited to the band edge emission and the external imperfections arising from iron vacancy at the tetrahedral site. Surfactants capped Fe3O4 NPs depict sharp emission which deters NPs from energy exchange with the neighboring atoms and disrupts the expansion of the wave function of an electron. This phenomenon is owing to the effect of quantum confinement and the surface state in nanometers and biomolecule interactions. Biogenic capping agents enable control over particle size thereby enhancing intense fluorescence and in biological applications, it aids biofunctionalization for better adherence of biomolecules and drugs. During the explosion process, the emission is an indication of the production of ions. Some of these ions get stuck to the exterior part of NPs and produce emissive centers. Supposed fluorescence is caused by the interface between emissive centers and the interface surrounding. Some allowed transitions are permitted through this emission superficial interface energy bands. Symbolically, semiconductor NPs holding photoexcited charge carriers possess a crucial part in medical and environmental arenas. Especially, for localized cancer treatment and wastewater decontamination, emission-bound excitation in NPs makes them competing materials37,42.

Figure 4
figure 4

Fluorescence spectrum of R. intricata aqueous extract-mediated Fe3O4 NPs.

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Structural properties of R. intricata mediated Fe3O4 NPs

FTIR analysis

Table 2 and Fig. 5 depict the FTIR spectrum data of the seaweed aqueous extract and R. intricata-mediated Fe3O4 NPs. Strong absorption around 3430 cm−1 and 3420 cm−1 is due to the existence of O–H groups from the polysaccharides and amino acids, inherent to seaweeds. These electron-rich biomolecules with hydroxyl (OH–) groups arising from amino acids and polysaccharides have the efficacy of reducing iron ions (Fe3+ and Fe2+) from their trivalent and divalent oxidation states into metallic Fe0. Further, the chemical activity aided by these functional groups converts zero-valent iron into magnetite NPs5. The short peak at 2927 cm−1 validates the existence of a C–H stretch of aliphatic hydrocarbons43. Peaks at 2027 cm−1 and 2026 cm−1 are due to the C–H stretching vibrations5. Strong bands at 1634 cm−1 and 1630 cm−1 denoted C=O, N=O stretch signifying a mark of ester and amide causative for distinguishing essence of seaweed and also confirming the presence of lignin with (C=C) group44. The peak at 1458 cm−1 is due to the C–N stretching vibration and C–H bending mode of alkanes5. Peaks around 1355 cm−1, 1385 cm−1, and 1345 cm−1 signify the C–O stretching and O–H bending vibrations5. Peaks overhead 1271 cm−1 denote the phenol groups which are characteristic of seaweeds with excess antioxidants, that could be employed in the progress of efficient food and their extract could be a potential candidate in nutraceutical preparation. This peak denotes the C–O stretching of ring oxygen5,45. Peaks at 1119 cm−1, 1016 cm−1, 1119 cm−1 and 1020 cm−1 confirmed the C–N stretch of aliphatic amines46. Feeble absorption at 620 cm−1 is indicative of S=O, C–S, and C=S modes of sulfates existing in the seaweed47. R. intricata possesses polysaccharides, amino acids, aliphatic hydrocarbons, alkanes, aliphatic amines, and sulfides, which are the major biomolecules present in it. It is believed that aquatic macroalgae produce a varied diversity of organic constituents that are accountable for their distinctive essence and freshness48. Peaks at 613 cm−1 and 438 cm−1 of R. intricata-mediated Fe3O4 NPs denote the Fe–O stretch of ferrites36.

Table 2 FTIR analysis of functional group assignment.
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Figure 5
figure 5

FTIR spectra of aqueous extract of R. intricata and its aqueous extract-mediated Fe3O4 NPs.

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XRD analysis

XRD is a suitable method to analyze the crystalline property of a material49. Figure 6 denoted the XRD pattern of R. intricata aqueous extract-mediated Fe3O4 NPs. Broad peaks at 2θ = 30.42°, 35.44°, 43.11°, 57.02°, and 63.15° resemble (220), (311), (400), (511) and (440) planes of magnetite Fe3O4 with an inverse cubic spinel structure (JCPDS file no. 89–2355)36.

Figure 6
figure 6

XRD spectra of R. intricata aqueous extract-mediated Fe3O4 NPs.

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The crystallite size was appraised to be 14.36 nm using Scherrer’s equation.

DLS analysis

Hydrodynamic particle size distribution was analyzed with a light scattering technique revealing that the average particle size of Fe3O4 NPs in colloidal dispersion is measured to be 84.6 ± 4.6 nm (Fig. 7) with a polydispersity index (PDI) of 0.479. It is observed that the size distribution of Fe3O4 NPs is much unvarying with a narrow distribution range36.

Figure 7
figure 7

DLS plot of R. intricata aqueous extract-mediated Fe3O4 NPs.

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Morphological properties of R. intricata mediated Fe3O4 NPs

SEM with EDX analysis

HRSEM analysis (Fig. 8a and b) confirmed the irregular shape with minor agglomerates with a rough surface. HRSEM coupled with EDX confirmed the presence of iron at 0.8, 6.3, and 6.9 keV in higher levels with minimum levels of carbon, chlorine, and sodium from the seaweed extract50. Peak at 0.5 keV signifies the occurrence of oxygen with a weight percent of 19.74% (Fig. 9a and b). The presence of iron and oxygen to maximum denotes the successful synthesis of Fe3O4 NPs.

Figure 8
figure 8

(a) and (b) HRSEM images of R. intricata aqueous extract-mediated Fe3O4 NPs.

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Figure 9
figure 9

(a) EDX spectrum and (b) Bar graph of elemental constituents of R. intricata aqueous extract-mediated Fe3O4 NPs.

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TEM with SAED analysis

HRTEM investigation proved the uniform size distribution, shape, and defects in the structure. HRTEM images (Fig. 10a and b), confirm the spherical-shaped NPs with a size of 125 nm and lattice fringe of 0.19 nm denoting the plane (400). The mean particle size distribution is 125 nm (Fig. 10c). SAED pattern (Fig. 10d) displays well-defined concentric bright circles aligned to the planes obtained from XRD data. Concentric bright circles consisting of small dots confirmed that the R. intricata-mediated Fe3O4 is highly crystalline. The existence of distinctive rings with tiny spots denoted the presence of planes conforming magnetite36,51.

Figure 10
figure 10

(a) TEM image and (b) HRTEM image (c) Particle size histogram and d) SAED pattern of R. intricata aqueous extract-mediated Fe3O4 NPs.

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Magnetic properties of R. intricata-mediated Fe3O4 NPs

VSM analysis

VSM hysteresis loop (Fig. 11) discloses that the synthesized material is superparamagnetic with saturation magnetization (Ms), remanent magnetization (Mr), and coercivity (Bc) values as 0.90007 emu/g, 0.17876 emu/g, and 335.72 G. Reduced magnetization (Ms) arises from spherical form, smaller size, exterior anisotropy, and disordered spins at NPs exterior layer37. The squareness ratio is estimated to be 0.1986. A squareness ratio less than 0.5 is a symbolic sign of small single-domain, randomly arranged spherical particles. Superparamagnetic nature aids in targeted drug delivery in cancer treatment36,37.

Figure 11
figure 11

VSM hysteresis curve of R. intricata aqueous extract mediated Fe3O4 NPs.

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Anticancer properties of R. intricata mediated Fe3O4 NPs

MTT assay

Cytotoxicity of R. intricata mediated Fe3O4 NPs studied against Hep3B and PANC1 human liver and pancreatic cancer cells exhibited dose-dependent effect. The impact of R. intricata-mediated Fe3O4 NPs on live cells is expressed (Figs. 12 and 13; Table 3). From linear regression, IC50 values are calculated as 311.7 µg/mL against Hep3B cancer cells and 460.5 µg/mL for PANC1 cancer cells. As the concentration of R. intricata-mediated Fe3O4 NPs upsurges, cell viability declines. Further, cytotoxicity is predominant against Hep3B liver cancer cells while related to PANC1 pancreatic cancer cells36 (Table 4).

Figure 12
figure 12

In vitro, cytotoxic activity of liver Hep3B cancer cells treated with various R. intricata aqueous extract concentrations mediated Fe3O4 NPs (Negative control = untreated cells; positive control = Doxorubicin 3.87 μg/mL). Results were stated as mean relative expression ± standard error of three replicates. Results were considered significant for ***P ≤ 0.001 and ns—non-significant.

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Figure 13
figure 13

In vitro cytotoxic activity of pancreatic PANC1 cancer cells treated with various R. intricata aqueous extract concentrations mediated Fe3O4 NPs (Negative control = untreated cells; positive control = Doxorubicin 8 μg/mL). Results were stated as mean relative expression ± standard error of three replicates. Results were considered significant for ***P ≤ 0.001 and ns—non-significant.

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Table 3 Statistical analysis of MTT cell proliferation assay results against Hep3B cancer cells.
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Table 4 Statistical analysis of MTT cell proliferation assay results against PANC1 cancer cells.
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% cell viability of PANC1 cancer cells

Cancer cell morphology analysis

Morphological changes of R. intricata mediated Fe3O4 NPs treated Hep3B and PANC1 cancer cells were visualized under a phase contrast microscope (Figs. 14 and 15). Control cells (untreated) without NPs displayed no alterations in structure with pure and monodispersed and fine matured cells (Figs. 14a and 15a). But the cancer cells treated with Rosenvingea intricata mediated Fe3O4 NPs (Figs. 14b–f and 15b–f) showed important morphological alterations such as asymmetrical morphology, engorged cells, and cell agglomeration thus confirming the reduction in the number of viable cells, detached cells and changed the morphology of Hep3B and PANC1 cancer cells37,38.

Figure 14
figure 14

Cell morphological analysis of different concentrations of R. intricata aqueous extract mediated Fe3O4 NPs {(a) 0 μg/mL, (b) 25 μg/mL, (c) 50 μg/mL, d) 100 μg/mL, (e) 250 μg/mL and (f) 500 μg/mL} against Hep3B human liver cancer cell line.

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Figure 15
figure 15

Cell morphological analysis of different concentrations of R. intricata aqueous extract mediated Fe3O4 NPs {(a) 0 μg/mL, (b) 25 μg/mL, (c) 50 μg/mL, (d) 100 μg/mL, (e) 250 μg/mL and (f) 500 μg/mL} against PANC1 human pancreatic cancer cell line.

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Morphological analysis of PANC1 cancer cells

Apoptotic analysis

Obtained results depicted exceptional apoptotic effect after 24 h treatment with R. intricata mediated Fe3O4 NPs. The yellow color cells designated the early apoptotic cells in treated Hep3B and PANC1 cancer cells and colorless cells visualized in control along with green color cells representing healthy cancer cells (Figs. 16 and 17). Apoptotic efficacy of R. intricata mediated Fe3O4 NPs in Hep3B liver cancer cells and PANC1 pancreatic cancer cells suggestively improved the cell mortality over apoptosis, not necrosis52. Normally, apoptosis is characterized by fluorescence intensity after the variations in structural features such as reduced or split chromatin with orange (early apoptosis) and red color (late apoptosis) fluorescence53. The above results support the benefit of R. intricata-mediated Fe3O4 NPs in nanobiology for improved cancer cure.

Figure 16
figure 16

Morphological changes analyzed by phase contrast microscope and apoptotic effect of R. intricata aqueous extract mediated Fe3O4 NPs (311.7 µg/mL) against Hep3B human liver cancer cell line.

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Figure 17
figure 17

Morphological changes analyzed by phase contrast microscope and apoptotic effect of R. intricata aqueous extract mediated Fe3O4 NPs (460.5 µg/mL) against PANC1 human pancreatic cancer cell line.

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Apoptotic effect on PANC1 cancer cells

From Figs. 16 and 17. it was clear that R. intricata mediated Fe3O4 NPs showed dose-dependent cytotoxicity against Hep3B and PANC1 cancer cells. Different properties like superparamagnetism (Ms = 0.90007 emu/g), smaller size (125 nm), spherical morphology, and surface adherence of biomolecules from R. intricata have enabled the cytotoxicity through targeted activity, and easy release of metal ions. Superparamagnetism aids targeted anticancer efficacy due to the magnetic field. This advantage makes them adaptable in targeted drug delivery with fewer side effects. VSM studies found that the R. intricata-mediated Fe3O4 NPs are superparamagnetic, augmenting the prospective utilization for targeted drug delivery without affecting the healthy cells26. Amidst different shapes of nanomaterials, spherical morphology has notable advantages like uniform surface functionalization, thus excess drugs can be efficiently loaded on the exterior portion of nanomaterials for better biological applications. It is also assumed that the total cell uptake in the drug release system is due to the smaller size of the nanomaterials. Smaller the size of nanomaterials better circulation in the human body with superior penetration via tiny capillaries. Functional groups adhered to the nanomaterials often enhance the biological properties with minimal toxicity to the human body. In the present research, the use of aqueous extract of R. intricata has endorsed dose-dependent cytotoxic effects owing to the biomolecules present in it36,37.

Mechanism of anticancer activity

Based on the review of literature, the hypothesis on the mechanism of anticancer activity by Fe3O4 NPs (Fig. 18), suggests that iron ions released generated reactive oxygen species (ROS) into the cancer cell which destructs the mitochondria through oxidative stress. Thereby, inhibiting the replication of DNA resulting in cell death. Nanomaterials with magnetic properties pierce into the nuclear membrane and cause damage to DNA. ROS at excess levels leads to hydrogen bond breakage in the structure of DNA36,37.

Figure 18
figure 18

Schematic depiction of the credible mechanism of anticancer activity of R. intricata-mediated Fe3O4 NPs.

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Mechanism of anticancer activity by R. intricata-mediated Fe3O4 NPs

Comparative studies. The anticancer efficacy of different NPs against various cancer cells is compared with previous reports as given in Table 5.

Table 5 Comparative analysis of anticancer efficacy of different NPs.
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Conclusion

This is the first report on the usage of marine brown seaweed R. intricata for the synthesis of Fe3O4 NPs. The use of ferric chloride hexahydrate and aqueous extract of R. intricata at moderate conditions produced Fe3O4 NPs. Herein, the direct optical band gap was estimated to be 2.30 eV from UV–Vis DRS analysis. From fluorescence studies, band edge emission is observed with a sharp peak at 660 nm. The biomolecules present in the aqueous extract of R. intricata played multiple roles as reducing and capping agents for the synthesis of Fe3O4 NPs which was proven from FTIR. Crystalline phases were confirmed from XRD with inverse cubic spinel structure. Hydrodynamic size distribution from DLS proved the smaller size of Fe3O4 NPs. Irregular morphology with slight aggregates was visualized from HRSEM. Iron and oxygen were present to be maximum from EDX analysis. Sphere-shaped with an average particle size of 125 nm and highly crystalline was witnessed from HRTEM and SAED techniques. The superparamagnetic nature with lesser saturation magnetization (0.90007 emu/g) was confirmed from VSM analysis. From these findings, it is concluded that Fe3O4 NPs with magneto-fluorescence properties overlay platform for fluorescent biosensor and magnetic hyperthermia applications in the future. Owed to the inimitable physicochemical properties of Fe3O4 NPs, this green synthesized nanomaterial has latent applications in the arena of environmental remediation and biomedicine for catalysis, pollutant detection, cell imaging, and hyperthermia treatment. A further advantage is that the superparamagnetic behavior of Fe3O4 NPs aids in the easy retrieval of catalyst from reaction solution with an exterior magnet. Likewise, in the field of biomedicine, this property enables targeted drug delivery. R. intricata-mediated Fe3O4 NPs augmented the anticancer efficiency against Hep3B and PANC1 cancer cells with IC50 values 311.7 and 460.5 µg/mL, correspondingly. Cell morphological analysis and dual staining assay revealed the characteristic apoptotic changes denoting cell death. These findings endorse that the synthesized Fe3O4 NPs can be an appropriate alternative for the treatment of human liver and pancreatic cancer. Also, the findings can be converted into technology with suitable testing and more trials in other human cell lines and testing in animal models.