Hydrothermal synthesis, characterization and enhanced photocatalytic activity and toxicity studies of a rhombohedral Fe2O3 nanomaterial

The present investigation focuses on the synthesis of metal oxide nanoparticles (MONPs) via a facile hydrothermal route. The material has been characterized by using X-ray diffractometry (XRD), X-ray fluorescence (XRF), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), high resolution transmission electron microscopy (HR-TEM), energy dispersive spectroscopy (EDS), photoluminescence (PL), atomic force microscopy (AFM) and Brunauer–Emmett–Teller (BET) techniques. However, the application of Fe2O3 metal oxide nanoparticles (MONPs) tied with their inimitable chemical and physical nature is thought to emphasize their exploitable medical and biological applications nowadays. Rhodamine-B (RB) was used for photocatalytic degradation studies by using rhombohedral Fe2O3, afterwards the material was recycled and utilized for toxicity assessments. Undeniably, a meticulous assessment is needed of the factors that influence the biocompatibility and is essential for the safe and sustainable development of the emerging chemically synthesized metal oxide nanoparticle (MONPs). The toxicity assessment of Fe2O3 is necessary to know the bioaccumulation and local or systemic toxicity associated to them. The aim of the present study is to investigate the effects of Fe2O3 and its histological alterations of the heart tissue of albino Wistar rat. The synthesized materials high dose was found to be highly stable and we found more toxicity against the skin melanoma cells (B16-F10), human embryonic kidney (HEK), 293 cells depending on dose. Finally, Escherichia coli, (MTCC 7410) bacterial cell wall damage studies were also conducted to provide a clear determination of rhombohedral nanomaterial behaviour. The fusion of these biocompatibility investigations paves a way for further applications in utilization of these materials in future eco-friendly applications.


Introduction
There is a growing interest about the strong associations between exposure to nanoparticles and adverse health effects in humans. 1 For this strong reason, environmental contamination by nanoparticles is attracting considerable and increasing global concern. 2-5 The behaviour and toxicity of particles mainly comes from studies on orally administrated MONPs. 6,7 (MONPs) may differ in reactivity and solubility and may interact with all kinds of endogenous proteins, lipids, polysaccharides and cells and series of tests were anticipated for systematic evaluation of the toxicity of (MONPs) used in drug delivery systems. 8,9 Medical applications of Fe 2 O 3 metal oxide nanoparticle (MONPs) are wider than others due to biocompatibility nature, high stability and ease of use and have a more applications in drug delivery. 10,11 Metal oxide photocatalysis is an enthralling application tool for solar energy conversion and dye degradation due to its prominent nature. 12 Because of widespread application of these particles in various industries human exposition to them increased, so investigation of this nanoparticle role in cell growth and survival has more importance. 13 Iron oxide is the most stable under ambient conditions to perform various applications with environment friendly label with a band gap of 2.1 eV with tuneable nature, [14][15][16] there are many methods used for the synthesis of Fe 2 O 3 metal oxide nanoparticle (MONPs) like sol-gel method, 17 hydrothermal and solvothermal synthesis, 14 thermal breakdown, 18 and sonochemical synthesis, 19 have been used for synthesis of Fe 2 O 3 with different morphologies and concerned nanostructures. Human skin, heart, lungs and digestive system are the most common entry routes for nanoparticles and its pathogenicity. 20 The airborne nanoparticles have high mobility and can be inhaled into the respiratory system easily. 21 One of the most common damaging effects of nanoparticles are rise of reactive oxygen species (ROS) that refer to oxidative stress in human and animal tissues. It's conrmed that almost all of the studied nanoparticle produce reactive oxygen species and it is the main mechanism for nanoparticle toxicity that can lead to inammation and apoptosis. 22 The unique properties and interactions between nanomaterials with biological system are essential. 23, 24 Cell line toxicity of human cell lines by using several synthesized metal oxides are more attractive in understanding the relationship between nanoparticle-cell interaction as well as in biocompatibility studies. 25,26 Furthermore, biocompatibility must be conducted with particular focus on the environment in which the nanomaterial will be placed in. 27 Size, shape, and morphology of nanoparticles, play a vital role in mammalian cell culture medium and the nanoparticle are much more toxic than the hydrothermally synthesized nanocomposite semiconductor. 28 The drug delivery, evaluation of metal oxide nanomaterial biocompatibility is essential to secure drug release with low cytotoxicity. 29,30 The biocompatibility evaluation of structured nanoparticles for drug delivery applications has been expanded from being primarily investigated in a laboratory setting to being applied in the multi-billion-dollar pharmaceutical industry. [31][32][33][34] The rhombohedral Fe 2 O 3 (MONPs) are effectively utilized for degradation of rhodamine-B (RB-B) degradation process nanomaterial was successfully recycled and utilized for toxicity assessment potentials. Aer the degradation studies, it is a researcher duty to assess the toxicity of material before releasing to environmental systems, in the point of view, we have successfully conducted the in vivo consequences of oral administration and histological characterization of the heart tissues due to Fe 2 O 3 (MONPs) has not been documented and identied before and attempt has been made to characterize the possible histological alterations in the heart tissues aer oral administration of (MONPs). Additionally, we have focused on exploiting and utilizing Fe 2 O 3 for biocompatibility studies of heart tissue of albino Wistar rat and melanoma cells (B16-F10), human embryonic kidney (HEK), 293 cells, and Escherichia coli, (MTCC 7410) bacterial cell wall damage studies were conducted to synthesized material action (Fig. 1).

Hydrothermal preparation of Fe 2 O 3
The rhombohedral Fe 2 O 3 were synthesized by microwave assisted hydrothermal method with slight modication in our earlier report, 25 to get the perfect rhombohedral geometry, in the point of assessment of toxicity in various biological experiments. FeCl 3 and NaOH, Ammonia (Sigma Aldrich, India) 10.14 g (37.5 mol) FeCl 3 $6H 2 O and 7.45 g (37.5 mmol) FeCl 2 -$4H 2 O were dissolved into 25 ml of H 2 O. 25 ml of 30% liquid ammonia was slowly added to the salt solution under stirring condition at 1000 rpm for 5 minutes, aer 20 ml of combination was put into a Teon-lined stainless Morey autoclave, and the autoclave be heated to 180 C in an oven and maintained at 10 hour reaction time. Autoclave was naturally cooled to room temperature; the end products were dried under conventional microwave unit at 60 C and material is ready for characterization.

Photocatalytic experiments of degradation of dyes
The photocatalytic behaviour of the synthesized rhombohedral Fe 2 O 3 were evaluated by the degradation of rhodamine-B (RB) dyes with slight modication to increase the photocatalytic activity. The experiment performed and the catalytic reading taken with the help of Heber-immersion type photoreactor. 35 In this experiment a specically weighted amount of photocatalysts was systematically added in the ask with adjusting pH with NaOH and HCl in the similar type of ask at pH 2 to 7. The ask was sited under Heber-immersion type photoreactor, by steady shaking by magnetic stirrer. The experimental trials were performed with an initial rhodamine-B (RB-B) concentration of 9 mM, catalyst concentration of 0.75 g L À1 .

Animal and treatment studies
An adult male Wistar rats (weight nearly about 130-160 g) were systematically used for the study. The animals were kept in inox steel cages and maintained in a 22 AE 2 C, 50-80% relative humidity area with 12 hour light/dark pulse. The rats were maintained on a commercial pellet diet and allowed to access deionized water and libitum. All animals were cared for according to the standards of the Guide for the Care and Use of Laboratory Animals. Body weight, water intake, and dietary consumption were monitored daily. Aer one-week acclimation, the rats were randomly divided into two groups: a control group (n ¼ 10), a Fe 2 O 3 (MONPs) treated group (n ¼ 10). Oral administrations of Fe 2 O 3 nanoparticle were given at dose of 20 mg kg À1 respectively, 36,37 the control rat treated with physiological saline. Aer 3, 6, 12, 24 and 36 hours treated animals were sacriced by cervical dislocation. From each animal's heart tissue was studied, the rat tissue was preserved in Bouin's xative and tissues blocks were embedded with paraffin and sliced into 5 mm thick section, then stained using haematoxylin and eosin.  The B16-F10 cell culture dose-dependent response curve was plotted using propagation and MTT [(2-(3,5-diphenyltetrazol-2ium-2-yl)-4,5-dimethyl-1,3-thiazole bromide)] assay, aer 72 hours of exposure was determined according to, 41,42 HEK-293 (human embryonic kidney), cell lines were gotten from the American Type Culture Collection (ACC), USA. The methodology involved in HEK-293 cell line IC 50 assessment, parallelly the test Fe 2 O 3 nanomaterial inhibits the 50% of the proliferating the normal cells as per above environmental conditions for 48 hours. 43,44 This procedure is non-hazardous, uses a thermally stable reagent and shows good correlation with constant proportion. 45 The experiments were performed in triplicate and three independent repetitions.

Characterizations and measurements
The structure and composition of Fe 2 O 3 (MONPs) were characterized by a various tools; Brunauer-Emmett-Teller (BET) specic surface area, pore volume, and pore size distribution of the samples were determined by N 2 sorption at 77 K using a Micromeritics ASAP-2020, Ultima-III Series, RIGAKU, TSX System, Japan, was used to assess the crystallinity with wideangle X-ray diffraction (XRD) patterns, high resolution transmission electron microscopic (HR-TEM) images of Fe 2 O 3 (MONPs) was captured using Jeol/JEM 2100, LaB 6 , 80-2000 mm with 200 kV acceleration voltages. The tissue section examined with the help of Carl Zeiss, Axio image A 2 M, advanced polarizing microscope, USA. The Energy Dispersive Spectroscopy (EDS) analysis was carried out using HITACHI (Noran System 7, USA) system attached to the Zeiss Supra 55VP, FE-SEM is an extremely soaring resolution eld emission scanning electron microscope for the detection of composite nanoparticles. Fourier-transformed infrared (FT-IR) spectra were obtained on a Thermo Scientic Nicolet, 6700 Analytical FT-IR spectrometers. Zeta potential measurements of the attenuate dispersions (0.1 mg mL À1 ) of the nanocomposites were conducted using a Brookhaven Nano-Brook Omni Instrument at 25 C. The particle size distribution (PDS) nanocomposite was monitored by using Microtrac (USA) particle size analyser. The analyser provides the size measurement, which conrms the particle size distribution. The measurement of the specic surface area of materials by using Brunauer-Emmett-Teller (BET), BEL:2 SORP, Japan and atomic force microscopy (AFM), APE Research, AFM, 100, ITALY used to determine the resolution on the order of fractions of a nanometre of a synthesized material. The photoluminescence results were taken from photoluminescence spectrometer, FLS-1000, the identication of rhodamine-B (RB) degradation intermediate products were measured by a liquid chromatography-mass spectrometer (LC-MS) model, (Waters-USA, Synapt G-2 HDMS). Microscopic with camera Olympus, (USA, 2000) used to take the photograph of HEK, 293 control and effected cell lines. The responding data replicates (three) were analysed for every attempt and for every analysis of discrepancy (ANOVA) using SPSS-Inc. 22.0. Trivial effects of treatments were resolved by F values (p # 0.05).

Characterization results of synthesized rhombohedral Fe 2 O 3 nanomaterial
The X-ray uorescence is recorded with ne focus X-ray tube, MO target of multi-layer monochromator of 17.5 keV. 46 The XRF spectra of Fe 2 O 3 clearly indicate that the presence of Fe 3+ . XRF pattern of Fe 2 O 3 nanomaterial are shown in Fig. 2. The diffraction peaks in 6.2 to 7.2 keV and the concentration of the compound of Fe-KA is 153 256.1 cps are perfectly aligned to the rhombohedral phase in Fe 2 O 3 .
In Fig. 3, shows XRD patterns of the samples obtained at 180 C in 10 hour hydrothermal reaction. According to the gure, it clearly shows that, the position of the diffraction peaks is in a good argument with those of rhombohedral Fe 2 O 3 The properties of Nano-structured materials habitually depend on their morphology and size. Table S1 in ESI † shows FT-IR spectrums of Fe 2 O 3 nanoparticles in the range 400-4000 cm À1 , nanoparticles show the spectral absorption in 3420, 3192, 1603, 1388, 1114, and 454 cm À1 . The absorption peaks at 3420 and 1603 cm À1 can be assigned to the stretching vibration and bending vibration of OH groups of H 2 O. 49 The bands at 1114 and 454 cm À1 can be attributed to the Fe-O stretching vibration modes in Fe 2 O 3 . This FTIR results indicate the formation of Fe 2 O 3 in the present outcome, which are reliably matched with XRD patterns. It is evident that, the FTIR peaks of Fe 2 O 3 nanoparticles have higher frequency to compare with already reported morphologies. 50,51 High resolution-TEM of synthesized rhombohedral shaped Fe 2 O 3 nanoparticles was shown in Fig. 4; from the images it evidently showed that, the high-quality geometry, in addition, the material results in the formation of mesopores between the particles (Fig. S6 in ESI †), which clearly conrmed by BET surface area and pore volume measurements. The size of both the individual nanoparticles was between 10 to 20 nm which are in concordant with the results of XRD, DLS and also FT-IR, and those results are in a good argument with Debye-Scherer formula in the above stated results. It was also observed that, the d-spacing measurements of twin domains were measured to be approximately 0.2Å to 0.4Å nm and corresponding to (110) to (220) planes of Fe 2 O 3 , respectively, the interplanar spacing of $0.25 nm agrees well with the spacing between (110) planes of rhombohedral iron oxide crystals (0.25 nm) and SAED patterns are the good proof for compact arrangement of the nanoparticles.
Energy dispersive spectroscopy (EDS) analysis conrmed the phase transparency of the Fe 2 O 3 as shown in Fig. 5. The characteristic peaks of Fe and O appear in the spectrum of Fe 2 O 3 and conrming its successive formation and purity (Table S1 and Fig. S7 in ESI †). The peak of (C) carbon seems due to the carbon tape used sample container in the energy dispersive unit. The particles histogram using DLS of Fe 2 O 3 , distribution of particle sizes when immersed in the solvent have a range from 10-20 nm. Zeta potential measurement is the reliable method for assessing material interaction with other biological system. The particles examined presently possessed a zeta potential for Fe 2 O 3 as -13.9 mV (Fig. S6 and S7 in ESI †), indicating safe to environmental as well as biological utilization.
The surface area is an important tool to determine the biological adoptability of nanoparticles and nanomaterial with a high surface area is easily attracting the biological material at   Paper the faster rate due to his inert nature. The superior specic surface area provides more surface-active sites and wellorganized transport lanes to reactant molecules and products in environment as well in biological systems. BET surface area as a function of pore volume and the size of the synthesized samples present in similar (type-II). 10,52,53 The BET surface areas reading of Fe 2 O 3 is 5.676 m 2 g À1 (Fig. 6 and Table S2 in ESI †). According to the hysteresis loop in the relative pressure region approximately 0.4-0.9, the nitrogen adsorption/desorption isotherms showed that the Fe 2 O 3 exhibited a similar (type-IV) curve and it is in nanoporous structure, with an increase in the surface area and decrease in the pore size, as well as the large total pore volume, of Fe 2 O 3 is expected to have a high biological hold up.
UV-Vis diffuse reectance spectra (DRS) and band-gap energy of Fe 2 O 3 are shown in Fig. 7. DRS were used to investigate the light-harvesting nature of the Fe 2 O 3 photocatalyst, but in this study we are effectively adopting to determine the material environment for biological susceptibility. Conduction-band minimum (CBM) and valence-band maximum (VBM) is play a vital understanding to mechanism absorption on biological materials. To investigate the CBM and VBM of the Fe 2 O 3 . UV-DRS spectra were used to record the spectrum. The associated band-gap values were calculated using the equation in ref. 54. Which are consistent with the similar results obtained from the related work. 55,56 Evidently calculated band-gaps of rhombohedral Fe 2 O 3 were found to be 1.96, this reading is important, because of nanomaterial can be photo excited to generate more electron-hole pair under visible-light irradiation, it's results have higher catalytic performance. This mechanism leads to more impact on natal material effect (Table S3 in ESI †).
PL spectra of Fe 2 O 3 are evidently shown in Fig. 8. The PL emission is light source emission from any form of matter aer the absorption of photons. The peaks are observed at 416 nm and 440 nm. In general photoluminescence positive energy can be viewed as an indication of electron populated an exited state associated with transition energy. [56][57][58] The true atoms are the similar systems, correlations and many more multifaceted trends and also act as starting place for photoluminescence in catalytic and also many body systems.
The atomic force microscopy (AFM) image shows synthesized material in a high quality, it is essential to execute corrections of the sample slope and background. 59,60 Apropos the instabilities occurred during scanning, especially in the ranges under 4 mm, the AFM used to assess the particle size and morphology and thus to explain the colour difference. The iron oxide nanoparticles with the size of 10-20 nm and with the morphology corresponding to very thin rhombohedral geometric structures, particles of metal oxide nanomaterials prepared by hydrothermal route method, just the different values of vertical dimensions, which are accountable for dissimilar colour excellence, cannot be effortlessly found by using other techniques together with HR-TEM.
Surface roughness (R a ) value, the R a value indicates average of a set of individual measurements of a surface's peaks or valleys. The R a value for rhombohedral Fe 2 O 3 was 27.2 nm that shows the evenness of nanomaterial (Fig. 9). Due to rough surface texture of synthesized nanoparticle, there was well   absorption in heart muscles caused inammation like increased neutrophil count was observed in this study.
In the photocatalytic activity of nanomaterials effect of pH play a vital role. 61 The forethought of rhodamine-B (RB-B) (Fig. S9 in ESI †) and catalyst dosage were xed at 9 mM rhodamine-B (RB-B) and 0.75 g L À1 (rhombohedral Fe 2 O 3 ), respectively. The outcome of pH on the photodegradation of rhodamine-B (RB-B) is revealed in Fig. S2 in ESI †. pH zero-point charge (zpc), the external play an important role in dye degradation and mineralization studies. 62,63 The zpc values of rhombohedral Fe 2 O 3 is 7.4 respectively, this clearly below pH the surface of material is positively charge and dye was simultaneously degraded and nontoxic small molecular intermediates was obtained (  Fig. S3 in ESI †. The Langmuir-Hinshelwood model. 12,64 was efficiently used to gure out the degradation kinetics of rhombohedral Fe 2 O 3 photodegradation. Fig. 10 explains the apparent logarithmic plot of rhodamine-B (RB-B) concentration as a utility of irradiation time. The pseudo-rst-order rate kinetics explains Fe 2 O 3 is 2.65 Â 10 À2 s À1 is appreciably higher than of TiO 2 (5.12 Â 10 À3 s À1 ) and CuO 2 (7.38 Â 10 À3 s À1 ). For this reason, the activity of the Fe 2 O 3 rhombohedral material is about 2.7 percent superior than that of other examined material and the ZnO (2.01 Â 10 À3 S À1 ) shows the triing photocatalytic activity to compare to other efficient materials in various studies so far.
The photodegradation intermediates in the reaction process were help to predict the actual reaction process. 65,66 The tiny molecules during mineralization process are responsible for CO 2 and H 2 O, the COD and TOC (Table S4 in ESI †), results evident to photodegradation process (Fig. 11). The two spirited actions have occurred at the same time during the photoreaction: N-deethylation and destruction of dye chromophore structure, 67 The negative correlation was observed in COD and TOC in respective time consider for the study of degradation ( Fig. S9 and S10 in ESI †). Interpermeates of photocatalytic degradation of rhodamine-B (RB) were performed using LC-MS and results were hypothesized in ESI (Table S7 in ESI †).
The recovery of a photocatalytic nanomaterial is the most important for eco-friendly utilization of product. 68,69 In order to examine the constancy and stability of the Fe 2 O 3 nanomaterial is essential aer the photodegradation of rhodamine-B (RB-B). The photodegradation proportion of ve consecutive cycles is 76.23%, 73.78%, 72.56%, 71.09% and 71.43%, respectively, and showed in Fig. 12.   The rhombohedral Fe 2 O 3 nanomaterial exhibits an average of 73.18% of solar driven activity aer ve successive cycles. In addition, there is slight change observed in the XRD pattern of Fe 2 O 3 , due to absorption of small molecular intermediates organic material present in dye (Fig. 13), aer ve cycles.
The systematic hydrogen production by photo-induced water break-down was more captivating and give a suitable solution for energy conversion and conversional problems. 65,70 H 2 generation methods and photocatalytic water excruciating using visible light spectrum with low cost and more sustainability features in the photo reaction system are more popular. This radical piercing process makes a crucial role in the degradation of dyes rhodamine-B (RB-B) concentration. In our experiment, we are proposed the photo-reaction efficiency of the adsorption of untreated organic dye on the by rhombohedral Fe 2 O 3 and the progression of splitting photo-twisted electron-hole pairs. The holes can either react through rhombohedral pore surface, and form hydroxyl to hydroxy radicals. Consequently, the degradation was occurred to allowing dye molecules to move from solitary explication to the material interface and to subsequently carbon dioxide (CO 2 ), water (H 2 O) and other small molecular intermediates are the byproducts of redox reactions as clearly showed in Fig. 14.

Biological toxicity results of synthesized rhombohedral Fe 2 O 3 nanomaterial
The epidemiological studies have clearly recommended that, health effects of nano and micro particles dependent one and each other. 71,72 An earlier toxicity studies showed that nanoparticle was made specic acute effects while micro particles induced chronic effects. [73][74][75] In this study, our study explains the environmental and health concern by using biological specimens mimic oral exposure. We found that structured Fe 2 O 3 nanoparticles entered the body via oral administration; they became systemically and cause toxic effects in cardiovascular system. These actions were measured to be the essential mechanisms of organ damage. The metal oxide nanoparticles (MONPs), due to his high calibre in their size, they are probably transported from the oral way into body circulation, and reach cardiovascular system and made remarkable hazard. Metal oxide nanoparticles (MONP's) by products from industrial as well as biomedical output will in aquatic systems. However, some studies have shown that the solubility of metal oxide nanoparticles in biological liquids is in addition one of the factor that, determines legatee velocity of penetration into blood streams, 76 especially Fe 2 O 3 nanoparticles has been shown to dissolve in normal environment and easily get in to living systems. [77][78][79] Simultaneously, musculus skin   melanoma cells (B16-F10), human embryonic kidney (HEK), 293 cells, and Escherichia coli, (MTCC 7410) bacterial cell wall impairment also carryout (Fig. 15).
The result of anti-bacterial activity showed that, there exists a signicant zone of inhibition against test pathogens, many studies have proved that the photo-generated holes and $OH are the main oxidative agents for Escherichia coli suppression. It should be noted that, the test samples, due to its large surface area Fe 2 O 3 nanoparticle, Escherichia coli, (MTCC 7410) were used for study with different concentrations of material were added to the disc plate containing a nutrient agar media (NA), yields the maximum inhibition zones 20.60 AE 0.24 at 1.0 mg, followed by 0.6 mg is 18.35 AE 0.08, and 0.3 mg was 15.81 AE 0.15 (Table S5 in ESI †), respectively (Fig. 16). We did not observe any zone of inhibition in SDW (Sterile Distilled Water) diffused discs against test pathogens. This revise clearly suggests that, Fe 2 O 3 material inhibit the E. Coli (MTCC 7410) bacterial pathogens by rupturing the outer and inner wall of the cell which leads to toxic and damage the cell membrane. The uorescent microscopic images (Fig. 17) are agreed the clear evident of bacterial death.
In Fig. 18a, normal rat demonstrating normal heart muscle, the rhombohedral Fe 2 O 3 treated rat received 20 mg kg À1 of 10 nm particles for 0 to 6 hours ( Fig. 18b and c) demonstrating benign normal looking heart muscle with normal muscle direction and fascicles with no pathological effects. The metal oxide nanoparticles (MONP's) for 6 to 12 hours (Fig. 18d and e), demonstrating spread in the extravasations of red blood cells,   Paper packed dilated blood vessels, prominent focus of tiny lymphocytic inltrate associated with focus of muscle hyalinosis, troubled muscle fascicles and scattered red blood cells. In the view of 24 to 36 hours (Fig. 18f), demonstrating dense prominent focuses of inammatory cells inltrate of minute lymphocytes and few plasma cells and prominent congested dilated, blood vessels and few scattered extravasation red blood cells. Histological studies induced in the heart tissue exposure could be an indication of congested cardiovascular system with extravasations, red blood cells and vessels, heart muscle hyalinises, troubled muscle fascicles, inammatory cells inltrate by small lymphocytes and plasma cells, due to higher dose Fe 2 O 3 toxicity. The nano residue accumulated heart muscle leads to metabolic and structural changes. The increased neutrophil counts have been indicated in the red coloured square boxes in the respective images.
The projecting anti-proliferative outcome of functionalized Fe 2 O 3 on HEK-293 as exposed by its IC 50 built on XTT assay was instigate to be 202.14 AE 0.14 equated with the standard drug cisplatin >500 (Table S6 and Fig. S8 in ESI †). Consequently, the hydrothermally synthesized material is the promising aspirant for hopeful drug to anti-cancer with its toxicity. However, the synthesis methodology gives a nontoxic nature to rhombohedral Fe 2 O 3 . The plasma semi-penetrable membrane allows freely dissemination of tiny and non-polar molecules available in the synthesized material were uptake the endocytosis. Most of chemically worked Fe 2 O 3 are easily taken up by the cells through endocytic mechanisms, and persist in endosome vesicles, and become incapable of reaching the cytosol system. The prociency mainly depends on nanomaterial size, shape, physical, chemical and its dispersive nature. Finally, the hydrothermally synthesized rhombohedral metal oxides can be used effectively at a very low concentration against different types of cell lines for the drug delivery procedures and also, pharmaceutical applications. The efficient permeability effects (EPR) of nanoparticle play an vital role with a solution for frequent toxicity assessment strategies through cell line, the targeting efficiency via a wide spectrum of nanomaterial based on various engineering methodologies, [80][81][82][83] up till now, ample of immunotherapy strategies against cell line toxicity have undergone extensive development and clinical testing, from. 84,85 HEK-293 cells have been extensively utilized in biological research for many years, for the reason of consistent development and penchant. The therapeutic effect of human embryonic kidney (HEK), 293 cell line efficacies by rhombohedral Fe 2 O 3 in vivo toxicity effect were inoculated in the right ank of mice and applied treatments 7 days later. The cell line volume was recorded in the following two systematic weeks. Fe 2 O 3 exhibited the most excellent on cell line growth with was compared with free drugs loaded vessel systematically (Fig. 19a). Obviously, there is effect in the photograph of the normal cell line, the staining pictures of cell line sections from each cluster were dependable with the results like incompact cells, shrunk nuclei and damaged morphology were observed in both (Fig. 19b and c), which reected more advanced cell apoptosis was induced by Fe 2 O 3 , aer systemic administration. We deem the conception of this toxicity combined approach could also be applicable for other proper anti-cancer drugs and immune-stimulating agents to rescue the liposomes in future.  The cell viability is the major biocompatibility tool now a days. [86][87][88][89] We found out the toxicity of the Fe 2 O 3 using musculus skin melanoma cells using numerous concentrations (5-500 g mL À1 ). The results clearly suggested that the viability of B16-F10 cells have decreased in a dose-dependent manner in each of the sample. Fig. 20 clearly showed that, 5 to 50 g mL À1 concentration, the lowest concentration of three respective samples used in the assay did not reduce the cell viability noticeably as compared to other increased concentrations, therefore, Fe 2 O 3 in lower dose is the safest and the superlative alternative in terms of toxicity in biological application.

Conclusions
In this experimental work, a unique a rhombohedral Fe 2 O 3 has been prepared by a simple, eco-friendly and low-cost hydrothermal method for multi-purpose application like rhodamine-B (RB-B) and biological toxicity testing by using cardiovascular muscle of rats, human embryonic kidney (HEK), 293 cells, viability cell testing in melanoma cells (B16-F10) and Escherichia coli, (MTCC 7410). The lower doses of synthesized material were found to be highly stable, low toxic in nature, in the case of increasing the dose level rhombohedral Fe 2 O 3 was cause the hazardable toxicity to the various experimental models. Our material gives excellent photocatalytic activity and less toxicity to compare with single, bi and tri-composite nanomaterials. This hydrothermal method synthesis and exposure to biological models tells fate of safe utilization material and the results are hopeful can be utilized these materials to biomedical eld and environmental conversion systems.

Live subject statement
The animal welfare and the experimental procedures were carried out in accordance with the regulations for the administration of affairs concerning experimental animals, and the Ethical Regulations on the care and use of Laboratory animals from University of Mysore, Karnataka, India, approved by university committee for animal experiments.

Conflicts of interest
The authors declare no competing nancial interest.