Preparation of α-Tocopherol based nanoemulsion for efficacious delivery of Methotrexate

Abstract The present work dwells upon developing and assessing the efficacy of nanoemulsion (NEm) for Methotrexate (MTX) delivery. Herein, MTX-loaded NEm was prepared using the ultrasonic nano emulsification method by exploiting biocompatible excipients such as α-Tocopherol, Miglyol 812, Tween 80, and ethanol. Employing ultrasonic nano emulsification processes leads to the preparation of NEm with smaller droplet sizes and uniform distribution. The physicochemical characterization by transmission electron microscopy confirmed that the blank NEm and MTX-loaded NEm possesses spherical-shaped nano range particles and drug might be present within the droplet or on the surface of the nanoemulsion. The encapsulation efficiency of MTX in NEm was estimated as 52 ± 0.6%. The recorded cumulative in-vitro release was 54.74 ± 2.73% in 24 h and using kinetic modeling, its Fickian diffusion release was validated. Higher stability (94.16 ± 1.51%) and bioaccessibility (96.06 ± 1.01%) of MTX-loaded NEm were also confirmed. The hemolysis profile was under the tolerance limit (<8%) for all the concentrations of the formulation. The cell viability assay of NEm loaded with MTX showed significant toxicity against the MCF-7 and HeLa cell lines which validated MTX-loaded NEm as a biosafety vehicle. Graphical Abstract


Introduction
The development of numerous novel therapeutic systems has gained unprecedented attention for the effective delivery of therapeutics. The main focus has been on the drug delivery vehicles that can delay the release of the drug, minimize the daily dosage, improve the effectiveness, and reduce the drug's side effects after its administration. [1] However, a large number of delivery vehicles such as metallic (silver and gold) and metal oxide (TiO 2, ZnO and CuO) nanoparticles meet with failure at a very advanced stage of drug development owing to their poor bioavailability, increased toxicity, and less stability. [1,2] Both silver and gold nanoparticles have some disadvantages, in particular the possibility of releasing the metal ions to the environment, which affects their cytotoxicity. [2] The toxicity of the metal oxide nanoparticles may be attributed to the oxidative stress exerted by the photocatalysis of metal oxide nanoparticles. [2] In recent times, monodisperse systems like nanoemulsion (NEm) have come forth as a respite that offered improved therapeutic effect, good tolerability, higher bioavailability, and enhanced targeting of drugs. NEm is a kinetically stable mixture of two immiscible liquids having a droplet size of 100 nm that offers numerous advantages like increased rate of adsorption, protection from the environment, and enhanced lipophilic drug delivery after solubilization over other dosages. It is a non-equilibrium system that requires some input of energy in the form of agitation to assist the emulsification process by reducing the interfacial tension and droplet size. Several techniques such as high-speed homogenization and high energy agitations are preferred for NEm preparation. However, utilization of these techniques requires high energy and does not regulate the size and stability of NEm. While, the method of ultra-sonication is found to be an efficient technique for NEm preparation being simple, eco-friendly, and capable of regulating the size and stability of the NEm. [3] There are several reports [4] wherein, ultrasound-assisted preparation of NEm has proven to be a better candidate for the encapsulation and delivery of bioactive agents. In some recent reports [5,6] such formulation has been used in the delivery of several anticancer drugs like doxorubicin and paclitaxel.
Methotrexate (MTX), a folate antimetabolite drug, is commonly employed for cancer treatment. The conventional therapy of MTX is challenged by multiple drug resistance properties that hinder its therapeutic efficiency. [7] The clinical efficiency of the MTX is compromised by its low bioavailability, short half-life span, and its side effects. Rajitha and coworkers [8] prepared MTX loaded chaulmoogra oilbased NEm of 34.52 ± 2.05 nm size by emulsification technique and ex-vivo skin permeation studies were performed by employing porcine skin. The results showed the enhanced skin permeation and retention of drugs in the deep skin layers in comparison to the control drug solution. The developed NEm formulation was found to be cytocompatible toward the L929 cell line and was stable for 3 months. However, MTX release from chaulmoogra oilbased NEm was slow and in a controlled manner, but it was not a sustained release. NEm system can itself behave as an antioxidant agent and might display symbiotic action with other anticancerous molecules utilized in drug delivery. Therefore, the development of a better formulation that offers higher solubility, increased bioavailability, and sustained release of anticancer drugs stays in demand in the era of nanomedicine research.
In the present report, we focused on the preparation and assessment of NEm for the delivery of MTX. NEm was formulated by the ultrasonic nano emulsification method using GRAS excipients such as a-Tocopherol and Miglyol 812 (oil phase), Tween 80 (surfactant), and ethanol (co-surfactant). Physicochemical aspects were investigated using dynamic light scattering, transmission electron microscopy (TEM), zeta potential, viscosity, refractive index, and surface tension techniques. Furthermore, the interaction of MTX with the optimized NEm was studied using Fourier-transform infrared spectroscopy (FTIR). The encapsulation efficiency (EE) and in-vitro release were also determined for the NEm loaded with MTX (NEm-MTX). In-vitro cell viability test on Hela and MCF-7 cell lines was carried out to study the cytotoxicity of MTX, NEm, and NEm-MTX formulations.
Overall, a NEm formulation is developed which is hemocompatible and can be employed in MTX delivery.

Solubility study of MTX in different oils
The solubility of MTX in different oils (clove oil, a-Tocopherol, corn oil, lemon oil, Miglyol 812, and in a mixture of a-Tocopherol and Miglyol 812) was carried out by the shake flask method. MTX (in excess amount) was added into 1.5 mL of each oil and shaken at 100 rpm maintaining 25 C temperature for 24 h. [9] Further, the sample was centrifuged at 9391 RCF for 10 min to remove the undissolved drug. Thereafter, after the collection of supernatants, the concentration of MTX was measured at 303 nm wavelength using UV-vis spectroscopy. [8]

Preparation of NEm
The ultrasonic nano emulsification method was employed for the preparation of o/w NEm. [10] In brief, the oil phase (a Tocopherol: Miglyol 812 ¼ 1) and surfactant mixture (Ethanol/Tween 80 ¼ 0.3) was mixed at 65 C by stirring the mixture at 1000 rpm for 15 min. Later, the dropwise addition of water was done to the oil phase with continuous stirring at 1000 rpm. The components were mixed in 96:1:3 w/w % of water: oil: surfactant, respectively. The formed mixture was stirred at 40 C for 30 min and thereafter, ultra-sonicated for 5 min. The effect of the duration of the sonication time on the average droplet size of NEm was also studied in different time frames. Further, the effect of variation of concentration of oil phase (a -Tocopherol and Miglyol 812), while keeping the concentration of the surfactant fixed was carried out. Finally, the prepared NEm was stored at 25 C and utilized for further characterization. For the preparation of the NEm-MTX, MTX (0.7 mg/mL) was added in the oil phase.

Preliminary physicochemical characterization
The hydrodynamic diameter of the prepared formulation was analyzed using the Malvern Zeta Nano S90. Before measurements, the formulation was filtered using a 0.22 lm membrane PTFE filter. Thereafter, a disposable cuvette having 2 mL of the formulation was exposed to laser light diffraction to measure droplet size. Zeta potential of the NEm and NEm-MTX was measured using the Malvern Zeta Nano S 90 at room temperature. The optical clarity of the prepared formulations was examined using Abbemat 500 refractometer (Anton Paar). The surface tension was measured using the Kruss K20 tensiometer and the viscosity of the samples was determined using Brookfield DV-II þ Pro Extra Rheometer. Experiments were repeated thrice for the authenticity of the results.
The interaction of NEm, MTX, and NEm-MTX was assessed using the Thermo-Scientific Nicolet iS-50 Fouriertransform infrared (FTIR) spectrometer having 4 cm À1 spectral resolution and operating in 4000-500 cm À1 frequency range. The samples were loaded directly into the sampling compartment. A beam of infrared light is passed through the sample, then the transmitted light reveals how much energy was absorbed in each wavelength and information appeared as the FTIR spectrum after the Fourier transformations.

Physical stability
NEm's stability was examined by noting down the deviations in the hydrodynamic diameter during storage at 25 C. The hydrodynamic diameter of the NEm has been noted down for a period of 3 months using the Malvern Zeta Nano S90.

Transmission electron microscopy (TEM) analysis
For the morphological evaluation of NEm and NEm-MTX formulations, TEM imaging was done on JEOL JEM 2100 PLUS. A small amount of the prepared sample was added to the copper grid, excess liquid was removed and the grid was air-dried. Thereafter, the grid was viewed and photographed under the electron microscope at operating voltage 200kV at a suitable magnification. A beam of an electron is passed through the specimen and a transmitted beam is projected on a fluorescent screen on which the final image of the sample is obtained.

Entrapment efficiency (EE)
The EE of the NEm was measured using the dialysis bag method. [10] MTX was added into the oil phase of the NEm to prepare the NEm-MTX. Briefly, 2 mL of NEm-MTX was taken in a cellulose tube and both sides were clipped. Thereafter, the clipped bag was dropped into the doublewalled thermal jacket attached to the thermostat maintained at 25 C. Phosphate buffer saline (PBS) of pH 7.4 was prefilled in the jacket. After 24 h, the sample was withdrawn and the absorbance was measured using the UV-vis spectrophotometer by Equation (1): where C total denotes the concentration of MTX in NEm before dialysis and C out refers to MTX concentration after 24 h dialysis.

In-vitro release study
In-vitro release study was also carried out using the dialysis bag method with the only difference in temperature which was kept constant at 37 C, and at a pre-set time interval, 2 mL of the formulation was taken out and replaced with the 2 mL PBS by keeping the net volume constant. [11] The sample's concentration was determined spectrophotometrically at the characteristic wavelength of MTX.

In-vitro bioaccessibility of MTX
The bioaccessibility and stability of MTX after digestion were carried out using an earlier reported in-vitro digestion model. [12] All three phases i.e., simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared with slight modifications in the reported method. [13] NEm-MTX along with the prepared stimulated phases was preheated to 37 C. Firstly, NEm-MTX and SSF phases were mixed in 1:1 (w/w ratio) and pH was maintained to 6.8. The prepared system was incubated for 10 min. Bioaccessibility was measured after the collection of the digest. Secondly, the SGF phase formed using calcium chloride, magnesium chloride, potassium chloride, potassium hypophosphate, and sodium chloride (at pH 4.0) and incubated for 30 min in dark at 100 rpm stirring. Further, HCl was added to adjust the 2.0 pH and the sample was again incubated for 30 min. Finally, the SIF phase was created by using bile salts, porcine, lipase, and pancreatic enzymes. The pH of the solution was maintained as 7.0 and incubated for 2 h at 100 rpm continuous stirring. At this phase, the raw digest was collected and centrifuged to collect the plasma solubilized with MTX. Thereafter, dilution was done with ethanol and analyzed to determine the bioaccessibility. [12,13] 2.8. Hemolytic assay Hemolytic activity of NEm and NEm-MTX was measured by doing slight modification in the earlier reported hemolytic assay. [14,15] Vacutainer heparin tubes were utilized for the fresh blood sample collection (3 mL) from the healthy rats. Thereafter, 10 min centrifugation at 1000 g stirring was done for 10 min, and separated plasma was removed. Cells were washed with PBS of pH 7.4 three times and further, the suspension of washed RBCs was done into PBS. For the hemolytic assay, diluted RBCs (190 mL) were mixed with 10 mL of NEm and NEm-MTX to obtain the final concentration of 5, 25, 50, 100 mg/mL of NEm. Triton X-100 (1% v/v, positive control) and PBS (negative control) were used in the study. After mixing the diluted RBCs with the formulations, the samples were incubated at 37 C with intermittent shaking for 30 min. Further, 10 min centrifugation at 1000 g stirring was performed and the 96 well plates were utilized for the supernatant collection. The absorbance using a microplate reader was recorded at 540 nm for measuring hemoglobin released from hemolytic RBC. The hemolytic assay was repeated two times for the authenticity of the results. Hemolysis for each sample was calculated using the given formula in Equation (2): where A S denotes the sample's absorbance, and A C refers to the control's absorbance.

Cell viability assay
Michigan Cancer Foundation-7 (MCF-7) and Henrietta Lacks (HeLa) cell lines were employed for carrying out the cell viability assay of MTX, NEm, and NEm-MTX. MCF-7 cells and HeLa cell lines were grown as a monolayer in Eagle's minimal essential medium (EMEM) and mammary epithelial cells (MEC) media, respectively. The media were added with 2 mM a-glutamine and Earle's BSS adjusted to contain 1.5 g/L Na bicarbonate, 1 mM Na-pyruvate, 0.1 mM non-essential amino acids, 1% pen-strap and10% PBS. The culture condition was preserved at 37 C with 5% CO 2 in a CO 2 incubator. MCF-7 cells (passage no 29) and HeLa cells (passage no 9) having 90% confluence were employed for the in-vitro cell viability studies. The cell viability assay of MTX, NEm, and NEm-MTX on MCF-7 and HeLa cell lines was carried out using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay. For the assay, both the cancerous cells were trypsinized and cells were seeded separately in 96 well plates (at a cell density of 1-1.5 Â 10 4 cells) in 100 mL in their respective media and were incubated overnight at 37 C. The MTT reduction assay was carried out in triplicate and samples including MTX, NEm, and NEm-MTX with concentrations (2, 10, 25, 50, and 100 mg/mL) were added into each well and incubated at 37 C for 24 h. After 24 h incubation, the old media was removed and 100 mL of media containing MTT (concentration of 0.5 mg/mL) was incubated for 4 h after addition to each well. MTT has been absorbed by the cells and reduces to insoluble formazan (a purple-colored compound due to metabolic activity of the viable cells). The supernatant was discarded and further, to solubilize the purple formazan crystals, 100 mL of DMSO was added. The color was allowed to develop by complete solubilization of formazan with intermittent shaking. The absorbance was recorded at 590 nm by the microplate reader. The untreated cells (with 100% viability) were used as the control to compare the relative cell viability in the test wells.

Preliminary optimization
MTX solubility in the oil phase is an essential criterion to formulate a stable NEm. If the drug is found to be less soluble then there are chances of its precipitation after the dilution step. A higher drug's solubility in the oil phase allows higher drug loading within the NEm. [9] MTX solubility in a variety of oils like clove oil, corn oil, lemon oil, a-Tocopherol, Miglyol 812 was assessed using UV-vis spectroscopy. The calculated extinction coefficient (e) of the drug was 0.0517 lg À1 mL cm À1 . The concentration of MTX was determined using e. Out of all oils, MTX showed the highest solubility in the a-Tocopherol oil (70.328 lg/mL) ( Table 1). Apart from its high solubility, a-Tocopherol is the main constituent of vitamin E and exhibits strong antiproliferative activity against cancerous cell lines. [10,16,17] Hence, a-Tocopherol has been selected as the primary oil in the NEm. Furthermore, Miglyol 812 is a mixture of medium-chain fatty acid triglycerides that offers reduced particle crystallinity, good stability, and controlled release for the efficacious MTX administration. [18,19] Hence, Miglyol 812 has been selected as the oil phase in combination with a-Tocopherol, as it is reported to increase the drug solubility and adsorption. When the solubility of the combined oil phase was checked, it showcased the highest solubility of MTX (74.086 lg/mL) in it.

Effect of sonication time
The effect of the duration of sonication time on the average droplet size of the pure NEm was examined. Six identical samples were prepared using the same excipients and thereafter, ultrasonicated for 9 min in the different time frames (1, 3, 5, 7, 9 min) The results are depicted in Figure 1.
With the increase in the sonication time, a reduction in the droplet size of NEm to 30 nm was observed. [3] However, after 5 min of sonication, no change in the average size of NEm was seen. Therefore, the 5 min time frame was optimized for the sonication.

Effect of variation of the oil phase
Thereafter, to optimize the concentration of the oil phase, the concentration of the combined phase (a-Tocopherol and  Miglyol 812) was varied while keeping the concentration of the surfactant fixed. Five NEms were prepared (Table 2), however, formulation c (1:1 ratio), a translucent NEm, was stable. While the remaining formulations (a, b, d, e) were unstable as phase separation occurs after some time. Hence, based on the stability, formulation c was selected and utilized for the delivery of MTX. In the given formulation, flavored oil (Miglyol 812) might act as a carrier oil that can reduce the viscosity of a-Tocopherol and stabilize the formulation. [19]

Physicochemical characterization
The prepared NEm and NEm-MTX formulations were examined w.r.t. hydrodynamic diameter, surface tension, viscosity, and refractive index characteristics. The hydrodynamic diameter of NEm and NEm-MTX came out to be 22 ± 4 and 30 ± 5 nm, respectively with a low polydispersity index of <0.5. The enhancement in hydrodynamic diameter of NEm-MTX might be due to the adsorption of drugs on NEm's surface. The viscosity for the NEm was around 1.50 ± 0.4 mPas while it came out to be 1.45 ± 0.5 mPas for NEm-MTX. Although the change in the viscosity is not much, this is in coherence with the trend reported in the literature [20] which indicates that the viscosity of the NEm decreases when the mean droplet size of the NEm increases. The surface tension of the NEM increased from 32.45 ± 0.14 to 34.45 ± 0.24 mN/m when MTX was encapsulated in the NEM, as the NEm's droplet size increased on encapsulation of the MTX. [15] Furthermore, the good optical clarity of the formulations was reported where the refractive index for the NEm was obtained as 1.337 ± 0.03 and for the NEm-MTX was 1.338 ± 0.05.

Stability study
The prepared NEm was stored at 25 C to evaluate the formulation's stability. NEm was stable for up to two months ( Figure 2) which indicated the longer shelf life of the prepared NEm. The employed sonication method acts as an effective tool to prepare the NEm having uniform hydrodynamic diameter distribution with good stability. DLS measurements indicated that hydrodynamic diameter remained constant for almost 23 days. Thereafter, a minor increase in droplet size takes place up to 63 days. Further, after a month, the observed size increases up to $56 ± 2 nm, which might be due to the aggregation of the particles upon storage. Further, the formulation's stability was investigated by examining zeta potential. NEm and NEm-MTX showcased À11.8 ± 0.2 and À12.0 ± 0.3 zeta potential values respectively. The negative charge presence on the NEm is due to the polysorbate coating which acts as a stabilizing agent. [11] No significant change in the zeta potential of NEm and NEm-MTX was observed that validates the NEm's stability after MTX encapsulation.  Figure 3 shows the FTIR spectra for MTX, NEm, and NEm-MTX formulation. In MTX, typically three characteristic fingerprints are observed at 1636, 1300, and 1400 cm À1 that indicate the C ¼ O and C-O stretching bands, and O-H bending in the carboxylic acid group. [21,22] The FTIR spectra of NEm and NEm-MTX were found exactly similar and the characteristic peaks for the pure MTX were not seen in NEm-MTX that confirmed the successful dispersion of MTX in the NEm.

Microscopic analysis
TEM analysis was used for analyzing the morphology and particle size of the formulations. TEM examination of NEm and MTX-NEm while confirming the spherical shape of particles, depicted that NEm and NEm-MTX are 10 ± 1 and 17 ± 3 nm-sized, respectively. The particle size of the formulations was determined by plotting histograms employing Image J software using n ¼ 20 ( Figure 4c) and n ¼ 17 (Figure 4d) readings.
TEM analysis was found smaller in contrast to DLS measurements as in TEM analysis, the size refers to the exact diameter of the particle whereas, in DLS, the size corresponds to the hydrodynamic diameter of the particle.

Encapsulation efficiency (EE %)
An ideal drug delivery wagon should have the ability to carry a significant amount of drugs to the specific site. In the present case, the EE was found to be 52 ± 0.6%. The EE % came out to be comparatively higher than other nano assemblies like PEG capped MTX silver nanoparticles exhibit 39.6% EE and CTAB modified sodium montmorillonite clay (Na-MMT) vehicle reports 29.17% EE for MTX. [23,24] 3.9. In-vitro release study In-vitro release study is useful to envisage the in-vivo behavior of the pharmaceutical compound. Figure 5 depicts the MTX release from the NEm as determined by the dialysis method.
When MTX was loaded in the NEm, the amount of the cumulative release came out to be 54.74% in 24 h. The prepared system exhibited the release of MTX in three phases, starting with an initial burst release (35.77%) in 125 min, followed by a steady release phase. In this phase (46.58%) release was observed in 275 min. In the third and final phase, a very sustained release was observed (54.74%) in 24 h. However, to diminish the toxicity of MTX, the controlled release of MTX is required for its administration. [25] The prepared system has showcased the slow and sustained release of MTX throughout 24 h has compared to the previous studies. [8,26,27] Rajitha et al. [8] prepared Chaulmoogra-based NEm which showed 98.27% release of MTX in 12 h. Jang et al. [26] formulated a water-in-oil-inwater (W/O/W) double emulsion for Methotrexate delivery. The prepared nanoemulsion showcased almost 70% release within 75 min at pH 7.4. Sheikh Abdur Rashid et al. [27] formulated nanoemulsion composed of olive oil for the Methotrexate delivery and reported $96% drug release in 20 h.
The slow and sustained release of MTX might be due to the interaction of the drug (either electrostatic or van der Waals) with the oil phase as the release from the NEm is controlled by the interactions between the drug, surfactant, and partitioning of drugs between the phases. [28] The small size and higher surface area of NEm permit the release of drugs in a controlled manner. Vadia et al. [29] in their study on the commercial formulation of Methotrexate found 26% release of the drug in 10 min and 85% in 30 min. In our system, the prepared formulation showed a comparable release profile of MTX in the first 10 min. However, a slow and  sustained release was observed from the prepared nanoemulsion over the further period of 24 h.
Several mathematical drug models [30] were employed on NEm-MTX to understand the MTX release mechanism from the NEm. When the correlation coefficient was calculated after kinetic modeling, it was found that the release profile could be best described by the Korsmeyer Peppas method with n ¼ 0.261, thereby indicating Fickian diffusion release behavior (Table S1, Supporting Information)

Bioaccessibility study
A bioaccessibility study has been carried out to study the fate of formulation in gastrointestinal conditions. When the NEm-MTX formulated system was passed in the gastrointestinal tract (through the replica simulated conditions) initially starting from the mouth then to the stomach and followed by the intestine. MTX is hydrophobic and exhibits low solubility. Therefore, the delivery system should be capable to  solubilize the MTX and increase its absorption in the body. NEm is an effective tool for increasing the bioaccessibility of bioactive agents. [31] Therefore, the stability and bioaccessibility of MTX were determined in NEm and it was 94.16 ± 1.51% and 96.06 ± 1.01%, respectively. The EB after the collection of digesta was found to be 92.29 ± 2.50%. The increase in bioaccessibility can be attributed to the protective action of the Tween 80 in the gastric phase that prevents the degradation of NEm-MTX. [32] The EB calculated for the NEm-MTX was relatively high, which corresponds to the higher compatibility of MTX with the NEm. Bioaccessibility study indicates NEm as a promising strategy to improve the stability and bioaccessibility of the MTX.

Hemolytic assay
Hemolytic analysis of erythrocytes was performed to evaluate the biocompatibility of the developed formulation. Figure 6 represents the quantitative hemolysis results for different concentrations of NEm and NEm-MTX.
It can be seen that NEm and NEm-MTX did not induce any undesirable hemolytic effect on red blood cells at all the concentrations varied from 5, 25, 50, 100 mg/mL. The compounds having <10% hemolysis are considered to be nonhemolytic whereas the ones with >25% hemolysis are known to be toxic. [14] In the present study, developed NEm with or without drug showcased less than 8% hemolysis at all tested concentrations and it is considered to be safe for therapeutic development

Cell viability assay
The in-vitro cell viability assay for MTX, NEm, and NEm-MTX was measured using an MTT reduction assay. [33] MCF-7 and HeLa cell lines were employed for cell viability assay. Figures 6B and 7A showed higher cytotoxic activity of MTX against both cell lines. When the concentration of NEm increases from 25 to 100 mg/mL, the NEm turns slightly toxic to both HeLa and MCF-7 cell lines. This might be due to the antiproliferative activity of a-Tocopherol. It inhibits cancer cell proliferation by cytotoxicity-mediated induction of apoptosis. [10,17] At a higher concentration (100 mg/mL) of NEm, 30% of cells (MCF-7 and HeLa cells) were killed. When NEm-MTX was studied at different concentrations (2, 10, 25, 50, and 100 mg/mL) the higher cytotoxicity could be seen in NEm-MTX as compared to NEm. The higher cytotoxicity of NEm-MTX in comparison to NEm is attributed to the slow release of MTX from the NEm-MTX in 24 h. As discussed in the in-vitro release study, the cumulative release of MTX from NEm-MTX came out to be 54.74 ± 2.73% in 24 h. At higher concentration (100 mg/mL), NEm-MTX killed almost 43% of HeLa cells and 41% MCF-7 cells. In comparison to the literature report [33] where PEGAylated graphene oxide/ superparamagnetic nanocomposite loaded with MTX showcased 35% cell death on MCF-7 cell line and almost 40% on HeLa cell lines at 24 h incubation period. The obtained results of NEm-MTX on MCF-7 and HeLa cell lines revealed the high potential of the synthesized system in  biomedical research. The optimized NEm-MTX can offer great potential as a new delivery system for MTX and might be exploited as an effective approach and targeted delivery system in cancer treatment.

Conclusions
In the present article, a successful attempt was made to formulate MTX within the a-Tocopherol, Miglyol 812, Tween 80, and water-based NEm using the ultrasonic nano emulsification method. To the best of our knowledge, this is the first attempt toward the preparation of a-Tocopherol and Miglyol 812 based Methotrexate encapsulated nanoemulsion which gives a complete amalgamation of physicochemical characterization tools as well as their in vitro toxicological studies to confirm the safety of this system for drug delivery. The ultra-sonication assisted technique was proved to be an effective technique in the preparation of NEm having uniform particle size distribution with long-term stability. Microscopic analysis of the formulation confirmed the spherical shape of the prepared formulation. The obtained result showed NEm as a better nano wagon having longterm stability of around 60 days, good EE, and a sustained release profile (54.74 ± 2.73% in 24 at pH 7.4). The hemolysis profile of formulations examined at different concentrations was under the tolerance limit (<8%). The MTT assay was performed using cancerous (HeLa and MCF-7) cell lines, the NEm-MTX depicted increased cytotoxicity which is attributed to the slow release of MTX over a period of time. Based on the positive results, the developed assembly is expected to overcome the hurdles associated with other challenging drugs. Further, in vivo studies using animal models is necessary to explore the effectiveness of the NEm-MTX formulation.