Development of nanostructured lipid carriers as a promising tool for methotrexate delivery: physicochemical and in vitro evaluation

Abstract The aim of the present study is to fabricate the stable nanostructured lipid carriers (NLCs) using biocompatible excipients for the encapsulation of Methotrexate (MTX), a chemotherapeutic agent for breast cancer treatment. MTX has restricted clinical applications owing to its low solubility, non-specific targeting and adverse side effects. Glyceryl Monostearate (GMS) and Miglyol 812 (MI1) were chosen as solid and liquid lipids, respectively, for the fabrication of NLCs, and the influence of variation of solid and liquid composition was investigated. The prepared NLCs exhibited long-term stability and spherical shape morphology as characterized by electron microscopy. The internal structure of fabricated NLCs was arranged into cubic crystalline as confirmed by small-angle X-ray scattering (SAXS) analysis. MTX's encapsulation efficiency of ∼85 ± 0.9%. and sustained in vitro release of MTX ∼ 52% ± 3.0 in 24 h was achieved. Classical molecular dynamics (MD) simulations were performed to study the structural stability of the MTX encapsulated NLCs. Hemolysis carried out on the NLCs showcased the biosafety of the formulation under the tolerance limit (<10%). Further, the MTT assay demonstrates that MTX-loaded NLCs exhibited toxicity against HeLa and MCF-7 cell lines as compared to blank NLCs. The finding demonstrates NLCs as promising vehicles for MTX delivery to address cancer. Communicated by Ramaswamy H. Sarma Graphical abstract


Introduction
In recent years, the development of oral anticancer drug delivery modules has gained significant attention owing to their numerous advantages such as patient acceptance and diminishing financial costs as compared to the intravenous administration (Mazzaferro et al., 2013). Among various drug delivery carriers, nanostructured lipid carriers (NLCs) are utilized as an efficacious tool for oral drug administration owing to their excellent compatibility, biodegradability, and ability to enhance drug payload and improved bioavailability of the bioactive agents (Aditya et al., 2013).
Methotrexate (MTX) is the first drug employed as singleagent therapy to cure cancer and is used to treat different types of cancers. MTX is structural analog to folic acid (vitamin B9) and inhibits enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS) activity involved in the synthesis of purines and pyrimidines, the building blocks for RNA and DNA thereby, interfering with the DNA/RNA synthesis, repair and cellular replication (Karasulu et al., 2007;Ko zmi nski et al., 2020;Wessels et al., 2008). The methyl-tetrahydrofolate (THF) acts as a proximal methyl donor in various methylation reactions of RNAs, DNAs, proteins, phospholipid and amino acid synthesis and further, the inhibition of THF production by MTX results in disruption of cellular functions and metabolic imbalance (Karasulu et al., 2007;Ko zmi nski et al., 2020;Wessels et al., 2008).
However, MTX is not bioavailable due to its solubility, stability, and permeability. So, it has little chance to get into the blood system and reach the tumor site (Karasulu et al., 2007;Ko zmi nski et al., 2020;Wessels et al., 2008). So, to overcome the shortcomings associated with MTX on its delivery, NLCs have been formulated which are composed of two lipids namely, glyceryl monostearate (GMS) and Migylol 812 (Ml1). The oral bioavailability of a drug can be altered by the administration in a lipid vesicle. Triglycerides and their composite fatty acid of lipids are known to alter the adsorption by modifying the physiological parameters such as gastric emptying rate, bile salt solubilization, membrane permeability and lymphatic absorption (Woolfrey et al., 2011). The effect of the lipid vehicle depends on both the physicochemical characterizations of the drug and the composition of the oil phase. Among several drug delivery carriers, NLCs are utilized as an efficacious tool for oral drug administration owing to their excellent compatibility and biodegradability. NLCs are used to enhance the drug loading efficiency and prolong exposure of tumor cells to the antitumor drug which enhances the permeability and retention (EPR) effect that improves the drug concentration in tumor tissue after the systematic administration and subsequently increases the therapeutic effect (Barkat et al., 2019;Pinto et al., 2014). Recently, Pinto and coworkers (Barkat et al., 2019;Pinto et al., 2014) formulated NLCs of size 274-298 nm using the hot homogenization-assisted ultra-sonication method for MTX's encapsulation. Higher entrapment efficiency ($65%) and in vitro fast release of MTX ($70% in 2 h) from NLCs were obtained. Garg et al. (Barkat et al., 2019;Pinto et al., 2014) developed NLCs and hydrogel for the efficient transdermal delivery of the MTX. The developed system demonstrated better therapeutic efficiency formulation over the conventional gel. However, the currently developed systems are undesirable due to their drawbacks, such as low loading efficiency, expensive reagents and rigorous reaction conditions. In our recent work, we have fabricated a biocompatible nanoemulsion for MTX drug delivery, which offers excellent stability and sustained MTX release (under communication). However, the low entrapment efficiency of MTX in formulated nanoemulsion and less cytotoxicity toward the cancerous cell line was observed. Thus, to increase the entrapment efficiency and to find a better candidate for the MTX delivery, we have tried to formulate the NLCs using the GRAS excipients. GMS was selected as a solid lipid due to its less ordered crystal structure, higher accommodation of drug molecules within the lattice, and improved stability and higher entrapment efficiency (Garg et al., 2016). MI1 was chosen as a liquid lipid-based owing to its advantages like better stability and sustained release for the efficacious MTX's administration . Even though several lipid nanoparticles were utilized for MTX's administration, no efforts have been made to fabricate a lipid-based structure composed of the GMS and MI1 encapsulated with MTX as a delivery agent for cancerous cell lines, to the best of our knowledge.
In the present report, NLCs were fabricated using the hotdispersion method by exploiting biocompatible excipients like GMS, MI1 and Tween 80 (emulsifier). The developed nanocarrier with higher biocompatibility was used to increase MTX's encapsulation efficiency and obtained the slow and sustained MTX's release. MTX-loaded NLCs (NLCs-MTX) were structurally evaluated by employing transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) techniques. NLCs-MTX were also evaluated on cancerous cell lines for the in vitro cell viability study. The obtained information may facilitate the fabrication of delivery systems that improve the bioactive agents' stability and bioavailability.
In addition to the experimental studies, molecular dynamics (MD) simulation was also performed to understand the formation of NLCs as well as MTX-loaded NLCs. We have investigated the structural dynamics of MTX encapsulated in the NLCs at the atomistic level. The aim of the computational study was to get the detailed information about the internal structure of NLCs and microscopic understanding of drug-NLCs interactions.

Materials
GMS (purity ! 98%), Tween 80 (purity ! 98%) were purchased from Sigma Aldrich. Methotrexate (purity ! 98%) was obtained from the R and D Laboratories. Miglyol 812 (MI1) was taken from the Omega Chrom. Double distilled water was utilized in experiments. The employed chemicals are generally recognized as safe (GRAS) excipients.

Fabrication of NLCs
The hot-dispersion method was used for the preparation of NLCs (Fadda et al., 2013;Kharkar et al., 2020;Neves et al., 2013). Briefly, GMS lipid (0.50 g), and MI1 (0.50 g) were heated at 60 C for 15 min. MTX (0.7 mg/mL) was added to the lipid phase. After the complete melting of the lipid phase, a preheated aqueous phase composed of Tween 80 and water was added into the lipid phase. Thereafter, hot dispersion was added to the cold water at 4 C and stirred at 500 rpm for 15 min. Further, centrifugation was done at 8000 rpm for the 10 min to precipitate out the particle aggregation. The prepared formulation was stored at 25 C for further studies. Blank NLCs were prepared in the same manner without the drug.

Morphological analysis
The morphological and inner structure analysis of NLCs and NLCs-MTX samples, FE-SEM imaging was mapped on Hitachi-SU 8010 operating at 15 kV voltage on carbon tape. FE-SEM sample was made on the glass slide by adding a tiny droplet of the formulation which was dried overnight. HRTEM analysis was done on JEOL JEM 2100 PLUS at an operating voltage of 200 kV. The sample preparation of the TEM examination was carried out by adding a drop of the fabricated system on the copper grid which was kept for drying to remove excess liquid.

Physicochemical characterization of the NLCs
The hydrodynamic diameter of NLCs and NLCs-MTX was measured by the Malvern Zeta Nano S 90. In brief, 2 mL of the NLCs and NLCs-MTX were individually taken into polystyrene cuvette, and hydrodynamic diameter and polydispersity index (PDI) were recorded at a fixed 90 angle at 25 ± 0.1 C. The surface tension of NLCs and NLCs-MTX was determined at 25 C± 0.1 C using the Kruss K20 instrument. The optical clarity of the prepared NLCs and NLCs-MTX was measured using Anton Paar Abbemat 500 refractometer. NLCs and NLCs-MTX were further examined by Fourier transform infrared spectroscopy (FTIR) using Spectrum 400 FTIR Spectrometer of 0.4 cm À1 spectral resolution and working in 4000-400 cm À1 range.

SAXS analysis
SAXS studies were carried out using line collimation on the SAXS ess instrument (Anton Parr, Austria) at room temperature for a 30 min exposure time. The sample collection was done in the capillary tube and later on, the tube was placed in the evacuated chamber. The scattered intensities were measured at a 307 nm distance from the sample by the Eiger detector. The scattering intensity I (q) and scattering vector q was examined to gain quantitative information. The net scattering intensity of the particles (I p(q) ) was extracted by employing Equation (1): where I (b) refers to HPLC water-filled capillary's scattering intensity (Sunaina et al., 2019).

MTX-loaded NLCs
2.6.1. Encapsulation efficiency (EE) and in vitro release analysis of NLCs EE was measured with the dialysis method (Kanwar et al., 2019). NLCs-MTX was taken in a cellulose tube (having 33 mm flat width) and clipped at both sides. After that, the dialysis bag was taken into the solution of phosphate buffer saline (pH 7.4) placed inside a double-walled jacket connected with a thermostat (25 C± 0.1 C) at 100 rpm stirring. Later on, the sample was taken out after 24 h, and analyzed using UV-vis spectrophotometer by Equation (2): where A 1 is the total drug concentration in NLCs and A 2 denotes the unentrapped drug concentration. The dialysis tube diffusion method was utilized for carrying out the release study (Kanwar et al., 2019). Likewise, the dialysis bag method employed for calculating the EE, 2 mL of the sample was taken out at a specific interval from the double-walled thermal jacket (containing the dipped dialysis bag having the 2 mL (NLCs-MTX)into 50 mL phosphate buffer of pH 7.4) and changed with the same amount of buffer throughout the experiment. Each sample's concentration was measured at 303 nm.

Computational methods
The classical MD simulation was performed to determine the stability of MTX inside the NLCs and the internal arrangement of the (NLCs) using the NAMD software package (Bhatt & Ali, 2021;Bisht et al., 2019;Khalkhali et al., 2019;Kour et al., 2020). To mimic the in vitro assay, the cluster of 20 molecules of GMS and 20 molecules of MI1 (in 1:1 ratio) were considered to prepare the NLCs. One MTX drug molecule was added randomly to the matrix. The structural and electronic properties of the Individual lipid and drug molecules were investigated applying density-functional theory (DFT) (Becke, 1993). The molecular geometries were initially optimized and charge density and population analysis were performed adopting the B3LYP/6-311G(d,p) method (Rives & Jorgensen, 2008). The optimized molecules and the computed charge distributions were used to prepare force field parameters for GMS, MI1 and MTX for the atomistic MD simulations. The initial nanostructure of lipids (NLCs) was prepared from the optimized geometries of GMS and MI1 with a total of 40 molecules using the Packmol software (Martinez et al., 2009) to make the assembly of the NLCs. AMBER FF14SB force fields were used to generate the force fields of GMS and MI1 (Maier et al., 2015). The force field for the nonstandard residue/MTX was prepared with the help of GAFF2 (General Amber Force Field) (Wang et al., 2004). The System (NLCs þ MTX) was solvated in the cubic water box maintaining the minimal distance between box boundary and NLCs at 15 Å. The TIP3P (transferable intermolecular potential with three points) water model was utilized to solvate the simulation system (Price & Brooks, 2004). The system was centered in a periodic box of dimensions 79.11 Â 87.77 Â 88.53 Å containing 15,689 water molecules. A cutoff value of 10 Å was used for the short-range non-bonded interactions while the long-range electrostatic interactions were determined with the Partial Mesh Ewald (PME) method. The system was minimized at 0 K for 0.1 ns keeping the heavy atoms atom fixed, then the system was gradually heated by increasing the temperature from 0 to 300 K. Further, the system was equilibrated at a constant temperature, i.e., at 300 K and 1 atm pressure for 0.25 ns. A simulation was carried out under NVT ensembles for 200 ns. The integration time step is 2 fs for the simulation. The initial 50 ns were performed as equilibration at a finite temperature of 300 K by removing the constraint force and allow the free movement of the atoms. Subsequently, 200 ns were treated as a production dynamic for the structure analysis. The resulting trajectories were visualized and analyzed by using the VMD software.

Bioaccessibility study
The bioaccessibility of MTX was calculated as described in the literature (Aditya et al., 2015;Liu et al., 2018). NLCs-MTX along with the prepared simulated phases were preheated to 37 C± 0.1 C. First, NLCs-MTX and simulated saliva fluid (SSF) phase (consist of a mixture of mucin and salts) were mixed in 1:1 (mole ratio) and the pH of the system was maintained to 6.8. The prepared system was incubated on the shaker for 10 min. Digesta was collected at the end of all three phases and analyzed to determine bioaccessibility. Further, pH of the simulated gastric fluid (SGF) phase (correspond to stomach phase) formed using various electrolytes (calcium chloride (0.15%), magnesium chloride (0.07%), potassium chloride (0.11%), potassium hypophosphate (0.05%) and sodium chloride (0.30% in water) was maintained to 4.0 using 1 M HCl and the sample was incubated for the 30 min in dark at 100 rpm stirring. Thereafter, again pH of the solution was reduced to 2.0 and the sample was incubated for 30 min. Finally, the simulated intestinal fluid (SIF) phase was created by using bile salts, porcine, lipase and pancreatic enzymes. The pH of the solution was maintained to 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 thebioaccessibility (Liu et al., 2018).

Hemolysis study
The biocompatibility of the nanovehicle is determined by employing a hemolysis assay. Briefly, 3 mL freshly healthy rats' blood sample was taken in the vacutainer heparin tubes and centrifuged. After centrifugation, the collected red blood pallet was washed with the sterile isotonic phosphate buffer saline (pH 7.4) to get clear red blood cells (RBCs). RBCs' suspensions were made in Triton X-100 and sterile PBS. Hemolytic activity of the NLCs and NLCs-MTX was carried out as reported in the literature with slight modification (Pereira et al., 2016). The hemolysis assay was done by mixing diluted RBCs with equal volumes of test solutions and further incubation for 30 min at 37 C ± 0.1 C was done with shaking for reaction completion. After that, the collected supernatants were transferred to 96-well plates and quantified at 540 nm. Each sample's hemolysis was measured using Equation (3): where A 3 is the sample's absorbance and A 4 is the control's absorbance. The assay was repeated twice for the authenticity of the results.

Cell viability assay
The cytotoxicity studies of MTX, NLCs and NLCs-MTX, were done by studying the cell growth inhibition using 3-(4,5-dimethlythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MTT assay is widely adopted as a standard method to determine cell viability. It has been extensively studied and characterized, and its precision makes it an excellent tool for assessing the efficacy of new drug compounds and drug formulations. The color shifts of the MTT molecule when it is exposed to viable cells is the basis for this viability assay, viable cell reduces MTT to insoluble purple color compound formazan. The ability to determine the cell growth inhibition capability of the compound is done by measuring the absorbance, which is proportional to the number of viable cells, and comparing it to untreated controls. A 96-well plate format is used for the MTT colorimetric assay. Two cancerous cell lines (MCF-7 and HeLa) were used for performing cell viability studies of MTX, NLCs and NLCs-MTX formulations. Both cell lines were grown in form of a monolayer in Eagle's minimal essential medium (EMEM) and mammary epithelial cells (MEC) media. The cells (MCF-7 and HeLa cells) were harvested by using trypsin and subculture in 96-well plates at 1-1.5 Â 10 4 cells density and further incubated at 37 C for overnight. After the addition of test samples into each well, the cells were reserved at 37 ± 0.1 C for 24 h. The previous media was removed, and fresh media (100 mL) having MTT of 0.5 mg/mL concentration was incubated for 4 h after its addition in each well. The supernatant was removed and 100 mL of DMSO was added to each well to solubilize the purple formazan crystals. The color was allowed to develop by complete solubilization of formazan with intermittent shaking. The absorbance was recorded at 590 nm using a Biorad microplate reader. The absorbance was compared with the well in which no formulation was added, untreated cells were taken as control with 100% viability that was used to compare the relative cell viability in the test wells. The MTT reduction assay was carried out in triplicate.
The test solutions of MTX, NLC and NLCs-MTX with different concentrations (2, 10, 25, 50 and 100 mg/mL) were prepared where the concentration of MTX in NLCs-MTX formulation was the same as that of the pure drug. The concentration of different compositions of NLCs in blank was kept equivalent to that of NLCs-MTX to compare the observed effects.  Supplementary Table S1. Based on the observation, formulation C of 1:1 composition (having GMS and MI1 in (0.50 w/w %)) has been selected for the preparation of NLCs as it was found to be long-term stable. GMS is an effective emulsifier for the solubilization and incorporation of the lipophilic components within the oil phase (Park et al., 2018). Tween 80 as an emulsifier is utilized to provide steric stability to the formulation. Employed hot-dispersion method has numerous advantages such as greater homogeneity and reduced nanoparticle's dimension as compared to other methods.

Structural characterization of NLCs formulation
The hydrodynamic diameter determined using the particle size analyzer depicted that the NLCs were of 300-400 nm size range along with smaller structures of around 10-20 nm. As reported by our group ( FE-SEM and HRTEM analysis revealed the spherical shape morphology for the NLCs and NLCs-MTX formulations ( Figure  1(a,b)). The spherical morphology of the formulation was maintained even after the encapsulation of MTX. HRTEM investigation further confirmed the spherical morphology and uniform compact structure of NLCs without any visible drug crystal (Figure 1(c,d)) having a particle size of about 90 ± 10 nm (NLCs) and 95 ± 10 nm (NLCs-MTX). Moreover, the presence of small spherical particles in the range of 15-25 nm in HRTEM images can be due to micelles formation by Tween 80 (Kaur et al., 2015;Tetyczka et al., 2019).

SAXS analysis
The shape and inner structural organizations of the NLCs play a crucial role in controlled release and interaction with cells and biological fluids. The main feature of the formulations was determined using SAXS, where MTX's loading effect on the blank NLCs structures was obtained. SAXS curves of the NLCs and NLCs-MTX (Figure 2(a)) exhibited no obvious difference which demonstrates that the MTX's addition did not alter the NLCs' internal structures (Lv et al., 2016).
Further, SAXS has been employed for a better understanding of the specific arrangement of the lipid that forms the core of the NLCs. Figure 2(b,c) indicates the scattering intensity curve as a function of q vector for the NLCs formulations. Four peaks (q 1 ¼0.34, q 2 ¼0.42, q 3 ¼0.56 and q 4 ¼0.74 nm À1 for NLCs and q 1 ¼0.36, q 2 ¼0.43, q 3 ¼0.57 and q 4 ¼0.75 nm À1 for NLCs-MTX could be observed with repeating intervals. The ratio of SAXS peaks on the q-scale obey the relationship of ͱ1: ͱ2: ͱ3: ͱ4 … , which revealed the cubic symmetry (Chen et al., 2014). Therefore, the internal phase of the formulation was arranged into the cubic symmetry with d spacing of 3.02 nm (NLCs) and 3.37 nm (NLCs-MTX). The addition of the MTX did not alter the internal cubic arrangement of the blank NLCs. The d spacing of the NLCs-MTX was found to be slightly higher than the NLCs which might contribute to the successful loading of the MTX inside the NLCs. The microscopic structure of the NLCs was further studied by analyzing the low q part of SAXS data in a slope approximation (I (q) vs q Àa ). From Figure 2(b,c), the a was found to be between 3 (surface fractal) and 4 (smooth fractal) that showcased the dense particle cores' existence and also the rough interference between the solvent and particles (Lv et al., 2016). For blank NLCs, the slope was found to be 3.87 that indicates the surface fractal. Also, the addition of MTX into NLCs did not change the slope significantly which confirmed the successful loading of the MTX inside the NLCs successfully.   of the MTX peaks that confirmed the MTX molecules embedding within the lipid matrix (Elmowafy et al., 2018).

EE and in vitro release study
EE is a crucial parameter analyzed during formulation design owing to its impact on the drug release and the cost-effectiveness of the formulation (Khosa et al., 2018). Fabricated NLCs-MTX system showcased higher EE, i.e. 85 ± 0.9%. In NLCs, liquid lipids' presence enhances the crystal structure imperfections and leaves more space to encapsulate the drug. Here, less ordered crystal lattices of GMS lipids are responsible for the higher EE that favors the accommodation of more drug molecules.
Drug incorporation within the lipid matrix of lipid nanoparticles occurs either in the dissolved form or in a dispersed phase (Barkat et al., 2019;Pinto et al., 2014). Throughout 3 h, initial burst release (40%) was observed (Figure 4). Surface accumulation of MTX on the NLCs might be responsible for the initial burst release (Malvajerd et al., 2019). Further, a slow and sustained MTX's release (52 ± 3%) was observed up to 24 h that might be due to the presence of MTX within the lipid core (Khosa et al., 2018).
To reduce MTX's toxicity, the controlled release of MTX is required for the administration. NLCs has been primarily utilized for drug administration to increase oral bioavailability by enhancing the drug uptake using micro fold cells in the intestinal membrane (Lin et al., 2017). Using NLC, we achieved a better nano wagon for the encapsulation of the MTX as compared to the literature (Barkat et al., 2019;Pinto et al., 2014), where NLCs-MTX system showed an initial fast release of 64 ± 16% for NLC-P60 and 64 ± 10% for NLC-P80 and approximate 99% drug was released within 8 h.
The higher value of EE % further proves that the drug is stable within the nanocarrier and MTX is successfully encapsulated within the NLCs which was also supported by the computational study.

MD simulations
The simulated trajectory of 200 ns long is analyzed to understand the structural dynamics and molecular interactions between NLCs and MTX drug molecules. The root-meansquare deviation (RMSD) and Radius of Gyration (Rg) for the NLCs and MTX drug molecule are compared meticulously. The small average RMSD value and featureless regular fluctuations in RMSD are observed for the MTX along the entire trajectory. This indicates that no structural deformation occurs due to the encapsulation of the MTX in the NLCs, this signifies the MTX is quite stable inside the NLCs cavities ( Figure 5).
Further, we performed the radius of gyration (Rg) that provides information about the structural compactness and the binding pattern of the drug molecule. When a drug molecule binds to the NLCs, a change in its conformation would change the radius of gyration. As shown in Figure 6, the radius of gyration remains stable at an approximate value of 6 Å throughout the trajectory, while the radius of NLCs reached high because of assembly and disassembly of the lipids inside the water medium. The relative compactness of the NLCs was not affected by the binding of the MTX drug molecule. The interaction between MTX drug molecules and NLCs was shown in Figure 7.
Next, we have calculated the Radial distribution function g(r) which represents the probability of finding an atom or molecule at some distance away from the reference molecule. RDF, g(r), provides an estimate of spatial conformation of atoms or molecules relative to a focal point and demonstrates a long-range arrangement in the structure or system. Figure 8 showed the maximum interaction of the MTX drug molecule with the GMS lipid carrier. Around 5-10 Å the welldefined peaks with MTX were observed, showing its first interaction in the shortest distance. A peak around 5 Å was observed with the MI1 molecules.
Finally, it was observed that the internal structure of the NLCs did not change significantly in the presence of

Bioaccessibility study
In vitro, the bioaccessibility of encapsulated MTX was determined using replicated simulated gastrointestinal conditions to evaluate the effectiveness of the NLCs for delivering the bioactive agents. MTX is a hydrophobic moiety that exhibits low bioaccessibility owing to its low water solubility. Hence, a drug Figure 7. Snapshots of different conformations of NLCs (GMS (green), MI1 (magenta)) with MTX (cyan for carbon, blue for nitrogen and red for oxygen) drug molecule obtained along the 200 ns MD simulation trajectory. The 0 ns was assigned for the initial structure which was captured after performing the 50 s of equilibrium, where the MTX was far away from the NLCs and lipid molecules are disaggregated. As the simulation progressed, the MTX molecule interacts with the GMS. At around 80 ns the GMS molecule aggregated with each other and MI1 also bind with GMS and make an assembly. delivery vehicle should have the potential to solubilize MTX and enhance its absorption in the body. The bioaccessibility of MTX using NLCs was quantified and was found to be 93.6 ± 1.92% and 93.4 ± 2.92%. Tween 80 protective action during the gastric passage is responsible for the higher bioaccessibility as it prevents the degradation of the drug in an acidic environment and from the action of pepsin. When the lipid carriers reach the intestine, they will get digested by the intestinal enzyme complex into mixed micelles, which are made up of lipid degradation products, fatty acids, and phospholipids that can enhance its bioaccessibility and bioavailability. The effective bioaccessibility after the collection of digesta was 87.5 ± 3.27%, demonstrating the compatibility between the carrier and the MTX (Park et al., 2018). Furthermore, acid-stable GMS was employed as the wall material for NLCs' fabrication that might have contributed to the stability of NLCs in the simulated gastric phase.

Hemolytic assay
The prepared formulation was assessed as a safe delivery vehicle by studying the hemolysis profile of the prepared formulations in vitro (Figure 9) at different concentrations (5-100 mg/mL) of NLCs and NLCs-MTX formulations (Raza et al., 2016).
The percent hemolysis at all the tested concentrations was found to be less than 10% that is considered to be safe for further therapeutic development. The critical limit of 25% given in the guidelines was not exceeded by any of the formulations that showcased the safety of the nanowagon for the delivery of MTX's without harming red blood cells. (Amin & Dannenfelser, 2006).

Cell viability assay
MTT reduction assay was employed to carry out the in vitro cell viability studies (Abdollahi et al., 2019) using MCF-7 and HeLa cancer cell lines. The cell viability in both cell lines was examined after 24 h of incubation at several concentrations (2-100 mg/mL of MTX, NLCs, and NLCs-MTX, respectively). As expected, cytotoxicity of MTX against both HeLa and MCF-7 cell lines was observed ( Figure 10). Figure 10(a,b) showcased Figure 8. Radial Distribution Function of MTX with the NLCs in a solvated medium. MTX is the reference molecule about GMS lipids which showed the maximum interaction. While with the MI1 the discrete peak was observed at a distance of 5 Å with less probability. It was observed that the curves of greater intensity were obtained by interaction with GMS.  that in HeLa and MCF-7 cell lines, NLCs were not toxic at lower concentrations, whereas slight toxicity of NLCs to cancerous cell lines was observed at higher concentrations which can be attributed to the employed surfactant (Tween 80) in the formulation. When the NLCs-MTX was examined at various concentrations, the toxicity of NLCs-MTX was found to be higher as compared to NLCs owing to the MTX's release from NLCs-MTX formulation. NLCs-MTX at 100 mg/mL concentration killed approximately 55% HeLa cells and 40% of MCF-7 cells. Greater cytotoxicity effects were observed in the NLCs-MTX as compared to blank NLCs is because of the presence of surfactant as well as the drug. The lower cell viability for the NLCs-MTX as compare to MTX at the same concentration is attributed to the encapsulation and sustained release of MTX from NLCs over some time. In conclusion, the percentage of cell viability was reduced greatly with the increasing concentration of NLCs-MTX.

Conclusions
NLCs-MTX using GMS (as solid), MI1 (liquid lipid) and Tween 80 (surfactant) were developed by the hot-dispersion method. FE-SEM and HRTEM analysis revealed spherical shape morphology of NLCs and NLCs-MTX formulations. As per SAXS findings, MTX did not change the internal cubic structure of NLCs. Classical MD simulation was utilized to investigate the stability of the NLCs and the molecular interaction of MTX with the NLCs in an aqueous medium. It was observed that MTX is quite stable inside the system and showed the binding with NLCs. The structural stability and fluctuation of the NLCs did not change with the incorporation of the MTX molecule. The positive outcomes for the prepared NLCs like higher entrapment efficiency ($85 ± 0.4), slow and sustained release ($52 ± 2.3%) profile, good stability under gastric conditions, increased cytotoxicity against cancer cell lines (MCF-7 and HeLa) etc., forms the basis for the further utilization of NLCs for MTX delivery in the pharmaceutical industry.
The NLCs because of their biocompatible characteristics are known to have diverse applications and can be administered via. different routes such as oral, cutaneous, ocular and pulmonary. Among these, the oral delivery is the most accepted and economical route for drug administration and co-formulated NLCs with mutually synergistic drugs, may lead to substantial reduction in dosing frequency. Consequently, the prepared assembly will be fabricated in the liquid form and will be administrated. We will further pursue more studies on the distribution, metabolism and excretion of the MTX from the NLCs in our future work.
NLCs being an alternative to first generation nanoparticles can be tailored by varying different lipids, surfactants as well as co-surfactants to encapsulate different drugs of variable amphiphilicity. For instance, bioactive small molecules such as Toll like receptor (TLR) agonists and Indole amine 2,3-dioxygenase (IDO) inhibitors will be encapsulated in the NLCs for their possible applications as novel cancer immunotherapeutic agents. The application of NLCs as vaccine adjuvants will also be investigated in near future. Improved drug loading capacity and drug stability, controlled as well as targeted drug release, biocompatible composition for easy regulatory approval, amenable physicochemical properties and ease of production are the prominent features of the NLCs. Till date, mainly the research has been focused on using NLCs as chemotherapeutic agents comprising of low molecular weight drugs. Therefore, there is a need to expand the spectrum of their applications to include high molecular weight therapeutics such as peptides, proteins and other macromolecules.