A system for the production of methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for the treatment of cancer

Abstract

A system for the production of methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for the treatment of methotrexate-resistant breast cancer cells, consisting of: a) a conjugate synthesis unit configured to synthesize methotrexate-amino acid methyl ester conjugates selected from the group consisting of methotrexate-alanine methyl ester (MAME), methotrexate-cysteine methyl ester (MCME), and methotrexate-aspartic acid methyl ester (MADE) conjugates; b) a nanocapsule manufacturing unit used to formulate lipid nanocapsules (LNCs) derived from MCME and MADE conjugates; c) a homogenization unit configured to process the lipid acid nanocapsules to obtain a uniform dispersion at the nanoscale; and d) a purification unit configured to purify the lipid nanocapsules.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for producing methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for cancer treatment of methotrexate-resistant breast cancer cells. BACKGROUND OF THE INVENTION

[0002] Methotrexate (MTX) faces significant challenges as a chemotherapeutic agent against triple-negative breast cancer (TNBC, MDA-MB-231) due to innate and acquired drug resistance mechanisms. Overexpression of breast cancer-resistant proteins reduces MTX transport to resistant cells and decreases MTX polyglutamate levels. Elevated dihydrofolate reductase (DHFR) levels and reduced DHFR binding affinity for MTX further contribute to the resistance phenotype.

[0003] Current approaches to overcome this resistance include conjugating MTX with various organic moieties to improve biocompatibility, pharmacological activity, and target specificity. Previous strategies have explored conjugation with oligosaccharides, proteins, dendrimers, polymers, and cell-penetrating peptides, with varying degrees of success in targeting MTX-resistant breast cancer cells.

[0004] Lipid nanocapsules (LNCs) represent a promising platform for drug delivery for targeted tumor control. Compared to other lipid-based carrier systems, they offer improved physical stability, selectivity, therapeutic efficacy, and reduced cellular toxicity. The development of LNCs with MTX-amino acid methyl ester conjugates represents a novel approach to enhance cytotoxic efficacy against MTX-resistant breast cancer cells through improved cellular uptake and resistance-reversal mechanisms.

[0005] In view of the foregoing discussion, the present invention provides a system for producing methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for cancer treatment of methotrexate-resistant breast cancer cells. Summary of the invention

[0006] The present disclosure relates to a system for producing methotrexate-amino acid methyl ester-loaded lipid nanocapsules (LNCs) for the treatment of methotrexate-resistant breast cancer cells. The system consists of several integrated units that cooperate to synthesize methotrexate-amino acid methyl ester conjugates, encapsulate them into lipid nanocapsules, process these nanocapsules to obtain uniform nano-sized dispersions, and purify the final product for storage and evaluation. The invention particularly focuses on methotrexate-cysteine methyl ester (MCME) and methotrexate-aspartic acid methyl ester (MADE) conjugates, which exhibit increased selectivity and cytotoxicity toward MTX-resistant breast cancer cells.

[0007] The present disclosure aims to provide a system for producing methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for cancer treatment of methotrexate-resistant breast cancer cells. The system comprises: a) a conjugate synthesis unit for synthesizing methotrexate amino acid methyl ester conjugates selected from the group consisting of methotrexate alanine methyl ester (MAME), methotrexate cysteine methyl ester (MCME), and methotrexate aspartic acid methyl ester (MADE); b) a nanocapsule production unit for formulating lipid nanocapsules (LNCs) from MCME and MADE conjugates; c) a homogenization unit for processing the lipid nanocapsules to obtain a uniform nanoscale dispersion; and d) a purification unit configured to purify the lipid nanocapsules.

[0008] An object of the present disclosure is to provide a system for producing methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for cancer treatment of methotrexate-resistant breast cancer cells.

[0009] Another object of the present disclosure is to develop a system for synthesizing and formulating methotrexate-amino acid methyl ester conjugate-loaded lipid nanocapsules capable of overcoming the resistance of methotrexate-resistant breast cancer cells.

[0010] Another object of the present disclosure is to provide a comprehensive system for producing stable, uniform nanoparticle lipid nanocapsules with methotrexate-cysteine methyl ester (MCME) and methotrexate-aspartic acid methyl ester (MADE) conjugates with improved anticancer efficacy.

[0011] Another object of the present disclosure is to provide an integrated system for the synthesis of methotrexate-amino acid methyl ester conjugates and their incorporation into lipid nanocapsules with improved physical stability, selectivity and therapeutic efficacy against triple negative breast cancer cells.

[0012] To further clarify the advantages and features of the present disclosure, the invention will be explained in more detail with reference to specific embodiments illustrated in the accompanying drawings. These drawings illustrate only typical embodiments of the invention and are therefore not to be considered as limiting its scope. The invention will be described and explained in more detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE CHARACTERS

[0013] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Fig. 1 shows a block diagram of a system for producing methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for cancer treatment of methotrexate-resistant breast cancer cells according to an embodiment of the present disclosure; Fig. Figure 2a illustrates a detailed synthetic route for the synthesis of methotrexate-amino acid methyl ester conjugates according to an embodiment of the present disclosure; Fig. Figure 2b illustrates a concise synthetic pathway for the synthesis of methotrexate-amino acid methyl ester conjugates according to one embodiment of the present disclosure; Fig. Figure 3a illustrates the structure of the MAME (6a) conjugate according to one embodiment of the present disclosure; Fig. Figure 3b illustrates the structure of the MCME (6b) conjugate according to one embodiment of the present disclosure; and Fig. Figure 3c illustrates the structure of the MADE (6c) conjugate according to one embodiment of the present disclosure.

[0014] Those skilled in the art will also appreciate that the elements in the drawings are shown for convenience and are not necessarily to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, with respect to device construction, one or more components of the device may be represented in the drawings by conventional symbols. The drawings may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art from the present description. DETAILED DESCRIPTION:

[0015] To facilitate understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and will be clearly described. However, the scope of the invention is not limited thereby. Changes and further modifications to the illustrated system, as well as further applications of the principles of the invention, are possible, as would normally occur to one skilled in the art to which the invention pertains.

[0016] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0017] References in this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the language "in one embodiment," "in another embodiment," and similar language throughout this specification may or may not refer to the same embodiment.

[0018] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps may include not only those steps, but also additional steps not expressly listed or inherent in that process or method. Likewise, the statement "comprises" for one or more devices, subsystems, elements, structures, or components does not exclude, without further limitation, the existence of other devices, subsystems, elements, structures, components, or additional devices, subsystems, elements, structures, or components.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The systems, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0021] Fig. 1 shows a block diagram of a system (100) for producing methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for cancer treatment of methotrexate-resistant breast cancer cells according to an embodiment of the present disclosure.

[0022] Referring to Fig. 1, the system (100) comprises: a) a conjugate synthesis unit (102) configured to synthesize methotrexate-amino acid methyl ester conjugates selected from the group consisting of methotrexate-alanine methyl ester (MAME), methotrexate-cysteine methyl ester (MCME), and methotrexate-aspartic acid methyl ester (MADE) conjugates; b) a nanocapsule manufacturing unit (104) configured to produce lipidic nanocapsules (LNCs) obtained from MCME and MADE conjugates; c) a homogenization unit (106) configured to homogenize the lipidic nanocapsules to obtain a uniform dispersion at the nanoscale; and d) a purification unit (108) configured to purify the lipidic nanocapsules.

[0023] In one embodiment, the conjugate synthesis unit (102) comprises: a) a reaction vessel (102a) configured to mix methotrexate (MTX), N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxy-1,2,3-benzotriazole (HOBt), dimethylformamide, triethylamine (TEA), and L-amino acid methyl ester; and b) a purification component (102b) configured to purify the synthesized conjugates by column chromatography.

[0024] In one embodiment, the nanocapsule manufacturing unit (104) is configured to mix the methotrexate-amino acid methyl ester conjugates with virgin coconut oil and soy lecithin in a specific molar ratio.

[0025] In one embodiment, the nanocapsule manufacturing unit (104) further comprises: an organic solution preparation vessel (104a) configured to enable the preparation of a formulation of MCME and MADE lipid nanocapsules (LNCs), wherein the aqueous phase preparation unit enables the mixing of 100 mg of MCME or MADE conjugate, ethanol, virgin coconut oil, and soy lecithin; and a second mixing vessel (104b) configured to enable the mixing of the prepared organic solution in the organic solution preparation vessel with the aqueous phase of glycerol and Poloxamer 407 to form lipid nanocapsules (LNCs).

[0026] In one embodiment, the homogenization unit (106) connected to the nanocapsule manufacturing unit (104) is configured to subject the lipidic nanocapsules to several cycles of high pressure homogenization at pressures between 250 and 600 bar.

[0027] In one embodiment, the cleaning unit (108) connected to the nanocapsule manufacturing unit (104) comprises: a) a centrifugation component (108a) configured to separate the lipid nanocapsules from the dispersion; and b) a washing component (108b) configured to rinse the separated lipid nanocapsules with distilled water.

[0028] In one embodiment, the system (100) further comprises: a lyophilization unit (110) connected to the nanocapsule manufacturing unit and configured to freeze-dry the purified lipid capsules. Nanocapsules using a cryoprotectant, wherein the freeze-drying unit is configured to use sucrose as a cryoprotectant.

[0029] In one embodiment, the system (100) further comprises: a storage unit (112) configured to store the lyophilized lipid nanocapsules at 4°C in airtight containers.

[0030] The present invention relates to a system for the production of methotrexate amino acid methyl ester-loaded lipid nanocapsules with enhanced efficacy against methotrexate-resistant breast cancer cells. The system consists of a series of integrated units designed to operate sequentially to produce the final therapeutic formulation. The conjugate synthesis unit initiates the process by facilitating the reaction between methotrexate and selected amino acid methyl esters (alanine, cysteine, and aspartic acid) using a DCC / HOBt / TEA-based amidation chemistry. This unit contains a reaction vessel with precise temperature control, initially at 0 °C during the activation phase and subsequently at room temperature for the conjugation reaction.The conjugate synthesis unit also contains a thin-layer chromatography monitoring system and a column chromatography component for purifying the synthesized conjugates (MAME (6a), MCME (6b), MADE (6c)) using a chloroform / methanol solvent system. The nanocapsule production unit processes the purified conjugates, specifically MCME and MADE, by dissolving them in ethanol and incorporating them into a lipid phase containing virgin coconut oil and soy lecithin in a specific molar ratio (90:2.5). This lipid phase is then combined with an aqueous solution of glycerol (2.25% w / v) and Poloxamer 407 (5% w / v) under controlled temperature and stirring conditions. The unit is continuously stirred overnight to ensure complete ethanol removal and nanocapsule formation.The homogenization unit further processes the resulting nanocapsules through a high-pressure homogenizer, which subjects the formulation to multiple cycles (up to 35) at pressures between 250 and 600 bar. This critical step ensures the production of uniform lipid nanocapsules with optimal physical properties for improved cellular uptake and therapeutic efficacy. The purification unit then separates the lipid nanocapsules from the dispersion by centrifugation at 10,000 rpm for 35 minutes, followed by washing with distilled water to remove any residual components. The purified nanocapsules are then processed in the freeze-drying unit, which freeze-dries the formulation using sucrose (5% w / v) as a cryoprotectant to maintain structural integrity during the freeze-drying process.The resulting freeze-dried product is stored in airtight containers at 4°C, thus preserving its stability and therapeutic potential until further evaluation or use. The entire system operates as an integrated platform, with each unit performing specific functions that contribute to the overall goal of producing methotrexate-amino acid methyl ester-loaded lipid complexes. Nanocapsules capable of overcoming resistance mechanisms in triple-negative breast cancer cells, thereby potentially improving therapeutic outcomes in resistant cancer populations. Fig. Figure 2a illustrates a detailed synthetic route for the synthesis of methotrexate-amino acid methyl ester conjugates according to an embodiment of the present disclosure; Fig. Figure 2b illustrates a concise synthetic pathway for the synthesis of methotrexate-amino acid methyl ester conjugates according to one embodiment of the present disclosure; Fig. Figure 3a illustrates the structure of the MAME (6a) conjugate according to one embodiment of the present disclosure; Fig. Figure 3b illustrates the structure of the MCME (6b) conjugate according to one embodiment of the present disclosure; and Fig. Figure 3c illustrates the structure of the MADE (6c) conjugate according to one embodiment of the present disclosure.

[0031] In one embodiment, the conjugate synthesis unit is configured to synthesize the conjugates as described in Fig. 2a and Fig. 2b. To prepare the conjugates, MTX (1 mmol), HOBt (2 mmol), triethylamine (2 mmol), and DCC (3 mmol) were combined in 30 mL of dimethylformamide in a round-bottom flask. The mixture was stirred for 1 h under ice-cold conditions (0 °C). Subsequently, L-amino acid methyl ester (2 mmol) was added, and the reaction continued for 24 h at room temperature. The progress was monitored by TLC using a chloroform:methanol (7:3) solvent system. Upon completion, the mixture was dissolved in ethyl acetate and filtered to remove N,N'-dicyclohexylurea (DCU). The organic layer was washed successively with 60 mL of 0.1 N HCl, 60 mL of saturated aqueous NaHCO3 solution, 40 mL of brine, and deionized water, and then dried over activated sodium sulfate. The solvent was removed using a rotary evaporator.The crude product was then purified by column chromatography on silica gel using a chloroform-methanol mixture (7:3 v / v) as eluent. The conjugate synthesis unit synthesizes three conjugates: MAME, MCME, and MADE (6a-6c), as described in . Fig. 3a, Fig. 3b and Fig. 3c, where the conjugates MAME (6a), MCME (6b) and MADE (6c) were developed by reacting the respective L-amino acid methyl esters (L-alanine methyl ester, L-cysteine methyl ester and L-aspartic acid dimethyl ester) with carboxylic acid functions in MTX.

[0032] A simple route for the synthesis of MTX-α,γ -bis(amide)-amino acid methyl ester conjugates is described in Fig. 2a and Fig. 2b. The carboxyl groups of MTX (1) were first activated with N,N'-dicyclohexylcarbodiimide (DCC, 2), leading to the formation of an O-acylisourea intermediate (3). This intermediate then reacted with 1-hydroxy-1,2,3-benzotriazole (HOBt) to generate the corresponding HOBt esters (4). In this context, HOBt acted as both a coupling reagent and a racemization inhibitor. The MTX-HOBt esters (4) were then coupled with L-amino acid methyl esters (5) in the presence of triethylamine (TEA), leading to the formation of MTX-α,γ-bis(amide)-amino acid methyl ester conjugates (BAME) (6). In this approach, MTX was individually reacted with L-alanine methyl ester, L-cysteine methyl ester, and L-aspartic acid dimethyl ester to obtain MAME (6a), MCME (6b), and MADE (6c). Each of these conjugates was purified by column chromatography. Their successful formation was confirmed by various analytical techniques, including FT-IR, proton NMR ( 1H-NMR) and mass spectrometry. The spectroscopic results were consistent with the proposed chemical structures. The structures of the synthesized conjugates (MAME, MCME, and MADE, (6a-6c)) are shown in Fig. 3a, Fig. 3b and Fig. 3c. The yields of the MAME, MCME, and MADE conjugates were 90%, 87%, and 84%, respectively.

[0033] The structural and functional properties of the synthesized MAME, MCME, and MADE conjugates were analyzed using Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR), and mass spectrometry. FTIR spectra were recorded using a Bruker spectrophotometer over a wavenumber range of 600 to 4000 cm -1 to identify the presence of important functional groups. To gain insight into the hydrogen bonding patterns within the molecular framework, 1H NMR spectra were recorded on a Bruker AC spectrometer at 400 MHz using DMSO as the deuterated solvent and tetramethylsilane as an internal reference. Chemical shift values (δ) were expressed in parts per million (ppm). Additionally, the molecular masses of MAME, MCME, and MADE were confirmed by LC-MS using a Shimadzu QP2010 instrument, which provided data on molecular weight changes upon conjugation.

[0034] To understand the interaction of these conjugates with biological targets, molecular docking studies were conducted in one embodiment. Human dihydrofolate reductase (hDHFR) with PDB ID 1KMV was selected as the target enzyme. The synthesized conjugates—MAME (6a), MCME (6b), and MADE (6c)—were converted from MDL-MOL format to PDB format using OpenBabel. Docking was performed using AutoDock software to evaluate the binding interactions between hDHFR and the conjugates. The structural binding conformations and interactions were analyzed and interpreted using visualization software such as CHIMERA.

[0035] In one embodiment, the stability and dynamic behavior of the docked complexes were investigated using molecular dynamics simulation (MDS). This was performed using the GROMACS 2018.3 software integrated with the GROMOS96 54a7 force field on a CentOS 7.0 platform. The crystal structure of hDHFR bound to the experimental inhibitor Sri-9439 (PDB ID: 1KMS) served as reference. Topology files for the conjugates (6a-6c) and the control inhibitor were generated via the PRODRG server. Each complex was centered in a cubic simulation box with a distance of 1.0 nm from the box edge and solvated with SPC216 water molecules. Counterions were introduced to neutralize the systems. The steepest descent method was used to minimize the energy for 50,000 steps at 300 K, using periodic boundary conditions (PBC) to eliminate unfavorable atom contacts. After energy minimization, the systems were equilibrated in two steps.In the first phase, a V-rescale thermostat was used to maintain a temperature of 300 K for 100 picoseconds. In the second phase, the Parrinello-Rahman barostat maintained the pressure at 1 bar for one nanosecond under NPT conditions. The LINCS algorithm was used to confine all hydrogen-containing bonds. Long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method, with cutoffs of 1.2 nm and 1.4 nm for Coulomb and van der Waals forces, respectively. To evaluate conformational behavior and system stability, molecular dynamics simulations were extended to 100 nanoseconds. Periodic boundary conditions and all constraints were lifted during this simulation to avoid edge effects. Structural trajectory files for the complexes and the control system were created and analyzed using GROMACS utilities such as gmx.rms, gmx rmsf, gmx radius of gyration, and gmx hydrogen bonding were used to determine the root mean square deviation (RMSD), root mean square fluctuations (RMSF), radius of gyration, and hydrogen bond profiles. Additionally, the druglikeness and pharmacokinetic potential of MAME, MCME, and MADE were predicted using MTX as a reference compound. ADMET properties, including absorption, distribution, metabolism, excretion, and toxicity, were evaluated using the ADMETsar2 server. To assess druglikeness based on Lipinski's rule of five, calculations of molecular properties such as hydrogen bond donors and acceptors, polar surface area, Log P, and bioactivity value were performed using the online platform Molinspiration.

[0036] In one embodiment, the nanocapsule formulation unit is configured to contain the prepared conjugates for the synthesis of the formulation of MCME (6b) and MADE (6c) lipid nanocapsules. Lipid nanocapsules (LNCs) encapsulating MCME (6b) and MADE (6c) were prepared using slight modifications of the method described by Ahmad et al. Initially, 100 mg of each conjugate was precisely weighed and dissolved separately in 2.5 ml of ethanol. A mixture of virgin coconut oil and soy lecithin in a molar ratio of 90:2.5 was incorporated into this solution while maintaining the temperature at 55-60 °C to ensure thorough mixing. The resulting organic phase was added dropwise to an aqueous phase containing glycerol (2.25% w / v) and Poloxamer 407 (5% w / v) under continuous stirring at 2500 rpm. Stirring was continued overnight to allow evaporation of ethanol and formation of LNCs.To achieve uniform dispersion at the nanoscale, the LNCs were subjected to high-pressure homogenization using an APV-1000 homogenizer (SPX, Denmark) for up to 35 cycles at a pressure range of 250 to 600 bar. The nanocapsules were then separated and purified by centrifugation at 10,000 rpm for 35 minutes at room temperature. The supernatant was discarded, and the pellet was washed with distilled water. The purified LNCs were redispersed in distilled water and subsequently lyophilized with sucrose (5% w / v) as a cryoprotectant using an Alpha 2-4 LSC plus freeze-dryer (CHRIST). The lyophilized samples were stored in airtight containers at 4 °C for subsequent analysis. In this invention, LNCs are synthesized from MCME (6b) and MADE conjugates (6c) with the proposed system using a high-pressure homogenization technique.Poloxamer 407, a hydrophilic nonionic surfactant, was included in the formulation due to its excellent biocompatibility. Virgin coconut oil was also used to improve the patient's quality of life. The inherent cytotoxic effect of soy lecithin also enhances its antitumor effect. Lyophilization of the LNCs also conferred physicochemical stability and preserved the structural integrity of the conjugate through the addition of sucrose as a cryoprotectant.

[0037] In one embodiment, high-performance liquid chromatography (HPLC) was performed using a Shimadzu Prominence Modular system to determine the MTX content in the MCME and MADE LNCs. The setup included a C18 column (4.6 mm inner diameter, 250 mm length, 5 µm particle size, coated with silyloctadecyl). The mobile phase consisted of a 6:3 mixture of acetonitrile and 50 mM sodium acetate buffer at pH 3.6. The flow rate was maintained at 1.0 mL / min, with an injection volume of 20 µL and a column temperature of 30 °C. For drug-loading studies, LNC samples (5 mL) were mixed with an equal amount of ethanol and vortexed for 5 minutes to disrupt the nanocapsule structure. Each sample was then diluted with 10 mL of distilled water and injected into the HPLC system. All measurements were performed in triplicate, and MTX loading was expressed as micrograms per milligram of LNCs.Loading efficiency was calculated based on the proportion of encapsulated drug relative to the initially added amount. Particle size, polydispersity index (PDI), and zeta potential were measured using a dynamic light scattering analyzer after diluting the LNCs 1:10 in water. Surface features such as morphology, size, aggregation, and structural integrity were examined by transmission electron microscopy (TEM) using a Jeol JEM 2100 plus microscope.

[0038] The in vitro drug release profile of MTX from MAME (6a), MCME (6b), and MADE (6c) conjugates, as well as from MCME and MADE LNCs, was evaluated using the dialysis bag method. A dialysis membrane with a molecular weight cutoff of 10 kDa was pretreated according to HiMedia's instructions and soaked overnight in phosphate-buffered saline (PBS, pH 7.4). Each test sample was reconstituted in PBS to achieve a concentration of 10 mg / mL, and 2 mL of the dispersion was added to the dialysis bag, which was tightly sealed. The dialysis bag was immersed in 250 mL of PBS (pH 7.4) containing Tween 80 (0.2% v / v) to maintain sink conditions. The system was stirred at 150 rpm, and samples were collected at scheduled time points, centrifuged, filtered, and analyzed by HPLC at 303 nm. The collected medium was replaced with fresh PBS, and all release studies were performed in triplicate.Results were expressed as mean ± standard deviation. To evaluate the stability of the LNCs, lyophilized MCME and MADE samples were sealed in glass vials and stored for six months in a Thermo Scientific™ 3911 Stability Chamber at 4 ± 1 °C. Samples were analyzed both before and after storage by redispersing in distilled water, and changes in particle size, PDI, and zeta potential were determined.

[0039] The anticancer potential and cytocompatibility of LNCs and their conjugates were investigated in in vitro studies. Cell lines used included MTX-resistant breast cancer (MDA-MB-231), lung carcinoma (A-549), human skin epidermoid carcinoma (A-431), and normal human lung fibroblasts (WI-38), all obtained from the National Centre for Cell Science (NCCS), Pune, India. These were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, and 100 µg / ml streptomycin. Incubation was performed at 37°C in a humidified environment with 5% CO2. For the determination of the IC 50 -values of MTX, MAME, MCME and MADE (6a-6c), cells were cultured in 96-well plates at a density of 6×10 5Cells were seeded per well and incubated for 24 hours. They were then treated with different concentrations (31.25–1000 µg / ml) of MTX and the conjugates for 48 hours. After treatment, MTT (0.5 mg / ml) was added and incubated for 4 hours. The medium was removed, and DMSO was used to dissolve the formazan crystals. Absorbance was measured at 570 nm using an Infinite M200 PRO UV-VIS spectrophotometer (Tecan). Both cytotoxicity and biocompatibility studies were performed using this setup.

[0040] Further analyses of the cell cycle distribution and apoptotic activity of MCME and MADE LNCs (6b, 6c) were performed by flow cytometry. MTX-resistant MDA-MB-231 cells were cultured in 6-well plates at a density of 4.6×10 5Cells were seeded per well and incubated for 24 hours, then treated with MCME, MADE-LNCs, or MTX (10 µg / ml) for another 24 hours. After trypsinization, the cells were fixed in 70% cold ethanol for 1 hour and incubated in the dark at 37°C with 5 µl of ribonuclease (10 mg / ml) and 10 µl of propidium iodide (PI) (10 mg / ml). The samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences, NJ). The apoptotic potential was determined using an Annexin V-FITC / PI kit. After 24 hours of incubation, the treated cells were stained with 2 µl each of Annexin V-FITC and PI and analyzed by flow cytometry. The Annexin V-FITC / PI kit (BD, Biosciences, NJ) was used to determine the apoptosis-inducing potential of (6b, 6c) LNCs and MTX. MTX-resistant MDA-MB-231 cells were cultured in 6-well plates at a density of 4.6 × 10 5Cells were seeded / well and incubated for 24 hours. Subsequently, the cells were treated with the respective LNCs (6b, 6c) and MTX (according to their IC50 values) for 24 hours. Subsequently, the cells were collected by subsequent washing and stained with Annexin V-FITC (2 µl) and propidium iodide (PI) (2 µl) for 15 minutes. Finally, cell analysis was performed using an instrument. Fluorescence microscopy was used to examine the cellular uptake of MCME, MADE (6b, 6c) LNCs compared to MTX. MTX-resistant MDA-MB-231 cells were seeded at a density of 3.3 × 105 cells / well in 6-well plates on 18-mm coverslips and cultured for 24 hours. FITC-labeled MCME, MADE (6b, 6c), LNCs, and MTX (10 µg / ml) were added to the cell culture, and the cells were incubated for 5 hours at 37 °C. Subsequently, the cells were washed with PBS and then trypsinized.The cells were then resuspended in PBS and further processed for fixation in methanol. Internalization of FITC-labeled MCME, MADE (6b, 6c) LNCs, and MTX in Resis breast cancer cells (MDA-MB-231) was analyzed using the mean relative fluorescence intensity of the cell population. All images were acquired using an Olympus fluorescence microscope. The DNA cleavage potential of the LNCs and MTX was determined by agarose gel electrophoresis using supercoiled pUC18 plasmid DNA (1000 ng). DNA was treated with different concentrations (25, 50, 75, and 100 µg / ml) of MCME and MADE LNCs (6b, 6c) and MTX in the presence of H2O2 (200 µM) and Tris-HCl / DMSO buffer (9:1, 10 mM, pH 7.2) and then incubated for 90 minutes at 37 °C. DNA was applied to a 1% agarose gel containing ethidium bromide and electrophoresed for two hours in TAE buffer at 80 V.The bands were visualized under UV light, and images were acquired using the imaging system (BioRad). The extent of DNA cleavage was determined by the conversion from the supercoiled form (I) to the nicked circular (II) and linear forms (III).

[0041] Histotoxicological studies of MCME and MADE (6b, 6c) LNCs and free MTX were conducted on juveniles of the freshwater fish Cirrhinus mrigala, obtained from the State Fishery Farm in Satara, Maharashtra, India. Fish weighing approximately 5 ± 1 g and measuring 5 ± 1 × 2 ± 1 cm were divided into groups of five. Each group was kept in a 10 dm 3The fish were kept in a large glass tank containing dechlorinated water (pH 7.2 ± 1; temperature 25 ± 1 °C; DO 7.2 mg / L) under continuous aeration and a 12-hour light / dark cycle. The negative control group received only tap water, while the positive control group received 0.15 mg / L MTX. The MCME and MADE LNC treatment groups were administered the same MTX-equivalent concentration. After a 96-hour exposure period, the fish were euthanized, and gill and liver tissues were fixed in neutral buffered formalin (NBF) for 24 hours, followed by a water wash. The tissues were alcohol dehydrated, cleared with xylene, and embedded in paraffin. The 5 µm-thick sections were cut with a rotary microtome, rehydrated, and stained with hematoxylin and eosin. They were then mounted with DPX and examined under a light microscope at 400x magnification. Images were captured with an attached camera.All procedures complied with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and the APHA 2012 standards for water quality monitoring.

[0042] The present invention provides a system for the synthesis of methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for the treatment of methotrexate-resistant breast cancer cells. The system consists of various units and components. First, it enables the synthesis of three conjugates: MAME (6a), MCME (6b), and MADE (6c). For this purpose, MTX is linked to L-alanine methyl ester, L-cysteine methyl ester, and L-aspartic acid dimethyl ester, respectively. The system then utilizes the MCME (6b) and MADE (6c) conjugates to synthesize lipid nanocapsules of the conjugates used. The synthesized conjugates (6a-6c) exhibited strong binding affinity to the active sites of human dihydrofolate reductase (hDHFR, PDB ID: 1KMV), as confirmed by molecular docking studies.The stability of the conjugate-protein interactions was verified by molecular dynamics simulations (MDS), while in silico ADMET profiling, physicochemical evaluations, and bioactivity assessments supported their drug-like behavior. Among the synthesized conjugates—MAME (6a), MCME (6b), and MADE (6c)—MCME and MADE demonstrated superior cytotoxicity against MTX-resistant MDA-MB-231 cells while exhibiting low toxicity against normal human WI-38 lung fibroblasts. Based on this performance, MCME (6b) and MADE (6c) were selected for incorporation into LNCs to further enhance their efficacy. The operation of the system involved the combination of soy lecithin, virgin coconut oil, Poloxamer 407, and glycerol using a high-pressure homogenizer, followed by freeze-drying to obtain the LNC formulations.These LNCs exhibited a spherical shape, a narrow particle size distribution, a stable zeta potential, and a low polydispersity index (PDI). The nanocapsules remained stable during six months of storage at 4 ± 1 °C and exhibited a controlled MTX release profile. Biological evaluation of LNCs 6b and 6c showed significantly enhanced cytotoxicity against MTX-resistant MDA-MB-231 cells, with pronounced effects on cell cycle arrest (particularly in the sub-G1 phase), cellular uptake, and apoptotic activity, as observed by MTT assays, flow cytometry, and Annexin V-FITC / PI staining. Both nanocapsules also exhibited moderate ability to promote oxidative DNA cleavage and converted pUC18 plasmid DNA into circular and linear forms.Histotoxicological studies with Cirrhinus mrigala fingerlings showed that MCME (6b) LNCs exhibited a safer ecotoxicological profile compared to MADE (6c) LNCs and free MTX, as evidenced by lower toxic effects on gill and liver tissue. The results suggest that the enhanced solubility provided by the amino acid methyl ester and the improved permeability provided by the lipid core contributed to the enhanced anticancer effect of the LNCs. Among them, MCME (6b) LNCs emerged as particularly promising candidates for the treatment of MTX-resistant breast cancer, combining therapeutic efficacy with a favorable safety profile for biological systems and the environment.

[0043] The drawings and the foregoing description illustrate examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements of one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the actions of a flowchart need not be performed in the order shown; nor do all actions need to be performed. Also, actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and material use, are possible.

[0044] The scope of the embodiments is at least as broad as indicated in the following claims.

[0045] Advantages, further benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, advantages, solutions to problems, and any components that may result in or enhance an advantage, advantage, or solution are not to be construed as critical, required, or essential features or components of any or all of the claims. REFERENCES 100 A system for the preparation of methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for cancer treatment of methotrexate-resistant breast cancer cells. 102 Conjugate synthesis unit 102a Reaction vessel 102b Cleaning component 104 Nanocapsule Manufacturing Unit 104a Containers for the preparation of organic solutions 104b second mixing tank 106 homogenization unit 108 cleaning unit 108a Centrifuge component 108b Washing component 110 Freeze-drying unit 112 storage unit

Claims

[1] A system for the production of methotrexate amino acid methyl ester-loaded lipid nanocapsules (LNCs) for the cancer treatment of methotrexate-resistant breast cancer cells, consisting of: a) a conjugate synthesis unit configured to synthesize methotrexate-amino acid methyl ester conjugates selected from the group consisting of methotrexate-alanine methyl ester (MAME), methotrexate-cysteine methyl ester (MCME), and methotrexate-aspartic acid methyl ester (MADE) conjugates; b) a nanocapsule manufacturing unit used to formulate lipid nanocapsules (LNCs) derived from MCME and MADE conjugates; c) a homogenization unit configured to process the lipid acid nanocapsules to obtain a uniform dispersion at the nanoscale; and d) a purification unit configured to purify the lipid nanocapsules. [2] The system of claim 1, wherein the conjugate synthesis unit comprises: a) a reaction vessel allowing the mixing of methotrexate (MTX), N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxy-1,2,3-benzotriazole (HOBt), dimethylformamide, triethylamine (TEA) and L-amino acid methyl ester; and b) a purification component configured to purify the synthesized conjugates by column chromatography. [3] The system according to claim 1, wherein the nanocapsule manufacturing unit is configured to mix the methotrexate-amino acid methyl ester conjugates with virgin coconut oil and soy lecithin in a specific molar ratio. [4] The system according to claim 1, wherein the nanocapsule manufacturing unit further comprises: an organic solution preparation container configured to enable the preparation of the formulation of MCME and MADE lipidic nanocapsules (LNCs), wherein the water phase preparation unit enables the mixing of 100 mg of MCME or MADE conjugate, ethanol, virgin coconut oil, and soy lecithin; and a second mixing vessel configured to allow mixing of the prepared organic solution in the organic solution preparation vessel with the aqueous phase of glycerol and Poloxamer 407 to form lipid nanocapsules (LNCs). [5] The system according to claim 1, wherein the homogenization unit connected to the nanocapsule manufacturing unit is configured to subject the lipidic nanocapsules to several cycles of high pressure homogenization at pressures between 250 and 600 bar. [6] The system of claim 1, wherein the purification unit connected to the nanocapsule manufacturing unit comprises: a) a centrifugation component configured to separate the lipid nanocapsules from the dispersion; and b) a washing component configured to wash the separated lipid nanocapsules with distilled water. [7] The system of claim 1, further comprising a lyophilization unit connected to the nanocapsule manufacturing unit and configured to freeze-dry the purified lipid capsules. Nanocapsules using a cryoprotectant, wherein the freeze-drying unit is configured to use sucrose as a cryoprotectant. [8] The system of claim 1, further comprising a storage unit configured to store the lyophilized lipid solution nanocapsules at 4°C in airtight containers.

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