The potential of MTX-loaded polymeric NPs has been exploited for various outcomes. MTX was loaded into methoxy poly(ethylene glycol) (MPEG)-grafted chitosan (chitoPEG) copolymer (Seo et al., 2009). The mean size of the NPs was 50–300 nm with spherical shape. ¹H nuclear magnetic resonance (NMR) study demonstrated that MTX interacted with chitosan being embeeded in the inner core, and the outer shell of the NPs was composed of MPEG (Figure 2). The loading efficiency of MTX was demonstrated to be between 65.2% and 94%. The cytotocicity of the developed MTX-loaded NPs was evaluated on the B16F10 melanoma cells, and the outcomes revealed that MTX-loaded NPs presented less cytotoxicity compared to free MTX. In another study, MTX was enclosed into poly(lactide-co-glycolide) (PLGA) NPs by the process of solvent evaporation from a double emulsion (Jang et al., 2019).

The average size of the NPs was demonstrated to be 163.7 nm with an encapsulation efficiency of 93.3%, and the drug was released according to sustained kinetics (Figure 2). The cytotoxicity studies showed that the NPs presented higher antiproliferative activity against the cancerous cells, MFC-7 and CWR22Rv, in comparison with the free MTX. Moreover, the NPs were evaluated for properties of the pharmacokinetics and consequences of lymphatic delivery. The in vivo studies revealed high lymphatic efficiency and low accumulation of MTX in the kidney and liver. Furthermore, the fabricated NPs can be investigated by applying different administration routes, and MTX was still detectable in the blood for a long time in comparison with free MTX. To evaluate the anti-breast cancer activity, polymeric NPs comprised of PLGA and PEG were developed by the double emulsion solvent-evaporation method. The PLGA-PEG NPs were co-loaded with MTX and trapoxin A (TPX) (Akbari et al., 2021a). The developed PLGA-PEG NPs exhibited smooth morphology and nanosize particle distribution. Moreover, the NPs presented promising results against MCF-7 breast cancerous cells. The co-loaded NPs elicited an effective inhibition of MCF-7 cells. Also, the flow cytometry analysis demonstrated cell growth inhibition by induction of apoptosis. Furthermore, the cell cycle analysis was also done, which revealed that there was a stopped cell cycle at G1. The dual drug-loaded PLGA-PEG NPs also exhibited the mitochondrial apoptosis. Altogether, the developed NPs proved to be promising in breast cancer therapy. To improve the effective concentration of MTX at the target site for cancer therapy, a novel system was developed that combined physical targeting along with chemical targeting (Lin et al., 2015). Poly(lactic acid) (PLA) and MPEG were used to encapsulate MTX in the nanobacilli-shaped carriers, and the dye Cy5.5 was conjugated with them. The nanobacilli had improved intratumoral uptake in the H22 xenograft mouse model. Moreover, the MPEG-PLA- MTX-Cy5.5 nanospheres released the drug into the tumour cells in a controlled manner to inhibit tumour growth and improve therapeutic efficiency. Similarly, another MTX loaded PLGA-beta cyclodextrin nanosystem was developed using double emulsion method to improve drug atability and solubility (Gorjikhah et al., 2017). The mean particle size ranged from 70 to 200 nm with an entrapment of 80%. The PLGA-β cyclodextrin NPs increased the cytotoxicity after releasing the drug in T47D breast cancer cell lines. In another study, PLA-PEG-PLA triblock polymeric NPs were synthesized to deliver MTX in a controlled way to the cancer cells. The double emulsion method was adopted, and the prepared polymeric NPs were then covered with bovine serum albumin to enhance their biocompatibility. The average size of the NPs was reported to be in the range of 100–157 nm with an entrapping efficiency of 80.4% (Massadeh et al., 2016). In another study, biodegradable MPEG-PCL copolymer-based NPs were designed for their activity on breast cancer cells. MTX and curcumin were co-loaded in the NPs to improve the therapeutic activity against cancer cell lines. The nanoprecipitation method was adopted to develop self-assembled micelles. The average micelle size was found to be 88 nm. The cancer cell growth was inhibited with an increase in the concentration of the micelles in the treatment groups. The micelles improved the cytotoxicity against MCF-7 cancer cell lines (Saraei et al., 2019). Anti-inflammatory effects of MTX can also be relevant to the field of cancer treatment as some correlations were suggested between cancer development and persistent inflammation states. In this regard, MTX-loaded spherical chitosan and Pluronic F-127 NPs were developed using the ionic gelation technique and self-micellization for treating rheumatoid arthritis. The average size of the NPs was 181–417 nm, with a loading efficiency of 80.14%. The NPs prolonged the release of MTX for 48 h and improved the pharmacokinetic profile. There was an improved therapeutic activity with 3.48 times higher residence time in the inflamed tissues. The haematological study revealed that the NPs showed no adverse effects on the therapeutic activity. Chitosan was also employed to develop NPs and fucoidan to treat skin-related inflammatory issues. MTX was enclosed in the NPs with an entrapment efficiency of 80%–96% with a mean size of 300–500 nm. The NPs exhibited low cytotoxicity to the human keratinocytes and fibroblasts. In vivo studies showed that there was a significant reduction in the pro-inflammatory cytokines. Moreover, the NPs improved the permeation of MTX through the pig ear skin barrier as compared to free MTX. Altogether, this MTX topical delivery using this approach demonstrated promising anti-inflammatory activity (Barbosa et al., 2019). Polycaprolactone and polyethylene glycol (PCL-PEG) were used to develop NPs co-loaded with MTX and cisplatin for an effective treatment for non-small lung cancer (Zhang et al., 2017). The NPs were of spherical morphology and induced apoptosis in the tumour tissues as revealed by DAPI analysis. The PCL-PEG NPs improved the cytotoxicity against A549 cells by reducing bcl2 and cyclin D1 gene expression.

Iron oxide magnetic NPs (110–130 nm) were developed based on lignin as a natural polymeric-magnetic-nano-system via anti-solvent precipitation and ultrasonication for the targeted delivery of MTX. The drug loading and encapsulation efficiencies were calculated to be about 66.06% and 64.88%, respectively. The NPs, as a non-hemolytic material, exhibited a concentration-dependent release of MTX for the initial 24 h. MTT assay for NPs revealed higher cytotoxicity against breast cancer and macrophage cell lines in comparison to the free form of the drug by increasing caspase-3 activity and decresing glutathionelevels (Pathania et al., 2023).

Magnetic alginate beads coated with glutaraldehyde cross-linked chitosan were prepared to decrease the side effects and improve stability due to biodegradable and biocompatible nature of the NPs, resulting in an encapsulation efficiency of 75% and showing acceptable controlled release behavior. Cytotoxity assay based on MTT revealed higher anticancer effect compared to the free MTX by increasing expression of Bax and decreasing the expressions of Bcl-2 and TNF-α in MCF-7 cells (Taran et al., 2023). β-CD/MTX complex was fabricated to enhance the solubility and stability of MTX in aqueous solvents. β-CD as a nanocarrier had a good capacity for loading and releasing MTX. MTT assay exhibited improvement of the MTX performance (Hadi et al., 2023). Arrow et al. (2023) prepared poly(lactide-co-glycolide) complexes to load MTX, which demonstrated inhibitory effects on human PVR-cells. Samani et al. (2023) synthesized folic acid-functionalized MTX-loaded perfluorohexane nanodroplets (NDs) with an alginate shell (FA-MTX/PFH@alginate NDs) as a tool for smart ultrasound-guided chemoradiotherapy with controlled release. This composite could enhance the anticancer activity of chemotherapeutics and reduce side effects to normal tissue during breast cancer treatment in vitro [cancer cell (4T1)] and in vivo. In addition, MTX-loaded nanostructures with 26.7 ± 1.5 nm showed high inhibitory effects on tumor growth. Furthermore this nanostructure had no significant hemolytic activity and organ toxicity in combination with ultrasound exposure and X-ray irradiation. Lastly, FA-functionalized MTX/PFH@alginate NDs were accumulated selectively in the tumor region (Samani et al., 2023).

Various polymeric NPs have been developed to enhance the effective therapeutic concentration of the drug at the target site (Lodhi et al., 2022; Shahrousvand et al., 2022; Yoosefi, 2022). Delivery approaches such as stimuli-responsive NPs are being exploited. Such stimuli include chemical and physical changes, and the chemical stimuli are divided into pH, enzyme, and redox activity. In contrast, the physical stimuli can include light, magnetic field or temperature. Hence, using stimuli-responsive approaches can be advantageous in the targeted delivery of tumour cells and other disorders (Li et al., 2013a).

Asad et al. (2021) demonstrated the effectiveness of pH-responsive MTX NPs loaded into chitosan hydrogel to treat psoriasis. Eudragit-100 (E100) was used to make the hydrogel stimuli-responsive (pH-sensitive). The adopted method was the o/w emulsion solvent evaporation method (Figure 2). The average size of the NPs was 256 nm, with a loading efficiency of 86%. The ex-vivo permeation study showed that 81% of MTX was retained in the epidermis following the hydrogel application. No significant symptoms of hyperkeratosis and parakeratosis were observed. Moreover, the studies on the imiquimod-induced psoriasis model exhibited promising therapeutic activity. Histopathological studies showed reduction of inflammation and redness in the MTX-treated groups following topical administration.

In another research, chitosan was employed with fucoidan to assemble MTX-loaded NPs to study their effectiveness in lung carcinoma. It was shown that NPs with an average size of 300 nm were stable in the stomach acidic environment and improved the oral bioavailability in lung cancer. The NPs induced apoptosis of the lung cancer cells and were safe toward fibroblasts. The NPs exhibited biological activity and could suppress the growth and proliferation of lung carcinoma cells about 7-fold more as compared to free drug. Altogether, the nanosystem was found to be mucoadhesive and pH-responsive for the MTX oral delivery (Coutinho et al., 2020). In a novel study, Ganoderma lucidum polysaccharide (GLP) was utilized because of its antioxidant and antibacterial activity to develop a pH-sensitive nanosystem for the delivery of hydroxycamptothecin (HCPT) and MTX for cancer therapy. The developed NPs had a size of 190 nm and exhibited good loading capacity, i.e., 21.5% for MTX and 22.6% for HCPT. GLP NPs released the drugs under the acidic conditions of the tumour cells; however, the NPs remained stable at the other physiological pH. Moreover, tumour suppression was observed following the in vivo administration (Nezhad-Mokhtari et al., 2019). Similarly, an ultraviolet (UV) light and pH dual-responsive polymeric nanosystem was developed to deliver MTX in tumour therapy. Supramolecular polymeric NPs were fabricated from acetal-modified β-cyclodextrin-azobezene and poly(α-cyclodextrin) (Dai and Zhang, 2018).

Supramolecular polymeric NPs were discovered to have an average size of 85 nm, with an encapsulation efficiency of 21.3%. pH responsiveness was due to acetal group, and UV responsiveness was due to azobenzene, which was attributed to the controlled release of MTX upon UV irradiation. Based on the function of pH, over 65% of MTX was released in 12 h according to fast delivery kinetics. Upon UV irradiation, the drug was released in a slow manner after the initial burst release at the tumor site. A derivative of chitosan, thiolated carboxymethyl chitosan, was used to develop another kind of NPs. The polymer self-assembled via disulfide linkage in deionized water to synthesize NPs. NPs were discovered to have an average size of 160 nm, with an encapsulation efficiency of 43.4%. The slow and sustained release of MTX from the polymeric NPs was beneficial for tumour suppression. The inhibition rate of the MTX-loaded NPs was estimated to be 90% against HeLa cells. Owing to the existence of the carboxyl group on the polymer, the NPs were swelled and released the drug in the acidic environment based on the function of the surface area of NPs in contact with water and pH (Gao et al., 2014). A pH-responsive nanohydrogel was also established for MTX delivery in cancer therapy. In this regard, poly(hydroxyethyl methacrylate) (PHEMA) was crosslinked with poly(ethyleneglycol) diacrylate (PEG-DA) via chain transfer polymerization technique. The mean size of MTX-loaded nanohydrogel was 194 nm. MTX was coupled with polymeric structure by a pH-reponsive esteric bond, facilitating the drug release in acidic pH. PHEMA and PEG-DA conjugated nanohydrogel induced cytotoxicity in the MCF-7 cancer cells. pH-responsive conjugate enhanced the toxicity and cellular uptake in a cancer animal model (Farzanfar et al., 2021).

To successfully deliver MTX and curcumin for treating colorectal cancer, three-block copolymers polycaprolactone-poly(ethylene glycol)-poly(caprolactone) (PCL-PEG-PCL) were proposed (Zhang et al., 2021). PCL-PEG-PCL NPs presented good biocompatibility, low toxicity, good stability, and amphiphilicity. These NPs exhibited an encapsulation efficiency of 77%–95%, with an average particle size ranging from 95 to 135 nm. PCL-PEG-PCL NPs demonstrated dose-dependent toxicity to CL40 and SW1417 cells. The hybrid NPs showed improved cellular absorbance in the cancer cells and induced apoptosis, as revealed by the florescence staining assay.

In a novel hybrid approach, chitosan NPs loaded with MTX were functionalized with photocatalytic TiO₂ NPs. The photocatalytic controlled release of MTX was facilitated through the developed nanosystem based on the function of TiO₂. Upon irradiation with UV, the fabricated drug delivery system released MTX as TiO₂ triggered the polymeric bond rupturing in the presence of light. The entrapment efficiency for MTX was around 75%, with a particle average size of 240 nm. CS NPs without TiO₂ NPs coating did not release a significant percentage of a drug upon UV irradiation, whereas TiO₂ NPs improved the release of MTX up to 40% in the presence of UV light. The cytotoxicity data confirmed that the developed nanosystem was cytotoxic against MCF-7 cells, hence showing promise in cancer therapy (Al-Nemrawi et al., 2022). In order to pass through the blood–brain barrier (BBB), which is as physical/biochemical barrier, the transactivator of transcription (TAT) peptide was used to produce mesoporous silica nanoparticles-linked TAT (MSN-NH-TAT) with the aim of improving MTX (MTX-loaded MSN-NH-TAT) penetration into the brain. As a result, brain-to-plasma concentration ratio and the drug blood terminal half-life was improved for delivery of MTX into the brain as compared with the use of free MTX (Shadmani et al., 2023). Poly(3-hydroxybutyrate)/chitosan-graft poly(acrylic acid)-conjugate hyaluronate was fabricated and used for targeted delivery of MTX to colon cancer cell lines (Caco-2). The overall copolymeric system showed the maximum drug loading efficiency about 69.7% ± 2.7% (4.65 mg/g) and the release about 98.6% ± 1.12%, at pH 7.4. The results of cytotoxicity exhibited IC₅₀ of 11.7 μg/mL and enhanced level of DNA breakage as well as high reactive oxygen species (Hanna et al., 2023).

Biotin-PEG-conjugated nanogels of carboxymethyl polyethyleneimine (Biotin-PEG-CMPEI) were synthesized with a size of 100 nm and ζ-potential of +15 mV (pH = 7.4) for active targeted delivery of MTX in triple negative breast cancer (TNBC). This nano-system exhibited zero-order release kinetics of MTX at pH = 7.5 and a swelling-controlled release at pH = 5.5. The IC₅₀ of the MTX-loaded Biotin-PEG-CMPEI was about 10 folds lower than the free drug, while the unloaded nanogels had no significant toxicity. In a mouse model, the volume of tumor, animal mortality rate, lung metastasis and glomerular damage were lower after treatment with the MTX-loaded Biotin-PEG-CMPEI as compared to the group receiving the free drug (Abolmaali et al., 2022).

ROS-responsive hyaluronic acid–MTX was prepared combining chemotherapy with photothermal therapy (PTT) for cancer treatment. The synthesized thioketal moiety linker (TK) of MTX and hyaluronic acid (HA) (HA-TK-MTX (HTM)) in the presence of the photothermal agent IR780 (IHTM NPs) allowed reaching a tumor inhibition rate of 70.95% for 4T1 tumor-bearing mice due to ROS-responsive NPs (Yun et al., 2022). Amphiphilic cross-linked poly(vinyl alcohol-methyl methacrylate) (PVA-PMMA) copolymer NPs (NPs) as pH-responsive system were fabricated to load MTX. It was shown that 47.24% and 65.39% of the drug were released at pH = 7.4 and pH = 5.8, respectively, after 96 h. MTT assay confirmed the effectiveness of NPs against MCF-7 cells (Ziyaee et al., 2023).

Lipid-based NPs are the most versatile drug delivery vehicles being exploited for their potential to treat various disorders, including cancer. Owing to the nature of lipids, these carriers can overcome the biological barriers. In one study, solid lipid NPs (SLNs) were developed using the fatty acid coacervation technique. Transferrin or insulin served as anchors, and they were stuffed with an MTX active lipophilic agent (Figure 4). Transferrin or insulin was selected because their proteins are overabundantly expressed on the blood-brain barriers. For the functionalization of proteins on SLNs, a PEG spacer was used. These developed SLNs presented higher cytotoxicity against glioblastoma cells and improved bioavailability in Wistar rats. Significant levels of SLNs were found in the tumour cells and suppressed the tumour growth in glioma models (Muntoni et al., 2019).

For the treatment of psoriasis systemic therapy, targeted therapy was developed using lipid NPs. MTX and etanercept were co-loaded into lipid NPs and mediated by carbopol hydrogel to deliver a drug into the skin with minimal transdermal permeation. The SNLs did not elicit cytotoxicity to the human keratinocytes and fibroblasts. Therapeutic amounts of drugs were deposited across the pig ear with reduced transdermal permeation. Cetyl palmitate was used as a lipid component, and the drugs were conjugated to the developed SLNs. The SLN size varied from 292 to 356 nm. The appearance of the SLN-loaded hydrogel was transparent, uniform, and glossy. The mucoadhesive characteristic of the hydrogel improved the MTX retention and reduced the systemic circulation in psoriasis (Ferreira et al., 2017). In a similar approach also addressed to MTX delivery, a pegylated lipid core containing strearic acid was created in order to overcome the limitation of low bioavailability of the drug. The pegylated SLN had an average size of 130 nm with limited size dispersion and spherical morphology. The developed SLNs were compatible with the blood cells and improved the pharmacokinetics profile in tumour models. SLNs improved the blood circulation profile with efficient tumour cell uptake, as evident through SPECT imaging. Because of the presence of stearic acid, the bioavailability of the SLNs was effectively improved in BALB/c mice (Kakkar et al., 2015). Another topical treatment approach for psoriasis was developed using MTX-loaded nanostructured lipid carriers incorporated into the gel. The solvent diffusion technique was utilized to synthesize the nanostructured lipid carriers, which were later loaded into the carbopol gel following optimization. The deposition studies of the nanostructured lipid carriers were carried out on human cadaver skin which exhibited significant deposition across the biological barrier (71%) and resulted in the reduction of the psoriasis stmptoms. The amount of pro-inflammatory cytokines were reduced following the gel application in the BALB/c mic, as compared to the marketed MTX gel (Agrawal et al., 2020). Lipid NPs have also shown promising results in osteosarcoma, where the traditional drug delivery systems could not slow the progression of the metastasis. MTX and edelfosine were co-loaded into the lipid NPs to synergistically improve the activity against osteosarcoma. Edelfosine suppressed the metastasis in the cancer cells, and MTX reduced the growth of osteosarcoma. Lipid NPs were found to be internalized in high concentration inside the cells irrespective of the expression of dihydrofolate reductase (González-Fernández et al., 2015). In another dual drug delivery approach, SLNs were fabricated to deliver MTX and calcipotriol for the topical therapy of psoriasis. Precirol ATO 5 and squalene were used in different ratios to synthesize the SLNs. The average particle size was in the range of 270–320 nm; however, the particle size increased with increasing concentration of Precirol. The developed SLNs improved the permeation of the loaded drugs across the skin (2.4–4.4 times higher in comparison with control). Confocal laser scanning microscopy (CLSM) exhibited improved permeation following the internalization of SLNs across the biological membrane. The promising outcome of the study was that drugs with different polarities were successfully embedded into one system without using any complex methodology. SLNs did not reveal any irritation to the skin and served as a potential vehicle for the efficient treatment of psoriasis (Lin et al., 2010).

SLNs have also been developed in a hybrid approach by anchorage with moieties such as fucose to target breast cancer. Gelucire 50/30 and stearic acid served as core, whereas stearyl amine and phospholipid 90 served as a coating. Fucose was anchored to the SLNs to target the breast cancer cells precisely while the off-site drug loading was minimized. SLNs demonstrated enhanced cellular absorbance in the breast cancerous cell with high drug release inside the cells and improved bioavailability. SLNs presented higher cytotoxicity at a lower IC₅₀ value and facilitated programmed cell death, i.e., apoptosis. Moreover, the permeation profile was very promising, with improved anti-cancer activity due to the higher biodistribution of SLNS at the target site (Garg et al., 2016).

In order to prepare stable nanostructured lipid carriers (NLCs) for the encapsulation of MTX, glyceryl monostearate (GMS as solid lipids) and Miglyol 812 (MI1 as liquid lipids) were used to reduce adverse side effects and increase the low solubility and non-specific targeting of MTX. The nanoystems exhibited long-term stability and spherical morphology along with an internal cubic crystalline structure, as revealed by small-angle X-ray scattering (SAXS) analysis. MTX was encapsulated at 85 ± 0.9% and in vitro release of MTX was 52% ± 3.0 in 24 h. Furthermore, the MTT assay showed that MTX-loaded NLCs exhibited more toxicity against HeLa and MCF-7 cell lines as compared to free drug. In addition, hemolysis was assessed to be <10% (Li et al., 2019).

Dendritic nanocarriers such as lysine dendrimers were used for drug delivery owing to their high drug-loading capacity and high resistance to proteolytic degradation. The poly-lysine dendritic nanocarrier (P₄LDN) for targeted chemotherapy was prepared and loaded with an epidermal growth factor receptor (EGFR)-specific short peptide E2 (ARSHVGYTGAR) and MTX. MTX was linked to the nanocarrier by using cathepsin B cleavable spacer (glycine–phenylalanine–leucine–glycine (GFLG)). The results showed that MTX released from the nanosystem decreased the percentage of cells in the G2/M phase and enhanced the percentage of cells in the S and G0 phase in both MDA-MB-231 (EGFR overexpressed) and SiHa (EGFR negative) cells (Narayanan et al., 2022).

Currently, polymer and lipid-based hybrid NPs are being explored for their potential to deliver MTX in various disorders. Such hybrid NPs have high (Kumar et al., 2022) biocompatibility with improved blood circulation time (Surve et al., 2022). Also, these systems can further be modified in response to specific stimuli, including redox potential, pH, and temperature (Teixeira et al., 2017; Kumar et al., 2022; Chang et al., 2023).

Kumar et al. synthesized MTX-loaded nano-lipid polymeric system of poly(lactic-co-glycolic acid) (PLGA) in phosphatidylcholine (MTX-NLPHS) to release MTX through endocytosis against MCF-7 cell lines (encapsulation efficiency about 86.48% ± 0.31%). This system was not agglomerated due to highly negative zeta potential. In vitro release of MTX reached 100% in the long time. In a MTT study, the cell toxicity of MTX-NLPHS was lower with decreasing concentration of MTX. Meanwhile, at the higher concentration of MTX, toxicity was enhanced as compared to free MTX. ROS was also increased in the cells exposed to MTX-NLPHS, resulting from the controlled transport of drugs into the cells by PLGA (Kumar et al., 2023). In another study, NPs based on hyaluronanm (HA)-coated lipid with low molecular weight (<10 kDa) and high molecular weight (700 kDa) were fabricated and tested for tumor targeting towards the HA receptor (CD44). In vitro results showed that NPs with high molecular weight influenced significantly the affinity towards CD44 on B16F10 murine melanoma cells. In addition, low-molecular-weight NPs had weak binding on B16F10 murine melanoma cell lines. Lastly, MTX as a chemotherapy drug was loaded into high-molecular-weight HA–tsNPs, resulting in slow drug diffusion with a half-life of 13.75 days (Mizrahy et al., 2014). Lipid polymer hybrid NPs were also tested to co-deliver two therapeutic drugs (Teixeira et al., 2017). Fucose-anchored lipid-polymer hybrid nanoparticles (Fu-LPHNPs) were fabricated to co-encapsulate MTX and aceclofenac (ACL) for targeted/controlled delivery purposes against MCF-7 and MDA-MB-231 cells. In vitro drug release studies showed that the release of ACL and MTX was rapid in the early 12 h (over 25%), then slower and at last the release was sustained over 168 h. The drug bioavailability when LPHNP was used as carrier was greater than free MTX and ACL. It was reported that the therapeutic activity of MTX was improved by ACL and the inhibitory effect of MTX/ACL-loaded LPHNPs on cancer growth in a breast cancer mouse model was greater as compared to the simple combination of free MTX and ACL or MTX (Teixeira et al., 2017; Rui et al., 2022).