Zein nanoparticles stabilized by hydrophilic small molecule stabilizer matrine deliver curcumin effectively

Matrine (MAR), a quinolone alkaloid, was employed to augment the stability of zein nanoparticles. The incorporation of MAR into the hydrophobic shell of zein nanoparticles was primarily achieved through hydrogen bonding. Curcumin (CUR), a hydrophobic active substance, was encapsulated in the hydrophobic core of zein/matrine nanoparticles (ZMNPs). The preparation of ZMNPs and curcumin-loaded zein/matrine nanoparticles (CZMNPs) was accomplished using an antisolvent precipitation method. The encapsulation efficiency of curcumin in ZMNPs (zein/MAR = 8:1, 20 mg zein and 2.5 mg matrine) was significantly greater (52.64%) than that of nanoparticles produced from a single zein (2.50%). CZMNPs demonstrated a notable encapsulation efficiency and loading capacity (88.30% and 7.84%, respectively) upon the addition of 2 mg of curcumin, and were capable of sustained and gradual release of curcumin in simulated intestinal fluid. Furthermore, the stability of ZMNPs was observed to be favorable across a range of environmental conditions, including pH levels of 2–4 and 6–9, salt concentrations of ≤150 mM, temperatures of ≤90 °C, and storage at room temperature for a duration of 30 days. Additionally, the inherent anti-cancer properties of MAR make CZMNPs a more efficacious inhibitor of tumor cell proliferation in vitro. Moreover, the uptake of CZMNPs by A549 cells was significantly enhanced, potentially through the process of endocytosis. Therefore, the incorporation of matrine in zein-based nanoparticles confers anticancer properties to the resulting ZMNPs. These nanoparticles can serve as encapsulating agents for bioactive compounds in pharmaceutical formulations and as a novel delivery strategy for long-term cancer care. Specifically, matrine is anticipated to function as a potential stabilizer for other nanosystems.


Introduction
The effect of various nano-delivery systems for encapsulating hydrophobic bioactive substances, including composite nanoparticles, nano-liposomes, polymer colloids, and mesoporous silica, have been evaluated [1][2][3][4]. Zein has recently attracted significant attention owing to its self-assembling properties. Zein is a major storage protein in maize and a characteristic class of proteins known as 'prolamine'. The high abundance of non-polar amino acid residues and the lack of basic and acidic amino acids contribute to the low solubility of zein; however, its dissolution can be achieved at high ethanol concentrations in aqueous solutions [5][6][7][8][9]. Therefore, using an anti-solvent precipitation method, zein can form nanoparticles (NPs) to encapsulate hydrophobic substances such as curcumin (CUR) [6,8], resveratrol [7], quercetin [10] and ɑ-tocopherol [11]. However, the applications of zein are limited by its poor stability. The isoelectric point (IEP) of zein is 6 [12]. The high hydrophobicity of zein nanoparticle surfaces and low net charge near the isoelectric point enhances the susceptibility of single zein nanoparticles (ZNPs) to physical instability and aggregation [13]. There has been interested in the fabrication of simulated gastrointestinal medium. We further investigated the anti-proliferative activity of the composite nano-system on tumour cells as well as comparing the cellular uptake of free curcumin and composite nanoparticles by tumour cells. Figure 1 shows the general idea of this study and the potential self-assembly mechanism. Hydrophobic zein escapes water molecules and spontaneously form spherical particles with a shell structure. When MAR is present, it acts as a protein stabilizer by being embedded in the zein shell, mainly through hydrogen-bonding interactions. Nonpolar CUR was encapsulated in the hydrophobic core of zein via electrostatic and hydrophobic interactions. As a result, CZMNPs exhibit higher packaging and loading performance [13]. During the formation of nanoparticles, non-covalent interactions, such as hydrogen bonding and electrostatic and hydrophobic interactions, drive self-assembly.

Materials and cell lines
Matrine (MAR, 98.6%) was procured from Angsheng Biological medicine (Shaanxi, China). Zein was purchased from Yuanye Biotechnology (Shanghai, China). Ethanol (99.9%) was obtained from Sichuan Xilong Science (Sichuan, China). Curcumin (CUR) and phosphotungstic acid hydrate was obtained from Energy Chemical The human lung cancer A549 cells, the human breast cancer MCF-7 cells, the human cervical cancer HeLa cells and the human hepatocellular carcinoma HepG2 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). A549 and HeLa grow in Roswell Park Memorial Institute (RPMI)−1640 (Gibco, China) supplemented with 10% fetal bovine serum (FBS; Gibco/Thermo Fisher Scientific, Waltham, MA, USA). HepG2 and MCF-7 grow in Dulbecco's Modified Eagle's Medium (DMEM) media (Gibco/Thermo Fisher Scientific) supplemented with 10% FBS. All cells were maintained at 37°C in a humidified incubator containing 5% CO 2 .

Preparation of composite nanoparticles
Zein/MAR composite nanoparticles (ZMNPs) were prepared as previously described [17] with some modifications. Zein (150 mg) was dissolved in 80% ethanol solution (30 ml) for 1 h under magnetic stirring at 800 rpm until completely dissolved. MAR (1, 2.5, 5, 7.5, 10, 15, and 20 mg) was stirred in 16 ml pure water for 15 min until it was completely dissolved. Next, the zein stock solution (4 ml) was slowly added dropwise to the MAR solution via a syringe and stirred for 1 h to obtain ZMNPs with varying mass ratios of 20:1, 8:1, 4:1, 8:3, 2:1, 4:3 and 1:1 (w/w) of zein to MAR. A rotary evaporator (50 ℃, −0.1 MPa) was used to remove ethanol, after which the sample was centrifuged for 20 min at 1000 × g to remove large particles. Finally, the samples were stored at 4 ℃ until further analysis. Curcumin-loaded zein/MAR composite nanoparticles (CZMNPs) were prepared by a similar procedure. First, curcumin (20 mg) was dissolved in anhydrous ethanol (10 ml) in the dark using an ultrasound. Subsequently, an appropriate amount of curcumin solution was quickly added dropwise to the ZMNPs, using a syringe. After stirring for 1 h, CZMNPs were obtained. Thereafter, we followed the steps of the anti-solvent process mentioned earlier.
2.3. Assessments of polydispersity indices (PDI), particle size and zeta-potential Particle size, PDI, and zeta potential of the NPs were measured using a Zetasizer Nano ZS90 system (Malvern Instruments, UK) at 25 ℃ with an equilibration time of 150 s. Samples were diluted 40 times with deionized water to eliminate multiple scattering effects. The sample measurements were conducted three times, and the mean values were used for analysis.
To refine the loading capacity of CUR, the mass of CUR added was controlled at 0.25, 0.5, 0.75, 1, 1.5, 2 and 2.5 mg, respectively. The amount of MAR was the result of the above optimization.

Transmission electron microscopy (TEM)
The morphologies of the ZMNPs and CZMNPs were assessed by TEM (HT-7700, Hitachi, Japan). The dispersion of the sample was diluted with water and dropped onto a copper grid filter paper to remove excess dispersion, after which the copper mesh was stained for 1 min with phosphotungstic acid (1% (w/v)) [41]. The morphologies of the freshly prepared samples were observed.

Fourier transform infrared spectroscopy (FTIR)
The chemical structures of the native (zein, CUR, and MAR) and freeze-dried (ZMNPs and CZMNPs) samples were determined to use FTIR spectroscopy. The powder sample (2 mg) was mixed with KBr (198 mg) and pressed into a clear tablet, and FTIR spectra was obtained from 400 to 4000 cm −1 at a resolution of 4 cm −1 in 32 scans.

X-ray diffraction (XRD)
The crystalline characteristics of the samples were determined by XRD (40 kV and 40 mA, D8 Advance, Bruker, Germany). The 2θ angles were in the range 5-35°at a rate of 2°min −1 .
2.8. Physicochemical stability of ZMNPs at various conditions 2.8.1. Effects of pH The pH of fresh ZMNPs was attuned to 2.0-9.0 using 1 M HCl or NaOH solutions to assess their stability at different pH levels. To avoid the influence of salt ions, only one solution (HCl or NaOH) was added to adjust the pH. The samples were then maintained for 24 h at room temperature ((RT) at 25 ℃) and characterized. Particle size, PDI, and zeta potential were determined at various the pH points (2.0-9.0).

Effects of salt
The original NaCl solution (1 M) was diluted to obtain salt solutions with various concentrations (0-200 mM NaCl). The effects of salt concentration on the stability of ZMNPs at pH 7.0 were investigated. Freshly prepared dispersion samples of ZMNPs (10 ml) were mixed with an equal volume of saline solution, vortexed, and stored for 24 h at RT (25 ℃). Particle size, PDI, and zeta potential were determined at various the NaCl solution points (0-200 mM NaCl).

Storage stability
To assess storage stability, fresh ZMNPs were stored for 30 d at 25 ℃. Particle size, PDI, and zeta potential were determined at various time points (0, 7, 15, and 30 days).

In vitro simulated gastrointestinal digestion
This assay was conducted as previously reported, with minor modifications [14,42]. The release properties of CUR from ZMNPs, CZMNPs, and free CUR were investigated under static in vitro conditions. First, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were mixed with ethanol (2:1 v/v) to prepare the release medium. A 4 ml sample was sealed in a dialysis bag with a 10,000 Da molecular cut-off and incubated in a flask containing 30 ml SGF release medium while shaking gently at 37 ℃ for 2 h. The dialysis bag containing the sample was then transferred to a flask containing 30 ml SIF release medium, followed by incubation for 4 h at 37 ℃. Curcumin levels in 200 μl of the release medium were determined to use a Synergy H1 Hybrid Reader (Biotek, Winooski, VT, USA), and an equal amount of fresh release medium was added to the flask to a constant volume.

MTT assay
The present study evaluated the cytotoxic effects of curcumin-loaded nanoparticles through the implementation of the MTT assay. Specifically, A549, MCF-7, HeLa, and HepG2 cells were seeded in 96-well plates at a density of 15 × 10 3 cells/well and subsequently treated with CZMNPs, ZMNPs, matrine (MAR), curcumin (CUR), and a physical mixed group of equal concentration matrine and curcumin (MAR+CUR) at varying concentrations (2.5, 5, 10, 20, 40, or 80 μg ml −1 ) for a duration of 48 h. Following the removal of the post-treatment media, the cells underwent treatment with MTT (5 mg mL −1 ) and were subsequently incubated in a CO 2 environment at 37 ℃ for a duration of 4 h, resulting in the formation of dark blue formazan crystals. The crystals were then dissolved in 150 μl of DMSO and gently shaken for a period of 15 min The absorbance was measured at 570 nm utilizing a multiplate reader [7].

Cellular uptake of CZMNPs in A549 cells
The cellular uptake of free curcumin (CUR) and curcumin-loaded zein/matrine nanoparticles (CZMNPs, Zein/ MAR = 8:1, CUR = 2 mg) was assessed using fluorescence microscopy (Olympus szx16, Olympus, Tokyo, Japan). A549 cells were seeded at a density of 2 × 10 5 cells/well in 6-well cell culture plates and incubated at 37 ℃ in a 5% CO 2 incubator for 24 h. Once cell confluence reached approximately 80%, the original medium was removed from the wells. Subsequently, an equivalent volume of serum-containing medium was introduced to each cell well, comprising CZMNP solution or free curcumin solution, or a blank control group with fresh medium, with a curcumin concentration of 20 μg ml −1 in both solutions. The cells were then incubated at 37 ℃ and 5% C0 2 for 4 and 8 h, respectively, following which the medium was eliminated and washed thrice with PBS under light-proof conditions. Thereafter, the cells were subjected to staining with Hoechst 33258 (10 μg ml −1 ) for 10 min at room temperature while being shielded from light. The cells were then washed with PBS and fixed with 4% paraformaldehyde for 15 min Following this, the cells were washed again with PBS and antifluorescence quencher was added dropwise. Ultimately, the cells were observed under a fluorescent microscope and captured in photographs.

Statistical analysis
The results were expressed as the mean ± standard deviation (SD) for n = 3. The data were analyzed with oneway analysis of variance (ANOVA) and Tukey's multiple comparisons. P < 0.05 was considered statistically significant.

Results and discussion
3.1. Zein/MAR composite nanoparticles The particle size, PDI, zeta potential, and changes in the appearance of zein nanoparticles and zein/MAR composite nanoparticles (ZMNPs) with various mass ratios of zein to MAR are shown in figure 2. Increasing the mass ratio of ZMNPs was associated with a decrease in the average particle size. In figure 2(A) (P < 0.05), when the mass ratio of zein to MAR was 20:1, mean size of the composite nanoparticles were 140.6 ± 3.9 nm, which was much lower than particle size of single zein dispersion (181.6 ± 4.9 nm). The surface charge of MAR was 0, while the zeta potential of the composite nanoparticles (−13.7 ± 1.3 mV) was not reduced and was comparable to that of a single zein dispersion (−13.5 ± 3.7 mV) (figure 2(B)) (P < 0.05). These findings imply that MAR acts within zein to stabilize it and is not exposed on the surfaces of the zein nanoparticles. This is in contrast to most anionic polysaccharides that are coated on zein nanoparticle surfaces via electrostatic interactions [17]. The addition of MAR also helps overcome the aggregation caused by the lack of electrostatic repulsion of the zein nanoparticles. At this point, although the lumpy polymer disappeared, there was a small amount of milky white floc in the suspension and the turbidity deepened (figure 2(C)). This indicates that the aggregation of zein nanoparticles was alleviated, but not enough MAR was adsorbed by nanoparticles.
As the mass ratio increased to 8:1, the size of the composite nanoparticles rapidly decreased again to 110.8 ± 2.3 nm. Zeta-potential increased slightly to −16.6 ± 2.5 mV and the flocs were eliminated. Since then, the turbidity has become increasingly low. Therefore, an appropriate amount of MAR enhances the strength of the interaction between MAR and zein.
At a mass ratio of 4:1, the average particle size was 106.9 ± 4.5 nm, which was comparable to the mass ratio of 8:1. The zeta potential slightly changed to −15.8 ± 6.3 mV, but began to show some instability. We postulate that MAR is adsorbed on the surfaces of some composite nanoparticles or encapsulated in their cavities.
When the mass ratio of zein to MAR was in the range of 8:3 to 1:1, the average zeta potential and particle size of the composite nanoparticles decreased, and the PDI value exhibited an increasing trend. This can be explained by the following factors: First, the presence of excess hydrophilic MAR reduces interfacial tension and maintains the stability of the small-sized nano-system. Secondly, zein nanoparticles are negatively charged when the pH is greater than their isoelectric point (∼6.0). ZMNPs (zein/MAR = 8:1) showed a negative charge similar to zein nanoparticles at the original pH (∼7.0); The zeta potential of ZMNPs (zein/MAR = 2:1) was −30 ± 4.9 mV at the original pH (∼8.4), which was much lower than that of zein nanoparticles. Excess MAR, with a mass ratio >8:1, leads to an increase in basicity of the composite nano-system, thereby decreasing the zeta-potential and increasing the solubility and stability of zein.
However, when the mass ratio of zein to MAR was 1:1, the PDI significantly increased to 0.36, indicating that excess MAR led to a change in the polarity of the solvent environment [43], which is not conducive to the formation of a relatively stable composite nanosystem. At all other ratios, the PDI value was low (PDI 0.20 ; figure 2(A)), indicating that all the formed nano-systems were stable. The particle size distributions (figure 2(D)) exhibited bimodal distributions with particles >1000 nm in diameter when the mass ratios of zein to MAR were 2:1 and 4:3. This is because particles <100 nm tend to aggregate into micelles [44]. When the mass ratio of zein to MAR was in the range of 20:1 to 8:3, a narrow and high single peak was observed, indicating the formation of stable and uniformly sized composite nanoparticles.

Encapsulation and loading properties
The LC and EE were used to assess the basic characteristics of the delivery system. The encapsulation capacities of zein-based composite nanoparticles produced by the anti-solvent precipitation approach were normal at a zein/CUR ratio of 10:1 (w/w) [20,45]. Differences in the encapsulation capacities between ZMNPs and single zein dispersion were better visualized when the zein/CUR ratio was set to an excess value. Thus, the zein/MAR ratio was assessed based on the zein/CUR ratio of 5:1 (w/w). As shown in figure 3, the EE of CZNPs was 2.50% (figure 3(A)) (P < 0.05) and insoluble precipitates was present (figure 3(E)). However, when the zein/MAR ratio was 20:1, the EE of the nanoparticles rapidly increased to 45.84% and the precipitation disappeared. The EE of CZMNPs continued to increase to 52.64% as the ratio increased from 20:1 to 8::1 (w/w). These findings suggest that MAR acts as a stabilizer for CZNPs. As the MAR mass was increased (CZMNPs4:1 to CZMNPs1:1), changes in EE were insignificant, indicating that CZMNPs8:1 enhanced the stability of the system. The corresponding LC values are shown in figure 3(B) (P < 0.05). The presence of MAR significantly increased LC. For CZMNPs4:1 to CZMNPs1:1, there was a marked decrease in the LC. This is because embedded CUR remains essentially unchanged; however, an increase in MAR mass reduces this ratio. Considering these findings and economic principles, a zein/MAR ratio of 8:1 (w/w) was used in the follow-up study.
Owing to the particle size and space limitations of nanoparticles, the addition of excess drugs does not increase the loading rate and may reduce the stability of the delivery system. To determine the optimal loading capacity of CZMNPs8:1, the number of CUR was screened. In figure 3(C) (P < 0.05), differences in EE value when the mass of CUR was increased from 0.25 mg to 2 mg was insignificant, whereas EE significantly decreased when the mass of CUR was 2.5 mg. These findings show that the optimum loading capacity of the system for CUR was 2 mg. Moreover, for samples treated with varying doses of CUR ( figure 3(D)), the LC value increased significantly until the mass of CUR reached 2 mg. This is because with increasing amounts of CUR, the amount of encapsulated CUR increased, while the protein and stabilizer masses remained the same, resulting in an increasing ratio. Based on the above findings, the optimal amount of CZMNPs loaded with CUR was determined to be 2 mg for the subsequent experiments. The size of the CZMNPs was 105.4 ± 4.5 nm while the Figure 3. The encapsulation efficiency (A) and loading capacity (B) of CZMNPs with a Zein/curcumin ratio of 5:1 (w/w) across varying Zein-to-MAR ratios. Additionally, the investigation evaluates the encapsulation efficiency (C) and loading capacity (D) of CZMNPs with different mass ratios of zein to curcumin, based on a fixed Zein/MAR ratio of 8:1 (w/w). The research also analyzes the appearance changes (E) of CZMNPs with a Zein/curcumin ratio of 5:1 (w/w) across different Zein-to-MAR ratios. Finally, the study examines the size distributions (F) of zein/matrine nanoparticles (ZMNPs; Zein/MAR = 8:1) and curcumin-loaded zein/matrine nanoparticles (CZMNPs; Zein/MAR = 8:1; CUR = 2 mg). The data presented are the mean ± SD (n = 3), with error bars representing standard deviation (n = 3). The use of Tukey's multiple comparisons indicates significant differences (p < 0.05) between groups, as indicated by the different letters.
PDI was 0.132. The particle size distribution graph (figure 3(F)) shows that the addition of CUR had no marked effect on the particle size distribution of the ZMNPs, implying that MAR and zein formed a stable system. Zeta potential for ZMNPs and CZMNPs were −16.6 ± 2.5 mV and −24.7 ± 3.5 mV, respectively, suggesting that the higher colloidal stability of CZMNPs may be due to enhanced interactions upon encapsulation, as discussed in section 3.4 for the specific interaction analysis. The EE and LC values of CZMNPs were 88.30% and 7.84%, respectively.
Previously, rhamnoilpid/zein complex nanoparticles were created to load CUR using an anti-solvent coprecipitation approach with an LC < 7% [46]. Zein/carboxymethyl dextrin nanoparticles prepared by antisolvent precipitation showed an LC of less than 3% for CUR [14]. A zein/xanthan gum (heteropolysaccharide) composite nano-system was fabricated by anti-solvent precipitation to load CUR with an LC < 2.5% [17]. Zeinbased ternary systems, such as zein/K-carrageenan/Tween80 NPs and zein/chondroitin sulfate/Spl NPs obtained by anti-solvent precipitation, exhibit low LC [47,48]. The CZMNPs fabricated in this study using MAR as a stabilizer exhibited better encapsulation properties than the zein composite nanoparticles used to produce them, as described above.

TEM
The micromorphology of the samples was assessed by TEM ( figure 4). TEM images of the ZMNPs and CZMNPs prepared to use the inverse solvent precipitation approach showed smooth spherical nanoparticles with no changes in the basic structures of the ZMNPs after encapsulation. Moreover, the average particle size of ZMNPs and CZMNPs measured in TEM field was 95.66 nm and 91.50 nm respectively. The sizes of the ZMNPs and CZMNPs were comparable, which is consistent with the particle size measurements using DLS ( figure 3(D)). Nevertheless, the sizes of the particles in the TEM images were slightly smaller than those determined by DLS, which may be ascribed to nanoparticle swelling in the solution. The size measured by TEM was that of the nanoparticles after natural air drying [49].

FTIR
FTIR is an analytical method for the efficient identification of chemical molecules in a sample by detecting specific vibrations in the chemical bonds of polymers [19]. Therefore, we investigated possible interactions during the formation of ZMNPs and CZMNPs using FTIR spectroscopy. The FTIR spectra of zein, MAR, CUR, CZMNPs, and ZMNPs are shown in figure 5(A).
In the FTIR spectra of MAR, aliphatic C-H stretching and bending vibrations is represented by bands at 2937 and 1440 cm −l , respectively. The C=O stretching vibration typical of lactam functional groups is observed at 1647 cm −l , while bands at 1340 cm −l and 1254 cm −l indicate the presence of characteristic C-N stretching vibrations, in agreement with previous reports [33]. However, after the formation of ZMNPs, the characteristic peaks of pure MAR were not observed in the ZMNPs spectra, and these hidden characteristic peaks suggest that MAR acts between zein or is encapsulated in the nanoparticles. The hydrophilic O-H group stretching band of zein occurs at 3291 cm −1 and represents the O-H stretching vibration. After the formation of ZMNPs, the O-H group stretching band shifted from 3291 cm −1 to 3440 cm −1 , implying a strong hydrogen bond between zein and MAR. When CUR was encapsulated, the O-H stretching vibration of the CZMNPs remained at 3440 cm −1 . These findings show that hydrogen bonding is one of the interactions between zein, MAR, and CUR; hydrogen bonding between zein and MAR is the main driving force for the formation of stable colloids from ZMNPs and CZMNPs.
The characteristic peak of zein at 2925 cm −1 is due to C-H stretching [18]. After the formation of ZMNPs and CZMNPs, the hydrophobic C-H was transferred to 2957 cm −1 and 2959 cm −1 , respectively, with similar peaks. MAR mainly acts between zeins, reducing the hydrophobic interactions between zeins and improving the aggregation of nanoparticles owing to the high hydrophobicity of zeins. Both CUR and zein are hydrophobic compounds; therefore, hydrophobic associations existed between them. The characteristic peaks of zein at 1656 cm −1 and 1542 cm −1 correspond to the amide I and II bands, respectively. The amide I band is highly associated with C-O stretching, whereas the amide II band is highly correlated with C-N stretching and N-H bending [15]. The peak of the amide II band of ZMNPs did not shift, indicating that there were no electrostatic interactions between zein and MAR, which was due to the zero surface charge of MAR. The shift in the amide II band of CZMNPs from 1541 cm −1 to 1594 cm −1 implies the formation of stronger electrostatic interactions between CUR and ZMNPs, indicating enhanced interactions after encapsulation and the formation of a more stable colloid, as discussed in section 3.2, as shown by the increase in the absolute value of the zeta potential after encapsulation.
In the FTIR spectrum of CUR, the peak at 3506 cm −1 was due to the stretching of the O-H of phenol. The stretching vibrations of C=O and C=C were observed at 1628 cm −1 and 1603 cm −1 , respectively. The peak at 1429 cm −1 is associated with C-C-C stretching vibrations; 1284 cm −1 and 1155 cm −1 are specific peaks for chain stretching vibrations between aromatic rings and ketones, respectively; and the peak at 1510 cm −1 is due to mixing vibrations [20]. These peaks disappeared after encapsulation, implying that CUR was effectively encapsulated in the ZMNPs [14]. Furthermore, the primary structures of both the ZMNPs and CZMNPs were unchanged compared to the zein spectra, implying the presence of non-covalent interactions.

XRD
The crystal diffraction information was obtained using XRD. Figure 5(B) shows the XRD patterns of zein, MAR, CUR, ZMNPs, and CZMNPs nanoparticles. There are two broad diffraction peaks for zein at 9°and 19°, indicating the amorphous nature of zein. Compared to zein, the peaks of ZMNPs and CZMNPs nanoparticles at 9°almost disappeared, and the intensity of the peak at 19°was markedly low. This finding suggests that there are non-covalent interactions between zein, MAR, CUR, and maize proteins [14], which was also confirmed by FTIR analysis. Moreover, the sharp diffraction peaks of MAR and CUR indicate a high degree of crystallization. Nevertheless, these peaks disappeared after self-assembly, indicating a transition from crystalline to noncrystalline MAR and CUR. For control purposes, physical mixing of CUR and CZMNPs was studied, and the constituent masses of CUR and CZMNPs were equal. Physical mixing revealed a crystalline peak for CUR, indicating that self-assembly transformed the crystals.

Controlled release of CUR
The release properties of CUR are shown in figure 5(C). Approximately 54.6% of free CUR was explosively and rapidly released after 2 h of SGF digestion, and 35.2% that remained after 4 h of incubation was rapidly released into the SIF. However, the release rate of the encapsulated CUR was significantly low. In particular, 27.3% and 33.8% of CZNPs and CZMNPs, respectively, were slowly released from CUR after 2 h of digestion in the SGF. These findings indicated a controlled release rate after encapsulation. This may be attributed to electrostatic interactions, hydrogen bonding, and strong hydrophobic interactions between CUR and the transfer carrier [40,50]. CZNPs and CZMNPs showed similar trends in CUR release in SGIF, with CZMNPs showing a significantly higher cumulative release rate (66.6%) than CZNPs (59.2%). When a sample does not contain lipids, mixed micelles or vesicles are composed of peptides, bile salts, phospholipids, and undigested soluble proteins [51]. The soluble alkaloid MAR may be involved in micelle or vesicle formation, and may protect CUR in the gastrointestinal environment. Therefore, CZMNPs contribute to the release of CUR with a higher bioavailability. These findings suggest that ZMNPs are effective delivery vehicles for hydrophobic active compounds.

Effects of pH
Composite nanoparticle dispersion is subjected to varying pH levels during processing, storage, and passage through the human gastrointestinal tract. Therefore, we investigated the changes in particle size, PDI, and zeta potential of the nanoparticle dispersion over the entire pH range (2.0-9.0). As shown in figure 6, at pH value of 2.0-3.0 and 7.0-9.0, the particle size of fresh ZMNPs ranged from to 110-130 nm (figure 6(A)) (P < 0.05), the absolute value of zeta potential was higher than 17 mV (figure 6(B)) (P < 0.05), and the PDI was very small (PDI < 0.14), indicating that fresh ZMNPs were colloidal stable in the pH range of 2.0-3.0 and 7.0-9.0. In addition, because the IEP of zein is approximately 6, zein nanoparticles do not form stable dispersion near pH 6.0. Similarly, during the study, as fresh ZMNPs were adjusted to pH 5, it was observed that the dispersion rapidly aggregated into milky flocs deposited at the bottom of the vessel (figure 6(C)) (P < 0.05), resulting in the inability to measure the particle size, PDI, and zeta potential of the nanoparticle dispersion at this pH. This suggests that the IEP of the ZMNPs was 5, which is attributed to the fact that MAR acts between the zein and the surface charge of ZMNPs changes under different pH conditions, thus failing to prevent instantaneous aggregation of nanoparticles caused in the isotropic situation. Notably, the ZMNPs had PDI values of 0.236 and 0.174 at pH 4.0 and 6.0, with a marked increase in particle size (∼250 nm) and a significant increase in dispersion turbidity. Although their zeta potentials were close to zero, they still formed relatively stable nanoparticles. This could be explained by two factors. First, MAR acts as a stabilizer, and the strong hydrogen bonding between MAR and zein overcomes aggregation owing to insufficient electrostatic repulsion among the nanoparticles. Secondly, the addition of MAR resists the aggregation of zein because of its high hydrophobicity caused by the deficiency of basic amino acids, thus promoting the stability and homogeneity of the dispersion.

Effects of salt concentrations
The ionic strength stability of a drug delivery system is an important indicator for evaluating the stability of nanoparticles. We investigated the ionic strength stability of the ZMNPs. The particle size, PDI (figure 6(D)) (P < 0.05), zeta potential (figure 6(E)) (P < 0.05), and appearance (figure 6(F)) (P < 0.05) are shown in figure 5. NaCl concentrations in the range of 0-25 mM had no significant effect on the particle size, PDI, or zeta potential of the ZMNPs. When the concentration of NaCl was in the range of 25-150 mM, the particle size increased (110-320 nm), whereas PDI and zeta potential decreased. However, they still had smaller particle size (145.2 ± 10.6 nm), PDI, and zeta potential (0.078 ± 0.014, −20.3 ± 1.4 mV) when the NaCl concentration was 75 mM. This is because the hydrophilicity of ZMNPs decreases in this ionic strength range [49]. During this process, the turbidity of the solution gradually increases. However, after increasing the NaCl concentration to 200 mM, the particle size of ZMNPs increased to 386.5 ± 18.2 nm, the absolute value of the zeta potential decreased to −15 mV, and the PDI increased (PDI > 0.2). Small deposits were attached to the vessel walls and the nanoloaded system became unstable. Electrostatic interactions between zein molecules are involved in the formation of ZMNPs. When the ionic strength increases to a certain value, the ionic bonds between the polymer chains are broken by shielding effects created by the small-molecular-weight ions, which prevents them from forming stable dispersion [42]. This was confirmed by zeta potential results. The ZMNPs exhibited ionic strength stability at concentrations less than 200 mM.

Effects of temperature
Nanoparticles are frequently exposed to high temperatures during preparation or processing; therefore, the effects of heat treatment on the stability of the ZMNPs were investigated (figure 6). During the heat treatment at 30-90 ℃ for 30 min, the size of the nanoparticles increased with increasing temperature, whereas the average particle size of the composite nanoparticles ranged between 100 and 130 nm. This is because during heat treatment, molecules begin to expand, and the heat causes more molecular collisions and reorganization. The PDI was approximately 0.1, indicating that the nano-system had a good dispersion (figure 6(G)) (P < 0.05). This was due to thermal denaturation of thermodynamically denatured protein at approximately 100 ℃ [39]. Furthermore, the effects of temperature on the zeta potential were insignificant (figure 6(H)) (P < 0.05). In summary, the nano-system prepared to use zein and MAR exhibited a high stability.

Effects of storage
In commercial applications, conveying systems must maintain stability during the shelf life of the products. Thus, we assessed the long-term storage stability of the ZMNPs. Figure 7 (P < 0.05) shows the changes in particle size, PDI, and zeta potential of the composite nanoparticle dispersion with zein and MAR mass ratios of 8:1 and 8:3 after 30 days of storage at room temperature. The particle size of the ZMNPs with zein and MAR mass ratios of 8:1 and 8:3 did not change significantly throughout the storage period (figure 7(A)) (P < 0.05), and there was no precipitation or flocculation. The PDI of zein and MAR mass ratio of 8:1 group was lower during storage than that of the mass ratio of the 8:3 group (figure 7(B)) (P < 0.05), indicating a more uniform particle size distribution and that their PDI was less than 0.2. Therefore, ZMNPs remained stable over time. In addition, zeta potential of zein and MAR mass ratio of 8:1 group was more stable during storage than zeta potential of the mass ratio of the 8:3 group, both of which had absolute charge value greater than 16 mV. This is consistent with the analysis and discussion of the zeta potential in section 3.1. Overall, the groups with zein and MAR mass ratios of 8:1 and 8:3 showed good storage stability; however, the group with an 8:1 ratio performed better during prolonged storage. The ZMNPs can be stored at room temperature to save energy and protect the environment.

In vitro anticancer activities
In order to evaluate the anticancer potential of nanoparticles, four distinct cancer cell lines (A549, MCF-7, HeLa, and HepG2) were subjected to varying concentrations of CZMNPs, ZMNPs, matrine, curcumin, or a physical mixture of matrine and curcumin at equal concentrations (2.5, 5, 10, 20, 40, and 80 μg ml −1 ), and their viability was assessed. The results of the viability assay after 48 h of treatment are presented in figure 8. Notably, treatment with CZMNPs and ZMNPs demonstrated a significantly reduced viability of cancer cells in comparison to matrine, curcumin, and their mixed physical group, with CZMNPs exhibiting greater efficacy than ZMNPs. The utilization of MAR in the stabilization of zein nanoparticles resulted in the acquisition of anticancer properties by the nanoparticles. Consequently, the co-delivery of CUR with ZMNPs exhibited enhanced efficacy in inducing cytotoxicity in vitro against tumor cells. The half-inhibitory concentrations of A549, MCF-7, HeLa, and HepG2 cell lines treated with ZMNPs were 22.06, 63.80, 51.54, and 57.25 μg ml −1 , respectively. Meanwhile, the half-inhibitory concentrations of A549, MCF-7, HeLa, and HepG2 cell lines treated with CZMNPs were 5. 26, 30.78, 19.45, and 20.63 μg ml −1 , respectively. The anti-proliferative effect of CZMNPs on A549 cells was found to be significantly stronger than that observed in the other three tumour cell lines, suggesting that CZMNPs exhibit selectivity towards A549 cells. Treatment with CZMNPs or ZMNPs for 48 h resulted in a concentration-dependent inhibition of A549 cell viability.

Cellular uptake of A549 cells
In order to investigate the cellular uptake of free curcumin and CZMNPs by A549 cells, the intensities of intracellular curcumin fluorescence were examined via fluorescence microscopy. The resulting fluorescence micrographs, depicted in figure 9, illustrate the absence of fluorescence in the control group treated for 0 h. Conversely, a slight increase in fluorescence intensity was observed in cells treated with free curcumin (20 μg ml −1 ) at the 4 and 8 h time points, with the majority of curcumin uptake occurring at the 4 h mark. Within a 4-hour timeframe of CZMNPs exposure to A549 cells, a gradual accumulation of the nanoparticles was observed, manifesting as a limited amount of speckled green fluorescence. However, after 8 h of CZMNPs treatment, a substantial number of converging green fluorescence spots emerged, and the fluorescence intensity increased significantly. This phenomenon implies that the cells may internalize CZMNPs via endocytosis, leading to the formation of intracellular vesicles, which in turn generate small green patches of light. Consequently, the uptake of curcumin via CZMNPs is slower than that of free curcumin. Significantly, the fluorescence intensity of CZMNPs on A549 cells after an 8-hour exposure was observed to be stronger in comparison to free curcumin substrates, indicating a higher accumulation of CZMNPs in A549 cells. Consequently, CZMNPs exhibit enhanced cytotoxicity in contrast to free curcumin.

Conclusion
Spherical ZMNPs of regular shape were successfully synthesized using MAR-stabilized zein. At a zein/MAR ratio of 8:1 (w/w), the ZMNPs exhibited physicochemical stability under varying conditions, including pH ranges of 2.0-4.0 and 6.0-9.0, ionic strengths of 0-150 mM, temperatures of 30 ℃-90 ℃, and storage for 30 days at room temperature. However, the ZMNPs displayed a tendency to precipitate rapidly at pH 5.0, which poses certain limitations and requires further investigation. Notably, the MAR was incorporated into the ZMNPs' shells. The mechanism by which most polysaccharides interact with the surface of zein nanoparticles via electrostatic interactions differs from the process observed in this study. The formation of ZMNPs is primarily driven by hydrogen bonding and hydrophobic interactions. A mere 2.5 mg of matrine is adequate to stabilize 20 mg of zein, which is a significantly lower amount compared to other conventional stabilizers. This results in the formation of small particle size composite nanoparticles. The complex formed by the addition of 2 mg of CUR to the stabilized zein nanoparticles (CZMNPs) exhibits stronger electrostatic interactions. CZMNPs, acquired through anti-solvent precipitation, exhibited superior encapsulation efficacy and loading capacity values of 88.30% and 7.84%, respectively, in comparison to conventional stabilizers for zein. Furthermore, CZMNPs were found to delay the release of CUR in simulated gastrointestinal fluids. Remarkably, CZMNPs demonstrated exceptional anti-proliferative properties against four types of cancer cells, with a selective and potent cytotoxic effect on A549 cancer cells, and a concentration-dependent reduction in cancer cell viability. It is noteworthy that cytosolic uptake may facilitate the uptake of CZMNPs, leading to significant accumulation in A549 cells within a relatively short period of 8 h. This observation underscores the potential of ZMNPs as a promising vehicle for the encapsulation and delivery of hydrophobic reactive substances, thereby expanding the scope of novel stabilizers such as MAR in the realm of delivery systems.

Declaration of competing interest
There are no conflicts of interest to declare.

Ethical compliance
All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.