CN114129538B

CN114129538B - Preparation method and application of crocin-loaded zein nanoparticles

Info

Publication number: CN114129538B
Application number: CN202111250924.3A
Authority: CN
Inventors: 龚金炎; 刘家源; 宋恭帅; 王淇; 王丹丽; 濮发祥; 楚秉泉; 李玲; 肖功年
Original assignee: Zhejiang Lover Health Science and Technology Development Co Ltd
Current assignee: Zhejiang Lover Health Science and Technology Development Co Ltd
Priority date: 2021-10-26
Filing date: 2021-10-26
Publication date: 2022-12-09
Anticipated expiration: 2041-10-26
Also published as: CN114129538A

Abstract

The invention relates to a preparation method of crocin-loaded zein nanoparticles, which comprises the following steps of: 1) Preparing zein stock solution; 2) Preparing crocin-loaded hollow zein nanoparticles; 3) And (3) preparation of the crocin-loaded LbL hollow DS/CH-coated zein nanoparticles. The crocin-loaded hollow dextran sulfate/chitosan (DS/CH) -coated zein nanoparticles are manufactured by a layer-by-layer self-assembly technology, so that the crocin-loaded hollow dextran sulfate/chitosan (DS/CH) -coated zein nanoparticles have better controlled-release behavior and stronger antioxidant activity, and in addition, the LbL self-assembled crocin-loaded hollow DS/CH-coated zein NPs can be developed into a promising delivery system of an effective AD treatment method, and a novel practical technology is provided for delivering bioactive components.

Description

Preparation method and application of crocin-loaded zein nanoparticles

Technical Field

The invention belongs to the technical field of functional composite nanoparticle preparation, and particularly relates to a preparation method and application of crocin-loaded zein nanoparticles.

Background

Gardenia yellow pigment extracted from gardenia fruits is one of the main natural colorants, and is widely used as a food colorant worldwide, particularly in east asian areas. The research finds that the crocin is the main component of the gardenia yellow pigment. Crocin is a unique water-soluble carotenoid, is considered as one of the main bioactive substances, has remarkable beneficial functions, and mainly comprises the effects of resisting diabetes, easing pain, reducing blood pressure, resisting toxin, reducing blood sugar and resisting oxidation. In addition, some studies report that crocin has the potential to enhance memory and learning, as well as protect brain cells. Crocin is considered to be an ingredient effective in preventing or treating Alzheimer's Disease (AD). AD is a dangerous neurodegenerative and age-related chronic disease that involves primarily beta amyloid (A β) in the brain _1-42 ) And abnormal clearance and accumulation of various proteins such as tau protein.

Despite these active functions, crocin is still limited in its use due to its high instability and sensitivity to processing conditions. Crocin has low stability to pH changes, oxidative and thermal stress, poor absorption and low bioavailability due to hydrolysis and rapid elimination of endogenous β -glucosidase after oral administration. Recently, many studies have been made on encapsulation of bioactive substances. For example, NPs for encapsulation have been widely used in the food industry, mainly including bioactive carrier systems, fat substitutes, and the like. NPs are generally in the size range of 50-150nm and can easily enter cells. Layer-by-Layer self-assembly (LbL) technology, heteropolymerization and direct mixing technology are three common methods for preparing nanoparticles. Among them, the LbL technique has been approved. This technique, formed by electrostatic attraction between polycations and polyanions, can be used to control the composition, permeability, and thickness of protein-polysaccharide membranes. A multilayer film is formed on the surface of the nanoparticles by covalent binding of the polysaccharide to the protein supported on the oppositely charged droplets. NPs obtained by LbL self-assembly have superior properties in terms of chemical stability, rheological behavior and microstructure.

Food proteins (e.g., animal-derived proteins and plant-derived proteins) are widely used as bioactive ingredient delivery materials due to their remarkable functional properties. Plant-derived proteins are immunogenic and less expensive than animal-derived proteins. Hydrophobic plant-derived proteins (e.g., gliadins and zeins) are reported to have the ability to produce sustained release particle carriers, suggesting that nanoparticle formation may be possible without the use of toxic chemical crosslinkers. Zein is a prolamin extracted from corn and has been widely used in encapsulation and delivery systems for bioactive lipids, food colors, essential oils, and other functional ingredients. The zein-based nanoparticles appear to be uniformly spherical in the aqueous phase due to unique self-assembly properties and hydrophobicity. Wang et al prepared citral and lime flavored loaded nanoparticles via self-assembled core-shell structures of zein, which is critical for encapsulation purposes in the cosmetic, pharmaceutical and food industries. However, encapsulation and controlled release of crocin-loaded hollow zein NPs have not been reported.

Disclosure of Invention

Aiming at the problems in the prior art, the research of the invention aims to prepare the crocin-loaded hollow zein nanoparticles by an LbL self-assembly technology. The prepared nanoparticles were then characterized by particle size, zeta potential, structure and morphology.

Another objective of the present invention is to evaluate the antioxidant activity and controlled release properties of crocin loaded nanoparticles during in vitro digestion, respectively, and to evaluate the effect of crocin containing nanoparticles on AD cell models.

The invention is realized by the following technical scheme:

the preparation method of the crocin-loaded zein nano-particles is characterized by comprising the following steps of:

1) Preparing zein stock solution: dispersing zein powder into 70% ethanol water solution, heating under continuous magnetic stirring, centrifuging, and filtering supernatant to obtain zein stock solution;

2) Preparing the crocin-loaded hollow zein nano particles: adding crocin powder and the zein stock solution prepared in the step 1) into 70% ethanol aqueous solution according to a certain mass ratio, stirring for 30min at room temperature to obtain mixed solution, slowly injecting sodium carbonate ethanol aqueous solution into the mixed solution, continuously stirring for 30min at room temperature to coat zein with sodium carbonate particles to obtain reaction solution, and then mixing the reaction solution with Milli-Q water for reaction for 10min to prepare the hollow zein nano particles loaded with crocin;

3) Preparation of crocin-loaded LbL hollow DS/CH coated zein nanoparticles: mixing the DS aqueous solution with the crocin-loaded hollow zein nanoparticles prepared in the step 2), and continuously magnetically stirring for 20min at room temperature to obtain a DS-coated zein nanoparticle mixed solution; then mixing the DS-coated zein nanoparticle mixed solution with the CH solution, continuously magnetically stirring for 20min at room temperature, and repeating the same procedure twice to prepare two bilayers on the surface of the crocin-loaded LbL hollow zein nanoparticle;

4) Vacuum freeze drying the crocin-loaded LbL hollow DS/CH-coated zein nanoparticles prepared in the step 3) in an aqueous solution to obtain crocin-loaded zein nanoparticles.

Further, in the step 1), the feed-liquid ratio of the zein powder to the ethanol water solution is 1g/20ml, the heating temperature is 70 ℃, the heating time is 30min, and the centrifugation conditions are as follows: centrifuge at 1000rpm for 20min.

Further, the certain mass ratio in the step 2) is that the mass ratio of the crocin powder to the zein powder is 1.

Further, the mass concentration of the sodium carbonate ethanol aqueous solution in the step 2) is 2%, and the addition amount of the sodium carbonate ethanol aqueous solution is that the volume ratio of the 2% sodium carbonate ethanol aqueous solution to the zein stock solution is 1.

Further, the mixing mass ratio of the DS aqueous solution to the crocin-loaded hollow zein nanoparticles in the step 3) is as follows: the mass ratio of the zein powder to the dextran sulfate is 1-4.

Further, the DS aqueous solution in the step 3) is mixed with the crocin-loaded hollow zein nanoparticles under the condition that the pH value of the mixed solution is 4.0.

Further, the concentration of the CH solution in the step 3) is 1.0mg/mL, the pH value is 4.0, and the using amount of the CH solution is as follows: the mass ratio of chitosan to dextran sulfate is 1.

The crocin-loaded LbL hollow DS/CH-coated zein nano-particles are applied to the treatment of Alzheimer's disease.

The preparation method can realize effective embedding of the crocin, and the hollow DS/CH-coated zein nano-particles loaded with the crocin are successfully prepared by LbL self-assembly based on electrostatic interaction, hydrogen bond and hydrophobic interaction by using sodium carbonate as a sacrificial template. Under the optimal mass ratio of zein/DS 2.

Drawings

FIG. 1 is a graph of the effect of zein to DS mass ratio on EE, PDI and particle size (A) and zeta potential (B) of hollow DS/CH coated zein NPs;

fig. 2 is a 3D view of TEM (a, B), AFM (C, D) and AFM (E, F) images of crocin-loaded hollow zein NPs and crocin-loaded LbL hollow zein NPs;

FIG. 3 is FTIR spectra of CH (A), DS (B), zein (C), free crocin (D), crocin-loaded hollow zein NPs (E) and crocin-loaded LbL hollow zein NPs (F);

FIG. 4 is TEM images of nanoparticles in gastric (A) and intestinal (B) simulations, and the release rate of nanoparticles in the simulated gastrointestinal environment (C);

FIG. 5 shows OH clearance (%), fe2+ chelating activity (%) and DPPH clearance (%) of crocin-loaded LbL NPs. Different letters indicate significant difference (p < 0.05);

figure 6 is the cell viability of SH-SY5Y cells treated with different concentrations of free crocin, crocin loaded NPs and crocin loaded LbL NPs. Different letters indicate significant difference (p < 0.05);

FIG. 7 shows SH-SY5Y cell morphology after different sample treatments ((magnification 200X. A, untreated SH-SY5Y cells; B, ATRA differentiated SH-SY5Y cells; C, ATRA + BDNF differentiated SH-SY5Y cells; D, A β 1-42 treated differentiated SH-SY5Y cells; E, donepezil treated differentiated SH-SY5Y cells as a positive drug control group; F, free crocin treated differentiated SH-SY5Y cells; G, differentiated SH-SY5Y cells treated with crocin-loaded NPs; H, differentiated SH-SY5Y cells treated with LbL NPs-loaded crocin, I, PBS treated differentiated SH-SY5Y cells as a negative control group;

FIG. 8 shows the levels of A.beta.1-42 in differentiated SH-SY5Y cells treated with different samples. Different letters indicate significant differences (p < 0.05). P control: a positive control group; n control, negative control group; NPs, crocin-laden NPs; lbL NPs, crocin-loaded LbL NPs.

Detailed Description

The present invention will be further described with reference to the following specific examples to better understand the technical solution.

Materials and reagents: saffron protein (A)>99%), zein, dextran sulfate (DS, 500 kDa), porcine gastric protein from the gastric mucosaEnzyme (enzyme activity 250U/mg protein), all-trans retinoic acid (ATRA), brain-derived neurotrophic factor (BDNF)), and Abeta _1-42 Donepezil, pancreatin from porcine pancreas (enzyme activity of 24U/mg lipase), uranyl acetate, hydrogen peroxide, and Phosphate Buffered Saline (PBS) were purchased from Sigma Chemical co. SH-SY5Y human neuroblastoma cells were supplied by BenA Culture Collection, beijing, china. Chitosan (CH, molecular weight 190-310kDa, viscosity potential 200-800cP, degree of deacetylation 85%), sodium carbonate, salicylic acid-ethanol, ferrous chloride, ethanol, naOH, HCl and analytically pure acetic acid were provided by the national pharmaceutical group chemical agents, inc. (Shanghai, china). Dulbecco's Modified Essential Medium (DMEM), fetal Bovine Serum (FBS), bovine Serum Albumin (BSA), penicillin and streptomycin were supplied by Thermo Fisher Scientific (SanFrancisco, calif., USA). Ultrapure water (18.2 M.OMEGA.. Multidot.cm) was produced by a Milli-Q system (Millipore, MA, US).

Example 1: preparation of hollow zein nanoparticles

Zein stock solution is prepared by dispersing 5g protein powder into 100mL 70% (v/v) ethanol aqueous solution, heating at 70 deg.C for 30min under continuous magnetic stirring, and centrifuging at 10,000rpm for 20min. Filtering the obtained supernatant with 0.45 μm syringe filter to remove insoluble particles, i.e. zein stock solution. Using 2% sodium carbonate ethanol water solution as a sacrificial template, slowly injecting sodium carbonate solution into the zein stock solution according to the volume ratio of 1. Then, the reaction solution was mixed with Milli-Q water at room temperature for 10min. Water can dissolve the sodium carbonate and produce hollow zein nanoparticles.

Example 2: preparation of crocin-loaded hollow zein nanoparticles

Adding crocin powder into 70% (v/v) ethanol water solution according to the mass ratio of crocin/zein of 1. Then, a sodium carbonate solution was slowly injected into the above solution, and stirred at room temperature for 30min to coat the zein on the sodium carbonate particles. Then, the reaction solution was mixed with Milli-Q water at room temperature for 10min to prepare crocin-supported hollow zein nanoparticles.

Example 3: preparation of crocin-loaded LbL hollow zein nanoparticles

The DS aqueous solution (1.0 mg/mL) was continuously magnetically stirred at room temperature for 20min with different zein/dextran sulfate mass ratios (4. To prepare the first layer of LbL self-assembly, the solution pH was adjusted to 4.0 by 1.0M HCl. The mixture was then mixed with CH solution (1.0 mg/mL, pH 4.0) at a mass ratio of 1. Two bilayers of the surface of crocin-loaded LbL hollow zein NPs were prepared by repeating the same procedure twice. The prepared LbL hollow zein NPs carrying the crocin are frozen and dried into powder in water solution in vacuum.

Test examples

Encapsulation efficiency: encapsulation Efficiency (EE) of the crocin-loaded hollow zein NPs, free crocin in the supernatant is obtained by high speed centrifugation and low temperature (16,000rpm, 4 ℃) for 30min. The crocin content was analyzed at 440nm using a UV-visible spectrometer (Infine 200PRO, TECAN, switzerland). The calculation formula of EE is:

particle size and zeta potential: the mean particle size, polydispersity index (PDI) and zeta potential of the samples were measured under dynamic light scattering using a Malvern zetasizer (Malvern Instruments ltd., malvern, UK). Each measurement was performed in triplicate.

Fourier Transform Infrared (FTIR) spectroscopy: FTIR of the samples was performed by a spectrophotometer (Nicolet 5700, thermo Electron Co., waltham, mass., USA) by KBr particle technology. The spectrophotometer was purged with a continuous flow of dry air. The scanning speed is 64 times per second, and the scanning range is 4000 to 400cm ^-1 。

Projection Electron microscope (TEM) morphological analysis of crocin-loaded hollow zein NPs was performed using TEM (JEOL Jem-2100F, thermo scientific, MA, USA). A drop of the NPs suspension was placed on a copper mesh and dried by blotting with blotter paper. The sediment was negatively stained with 1% (w/w) uranyl acetate solution for 30s and then dried again. Excess acetate solution was removed and the stained sample was observed.

Atomic Force Microscopy (AFM): the surface morphology of the nanoparticles was observed by AFM images using a digital multimode V scanning probe microscope (Veeco Metrology, USA). The samples were sonicated, deposited on freshly cut mica substrates and dried in air. Using Si ₃ N ₄ The probe was scanned by AFM over different areas of the mica.

In vitro simulated digestion: the kinetic release profile of crocin was studied in vitro in mimicking the gastrointestinal tract, and the prepared NPs were adjusted to pH 2.0 by addition of HCl (1.0M) and incubated at 37 ℃ for 30min under continuous magnetic stirring. Then, an aqueous solution of porcine pepsin (15 mg/mL) was added to the above mixture as Simulated Gastric Fluid (SGF). After digestion for 2h, pancreatin aqueous solution (30 mg/mL) was added to the gastric digestive juice as Simulated Intestinal Fluid (SIF). The pH of the mixture was adjusted to 7.2 with NaOH (1.0M) and reacted at 37 ℃ for 120min. Samples were collected at designed time intervals (10, 20, 30, 60, 90 and 120 min) for SGF and SIF, respectively. The sample was then removed in volume and replaced with an equal volume of fresh simulated fluid. The release rate of crocin during in vitro digestion was calculated using the equation.

Potential biofunctional activity assay for NPs

Antioxidant activity: based on the in vitro simulated digestion test, the antioxidant activity of the crocin-loaded LbL hollow zein NPs in the 120min reaction process at 37 ℃ in the SIF environment is determined. The reaction solution was removed at a series of time intervals (30, 60, 90 and 120 min) for analysis. In the present study, three methods were used to measure the antioxidant activity of nanoparticles, namely hydroxyl radical scavenging activity, DPPH scavenging activity and ferrous ion chelating activity.

Hydroxyl radical scavenging activity: hydroxyl radical (. OH) scavenging activity was measured according to the reported method [22]. Briefly, the sample was mixed with salicylic acid-ethanol (6 mM) and ferrous chloride (9 mM), then hydrogen peroxide (8.8 mM) was added to start the reaction and incubated at 37 ℃ for 30min. The absorbance of the solution was measured at 510 nm. Hydroxyl radical scavenging activity is expressed as clearance rate and calculated using equation 3:

DPPH scavenging activity: superoxide anion radical (. O) was measured _2- ) The activity was cleared by mixing the sample with a DPPH solution (0.1 mM). The mixture was then incubated in the dark at 37 ℃ for 30h and the absorbance was measured at 517 nm. The radical scavenging activity was calculated by the formula:

iron ion chelating activity: the iron-reducing ability of the sample was determined [24 ]]. The samples were mixed with ferrous chloride (20 mM) and incubated at 25 ℃ for 30min. Then, ferriazine (5 mM) was added to the above mixture and incubated at 25 ℃ for 10min. Then, the absorbance of the solution was measured at 560 nm. Fe ²⁺ Chelation activity was calculated using the equation:

in vitro cytotoxicity assay: SH-SY5Y cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS and 100U/ml penicillin/streptomycin. Cells are placed at 5% CO ₂ And humidified incubator (MCO-15AC, sanyo Electric Co., osaka, japan) at 37 ℃. Cytotoxicity of prepared samples was determined and evaluated by methylthiazolyl diphenyl tetrazolium bromide (MTT)And (4) sex. Three sample solutions (free crocin, empty hollow zein NPs and crocin loaded hollow zein NPs) were prepared in supplemented DMEM medium and cytotoxicity was measured at 10, 30, 50, 70 and 90 μ g/mL crocin SH-SY5Y cells (approx. 2X 10 ⁴ Cells/well) were placed in 96-well plates for 24h. After removal of the medium, the sample was added and incubated for 24h. The medium was discarded and the cells were washed 3 times with PBS. Thereafter, MTT solution (100 μ L,0.5 mg/mL) was added to each well and incubated at 37 ℃ for 4h to form purple formazan crystals. The solution was then removed and the formazan crystals formed dissolved by addition of DMSO (100 μ L,0.5 mg/mL) and incubated for 20min with continuous magnetic stirring. The absorbance was measured at 570 nm. The percentage of cell viability was calculated by the equation:

AD model cell morphology change: differentiation was initiated when SH-SY5Y cells reached 70% -80%. Untreated controls were harvested at this stage, while differentiated groups were treated with 10mM ATRA in medium (0 d). The medium was changed and ATRA was replenished every two days. At 5d, cells were washed 3 times with PBS and incubated in serum free medium supplemented with BDNF (50 ng/mL). BDNF stock solution (100. Mu.g/mL dissolved in 0.1% BSA solution was prepared (de Medeiros et al, 2019). Medium was changed every two days to supplement BDNF. Cell differentiation was completed at 11d and a neuron-like phenotype was obtained, the differentiated cells were treated with A.beta. _1-42 (2.0 mM) for 24h and collected as AD model cells.

According to the above cytotoxicity results, three sample solutions (free crocin, hollow zein NPs and crocin-loaded hollow zein NPs, positive drug control group (donepezil) and negative control group (PBS) were prepared, respectively, the obtained AD model cells were further treated with three samples in the medium for 24h before cell collection, morphological changes of SH-SY5Y cells at the culture and differentiation stages were observed by Zeiss Axiovert 200 inverted fluorescence microscope (Carl Zeiss, oberkochen, germany),and recorded using AxioVisionLE (Carl Zeiss). In addition, SH-SY5Y cells were assayed for A.beta.using an ELISA kit according to the instructions _1-42 And (4) concentration.

Statistical analysis: data were processed using Microsoft office software, origin 8.0, and IBM SPSS Statistics (version 21.0). Statistical analysis was performed by analysis of variance (ANOVA). All the above experiments were repeated at least three times.

Test results

Preparation of hollow DS/CH-coated zein NPs As shown in FIG. 1, the changes of EE, PDI, particle size and zeta potential of crocin-loaded hollow DS/CH-coated zein NPs at different zein/DS mass ratios can be seen. First, the particle size of NPs increased significantly due to the increased DS and CH loading on the surface of zein NPs (FIG. 1A). After the highest value (about 456 nm) was obtained at a zein to DS mass ratio of 4, a sharp drop occurred when the DS and CH mass were further increased, probably due to steric hindrance, coulombic force and electrostatic repulsion. In addition, the same trend of variation can be seen in PDI. At a zein/DS mass ratio ranging from 4 to 1, the NPs have PDI values less than 20%, indicating that the particle size of the NPs is uniform. The EE change of NPs was relatively significant (p < 0.05) with increasing DS and CH quality. When the mass ratio of the zein to the DS is 2. The results show that crocin encapsulated within hollow zein nanoparticles is stable. Due to thermal denaturation at 70 ℃ for 30min, native zein unfolds some of the alpha-helical structure by breaking hydrogen bonds and forms a beta-sheet conformation by rearrangement and recombination, which may provide a complexing site for crocin.

It was found that electrostatic attraction is essential for the formation of protein/polysaccharide complexes. As shown in FIG. 1B, the zeta potential values of NPs are all positive. The anionically charged DS was used as the first layer because it can interact electrostatically with cationic zein surfaces and form a negative charge density on the uncoated zein NPs surfaces. Then, the cation CH is used to create a second layer due to electrostatic attraction. The outermost layer of crocin-loaded hollow DS/CH-coated zein NPs is generated by CH, which determines that the charge of NPs is positive. CH has good adhesion and permeability due to the presence of primary amino groups with cationic properties. As the mass of DS increases, the zeta potential of the NPs decreases, indicating that anionic DS is deposited on the surface of the NPs. The results show that the successful preparation of LbL DS/CH coatings of zein nanoparticles facilitates electrostatic interactions between anionic DS and cationic CH. Based on the analysis results, hollow DS/CH coated zein NPs with a zein/DS mass ratio of 2 were selected for the following study.

And (3) morphological observation: TEM and AFM images were used to assess whether the crocin-loaded hollow zein (crocin-loaded NPs) coated DS/CH and the microstructure differences between the crocin-loaded NPs and the crocin-loaded LbL hollow zein NPs (crocin-loaded LbL NPs), as shown in fig. 2. The crocin loaded NPs (fig. 2A) show dark rings, representing the capsule shell. The results are consistent with those reported for hollow zein NPs. After coating with DS and CH (fig. 2B), the core-shell structure is clearly visible from the dark edge corresponding to the vesicles. The NPs shell thickness and particle size were both large compared to crocin loaded NPs due to hydrodynamic swelling of the layers in aqueous solution.

From atomic force microscope images, by comparing surface roughness (R) _ms ) The surface roughness of the nanoparticles was observed. As shown in FIG. 2C, the surface of the crocin-loaded NPs is relatively smooth, R _ms =6.98nm. After coating with DS/CH, the surface roughness of crocin-loaded LbL NPs is increased (R) _ms =21.70 nm). Fig. 2F is a 3D view of an AFM image. From the images, the average height of the crocin-loaded LbL NPs was about 140.80nm, corresponding to 3.88 times the thickness (36.20 nm) of the crocin-loaded NPs. NPs are relatively well distributed, consistent with the reference to LbL NPs profile. Morphological observation and analysis results preliminarily show that the crocin-loaded LbL hollow zein NPs are prepared.

FTIR analysis: FTIR spectroscopy is used to explore the vibrations of functional groups present in macromolecules as well as molecular structural changes and secondary structures of substances. CH (sample A), DS (sample B), zein (sample B)C) Characteristic transmission bands (sample F) of free crocin (sample D), crocin-loaded hollow zein NPs (sample E) and crocin-loaded LbL hollow zein NPs are 4000-400cm ^-1 The range was plotted in FIG. 3. The spectrum of CH was 1655cm according to the previous study ^-1 、1590cm ^-1 、1415cm ^-1 、 ^-1 ,1375cm ^-1 And 1150cm ^-1 Respectively due to stretching vibration, N-H bending vibration, C-O-C, CH of amide I ₃ And C-N and C-O are stretching vibrations due to the presence of carbonyl and aliphatic groups. By analyzing the spectrum of DS (FIG. 3B), at 1230cm ^-1 、1020cm ^-1 、980cm ^-1 And 815cm ^-1 With SOO ^- And S-O-S, asymmetric and symmetric stretching vibrations. For natural zein, the characteristic band is 1650cm from literature report ^-1 (stretching vibration of amide I) and 1535cm ^-1 (NH bending and stretching CN from amine II.) FTIR spectra of free crocin at 1700cm ^-1 、1220cm ^-1 And 1080cm ^-1 There are characteristic bands corresponding to C-O stretching vibration of the ester or acid groups, as well as C-O and O-H, which are consistent with the Rahaiee et al report.

The spectrum of sample E did not change significantly compared to the FTIR spectrum of natural zein, indicating that the structure of zein was not significantly altered by free crocin. This may prove that free crocin is complexed in the hydrophobic region of zein. After formation of LbL NPs (sample F), the hydrogen band was 3600cm due to the interaction between hydroxyl groups in DS and CH and amide groups in glutamine ^-1 To 3250cm ^-1 A slight shift in range occurred. Sample F at 1020cm due to the coating of DS and CH ^-1 And 805cm ^-1 There is a characteristic band. Furthermore, 1250cm ^-1 The band strength (due to C-N stretching vibration) is enhanced, suggesting that the interaction of DS and CH may be related to the carboxyhydroxy group in CH, the hydroxy group in crocin, and the carbonyl group in amide bond in zein. It has been reported that the combination of CH and zein through electrostatic interaction leads to the appearance of characteristic peaks of amide IA blue shift was apparent. The results reported are consistent with the results of this study. In this study, FTIR analysis results further confirmed that the surface of the crocin-loaded LbL hollow zein NPs was due to the deposition of polyelectrolytes.

Controlled release of crocin: in oral nanocarrier delivery systems, the release profile of the active substance from the biodegradable NPs is generally biphasic, since the release rate depends on the extent of penetration of water into the polymer matrix. It was found that a small fraction of the material adhered to the shell surface rather than being supported in the core. Morphological changes of crocin-loaded LbL NPs during simulated gastrointestinal digestion were observed by TEM images. As shown in fig. 4A, the SGF digested NPs were much larger in particle size, the structure became fluffy, while the core-shell structure was still visible. After SIF digestion (FIG. 4B), the core-shell structure of NPs could not be observed, the particle size was small, and NPs were significantly digested. The size of the digested NPs was about 75nm, which is consistent with the reported conclusion that the size of the nanoparticles is typically in the range of 50-150nm and can be easily accessed into cells. The results may be due to the influence of the pH environment during simulated gastrointestinal digestion. The morphology of the nanoparticles was also found to change significantly in the simulated digestive environment by wear et al. The in vitro release profile of the crocin loaded LbL nanoparticles is shown in figure 4C. It can be seen that the in vitro release pattern of crocin is a rapid burst followed by a controlled release. During gastric mimicking, 60.75% of free crocin was dispersed into SGF due to the rehydration process of the hydrophilic compound, the rest was completely released into SIF, which is consistent with previous reports. The rate of NPs release is slower than free crocin due to the weaker interaction between biopolymer and crocin. Compared to crocin loaded NPs, crocin loaded LbL NPs were released at lower rates (p < 0.05). As the combination of zein encapsulation and DS/CH coating can improve the availability of crocin. The presence of DS/CH in NPs buffers the pepsin degradation of zein, which helps to delay the release of crocin. Furthermore, the controlled release behavior of crocin-loaded LbL NPs was superior during intestinal simulation than during gastric simulation, probably due to the increased electrostatic interaction between DS/CH and zein as pH increased. The results indicate that DS/CH coated zein NPs are effective in increasing the bioavailability of crocin and achieving targeted release before delivery to the gut by limiting digestive disruption in the stomach.

In vitro antioxidant activity: scavenging activity with OH and DPPH free radicals and Fe ²⁺ Chelation capacity the in vitro antioxidant activity of crocin-loaded LbL NPs was evaluated. As shown in fig. 5, the antioxidant activity of free crocin against OH and DPPH radicals decreases with the time of digestion due to its easy degradation and instability in simulated digestion environment. At the beginning of the SIF environment, the free crocin scavenging of OH and DPPH free radicals was 75.75% and 62.91%, respectively, corresponding to 6.17 and 3.25 times the LbL NPs loaded with crocin. The clearance of the crocin-loaded NPs is slightly higher than that of the crocin-loaded LbL NPs (p)<0.05). In contrast, the antioxidant activity of NPs against. OH and DPPH. Free radicals increases and then decreases. After reacting for 90min, the oxidation resistance activity of the LbL NPs loaded with crocin is the highest. The result shows that the crocin can be decomposed in SIF environment, and the DS/CH layer has obvious protective effect on the saffron. In addition, FRAP has a similar trend of change. After 120min of digestion, FRAP values for free crocin decreased from 68.07% to 46.49%, while FRAP values for crocin-loaded NPs and crocin-loaded LbL NPs increased from 14.27% to 59.17% and 16.35% to 61.71%, respectively. According to previous reports, the difference in antioxidant activity between NPs and free crocin may be due to the exposure of many aromatic amino acids such as tryptophan, phenylalanine and tyrosine in zein by heat induction technology, which enhances the antioxidant capacity of proteins. In addition, it was found that the active amino and hydroxyl groups in CH may be involved in free radical scavenging and contribute to antioxidant activity.

Cytotoxicity: the potential cytotoxicity of crocin-loaded LbL NPs was assessed using MTT method. We selected SH-SY5Y cells as an in vitro AD model. Currently, there are many common in vitro models available for AD studies. Such as cell lines that lack the proper neurite structure and many of the features that define neurons (e.g., mature neuronal markers). However, differentiated SH-SY5Y cells can overcome this limitation. SH-SY5Y cells originate from a neuronal lineage in the sympathetic nervous system at an immature stage and express human proteins. As shown in figure 6, cell viability after treatment with free crocin, crocin-loaded NPs and crocin-loaded LbL NPs was dose dependent. With increasing concentration, cell viability gradually decreased. Of the three samples tested, free crocin was the most viable (88.21%), followed by crocin-loaded LbL NPs (86.75%) and crocin-loaded NPs (84.96%). The difference in cell viability between free crocin and crocin loaded LbL NPs and crocin loaded NPs was significant (p < 0.05). In addition, the positive charges of the three samples readily bound to negatively charged SH-SY5Y cells due to electrostatic adsorption. CH is recognized as a common ingredient in foods with safety and good biocompatibility. The cytotoxicity found in the higher concentration samples may be due to interactions upon contact with SH-SY5Y cells. Nunes et al found that the cytotoxicity of SH-SY5Y cells could increase with increasing sample concentration due to the absence of a protective layer. Therefore, further studies were carried out using the crocin-loaded LbL NP at a concentration of 10. Mu.g/mL

Morphological characteristics of AD cell models: studies have demonstrated that SH-SY5Y cells can differentiate and acquire mature neuronal-like characteristics, which can be used as an in vitro model for neurodegenerative disease studies (e.g., AD). Expression of mature neuron markers, decrease in proliferation rate, formation and expansion of neuronal synapses, etc. in differentiated SH-SY5Y cells are developed. The combination of ATRA and BDNF is a common neuronal differentiation method. It was reported that by adding ATRA, the growth of the neurosynaptic and the expression of the neuronal markers were enhanced. In addition, many studies have found BDNF to be critical for the differentiation of cholinergic phenotypes in brain development. In this study, SH-SY5Y cells treated with the sample were imaged using a Zeiss Axiovert 200 inverted microscope. FIG. 7A is an image of untreated SH-SY5Y cells. After treatment of 5d with ATRA, SH-SY5Y cells (fig. 7B) had a reduced proliferation rate and more polar morphology, as the cell bodies extended longer and the network began to form. Upon further maturation with BDNF, the cells (fig. 7C) migrated into clusters, and the cell network became increasingly complex, resembling mature neurons.

These mature neurons are further treated with a β _1-42 Processed to obtain an AD model. According to the existing report, the optimal A beta for inducing AD cell model _1-42 The concentration was 2.0. Mu.M. As shown in FIG. 7D, the cell morphology data also shows that A.beta. _1-42 The treated group had small cell volume, reduced neurosynaptic withdrawal, low density, and irregular shape. In contrast, the cell density, size and shape after free crocin treatment (fig. 7F) were similar to the positive control (fig. 7E), indicating that crocin has similar AD pathology improvement effect as donepezil. The results initially indicate that free crocin and nanoparticles can alleviate the pathology of AD, but the effect of crocin-loaded NPs and crocin-loaded LbL NPs on AD is relatively low. This may be due to the controlled release of crocin by the DS/CH coated zein NPs, as well as the potential cytotoxicity of the DS/CH layer.

As shown in FIG. 8, A.beta.in cell samples _1-42 The difference in concentration is significant (p)<0.05). A beta of cells after donepezil and free crocin treatment _1-42 The lowest concentration (101.72 pg/mg and 123.54pg/mg, respectively) indicates that crocin can induce A beta _1-42 And (4) degrading. Furthermore, crocin-loaded LbL NPs have been shown to have potential effects in alleviating AD progression as a co-targeted drug delivery system. Although the results do not show a great difference between crocin loaded LbL NPs and crocin loaded NPs in alleviating AD pathology, the controlled release, in vitro antioxidant activity and cytotoxic properties of crocin loaded LbL NPs are superior.

Claims

1. A preparation method of crocin-loaded zein nanoparticles is characterized by comprising the following steps:

2) Preparing the crocin-loaded hollow zein nano particles: adding crocin powder and the zein stock solution prepared in the step 1) into 70% ethanol aqueous solution according to a certain mass ratio, stirring for 30min at room temperature to obtain mixed solution, slowly injecting the sodium carbonate ethanol aqueous solution into the mixed solution, continuously stirring for 30min at room temperature to coat zein with sodium carbonate particles to obtain reaction solution, and then mixing the reaction solution with Milli-Q water for reaction for 10min to prepare hollow zein nanoparticles loaded with crocin;

3) Preparation of crocin-loaded LbL hollow dextran sulfate/chitosan-coated zein nanoparticles: mixing a dextran sulfate aqueous solution with the crocin-loaded hollow zein nanoparticles prepared in the step 2), and continuously magnetically stirring for 20min at room temperature to obtain a dextran sulfate-coated zein nanoparticle mixed solution; then mixing the zein nano-particle mixed solution coated by the dextran sulfate with the chitosan solution, continuously magnetically stirring for 20min at room temperature, and repeating the same procedure twice to prepare two double layers on the surfaces of the crocin-loaded hollow zein nano-particles;

4) Vacuum freeze drying the crocin-loaded LbL hollow dextran sulfate/chitosan-coated zein nanoparticles prepared in the step 3) in an aqueous solution to obtain crocin-loaded zein nanoparticles.

2. The method for preparing the crocin-loaded zein nanoparticles as claimed in claim 1, wherein the feed-to-liquid ratio of the zein powder to the ethanol aqueous solution in step 1) is 1g/20ml, the heating temperature is 70 ℃, the heating time is 30min, and the centrifugation conditions are as follows: centrifuge at 1000rpm for 20min.

3. The method for preparing the crocin-loaded zein nanoparticles as claimed in claim 1, wherein the certain mass ratio in step 2) is 1.

4. The method according to claim 1, wherein the weight concentration of the sodium carbonate ethanol aqueous solution in step 2) is 2%, and the addition amount of the sodium carbonate ethanol aqueous solution in the 2% sodium carbonate ethanol aqueous solution to the zein stock solution is 1.

5. The method of claim 1, wherein the concentration of the aqueous solution of dextran sulfate in step 3) is 1.0mg/mL.

6. The method according to claim 1, wherein the mixing reaction condition of the dextran sulfate aqueous solution and the crocin-loaded hollow zein nanoparticles in the step 3) is that the pH value of the mixed solution is 4.0.

7. The method according to claim 1, wherein the concentration of the chitosan solution in step 3) is 1.0mg/mL, the pH value is 4.0, and the mass ratio of chitosan to dextran sulfate is 1.

8. The use of crocin-loaded zein nanoparticles prepared by the preparation methods of claims 1 to 7 in the preparation of medicaments for the treatment of alzheimer's disease.