Preparation method of food-based micro-nano motor with ros responsiveness and application thereof
By preparing chitosan and fucoidan-based micro/nanomotors and using allicin and allyl propyl sulfide as ROS-responsive groups, the delivery problem of β-carotene in food was solved, achieving efficient, stable, and targeted delivery, and significantly improving its bioavailability and antioxidant capacity.
Patent Information
- Application Number
- CN202610384009.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-26
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies have difficulty effectively delivering β-carotene into the gastrointestinal tract, and the hydrophobicity and instability of β-carotene limit its application in food. Existing micro-nano motor carriers have low binding rates and poor bioavailability.
Using chitosan and fucoidan as raw materials, a polysaccharide-based microtubule structure was constructed through layer-by-layer self-assembly technology. Allicin and allyl propyl sulfide were introduced as ROS-responsive groups to prepare micro-nanomotors with autonomous movement capabilities for loading and delivering β-carotene.
It achieved precise release and targeted delivery of β-carotene under high ROS conditions, significantly improving its stability and bioavailability, alleviating symptoms of ulcerative colitis, and enhancing cellular antioxidant capacity.
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Figure CN122096416A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of food science, specifically relating to a method for preparing food-based micro / nano motors with ROS responsiveness and their applications. Background Technology
[0002] Food active ingredients refer to bioactive compounds present in food that promote health, including essential nutrients such as vitamins, fatty acids, and amino acids; chemical substances from plants such as terpenes, polyphenols, phytosterols, and alkaloids; and bioactive compounds produced through food processing, such as bioactive peptides. β-Carotene (β-CA) is one of the most bioactive carotenoids, widely found in various fruits and vegetables. Due to the presence of unsaturated bonds, β-carotene possesses excellent antioxidant properties, effectively scavenging reactive oxygen species and regulating inflammatory signaling pathways, making it an ideal food-grade active ingredient. However, due to its unsaturated structure, it has low solubility in water, high sensitivity to light and heat, and a tendency to be oxidized and decomposed. These characteristics severely limit the application of β-carotene in food. This is also a limitation shared by most food active ingredients. Solutions typically utilize traditional delivery systems, mainly including microemulsions, liposomes, micelles, and hydrogels, for example, microemulsions, relying on the passive diffusion transport of the carrier and active ingredient, can be used to encapsulate food active ingredients, preventing their degradation in the gastrointestinal environment and improving tolerability. However, microemulsions are prone to problems such as Auschwitz curing and flocculation during storage, resulting in poor stability, incomplete encapsulation, and the inability to achieve the slow release of food active ingredients at specific sites in the body.
[0003] Currently, nanoemulsions with small and uniform particle sizes have been developed using high-pressure microfluidics and high-pressure homogenization equipment, significantly improving the encapsulation efficiency of food active ingredients. Researchers have prepared oil-in-water nanoemulsions loaded with astaxanthin by high-pressure homogenization of sodium caseinate and modified lecithin. These nanoemulsions exhibited good chemical and physical stability over a wide range of temperature, pH concentrations, and during storage. However, the small contact area between the aqueous and oil phases led to structural changes in sensitive food active ingredients, reducing chemical stability. Furthermore, overloading of fat-soluble active ingredients can produce toxicity. In addition, delivery systems developed in recent years, such as metal-organic frameworks (MOFs) and hydrogels, have provided new methods for achieving precise delivery. For example, MOFs encapsulate curcumin, achieving high loading efficiency and stable release in the gastrointestinal tract. However, the raw materials and chemical cross-linking processes used in MOF preparation limit their application in food due to the presence of reagents harmful to bioactivity.
[0004] Micro-nanomotors are dynamic machines that utilize various energy sources, such as chemical energy, light energy, and thermal energy, to convert into mechanical kinetic energy to achieve autonomous movement. It has been reported that micro-nanomotors have broad application potential in multiple fields, including drug delivery, environmental monitoring, and biomedical imaging; however, their application in the food industry requires further development and expansion. Currently, micro-nanomotors are used to deliver curcumin, but the main challenge in delivering β-carotene using micro-nanomotors lies in the presence of the conjugated double bonds and terminal ionone ring in β-carotene. Their hydrophobicity hinders their dispersion in the gastrointestinal environment and absorption by small intestinal epithelial cells, resulting in lower bioavailability than curcumin. Furthermore, the hydrophobic structure has a weak binding rate to the carrier, and the conjugated polyene structure is unstable. Summary of the Invention
[0005] The purpose of this invention is to prepare micro-nano motors with ROS-responsive driving capabilities using a simple, fast, and pollution-free method, and to propose a novel strategy for delivering β-carotene using micro-nano motors based on food polysaccharides as carriers.
[0006] The first objective of this invention is to provide a method for preparing a polysaccharide-based responsive micro / nano motor. This micro / nano motor uses chitosan and fucoidan as raw materials, constructs a polysaccharide-based microtubule structure through layer-by-layer self-assembly technology, and introduces two ROS-responsive groups—allicin and allyl propyl sulfide—through chemical cross-linking to form a micro / nano motor with autonomous movement capability.
[0007] In one embodiment of the present invention, the method for preparing a polysaccharide-responsive micro / nano motor includes: (1) Using polycarbonate membrane as template, chitosan and fucoidan are alternately assembled on the template by pressure assembly method, and then the template is washed away to obtain chitosan-fucoidan microtubules (CFMTs). (2) Then, allicin (AL) and allyl propylsulfide (APS) were added respectively, and the mixture was shaken, centrifuged and washed to remove free AL and APS, so as to obtain two polysaccharide-responsive micro-nano motors (Chitosan fucoidan micro-nano motor, CFNMs-AL and CFNMs-APS).
[0008] In one embodiment of the present invention, in step (1), the concentration of chitosan solution is 1 mg / mL and the concentration of fucoidan solution is 0.5 mg / mL.
[0009] In one embodiment of the present invention, step (1), the method for preparing chitosan-fucose-based microtubules specifically includes: A polycarbonate membrane with a diameter of 25 mm, a thickness of 9 nm, and a pore size of 0.6 μm was placed on a sintered glass filter. 1 mL of a 1 mg / mL chitosan solution was dropped onto the surface of the polycarbonate membrane, and the membrane was filtered completely. The polycarbonate membrane was then rinsed three times with deionized water and dried. Next, 1 mL of a 0.5 mg / mL fucoidan solution was dropped onto the surface of the polycarbonate membrane. After the fucoidan solution was completely filtered, the membrane was rinsed three times with deionized water and dried. This process was repeated eight times. The polycarbonate membrane was then laid flat on a petri dish and moistened with anhydrous ethanol. Both sides of the polycarbonate membrane were then repeatedly rubbed with cotton swabs, and finally rinsed again with deionized water. The uniformly polished polycarbonate membrane was placed in a 2 mL microcentrifuge tube and dissolved in 1 mL of N,N-dimethylformamide. The membrane was then washed 4 times with N,N-dimethylformamide, 3 times with anhydrous ethanol, and 2 times with deionized water to obtain food-based microtubules dispersed in 1 mL of water, namely chitosan-fucoidan microtubules (CFMTs).
[0010] In one embodiment of the present invention, step (2) of the preparation method of the polysaccharide-responsive micro / nano motor specifically includes: 4 μL of allicin (AL) and allyl propylsulfide (APS) were added separately, mixed and shaken for 2 h, centrifuged with deionized water at 10000 rpm for 5 min, and washed 3 times to remove free AL and APS, to obtain two polysaccharide-responsive micro-nano motors (Chitosan fucoidan micro-nano motor, CFNMs-AL and CFNMs-APS).
[0011] A second objective of this invention is to provide a polysaccharide-based responsive driven micro / nano motor prepared by the method described above.
[0012] A third objective of this invention is to provide a polysaccharide-responsive micro / nano motor loaded with β-carotene.
[0013] In one embodiment of the present invention, micro / nano motors (CFNMs-AL and CFNMs-APS) were dispersed in 1 mL of a 0.1 mg / mL β-carotene solution and shaken separately at a constant temperature and in the dark to achieve loading. After centrifugation at 10000 rpm for 5 min, the mixture was vortexed and centrifuged again, and washed twice with deionized water to obtain β-carotene-loaded polysaccharide-based ROS-responsive micro / nano motors (β-CA@CFNMs-AL and β-CA@CFNMs-APS).
[0014] The beneficial effects of this invention are as follows: (1) Using chitosan and fucoidan as raw materials, micro-nanotubes were constructed through supramolecular layer-by-layer self-assembly, and then crosslinked with allicin and allyl propyl sulfide to successfully prepare food-based reactive oxygen species responsive micro-nanomotors. The motor has a hollow tubular structure with good size uniformity, which not only preserves the structural integrity of the carrier, but also endows the micro-nanomotors with reactive oxygen species responsive characteristics. It has excellent biocompatibility in vivo and no obvious biotoxicity, providing ample space for subsequent loading and targeted delivery of active substances.
[0015] (2) Compared with existing food-based β-carotene delivery systems, the motor prepared in this invention is hollow inside, providing a hydrophobic environment for β-carotene. Compared with existing polysaccharide systems, the fucoidan selected in this invention not only enhances the hydrophobic binding capacity of β-carotene, but also has specific cleavage characteristics under high ROS conditions (utilizing the specific cleavage characteristics of the sulfate ester bond of fucoidan under high ROS conditions to achieve precise control of the release of ROS-responsive motor-driven active ingredients). This invention screened out two edible compounds containing ROS-responsive groups, allicin and allyl propyl sulfide, further ensuring the targeting and release characteristics of the motor in vivo, effectively alleviating the symptoms of ulcerative colitis.
[0016] (3) Antioxidant-encapsulated β-carotene was used to prepare β-CA@CFNMs loading systems (β-CA@CFNMs-AL and β-CA@CFNMs-APS). CFNMs-AL reached its optimal loading state after 4 h of loading, with a β-CA loading rate of 25.82 μg / mg; CFNMs-APS achieved its optimal loading at 2 h of loading, with a loading rate of 20.04 μg / mg. This loading system can effectively reduce the damage of β-CA to external factors, significantly improve its stability, and with the increase of H2O2 concentration, the trajectory and cumulative distance of the loading system are longer, which also provides a guarantee for the targeted delivery of β-CA in the intestine.
[0017] (4) Finally, the functionality of β-CA@CFNMs was explored using cellular oxidative stress and mouse colitis models. This system significantly improved cellular antioxidant levels, reduced the accumulation of lipid peroxidation products, and effectively alleviated H2O2-induced oxidative damage. The total cellular antioxidant capacity reached 0.96 mmol / g prot, which was significantly better than that of the free β-CA group. At the same time, the DAI scores of the β-CA@CFNMs-AL group and the β-CA@CFNMs-APS group were significantly lower than those of the DSS model group and the free β-CA group, at 2.4 and 2.7, respectively. It can significantly improve colitis symptoms such as diarrhea, bloody stools, and weight loss in mice, reduce colonic shortening and mucosal damage, and further significantly alleviate DSS-induced colonic oxidative stress damage by enhancing the body's overall antioxidant capacity and inhibiting lipid peroxidation. At the same time, it upregulates the expression of tight junction proteins such as Occludin and ZO-1, regulates the abundance and diversity of intestinal flora, and maintains intestinal microecological homeostasis. Attached Figure Description
[0018] Figure 1 Bright-field microscopy and scanning electron microscopy images of polysaccharide-based ROS-responsive micro / nanomotors.
[0019] Figure 2 This is the energy spectrum of a polysaccharide-based ROS-responsive micro / nanomotor.
[0020] Figure 3 Infrared spectrum of polysaccharide-based ROS-responsive micro / nanomotor.
[0021] Figure 4 Potentiogram of polysaccharide-based ROS-responsive micro / nanomotor.
[0022] Figure 5 Bright-field and fluorescence images of the polysaccharide-based ROS-responsive micro / nanomotor loading system.
[0023] Figure 6 The loading rate of β-carotene for polysaccharide-based ROS-responsive micro / nanomotors.
[0024] Figure 7 The photostability of the polysaccharide-based ROS-responsive micro / nano motor loading system.
[0025] Figure 8 Thermal stability of the polysaccharide-based ROS-responsive micro / nano motor load system.
[0026] Figure 9 To assess the stability and bioaccessibility of polysaccharide-based ROS-responsive micro / nanomotor loading systems in simulated digestive fluids. Figure 10 This demonstrates the scavenging ability of the polysaccharide-based ROS-responsive micro / nano motor loading system for DPPH free radicals.
[0027] Figure 11 Analysis of the self-driving behavior of a polysaccharide-based ROS-responsive micro / nano motor load system.
[0028] Figure 12 To analyze the toxicity of polysaccharide-based ROS-responsive micro / nanomotor loading systems to RAW264.7 macrophages.
[0029] Figure 13 These are biochemical indicators in mouse serum.
[0030] Figure 14 H&E sections of mouse tissue.
[0031] Figure 15 The effect of H2O2 on the survival rate of RAW264.7 cells.
[0032] Figure 16 ROS imaging of RAW264.7 cells after different treatments.
[0033] Figure 17 Indicators of cellular oxidative stress after different treatments.
[0034] Figure 18 This is a live fluorescence imaging image of a mouse.
[0035] Figure 19 The values represent changes in mouse body weight and DAI values.
[0036] Figure 20 H&E sections of mouse tissue.
[0037] Figure 21 This is an indicator of oxidation in mouse serum.
[0038] Figure 22 This is an indicator of colonic oxidation in mice.
[0039] Figure 23 Immunofluorescence staining of mouse colon tissue.
[0040] Figure 24 Alpha diversity analysis of mouse gut microbiota.
[0041] Figure 25 Venn diagram analysis of mouse gut microbiota.
[0042] Figure 26 Analysis of the composition of the intestinal flora in mice.
[0043] Figure 27 The image shown is a scanning electron microscope (SEM) image of allyl isothiocyanate cross-linked micro / nanotubes for Comparative Example 1.
[0044] Figure 28The image shown is a scanning electron microscope image of sulforaphane-crosslinked micro-nanotubes for Comparative Example 2.
[0045] Figure 29 The image shown is a scanning electron microscope image of the allicin-crosslinked micro-nanotubes in Comparative Example 3.
[0046] Figure 30 The image shows a scanning electron microscope (SEM) image of the cross-linked micro / nanotubes of allylpropyl sulfide, which is a comparative example. Detailed Implementation
[0047] The testing methods used in this invention: The morphological characteristics of the samples from Example 1 were characterized using a fluorescence inverted microscope. The specific procedure was as follows: 10 μL of the micro / nano motor solution was dropped onto a coverslip, the coverslip was closed and fixed, and the sample was observed and recorded under bright-field conditions using a 20x fluorescence inverted microscope. The morphological characteristics of the micro / nano motors were characterized using a JSM-7800F scanning electron microscope and an X-Max50 energy dispersive spectroscopy (EDS) spectrometer. The specific procedure was as follows: 5 μL of the micro / nano motor solution was dropped onto a 3 mm × 3 mm silicon wafer, allowed to air dry, and then platinum was sputtered onto it using an ion sputtering instrument. Electron microscopy and EDS scanning were then performed under accelerating voltage.
[0048] The morphological characteristics of the sample in Example 1 were characterized using Fourier transform infrared spectroscopy. The specific operation was as follows: the micro-nano motor was dispersed in 1 mL of anhydrous ethanol solution, and the motor solution was repeatedly coated onto a potassium bromide window using a glass rod. The sample was then quickly placed into the instrument for full-band scanning to acquire the vibrational spectrum of the micro-nano motor.
[0049] The morphological characteristics of the sample from Example 1 were characterized using a Zeta potentiometer. The specific procedure was as follows: 1 mL of sample was drawn into a disposable syringe and slowly injected into the potentiometer cell. The sample was then placed in the potentiometer for potential detection, and this process was repeated three times.
[0050] Example 1 A polycarbonate membrane with a diameter of 25 mm, a thickness of 9 nm, and a pore size of 0.6 μm was placed on a sintered glass filter. 1 mL of a 1 mg / mL chitosan solution was dropped onto the surface of the polycarbonate membrane, and the membrane was filtered completely. The polycarbonate membrane was then rinsed three times with deionized water and dried. Next, 1 mL of a 0.5 mg / mL fucoidan solution was dropped onto the surface of the polycarbonate membrane. After the fucoidan solution was completely filtered, the membrane was rinsed three times with deionized water and dried. This process was repeated eight times. The polycarbonate membrane was then laid flat on a petri dish and moistened with anhydrous ethanol. Both sides of the polycarbonate membrane were then repeatedly rubbed with cotton swabs, and finally rinsed again with deionized water. The uniformly polished polycarbonate membrane was placed in a 2 mL microcentrifuge tube and dissolved in 1 mL of N,N-dimethylformamide. The membrane was then washed 4 times with N,N-dimethylformamide, 3 times with anhydrous ethanol, and 2 times with deionized water to obtain food-based microtubules dispersed in 1 mL of water, namely chitosan-fucoidan microtubules (CFMTs).
[0051] Example 2 4 μL of allicin (AL) and allylpropyl sulfide (APS) were added to Example 1, respectively. The mixture was stirred and shaken for 2 h. The mixture was then centrifuged with deionized water at 10,000 rpm for 5 minutes and washed 3 times to remove free AL and APS, resulting in two polysaccharide-responsive micro-nanomotors (Chitosan fucoidan micro-nanomotor, CFNMs-AL and CFNMs-APS).
[0052] Example 3 Micro- and nano-motors (CFNMs-AL and CFNMs-APS) were dispersed in 1 mL of 0.1 mg / mL β-carotene solution and shaken separately in the dark at a constant temperature to achieve loading. After centrifugation at 10,000 rpm for 5 min, the mixture was vortexed and centrifuged again, and washed twice with deionized water to obtain β-carotene-loaded polysaccharide-based ROS-responsive micro- and nano-motors (β-CA@CFNMs-AL and β-CA@CFNMs-APS).
[0053] Test example: (1) Microscopic bright-field image, scanning electron microscope image and energy dispersive spectroscopy (EDS) spectrum The samples prepared in Examples 1 and 2 were first dispersed by low-frequency ultrasonication, and 10 μL was dropped onto a 3 mm × 3 mm silicon wafer and dried at room temperature. Gold plating was then performed using an ion sputtering system, followed by electron microscopy and energy dispersive spectroscopy at an accelerating voltage. The results are shown below. Figure 1-2As shown, the basic morphology of CFMTs and CFNMs prepared in Examples 1 and 2 can be seen through the light field diagrams obtained by optical microscopy. The length of the nanotubes is approximately 9 μm. Since both matrix materials are viscous polysaccharides, the microtube pores are prone to cross-linking and aggregation after polishing, resulting in some adhesion. Scanning electron microscopy results can more clearly observe the hollow tubular structure of CFNMs. Its length can be determined to be 9 μm and its diameter to be approximately 600 nm. The length of the microtubes is relatively uniform and consistent with the template size. Energy dispersive spectroscopy (EDS) spectra show that AL and APS successfully modified CFMTs.
[0054] (2) Infrared spectroscopy and potential analysis The molecular structures of CFMTs and CFNMs were characterized using infrared spectroscopy. The samples prepared in Examples 1 and 2 were dispersed in ethanol and dropped into a sample plate using a glass rod. After air drying, the samples were scanned using an infrared spectrometer to analyze changes in functional groups and determine the interaction forces between the components. The results are as follows: Figure 3 As shown in the figure, the chemical composition and molecular structure of carbon nanotubes were analyzed. CO content in the 1000-1200 cm⁻¹ range... -1 The presence of strong absorption peaks in the wavenumber range indicates that the carbon nanotube structure of the polysaccharide portion has been basically formed; the amino (-NH2) peak of the carbon nanotubes has shifted to 3200-3500 cm⁻¹. -1 Strong absorption peaks appeared in the range of 2800-3000 cm⁻¹, indicating electrostatic adsorption and further confirming the formation of CFMTs. CFNMs-AL and CFNMs-APS showed strong absorption peaks in the 2800-3000 cm⁻¹ range. -1 Strong absorption peaks were observed in all wavelength ranges, which are caused by the stretching vibration of CH. This indicates the presence of alkyl groups and confirms the successful crosslinking of AL and APS. Simultaneously, at 1600 cm⁻¹... -1 The enhanced absorption peaks in the vicinity indicate the presence of C=C bonds in the allyl structure, confirming the successful introduction of allicin and allyl propyl sulfide. The infrared spectra of CFNMs-AL and CFNMs-APS are close to those of CFMTs, indicating that the incorporation of AL and APS did not generate new chemical bonds or functional groups and would not significantly affect the main structure of CFMTs. From the Zeta potential results ( Figure 4 It can be seen that the potential of CFMT is 11.30±1.339 mV, the potential of CFNMs-AL is 13.74±3.466 mV, and the potential of CFNMs-APS is... The value of 8.95 ± 0.344 mV indicates that AL and APS have successfully crosslinked.
[0055] (3) Load capacity β-CA solutions with different dilution ratios were prepared, and standard curves were plotted. Micro-nano motors (CFNMs-AL and CFNMs-APS) were dispersed in 1 mL of 0.1 mg / mL β-carotene solution and incubated in the dark at constant temperature for 1, 2, 4, 6, and 12 h to achieve gradient loading. The mixture was centrifuged at 10000 rpm for 5 min, vortexed, and centrifuged again. The mixture was washed twice, and the supernatants were collected and combined (keeping the supernatant volume consistent). The absorbance was measured at 453 nm. The β-carotene loading was determined according to the standard curve.
[0056] Figure 5-6 The images show the fluorescence spectra of β-CA loaded onto polysaccharide-based micro / nanomotors and the β-CA loading at different reaction times. Figure 6 As can be seen, after AL and APS loading, the loading reached 25.82 μg mg. -1 The loading is relatively low. This may be because the particles on the CFMT surface occupy the binding sites of β-CA and CFMT, i.e., the number of active sites is reduced. As the reaction time increases, depolymerization occurs, and the loading decreases.
[0057] (4) Light stability and thermal stability Add 1 mL of β-CA solution (0.1 mg / mL) -1 The solution (dissolved in anhydrous ethanol) and an equal volume of β-CA@CFNMs were added to a transparent glass bottle, and the bottle was irradiated with ultraviolet light for 90 min. Samples were taken and recorded sequentially at 0, 15, 30, 45, 60, 75, and 90 min, avoiding light exposure. The retention rate was calculated based on the standard curve.
[0058] Free β-CA was used as the control group, and β-CA@CFNMs were used as the sample group. They were placed in transparent glass vials and heated at 85 °C. Samples were collected at 30, 60, 90, 120, 150, and 180 min, and immediately cooled to room temperature. The absorbance was then measured at 453 nm. Retention rates were calculated based on the standard curve.
[0059] Figure 7-8 Experiments were conducted to investigate the photostability and thermal stability of curcumin-loaded micro / nano motors. Figure 7 It can be seen that the retention rate of β-CA decreases with increasing irradiation time, while the retention rate of free β-CA decreases significantly. At 90 min, the retention rate of AL is 67.83%, the retention rate of APS is 76.49%, and the retention rate of free β-CA is only 24.72%, indicating that CFNMs can effectively protect β-CA. When the sample is heat-treated at 85 °C, Figure 8The β-CA retention rate decreased with increasing heating time. After heating for 180 min, the retention rate of free β-CA was 21.23%, while AL and APS retained 75.09% and 69.36%, respectively. This indicates that CFNMs significantly protect β-CA from degradation. Therefore, CFNMs exhibit good protective effects on β-CA under high temperature and light conditions, making them an ideal carrier that can significantly improve the anti-degradation ability of β-CA and show promising application prospects in food bioactive ingredient delivery systems.
[0060] (5) Simulated digestion stability The samples were first equilibrated at 37 °C for 10 min, and then processed sequentially through three stages: oral cavity, stomach, and intestine. Take 5 mL of β-carotene, β-CA@CFNMs-AL, and β-CA@CFNMs-APS dispersions preheated to 37 °C, mix with an equal volume of artificial saliva (pH 6.8), and shake at 37 °C and 100 rpm for 10 min. Then add 10 mL of simulated gastric juice, adjust the pH to 2.5 with 1 mol / L HCl, and continue stirring at 37 °C for 2 h. Take 10 mL of gastric digestive fluid, add 1.5 mL of small intestinal fluid and 62.5 mg of bile salts, adjust the pH to 7.4 with 0.5 mol / L NaOH, and continue stirring at 37 °C for 2 h. Samples were taken immediately before and after the oral phase, and then 1 mL of digestive fluid was collected every 30 min. The mixture was immediately mixed with an equal volume of dichloromethane, vortexed for 2 min, and allowed to stand for 10 min. The lower organic phase was collected, and the absorbance of β-carotene was measured at 453 nm.
[0061] Figure 9 As can be seen, the samples entered the simulated gastric digestion stage. The release rate of free β-CA was 36.84%, while the release rates of β-CA encapsulated in CFNMS-AL and CFNMS-APS were 15.45% and 13.05%, respectively. The release rate of free β-CA was twice or even higher than that of encapsulated β-CA, indicating that the physical barrier of CFNM can effectively protect β-CA from the acidic environment and achieve sustained release. During the 2-hour simulated intestinal digestion process, the β-CA release rate of both loading systems showed a rapid upward trend, but remained lower than the release rate of free β-CA.
[0062] (6) Free radical scavenging ability and stability Accurately weigh 0.040 g of DPPH and dilute to 100 mL with anhydrous ethanol to obtain a 0.4 mmol / L DPPH-ethanol solution. Take 1 mL of β-carotene, β-CA@CFNMs-AL, and β-CA@CFNMs-APS respectively and mix them rapidly with 1 mL of DPPH-ethanol solution (1:1, V / V), designating this as sample group A1. After standing at room temperature in the dark for 30 min, measure the absorbance at 517 nm. Also, prepare a blank group A0 (DPPH-ethanol solution) and a control group A2 (1 mL of sample mixed with 1 mL of anhydrous ethanol). To determine the stability of the free radical scavenging ability, the same batch of samples was placed under natural light indoors for 24 h and then measured again to calculate the free radical scavenging rate.
[0063] The results are shown in Figure 10 In the study, the radical scavenging rate of β-CA encapsulated in CFNMs was higher than that of free β-CA. Furthermore, after 24 hours of exposure to an indoor environment, the radical scavenging rate of free β-CA decreased by 4.44%, while the radical scavenging abilities of β-CA@CFNMs-AL and β-CA@CFNMs-APS decreased by only 0.78% and 2.37%, respectively. This indicates that CFNMs-AL and CFNMs-APS effectively protected β-CA, preventing its decomposition and inactivation.
[0064] (7) Driving motion trajectory diagram and mean square displacement Equal amounts of β-CA@CFNMs-AL and β-CA@CFNMs-APS were added to 5%, 10%, and 15% H2O2 solutions, respectively. The motion trajectories of the micro / nanomotors within the same time period were recorded using a fluorescence microscope. Figure 11 As shown, with increasing H2O2 concentration, the trajectories and cumulative distances of CFNMs-APS and CFNMs-AL become longer, exhibiting autonomous movement towards higher concentrations. Simultaneously, the mean square displacement increases very rapidly, indicating that the particles exhibit strong directional motion rather than random Brownian motion.
[0065] (8) Safety evaluation Cell viability was determined using the CCK-8 assay, and RAW264.7 cells in the logarithmic growth phase were selected for in vitro toxicity testing.
[0066] RAW 264.7 macrophages were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and maintained at 37 ℃, 5% CO2, and saturated humidity. RAW264.7 macrophages in good condition and in the logarithmic growth phase were selected for in vitro evaluation of the toxicity of micro / nanomotors. When the RAW264.7 cell density reached 80%, 1×10⁻⁶ cells were injected with the micromotors. 4100 μL of CFNMs-AL and CFNMs-APS were seeded per well in a 96-well plate and incubated at 37 °C with 5% CO2 for 24 h to ensure complete adhesion. The old medium was discarded, and serum-free high-glucose DMEM was replaced with a gradient concentration of micro / nanomotors: CFNMs-AL and CFNMs-APS at concentrations of 10, 20, 50, 100, 200, and 400 μg / mL, with six parallel wells per concentration. The plates were then incubated for another 24 h. Subsequently, 10 μL of LCK-8 reagent was added to each well, and the reaction was carried out in the dark for 2 h. The absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 12 As shown.
[0067] Thirty four-week-old BALB / c mice were divided into three groups. In the experiment, the mice were housed individually in cages with free access to food and water. The mice's mental state and diet were observed on days 0, 7, and 14. Blood samples were collected from the mice's eyes and centrifuged at 4000 r / min for 15 min to obtain serum. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid (UA), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-CHO) were measured using a kit. The results are as follows: Figure 9 As shown. Heart, liver, spleen, lung, kidney, and stomach tissues were collected from euthanized mice. The mouse tissues were fixed in formaldehyde solution, processed into slides, and stained with hematoxylin and eosin (HE) to observe for any lesions or abnormalities, such as... Figure 13-14 As shown.
[0068] Safety evaluation diagrams of the polysaccharide-based micro / nanomotors prepared in Examples 1 and 2 are shown below. Figure 12-14 As shown, Figure 12 As shown, after treatment with 400 μg / mL CFNMs for 24 h, the cell viability remained above 90%, indicating that CFNMs-AL and CFNMs-APS have good in vitro safety.
[0069] Fourteen days after gavage, blood samples were collected for biochemical analysis, and the results were as follows: Figure 13As shown in the figure, there were no statistically significant differences in serum ALT, AST, UA, HDL-C, and T-CHO levels among the sample groups and the blank control group (p>0.05), indicating that CFNMs did not damage liver and kidney function. HE staining revealed clear gastric tissue structures in all groups, with tightly packed gastric mucosa and fundic glands, and no bleeding, detachment, or inflammatory cell infiltration. Liver and kidney microscopy showed no abnormalities in the kidneys of both the normal control group and the sample group; no abnormal changes were observed in the glomerular capillaries of the renal cortex; the morphology of tubular epithelial cells and tubular wall cells was normal; and the renal corpuscle boundaries were clear. Under the microscope, the liver lobule structure of both the normal control group and the sample group was intact and normal; the outlines of liver parenchymal cells were clear and normal; the distribution of nuclei and cytoplasmic cells was uniform; and the hepatocytes were arranged in an orderly manner. Spleen tissue sections showed clear splenic follicular structures; the spleen tissue was normal; and no obvious damage was observed in the cardiac tissue. Figure 14 The results of cytotoxicity tests and serum biochemical assays indicate that CFNMs have good biocompatibility and low toxicity, suggesting that CFNMs can serve as non-toxic carriers for bioactive substances.
[0070] (9) In vitro antioxidant activity RAW264.7 cells were fed at a concentration of 1×10⁻⁶. 5 mL -1 The following groups were seeded at a density in 12-well plates and cultured until adherent: a blank control group (Control); an oxidative damage model group (H2O2, 500 μmol / L); a free β-CA group; a micro / nanotube group (CFMTs); a β-CA@CFNMs-AL group; and a β-CA@CFNMs-APS group. The final β-CA concentration in groups ③–⑥ was uniformly 100 μg / mL. After pre-incubation for 24 h according to the groupings, H2O2 solution was added to all wells except the blank group, and incubation continued for 6 h to establish the oxidative stress model. The culture medium was then discarded, and the DCFH-DA fluorescent probe was diluted to 10 μmol / L with serum-free DMEM and incubated at 37 ℃ in the dark for 20 min. The plates were then gently rinsed twice with phosphate-buffered saline (PBS, pH 7.4) to remove unbound probe. Images were immediately acquired randomly using a fluorescence microscope, and the average fluorescence intensity was calculated using ImageJ.
[0071] RAW264.7 cells in logarithmic phase were loaded with 2 × 10⁻⁶ cells. 5Cells were seeded at a density of 1 / mL in 6-well plates, and cultured under the same conditions and groupings as described above. After the sample-oxidative stress treatment, the cells were washed twice with pre-cooled PBS, collected with a scraper, centrifuged at 4 °C and 1200 rpm for 5 min, the supernatant was discarded, and 150 μL of lysis buffer (Western / PI: PMSF = 100:1, V / V) was added to the precipitate. The cells were lysed on ice for 3-15 min, centrifuged at 12000 rpm and 4 °C for 10-20 min, and the supernatant was collected as the intracellular extract. After determining the total protein concentration using the BCA method, the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) activity were determined sequentially according to the kit instructions.
[0072] Figure 15-17 To evaluate the in vitro antioxidant properties of the β-CA@CFNMs system. Figure 16 (a) Intracellular ROS imaging in RAW264.7 cells after different treatments; (b) Average fluorescence intensity results after software analysis. As shown in the figure, fluorescence intensity analysis revealed that the H2O2-induced model group exhibited the highest fluorescence intensity. The fluorescence intensities of β-CA@CFNMs-AL and β-CA@CFNMs-APS were 40.05 au and 33.15 au, respectively, while the fluorescence intensity of free β-CA was 48.57 a.u. This indicates that β-CA@CFNMs can effectively scavenge ROS, reduce intracellular ROS release, and improve cell survival. Figure 17(a) Malondialdehyde content (b) Superoxide dismutase activity (c) Catalase activity (d) Glutathione peroxidase activity (e) Total antioxidant capacity. As shown in the figure, after H2O2 treatment, the SOD and CAT enzyme activities of β-CA@CFNMs-AL decreased by 7.96% and 47.5% respectively compared with the control group, while the SOD and CAT enzyme activities increased by 28.89% and 60.83% respectively compared with the control group. The protein content was 57.19 U / mgprot and 45.98 U / mgprot, respectively. The SOD and CAT enzyme activities of β-CA@CFNMs-APS increased by 18.04% and 41.89% respectively compared with the control group, and the protein content was 52.38 U / mgprot and 40.57 U / mgprot, respectively. This indicates that β-CA@CFNMs-AL and β-CA@CFNMs-APS can improve the activity of antioxidant enzymes SOD and CAT. GSH-PX activity was significantly decreased in the H2O2 group, while the GSH-PX enzyme activity in the β-CA@CFNMs-AL and β-CA@CFNMs-APS groups was significantly increased compared to the control group, increasing by 64.52% and 24.85% respectively compared to the free β-CA group. This indicates that β-CA@CFNMs-AL and β-CA@CFNMs-APS can alleviate oxidative damage. The MDA levels of β-CA@CFNMs-AL and β-CA@CFNMs-APS were 2.69 mmol / mgprot and 2.31 mmol / mgprot respectively, significantly lower than those in the control group and the free β-CA group. These findings suggest that β-CA@CFNMs-AL and β-CA@CFNMs-APS can effectively alleviate cellular oxidative stress damage and more effectively protect β-CA.
[0073] (10) Mouse in vivo imaging Nine 7-week-old SPF-grade male BALB / c-nu nude mice were selected and acclimatized for 7 days under a 12-hour light / dark cycle with free access to food and water. They were then randomly divided into three groups (n = 3) based on body weight. The same rearing environment was maintained throughout the experiment, and all feed, water, and bedding were sterilized. The experimental groups were: free Nile Red (NR), NR@CFNMs-AL, and NR@CFNMs-AL. NR replaced β-CA to complete the cross-linking with the micro-nanomotors. After 12 hours of fasting, mice were administered 5 µg NR / 200 µL via gavage, with the timer set to 0 h. Anesthesia (2% isoflurane, 0.2 L / min) was repeated at 0, 2, 4, 6, 8, and 12 hours, and the patient was taken in a prone position. After each imaging session, the patient was immediately returned to the SPF environment to resume feeding. After the last imaging session at 12 hours, the patient was euthanized by cervical dislocation, and the heart, liver, spleen, lungs, kidneys, stomach, and intestines were completely removed. After rinsing off residual blood with PBS, the patient was laid flat on a black ruler plate and subjected to ex vivo imaging with the same parameters.
[0074] Figure 18 The images show in vivo imaging of mice. The results indicate that in NR@CFNMs-AL and NR@CFNMs-APS, as well as in the free NR group, NR began to appear in the intestine 6 hours after gastric digestion. The decreased content of free NR in the intestine indicates that free NR had been digested, while only a small amount of CFNMs encapsulating NR was digested, suggesting that CFNMs effectively protect NR from degradation.
[0075] (11) Antioxidant activity in vivo Fifty 8-week-old SPF-grade male BALB / c mice were selected and acclimatized for 7 days under a 12-hour light / dark cycle with free access to food and water. They were then randomly divided into 5 groups (n = 10) based on body weight, individually labeled, and kept in the same environment throughout the experiment. Feed, water, and bedding were all sterilized. Grouping consisted of: a blank control (Control), a model control (DSS), a positive control (β-CA), and two sample groups (β-CA@CFNMs-AL and β-CA@CFNMs-APS). The experiment lasted 21 days: From days 1 to 14, the positive control and sample groups were administered the corresponding sample via oral gavage at 2 mg / kg body weight daily, while the blank control and model groups received an equal volume of solvent. From days 15 to 21, except for the blank control group, all other groups had free access to 3.5% (w / v) sodium dextran sulfate (DSS) solution, while simultaneously continuing the sample intervention via gavage. Body weight, fecal characteristics, and occult blood were recorded daily at fixed times, and the Disease Activity Index (DAI) was calculated. Two hours after gavage on day 21, blood was collected from the eyeballs of mice, and they were euthanized by cervical dislocation. The serum was aliquoted and stored at -80 °C. The contents of the heart, liver, spleen, lungs, kidneys, stomach, colon, and intestines were aseptically collected. A portion of the colon was fixed in paraformaldehyde for histological analysis, and the remaining colon and contents were flash-frozen in liquid nitrogen and stored at -80 °C.
[0076] Collected mouse organs were fixed in paraformaldehyde, routinely dehydrated, embedded in paraffin, and sectioned for hematoxylin and eosin staining. The tissue structure was systematically observed under a microscope to assess the presence of inflammatory cells and the normality of crypts and goblet cells in the colon tissue, among other pathological changes. Mouse serum, frozen at -80 °C, was slowly thawed at 4 °C in the dark. Malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) activity were measured sequentially according to the kit instructions.
[0077] Accurately weigh the colon tissue and add pre-cooled physiological saline at a weight (g) to volume (mL) ratio of 1:9. Grind thoroughly using a tissue homogenizer in an ice bath to prepare a 10% tissue homogenate. Centrifuge at 10,000 rpm for 15 min at 4 ℃, collect the supernatant, and store at -20 ℃. Determine the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) activity according to the kit instructions.
[0078] After heat retrieval with sodium citrate for 15 min, permeabilization with 0.3% Triton X-100, and blocking with 5% goat serum at room temperature for 1 h, sections were incubated overnight at 4 °C with ZO-1 and Occludin primary antibody. The next day, the sections were washed with PBS, incubated with the corresponding fluorescent secondary antibody in the dark for 1 h, counterstained with DAPI for 5 min, and mounted as usual. To reduce non-specific binding, 1% BSA was added to the secondary antibody dilution buffer. All procedures were performed in the dark. After mounting, the sections were stored at 4 °C and confocal scanning was performed within 24 h to ensure stable fluorescence signals.
[0079] Figure 19-23 For in vivo antioxidant experiments, Figure 19 Mouse weight changes and DAI values. Figure 20 H&E sections of mouse tissue. Figure 21 The following are indicators of oxidation in mouse serum: (a) malondialdehyde content, (b) superoxide dismutase activity, (c) catalase activity, (d) glutathione peroxidase activity, and (e) total antioxidant capacity. Figure 22 The following are indicators of colonic oxidation in mice: (a) malondialdehyde content, (b) superoxide dismutase activity, (c) catalase activity, and (d) glutathione peroxidase activity. Figure 23 Immunofluorescence staining of mouse colon tissue was performed. The most significant weight loss was observed in the DSS group, with a reduction of 14.07 g, which was 4.68 g and 3.39 g greater than the weight losses in β-CA@CFNMs-AL and β-CA@CFNMs-APS, respectively. The control group showed a weight gain of 1.94 g. (DAI, the results indicate that both β-CA@CFNMs-AL and β-CA@CFNMs-AL are core indicators for assessing colitis in mice. H&E staining) Figure 20The results showed that control mice maintained intact crypt structures, abundant goblet cells, and a preserved epithelial barrier. In contrast, the DSS group exhibited severe damage to the colonic mucosal structure, loss of crypts, partial epithelial shedding, dense infiltration of inflammatory cells accompanied by congestion and edema, and a reduced number of goblet cells, confirming the successful establishment of the model. Compared with the DSS group, the β-CA@CFNMs-AL and β-CA@CFNMs-APS groups showed intact colonic mucosal structure, regular crypt arrangement, and similar length and morphology to the control group, with significantly reduced inflammatory infiltration (although a small number of inflammatory cells remained). These results indicate that β-CA@CFNMs-AL and β-CA@CFNMs-APS can significantly enhance the colonic protective effect of β-CA. Figure 21-22 The activities of antioxidant enzymes SOD, CAT, and GSH-PX, total antioxidant capacity (T-AOC), and MDA content in mouse colonic tissue and serum were measured. The results showed that, compared with the control group, the DSS group had significantly increased MDA content in serum and colonic tissue, and significantly decreased SOD, CAT, and GSH-PX activities, as well as significantly reduced serum T-AOC. This indicates that DSS successfully induced systemic and colonic oxidative stress damage in mice. After intervention with β-CA@CFNMs-AL and β-CA@CFNMs-APS, MDA levels in serum and colonic tissue decreased, and antioxidant enzyme activities recovered to varying degrees. Subsequent H&E staining further confirmed that the recovery of tight junction proteins was highly consistent with the reduction of colonic mucosal structural damage, indicating that β-CA@CFNMs-AL and β-CA@CFNMs-APS exert significant anti-colitis effects by protecting tight junction proteins and rebuilding the intestinal epithelial barrier.
[0080] (12) Intestinal flora analysis Fresh feces were collected from mice within 30 minutes of excretion under aseptic conditions. Two to three samples were placed in 2 mL sterile cryovials, immediately flash-frozen in liquid nitrogen, and then transferred to an ultra-low temperature freezer at -80 °C for storage in the dark. Subsequently, a series of steps were performed to analyze the gut microbiota information, including genomic DNA extraction, PCR amplification, quantitative fluorescence, library construction, and sequencing.
[0081] Figure 24-26 This is a diagram showing the gut microbiota analysis of mice. Figure 24 Alpha diversity analysis of mouse gut microbiota: (a) Chao1 index (b) Shannon index. Figure 25 Venn diagram analysis of mouse gut microbiota. Figure 26The analysis of mouse gut microbiota composition included (a) the ratio of Firmicutes to Bacteroidetes, (b) the relative abundance of gut microbiota at the phylum level, (c) the relative abundance of gut microbiota at the order level, and (d) a hierarchical clustering heatmap of bacterial abundance at the genus level. The Chao 1 index and Shannon index were significantly reduced in the DSS group, indicating that DSS treatment severely disrupted gut microbial diversity. At the phylum level (… Figure 26 ), β-CA@CFNMs-AL and β-CA@CFNMs-APS treatments effectively normalized the Firmicutes / Bacteroidetes ratio to a level indistinguishable from the control group, accompanied by a relative increase in Bacteroidetes abundance and a relative decrease in Firmicutes abundance. DSS treatment reduced Bacteroidetes abundance while increasing the abundance of potentially pathogenic flora. β-CA@CFNMs-AL, β-CA@CFNMs-APS and the control group clustered together, indicating that β-CA@CFNMs treatment remodeled the gut microbiota into a composition highly similar to that of a healthy state.
[0082] Comparative Example 1 The preparation method is the same as in Example 2, except that an equal amount of allyl isothiocyanate is added to Example 1.
[0083] Comparative Example 2 The preparation method is the same as in Example 2, except that an equal amount of sulforaphane is added to Example 1.
[0084] like Figure 27-28 As shown, allyl isothiocyanate and sulforaphane cannot be successfully cross-linked, and even micro-nanotubes cannot be observed, thus failing to provide evidence for the successful cross-linking of allyl isothiocyanate and sulforaphane. In contrast, the tubular structure observed in this invention using allicin and allyl propyl sulfide is clearly visible.
[0085] Comparative Example 3 The preparation method is the same as in Example 2, except that 1 mL of allicin and allyl propyl sulfide were added to Example 1 respectively. Allicin and allyl propyl sulfide were prepared at a volume ratio of 1:9 between the original solution and deionized water. The mixture was stirred and shaken for 4 hours, centrifuged with deionized water at 10,000 rpm for 5 minutes, and washed 3 times to obtain the polysaccharide-based responsive micro / nano motor.
[0086] The results are as follows Figures 29-30 The results showed that allicin and allyl propyl sulfide exhibited tubular structures, but they were severely fragmented, and their morphology differed somewhat from that of microtubules.
[0087] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.
Claims
1. A method for preparing a polysaccharide-responsive micro / nano motor, characterized in that, This micro-nano motor uses chitosan and fucoidan as raw materials to construct a polysaccharide-based microtubule structure through layer-by-layer self-assembly technology, and introduces two ROS-responsive groups—allicin and allyl propyl sulfide—through chemical cross-linking to form a micro-nano motor with autonomous movement capabilities.
2. The preparation method according to claim 1, characterized in that, The preparation method of polysaccharide-responsive micro / nanomotors specifically includes the following steps: (1) Using a polycarbonate membrane as a template, an alternating assembly membrane layer of chitosan and fucoidan is formed on the template by pressure assembly method. Then the template is washed away to obtain chitosan-fucoidan-based microtubules. (2) Then, allicin and allyl propyl sulfide were added, mixed, centrifuged, and washed to remove free allicin and allyl propyl sulfide, to obtain two polysaccharide-based response-driven micro-nano motors.
3. The preparation method according to claim 2, characterized in that, In step (1), the concentration of chitosan solution is 1 mg / mL and the concentration of fucoidan solution is 0.5 mg / mL.
4. The preparation method according to claim 2, characterized in that, In step (1), the preparation method of chitosan-fucose-based microtubules specifically includes: A polycarbonate membrane with a diameter of 25 mm, a thickness of 9 nm, and a pore size of 0.6 μm was placed on a sand filter. 1 mL of 1 mg / mL chitosan solution was dropped onto the surface of the polycarbonate membrane and filtered until the solution was completely filtered out. The polycarbonate membrane was washed and dried. Then, 1 mL of 0.5 mg / mL fucoidan solution was dropped onto the surface of the polycarbonate membrane. After the fucoidan solution was completely filtered out, it was rinsed and dried. This process was repeated 8 times. The polycarbonate membrane was then laid flat on a petri dish and moistened with anhydrous ethanol. The front and back sides of the polycarbonate membrane were then repeatedly abraded with cotton swabs. It was rinsed again with deionized water. The abraded polycarbonate membrane was placed in a microcentrifuge tube and dissolved in N,N-dimethylformamide. After washing, chitosan-fucose-based microtubules were obtained.
5. The preparation method according to claim 2, characterized in that, In step (2), the preparation method of the polysaccharide-responsive micro / nanomotor specifically includes: Add 4 μL of allicin and allyl propyl sulfide respectively, mix and shake for 2 h, centrifuge with deionized water at 10000 rpm for 5 min, and continue washing to remove free allicin and allyl propyl sulfide to obtain two polysaccharide-based responsive micro / nano motors.
6. The polysaccharide-based responsive driven micro / nano motor prepared by the method described in any one of claims 1-5.
7. A polysaccharide-responsive driven micro / nano motor loaded with β-carotene, characterized in that, The glycosyl-responsive driven micro / nano motor described in claim 6 is used as a carrier for delivering β-carotene.
8. The β-carotene-loaded polysaccharide-responsive driven micro / nano motor according to claim 7, characterized in that, The micro-nano motors were dispersed in 1 mL of 0.1 mg / mL β-carotene solution and shaken separately in the dark and at a constant temperature to achieve loading.