Preparation of a hydrophobic compound high-encapsulation protein carrier and use thereof

Abstract

This invention discloses the preparation and application of a hydrophobic compound high-encapsulation protein carrier, belonging to the field of bionanomaterials and food functional factor delivery technology. This invention provides a hydrophobic lumen-modified ferritin mutant rXHF, obtained by mutating polar residues at positions 68, 76, 141, and 148 on the lumen surface of human heavy chain ferritin rHuHF to tryptophan. Using this mutant as a carrier, lycopene is compounded with lycopene at a molar ratio of 1:200 to prepare the lycopene nanodelivery system rXHF-LYC. This system has a lycopene encapsulation efficiency of 17.8% and a DPPH clearance rate of 30%. Through transferrin receptor-mediated endocytosis, it crosses the blood-brain barrier, delivering lycopene to the hippocampus, inhibiting oxidative stress and neuroinflammation, restoring cholinergic function, and improving age-related cognitive impairment. This invention provides a new carrier for brain-targeted delivery of hydrophobic functional factors and offers a new strategy for the intervention of age-related neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanomaterials and food functional factor delivery technology, specifically relating to the preparation and application of a protein carrier with high encapsulation rate of hydrophobic compounds. Background Technology

[0002] Age-related cognitive impairment is a common neurodegenerative disease in the elderly, and its pathogenesis is closely related to oxidative stress, chronic neuroinflammation, and synaptic plasticity damage. Lycopene, as a highly active natural antioxidant, has a free radical scavenging capacity twice that of β-carotene and 100 times that of vitamin E, and has potential value in alleviating neuronal oxidative damage and improving cognitive function.

[0003] However, the clinical application of lycopene faces significant bottlenecks: its highly unsaturated linear structure leads to poor stability, making it susceptible to degradation by light, heat, and oxygen; its extreme lipid solubility (water solubility <0.1 μg / mL) results in a gastrointestinal absorption rate of <10%; and its poor blood-brain barrier penetration prevents effective accumulation in the central nervous system to exert its effects. To address these issues, researchers have developed nanodelivery systems such as liposomes and polymer nanoparticles, but these carriers suffer from poor biocompatibility and insufficient brain targeting.

[0004] Human heavy chain ferritin (rHuHF) is a natural 24-mer hollow nanocage protein with good biocompatibility and degradability. It can cross the blood-brain barrier via transferrin receptor-mediated endocytosis, making it an ideal carrier for targeted delivery to the central nervous system. However, the lumen surface of natural ferritin is rich in polar amino acid residues, creating a hydrophilic microenvironment. This results in weak affinity for strongly hydrophobic substances such as lycopene, with an encapsulation efficiency of only about 6%, far below its theoretical loading potential, severely limiting its application in the delivery of hydrophobic functional factors.

[0005] Currently, modifications to ferritin mainly focus on external surface channel modification or surface coupling, with no reports on hydrophobic modifications to the ferritin lumen to improve the encapsulation efficiency of hydrophobic substances. Therefore, by rationally designing protein molecules to hydrophobically modify the ferritin lumen, thereby enhancing its lycopene loading capacity and delivery efficiency while maintaining its nanocage structure and bioactivity, it is crucial to solving the problem of brain-targeted lycopene delivery. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a hydrophobic lumen-modified ferritin mutant rXHF, and to prepare a high-load-efficiency lycopene nanodelivery system rXHF-LYC using this mutant as a carrier. The invention also discloses its application in improving age-related cognitive impairment, providing a new carrier for the brain-targeted delivery of hydrophobic functional factors and a new strategy for the intervention of age-related neurodegenerative diseases.

[0007] To achieve the above objectives, the present invention first provides a hydrophobic lumen-modified ferritin mutant rXHF, which is obtained by using the natural human heavy chain ferritin rHuHF shown in SEQ ID No.1 as the starting amino acid sequence and mutating the glutamic acid at position 68, position 76, position 141, and position 148 on its lumen surface to tryptophan.

[0008] The present invention also provides a gene encoding the mutant rXHF.

[0009] The present invention also provides recombinant microbial cells expressing the mutant rXHF.

[0010] The present invention also provides products containing the said mutant rXHF.

[0011] In one embodiment of the present invention, the product includes food, medicine, or health products.

[0012] The present invention also provides a lycopene nanodelivery system rXHF-LYC containing the above-mentioned ferritin mutant rXHF. The delivery system is assembled by rXHF and lycopene through hydrophobic interactions and π-π stacking interactions. The lycopene is encapsulated in the hydrophobic cavity of rXHF. The molar ratio of mutant rXHF to lycopene is 1:(100~400).

[0013] In one embodiment of the present invention, the delivery system is prepared as follows: S1. Utilizing the pH-responsive depolymerization-reassembly properties of ferritin, rXHF is depolymerized into subunits under alkaline conditions, mixed with lycopene, and reassembled under neutral conditions, so that lycopene is encapsulated in rXHF. S2. The reaction product of S1 was dialyzed using a dialysis bag with a molecular weight <100 Da to remove free lycopene. The solution was then freeze-dried to obtain the lycopene nanodelivery system rXHF-LYC.

[0014] In one embodiment of the present invention, step S1 specifically includes the following steps: (1) Prepare a solution of the mutant rXHF described in claim 1 and adjust the pH to >10.6; (2) Add lycopene solution to the solution obtained in step (1) to obtain a mixture; (3) Adjust the pH of the mixture obtained in step (2) to 6-8, stir and then protect it from light for at least 2 hours.

[0015] In one embodiment of the present invention, in the mixture of step (2), the molar ratio of mutant rXHF and lycopene is 1:(100~200).

[0016] In one embodiment of the present invention, in step S2, dialysis is performed at 4°C for at least 18 hours. The dialyzed solution is then freeze-dried to obtain the lycopene nanodelivery system rXHF-LYC.

[0017] The present invention also provides the application of the above-mentioned ferritin mutant rXHF or the above-mentioned lycopene nanodelivery system rXHF-LYC in the preparation of a medicine for improving age-related cognitive impairment.

[0018] In one embodiment of the present invention, the improvement of age-related cognitive impairment is achieved through at least one of the following mechanisms: clearing intracellular ROS and restoring the oxidative-antioxidant balance in the brain and liver; reducing serum pro-inflammatory factor levels and inhibiting systemic and neuroinflammatory inflammation; upregulating the BDNF / TrkB signaling pathway and restoring synaptic plasticity; increasing hippocampal acetylcholine content and restoring cholinergic neuronal function; and inhibiting hippocampal neuronal aging and morphological damage.

[0019] In one embodiment of the present invention, the lycopene nanodelivery system rXHF-LYC can cross the blood-brain barrier through transferrin receptor 1-mediated endocytosis, thereby achieving brain-targeted delivery of lycopene.

[0020] In one embodiment of the present invention, the product is a food functional factor preparation, a health product, or a biopharmaceutical preparation.

[0021] The present invention also provides the application of the mutant rXHF in improving the lycopene loading capacity of ferritin.

[0022] Beneficial effects: This invention utilizes rational protein molecular design to prepare a hydrophobic lumen-modified ferritin mutant, rXHF. This mutant retains the 24-mer nanocage structure, colloidal stability, and biocompatibility of natural ferritin, while significantly enhancing the hydrophobicity of the lumen. The lycopene nanodelivery system rXHF-LYC, prepared using this mutant as a carrier, increases the encapsulation efficiency and loading rate of lycopene to 75% and 17.9%, respectively, with significantly enhanced antioxidant activity and a DPPH free radical scavenging rate of 30%. Furthermore, the rXHF-LYC nanodelivery system can efficiently cross the blood-brain barrier via receptor-mediated endocytosis, achieving brain-targeted delivery of lycopene. Compared to wild-type ferritin, the mutant rXHF exhibits a 1.3-fold increase in encapsulation efficiency and a 2.6-fold increase in lycopene loading rate.

[0023] The rXHF-LYC delivery system prepared in this invention can improve spatial learning and memory impairment induced by D-galactose in aging mice in a dose-dependent manner through multiple target mechanisms, such as clearing ROS, inhibiting oxidative stress, reducing pro-inflammatory factor levels, inhibiting neuroinflammation, upregulating the BDNF / TrkB pathway, restoring synaptic plasticity, increasing acetylcholine content, and restoring cholinergic neuronal function. It can also inhibit hippocampal neuronal aging and morphological damage, protect PC12 cells from oxidative stress damage, enhance cell viability, and inhibit apoptosis.

[0024] This invention provides a novel protein nanocarrier for the brain-targeted delivery of hydrophobic food functional factors / drugs, and also provides a new candidate formulation for the intervention of neurodegenerative diseases such as age-related cognitive impairment, with good prospects for food and biopharmaceutical applications. Attached Figure Description

[0025] Figure 1 The structural and physicochemical characterization of the hydrophobic lumen-modified ferritin mutant rXHF is shown in the following figures: A shows the site-directed mutation sites and lycopene molecular docking results of rHuHF and rXHF; B shows the sodium dodecyl sulfate polyacrylamide gel electrophoresis (left) and non-denaturing polyacrylamide gel electrophoresis (right) of rHuHF and rXHF; C shows the morphology of rXHF under a transmission electron microscope.

[0026] Figure 2 A shows the fluorescence spectra of rHuHF and rXHF; B shows the dynamic light scattering particle size distribution of rHuHF and rXHF.

[0027] Figure 3 Figure 1 shows the loading performance and antioxidant activity of the lycopene nanodelivery system rXHF-LYC: A is the encapsulation efficiency of rXHF-LYC at different molar ratios; B is the loading rate of rXHF-LYC at different molar ratios; C is the DPPH radical scavenging rate of rXHF-LYC, rHuHF-LYC and free lycopene; D is the dynamic light scattering particle size distribution of rXHF-LYC at different molar ratios.

[0028] Figure 4 The effect of rXHF-LYC on cognitive function and hippocampal neuronal aging in aging mice (swimming trajectory of mice during the Morris water maze exploration period).

[0029] Figure 5 The escape latency period of mice during the Morris water maze training period.

[0030] Figure 6 A shows the results of SA-β-gal staining in the hippocampus; B shows the results of H&E staining in the hippocampus; C shows the quantitative analysis of the proportion of SA-β-gal positive cells; and D shows the overall flowchart of the animal experiment.

[0031] Figure 7 The effect of rXHF-LYC on oxidative stress, inflammation, synaptic plasticity and cholinergic function in aging mice (quantitative analysis of immunofluorescence intensity of NeuN, BDNF, TrkB and SYP in the hippocampus).

[0032] Figure 8 AC represents the serum levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β; D represents the hippocampal ACh content.

[0033] Figure 9 A represents an indicator of oxidative stress in the hippocampus; B represents an indicator of oxidative stress in the liver.

[0034] Figure 10 The diagram illustrates the protective effect of rXHF-LYC against oxidative damage in PC12 cells: A shows the intracellular ROS fluorescence staining results; B shows the cellular uptake and localization results of rXHF-LYC, with the upper figure showing co-localization analysis of FITC-rXHF-LYC and LysoTracker, and the lower figure showing co-localization analysis of control free FITC and LysoTracker; C shows the cell viability detection results; D shows the quantitative analysis of cell apoptosis and mitochondrial membrane potential by flow cytometry; E shows the intracellular ACh content detection results.

[0035] Figure 11 A schematic diagram illustrating the mechanism by which mutant human heavy chain ferritin rXHF loaded with lycopene exerts its neuroprotective effect. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Statistical analyses involved in the following examples: all experiments were performed at least three times. GraphpadPrism 10.0.3 software was used for data analysis.

[0038] Example 1: Preparation of hydrophobic lumen-modified ferritin mutant rXHF 1. Construction of site-directed mutagenesis vectors Human heavy chain ferritin rHuHF mRNA (as shown in SEQ ID No. 2) was converted into cDNA. Using the cDNA as a template, site-directed mutagenesis primers were designed targeting amino acids at positions 68 (E to W), 76Q to W), 141 (E to W), and 148 (E to W). Figure 1A) The upstream primer sequence was 5'-GTTTAACTTTAAGAAGGAGATATACATATGACAACAGCATCAACCTCACAAGTAAGAC-3', and the downstream primer sequence was 5'-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCAACTTTCATTATCGCTATC-3'. The mutant gene rXHF was obtained by PCR amplification. The mutant gene was double-digested and ligated with the pET-21a(+) vector, transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and cultured at 37°C for 12 h. Single clones were picked and sequenced for verification, constructing the mutant engineered strain BL21-pET21a-rXHF. The unmutated cDNA was ligated into the pET-21a(+) vector and transformed into E. coli to construct the wild-type engineered strain BL21-pET21a-rHuHF.

[0039] 2. Protein-induced expression The verified wild-type and mutant engineered strains were inoculated into 50 mL LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37 °C and 200 rpm until OD600 = 0.6–0.8. IPTG was then added to a final concentration of 0.1 mM, and expression was induced for another 9 h at 37 °C and 200 rpm. After induction, the bacterial cells were collected by centrifugation at 8000 rpm and 4 °C for 10 min.

[0040] 3. Protein purification The bacterial cells were resuspended in 20 mM Tris-HCl buffer (pH 7.4) and sonicated on ice (300 W, 3 s operation, 5 s interval, total 30 min). The lysate was heat-precipitated in a 60 °C water bath for 10 min, and centrifuged at 12000 rpm and 4 °C for 20 min to remove contaminating proteins. The supernatant was collected. The supernatant was loaded onto a DEAE-Sepharose Fast Flow weak anion exchange chromatography column equilibrated with 20 mM Tris-HCl buffer (pH 7.4) and eluted with a linear gradient of 0–0.5 M NaCl. The elution peak containing rXHF was collected. The collected solution was concentrated and loaded onto a Sephacryl S-300 HR gel filtration chromatography column equilibrated with 20 mM Tris-HCl buffer (pH 7.4). The main peak was collected, yielding the hydrophobic lumen-modified ferritin mutant rXHF and the wild-type protein rHuHF with a purity >95%. The protein concentration was determined by the BCA method and stored at -80 °C for later use.

[0041] 4. Structural verification of rXHF Molecular docking simulations showed that the LibDockScore (117.93) for lycopene binding to rXHF was slightly higher than that for rHuHF (115.233), indicating that the overall binding affinity between ferritin and lycopene did not change significantly. However, a fundamental shift in the binding interface was observed: lycopene was primarily located in the outer A / C helix region of rHuHF, while when binding to rXHF, it was specifically located on the inner B / D helix surface. Figure 1 A). SDS-PAGE and Native-PAGE confirmed that the subunit molecular weight of rXHF is approximately 20 kDa, and the 24-mer structure is approximately 440 kDa. Figure 1 B); its spherical hollow nanocage morphology was observed by transmission electron microscopy. Figure 1 C); The enhanced hydrophobicity of the cavity was verified by fluorescence spectroscopy, and the results showed that rXHF maintained the complete 24-polymer nanocage structure and the hydrophobicity of the cavity was significantly enhanced. Figure 2 A); The hydrodynamic diameter distribution of the mutant protein is similar to that of the wild-type protein, indicating that the mutation did not cause protein aggregation or disassembly, and the nanoparticles maintained a uniform and stable dispersion in the solution. Figure 2 B).

[0042] Example 2: Preparation of the lycopene nanodelivery system rXHF-LYC 1. Reagent preparation Lycopene (LYC) stock solution: Take lycopene standard (purity ≥98%), dissolve it in DMSO to prepare a 10mM stock solution, and store it at -20℃ protected from light; rXHF protein solution: Prepare a 1mg / mL protein solution from the rXHF prepared in Example 1 using 20mM Tris-HCl buffer (pH 7.4).

[0043] 2. Preparation of rXHF-LYC Take 10 mL of rXHF protein solution and slowly adjust the pH to 10.8 with 1 M NaOH. Stir at room temperature for 10 min to depolymerize rXHF into subunits. Under light-protected, magnetically controlled, and gently stirring conditions (100-120 rpm), slowly add lycopene stock solution dropwise to the depolymerized rXHF solution to achieve different molar ratios (rXHF:lycopene = 1:100, 1:200, 1:400, corresponding to...). Figure 3The rXHF-LYC-1, rXHF-LYC-2, and rXHF-LYC-3 groups were added dropwise and incubated at room temperature for 15 min. The pH of the mixed solution was slowly adjusted back to 7.4 with 1M HCl, and stirred at room temperature for 20 min to allow the rXHF subunits to reassemble and encapsulate lycopene. The mixed solution was aged in a 4°C refrigerator in the dark for 2 h, followed by centrifugation at 10000 rpm and 4°C for 10 min to remove unencapsulated lycopene precipitate. The supernatant was transferred to a 100 kDa dialysis bag, and dialyzed at 4°C for 18 h with 20 mM Tris-HCl buffer (pH 7.4) as the external dialysis solution. The external dialysis solution was changed every 6 h to remove free lycopene and DMSO, resulting in the lycopene nanodelivery system rXHF-LYC, which was stored in the dark at 4°C for later use.

[0044] Using the same method, the lycopene nanodelivery system rHuHF-LYC was prepared with the wild-type protein rHuHF prepared in Example 1 (the molar ratio of rHuHF to lycopene was 1:100, corresponding to the rHuHF group in the figure).

[0045] 3. Performance testing of rXHF-LYC The lycopene content was determined at 360 nm using ultraviolet-visible spectrophotometry, and the encapsulation efficiency and loading rate were calculated (encapsulation efficiency (%) = encapsulated lycopene content / total lycopene content × 100%, loading rate (%) = encapsulated lycopene content / ferritin content × 100%).

[0046] The results are as follows Figure 3 A and Figure 3 As shown in B, under the condition that the molar ratio of heavy chain ferritin to lycopene is 1:100 (rHuHF group and rXHF-LYC-1 group), the encapsulation efficiency of the wild type is 58.1±1.3%, and the loading rate is 6.97±0.24%; the encapsulation efficiency of the mutant is 75.5±0.5%, and the loading rate is 9.09±0.1%. Compared with the wild type, the encapsulation efficiency and loading rate of the mutant are both improved. To further explore the cavity capacity limit of the mutant ferritin, experiments were conducted with molar ratios of 1:200 and 1:400 (rXHF-LYC-2 and rXHF-LYC-3 groups). At a molar ratio of 1:200, the encapsulation efficiency of rXHF-LYC is 75±3%, and the loading rate is 17.9±0.7%.

[0047] Wild-type and mutant lycopene nanodelivery systems rHuHF-LYC and rXHF-LYC were prepared at a heavy chain ferritin to lycopene molar ratio of 1:100. Their antioxidant activity was determined by DPPH free radical scavenging assay. The initial addition amount of heavy chain ferritin was 10 mL and 1 mg / mL, respectively, and the nanodelivery system was prepared according to the method in step 2. 100 μL of dialyzed lycopene nanodelivery system solution (2 μM, pH 7.5) was added to 100 μL of ethanol-dissolved DPPH free radical solution, and the mixture was incubated in a 96-well plate at 37 ℃. The absorbance was measured at 520 nm at 30 min intervals for 1.5 h. An equivalent dose of unencapsulated lycopene (LYC) was used as a control. DPPH free radical scavenging rate (%) = (AB - AE) / AB × 100%, where AB is the OD520 nm of the blank group; AE is the OD520 nm of the sample group.

[0048] The results showed that its DPPH scavenging rate at 90 min was 30±1.2%, significantly higher than that of the free lycopene group (25.6±0.5%) and the rHuHF-LYC group (27.4±0.4%). Figure 3 C); Dynamic light scattering was used to determine its particle size and zeta potential, and the results showed that its hydrated particle size was approximately 12 nm, with a uniform particle size distribution and no obvious aggregation. Figure 3 D).

[0049] Example 3: In vivo experiment on the improvement of D-galactose-induced cognitive impairment in aging mice by rXHF-LYC 1. Animal model establishment and grouping Sixty 16-week-old SPF-grade male C57BL / 6 mice were selected and, after 7 days of acclimatization, randomly divided into 5 groups (n=12): normal control group (Control, saline), D-galactose model group (D-gal, 250 mg / kg D-galactose), low-dose rXHF-LYC group (LD, 15 mg / kg rXHF-LYC + 250 mg / kg D-galactose), high-dose rXHF-LYC group (HD, 45 mg / kg rXHF-LYC + 250 mg / kg D-galactose), and free lycopene group (LYC, lycopene equivalent of high-dose group + 250 mg / kg D-galactose). The doses in the LD and HD groups were based on the mass of ferritin. The D-gal, LD, HD, and LYC groups were intraperitoneally injected with 250 mg / kg D-galactose daily for 2 weeks to establish the aging model; the Control group was intraperitoneally injected with an equal volume of saline. Starting from week four, while continuing intraperitoneal injections of D-gal, a treatment intervention experiment was added. The LD and HD groups received daily tail vein injections of the corresponding dose of rXHF-LYC, the LYC group received a tail vein injection of the same dose of free lycopene as the HD group, and the Control and D-gal groups received a tail vein injection of the same volume of normal saline. The intervention continued for 5 weeks, ending in week 8. Figure 6 D).

[0050] 2. The Morris water maze experiment assesses cognitive function. Starting from week 8 of intervention, the Morris water maze experiment was conducted: During the training period (days 1-4), mice were placed into the water maze from different entry points daily, and the escape latency to find the hidden platform within 60 seconds was recorded; during the exploration period (day 5), the hidden platform was removed, and the time spent in the target quadrant and swimming trajectory of the mice within 60 seconds were recorded. Results showed that the escape latency of mice in the D-gal group was significantly longer than that in the Control group, and the time spent in the target quadrant was significantly shorter, indicating significant spatial learning and memory impairment; there was no significant difference between the free lycopene group and the D-gal group; however, the escape latency of the low- and high-dose rXHF-LYC groups was significantly shortened, and the time spent in the target quadrant was significantly prolonged, showing a dose-dependent effect. The high-dose group was close to the Control group, indicating that rXHF-LYC can significantly improve the cognitive function of aging mice. Figure 4 , Figure 5 ).

[0051] 3. Hippocampal neuronal aging and morphology detection After the behavioral experiment, mice were sacrificed at week 8, and hippocampal tissue was collected for SA-β-gal and H&E staining. SA-β-gal staining results showed that the proportion of senescent positive cells in the CA1 and CA3 regions of the hippocampus in the D-gal group (5.05±0.8%) was significantly higher than that in the Control group (0.33±0.35%), while the proportion of senescent positive cells in the rXHF-LYC treatment group was significantly reduced (0.71% in the HD group and 1.11% in the LD group), showing a dose-dependent effect. Figure 6 A, Figure 6 C); H&E staining results showed that hippocampal neurons in the D-gal group were loosely arranged and some neurons were shrunken, while neurons in the high-dose rXHF-LYC group were tightly arranged and morphologically intact, with no significant difference from the Control group, indicating that rXHF-LYC can inhibit hippocampal neuronal senescence and improve neuronal morphological damage (C). Figure 6 B).

[0052] 4. Detection of oxidative stress and inflammatory markers Hippocampal and liver tissues were collected from mice, and oxidative stress indicators were measured: The D-gal group showed significantly increased MDA content in the hippocampus and liver, and significantly decreased SOD and GSH-Px activities (hippocampal MDA, SOD, and GSH-Px were 11.59%, 6.02%, and 25.56%, respectively). The rXHF-LYC treatment group significantly reduced MDA content, increased SOD and GSH-Px activities, and restored the oxidative-antioxidant balance, with higher doses showing more significant effects (hippocampal MDA, SOD, and GSH-Px were 5.67%, 21.24%, and 56.22%, respectively). Figure 9 Mouse serum was collected, and pro-inflammatory factors were measured using ELISA. In the D-gal group, serum levels of TNF-α, IL-6, and IL-1β were significantly increased (47.83%, 65.75%, and 69.78%, respectively). The high-dose rXHF-LYC group significantly reduced the levels of these pro-inflammatory factors and inhibited systemic inflammation; TNF-α, IL-6, and IL-1β levels were 12.38%, 52.34%, and 28.07%, respectively, indicating that rXHF-LYC exerts its antioxidant and anti-inflammatory effects through a synergistic effect between the peripheral and central nervous systems. Figure 8 ).

[0053] 5. Synaptic plasticity and cholinergic function testing Hippocampal tissue from mice was collected for immunofluorescence staining to detect the expression of NeuN, BDNF, TrkB, and synaptophysin SYP. The results showed that the fluorescence intensity of these proteins in the D-gal group was significantly lower than that in the Control group; the rXHF-LYC treatment group significantly upregulated the expression of BDNF / TrkB pathway and SYP, restored synaptic plasticity, and showed a dose-dependent effect. Figure 7The number of NeuN-immunopositive cells increased significantly, and NeuN expression levels rebounded markedly. Measurements of hippocampal acetylcholine levels showed that the D-gal group had significantly decreased hippocampal acetylcholine levels; the rXHF-LYC treatment group significantly increased acetylcholine levels and restored cholinergic neuronal function, with the high-dose group approaching the control group. Figure 8 D).

[0054] Example 4: In vitro experiment of rXHF-LYC protecting D-galactose-induced oxidative damage in PC12 cells 1. Cytotoxicity assay After PC12 cells adhered to the culture medium, they were incubated with D-gal at final concentrations ranging from 100 to 500 mM for 4 hours. After incubation, a CCK-8 assay was performed. The culture medium was discarded, and CCK-8 reagent was added for incubation for 2 hours. The absorbance was measured at 450 nm after incubation. The results showed that 500 mM D-gal reduced the viability of PC12 cells to approximately 50% of the normal control group. Figure 10 C).

[0055] After PC12 cells adhered to the culture medium, rXHF-LYC at final concentrations of 50-800 nM was added and incubated overnight. The culture medium was then discarded, and the cells were incubated with prepared CCK-8 reagent for 2 hours. After incubation, absorbance was measured at 450 nm. The results showed that all concentrations of rXHF-LYC had no significant effect on cell viability, indicating that it was non-toxic to cells. Figure 10 C).

[0056] 2. Cell model establishment and grouping PC12 cells were cultured and divided into several groups: a normal control group, a D-gal model group (500 mM D-gal), a low-dose rXHF-LYC group (10 nM rXHF-LYC + 500 mM D-gal, LD), a medium-dose group (50 nM rXHF-LYC + 500 mM D-gal, MD), a high-dose group (100 nM rXHF-LYC + 500 mM D-gal, HD), and a free lycopene group (containing 100 nM rXHF-LYC at the same lycopene dose + 500 mM D-gal). After cell adhesion, cells were pretreated by incubating in medium containing the appropriate dose of rXHF-LYC or free lycopene for 24 h. The medium was then replaced with fresh medium containing the appropriate dose of rXHF-LYC or free lycopene, and a final concentration of 500 mM D-gal was added for 4 h to establish an oxidative stress injury model. The normal control group received the same dose of medium throughout the treatment.

[0057] 3. Cellular ROS detection PC12 cells were cultured stably in 6-well plates for 24 h, and the model was established by stimulation as described above. After the experiment, ROS in PC12 cells were stained using the DCFH-DA fluorescent probe. The DCFH-DA fluorescence staining results showed that the intracellular ROS fluorescence intensity was significantly increased in the D-gal group; rXHF-LYC could reduce the ROS fluorescence intensity in a concentration-dependent manner, clear excess ROS in cells, and inhibit oxidative stress damage. Figure 10 A).

[0058] 4. Apoptosis and mitochondrial function detection The experiment was conducted following the method in step 2. After the experiment, PC12 cells were collected for analysis. Double staining with Mito-Tracker Red CMXRos (labeling mitochondrial membrane potential) and Annexin V-FITC (labeling early apoptosis) showed that the proportion of apoptotic cells in the D-gal model group was significantly increased (42.3±0.7%). While the proportion of apoptotic cells in the free LYC group was slightly reduced, it remained at a high level. rXHF-LYC treatment reduced the proportion of apoptotic cells in a concentration-dependent manner, with the high-dose group showing the best effect (13.1±0.48%). Figure 10 D).

[0059] 5. Cell uptake and localization detection rXHF-LYC was mixed with FITC (100 times the molecular weight) in 20 mM PBS (pH 7.4) and incubated overnight at 4°C to label rXHF-LYC. Free FITC was removed by dialysis. FITC-labeled rXHF-LYC (200 nM) was added to cell culture medium, and the control group was added with the same concentration of free FITC. After co-incubation with PC12 cells cultured for 24 h for 1 h, the cells were stained with LysoTracker Red lysosomal probes, and observed under a laser confocal microscope. Figure 10 B): FITC-labeled rXHF-LYC can efficiently enter PC12 cells and is highly colocalized with lysosomal probes, indicating that rXHF-LYC enters cells through receptor-mediated endocytosis, is located in lysosomes, and depolymerizes to release lycopene in an acidic microenvironment.

[0060] 6. Detection of acetylcholine content The experiment was conducted following the method in step 2. Afterwards, the intracellular acetylcholine content was measured. The results showed that ( Figure 10 E): The intracellular acetylcholine content in the D-gal group was significantly reduced (168±10 ug / mgprot), while rXHF-LYC could increase the acetylcholine content in a concentration-dependent manner (285±11 ug / mgprot), restoring the acetylcholine synthesis function of cholinergic neurons, which was consistent with the results of in vivo experiments.

[0061] In summary, this invention constructed a lumen-modified hydrophobic ferritin mutant, rXHF, and successfully prepared a lycopene-encapsulated nanodelivery system, rXHF-LYC, using a pH-controlled reassembly technique. In vitro and in vivo experiments confirmed that rXHF-LYC effectively improves D-galactose-induced age-related cognitive impairment through multiple mechanisms, including antioxidant, anti-inflammatory, inhibition of neuronal senescence, and restoration of synaptic plasticity and cholinergic function, with significantly better effects than free lycopene and natural ferritin encapsulation systems.

[0062] 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 hydrophobic lumen-modified ferritin mutant rXHF, characterized in that, Based on the amino acid sequence shown in SEQ ID No. 1, glutamic acid at position 68 was mutated to tryptophan, glutamine at position 76 was mutated to tryptophan, glutamic acid at position 141 was mutated to tryptophan, and glutamic acid at position 148 was mutated to tryptophan.

2. The gene encoding the mutant rXHF of claim 1.

3. Recombinant microbial cells expressing the mutant rXHF of claim 1.

4. A product containing the mutant rXHF as described in claim 1.

5. A lycopene nanodelivery system, characterized in that, The product is prepared by loading lycopene onto the mutant rXHF described in claim 1; in the delivery system, the molar ratio of mutant rXHF to lycopene is 1:(100~400).

6. A method for preparing the delivery system of claim 5, characterized in that, Includes the following steps: (1) Prepare a solution of the mutant rXHF described in claim 1 and adjust the pH to >10.6; (2) Add lycopene solution to the solution obtained in step (1) to obtain a mixture; (3) Adjust the pH of the mixture obtained in step (2) to 6-8, stir and then protect it from light for at least 2 hours.

7. The method according to claim 6, characterized in that, In step (2), the molar ratio of mutant rXHF to lycopene in the mixture is 1:(100~200).

8. The method according to claim 7, characterized in that, After avoiding light for at least 2 hours in step (3), dialysis is performed; the dialysis is performed using a dialysis bag with a molecular weight <100 Da for at least 18 hours.

9. The application of the mutant rXHF of claim 1 in improving the lycopene loading capacity of ferritin.

10. The use of the lycopene nanodelivery system of claim 5 in the preparation of a medicament for improving age-related cognitive impairment.

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