Liver-targeted lycopene liposome microspheres and application thereof

Abstract

The present application relates to a kind of liver targeted lycopene plastosome globule and its application, the nanoscale structure of lycopene wrapped by the plasma membrane of plastosome globule can solve the problems such as poor water solubility of lycopene, easy oxidation degradation, as a kind of natural delivery system, plastosome globule can improve the water solubility of lycopene and the resistance to ion, acid, heat and other conditions, with good gastrointestinal stability and intestinal permeability, can effectively deliver lycopene to blood and liver, improve the bioavailability of lycopene, can be applied to the field such as biological medicine and functional food.In addition, the plastosome globule is derived from natural edible plant tissue, can meet the pursuit of consumer to health without side effect, has broad development prospect.

Description

Technical Field

[0001] This invention belongs to the fields of functional foods and biotechnology, specifically relating to a liver-targeting lycopene microsphere and its application. Background Technology

[0002] Lycopene, an isoprene compound derived from plant-based foods, possesses potent antioxidant activity—100 times that of vitamin E and twice that of β-carotene. It can slow down the oxidation of proteins, lipids, and DNA, regulate the cell cycle and intercellular connections, and shows significant effects in preventing and treating non-alcoholic fatty liver disease, inhibiting tumor cell proliferation, and preventing cardiovascular and cerebrovascular diseases. However, lycopene has extremely poor water solubility and stability; it is insoluble in water and easily degraded by environmental factors such as light and heat, as well as by internal factors such as extreme pH in the stomach, gastrointestinal digestive enzymes, and physical peristalsis. This degradation disrupts the lycopene structure, resulting in extremely low bioavailability and severely hindering its application. Therefore, improving the bioavailability of lycopene is a key issue in its efficient utilization.

[0003] Lycopene is almost insoluble in solvents such as water and ethanol, but has high solubility in oily substances. Therefore, the main approach to improving the bioavailability of lycopene is currently to increase its water solubility. However, improving the water solubility of lycopene often requires multiple composite materials to help it dissolve in water, which essentially involves adding a water-soluble shell. This not only increases the complexity of industrial production steps but also raises production costs. On the other hand, the composite materials used are often non-biodegradable chemical materials, which may cause sensitization reactions and chemical residues during consumer contact, potentially posing a significant threat to consumer health. Furthermore, the in vivo stability of this method of increasing lycopene water solubility remains questionable. For example, patent CN109419775B authorizes a water-soluble lycopene, its preparation method, and its application. Its formula consists of 1 part by weight of lycopene oil, 1-5 parts by weight of a solid dispersion carrier material mixture, and 1-5 parts by weight of cyclodextrin. The solid dispersion carrier material mixture is a mixture of polyethylene glycol 6000 and polyvinylpyrrolidone K30 in a 3:2 mass ratio. The prepared lycopene system significantly improves the water solubility of lycopene; however, the formulation requires strict and precise control of the ratio of solid dispersion carrier materials and lycopene oils, resulting in substantial costs and risks in actual production. Therefore, developing a novel, highly stable, and low-risk water-soluble lycopene system is crucial for promoting the application of lycopene in various fields. Plastoglobules are specialized organelle structures derived from chloroplasts in higher plants. In mature plant fruits, plastoglobules are the main metabolic site for carotenoids. They are mostly spherical in shape, with a particle size between 30-500 nm, encapsulated by a single plasma membrane, and stably exist within the plastid matrix. Therefore, in plants such as tomatoes and watermelons, plastoglobules are rich in lycopene and exist in the form of spherical nanoparticles, with lycopene encapsulated within the plasma membrane, representing a natural lycopene encapsulation system. The nanoscale size of the plastospheres allows them to easily penetrate the intestinal mucosa and intestinal wall, enabling uptake by the intestines and further entry into the systemic circulation, thus achieving efficient delivery of lycopene. Furthermore, the amphiphilic plasma membrane of the plastospheres can encapsulate lycopene, significantly improving its water solubility and facilitating absorption by the liver. These characteristics make plastospheres excellent in improving the stability, water solubility, and biodegradability of lycopene, and they hold promise as a potential natural nanodelivery system for liver-targeted delivery applications. Currently, there are almost no precedents in published or granted patents involving natural nanosystems loaded with carotenoids for liver-targeted delivery applications. Therefore, this invention has broad application prospects. Summary of the Invention

[0004] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a liver-targeting natural lycopene plasmid. This plasmid is green and safe, can improve the water solubility of lycopene and its resistance to adverse external factors, has good gastrointestinal stability and intestinal permeability, can effectively deliver lycopene to the blood and liver, improve the in vivo bioavailability of lycopene, and can be applied to the fields of biomedicine and functional foods.

[0005] Technical solution: A liver-targeting lycopene plasmid structure, wherein the plasmid structure consists of a plasma membrane encapsulating lycopene.

[0006] Furthermore, the plastid spheres are derived from plant fruits rich in lycopene.

[0007] Furthermore, the plant fruits rich in lycopene include tomatoes, watermelons, peppers, grapefruits, and guavas.

[0008] A method for preparing liver-targeted lycopene microspheres includes the following steps:

[0009] S1: After rinsing the cut lycopene-rich plant fruits with pre-cooled extraction buffer, add pre-cooled extraction buffer at a mass-to-volume ratio of 1:(3-7), homogenize and stir to obtain crude juice;

[0010] S2: After filtering the crude fruit juice, collect the filtrate, centrifuge the filtrate and collect the precipitate. Add extraction buffer to the precipitate at a mass-to-volume ratio of 1:(1-4) to reconstitute it. After reconstitution, sonicate to obtain a suspension.

[0011] S3: Add the suspension, 35-38 wt.% sucrose solution, 18-20 wt.% sucrose solution, 12-15 wt.% sucrose solution and 3-5 wt.% sucrose solution to a centrifuge tube and centrifuge. Collect the top layer of material, which is the lycopene granules.

[0012] Furthermore, the extraction buffer in step S1 comprises 20 mM Tricine-KOH, 450 mM sorbitol, 10 mM EDTA-Na2, 10 mM NaHCO3 and 0.5 wt.% bovine serum albumin.

[0013] Furthermore, in step S1, the homogenization and stirring are performed every 1-3 seconds for a total of 1-5 times.

[0014] Furthermore, in step S2, the filtering material consists of 2-5 layers of degreased cotton gauze and a 60-80μm filter screen, and the centrifugation conditions are 1200g, 4℃, 10-15min, and the ultrasonic treatment frequency is 60-80Hz, 1-3min.

[0015] Furthermore, in step S3, the ratio of the suspension, 35-38 wt.% sucrose solution, 18-20 wt.% sucrose solution, 12-15 wt.% sucrose solution, and 3-5 wt.% sucrose solution is 3:3:3:2:4.

[0016] Furthermore, the centrifugation conditions in step S3 are 80,000-120,000g, 4℃, 2-4h.

[0017] This invention provides an application of liver-targeted lycopene microspheres in biomedicine and functional foods.

[0018] Beneficial effects:

[0019] 1. This invention uses a gentle pulping speed to crush fruit pieces, preserving the integrity of plastid spheres to the greatest extent. Extraction is carried out using ultrasonic treatment and density gradient centrifugation. The prepared plastid spheres have a complete structure and contain abundant carotenoids, especially lycopene, which improves the retention rate and stability of lycopene under different salt ion concentrations, pH, and temperature conditions.

[0020] 2. The plastid microspheres of the present invention, as a lycopene delivery system, have good gastrointestinal stability and good intestinal permeability. Their permeability coefficients in the duodenum, jejunum and ileum are 32.00 times, 39.00 times and 143.00 times that of free lycopene, respectively.

[0021] 3. The lycopene plastid microspheres of the present invention can increase the bioavailability of lycopene in vivo. Compared with free lycopene, the time to peak plasma concentration of lycopene in the plastid microspheres is delayed by 2 hours, the peak plasma concentration is 1.96 times higher, and the bioavailability is increased by 1.71 times.

[0022] 4. The plastosphere delivery method of this invention significantly enhances lycopene accumulation in the liver. Compared to free lycopene, the peak liver concentration of lycopene delivered via plastospheres is delayed by 7 hours, the peak liver concentration is 4.90 times higher, and the bioavailability is increased by 7.28 times. The plastospheres, with an average particle size of approximately 200 nm, can passively accumulate in the liver through the sinusoidal space, achieving liver-targeted delivery of lycopene and providing theoretical support for research on liver-targeted delivery in biomedicine and food.

[0023] 5. The present invention extracts plastid microspheres directly from plants, which is simpler and less costly than extracting lycopene and then encapsulating or loading it. Moreover, the plastid microspheres are derived from natural edible plant tissues, making them green and environmentally friendly. Compared with other delivery systems, they are safer and can meet consumers' pursuit of health and no side effects. Attached image description:

[0024] Figure 1This is a diagram showing the separation of crude extracts at different density gradients after density gradient centrifugation in Example 1.

[0025] Figure 2 This is a Western blot image of the top and second-layer materials collected after density gradient centrifugation in Example 2.

[0026] Figure 3 This is a TEM transmission electron microscope image of the top layer material collected after density gradient centrifugation in Example 3;

[0027] Figure 4 This is a CLSM laser confocal fluorescence microscope image of the plasmonic spheres in Example 3 after being excited by a single channel at 488 nm.

[0028] Figure 5 This is a CLSM laser confocal fluorescence microscope image of the plasmonic spheres in Example 3 after being excited by a dual-channel 488 / 543nm laser.

[0029] Figure 6 This is a particle size distribution diagram of the plasmonic spheres in Example 3;

[0030] Figure 7 The graph shows the lycopene retention rate of the plastid microspheres in Example 4 under different salt ions, pH, and temperature conditions.

[0031] Figure 8 This is a graph showing the lycopene retention rate during the in vitro simulated digestion process in Example 4;

[0032] Figure 9 Transwell permeability diagram of intestinal mucus in Example 5;

[0033] Figure 10 This is a permeability coefficient diagram of the isolated intestine in Example 5;

[0034] Figure 11 This is a CLSM laser confocal fluorescence microscopy image of intestinal permeation in Example 5. A is free lycopene, and B is plastid spheres.

[0035] Figure 12 This is a graph showing the thickness of the mucus layer at the maximum fluorescence intensity for different treatment times in Example 5;

[0036] Figure 13 This is a graph showing the change in lycopene concentration in blood over time in Example 6;

[0037] Figure 14 This is a graph showing the change in lycopene concentration in the liver over time in Example 6. Detailed Implementation

[0038] To provide a clearer and more thorough understanding of the technical solution of this invention, the invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the invention, but the invention is not limited to these embodiments:

[0039] Example 1

[0040] Extraction of plasmids:

[0041] Preparation of pre-cooled extraction buffer: The extraction buffer contains: 20 mM Tricine-KOH (pH 8.4), 450 mM sorbitol, 10 mM EDTA-Na2, 10 mM NaHCO3, and 0.5% wt.% bovine serum albumin.

[0042] Wash fresh, ripe tomatoes, remove the seeds, and dice them. Rinse the diced tomatoes with pre-cooled extraction buffer. Add pre-cooled extraction buffer at a ratio of 1:5 (w / v), and homogenize using a multi-functional juicer. Stir intermittently at low speed every 2 seconds for 2 seconds each time, for a total of 4 times. Filter the mixture through four layers of absorbent cotton gauze and a 60μm filter, and collect the filtrate. Centrifuge the filtrate at 1200g and 4℃ for 10 minutes, collect the precipitate, and reconstitute it with extraction buffer at a ratio of 1:4 (w / v).

[0043] Extraction was performed using ultrasonic treatment and density gradient centrifugation. The reconstituted suspension was ultrasonicated at 60 Hz for 3 min. In a 30 mL transparent ultracentrifuge tube, 6 mL of the suspension, 6 mL of 38 wt.% sucrose solution, 6 mL of 20 wt.% sucrose solution, 4 mL of 15 wt.% sucrose solution, and 8 mL of 5 wt.% sucrose solution were added sequentially to the bottom. The resulting density gradient centrifuge tube was then centrifuged at 100,000 g at 4 °C for 2 h. The results after density gradient centrifugation are shown below. Figure 1 As shown, use a pipette to collect the top layer and the next layer containing plasmid spheres.

[0044] Example 2

[0045] Identification of plasmonic spheres:

[0046] The top and second-layer samples were used for Western blot analysis. The extracted top and second-layer samples were vortexed for 3 min in an extraction buffer containing: 200 mM Tris (pH 8.0), 150 mM NaCl, 2.5 mL of a protease inhibitor mixture per 1 mL of extraction buffer, 1 mM PMSF, and 0.125% (v / v) Triton X-100. The mixture was then centrifuged at 13000 g for 15 min at 4 °C, and the supernatant was collected. After 12% SDS-PAGE, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The transferred membrane was incubated with 1:2500 diluted antibodies against Arabidopsis thaliana AtPGL35 protein (a protein specific to the surface of plastid globules) and Arabidopsis thaliana AtToC75 protein (a protein specific to the surface of chromoplasts), followed by incubation with an anti-rabbit IgG alkaline phosphatase conjugate (1:80000 dilution). Finally, the transfer membrane was developed using an alkaline phosphatase substrate, consisting of 66 mL of nitroblue tetrazolium (50 mg / mL) and 33 mL of 5-bromo-4-chloro-3-indole phosphate (50 mg / mL). The substrate was dissolved in 10 mL of buffer solution containing 100 mM Tris-HCl, 100 mM NaCl, and 5 mM MgCl2 at pH 9.5. The treated transfer membrane was then subjected to chemiluminescence imaging. The results are as follows: Figure 2 As shown, AtPGL35 antibody is an antibody against the characteristic membrane protein of plasmids, and ToC75 antibody is an antibody against the characteristic membrane protein of chromoplasms. After imaging with a chemiluminescence imager, different bands appeared on the transfer membranes of the top and second layers. Looking at the top layer, the band on the transfer membrane incubated with PGL35 antibody was located around 35 kDa, indicating the presence of plasmids, meaning plasmids were present in the top layer. The transfer membrane incubated with ToC75 antibody did not show a band, indicating the absence of chromoplasm membranes in the top layer. On the other hand, the transfer membrane incubated with PGL35 antibody also showed a band around 35 kDa, indicating the presence of plasmids in the second layer as well. The transfer membrane incubated with ToC75 antibody showed a distinct band, indicating the presence of chromoplasm membranes in the second layer. In summary, after density gradient centrifugation, plasmids were collected from the top layer, and no residue of chromoplasm membranes or other substances was found, indicating that the extraction process successfully extracted plasmids with high purity.

[0047] Example 3

[0048] Characterization of plasmonic spheres:

[0049] (1) Morphology and structure of plasmonic spheres

[0050] One drop of the extracted plasmid sample suspension was adsorbed onto a copper grid for 1 min, then the sample on the copper grid was stained with 2 wt.% uranium acetate solution for 30 seconds, allowed to air dry, and the morphology of the extracted plasmids was observed using a TEM transmission electron microscope. Figure 3 As shown, the extracted plasmids were observed to be spherical in TEM, with a particle size of approximately 100 nm.

[0051] (2) Observation by CLSM laser confocal fluorescence microscopy

[0052] To further observe the CLSM, since plastospheres potentially contain lycopene, and lycopene exhibits autofluorescence, the CLSM experiment was designed to observe the autofluorescence of lycopene. However, because plastospheres contain a lipid membrane, and lipids do not exhibit autofluorescence, the membrane needs to be stained with a lipid-soluble dye before observation. Nile red dye was added to the extracted plastosphere sample at a ratio of 1:100 and incubated for 20 min. The sample was then fixed on a microscope slide and observed using a CLSM at single-channel excitation at 488 nm and dual-channel excitation at 488 nm and 543 nm, respectively. Images of 1024 × 1024 pixels were collected.

[0053] pass Figure 4 It can be seen that CLSM successfully captured the autofluorescent structure of lycopene in single-channel excitation at a wavelength of 488 nm. Its morphology is a nanoscale spherical shape, consistent with the spherical structure of plastospheres, therefore lycopene may be encapsulated within plastospheres; through... Figure 5 As can be seen, the co-localization results of the two channels can be captured in the plasmid sample treated with Nile Red dye. The red represents Nile Red dye excited at a wavelength of 543 nm, while the green represents lycopene excited at a wavelength of 488 nm. The co-localization shows that the Nile Red dye channel and the lycopene channel are superimposed, indicating that the plasmid encapsulates lycopene through the plasma membrane, which can serve as a potential natural nanodelivery system.

[0054] (3) Particle size analysis

[0055] The particle size of the plasmids was determined using a laser particle size analyzer. 200 μL of the prepared plasmid suspension was slowly added dropwise to the inlet of the particle size analyzer until the light-blocking ratio reached 8-15%, at which point the measurement could begin. Each sample was scanned three times, and the average value was taken. The sample refractive index was 1.476, and the dispersive medium was deionized water with a refractive index of 1.333.

[0056] Depend on Figure 6It can be seen that the average particle size of the plasmoids is 204.4±2.05nm, and the particle size shows a single-peak distribution. The PDI is 0.475, indicating that the particle size is uniform and the plasmoids have good stability.

[0057] (4) Lycopene content in plastid globules

[0058] Sample preparation: 1 mL of plasmid suspension was thoroughly mixed with 3 mL of extraction solvent (methanol / ethyl acetate / petroleum ether, 1:1:1, v / v / v) containing 0.1 g / L BHT. The mixture was extracted using an ultrasonic extractor at 70% amplitude for 30 min, followed by centrifugation at 1320 g and 4 °C for 3 min. The supernatant organic phase was collected, and the residue was extracted twice more using 2 mL of the same solvent and conditions. The combined supernatant organic phases were dried under nitrogen, dissolved in 1 mL of dichloromethane, and filtered through a 0.22 μm PTFE membrane. The filtrate was analyzed by high-performance liquid chromatography (HPLC). The lycopene content was calculated using a standard curve.

[0059] High performance liquid chromatography (HPLC) detection conditions: detection wavelength 472 nm, chromatographic column C18, mobile phase a mixture of methanol, acetonitrile and dichloromethane (20:75:5), flow rate 1.0 mL / min, injection volume 20 μL.

[0060] The results showed that the main substance in the plastid spheres was lycopene, and the concentration of lycopene was 50.45 μg / mL, indicating that the plastid spheres were loaded with lycopene.

[0061] Example 4

[0062] Lycopene stability test:

[0063] (1) Salt ion stability analysis: The plasmid suspension was mixed with 0mM, 50mM, 100mM, 150mM and 200mM NaCl solutions at a ratio of 1:1 (v / v). The mixture was stirred at 300rpm for 1h at room temperature (25℃). 1mL of the mixture was taken from each sample and centrifuged at 5000g and 25℃ for 10min. The supernatant was taken. The same mass of free lycopene suspension was used as a control.

[0064] (2) pH stability analysis: The plasmid suspension was mixed with solutions of pH 3.0, pH 6.0, pH 9.0 and pH 12.0 at a ratio of 1:1 (v / v). The mixture was stirred at 300 rpm for 1 h at room temperature (25℃). 1 mL of the mixture was taken from each sample and centrifuged at 5000 g and 25℃ for 10 min. The supernatant was collected. The same mass of free lycopene suspension was used as a control.

[0065] (3) Temperature stability analysis: The plasmid suspension was mixed with distilled water at a ratio of 1:1 (v / v) and stirred at 300 rpm for 1 h at 27℃, 37℃, 47℃, 57℃, 67℃, 77℃, 87℃ and 100℃ respectively. 1 mL of the mixture was taken from each sample and centrifuged at 5000 g and 25℃ for 10 min. The supernatant was taken. The same mass of free lycopene suspension was used as a control.

[0066] (4) In vitro simulated digestion stability analysis

[0067] The in vitro simulated digestion stability of plastospheres was analyzed using the INFOGEST 2.0 method. In short, firstly, simulated gastric fluid containing 2000 U / mL pepsin and 60 U / mL gastric lipase was prepared and incubated at 37°C. Then, plastosphere samples were added to the simulated gastric fluid at a 1:1 (v / v) ratio, the pH was adjusted to 3.0, and the mixture was stirred for 2 h. Subsequently, an equal volume of simulated intestinal fluid containing 200 U / mL trypsin was added. The mixture was stirred for another 2 h at pH 7.0 and 37°C. Initial lycopene content determination: At 0 min, 100 μL of sample solution was taken and mixed with 900 μL of ethyl acetate. After centrifugation at 10000g and 4°C for 10 min, the supernatant was collected for analysis. Lycopene retention determination: 200 μL of sample solution was collected at 30, 60, 90, 120, 150, 180, 210, and 240 min and centrifuged. Take 100 μL of supernatant and repeat the sample processing steps in the structure test (5) HPLC high-performance liquid chromatography for extraction. The lycopene content is determined by an enzyme-linked immunosorbent assay (ELISA) reader, and the retention rate is calculated according to the following formula. Free lycopene is used to replace plasmids in the above experiment at the same concentration.

[0068] The extracted lycopene was analyzed for lycopene content using a microplate reader at a wavelength of 472 nm. The lycopene retention rate was calculated using the following formula:

[0069]

[0070] Depend on Figure 7 It was found that plastospheres significantly improved the water solubility of lycopene under different salt ion concentrations, pH, and temperatures. Loading with plastospheres improved the retention rate of lycopene, with retention rates exceeding 70% at different salt ion concentrations, and also exhibiting high retention rates under alkaline and high-temperature conditions. These results demonstrate that plastospheres maintain good stability under environmental changes (salt ion concentration, pH, and temperature), further confirming their potential application as a natural delivery system resistant to harsh environments.

[0071] pass Figure 8It can be seen that, during the simulated gastrointestinal digestion process, plastids can significantly resist the harsh environment of the gastrointestinal tract, with a retention rate of over 60% in the stomach and over 40% in the intestine. The stability of plastids in the gastrointestinal tract indicates that they can be a potential natural delivery system.

[0072] Example 5

[0073] Intestinal osmotic behavior test:

[0074] (1) Transwell intestinal mucus permeability assessment

[0075] The permeability of plastosomes and free lycopene in intestinal mucus was compared using a Transwell intestinal mucus permeability assay. 50 mg of porcine small intestinal mucus was weighed and spread evenly in the upper chamber of a Transwell plate. 600 μL of 0.01 M PBS was added to the lower chamber of the Transwell plate beforehand. The upper chamber was then placed back into the Transwell plate, and 200 μL of plastosomes (0.1 mg / mL lycopene) and 200 μL of free lycopene were added to the upper chamber. The plates were incubated at 37°C for 0.5, 1, 2, and 4 h, respectively. At each time point, 200 μL of sample was taken from the lower chamber of the Transwell plate. Lycopene was extracted, and its content was determined using a microplate reader at 472 nm. The lycopene permeability was calculated using the following formula.

[0076]

[0077] Experimental results are as follows Figure 9 As shown in the figure, the permeability of free lycopene is extremely low with changes in incubation time, showing no significant change between incubation times. However, the permeability of plastospheres gradually increases, reaching 37% at 4 hours, which is significantly higher than that of free lycopene. This is because the natural system of plastospheres can enhance the water solubility of lycopene, and their particle size is at the nanoscale, allowing them to penetrate the intestinal mucus layer dominated by the mucin network. This provides preliminary evidence that plastospheres can serve as a natural nanodelivery system.

[0078] (2) Evaluation of permeability of isolated intestine

[0079] Duodenum, jejunum, and ileum were harvested from 7-week-old Sprague Dawley rats and quickly placed in Krebs-Ringer buffer. Four-cm segments of each intestine were taken and rinsed with Krebs-Ringer buffer for later use. 0.4 mL of plastid globules (0.1 mg / mL lycopene concentration) and free lycopene were added to each intestinal segment. The intestinal opening was sealed, and the sample was placed in 3 mL of 0.01 M PBS and incubated at 37°C for 2 hours using a shaker. 200 μL of the external PBS was then used for extraction. The lycopene content was determined using a microplate reader, and the permeability coefficient was calculated using the following formula.

[0080]

[0081] Among them, P app dQ / dt represents the permeability coefficient, dQ / dt represents the lycopene permeation rate (mg / s), and A represents the intestinal surface area (cm²). 2 C represents the initial lycopene concentration (mg / cm³). 3 ).

[0082] from Figure 10 As can be seen, for the duodenum, jejunum, and ileum, the intestinal permeability coefficient of plastid globules is 32.00 times, 39.00 times, and 143.00 times higher than that of free lycopene, respectively. This further confirms the results of Transwell's intestinal mucus permeability evaluation, indicating that in the real system, plastid globules are still more likely to permeate the intestine than free lycopene. This also further proves the ability of plastid globules as a natural delivery system.

[0083] (3) Observation by CLSM laser confocal fluorescence microscopy

[0084] To further observe the permeability of plastosomes and free lycopene in intestinal mucus, CLSM laser confocal fluorescence microscopy was used. 20 μL of 10 μg / mL Alexa Fluor 555-WGA dye solution was added to the intestinal mucus, followed by 200 μL of free lycopene sample and extracted plastosome sample, and incubated at 37℃ for 0, 10, 30, and 60 min, respectively. Finally, the samples were observed after dual-channel excitation at 488 nm and 553 nm wavelengths using CLSM. Images with a z-axis resolution of 20 μm and a resolution of 1024 × 1024 pixels were collected.

[0085] pass Figure 11 It can be seen that both free lycopene and lycopene in plastid globules are excited and exhibit green light, while mucin in intestinal mucus is excited to red light after being stained with Alexa Fluor 555-WGA dye. And from... Figure 11As can be seen from A, the free lycopene did not undergo significant osmosis even with prolonged incubation. For Figure 11 For sample B, the plasmids significantly penetrated into the mucus layer as incubation time increased. The maximum fluorescence pattern in the figure below represents the mucus thickness at which the maximum fluorescence intensity was observed in both samples over time. Figure 12 As shown, the maximum fluorescence of free lycopene remained at a mucus thickness of 2 μm over time, indicating that free lycopene did not significantly permeate over time. In contrast, the maximum fluorescence intensity of plastospheres increased from 2 μm to 14 μm over time, suggesting that plastospheres continuously permeated the mucus layer. This further demonstrates that plastospheres can pass through the intestines and enter the body for further lycopene delivery, proving that plastospheres can serve as a natural delivery system.

[0086] Example 6

[0087] Metabolic kinetics test:

[0088] (1) Blood metabolism test

[0089] Sample preparation: To determine the metabolic and delivery states of free lycopene and plastid samples in a real system, and to determine pharmacokinetics. In summary, 7-week-old C57BL / 6J mice were administered the same dose (0.6 mg / kg) of free lycopene and plastid samples via gavage. Mice were sacrificed at 0, 0.5, 1, 2, 4, 8, 12, and 24 hours. Serum was collected in 1.5 mL anticoagulant tubes and immediately centrifuged at 5000g for 5 min. 100 μL of the supernatant was collected, and 100 μL of ethanol was added. After mixing, 200 μL of n-hexane was added, and the mixture was centrifuged at 3000g at 4°C for 10 min. The supernatant was collected, and this process was repeated three times with the addition of another 200 μL of n-hexane. The collected supernatant was dried under nitrogen, dissolved in 200 μL of dichloromethane, and filtered through a 0.22 μm PTFE membrane. The filtrate was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Results were calculated using a lycopene standard curve.

[0090] LC-MS / MS determination: C18 column, column temperature 40℃, flow rate 0.2 mL / min, injection volume 3 μL, mobile phase A was a mixture of methanol, acetonitrile, and dichloromethane in a ratio of 25:60:15, mobile phase B was methanol. LC conditions are shown in Table 1. Mass spectrometry conditions: m / z 536.3 → 444.4, ion source ESI, positive ion mode, Dwell set to 0.015 s, Cone voltage 50 V, Collision voltage 10 V.

[0091] Table 1 Liquid phase conditions

[0092] 0min 60:40 10min 80:20

[0093] like Figure 13 As shown, the maximum plasma concentrations (C0) of free lycopene and plastid globules were statistically analyzed. max ), time to reach maximum blood drug concentration (T) max ) and use the area under the curve (AUC) 0-t The values ​​() represent the relative bioavailability in blood (Table 2). Plastidomers significantly improved the oral utilization of lycopene in mice, increasing its blood concentration by 1.96 times, delaying the time to reach maximum blood concentration by 2 hours, and increasing the relative bioavailability in blood by 1.71 times. This indicates that plasmidomers can both improve the bioavailability of lycopene in mice and have a sustained-release effect, demonstrating that plasmidomers can serve as a natural delivery system.

[0094] Table 2. Pharmacokinetic characteristics of free lycopene and plastids in mouse serum.

[0095]

[0096]

[0097] (2) Hepatic metabolic kinetics test

[0098] Sample preparation: To determine the hepatic delivery of free lycopene and plastid samples, hepatic pharmacokinetics were determined. In short, 7-week-old C57BL / 6J mice were used. The mice were administered the same dose (0.6 mg / kg) of free lycopene and plastid samples via gavage. Mice were sacrificed at 0, 0.5, 1, 2, 4, 8, 12, and 24 hours. Their livers were collected in cryovials, lyophilized, and ground into powder. 40 mg of the powder was weighed and added to 100 μL of 300 mM vitamin C solution. After mixing, 200 μL of n-hexane was added, and the mixture was sonicated for 5 min. The mixture was then centrifuged at 10000 g at 4°C for 10 min. The supernatant was collected, and another 200 μL of n-hexane was added. This process was repeated three times. The collected supernatant was dried under nitrogen, then dissolved in 200 μL of dichloromethane. The solution was filtered through a 0.22 μm PTFE membrane, and the filtrate was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results were calculated using a lycopene standard curve.

[0099] like Figure 14 As shown, the maximum hepatic drug concentrations (C0) of free lycopene and plastid globules were statistically analyzed. max ), time to reach maximum hepatic drug concentration (T) max ) and use the area under the curve (AUC) 0-tThe values ​​() represent the relative bioavailability in the liver (Table 3). Plastidospheres significantly increased the accumulation of lycopene in mouse liver, increasing its hepatic concentration by 4.90 times, delaying the time to reach maximum hepatic concentration by 7 hours, and increasing the relative bioavailability in the liver by 7.28 times. This indicates that plastidospheres can both improve the bioavailability of lycopene in mouse liver and have a sustained-release effect, demonstrating that plastidospheres can serve as a natural delivery system for liver-targeted delivery.

[0100] Table 3. Pharmacokinetic characteristics of free lycopene and plastids in mouse liver.

[0101] Free lycopene 3.43±0.28 1 37.10±1.89 plastospheres 16.80±2.29 8 270.18±5.66

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. The use of lycopene microspheres in the preparation of lycopene nanodelivery systems, characterized in that, The plastosphere structure is a natural nanoscale spherical structure in which lycopene is encapsulated by a plasma membrane; the preparation method of the plastosphere includes the following steps: S1: After rinsing the chopped tomatoes with pre-cooled extraction buffer, add the pre-cooled extraction buffer at a mass-to-volume ratio of 1:(3-7), homogenize and stir to obtain crude juice. The solutes in the extraction buffer consist of Tricine-KOH, sorbitol, EDTA-Na2, NaHCO3, and bovine serum albumin. The concentrations of the solutes Tricine-KOH, sorbitol, EDTA-Na2, NaHCO3, and bovine serum albumin in the extraction buffer are 20 mM, 450 mM, 10 mM, 10 mM, and 0.5 wt.%, respectively. S2: After filtering the crude fruit juice through 2-5 layers of degreased cotton gauze and a 60-80μm filter, collect the filtrate. Centrifuge the filtrate at 1200g for 10-15min at 4℃ and collect the precipitate. Add extraction buffer to the precipitate at a mass-to-volume ratio of 1:(1-4) for reconstitution. After reconstitution, sonicate for 1-3min at a frequency of 60-80Hz to obtain a suspension. S3: Add the suspension, 35-38 wt.% sucrose solution, 18-20 wt.% sucrose solution, 12-15 wt.% sucrose solution and 3-5 wt.% sucrose solution to a centrifuge tube in a ratio of 3:3:3:2:

4. Centrifuge at 80,000-120,000 g for 2-4 hours at 4°C. Collect the top layer of material, which is the tomato red granule.

2. The use according to claim 1, characterized in that: The homogenization and stirring described in S1 involves stirring for 1-3 seconds every 1-3 seconds, for a total of 1-5 times.

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