Preparation of response targeting type xanthophyll base material powder and application of response targeting type xanthophyll base material powder in relieving blue light damage direction

Abstract

The invention discloses preparation of response targeting type xanthophyll base material powder and application of the response targeting type xanthophyll base material powder in relieving the blue light damage direction, and belongs to the technical field of nanotechnology. According to the invention, lutein is preliminarily embedded by a Maillard reaction compound modified by (5-carboxyl amyl) triphenylphosphonium bromide, and then an outer layer structure is formed by complexing a compound of 3-aminophenylboronic acid and sodium alginate through a phenyl boron ester bond, so that a base material powder system with pH response, ROS response and mitochondrial targeting functions is constructed. The system can escape gastric acid, reduce degradation of xanthophyll, realize controllable release of xanthophyll under stimulation of ROS, and can gather in mitochondria, so that targeted delivery is realized, and the bioavailability of xanthophyll is remarkably improved. In addition, the raw materials adopted by the method are low in cost, the embedding process is mild, and the method has a good large-scale production characteristic and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of nanotechnology, and more specifically, to the preparation of a responsive targeted lutein-based powder and its application in alleviating blue light damage. Background Art

[0002] Blue light is a high-energy short-wavelength light in the visible spectrum, with a wavelength of about 380 - 500 nm. Blue light has relatively high energy and can penetrate the eye and be absorbed by the retina, which may cause oxidative damage to photoreceptors in the retina. With the popularization of electronic products and the increase in usage time, the potential damage of blue light to the eyes has indeed become the focus of attention of more and more people.

[0003] Lutein is a kind of carotenoid with strong antioxidant ability. Lutein is a key nutrient for maintaining eye health, especially in the macular area. It can help filter blue light, protect the retina from oxidative damage, and prevent eye diseases such as age-related macular degeneration. Lutein is sensitive to factors such as heat, light, oxygen, intestinal pH value, and temperature, which makes lutein prone to losing its biological activity during storage, processing, and use, thus limiting its bioavailability.

[0004] Nanodelivery carriers are an effective method to improve the bioavailability of poorly water-soluble compounds. Responsive targeted delivery systems are a type of carrier system that can release the loaded substances or target specific locations under specific stimuli (such as pH value, ROS, temperature, enzymes, light, etc.). Compared with traditional delivery systems, they can better improve the bioavailability of the loaded substances.

[0005] Sodium alginate (SA) is a natural polysaccharide widely used in the field of drug delivery. In the acidic environment of the stomach, the protonation of sodium alginate helps it maintain relatively low solubility and a relatively compact structure, reducing the degradation of the loaded substances and achieving pH responsiveness. The generation of ROS is one of the core mechanisms of blue light damage. Phenylboronic ester bonds have broad application potential in ROS-responsive drug delivery and intelligent materials. Phenylboronic ester bonds break through oxidation under the action of excessive ROS, enabling the directional release and intelligent response of the loaded substances. Blue light damage directly destroys the function and structural integrity of mitochondria by increasing oxidative stress. Therefore, mitochondria become one of the main targeted organelles of blue light damage. As a delocalized lipophilic cation, TPP can accurately direct bioactive substances to mitochondria through charge-driven means, thus achieving precise therapeutic effects in specific cells or tissues. Therefore, a base powder with pH, ROS responsiveness, and mitochondrial targeting may be an effective strategy for treating blue light damage, and there is currently no technology that combines the above three to prepare a delivery system. Summary of the Invention

[0006] Technical problem

[0007] Lutein plays an important role in protecting the retina, alleviating blue light damage, and maintaining eye health. However, due to its extreme sensitivity to heat, light, oxygen, pH value, etc., taking lutein alone often has poor effects. Therefore, a special delivery system needs to be added to further improve the bioavailability of lutein and exert the effect of lutein. In addition, in terms of alleviating blue light damage, the delivery system needs to have the functions of pH, ROS response, and mitochondrial targeting. Therefore, how to prepare a delivery system with pH, ROS response, and mitochondrial targeting to improve the bioavailability of lutein is a technical problem that urgently needs to be solved.

[0008] Technical content

[0009] The present invention aims to overcome the limitations of traditional carrier systems, and proposes a preparation method of a functional factor delivery system with pH, ROS responsiveness, and mitochondrial targeting constructed by boric acid esterification complexation technology, and explores its application in retinal blue light damage. This innovative method effectively solves the problem of the instability of lutein in the external environment, protects lutein from being damaged by extreme environments such as gastric acid, and thus improves its stability and bioavailability.

[0010] The preparation method of the above-mentioned responsive targeted lutein-based powder comprises the following steps:

[0011] Step (1): Mix casein and mannose in water, adjust the pH to alkaline, heat, cool, and then perform freeze-drying to obtain a Maillard reaction complex;

[0012] Step (2): Dissolve TPP, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in dimethyl sulfoxide, stir to obtain a TPP carboxyl activation solution; dissolve the Maillard reaction complex prepared in step (1) in water to obtain a Maillard reaction complex solution, then pour it into the TPP carboxyl activation solution and stir, then perform dialysis, and freeze-dry the dialyzed solution to obtain a TPP-modified Maillard reaction complex;

[0013] Step (3): Dissolve the TPP-modified Maillard reaction complex in water to obtain a TPP-modified Maillard reaction complex solution, dissolve lutein in absolute ethanol to obtain a lutein solution, stir and mix the Maillard reaction complex solution and the lutein solution, then homogenize, centrifuge, rotary evaporate, and freeze-dry to obtain lutein-based powder;

[0014] Step (4): Dissolve sodium alginate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in water to obtain a sodium alginate carboxyl activation solution; dissolve 3-aminophenylboronic acid in an ethanol solution, then stir and mix it with the sodium alginate carboxyl activation solution, followed by dialysis, and then freeze-dry to obtain a 3-aminophenylboronic acid-sodium alginate complex; add the 3-aminophenylboronic acid-sodium alginate complex solution to the lutein base powder solution and stir to obtain the responsive targeted lutein base powder.

[0015] Further, in step (1), the mass ratio of casein to mannose is 1:1 to 6:1.

[0016] Further, in step (1), the mass ratio of casein to mannose is 4:1 to 6:1.

[0017] Further, in step (1), the mass ratio of casein to water is 1:15 to 1:30.

[0018] Further, in step (1), the adjusted pH is 8 to 10.

[0019] Further, in step (1), the heating is carried out at 70 to 95 °C for 1 to 5 h.

[0020] Further, in step (2), the mass ratio of TPP to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:0.6 to 1:1.2.

[0021] Further, in step (2), the mass ratio of TPP to N-hydroxysuccinimide is 1:0.3 to 1:0.7.

[0022] Further, in step (2), the mass ratio of TPP to dimethyl sulfoxide is 1 mg:0.8 g to 1 mg:1.2 g.

[0023] Further, in step (2), the stirring for preparing the TPP carboxyl activation solution is carried out at 20 to 30 °C for 5 to 7 hours.

[0024] Further, in step (2), the concentration of the Maillard reaction complex solution is 5 to 15 mg / mL.

[0025] Further, in step (2), the volume ratio of the Maillard reaction complex solution to the TPP carboxyl activation solution is 3:1 to 7:1.

[0026] Further, in step (2), the stirring after pouring the Maillard reaction complex solution into the TPP carboxyl activation solution is carried out at 20 to 30 °C for 20 to 30 hours.

[0027] Further, in step (2), dialysis is performed using a dialysis bag with a molecular weight cut-off of 500-1000 Da for 24-48 h.

[0028] Further, in step (3), the concentration of the TPP-modified Maillard reaction complex solution is 5-15 mg / mL.

[0029] Further, in step (3), the concentration of the lutein solution is 1-10 mg / mL.

[0030] Further, in step (3), the volume ratio of the Maillard reaction complex solution to the lutein solution is 5:1-10:1.

[0031] Further, in step (3), the homogenization time is 10-30 minutes.

[0032] Further, in step (4), the mass ratio of sodium alginate to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1-1:2.

[0033] Further, in step (4), the mass ratio of sodium alginate to N-hydroxysuccinimide is 1:0.5-1:1.5.

[0034] Further, in step (4), the mass ratio of sodium alginate to water is 1:100-1:200.

[0035] Further, in step (4), the ethanol solution is an ethanol aqueous solution with a volume fraction of 40-60%.

[0036] Further, in step (4), the mass ratio of 3-aminophenylboronic acid to the volume of the ethanol solution is 60-90 mg:10 mL.

[0037] Further, in step (4), the volume ratio of the sodium alginate carboxyl activation solution to the ethanol solution is 1-1.5:1.

[0038] Further, in step (4), the stirring of the ethanol solution dissolved with 3-aminophenylboronic acid and the sodium alginate carboxyl activation solution is carried out at 20-30 °C for 20-30 hours.

[0039] Further, in step (4), dialysis is performed using a dialysis bag with a molecular weight cut-off of 3-4 kDa for 24-48 h.

[0040] Further, in step (4), the concentration of the 3-aminophenylboronic acid-sodium alginate complex solution is 1-2 mg / mL.

[0041] Further, in step (4), the concentration of the lutein nanoparticle solution is 1-2 mg / mL.

[0042] Further, in step (4), the volume ratio of the 3-aminophenylboronic acid-sodium alginate complex solution to the lutein nanoparticle solution is 1:0.25 to 1:4.

[0043] Further, in step (4), the volume ratio of the 3-aminophenylboronic acid-sodium alginate complex solution to the lutein nanoparticle solution is 1:0.75 to 1:1.25.

[0044] Further, the stirring in step (4) of the 3-aminophenylboronic acid-sodium alginate complex solution and the lutein base powder solution is carried out at 20-30 °C for 4-8 hours.

[0045] The present invention provides a response-targeted lutein base powder prepared according to the above method.

[0046] Further, the response-targeted lutein base powder can have pH response, ROS response and mitochondrial targeting effect.

[0047] Application of the response-targeted lutein base powder prepared by the present invention in retinal blue light damage.

[0048] Application of the response-targeted lutein base powder prepared by the above preparation method of the present invention in the preparation of health foods or drugs for alleviating retinal blue light damage.

[0049] The beneficial effects of the present invention are:

[0050] 1. The present invention uses the Maillard reaction complex modified by TPP as a carrier to deliver lutein. The 3-aminophenylboronic acid-sodium alginate complex is complexed on the outer layer through phenylborate bonds to prepare a response-targeted lutein delivery system. This system can escape gastric acid through protonation, has pH response, and can release lutein under ROS stimulation, and realizes the aggregation of the targeted delivery system in mitochondria.

[0051] 2. The delivery system prepared by the present invention is spherical and has a nanoscale particle size. It can escape gastric acid through protonation and achieve controlled release of lutein under ROS stimulation. Intracellular co-localization and in vivo imaging experiments reveal the distribution of the lutein delivery system in mice. Pharmacokinetics shows that the retention rate of the lutein delivery system in plasma, eyeballs and livers is higher. This lutein delivery system can improve the thickness of the retina of blue light-damaged mice and reduce the inflammation level. Description of the Drawings

[0052] Figure 1 is the transmission electron micrograph of the response-targeted lutein base powder prepared in Example 1 of the present invention;

[0053] Figure 2 is the transmission electron micrograph of the mitochondria-targeted lutein base powder prepared in Comparative Example 1 of the present invention;

[0054] Figure 3 It is a diagram showing the in vitro release of the response-targeted lutein base powder prepared in Example 1 of the present invention under different concentrations of ROS;

[0055] Figure 4 It is a diagram showing the distribution of the Nile red base powder prepared in Comparative Example 3 in the mitochondria of ARPE-19 retinal epithelial cells;

[0056] Figure 5 It is a diagram showing the distribution of the mitochondrially targeted Nile red base powder prepared in Comparative Example 2 in the mitochondria of ARPE-19 retinal epithelial cells;

[0057] Figure 6 It is an in vivo imaging diagram of the mitochondrially targeted Nile red base powder prepared in Comparative Example 2 and the response-targeted Nile red base powder prepared in Example 2 in mice;

[0058] Figure 7 It is a pharmacokinetic diagram of the mitochondrially targeted lutein base powder prepared in Comparative Example 1 and the response-targeted lutein base powder prepared in Example 1 in the plasma, eyeballs and livers of mice;

[0059] Figure 8 It is a diagram showing the results of the retinal thickness of mice after blue light damage by the mitochondrially targeted lutein base powder prepared in Comparative Example 1 and the response-targeted lutein base powder prepared in Example 1;

[0060] Figure 9 It is a diagram showing the detection results of interleukin-1β after blue light damage by the mitochondrially targeted lutein base powder prepared in Comparative Example 1 and the response-targeted lutein base powder prepared in Example 1;

[0061] Figure 10 It is a diagram showing the detection results of interleukin-6 after blue light damage by the mitochondrially targeted lutein base powder prepared in Comparative Example 1 and the response-targeted lutein base powder prepared in Example 1. Detailed Embodiments

[0062] This application describes in detail the preferred implementation schemes of the present invention, and is not limited to the specific conditions and details in the following implementation schemes. Without contradiction, various specific technical features can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe all possible combination methods. Any person skilled in the art can make simple modifications and substitutions within the technical scope described in the present invention according to their own circumstances, and these simple modifications all fall within the protection scope of the present invention. In practicing the present invention, various alternative schemes of the implementation schemes of the present invention described in this application can be used. The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, aiming to explain the present invention, describe the technical scheme clearly and completely, and do not limit the reagents or instruments used in the invention.

[0063] Raw material source

[0064] Lutein, triphenylphosphonium bromide (TPP), and sodium alginate were purchased from Shanghai Macklin Biochemical Co., Ltd.; casein and 3-aminophenylboronic acid were purchased from Aladdin Industrial Corporation in Shanghai, China; mannose was purchased from Sigma-Aldrich in the United States.

[0065] Detection process

[0066] Morphology characterization of the base powder: The morphology of the base powder was observed using a JEM-2100UHR transmission electron microscope.

[0067] Detection of lutein release: The response-targeted lutein base powder was dissolved in PBS buffer with different H 2 O 2 concentrations (0, 0.5, 2, and 8 mM) at pH 7.4, and the solution was placed in a dialysis bag (MWCO 3500 Da), then the dialysis bag was placed in PBS buffer and stirred under dark conditions. Samples of the release medium were collected at predetermined time points, and the cumulative release of lutein was measured at 444 nm using a microplate reader.

[0068] Mitochondrial targeting experiment: ARPE-19 cells were seeded in 12-well plates at a density of 1×105 cells per well. After 24 h of culture, the medium was replaced with fresh medium containing the base powder of Comparative Example 2 and Comparative Example 3 and incubated for 4 h. Subsequently, the medium was removed, and 100 nM Mito-tracker was added and incubated for 30 min. After incubation, the cells were washed three times with PBS buffer, the samples were collected, and fluorescence images were obtained using a fluorescence inverted microscope. Finally, image analysis and result calculation were performed using NIH Image J software (Bethesda, MD, USA).

[0069] In vivo distribution experiment: After fasting for 24 h, mice were intragastrically administered the base powder of Comparative Example 2 and Example 2. The mice were anesthetized and photographed at 2, 4, 8, 12, and 24 h using a MIIS XFP-BIX imaging analysis system (Molecular Device Corporation, Sunnyvale, CA, USA) to observe the distribution of fluorescence signals.

[0070] Pharmacokinetic experiment: SD rats were gavaged with the base powder of Comparative Example 2 and Example 2, respectively, and blood was collected using anticoagulant tubes at 2, 4, 8, 12, and 24 hours, and plasma was collected by centrifugation (10000rpm, 10min). For the lutein content in the tissue, the SD rats were killed 8 hours after gavage with the base powder of Comparative Example 2 and Example 2, and the eye and liver tissues were collected. The eye and liver tissues were mixed and homogenized with normal saline and stored on ice. The sample was mixed with a solution of methanol and chloroform (2:1, v / v), vortexed for 1min, and then n-hexane was added and centrifuged for 10min. The organic layer was evaporated with nitrogen and resuspended with 200μL of dichloromethane. The resuspension was filtered with a filter membrane (0.22μm) and analyzed by high performance liquid chromatography.

[0071] Retinal HE staining: Animal experiments were conducted in accordance with the National Standard of the People's Republic of China GB / T 35892-2018 "Ethical Review of Laboratory Animal Welfare" and approved by the Animal Ethics Committee of Dalian University of Technology. For 14 days, mice were gavaged with the base powder of Comparative Example 1 and Example 1 every day and irradiated with 445nm blue light at 7000lux for 2h every day. The mice were killed, and the collected mouse eyeballs were fixed with 4% (w / v) paraformaldehyde, stained with hematoxylin and eosin (H&E), and histopathological changes were observed.

[0072] The levels of mouse interleukins IL-6 and IL-1β were analyzed using enzyme-linked immunosorbent assay kits from Shanghai Keaibo Biotechnology Co., Ltd. (Shanghai, China).

[0073] Example 1

[0074] Step 1: Weigh 4 g of casein and 1 g of mannose in 100 mL of deionized water, mix thoroughly, stir overnight at room temperature, adjust the pH to 9, heat at 90° C. for 3 h, place in an ice bath, cool to room temperature, and freeze-dry to obtain a Maillard reaction complex.

[0075] Step 2: Weigh 12.87 mg of TPP, 11.38 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 6.91 mg of N-hydroxysuccinimide, dissolve in 10 mL of dimethyl sulfoxide, and stir for 6 hours to prepare a TPP carboxyl activation solution. Subsequently, weigh 500 mg of the Maillard reaction complex prepared in step 1, dissolve in 50 mL of deionized water, and then pour it into the prepared TPP carboxyl activation solution, and continue to stir and react for 24 hours. After the reaction is completed, dialyze for 48 hours using a dialysis bag with a molecular weight cutoff of 500 Da, and freeze-dry the dialyzed solution to obtain a TPP-modified Maillard reaction complex.

[0076] Step 3: Dissolve the TPP-modified Maillard reaction complex in deionized water to prepare a TPP-modified Maillard reaction complex solution with a concentration of 10 mg / mL. Dissolve lutein in anhydrous ethanol to prepare a lutein solution with a concentration of 6 mg / mL. Mix the TPP-modified Maillard reaction complex solution with the lutein solution at a volume ratio of 5:1, disperse it with a high-speed stirrer at 10,000 rpm for 2 minutes, further homogenize it with ultrasonic homogenization in an ice bath for 15 minutes, centrifuge it at 2,000 rpm for 2 minutes, and then use a rotary evaporator to remove the anhydrous ethanol, and finally freeze-dry it to prepare a mitochondrial-targeted lutein base powder.

[0077] Step 4: Take 80 mg of sodium alginate and dissolve it in 12 mL of deionized water, add 122.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 73.6 mg of N-hydroxysuccinimide, and stir for 6 hours to prepare a sodium alginate carboxyl activation solution. Dissolve 86.4 mg of 3-aminophenylboronic acid in 10 mL of 50% (v / v) ethanol aqueous solution, gradually add it dropwise to the sodium alginate carboxyl activation solution, react at room temperature for 24 hours, dialyze with 3.5 kDa dialysis bag water for 48 hours, and freeze-dry the dialyzed solution to obtain a 3-aminophenylboronic acid-sodium alginate complex. The 3-aminophenylboronic acid-sodium alginate complex was prepared into a 1 mg / mL 3-aminophenylboronic acid-sodium alginate complex solution, which was then gradually added into the mitochondrial targeted lutein base powder solution (1.5 mg / mL) at a volume ratio of 1:1. The mixture was stirred continuously for 6 hours and freeze-dried to obtain the responsive targeted lutein base powder.

[0078] Example 2

[0079] In this Example 2, Nile red was used to fluorescently label the prepared functional factor delivery system.

[0080] Step 1: Weigh 4 g of casein and 1 g of mannose in 100 mL of deionized water, mix thoroughly, stir overnight at room temperature, adjust the pH to 9, heat at 90° C. for 3 h, place in an ice bath, cool to room temperature, and freeze-dry to obtain a Maillard reaction complex.

[0081] Step 2: Weigh 12.87 mg of TPP, 11.38 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 6.91 mg of N-hydroxysuccinimide, dissolve them in 10 mL of dimethyl sulfoxide, stir for 6 h to prepare the TPP carboxyl activation solution. Subsequently, weigh 500 mg of the Maillard reaction complex prepared in Step 1, dissolve it in 50 mL of deionized water, then pour it into the prepared TPP carboxyl activation solution, and continue to stir and react for 24 h. After the reaction is completed, dialyze with a dialysis bag with a molecular weight cut-off of 500 Da for 48 h, and freeze-dry the dialyzed solution to obtain the Maillard reaction complex modified with TPP.

[0082] Step 3: Dissolve the TPP-modified Maillard reaction complex in deionized water to prepare a TPP-modified Maillard reaction complex solution with a concentration of 10 mg / mL. Dissolve nile red in absolute ethanol to prepare a nile red solution with a concentration of 6 mg / mL. Mix the TPP-modified Maillard reaction complex solution and the nile red solution according to a volume ratio of 5:1, disperse them with a high-speed stirrer at 10000 rpm for 2 min, further homogenize them with an ultrasonic homogenizer in an ice bath for 15 min, centrifuge at 2000 rpm for 2 min, then remove the absolute ethanol using a rotary evaporator, and finally freeze-dry to prepare the mitochondrion-targeted nile red base powder.

[0083] Step 4: Dissolve 80 mg of sodium alginate in 12 mL of deionized water, add 122.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 73.6 mg of N-hydroxysuccinimide, stir for 6 h to prepare the sodium alginate carboxyl activation solution. Dissolve 86.4 mg of 3-aminophenylboronic acid in 10 mL of 50% (v / v) ethanol aqueous solution, gradually add it dropwise to the sodium alginate carboxyl activation solution, react at room temperature for 24 h, then dialyze with a 3.5 kDa dialysis bag in water for 48 h, and freeze-dry the dialyzed solution to obtain the 3-aminophenylboronic acid-sodium alginate complex. Prepare a 3-aminophenylboronic acid-sodium alginate complex solution with a concentration of 1 mg / mL, and then gradually add it to the nile red base powder solution (1.5 mg / mL) at a volume ratio of 1:1, and continuously stir for 6 h to obtain the response-targeted nile red base powder.

[0084] Comparative Example 1

[0085] Step 1: Weigh 4 g of casein and 1 g of mannose, mix them well in 100 mL of deionized water, stir overnight at room temperature, adjust the pH to 9, heat at 90 °C for 3 h, place it in an ice bath, cool to room temperature, and freeze-dry to obtain the Maillard reaction complex.

[0086] Step 2: Weigh 12.87 mg of TPP, 11.38 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 6.91 mg of N-hydroxysuccinimide, dissolve them in 10 mL of dimethyl sulfoxide, stir for 6 h to prepare the TPP carboxyl activation solution. Subsequently, weigh 500 mg of the Maillard reaction complex prepared in Step 1, dissolve it in 50 mL of deionized water, then pour it into the prepared TPP carboxyl activation solution, and continue stirring and reacting for 24 h. After the reaction, dialyze with a dialysis bag with a molecular weight cut-off of 500 Da for 48 h, and lyophilize the dialyzed solution to obtain the Maillard reaction complex modified with TPP.

[0087] Step 3: Dissolve the Maillard reaction complex modified with TPP in deionized water to prepare a Maillard reaction complex modified with TPP with a concentration of 10 mg / mL. Dissolve lutein in absolute ethanol to prepare a lutein solution with a concentration of 6 mg / mL. Mix the Maillard reaction complex solution modified with TPP and the lutein solution according to a volume ratio of 5:1, disperse them with a high-speed stirrer at 10000 rpm for 2 min, further homogenize them in an ice bath using ultrasonic homogenization for 15 min, centrifuge at 2000 rpm for 2 min, then remove the absolute ethanol using a rotary evaporator, and finally lyophilize to prepare the mitochondrial-targeted lutein base powder.

[0088] Comparative Example 2

[0089] Step 1: Weigh 4 g of casein and 1 g of mannose, mix them well in 100 mL of deionized water, stir overnight at room temperature, adjust the pH to 9, heat at 90 °C for 3 h, place it in an ice bath, cool to room temperature, and lyophilize to obtain the Maillard reaction complex.

[0090] Step 2: Weigh 12.87 mg of TPP, 11.38 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 6.91 mg of N-hydroxysuccinimide, dissolve them in 10 mL of dimethyl sulfoxide, stir for 6 h to prepare the TPP carboxyl activation solution. Subsequently, weigh 500 mg of the Maillard reaction complex prepared in Step 1, dissolve it in 50 mL of deionized water, then pour it into the prepared TPP carboxyl activation solution, and continue stirring and reacting for 24 h. After the reaction, dialyze with a dialysis bag with a molecular weight cut-off of 500 Da for 48 h, and lyophilize the dialyzed solution to obtain the Maillard reaction complex modified with TPP.

[0091] Step 3: Dissolve the TPP-modified Maillard reaction complex in deionized water to prepare a TPP-modified Maillard reaction complex solution with a concentration of 10 mg / mL. Dissolve nile red in absolute ethanol to prepare a nile red solution with a concentration of 6 mg / mL. Mix the TPP-modified Maillard reaction complex solution and the nile red solution according to a volume ratio of 5:1, disperse them with a high-speed stirrer at 10000 rpm for 2 min, further homogenize them using ultrasonic homogenization in an ice bath for 15 min, centrifuge at 2000 rpm for 2 min, then remove absolute ethanol using a rotary evaporator, and finally prepare the mitochondrion-targeted nile red base powder by freeze-drying.

[0092] Comparative Example 3

[0093] Step 1: Weigh 4 g of casein and 1 g of mannose, mix them well in 100 mL of deionized water, stir overnight at room temperature, adjust the pH to 9, heat at 90 °C for 3 h, place in an ice bath, cool to room temperature, and then prepare the Maillard reaction complex by freeze-drying.

[0094] Step 2: Dissolve the Maillard reaction complex in deionized water to prepare a Maillard reaction complex solution with a concentration of 10 mg / mL. Dissolve nile red in absolute ethanol to prepare a nile red solution with a concentration of 6 mg / mL. Mix the Maillard reaction complex solution and the nile red solution according to a volume ratio of 5:1, disperse them with a high-speed stirrer at 10000 rpm for 2 min, further homogenize them using ultrasonic homogenization in an ice bath for 15 min, centrifuge at 2000 rpm for 2 min, then remove absolute ethanol using a rotary evaporator, and finally prepare the nile red base powder by freeze-drying.

[0095] Analyze the microstructure, cell targeting, in vivo distribution in mice, and retinal thickness of the response-targeted base powders prepared in each of the above examples.

[0096] Figure 1 Transmission electron micrograph of the response-targeted lutein base powder prepared in Example 1. The base powder has a spherical structure and good dispersibility.

[0097] Figure 2 Transmission electron micrograph of the mitochondrion-targeted lutein base powder prepared in Comparative Example 1. The base powder has a spherical structure and good dispersibility.

[0098] Figure 3 Lutein release amount of the response-targeted lutein base powder prepared in Example 1 at pH 7.4. The release amount at 48 h is about 21.12 ± 0.77%, and when the concentration of H 2 O 2 is 0.5 mM, the lutein release rate slightly increases, about 26.73 ± 0.69%, and at 2 mM H 2O 2 The lutein release rate was 33.01 ± 1.11% under 2 O 2 conditions, and the lutein release rate was 42.99 ± 1.68% under 10 mM H 2 O 2 conditions. In the presence of high-concentration H

[0099] Figure 4 For Comparative Example 3, the overlap of the red fluorescence of Nile red from the base powder and the green fluorescence from Mito-Tracker, and the Pearson correlation coefficient at 4 h was 0.82.

[0100] Figure 5 For Comparative Example 2, the overlap of the red fluorescence of Nile red from the base powder and the green fluorescence from Mito-Tracker, and the Pearson correlation coefficient at 4 h was 0.88, which was higher than the Pearson coefficient of Comparative Example 3, indicating that the mitochondrion-targeted base powder effectively accumulated in mitochondria.

[0101] Figure 6 For Comparative Example 2 and Example 2, the biodistribution in mice. The fluorescence intensity of Example 2 was higher than that of Comparative Example 2, indicating that the responsive-targeted base powder had a better protective effect on functional factors and could prolong the in vivo circulation time.

[0102] Figure 7 For the lutein concentrations in the plasma, liver, and eyeballs of mice of free lutein, Comparative Example 1, and Example 1 analyzed by pharmacokinetics. The highest plasma drug concentration of lutein in the Example 1 group was 589.01 ± 20.27 ng / mL, which was significantly higher than that in the Comparative Example 1 group (487.23 ± 12.40 ng / mL) and the free lutein group (408.35 ± 15.63 ng / mL), indicating that the responsive-targeted lutein base powder could better protect lutein and improve the bioavailability of lutein.

[0103] Figure 8 For the experimental results of the protection of the animal model of blue light-induced retinal damage by the responsive-targeted lutein base powder. The H&E staining results of the control group showed that the retinal layers were intact, the cell morphology was normal, the arrangement was tight, the cell nuclei were enlarged, the chromatin was dense, and there were more pigment granules. Compared with the control group, retinal degeneration occurred in the blue light group, and the thickness of each layer was significantly reduced. The retinal thickness of the mice in the Example 1 group was higher than that of free lutein and Comparative Example 1, indicating that the responsive-targeted lutein base powder effectively alleviated blue light-induced retinal damage.

[0104] Figure 9 and Figure 10They are the test results of interleukin-1β and interleukin-6 in mouse serum respectively. The results show that the inflammatory factors in the serum of mice treated with the responsive targeted lutein base powder are significantly lower than those in the blue light group, indicating that the responsive targeted lutein base powder constructed in the present invention can significantly alleviate the blue light damage of mice and increase the bioavailability of lutein.

[0105] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a responsive targeted lutein base powder, characterized in that: The steps include: Step (1): mixing casein and mannose in water, adjusting the pH to alkaline, heating, cooling, and freeze-drying to obtain a Maillard reaction complex; Step (2): dissolving TPP, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide, and stirring to obtain a TPP carboxyl activation solution; dissolving the Maillard reaction complex obtained in step (1) in water to obtain a Maillard reaction complex solution, then pouring it into the TPP carboxyl activation solution and stirring, then dialyzing, and freeze-drying the dialyzed solution to obtain a TPP-modified Maillard reaction complex; Step (3): dissolving the TPP-modified Maillard reaction complex in water to obtain a TPP-modified Maillard reaction complex solution, dissolving lutein in anhydrous ethanol to obtain a lutein solution, stirring and mixing the Maillard reaction complex solution and the lutein solution, and then homogenizing, centrifuging, rotary evaporating, and freeze-drying to obtain a lutein base powder; Step (4): dissolving sodium alginate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in water to obtain a sodium alginate carboxyl activation solution; dissolving 3-aminophenylboric acid in an ethanol solution and stirring and mixing with the sodium alginate carboxyl activation solution, dialyzing, and then freeze-drying to obtain a 3-aminophenylboric acid-sodium alginate complex; adding the 3-aminophenylboric acid-sodium alginate complex solution to a lutein base powder solution, stirring, and obtaining a responsive targeted lutein base powder.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of casein to mannose is 1:1 to 6:1; the mass ratio of casein to mannose is 4:1 to 6:1; and the mass ratio of casein to water is 1:15 to 1:

30.

3. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of TPP to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:0.6 to 1:1.2; the mass ratio of TPP to N-hydroxysuccinimide is 1:0.3 to 1:0.7; and the mass ratio of TPP to dimethyl sulfoxide is 1 mg:0.8 g to 1 mg:1.2 g.

4. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the Maillard reaction complex solution is 5 to 15 mg / mL; the volume ratio of the Maillard reaction complex solution to the TPP carboxyl activation solution is 3:1 to 7:

1.

5. The preparation method according to claim 1, characterized in that: In step (3), the concentration of the TPP-modified Maillard reaction complex solution is 5 to 15 mg / mL; the concentration of the lutein solution is 1 to 10 mg / mL; and the volume ratio of the Maillard reaction complex solution to the lutein solution is 5:1 to 10:

1.

6. The preparation method according to claim 1, characterized in that: In step (4), the mass ratio of sodium alginate to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1 to 1:2; the mass ratio of sodium alginate to N-hydroxysuccinimide is 1:0.5 to 1:1.5; and the mass ratio of sodium alginate to water is 1:100 to 1:

200.

7. The preparation method according to claim 1, characterized in that: In step (4), the ethanol solution is an ethanol aqueous solution with a volume fraction of 40 to 60%; the ratio of the mass of 3-aminophenylboronic acid to the volume of the ethanol solution is 60 to 90 mg:10 mL; and the volume ratio of the sodium alginate carboxyl activation solution to the ethanol solution is 1 to 1.5:

1.

8. The preparation method according to claim 1, characterized in that: In step (4), the concentration of the 3-aminophenylboronic acid-sodium alginate complex solution is 1 to 2 mg / mL; the concentration of the lutein nanoparticle solution is 1 to 2 mg / mL; and the volume ratio of the 3-aminophenylboronic acid-sodium alginate complex solution to the lutein nanoparticle solution is 1:0.25 to 1:

4.

9. A response-targeted lutein base powder, characterized in that: The responsive targeted lutein base powder is prepared by the preparation method described in any one of claims 1 to 8 above.

10. Use of the responsive targeted lutein base powder as claimed in claim 9 in the preparation of health food or medicine for alleviating retinal blue light damage.

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