Chitosan-based nanoparticles capable of realizing targeted delivery of micrococcus oil mitochondria as well as preparation method and application of chitosan-based nanoparticles

Abstract

The invention discloses chitosan-based nanoparticles capable of realizing targeted delivery of micrococcus oil mitochondria as well as a preparation method and application of the chitosan-based nanoparticles, and belongs to the technical field of biomedical materials. The nano particles are obtained by taking fenugreek gum, xylooligosaccharide, hydroxypropyl chitosan, fucoidin, mannose and (3-propyl carboxyl) triphenylphosphonium bromide as carriers, adding a phycocyanin-lactoferrin compound, and wrapping micrococcus oil and rhamnolipid. Chitosan-based nanoparticles with high drug loading rate and steady state are prepared, the nanoparticles have specific recognition and binding capacity to cells and mitochondria, active substances can be released at fixed points and accurately delivered, real-time tracking can be carried out, a new way is provided for accurate delivery of active ingredients such as micrococcus oil, and the application prospect is broad. The technical support is provided for the visual research of the functional component action mechanism.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a chitosan-based nanoparticle capable of achieving mitochondrial targeting delivery of parafleuriella microsphaerica oil and a preparation method and application thereof. BACKGROUND

[0002] Chitosan, as a main derivative polysaccharide of by-products (shrimp and crab shells) of crustaceans, is an ideal skeleton material for encapsulation of bioactive ingredients. It has the following advantages: good biocompatibility and biodegradability, no biological toxicity, and rich in amino and hydroxyl groups in the molecular chain, which can realize efficient loading of active ingredients through electrostatic interaction, hydrogen bonding, etc. It can also be chemically modified to control the hydrophilicity and targeting of the carrier, thereby improving the delivery efficiency. At the same time, the molecular barrier can isolate external factors such as light and oxygen, reduce the degradation of active ingredients, and enhance the stability. In addition, chitosan is prepared from crustacean by-products, which not only reduces the cost of the carrier, but also realizes the high-value utilization of aquatic waste, alleviates environmental pressure, and provides a green and sustainable carrier solution for functional food and pharmaceutical fields, with both economic and ecological benefits.

[0003] Parafleuriella microsphaerica oil is rich in active ingredients such as polyunsaturated fatty acids (such as EPA, DHA), and has significant anti-inflammatory efficacy. It can regulate the release of inflammatory factors and inhibit inflammatory signaling pathways, and has application potential in functional food and pharmaceutical fields. However, it has obvious instability: it is easily affected by light, oxygen, and temperature, and is prone to oxidative degradation, and is easily enzymatically degraded in the digestive process, resulting in loss of activity and reduced bioavailability, limiting its practical application. Nanocapsulation technology can solve this problem: by constructing liposomes, polymer nanoparticles, and other carriers, a physical barrier is provided for parafleuriella microsphaerica oil to isolate external destructive factors and reduce oxidation; at the same time, its dispersibility and targeting delivery efficiency are optimized, improving the intestinal absorption effect after oral administration, and providing key technical support for the preservation of parafleuriella microsphaerica oil activity and efficacy.

[0004] Nanocapsulation technology provides an ideal solution for the design of carriers for bioactive ingredients (such as polyphenols, probiotics, functional peptides, etc.) due to its unique size effect and controllable structure. Its core advantages lie in two aspects: first, the high flexibility of carrier design, which can achieve efficient loading and targeted delivery of active ingredients by adjusting the composition, morphology, and surface properties of nanomaterials (such as liposomes, polymer nanoparticles, nanoemulsions, etc.). For example, polymer nanoparticles can be surface-modified to enhance adhesion to the intestinal mucosa, improving oral bioavailability. Second, it significantly improves the stability level of active ingredients. Nanocarriers can construct a physical barrier to effectively isolate external factors such as light, oxygen, temperature, and digestive enzymes, reducing the oxidative degradation and loss of active ingredients, and prolonging their shelf life and in vivo action time. This technology provides key technical support for the application of bioactive ingredients in functional food, medicine, and other fields.

[0005] Therefore, it is particularly important to develop a kind of nanometer particle of parachlorella oil with the characteristics of fluorescence tracing, which can improve the stability of parachlorella oil, solve the problem of more accurate control of targeted delivery of parachlorella oil and utilize nanotechnology. SUMMARY

[0006] The present application aims to provide a kind of chitosan-based nanoparticles that can achieve mitochondrial targeted delivery of parachlorella oil and its preparation method and application, to solve the problems existing in the prior art. The present application selects psyllium, xylo-oligosaccharide as the core component, integrates the biological adhesion of hydroxypropyl chitosan and fucoidan, and the cell targeting of mannose, mitochondrial targeting of (3-propylcarboxyl) triphenylphosphonium bromide, and the fluorescence of phycocyanin and other raw materials, to prepare chitosan-based nanoparticles with high drug loading rate and stability. The nanoparticles have specific recognition and binding capacity for cells and mitochondria, can release and accurately deliver active substances, and can also be tracked in real time, providing a new way for the accurate delivery of active ingredients such as parachlorella oil and providing technical support for the visualization of functional ingredient mechanism.

[0007] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0008] The present application provides a kind of chitosan-based nanoparticles that can achieve mitochondrial targeted delivery of parachlorella oil, which uses psyllium, xylo-oligosaccharide, hydroxypropyl chitosan, fucoidan, mannose and (3-propylcarboxyl) triphenylphosphonium bromide as the carrier, adds phycocyanin-lactoferrin complex, and then wraps parachlorella oil and rhamnolipid.

[0009] The present application also provides a kind of preparation method of the above-mentioned chitosan-based nanoparticles that can achieve mitochondrial targeted delivery of parachlorella oil, comprising the following steps:

[0010] S1, preparing double-targeted hydroxypropyl chitosan: mixing hydroxypropyl chitosan solution and carboxylated mannose, and then adding activated (3-propylcarboxyl) triphenylphosphonium bromide solution for reaction;

[0011] S2, preparing fucoidan-xylo-oligosaccharide conjugate: mixing fucoidan and xylo-oligosaccharide, and then reacting under the action of xylanase;

[0012] S3, preparing psyllium aldehyde derivative: treating psyllium with acid, and then reacting with sodium periodate;

[0013] S4, preparing phycocyanin-lactoferrin complex;

[0014] S5, preparing parachlorella oil-rhamnolipid nanoemulsion;

[0015] S6, mixing the double-targeted hydroxypropyl chitosan, the fucoidan-xylo-oligosaccharide conjugate, the fenugreek gum aldehyde derivative and the phycocyanin-lactoferrin complex to obtain an aqueous dispersion; after ultrasonic treatment of the aqueous dispersion and the drug nanoemulsion, the chitosan-based nanoparticles are obtained.

[0016] Further, in step S1, the concentration of the hydroxypropyl chitosan solution is 1.0-3.0 g / 200-400 mL; the activated (3-carboxyl) triphenylphosphonium bromide solution is a solution with 50% ethanol as solvent, and the concentration is 0.3-0.5 g / 30-50 mL;

[0017] The ratio of the hydroxypropyl chitosan solution, the carboxylated mannose and the activated (3-carboxyl) triphenylphosphonium bromide solution is 80-100 mL:0.4-0.8 g:30-50 mL;

[0018] The mixing is carried out under the action of EDC·HCl and NHS, the temperature is 30℃, the time is 6-8 h, and the rotation speed is 350-500 rpm;

[0019] The activated (3-carboxyl) triphenylphosphonium bromide is added at a speed of 1 mL / min;

[0020] The temperature of the reaction is 45℃, the time is 10-12 h, the rotation speed is 400-500 rpm; after the reaction is completed, dialysis is carried out for 96-120 h with a 10 kDa dialysis bag.

[0021] Further, in step S2, the mass ratio of the fucoidan and the xylo-oligosaccharide is 5-6:2-3; the addition amount of the xylanase is 2 U / mL; the temperature of the reaction is 45℃, the time is 8-10 h, and the rotation speed is 200-300 rpm; after the reaction is completed, dialysis is carried out for 48-72 h with a 3.5 kDa dialysis bag.

[0022] Further, in step S3, the acid is citric acid; when the acid treatment is carried out, the final concentration of the citric acid is 0.08 M; the temperature of the acid treatment is 55℃, the time is 5-6 h, and the rotation speed is 200-300 rpm; after the acid treatment is completed, dialysis is carried out for 48-72 h with a 3.5 kDa dialysis bag.

[0023] The mass ratio of the sodium periodate and the fenugreek gum is 0.3-0.35:1-2; the reaction is carried out in the dark, the temperature is 25℃, the time is 2-2.5 h, and the rotation speed is 200-300 rpm; after the reaction is completed, dialysis is carried out for 72-96 h with a 10 kDa dialysis bag.

[0024] Further, in step S4, the mass ratio of phycocyanin to lactoferrin is 0.15-0.3:0.02-0.03.

[0025] Further, in step S5, the preparation method of the *Micrococcus pluvialis* oil-rhamnolipin nanoemulsion is as follows: *Micrococcus pluvialis* oil and the rhamnolipin solution are mixed and ultrasonically obtained at a volume ratio of 1.5-3:13.5-27.

[0026] The concentration of the rhamnolipid solution is 1.2 vol%-2.4 vol%; the ultrasound conditions are 400-600 W, 5 seconds of operation, 3 seconds of interval, for a total of 15-20 minutes.

[0027] Further, in step S6, the ratio of the dual-targeted hydroxypropyl chitosan, the fucoidan-xylooligosaccharide conjugate, the fenugreek aldehyde derivative, and the phycocyanin-lactoferrin complex is 0.316-0.632g: 0.2-0.4g: 0.15-0.30g: 0.11-0.22g;

[0028] The mixing method is as follows: the dual-targeting hydroxypropyl chitosan and the fucoidan-xylooligosaccharide conjugate are mixed in water and stirred at 600-800 rpm for 2-3 hours at 25°C; the fenugreek aldehyde derivative is added and stirred at 350-500 rpm for 2-3 hours at 30°C; calcium chloride is added and stirred at 200-300 rpm for 30-40 minutes at 25°C; the phycocyanin-lactoferrin complex is added and stirred at 200-300 rpm for 40-50 minutes at 25°C.

[0029] The volume ratio of the aqueous dispersion to the drug nanoemulsion is 70-140:15-20;

[0030] After mixing the aqueous dispersion with the drug nanoemulsion, sodium citrate at 4 times the mass of hydroxypropyl chitosan is added, and the mixture is stirred at 600-700 rpm for 30-40 min, allowed to stand for 2-3 h, and then subjected to ultrasonic treatment. The ultrasonic conditions are: 300-400 W, 5 s operation time, 5 s interval, for a total of 10-15 min.

[0031] The present invention also provides the application of chitosan-based nanoparticles or chitosan-based nanoparticles prepared by the above preparation method in the preparation of functional foods.

[0032] The present invention also provides the application of chitosan-based nanoparticles or chitosan-based nanoparticles prepared by the above preparation method in the preparation of visual fluorescent tracer reagents.

[0033] The present invention discloses the following technical effects:

[0034] This invention addresses the problem of nutrient waste caused by the indiscriminate distribution of nutrients in chitosan-encapsulated active substances. It provides chitosan-based nanoparticles capable of mitochondrial-targeted delivery of *Chlorella vulgaris* oil, thus improving nutrient utilization efficiency. These nanoparticles are made with carefully selected fenugreek gum (FG) and xylooligosaccharide (XOS) as core components, integrating the bioadhesive properties of hydroxypropyl chitosan (HPCS) and fucoidan (Fu), as well as cell-targeting mannose (Man), mitochondrial-targeting (3-propanoyl)triphenylphosphine bromide (TPP), and fluorescent phycocyanin, providing support for the precise nanoscale delivery of active ingredients such as *Chlorella vulgaris* oil. This invention establishes a chitosan nanoparticle preparation technology with high drug loading rate and stable state. By introducing targeted ligands through structural modification, the nanoparticles' ability to specifically recognize and bind to specific cells and tissues is enhanced, thus preparing a targeted delivery system capable of site-specific release and precise delivery of active substances. This provides a new approach for the precise delivery of active ingredients such as *Chlorella vulgaris* oil. By integrating fluorescent phycocyanin, the nanoparticles can be tracked in real time, providing technical support for the visualization study of the mechanism of action of functional components. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 An atomic force microscope image of *Chlorella vulgaris* oil nanoparticles;

[0037] Figure 2 The particle size distribution of the microalgae oil nanoparticles is shown.

[0038] Figure 3 Fourier transform infrared spectra of hydroxypropyl chitosan (HPCS), mannose (Man), and their binary complex (Man-HPCS);

[0039] Figure 4 Fourier transform infrared spectra of (3-propanoyl)triphenylphosphine bromide (TPP), carboxylated mannose-hydroxypropyl chitosan complex (Man-HPCS) and its ternary complex (Man-TPP-HPCS);

[0040] Figure 5 Fourier transform infrared spectra of fenugreek gum (FG), phycocyanin, hydroxypropyl chitosan (HPCS), and microalgae oil nanoparticles (NPs).

[0041] Figure 6 This is a peak diagram of elements in Man-HPCS.

[0042] Figure 7 The elemental peak diagram of Man-HPCS-TPP;

[0043] Figure 8 Zeta potential diagrams for hydroxypropyl chitosan (HPCS), fucoidan (Fu), carboxylated mannose-hydroxypropyl chitosan-(3-propanoyl)triphenylphosphine bromide complex (Man-HPCS-TPP), fucoidan-xylooligosaccharide complex (Fu-XOS) and its multi-component complex (MHT-FX).

[0044] Figure 9 Zeta potential diagrams for hydroxypropyl chitosan (HPCS), phycocyanin, and pseudomicroalgal oil nanoparticles (NPs).

[0045] Figure 10 The carbon NMR spectral characterization of fucoidan (Fu), xylooligosaccharide (XOS), and fucoidan-xylooligosaccharide conjugate (Fu-XOS);

[0046] Figure 11 The 1H NMR structural characterization of hydroxypropyl chitosan (HPCS), fenugreek gum (FG), multi-component complex (MHT-FX), and microalgae oil nanoparticles (NPs) are shown.

[0047] Figure 12 To improve the encapsulation efficiency and loading rate of *Chlorella vulgaris* oil nanoparticles;

[0048] Figure 13 Laser confocal microscopy image of phycocyanin colocalization in the mitochondria of RAW 264.7 cells; scale bar: 10 μm;

[0049] Figure 14 This is a laser confocal microscope image showing the co-localization of *Chlorella vulgaris* oil nanoparticles in the mitochondria of RAW 264.7 cells; scale bar is 10 μm. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0054] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0055] Example 1: Preparation of Microalgae-like Oil Nanoparticles

[0056] Preparation of S1 and HPCS solutions

[0057] Weigh 1.0 g of HPCS (50 kDa, degree of substitution 0.8) and add 100 mL of 0.05 M acetic acid. Stir magnetically (300 rpm) at 25 °C until completely dissolved. Adjust the pH to 6.0 with 0.1 M NaOH and bring the volume to 200 mL with deionized water. The HPCS solution concentration is now 1.0 g / 200 mL. Refrigerate at 4 °C for later use.

[0058] S2, Preparation of carboxylated mannose

[0059] Weigh 1.0 g of mannose and dissolve it in 30 mL of anhydrous ethanol. Add 20 mL of deionized water (volume ratio 3:2). Add 0.87 g of succinic anhydride (molar ratio of mannose to succinic anhydride 1:1.2), and add 0.1 mL of triethylamine dropwise. Stir at 50 °C (200 rpm) for 4 h. Pour the reaction solution into 200 mL of ice water, let it stand at 4 °C for 1 h, filter to obtain the precipitate, and wash it three times with deionized water. Place it in a 3.5 kDa dialysis bag and dialyze with deionized water for 72 h (changing the water every 12 h). Freeze-dry and store in a sealed container.

[0060] Activation of S3 and TPP-COOH

[0061] Weigh 0.3 g TPP and dissolve it in 30 mL of 50% ethanol. Add 0.15 g EDC・HCl and 0.09 g NHS, and stir at 25°C (250 rpm) for 30 min. Label this as TPP-COOH. Store for later use (within 1 hour).

[0062] Preparation of S4, Fu-XOS conjugates

[0063] Weigh 0.5 g Fu and dissolve it in 50 mL of deionized water. Add 0.2 g XOS (degree of polymerization 3-5). Add 2 U / mL xylanase and stir at 45 °C (200 rpm) for 8 h. Dialyze through a 3.5 kDa dialysis bag for 48 h, then freeze-dry and store in the dark.

[0064] S5. Preparation of fenugreek gum aldehyde derivatives (acid hydrolysis instead of enzymatic hydrolysis)

[0065] Weigh 1.0 g of fenugreek gum (80 kDa) and dissolve it in 100 mL of 0.1 M PBS (pH 6.0). Add citric acid to a final concentration of 0.08 M. Stir magnetically at 55 °C (200 rpm) for 5 h, and adjust the pH to 6.0 with 0.1 M NaHCO3. Transfer the solution to a 3.5 kDa dialysis bag and dialyze with deionized water for 48 h (changing the water every 8 h). Add 0.3 g of sodium periodate and stir at 25 °C for 2 h in the dark. Stop the reaction by adding 0.2 mL of ethylene glycol. Dialyze with a 10 kDa dialysis bag for 72 h, and freeze-dry for later use.

[0066] S6. Preparation of *Microcystis aeruginosa* oil-rhamnolipin nanoemulsion

[0067] Weigh 1.5 mL of *Micrococcus pseudocarpa* oil and add 13.5 mL of deionized water containing 1.2% (v / v) rhamnolipids. Sonicate the mixture (400 W, 5 s on, 3 s off) for 15 min to form an emulsion with a particle size of 80 ± 10 nm. Store at 4 °C.

[0068] S7. Preparation of phycocyanin-lactoferrin complex

[0069] Weigh 0.15g of phycocyanin and dissolve it in 15mL of PBS (pH 7.4), then add 0.02g of lactoferrin. Stir at 25℃ (150rpm) for 30min to obtain the final product (prepare immediately before use).

[0070] S8, Preparation of Dual-Targeted HPCS (Man-TPP-HPCS)

[0071] Weigh 80 mL of the HPCS solution from step S1, add 0.4 g of carboxylated mannose from step S2, then add 0.24 g of EDC·HCl and 0.14 g of NHS. Stir at 30°C (350 rpm) for 6 h, then add 30 mL of TPP-COOH activation solution from step S3 dropwise at a rate of 1 mL / min. Stir at 45°C (400 rpm) for 10 h, transfer to a 10 kDa dialysis bag, and dialyze with 0.01 M PBS for 96 h (changing water every 8 h). Add 5% (w / v) mannitol (lyophilization protectant) to the dialyzed Man-TPP-HPCS solution, stir to dissolve, and then lyophilize. The protectant reduces hydrophobic interactions between molecules during lyophilization, inhibiting aggregation. The lyophilized product is a powder.

[0072] S9. Assembly of nanoparticles (stepwise continuous operation)

[0073] Weigh 0.316 g of Man-TPP-HPCS from step S8 and dissolve it in 60 mL of deionized water. Stir at 35°C (300 rpm) for 40 min. Add 0.2 g of fucoidan-XOS conjugate from step S4 and stir at 25°C (600 rpm) for 2 h. Add 0.15 g of fenugreek aldehyde derivative from step S5, adjust the pH to 6.8, and stir at 30°C (350 rpm) for 2 h. Add 0.03 g of calcium chloride and stir at 25°C (200 rpm) for 30 min. Slowly add 10 mL of phycocyanin-lactoferrin complex from step S7 and stir at 25°C (200 rpm) for 40 min to obtain aqueous dispersion A.

[0074] Oil phase encapsulation and final assembly: Add 15 mL of the nanoemulsion from step S6 to the aqueous dispersion A, and stir at 25°C (800 rpm) for 15 min. Add 2 mg / mL sodium citrate (sodium citrate to HPCS mass ratio of 1:4), and stir at 600 rpm for 30 min. Let stand at 4°C for 2 h, and sonicate (300 W, 5 s working time / 5 s interval) for 10 min. Centrifuge at 4°C and 15000 rpm for 20 min, and resuspend the precipitate in PBS to 1 mg / mL to obtain the suspension of *Chlorella pseudo-microalgae* oil nanoparticles.

[0075] Example 2 Characterization of the properties of the pseudo-microalgae oil nanoparticles prepared in Example 1

[0076] 1. Morphology and size of microalgae oil nanoparticles

[0077] like Figure 1 As shown, the surface morphology of the pseudo-microalgae oil nanoparticles (NPs) is nearly spherical, as can be observed under an atomic force microscope.

[0078] like Figure 2 As shown, the size of the microalgae oil nanoparticles is around 1.5 nm, and the dispersion effect is quite ideal.

[0079] 2. Fourier transform infrared spectra of hydroxypropyl chitosan (HPCS), mannose (Man), and their binary complex (Man-HPCS).

[0080] like Figure 3 As shown. The preparation method of the complex (Man-HPCS) is as follows: weigh 80 mL of HPCS solution from step S1, add 0.4 g of carboxylated mannose from step S2, and stir at 30°C (350 rpm) for 6 h.

[0081] It can be seen that the amino groups of hydroxypropyl chitosan are at 1600 cm⁻¹ -1 (NH bending vibration), hydroxyl group at 3400 cm -1 (OH stretching vibration) has absorption; after carboxylation, mannose was observed to have a 1700 cm⁻¹ absorption in the complex. -1 The remaining carboxyl peak after the amide reaction is complete is present. The complex shows a peak at 1650 cm⁻¹. -1 (Amide I band, C=O stretching vibration) and 1540cm -1 At the (amide II band, NH bending vibration and CN stretching vibration) position, a shift can be observed due to the participation of some amino groups in the reaction.

[0082] 3. Fourier transform infrared spectra of (3-propanoyl)triphenylphosphine bromide (TPP), carboxylated mannose-hydroxypropyl chitosan complex (Man-HPCS), and its ternary complex (Man-TPP-HPCS).

[0083] like Figure 4 As shown, (3-propanoyl)triphenylphosphine bromide can be observed at 1580 cm⁻¹. -1 and 1710cm -1 It exhibits unique aromatic ring skeletal vibration peaks (C=C stretching vibration) and carboxyl peaks, which can be used as characteristic signals introduced by TPP. Carboxylated Man combines with HPCS to form Man-HPCS. Man-HPCS-TPP peaks at 1710 cm⁻¹ -1 The disappearance of the carboxyl peak indicates that an amide reaction occurred between TPP and HPCS. (At 1650 cm⁻¹) -1 (Amide I band) and 1540cm -1 The (amide II band) peak vibrated.

[0084] 4. Fourier transform infrared spectra of fenugreek gum (FG), phycocyanin, hydroxypropyl chitosan (HPCS), and microalgae oil nanoparticles (NPs).

[0085] like Figure 5 As shown, after the hydroxyl group (-OH) of fenugreek gum and the amino group (-NH2) of phycocyanin form a hydrogen bond, the peak position shifts to 3280 cm⁻¹. -1 Simultaneously, the negatively charged phycocyanin interacts electrostatically with the positively charged HPCS, causing the pseudo-microsphere algal oil nanoparticles to disperse at 1600 cm⁻¹. -1 and 1540cm -1 The peak showed signs of weakening and shifting.

[0086] 5. Elemental peak analysis using energy-dispersive X-ray spectroscopy (EDS)

[0087] Figure 6 This is the elemental peak diagram of Man-HPCS. Figure 7 The image shows the elemental peak diagrams for Man-HPCS-TPP. It can be seen that the P element signal is stronger in the X-ray energy dispersive spectroscopy (EDS) elemental mapping diagrams of Man-HPCS and Man-HPCS-TPP, indicating that the reaction in TPP and Man-HPCS proceeded successfully.

[0088] 6. Zeta potential analysis

[0089] Figure 8 Zeta potential diagrams for hydroxypropyl chitosan (HPCS), fucoidan (Fu), carboxylated mannose-hydroxypropyl chitosan-(3-propanoyl)triphenylphosphine bromide complex (Man-HPCS-TPP), fucoidan-xylooligosaccharide complex (Fu-XOS) and its multi-component complex (MHT-FX). Figure 9 Zeta potential diagrams for hydroxypropyl chitosan (HPCS), phycocyanin, and pseudomicrosphere oil nanoparticles (NPs).

[0090] The preparation method of the multi-component complex (MHT-FX) is as follows: 0.316 g of Man-TPP-HPCS from step S8 is weighed and dissolved in 60 mL of deionized water, and stirred at 35 °C (300 rpm) for 40 min. 0.2 g of Fu-XOS conjugate from step S4 is added, and stirred at 25 °C (600 rpm) for 2 h.

[0091] from Figure 8 and Figure 9It can be seen that the Zeta potential of hydroxypropyl chitosan is +48mV, while that of fucoidan is -46mV, indicating electrostatic interaction between the two. Similarly, the potential of Man-HPCS-TPP is +11mV, the potential of the fucoidan-xylooligosaccharide complex is -43mV, and the potential of the multi-component complex is -24mV.

[0092] 7. Structural characterization and analysis using carbon and hydrogen nuclear magnetic resonance spectra.

[0093] Figure 10 It consists of fucoidan (Fu), xylooligosaccharide (XOS), and fucoidan-xylooligosaccharide conjugate (Fu-XOS). Figure 11 The 1H NMR structural characterization of hydroxypropyl chitosan (HPCS), fenugreek gum (FG), multi-component complex (MHT-FX), and microalgae oil nanoparticles (NPs) are shown.

[0094] from Figure 10 and Figure 11 It can be seen that the chemical shift of the characteristic peak of Fu is 95-105 ppm, and the chemical shift of the characteristic peak of XOS is 98-108 ppm. After the formation of the glycosidic bond, the electronic environment of the two changes, and a new composite characteristic peak appears at 98 ppm. After acid hydrolysis of FG to expose the hydroxyl group, sodium periodate oxidation introduces an aldehyde group (-CHO), which forms a Schiff base bond (dynamic covalent bond) with the amino group (-NH2) of HPCS. The imine hydrogen (-C=NH) of the Schiff base bond shows a characteristic peak at δ8.4 ppm.

[0095] 8. Encapsulation efficiency (EE) and loading capacity (LC) of microalgae oil nanoparticles

[0096] like Figure 12 As shown. The encapsulation efficiency of the carrier for *Chlorella pseudocaryophylla* oil in NPs was calculated to be 84.61 ± 2.32% and the loading rate was 45.61 ± 1.25%, indicating that the chitosan-based nanoparticles of the present invention have a good transport effect on *Chlorella pseudocaryophylla* oil.

[0097] 9. Study on the targeted delivery characteristics of *Chlorella vulgaris* oil nanoparticles to RAW264.7 macrophages

[0098] RAW264.7 cells (density 1×10⁻⁶) were used. 5RAW 264.7 cells (cells / mL) were seeded in two confocal culture dishes and incubated overnight in DMEM, after which the medium was removed. Group 1: 2 mL of medium containing 0.2 mg phycocyanin was added; Group 2: 2 mL of medium containing 0.2 mg *Plasmodium styracifolium* oil nanoparticles was added. After incubation for 7 hours, RAW 264.7 cells were labeled with blue fluorescence using 2 mL of phosphate-buffered saline (PBS) containing mitochondrial tracer MitoTracker Green (74 nM) and Hoechst-33342 (10 µg / mL) for 40 minutes. The cells were washed three times with phosphate-buffered saline, and intracellular fluorescence signals were captured using a laser confocal microscope.

[0099] Figure 13 This is a laser confocal microscope image showing the colocalization of phycocyanin in the mitochondria of RAW 264.7 cells. Figure 14 This is a laser confocal microscope image showing the co-localization of *Chlorella vulgaris* oil nanoparticles in the mitochondria of RAW 264.7 cells.

[0100] from Figure 13 and Figure 14 As can be seen, due to the inclusion of phycocyanin in the NPs, and the fact that phycocyanin exhibits autofluorescence (green), the NPs possess visual fluorescent tracking properties. RAW 264.7 cells co-incubated with NPs and Mito-Tracker Red showed varying degrees of fluorescence co-localization. The Pearson coefficient for fluorescence co-localization of phycocyanin and Mito-Tracker Red in the mitochondria was 48%, while the Pearson coefficient for fluorescence co-localization of NPs and Mito-Tracker Red in the mitochondria was 71%, a significant increase of 23%.

[0101] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A chitosan-based nanoparticle enabling mitochondria-targeted delivery of Nannochloropsis oil, characterized in that, The nanoparticles are obtained by using fenugreek gum, xylo-oligosaccharide, hydroxypropyl chitosan, fucoidan, mannose and (3-carboxypropyl)triphenylphosphonium bromide as carriers, adding phycocyanin-lactoferrin complex, and wrapping Nannochloropsis oil and rhamnolipid.

2. A method for preparing chitosan-based nanoparticles capable of mitochondria-targeted delivery of Nannochloropsis oil according to claim 1, characterized by, The method comprises the following steps: S1, preparing double-targeted hydroxypropyl chitosan: mixing hydroxypropyl chitosan solution and carboxylated mannose, and then adding activated (3-carboxypropyl)triphenylphosphonium bromide solution for reaction; S2, preparing fucoidan-xylo-oligosaccharide conjugate: mixing fucoidan and xylo-oligosaccharide, and then reacting under the action of xylanase; S3, preparing aldehyde derivative of fenugreek gum: treating fenugreek gum with acid, and then reacting with sodium periodate; S4, preparing phycocyanin-lactoferrin complex; S5, preparing Nannochloropsis oil-rhamnolipid nanoemulsion; S6, mixing the double-targeted hydroxypropyl chitosan, the fucoidan-xylo-oligosaccharide conjugate, the aldehyde derivative of fenugreek gum and the phycocyanin-lactoferrin complex to obtain an aqueous dispersion; and then treating the aqueous dispersion and the drug nanoemulsion by ultrasonic treatment to obtain the chitosan-based nanoparticles.

3. The production method according to claim 2, characterized by, In step S1, the concentration of the hydroxypropyl chitosan solution is 1.0-3.0 g / 200-400 mL; and the activated (3-carboxypropyl)triphenylphosphonium bromide solution is a solution with a concentration of 0.3-0.5 g / 30-50 mL and 50% ethanol as solvent; The ratio of the hydroxypropyl chitosan solution, the carboxylated mannose and the activated (3-carboxypropyl)triphenylphosphonium bromide solution is 80-100 mL:0.4-0.8 g:30-50 mL; The mixing is carried out under the action of EDC·HCl and NHS at a temperature of 30℃ for 6-8 h at a rotation speed of 350-500 rpm; The activated (3-carboxypropyl)triphenylphosphonium bromide is added at a speed of 1 mL / min; The reaction is carried out at a temperature of 45℃ for 10-12 h at a rotation speed of 400-500 rpm; and after the reaction is completed, the dialysis is carried out for 96-120 h with a 10 kDa dialysis bag.

4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of the fucoidan to the xylo-oligosaccharide is 5-6:2-3; the addition amount of the xylanase is 2 U / mL; the reaction is carried out at a temperature of 45℃ for 8-10 h at a rotation speed of 200-300 rpm; and after the reaction is completed, the dialysis is carried out for 48-72 h with a 3.5 kDa dialysis bag.

5. The preparation method according to claim 2, characterized in that, In step S3, the acid is citric acid; during the acid treatment, the final concentration of citric acid is 0.08 M; the acid treatment is carried out at a temperature of 55℃ for 5-6 h at a rotation speed of 200-300 rpm; and after the acid treatment is completed, the dialysis is carried out for 48-72 h with a 3.5 kDa dialysis bag; The mass ratio of the sodium periodate to the fenugreek gum is 0.3-0.35:1-2; the reaction is carried out in the dark at a temperature of 25℃ for 2-2.5 h at a rotation speed of 200-300 rpm; and after the reaction is completed, the dialysis is carried out for 72-96 h with a 10 kDa dialysis bag.

6. The preparation method according to claim 2, characterized in that, The mass ratio of the phycocyanin and the lactoferrin in step S4 is 0.15-0.3:0.02-0.

03.

7. The preparation method according to claim 2, characterized in that, In step S5, the preparation method of the Nannochloropsis sp. oil-rhamnolipid nanoemulsion is as follows: Nannochloropsis sp. oil and the rhamnolipid solution are mixed at a volume ratio of 1.5-3:13.5-27, and then ultrasonic treatment is performed. The concentration of the rhamnolipid solution is 1.2vol%-2.4vol%, and the ultrasonic treatment is performed at 400-600W for 5s, with an interval of 3s, for a total of 15-20min.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of the double-targeted hydroxypropyl chitosan, the fucoidan-xylo-oligosaccharide conjugate, the trigonella foenum-graecum gum aldehyde derivative, and the phycocyanin-lactoferrin complex is 0.316-0.632:0.2-0.4:0.15-0.30:0.11-0.

22. The mixing method is as follows: the double-targeted hydroxypropyl chitosan and the fucoidan-xylo-oligosaccharide conjugate are mixed in water, and then stirred at 25°C at 600-800rpm for 2-3h; the trigonella foenum-graecum gum aldehyde derivative is added, and then stirred at 30°C at 350-500rpm for 2-3h; calcium chloride is added, and then stirred at 25°C at 200-300rpm for 30-40min; the phycocyanin-lactoferrin complex is added, and then stirred at 25°C at 200-300rpm for 40-50min. The volume ratio of the aqueous dispersion and the drug nanoemulsion is 70-140:15-20. After the aqueous dispersion and the drug nanoemulsion are mixed, sodium citrate is added in an amount of 4 times the mass of hydroxypropyl chitosan, and then stirred at 600-700rpm for 30-40min, and left to stand for 2-3h, and then subjected to ultrasonic treatment; the ultrasonic treatment is performed at 300-400W for 5s, with an interval of 5s, for a total of 10-15min.

9. Use of the chitosan-based nanoparticles of claim 1 or the chitosan-based nanoparticles prepared by the method of any one of claims 2-8 in the preparation of functional food.

10. Use of the chitosan-based nanoparticles of claim 1 or the chitosan-based nanoparticles prepared by the method of any one of claims 2-8 in the preparation of visual fluorescent tracer reagents.

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