WO2022116356A1 - 一种基于海洋多糖载体的花色苷纳米粒子及其制备方法和在靶向递送中的应用 - Google Patents

一种基于海洋多糖载体的花色苷纳米粒子及其制备方法和在靶向递送中的应用 Download PDF

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WO2022116356A1
WO2022116356A1 PCT/CN2020/142199 CN2020142199W WO2022116356A1 WO 2022116356 A1 WO2022116356 A1 WO 2022116356A1 CN 2020142199 W CN2020142199 W CN 2020142199W WO 2022116356 A1 WO2022116356 A1 WO 2022116356A1
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anthocyanin
nanoparticles
marine polysaccharide
fluorescent
dmf
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French (fr)
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谭明乾
赵雪
张雪迪
铁珊珊
曹纪鋆
李运
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大连工业大学
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    • A—HUMAN NECESSITIES
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    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
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    • A61K31/33—Heterocyclic compounds
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    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
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    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
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    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
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    • A61K49/00—Preparations for testing in vivo
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of nanotechnology, and more particularly, to an anthocyanin nanoparticle based on a marine polysaccharide carrier and its preparation and application in fluorescence tracing and colon-targeted delivery.
  • Anthocyanins are biologically active compounds belonging to flavonoids, which exist in human nutrition through plant foods.
  • the fruit components of bilberry are rich in anthocyanins. With strong antioxidant properties, regular consumption has health benefits.
  • environmental conditions such as pH and temperature have a great influence on the color stability and properties of anthocyanins.
  • the anthocyanins prepared in the prior art have poor stability, especially the sodium alginate microspheres prepared by the emulsification method have insufficient residence time in the upper digestive tract, resulting in low bioavailability and need to be improved.
  • Nanoencapsulation technology is defined as the process by which biologically active ingredients are encapsulated by a coated carrier material and delivered to the core at the right time and place.
  • nano-encapsulation in addition to being biologically active, nano-encapsulation can also increase its surface-to-volume ratio by reducing the particle size to the nanometer range (usually less than 1 ⁇ m), which is further improved compared to microspheres prepared by general embedding methods Its bioavailability can better control the sustained release, and has a better prospect of utilization.
  • an anthocyanin nanoparticle that utilizes nanotechnology to further improve the stability of anthocyanins, solves the problem of more precise control of sustained release and fixed-point release of anthocyanins, and has fluorescent tracer properties.
  • the invention provides an anthocyanin nanoparticle based on a marine polysaccharide carrier and its preparation and application in fluorescence tracing and colon targeted delivery, so as to solve the problem that the stability of bilberry anthocyanins in the prior art is poor and the functional active components are easily lost. and application of anthocyanin nanoparticle pretreatment in protection of lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages and alleviation of dextran sodium sulfate (DSS)-induced colonic inflammatory injury.
  • LPS lipopolysaccharide
  • DSS dextran sodium sulfate
  • the present invention proposes an anthocyanin nanoparticle based on a marine polysaccharide carrier, and the anthocyanin nanoparticle uses polysaccharide, food-derived fluorescent nanoparticle, and sodium hyaluronate as a composite carrier to combine with anthocyanin.
  • the polysaccharide includes sodium alginate.
  • a preparation method of anthocyanin nanoparticles based on marine polysaccharide carrier comprising the following steps:
  • the evaporation conditions in step S3 are a temperature of 25-40° C. and a vacuum degree range of 0.29-1.6 mbar.
  • the method for removing unreacted small molecules in step S4 includes washing with DMF and then dialysis with DMF; the method for removing DMF includes dialysis with deionized water.
  • the stirring speed in steps S1 and S2 is 500-800 r/min.
  • the preparation method of the food-derived fluorescent nanoparticles comprises the following steps:
  • the chromatographic column packing of the chromatography in step S2 comprises D101 macroporous adsorption resin.
  • the present invention adopts marine polysaccharide sodium alginate, food-derived fluorescent nanoparticles, and sodium hyaluronate as composite carrier materials of anthocyanins.
  • food-derived nanoparticles are used instead of chemical fluorescent additives to deliver anthocyanins. It is the first case.
  • the carrier designed in the present invention has a variety of advantages, and can further reduce the destruction of anthocyanin molecular structure caused by environmental factors such as temperature, pH and other conditions.
  • the present invention improves the stability of anthocyanins in salt solution, different pH, ultraviolet light irradiation and storage, wherein the retention rate of anthocyanins is increased by 8-15%, and has stronger stability;
  • the present invention also has the characteristic of targeted delivery, and can accurately control the slow release and fixed-point release of anthocyanins.
  • the present invention has a better protective effect on lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages, and the anthocyanin nanoparticles of the present invention can be delivered to the colon of BALB/c mice in a targeted manner, which can further alleviate the Colon inflammation injury induced by dextran sodium sulfate (DSS); the invention also has fluorescent tracer properties, has better photostability and biocompatibility than chemical fluorescent additives, and can also be used as a fluorescent marker for animals In vivo imaging of tissues and organs.
  • LPS lipopolysaccharide
  • DSS dextran sodium sulfate
  • Fig. 1 is the photograph of the scanning electron microscope (SEM) of the anthocyanin nanoparticles prepared in embodiment 1;
  • Fig. 2 is the spectrum of the XRD of the anthocyanin nanoparticles prepared in embodiment 1;
  • Fig. 3 is the ultraviolet and fluorescence spectra of the anthocyanin nanoparticles prepared in Example 1;
  • Fig. 4 is the fluorescence lifetime spectrum of anthocyanin nanoparticles prepared in Example 1;
  • Fig. 5 is the FT-IR spectrum of the anthocyanin nanoparticles prepared in Example 1;
  • Fig. 6 is the spectrum of the temperature stability of the anthocyanin nanoparticles prepared in Example 1;
  • Fig. 7 is the collection of illustrative plates of the NaCl stability of the anthocyanin nanoparticles prepared in Example 1;
  • Fig. 8 is the spectrum of pH stability of the anthocyanin nanoparticles prepared in Example 1;
  • Fig. 9 is the spectrum of UV photostability of the anthocyanin nanoparticles prepared in Example 1;
  • Fig. 10 is a spectrum of storage stability of anthocyanin nanoparticles prepared in Example 1;
  • Figure 11 is a fluorescence imaging image of lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages;
  • LPS lipopolysaccharide
  • Figure 12 is a fluorescence imaging image of the protective effect of unencapsulated anthocyanins on lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages;
  • LPS lipopolysaccharide
  • Figure 13 is a fluorescence imaging image of the protective effect of anthocyanin nanoparticles prepared in Example 1 on lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages;
  • LPS lipopolysaccharide
  • Figure 14 is a pseudo-color image of the distribution of anthocyanin nanoparticles prepared in Example 1 for fluorescently labeling mouse tissues and organs;
  • FIG. 15 is a graph showing that the anthocyanin nanoparticles prepared in Example 1 alleviate the damage of colon inflammation induced by DSS.
  • Hyaluronic acid can be further conjugated to the skeleton of alginate by esterification, using diisopropylcarbodiimide (DIC) as a coupling agent and dimethylaminopyridine (DMAP) as a catalyst.
  • DIC diisopropylcarbodiimide
  • DMAP dimethylaminopyridine
  • 40-60 mg of the fluorescent-alginic acid complex was added to 4-6 ml of a mixture of DMF and DCM with a volume ratio of 1:1-1.5, 8-16 mg of DIC and 25-50 mg of DMAP were added thereto, and at room temperature Stir for 1 ⁇ 1.5h, stirring speed 500-800r/min;
  • anthocyanins can form electrostatic interactions with fluorescent-alginic acid complexes through electrostatic self-assembly, and anthocyanins themselves have phenolic hydroxyl groups and can also produce esterification with the carboxyl groups of alginic acid;
  • step S2 40 mg of the fluorescence-alginic acid complex prepared in step S1 was added to 4 ml of a 1:1 mixture of dimethylformamide DMF and DCM by volume. Subsequently, DIC (8.1 mg) and DMAP (25 mg) were added thereto. The reaction mixture was allowed to stir gently at room temperature for 1 h to activate the carboxyl groups of the alginic acid.
  • the sample was washed three times with DCM, and the solvent was removed by rotary evaporation within the vacuum degree range of 0.29-1.6 mbar at 25°C.
  • the resulting product was washed with DMF and dialyzed against DMF for 24 hours to remove unreacted small molecules. DMF was then removed by dialysis against deionized water for 72 h. Finally, the obtained sample was pre-cooled at -80°C for 2h, and then freeze-dried at -50°C under a vacuum of 40Pa for 48h.
  • Figure 1 is a scanning electron microscope (SEM) photograph of anthocyanin nanoparticles. The results show that the prepared nanoparticles are strawberry-like in shape, uniformly distributed, and have a particle size of about 30 nm.
  • Figure 3 is the ultraviolet and fluorescence spectra of anthocyanin nanoparticles.
  • the ultraviolet spectral peak appears at 270 nm, which is presumed to be the characteristic absorption peak of n ⁇ * transition. From the fluorescence spectrum of anthocyanin nanoparticles, it can be seen that there is an obvious red-shift phenomenon with the increase of wavelength, and the maximum excitation wavelength of the nanoparticles appears at 320 nm.
  • Figure 4 is a fluorescence lifetime map of anthocyanin nanoparticles.
  • the anthocyanin nanoparticle aqueous solution of 1 mg/mL was prepared, excited under the excitation light of 320 nm, and the maximum emission peak was emitted at 420 nm, and the fluorescence lifetime was measured.
  • Figure 5 is an FT-IR spectrum of anthocyanin nanoparticles. The results in the figure show that the surface of anthocyanin nanoparticles contains C-C, C-O, C-N, C-H and other functional groups.
  • Figure 6 is a graph of the thermal stability of anthocyanin nanoparticles.
  • a 1 mg/mL aqueous solution of anthocyanin nanoparticles was prepared and stored in an incubator at 55°C for 12 hours continuously, with monitoring every 2 hours in between. The results showed that compared with the thermal stability of unencapsulated anthocyanins, the thermal stability of anthocyanin nanoparticles was better.
  • Figure 7 is a graph of the NaCl stability of anthocyanin nanoparticles. Take 1 mg of lyophilized powder of anthocyanin nanoparticles, and monitor the retention rate of anthocyanins at different concentrations of NaCl solution (0.2M, 0.4M, 0.6M, 0.8M and 1M). It can be observed that anthocyanin nanoparticles are more stable in NaCl solution compared to the NaCl stability of unencapsulated anthocyanins.
  • Figure 8 is a pH stability profile of anthocyanin nanoparticles.
  • Figure 9 is a graph of UV stability of anthocyanin nanoparticles.
  • a 1 mg/mL aqueous solution of anthocyanin nanoparticles was prepared, and UV light was irradiated under the condition of excitation light wavelength of 365 nm for 12 hours continuously, with monitoring every 2 hours in between.
  • the results showed that compared with the UV stability of unencapsulated anthocyanins, the UV stability of anthocyanin nanoparticles was better.
  • Figure 10 is a graph of the storage stability of anthocyanin nanoparticles.
  • the anthocyanin nanoparticle aqueous solution of 1 mg/mL was prepared, and the storage stability was tested at room temperature for 70 d, which was monitored every 10 d. It can be observed that the storage stability of anthocyanin nanoparticles is better than that of unencapsulated anthocyanins during storage.
  • Example 4 Protective effect of anthocyanin nanoparticles on lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages
  • the cells were seeded in a 96-well plate at a density of 5 ⁇ 10 4 /well, incubated in a 5% CO 2 incubator for 12 h, and then unencapsulated anthocyanins and anthocyanins at a final concentration of 40 ⁇ g/mL were added to the medium. After anthocyanin nanoparticles were cultured for another 12 h, 50 ⁇ L of LPS (1 ⁇ g/mL) was added to each well to react for 12 h, followed by cell morphology observation and in situ fluorescence detection of adherent cells.
  • In situ fluorescence detection of adherent cells use Annexin V-FITC and propidium iodide (PI) staining solution for cell staining, the green fluorescence in the figure is Annexin V-FITC positive cells (cytoplasm), and the red fluorescence is propidium iodide pyridine staining positive cells (nuclei). Apoptotic cells were only stained by green fluorescence (with bright spots), necrotic cells were double-stained with green and red fluorescence (more bright spots), and normal cells were not stained by fluorescence (no bright spots).
  • Figure 11 is a fluorescence imaging image of lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages.
  • LPS lipopolysaccharide
  • Figure 12 is a fluorescence imaging image of the protective effect of unencapsulated anthocyanins on lipopolysaccharide (LPS)-mediated apoptosis in RAW 264.7 macrophages.
  • Figure 13 is a fluorescence imaging image of the protective effect of anthocyanin nanoparticles prepared in Example 1 on lipopolysaccharide (LPS)-mediated apoptosis of RAW 264.7 macrophages.
  • Figure 11 shows that LPS has a pro-apoptotic effect on RAW 264.7 macrophages. From Figure 12, it can be observed that unencapsulated anthocyanins have a certain protective effect on RAW 264.7 macrophages. From Figure 13, anthocyanins can be observed The protective effect of nanoparticles on RAW 264.7 macrophages is better than that of unencapsulated anthocyanins, which shows the necessity of anthocyanin encapsulation.
  • Example 5 Colon-targeted delivery of anthocyanin nanoparticles in mice and alleviation of DSS-induced colon inflammatory injury
  • mice were randomly divided into 5 groups, normal control group, DSS injury group, anthocyanin-free nanocarrier group, unencapsulated anthocyanin group, and anthocyanin nanoparticle group.
  • mice in the normal control group were given continuous free drinking water (deionized water) for 12 days;
  • the anthocyanin-free nanocarrier group continuously drank water (deionized water) freely for 12 days, and the anthocyanin-free nanocarriers were administered by gavage every day at a dose of 10 mg/kg. From the 7th day, DSS ( 5%, w/v);
  • the unencapsulated anthocyanins group continuously drank water (deionized water) freely for 12 days, and the unencapsulated anthocyanins were administered orally every day at a dose of 10 mg/kg. From the 7th day, DSS (5%, w /v);
  • the anthocyanin nanoparticle group continuously drank water (deionized water) freely for 12 days, and the anthocyanin nanoparticles were administered orally every day at a dose of 10 mg/kg. From the 7th day, DSS (5%, w/v) was added to the drinking water. ).
  • Figure 14 is a pseudo-color image of the distribution of anthocyanin nanoparticles for fluorescently labeling mouse tissues and organs. It can be observed from the figure that anthocyanin nanoparticles are concentrated in the colon, which can achieve colon-targeted delivery.
  • Figure 15 is a graph of anthocyanin nanoparticles attenuating DSS-induced colon inflammatory injury. The results showed that the anthocyanin nanoparticle group could effectively alleviate the damage of colitis induced by DSS, and had an effective protective effect on the colon of mice, and the protective effect was better than that of the unencapsulated anthocyanin group.

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Abstract

本发明提供一种基于海洋多糖载体的花色苷纳米粒子，以海洋多糖、食源性荧光纳米粒子、透明质酸钠作为复合载体与花色苷结合，制备方法为：S1、将海藻酸钠溶解在磷酸盐缓冲盐水中，依次加入的EDC和HOBt及食源性荧光纳米粒子，得到荧光-海藻酸复合物；S2、将所述荧光-海藻酸复合物加入到DMF和DCM混合物中，再加入DIC和DMAP；S3、加入花色苷和所述透明质酸后除去溶剂；S4、除去未反应的小分子，并冷冻干燥。本发明可提高花色苷稳定性，控制花色苷缓慢释放，具有荧光示踪特性，可作为荧光标记用于动物组织和器官等活体成像，还可以靶向递送至动物结肠部位，缓解由DSS诱导的结肠炎症损伤。

Description

一种基于海洋多糖载体的花色苷纳米粒子及其制备方法和在靶向递送中的应用 技术领域
本发明涉及纳米技术领域，更具体地说，涉及一种基于海洋多糖载体的花色苷纳米粒子及其制备和在荧光示踪与结肠靶向递送中的应用。
背景技术
花色苷是一种属于类黄酮类的生物活性化合物，通过植物性食品存在于人体营养中，越橘的果实成分中含有丰富的花色苷类物质。具有较强的抗氧化特性，经常食用具有健康益处。但是pH、温度等环境条件对花色苷的颜色稳定性和性质有很大的影响。现有技术制备的花色苷稳定性差，尤其是乳化法制备的海藻酸钠微球，在上消化道的停留时间不足，导致生物利用率低，需要改善。
纳米技术作为生物活性化合物的纳米封装的主要应用，在食品工业等多种领域中迅速发展。纳米封装技术定义为生物活性成分被涂层载体材料封装并在正确的时间和位置递送核心的过程。在纳米封装过程中，除了具有生物活性外，纳米封装还可以通过将颗粒尺寸减小到纳米范围(通常小于1μm)而提高其表面体积比，相较于一般包埋法制备的微球进一步提高其生物利用度，能够更好的控制缓释，具有较好的利用前景。
因此发明一种利用纳米技术进一步提高花色苷稳定性、解决更精准的控制花色苷缓释和定点释放的难题同时具有荧光示踪特性的花色苷纳米粒子尤为重要。
发明内容
本发明提供一种基于海洋多糖载体的花色苷纳米粒子及其制备和在荧光示踪、结肠靶向递送中的应用，以解决现有技术中越橘花色苷稳定性差，且功能活性成分易受到损失的问题，以及花色苷纳米粒子预处理对脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的保护和缓解由葡聚糖硫酸钠(DSS)诱导的结肠炎症损伤方面的应用。
为实现上述目的，本发明提出一种基于海洋多糖载体的花色苷纳米粒子，所述花色苷纳米粒子以多糖、食源性荧光纳米粒子、透明质酸钠作为复合载体 与花色苷结合。
优选的，所述多糖包括海藻酸钠。
一种基于海洋多糖载体的花色苷纳米粒子的制备方法，包括以下步骤：
S1、将海藻酸钠溶解在pH值为4.75～6磷酸盐缓冲盐水中，依次加入的EDC和HOBt，室温下搅拌60～80min后，加入所述食源性荧光纳米粒子，搅拌24～30h，再用去离子水透析24～72h并冷冻干燥，得到荧光-海藻酸复合物；
S2、将所述荧光-海藻酸复合物加入DMF和DCM的混合物中，向其中加入按体积比1：1～1.5混合的DIC和DMAP，在室温下搅拌1～1.5h；
S3、再加入花色苷和所述透明质酸，在室温下反应24～30h，用DCM洗涤3～5次，并蒸发除去溶剂；
S4、用DMF洗涤，并用DMF透析20-30h除去未反应的小分子和DMF，在-80℃预冷2～3h，在-50～-55℃、真空度35～45Pa、冷冻干燥24～72h。
优选的，步骤S3中所述蒸发条件为温度25～40℃、真空度范围0.29～1.6mbar。
优选的，步骤S4中所述除去未反应的小分子的方法包括先用DMF洗涤再用DMF透析；所述除去DMF的方法包括用去离子水透析。
优选的，步骤S1、S2中所述搅拌转速500-800r/min。
优选的，所述食源性荧光纳米粒子的制备方法，包括以下步骤：
S1、将猪五花肉切块，在150～320℃条件下烤制15～40min，载浸泡在无水乙醇中，持续搅拌12～36h，选取过滤后的可溶性部分除去乙醇；
S2、以三氯甲烷:水＝3:1配置溶液将S1中除去乙醇的所述可溶性部分复溶，添加三氯甲烷萃取反复脱脂至油相澄清，选取澄清水相部分经过层析，将荧光部分进行-80℃预冷2～3h，在-50℃、真空度40Pa、冷冻干燥24～72h。
优选的，步骤S2中所述层析的色谱柱填料包括D101大孔吸附树脂。
本发明的有益效果是：
本发明采用海洋多糖海藻酸钠、食源性荧光纳米粒子、透明质酸钠作为花色苷的复合载体原料，此载体构建的发明中利用食源性纳米粒子代替化学荧光添加剂在递送花色苷的载体中尚属首例。相较于现有技术，本发明中设计的载体兼具多种优点，能进一步减少因环境因素如温度、PH等条件造成花色苷分子结构的破坏，本方法制备的海藻酸钠纳米粒子相对于其它方法制备的载体而言，本发明提高了花色苷在盐溶液、不同酸碱度、紫外光照射和贮藏稳定性，其中，花色苷的保留率提高了8～15％，拥有更强的稳定性；相较于乳化法制备的海藻酸钠微球，本发明还具有靶向递送的特性，能做到精准控制花色苷的缓慢释放 和定点释放。
本发明对于脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡具有更好地保护作用，并且本发明的花色苷纳米粒子可靶向递送至BALB/c小鼠结肠部位，能够进一步缓解由葡聚糖硫酸钠(DSS)诱导的结肠炎症损伤；本发明同时具有荧光示踪特性，相较于化学荧光添加剂有更好的光稳定性和生物相容性，还可作为荧光标记用于动物组织和器官等活体成像。
附图说明
图1是实施例1中制备的花色苷纳米粒子的扫描电镜(SEM)的照片；
图2是实施例1中制备的花色苷纳米粒子的XRD的图谱；
图3是实施例1中制备的花色苷纳米粒子的紫外、荧光光谱；
图4是实施例1中制备的花色苷纳米粒子的荧光寿命图谱；
图5是实施例1中制备的花色苷纳米粒子的FT-IR图谱；
图6是实施例1中制备的花色苷纳米粒子的温度稳定性的图谱；
图7是实施例1中制备的花色苷纳米粒子的NaCl稳定性的图谱；
图8是实施例1中制备的花色苷纳米粒子的pH稳定性的图谱；
图9是实施例1中制备的花色苷纳米粒子的UV光稳定性的图谱；
图10是实施例1中制备的花色苷纳米粒子的贮藏稳定性的图谱；
图11是脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的荧光成像图；
图12是未封装的花色苷对脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的保护作用的荧光成像图；
图13是实施例1中制备的花色苷纳米粒子对脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的保护作用的荧光成像图；
图14是实施例1中制备的花色苷纳米粒子用于荧光标记小鼠组织和器官的分布成像伪彩图；
图15是实施例1中制备的花色苷纳米粒子缓解由DSS诱导的结肠炎症损伤图。
具体实施方式
为了使本技术领域的人员更好的理解本发明，下面结合具体实施方式对本发明进一步说明。
1、花色苷纳米粒子的制备
S1、将5～30mg海藻酸钠溶解在pH 4.75～6磷酸盐缓冲盐水中，依次加入35～70mg的EDC和25～50mg的HOBt，室温中在搅拌转速500～800r/min条件下进行磁力搅拌60～80min后，加入1～5mg的所述食源性荧光纳米粒子，室温中在搅拌转速500～800r/min条件下进行磁力搅拌24～30h，再用去离子水透析24～72h并冷冻干燥，得到荧光-海藻酸复合物；
S2、透明质酸可进一步通过酯化反应共轭到藻酸盐的骨架上，使用二异丙基碳二亚胺(DIC)作为偶联剂，二甲氨基吡啶(DMAP)作为催化剂，在酯化反应过程中消除产生的水。将40～60mg所述荧光-海藻酸复合物加入到4～6ml体积比为1：1～1.5的DMF和DCM混合物中，向其中加入8～16mg的DIC和25～50mg的DMAP，在室温下搅拌1～1.5h，搅拌转速500-800r/min；
S3、再加入1～5mg花色苷和8～12mg所述透明质酸，在室温下反应24～30h，用DCM洗涤样品3～5次，并在25～40℃真空度范围0.29～1.6mbar之内，蒸发除去溶剂；花色苷可与荧光-海藻酸复合物通过静电自组装形成静电相互作用，并且花色苷本身带有酚羟基也可与海藻酸的羧基产生酯化反应；
S4、用DMF洗涤，并用DMF透析20～30小时除去未反应的小分子，然后用去离子水透析24～72h除去DMF，在-80℃预冷2～3h，在-50～-55℃、真空度35～45Pa、冷冻干燥24～72h。
2、食源性荧光纳米粒子的制备
S1、将猪五花肉切分为小块，在150～320℃条件下烤制15～40min，载浸泡在1～4L的无水乙醇中，持续搅拌在室温下12～36h，搅拌转速500～800r/min，选取过滤后的可溶性部分除去乙醇；
S2、以三氯甲烷:水＝3:1配置溶液将S1中除去乙醇的所述可溶性部分复溶，添加三氯甲烷萃取反复脱脂至油相澄清，选取澄清水相部分经过层析，将荧光部分进行-80℃预冷2～3h，在-50℃、真空度35～45Pa、冷冻干燥24～72h。
实施例1：花色苷纳米粒子的制备
S1：将海洋多糖海藻酸钠(30mg)溶解在磷酸盐缓冲盐水(pH 4.75)中，依次加入EDC(35mg)和HOBt(25mg)。室温下搅拌1h后，加入食源性荧光纳米粒子(5mg)，搅拌24h。之后，将产物用去离子水透析24h并冷冻干燥，得到荧光-海藻酸复合物。
S2：将40mg步骤S1中制备的荧光-海藻酸复合物加入到4ml按体积比1:1混合的二甲基甲酰胺DMF和DCM混合物中。随后，向其中加入DIC(8.1mg) 和DMAP(25mg)。让反应混合物在室温下温和搅拌1h，以活化海藻酸的羧基。
S3、向S2制备的混合物中缓慢加入1mg花色苷和8mg透明质酸，反应在室温下进一步进行24h。
S4、用DCM洗涤样品3次，并在25℃真空度范围0.29～1.6mbar之内，通过旋转蒸发除去溶剂。所得产物用DMF洗涤，用DMF透析24小时，除去未反应的小分子。然后用去离子水透析72h除去DMF。最后，所得样品在-80℃预冷2h，然后在-50℃、真空度40Pa、冷冻干燥48h。
实施例2：食源性荧光纳米粒子的制备
将1kg的猪五花肉均匀切分为1×1×1cm 3的小块，在温度为280℃条件下烤制30min，将烤制完成的肉浸泡在3L的无水乙醇中，持续搅拌12h，选取经过三层滤纸过滤后的可溶性部分利用旋转蒸发仪除去乙醇，再以三氯甲烷:水＝3:1配置溶液将处理好的所述可溶性部分复溶，添加三氯甲烷萃取进行反复脱脂直至油相澄清，选取澄清水相部分经过D101大孔吸附树脂柱层析，将荧光部分进行-80℃预冷2h，然后在-50℃、真空度40Pa、冷冻干燥48h。
实施例3：花色苷纳米粒子性质的表征
S1、花色苷纳米粒子的形貌及大小尺寸
图1是花色苷纳米粒子的扫描电镜(SEM)的照片，结果显示，制备的纳米粒子形态似草莓形，均匀分布，粒径大小约在30nm左右。
S2、花色苷纳米粒子的X射线光电子衍射(XRD)实验
图2是花色苷纳米粒子的XRD的图谱，显示在2θ＝21.8°处有一个很宽的中心衍射峰，在其他位置未发现波峰，这显示的是纳米粒子无定形态的特征峰。
S3、花色苷纳米粒子的紫外光谱和荧光光谱特征
图3是花色苷纳米粒子的紫外、荧光光谱。
紫外光谱峰出现在270nm处，推测为n→π*跃迁的特征吸收峰。由花色苷纳米粒子的荧光光谱可见随着波长增加出现了明显的红移现象，纳米粒子的最大激发波长出现在320nm处。
S4、花色苷纳米粒子的荧光寿命
图4是花色苷纳米粒子的荧光寿命图谱。
配置1mg/mL的花色苷纳米粒子水溶液，在320nm的激发光下激发，最大发射峰在420nm处发射，测得荧光寿命，经拟合计算烤肉纳米粒子的荧光寿命是4.54ns。
S5、花色苷纳米粒子的傅立叶变换红外光谱表征
图5是花色苷纳米粒子的FT-IR图谱。图中结果表明花色苷纳米粒子表面含有C-C、C-O、C-N、C-H等官能团组成。
S6、花色苷纳米粒子的温度稳定性实验
图6是花色苷纳米粒子的热稳定性的图谱。配置1mg/mL的花色苷纳米粒子水溶液，在55℃条件下的培养箱中进行保存，连续放置12h，中间每间隔2h监测一次。结果显示：与未封装的花色苷的热稳定性相比较，花色苷纳米粒子的热稳定性更好。
S7、花色苷纳米粒子的NaCl稳定性实验
图7是花色苷纳米粒子的NaCl稳定性的图谱。取1mg的花色苷纳米粒子冻干粉末，在不同NaCl溶液浓度(0.2M、0.4M、0.6M、0.8M和1M)下监测花色苷的保留率。图中可以观察到与未封装的花色苷的NaCl稳定性相比较，花色苷纳米粒子在NaCl溶液中稳定性更好。
S8、花色苷纳米粒子的pH稳定性实验
图8是花色苷纳米粒子的pH稳定性图谱。配置不同pH(1～6)的B-R缓冲溶液，取花色苷纳米粒子分别加入不同的pH溶液中(1mg/mL)，监测花色苷的保留率。图中可以看出在pH＝1～4左右处的极酸条件下，与未封装的花色苷相比较，花色苷纳米粒子的pH稳定性更好。
S9、花色苷纳米粒子的UV稳定性实验
图9是花色苷纳米粒子的UV稳定性的图谱。配置1mg/mL的花色苷纳米粒子水溶液，在激发光波长在365nm条件下进行紫外光照射，连续照射12h，中间每间隔2h监测一次。结果显示：与未封装的花色苷的UV稳定性相比较，花色苷纳米粒子的UV稳定性更好。
S10、花色苷纳米粒子的贮藏稳定性实验
图10是花色苷纳米粒子的贮藏稳定性的图谱。配置1mg/mL的花色苷纳米粒子水溶液，置于室温进行贮藏稳定性测试70d，每10d监测一次。图中可观察到在贮藏过程中，花色苷纳米粒子的贮藏稳定性优于未封装的花色苷。
实施例4：花色苷纳米粒子对脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的保护作用
选用RAW 264.7巨噬细胞和含有10％(V胎牛血清/V DMEM培养基＝1/9)的胎牛血清的DMEM培养基。将细胞以5×10 4/孔的密度接种于96孔板，在体积分数为5％的CO 2培养箱中孵育12h，然后在培养基中添加终浓度为40μg/mL的未封装花色苷和花色苷纳米粒子再培养12h后，每孔加入50μL的LPS (1μg/mL)反应12h后进行细胞形态学观察和贴壁细胞的原位荧光检测。
贴壁细胞的原位荧光检测：利用Annexin V-FITC和碘化丙啶(PI)染色液进行细胞染色，图中绿色荧光为Annexin V-FITC染色阳性细胞(细胞质)，红色荧光为碘化丙啶染色阳性细胞(细胞核)。仅被绿色荧光染色(有亮点处)的为凋亡细胞，被绿色和红色荧光双染(亮点较多处)的是坏死细胞，未被荧光染色(无亮点)的为正常细胞。图11是脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的荧光成像图。图12是未封装的花色苷对脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的保护作用的荧光成像图。图13是实施例1中制备的花色苷纳米粒子对脂多糖(LPS)介导的RAW 264.7巨噬细胞凋亡的保护作用的荧光成像图。图11显示了LPS对于RAW 264.7巨噬细胞具有促凋亡作用，从图12中可以观察到未封装的花色苷对于RAW 264.7巨噬细胞具有一定的保护作用，从图13中可以观察到花色苷纳米粒子对于RAW 264.7巨噬细胞的保护作用优于未封装的花色苷，可见对于花色苷封装的必要性。
实施例5：花色苷纳米粒子在小鼠体内的结肠靶向递送及缓解由DSS诱导的结肠炎症损伤
肠炎小鼠模型的构建：小鼠随机分为5组，正常对照组小鼠、DSS损伤组、不含花色苷的纳米载体组、未封装的花色苷组，花色苷纳米粒子组。
(1)正常对照组小鼠灌连续自由饮水(去离子水)12d；
(2)DSS损伤组连续自由饮水(去离子水)12d，从第7天起在饮水中添加DSS(5％，w/v)；
(3)不含花色苷的纳米载体组连续自由饮水(去离子水)12d，并且每天灌胃不含花色苷的纳米载体，剂量为10mg/kg，从第7天起在饮水中添加DSS(5％，w/v)；
(4)未封装的花色苷组连续自由饮水(去离子水)12d，并且每天灌胃未封装的花色苷，剂量为10mg/kg，从第7天起在饮水中添加DSS(5％，w/v)；
(5)花色苷纳米粒子组连续自由饮水(去离子水)12d，并且每天灌胃花色苷纳米粒子，剂量为10mg/kg，从第7天起在饮水中添加DSS(5％，w/v)。
图14是花色苷纳米粒子用于荧光标记小鼠组织和器官的分布成像伪彩图。从图中可以观察到，花色苷纳米粒子集中分布于结肠部位，可实现结肠靶向递送。图15是花色苷纳米粒子缓解由DSS诱导的结肠炎症损伤图。结果显示：花色苷纳米粒子组可有效缓解由DSS诱导结肠炎损伤，对于小鼠的结肠部位起到有效的保护作用，保护效果优于未封装的花色苷组。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种基于海洋多糖载体的花色苷纳米粒子,其特征在于，所述花色苷纳米粒子以多糖、食源性荧光纳米粒子、透明质酸钠作为复合载体与花色苷结合。
  2. 根据权利要求1所述基于海洋多糖载体的花色苷纳米粒子,其特征在于，所述多糖包括海藻酸钠。
  3. 一种权利要求1所述基于海洋多糖载体的花色苷纳米粒子的制备方法，其特征在于，包括以下步骤：
    S1、将海藻酸钠溶解在pH值为4.75～6磷酸盐缓冲盐水中，依次加入EDC和HOBt，室温下搅拌60～80min后，加入所述食源性荧光纳米粒子，继续搅拌24～30h，再用去离子水透析24～72h并冷冻干燥，得到荧光-海藻酸复合物；
    S2、将所述荧光-海藻酸复合物加入按体积比1：1～1.5混合的DMF和DCM中，再向其中加入DIC和DMAP，在室温下搅拌1～1.5h；
    S3、加入花色苷和所述透明质酸，在室温下反应24～30h，用DCM洗涤3～5次后除去溶剂；
    S4、除去未反应的小分子和DMF，在-20～-80℃预冷2～3h，在-50～-55℃、真空度35～45Pa、冷冻干燥24～72h，即得。
  4. 根据权利要求3所述基于海洋多糖载体的花色苷纳米粒子的制备方法，其特征在于，步骤S3中所述除去溶剂的方法包括蒸发，所述蒸发条件为温度25～40℃、真空度范围0.29～1.6mbar。
  5. 根据权利要求3所述基于海洋多糖载体的花色苷纳米粒子的制备方法，其特征在于，步骤S4中所述除去未反应的小分子的方法包括先用DMF洗涤再用DMF透析；所述除去DMF的方法包括用去离子水透析。
  6. 根据权利要求3所述基于海洋多糖载体的花色苷纳米粒子的制备方法，其特征在于，步骤S1、S2中所述搅拌转速500～800r/min。
  7. 根据权利要求1所述基于海洋多糖载体的花色苷纳米粒子，其特征在于，所述食源性荧光纳米粒子的制备方法，包括以下步骤：
    S1、将肉切块，在150～320℃条件下烤制15～40min，浸泡在无水乙醇中持续搅拌12～36h，选取过滤后的可溶性部分除去乙醇；
    S2、以三氯甲烷:水＝3:1配置溶液将S1中除去乙醇的所述可溶性部分复溶，添加三氯甲烷萃取反复脱脂至油相澄清，选取澄清水相部分经过层析，将荧光部分进行-20～-80℃预冷2-3h，再在-45～-55℃、真空度35～45Pa、冷冻干燥24～72h。
  8. 根据权利要求7所述食源性荧光纳米粒子的制备方法，其特征在于，步 骤S2中所述层析的色谱柱填料包括D101大孔吸附树脂。
  9. 一种基于海洋多糖载体的花色苷纳米粒子的应用，其特征在于，用于脂多糖介导的RAW264.7巨噬细胞的保护。
  10. 一种基于海洋多糖载体的花色苷纳米粒子的应用，其特征在于，用于缓解由DSS诱导结肠炎损伤。
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