WO2023193389A1 - Resveratrol-lecithin nanoparticle, method for preparing same, and use thereof - Google Patents

Resveratrol-lecithin nanoparticle, method for preparing same, and use thereof Download PDF

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WO2023193389A1
WO2023193389A1 PCT/CN2022/114615 CN2022114615W WO2023193389A1 WO 2023193389 A1 WO2023193389 A1 WO 2023193389A1 CN 2022114615 W CN2022114615 W CN 2022114615W WO 2023193389 A1 WO2023193389 A1 WO 2023193389A1
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resveratrol
lecithin
rsv
lec
nanoparticles
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姚燕丹
蔡佩娥
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中山大学孙逸仙纪念医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—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 non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • 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
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the present invention relates to the field of medical technology, and more specifically, to resveratrol lecithin nanoparticles and their preparation methods and uses.
  • Resveratrol (3,5,4-trihydroxystilbene, RSV) is a natural polyphenolic flavonoid that is rich in grapes, red wine, mulberries, peanuts, rhubarb and other plants. RSV is known to have anti-inflammatory, anti-obesity, and protective effects on the heart and brain. In neurological diseases, resveratrol has been found to reduce neuronal oxidation and apoptosis and has neuroprotective effects. Currently, a common method of systemic administration of resveratrol is intravenous injection.
  • resveratrol has poor water solubility and is chemically unstable. It will be isomerized and degraded when exposed to high temperatures, pH changes, UV light or certain types of enzymes, which is also not conducive to the realization of resveratrol. Storage and use of alcohol.
  • PEG Polyethylene glycol
  • the existing technology urgently needs a drug delivery system or corresponding preparations and drugs that can improve the storage performance and drug action performance of resveratrol and have high safety, and provide its therapeutic use in related diseases.
  • the present invention aims to overcome at least one of the deficiencies of the above-mentioned prior art, provide resveratrol lecithin nanoparticles and preparation methods and uses thereof, and overcome the difficulty of storing resveratrol, low bioavailability, and curative effect in the prior art. It has limited defects and is used in anti-oxidation and anti-cancer as a nano drug delivery system to provide alternative, effective and safe treatment strategies for corresponding diseases.
  • the technical solution adopted by the present invention is a resveratrol lecithin nanoparticle, which includes resveratrol and lecithin wrapped and/or coupled to resveratrol.
  • Resveratrol lecithin nanoparticles are hereinafter referred to as Lec(RSV).
  • the lecithin is a liposome extracted from soybeans.
  • the present invention uses lecithin and resveratrol to form a completely natural nano-delivery drug delivery and treatment system with high safety. And the use of nano-preparations can make the shelf life of resveratrol longer. In more than one embodiment of the present invention, it can be stably maintained at room temperature and 4°C for twelve months.
  • resveratrol and lecithin are also beneficial to improving the drug delivery effect.
  • resveratrol is sustained-released in the nanoparticles, which can overcome the problem of resveratrol in the prior art.
  • the defect of low bioavailability of intravenous injection of retitol increases the duration of action in the body; on the other hand, in more than one embodiment of the present invention, the resveratrol lecithin nanoparticles provided by the application can significantly improve the antioxidant and anti-oxidation effects.
  • resveratrol lecithin nanoparticles achieve a synergistic effect between the two, and the effect is significant.
  • the resveratrol lecithin nanoparticles provided by the present invention can overcome the shortcomings of the existing technology, improve the storage performance and bioavailability of resveratrol, and provide anti-oxidation and anti-cancer uses, which is a new addition to the existing technology.
  • Antioxidant diseases and cancers need to provide new treatment strategies and drugs, especially for cancer treatment, which can provide an alternative, cost-effective, low-side-effect anti-cancer therapy.
  • the resveratrol is wrapped between the inner and outer layers of lecithin.
  • the mass ratio of resveratrol and lecithin is (9-11):1.
  • the mass ratio of resveratrol to lecithin is 10:1.
  • the particle size range of the nanoparticles is 128 to 174 nm. Nanoparticles with this particle size are not only stable, but also have excellent loading capacity and good tumor penetration ability. As an anti-cancer drug with potential for clinical application, stability plays a crucial role in its storage and delivery capabilities.
  • the size of nanoparticles significantly affects their therapeutic and diagnostic applications. Treatment aspects, including drug half-life, targeting ability, cellular uptake and tumor penetration, are all affected by the size of nanoparticles; nanoparticles must be larger than 10nm in order to Avoid renal filtration, but the diameter should not be too large. For example, particles larger than 200nm will activate the complement system and be quickly removed from the blood and accumulate in the liver and spleen.
  • the particle size of nanoparticles will directly affect the effect of nanoparticles, and being too large or too small is not conducive to exerting its effect.
  • the particle size range of the resveratrol lecithin nanoparticles is 128-174nm, and the average particle size is 151nm, which can appropriately prevent the nanoparticles from being too small or too large, while ensuring their load. Abilities and effects.
  • the above-mentioned resveratrol lecithin nanoparticles are prepared and formed by a nanoprecipitation method.
  • Another object of the present invention is to provide the use of the above-mentioned resveratrol lecithin nanoparticles in the preparation of drugs for scavenging free radicals and/or killing tumor cells.
  • the resveratrol lecithin nanoparticles described in this application have a significant killing effect on tumor cells. Compared with resveratrol directly applied to tumor cells, resveratrol Alcohol lecithin nanoparticles have a synergistic killing effect and the killing ability is more significant.
  • tumor cells include breast cancer cells.
  • the tumor cells used are breast cancer cells.
  • Another object of the present invention is to provide a tumor cell killing drug, including the above-mentioned resveratrol lecithin nanoparticles.
  • Another object of the present invention is to provide a method for preparing the above-mentioned resveratrol lecithin nanoparticles, which includes the steps:
  • A1 Dissolve resveratrol and lecithin in an organic solvent miscible with water to obtain a resveratrol organic phase and a lecithin organic phase. Add the lecithin organic phase to the resveratrol organic phase. Then add organic solvent to obtain a mixed solution;
  • the organic solvent is dimethyl sulfoxide.
  • the mass ratio of resveratrol and lecithin in the mixed solution is (9-11):1;
  • the concentration of resveratrol in the mixed solution is 0.9-1.1 mg/ml
  • volume ratio of the mixed solution to the water phase is (0.05-0.15):10.
  • volume ratio of the mixed liquid to the water phase is 0.1:10.
  • step A3 the nanosuspension is added to the Amincon filter and centrifuged to remove the organic solvent.
  • step A3 includes:
  • A31 Centrifuge the nanosuspension for the first time to separate the organic solvent
  • step A33 Repeat step A32 two or more times to obtain resveratrol lecithin nanoparticles.
  • Another object of the present invention is to provide the use of resveratrol and/or lecithin in the preparation of drugs for scavenging free radicals and/or killing tumor cells.
  • resveratrol or lecithin alone also has certain free radical scavenging and tumor killing effects, and resveratrol lecithin nanoparticles containing resveratrol and lecithin simultaneously Have more significant synergistic effects.
  • the beneficial effects of the present invention are: the components of the nanoparticles in this application are of natural origin and are safe, and the nano-delivery system formed by resveratrol and lecithin has sustained-release characteristics, which is not only convenient for It improves the bioavailability of resveratrol in the body and enhances the duration of its action in the body. Moreover, the lecithin coating also facilitates the storage of the main drug resveratrol when it is not in use. More importantly, the formed resveratrol lecithin nanoparticles have significant effects in tumor killing.
  • the nanodelivery system facilitates the improvement of resveratrol bioavailability and promotes the targeting of tumor tissues, improving tumor cell Killing effect.
  • the nanoparticles formed by resveratrol and lecithin also have a synergistic killing effect, and the anti-tumor and antioxidant effects are more significant. They are expected to be used in tumor treatment to provide new therapeutic drugs and treatment strategies. , to delay the survival of cancer patients.
  • Lec(RSV) directly killing tumor cells Lec(RSV) also has more stable properties and is easy to store, overcoming the storage problems caused by the instability of resveratrol in the existing technology. question.
  • Figure 1 shows the Lec(RSV) synthesis schematic and characteristics.
  • A Schematic diagram of using lecithin and resveratrol to form Lec(RSV) nanoparticles;
  • B Nanoprecipitation method to obtain soluble Lec(RSV).
  • Figure 2 shows the sizes of empty Lec nanoparticles and Lec(RSV) nanoparticles.
  • A Transmission electron microscopy analysis of empty Lec nanoparticles;
  • B Transmission electron microscopy analysis of Lec(RSV) nanoparticles;
  • C The average size of the nanoparticles is (Lec(RSV 151.0 ⁇ 22.93nm vs. empty Lec 128.4 ⁇ 19.88nm) );
  • D RSV encapsulation effect.
  • Figure 3 shows the stability of Lec and Lec(RSV) nanoparticles.
  • A The particle size of lecithin nanoparticles and Lec(RSV) nanoparticles does not change significantly within 48 hours at room temperature;
  • B The particle size of lecithin nanoparticles and Lec(RSV) nanoparticles within 60 days at 4°C There is no obvious change;
  • C Lec(RSV) nanoparticles were placed for 3, 6, 9, and 12 months respectively, and no obvious turbidity and sediment were found;
  • D In vitro release test of Lec(RSV), the accumulation within 12 hours Release 55%.
  • Figure 4 shows the in vitro absorption capacity and cytotoxicity of Lec and Lec(RSV) nanoparticles.
  • A-B BT474 breast cancer cells take up Lec and Lec(RSV) wrapped with FITC;
  • C In vitro cytotoxicity test to observe the killing ability of Lec, RSV and Lec(RSV) against cancer cells respectively;
  • D Lec, RSV and ROS scavenging ability of Lec(RSV).
  • Figure 5 shows the effect of Lec(RSV) on tumor uptake in vivo.
  • the BT474 tumor-bearing model was used to conduct in vivo tumor uptake experiments.
  • test samples and test processes used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions, or in accordance with the conditions recommended by the reagent company; the conditions used in the following examples Reagents, consumables, etc., can be obtained from commercial sources unless otherwise specified).
  • Resveratrol purchased from Aladdin (Shanghai, China); lecithin, purchased from Sigma Aldrich (St. Louis, MO, USA), FITC, purchased from Sigma Aldrich (St. Louis, MO, USA), Alma Blue kit, purchased from Organic solvents (DMF, DMSO, 75% alcohol) were purchased from Sigma Aldrich (St. Louis, Missouri, USA).
  • the experimental water was treated with Millipore and Milli-Q systems. The remaining substances were All are using analysis level.
  • Lec(RSV) was prepared using nanoprecipitation method.
  • DMSO was used as the solvent to prepare resveratrol (RSV) (100 mg/mL) and lecithin (Lec) (100 mg/mL) solutions.
  • RSV resveratrol
  • Lec lecithin
  • Take a clean glass vial add 10 mL of ultrafiltered water, and stir continuously at 12,000 rpm at room temperature. The mixed solution was slowly added into the vial and the nanosuspension was obtained after stirring for five minutes.
  • the solution was transferred to an Amicon filter (Merck, Kenilworth, NJ, USA) and centrifuged at 4000 rpm for 15 minutes. The remaining solution was washed with 5 ml of water and centrifuged again at 4000 rpm for 10 min; repeat twice to remove any remaining DMSO. Lec(RSV) was stored at 4°C until further use.
  • the mass ratio of resveratrol to lecithin in the mixed solution is 10:1
  • the concentration of resveratrol in the mixed solution is 1 mg/ml
  • the volume ratio of the mixed solution to the water phase is 0.1:10.
  • the particle size and zeta potential of Lec and Lec(RSV) particles were determined by dynamic light scattering (DLS) analysis at Malvern Panalytical (MA, USA). Each sample data comes from 3 repeated experiments.
  • Lec(RSV) (10mg/mL) was dropped on the TEM grade carbon mesh copper grid. Place the pellets on the grid and leave at ambient temperature for 5 minutes. Wash each compartment 5 times with distilled water. Specimens were then negatively stained with 2% uranyl acetate and left at ambient temperature for 2 minutes. Then rinse with distilled water three times and air dry. Specimens were observed with a TECNAI F20 electron microscope (Philips Electronic Instruments Corp, Mahwah, 103NJ).
  • the encapsulated amount of RSV in Lec was determined using UV-visible spectrophotometry. After Lec(RSV) purification, samples were rehydrated in PBS to a volume of 1 ml. Take 10 ⁇ l of Lec(RSV) sample and add it to 90 ⁇ l DMSO. Take 10 ⁇ l of the original resveratrol-liposome working solution as a positive control group. RSV has specific UV absorbance at 330nm, which can be used to determine RSV concentration. In order to determine the encapsulation efficiency (EE) of RSV, the inventors calculated the encapsulation amount of RSV in the Lec(RSV) conjugate compared with the encapsulation amount of RSV initially used, and obtained the following formula:
  • Lec and Lec(RSV) were synthesized at a concentration of 10 mg/mL. Lec and Lec(RSV) particles were then stored in closed vials at 37°C. At preset time points (1, 2, 4, 8, 12, 24, 48 h), the DLS method was used to determine the Lec and Lec(RSV) particle sizes and record them.
  • Human breast cancer cells BT474 were purchased from ATCC and cultured according to the provided procedure. The temperature is 37°C. Use RPMI-1640/F-12K medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 g/mL streptomycin, and culture in a humidified cell culture room with 5% CO2.
  • FBS fetal bovine serum
  • Lec was fluorescently labeled with FITC at half the mass of the liposomes.
  • BT474 cells were grown on glass coverslips (12 ⁇ 12 mm; Fisher Scientific, Texas, USA) to ⁇ 80%. Before adding 200 g/mL Lec (FITC)-containing medium, the cells were incubated with serum-free medium for 1 hour, washed twice with PBS, and fixed with 4% (w/v) paraformaldehyde (PFA). Fixed cells were mounted on glass slides containing Dako mounting medium and detected using an Olympus Fluoview 1000 confocal microscope (Olympus Imaging Co, Tokyo, Japan).
  • Lec(RSV) was synthesized as a synergistic anti-tumor therapeutic platform ( Figure 1).
  • Resveratrol is soluble in organic solvents but not in water or buffers, a property that greatly limits the bioavailability of resveratrol.
  • Figure 1B (left) simply mixing lecithin with resveratrol does not automatically encapsulate RSV and form nanoparticles. Only through the nanoprecipitation method described above, Lec(RSV) nanoparticles can be prepared, and a transparent Lec(RSV) solution can be observed, as shown in Figure 1B (right).
  • nanoparticles significantly affects their therapeutic and diagnostic applications; therapeutic aspects including drug half-life, targeting ability, cellular uptake and tumor penetration are all affected by the size of nanoparticles; nanoparticles must be larger than 10nm to Avoid renal filtration, but the diameter should not be too large. For example, particles larger than 200nm will activate the complement system and be quickly removed from the blood and accumulate in the liver and spleen. Therefore, only nanoparticles of appropriate size can achieve the technical effect to be achieved by this application.
  • the particle size range of Lec(RSV) nanoparticles is 128 ⁇ 174nm, and the average size is 151nm, which is close to the ideal size and stable. , large load capacity, and good tumor penetration ability.
  • Lec(RSV) The stability of nanoparticles in nanodelivery systems is critical because nanoparticles need to circulate in the body for long periods of time. Both Lec and Lec(RSV) nanoparticles were stable for 48 hours.
  • Lec(RSV) the inventor measured the size of Lec(RSV) particles on days 1/4/8/5/30/60. As shown in Figure 3B, the size of Lec(RSV) was measured under DLS. Within 60 days, the nanometer size fluctuated very little, ranging from 100 to 200 nanometers, proving that Lec(RSV) has good stability.
  • Lec(RSV) can be stored at 4°C for a long time, proving that this nanosystem has good stability. Furthermore, RSV can achieve slow release after being wrapped by Lec, with only 55% of the cumulative release amount within 12 hours.
  • stability plays a crucial role in its storage and delivery capabilities.
  • the nanoparticle delivery system requires nanoparticles to circulate in the body for a long time, so the stability of Lec(RSV) plays a key role.
  • Lec(RSV) size was measured using DSL and found only slight size changes over 60 days. It is proved that Lec(RSV) has good stability. Lec(RSV) can be stored for 12 months at 4°C without aggregation or coagulation, which also shows that the nanoplatform has good stability. Combined with the aforementioned characteristics of Lec(RSV), Lec(RSV) shows greater advantages than other drugs or nano-delivery systems, and also has the potential for clinical application to treat related diseases.
  • Lec(RSV) was encapsulated into Lec and Lec(RSV) particles, and they were added to BT474 cells and cultured for 4 hours. After 4 hours of co-incubation, both Lec and Lec(RSV) showed significant uptake into the cytoplasm ( Figures 4A and 4B).
  • RSV treatment was highly toxic to cells (IC50 ⁇ 18 ⁇ M). As an antioxidant, Lec also has inherent tumor killing ability, but it is not as significant as RSV (IC50>40 ⁇ M).
  • Lec compared with using Lec or RSV alone, Lec (RSV) has a significant synergistic effect on the killing ability of tumor cells (IC50 ⁇ 11 ⁇ M). Similarly, compared with the control group or Lec, RSV had a significant scavenging effect on ROS (p ⁇ 0.001), and encapsulating RSV did not affect the biological activity of RSV (Figure 4D).
  • RSV As an ideal anti-cancer drug, in addition to the aforementioned particle size and stability, another important indicator is tumor cell enrichment ability and cytotoxicity.
  • the inventors used natural lecithin to encapsulate resveratrol to form nanoparticles, and tested its cellular uptake and cytotoxicity on BT474 breast cancer cells in vitro. After 4 hours of co-culture, both Lec and Lec(RSV) were obviously absorbed into the cytoplasm of the cells. Then, the inventors conducted an in vitro cytotoxicity test to observe the killing ability of RSV on tumor cells, and found that RSV (IC 50 , 11 ⁇ M) has obvious toxicity to cells. As an antioxidant, Lec also has a certain tumor-killing ability, but it is not as obvious as RSV (IC 50 , 40 ⁇ M).
  • Lec and RSV when Lec and RSV are combined to form nanoparticles, they have better tumor killing effect than Lec alone or RSV alone. The same is true in terms of antioxidants.
  • Lec(RSV) has a synergistic effect between components and has a significant effect.
  • encapsulating RSV will not change its biological activity, which further demonstrates the stability of the nanoparticles of the present application for encapsulating the main drug.
  • Lec(RSV) has excellent stability and biocompatibility, has an inhibitory effect on BT474 breast cancer cells, has less cytotoxicity, and is safe. Lec(RSV) is expected to be used clinically to become an effective clinical anti-tumor drug.
  • the inventor in the process of preparing resveratrol lecithin nanoparticles using nanoprecipitation method, the inventor also used DMSO to prepare resveratrol (RSV) (100 mg/mL) and lecithin (Lec) (100 mg/mL). ) solution, and add 9 ⁇ L or 11 ⁇ L of Lec to 1 ⁇ L of RSV, add DMSO to 100 ⁇ L, and use the same steps to prepare the white quinoa under the condition that the volume ratio of the mixed solution to the water phase is (0.05 ⁇ 0.15):10. Retinol lecithin nanoparticles.
  • RSV resveratrol
  • Lec lecithin

Abstract

Provided is a resveratrol-lecithin nanoparticle comprising resveratrol and lecithin encapsulating and/or conjugated to resveratrol. The resveratrol-lecithin nanoparticle features safety and sustained release, and can improve the in-vivo bioavailability of resveratrol, enhance the time of in-vivo exposure, promote the targeting performance to tumor tissues, and improve the killing effect on tumor cells, thus having more significant anti-tumor and antioxidant effects.

Description

一种白藜芦醇卵磷脂纳米粒及其制备方法、用途Resveratrol lecithin nanoparticles and preparation method and use thereof 技术领域Technical field
本发明涉及医药技术领域，更具体地，涉及一种白藜芦醇卵磷脂纳米粒及其制备方法、用途。The present invention relates to the field of medical technology, and more specifically, to resveratrol lecithin nanoparticles and their preparation methods and uses.
背景技术Background technique
白藜芦醇(3，5，4-三羟基二苯乙烯，RSV)是一种天然的多酚类黄酮，富含于葡萄、红酒、桑葚、花生、大黄等植物中。RSV已知具有抗炎、抗肥胖和保护心脏、大脑的作用。在神经***疾病中，发现白藜芦醇具有减少神经元氧化及凋亡的作用，有神经保护作用。目前，白藜芦醇的一种常见***给药方式是静脉注射，然而，白藜芦醇进入体内后的低生物利用度限制了白藜芦醇对相应病症的治疗效果；如，RSV在肝脏和肠道上皮细胞中迅速(在2小时内)代谢为葡萄糖醛酸和硫酸酚基结合物，然后被消除。因此，RSV的低生物利用度限制了其生物和药理作用，治疗效果不显著。另一方面，白藜芦醇水溶性差，在化学上不稳定，当暴露于高温、pH值变化、紫外光或某些类型的酶时，会被异构化降解，同样不利于实现白藜芦醇的保持和使用。Resveratrol (3,5,4-trihydroxystilbene, RSV) is a natural polyphenolic flavonoid that is rich in grapes, red wine, mulberries, peanuts, rhubarb and other plants. RSV is known to have anti-inflammatory, anti-obesity, and protective effects on the heart and brain. In neurological diseases, resveratrol has been found to reduce neuronal oxidation and apoptosis and has neuroprotective effects. Currently, a common method of systemic administration of resveratrol is intravenous injection. However, the low bioavailability of resveratrol after entering the body limits the therapeutic effect of resveratrol on corresponding diseases; for example, RSV in the liver and rapidly (within 2 hours) metabolized in intestinal epithelial cells to glucuronic acid and phenolic sulfate conjugates, which are then eliminated. Therefore, the low bioavailability of RSV limits its biological and pharmacological effects, and the therapeutic effect is not significant. On the other hand, resveratrol has poor water solubility and is chemically unstable. It will be isomerized and degraded when exposed to high temperatures, pH changes, UV light or certain types of enzymes, which is also not conducive to the realization of resveratrol. Storage and use of alcohol.
现有技术中，纳米传递***常用于携带并实现药物的***传递，通常采用聚乙烯乙二醇(PEG)封装小分子药物为纳米大小的球形颗粒，再通过该球形颗粒进行小分子药物的给药。通过该方式虽然能一定程度上提高封装在内的小分子药物体内存在持久性，但是，该类方式存在一定的安全隐患。具体的，PEG是一种广泛用于化妆品的石油基化合物，例如作为化妆品的增稠剂、溶剂、软化剂、保湿载体等，同时也作为泻药使用，但有研究提示(如，Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compounds；Low molecular weight polyethylene glycol induces chromosome aberrations in Chinese hamster cells cultured in vitro.Mutagenesis)，PEG复合物及产生的污染废料有基因毒性，可以在某些情况下引起基因突变或***中毒，即采用PEG为主体的纳米传递***安全性不能得到保障。In the existing technology, nano-delivery systems are often used to carry and achieve systemic delivery of drugs. Polyethylene glycol (PEG) is usually used to encapsulate small molecule drugs into nano-sized spherical particles, and then the small molecule drugs are administered through the spherical particles. medicine. Although this method can improve the persistence of encapsulated small molecule drugs in the body to a certain extent, there are certain safety risks in this method. Specifically, PEG is a petroleum-based compound widely used in cosmetics, such as as a thickener, solvent, softener, moisturizing carrier, etc. in cosmetics. It is also used as a laxative, but there are studies suggesting that (e.g., Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compounds; Low molecular weight polyethylene glycol induces chromosome aberrations in Chinese hamster cells cultured in vitro.Mutagenesis), PEG complexes and the polluted waste produced are genotoxic and can cause gene mutations or system poisoning in some cases , that is, the safety of nano-delivery systems using PEG as the main body cannot be guaranteed.
因此，现有技术亟需一种能提高白藜芦醇存储性能、药物作用性能且具有高安全性的给药***或相应制剂、药物，并提供其在相关病症上的治疗用途等。Therefore, the existing technology urgently needs a drug delivery system or corresponding preparations and drugs that can improve the storage performance and drug action performance of resveratrol and have high safety, and provide its therapeutic use in related diseases.
发明内容Contents of the invention
本发明旨在克服上述现有技术的至少一种不足，提供一种白藜芦醇卵磷脂纳米粒及其制备方法、用途，克服现有技术白藜芦醇不易存储、生物利用度低、疗效有限的缺陷，并用于抗氧化、抗癌中，作为纳米给药传递***以提供相应病症上替代的、有效的、安全的治疗策略。The present invention aims to overcome at least one of the deficiencies of the above-mentioned prior art, provide resveratrol lecithin nanoparticles and preparation methods and uses thereof, and overcome the difficulty of storing resveratrol, low bioavailability, and curative effect in the prior art. It has limited defects and is used in anti-oxidation and anti-cancer as a nano drug delivery system to provide alternative, effective and safe treatment strategies for corresponding diseases.
本发明采取的技术方案是，一种白藜芦醇卵磷脂纳米粒，包括白藜芦醇以及包裹和/或偶联于白藜芦醇上的卵磷脂。以下简称白藜芦醇卵磷脂纳米粒为Lec(RSV)。所述卵磷脂是从大豆中提取的一种脂质体，本发明采用卵磷脂与白藜芦醇配合，能形成一个完全天然的纳米传递给药、治疗***，安全性高。且使用纳米制剂，可使得白藜芦醇的保质期更长，在本发明的一个以上实施例中，室温中及4℃下能稳定保持十二个月。白藜芦醇与卵磷脂形成的纳米粒还有利于提高给药效果，一方面在本发明的一个以上实施例中，白藜芦醇在纳米颗粒中缓释，能够克服现有技术中白藜芦醇静脉注射生物利用度低的缺陷，增加体内作用时长；另一方面，在本发明的一个以上实施例中，通过本申请提供的白藜芦醇卵磷脂纳米粒能够显著提高抗氧化、抗癌治疗效果，且相比于单独白藜芦醇或卵磷脂自身的抗氧化、抗癌效果，白藜芦醇卵磷脂纳米粒实现了两者的协同作用，作用效果显著。本发明提供的白藜芦醇卵磷脂纳米粒，能够克服现有技术的不足，提高白藜芦醇存储性能和生物利用度，并提供了在抗氧化、抗癌上的用途，为现有技术需抗氧化疾病、癌症提供新的治疗策略和药物，尤其体现于癌症治疗，能提供一种替代的、经济有效的、低副作用的抗癌疗法。The technical solution adopted by the present invention is a resveratrol lecithin nanoparticle, which includes resveratrol and lecithin wrapped and/or coupled to resveratrol. Resveratrol lecithin nanoparticles are hereinafter referred to as Lec(RSV). The lecithin is a liposome extracted from soybeans. The present invention uses lecithin and resveratrol to form a completely natural nano-delivery drug delivery and treatment system with high safety. And the use of nano-preparations can make the shelf life of resveratrol longer. In more than one embodiment of the present invention, it can be stably maintained at room temperature and 4°C for twelve months. The nanoparticles formed by resveratrol and lecithin are also beneficial to improving the drug delivery effect. On the one hand, in more than one embodiment of the present invention, resveratrol is sustained-released in the nanoparticles, which can overcome the problem of resveratrol in the prior art. The defect of low bioavailability of intravenous injection of retitol increases the duration of action in the body; on the other hand, in more than one embodiment of the present invention, the resveratrol lecithin nanoparticles provided by the application can significantly improve the antioxidant and anti-oxidation effects. Cancer treatment effect, and compared with the antioxidant and anti-cancer effects of resveratrol or lecithin alone, resveratrol lecithin nanoparticles achieve a synergistic effect between the two, and the effect is significant. The resveratrol lecithin nanoparticles provided by the present invention can overcome the shortcomings of the existing technology, improve the storage performance and bioavailability of resveratrol, and provide anti-oxidation and anti-cancer uses, which is a new addition to the existing technology. Antioxidant diseases and cancers need to provide new treatment strategies and drugs, especially for cancer treatment, which can provide an alternative, cost-effective, low-side-effect anti-cancer therapy.
进一步地，包括内外层卵磷脂，所述白藜芦醇包裹于内外层卵磷脂之间。Further, it includes inner and outer layers of lecithin, and the resveratrol is wrapped between the inner and outer layers of lecithin.
进一步地，白藜芦醇与卵磷脂的质量比为(9～11)∶1。Further, the mass ratio of resveratrol and lecithin is (9-11):1.
进一步地，白藜芦醇与卵磷脂的质量比为10∶1。Further, the mass ratio of resveratrol to lecithin is 10:1.
进一步地，纳米粒子粒径范围为128～174nm。该粒径下的纳米粒子不仅稳定，且具有优秀的负载能力，良好的肿瘤穿透能力。而作为一种有望临床应用的抗癌药物，稳定性对其储存及传递能力起至关重要的作用。纳米颗粒的大小显著影响其治疗和诊断的应用，包括药物半衰期、靶向能力、细胞摄取和肿瘤穿透性在内的等等治疗方面都受到纳米颗粒大小的影响；纳米颗粒必须大于10nm，以避免肾过滤作用，但直径又不能过大，如：大于200nm的颗粒会激活补体***，并迅速从血液中被移除，在肝脏和脾脏中积累。因此，纳米粒的粒径会直接影响到纳米粒作用效果，且过大或者过小均不利于发挥其效果。而本发明中，所述白 藜芦醇卵磷脂纳米粒的粒径范围为128～174nm，平均粒径更是为151nm，能够适当的避免纳米粒过小或过大，同时又保障了其负载能力和作用效果。Further, the particle size range of the nanoparticles is 128 to 174 nm. Nanoparticles with this particle size are not only stable, but also have excellent loading capacity and good tumor penetration ability. As an anti-cancer drug with potential for clinical application, stability plays a crucial role in its storage and delivery capabilities. The size of nanoparticles significantly affects their therapeutic and diagnostic applications. Treatment aspects, including drug half-life, targeting ability, cellular uptake and tumor penetration, are all affected by the size of nanoparticles; nanoparticles must be larger than 10nm in order to Avoid renal filtration, but the diameter should not be too large. For example, particles larger than 200nm will activate the complement system and be quickly removed from the blood and accumulate in the liver and spleen. Therefore, the particle size of nanoparticles will directly affect the effect of nanoparticles, and being too large or too small is not conducive to exerting its effect. In the present invention, the particle size range of the resveratrol lecithin nanoparticles is 128-174nm, and the average particle size is 151nm, which can appropriately prevent the nanoparticles from being too small or too large, while ensuring their load. Abilities and effects.
进一步地，通过纳米沉淀法制备获得。在本发明的一个实施例中，通过纳米沉淀法制备形成上述白藜芦醇卵磷脂纳米粒。Further, it was prepared by nanoprecipitation method. In one embodiment of the present invention, the above-mentioned resveratrol lecithin nanoparticles are prepared and formed by a nanoprecipitation method.
本发明的另一目的在于提供一种上述白藜芦醇卵磷脂纳米粒在制备清除自由基和/或杀伤肿瘤细胞的药物中的用途。在本发明的一个以上实施例中，本申请所述的白藜芦醇卵磷脂纳米粒，对肿瘤细胞具有显著的杀伤作用，相比于直接施加于肿瘤细胞的白藜芦醇，白藜芦醇卵磷脂纳米粒具有协同杀伤效果，杀伤能力更为显著。Another object of the present invention is to provide the use of the above-mentioned resveratrol lecithin nanoparticles in the preparation of drugs for scavenging free radicals and/or killing tumor cells. In more than one embodiment of the present invention, the resveratrol lecithin nanoparticles described in this application have a significant killing effect on tumor cells. Compared with resveratrol directly applied to tumor cells, resveratrol Alcohol lecithin nanoparticles have a synergistic killing effect and the killing ability is more significant.
进一步地，肿瘤细胞包括乳腺癌细胞。在本发明的一个以上实施例中，采用的肿瘤细胞为乳腺癌细胞。Further, tumor cells include breast cancer cells. In more than one embodiment of the invention, the tumor cells used are breast cancer cells.
本发明的再一目的在于提供一种肿瘤细胞杀伤药物，包括上述白藜芦醇卵磷脂纳米粒。Another object of the present invention is to provide a tumor cell killing drug, including the above-mentioned resveratrol lecithin nanoparticles.
进一步地，还包括药学上可接受的辅料。Furthermore, pharmaceutically acceptable excipients are also included.
本发明的再一目的在于提供上述白藜芦醇卵磷脂纳米粒的制备方法，包括步骤：Another object of the present invention is to provide a method for preparing the above-mentioned resveratrol lecithin nanoparticles, which includes the steps:
A1、将白藜芦醇、卵磷脂分别溶于能与水混溶的有机溶剂中获得白藜芦醇有机相、卵磷脂有机相，将卵磷脂有机相加至白藜芦醇有机相中，然后补充有机溶剂，获得混合液；A1. Dissolve resveratrol and lecithin in an organic solvent miscible with water to obtain a resveratrol organic phase and a lecithin organic phase. Add the lecithin organic phase to the resveratrol organic phase. Then add organic solvent to obtain a mixed solution;
A2、搅拌条件下将混合液加入至水相中，获得纳米混悬液；A2. Add the mixed solution to the water phase under stirring conditions to obtain a nanosuspension;
A3、离心过滤去除有机溶剂，获得白藜芦醇卵磷脂纳米粒。A3. Remove the organic solvent by centrifugal filtration to obtain resveratrol lecithin nanoparticles.
进一步地，有机溶剂为二甲基亚砜。Further, the organic solvent is dimethyl sulfoxide.
进一步地，混合液中白藜芦醇与卵磷脂的质量比为(9～11)∶1；Further, the mass ratio of resveratrol and lecithin in the mixed solution is (9-11):1;
进一步地，混合液中白藜芦醇浓度为0.9～1.1mg/ml；Further, the concentration of resveratrol in the mixed solution is 0.9-1.1 mg/ml;
进一步地，混合液与水相的体积比为(0.05～0.15)∶10。Further, the volume ratio of the mixed solution to the water phase is (0.05-0.15):10.
进一步地，混合液与水相的体积比为0.1∶10。Further, the volume ratio of the mixed liquid to the water phase is 0.1:10.
进一步地，步骤A3中将纳米混悬液加入至Amincon过滤器离心去除有机溶剂。Further, in step A3, the nanosuspension is added to the Amincon filter and centrifuged to remove the organic solvent.
进一步地，步骤A3包括：Further, step A3 includes:
A31、将纳米混悬液进行初次离心，初步离心分离出有机溶剂；A31. Centrifuge the nanosuspension for the first time to separate the organic solvent;
A32、洗涤剩余有机溶剂，再次离心，去除剩余有机溶剂；A32. Wash the remaining organic solvent and centrifuge again to remove the remaining organic solvent;
A33、重复步骤A32两次以上，获得白藜芦醇卵磷脂纳米粒。A33. Repeat step A32 two or more times to obtain resveratrol lecithin nanoparticles.
本发明的再一目的在于提供白藜芦醇和/或卵磷脂在制备清除自由基和/或杀伤肿瘤细胞的药物中的用途。在本发明的一个以上实施例中，单独的白藜芦醇或卵磷脂也具有一定的 自由基清除、肿瘤杀伤作用，而同时包含白藜芦醇、卵磷脂的白藜芦醇卵磷脂纳米粒具有更为显著的协同作用。Another object of the present invention is to provide the use of resveratrol and/or lecithin in the preparation of drugs for scavenging free radicals and/or killing tumor cells. In more than one embodiment of the present invention, resveratrol or lecithin alone also has certain free radical scavenging and tumor killing effects, and resveratrol lecithin nanoparticles containing resveratrol and lecithin simultaneously Have more significant synergistic effects.
与现有技术相比，本发明的有益效果为：本申请中纳米粒的组分来源天然，具有安全性，且白藜芦醇与卵磷脂形成的纳米传递***，具有缓释特性，不仅便于提高白藜芦醇的体内生物利用度，增强体内作用时长，且卵磷脂的包裹还利于主药白藜芦醇在非使用状态时的存放。更重要的是，形成的白藜芦醇卵磷脂纳米粒在肿瘤杀伤方面具有显著效果，一方面该纳米传递***便于提高白藜芦醇生物利用度并促进对肿瘤组织的靶向，提高肿瘤细胞杀伤效果，另一方面，所述白藜芦醇与卵磷脂形成的纳米粒还具有协同杀伤效果，抗肿瘤、抗氧化效果更为显著，有望应用于肿瘤治疗中提供新的治疗药物、治疗策略，以延缓肿瘤患者生存期。此外，除了Lec(RSV)直接对肿瘤细胞杀伤而所体现的特性外，Lec(RSV)还具有更为稳定的性质，便于存放，克服现有技术中白藜芦醇不稳定而导致的存放等问题。Compared with the existing technology, the beneficial effects of the present invention are: the components of the nanoparticles in this application are of natural origin and are safe, and the nano-delivery system formed by resveratrol and lecithin has sustained-release characteristics, which is not only convenient for It improves the bioavailability of resveratrol in the body and enhances the duration of its action in the body. Moreover, the lecithin coating also facilitates the storage of the main drug resveratrol when it is not in use. More importantly, the formed resveratrol lecithin nanoparticles have significant effects in tumor killing. On the one hand, the nanodelivery system facilitates the improvement of resveratrol bioavailability and promotes the targeting of tumor tissues, improving tumor cell Killing effect. On the other hand, the nanoparticles formed by resveratrol and lecithin also have a synergistic killing effect, and the anti-tumor and antioxidant effects are more significant. They are expected to be used in tumor treatment to provide new therapeutic drugs and treatment strategies. , to delay the survival of cancer patients. In addition, in addition to the characteristics of Lec(RSV) directly killing tumor cells, Lec(RSV) also has more stable properties and is easy to store, overcoming the storage problems caused by the instability of resveratrol in the existing technology. question.
附图说明Description of the drawings
图1显示Lec(RSV)合成原理图及特点。(A)利用卵磷脂和白藜芦醇形成Lec(RSV)纳米颗粒的示意图；(B)纳米沉淀法获得可溶性Lec(RSV)。Figure 1 shows the Lec(RSV) synthesis schematic and characteristics. (A) Schematic diagram of using lecithin and resveratrol to form Lec(RSV) nanoparticles; (B) Nanoprecipitation method to obtain soluble Lec(RSV).
图2显示空Lec纳米颗粒和Lec(RSV)纳米颗粒的大小。(A)空Lec纳米颗粒的透射电镜分析；(B)Lec(RSV)纳米粒子的透射电镜分析；(C)纳米粒子的平均尺寸为(Lec(RSV 151.0±22.93nm vs空Lec 128.4±19.88nm)；(D)RSV的包封效果。Figure 2 shows the sizes of empty Lec nanoparticles and Lec(RSV) nanoparticles. (A) Transmission electron microscopy analysis of empty Lec nanoparticles; (B) Transmission electron microscopy analysis of Lec(RSV) nanoparticles; (C) The average size of the nanoparticles is (Lec(RSV 151.0±22.93nm vs. empty Lec 128.4±19.88nm) ); (D) RSV encapsulation effect.
图3显示Lec和Lec(RSV)纳米颗粒的稳定性。(A)室温下卵磷脂纳米颗粒及Lec(RSV)纳米颗粒48小时内的粒径大小没有明显变化；(B)在4℃下卵磷脂纳米颗粒及Lec(RSV)纳在60天内粒径大小没有明显变化；(C)分别放置3、6、9、12个月的Lec(RSV)纳米颗粒，未见明显浑浊及沉积物；(D)Lec(RSV)的体外释放试验，12小时内累计释放55％。Figure 3 shows the stability of Lec and Lec(RSV) nanoparticles. (A) The particle size of lecithin nanoparticles and Lec(RSV) nanoparticles does not change significantly within 48 hours at room temperature; (B) The particle size of lecithin nanoparticles and Lec(RSV) nanoparticles within 60 days at 4°C There is no obvious change; (C) Lec(RSV) nanoparticles were placed for 3, 6, 9, and 12 months respectively, and no obvious turbidity and sediment were found; (D) In vitro release test of Lec(RSV), the accumulation within 12 hours Release 55%.
图4显示Lec和Lec(RSV)纳米颗粒的体外吸收能力和细胞毒性。(A-B)BT474乳腺癌细胞摄取包裹FITC的Lec和Lec(RSV)；(C)体外细胞毒性试验，分别观察Lec、RSV和Lec(RSV)对癌细胞的杀伤能力；(D)Lec、RSV和Lec(RSV)的ROS清除能力。Figure 4 shows the in vitro absorption capacity and cytotoxicity of Lec and Lec(RSV) nanoparticles. (A-B) BT474 breast cancer cells take up Lec and Lec(RSV) wrapped with FITC; (C) In vitro cytotoxicity test to observe the killing ability of Lec, RSV and Lec(RSV) against cancer cells respectively; (D) Lec, RSV and ROS scavenging ability of Lec(RSV).
图5显示Lec(RSV)在体内肿瘤摄取效果。利用BT474荷瘤模型检测进行体内肿瘤摄取实验，一组雌性Balb/C裸鼠(n＝3)用Lec(RSV+FITC)NPs治疗1h，另一组(n＝3)相同条件治疗4h。Figure 5 shows the effect of Lec(RSV) on tumor uptake in vivo. The BT474 tumor-bearing model was used to conduct in vivo tumor uptake experiments. One group of female Balb/C nude mice (n=3) were treated with Lec(RSV+FITC) NPs for 1 hour, and the other group (n=3) were treated with the same conditions for 4 hours.
具体实施方式Detailed ways
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary skill in the art to which this application belongs.
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular forms are also intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, it will be understood that when the terms "comprises" and/or "includes" are used in this specification, they indicate There are features, steps, operations, means, components and/or combinations thereof.
现结合具体实例对本发明作进一步的说明，以下实施例仅是为了解释本发明，但不构成对本发明的限制。在以下实施例中所用到的试验样本及试验过程包括以下内容(如果实施例中未注明的实验具体条件，通常按照常规条件，或按照试剂公司所推荐的条件；下述实施例中所用的试剂、耗材等，如无特殊说明，均可从商业途径得到)。The present invention will be further described with reference to specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions, or in accordance with the conditions recommended by the reagent company; the conditions used in the following examples Reagents, consumables, etc., can be obtained from commercial sources unless otherwise specified).
实施例1Example 1
一、材料及方法1. Materials and methods
1、材料1. Material
白藜芦醇，购于Aladdin(上海，中国)；卵磷脂，购于Sigma Aldrich(密苏里州圣路易斯，美国)，FITC，购于Sigma Aldrich(密苏里州圣路易斯，美国)，阿尔玛蓝试剂盒，购于赛默飞(沃尔瑟姆，美国)，有机溶剂(DMF，DMSO，75％酒精)，购于Sigma Aldrich(密苏里州圣路易斯，美国)，实验用水使用Millipore，Milli-Q***处理，其余物质均是使用分析等级。Resveratrol, purchased from Aladdin (Shanghai, China); lecithin, purchased from Sigma Aldrich (St. Louis, MO, USA), FITC, purchased from Sigma Aldrich (St. Louis, MO, USA), Alma Blue kit, purchased from Organic solvents (DMF, DMSO, 75% alcohol) were purchased from Sigma Aldrich (St. Louis, Missouri, USA). The experimental water was treated with Millipore and Milli-Q systems. The remaining substances were All are using analysis level.
2、合成Lec(RSV)2. Synthesize Lec(RSV)
使用纳米沉淀法制备Lec(RSV)。DMSO作为溶剂制备白藜芦醇(RSV)(100mg/mL)及卵磷脂(Lec)(100mg/mL)溶液。将10μL的Lec溶液加入到1μL的RSV溶液中，将89μL的DMSO加入至11μL的Lec、RSV混合溶液中，得到100μL的混合液。取干净的玻璃小瓶加入10mL的超滤水，在室温下以12000rpm转速持续搅拌。将混合溶液缓慢加入小瓶中，搅拌五分钟后获得纳米混悬液。将溶液转移至Amicon过滤器(Merck，Kenilworth，NJ，USA)，在4000rpm条件下，离心15分钟。剩余的溶液用5ml水洗涤，再次以4000rpm离心10分钟；重复两次以去除所有剩余的DMSO。Lec(RSV)保存至4℃，待进一步使用。在本实施例中，混合液中 白藜芦醇与卵磷脂的质量比为10∶1，混合液中白藜芦醇浓度为1mg/ml，混合液与水相的体积比为0.1∶10。Lec(RSV) was prepared using nanoprecipitation method. DMSO was used as the solvent to prepare resveratrol (RSV) (100 mg/mL) and lecithin (Lec) (100 mg/mL) solutions. Add 10 μL of Lec solution to 1 μL of RSV solution, and add 89 μL of DMSO to 11 μL of Lec and RSV mixed solution to obtain 100 μL of mixed solution. Take a clean glass vial, add 10 mL of ultrafiltered water, and stir continuously at 12,000 rpm at room temperature. The mixed solution was slowly added into the vial and the nanosuspension was obtained after stirring for five minutes. The solution was transferred to an Amicon filter (Merck, Kenilworth, NJ, USA) and centrifuged at 4000 rpm for 15 minutes. The remaining solution was washed with 5 ml of water and centrifuged again at 4000 rpm for 10 min; repeat twice to remove any remaining DMSO. Lec(RSV) was stored at 4°C until further use. In this embodiment, the mass ratio of resveratrol to lecithin in the mixed solution is 10:1, the concentration of resveratrol in the mixed solution is 1 mg/ml, and the volume ratio of the mixed solution to the water phase is 0.1:10.
3、纳米大小分布及电势3. Nano size distribution and electric potential
Lec和Lec(RSV)颗粒的粒径和zeta电位由Malvern Panalytical(MA，USA)动态光散射(DLS)分析确定。每个样本数据来自3个重复实验。The particle size and zeta potential of Lec and Lec(RSV) particles were determined by dynamic light scattering (DLS) analysis at Malvern Panalytical (MA, USA). Each sample data comes from 3 repeated experiments.
4、透射电子显微学分析4. Transmission electron microscopy analysis
Lec(RSV)(10mg/mL)滴在TEM级碳网铜网格上。将颗粒放在网格上，在环境温度下放置5分钟。每格用蒸馏水洗涤5次。然后用2％的醋酸铀酰对标本进行负染色，并在环境温度下放置2分钟。然后用蒸馏水冲洗三次，风干。用TECNAI F20电子显微镜(Philips Electronic Instruments Corp，Mahwah，103NJ)观察标本。Lec(RSV) (10mg/mL) was dropped on the TEM grade carbon mesh copper grid. Place the pellets on the grid and leave at ambient temperature for 5 minutes. Wash each compartment 5 times with distilled water. Specimens were then negatively stained with 2% uranyl acetate and left at ambient temperature for 2 minutes. Then rinse with distilled water three times and air dry. Specimens were observed with a TECNAI F20 electron microscope (Philips Electronic Instruments Corp, Mahwah, 103NJ).
5、RSV包载率5. RSV loading rate
用紫外-可见分光光度法测定Lec中RSV的包封量。在Lec(RSV)纯化后，样品在PBS中再水化到1ml的体积。取Lec(RSV)样品10μl，加入90μl DMSO中。取10μl原始的白藜芦醇-脂质体工作液作为阳性对照组。RSV在330nm处具特异的紫外吸光度，可用于RSV浓度的测定。为了确定RSV的包封效率(EE)，本发明人计算了Lec(RSV)偶联物中RSV的包封量与最初使用的RSV的包封量的比较，得到如下公式：The encapsulated amount of RSV in Lec was determined using UV-visible spectrophotometry. After Lec(RSV) purification, samples were rehydrated in PBS to a volume of 1 ml. Take 10μl of Lec(RSV) sample and add it to 90μl DMSO. Take 10 μl of the original resveratrol-liposome working solution as a positive control group. RSV has specific UV absorbance at 330nm, which can be used to determine RSV concentration. In order to determine the encapsulation efficiency (EE) of RSV, the inventors calculated the encapsulation amount of RSV in the Lec(RSV) conjugate compared with the encapsulation amount of RSV initially used, and obtained the following formula:
Figure PCTCN2022114615-appb-000001
Figure PCTCN2022114615-appb-000001
6、RSV及Lec(RSV)的稳定性6. Stability of RSV and Lec(RSV)
为了确定Lec和Lec(RSV)的稳定性，合成了浓度均为10mg/mL的上述Lec、Lec(RSV)。然后将Lec和Lec(RSV)颗粒保存在封闭的小瓶中，温度37℃储存。在预先设定的时间点(1、2、4、8、12、24、48h)，采用DLS法测定Lec和Lec(RSV)颗粒大小并记录。In order to determine the stability of Lec and Lec(RSV), the above-mentioned Lec and Lec(RSV) were synthesized at a concentration of 10 mg/mL. Lec and Lec(RSV) particles were then stored in closed vials at 37°C. At preset time points (1, 2, 4, 8, 12, 24, 48 h), the DLS method was used to determine the Lec and Lec(RSV) particle sizes and record them.
7、Lec(RSV)纳米体外释放曲线7. Lec(RSV) nano in vitro release curve
Lec(RSV)(n＝3，纯化后)分散于1ml PBS(pH 7.4)中，然后转移到Float-a-lyzer G2透析装置(MWCO 100kD，Spectrum Lab)，该透析装置浸于37℃的PBS(pH 7.4)中。在预定的间隔时间(1，2，4，8，12，24，48，72，96小时)，从透析装置内取出5μL NP溶液，与95L DMSO混合。充分混合后，用Synergy HT多模酶标仪测定各孔中LN的分光光度。Lec(RSV) (n=3, purified) was dispersed in 1 ml PBS (pH 7.4) and then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100kD, Spectrum Lab), which was immersed in PBS at 37°C (pH 7.4). At predetermined intervals (1, 2, 4, 8, 12, 24, 48, 72, 96 hours), 5 μL of NP solution was taken out of the dialysis device and mixed with 95 L of DMSO. After thorough mixing, use a Synergy HT multi-mode microplate reader to measure the spectrophotometry of LN in each well.
8、细胞培养8. Cell culture
人乳腺癌细胞BT474购自ATCC，且按所提供的流程培养。温度为37℃。使用含10％ 胎牛血清(FBS)、100U/mL青霉素及100g/mL链霉素的RPMI-1640/F-12K培养基，置于5％CO2的湿化细胞培养室中培养。Human breast cancer cells BT474 were purchased from ATCC and cultured according to the provided procedure. The temperature is 37℃. Use RPMI-1640/F-12K medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 g/mL streptomycin, and culture in a humidified cell culture room with 5% CO2.
9、体内摄入实验9. In vivo intake experiment
为了观察Lec的体外摄取和内在化情况，我们将FITC代替RSV封装在Lec中。用脂质体一半质量的FITC荧光标记Lec。BT474细胞在玻璃盖(12×12mm；Fisher Scientific，Texas，USA)上生长至～80％。在加入200g/mL含有Lec(FITC)培养基之前，先用无血清培养基培养基孵育1小时后，用PBS洗涤细胞2次，用4％(w/v)多聚甲醛(PFA)固定。将固定细胞安装在装有Dako固定介质的载玻片上，使用Olympus Fluoview 1000共聚焦显微镜(Olympus Imaging Co，Tokyo，Japan)进行检测。In order to observe the uptake and internalization of Lec in vitro, we encapsulated FITC in Lec instead of RSV. Lec was fluorescently labeled with FITC at half the mass of the liposomes. BT474 cells were grown on glass coverslips (12×12 mm; Fisher Scientific, Texas, USA) to ∼80%. Before adding 200 g/mL Lec (FITC)-containing medium, the cells were incubated with serum-free medium for 1 hour, washed twice with PBS, and fixed with 4% (w/v) paraformaldehyde (PFA). Fixed cells were mounted on glass slides containing Dako mounting medium and detected using an Olympus Fluoview 1000 confocal microscope (Olympus Imaging Co, Tokyo, Japan).
10、肿瘤的体外抗癌特性10. Anti-cancer properties of tumors in vitro
为了探究Lec(RSV)的抗癌特性，将BT474细胞种植在96孔板中(5000个/孔)，将不同浓度(10μM到40μM)的Lec、RSV、Lec(RSV)加入BT474中，在含5％的细胞培箱中培养24小时。24小时后每孔加入阿拉玛染色剂10μl，避光反应2h后，在荧光分光光度计下读数(Ex/Em＝500/525nm)。In order to explore the anti-cancer properties of Lec(RSV), BT474 cells were planted in 96-well plates (5000 cells/well), and different concentrations (10 μM to 40 μM) of Lec, RSV, and Lec(RSV) were added to BT474. 5% cell culture incubator for 24 hours. After 24 hours, add 10 μl of Alama dye to each well, protect from light and react for 2 hours, then read on a fluorescence spectrophotometer (Ex/Em=500/525nm).
11、Lec(RSV)体外清除ROS的能力11. Lec(RSV)’s ability to scavenge ROS in vitro
用ROS检测试剂盒检测BT474上Lec(RSV)的ROS清除能力。简单地说，将细胞(1×10 5/孔)置于6孔板中，在5％CO2的培养箱中培养24小时。用含有不同浓度的RSV和Lec(RSV)的新鲜培养基培养24h，弃去培养液后加入10μmol/L DCFH-DA的PBS溶液100μL在细胞培养箱中避光孵育30分钟。反应完毕后转移至96孔黑色酶标板，细胞在5％CO2培养箱中培养30分钟。结束后转移到96孔黑色酶标板中，用多功能酶标仪在Ex/Em＝500/525nm下检测。 Use a ROS detection kit to detect the ROS scavenging ability of Lec(RSV) on BT474. Briefly, cells (1 × 10 5 /well) were placed in 6-well plates and cultured in a 5% CO2 incubator for 24 hours. Incubate for 24 hours with fresh culture medium containing different concentrations of RSV and Lec(RSV). After discarding the culture medium, add 100 μL of 10 μmol/L DCFH-DA in PBS and incubate in a cell culture incubator in the dark for 30 minutes. After the reaction is completed, transfer to a 96-well black microplate, and culture the cells in a 5% CO2 incubator for 30 minutes. After completion, transfer to a 96-well black microplate and detect with a multifunctional microplate reader at Ex/Em=500/525nm.
12、Lec(RSV)体内肿瘤浓聚试验12. Lec(RSV) in vivo tumor concentration test
将1.2×10 7/0.2mL的BT-474细胞注入免疫缺陷小鼠体内建立乳腺肿瘤模型。当肿瘤生长至120～200nm 3时，用于检测Lec(RSV)的肿瘤靶向能力。小鼠被分2组(n＝3)，一组注射Lec(RSV)1小时后检测，另一组注射Lec(RSV)3小时后检测。将提纯的Lec(RSV)重新悬于PBS中，每只小鼠行尾静脉注射0.2ml的Lec(RSV)混悬液(RSV 2mg/Kg)待1小时、3小时后在活体成像仪下检测。 Breast tumor model was established by injecting 1.2×10 7 /0.2mL BT-474 cells into immunodeficient mice. When the tumor grows to 120~ 200nm3 , it is used to detect the tumor targeting ability of Lec(RSV). Mice were divided into 2 groups (n=3), one group was injected with Lec(RSV) and tested 1 hour later, and the other group was injected with Lec(RSV) and tested 3 hours later. The purified Lec(RSV) was resuspended in PBS, and each mouse was injected with 0.2 ml of Lec(RSV) suspension (RSV 2mg/Kg) into the tail vein. Wait for 1 hour and 3 hours before detecting under the live imager. .
二、结果2. Results
1、Lec(RSV)的特性1. Characteristics of Lec(RSV)
本发明人的最终目标是开发一个纯天然、有天然抗癌特性的纳米粒子。具体地，合成了Lec(RSV)，作为一个同抗肿瘤治疗平台(图1)。白藜芦醇可以溶于有机溶剂但不能溶于水或缓冲液，这种特性大大限制了白藜芦醇的生物利用率。如图1B(左)所示，简单地将卵磷脂与白藜芦醇混合并不能自动封装RSV并形成纳米颗粒。只有通过上文所述的纳米沉淀方法，才能制备出Lec(RSV)纳米粒，并观测到透明的Lec(RSV)溶液，如图1B(右)所示。The inventor's ultimate goal is to develop a purely natural nanoparticle with natural anti-cancer properties. Specifically, Lec(RSV) was synthesized as a synergistic anti-tumor therapeutic platform (Figure 1). Resveratrol is soluble in organic solvents but not in water or buffers, a property that greatly limits the bioavailability of resveratrol. As shown in Figure 1B (left), simply mixing lecithin with resveratrol does not automatically encapsulate RSV and form nanoparticles. Only through the nanoprecipitation method described above, Lec(RSV) nanoparticles can be prepared, and a transparent Lec(RSV) solution can be observed, as shown in Figure 1B (right).
2、Lec和Lec(RSV)NPs的性质2. Properties of Lec and Lec(RSV)NPs
为了确定Lec和Lec(RSV)的大小和形态，进行了TEM和DLS分析。如图2A所示，空载的卵磷脂纳米表现为球形，而包载白藜芦醇的LEC(RSV)有相似的结构。虽然两组的形态相似，但Lec(RSV)的平均大小更大。这一观察结果表明，RSV的包封是成功的，与对照组相比(151.0±22.93nm vs 128.4±19.88nm)，它确实增加了纳米颗粒的平均尺寸(图2C)。RSV的包封率计算在85.16％左右(图2D)。To determine the size and morphology of Lec and Lec(RSV), TEM and DLS analyzes were performed. As shown in Figure 2A, unloaded lecithin nanoparticles appear spherical, while resveratrol-loaded LEC(RSV) has a similar structure. Although the morphology of the two groups was similar, the average size of Lec(RSV) was larger. This observation indicates that encapsulation of RSV is successful and it indeed increases the average size of the nanoparticles compared to the control group (151.0±22.93nm vs 128.4±19.88nm) (Figure 2C). The encapsulation efficiency of RSV was calculated to be around 85.16% (Figure 2D).
纳米颗粒的大小显著影响其治疗和诊断的应用；包括药物半衰期、靶向能力、细胞摄取和肿瘤穿透性在内的等等治疗方面都受到纳米颗粒大小的影响；纳米颗粒必须大于10nm，以避免肾过滤作用，但直径又不能过大，如：大于200nm的颗粒会激活补体***，并迅速从血液中被移除，在肝脏和脾脏中积累。因此，合适大小的纳米粒才能达到本申请所要达到的技术效果，在本实施例中，Lec(RSV)纳米颗粒的粒径范围为128～174nm，平均尺寸为151nm，是接近理想的尺寸，稳定、负载能力大，且具有良好的肿瘤穿透能力。The size of nanoparticles significantly affects their therapeutic and diagnostic applications; therapeutic aspects including drug half-life, targeting ability, cellular uptake and tumor penetration are all affected by the size of nanoparticles; nanoparticles must be larger than 10nm to Avoid renal filtration, but the diameter should not be too large. For example, particles larger than 200nm will activate the complement system and be quickly removed from the blood and accumulate in the liver and spleen. Therefore, only nanoparticles of appropriate size can achieve the technical effect to be achieved by this application. In this embodiment, the particle size range of Lec(RSV) nanoparticles is 128~174nm, and the average size is 151nm, which is close to the ideal size and stable. , large load capacity, and good tumor penetration ability.
3、Lec和Lec(RSV)的稳定性3. Stability of Lec and Lec(RSV)
在纳米传递***中纳米颗粒的稳定性至关重要，因为纳米颗粒需要在体内长时间循环。Lec和Lec(RSV)纳米颗粒在48小时内均稳定存在。为证明Lec(RSV)的稳定性，发明人在第1/4/8/5/30/60天分别测量Lec(RSV)粒子的大小。如图3B所示，DLS下测量Lec(RSV)大小，在60天内纳米大小波动非常小，范围在100到200纳米之间，证明Lec(RSV)有很好的稳定性。值得注意的是，即使存放在4℃下12个月后，仍然没有沉积物，即Lec(RSV)在4℃下可以长期保存，证明此纳米***有很好的稳定性。再且，RSV被Lec包裹后能实现缓慢释放，在12小时内只有55％的累计释放量。除了纳米粒径所体现的性能外，作为一种有望应用于临床上的抗癌药物，稳定性对其储存及传递能力起着至关重要的作用。而且，纳米粒子传递***需要纳米粒子在体内长时间循环，所以Lec(RSV)的稳定性起关键作用。在本研究中，使用DSL测量Lec(RSV)大小发现其在60天内只有轻微的大小变化。证明了Lec(RSV)有很好的稳定性。Lec(RSV)能在4℃中长期保存12个月而没有发生聚集、凝结，也表明该纳米平台 具有良好的稳定性。结合前述Lec(RSV)所体现的特性，Lec(RSV)显示出较其他药物或纳米传递***更大的优势，也具有临床应用以治疗相关病症的潜力。The stability of nanoparticles in nanodelivery systems is critical because nanoparticles need to circulate in the body for long periods of time. Both Lec and Lec(RSV) nanoparticles were stable for 48 hours. To prove the stability of Lec(RSV), the inventor measured the size of Lec(RSV) particles on days 1/4/8/5/30/60. As shown in Figure 3B, the size of Lec(RSV) was measured under DLS. Within 60 days, the nanometer size fluctuated very little, ranging from 100 to 200 nanometers, proving that Lec(RSV) has good stability. It is worth noting that even after 12 months of storage at 4°C, there is still no sediment, that is, Lec(RSV) can be stored at 4°C for a long time, proving that this nanosystem has good stability. Furthermore, RSV can achieve slow release after being wrapped by Lec, with only 55% of the cumulative release amount within 12 hours. In addition to the performance reflected by the nanoparticle size, as an anti-cancer drug that is expected to be used clinically, stability plays a crucial role in its storage and delivery capabilities. Moreover, the nanoparticle delivery system requires nanoparticles to circulate in the body for a long time, so the stability of Lec(RSV) plays a key role. In this study, Lec(RSV) size was measured using DSL and found only slight size changes over 60 days. It is proved that Lec(RSV) has good stability. Lec(RSV) can be stored for 12 months at 4°C without aggregation or coagulation, which also shows that the nanoplatform has good stability. Combined with the aforementioned characteristics of Lec(RSV), Lec(RSV) shows greater advantages than other drugs or nano-delivery systems, and also has the potential for clinical application to treat related diseases.
4、Lec和Lec(RSV)纳米颗粒的体外吸收能力和细胞毒性4. In vitro absorption capacity and cytotoxicity of Lec and Lec(RSV) nanoparticles
接下来本发明人证实了Lec(RSV)的体外吸收能力。将FITC包裹进Lec及Lec(RSV)粒子中，分别将他们加入BT474细胞中培养4小时。共孵育4小时后，Lec和Lec(RSV)均显示显著摄取到细胞质中(图4A和4B)。接下来，我们进行了体外细胞毒性实验，观察RSV对癌细胞的杀伤能力。如图4C所示，RSV处理对细胞(IC50～18μM)具有高毒性。作为抗氧化剂，Lec也具有固有的肿瘤杀伤能力，但不如RSV(IC50＞40μM)显著。值得注意是，与单独使用Lec或RSV相比，Lec(RSV)对肿瘤细胞的杀伤能力具有显著的协同作用(IC50～11μM)。同样，与对照组或Lec相比，RSV对ROS的清除效果显著(p＜0.001)，封装RSV不影响RSV的生物活性(图4D)。作为一种理想的抗癌药物，除了前述的粒径、稳定性外，另外还一个重要的指标是肿瘤细胞富集能力和细胞毒性。在本实施例中，本发明人使用来源天然的卵磷脂封装白藜芦醇来构成纳米粒子，并在体外测试其对BT474乳腺癌细胞的细胞摄取和细胞毒性。在经过4小时的共培养后，Lec和Lec(RSV)均明显地被吸收进入细胞的细胞质。然后，本发明人进行了体外的细胞毒性试验，观察RSV对肿瘤细胞的杀伤能力，发现RSV(IC 50，11μM)对细胞有明显毒性。作为一种抗氧化剂，Lec也有一定的肿瘤杀伤能力，但不如RSV明显(IC 50，40μM)。值得注意的是，当Lec与RSV组合在一起形成纳米粒，较之单独的Lec或单独的RSV有更好的肿瘤杀伤作用。在抗氧化方面亦是如此，Lec(RSV)具有组分之间的协同作用，效果显著。此外，包封RSV并不会改变它的生物活性，进一步体现了本申请纳米粒对包载主药而言具有稳定性。 Next, the inventors confirmed the in vitro absorption capacity of Lec(RSV). FITC was encapsulated into Lec and Lec(RSV) particles, and they were added to BT474 cells and cultured for 4 hours. After 4 hours of co-incubation, both Lec and Lec(RSV) showed significant uptake into the cytoplasm (Figures 4A and 4B). Next, we conducted in vitro cytotoxicity experiments to observe the killing ability of RSV on cancer cells. As shown in Figure 4C, RSV treatment was highly toxic to cells (IC50~18 μM). As an antioxidant, Lec also has inherent tumor killing ability, but it is not as significant as RSV (IC50>40μM). It is worth noting that compared with using Lec or RSV alone, Lec (RSV) has a significant synergistic effect on the killing ability of tumor cells (IC50 ~ 11 μM). Similarly, compared with the control group or Lec, RSV had a significant scavenging effect on ROS (p<0.001), and encapsulating RSV did not affect the biological activity of RSV (Figure 4D). As an ideal anti-cancer drug, in addition to the aforementioned particle size and stability, another important indicator is tumor cell enrichment ability and cytotoxicity. In this example, the inventors used natural lecithin to encapsulate resveratrol to form nanoparticles, and tested its cellular uptake and cytotoxicity on BT474 breast cancer cells in vitro. After 4 hours of co-culture, both Lec and Lec(RSV) were obviously absorbed into the cytoplasm of the cells. Then, the inventors conducted an in vitro cytotoxicity test to observe the killing ability of RSV on tumor cells, and found that RSV (IC 50 , 11 μM) has obvious toxicity to cells. As an antioxidant, Lec also has a certain tumor-killing ability, but it is not as obvious as RSV (IC 50 , 40 μM). It is worth noting that when Lec and RSV are combined to form nanoparticles, they have better tumor killing effect than Lec alone or RSV alone. The same is true in terms of antioxidants. Lec(RSV) has a synergistic effect between components and has a significant effect. In addition, encapsulating RSV will not change its biological activity, which further demonstrates the stability of the nanoparticles of the present application for encapsulating the main drug.
5、Lec(RSV)体内肿瘤摄取试验5. Lec(RSV) in vivo tumor uptake test
在前述体外实验中纳米粒的显著效果基础上，本发明人进一步进行体内肿瘤摄取试验，具体地，在BT474植瘤模型行体内肿瘤摄取实验。两组小鼠(n＝3)给予Lec(RSV+FITC)分别等待1小时和4小时。如图5所示，可以清晰看到相比于1小时组，4小时组Lec(RSV)累积量明显上升。表明Lec(RSV)作为一种肿瘤给药***是可靠的。Based on the significant effects of nanoparticles in the aforementioned in vitro experiments, the inventors further conducted in vivo tumor uptake experiments. Specifically, in vivo tumor uptake experiments were performed on the BT474 tumor transplant model. Two groups of mice (n=3) were given Lec (RSV+FITC) and waited for 1 hour and 4 hours respectively. As shown in Figure 5, it can be clearly seen that the accumulation of Lec(RSV) in the 4-hour group increased significantly compared with the 1-hour group. It shows that Lec(RSV) is reliable as a tumor drug delivery system.
总体上，在本实施例中，至少体现有：Lec(RSV)具有优秀的稳定性、生物相容性，对BT474乳腺癌细胞有抑制作用，较小的细胞毒性，安全。Lec(RSV)有望应用于临床中以成为有效的临床抗肿瘤药物。Generally speaking, in this embodiment, it is at least reflected that Lec(RSV) has excellent stability and biocompatibility, has an inhibitory effect on BT474 breast cancer cells, has less cytotoxicity, and is safe. Lec(RSV) is expected to be used clinically to become an effective clinical anti-tumor drug.
具体地，本发明人在应用纳米沉淀法制备白藜芦醇卵磷脂纳米粒过程中，还采用DMSO 制备有白藜芦醇(RSV)(100mg/mL)及卵磷脂(Lec)(100mg/mL)溶液，并加入9μL或11μL的Lec至1μL的RSV中，补足DMSO至100μL，并在混合液与水相体积比为(0.05～0.15)∶10的条件下，采用相同的步骤制备获得白藜芦醇卵磷脂纳米粒。并进行有上述稳定性、肿瘤细胞杀伤实验、摄取实验，实验表明，其具备等同显著的稳定性、协同杀伤效果、体内摄取特性。Specifically, in the process of preparing resveratrol lecithin nanoparticles using nanoprecipitation method, the inventor also used DMSO to prepare resveratrol (RSV) (100 mg/mL) and lecithin (Lec) (100 mg/mL). ) solution, and add 9 μL or 11 μL of Lec to 1 μL of RSV, add DMSO to 100 μL, and use the same steps to prepare the white quinoa under the condition that the volume ratio of the mixed solution to the water phase is (0.05~0.15):10. Retinol lecithin nanoparticles. The above-mentioned stability, tumor cell killing experiments, and uptake experiments were conducted. The experiments showed that it has equally significant stability, synergistic killing effect, and in vivo uptake characteristics.
显然，本发明的上述实施例仅仅是为清楚地说明本发明技术方案所作的举例，而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。Obviously, the above-mentioned embodiments of the present invention are only examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (10)

  1. 一种白藜芦醇卵磷脂纳米粒，其特征在于，包括白藜芦醇以及包裹和/或偶联于白藜芦醇上的卵磷脂。A resveratrol lecithin nanoparticle is characterized by including resveratrol and lecithin wrapped and/or coupled to the resveratrol.
  2. 根据权利要求1所述的白藜芦醇卵磷脂纳米粒，其特征在于，包括内外层卵磷脂，所述白藜芦醇包裹于内外层卵磷脂之间。Resveratrol lecithin nanoparticles according to claim 1, characterized in that they include inner and outer layers of lecithin, and the resveratrol is wrapped between the inner and outer layers of lecithin.
  3. 根据权利要求1或2所述的白藜芦醇卵磷脂纳米粒，其特征在于，白藜芦醇与卵磷脂的质量比为(9～11)∶1。The resveratrol lecithin nanoparticles according to claim 1 or 2, wherein the mass ratio of resveratrol to lecithin is (9-11):1.
  4. 权利要求1～3任一项所述白藜芦醇卵磷脂纳米粒在制备清除自由基和/或杀伤肿瘤细胞的药物中的用途。The use of the resveratrol lecithin nanoparticles described in any one of claims 1 to 3 in the preparation of drugs for scavenging free radicals and/or killing tumor cells.
  5. 根据权利要求4所述的用途，其特征在于，肿瘤细胞包括乳腺癌细胞。The use according to claim 4, wherein the tumor cells include breast cancer cells.
  6. 一种肿瘤细胞杀伤药物，其特征在于，包括权利要求1～3任一项所述白藜芦醇卵磷脂纳米粒。A tumor cell killing drug, characterized by comprising the resveratrol lecithin nanoparticles described in any one of claims 1 to 3.
  7. 权利要求1所述白藜芦醇卵磷脂纳米粒的制备方法，其特征在于，包括步骤：The preparation method of resveratrol lecithin nanoparticles according to claim 1, characterized in that it includes the steps:
    A1、将白藜芦醇、卵磷脂分别溶于能与水混溶的有机溶剂中获得白藜芦醇有机相、卵磷脂有机相，将卵磷脂有机相加至白藜芦醇有机相中，然后补充有机溶剂，获得混合液；A1. Dissolve resveratrol and lecithin in an organic solvent miscible with water to obtain a resveratrol organic phase and a lecithin organic phase. Add the lecithin organic phase to the resveratrol organic phase. Then add organic solvent to obtain a mixed solution;
    A2、搅拌条件下将混合液加入至水相中，获得纳米混悬液；A2. Add the mixed solution to the water phase under stirring conditions to obtain a nanosuspension;
    A3、离心过滤去除有机溶剂，获得白藜芦醇卵磷脂纳米粒。A3. Centrifugal filtration to remove the organic solvent and obtain resveratrol lecithin nanoparticles.
  8. 根据权利要求7所述的制备方法，其特征在于，有机溶剂为二甲基亚砜。The preparation method according to claim 7, characterized in that the organic solvent is dimethyl sulfoxide.
  9. 根据权利要求7所述的制备方法，其特征在于，混合液中白藜芦醇与卵磷脂的质量比为(9～11)∶1；和/或，混合液中白藜芦醇浓度为0.9～1.1mg/ml；和/或，混合液与水相的体积比为(0.05～0.15)∶10。The preparation method according to claim 7, characterized in that the mass ratio of resveratrol to lecithin in the mixed liquid is (9-11):1; and/or the concentration of resveratrol in the mixed liquid is 0.9 ~1.1mg/ml; and/or, the volume ratio of the mixed solution to the aqueous phase is (0.05~0.15):10.
  10. 白藜芦醇和/或卵磷脂在制备清除自由基和/或杀伤肿瘤细胞的药物中的用途。Use of resveratrol and/or lecithin in the preparation of drugs that scavenge free radicals and/or kill tumor cells.
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