CN114681430A - Resveratrol lecithin nanoparticles and preparation method and application thereof - Google Patents
Resveratrol lecithin nanoparticles and preparation method and application thereof Download PDFInfo
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- CN114681430A CN114681430A CN202210367902.3A CN202210367902A CN114681430A CN 114681430 A CN114681430 A CN 114681430A CN 202210367902 A CN202210367902 A CN 202210367902A CN 114681430 A CN114681430 A CN 114681430A
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- lecithin
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- 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
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- 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
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Abstract
A resveratrol lecithin nanoparticle comprises resveratrol and lecithin wrapped and/or coupled with resveratrol. The nanoparticles have natural component sources and safety, and a nano delivery system formed by the resveratrol and the lecithin has a slow release characteristic, so that the in-vivo bioavailability of the resveratrol is improved, the in-vivo action is prolonged, and the coating of the lecithin is also beneficial to the storage of the main drug resveratrol in a non-use state. More importantly, the formed resveratrol lecithin nanoparticles have a remarkable effect on tumor killing, on one hand, the nano delivery system is convenient for improving the bioavailability of resveratrol, promoting the targeting of tumor tissues and improving the killing effect of tumor cells, and on the other hand, the nanoparticles formed by resveratrol and lecithin also have a synergistic killing effect, have more remarkable anti-tumor and anti-oxidation effects, are expected to be applied to tumor treatment to provide new treatment medicines and treatment strategies so as to delay the life cycle of tumor patients.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to resveratrol lecithin nanoparticles and a preparation method and application thereof.
Background
Resveratrol (3,5, 4-trihydroxy stilbene, RSV) is a natural polyphenol flavone, and is rich in grape, red wine, mulberry, peanut, rhubarb, etc. RSV is known to have anti-inflammatory, anti-obesity and cardio-and cerebro-protective effects. In nervous system diseases, resveratrol is found to have the effects of reducing oxidation and apoptosis of neurons and protecting nerves. At present, a common systemic administration mode of resveratrol is intravenous injection, however, the low bioavailability of resveratrol after entering the body limits the treatment effect of resveratrol on corresponding diseases; for example, RSV is rapidly (within 2 hours) metabolized in liver and 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 is poorly water soluble, chemically unstable, and is subject to isomerization degradation when exposed to high temperatures, pH changes, ultraviolet light, or certain types of enzymes, and is also not conducive to achieving retention and use of resveratrol.
In the prior art, a nano delivery system is commonly used for carrying and realizing the systemic delivery of drugs, and polyethylene glycol (PEG) is usually adopted to encapsulate small-molecule drugs into spherical particles with nano sizes, and then the administration of the small-molecule drugs is carried out through the spherical particles. Although the persistence of the encapsulated micromolecular drugs in vivo can be improved to a certain extent by the method, the method has certain potential safety hazard. Specifically, PEG is a petroleum-based compound widely used in cosmetics, such as a thickener, a solvent, a softener, a moisturizing carrier, etc. of cosmetics, and also used as a laxative, but studies have suggested (e.g., Mouse lysohoma L5178Y thionine kinase assays of 50 compounds; Low molecular weight polyethylene glycol indexes and chromosom assays in Chinese hams cells concentrated in vitro. Mutagenesis), and PEG complexes and contaminated waste materials generated therefrom have genotoxicity, and may cause gene mutation or systemic poisoning in some cases, i.e., the safety of nano-delivery systems using PEG as a main body cannot be secured.
Therefore, the prior art needs a drug delivery system or a corresponding preparation and a drug which can improve the storage performance and the drug action performance of the resveratrol and have high safety, and provides the therapeutic application of the resveratrol in related diseases.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art, provides resveratrol lecithin nanoparticles, a preparation method and application thereof, overcomes the defects of difficult storage, low bioavailability and limited curative effect of resveratrol in the prior art, is used for antioxidation and anticancer, and is used as a nano drug delivery system to provide an effective and safe treatment strategy for replacing corresponding diseases.
The technical scheme adopted by the invention is that the resveratrol lecithin nanoparticles comprise resveratrol and lecithin wrapped and/or coupled on the resveratrol. The resveratrol lecithin nanoparticle is called Lec (RSV) in the following for short. The lecithin is a liposome extracted from soybean, and the invention adopts the combination of the lecithin and resveratrol, can form a completely natural nano delivery drug delivery and treatment system, and has high safety. And the use of the nano preparation can ensure that the shelf life of the resveratrol is longer, and in more than one embodiment of the invention, the resveratrol can be stably kept for twelve months at room temperature and 4 ℃. The nanoparticles formed by the resveratrol and the lecithin are also beneficial to improving the administration effect, on one hand, in more than one embodiment of the invention, the resveratrol is slowly released in the nanoparticles, so that the defect of low bioavailability of the resveratrol in intravenous injection in the prior art can be overcome, and the in-vivo action time is prolonged; on the other hand, in more than one embodiment of the invention, the resveratrol lecithin nanoparticles provided by the application can obviously improve the anti-oxidation and anti-cancer treatment effects, and compared with the anti-oxidation and anti-cancer effects of single resveratrol or lecithin, the resveratrol lecithin nanoparticles realize the synergistic effect of the resveratrol and the lecithin, and the action effect is obvious. The resveratrol lecithin nanoparticles provided by the invention can overcome the defects of the prior art, improve the storage performance and bioavailability of resveratrol, provide the applications in oxidation resistance and cancer resistance, provide new treatment strategies and medicines for diseases and cancers needing oxidation resistance in the prior art, particularly reflect cancer treatment, and provide a substitute, economic and effective anticancer therapy with low side effect.
Further, the resveratrol-containing food comprises inner and outer layers of lecithin, and the resveratrol is wrapped between the inner and outer layers of lecithin.
Further, the mass ratio of the resveratrol to the lecithin is (9-11) to 1.
Further, the mass ratio of the resveratrol to the lecithin is 10: 1.
Furthermore, the particle size range of the nanoparticles is 128-174 nm. The nano particles with the particle size are not only stable, but also have excellent loading capacity and good tumor penetration capacity. As an anticancer drug expected to be clinically applied, the stability plays a crucial role in the storage and transmission capacity of the anticancer drug. The size of the nanoparticles significantly affects the therapeutic and diagnostic applications, and therapeutic aspects including drug half-life, targeting ability, cellular uptake and tumor penetration are affected by the size of the nanoparticles; the nanoparticles must be larger than 10nm to avoid renal filtration, but not too large in diameter, such as: particles larger than 200nm activate the complement system and are rapidly removed from the blood, accumulating in the liver and spleen. Therefore, the particle size of the nanoparticles can directly influence the effect of the nanoparticles, and the effect of the nanoparticles cannot be exerted easily when the particle size is too large or too small. In the invention, the particle size range of the resveratrol lecithin nanoparticles is 128-174 nm, the average particle size is 151nm, the nanoparticles can be properly prevented from being too small or too large, and the loading capacity and the action effect of the resveratrol lecithin nanoparticles are ensured.
Further, the compound is prepared by a nano precipitation method. In one embodiment of the invention, the resveratrol lecithin nanoparticles are prepared and formed by a nano-precipitation method.
The invention also aims to provide application of the resveratrol lecithin nanoparticles in preparation of medicines for removing free radicals and/or killing tumor cells. In more than one embodiment of the invention, the resveratrol lecithin nanoparticles have a remarkable killing effect on tumor cells, and have a synergistic killing effect and more remarkable killing capability compared with resveratrol directly applied to tumor cells.
Further, the tumor cells include breast cancer cells. In one or more embodiments of the invention, the tumor cells employed are breast cancer cells.
The invention also aims to provide a tumor cell killing medicament, which comprises the resveratrol lecithin nanoparticles.
Further, pharmaceutically acceptable auxiliary materials are also included.
The invention also aims to provide a preparation method of the resveratrol lecithin nanoparticles, which comprises the following steps:
a1, respectively dissolving resveratrol and lecithin in organic solvents which can be mixed with water to obtain a resveratrol organic phase and a lecithin organic phase, adding the lecithin organic phase into the resveratrol organic phase, and then supplementing the organic solvents to obtain a mixed solution;
a2, adding the mixed solution into the water phase under the stirring condition to obtain a nano suspension;
a3, centrifuging and filtering to remove the organic solvent, and obtaining the resveratrol lecithin nanoparticles.
Further, the organic solvent is dimethyl sulfoxide.
Further, the mass ratio of the resveratrol to the lecithin in the mixed solution is (9-11) to 1;
further, the concentration of the resveratrol in the mixed solution is 0.9-1.1 mg/ml;
furthermore, the volume ratio of the mixed liquid to the water phase is (0.05-0.15) to 10.
Further, the volume ratio of the mixed solution to the aqueous phase was 0.1: 10.
Further, in step a3, the nanosuspension was added to an Amincon filter and the organic solvent was removed by centrifugation.
Further, step a3 includes:
a31, carrying out primary centrifugation on the nano suspension, and carrying out primary centrifugation to separate out an organic solvent;
a32, washing the residual organic solvent, centrifuging again, and removing the residual organic solvent;
a33, repeating the step A32 for more than two times to obtain the resveratrol lecithin nanoparticles.
Still another object of the present invention is to provide the use of resveratrol and/or lecithin in the preparation of a medicament for scavenging free radicals and/or killing tumor cells. In more than one embodiment of the invention, the single resveratrol or lecithin also has certain functions of scavenging free radicals and killing tumors, and the resveratrol lecithin nanoparticles containing the resveratrol and the lecithin have more obvious synergistic effect.
Compared with the prior art, the invention has the beneficial effects that: the nanoparticles have natural component sources and safety, and a nano delivery system formed by the resveratrol and the lecithin has a slow release characteristic, so that the in-vivo bioavailability of the resveratrol is improved conveniently, the in-vivo action is prolonged, and the coating of the lecithin is also beneficial to the storage of the main drug resveratrol in a non-use state. More importantly, the formed resveratrol lecithin nanoparticles have a remarkable effect on tumor killing, on one hand, the nano delivery system is convenient for improving the bioavailability of resveratrol, promoting the targeting of tumor tissues and improving the killing effect of tumor cells, and on the other hand, the nanoparticles formed by resveratrol and lecithin also have a synergistic killing effect, have more remarkable anti-tumor and anti-oxidation effects, are expected to be applied to tumor treatment to provide new treatment medicines and treatment strategies so as to delay the life cycle of tumor patients. In addition, in addition to the characteristics of Lec (RSV) which is directly killed to tumor cells, Lec (RSV) has more stable property, is convenient to store, and overcomes the problems of storage and the like caused by instability of resveratrol in the prior art.
Drawings
FIG. 1 shows the schematic diagram and features of the synthesis of Lec (RSV). (A) Schematic representation of the formation of lec (rsv) nanoparticles using lecithin and resveratrol; (B) the soluble Lec (RSV) is obtained by a nano precipitation method.
Fig. 2 shows the size 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 was (Lec (RSV 151.0 + -22.93 nm vs. empty Lec128.4 + -19.88 nm); D) the encapsulation effect of RSV.
Figure 3 shows the stability of Lec and Lec (rsv) nanoparticles. (A) The particle sizes of the lecithin nanoparticles and the Lec (RSV) nanoparticles are not obviously changed within 48 hours at room temperature; (B) the particle size of the lecithin nano-particles and the Lec (RSV) nano-particles is not obviously changed within 60 days at 4 ℃; (C) lec (RSV) nano-particles are respectively placed for 3, 6, 9 and 12 months, and no obvious turbidity and sediment are seen; (D) in vitro release assay for lec (rsv), 55% release is accumulated over 12 hours.
Fig. 4 shows the in vitro absorptive capacity and cytotoxicity of Lec and Lec (rsv) nanoparticles. (A-B) BT474 breast cancer cells take in Lec and Lec (RSV) encapsulated in FITC; (C) in vitro cytotoxicity test, observing the killing ability of Lec, RSV and Lec (RSV) on cancer cells respectively; (D) lec, RSV and Lec (RSV) ROS scavenging ability.
Fig. 5 shows the effect of lec (rsv) on tumor uptake in vivo. In vivo tumor uptake experiments were performed using the BT474 tumor-bearing model assay, one group of female Balb/C nude mice (n ═ 3) were treated with Lec (RSV + FITC) NPs for 1h, and the other group (n ═ 3) was treated under the same conditions for 4 h.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not to be construed as limiting the invention. The test samples and test procedures used in the following examples include the following (generally, according to the conventional conditions or according to the conditions recommended by the reagent company if the specific conditions of the experiment are not specified in the examples; and reagents, consumables and the like used in the following examples are commercially available unless otherwise specified).
Example 1
Materials and methods
1. Material
Resveratrol, purchased from Aladdin (shanghai, china); lecithin, purchased from Sigma Aldrich (st louis, mo), FITC, purchased from Sigma Aldrich (st louis, mo), alamar blue kit, purchased from zermer fly (waltham, u.s.a.), organic solvents (DMF, DMSO, 75% alcohol), purchased from Sigma Aldrich (st louis, mo., u.s.a.), experimental water treated using Millipore, Milli-Q system, and the rest using analytical grade.
2. Synthesis of Lec (RSV)
Lec (RSV) was prepared using a nanoprecipitation method. Solutions of Resveratrol (RSV) (100mg/mL) and lecithin (Lec) (100mg/mL) were prepared using DMSO as the solvent. mu.L of the Lec solution was added to 1. mu.L of the RSV solution, and 89. mu.L of DMSO was added to 11. mu.L of the mixed solution of Lec and RSV, to obtain 100. mu.L of a mixed solution. A clean glass vial was charged with 10mL of ultrafiltration water and stirred continuously at 12000rpm at room temperature. The mixed solution was slowly added to the vial, and after stirring for five minutes, a nanosuspension was obtained. The solution was transferred to an Amicon filter (Merck, Kenilworth, NJ, USA) and centrifuged at 4000rpm for 15 minutes. The remaining solution was washed with 5ml of water and centrifuged again at 4000rpm for 10 minutes; this was repeated twice to remove all remaining DMSO. Lec (rsv) was stored to 4 ℃ for further use. In this example, the mass ratio of resveratrol to lecithin in the mixed solution was 10: 1, the concentration of resveratrol in the mixed solution was 1mg/ml, and the volume ratio of the mixed solution to the aqueous phase was 0.1: 10.
3. Nano size distribution and potential
The particle size and zeta potential of Lec and Lec (rsv) particles were determined by Malvern Panalytical (MA, USA) Dynamic Light Scattering (DLS) analysis. Each sample data was from 3 replicate experiments.
4. Transmission electron microscopy analysis
Lec (RSV) (10mg/mL) was dropped onto a TEM grade carbon mesh copper grid. The particles were placed on a grid and left at ambient temperature for 5 minutes. Each compartment was washed 5 times with distilled water. The specimens were then negatively stained with 2% uranyl acetate and allowed to stand at ambient temperature for 2 minutes. Then rinsed three times with distilled water and air dried. The specimens were observed with a TECNAAI F20 electron microscope (Philips Electronic Instruments Corp, Mahwah,103 NJ).
5. RSV loading rate
The encapsulated amount of RSV in Lec was determined by uv-vis spectrophotometry. After lec (rsv) purification, the samples were rehydrated in PBS to a volume of 1 ml. Lec (RSV) samples were taken in 10. mu.l and added to 90. mu.l DMSO. Taking 10 μ l of original resveratrol-liposome working solution as a positive control group. The RSV has specific ultraviolet absorbance at 330nm, and can be used for determining the concentration of the RSV. To determine the Encapsulation Efficiency (EE) of RSV, the present inventors calculated a comparison of the encapsulated amount of RSV in the lec (RSV) conjugate to the encapsulated amount of RSV initially used, resulting in the following formula:
6. stability of RSV and Lec (RSV)
To determine the stability of Lec and Lec (RSV), the aforementioned Lec and Lec (RSV) were synthesized at a concentration of 10mg/mL each. The Lec and Lec (rsv) particles were then stored in closed vials at 37 ℃. At predetermined time points (1,2,4,8,12,24,48 h), Lec and Lec (rsv) particle sizes were determined by DLS and recorded.
7. Lec (RSV) in vitro Release profiles of nanoparticles
Lec (rsv) (n ═ 3, after purification) was dispersed in 1ml PBS (pH 7.4), and then transferred to a Float-a-lyzer G2 dialysis apparatus (MWCO 100kD, Spectrum Lab) immersed in PBS (pH 7.4) at 37 ℃. At predetermined intervals (1,2,4,8,12,24,48,72,96 hours), 5 μ L of NP solution was removed from the dialysis set and mixed with 95L DMSO. After mixing well, the spectrophotometry of LN in each well was measured using a Synergy HT multimode microplate reader.
8. Cell culture
Human breast cancer cells BT474 were purchased from ATCC and cultured according to the protocol provided. The temperature was 37 ℃. Using RPMI-1640/F-12K medium containing 10% Fetal Bovine Serum (FBS), 100U/mL penicillin and 100g/mL streptomycin, the cells were cultured in a humidified cell culture chamber containing 5% CO 2.
9. In vivo intake experiment
To observe the uptake and internalization of Lec in vitro, we encapsulated Lec with FITC instead of RSV. Lec was fluorescently labeled with FITC, half the mass of the liposomes. BT474 cells were grown to-80% on a glass lid (12X 12 mm; Fisher Scientific, Texas, USA). After incubation for 1 hour with serum-free medium prior to addition of 200g/mL of medium containing Lec (FITC), the cells were washed 2 times with PBS and fixed with 4% (w/v) Paraformaldehyde (PFA). The fixed cells were mounted on glass slides filled with Dako fixing medium and detected using an Olympus Fluoview 1000 confocal microscope (Olympus Imaging Co, Tokyo, Japan).
10. In vitro anti-cancer Properties of tumors
To explore the anticancer properties of Lec (RSV), BT474 cells were seeded in 96-well plates (5000 per well), and various concentrations (10 μ M to 40 μ M) of Lec, RSV, Lec (RSV) were added to BT474 and cultured in a 5% cell incubator for 24 hours. After 24 hours, 10. mu.l of the alamar stain was added to each well, and after 2 hours of reaction in the dark, the reaction was read on a fluorescence spectrophotometer (Ex/Em. 500/525 nm).
11. Lec (RSV) ability to scavenge ROS in vitro
The ROS-scavenging ability of lec (RSV) on BT474 was tested using the ROS detection kit. Briefly, cells (1X 10)5/well) were placed in 6-well plates and incubated in an incubator with 5% CO2 for 24 hours. Culturing with fresh medium containing different concentrations of RSV and Lec (RSV) for 24 hr, discardingAfter the culture solution, 100. mu.L of 10. mu. mol/L DCFH-DA PBS solution was added and incubated in a cell incubator for 30 minutes in the absence of light. After the reaction, the cells were transferred to a 96-well black plate and cultured in a 5% CO2 incubator for 30 minutes. After completion, the cells were transferred to a 96-well black plate and detected by a multifunctional microplate reader at 500/525nm (Ex/Em).
12. Lec (RSV) in vivo tumor concentration assay
Mixing 1.2X 107Injecting 0.2mL of BT-474 cells into an immunodeficient mouse to establish a breast tumor model. When the tumor grows to 120-200 nm3To test the tumor targeting ability of lec (rsv). Mice were divided into 2 groups (n-3), one group was tested 1 hour after lec (rsv) injection, and the other group was tested 3 hours after lec (rsv) injection. Purified lec (RSV) was resuspended in PBS and each mouse was injected tail vein with 0.2ml of a suspension of lec (RSV) (2 mg/Kg) for 1 hour, 3 hours before examination under a live imager.
Second, result in
1. Characterization of Lec (RSV)
The ultimate goal of the present inventors was to develop a purely natural nanoparticle with natural anti-cancer properties. Specifically, lec (rsv) was synthesized as a co-antitumor therapeutic platform (fig. 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 fig. 1B (left), simply mixing lecithin with resveratrol does not self-encapsulate RSV and form nanoparticles. Only by the nano-precipitation method described above, lec (rsv) nanoparticles could be prepared and a clear lec (rsv) solution was observed, as shown in fig. 1B (right).
2. Properties of Lec and Lec (RSV) NPs
To determine the size and morphology of Lec and Lec (rsv), TEM and DLS analyses were performed. As shown in fig. 2A, unloaded lecithin nanoparticles appear spherical, while resveratrol-loaded lec (rsv) has a similar structure. Although the morphology was similar in both groups, the average size of lec (rsv) was larger. This observation indicates that encapsulation of RSV was successful, it does increase the average size of the nanoparticles compared to the control (151.0 ± 22.93nm vs 128.4 ± 19.88nm) (fig. 2C). The encapsulation efficiency of RSV was calculated around 85.16% (fig. 2D).
The size of the 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 nanoparticle size; the nanoparticles must be larger than 10nm to avoid renal filtration, but not too large in diameter, such as: particles larger than 200nm activate the complement system and are rapidly removed from the blood, accumulating in the liver and spleen. Therefore, the nanoparticles with a suitable size can achieve the technical effect to be achieved by the present application, in this embodiment, the range of the particle size of the lec (rsv) nanoparticles is 128-174 nm, the average size is 151nm, the lec (rsv) nanoparticles are close to an ideal size, are stable, have a large loading capacity, and have a good tumor penetration capacity.
3. Stability of Lec and Lec (RSV)
The stability of nanoparticles in a nano-delivery system is of critical importance because nanoparticles need to be circulated in vivo for a long time. Both Lec and Lec (rsv) nanoparticles were stable for 48 hours. To demonstrate the stability of lec (rsv), the inventors measured the size of lec (rsv) particles on day 1/4/8/5/30/60, respectively. As shown in fig. 3B, the size of lec (rsv) measured under DLS fluctuates very little in size in nanometers within 60 days, ranging from 100 to 200 nanometers, demonstrating that lec (rsv) has good stability. It is noteworthy that even after 12 months at 4 ℃, there was no deposit, i.e. lec (rsv) could be stored for a long period of time at 4 ℃, demonstrating the excellent stability of this nanosystem. Furthermore, the slow release of RSV after encapsulation by Lec was achieved with only 55% of the total release over 12 hours. Besides the performance of the nano-particle size, the nano-particle size is expected to be applied to clinical anticancer drugs, and the stability plays a crucial role in the storage and delivery capacity of the nano-particle size. Furthermore, the nanoparticle delivery system requires long circulation of the nanoparticles in vivo, so the stability of lec (rsv) plays a key role. In this study, the lec (rsv) size was measured using DSL and found to vary only slightly in size within 60 days. The stability of Lec (RSV) was demonstrated to be very good. Lec (RSV) can be stored for 12 months at 4 ℃ without aggregation and coagulation, and also shows that the nano platform has good stability. In combination with the properties of lec (rsv) as described above, lec (rsv) shows greater advantages over other drugs or nanosupport systems and also has potential for clinical use in the treatment of related disorders.
4. Lec and Lec (RSV) nanoparticle in vitro uptake capacity and cytotoxicity
Next, the inventors confirmed the in vitro absorptive capacity of Lec (RSV). FITC was encapsulated in Lec and Lec (rsv) particles, which were added to BT474 cells and cultured for 4 hours, respectively. After 4 hours of co-incubation, both Lec and Lec (rsv) showed significant uptake into the cytoplasm (fig. 4A and 4B). Next, we performed in vitro cytotoxicity experiments to observe the killing ability of RSV against cancer cells. As shown in FIG. 4C, RSV treatment was highly toxic to cells (IC 50-18. mu.M). Lec also has inherent tumor killing ability as an antioxidant, but is inferior to RSV (IC 50)>40 μ M) was significant. Notably, Lec (RSV) has a significant synergistic effect (IC 50-11 μ M) on the killing ability of tumor cells compared to Lec or RSV alone. Similarly, RSV cleared ROS significantly (p) compared to control or Lec<0.001), encapsulating RSV did not affect the biological activity of RSV (fig. 4D). As an ideal anticancer drug, besides the aforementioned particle size and stability, an important index is tumor cell enrichment capacity and cytotoxicity. In this example, the present inventors encapsulated resveratrol using naturally derived lecithin to construct nanoparticles and tested it for cellular uptake and cytotoxicity against BT474 breast cancer cells in vitro. After 4 hours of co-culture, both Lec and Lec (rsv) were apparently taken up into the cytoplasm of the cells. Then, the present inventors carried out an in vitro cytotoxicity test to observe the ability of RSV to kill tumor cells and found that RSV (IC)5011. mu.M) was significantly toxic to the cells. As an antioxidant, Lec also has some tumor killing ability, but not as pronounced as RSV (IC)5040. mu.M). It is noteworthy that when Lec is combined with RSV to form nanoparticles, there is better tumor killing than Lec alone or RSV alone. The same is true in the aspect of antioxidation, and Lec (RSV) has a synergistic effect among components and has an obvious effectIt is well known that. In addition, the RSV is encapsulated without changing the biological activity thereof, and the stability of the nanoparticle for encapsulating the main drug is further embodied.
5. Lec (RSV) in vivo tumor uptake assay
Based on the significant effect of the nanoparticles in the in vitro experiments, the inventors further performed in vivo tumor uptake experiments, specifically, in vivo tumor uptake experiments in the BT474 tumor-implanted model. Two groups of mice (n ═ 3) were given Lec (RSV + FITC) waiting 1 and 4 hours, respectively. As shown in fig. 5, it is clear that the 4-hour group lec (rsv) accumulation is significantly increased compared to the 1-hour group. It was shown that lec (rsv) is reliable as a tumor delivery system.
In general, in the present embodiment, at least: lec (RSV) has excellent stability and biocompatibility, has an inhibiting effect on BT474 breast cancer cells, and is less in cytotoxicity and safe. Lec (RSV) is expected to be applied to clinic to become an effective clinical antitumor drug.
Specifically, in the process of preparing resveratrol lecithin nanoparticles by using a nano-precipitation method, the inventor also adopts DMSO to prepare a Resveratrol (RSV) (100mg/mL) and lecithin (Lec) (100mg/mL) solution, adds 9 mu L or 11 mu L of Lec to 1 mu L of RSV, complements DMSO to 100 mu L, and prepares the resveratrol lecithin nanoparticles by adopting the same steps under the condition that the volume ratio of the mixed solution to the water phase is (0.05-0.15): 10. And the stability, tumor cell killing experiment and the ingestion experiment are carried out, and the experiment shows that the composition has the characteristics of equivalent and remarkable stability, synergistic killing effect and in-vivo ingestion.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (10)
1. A resveratrol lecithin nanoparticle is characterized by comprising resveratrol and lecithin wrapped and/or coupled on the resveratrol.
2. Resveratrol lecithin nanoparticles according to claim 1, comprising inner and outer layers of lecithin, wherein the resveratrol is encapsulated between the inner and outer layers of lecithin.
3. The resveratrol lecithin nanoparticle according to claim 1 or 2, wherein the mass ratio of resveratrol to lecithin is (9-11) to 1.
4. Use of the resveratrol lecithin nanoparticles as claimed in any one of claims 1-3 in the preparation of medicaments for scavenging free radicals and/or killing tumor cells.
5. The use of claim 4, wherein the tumor cells comprise breast cancer cells.
6. A tumor cell killing drug, which is characterized by comprising the resveratrol lecithin nanoparticles as claimed in any one of claims 1-3.
7. The method for preparing resveratrol lecithin nanoparticles as claimed in claim 1, which is characterized by comprising the steps of:
a1, respectively dissolving resveratrol and lecithin in organic solvents which can be mixed with water to obtain a resveratrol organic phase and a lecithin organic phase, adding the lecithin organic phase into the resveratrol organic phase, and then supplementing the organic solvents to obtain a mixed solution;
a2, adding the mixed solution into the water phase under the stirring condition to obtain a nano suspension;
a3, centrifuging and filtering to remove the organic solvent, and obtaining the resveratrol lecithin nanoparticles.
8. The method according to claim 7, wherein the organic solvent is dimethyl sulfoxide.
9. The preparation method of claim 7, wherein the mass ratio of the resveratrol to the lecithin in the mixed solution is (9-11) to 1; and/or the concentration of the resveratrol in the mixed solution is 0.9-1.1 mg/ml; and/or the volume ratio of the mixed solution to the water phase is (0.05-0.15) to 10.
10. Use of resveratrol and/or lecithin in the preparation of a medicament for scavenging free radicals and/or killing tumor cells.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023193389A1 (en) * | 2022-04-08 | 2023-10-12 | 中山大学孙逸仙纪念医院 | Resveratrol-lecithin nanoparticle, method for preparing same, and use thereof |
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| WO2023193389A1 (en) | 2023-10-12 |
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