CN114917356B - Nano-composite for regulating estrogen and preparation method and application thereof - Google Patents
Nano-composite for regulating estrogen and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano-composite for regulating estrogen and a preparation method and application thereof. The preparation method of the nano composite comprises the following steps: the naringenin compound, the diblock polymer formed by the hydrophilic end and the hydrophobic end, the cell penetrating peptide and the galactose are self-assembled to form stable spherical nanoparticles, namely the nano composite for regulating the estrogen. The nano composite prepared by the invention increases the water solubility and stability of naringenin by wrapping two-block polymer, namely polyethylene glycol-b-polycaprolactone, formed by a hydrophilic end and a hydrophobic end, and simultaneously, combines intestinal barrier penetrating peptide to assist naringenin compounds to pass through the intestinal barrier, thereby improving the blood entry concentration of naringenin; in addition, modification of galactose confers its liver targeting ability, further increasing the bioavailability of naringenin, and thus, it may be useful for the treatment of diseases associated with imbalance in estrogen homeostasis.
Description
Technical Field
The invention belongs to the technical field of nano-drugs, and particularly relates to a nano-composite for regulating estrogen, and a preparation method and application thereof.
Background
Estrogens are steroid hormones, mainly in the form of estradiol, estrone, estriol, etc. Estrogens have wide biological activity, play an important role in the physiological development of women and the occurrence and development of certain diseases, and mainly combine with estrogen receptors to affect various aspects of reproductive system, cardiovascular system, central nervous system, islet function, bone metabolism and the like. Unbalanced and prolonged exposure to supraphysiological concentrations of estrogen can bind to estrogen receptors, mediate the over-expression of various growth factors, promote cell growth and proliferation, while various metabolites of estrogen can form adducts with DNA, induce gene mutations, and produce direct genotoxicity. Clinically, estrogen-dependent diseases such as breast cancer, ovarian cancer and other gynecological tumor diseases are closely related to the increase of the levels of estrogen and toxic metabolites thereof.
Naringenin (NAR, molecular formula: C) 15 H 12 O 5 Molecular weight: 272.25 g/mol) is an aglycone of naringin, belonging to the flavanoid family, found in plants of the rosaceous, rutaceae and citrus genera. The traditional Chinese medicines such as dried orange peel, bitter orange, immature bitter orange and the like all contain naringenin. License plateIn vivo and in vitro experimental studies show that naringenin has pharmacological activities of resisting bacteria, reducing blood fat, resisting inflammation, resisting tumors and the like, has strong medicinal potential, but the application of naringenin in the field of estrogen regulation and control is still to be further explored.
Chemotherapeutic drugs are currently still important in the treatment of various diseases. However, the chemical drugs have poor water solubility, so that the chemical drugs have toxic and side effects on other body organs, cannot play a targeting role, have drug resistance and the like, and greatly limit the clinical application of the chemical drugs. However, with the development of new supramolecular self-assembly nano materials in modern medicine, the supramolecular self-assembly nano materials are widely applied as carriers of therapeutic drugs. The water solubility of the medicine can be greatly improved through the mediation effect of the self-assembled nano material, the bioavailability of the medicine is improved, and the controllability of the targeting property and the drug-loading capacity of the medicine is realized. The naringenin has the problems of low bioavailability and incapability of crossing intestinal barriers in oral administration in vivo, so that the development of a medicament with targeting property, high bioavailability and small toxic and side effects has important significance for treating various estrogen-related diseases.
Disclosure of Invention
The primary object of the present invention is to overcome the disadvantages and drawbacks of the prior art and to provide a method for preparing a nanocomposite for modulating estrogen.
Another object of the present invention is to provide the nano-composite for regulating estrogen prepared by the method.
It is still another object of the present invention to provide the use of the nanocomposite for modulating estrogens.
The purpose of the invention is realized by the following technical scheme:
a method for preparing a nanocomposite for modulating estrogen comprising the steps of: naringenin compounds, a diblock polymer formed by a hydrophilic end and a hydrophobic end, cell Penetrating Peptides (CPPs) and Galactose (GA) are self-assembled to form stable spherical nanoparticles, namely the nano-composite for regulating estrogen.
The two-block polymer formed by the hydrophilic end and the hydrophobic end is preferably polyethylene glycol-b-polycaprolactone (PEG-PCL).
The nano-composite can be prepared by any one of an anti-solvent precipitation method, a solvent evaporation method, a salting-out emulsification diffusion method, a nano-deposition method and a supercritical fluid technology.
The preparation method of the nano-composite for regulating estrogen specifically comprises the following steps:
(1) Dissolving polyvinylpyrrolidone (PVP) in water to obtain polyvinylpyrrolidone solution as water phase;
(2) Dissolving polyethylene glycol-b-polycaprolactone (PEG-PCL), naringenin compound, galactose-polyethylene glycol-b-polycaprolactone (GA-PEG-PCL) and penetration peptide-polyethylene glycol-b-polycaprolactone (CPP-PEG-PCL) in acetone to obtain mixed solution as organic phase;
(3) And injecting the organic phase into the aqueous phase under the stirring condition to obtain the nano-composite for regulating the estrogen.
The polyvinylpyrrolidone in the step (1) is preferably polyvinylpyrrolidone K29/32.
The concentration of the polyvinylpyrrolidone solution in the step (1) is 0.2-0.3 mg/mL; preferably 0.2mg/mL.
The naringenin compound in the step (2) has a structure shown in a formula I:
wherein R is-H, -OH or-CH 3.
R is preferably-OH, namely the naringenin compound is preferably Naringenin (NAR).
The molecular weight of the polyethylene glycol in the polyethylene glycol-b-polycaprolactone in the step (2) is 1000-4000 (preferably 2000), and the molecular weight of the polycaprolactone is 4000-16000 (preferably 6000).
The molecular weight of the polyethylene glycol in the galactose-polyethylene glycol-b-polycaprolactone in the step (2) is 1000-4000 (preferably 2000), and the molecular weight of the polycaprolactone is 4000-16000 (preferably 6000).
The molecular weight of the polyethylene glycol in the penetrating peptide-polyethylene glycol-b-polycaprolactone in the step (2) is 1000-4000 (preferably 2000), and the molecular weight of the polycaprolactone is 4000-16000 (preferably 6000).
The concentration of the polyethylene glycol-b-polycaprolactone (PEG-PCL) in the mixed solution in the step (2) is 18-20 mg/ml (preferably 18-19 mg/ml; more preferably 18 mg/ml), the concentration of the naringenin compound is 10-15 mg/ml (preferably 15 mg/ml), the concentration of the galactose-polyethylene glycol-b-polycaprolactone (GA-PEG-PCL) is 0.5-1 mg/ml (preferably 1 mg/ml), and the concentration of the penetrating peptide-polyethylene glycol-b-polycaprolactone (CPP-PEG-PCL) is 0.5-1 mg/ml (preferably 1 mg/ml).
The mass ratio of the polyethylene glycol-b-polycaprolactone (PEG-PCL), the naringenin compound, the galactose-polyethylene glycol-b-polycaprolactone (GA-PEG-PCL) and the penetration peptide-polyethylene glycol-b-polycaprolactone (CPP-PEG-PCL) in the step (2) is 18-20: 10-15: 0.5 to 1:0.5 to 1.
The volume ratio of the organic phase to the aqueous phase in the step (2) is 1-50; preferably 1.
The stirring speed in the step (2) is 800-1000 rpm; preferably 1000rpm.
A nanocomposite for regulating estrogen, prepared by the method of any one of the above.
The application of the nano-composite for regulating the estrogen in preparing the medicines for preventing and/or treating the diseases related to the imbalance of the estrogen homeostasis.
The diseases related to imbalance of estrogen homeostasis comprise benign diseases dependent on estrogen and malignant diseases dependent on estrogen.
The benign disease of estrogen dependence is at least one of hyperplasia of mammary glands, polycystic ovary syndrome, hysteromyoma, endometriosis, preeclampsia, osteoporosis, climacteric syndrome, premenstrual syndrome and infertility.
The estrogen-dependent malignant disease is at least one of breast cancer, colorectal cancer, prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and cervical cancer; preferably breast cancer.
The use of the nanocomposite for modulating estrogen in the manufacture of a medicament for modulating estrogen levels.
The nanometer compound for regulating estrogen is applied to preparing the medicine for inhibiting tumor proliferation and metastasis.
The tumor is at least one of breast cancer, colorectal cancer, prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and cervical cancer; preferably breast cancer.
The tumor proliferation and metastasis are preferably breast cancer cell proliferation and metastasis caused by estrogen E2.
The medicine contains one or more pharmaceutically acceptable carriers or auxiliary materials.
The medicine can be further prepared into a pharmaceutical preparation by adopting a conventional method in the field; such as oral dosage forms, etc.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention provides a nano-composite for regulating estrogen, which is composed of Naringenin (NAR), a diblock polymer (PEG-PCL) consisting of a hydrophilic end and a hydrophobic end, cell Penetrating Peptides (CPPs), galactose (GA) and the like, and is self-assembled by methods such as an anti-solvent precipitation method and the like to form stable spherical nano-particle NAR/CPP/GA NPs, namely the nano-composite of the invention constructs the naringenin nano-composite with intestinal penetration capability and liver targeting capability by amphiphilic polymer and modifying the CPPs and the GA on the amphiphilic polymer; wherein NAR (active ingredient with effective dose) has the function of promoting estrogen metabolism, CPPs can improve the penetration capacity of the nano-composite to intestinal barriers, GA has targeting specificity to liver cells, and the accumulation of the nano-composite in the liver cells is increased.
2. The nano-composite prepared by the invention has the characteristics of liver targeting and intestinal barrier penetration capacity, thereby promoting liver estrogen metabolism, and being used for treating diseases related to estrogen homeostatic imbalance, such as breast cancer, hyperplasia of mammary glands, polycystic ovary syndrome, uterine fibroids and the like; meanwhile, the nano-composite can be stable for more than two weeks in a pH 7.35 environment and stable for more than 2 hours in a pH 1.5 environment (pH 7.35 is approximate to pH value in blood, pH 1.5 is approximate to pH value in gastric acid); in addition, cell penetrating peptides (CPP, rrrrrrrrrrrrrr) can help nanoparticles easily cross intestinal epithelial cells into the blood, overcoming intestinal barrier barriers; galactose (Galactose, GA) has the effect of targeting the liver, and ensures that the naringenin can reach the effective concentration in the liver.
3. The problems of poor water solubility and low in-vivo bioavailability of naringenin can be solved by constructing the nano-composite, and meanwhile, the problem that intestinal barriers cannot be crossed by oral administration is solved by modifying cell penetrating peptide on the surface of the nano-composite, so that the nano-composite with liver targeting property is constructed; in addition, the naringenin nano-drug can directly reduce the estrogen concentration in the liver in a targeted manner, and a new strategy is provided for breast cancer estrogen targeted therapy.
4. According to the invention, the water solubility and stability of naringenin are increased by wrapping polyethylene glycol-b-polycaprolactone, and meanwhile, the naringenin is assisted to pass through an intestinal barrier by combining intestinal barrier penetrating peptide, so that the blood entry concentration of the naringenin is increased; in addition, modification of galactose endows the liver with targeting ability, so that the bioavailability of naringenin is further improved, and a new way and a new tool are provided for endocrine treatment.
5. The invention develops a treatment strategy and a preparation method of a novel estrogen regulating medicament, has the characteristics of good activity, targeting property, small toxic and side effects and convenient and fast oral administration, can be used for treating various estrogen-related diseases (including benign diseases depending on estrogen and malignant diseases depending on estrogen), can ensure that the estrogen level in a body reaches a steady state after administration, has no systemic side effect and does not generate toxicity.
6. The breast cancer zebra fish model proves that when the dose of naringenin in the naringenin nano compound is 100nM, the compound has an obvious inhibiting effect on the development of estrogen-induced zebra fish breast tumors, and has no obvious toxicity on the zebra fish; meanwhile, in-vitro activity experiments prove that the naringenin nano-drug has the characteristic of promoting estrogen metabolism and liver targeting, and can be used for preparing drugs for treating diseases related to estrogen homeostasis imbalance, such as breast cancer.
Drawings
FIG. 1 is a particle size distribution diagram of NAR/GA/CPP NPs of the present invention.
FIG. 2 is a Zeta potential diagram of the NAR/GA/CPP NPs of the present invention.
FIG. 3 is a transmission electron micrograph of NAR/GA/CPP NPs of the present invention.
FIG. 4 is a graph showing the results of the stability test of NAR/GA/CPP NPs of the present invention; wherein, A is the stability experiment result of the solute of NAR/GA/CPP NPs at pH = 1.5; b is the result of the stability experiment of the solute at pH =7.35 for NAR/GA/CPP NPs.
FIG. 5 is a graph showing the results of the cytotoxicity test of NAR/GA/CPP NPs of the present invention; wherein, A is LO2 cell; b is Caco2 cell; c is RAW264.7 cells; d is Mcf-7 cells.
FIG. 6 is a graph showing the results of experiments on dual-luciferase reporter genes in which NAR/GA/CPP NPs of the present invention promote the expression of estrogen phosphotransferase EST in hepatocytes LO2 (the results show that NAR/GA/CPP NPs have superior effects compared to NAR monomers at the same dose).
FIG. 7 is a graph showing the results of experiments in which NAR/GA/CPP NPs of the present invention target hepatocytes; wherein, A is the uptake result of NAR/GA/CPP NPs in different cells (the result shows that the accumulation of NAR/GA/CPP NPs in the liver cells is obviously higher than that of other cells in all tested cells, which indicates that the NAR/GA/CPP NPs have the tendency of targeting the liver cells); b is the uptake result of NAR/GA/CPP NPs in LO2 cells before and after adding free GA (the result shows that the targeting of NAR/GA/CPP NPs to liver cells is influenced after adding free GA, which indicates that the liver targeting of NAR/GA/CPP NPs is mediated by GA).
FIG. 8 is a graph of experimental results of the inhibition of proliferation and metastasis of breast cancer in zebra fish by NAR/GA/CPP NPs (results show that estrogen can significantly promote proliferation and metastasis of breast cancer cells, and has a superior effect in inhibiting proliferation and metastasis of breast cancer compared to NAR monomer NAR/GA/CPP NPs); wherein A is the result of inhibiting the proliferation and metastasis of the breast cancer tumor of the zebra fish by NAR/GA/CPP NPs; B. c is tumor fluorescence and metastasis statistical map respectively.
Detailed Description
The present invention will be described in further detail with reference to examples, but it should be understood that these examples are for illustrative purposes only and are not intended to limit the present invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
Materials referred to in the following examples:
polyethylene glycol-b-polycaprolactone (PEG-PCL, PEG: mw =2000 PCL, mw = 6000), galactose-polyethylene glycol-b-polycaprolactone (GA-PEG-PCL, PEG: mw =2000 PCL Mw = 6000) and the penetrating peptide-polyethylene glycol-b-polycaprolactone (CPP-PEG-PCL, PEG: mw =2000 PCL Mw = 6000) were purchased from sienna ruixi biotechnology company. Naringenin (NAR): purchased from Nantong biological medicine company, with a purity of more than or equal to 99 percent.
Human normal hepatocytes LO2 and mouse breast cancer cells E0771 were purchased from weijia, guangzhou; human colorectal adenocarcinoma cells Caco2, mouse monocyte macrophage RAW264.7, human breast cancer cells Mcf-7, human umbilical vein endothelial cells HUVEC, and human breast cells HBL-100 were purchased from Kaikyi.
EXAMPLE 1 preparation of nanocomposites
(1) Preparing aqueous and organic phases
(1) Preparing a water phase: dissolving polyvinylpyrrolidone (PVP) K29/32 in water to obtain a polyvinylpyrrolidone K29/32 mother solution with the concentration of 0.2mg/ml;
(2) preparing an organic phase: dissolving polyethylene glycol-b-polycaprolactone (PEG-PCL), naringenin (NAR), galactose-polyethylene glycol-b-polycaprolactone (GA-PEG-PCL) and penetration peptide-polyethylene glycol-b-polycaprolactone (CPP-PEG-PCL) in acetone to obtain mixed solution of PEG-PCL, NAR, GA-PEG-PCL and CPP-PEG-PCL; wherein the concentration of PEG-PCL is 18mg/ml; NAR concentration is 15mg/ml; the concentration of GA-PEG-PCL is 1mg/ml; the concentration of CPP-PEG-PCL is 1mg/ml.
(2) Preparation of the nanocomposite by anti-solvent precipitation: before injection, the water phase is placed on a magnetic stirrer, the magnetic stirrer is turned on, the rotating speed is 1000rpm, 0.2mL of organic phase is injected into 10mL of water phase by using a 1mL sterile injector, and then the stirrer is turned off, so that NAR/GA/CPP NPs are obtained.
Example 2 characterization test
(1) The NAR/GA/CPP NPs prepared in step (2) of example 1 were subjected to particle size and potential measurements while NAR monomer was dissolved in acetone as a control at a concentration of 15mg/ml. The instrument used was a nanobook 90Plus PALS (brook hain instruments, usa). The results are shown in FIGS. 1 and 2: NAR monomer in water, average particle size is 167838nm, electric potential is-13.943 mV; NAR/GA/CPP NPs have a particle size of 142.898nm in water and a potential of-18.83 mV.
(2) Transmission electron microscopy tests were performed on the morphology of the NAR/GA/CPP NPs prepared in step (2) of example 1. The results are shown in FIG. 3: the transmission electron microscope image shows that the prepared nanoparticles are round.
(3) The stability of NAR/GA/CPP NPs prepared in step (2) of example 1 was tested: suspending NAR/GA/CPP NPs in a solute (1 mol/ml dilute hydrochloric acid is taken, water is added for dilution, the pH is adjusted to 1.5, 1g of pepsin is added into each 100ml of solution, the mixture is uniformly mixed, and a sterile filter head with the particle size of 0.2 mu m is used for filtration) with the pH value of 1.5 according to the mass ratio of 1; and NAR/GA/CPNPs were suspended in a solute at pH 7.35 (1: 19ml of 0.2mol/L NaH was taken 2 PO 4 And 81ml of 0.2mol/L Na 2 HPO 4 ·12H 2 O, mixed well, filtered through a 0.2 μm sterile filter head for use), and the change in particle size was measured on 1, 3, 5, and 7 days. As shown in FIG. 4, NAR/GA/CPP NPs showed no significant change in particle size within 2 hours in the solute with pH = 1.5; in the solute with pH =7.35, there was no significant change in particle size within 14 days.
EXAMPLE 3 cell culture
Human normal liver cells (LO 2), human colorectal adenocarcinoma cells (Caco 2), mouse mononuclear macrophages (RAW 264.7) andhuman breast cancer cells (Mcf-7) were inoculated into DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 1% penicillin (100U/mL) and streptomycin (100. Mu.g/mL), and then the cells were placed at 37 ℃ and 5% CO 2 And 95% relative humidity. The culture medium was replaced every 24 hours and the cells were subcultured by observing their growth under a microscope to 70-80%.
Example 4 evaluation of safety of NAR/GA/CPP NPs at cellular level
To demonstrate the safety of NAR/GA/CPP NPs at the cellular level, their cell viability was determined by MTT assay. The cells obtained in the subculture of example 3 were each 5X 10 3 The cells/well were seeded in 96-well culture plates, the cells in the well plates were first incubated for 24 hours, the medium was removed, then different sets (free NAR and NAR/GA/CPP NPs, respectively) were set to incubate in the well plates, setting different concentrations in the range of 0, 10, 20, 40, 80, 160, 320 μ M. After an additional 24 hours of incubation, 10. Mu.L of MTT (5 mg/mL in PBS) was added to each well and after 4 hours, 150. Mu.L of dimethyl sulfoxide (DMSO) was added to each well in place of the medium. The absorbance value at 570nm (A570 nm) was read using a microplate reader measurement. Cell viability was expressed as the percentage of the administered group at a570nm relative to the control group. The results are shown in FIG. 5: in the concentration range of 10-320 mu moL/L, the NAR/GA/CPP NPs group has no obvious difference in cell viability no matter compared with the NAR group or the control group.
Example 5 Dual luciferase reporter genes
The effect of NAR/GA/CPP NPs prepared in step (2) of example 1 in regulating EST enzyme expression in hepatocytes was examined. LO2 cells were seeded into 6-well plates, 2X 10 5 Each cell per well. mu.L of the transfection reagent was added to 450. Mu.L of complete medium (10% (v/v) Fetal Bovine Serum (FBS) +90% (v/v) DMEM medium) (no diabody), 150. Mu.L of SULT1E1 promoter cloning plasmid (GeneCopoeia) was added to 350. Mu.L of complete medium (10% (v/v) Fetal Bovine Serum (FBS) +90% (v/v) DMEM medium) (no diabody), the two were mixed, left at room temperature for 30min, 200. Mu.L of the mixture was added to 1.8mL of complete medium (10% (v/v) Fetal Bovine Serum (FBS) +90% (v/v) DMEM medium) (no diabody), and added to a 6-well plate. After 24 hours, the rotor will be rotatedSuccessfully stained LO2 cells were plated in 96-well plates at 5X 10 3 Individual cells/well. After the cells are attached to the wall, NAR and NAR/GA/CPP NPs with different concentration ranges are respectively given for intervention, so that the final concentration of NAR is 0, 1, 5, 10, 50, 100 and 200nM. After 24 hours of intervention, cell culture medium was taken. Dividing the cell culture medium into two parts, wherein one part is used for Gluc detection, the other part is used for Seap detection, and adding Gluc working solution and Seap working solution according to the using instruction according to the time, and placing the mixture into an instrument for detection.
The results are shown in FIG. 6: the results show that NAR/GA/CPP NPs have better effect compared with NAR monomer under the same dosage.
Example 6 targeting of hepatocytes
The effect of targeting the hepatocytes by the NAR/GA/CPP NPs prepared in step (2) of example 1 was examined.
Evaluation of NAR/GA/CPP NPs liver targeting ability in different cells: place the slide on the bottom of the well plate, and mix LO2, caco2, RAW264.7, mcf-7, HBL-100, HUVEC cells at 1X 10 5 Density per well was seeded in 12-well plates. At 37 ℃ C, 5% CO 2 Incubate for 24 hours. The medium was replaced with DMEM (Gibco) containing NAR/GA/CPP NPs at 100nM, incubated in the dark for 30min with NAR (100 nM) in DMEM as a control, and the extracellular NAR/GA/CPP NPs were washed out with ice PBS buffer, and kept in the dark for the following procedures. PBS was removed, 500. Mu.L of ice paraformaldehyde was added to each well, and incubation was performed for 10min. Gently washed with PBS buffer for 5min, PBS was aspirated off, 200. Mu.L of DAPI was added to each well, and incubated for 20min. Extracellular DAPI was aspirated off with PBS buffer and washed 3 times for 5min each. Sealing the film, observing and recording the fluorescence intensity by a confocal microscope. The experiment was repeated 3 times, and fluorescence values were collected three times and averaged.
After addition of free Galactose (GA), NAR/GA/CPP NPs were evaluated for liver targeting ability: place the slide on the bottom of the well plate and place the LO2 cells at 1X 10 5 Density per well was seeded in 12-well plates. At 37 ℃ C, 5% CO 2 And culturing for 24 hours in an incubator. The free GA intervention group was supplemented with 100mM GA 30min in advance, followed by replacement of the original medium (freeGA + NAR/GA/CPNPs) with DMEM (Gibco) containing 100nM NAR/GA/CPNPs, and the control group was directly treated with the medium containing NAR (100 nM) or NA without GA interventionDMEM medium of R/GA/CPP NPs (100 nM) is used for replacing the original medium, after incubation for 30min in the dark, the extracellular NAR/GA/CPP NPs are washed off by using ice PBS buffer solution, and the following operations are carried out in the dark. PBS was aspirated off, 500. Mu.L of ice paraformaldehyde was added to each well, and incubation was performed for 10min. Gently washed with PBS buffer for 5min, PBS was aspirated off, 200. Mu.L of DAPI was added to each well, and incubated for 20min. Extracellular DAPI was aspirated off with PBS buffer and washed 3 times for 5min each. Sealing the film, observing and recording the fluorescence intensity by a confocal microscope. The experiment was repeated 3 times, and fluorescence values were collected three times and averaged.
The results are shown in FIG. 7: FIG. 7A is the results of the uptake of NAR/GA/CPP NPs in different cells, showing that the accumulation of NAR/GA/CPP NPs in hepatocytes (LO 2) was significantly higher in all tested cells than in other cells, indicating that NAR/GA/CPP NPs have a tendency to target hepatocytes; FIG. 7B is a comparison of the uptake of NAR and NAR/GA/CPP NPs in LO2 cells, and the results of the addition of NAR/GA/CPP NPs 30min after free GA intervention, and the uptake of NAR/GA/CPP NPs in LO2 cells. Comparing the NAR with the NAR/GA/CPP NPs shows that the NAR/GA/CPP NPs are accumulated more in LO2 cells, and comparing the NAR/GA/CPP NPs with freeGA + NAR/GA/CPP NPs, the results show that the targeting of the NAR/GA/CPP NPs to the liver cells is influenced in the prognosis of free GA stem, and the liver targeting of the NAR/GA/CPP NPs is mediated by GA.
Example 7 evaluation of the in vivo efficacy of Zebra fish
The effect of NAR/GA/CPP NPs prepared in step (2) of example 1 on inhibiting proliferation and metastasis of breast cancer in zebra fish was examined.
In order to test the effect of naringenin on proliferation and metastasis of breast cancer in vivo, a zebra fish breast cancer xenograft model was constructed using mouse breast cancer cell E0771. E0771 cells were stained with 5. Mu.M of the membrane orange-red fluorescent probe DiI. After 48 hours of fertilization, 200 Dil stained E0771 cells were injected into each wild type zebrafish embryo and observed microscopically. Zebrafish carrying E0771 cells were placed in 6-well plates (10 fish per well) and drug intervention was added separately, with a final concentration of 40 μ M estrogen E2 and 100nM naringenin, and NAR monomers were dissolved in acetone (diluted to a final concentration of 100 nM) as a control, and triplicates were set with no drug added as a blank control (Conrtol). After 24 hours, the growth and transfer of E0771 cells in zebrafish was monitored by inverted fluorescence microscopy. And counted using Image J.
The results are shown in FIG. 8: the result shows that the estrogen E2 can obviously promote the proliferation and metastasis of the breast cancer cells, and compared with an NAR monomer, the NAR/GA/CPP NPs have better effect on inhibiting the proliferation and metastasis of the breast cancer.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for preparing a nanocomposite for modulating estrogen comprising the steps of:
(1) Dissolving polyvinylpyrrolidone in water to obtain polyvinylpyrrolidone solution as water phase;
(2) Dissolving polyethylene glycol-b-polycaprolactone, naringenin, galactose-polyethylene glycol-b-polycaprolactone and penetration peptide-polyethylene glycol-b-polycaprolactone in acetone to obtain a mixed solution as an organic phase;
(3) And injecting the organic phase into the aqueous phase under the stirring condition to obtain the nano-composite for regulating the estrogen.
2. The method for preparing a nanocomposite for modulating estrogens according to claim 1, wherein:
the polyvinylpyrrolidone in the step (1) is polyvinylpyrrolidone K29/32.
3. The method for preparing a nanocomposite for modulating estrogens as claimed in claim 1, wherein:
the concentration of the polyvinylpyrrolidone solution in the step (1) is 0.2-0.3 mg/mL;
the molecular weight of polyethylene glycol in the polyethylene glycol-b-polycaprolactone in the step (2) is 1000 to 4000, and the molecular weight of polycaprolactone is 4000 to 16000;
the molecular weight of polyethylene glycol in the galactose-polyethylene glycol-b-polycaprolactone in the step (2) is 1000 to 4000, and the molecular weight of polycaprolactone is 4000 to 16000;
the molecular weight of polyethylene glycol in the penetrating peptide-polyethylene glycol-b-polycaprolactone in the step (2) is 1000 to 4000, and the molecular weight of polycaprolactone is 4000 to 16000;
the concentration of the polyethylene glycol-b-polycaprolactone in the mixed solution in the step (2) is 18-20 mg/ml, the concentration of naringenin is 10-15 mg/ml, the concentration of the galactose-polyethylene glycol-b-polycaprolactone is 0.5-1 mg/ml, and the concentration of the penetrating peptide-polyethylene glycol-b-polycaprolactone is 0.5-1 mg/ml;
the volume ratio of the organic phase to the aqueous phase in the step (2) is 1 to 50.
4. The method for preparing a nanocomposite for modulating estrogens as claimed in claim 3, wherein:
the concentration of the polyvinylpyrrolidone solution in the step (1) is 0.2mg/mL;
the concentration of the polyethylene glycol-b-polycaprolactone in the mixed solution in the step (2) is 18mg/ml, the concentration of naringenin is 15mg/ml, the concentration of the galactose-polyethylene glycol-b-polycaprolactone is 1mg/ml, and the concentration of the penetrating peptide-polyethylene glycol-b-polycaprolactone is 1mg/ml;
the volume ratio of the organic phase to the aqueous phase in the step (2) is 1.
5. A nanocomposite for modulating estrogen, comprising: prepared by the method of any one of claims 1 to 4.
6. Use of a nanocomposite for modulating estrogens according to claim 5 for the manufacture of a medicament for the prevention and/or treatment of disorders associated with imbalances in estrogen homeostasis, wherein:
the diseases related to the imbalance of estrogen homeostasis comprise benign diseases depending on estrogen and malignant diseases depending on estrogen;
the benign disease of estrogen dependence is at least one of hyperplasia of mammary glands, polycystic ovary syndrome, hysteromyoma, endometriosis, preeclampsia, osteoporosis, climacteric syndrome, premenstrual syndrome and infertility;
the estrogen-dependent malignant disease is at least one of breast cancer, colorectal cancer, prostate cancer, uterine cancer, ovarian cancer and cervical cancer.
7. Use according to claim 6, characterized in that: the uterine cancer is endometrial cancer.
8. Use of a nanocomposite for modulating estrogen according to claim 5 in the manufacture of a medicament for modulating estrogen levels.
9. Use of the nanocomposite for modulating estrogens according to claim 5 in the manufacture of a medicament for inhibiting tumor proliferation and metastasis, wherein:
the tumor is at least one of breast cancer, colorectal cancer, prostate cancer, uterine cancer, ovarian cancer and cervical cancer.
10. Use according to claim 9, characterized in that: the uterine cancer is endometrial cancer.
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