CN115804762A - Macrophage membrane coated ectopic endometrium targeted nanoparticle, preparation method and application - Google Patents
Macrophage membrane coated ectopic endometrium targeted nanoparticle, preparation method and application Download PDFInfo
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Abstract
The invention discloses an ectopic endometrium targeting nanoparticle coated by a macrophage membrane, a preparation method and application, belonging to the technical field of pharmaceutical preparations, wherein the targeting nanoparticle comprises an inner core and an M1 type macrophage membrane coated outside the inner core; the inner core is a drug nanoparticle or a drug lipid nanoparticle, and the drug is selected from estrogen antagonist and/or estrogen synthesis inhibitor. The targeted nanoparticles utilize the natural targeting function of a CCL2-CCR2 mechanism to deliver estrogen antagonist and/or estrogen synthesis inhibitor to the focus of ectopic endometrium in a targeted manner, play a role of locally antagonizing estrogen or locally reducing the estrogen level, antagonize the growth promotion effect of estrogen on the ectopic endometrium, ensure that the ectopic endometrium is atrophied and necrotized, and achieve the aim of treating endometriosis.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an ectopic endometrium targeted nanoparticle coated by a macrophage membrane, a preparation method and application thereof.
Background
Endometriosis (EMs) refers to the condition that endometrial tissue with growth function appears in body parts outside uterine cavity, such as abdominal cavity, ovary and the like, and is a common gynecological disease which seriously affects the physical and mental health and the life quality of women, and the incidence rate of women of childbearing age is more than 20%. The clinical manifestations of infertility, chronic pelvic pain, dysmenorrhea, menstrual disorder and the like are benign lesions, but have malignant biological behaviors of planting, transferring, infiltrating, relapse and the like, seriously affect the life quality of patients and are also difficult to treat symptoms which trouble patients and doctors.
At present, the endometriosis is mainly treated by adopting operations and medicines clinically, the disease cannot be completely eradicated by excising the ectopic intima through operations, the symptoms of a patient can only be relieved, complications of urinary tracts, intestinal tracts and blood vessel parts can be caused, and the postoperative pain and the recurrence rate of the ectopic intima focus are still very high.
Drug therapy dominates long-term treatment of endometriosis, of which hormone-related therapies are well-established as effective treatments. The ectopic endometrium is in a local high estrogen state, and estrogen can stimulate the proliferation of ectopic endometrium cells, promote the growth of the ectopic endometrium and accelerate the development of endometriosis. It is well recognized that effective hormonal therapy is the treatment of endometriosis by lowering the estrogen levels in the patient, and that inhibition of sex hormones is used. The purpose of treating endometriosis is achieved by reducing the estrogen level of the whole body of a patient and causing atrophy, degeneration and necrosis of ectopic endometrium. However, the blood estrogen level is reduced, the physiological function of the hormone dependent organs of the whole body is interfered, the side effect is obvious, and the vasomotor symptoms such as hot flushes, night sweats, vaginal dryness, nausea, headache and the like and the adverse reactions such as irreversible bone loss and the like can appear. Long-term hypoestrogenemia can cause primordial follicle reduction and follicular atresia, cause damage to ovarian function, cause infertility, simultaneously cause symptoms such as osteoporosis and dyslipidemia, and influence the life quality of patients.
In addition, chinese patent publication No. CN106539790A discloses a compound for treating endometriosis, which has the following structure:
the compound can mainly improve pelvic local blood circulation, relieve pelvic adhesion, regulate disordered cellular immune function, improve existence and abnormality of autoimmune reaction, but only relieve endometriosis; chinese patent documents CN103169918A, CN104083551A and the like also disclose a traditional Chinese medicine composition for treating endometriosis, but the treatment mechanism is not clear, and the curative effect needs to be further verified.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an ectopic endometrium targeting nanoparticle coated by a macrophage membrane, the nanoparticle utilizes the natural targeting function of a CCL2-CCR2 mechanism to deliver a medicament to an ectopic endometrium focus in a targeting manner, so that the local estrogen level is reduced, and the treatment of endometriosis can be realized under the condition of not reducing the life quality of a patient.
The technical scheme is as follows:
an ectopic endometrium targeted nanoparticle coated by a macrophage membrane comprises an inner core and an M1 type macrophage membrane coated outside the inner core; the inner core is a drug nanoparticle or a drug lipid nanoparticle, and the drug is selected from estrogen antagonist and/or estrogen synthesis inhibitor.
The M1 type macrophage high expression chemokine receptor CCR2 and the ectopic endometrium high expression chemokine ligand CCL2 recruit CCR2 high expression cells to move to ectopic endometrium positions through a CCL2-CCR2 mechanism, and after the M1 type macrophage membrane is coated with the drug nanoparticles or the drug lipid nanoparticles, the M1 type macrophage membrane can deliver drugs to the ectopic endometrium in a targeted mode to play a natural targeting role.
M1 type macrophage is obtained by inducing and differentiating M0 type macrophage, and M1 type macrophage membrane is extracted from M1 type macrophage; alternatively, the M1 type macrophage is formed by inducing and differentiating mouse mononuclear macrophage leukemia cell RAW264.7 by lipopolysaccharide stimulation.
The inner core can be a nanoparticle consisting of the drug alone or a drug lipid nanoparticle formed by lipid encapsulation of the drug, and preferably, the weight ratio of the drug to the lipid is 1:4-10.
The lipid comprises at least one of monoglyceride, lecithin and stearic acid, preferably, the mass ratio of monoglyceride, lecithin and stearic acid in the lipid is 1-10:0-5:0-10.
Preferably, the particle size of the drug nanoparticles or the drug lipid nanoparticles is 50-200nm.
Estrogen antagonists exert a therapeutic effect by antagonizing the effects of estrogen at ectopic endometrial sites, preferably bazedoxifene; an estrogen synthesis inhibitor for lowering local estrogen level by inhibiting estrogen synthesis in ectopic endometrial sites for therapeutic effect, preferably anastrozole.
Preferably, the mass ratio of total protein to drug in the membrane of the M1-type macrophage is 1:0.1-1, the medicine loading amount under the parameters is 5% -50%, and the in vivo targeting of the mouse model with endometriosis can be effectively realized.
The invention also provides a preparation method of the ectopic endometrium targeted nanoparticle coated by the macrophage membrane, which specifically comprises the following steps:
(1) Preparing the drug or the mixture of the drug and lipid by a precipitation method to obtain drug nanoparticles or drug lipid nanoparticles;
(2) The M1 type macrophage membrane and the drug nanoparticles, or the M1 type macrophage membrane and the drug lipid nanoparticles are prepared into the ectopic endometrium targeting nanoparticles coated by the macrophage membrane by an ultrasonic method, an extrusion method or a microfluidic method.
Preferably, the preparation method of the drug nanoparticles comprises the following steps: dissolving anastrozole in ethanol to prepare a drug solution with the concentration of 10-50mg/ml, rapidly injecting the ethanol solution of anastrozole into water with the volume 10 times that of the drug solution under the condition of heating and stirring at 60 ℃, continuously stirring for 0-5min, and cooling to room temperature to obtain the drug nanoparticles.
Preferably, the preparation method of the drug lipid nanoparticle comprises the following steps: dissolving bazedoxifene and lipid in ethanol according to a certain proportion under the condition of heating, heating at 60 ℃ and stirring, quickly injecting the ethanol solution of bazedoxifene and lipid into water with the volume of 10 times of that of the solution, continuously stirring for 0-5min, and cooling to room temperature to obtain the drug lipid nanoparticles.
The invention also provides application of the heterotopic endometrium targeted nanoparticle coated by the macrophage membrane in preparing a medicinal preparation for treating endometriosis.
Preferably, the pharmaceutical preparation for treating endometriosis is an injection preparation, and the dosage is 1.5mg/kg.
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts an ectopic endometrium targeted M1 type macrophage membrane, utilizes the natural targeting function of a CCL2-CCR2 mechanism to deliver estrogen receptor antagonist bazedoxifene or estrogen synthetase inhibitor anastrozole to the ectopic endometrium focus in a targeted manner, plays a role in locally antagonizing estrogen or locally reducing the estrogen level, antagonizes the growth promoting effect of estrogen on the ectopic endometrium, leads the ectopic endometrium to be atrophied and necrotized, and achieves the aim of treating endometriosis. The invention avoids the prior hypothalamus-pituitary-ovary axis systemic hormone therapy, avoids the side effects caused by systemic hormone level change caused by the systemic hormone therapy, and improves the life quality of patients.
Drawings
FIG. 1 is a transmission electron microscope image of an ectopic endometrium targeted nanoparticle coated by a macrophage membrane vesicle and a macrophage membrane, wherein A is M0-CNVs, B is M1-CNVs, and C is M1-CNVs/BAZ-SLN.
FIG. 2 is a fluorescent photograph of the uptake of migrating M0-CNVs and M1-CNVs by Ishikawa cells.
FIG. 3 is the in vivo fluorescence distribution diagram of Dil labeled lipid nanoparticles SLN, M0-CNVs and M1-CNVs in endometriosis model animals.
FIG. 4 is a graph showing growth inhibition of model cells by bazedoxifene in an experimental group and a control group.
Detailed Description
The invention will be further elucidated with reference to the embodiments and the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 preparation of M1-type macrophage membrane
Mouse mononuclear macrophage leukemia cell RAW264.7 cell is adopted, 100ng/mL lipopolysaccharide is added into culture solution to be incubated with the cell for 12h, and M1 type macrophage is induced and formed. Respectively scraping induced M1 type macrophages and uninduced M0 type macrophages with a cell scraper, suspending in hypotonic solution, repeatedly freezing and thawing in a liquid nitrogen tank and a 37 ℃ water bath kettle for 3 times, centrifuging at 10000g for 20min, discarding precipitate, and collecting supernatant; ultracentrifuging the supernatant for 100000g 1h, and collecting the precipitate; after resuspension of PBS, 100000g of centrifugation were repeated twice. And (4) resuspending the PBS to obtain M0 type and M1 type macrophage membranes respectively. The protein concentration of the collected cell membranes was measured by the Bradford protein quantification kit and was 6.9mg/ml and 7.7mg/ml, respectively.
Repeatedly extruding the collected macrophage membranes for 20 times through 400nm and 200nm filter membranes respectively by using a liposome extruder to obtain M0 type macrophage membrane vesicles (M0-CNVs) and M1 type macrophage membrane vesicles (M1-CNVs), preparing a sample, dyeing by phosphotungstic acid, observing by a transmission electron microscope, wherein the forms of the prepared sample are respectively shown as A and B in the attached drawing 1, and the prepared sample is dried and has no three-dimensional form, so that the prepared sample is not spherical in the electron microscope but irregular; the particle size of the nano vesicle is about 50nm through electron microscope observation.
Example 2 preparation of macrophage membrane-coated ectopic endometrial targeted nanoparticles
Dissolving anastrozole in ethanol to obtain a 10mg/ml drug solution, quickly injecting the ethanol solution of anastrozole into 10 times of water by volume under the condition of heating and stirring at 60 ℃ by adopting an aqueous solvent diffusion method, continuously stirring, and cooling to room temperature to prepare anastrozole nanoparticle suspension.
The M1 type macrophage membrane and anastrozole nanoparticle suspension prepared in the example 1 is fully mixed according to the mass ratio of total protein in cell membranes to medicament 1.
Example 3 preparation of macrophage membrane-coated ectopic endometrial targeted nanoparticles
The bazedoxifene is dissolved in ethanol, monoglyceride with the mass being 10 times that of the bazedoxifene is added to obtain a mixed solution of the bazedoxifene and the lipid with the bazedoxifene concentration being 10mg/ml, the mixed solution is heated and mixed evenly at 60 ℃, the mixed solution of the bazedoxifene and the lipid is injected into hot water at 60 ℃ by adopting an aqueous solvent diffusion method, the mixture is continuously stirred and cooled to room temperature, and the bazedoxifene lipid nanoparticle suspension is prepared.
The M1 type macrophage membrane and bazedoxifene lipid nanoparticle suspension prepared in the embodiment 1 is fully mixed according to the mass ratio of total protein in cell membrane to medicament of 1.
Example 4 preparation of macrophage membrane-coated ectopic endometrial targeting nanoparticles
The difference between the embodiment and the embodiment 2 is only that the M1 type macrophage membrane prepared in the embodiment 1 and the anastrozole nanoparticle suspension are fully mixed according to the mass ratio of total protein in a cell membrane to drug of 1.1 to prepare the ectopic endometrium targeting nanoparticle (M1-CNVs/ANA) coated by the macrophage membrane.
Example 5 preparation of macrophage membrane-coated ectopic endometrial targeting nanoparticles
The difference between the present example and example 3 is that, in the preparation process of bazedoxifene lipid nanoparticles, the weight ratio of the drug to the lipid is 1; in the preparation process of the macrophage membrane coated ectopic endometrium targeted nanoparticle, the M1 type macrophage membrane prepared in the example 1 and bazedoxifene lipid nanoparticle suspension are fully mixed according to the mass ratio of total protein in a cell membrane to medicament of 1.2 to prepare the macrophage membrane coated ectopic endometrium targeted nanoparticle (M1-CNVs/BAZ-SLN) loaded with the bazedoxifene lipid nanoparticle.
EXAMPLE 6 in vitro model determination of ectopic endometrial targeting by M1-CNVs
Adopting a Transwell chamber with a membrane aperture of 0.4 mu M, planting endometrial cancer Ishikawa cells in a lower chamber, adding CCL2 chemotactic factor with the concentration of 200 mu g/L after the cells are attached to the wall, adding Dil-labeled M1-CNVs in an upper chamber, putting the cells into a cell culture box, fixing the cells by formaldehyde after 0.5h, 1h and 4h, staining cell nuclei by Hoechst 33342, and observing the transfer of the M0-CNVs and the M1-CNVs by a laser confocal microscope. The results are shown in FIG. 2, the fluorescence intensity of M1-CNVs group migrating to the lower part of the cell is higher than that of M0-CNVs, which indicates that M1-CNVs can actively migrate under the chemotaxis of CCL2 and have targeting property.
EXAMPLE 7 endometriosis model mouse Targeted distribution Studies of M1-CNVs
Establishing an endometriosis model animal: and (3) anesthetizing a female adult mouse by pentobarbital sodium, performing an operation on the lower right abdomen for abdomen opening, cutting off the right uterus outside a position 0.5cm away from the junction of the uteruses, longitudinally cutting the uterus, cutting the uterus into 4 equal-length segments, and soaking the segments in a DMEM/12 culture solution. Wherein the two endometrium surfaces are sewed on the right peritoneum of the mouse, small endometrium pieces are respectively placed from the incision of the abdomen to the left subcutaneous side, and the abdomen is closed by a conventional operation. After 3 weeks, the subcutaneous tissue and the peritoneal tissue of the mouse have nodules, and the microscopic histological observation shows that if the nodules contain endometrial glands and stroma which are consistent in shape and in-place intima, the modeling is successful.
The cell membrane infrared fluorescent probe DiR marks M1-CNVs, M0-CNVs and blank lipid nanoparticles SLN, tail vein injection is carried out into the model animal body, and the whole body fluorescence distribution condition of the model animal at different time is observed by adopting a small animal living body imaging instrument. The excitation wavelength was set at 704nm, the emission wavelength ranged from 740nm to 950nm, and the filter was NIR. The in vivo fluorescence distribution graph of the model animal of 48h is shown in figure 3, lipid nanoparticles and M0-CNVs mainly accumulate in the liver part, and M1-CNVs can migrate to ectopic focus of endometrium, which indicates that M1-CNVs have focus tropism in vivo and can be actively targeted to the ectopic endometrium.
Example 8 study of the cellular pharmacodynamics of endometriosis model of M1-CNVs/BAZ-SLN
The primary ectopic endometrial cells are difficult to extract and culture, while the endometrial cancer Ishikawa cells are immortalized in vitro and highly express estrogen receptors, and estrogen can stimulate the Ishikawa to rapidly proliferate. Therefore, primary ectopic endometrial cells were simulated in vitro by using endometrial cancer Ishikawa cells, and the growth inhibitory effects of bazedoxifene lipid nanoparticles BAZ-SLN, M0-CNVs/BAZ-SLN and M1-CNVs/BAZ-SLN of example 3 on ectopic endometrial cells were examined.
Ishikawa cells in the logarithmic growth phase were seeded at a density of 1 ten thousand cells per well in a 96-well plate and incubated overnight at 37 ℃ in a cell incubator to allow the cells to adhere to the wall. Setting a group: blank group, estrogen + bazedoxifene lipid nanoparticles BAZ-SLN, estrogen + M0-CNVs/BAZ-SLN, estrogen + M1-CNVs/BAZ-SLN, the concentration of estrogen is 2 mu g/ml, the concentration of bazedoxifene is 10 mu mol/L, and each sample has three multiple wells. After the drug was added, the cells were further cultured in a cell incubator for 24h. Adding 20 mu L/hole MTT solution of 10mg/ml, continuously incubating for 4h, discarding the culture solution, dissolving the purple precipitate with dimethyl sulfoxide, and measuring the absorbance at 560nm with an enzyme-labeling instrument. The results of comparing the growth conditions of different groups of cells are shown in figure 4, and the bazedoxifene lipid nanoparticles BAZ-SLN, M0-CNVs/BAZ-SLN and M1-CNVs/BAZ-SLN can antagonize the growth promotion effect of estrogen on model cells Ishikawa and play the function of antagonizing estrogen.
The technical solutions of the present invention are described in detail in the above embodiments, it should be understood that the above embodiments are only specific examples of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. An ectopic endometrium targeted nanoparticle coated by a macrophage membrane is characterized by comprising an inner core and an M1 type macrophage membrane coated outside the inner core; the inner core is a drug nanoparticle or a drug lipid nanoparticle, and the drug is selected from estrogen antagonist and/or estrogen synthesis inhibitor.
2. The macrophage membrane-coated ectopic endometrium-targeted nanoparticle according to claim 1, wherein the M1-type macrophage is induced and differentiated from the M0-type macrophage, and the M1-type macrophage membrane is extracted from the M1-type macrophage.
3. The macrophage membrane-coated ectopic endometrium-targeted nanoparticle according to claim 1, wherein the drug lipid nanoparticle is formed by lipid-encapsulating the drug, and the weight ratio of the drug to the lipid is 1:4-10.
4. The macrophage membrane-coated ectopic endometrial targeting nanoparticle according to claim 1, wherein the particle size of the drug nanoparticle or the drug lipid nanoparticle is 50-200nm.
5. The macrophage membrane-coated ectopic endometrial-targeted nanoparticle according to claim 1, wherein the estrogen antagonist is bazedoxifene.
6. The macrophage membrane-coated ectopic endometrium-targeted nanoparticle according to claim 1, wherein the estrogen synthesis inhibitor is anastrozole.
7. The macrophage membrane-coated ectopic endometrium-targeted nanoparticle according to claim 1, wherein the mass ratio of total protein to drug in the M1-type macrophage membrane is 1:0.1-1.
8. The preparation method of the macrophage membrane-coated ectopic endometrium targeted nanoparticle according to any one of claims 1-7 is characterized by comprising the following steps:
(1) Preparing the drug or the mixture of the drug and lipid by a precipitation method to obtain drug nanoparticles or drug lipid nanoparticles;
(2) The M1 type macrophage membrane and the drug nanoparticles, or the M1 type macrophage membrane and the drug lipid nanoparticles are prepared into the ectopic endometrium targeting nanoparticles coated by the macrophage membrane by an ultrasonic method, an extrusion method or a microfluidic method.
9. Use of an ectopic endometrium-targeted nanoparticle coated with a macrophage membrane according to any one of claims 1-7 for the preparation of a pharmaceutical formulation for the treatment of endometriosis.
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