Targeting MMP-9 magnetic resonance imaging nano probe and preparation method thereof

Abstract

The invention discloses a magnetic resonance imaging nano probe targeting MMP-9 and a preparation method of the magnetic resonance imaging nano probe. The method comprises the following steps: firstly, respectively synthesizing NaGdF4 (at) PEG (Polyethylene Glycol)-cholesterol, a cholesterole-PEG-MMP-9 targeting peptide and a Cy5.5-MMP-9 targeting peptide, and then adding the cholesterole-PEG-MMP-9 targeting peptide and the Cy5.5-MMP-9 targeting peptide into a NaGdF4 (at) PEG-cholesterol solution, so as to prepare the NaGdF4 (at) PEG-cholesterol (at) MMP-9 targeting peptide-Cy5.5. As a molecular imaging probe, the NaGdF4 (at) PEG-cholesterol (at) MMP-9 targeting peptide-Cy5.5 can specifically target adenomyosis, after injection, magnetic resonance scanning shows that the relaxation value of a focus T1 of the adenomyosis is obviously increased, the detection rate of the focus of the adenomyosis can be increased, an MMP-9 high-expression region caused by endometriosis can be developed in a targeted manner, and the application prospect is wide. The method has a wide application prospect in magnetic resonance imaging for displaying the focus boundary of adenomyosis.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials and relates to a magnetic resonance imaging nanoprobe targeting MMP-9 and a preparation method thereof. Background Art

[0002] Adenomyosis refers to the invasion of endometrial glandular structures and interstitial cells into the myometrium, which, under the action of hormones in the body, causes periodic bleeding in the myometrium, thereby causing proliferation of surrounding smooth muscle fibers. Adenomyosis is a common disease in women of childbearing age, with an incidence of about 20%-30%, and the incidence has been on the rise in recent years. The main clinical symptoms of adenomyosis include dysmenorrhea, dyspareunia, infertility, and miscarriage. Studies have shown that adenomyosis destroys the ability of the uterus to transport sperm in a directional manner and the function of the ovaries, thereby causing infertility. At the same time, the success rate of artificial insemination in patients with adenomyosis is lower than that in infertile patients without adenomyosis, and the miscarriage rate after conception is also higher than that in the non-adenomyosis group. Dysmenorrhea increases the pain of patients with adenomyosis, greatly reducing the quality of life of patients; infertility and miscarriage not only affect the quality of life of patients, but also affect the harmony of family relationships, and have a great negative impact on the physical and mental health of patients.

[0003] At present, the clinical treatment methods for adenomyosis mainly include surgical treatment and drug treatment. Although surgical removal of the uterus can control the disease and alleviate the patient's symptoms, it will cause the patient to lose fertility. Changes in pelvic hemodynamics will also affect ovarian function, cause some side effects related to endocrine disorders, and affect the patient's quality of life. Although partial adenomyosis resection preserves the patient's uterus to a certain extent, it increases the miscarriage rate of postoperative pregnancy and the risk of asymptomatic uterine rupture. Although drug treatment can control the symptoms related to adenomyosis to a certain extent, recurrence will occur after discontinuation of the drug, and even increase the chance of uterine bleeding.

[0004] However, since the boundaries of adenomyosis lesions are often unclear on magnetic resonance images, this limits the scope of interventional and surgical treatments before or during surgery, which affects the effectiveness of treatment. Therefore, there is an urgent need for a magnetic resonance nanoprobe that can target and image adenomyosis lesions.

[0005] During the development of adenomyosis, the implantation of ectopic endometrium needs to go through three steps: adhesion, implantation, and angiogenesis. Matrix metalloproteinase-9 (MMP-9) promotes angiogenesis by specifically degrading the main components of the extracellular matrix, type V, type IV collagen, and gelatin, and promotes the colonization of ectopic endometrium or decidual cells, which is one of the important reasons for the invasiveness of ectopic endometrium. Therefore, the high expression of local MMP-9 caused by ectopic endometrium is a key factor leading to the survival and recurrence of endometriotic foci. Therefore, MMP-9 can be used as an effective target for targeted imaging of adenomyosis lesions, thereby displaying the effective range of the lesions in real time on magnetic resonance imaging, and on this basis, a personalized treatment plan can be formulated to obtain better efficacy.

[0006] In the previous studies targeting MMP-9, broad-spectrum MMP molecular imaging tracers (J Med Chem 2012; 55(10):4714-27, Curr Pharm Des 2013; 19(25):4647-72) were mostly used, which can target a variety of MMPs, including gelatinases, interstitial collagenases, and broad-spectrum matrix lysins; or novel radioligands targeting MMP2 / 9 (N-[ 123 I] iodobenzenesulfonyl iminodiacetyl monohydroxamate (PLoS One, 2017Nov 30; 12(11): e0187767.). The role of MMP-9 in adenomyosis is relatively unique. Although other matrix metalloproteinases (such as MMP-2) also play a role in tissue remodeling, studies have shown that MMP-9 contributes more significantly to inflammatory response, matrix degradation, and endometrial invasion. Therefore, choosing MMP-9 as a target can more specifically reflect pathological changes and avoid inaccurate diagnosis or information interference caused by nonspecific probes. However, there are few reports on single targeted imaging agents for MMP-9. The reports retrieved so far are that gold nanoparticles are radiolabeled with 125I and 111In, and functionalized with MMP-9 cleavable peptides as SPECT imaging contrast agents targeting MMP-9 (Nanoscale. 2015Jan 14; 7(2): 440-4.), which are not MRI contrast agents. Summary of the invention

[0007] The purpose of the present invention is to provide a magnetic resonance imaging nanoprobe with strong specificity targeting MMP-9 and a preparation method thereof.

[0008] The technical solution for achieving the purpose of the present invention is as follows:

[0009] The method for preparing a magnetic resonance imaging nanoprobe targeting MMP-9 comprises the following steps:

[0010] (1) Preparation of NaGdF4@PEG-cholesterol:

[0011] Add ethanol to the cyclohexane dispersion of NaGdF4 to precipitate, collect the precipitate by centrifugation, redisperse in chloroform, add NHS-PEG2000-cholesterol, stir overnight at room temperature, then add ethanol, remove chloroform by rotary evaporation, disperse with water, then dialyze to remove free PEG, add water to obtain NaGdF4@PEG-cholesterol and store at low temperature;

[0012] (2) Preparation of cholesterol-PEG-MMP-9 targeting peptide:

[0013] The NHS-PEG2000-cholesterol was completely dissolved in dimethyl sulfoxide (DMSO), and then the MMP-9 targeting peptide was dissolved in water and added to the DMSO solution of the NHS-PEG2000-cholesterol, and the reaction was stirred at room temperature to obtain the cholesterol-PEG-MMP-9 targeting peptide, which was stored at low temperature;

[0014] (3) Preparation of Cy5.5-MMP-9 targeting peptide:

[0015] Dissolve Cy5.5-NHS in DMSO, add MMP-9 targeting peptide, stir and react at room temperature to obtain Cy5.5-MMP-9 targeting peptide and store at low temperature;

[0016] (4) Preparation of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5:

[0017] Cholesterol-PEG-MMP-9 targeting peptide and Cy5.5-MMP-9 targeting peptide were added to NaGdF4@PEG-cholesterol solution to react and obtain NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5, which was dispersed in water and stored at low temperature.

[0018] Preferably, in step (1), the centrifugation condition is 10000 rpm for 10 min.

[0019] Preferably, in step (1), the molecular weight cut-off of the dialysis bag is 7000Da.

[0020] Preferably, in step (2) or (3), the reaction time is 24 h.

[0021] Preferably, in step (2) or (3), the amino acid sequence of the MMP-9 targeting peptide is CTTHWGFTLC (SEQ ID No. 1).

[0022] The present invention also provides a magnetic resonance imaging nanoprobe targeting MMP-9 prepared by the above preparation method.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The present invention uses PEG-cholesterol as a raw material. On the one hand, it utilizes the hydrophobicity of the cholesterol end. On the other hand, cholesterol has biomembrane affinity, which can promote the enrichment of nanoparticles in cell membranes or specific tissues (such as tumors or inflammatory sites). The cholesterol part can be inserted into the cell membrane or vascular endothelial cells to enhance its enrichment ability in the target area. Therefore, in NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5, PEG is not only a simple modification of hydrophilicity, but PEG-cholesterol is used as an intermediate layer to stably connect NaGdF4 and MMP-9 targeting peptide, and the functions of both are kept intact. The length and structure of PEG can be flexibly adjusted to adapt it to different functional requirements and ensure the stability and activity of the probe. Although ordinary PEG has good biocompatibility and the effect of prolonging blood circulation time, it lacks the membrane affinity of cholesterol, and is therefore weak in cell or tissue-specific enrichment, resulting in a low concentration of the targeting peptide at the target site, affecting its binding efficiency with the MMP-9 receptor.

[0025] (2) After the NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 of the present invention was injected as a molecular imaging probe, the T1 relaxation value of adenomyosis lesions was significantly increased, and the boundary with the surrounding normal tissues was clear, indicating that it has broad application prospects in magnetic resonance imaging of adenomyosis lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Transmission electron microscopy (a), X-ray energy spectrum analysis (b), Fourier transform infrared spectrum (c) and hysteresis loop analysis (d) of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5;

[0027] Figure 2 The results of the cell activity detection of hEM15A by NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 probe with different Gd concentrations;

[0028] Figure 3 Laser confocal microscopy image of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 in endometriotic cells;

[0029] Figure 4 Magnetic resonance T1-weighted scans of adenomyosis lesions before and after intravenous injection of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. In the following examples, if no specific technology or conditions are indicated, the technology or conditions described in the literature in this area or the product instructions are used, and the reagents or raw materials used can be purchased commercially or synthesized according to the literature in this area.

[0031] In the following examples, the MMP-9 targeting peptide used is the MMP-9 targeting polypeptide CTTHWGFTLC.

[0032] Example 1

[0033] 1. Synthetic molecular imaging probe NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5

[0034] By connecting the MMP-9 targeting peptide to the gadolinium-based nanomaterial NaGdF4, the NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 molecular imaging probe was synthesized. The specific steps are as follows.

[0035] 1. Preparation of NaGdF4:

[0036] (1) GdCl3 powder was dissolved in deionized water, and a certain proportion of oleic acid and octadecene were added to a three-necked flask and stirred at room temperature. The air in the flask was removed by passing argon gas, and then the system was dehydrated: first the temperature was raised to 80°C and maintained for 1.5 hours; then the temperature was raised to 120°C and maintained for 1 hour, and then the temperature was raised to 150°C and maintained for 1 hour, and finally a yellow clear liquid was obtained. The heating was stopped and the system was allowed to cool naturally to room temperature;

[0037] (2) preparing NaOH and NHF4, dissolving them in 10 mL of methanol, accelerating dispersion by ultrasound, adding them to the above system, and stirring them at room temperature overnight to promote the exchange of ions and the formation of precursors;

[0038] (3) Removal of methanol: First, pass argon for 5 minutes, then gradually increase the temperature and maintain it for a certain period of time: 70°C for 1 hour, 80-100°C for 1 hour, 140-150°C for 1 hour, then connect the condenser, start to increase the temperature, stabilize it at 245°C, maintain it for 20 minutes, then naturally drop to room temperature and disperse it in cyclohexane.

[0039] 2. Preparation of NaGdF4@PEG-cholesterol:

[0040] Take the NaGdF4 solution dispersed in cyclohexane, add ethanol to precipitate, centrifuge at 10000rpm for 10min, redisperse in chloroform, add NHS-PEG2000-cholesterol, stir overnight at room temperature, then add ethanol, remove chloroform by rotary evaporation, add deionized water to disperse, then dialyze to remove free PEG (dialysis bag molecular weight cutoff: 7000Da), add deionized water, and store at low temperature.

[0041] 3. Preparation of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5:

[0042] (1) Preparation of cholesterol-PEG-MMP-9 targeting peptide: NHS-PEG2000-cholesterol was completely dissolved in DMSO, and then the MMP-9 targeting peptide was dissolved in water and added to the above-mentioned NHS-PEG2000-cholesterol DMSO solution. The mixture was stirred at room temperature for 24 hours and stored at low temperature for later use.

[0043] (2) Preparation of Cy5.5-MMP-9 targeting peptide: Cy5.5-NHS was dissolved in DMSO, and MMP-9 targeting peptide was added. The mixture was stirred at room temperature for 24 h and stored at low temperature for later use.

[0044] (3) Preparation of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5: Cholesterol-PEG-MMP-9 targeting peptide and Cy5.5-MMP-9 targeting peptide were added to NaGdF4@PEG-cholesterol solution to prepare NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5, which was dispersed in deionized water and stored at low temperature.

[0045] 2. Materials Characterization and Biological Studies

[0046] 1. Material composition / structure characterization:

[0047] Transmission electron microscopy, X-ray energy spectrum analysis, Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, dynamic light scattering technology, hysteresis loop analysis, etc. were used to characterize the morphology, size, composition, crystal phase, surface modification and magnetic properties of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 nanoprobes. The results are as follows Figure 1 shown.

[0048] from Figure 1Figure a shows the transmission electron microscopy of the nanomaterials, which shows that the nanomaterials have stable morphology and uniform size. Figure b shows that the X-ray spectrum analysis shows that the spectrum of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 is consistent with that of NaGdF4, which has the crystal structure of NaGdF4 and the characteristic peaks of PEG-cholesterol. Figure c shows that NaGdF4, NaGdF4@PEG-cholesterol, and NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 all have N aGdF4 characteristic peak, NaGdF4@PEG-cholesterol, NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 all have the characteristic peak of PEG-cholesterol, indicating that the synthesis of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 has no effect on the composition of the material; d Figure shows that NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 has concentration-dependent T1 imaging characteristics, with a relaxation rate of 11.81 mM·s -1 .

[0049] 2. Cell-level biological research:

[0050] Human endometriotic epithelial cells (hEM15A) were used as toxicity evaluation cells, and the standard CCK8 assay was used to evaluate the cytotoxicity of the NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 nanoprobe. The results are shown in Figure 2 As shown, it can be seen that the NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 nanoprobe has excellent cell compatibility. At the same time, human blood samples were used to study the hemolytic / coagulation effects of the nanoprobe to evaluate its blood compatibility. Endometriotic cells with good growth status were co-cultured with NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 and NaGdF4@PEG-cholesterol-Cy5.5 without targeting labeling. At the same time, endometriotic cells pretreated with MMP-9 targeting peptide were co-cultured with NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 to observe whether the molecular probe can enter the endometriotic cells. The results are shown in Figure 2. Figure 3 As shown, NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5 was able to enter endometriotic cells, showing excellent targeting.

[0051] 3. Study on biological effects at the in vivo level:

[0052] A rat model of adenomyosis was established. After intravenous injection of the molecular probe, a magnetic resonance T1-weighted scan was performed, and it was found that the adenomyosis lesions were significantly enhanced and the boundaries were clear. The results are as follows Figure 4As shown, A is before injection of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5, and B is after injection of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5.

Claims

1. A method for preparing a magnetic resonance imaging nanoprobe targeting MMP-9, characterized in that: The following steps are involved: (1) Preparation of NaGdF4@PEG-cholesterol: Add ethanol to the cyclohexane dispersion of NaGdF4 to precipitate, collect the precipitate by centrifugation, redisperse in chloroform, add NHS-PEG2000-cholesterol, stir overnight at room temperature, then add ethanol, remove chloroform by rotary evaporation, disperse with water, then dialyze to remove free PEG, add water to obtain NaGdF4@PEG-cholesterol and store at low temperature; (2) Preparation of cholesterol-PEG-MMP-9 targeting peptide: Completely dissolve NHS-PEG2000-cholesterol in DMSO, then dissolve the MMP-9 targeting peptide in water and add it to the DMSO solution of NHS-PEG2000-cholesterol, stir and react at room temperature to obtain cholesterol-PEG-MMP-9 targeting peptide and store it at low temperature; (3) Preparation of Cy5.5-MMP-9 targeting peptide: Dissolve Cy5.5-NHS in DMSO, add MMP-9 targeting peptide, stir and react at room temperature to obtain Cy5.5-MMP-9 targeting peptide and store at low temperature; (4) Preparation of NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5: Cholesterol-PEG-MMP-9 targeting peptide and Cy5.5-MMP-9 targeting peptide were added to NaGdF4@PEG-cholesterol solution to prepare NaGdF4@PEG-cholesterol@MMP-9 targeting peptide-Cy5.5, which was dispersed in water and stored at low temperature.

2. The preparation method according to claim 1, characterized in that In step (1), the centrifugation condition is 10000 rpm for 10 min.

3. The preparation method according to claim 1, characterized in that In step (1), the molecular weight cut-off of the dialysis bag is 7000 Da.

4. The preparation method according to claim 1, characterized in that: In step (2) or (3), the reaction time is 24 h.

5. The preparation method according to claim 1, characterized in that: In step (2) or (3), the amino acid sequence of the MMP-9 targeting peptide is CTTHWGFTLC.

6. A magnetic resonance imaging nanoprobe targeting MMP-9 prepared according to the preparation method of any one of claims 1 to 5.

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