Preparation and application of MRI-based nanoparticles targeting ovarian cancer

Abstract

The present invention belongs to the field of medical technology and provides a preparation and application of MRI diagnosis and treatment integrated nanoparticles targeting ovarian cancer. 3+ The coordination between tetravalent platinum (Pt‑COOH) and gossypol has created a high-drug-loading, deliverable nanoparticle that does not require the addition of additional excipients. It is made by a simple nanoprecipitation method, has uniform size, is tumor-targeted, and has controlled release. It can achieve the synergistic co-delivery of platinum drugs and gossypol dual chemotherapy drugs, and can also be delivered by Fe 3+ It realizes the dual functions of ferroptosis and magnetic resonance imaging (MRI); overall, the method of the present invention is simple, has good treatment and imaging effects, is highly practical, and has good application prospects in the integrated diagnosis and treatment of ovarian cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the preparation and application of MRI diagnosis and treatment integrated nanoparticles targeting ovarian cancer. Background Art

[0002] Ovarian cancer has an insidious onset and is a malignant tumor with a very poor prognosis. More than 70% of cases are diagnosed in the late stage, and the overall 5-year survival rate is less than 30%. The lack of early diagnosis and effective intervention methods are the main reasons for the high mortality and low survival rate. Early detection and diagnosis of ovarian cancer can significantly improve the prognosis of patients. Ultrasound, CT and MRI are commonly used for preoperative diagnosis of ovarian cancer. Among these imaging techniques, MRI is one of the most powerful diagnostic tools with high spatial resolution and soft tissue contrast, and is widely used for diagnosis, especially differential diagnosis and preoperative treatment selection. Gadolinium contrast agents are widely used in clinical MRI, however, gadolinium contrast agents are associated with nephrogenic systemic fibrosis in patients with renal insufficiency. Therefore, the development of new contrast agents is a very urgent need. Fe 3+ It is a paramagnetic substance that can be used for non-invasive measurement using MRI, which makes Fe 3+ Become a promising candidate for safe contrast agent.

[0003] Cisplatin-based chemotherapy is the first-line treatment for cancer, and various forms of cisplatin-based nanomedicines have been widely studied. However, their application is always limited by their low loading capacity, which increases the dosage of cisplatin and leads to side effects, such as nephrotoxicity. The emergence of metal-based nanoparticles provides a new opportunity to solve this problem. Metal-based nanoparticles are connected by coordination bonds between transition metal ions and chemotherapeutic drugs and have become a hot topic in the field of nanomedicine. 3+ It is not only an essential element for living organisms, but also a good candidate material for magnetic resonance imaging. 3+ Metal-based nanoparticles represented by ferrous metals have attracted the interest of researchers. Currently, there are few studies on the combination of cisplatin prodrugs and iron-based metal polyphenol nanoparticles.

[0004] The results of searching domestic and foreign literature and patents show that: 3+ -Pt-COOH nanoparticles and their use in the integrated diagnosis and treatment of ovarian cancer through the targeting effect of hyaluronic acid has not been reported.

[0005] To this end, those skilled in the art have proposed the preparation and application of MRI diagnosis and treatment integrated nanoparticles targeting ovarian cancer to solve the problems raised by the background technology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a preparation and application technology of MRI diagnosis and treatment integrated nanoparticles targeting ovarian cancer. It achieves targeting of ovarian cancer cells through hyaluronic acid coated on the surface, and through Pt-COOH, cotton phenol and Fe 3+ The synergistic effect between them can kill ovarian cancer cells. 3+ The HA@PFG nanoparticles have good potential for clinical application, integrating diagnosis and treatment.

[0007] A method for preparing MRI diagnosis and treatment integrated nanoparticles targeting ovarian cancer, comprising the following steps:

[0008] Step 1: Preparation and verification of tetravalent cisplatin (Pt-COOH);

[0009] Step 2: Preparation of PFG nanoparticles;

[0010] Step 3: Preparation of HA@PFG nanoparticles coated with hyaluronic acid.

[0011] Preferably, the specific description of the preparation of tetravalent cisplatin (Pt-COOH) in step S1 is as follows:

[0012] S11. Synthesis of Cisplatin Oxidation:

[0013] (1) First, 300 mg of cisplatin was mixed with 2 mL of deionized water to form a uniform yellow suspension under ultrasonic conditions;

[0014] (2) Then, the suspension in (1) was added dropwise into 10 mL of 30% H2O2 solution;

[0015] (3) the mixed solution was reacted in a constant temperature water bath at 70° C. for 5 hours and then reacted in a refrigerator at 4° C. overnight to obtain a reaction product;

[0016] (4) Finally, the reaction product was filtered, and the residue was washed with cold water, ethanol, and ether three times respectively and then dried;

[0017] Synthesis of S12, Pt-COOH:

[0018] (1) First, 237 mg of oxidized cisplatin obtained in the first step and 140 mg of succinic anhydride were dissolved in 11.2 mL of DMSO and stirred in a 50° C. oil bath in the dark for 12 hours;

[0019] (2) Filter and collect the filtrate, place it in a -20°C refrigerator for freezing and precipitation for half an hour, and add ether for precipitation;

[0020] (3) then filtering, collecting the precipitate, and drying;

[0021] Structural verification: 2 mg of the dried second step product was dissolved in 600 μL DMSO-d6, and its purity was verified by nuclear magnetic resonance spectroscopy.

[0022] Preferably, the step S2 is specifically described as follows:

[0023] (1) First, weigh 5 mg of Pt-COOH and 5 mg of gossypol and dissolve them in 500 μL of DMSO solution;

[0024] (2) Under ultrasonic conditions, add (1) dropwise into 9.5 mL of 200 μM FeCl3;

[0025] (3) Then quickly add Tris solution;

[0026] (4) Then dialyze in a 3500Da dialysis bag for 12 hours;

[0027] (5) Finally, collect the nanoparticle liquid in the dialysis bag and store it in a refrigerator at 4°C.

[0028] Preferably, the preparation steps of the nanoparticles HA@PFG nanoparticles in step S3 are as follows:

[0029] (1) First, weigh 5 mg of Pt-COOH and 5 mg of gossypol and dissolve them in 500 μL of DMSO solution;

[0030] (2) Under ultrasonic conditions, add (1) dropwise into 9.5 mL of 200 μM FeCl3;

[0031] (3) Then quickly add Tris solution;

[0032] (4) then, under ultrasonic conditions, low molecular weight hyaluronic acid (HA) was added to (3) to a final concentration of 1 mg / mL;

[0033] (5) Then dialyze in a 3500Da dialysis bag for 12 hours;

[0034] (6) Finally, collect the nanoparticle liquid in the dialysis bag and store it in a refrigerator at 4°C.

[0035] Based on the above needs, we developed a novel nanostructured ... 3+HA@PFG nanoparticles, formed by coating metal-coordinated complex nanoparticles with hyaluronic acid (HA), exhibit specific targeting behavior, controllable drug release characteristics, and good contrast imaging capabilities both in vitro and in vivo. These characteristics indicate that HA@PFG nanoparticles are an excellent nanocarrier with good clinical application potential, integrating diagnosis and targeted tumor therapy.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention provides a metal polyphenol nanocomposite for multi-drug delivery in the process of tumor treatment. The complex can co-deliver the chemotherapy drug Pt-COOH that causes intracellular redox imbalance, the gossypol that causes tumor cell apoptosis, and the Fe 3+ At the same time, these three drugs have a synergistic effect. Pt-COOH consumes intracellular GSH and enhances Fe 3+ The inhibition of BCl-2 protein by gossypol enhanced the chemotherapeutic killing effect of Pt-COOH on tumor cells.

[0038] 2. The present invention adopts Fe 3+ A pure drug-deliverable nanoparticle was constructed with Pt-COOH and cottonypol without adding additional excipients. Fe 3+ , Pt-COOH, and polyphenol structures formed a coordination effect and self-assembled to form nanoparticles (HA@PFG) with dual functions of treatment and magnetic resonance imaging.

[0039] 3. The present invention achieves targeting of ovarian cancer by coating the surface of nanoparticles with hyaluronic acid, which specifically binds to CD44 highly expressed on the surface of ovarian cancer.

[0040] 4. The present invention synthesizes for the first time a nanocomplex with different action mechanisms and synergistic anti-cancer effects, which greatly facilitates synergistic drug delivery.

[0041] 5. The method of the present invention is simple, has good MR imaging and treatment effects, and is highly practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the synthesis and application of HA@PFG nanoparticles prepared in the present invention;

[0043] Figure 2 Schematic diagram of the synthesis of Pt-COOH in the present invention;

[0044] Figure 3UV spectra of Gosspo l, Pt-COOH and HA@PFG nanoparticles;

[0045] Figure 4 Transmission electron microscopy images (A and B) of PFG and HA@PFG nanoparticles prepared in the present invention;

[0046] Figure 5 The particle size, potential and PDI of PFG nanoparticles and HA@PFG nanoparticles prepared by the present invention;

[0047] Figure 6 Release profiles of HA@PFG nanoparticles prepared by the present invention in phosphate buffers of different pH values;

[0048] Figure 7 T1-weighted MR imaging and relaxivity of the HA@PFG nanoparticles prepared by the present invention under different pH conditions in vitro;

[0049] Figure 8 This is a T1-weighted MR imaging image of the HA@PFG nanoparticles prepared by the present invention in vivo;

[0050] Fig. 9 This is a diagram showing the targeting effect of the HA@PFG nanoparticles prepared by the present invention on ovarian cancer A2780 cells;

[0051] Fig.10 This is a cytotoxicity analysis diagram of the HA@PFG nanoparticles prepared in the present invention;

[0052] Fig.11 The graphs are the changes in relative tumor volume of mice in each group (A), the graphs of tumor entities of mice in each group (B), and the graphs of weight changes of mice in each group (C). DETAILED DESCRIPTION

[0053] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0054] As attached Figure 1 To Attachment Fig.11 As shown:

[0055] Embodiment: The present invention provides a preparation and application of MRI diagnosis and treatment integrated nanoparticles targeting ovarian cancer, comprising the following steps:

[0056] Step 1) The first step is the synthesis of oxidized cisplatin ( Figure 2): ① Mix 300 mg of cisplatin with 2 mL of deionized water to form a uniform yellow suspension under ultrasonic conditions; ② Add the suspension in ① dropwise into 10 mL of 30% H2O2 solution; ③ The mixed solution is reacted in a 70°C constant temperature water bath for 5 hours and then reacted in a 4°C refrigerator overnight; ④ The reaction product is filtered, and the filter residue is rinsed with cold water, ethanol, and ether for 3 times and then dried; The second step is the synthesis of Pt-COOH ( Figure 2 ): ① Dissolve 237 mg of oxidized cisplatin obtained in the first step and 140 mg of succinic anhydride in 11.2 mL of DMSO, and stir in a 50°C oil bath in the dark for 12 hours; ② Filter, collect the filtrate, place in a -20°C refrigerator to freeze and precipitate for half an hour, and add ether to precipitate; ③ Filter, collect the precipitate, and dry; Structural verification: Take 2 mg of the dried product of the second step, dissolve it in 600 μL DMSO-d6, and verify its purity by nuclear magnetic resonance spectroscopy;

[0057] Step 2) The preparation method of PFG nanoparticles is as follows: ① weigh 5 mg Pt-COOH and 5 mg gossypol, and dissolve them in 500 μL DMSO solution; ② under ultrasonic conditions, add ① dropwise to 9.5 mL of 200 μM FeCl3; ③ quickly add Tris solution; ④ dialyze in a 3500Da dialysis bag for 12 hours; ⑤ collect the nanoparticle liquid in the dialysis bag and store it in a refrigerator at 4°C;

[0058] Step 3) The preparation method of HA@PFG nanoparticles is as follows: ① Weigh 5 mg Pt-COOH and 5 mg gossypol, and dissolve them in 500 μL DMSO solution; ② Under ultrasonic conditions, add ① dropwise to 9.5 mL of 200 μM FeCl3; ③ Quickly add Tris solution; ④ Under ultrasonic conditions, add low molecular weight hyaluronic acid to ③ to a final concentration of 1 mg / mL; ⑤ Dialyze for 12 hours in a 3500Da dialysis bag; ⑥ Collect the nanoparticle liquid in the dialysis bag and store it in a refrigerator at 4°C.

[0059] Application example: HA@PFG nanoparticles exhibit specific targeting behavior, strong penetration, controllable drug release characteristics and good contrast imaging capabilities both in vitro and in vivo:

[0060] (1) Characterization experiment: First, we prepared integrated diagnosis and treatment nanoparticles targeting ovarian cancer. We found through UV spectrophotometer that the UV peak of the coordinated nanoparticles and the free drug changed significantly, confirming the occurrence of a new structure ( Figure 3 ). Transmission electron microscopy confirmed that it has a spherical and regular morphology ( Figure 4 ), the particle size was measured by Malvern particle size analyzer to be 105.47±0.28nm, PD I was 0.15±0.05, and the potential was -43.8±0.34mV ( Figure 5), and tested its drug release ability under different pH environments through release experiments. In the extracellular fluid environment of pH=7.4, the nanoparticles were basically not released. At pH6.5, nearly 40% of the nanoparticles could be released smoothly ( Figure 6 ); then the in vitro nuclear magnetic resonance imaging performance of the nanoparticles was determined, and the in vitro imaging effect was observed by nuclear magnetic resonance imaging technology, confirming that under the condition of pH = 6.5, HA@PFG nanoparticles have good in vitro imaging ability ( Figure 7 );

[0061] (2) In vitro experiments: First, fluorescence imaging was used to determine that the nanoparticles had good targeting properties for ovarian cancer cells ( Fig. 9 ); then the cell experiment was carried out and the in vitro anti-tumor effect was evaluated by MTT assay ( Fig.10 ), it was found that HA@PFG nanoparticles have higher toxicity than free gossypol and Pt-COOH, indicating that there is a synergistic effect between the components of HA@PFG nanoparticles;

[0062] (3) In vivo experiments: First, an ovarian cancer mouse model was prepared, and the integrated diagnostic and therapeutic nanoparticles targeting ovarian cancer were injected into the mice through the tail vein. Then, the distribution and imaging effects of the nanoparticles in the tumor site were observed using in vivo MRI technology. It was found that HA@PFG nanoparticles were enriched in the mouse tumor site about 6 hours after injection and had good T1-weighted MRI imaging capabilities ( Figure 8 During the treatment period, the effect and safety of nanoparticle therapy were evaluated by detecting changes in tumor volume and the effect on mouse body weight. Fig.11 A, B), the tumor volume of the PBS group was found to be 1348.04±409.15mm 3 The tumor volume of Pt-COOH was 983.50±433.53mm 3 The tumor volume of the cottonpol group was 604.74±283.86mm 3 The tumor volume of the cisplatin group was 294.33±110.82mm 3 The tumor volume of the PFG group was 575.69±164.29mm 3 , while the tumor volume of the HA@PFG group was 294.33±110.82mm 3 , confirming that HA@PFG nanoparticles have a good anti-tumor effect. In addition, there was no significant change in the body weight of mice in the HA@PFG group during treatment ( Fig.11 C), which also confirmed the safety of HA@PFG in vivo application.

[0063] As can be seen from the above, the nanoparticles can co-deliver the chemotherapy drug Pt-COOH, gossypol that causes tumor cell apoptosis, and Fe that causes tumor cell ferroptosis and has in vivo nuclear magnetic resonance imaging function. 3+ At the same time, these three drugs have a synergistic effect. Pt-COOH consumes intracellular GSH and enhances Fe 3+ The inhibition of BC l-2 protein by gossypol enhanced the chemotherapeutic killing effect of Pt-COOH on tumor cells.

[0064] In summary: in combination with the drawings and descriptions thereof, the present invention has the following beneficial effects:

[0065] (1) The present invention can co-deliver the chemotherapy drug Pt-COOH, and the gossypol that causes tumor cell apoptosis can cause ferroptosis of tumor cells, and the Fe 3+ At the same time, these three drugs have a synergistic effect. Pt-COOH consumes intracellular GSH and enhances Fe 3+ The ferroptosis effect caused by gossypol and the inhibition of BC l-2 protein enhanced the chemotherapeutic killing effect of Pt-COOH on tumor cells;

[0066] (2) The present invention combines Fe 3+ A pure drug-deliverable nanoparticle was constructed with Pt-COOH and cottonyphenol without the addition of additional excipients. Through a simple nanoprecipitation method, the iron formed a coordination effect with the polyphenol structure to self-assemble into nanoparticles (HA@PFG) with dual functions of treatment and imaging;

[0067] (3) The present invention achieves targeting of ovarian cancer by coating the surface of nanoparticles with hyaluronic acid, which specifically binds to CD44 highly expressed on the surface of ovarian cancer;

[0068] (4) The present invention synthesizes for the first time a nanocomplex with different action mechanisms and synergistic anti-cancer effects, which greatly facilitates synergistic drug delivery;

[0069] (5) The method of the present invention is simple, has good MR imaging and treatment effects, and is highly practical.

[0070] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing nanoparticles for integrated MRI diagnosis and treatment targeting ovarian cancer, Features: The following steps are involved: S1. First, prepare and verify Pt-COOH; S2, then preparing PFG nanoparticles; S3, finally preparing hyaluronic acid-coated nanoparticles HA@PFG; The specific description of the preparation of Pt-COOH in step S1 is as follows: S11. Synthesis of Cisplatin Oxidation: (1) First, 300 mg of cisplatin was mixed with 2 mL of deionized water to form a uniform yellow suspension under ultrasonic conditions; (2) Then, add the suspension in (1) dropwise into 10 mL of 30% H 2 O 2 in solution; (3) the mixed solution was reacted in a constant temperature water bath at 70° C. for 5 hours and then reacted in a refrigerator at 4° C. overnight to obtain a reaction product; (4) Finally, the reaction product was filtered, and the residue was washed with cold water, ethanol, and ether three times respectively and then dried; Synthesis of S12, Pt-COOH: (1) First, 237 mg of oxidized cisplatin obtained in the first step and 140 mg of succinic anhydride were dissolved in 11.2 mL of DMSO and stirred in a 50° C. oil bath in the dark for 12 hours; (2) Filter and collect the filtrate, place it in a -20°C refrigerator for freezing and precipitation for half an hour, and add ether for precipitation; (3) then filtering, collecting the precipitate, and drying; The specific description of the verification in step S1 is as follows: 2 mg of the dried product is dissolved in 600 μL DMSO-d6, and then its purity is verified by nuclear magnetic resonance spectroscopy; The step S2 is specifically described as follows: (1) First, weigh 5 mg of Pt-COOH and 5 mg of gossypol and dissolve them in 500 μL of DMSO solution; (2) Under ultrasonic conditions, add (1) dropwise to 9.5 mL of 200 μM FeCl 3 middle; (3) Then quickly add Tris solution; (4) Then dialyze in a 3500Da dialysis bag for 12 hours; (5) Finally, the nanoparticle liquid in the dialysis bag was collected and stored in a refrigerator at 4°C; The preparation steps of the nanoparticles HA@PFG nanoparticles in step S3 are as follows: (1) First, weigh 5 mg of Pt-COOH and 5 mg of gossypol and dissolve them in 500 μL of DMSO solution; (2) Under ultrasonic conditions, add (1) dropwise to 9.5 mL of 200 μM FeCl 3 middle; (3) Then quickly add Tris solution; (4) then adding low molecular weight hyaluronic acid to (3) under ultrasonic conditions to a final concentration of 1 mg / mL; (5) Then dialyze in a 3500Da dialysis bag for 12 hours; (6) Finally, collect the nanoparticle liquid in the dialysis bag and store it in a refrigerator at 4°C.

Images