Olaparib-copper nano complex as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of an olaparib-copper nano complex, which comprises the following steps: respectively dissolving copper chloride hydrate and olaparib in DMF (Dimethyl Formamide), then mixing, regulating the pH value to 8-9 by using sodium ethoxide, and finally adding trimethylamine for reaction to generate the olaparib-copper nano complex. The invention also discloses an application of the prepared olaparib-copper nano complex in preparation of drugs for treating breast cancer. The synthesized olaparib-copper nano complex has targeting and chemical kinetics effects, is controllable in size and low in effect dose, effectively induces killing of breast cancer cells, and is applied to treatment of tumors. The material shows strong superiority in tumor targeted drug delivery and treatment, and provides an effective development approach for development and design based on multifunctional biological materials.

Description

Technical Field

[0001] The invention relates to an olaparib-copper nanocomplex and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Nanotechnology shows great promise in addressing several important issues of conventional anticancer chemotherapy. A new generation of effective cancer therapies with great potential is expected to be developed to overcome the biological, biophysical and biomedical barriers of the human body to standard chemotherapy treatment. In general, nanostructures can protect buried drug molecules from degradation in the blood, allowing them to be delivered safely and unimpaired to specific target sites in the body. The unique features of these nanostructured materials make them suitable for the functionalization of small molecule drugs. Recently, oncologists and medicinal chemists have shown great interest in functionalizing nanostructured materials with anticancer metal drugs to ensure better drug delivery. Among them, nanometal complexes show great potential in facilitating the delivery of effective anticancer metal drugs.

[0003] Nanometal complexes are composed of metals and ligands. Depending on the metal center and the ligand, they exhibit different redox properties and biological activities. Nanometal complexes show great potential for cancer treatment due to their adjustable ligands, high biocompatibility, excellent photophysical and chemical properties, and easy modification. According to the needs of diagnosis or treatment, suitable organic ligands are selected, mainly including non-functional ligands and functional ligands. Nanometal complexes synthesized by non-functional ligands are mainly used as drug carriers, while functional ligands give them different properties, such as fluorescent diagnostic or therapeutic properties. By combining targeted small molecules in a covalent or non-covalent manner, the synthesized nanometal complexes can specifically reach the lesions, improve the accuracy of diagnosis and the targeting effect of treatment, and reduce the possible toxic side effects of the material. With the emergence of the concept of copper death, nanocopper complexes have also attracted more and more attention from scholars. Copper death is a copper-dependent death, in which cells die due to copper directly binding to the lipidated components of the tricarboxylic acid (TCA) cycle and aggregating enzymes, leading to proteotoxic stress and further inducing cell death. In addition, monovalent copper ions have also been shown to effectively catalyze hydrogen peroxide to generate hydroxyl radicals that damage DNA, while excess glutathione in tumor sites can reduce divalent copper ions to monovalent ones, thereby achieving a certain degree of chemical damage. Therefore, copper-containing nanomaterials have also become a hot topic in anti-tumor research.

[0004] Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) are a class of anti-tumor drugs whose mechanism of action relies on exploiting defective DNA repair pathways in breast cancer (BRCA) mutations and homologous recombination (HR) repair gene deficiency (HRD) cells, a set of key genes for double-strand breaks (DSBs) and interstrand crosslink (ICL) repair pathways, a process known as "synthetic lethality". Olaparib is a classic small molecule drug of poly (ADP-ribose) polymerase 1 (PARP 1) inhibitor, which works through a "synthetic lethality" mechanism because it inhibits the PARP enzyme, leading to the accumulation of DNA damage. However, in actual clinical applications, cancer cells can easily develop resistance to it, which contributes more to DNA fragmentation and apoptosis than olaparib alone, in addition or synergistically with the cytotoxicity imposed by chemotherapeutic agents on DNA. As a broad-spectrum targeted drug, cRGD peptide can target nucleolin on the surface of a variety of cancer cells, thereby achieving directional accumulation of drugs and achieving the effect of targeting tumors. Summary of the invention

[0005] The purpose of the present invention is to provide an olaparib-copper nanocomplex, which combines the olaparib anticancer drug with copper having a copper death effect, thereby achieving low-dose treatment of breast cancer and reducing the drug resistance of olaparib.

[0006] The technical solution adopted by the present invention is:

[0007] A method for preparing an olaparib-copper nanocomplex comprises dissolving hydrated copper chloride and olaparib in DMF respectively, then mixing them, adjusting the pH value thereof to be alkaline with sodium ethoxide, and finally adding trimethylamine for reaction to generate the olaparib-copper nanocomplex.

[0008] Preferably, the mass ratio of cupric chloride hydrate to olaparib is 2:1, and the molar ratio of trimethylamine to cupric chloride hydrate is 1:2.

[0009] Preferably, the reaction temperature is 70-90°C and the reaction time is 16-24h.

[0010] Preferably, the sodium ethoxide used to adjust the pH is a 0.1 M sodium ethoxide ethanol solution.

[0011] Preferably, the generated olaparib-copper nanocomplex is also modified with a maleimide group.

[0012] Preferably, maleimide group modification refers to adding a Py-PAA-PEG aqueous solution to an aqueous solution of an olaparib-copper nanocomplex, and reacting with ultrasound in an ice bath to obtain an olaparib-copper nanocomplex modified with a maleimide group. This step is also a PEG coating process. After PEG coats the olaparib-copper nanocomplex, its cytotoxicity is significantly reduced and its blood retention time is increased.

[0013] Preferably, the mass ratio of Py-PAA-PEG to Olaparib-copper nanocomplex is between 1:1 and 1:3.

[0014] Preferably, the ice bath ultrasonic reaction time is more than 2 hours, the ultrasonic frequency is 40KHZ, and after the ice bath ultrasonic reaction is completed, the precipitate is collected by centrifugation, which is the olaparib-copper nanocomplex modified with maleimide groups, and washed with deionized water.

[0015] Preferably, the small molecule polypeptide cRGD is also loaded on the olaparib-copper nanocomplex modified with a maleimide group.

[0016] Preferably, the specific method for loading the small molecule polypeptide cRGD is to dissolve the small molecule polypeptide cRGD in a PBS solution, add TCEP to activate the thiol group of the polypeptide, and then dropwise add it into an aqueous solution of olaparib-copper nanocomplex modified with a maleimide group to react.

[0017] The invention also discloses an olaparib-copper nanocomplex prepared by the preparation method.

[0018] And the use of the above-mentioned olaparib-copper nanocomplex in the preparation of drugs for treating breast cancer.

[0019] The olaparib-copper nanocomplex synthesized by the present invention has targeting and chemical dynamics effects, controllable size, low effective dose, effectively induces breast cancer cell killing, and is applied to the treatment of tumors. The material demonstrates strong superiority in tumor targeted drug delivery and treatment, and provides an effective development path for the development and design of multifunctional biomaterials.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) The present invention uses the stable coordination of the small molecule drug olaparib and copper ions to obtain an olaparib-copper nanocomplex with stable properties and chemical dynamic function using an oil bath method. The design method is simple and easy to operate and is suitable for large-scale synthesis.

[0022] (2) The material synthesized by the present invention has good tumor targeting effect and can be used as a targeted drug delivery system. It is safe and reliable and has a very broad application prospect in the field of tumor treatment.

[0023] (3) The material synthesized by the present invention enhances the efficacy of the chemotherapy drug Olaparib and copper ions, achieving an effect of one plus one being greater than two.

[0024] (4) The effective drug dose of the material synthesized by the present invention is much lower than the effective drug dose of Olaparib.

[0025] (5) The present invention relates to the modification of the material surface with organic molecules, such as polyethylene glycol and small molecule peptides, that is, Cu-Olaparib can be surface-modified with pyrene-polyacrylic acid-polyethylene glycol (Py-PAA-PEG) and coupled with the tumor targeting peptide cRGD.

[0026] (6) The material described in the present invention has chemical dynamics function and forms a targeted drug delivery system with the small molecule polypeptide cRGD to achieve breast cancer treatment in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a graph of the hydrated particle size of Cu-ola, Cu-ola-PEG and Cu-ola-PEG-cRGD of the present invention measured by dynamic light scattering (DLS).

[0028] Figure 2 It is a scanning electron microscope image (SEM) of Cu-ola in the present invention.

[0029] Figure 3 It is the ultraviolet-visible spectroscopic spectrum diagram of Cu-ola and Cu-ola-PEG of the present invention.

[0030] Figure 4 This is the elemental composition diagram of Cu-ola detected by X-ray photoelectron spectroscopy (XPS).

[0031] Figure 5 It is the thermogravimetric analysis (TGA) of Cu-ola and Olaparib of the present invention.

[0032] Figure 6 It is a UV-visible spectroscopic diagram using methylene blue to detect the generation of hydroxyl radicals.

[0033] Figure 7 It is a graph showing the cell viability of Cu-ola-PEG of the present invention at the 4T1 cell level.

[0034] Figure 8 The Cu-ola-PEG, Cu 2+ , Olaparib, Cu 2+ Cell survival rate of the +Olaparib group at the 4T1 cell level.

[0035] Fig. 9 This is a graph showing the expression of γ-H2AX protein after Cu-ola-PEG of the present invention acts on 4T1 cells.

[0036] Fig.10 This is a diagram showing the tumor inhibition effect of the Cu-ola-PEG of the present invention in a 4T1 breast cancer mouse model.

[0037] Fig.11 The figure is the tumor growth volume data of Cu-ola-PEG of the present invention in 4T1 breast cancer mouse model. Specific implementation methods

[0039] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0040] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0041] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0042] Example 1

[0043] Example 1 Synthesis of Olaparib-Copper Nanocomplex

[0044] Dissolve 4 mg of copper chloride dihydrate in 1 mL of DMF solution, and 2 mg of Olaparib in 1 mL of DMF. After they are fully dissolved, mix the two, add 1 ml of 0.1 M sodium ethoxide dissolved in ethanol, adjust its pH to 8, then add 100 μL of trimethylamine aqueous solution, stir evenly, and react at 70-90 ° C for 16-24 hours. After collecting the precipitate by centrifugation at 14000 rpm for 10 minutes, wash it three times with DMF, anhydrous ethanol and water respectively, and finally obtain Olaparib-copper nanocomplex, namely Cu-Olaparib, referred to as Cu-ola.

[0045] Example 2 Polyethylene glycol modified maleimide group and linked to targeting small molecule peptide cRGD

[0046] An aqueous solution (5 mL) containing 10 mg of Py-PAA-PEG (for the synthesis method, see Acta Biomater. 2024 Jan 15; 174: 372-385.) was added dropwise to an aqueous solution (5 mL) containing 10 mg of Cu-ola, and ultrasonicated on an ice bath for 2 hours to coat PEG on the surface of the Cu-ola material. The PEG-coated Cu-ola material was then collected by centrifugation and washed three times with deionized water to obtain Cu-Olaparib-PEG modified with a maleimide group, referred to as Cu-ola-PEG. Subsequently, 1 mg of small molecule peptide cRGD was dissolved in PBS solution, and 0.25 mg of tri(2-carboxyethyl)phosphine (TCEP) was added to activate the thiol group of the peptide. The mass ratio of the two was 4:1. After stirring at room temperature for 1 hour, Cu-ola-PEG aqueous solution (10 mg) was added dropwise, and stirring at room temperature was continued for 24 hours to complete the connection between cRGD and Cu-ola-PEG. Finally, centrifugation was performed to obtain the olaparib-copper nanocomplex Cu-Olaparib-PEG-cRGD loaded with the targeted small molecule peptide cRGD, referred to as Cu-ola-PEG-cRGD. It can be quantified by partial drying, that is, taking part of the solution and drying it in an oven at 55°C. The hydrated particle sizes of the three products were detected by a particle size analyzer to be approximately 118 nm, 126.9 nm and 154.4 nm ( Figure 1 ), and its morphology is spherical under scanning electron microscopy ( Figure 2 ), the absorbance curve detected by UV-visible spectrophotometer is shown in the figure, with an absorption peak at about 280nm ( Figure 3 ), XPS analysis showed that its elemental composition was mainly Cu, C and O ( Figure 4 ), TGA detected that Olaparib began to decompose at 400℃ ( Figure 5 ), methylene blue detects the generation of hydroxyl radicals. When Cu-ola reacts with hydrogen peroxide, hydroxyl radicals can be effectively generated, causing the absorption peak of methylene blue at 652nm to decrease ( Figure 6 ).

[0047] Example 3 Cell-level 4T1 breast cancer cell cytotoxicity assay

[0048] 4T1 cells were seeded in 96-well plates at a density of 10,000 cells per well. After overnight attachment, they were incubated with different concentrations of olaparib (0-200 μM calculated based on the concentration of olaparib and copper ions), copper ions, olaparib + copper ions, and Cu-ola-PEG for different time periods. After washing twice with PBS, CCK 8 solution (100 uL) was added and incubated at 37°C in the dark for 1 h. The absorbance of the cells was then detected at 450 nm, and the cell activity was calculated ( Figure 7 ).

[0049] Example 4 Fluorescence detection of γ-H2AX expression at the cell level

[0050] 4T1 cells (200,000 cells / well) were seeded in a confocal dish and treated with olaparib and Cu-ola-PEG (low concentration (15 μg / mL) and high concentration (30 μg / mL) respectively) for 24 hours, and the culture medium was discarded. 4% paraformaldehyde was added for fixation for 10 minutes, 0.2% Triton X-100 was permeabilized for 5 minutes, 1% BSA was blocked for 1 hour, γ-H2AX primary antibody was added for incubation overnight (4°C), and the secondary antibody was continued to be incubated at room temperature for 1 hour. Finally, DAPI anti-fluorescence quencher was added and the images were taken with a laser confocal scanning microscope ( Figure 8 ), as can be seen from the figure, the cell nuclei in the Cu-ola-PEG group were obviously shrunk, and the expression of γ-H2AX protein was higher than that of olaparib at the same concentration, causing more obvious damage to the DNA of 4T1 cells.

[0051] Example 5 Detection of γ-H2AX protein expression at the cell level

[0052] 4T1 cells (200,000 cells / well) were seeded in a 6-well plate and treated with olaparib and Cu-ola-PEG for 24 hours. The cells were collected and PIPA, PMSF and phosphatase inhibitors were added. The cells were lysed on ice for 30 minutes, then centrifuged at 12,000 g for 15 minutes. The supernatant protein solution was taken and detected with a BCA protein kit. A 10% separation gel was then prepared for protein electrophoresis. After blocking with 5% BSA, the primary antibody γ-H2AX was added and incubated overnight at 4°C. The secondary antibody was then added and incubated at room temperature for 2 hours for protein exposure ( Fig. 9 ). Sample grouping and implementation in the figure Figure 4 The results were consistent with those of the previous study, further confirming the serious damage to the DNA of 4T1 cells.

[0053] Example 6 Tumor Inhibition Effect of Cu-ola-PEG-cRGD

[0054] Balb / c mice with established 4T1 tumor model were used. When the 4T1 tumor volume reached 80 mm 3 The mice were injected with the drug through the tail vein three times, once every other day, for 14 consecutive days for efficacy testing. The experiment was divided into normal saline group, olaparib group (3 mg / kg), Cu-ola-PEG group (3 mg / kg), and Cu-ola-PEG-cRGD group (3 mg / kg), with 6 parallel mice in each group. The weight and tumor size of the mice were recorded every day during the period, and the tumor size was observed after dissection. The order of grouping was normal saline, olaparib, Cu-ola-PEG, Cu-ola-PEG-cRGD ( Figure 10-11). Experiments have shown that compared with olaparib, Cu-ola-PEG has a certain tumor inhibition effect, but the Cu-ola-PEG-cRGD group with the addition of the targeting peptide cRGD has the best effect and the most obvious tumor inhibition. However, from the perspective of cell level, PEG and cRGD themselves have no therapeutic effect on 4T1 tumors.

[0055] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an olaparib-copper nanocomplex, characterized in that: Copper chloride hydrate and olaparib are dissolved in DMF respectively, then mixed, the pH value is adjusted to 8-9 with sodium ethoxide, and finally trimethylamine is added to react to generate olaparib-copper nanocomplex.

2. The preparation method according to claim 1, characterized in that: The mass ratio of cupric chloride hydrate to olaparib is 2:1, and the molar ratio of trimethylamine to cupric chloride hydrate is 1:

2.

3. The preparation method according to claim 2, characterized in that: The reaction temperature is 70-90°C and the reaction time is 16-24h.

4. The preparation method according to claim 2, characterized in that: The sodium ethoxide used to adjust the pH is a 0.1 M sodium ethoxide ethanol solution.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The resulting olaparib-copper nanocomplex was also modified with maleimide groups.

6. The preparation method according to claim 5, characterized in that: Maleimide group modification refers to adding a Py-PAA-PEG aqueous solution to an aqueous solution of an olaparib-copper nanocomplex, and performing an ice bath ultrasonic reaction to obtain an olaparib-copper nanocomplex modified with a maleimide group.

7. The preparation method according to claim 6, characterized in that: The mass ratio of Py-PAA-PEG to olaparib-copper nanocomplex is between 1:1 and 1:

3.

8. The preparation method according to claim 6, characterized in that: The small molecule peptide cRGD was also loaded on the olaparib-copper nanocomplex modified with maleimide groups.

9. The olaparib-copper nanocomplex prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the olaparib-copper nanocomplex according to claim 9 in the preparation of a drug for treating breast cancer.

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