Olaparib-copper nano-complexes, methods of making and uses thereof

Abstract

This invention discloses a method for preparing olaparib-copper nanocomplexes. Hydrated copper chloride and olaparib are dissolved separately in DMF, then mixed, and the pH is adjusted to 8-9 with sodium ethoxide. Finally, trimethylamine is added to react and generate the olaparib-copper nanocomplexes. The application of the prepared olaparib-copper nanocomplexes in the preparation of drugs for treating breast cancer is also disclosed. The olaparib-copper nanocomplexes synthesized in this invention exhibit targeting and chemokinetic effects, controllable size, low effective dose, and effectively induce the killing of breast cancer cells, making them suitable for tumor treatment. This material demonstrates strong advantages in tumor-targeted drug delivery and therapy, providing an effective development pathway for the development and design of multifunctional biomaterials.

Description

Technical Field

[0001] This invention relates to an olaparib-copper nanocomplex, its preparation method and application, belonging to the field of biomedical technology. Background Technology

[0002] Nanotechnology shows great promise in addressing several key challenges of conventional anticancer chemotherapy. It is anticipated to develop a new generation of highly effective cancer therapies that overcome the biological, biophysical, and biomedical barriers to standard chemotherapy in the human body. Typically, nanostructures can protect embedded drug molecules from degradation in the bloodstream, allowing for their safe and undamaged delivery to specific target sites within the body. The unique characteristics 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 these, nanometal complexes show great potential in facilitating the delivery of effective anticancer metal drugs.

[0003] Nanoscale metal complexes, composed of metals and ligands, exhibit varying redox properties and bioactivities depending on the metal center and ligand. Due to their tunable ligands, high biocompatibility, excellent photophysical and chemical properties, and ease of modification, nanoscale metal complexes show great potential for cancer therapy. Depending on the diagnostic or therapeutic needs, suitable organic ligands are selected, mainly including non-functional and functional ligands. Nanoscale metal complexes synthesized with non-functional ligands primarily serve as drug carriers, while functional ligands endow them with different properties, such as fluorescent diagnostic or therapeutic properties. By covalently or non-covalently binding to targeted small molecules, synthesized nanoscale metal complexes can specifically reach lesions, improving diagnostic accuracy and therapeutic targeting, as well as reducing potential toxic side effects. With the emergence of the concept of copper death, nanoscale copper complexes have also attracted increasing attention from researchers. Copper death is a copper-dependent form of cell death in which cells die due to the direct binding of copper to lipid components of the tricarboxylic acid (TCA) cycle and the aggregation of enzymes, leading to protein toxicity stress and further inducing cell death. In addition, monovalent copper ions have been shown to effectively catalyze the generation of DNA-damaging hydroxyl radicals from hydrogen peroxide, while excessive glutathione at tumor sites can reduce divalent copper ions to monovalent copper ions, thereby achieving certain chemical damage. Therefore, copper-containing nanomaterials have become a research hotspot in anti-tumor studies.

[0004] Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) are a class of anti-tumor drugs whose mechanism of action relies on the defective DNA repair pathways in breast cancer (BRCA) mutations and homologous recombination (HR) repair genes (HRD), a set of key genes for double-strand breaks (dsb), and interstrand crosslinking (icl) repair pathways. This process is known as "synthetic lethality." Olaparib is a classic small molecule drug that inhibits PARP 1 (PARP 1) and works through a "synthetic lethality" mechanism because it inhibits PARP enzymes, leading to the accumulation of DNA damage. However, in actual clinical applications, cancer cells easily develop resistance to these drugs, and their cytotoxic effects on DNA, when combined with those of chemotherapy agents, are additive or synergistic, contributing more to DNA fragmentation and apoptosis than when olaparib is used alone. cRGD peptides, as a broad-spectrum targeted drug, can target nucleolins on the surface of various cancer cells, thereby achieving targeted drug accumulation and achieving a tumor-targeting effect. Summary of the Invention

[0005] The purpose of this invention is to provide an olaparib-copper nanocomplex, which combines the anticancer drug olaparib with copper, which has a copper death effect, to achieve low-dose treatment of breast cancer and reduce olaparib resistance.

[0006] The technical solution adopted in this invention is as follows:

[0007] A method for preparing olaparib-copper nanocomplexes involves dissolving hydrated copper chloride and olaparib separately in DMF, then mixing them, adjusting the pH to alkaline with sodium ethoxide, and finally adding trimethylamine to react and generate olaparib-copper nanocomplexes.

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

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

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

[0011] Preferably, the generated olaparib-copper nanocomplex is further modified with maleimide groups.

[0012] Preferably, the maleimide group modification involves adding a Py-PAA-PEG aqueous solution dropwise to an aqueous solution of the olaparib-copper nanocomplex, followed by an ice bath sonication reaction to obtain the olaparib-copper nanocomplex modified with maleimide groups. This step is also a PEG coating process. After PEG coating 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 and the ultrasonic frequency is 40 kHz. After the ice bath ultrasonication is completed, the precipitate is collected by centrifugation, which is the olaparib-copper nanocomplex modified with maleimide groups, and then washed with deionized water.

[0015] Preferably, the small molecule peptide cRGD is loaded onto the olaparib-copper nanocomplex modified with maleimide groups.

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

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

[0018] And the application of the aforementioned olaparib-copper nanocomplex in the preparation of drugs for treating breast cancer.

[0019] The olaparib-copper nanocomplex synthesized in this invention exhibits targeting and chemokinetic effects, controllable size, and low effective dose, effectively inducing the killing of breast cancer cells and thus applicable to tumor treatment. This material demonstrates significant advantages in tumor-targeted drug delivery and therapy, providing an effective development pathway for the development and design of multifunctional biomaterials.

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

[0021] (1) This invention utilizes the stable coordination of the small molecule drug olaparib and copper ions to obtain a stable olaparib-copper nanocomplex with chemokinetic function by using an oil bath method. This design method is simple and easy to operate and is suitable for large-scale synthesis.

[0022] (2) The material synthesized in this 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 in this invention enhances the efficacy of the chemotherapy drug olaparib and copper ions, achieving a synergistic effect greater than the sum of its parts.

[0024] (4) The effective drug dose of the material synthesized in this invention is much lower than that of olaparib.

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

[0026] (6) The material described in this invention has chemical kinetic function and forms a targeted drug delivery system with small molecule polypeptide cRGD to achieve breast cancer treatment in mice. Attached Figure Description

[0027] Figure 1 The hydrated particle size diagrams of Cu-ola, Cu-ola-PEG, and Cu-ola-PEG-cRGD of the present invention were determined using dynamic light scattering (DLS).

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

[0029] Figure 3 This is the UV-Vis spectra of Cu-ola and Cu-ola-PEG of the present invention.

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

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

[0032] Figure 6 It uses methylene blue to detect the ultraviolet-visible spectrophotometer of hydroxyl radicals.

[0033] Figure 7 This is a cell survival rate diagram of Cu-ola-PEG at the 4T1 cell level according to the present invention.

[0034] Figure 8 The present invention is Cu-ola-PEG, Cu 2+ Olaparib, Cu 2+ Cell viability diagram of the +Olaparib group at the 4T1 cell level.

[0035] Figure 9 This is a diagram showing the expression of γ-H2AX protein in 4T1 cells after Cu-ola-PEG of the present invention has been applied.

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

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

[0038] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Furthermore, while this document may provide examples of parameters containing specific values, it should be understood that the parameters need not be exactly equal to the corresponding values, but may approximate the corresponding values ​​within acceptable error tolerances or design constraints.

[0040] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0041] Example 1

[0042] Example 1 Synthesis of Olaparib-Copper Nanocomplexes

[0043] 4 mg of copper chloride dihydrate and 2 mg of olaparib were dissolved in 1 mL of DMF solution. After thorough dissolution, the two solutions were mixed, and 1 mL of 0.1 M sodium ethoxide dissolved in ethanol was added to adjust the pH to 8. Then, 100 μL of trimethylamine aqueous solution was added, and the mixture was stirred until homogeneous. The reaction was carried out at 70-90 °C for 16-24 h. The precipitate was collected by centrifugation at 14000 rpm for 10 min and washed three times with DMF, anhydrous ethanol, and water, respectively, to obtain the olaparib-copper nanocomplex, namely Cu-Olaparib, or Cu-ola for short.

[0044] Example 2: Polyethylene glycol-modified maleimide groups and linked to the targeted small molecule peptide cRGD

[0045] Add 5 mL of an aqueous solution containing 10 mg of Py-PAA-PEG (synthesis method see Acta Biomater. 2024 Jan 15; 174: 372-385.) dropwise to an aqueous solution containing 10 mg of Cu-ola. Sonicate in an ice bath for 2 hours to coat the surface of the Cu-ola material with PEG. Then collect the PEG-coated Cu-ola material by centrifugation and wash it three times with deionized water to obtain Cu-Olaparib-PEG modified with maleimide groups, abbreviated as Cu-ola-PEG. Subsequently, 1 mg of the small molecule peptide cRGD was dissolved in PBS solution, and 0.25 mg of tris(2-carboxyethyl)phosphine (TCEP) was added to activate the thiol groups of the peptide. The mass ratio of the two was 4:1. After stirring at room temperature for 1 hour, 10 mg of Cu-ola-PEG aqueous solution was added dropwise, and stirring was continued at room temperature for 24 hours to complete the connection between cRGD and Cu-ola-PEG. Finally, centrifugation yielded the olaparib-copper nanocomplex Cu-Olaparib-PEG-cRGD, abbreviated as Cu-ola-PEG-cRGD, loaded with the targeted small molecule peptide cRGD. Quantification can be performed by partial drying, i.e., a portion of the solution is dried in an oven at 55°C. The hydrated particle sizes of the three products were measured using a particle size analyzer and were approximately 118 nm, 126.9 nm, and 154.4 nm, respectively. Figure 1 ), its morphological characteristics observed by scanning electron microscopy are spherical ( Figure 2 The absorbance curve of the sample, measured by a UV-Vis spectrophotometer, is shown in the figure. It exhibits an absorption peak at approximately 280 nm. Figure 3 XPS analysis showed that its elemental composition was mainly Cu, C, and O. Figure 4 TGA analysis showed that olaparib began to decompose at 400℃. Figure 5 Methylene blue is used to detect the generation of hydroxyl radicals. When Cu-ola reacts with hydrogen peroxide, it can effectively generate hydroxyl radicals, causing the absorption peak of methylene blue at 652 nm to decrease. Figure 6 ).

[0046] Example 3: Cell-level 4T1 breast cancer cytotoxicity detection

[0047] 4T1 cells were seeded at a density of 10,000 cells per well in 96-well plates. After overnight adhesion, the cells were incubated for different durations with different concentrations (0-200 μM based on olaparib and copper ion concentrations), copper ions, olaparib + copper ions, and Cu-ola-PEG. After washing twice with PBS, the cells were incubated with 100 μL of CCK 8 solution at 37°C for 1 h in the dark. The absorbance of the cells at 450 nm was then measured, and cell viability was calculated. Figure 7 ).

[0048] Example 4: Fluorescence detection of γ-H2AX expression at the cellular level

[0049] 4T1 cells (200,000 cells / well) were seeded in confocal dishes and treated with olaparib and Cu-ola-PEG (low concentration (15 μg / mL) and high concentration (30 μg / mL), respectively) for 24 h, after which the culture medium was discarded. The cells were fixed with 4% paraformaldehyde for 10 min, infiltrated with 0.2% Triton X-100 for 5 min, blocked with 1% BSA for 1 h, incubated overnight with γ-H2AX primary antibody (4℃), and incubated with secondary antibody at room temperature for 1 h. Finally, a DAPI-containing antifluorescence quencher was added, and images were taken using a laser confocal scanning microscope. Figure 8 As shown in the figure, the cell nuclei of the Cu-ola-PEG group were significantly shrunken, and the expression level of γ-H2AX protein was higher than that of the same concentration of olaparib, resulting in more obvious DNA damage to 4T1 cells.

[0050] Example 5: Detection of γ-H2AX protein expression at the cellular level

[0051] 4T1 cells (200,000 cells / well) were seeded in 6-well plates and treated with olaparib and Cu-ola-PEG for 24 h. Cells were then collected, and PIPA, PMSF, and a phosphatase inhibitor were added. Cells were lysed on ice for 30 min, followed by centrifugation at 12000g for 15 min. The supernatant protein solution was collected and analyzed using a BCA protein assay kit. A 10% separating gel was then prepared for protein electrophoresis. After blocking with 5% BSA, primary antibody γ-H2AX was added, and the cells were incubated overnight at 4°C. Secondary antibody was then added, and the cells were incubated at room temperature for 2 h before protein exposure. Figure 9 The figure shows the sample grouping and implementation. Figure 4 The results were consistent, further confirming the severe DNA damage in 4T1 cells.

[0052] Example 6: Antitumor effect of Cu-ola-PEG-cRGD

[0053] Using Balb / c mice with an established 4T1 tumor model, when the 4T1 tumor volume reached 80 mm... 3 The mice were administered three times via tail vein injection, once every other day, for 14 consecutive days to assess efficacy. The experiment was divided into four groups: saline, olaparib (3 mg / kg), Cu-ola-PEG (3 mg / kg), and Cu-ola-PEG-cRGD (3 mg / kg), with six mice in each group. Mouse weight and tumor size were recorded daily, and tumor size was observed after dissection. The grouping order was saline, olaparib, Cu-ola-PEG, and Cu-ola-PEG-cRGD (3 mg / kg). Figures 10-11Experiments have shown that Cu-ola-PEG has a certain tumor-suppressive effect compared to olaparib, but the Cu-ola-PEG-cRGD group with added targeting peptide cRGD showed the best effect and the most significant tumor inhibition. However, cellular-level verification showed that PEG and cRGD themselves have no therapeutic effect on 4T1 tumors.

[0054] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing olaparib-copper nanocomplexes, characterized in that: Hydrated copper chloride and olaparib were dissolved separately in DMF, then mixed, and the pH was adjusted to 8-9 with sodium ethoxide. Finally, trimethylamine was added to react and generate olaparib-copper nanocomplex. The mass ratio of hydrated copper chloride to olaparib was 2:1, and the molar ratio of trimethylamine to hydrated copper chloride was 1:

2.

2. The preparation method according to claim 1, characterized in that, The reaction temperature is 70-90℃, and the reaction time is 16-24 h.

3. The preparation method according to claim 1, characterized in that, The sodium ethoxide solution used to adjust the pH is a 0.1M sodium ethoxide ethanol solution.

4. Olaparib-copper nanocomplexes prepared by the preparation method according to any one of claims 1-3.

5. A method for preparing maleimide-modified olaparib-copper nanocomplexes, characterized in that, Olaparib-copper nanocomplexes were prepared according to the preparation method of any one of claims 1-3, and the generated olaparib-copper nanocomplexes were modified with maleimide groups. The maleimide group modification refers to adding an aqueous solution of Py-PAA-PEG to an aqueous solution of olaparib-copper nanocomplex and reacting it with sonication in an ice bath to obtain olaparib-copper nanocomplex modified with maleimide groups. The mass ratio of Py-PAA-PEG to olaparib-copper nanocomplex is between 1:1 and 1:

3.

6. The maleimide-modified olaparib-copper nanocomplex prepared by the method of claim 5.

7. A method for preparing an olaparib-copper nanocomplex modified with maleimide groups and loaded with a small molecule polypeptide cRGD, characterized in that, The maleimide-modified olaparib-copper nanocomplex was prepared according to the preparation method of claim 5, and the small molecule polypeptide cRGD was loaded onto the maleimide-modified olaparib-copper nanocomplex.

8. The olaparib-copper nanocomplex loaded with maleimide groups and modified with maleic anhydride groups of the small molecule polypeptide cRGD, prepared by the method of claim 7.

9. The use of the maleimide-modified olaparib-copper nanocomplex loaded with the small molecule polypeptide cRGD as described in claim 8 in the preparation of a medicament for treating breast cancer.

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