A nano-probe targeting prostate cancer, its preparation method and use

Abstract

The application discloses a kind of nano-probes targeting prostate cancer, its preparation method and purposes.The expression of the nano-probe is AuNC@MOF / OLA-PSMA-617, wherein, olaparib is in gold nanocage, MOF is coated outside gold nanocage, PSMA-617 is combined with MOF to form surface modification;The mass ratio of the MOFs and the gold nanocage is 30-35:60, and the mass ratio of the olaparib and the gold nanocage is 5-10:60.The gold nanomaterial is combined with olaparib and metal organic framework material in the application, the metal organic framework material is targeted surface modification, and a radiotherapy sensitization nano-probe with clinical transformation application potential is constructed, which brings hope for the treatment or diagnosis of prostate cancer.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine, specifically to a method for preparing and applying a nanoprobe that can be used to enhance the radiosensitization of advanced castration-resistant prostate cancer, and more specifically to a nanoprobe targeting prostate cancer, its preparation method, and its uses. Background Technology

[0002] Radiotherapy is an important treatment for advanced prostate cancer, but side effects such as damage to surrounding tissues and organs, anemia, leukopenia, and pathological fractures limit its application. Due to the limitations imposed by the principles of external beam radiation therapy, the radiation tolerance dose of normal tissues remains an insurmountable limiting factor. Therefore, improving the radiation sensitivity of prostate cancer tumor tissue has become an important approach to enhancing the efficacy of radiotherapy.

[0003] Olaparib (OLA), an inhibitor of DNA repair enzymes, has been found to have a radiosensitizing effect in the treatment of various tumors. However, its clinical application is limited due to its lack of tumor targeting, hematologic toxicity, and poor water solubility. Summary of the Invention

[0004] The purpose of this invention is to address the clinical limitations of olaparib by combining gold nanomaterials with olaparib and metal-organic frameworks (MOFs) and performing targeted surface modification on the MOFs to construct a radiosensitizing nanoprobe, AuNC@MOF / OLA, with potential for clinical translational applications, targeting prostate cancer.

[0005] To achieve the above objectives, the present invention provides a nanoprobe targeting prostate cancer, with the expression AuNC@MOF / OLA-PSMA-617, wherein olaparib (OLA) is contained within a gold nanocage, MOF coats the AuNC nanocage, and PSMA-617 binds to the MOF to form a surface modification; the mass ratio of MOFs to the gold nanocage is 30-35:60, and the mass ratio of olaparib to the gold nanocage is 5-10:60.

[0006] Optionally, the mass ratio of PSMA-617 to the gold nanocage is 1-5:60.

[0007] Optionally, the MOF is a porphyrin MOF, prepared by reacting tetracarboxyphenylporphyrin with zirconium oxychloride.

[0008] The present invention also provides a method for preparing the above-described nanoprobe targeting prostate cancer, comprising:

[0009] Step S1: Tetracarboxyphenylporphyrin and zirconium dichloride are mixed in a gold nanocage suspension and subjected to hydrothermal reaction to obtain a gold nanocage suspension coated with MOFs.

[0010] Step S2: Olaparib is added to the gold nanocage suspension coated with MOFs, mixed, resuspended and stirred to load olaparib into the gold nanocage coated with MOFs, thus obtaining AuNC@MOF / OLA.

[0011] Step S3: Mix AuNC@MOF / OLA and PSMA-617 uniformly to obtain the AuNC@MOF / OLA-PSMA-617 complex.

[0012] Optionally, the mass ratio of olaparib to the gold nanocage is 5-10:60.

[0013] Optionally, the mass ratio of MOFs to the gold nanocage is 30-35:60.

[0014] Optionally, the mass ratio of PSMA-617 to the gold nanocage is 1-5:60.

[0015] The present invention also provides the use of the above-described nanoprobe targeting prostate cancer for the preparation of a drug for diagnosing and / or treating prostate cancer.

[0016] Optionally, under near-infrared light, the MOF with porphyrin structure in the nanoprobe excites H2O2 in the tumor microenvironment to generate singlet oxygen, thereby playing a photodynamic therapy role; the gold nanocage excitation plays a photothermal therapy role.

[0017] Optionally, the nanoprobe can release olaparib and gold nanocages under radiotherapy laser to enhance the effect of radiotherapy.

[0018] Compared with the prior art, the technical solution of the present invention has at least the following beneficial effects:

[0019] By constructing porphyrin MOFs, surface-modifying them with PSMA-617, and then combining them with gold nanocages to form AuNC@MOF / OLA-PSMA-617 nanoprobes, PSMA-617 is specifically and precisely delivered to prostate cancer tumor tissue. The porphyrin MOFs, possessing photosensitizing properties, are coated on the nanoprobes, generating photothermal and photodynamic therapeutic effects under near-infrared light excitation. Under the influence of the slightly acidic tumor microenvironment, the MOFs gradually degrade and release, at the tumor site, targeted gold nanocages and olaparib, which can be used for radiosensitization. The nanoprobes of this invention have potential for clinical translational applications, bringing hope for the diagnosis or treatment of prostate cancer. Furthermore, the preparation method of the nanoprobes of this invention is simple and suitable for industrial application. Attached Figure Description

[0020] Figure 1a Transmission electron microscopy image of the nanoprobe prepared in this invention;

[0021] Figure 1b This is a mapping elemental analysis diagram of the nanoprobe prepared in this invention.

[0022] Figure 2 This is a process flow diagram of a method for preparing a nanoprobe with radiosensitizing properties according to the present invention.

[0023] Figure 3 This is a schematic diagram of the synthesis route of a nanoprobe with radiosensitizing properties according to the present invention.

[0024] Figure 4 Photographs of cell apoptosis experiments under radiotherapy using nanoprobes prepared according to this invention.

[0025] Figure 5 This is a schematic diagram showing the condition of tumors after radiotherapy.

[0026] Figure 6 Photographs showing the biosafety assessment of the nanoprobes prepared for this invention as diagnostic and therapeutic agents. Detailed Implementation

[0027] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The “gold nanocages” mentioned in this article refer to hollow, porous gold nanoparticles, ranging in size from 10 to 150 nanometers, which have a hollow structure, are porous, and have a high loading capacity.

[0029] The “AuNC@MOF / OLA-PSMA-617” described in this article is a functionalized nanoprobe with targeted radiosensitization properties. AuNC represents a gold nanocage, MOF is a metal-inorganic framework material (in this invention, it is a porphyrin MOF), OLA is olaparib, and PSMA-617 is a prostate cancer-targeting ligand. In the AuNC@MOF / OLA-PSMA-617 composite material, olaparib (OLA) is loaded within the hollow structure of the gold nanocage AuNC, the MOF coats the outside of the gold nanocage AuNC, and PSMA-617 combines with the MOF to form a surface modification. The mass ratio of MOFs to the gold nanocage is 30-35:60, the mass ratio of olaparib to the gold nanocage is 5-10:60, and the mass ratio of PSMA-617 to the gold nanocage is 1-5:60. The electron microscopy image of this nanoprobe AuNC@MOF / OLA-PSMA-617 is shown below. Figure 1aAs shown, under near-infrared light excitation, MOF materials with porphyrin structures can stimulate H2O2 and other substances in the tumor microenvironment to generate singlet oxygen, thus producing a photodynamic therapy effect; under near-infrared light excitation, gold nanocages can produce a photothermal therapy effect. In the slightly acidic tumor microenvironment, with the decomposition of MOFs, gold nanocages and olaparib are released, which can enhance the therapeutic effect of radiotherapy.

[0030] like Figure 2 , Figure 3 As shown, the method for preparing the nanoprobe with radiosensitizing properties of the present invention includes:

[0031] S1, Tetracarboxyphenylporphyrin and zirconium dichloride are mixed in a gold nanocage suspension and subjected to hydrothermal reaction to obtain a gold nanocage suspension A coated with MOFs.

[0032] Tetracarboxyphenylporphyrin reacts with zirconium dichloride to generate porphyrin MOF. The porphyrin MOF material can be degraded in the acidic microenvironment of a tumor, and near-infrared laser light also has a promoting effect.

[0033] Gold nanomaterials can generate a photothermal effect under near-infrared light excitation, making them an excellent photothermal therapeutic agent. Furthermore, as a high atomic number material, gold nanomaterials can generate electrons through the photoelectric effect when irradiated with radiotherapy rays. These electrons can react with water molecules to produce cytotoxic free radicals, directly accelerating the breakage of DNA strands in the cell nucleus and thus killing tumor cells. This allows gold nanomaterials to exert a radiosensitizing effect. The gold nanomaterial used in this paper is a gold nanocage with a hollow, porous structure.

[0034] The gold nanocages are obtained by stirring silver nanocages with HAuCl4 and then undergoing a displacement reaction. The silver nanocages can be commercially available or prepared by conventional methods, such as reduction reaction using silver trifluoroacetate as a raw material and sodium hydrosulfide as a reducing agent.

[0035] S2, Olaparib is added to solution A, mixed, resuspended and stirred to load Olaparib into gold nanocages coated with MOFs, to obtain solution B.

[0036] Olaparib (OLA), an inhibitor of DNA repair enzymes, has been found to have a radiosensitizing effect in the treatment of various tumors. However, its clinical application is limited due to its lack of tumor targeting, hematologic toxicity, and poor water solubility.

[0037] Olaparib is a PARP inhibitor that acts on SW620 cells, inactivating PARP-1. PARP inhibitors can treat hereditary cancers that share the same "rogue gene," such as breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

[0038] Olaparib is typically administered orally in clinical practice, but these small molecule drugs have weak targeting specific tumor tissues. Furthermore, drug delivery to the tumor site via the bloodstream is inefficient. This invention utilizes surface-modified PSMA-617 to precisely target prostate cancer tumor sites. Additionally, the nanoprobe constructed in this invention leverages the EPR effect (enhanced permeability and retention effect) to deliver the drug to the tumor site efficiently and effectively.

[0039] Olaparib's hematologic toxicity refers to its toxic effects on the blood and lymphatic system, such as anemia. This nanoprobe avoids damage to the blood and lymphatic system by encapsulating OLA within MOF materials until the nanoprobe degrades in the slightly acidic microenvironment inside the tumor tissue, releasing the OLA.

[0040] Olaparib suffers from poor water solubility, meaning it easily precipitates in solution. This characteristic can affect drug delivery efficiency in vivo, thus impacting therapeutic efficacy. This invention overcomes this drawback by loading olaparib into gold nanocages and then encapsulating it with MOFs. Through resuspension and stirring, olaparib is loaded into the hollow structure of the gold nanocages via the porous structure of MOFs. Due to olaparib's poor water stability, once dissolved olaparib enters the hollow structure of the gold nanocages and precipitates, it is difficult for it to escape from the MOF-encapsulated gold nanocages, thus achieving loading of olaparib by the gold nanocages.

[0041] This invention first forms gold nanocages coated with MOFs through a hydrothermal reaction. Since both the gold nanocages and MoFs are porous structures, olaparib is loaded by resuspension and stirring. Compared with the method of first loading olaparib into gold nanocages and then forming MoFs coating through hydrothermal reaction, this method avoids the loss of efficacy of olaparib during the hydrothermal reaction process and the uncontrollable loading amount.

[0042] The mass ratio of olaparib to gold nanocages is 5-10:60. During preparation, olaparib is in excess, and the olaparib loading can be calculated by the reduction of olaparib in the solution.

[0043] S3, PSMA-617 is added to solution B and mixed thoroughly to obtain AuNC@MOF / OLA-PSMA-617 complex, which can be used as a nanoprobe and has photodynamic / photothermal therapy and radiosensitization properties under near-infrared light excitation.

[0044] PSMA-617 (Vipivotide tetraxetan) has the CAS number 1702967-37-0, and its structural formula is as follows:

[0045]

[0046] PSMA-617 is a potent inhibitor of prostate-specific membrane antigen (PSMA) and can selectively bind to PSMA.

[0047] The carboxyl bonds on the PSMA-617 structure can combine with incompletely paired zirconium clusters in MOF materials to form surface modifications.

[0048] Olaparib (OLA) is loaded within a hollow structure of gold nanocages (AuNC), with a metallofibrillated (MOF) coating the outside of the AuNC nanocages. PSMA-617 is used to specifically modify the MOF surface for prostate cancer targeting. Porphyrin-based MOFs possess photosensitizing properties; these functionalized nanoprobes, aggregated within the tumor, can progressively release olaparib and gold nanomaterials within the slightly acidic tumor microenvironment, exhibiting targeted and sustained-release effects. They can generate photodynamic / photothermal therapy and radiosensitization properties under in vitro near-infrared light excitation and radiotherapy, as needed. Under near-infrared laser excitation, photothermal and photodynamic therapy effects can be produced. Under radiotherapy, a radiosensitizing effect can be achieved. In the laboratory, the wavelength of near-infrared light is typically 808 nm; this wavelength is used as an example and is not a limitation.

[0049] The mass ratio of MOFs to the gold nanocages is 30-35:60.

[0050] The following detailed description is based on specific examples. All raw materials and reagents used in the examples are commercially available.

[0051] Example

[0052] Gold nanocages were prepared using a template-based gold-silver substitution reaction. 50 mL of ethylene glycol was added to a reaction flask, and after heating in an oil bath at 150 °C, 1 mL of a 2 mM sodium hydrosulfide solution in ethylene glycol was added. After heating for 2 min, 0.5 mL of a 2 mM hydrochloric acid solution and 15 mL of a 20 mg / mL polyvinylpyrrolidone (PVP) solution in ethylene glycol were added. After heating for another 2 min, 4 mL of a 282 mM silver trifluoroacetate solution in ethylene glycol was added, and the mixture was stirred for 90 min. The ethylene glycol was then washed away with acetone, and excess PVP was washed away with ultrapure water to obtain the silver nanocages.

[0053] Next, 100 ml of ethylene glycol solution of PVP (1 mg / ml) was placed in a reaction flask, heated to 100°C, and then 1 ml of silver nanocage suspension was added and gently stirred for 15 min. Then, 20 ml of HAuCl4 solution (0.1 mM) was slowly added dropwise while stirring continuously. After the gold-silver displacement reaction was complete, 2 ml of ammonia was added to remove the displaced AgCl, and the mixture was then washed with ultrapure water to obtain the gold nanocage suspension.

[0054] In a 10 ml centrifuge tube, 2.2 mg of tetracarboxyphenylporphyrin (H2TCPP) and 3.3 mg of zirconium dichloride (ZrOCl2) were added to DMF solution and dissolved completely using high-frequency sonication. 50 μl of the solution prepared in the above steps and 200 μl of gold nanocage solution were added to a 25 ml hydrothermal reactor, followed by the addition of DMF solution. The mixture was then sonicated to disperse and dissolve the solution before proceeding with the hydrothermal reaction. After the reaction was complete, the reaction solution was cooled to room temperature and centrifuged at high speed (12000 g, 20 min) with deionized water and ethanol (V:V = 4:1). The precipitate was collected. The precipitate was resuspended in ultrapure water and then resuspended and stirred in olaparib (OLA) (5 mg / ml) solution.

[0055] After adding PSMA-617 and stirring thoroughly for 24 hours, the mixture is centrifuged at high speed (12000g, 10min) to obtain the multifunctional nanoprobe AuNC@MOF / OLA-PSMA-617 constructed in this invention. Its transmission electron microscope image is shown below. Figure 1a As shown, the surface-modified PSMA-617 MOF material is coated on the outside of gold nanocages. The elemental analysis diagram of the nanoprobe AuNC@MOF / OLA-PSMA-617 is shown below. Figure 1b As shown, the structure of the nanoprobe can be confirmed, with a particle size of approximately 40–50 nm. Figure 1b In the diagram: a represents the overall distribution of elements in the nanoprobe, b represents the distribution of Au, c represents the distribution of Zr, d represents the distribution of O, e represents the distribution of N, and f represents the distribution of C.

[0056] The effectiveness of the nanoprobe of the present invention will be verified below.

[0057] Radiation therapy involves irradiating tumor cells with high doses of radiation, causing DNA damage and promoting apoptosis.

[0058] The animal radiotherapy method of the present invention is as follows:

[0059] Subcutaneous injection of approximately 10 7 PC-3 cells were injected subcutaneously into nude mice (purchased from the Shanghai Pudong Experimental Animal Center and housed in the SPF-grade animal laboratory of Shanghai Jiao Tong University) until the tumor volume reached 200 mm. 3At that time, 48 hours after intravenous injection of PBS (control) or AuNC@MOF / OLA-PSMA-617 suspension, X-ray irradiation (8 Gy) was performed for 8 minutes. Alternatively, X-ray irradiation (1 Gy) for 1 minute was sufficient.

[0060] Cell apoptosis experiment under radiotherapy

[0061] The subjects were divided into four groups for comparison.

[0062] PBS control group: PBS was injected via the tail vein only, without radiotherapy.

[0063] Nanoprobe control group: The nanoprobe of the present invention was injected only into the tail vein, without radiotherapy.

[0064] PBS + radiotherapy group: PBS was injected into the tail vein, followed by radiotherapy.

[0065] Nanoprobe + radiotherapy group: The nanoprobe of this invention is injected into the tail vein and then radiotherapy is performed.

[0066] Experimental results are as follows Figure 4 As shown, the proportion of apoptotic cells in tumor cells that have taken up the nanoprobe constructed in this invention increased significantly after radiotherapy (nanoprobe + radiotherapy group), which proves that the nanoprobe of this invention has radiosensitizing properties.

[0067] After treatment, tumor samples were excised and fixed with paraformaldehyde 21 days later. Immunohistochemical staining (Ki-67) was used to assess the tumor's response to radiotherapy and to evaluate the sensitization effect of the nanoprobe. Yellow-stained cells represented a positive result; therefore, fewer yellow-stained cells indicated a better treatment effect. Results are as follows... Figure 5 As shown, the treatment effect of the nanoprobe + radiotherapy group was significantly better than that of the PBS + radiotherapy group, with a significant reduction in yellow-stained cells, proving that the nanoprobe of the present invention significantly enhances the radiotherapy effect.

[0068] Biosafety assessment

[0069] Experimental method: Nanoprobe suspension was administered via tail vein to assess safety. The concentration was 20 mg / ml. One week after injection, major organs (heart, liver, spleen, lung, and kidney) were collected for HE staining to assess acute toxicity of the nanoprobe.

[0070] The heart, liver, spleen, lungs, and kidneys were scanned and photographed respectively. The experimental results are as follows: Figure 6 As shown, there were no significant changes in the heart, liver, spleen, lungs, and kidneys in the PBS-treated group and the nanoprobe group of the present invention, demonstrating the good biosafety of the nanoprobe of the present invention.

[0071] In summary, the method for preparing the nanoprobe provided by this invention first forms a porous metal framework material coated with gold nanocages through a hydrothermal reaction, and then loads olaparib onto it, avoiding the loss of efficacy and uncontrollable loading caused by the water instability of olaparib. Next, PSMA-16 is added to allow its surface carboxyl groups to bind with uncoordinated zirconium clusters in the MoFs, thus modifying the surface and enabling the nanoprobe to target prostate cancer. This invention constructs a radiosensitizing nanoprobe with potential for clinical translational applications by combining gold nanomaterials with olaparib and metal-organic framework materials, and by targeting the surface of the metal-organic framework material. This brings hope for the diagnosis or treatment of prostate cancer (especially advanced prostate cancer).

[0072] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A nanoprobe targeting prostate cancer, characterized in that, Its expression is AuNC@MOF / OLA-PSMA-617, where olaparib OLA is located in a gold nanocage, MOF is coated on the outside of the gold nanocage AuNC, and PSMA-617 is combined with MOF to form a surface modification; the mass ratio of MOF to the gold nanocage is 30-35:60, and the mass ratio of olaparib to the gold nanocage is 5-10:

60.

2. The nanoprobe targeting prostate cancer as described in claim 1, characterized in that, The mass ratio of PSMA-617 to the gold nanocage is 1-5:

60.

3. The nanoprobe targeting prostate cancer as described in claim 1, characterized in that, The MOF is a porphyrin MOF, prepared by reacting tetracarboxyphenylporphyrin with zirconium oxychloride.

4. A method for preparing a nanoprobe targeting prostate cancer according to claim 1, characterized in that, The method includes: Step S1: Tetracarboxyphenylporphyrin and zirconium dichloride are mixed in a gold nanocage suspension and subjected to hydrothermal reaction to obtain a gold nanocage suspension coated with MOF. Step S2: Olaparib is added to the suspension of gold nanocages coated with MOF, mixed, resuspended and stirred to load olaparib into the gold nanocages coated with MOF, thus obtaining AuNC@MOF / OLA; Step S3: Mix AuNC@MOF / OLA and PSMA-617 uniformly to obtain the AuNC@MOF / OLA-PSMA-617 complex.

5. The preparation method according to claim 4, characterized in that, The mass ratio of olaparib to the gold nanocage is 5-10:

60.

6. The preparation method according to claim 4, characterized in that, The mass ratio of MOF to the gold nanocage is 30-35:

60.

7. The preparation method according to claim 4, characterized in that, The mass ratio of PSMA-617 to the gold nanocage is 1-5:

60.

8. The use of a nanoprobe targeting prostate cancer according to any one of claims 1 to 3, characterized in that, It is used to prepare drugs for the diagnosis and / or treatment of prostate cancer.

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