Preparation method and application of a new composite nanomedicine based on rare earth materials

Abstract

The present invention discloses a method for preparing and applying a novel composite nanomedicine based on rare earth materials. The magnetic polypeptide nanocomposite diagnostic and therapeutic agent of the present invention, the general formula of the polypeptide nanocomposite is miPEP133-GC@GdPO4:Eu, comprising a carrier and an active polypeptide component loaded on the carrier; the carrier is GdPO4:Eu, and the active component is a hydrophilic anti-tumor miPEP133-GC peptide. The particle size of the nanomedicine is 140-160 nm. The molecular formula of the miPEP133-GC peptide is C76H125N25O26S, and the amino acid sequence is: Gly-Gln-Pro-Ser-Ser-Pro-Asp-Pro-Gly-Leu-Glu-Arg-Arg-Val-Ala-Ala-Pro-Cys, with an average molecular weight of 1837.01 g / mol. The present invention can not only realize the imaging and treatment of cancer, but also realize real-time monitoring of the therapeutic effect, which is helpful to realize the integration of diagnosis, treatment and efficacy monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and in particular relates to a preparation method and application of a novel composite nanomedicine based on rare earth materials. Background Art

[0002] Currently, common cancer treatments include surgery, radiotherapy, and chemotherapy. Chemotherapy-based combination therapy is often the primary treatment option for most patients with advanced cancer. However, chemotherapeutic drugs have low selectivity for tumor cells, are often associated with significant toxic side effects, and are prone to drug resistance, significantly diminishing the anti-cancer efficacy of chemotherapy. As important signaling molecules for many physiological functions, peptides hold enormous potential in biotherapy. Peptides generally refer to compounds consisting of fewer than 100 amino acids linked by peptide bonds. Their molecular weight lies between that of small molecules and proteins, characterized by low molecular weight, strong penetrability, and minimal toxic side effects. Tumors develop through the interaction of various molecular events across multiple factors and stages, ultimately involving the expression and regulation of oncogenes. Antitumor peptides can specifically bind to tumor-related genes and regulatory factors that affect tumors, thereby inhibiting tumor DNA synthesis, preventing angiogenesis, suppressing metastasis, and inducing apoptosis. However, drawbacks of peptide drugs, such as instability, short half-lives, and rapid clearance, limit their clinical application. Therefore, the rational design of peptide drugs, taking into account their advantages and disadvantages, is crucial.

[0003] Nanodelivery systems can encapsulate therapeutic molecules within nanoparticles or adsorb them onto their surfaces for selective delivery to lesions. This can increase the solubility of the loaded drug, improve drug absorption and bioavailability, and reduce toxic side effects. Gd-based materials have long been used clinically as magnetic resonance imaging (MRI) contrast agents. Combining Gd-based compounds with nanodelivery technologies not only enhances imaging contrast and mitigates toxicity and excretion issues, but also allows for tracking drug metabolic pathways via MRI, CT, and fluorescence imaging (FL), monitoring drug response and tumor progression during treatment. Furthermore, Gd's high atomic number (Z = 64) provides it with a larger X-ray photon capture cross section and Compton scattering, resulting in a radiation dose enhancement effect. After intravenous injection, gadolinium-containing radiosensitizers preferentially accumulate at tumor sites, leveraging the tumor's enhanced permeability and retention (EPR) effect. Ionizing radiation excites them to generate photoelectrons, Auger electrons, free radicals, and other substances. These substances can cause nanoscale ionization damage to tumor cells, enhancing the effectiveness of X-rays and sensitizing tumors to radiotherapy. The rare earth element Eu possesses excellent optical properties, including high luminescence efficiency and fast response time. It can upconvert low-energy near-infrared light into high-energy ultraviolet or visible light, minimizing background fluorescence interference, making it particularly suitable for biological imaging.

[0004] Currently, there are few reports on the synthesis and related applications of pure single-phase rare earth upconversion mesoporous or microcavity-structured materials. To facilitate large-scale synthesis and functional homogeneity, single-phase mesoporous rare earth nanoparticles with both fluorescent and magnetic properties have become attractive multifunctional imaging probes. Existing magnetic nanoparticles are mostly constructed from paramagnetic metals such as Fe and its oxides. Iron oxide nanoparticles are one of the most commonly used T2 contrast agents in clinical practice, but their clinical application is primarily limited by negative contrast effects and susceptibility artifacts. The generation of a dark signal can mislead clinical diagnoses based on T2-weighted magnetic resonance images and can easily be confused with signals from hemorrhage, calcification, or metal deposition. Furthermore, susceptibility artifacts often distort background images, limiting their application in magnetic resonance imaging. For these reasons, Gd-based T1 contrast agents offer advantages over T2 contrast agents in precise, high-resolution imaging.

[0005] Because conventional rare earth nanocrystals are solid structures lacking microcavities or mesoporous structures capable of carrying or loading functional molecules, combining rare earth nanoluminescent materials with other matrix materials with mesoporous microcavities to create rare earth composite nanoluminescent materials for multifunctional applications, including sustained drug release, has become a key approach in material design. Currently, the primary approach is to surface-modify or restructure fluorescent nanocrystals using materials with loading and coating capabilities, such as polymers and mesoporous SiO2. However, integrating multiple functionalities into one structure has made the construction of such multi-component composite nanostructures increasingly complex. The preparation of these composite materials is complex, often involving complex synthesis steps, the introduction of potentially toxic reagents such as surfactants, and extremely harsh synthesis conditions, hindering their biological applications and clinical translation. Furthermore, the composite shell can negatively impact the performance and morphology of the nanoluminescent core.

[0006] The purpose of synthesizing such composite materials is to achieve special functions through interactions such as fluorescence quenching and enhancement, and resonant energy transfer between carrier materials with special luminescent properties and guest molecules. However, in the above-mentioned luminescent core-mesoporous shell structure composite system, the interaction between the guest molecules loaded in the mesoporous shell and the luminescent center can often only occur at the interface of the core-shell structure, which greatly weakens the signal expression of the interaction between the two and greatly limits the application of the material.

[0007] The above-mentioned prior art still needs to be further improved. Summary of the Invention

[0008] In order to overcome the shortcomings of the existing technology, the purpose of the present invention is to provide a new type of hollow nanocarrier constructed based on rare earth gadolinium-based materials doped with Eu to encapsulate therapeutic polypeptides, and to construct a multifunctional mesoporous hollow nanodiagnostic and therapeutic preparation. This nanomaterial has the characteristics of magnetic resonance imaging and fluorescence imaging, and has a radiotherapy sensitization effect, thereby realizing the functions of tumor diagnosis, treatment and efficacy monitoring in one.

[0009] Another object of the present invention is to provide a directly synthesized nanoluminescent material with a microcavity or mesoporous structure. This type of material will greatly improve the surface connection between the luminescent center and the loaded guest, significantly improve its loading efficiency for guest molecules, increase signal expression, and improve sensitivity.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The present invention discloses a magnetic polypeptide nanocomposite diagnostic and therapeutic agent. The general formula of the polypeptide nanocomposite is miPEP133-GC@GdPO4:Eu, which includes a carrier and an active polypeptide component loaded on the carrier; the carrier is GdPO4:Eu, and the active component is a hydrophilic anti-tumor miPEP133-GC peptide.

[0012] Furthermore, in the method for preparing the magnetic polypeptide nanocomposite diagnostic and therapeutic agent, the particle size of the nanomedicine is 140-160nm.

[0013] Furthermore, in the preparation method of the magnetic polypeptide nanocomposite diagnostic and therapeutic agent, the molecular formula of the miPEP133-GC peptide is C76H125N25O26S, the amino acid sequence is: Gly-Gln-Pro-Ser-Ser-Pro-Asp-Pro-Gly-Leu-Glu-Arg-Arg-Val-Ala-Ala-Pro-Cys, and the average molecular weight is 1837.01 g / mol.

[0014] Furthermore, the preparation method of the magnetic polypeptide nanocomposite diagnostic and therapeutic agent is that the hydrophilic anti-tumor miPEP133-GC peptide structure is synthesized by a solid phase method using an fmoc synthesis strategy, and the method comprises the following steps:

[0015] ① After swelling the Fmoc-Cys(Trt)-OH resin, add 20% Piperidine (to remove the N-terminal Fmoc group) and react for 20 minutes. Then add ninhydrin-phenol solution and heat at 105℃-110℃ for 5 minutes. If the color turns dark blue, it indicates a positive reaction and the subsequent operation can be carried out.

[0016] ② Add HBTU (condensation reagent) + DIEA (activation reagent) and Fmoc-Pro-OH, react for 1 hour, and the color is colorless when tested with ninhydrin-phenol solution. Then add 20% Piperidine and react for 20 minutes. The color is blue when tested with ninhydrin-phenol solution.

[0017] ③Add HBTU+DIEA and Fmoc-Ala-OH and react for 1 hour. The color is colorless when tested with ninhydrin-phenol solution. Then add 20% piperidine and react for 20 minutes. The color is blue when tested with ninhydrin-phenol solution. Repeat this process until the last amino acid Cys is de-Fmocated.

[0018] ④ The resin was drained and cut to obtain a crude peptide; a cutting solution was added to the crude polypeptide, the mixture was shaken at a constant temperature, and centrifuged with ether to obtain a crude peptide;

[0019] ⑤ The crude peptide was purified by HPLC and freeze-dried to obtain the refined product.

[0020] Furthermore, in the method for preparing the magnetic polypeptide nanocomposite diagnostic and therapeutic agent, the carrier portion is a uniform, monodispersed porous hollow GdPO4:Eu nanocarrier that can simultaneously achieve MR imaging and fluorescence visibility;

[0021] The synthesis method comprises the following steps:

[0022] ①Synthesis of precursor Gd(OH)CO3:Eu solid spheres:

[0023] Gd(OH)CO3 colloidal microspheres were prepared by homogeneous precipitation method as follows:

[0024] Eu(NO3)3 and Gd(NO3)3 aqueous solutions were added to urea [CO(NH2)2] solution, and the resulting solution was stirred at room temperature for 2 hours, and then heated and stirred at 85°C for 2 hours; the resulting suspension was naturally cooled, centrifuged, washed with deionized water several times, and then collected;

[0025] ②Synthesis of sea urchin-shaped GdPO4 hollow spheres

[0026] The GdPO4 hollow carrier was prepared by hydrothermal method:

[0027] The Gd(OH)CO3:Eu solid spheres obtained in step ① were redispersed in deionized water by ultrasonication and stirring; NH4H2PO4 solution was added dropwise, CTAB was added, and the mixture was stirred; after stirring for 60 minutes, the resulting mixture was placed in a polytetrafluoroethylene bottle in a stainless steel autoclave, sealed, and reacted at 200°C for 12 hours. The mixture was naturally cooled to room temperature in the autoclave, and the precipitate (GdPO4:Eu hollow spheres) was separated by centrifugation, washed with deionized water and ethanol in sequence, and then dried at 80°C for 12 hours.

[0028] ③Carrier-peptide loading

[0029] The sea urchin-shaped GdPO4 was dispersed in water, and then the dissolved miPEP133-GC peptide solution was added and stirred at room temperature overnight; then centrifuged at 12000rpm for 15 minutes, and the supernatant solution was taken for protein quantification to measure the drug loading of the sea urchin-shaped GdPO4; the precipitate was washed twice with water and resuspended in water, and recorded as miPEP133-GC@GdPO4:Eu nano-diagnostic and therapeutic agent.

[0030] Furthermore, the preparation method of the magnetic polypeptide nanocomposite diagnostic and therapeutic agent adopts the inorganic rare earth material Gd as the main body, doped with luminescent Eu material to construct a nanoparticle delivery system, which can combine the nuclear magnetic imaging characteristics of Gd and the fluorescence imaging properties of Eu to achieve dual-modal imaging, and carry the miPEP133-GC peptide with anti-tumor effect to achieve integrated diagnosis and treatment; it can be applied to medical testing, clinical diagnosis, biological imaging, drug transportation and disease treatment.

[0031] Furthermore, the preparation method of the magnetic polypeptide nanocomposite diagnostic and therapeutic agent, the nano preparation is based on the high atomic number of Gd, has a radiotherapy sensitization effect, and can effectively improve radiosensitivity; it can use its own radiotherapy sensitization effect in combination with X-rays to achieve a synergistic treatment effect on tumor cells, and can also be used for the synergistic treatment of drug-resistant tumors.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial technical effects:

[0033] 1) The novel anti-tumor polypeptide of the present invention, namely miPEP133-GC peptide, adopts a magnetic nanocarrier delivery strategy to develop a GdPO4:Eu multifunctional nanocarrier with a fluorescent probe, and construct the @miPEP133-GC peptide@GdPO4:Eu multifunctional composite nano-diagnostic and therapeutic agent.

[0034] 2) The nanoparticles described in the present invention are composed of a drug transport carrier and an active polypeptide drug; the multifunctional nanosystem made of a gadolinium complex has a sea urchin-like hollow structure, which can carry the active miPEP133-GC anti-tumor peptide to accumulate in situ in the tumor and induce tumor cell apoptosis.

[0035] 3) The present invention directly utilizes gadolinium-based compounds, which are positive MRI imaging contrast agents, to provide clearer MR imaging images and diagnostic value compared to nanocarriers constructed using Fe, a negative imaging contrast agent.

[0036] 4) The nanomaterial of the present invention is made of Gd material with a high atomic number. The high atomic number of Gd (Z=64) gives it a larger X-ray photon capture cross-section and Compton scattering effect, has a radiation enhancement effect, and can effectively improve radiosensitivity; combined with X-rays, it can achieve a synergistic treatment effect on tumors.

[0037] 5) This nanomaterial is doped with the upconversion luminescent material Eu and can be used for highly specific molecular fluorescence imaging and treatment of tumors. While providing treatment methods, this nanomaterial also has accurate dual-modality imaging diagnosis, personalized magnetic targeted treatment, and drug release and metabolic monitoring after administration.

[0038] 6) The rare earth nanocrystals composed of this material have a hollow structure and can carry or load functional molecules. There is no need to incorporate new materials such as mesoporous SiO2 materials to construct a mesoporous structure or a cavity structure. The rare earth nanoluminescent material is compounded with other matrix materials with a mesoporous microcavity structure to obtain a rare earth composite nanoluminescent material. The raw materials are simple, and the synthesis components and steps of the multifunctional inorganic nanocarrier material are simplified, reducing the introduction of potentially toxic reagents such as surfactants, which is beneficial to clinical transformation and application and increases the safety of drugs.

[0039] 7) These Eu-doped gadolinium-based nanoparticles can be used for both magnetic resonance imaging and fluorescence imaging, enabling tumor imaging and real-time monitoring of treatment progress. They can also be combined with radiotherapy to achieve efficient and safe anti-tumor treatment, reducing toxic side effects and drug resistance, enabling efficient tumor treatment and early diagnosis. This provides a new approach for detailed research on highly effective nanoagents for cancer diagnosis and treatment. They have great potential and broad prospects for application in medical testing, clinical diagnosis, bioimaging, drug delivery, and disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shown is the MS spectrum of the miPEP133-GC peptide;

[0041] Figure 2 Shown is an electron microscopy schematic diagram of the miPEP133-GC@GdPO4:Eu nanotheranostic agent;

[0042] Figure 3 Shown is the particle size distribution of miPEP133-GC@GdPO4:Eu nanotheranostic agent;

[0043] Figure 4 Shown is the biocompatibility of miPEP133-GC@GdPO4:Eu nanotheranostic agent detected using normal human nasopharyngeal epithelial cells NP69;

[0044] Figure 5 Shown are the numbers of cell colony growth inhibited by miPEP133-GC@GdPO4:Eu nanotheranostic agents after tumor irradiation;

[0045] Figure 6 Shown is the ability of miPEP133-GC@GdPO4:Eu nanotheranostic agent to inhibit cell invasion after tumor radiation. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below through specific preferred embodiments combined with effect test examples, but the present invention is not limited to the following embodiments.

[0047] The present invention uses the magnetic rare earth material GdPO4 as the skeleton and gives it a functional structure (hollow, mesoporous, core-shell, etc.) to construct a sea urchin-shaped hollow nanocarrier, which is doped with rare earth luminescent material Eu and encapsulated with the anti-tumor active polypeptide miPEP133-GC peptide. While achieving MRI imaging and fluorescence imaging labeling, it can be used as a drug carrier for tumor treatment, thereby realizing imaging-guided anti-tumor treatment.

[0048] This study uses a magnetic Gd-based material as a hollow carrier framework, doped with a single-phase luminescent material, and synthesized mesoporous GdPO4:Eu sea urchin-like hollow nanocarriers via a simple homogeneous precipitation method combined with a hydrothermal method. These nanocarriers are used for drug delivery and can simultaneously load therapeutic peptides, integrating diagnostic probes and therapeutic agents into a single platform for cancer diagnosis and treatment. The entire carrier synthesis process requires no additional hollow mesoporous material, surfactants, or catalysts. The resulting product exhibits both magnetic resonance imaging and photoresponsive drug release properties as a fluorescent probe.

[0049] The present invention discloses a method for preparing and applying a novel rare earth material-based composite nanomedicine. The magnetic polypeptide nanocomposite diagnostic and therapeutic agent of the present invention has a general formula of miPEP133-GC@GdPO4:Eu and comprises a carrier and an active polypeptide component loaded on the carrier; the carrier is GdPO4:Eu, and the active component is a hydrophilic anti-tumor miPEP133-GC peptide. The nanomedicine has a particle size of 140-160 nm. The molecular formula of the miPEP133-GC peptide is C76H125N25O26S, and the amino acid sequence is Gly-Gln-Pro-Ser-Ser-Pro-Asp-Pro-Gly-Leu-Glu-Arg-Arg-Val-Ala-Ala-Pro-Cys, with an average molecular weight of 1837.01 g / mol.

[0050] The present invention can not only realize the imaging and treatment of cancer, but also realize the real-time monitoring of the treatment effect, which helps to realize the integration of diagnosis, treatment and efficacy monitoring.

[0051] The present invention includes the following parts:

[0052] 1) Solid-phase synthesis of peptides

[0053] The present invention provides a method for solid-phase synthesis of the miPEP133-GC polypeptide. The polypeptide, miPEP133-GC, has a molecular formula of C76H125N25O26S, an amino acid sequence of Gly-Gln-Pro-Ser-Ser-Pro-Asp-Pro-Gly-Leu-Glu-Arg-Arg-Val-Ala-Ala-Pro-Cys, and an average molecular weight of 1837.01 g / mol. The polypeptide can be used in the preparation of anti-tumor drugs.

[0054] This synthetic polypeptide, encoded by a miR-34a precursor, can target the mitochondrial chaperone HSP9A through protein-protein interactions, inhibiting the interaction between HSP9A and other proteins. This reduces mitochondrial mass and induces apoptosis in tumor cells. The invention is non-toxic, highly stable, and easily incorporated into other nanocarriers, demonstrating its potential for application.

[0055] The synthesis method of miPEP133-GC specifically comprises the following steps:

[0056] ① After swelling the Fmoc-Cys(Trt)-OH resin, add 20% Piperidine (to remove the N-terminal Fmoc group) and react for 20 minutes. Then add ninhydrin-phenol solution and heat at 105℃-110℃ for 5 minutes. If the color turns dark blue, it indicates a positive reaction and the subsequent operation can be carried out.

[0057] ② Add HBTU (condensation reagent) + DIEA (activation reagent) and Fmoc-Pro-OH, react for 1 hour, and the color is colorless when tested with ninhydrin-phenol solution. Then add 20% Piperidine and react for 20 minutes. The color is blue when tested with ninhydrin-phenol solution.

[0058] ③Add HBTU+DIEA and Fmoc-Ala-OH and react for 1 hour. The color is colorless when tested with ninhydrin-phenol solution. Then add 20% piperidine and react for 20 minutes. The color is blue when tested with ninhydrin-phenol solution. Repeat this process until the last amino acid Cys is de-Fmocated.

[0059] ④ Drain the resin and cut to obtain the crude peptide. Add cutting solution to the crude peptide, shake at constant temperature, and centrifuge with ether to obtain the crude peptide;

[0060] ⑤ The crude peptide was purified by HPLC and freeze-dried to obtain the refined product.

[0061] 2) Synthesis of GdPO4:Eu sea urchin-like hollow nanocarriers

[0062] The present invention provides a method for synthesizing a GdPO4:Eu sea urchin-shaped hollow nanocarrier, which can be used for drug delivery, MRI imaging and fluorescence imaging.

[0063] The method for synthesizing the carrier specifically comprises the following steps:

[0064] ①Synthesis of precursor Gd(OH)CO3:Eu solid spheres

[0065] Gd(OH)CO3 colloidal microspheres were prepared by homogeneous precipitation method as follows:

[0066] Eu(NO3)3 and Gd(NO3)3 aqueous solutions were added to urea [CO(NH2)2] solution, and the resulting solution was stirred at room temperature for 2 hours, and then heated and stirred at 85°C for 2 hours; the resulting suspension was naturally cooled, centrifuged, washed with deionized water several times, and then collected;

[0067] ②Synthesis of sea urchin-shaped GdPO4 hollow spheres

[0068] The GdPO4 hollow carrier was prepared by hydrothermal method:

[0069] The Gd(OH)CO3:Eu solid spheres obtained in step ① were redispersed in deionized water by ultrasonication and stirring; NH4H2PO4 solution was added dropwise, CTAB was added, and the mixture was stirred; after stirring for 60 minutes, the resulting mixture was placed in a polytetrafluoroethylene bottle in a stainless steel autoclave, sealed, and reacted at 200°C for 12 hours. The mixture was naturally cooled to room temperature in the autoclave, and the precipitate (GdPO4:Eu hollow spheres) was separated by centrifugation, washed with deionized water and ethanol in sequence, and then dried at 80°C for 12 hours.

[0070] ③Carrier-peptide loading

[0071] The urchin-shaped GdPO4 was dispersed in water, and then the dissolved miPEP133-GC peptide solution was added. The mixture was stirred at room temperature overnight. The mixture was then centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected for protein quantification and drug loading of the urchin-shaped GdPO4. The precipitate was washed twice with water and resuspended in water. This is designated as the miPEP133-GC@GdPO4:Eu nanotheranostic agent.

[0072] Example 1

[0073] The present invention provides a solid-phase synthesis method for a miPEP133-GC peptide with anti-tumor activity, which can be prepared by the following method:

[0074] 1) Solid-phase synthesis of miPEP133-GC peptide:

[0075] The present invention provides a method for solid-phase synthesis of the miPEP133-GC polypeptide. The polypeptide, miPEP133-GC, has a molecular formula of C76H125N25O26S, an amino acid sequence of Gly-Gln-Pro-Ser-Ser-Pro-Asp-Pro-Gly-Leu-Glu-Arg-Arg-Val-Ala-Ala-Pro-Cys, and an average molecular weight of 1837.01 g / mol. The polypeptide can be used in the preparation of anti-tumor drugs.

[0076] like Figure 1 As shown, the hydrophilic anti-tumor miPEP133-GC peptide structure can be synthesized by a solid phase method using an fmoc synthesis strategy, which includes the following steps:

[0077] The synthesis method of miPEP133-GC specifically comprises the following steps:

[0078] ① After swelling the Fmoc-Cys(Trt)-OH resin (3 g), add 20% Piperidine (to remove the N-terminal Fmoc group) and react for 20 minutes. Then add ninhydrin-phenol solution (300 μl - 1500 μl) and heat at 105°C - 1110°C for 5 minutes. If the color turns dark blue, it indicates a positive reaction and the subsequent steps can be performed.

[0079] ② Add HBTU (condensation reagent) (1.75 g) + DIEA (activation reagent) (2.79 ml) and Fmoc-Pro-OH (1.51 g), and react for 1 hour. The color test with ninhydrin-phenol solution is colorless. Then add 20% Piperidine (301 40 ml) and react for 20 minutes. The color test with ninhydrin-phenol solution is blue.

[0080] ③ Add HBTU + DIEA (1.75 g + 2.79 ml) and Fmoc-Ala-OH (1.4 g) and react for 1 hour. The color is colorless when tested with ninhydrin-phenol solution. Then add 20% Piperidine and react for 20 minutes. The color is blue when tested with ninhydrin-phenol solution. Repeat this process until the last amino acid Cys is de-Fmocated.

[0081] ④ Drain the resin and cut to obtain the crude peptide. Add cutting solution (TFA system cutting solution, 95% TFA, 2.5% H2O, 2.5% TIS. 20130ml) to the crude peptide, shake at constant temperature, and centrifuge with ether to obtain the crude peptide;

[0082] ⑤ The crude peptide was purified by HPLC and freeze-dried to obtain a fine product of miPEP133 peptide with a purity of more than 98%.

[0083] like Figure 1 As shown, the miPEP133-GC peptide prepared in Example 1 was subjected to ESI-MS mass spectrometry analysis. The results show that the molecular weight of the synthesized polypeptide is 1837.02, which is consistent with the theoretical value of the calculated polypeptide molecular weight, that is, the miPEP133-GC peptide required in this application.

[0084] Example 2

[0085] The present invention provides a method for synthesizing a mesoporous GdPO4:Eu sea urchin-shaped hollow nanocarrier using a magnetic Gd-based material as a hollow carrier framework and doped with a single-phase luminescent Eu material. Simultaneously, the miPEP133-GC peptide prepared in Example 1 is incorporated into the nanocarrier to produce a diagnostic and therapeutic nanoparticle with integrated tumor diagnostic and therapeutic properties.

[0086] The nano diagnostic and therapeutic agent can be prepared by the following method:

[0087] Synthesis of GdPO4:Eu sea urchin-like hollow nanocarriers:

[0088] The present invention provides a method for synthesizing a GdPO4:Eu sea urchin-shaped hollow nanocarrier, which can be used for drug delivery, MRI imaging and fluorescence imaging.

[0089] The method for synthesizing the carrier specifically comprises the following steps:

[0090] ①Synthesis of precursor Gd(OH)CO3:Eu solid spheres;

[0091] Gd(OH)CO3 colloidal microspheres were prepared by homogeneous precipitation method as follows:

[0092] Dissolve 1 ml of Eu(NO3)3 (1 M), 1 ml of Gd(NO3)3 (0.05 M) and 3 g of urea [CO(NH2)2] in water to a volume of 50 ml. Stir the resulting solution at room temperature for 2 h, then heat and stir at 85°C for 2 h. Cool the resulting suspension naturally, centrifuge, wash with deionized water several times, and collect.

[0093] ②Synthesis of sea urchin-shaped GdPO4 hollow spheres

[0094] The GdPO4 hollow carrier was prepared by hydrothermal method:

[0095] 1 mmol of Gd(OH)CO3:Eu solid spheres obtained in step ① were redispersed in deionized water by ultrasonication and stirring; 0.115 g of NH4H2PO4 was dispersed in water; NH4H2PO4 solution was dropwise added to the Gd(OH)CO3 dispersion, CTAB was added, and the mixture was stirred; after stirring for 60 minutes, the resulting mixture was sealed in a polytetrafluoroethylene bottle in a stainless steel autoclave and reacted at 200°C for 12 hours, naturally cooled to room temperature in the autoclave, and the precipitate (GdPO4:Eu hollow spheres) was separated by centrifugation, washed with deionized water and ethanol in sequence, and then dried at 80°C for 12 hours;

[0096] ③Carrier-peptide loading

[0097] 20mg of sea urchin-shaped GdPO4 was dispersed in water, followed by the addition of 10mg of the dissolved miPEP133-GC peptide solution. The mixture was stirred at room temperature overnight. The mixture was then centrifuged at 12,000 rpm for 15 minutes. The supernatant was collected for protein quantification, revealing a drug loading of 7.6% in the sea urchin-shaped GdPO4. The precipitate was washed twice with water and resuspended in water. This is designated as the miPEP133-GC@GdPO4:Eu nanotheranostic agent.

[0098] like Figure 3As shown, the miPEP133-GC@GdPO4:Eu nanodiagnostic agent prepared in Example 2 was taken for morphological analysis, and the transmission electron microscope observation was as follows: Figure 2 As shown, the nano-diagnostic agent is uniform and monodisperse hollow spheres under electron microscopy. The results of hydrated particle size distribution analysis show that the hydrated particle size of the nano-diagnostic agent is uniformly distributed, about 140-160nm.

[0099] The following is the content of the effect test example part.

[0100] Efficacy Test Example 1

[0101] In vitro experiments of miPEP133-GC@GdPO4:Eu nanotheranostics:

[0102] ① CCK8 method to detect the biocompatibility of the nano-diagnostic agent:

[0103] Normal nasopharyngeal epithelial cell line NP69 was used for detection.

[0104] Take nasopharyngeal epithelial cells in the logarithmic growth phase and adjust the viable cell concentration to 1×10 4 / mL was added to a 96-well culture plate, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. After attachment, 100 μL of the miPEP133-GC@GdPO4:Eu nanodiagnostic and therapeutic agent solution prepared in Example 2 above was added at different concentrations (50 μg / ml, 100 μg / ml, 1 mg / ml), and an equal volume of PBS solution and a blank carrier solution were used as controls.

[0105] Five replicate wells were set up for both the sample group and the control group, and the cells were cultured at 37°C and 5% CO2 for 48 h. Then 10 μl of CCK8 solution was added to each well, and the OD value was measured at a wavelength of 570 nm using a microplate reader 2 h later.

[0106] Calculate cell survival rate: Cell survival rate (%) = actual OD value of drug-treated well / OD value of control well; Cell inhibition rate (%) = 100% - cell survival rate.

[0107] The experimental results are shown in Figure 4 The miPEP133-GC@GdPO4:Eu nano-diagnostic and therapeutic agent of the present invention has good cell compatibility.

[0108] The radiosensitization effect of the nano-diagnostic and therapeutic agent was detected by plate cloning method using nasopharyngeal carcinoma cell line C666-1.

[0109] Take nasopharyngeal carcinoma cells in the logarithmic growth phase, adjust the viable cell concentration to 100 cells / mL and add them to a 6-well culture plate, 2 mL per well, and culture in a 37°C, 5% CO2 incubator for 24 hours. After attachment, the blank carrier and miPEP133-GC@GdPO4:Eu nano-diagnostic agent prepared in Example 2 were added, and the control was an equal volume of PBS solution. After incubation at 37°C, 5% CO2 for 48 hours, irradiate with 4Gy. After irradiation, continue to culture in a 37°C, 5% CO2 incubator for 2 weeks, fix, stain with crystal violet, and take pictures.

[0110] The experimental results are shown in Figure 5 As shown, the miPEP133-GC@GdPO4:Eu nano-therapeutic agent of the present invention has a good radiotherapy sensitization effect.

[0111] Efficacy test example 2

[0112] The Transwell method was used to detect the effect of the nano-diagnostic and therapeutic agent on the invasive ability of nasopharyngeal carcinoma.

[0113] The blank carrier prepared in Example 2 and the nasopharyngeal cancer cells treated with miPEP133-GC@GdPO4:Eu nanodiagnostic agent were collected, and the control group was treated with an equal amount of PBS. 2.5×10 6 0.2 mL of cell suspension was inoculated into the upper chamber of a Transwell. 600 μL of DMEM medium supplemented with 15% fetal bovine serum was added to the lower chamber. After incubation in a 5% CO2 incubator for 36 hours, the upper chamber was removed and cells on the inner surface of the filter membrane were gently wiped with a cotton swab. Cells that had invaded the outer surface of the filter membrane were fixed with methanol at room temperature for 20 minutes. After crystal violet staining, the number of stained cells was recorded in four random fields (40x magnification).

[0114] The experimental results are shown in Figure 6 As shown, the miPEP133-GC@GdPO4:Eu nano-diagnostic and therapeutic agent of the present invention has the effect of inhibiting the invasion ability of tumor cells.

[0115] The present invention is not limited to the above-mentioned embodiments. If various changes or modifications of the present invention do not depart from the spirit and scope of the present invention, and if these changes and modifications fall within the scope of the claims and equivalent technologies of the present invention, the present invention is also intended to include these changes and modifications.

Claims

1. A magnetic polypeptide nanocomposite diagnostic and therapeutic agent, characterized by: The general formula of the polypeptide nanocomplex is miPEP133-GC@GdPO4:Eu, which includes a carrier and an active polypeptide component loaded on the carrier; the carrier is GdPO4:Eu, and the active component is a hydrophilic anti-tumor miPEP133-GC peptide; The molecular formula of the miPEP133-GC peptide is C76H125N25O26S, the amino acid sequence is: Gly-Gln-Pro-Ser-Ser-Pro-Asp-Pro-Gly-Leu-Glu-Arg-Arg-Val-Ala-Ala-Pro-Cys, and the average molecular weight is 1837.01 g / mol; The synthesis method of the GdPO4:Eu nanocarrier comprises the following steps: ①Synthesis of precursor Gd(OH)CO3:Eu solid spheres: Gd(OH)CO3 colloidal microspheres were prepared by homogeneous precipitation method as follows: Eu(NO3)3 and Gd(NO3)3 aqueous solutions were added to urea [CO(NH2)2] solution, and the resulting solution was stirred at room temperature for 2 hours, and then heated and stirred at 85°C for 2 hours; the resulting suspension was naturally cooled, centrifuged, washed with deionized water several times, and then collected; ②Synthesis of sea urchin-shaped GdPO4 hollow spheres The GdPO4 hollow carrier was prepared by hydrothermal method: The Gd(OH)CO3:Eu solid spheres obtained in step ① were redispersed in deionized water by ultrasonication and stirring; NH4H2PO4 solution was added dropwise, CTAB was added, and the mixture was stirred; after stirring for 60 minutes, the resulting mixture was placed in a polytetrafluoroethylene bottle in a stainless steel autoclave, sealed, and reacted at 200°C for 12 hours. The mixture was naturally cooled to room temperature in the autoclave, and the precipitated GdPO4:Eu hollow spheres were separated by centrifugation, washed with deionized water and ethanol in sequence, and then dried at 80°C for 12 hours. ③Carrier-peptide loading The urchin-shaped GdPO4 was dispersed in water, and then the dissolved miPEP133-GC peptide solution was added and stirred at room temperature overnight. The mixture was then centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected for protein quantification and drug loading of the urchin-shaped GdPO4. The precipitate was washed twice with water and resuspended in water, which was designated as the miPEP133-GC@GdPO4:Eu nanodiagnostic agent. The hydrophilic anti-tumor miPEP133-GC peptide structure is synthesized by a solid phase method using an fmoc synthesis strategy, which includes the following steps: ① After swelling the Fmoc-Cys(Trt)-OH resin, add 20% Piperidine to remove the N-terminal Fmoc group and react for 20 minutes. Then add ninhydrin-phenol solution and heat at 105℃~110℃ for 5 minutes. If the color turns dark blue, it indicates a positive reaction and proceed to the subsequent steps. ② Add HBTU condensation reagent + DIEA activation reagent and Fmoc-Pro-OH, react for 1 hour, and the color is colorless when tested with ninhydrin-phenol solution. Then add 20% Piperidine and react for 20 minutes. The color is blue when tested with ninhydrin-phenol solution. ③Add HBTU+DIEA and Fmoc-Ala-OH and react for 1 hour. The color is colorless when tested with ninhydrin-phenol solution. Then add 20% piperidine and react for 20 minutes. The color is blue when tested with ninhydrin-phenol solution. Repeat this process until the last amino acid Cys is de-Fmocated. ④ The resin was drained and cut to obtain a crude peptide; a cutting solution was added to the crude polypeptide, the mixture was shaken at a constant temperature, and centrifuged with ether to obtain a crude peptide; ⑤ The crude peptide was purified by HPLC and freeze-dried to obtain the refined product.

2. The magnetic polypeptide nanocomposite diagnostic and therapeutic agent according to claim 1, wherein: The particle size of the nano drug is 140-160 nm.

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