Preparation and application of nuclear magnetic imaging / photo-thermal therapy diagnosis and treatment integrated preparation

Abstract

The invention relates to the technical field of biomedical materials, in particular to a gadolinium oxide-based nano preparation as well as a preparation method and application thereof. The preparation method of the preparation comprises the following steps: preparing gadolinium oxide nanoparticles through a hydrothermal method and amination modification, and dissolving the gadolinium oxide nanoparticles in deionized water to obtain a contrast agent solution; dropwise adding the nano-particles into a gold source solution under stirring at a certain temperature to obtain nano-particles with core-shell structures; and finally, slowly adding the synthesized dispersant solution of the water-soluble polymer into the core-shell nanoparticle solution to obtain a gadolinium oxide-based nano preparation solution. And performing post-treatment such as dialysis, freeze drying and the like to obtain the core-shell structure nanoparticles based on the gadolinium oxide nano preparation. The invention relates to a gadolinium oxide-based nano preparation for cancer diagnosis and treatment, gadolinium oxide is used as an MRI contrast agent, and a gold shell is grown on the periphery of the gadolinium oxide as a photo-thermal preparation; the hydrogel has the characteristics of good biocompatibility and the like, and has an important practical prospect in the field of preparation of diagnosis and treatment integrated medicines.

Description

Preparation and application of integrated MRI / photothermal therapy formulations Technical Field

[0001] This application belongs to the field of medical materials technology, specifically, it relates to a gadolinium oxide-based nanoparticle formulation with both imaging and photothermal effects, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease threatening human health and life. Its difficulty in treatment and high mortality rate stem from the challenge of early diagnosis—most patients are diagnosed at middle or late stages, making a complete cure extremely difficult. Furthermore, the pathological environment of middle and late-stage cancers is complex, limiting the effectiveness of current clinical treatments. Before diagnosis and treatment, precise diagnostic techniques are needed to clarify the phenotype and heterogeneity of cancer cells, providing a basis for treatment planning. This need, coupled with close integration with the specific treatment process, has driven the rapid development of integrated diagnostic and therapeutic technologies.

[0003] Among commonly used clinical cancer diagnostic methods, magnetic resonance imaging (MRI) has core advantages such as being radiation-free, having high soft tissue resolution, and being highly accurate in localization. It can clearly present the morphology, distribution, and relationship with surrounding tissues of lesions, providing reliable support for early lesion detection and cancer cell characteristic analysis. It is a key means of accurate cancer diagnosis, and is especially suitable for the evaluation of radiation-sensitive patients and deep lesions.

[0004] Photothermal therapy (PTT), as an emerging cancer treatment technology, has attracted much attention due to its convenient operation, non-invasiveness, strong local selectivity, low drug resistance, and mild side effects. Its working principle is based on the photothermal conversion effect of photothermal agents: after intravenous injection, the photothermal agent is preferentially recognized and accumulated by tumor tissue. Under precise irradiation with a specific wavelength light source, the absorbed light energy is efficiently converted into heat energy. This localized high temperature (usually >42℃) destroys the tumor cell structure, inducing apoptosis or necrosis, thus achieving targeted tumor ablation.

[0005] However, in traditional cancer diagnosis and treatment models, contrast agents used for MRI diagnosis and photothermal agents used for PTT therapy are independent systems. Their targeting selectivity, metabolic pathways, and accumulation efficiencies differ significantly in vivo, leading to a mismatch between the lesion located by diagnosis and the actual site of action of the therapeutic drug. This makes it impossible to monitor treatment effectiveness in real time through diagnosis, and also hinders the synergistic closed loop of "diagnosis-guided treatment and treatment feedback to diagnosis," directly limiting treatment accuracy and overall efficacy. This application aims to provide a novel integrated diagnostic and therapeutic formulation.

[0006] Currently, developing integrated formulations that combine high MRI contrast efficiency, excellent photothermal conversion performance, good biocompatibility, and targeted specificity has become a research hotspot in academia and industry. The inventors also aim to provide a new technical path for a full-chain solution of "precision diagnosis-targeted therapy-efficacy monitoring" for cancer. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, this application solves the problems mentioned in the background technology through the following technical solutions.

[0008] The primary objective of this application is to provide a method for preparing gadolinium oxide nanoparticles, comprising: linking an MRI contrast agent and an optical therapy drug via a connecting unit, and linking a water-soluble polymer to increase its dispersibility in an aqueous solution. One end of the connecting unit contains a first functional group for linking with the MRI contrast agent, and the other end contains a second functional group for linking with the optical therapy drug. Specifically, the method includes the following steps: S1, determining the MRI contrast agent and the optical therapy drug, and preparing a water-soluble polymer; S2, introducing the connecting unit into the MRI contrast agent to obtain an intermediate linker; S3, linking the intermediate linker to the optical therapy drug to obtain an intermediate linker-optical therapy drug conjugate; S4, linking the intermediate linker-optical therapy drug conjugate to the water-soluble polymer to obtain a therapeutic formulation with a core-shell structure, which simultaneously possesses both contrast imaging and photothermal therapeutic functions.

[0009] Preferably, the phototherapy drug is photosensitizing and has the effect of producing substances that induce apoptosis and / or death of cancer cells under irradiation with light of a specific wavelength. The phototherapy drug is selected from one or more of gold nanoshells, gold nanorods, copper sulfide nanoparticles, indocyanine green, and photothermal agents that can generate heat after photoinduction.

[0010] Preferably, the connecting unit satisfies at least one of the following: (1) the connecting unit is selected from dopamine or dopamine analogues; (2) the first functional group that directly reacts with the MRI contrast agent is selected from one or more of the functional groups containing hydroxyl groups and halogen atoms; (3) the second functional group containing a group that directly reacts with the phototherapy drug is selected from one or more of the functional groups containing amino groups, hydroxyl groups, carboxyl groups, halogen atoms, carbonyl groups, nitro groups, sulfonic acid groups, and mercapto groups.

[0011] Preferably, one end of the water-soluble polymer includes a functional group for connecting with the surface of nanoparticles. The functional group is a functional group containing a group that directly reacts with the phototherapy drug. The functional group containing a group that directly reacts with the phototherapy drug is selected from one or more of the following functional groups: amino group, hydroxyl group, carboxyl group, halogen group, carbonyl group, nitro group, sulfonic acid group, and mercapto group.

[0012] Preferably, the therapeutic formulation satisfies at least one of the following: (1) the linker is selected from dopamine hydrochloride (DH); (2) the water-soluble polymer is selected from lipoic acid-polyethylene glycol (LA-PEG); (3) the mass ratio of the water-soluble polymer to the intermediate linker-phototherapy drug conjugate is 2:1.

[0013] The second objective of this application is to provide a therapeutic formulation prepared by the above-described preparation method. The therapeutic formulation has a particle size of 150nm-209nm, includes an aminated gadolinium oxide nanoparticle core and an encapsulated optical therapeutic drug, and has a functionalized structure for both imaging and photothermal therapy.

[0014] Preferably, a therapeutic formulation is provided, wherein the phototherapy drug is selected from one or more of gold nanoshells and gold nanorods, and the therapeutic formulation has a core-shell structure with a particle size of 150nm-209nm, including an aminated gadolinium oxide nanoparticle core and an outer gold shell, and has a functional structure for both imaging and photothermal therapy.

[0015] A third objective of this application is to provide an integrated diagnostic and therapeutic formulation for use in the preparation of drugs for the prevention, diagnosis, or treatment of tumors. This integrated formulation possesses both tumor diagnostic and therapeutic functions. The tumor diagnostic function includes magnetic resonance imaging capability under specific conditions, and the therapeutic function includes the generation of localized high temperatures by the integrated formulation under laser irradiation of a specific wavelength.

[0016] Preferred application of therapeutic formulations in cervical cancer targeted drug delivery systems.

[0017] Compared with the prior art, this application has the following beneficial effects:

[0018] (1) The diagnostic and therapeutic nano-formulation of this application, with gadolinium oxide as the core imaging component, innovatively integrates the cancer diagnostic function mediated by gadolinium oxide with the therapeutic effect of anticancer drugs, constructing an integrated formulation system with both cancer diagnosis and targeted therapy efficacy. It aims to solve the drawbacks of the independent nature of diagnostic agents and anticancer drugs in traditional solutions, and avoid the problem of low treatment efficiency caused by the difference in selective enrichment and distribution in tumor tissues. Through functional synergy, it significantly improves the cancer treatment effect and provides a highly promising formulation option for novel anticancer diagnostic and therapeutic technologies. First, the diagnostic and therapeutic nano-formulation relies on the excellent imaging properties of gadolinium oxide to determine the nature of the tumor. If the diagnosis result is non-cancerous, no treatment procedure needs to be initiated, and there is no additional intervention effect on the body. If cancer is diagnosed, the treatment mode of the formulation can be triggered by laser irradiation to achieve precise targeted therapy to the lesion site, and achieve a synergistic closed loop of "diagnosis-guided treatment and treatment-response-diagnosis". Secondly, gadolinium oxide, as one of the core raw materials for MRI-positive contrast agents, not only has a high longitudinal relaxation rate but also excellent biocompatibility. Water-soluble polymers (such as polyethylene glycol) possess good biocompatibility, water solubility, and biodegradability. Their modification can effectively improve the dispersion stability of gold-shell structured formulations, preventing in vivo aggregation. Simultaneously, by introducing targeting groups, the formulation's tumor-specific enrichment ability can be enhanced, reducing damage to normal tissues. Furthermore, it can regulate the formulation's metabolic pathways in vivo, enabling precise matching between MRI-diagnostic lesions and photothermal therapy sites, achieving a synergistic closed loop of "diagnosis-guided treatment and treatment-feedback-diagnosis." The gold-shell structure in some formulations, with its unique plasma resonance effect, not only possesses extremely high photothermal conversion efficiency, enabling efficient tumor photothermal ablation, but also serves as an MRI contrast agent carrier, significantly improving the contrast relaxation rate and achieving integrated diagnostic and therapeutic functions. Finally, this integrated diagnostic and therapeutic nanoformulation has broad applicability, finding applications in the preparation of drugs for treating tumors and in numerous other fields.

[0019] (2) This application relates to a method for preparing a gadolinium oxide-based integrated diagnostic and therapeutic nanoparticle. This method uses clinically applied gadolinium oxide contrast materials as a substrate and employs a biocompatible surface functionalization modification strategy to precisely load photoresponsive therapeutic drugs, constructing a biomedical nanoparticle that combines magnetic resonance imaging (MRI) diagnostics with photo-induced targeted therapy. This preparation method achieves the organic integration of diagnostic function and photo-triggered therapeutic effect. Through the synergistic mode of "imaging guidance-photo-induced therapy," it improves the precision and efficacy of tumor treatment, providing a feasible preparation technology solution for the clinical translation of biomedical materials in the field of tumor diagnosis and treatment.

[0020] (3) This application synthesizes gadolinium oxide nanoparticles as MRI contrast agents in one step by a simple hydrothermal method, which has the advantages of small particle size, high relaxation rate and good biocompatibility; the photothermal material prepared by this invention has excellent photothermal conversion efficiency and can be used as a photothermal conversion reagent.

[0021] (4) This application solves the functional disconnect problem that gadolinium oxide cannot be directly connected to photothermal agents by linking aminated gadolinium oxide nanoparticles with photothermal nanoparticles, thereby achieving synergistic targeted enrichment of diagnostic and therapeutic units. It retains the high efficiency of core functions, while also possessing excellent biocompatibility and simplified preparation process, providing a practical solution for precision diagnosis and treatment of cancer, with significant potential for clinical translation. Attached Figure Description

[0022] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0023] Figure 1 is a schematic diagram of the preparation of (DH@Gd2O3)@AuShell@PEG@LA in Example 1 of this application;

[0024] Figure 2 is a schematic diagram of the results of Experimental Example 1 of this application. Figure 2(A) shows the results of LA, PEG, and LA@PEG in Example 1. 1 Schematic diagram of 1H NMR spectrum, Figure 2(B) shows the 1H NMR spectrum of TP, T and TPT in Example 3;

[0025] Figure 3 is a schematic diagram of the ultraviolet absorption spectra of (DH@Gd2O3)@AuShell and (DH@Gd2O3)@AuShell@PEG@LA in this application;

[0026] Figure 4(A) is a schematic diagram of the dynamic light scattering (DLS) spectrum of Gd2O3 in this application, and Figure 4(B) is a schematic diagram of the dynamic light scattering (DLS) spectrum of (DH@Gd2O3)@AuShell@PEG@LA.

[0027] Figure 5(A) is a schematic diagram of a transmission electron microscope (TEM) image of Gd2O3 in this application, and Figure 5(B) is a schematic diagram of a transmission electron microscope (TEM) image of (DH@Gd2O3)@AuShell@PEG@LA.

[0028] Figure 6 shows the elemental analysis (EDS) of (DH@Gd2O3)@AuShell@PEG@LA in this application;

[0029] Figure 7(A) shows the photothermal conversion curve of the therapeutic formulation (DH@Gd2O3)@AuShell@PEG@LA under constant power, and Figure 7(B) shows the photothermal conversion curve of (DH@Gd2O3)@AuShell@PEG@LA under constant concentration conditions.

[0030] Figure 8 shows the T1 and T2 values ​​of Gd2O3 and (DH@Gd2O3)@AuShell@PEG@LA at different concentrations in this application;

[0031] Figure 9 shows a comparison of T1-weighted imaging of Gd2O3 and (DH@Gd2O3)@AuShell@PEG@LA at different concentrations in this application;

[0032] Figure 10 is a schematic diagram of the dark toxicity (A) and phototoxicity (B) of the therapeutic formulation (DH@Gd2O3)@AuShell@PEG@LA in Example 1 of this application on HeLa cells and a schematic diagram of the dark toxicity (C) and phototoxicity (D) of SiHa cells. Detailed Implementation

[0033] The present application will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present application and should not be construed as limiting the scope of protection of the present application. Anything not described in detail in this patent application is considered common knowledge in the art.

[0034] The term "and / or" as used in this application includes any and all combinations of one or more of the associated listed items. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight, and all reagents used in the examples are commercially available or synthesized by conventional methods and are ready for use without further processing, as are the instruments used in the examples. All technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0035] In some embodiments, a gadolinium oxide-based therapeutic nanoformulation is provided. The formulation includes an MRI contrast agent and an optical therapy drug connected by connecting units, and a water-soluble polymer connected to the optical therapy drug to increase the dispersibility of the formulation in an aqueous solution. The preparation method of the formulation specifically includes:

[0036] S1. Determine MRI contrast agents and phototherapy drugs, and prepare water-soluble polymers.

[0037] Magnetic resonance imaging (MRI), as a core clinical diagnostic tool, possesses advantages such as no ionizing radiation, high soft tissue resolution, and the ability to perform multi-planar tomographic imaging of the whole body. It also boasts diagnostic sensitivity, specificity, and precise localization, comprehensively presenting the overall physiological and pathological state of the body and providing reliable imaging evidence for early screening and accurate diagnosis of lesions. Gadolinium oxide (Gd₂O₃), a classic T1-weighted MRI contrast agent, derives its core mechanism of action from gadolinium ions (Gd₂O₃). 3+ The high electron spin magnetic moment of the contrast agent can efficiently accelerate the longitudinal relaxation process of water protons in vivo, increase the relaxation rate (r1 value), and significantly enhance the signal contrast between tumor tissue and normal tissue, thus aiding in the precise localization and characterization of lesions. Existing technologies use a hydrothermal method to prepare contrast agents, followed by centrifugation to obtain the contrast agent product; however, the unmodified contrast agent lacks reactive sites on its surface and cannot participate in subsequent predetermined reaction processes. To ensure the smooth conduct of subsequent experiments, the contrast agent needs to be aminated; after amination, the contrast agent can serve as a linking medium, achieving specific binding with the photothermal agent and providing a structural basis for the orderly conduct of subsequent reactions. In one embodiment, the aminated contrast agent is dialyzed, freeze-dried, and stored for later use.

[0038] Photothermal therapy (PTT), as a highly efficient optical therapy technology, works by the following mechanism: When irradiated with near-infrared light of a specific wavelength, photothermal therapeutic drugs efficiently absorb light energy and convert it into localized heat energy, rapidly raising the temperature of the tumor lesion area to 42℃-48℃. This heat energy disrupts the tumor cell membrane structure, induces protein denaturation, and interferes with cell metabolism, ultimately leading to tumor cell apoptosis and necrosis. The inventors have specifically selected photothermal therapeutic drugs that are easily biocompatible and require photosensitivity and the ability to induce apoptosis and / or death of cancer cells under irradiation with specific wavelengths of light. These include, but are not limited to, gold nanoshells, gold nanorods, copper sulfide nanoparticles, indocyanine green, and photothermal preparations that generate heat after photoinduction.

[0039] Water-soluble polymers are used to increase the dispersibility of formulations in aqueous solutions. One end of the polymer contains a functional group for bonding with the surface of nanoparticles. The functional group is a functional group containing a group that directly reacts with the phototherapy drug. The functional group containing a group that directly reacts with the phototherapy drug is selected from one or more of the following functional groups: amino group, hydroxyl group, carboxyl group, halogen group, carbonyl group, nitro group, sulfonic acid group, and mercapto group.

[0040] In one embodiment, the water-soluble polymer is lipoic acid-polyethylene glycol (LA-PEG), and the preparation method is as follows: lipoic acid (LA), 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) are added sequentially to 10 mL of DMSO and sonicated for 30 min for later use; amino polyethylene glycol monomethyl ether (MPEG-NH2) (hereinafter referred to as PEG) is dissolved in 10 mL of DMSO and sonicated for 30 min for later use; the two are mixed and transferred to a flask, stirred, and triethylamine (TEA) is added. The mixture is stirred at room temperature under nitrogen for 24 h, and then sodium borohydride (NaBH4) aqueous solution is added and reacted for 24 h to carry out the ring-opening reaction. The mixture is dialyzed in deionized water for two days and then freeze-dried to obtain the water-soluble polymer.

[0041] S2. Prepare the connecting unit and introduce the connecting unit into the MRI contrast agent to obtain the intermediate linker.

[0042] Based on the structural characteristics of MRI contrast agents and phototherapy drugs, the inventors selected one or more reagents to form a connecting unit for binding the MRI contrast agent and the phototherapy drug. One end of the connecting unit contains a first functional group for bonding with the MRI contrast agent, and the other end contains a second functional group for bonding with the surface of the phototherapy drug nanoparticles. The selected connecting unit is then chemically bonded to the MRI contrast agent to obtain an intermediate linker.

[0043] The linker unit is selected from dopamine or dopamine analogs; in one embodiment, the linker unit is dopamine hydrochloride (DH). In one embodiment, the first functional group of the linker unit is selected from one or more functional groups containing a hydroxyl group and a halogen atom. In one embodiment, the second functional group is selected from one or more functional groups containing a group that directly reacts with the phototherapy drug, specifically selected from one or more functional groups containing an amino group, a hydroxyl group, a carboxyl group, a halogen atom, a carbonyl group, a nitro group, a sulfonic acid group, or a mercapto group.

[0044] In one embodiment, the linker is dopamine hydrochloride containing an amino group: Dopamine hydrochloride is first dissolved in deionized water to form a dopamine hydrochloride solution. Then, an MRI contrast agent is dispersed in deionized water and stirred at 800 rpm. The dopamine hydrochloride solution is slowly added, allowing the amino group of dopamine hydrochloride to bind with the MRI contrast agent. The mixture is stirred overnight at room temperature in the dark to obtain an intermediate linker. The intermediate linker is then purified by dialysis for three days, with the water changed every four hours. In another embodiment, when the photothermal therapy drug is a gold nanoshell, the purified intermediate linker is freeze-dried to obtain a solid intermediate linker.

[0045] S3. Obtaining intermediate linker-optical therapy drug conjugates by growing or connecting phototherapy drugs with intermediate linkers as the core.

[0046] An intermediate linker is chemically bonded to an optical therapeutic agent to obtain an intermediate linker-optical therapeutic agent conjugate. In one embodiment, a contrast agent solution of appropriate concentration is added dropwise to a stirred gold source solution at a certain temperature to obtain core-shell structured nanoparticles. In another embodiment, the linker unit is dopamine hydrochloride and the optical therapeutic agent is gold nanoparticles. The gold nanoparticles are bonded to the amino groups on dopamine hydrochloride, and a gold nanoshell is then grown around the gold nanoparticles to obtain the intermediate linker-optical therapeutic agent conjugate.

[0047] S4, Connecting intermediate linker - phototherapy drug conjugate and water-soluble polymer

[0048] A water-soluble polymer is linked to the aforementioned intermediate linker-phototherapy drug conjugate to obtain a therapeutic formulation with a core-shell structure that simultaneously possesses imaging and photothermal diagnostic functions. In one embodiment, the mass ratio of the water-soluble polymer to the intermediate linker-phototherapy drug conjugate is 2:1. In another embodiment, the water-soluble polymer is prepared as a dispersant solution and slowly added to a core-shell nanoparticle solution to obtain a gadolinium oxide-based nanoparticle formulation solution. This solution is then subjected to post-treatment processes such as dialysis and freeze-drying in deionized water to obtain core-shell structured nanoparticles based on the gadolinium oxide nanoparticle formulation.

[0049] The inventors have organically integrated gadolinium oxide contrast agents with phototherapy drugs through rational formulation design, constructing a multifunctional integrated nanosystem of "MRI diagnosis-photothermal therapy". This allows the same carrier to simultaneously possess MRI contrast function and PTT photothermal therapy performance: on the one hand, the MRI contrast unit in the formulation can accurately target tumors and generate strong contrast signals, realizing early and accurate localization and dynamic monitoring of lesions; on the other hand, after the photothermal unit is enriched in the diagnosed lesion area, it can efficiently exert photothermal therapy under light source excitation. At the same time, the MRI contrast agent can track changes in lesion size in real time, and the photo-induced local thermal effect can improve the targeting and efficacy of cancer treatment, providing a basis for dynamic adjustment of treatment plans.

[0050] In one embodiment, the therapeutic formulation obtained by the above preparation method has a core-shell structure with a particle size of 150nm-209nm, including an aminated gadolinium oxide nanoparticle core and an outer gold shell, and has a functional structure for both imaging and photothermal therapy.

[0051] In some embodiments, the above-mentioned gadolinium oxide therapeutic nanoformulations are provided for extended applications in the biomedical field, including applications in magnetic resonance imaging and in the preparation of drugs for the prevention, diagnosis, or treatment of tumors. The therapeutic nanoformulations possess both tumor diagnostic and therapeutic functions; specifically, the tumor diagnostic function includes magnetic resonance imaging capabilities under certain conditions, and the therapeutic function includes the generation of localized high temperatures under specific wavelength laser irradiation. In one embodiment, the application of the therapeutic nanoformulation is its use in a cervical cancer targeted drug delivery system.

[0052] Example 1

[0053] This embodiment provides a gadolinium oxide nanoparticle formulation (DH@Gd2O3)@AuShell@PEG@LA), wherein the MRI contrast agent is gadolinium oxide (Gd2O3), and the phototherapy drug is a photothermal formulation, specifically a gold shell (AuShell).

[0054] The preparation method is shown in Figure 1, and specifically includes the following steps:

[0055] S1. Determine MRI contrast agents, phototherapy drugs, and prepare water-soluble polymers.

[0056] Preparation of gadolinium oxide (Gd2O3) nanoparticles: Weigh 1.8215 g of gadolinium nitrate hexahydrate (Gd(NO3)3·6H2O) and dissolve it in 10 mL of diethylene glycol (DEG). Stir at 600 rpm at 100 °C until transparent. Dissolve 0.2 g of sodium hydroxide (NaOH) in 10 mL of DEG and sonicate until completely dissolved to form a NaOH solution. Transfer the gadolinium nitrate hexahydrate solution to a reaction vessel, then add the NaOH solution evenly. Place the vessel in a drying oven and heat to 140 °C for 1 h, then heat to 180 °C for 4 h. After the reaction is complete, turn off the drying oven and cool to room temperature. Transfer the solution to a centrifuge tube, add deionized water, and centrifuge (2000 rpm, 30 min). After centrifugation, remove the supernatant, add deionized water again, and centrifuge until the supernatant appears, thus obtaining gadolinium oxide nanoparticles.

[0057] Preparation of water-soluble polymer: 10.7 mg of LA, 17.225 mg of EDC, and 10.34 mg of NHS were added sequentially to 10 ml of DMSO and sonicated for 30 min. The mixture was then transferred to a round-bottom flask and stirred at 550 rpm. 200 mg of PEG was dissolved in 10 ml of DMSO and sonicated for 30 min. PEG was then added to the round-bottom flask, followed by 18.94 mg of TEA. The mixture was stirred under nitrogen atmosphere in the dark for 24 h. After 24 h, 0.76 ml of 10 mg / ml NaBH4 was slowly added to initiate a ring-opening reaction for 24 h. The mixture was then dialyzed for two days and freeze-dried to obtain LA@PEG.

[0058] S2. Introduce the connecting unit into the MRI contrast agent to obtain the intermediate connector.

[0059] 200 mg of gadolinium oxide nanoparticles were dispersed in 20 ml of deionized water and sonicated for 30 min to obtain a gadolinium oxide dispersion. 400 mg of dopamine hydrochloride (DH) was dissolved in 20 ml of deionized water to form an aqueous solution of dopamine hydrochloride. The aqueous solution of dopamine hydrochloride was slowly added to the gadolinium oxide dispersion and magnetically stirred (800 rpm) overnight. The next day, the mixture was dialyzed in deionized water for three days using a MW3500 dialysis bag. After freeze-drying, the intermediate linker DH@Gd2O3, i.e., aminated gadolinium oxide nanoparticles, was obtained.

[0060] S3, Connecting the intermediate linker and phototherapy drug

[0061] 100 mL of 0.17 mg / mL chloroauric acid (HAuCl4) aqueous solution was added to a round-bottom flask as the gold source solution, and heated to boiling with vigorous stirring. 75 mg of DH@Gd2O3 was dispersed in 75 mL of deionized water and sonicated for 30 min to obtain a DH@Gd2O3 dispersion. The DH@Gd2O3 dispersion was quickly added to the boiling chloroauric acid aqueous solution. After the solution color stopped changing (brownish-red), stirring was continued for 10 min. Then heating was stopped and the mixture was cooled to room temperature to obtain the intermediate linker-optical therapy drug conjugate.

[0062] S4. Connect the intermediate linker—the phototherapy drug conjugate—to the water-soluble polymer to obtain a therapeutic formulation.

[0063] 150 mg of PEG was dissolved in 10 ml of deionized water and slowly added to the intermediate linker-optical therapy drug conjugate solution prepared by S3. The mixture was stirred vigorously overnight and then freeze-dried to obtain the therapeutic formulation.

[0064] Example 2

[0065] This embodiment provides another gadolinium oxide nanoparticle formulation LA@PEG@(DH@Gd2O3)@CuSNPs, wherein the MRI contrast agent is gadolinium oxide (Gd2O3), and the phototherapy drug is a photothermal formulation, specifically copper sulfide nanoparticles (CuSNPs).

[0066] The preparation method specifically includes the following steps:

[0067] S1. Determine MRI contrast agents, phototherapy drugs, and prepare water-soluble polymers.

[0068] Preparation of gadolinium oxide (Gd2O3) nanoparticles: Same as in Example 1.

[0069] Preparation of phototherapy drugs: 0.1 mL of 1M sodium sulfide solution was added to 100 mL of an aqueous solution containing copper chloride (0.01345 g, 0.1 mmol) and sodium citrate (0.02 g, 0.068 mmol). The mixture was stirred at room temperature. The pale blue copper chloride solution turned dark brown immediately after the addition of sodium sulfide. After 5 min, the reaction mixture was heated to 90 °C and stirred for 15 min until a dark green solution was obtained. The solution was then transferred to ice water to obtain citric acid-coated photothermal preparation copper sulfide nanoparticles (CuSNPs), which were stored for later use.

[0070] Preparation of water-soluble polymer: Same as in Example 1, LA@PEG was obtained.

[0071] S2. Introduce the connecting unit into the MRI contrast agent to obtain the intermediate connector.

[0072] Similar to Example 1, the freeze-drying process was omitted to obtain the intermediate linker DH@Gd2O3 dispersion.

[0073] S3, Connecting the intermediate linker and phototherapy drug

[0074] The DH@Gd2O3 dispersion was slowly added to the citric acid-coated copper sulfide nanoparticle solution in S1 under vigorous stirring, and the reaction was carried out overnight to obtain the intermediate linker-optical therapy drug conjugate.

[0075] S4, Connecting intermediate linker - phototherapy drug conjugate and water-soluble polymer

[0076] 200 mg of PEG was dissolved in 10 ml of deionized water and slowly added to the intermediate linker-optical therapy drug conjugate solution prepared by S3. The mixture was stirred vigorously overnight, then dialyzed for two days and freeze-dried to obtain the integrated diagnostic and therapeutic formulation.

[0077] Example 3

[0078] This embodiment provides gadolinium oxide nanoparticles LA@PEG@(DH@Gd2O3)@TPTNPs, wherein the MRI contrast agent is gadolinium oxide (Gd2O3) and the phototherapy drug is a photothermal agent, specifically TPT nanoparticles (TPTNPs).

[0079] The preparation method specifically includes the following steps:

[0080] S1. Determine MRI contrast agents, phototherapy drugs, and prepare water-soluble polymers.

[0081] Preparation of gadolinium oxide (Gd2O3) nanoparticles: Same as in Example 1.

[0082] Preparation of phototherapy drugs: Weigh 500 mg of 3,4-diaminothiophene dihydrochloride and 524 mg of potassium acetate, and transfer them to a 100 ml round-bottom flask equipped with a magnetic stir bar; then add 1 ml of anhydrous ethanol and 5 ml of dichloromethane, stir at 350 rpm for 30 min, then add 34 µl of 2,3-butanedione, stir and reflux at 50 °C (320 rpm), and start TLC after 6 h; obtain TP monomer by rotary evaporation, extraction, column chromatography, drying and filtration. Weigh 500 mg of 2-(2-bromoethoxy)thiophene and dissolve it in 12.5 ml of chloroform; weigh 832 mg of NBS, add 105 ml of glacial acetic acid and sonicate until completely dissolved, add the NBS solution dropwise to the thiophene, then stir overnight at 30 °C under nitrogen protection and in the dark, and obtain monomer T by extraction, drying, filtration, rotary evaporation and column chromatography. 110 mg of monomer T and 45.9 mg of monomer TP were weighed and dissolved completely in 3 ml of toluene, and nitrogen was purged for 15 min. The catalyst was weighed and added, and nitrogen was purged for 10 min. The mixture was stirred at 110 °C (300 rpm) for 48 h. The photothermal formulation TPT was obtained by extraction, drying, filtration, rotary evaporation and column chromatography.

[0083] Preparation of water-soluble polymer: Same as in Example 1, LA@PEG was obtained.

[0084] S2. Introduce the connecting unit into the MRI contrast agent to obtain the intermediate connector.

[0085] Same as in Example 2, DH@Gd2O3 dispersion was obtained.

[0086] S3, Connecting the intermediate linker and phototherapy drug

[0087] Weigh 17.5 mg of TPT and dissolve it in 3 ml of tetrahydrofuran. Slowly add 17.5 ml of DH@Gd2O3 dispersion and stir overnight at 1000 rpm at 30°C to obtain the intermediate linker-optical therapy drug conjugate.

[0088] S4. Connect the intermediate linker—the phototherapy drug conjugate—to the water-soluble polymer to obtain a therapeutic formulation.

[0089] 35 mg of PEG was dissolved in 5 ml of deionized water and slowly added to the intermediate linker-optical therapy drug conjugate solution prepared by S3. The mixture was stirred vigorously overnight, dialyzed for two days, and then freeze-dried to obtain the integrated diagnostic and therapeutic formulation.

[0090] To further verify the technical solution of this application and its beneficial effects, Examples 1-3 were verified.

[0091] Test Example 1, Performance Testing

[0092] The water-soluble polymer LA@PEG prepared in Example 1 and the photothermal formulation TPT prepared in Example 3 were respectively subjected to... 1 ¹H NMR characterization results are shown in Figures 2(A) and 2(B), respectively. The results indicate that the ¹H NMR spectra in Figure 2, through comparative characterization, clarified the synthetic effectiveness of two key intermediates in the preparation of the integrated diagnostic and therapeutic formulation: In Figure 2(A), the characteristic proton peaks of LA and PEG coexist without any free redundant peaks, and the chemical shifts or integral ratios of the characteristic peaks show regular changes, confirming the formation of amide bonds and verifying the successful synthesis of the water-soluble polymer LA@PEG; In Figure 2(B), the characteristic proton peaks of TP and T monomers appear, and the peak shapes conform to copolymer characteristics. The proton peak integral ratio is consistent with the monomer feed ratio, confirming the synthesis and controllable composition of the photothermal formulation TPT nanoparticles. These results eliminate problems such as physical mixing and incomplete reactions, providing core structural support for subsequent steps such as coupling of intermediate linkers with phototherapy drugs and modification of water-soluble polymers, indirectly supporting the functional realization of the final integrated diagnostic and therapeutic formulation.

[0093] Test Example 2, Performance Testing

[0094] 1. Absorption spectroscopy experiments were performed on (DH@Gd2O3)@AuShell and (DH@Gd2O3)@AuShell@PEG@LA prepared in Example 1. The results are shown in Figure 3. Both products exhibited characteristic absorption peaks of gold nanoshells (AuShell) in the 500nm-900nm wavelength range, and the outer PEG@LA modification did not change the key properties of these characteristic absorption peaks (such as peak position and peak shape). This confirms that the gold shell has been successfully coated on the surface of the aminated gadolinium oxide intermediate linker, and the subsequent PEG@LA modification did not damage the structural integrity and photoresponse performance of the gold shell. The gold shell can still efficiently absorb near-infrared light, providing key performance verification for the subsequent photothermal conversion of the formulation and the realization of tumor photothermal therapy.

[0095] 2. The hydration diameter of (DH@Gd2O3)@AuShell@PEG@LA prepared in Example 1 was characterized, and the results are shown in Figure 4. It can be seen from the figure that the size of (DH@Gd2O3)@AuShell@PEG@LA has increased compared with Gd2O3, which proves the successful introduction of the contrast agent analog.

[0096] 3. The morphological characteristics of (DH@Gd2O3)@AuShell@PEG@LA prepared in Example 1 were characterized. The results are shown in Figure 5. The original Gd2O3 nanoparticles exhibited a single and uniform nanomorphic morphology. The (DH@Gd2O3)@AuShell@PEG@LA particles were uniformly dispersed without obvious agglomeration, confirming that each functional unit was stably assembled according to the design scheme. Moreover, the PEG@LA modification effectively improved the dispersibility, and the core-shell boundary was clear and the coating was complete, providing a reliable structural guarantee for the formulation to simultaneously achieve MRI imaging and photothermal therapy functions.

[0097] 4. Elemental analysis was performed on the (DH@Gd2O3)@AuShell@PEG@LA sample prepared in Example 1. As shown in Figure 6, key elements such as gadolinium (Gd), oxygen (O), gold (Au), carbon (C), hydrogen (H), and nitrogen (N) were clearly detected in the spectrum. The presence of each element corresponds completely to the layered structure design of the formulation. Gd and O are derived from the core MRI imaging unit gadolinium oxide (Gd2O3), Au is derived from the photothermal therapy unit gold shell (AuShell), and C, H, and N are derived from the intermediate linker dopamine hydrochloride (DH) and the outer modified water-soluble polymer PEG@LA (lipoic acid-amino polyethylene glycol monomethyl ether). The results directly confirm that the gadolinium oxide core, gold shell, connecting units, and water-soluble polymers have achieved chemical bonding and structural integration according to the design scheme, eliminating problems such as incomplete gold shell coating and ineffective connection of functional units. From the elemental composition level, the integrity of the core-shell structure of the formulation and the successful assembly of each functional unit are verified, providing a material basis for the formulation to have both MRI imaging and photothermal therapy functions.

[0098] Test Example 3, Performance Testing

[0099] The (DH@Gd2O3)@AuShell@PEG@LA prepared in Example 1 was subjected to photothermal testing. Figure 7 shows the photothermal conversion curves. Under irradiation with an 808nm laser, (DH@Gd2O3)@AuShell@PEG@LA exhibited good photothermal conversion properties. Figure 7(A) shows that under constant power, the higher the concentration of the formulation, the faster the solution temperature rises; Figure 7(B) shows that under constant concentration, the higher the laser intensity, the more significant the temperature rise rate. This result confirms that the gold-shell (AuShell) photothermal unit in the formulation can efficiently convert light energy into heat energy, enabling the lesion area to reach a local high temperature (42℃-48℃) that induces tumor cell apoptosis. This directly verifies the effectiveness and controllability of the photothermal therapeutic function of the formulation, providing key performance support for its application in tumor photothermal therapy.

[0100] Test Example 4, Performance Testing

[0101] Figures 8 (relaxation time curves) and 9 (T1-weighted imaging comparison) jointly verify the effectiveness of the MRI contrast function of the therapeutic formulation: Figure 8 shows that the T1 and T2 relaxation times of Gd2O3 and (DH@Gd2O3)@AuShell@PEG@LA are significantly shortened with increasing formulation concentration, and their relaxation rates (r1 values) are similar, with the R² values ​​of the fitted curves close to 1, indicating a good linear relationship between relaxation performance and concentration. Aminolation modification, gold shell coating, and PEG@LA modification do not destroy the magnetic activity of gadolinium oxide. Figure 9 visually shows that the T1-weighted imaging signal intensity gradually increases with increasing formulation concentration, and the imaging effect of the integrated formulation is comparable to that of pure Gd2O3. The combination of the two figures confirms that this formulation can achieve precise MRI contrast by accelerating water proton relaxation and improving signal contrast. Its contrast function is efficient and stable, providing key performance support for early tumor diagnosis and lesion localization.

[0102] Test Example 5, Performance Testing

[0103] In vitro HeLa and SiHa cell experiments were conducted on the (DH@Gd2O3)@AuShell@PEG@LA prepared in Example 1. The results are shown in Figure 10. After co-culturing the prepared therapeutic formulation with HeLa and SiHA cells, (DH@Gd2O3)@AuShell@PEG@LA showed good biocompatibility under laser-free conditions. However, after irradiation with an 808nm laser, (DH@Gd2O3)@AuShell@PEG@LA exhibited significant phototoxicity. At a concentration of 0.3 mg / ml, the cell survival rate dropped below 50% after photoirradiation; with further increases in concentration to 1 mg / ml, the survival rate was only about 20%. These results confirm that the formulation possesses "photoresponsive therapeutic specificity," exerting its tumor cell-killing effect only under laser excitation. This ensures biosafety in vivo circulation and verifies the effectiveness of its photothermal therapy function, providing crucial in vitro cellular-level support for the clinical application of photothermal therapy for tumors.

[0104] Based on the preferred embodiments of this application, and through the above description, those skilled in the art can make various changes and modifications without departing from the technical concept of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing gadolinium oxide-based nanoparticles, characterized in that, include: An MRI contrast agent and an optical therapy drug are connected via a connecting unit, and a water-soluble polymer is connected to increase its dispersibility in an aqueous solution. One end of the connecting unit contains a first functional group for connecting with the MRI contrast agent, and the other end contains a second functional group for connecting with the optical therapy drug. Specifically, the steps include: S1, determining the MRI contrast agent and the optical therapy drug, and preparing the water-soluble polymer. S2. Introduce the connecting unit into the MRI contrast agent to obtain an intermediate linker; S3. Connect the intermediate linker to the phototherapy drug to obtain an intermediate linker-phototherapy drug conjugate; S4. Connect the intermediate linker-phototherapy drug conjugate to the water-soluble polymer to obtain a therapeutic formulation, which has both contrast imaging and photothermal therapy functions.

2. The preparation method according to claim 1, characterized in that, The optical therapeutic drug is photosensitized and has the effect of producing substances that induce apoptosis and / or death of cancer cells under irradiation with light of a specific wavelength. The optical therapeutic drug is selected from one or more of gold nanoshells, gold nanorods, copper sulfide nanoparticles, indocyanine green, and photothermal agents that can generate heat after photoinduction.

3. The preparation method according to claim 1, characterized in that, The connecting unit satisfies at least one of the following: (1) the connecting unit is selected from dopamine or dopamine analogues; (2) the first functional group that directly reacts with the MRI contrast agent is selected from one or more of the functional groups containing hydroxyl groups and halogen atoms; (3) the second functional group containing a group that directly reacts with the phototherapy drug is selected from one or more of the functional groups containing amino groups, hydroxyl groups, carboxyl groups, halogen atoms, carbonyl groups, nitro groups, sulfonic acid groups, and mercapto groups.

4. The preparation method according to claim 1, characterized in that, One end of the water-soluble polymer includes a functional group for bonding with the surface of nanoparticles. The functional group is a functional group containing a group that directly reacts with the phototherapy drug. The functional group containing a group that directly reacts with the phototherapy drug is selected from one or more of the following functional groups: amino group, hydroxyl group, carboxyl group, halogen group, carbonyl group, nitro group, sulfonic acid group, and mercapto group.

5. The preparation method according to any one of claims 1-4, characterized in that, At least one of the following conditions must be met: (1) the linking unit is selected from dopamine hydrochloride (DH); (2) the water-soluble polymer is selected from lipoic acid-polyethylene glycol (LA-PEG); (3) the mass ratio of the water-soluble polymer to the intermediate linker-optical therapy drug conjugate is 2:

1.

6. A diagnostic and therapeutic preparation, characterized in that, Prepared by any one of the preparation methods described in claims 1-5, the integrated diagnostic and therapeutic formulation has a particle size of 150nm-209nm, comprising an aminated gadolinium oxide nanoparticle core and an encapsulated optical therapeutic drug, and simultaneously possesses a functionalized structure for both imaging and photothermal diagnostics.

7. A diagnostic and therapeutic preparation, characterized in that, Prepared by any one of the preparation methods described in claims 1-5, the optical therapy drug is selected from one or more of gold nanoshells and gold nanorods, and the integrated diagnostic and therapeutic formulation has a core-shell structure with a particle size of 150nm-209nm, including an aminated gadolinium oxide nanoparticle core and an outer gold shell, and has a functionalized structure for both imaging and photothermal diagnosis and treatment.

8. The use of a therapeutic formulation as described in claim 6 or 7 in the preparation of medicaments for the prevention, diagnosis, or treatment of tumors, characterized in that, The integrated diagnostic and therapeutic preparation has both tumor diagnostic and therapeutic functions.

9. The application according to claim 8, characterized in that, The tumor diagnostic function includes the ability of the integrated diagnostic and therapeutic preparation to perform magnetic resonance imaging under specific conditions, and the therapeutic function includes the generation of local high temperature by the integrated diagnostic and therapeutic preparation under laser irradiation of a specific wavelength.

10. The application according to claim 8, characterized in that, The application of the aforementioned integrated diagnostic and therapeutic formulation in a cervical cancer targeted drug delivery system.

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