Anti-tumor targeting nano material and preparation method thereof

Abstract

The invention provides an anti-tumor targeting nano material and a preparation method thereof, and belongs to the field of novel targeting nano drugs. The method comprises the following steps: stirring and mixing up-conversion nanoparticles, calcium peroxide nanoparticles, manganese chloride and a chemotherapeutic drug in a water phase to obtain a drug-loading core; cystamine and 3-aminophenylboronic acid are dissolved in water, EDC and NHS are added, a first amidation reaction is carried out, and a cross-linking agent is obtained; dispersing the drug-loading core in a polyacrylic acid solution, adding a cross-linking agent, and then carrying out a cross-linking reaction to obtain an intermediate product; activating carboxyl on the surface of the intermediate product, and then carrying out second amidation reaction with cRGD-PEG-NH2, so as to obtain the anti-tumor targeting nano material. According to the application, a multifunctional nano platform integrating targeted enrichment, double-response precise drug release, chemotherapy-chemical kinetics-immune collaborative treatment and up-conversion imaging tracing is constructed, and specific efficient killing of tumors and inhibition of metastasis and relapse of the tumors are realized.

Description

Technical Field

[0001] This application relates to the field of novel targeted nanomedicine technology, and in particular to an antitumor targeted nanomaterial and its preparation method. Background Technology

[0002] Malignant tumors are among the most serious diseases threatening human health. Chemotherapy, as a routine clinical treatment for tumors, lacks specificity and, while killing tumor cells, also causes severe damage to normal tissues, leading to systemic toxic side effects and easily inducing tumor drug resistance. In recent years, nanomedicine delivery systems have offered hope for solving this problem. By utilizing the high permeability and retention effect (EPR effect) of solid tumor tissue, nanomedicines can achieve a certain degree of enrichment at the tumor site, thereby improving efficacy and reducing side effects.

[0003] However, first-generation nanomedicines still face many serious challenges. First, passive targeting efficiency is limited, with most drugs still distributed in normal tissues such as the mononuclear phagocyte system. Second, traditional nanocarriers lack intelligent drug release capabilities; drugs are difficult to release on demand after reaching the tumor site, and leaked drugs can still lead to systemic toxicity. Furthermore, the tumor microenvironment is characterized by hypoxia, high concentrations of glutathione, and weak acidity. These characteristics are not only key factors in tumor progression and drug resistance but also constitute important barriers to drug delivery. Although some studies have attempted to design nanomaterials that respond to a single stimulus, their response specificity and sensitivity are insufficient in the complex in vivo environment, easily leading to false-positive releases.

[0004] More importantly, traditional treatment modalities (such as chemotherapy alone) are insufficient to completely eliminate tumors and inhibit their metastasis and recurrence. Chemokinetic therapy, as an emerging treatment strategy, converts endogenous hydrogen peroxide in tumors into highly toxic hydroxyl radicals through Fenton or Fenton-like reactions, but its efficacy is severely limited by insufficient hydrogen peroxide concentration within the tumor and excessive GSH consumption. Meanwhile, traditional nanotherapy systems often neglect the synergistic effect of the immune system. The tumor immunosuppressive microenvironment makes "cold tumors" difficult for immune cells to recognize and eliminate, leading to a high risk of recurrence and metastasis after treatment. Therefore, there is an urgent need in this field for a novel nanomedicine that can not only achieve precise drug delivery through active targeting and multiple stimulus responses, but also integrate multiple treatment modalities to overcome tumor microenvironment barriers and activate the body's immune system while killing tumors, thereby achieving long-term monitoring and elimination of tumors. Specifically, how to construct a multifunctional nanoplatform that integrates targeted enrichment, pH / GSH dual-response precise drug release, chemotherapy-chemokinetics-immunotherapy synergistic therapy, and upconversion imaging tracing to achieve specific and efficient killing of tumors and inhibit their metastasis and recurrence, while reducing systemic toxicity, has become a key technical problem that urgently needs to be solved in the field of nanobiomedicine. Summary of the Invention

[0005] This application provides an anti-tumor targeted nanomaterial and its preparation method to solve the following technical problem: how to construct a multifunctional nanoplatform that integrates targeted enrichment, pH / GSH dual-response precise drug release, chemotherapy-chemokinetics-immunotherapy synergistic therapy and upconversion imaging tracing, so as to achieve specific and efficient killing of tumors and inhibit their metastasis and recurrence, while reducing systemic toxicity.

[0006] In a first aspect, this application provides a method for preparing anti-tumor targeted nanomaterials, the method comprising the following steps:

[0007] S1. Upconversion nanoparticles, calcium peroxide nanoparticles, manganese chloride, and chemotherapeutic drugs are stirred and mixed in an aqueous phase to ensure that the calcium peroxide nanoparticles, the chemotherapeutic drugs, and Mn are mixed. 2+ Adsorbed onto the surface of the upconversion nanoparticles, the drug-loaded core is obtained after post-processing.

[0008] S2. Cystamine and 3-aminophenylboronic acid are dissolved in water, and EDC and NHS are added. Then, the first amidation reaction is carried out to obtain a crosslinking agent with boric acid groups at both ends.

[0009] S3. The drug-loaded core is dispersed in a polyacrylic acid solution, and the crosslinking agent is added. Then, a crosslinking reaction is carried out to form a dense polymer shell with both pH responsiveness and glutathione responsiveness on the outside of the drug-loaded core. After post-processing, an intermediate product is obtained.

[0010] S4. The surface carboxyl groups of the intermediate product are activated, and then a second amidation reaction is carried out with cRGD-PEG-NH2 to graft the targeting peptide onto the polymer shell through amide bonds. After post-treatment, anti-tumor targeting nanomaterials are obtained.

[0011] Optionally, in step S1, the mass ratio of the upconversion nanoparticles, the calcium peroxide, the chemotherapeutic drug (DOX), and the manganese chloride is 1:(0.5-2):(0.5-2):(0.1-0.5).

[0012] The upconversion nanoparticles are core-shell structured NaYF4:Yb,Er@NaYF4;

[0013] The chemotherapy drug is doxorubicin hydrochloride.

[0014] Optionally, in step S1, the stirring and mixing is carried out under light-protected conditions, at a temperature of 20–25°C, for a time of 12–24 hours.

[0015] Optionally, in step S2, the molar ratio of cystamine to 3-aminophenylboronic acid is 1:(2-2.2);

[0016] The molar ratio of EDC to 3-aminophenylboronic acid is (1.2-1.5):1;

[0017] The molar ratio of NHS to EDC is 1:1.

[0018] Optionally, in step S2, the first amidation reaction is carried out under light-protected conditions, at a reaction temperature of 20–25°C, and for a reaction time of 6–12 h.

[0019] Optionally, in step S3, the weight-average molecular weight of the polyacrylic acid is 1800-2500 Da;

[0020] The mass ratio of the drug-loaded core to the polyacrylic acid is 1:(1-5);

[0021] The molar ratio of the carboxyl groups on the polyacrylic acid chain to the boric acid groups on the crosslinking agent is 1:(0.2-0.5).

[0022] Optionally, in step S3, the crosslinking reaction is carried out at a temperature of 20–25°C for 2–4 hours.

[0023] Optionally, in step S4, the activation method of the surface carboxyl groups is as follows:

[0024] The intermediate product was dispersed in MES buffer solution with a pH of 5.5–6.5, and EDC and NHS were added. The mixture was then reacted at 20–25°C in the dark for 30–60 min to activate the surface carboxyl groups of the intermediate product.

[0025] The molar ratio of EDC to the carboxyl group to be activated is (1-2):1, and the molar ratio of NHS to EDC is 1:1.

[0026] Optionally, in step S4, the second amidation reaction is carried out in a PBS buffer with a pH of 7.5 to 8.5, at a reaction temperature of 20 to 25°C, and for a reaction time of 4 to 8 hours.

[0027] The mass ratio of the intermediate product to the cRGD-PEG-NH2 is 1:(0.5~2).

[0028] In a second aspect, this application provides an antitumor-targeting nanomaterial prepared by the method described in any one of the first aspects, wherein the nanomaterial has a core-shell targeting structure, comprising:

[0029] The drug-carrying core consists of upconversion nanoparticles, calcium peroxide nanoparticles adsorbed on the surface of the upconversion nanoparticles, chemotherapeutic drugs, and Mn. 2+ constitute;

[0030] A polymer shell encapsulating the drug-carrying core, the polymer shell being cross-linked from polyacrylic acid via a dual-responsive cross-linking agent containing disulfide bonds and phenylboronic acid groups;

[0031] cRGD-PEG targeting molecules are grafted onto the polymer shell via amide bonds.

[0032] The technical solutions provided in this application have the following advantages compared with the prior art:

[0033] This application provides a method for preparing anti-tumor targeted nanomaterials. Through stepwise construction and precise integration of functional modules, a nanoplatform integrating multiple functions is successfully created to achieve the core goals of precise tumor treatment and reduced toxicity.

[0034] In terms of targeted enrichment, step S4 activates the carboxyl groups on the surface of the intermediate product through EDC / NHS, and then undergoes a second amidation reaction with cRGD-PEG-NH2 to covalently graft the targeting peptide onto the polymer shell. With the help of cRGD's specific recognition of the integrin αvβ3 receptor highly expressed by tumor cells, the nanomaterial is guided to actively enrich itself at the tumor site, thereby improving the targeted delivery efficiency.

[0035] In terms of precise drug release with both pH and GSH responses, step S2 first synthesizes a dual-response crosslinking agent containing disulfide bonds (glutathione response) and phenylboronic acid groups (pH response). In step S3, the crosslinking agent is then used to crosslink polyacrylic acid to form a dense polymer shell that encapsulates the drug-carrying core. This shell remains stable in a normal physiological environment and only disintegrates under the dual stimulation of acidic conditions in the tumor microenvironment and high intracellular glutathione concentration, thereby achieving precise and controllable drug release at the target site and avoiding systemic toxicity caused by premature leakage.

[0036] In terms of synergistic therapy and imaging tracing, step S1 involves upconversion nanoparticles (imaging scaffold), calcium peroxide nanoparticles (self-oxygenated and chemokinetic precursors), chemotherapeutic drugs (directly killing tumors), and manganese chloride (Mn) 2+ The source is integrated into the drug delivery core, in which upconversion nanoparticles enable in vivo imaging and tracking, chemotherapy drugs directly exert their killing effect, and calcium peroxide decomposes in the acidic environment of the tumor to produce O2 and H2O2. O2 relieves tumor hypoxia to enhance the effect of chemotherapy, and H2O2 in Mn 2+ Under catalysis, a Fenton-like reaction occurs to generate highly toxic ·OH, achieving chemokinetic treatment, while Mn... 2+ By activating the innate immune pathway and inducing immunogenic death of tumor cells with chemotherapy drugs, the two work together to initiate an anti-tumor immune response, forming a triple synergy of "chemotherapy-chemokinetics-immunity". This achieves both highly efficient and specific killing of tumors and inhibition of tumor metastasis and recurrence through immune memory, ultimately achieving a unified approach of multifunctional integration and precise and efficient treatment. Attached Figure Description

[0037] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0038] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 A schematic flowchart illustrating a method for preparing antitumor targeted nanomaterials provided in this application embodiment;

[0040] Figure 2 This is a schematic diagram of the structure of the antitumor-targeting nanomaterial provided in the embodiments of this application;

[0041] Figure label:

[0042] 1-Drug-carrying core, 2-Polymer shell, 3-cRGD-PEG targeting molecule. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0044] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0045] Figure 1 This is a schematic flowchart illustrating a method for preparing anti-tumor targeted nanomaterials, as provided in an embodiment of this application.

[0046] like Figure 1 As shown, this application provides a method for preparing anti-tumor targeted nanomaterials, the method comprising the following steps:

[0047] S1. Upconversion nanoparticles, calcium peroxide nanoparticles, manganese chloride, and chemotherapeutic drugs are stirred and mixed in an aqueous phase to ensure that the calcium peroxide nanoparticles, the chemotherapeutic drugs, and Mn are mixed. 2+ Adsorbed onto the surface of the upconversion nanoparticles, the drug-loaded core is obtained after post-processing.

[0048] S2. Cystamine and 3-aminophenylboronic acid are dissolved in water, and EDC and NHS are added. Then, the first amidation reaction is carried out to obtain a crosslinking agent with boric acid groups at both ends.

[0049] S3. The drug-loaded core is dispersed in a polyacrylic acid solution, and the crosslinking agent is added. Then, a crosslinking reaction is carried out to form a dense polymer shell with both pH responsiveness and glutathione responsiveness on the outside of the drug-loaded core. After post-processing, an intermediate product is obtained.

[0050] S4. The surface carboxyl groups of the intermediate product are activated, and then a second amidation reaction is carried out with cRGD-PEG-NH2 to graft the targeting peptide onto the polymer shell through amide bonds. After post-treatment, anti-tumor targeting nanomaterials are obtained.

[0051] It should be noted that this application rationally designs a method for preparing anti-tumor targeted nanomaterials, thereby successfully constructing a multifunctional anti-tumor nanoplatform integrating active targeted enrichment, pH / GSH dual-response precise drug release, self-oxygenated enhancement of the synergistic effects of chemotherapy-chemokinetic therapy-immunotherapy, and upconversion imaging tracing. This achieves specific and efficient killing of tumor tissues and effectively inhibits their metastasis and recurrence, while significantly reducing systemic toxicity to normal tissues. The specific roles of each step are as follows:

[0052] (1) Preparation of the drug-loaded core (S1): Upconversion nanoparticles serve as the core scaffold, and their surfaces are naturally rich in polar functional groups such as hydroxyl or carboxyl groups. These functional groups provide stable binding sites for the integration of various active components. After dispersing upconversion nanoparticles, calcium peroxide nanoparticles, manganese chloride, and chemotherapeutic drugs in an aqueous phase, efficient loading is achieved through continuous vigorous stirring and utilizing multiple intermolecular interactions: calcium peroxide nanoparticles and chemotherapeutic drugs, due to their polar characteristics, are tightly adsorbed onto the surface of upconversion nanoparticles through electrostatic attraction, van der Waals forces, or hydrogen bonding; while the Mn released from manganese chloride... 2+ Through coordination, it binds to the hydroxyl and carboxyl groups on the surface of upconversion nanoparticles, as well as the amino and hydroxyl groups in chemotherapy drug molecules, forming stable coordination bonds and thus firmly anchoring itself in the core system. After stirring, the precipitate is collected by centrifugation and washed multiple times with deionized water to thoroughly remove unadsorbed free calcium peroxide particles, chemotherapy drug molecules, and Mn from the system. 2+This ensures that the final drug-loaded core has a uniform composition and a stable drug loading. The core value of this step lies in the precise integration of multiple functional modules: the upconversion nanoparticles serve as a physical scaffold supporting the entire core structure and also function as subsequent in vivo imaging tracers; calcium peroxide nanoparticles are the core source of subsequent self-oxygenation and chemokinetic precursors; the chemotherapy drug is the core component for directly killing tumor cells; and Mn... 2+ It simultaneously serves as a catalyst for chemokinetic therapy and an adjuvant for immune activation. These components achieve a high degree of spatiotemporal concentration through S1 loading, laying a solid foundation for the synchronous initiation of subsequent cascade therapies, ensuring that each active ingredient can be delivered together into tumor cells, and avoiding premature loss or misdirection of action of any single component.

[0053] (2) Synthesis of a dual-response crosslinking agent (S2): The structural characteristics of cystamine molecules are that both ends contain primary amino groups, and a disulfide bond is embedded in the middle of the molecular chain. 3-Aminophenylboronic acid, on the other hand, possesses both carboxyl and boric acid groups. The structural characteristics of these two raw materials provide an inherent condition for the integration of dual-response functions. After dissolving both in water, EDC and NHS are added as activating agents. EDC can specifically activate the carboxyl group in the 3-aminophenylboronic acid molecule, causing it to form an unstable active intermediate. Subsequently, NHS rapidly combines with this intermediate to generate an NHS ester derivative with higher stability and stronger reactivity with amino groups, effectively avoiding the side reaction of self-hydrolysis after carboxyl group activation. At this time, the primary amino groups at both ends of the cystamine molecule act as nucleophiles, specifically reacting with the NHS esters in the two activated 3-aminophenylboronic acid molecules, completing covalent linkage through amide bonds, and releasing small NHS molecules, ultimately generating the target crosslinking agent with boric acid groups at both ends and a disulfide bond in the middle. The synthesis of this crosslinking agent is a crucial prerequisite for the intelligent responsive release of the entire nanosystem. Its molecular structure integrates two functional units, each with its own function: the boric acid group forms a borate ester bond with the cis-diol structure in the subsequent polymer shell, and this chemical bond specifically breaks under the weakly acidic conditions unique to the tumor microenvironment (pH≈6.5), achieving a pH response; the intermediate disulfide bond is highly sensitive to the high concentration of glutathione in tumor cells (approximately 1000 times the concentration in normal tissue), and can be reduced and broken by glutathione, achieving a glutathione response. The synergistic existence of these two response units provides the core structural basis for the subsequent construction of a "dual-lock" logic-gated release system, making the crosslinking agent a key bridge connecting the polymer backbone and the environmental response function.

[0054] (3) Construction of the dual-response polymer shell (S3): The drug-loaded core prepared in S1 was dispersed in a polyacrylic acid solution. As a water-soluble polymer, polyacrylic acid has a large number of carboxyl groups on its molecular chain, giving it good hydrophilicity and reactivity. Under stirring, the polyacrylic acid molecular chains uniformly coat the surface of the drug-loaded core through electrostatic adsorption, hydrogen bonding, or physical entanglement, forming a loose polymer coating layer. Subsequently, the dual-response crosslinking agent synthesized in S2 was added. The borate groups at both ends of the crosslinking agent specifically bind to the adjacent cis-diol structure (or a cis-diol-like structure composed of carboxyl groups and ortho-hydroxyl groups) on the polyacrylic acid chain, forming stable borate ester bonds. Through the connection of numerous such crosslinking sites, the originally loose linear polyacrylic acid chains are crosslinked to form a three-dimensional network structure, ultimately constructing a dense polymer shell layer outside the drug-loaded core. In this process, the disulfide bonds in the crosslinking agent molecules are simultaneously integrated into the polymer network, so that the final shell layer has dual responsive characteristics of both borate ester bonds (pH response) and disulfide bonds (glutathione response). After centrifugation and washing purification, a core-shell structured intermediate product was obtained. The shell layer plays a role in two key dimensions: First, "sealing and protection." The dense polymer network can physically block the contact between the active components such as chemotherapeutic drugs and calcium peroxide in the drug-carrying core and the external environment during blood circulation, effectively avoiding systemic toxicity caused by premature leakage. At the same time, it protects the core components from degradation in the complex in vivo environment, ensuring that they can still maintain their activity when they reach the tumor site. Second, "intelligent release control." The "double-lock" logic gating characteristics of the shell layer make it stable in the normal physiological environment (pH≈7.4, low glutathione concentration). Only when the nanosystem enters the tumor microenvironment (weakly acidic) and is internalized by tumor cells (high glutathione concentration) will the borate ester bond and disulfide bond break successively, causing the polymer network to gradually disintegrate, and finally achieving precise and efficient release of the active components in the tumor cells.

[0055] (4) Grafting of Target Peptides (S4): The intermediate product prepared in S3 has a polymer shell formed by cross-linking of polyacrylic acid, thus retaining a large number of unreacted carboxyl groups. These carboxyl groups provide ample reaction sites for the grafting of target molecules. First, these surface carboxyl groups are activated by EDC and NHS. The active intermediate formed by the reaction of EDC and carboxyl groups is converted into a stable NHS ester under the action of NHS, significantly improving the specificity and efficiency of the reaction between carboxyl and amino groups. Subsequently, the cRGD-PEG-NH2 target molecule is added. One end of this molecule is a cRGD peptide, and the other end is a primary amino group. The terminal primary amino group undergoes an amidation reaction with the activated carboxyl group to form a stable covalent bond, thereby firmly grafting the cRGD-PEG molecule onto the surface of the nanoparticles. After the reaction is completed, the ungrafted free cRGD-PEG-NH2 molecules in the system are removed by multiple centrifugation and washing to ensure the purity of the final product and the uniformity of the grafting density of the target molecules. This step endows the entire nanosystem with dual characteristics of active targeting and long circulation: the cRGD peptide can specifically recognize and bind to the integrin αvβ3 receptor highly expressed on the surface of tumor neovascular endothelial cells and tumor cells, significantly improving the enrichment efficiency of nanomaterials at tumor sites and reducing non-specific distribution in normal tissues through receptor-mediated endocytosis; while after grafting with PEG segments, a hydration film can be formed on the surface of nanoparticles, effectively shielding them from recognition and phagocytosis by macrophages in the immune system, prolonging the blood circulation time of nanomaterials in vivo, providing a sufficient time window for their active targeting of tumor sites and achieving efficient enrichment, while further reducing the immunogenicity and systemic toxicity of nanomaterials.

[0056] More importantly, through the synergistic effect of each step, chemotherapy, chemokinetics and immunotherapy are deeply integrated, forming a synergistic therapeutic effect of "local killing - immune activation - systemic protection", which effectively inhibits tumor recurrence and metastasis.

[0057] (1) The synergy between S1 and S2 constitutes the supporting relationship between the "functional core" and the "response basis": S1, through the multi-component loading of the drug-carrying core, is the functional basis for the entire system to achieve chemotherapy, chemokinetic therapy and immunotherapy. However, these active components are easily inactivated or prematurely released due to environmental interference during in vivo circulation, and need to rely on specific response mechanisms to achieve precise release. The dual-response crosslinking agent synthesized by S2 provides the structural basis for this requirement. Its disulfide bonds and boric acid groups are the core "switch" for subsequent specific response of the tumor microenvironment. Without the synthesis of the crosslinking agent of S2, the intelligent response function of the subsequent shell is out of the question. If the drug-carrying core of S1 lacks the protection and control of the responsive shell, its therapeutic effect will be greatly reduced due to the loss of active components and off-target release.

[0058] (2) The synergy between S2 and S3 realizes the transformation from "responsive structure" to "practical function": The crosslinking agent synthesized by S2 is only a small molecule with dual-response characteristics and cannot be directly applied to the protection and release control of the drug delivery system; while S3, through a crosslinking reaction, combines this small molecule crosslinking agent with the polyacrylic acid polymer backbone to form a dense polymer shell that wraps around the drug delivery core, transforming the dual-response characteristics of the crosslinking agent into the macroscopic function of the entire shell. The borate ester bond formed by the borate groups at both ends of the crosslinking agent and the PAA chain, as well as the disulfide bond retained in the middle, together endow the shell with "AND" logic gated release capability. It will only disintegrate under the dual stimulation of the acidity of the tumor microenvironment and the high GSH in the cell. This synergy upgrades the single responsive molecular structure into an intelligent shell with practical application value, which not only achieves stable protection of the drug delivery core, but also ensures the precise release of the active components.

[0059] (3) The synergy between S3 and S4 maximizes the efficiency of "targeted delivery" and "intelligent release": S3's dual-response shell solves the problem of "how to release drugs at the correct location," ensuring that drugs are released only within tumor cells through physical barriers and response mechanisms; while S4's targeted peptide grafting solves the problem of "how to get more drugs to the correct location," significantly increasing the enrichment of nanomaterials at the tumor site through active target recognition. The synergistic effect of the two enables the nanosystem to reduce drug leakage and off-target damage during circulation, while increasing the effective drug concentration at the target site, realizing a closed loop of "precise delivery-intelligent release," significantly improving the efficiency and safety of treatment, and avoiding the inefficiency problems of single-target or single-response systems.

[0060] (4) The synergistic effect of the internal components of S1 and subsequent steps constructs a complete "cascade therapy-immune activation" system: the calcium peroxide loaded in S1 decomposes rapidly in the acidic environment of the tumor after the S3 shell disintegrates. The oxygen produced can alleviate the hypoxia of the tumor tissue, which can not only enhance the cytotoxicity of chemotherapy drugs (hypoxia reduces the sensitivity of chemotherapy), but also improve the tumor immunosuppressive microenvironment, creating favorable conditions for immunotherapy; at the same time, the H2O2 produced is the Mn loaded in S1. 2+ Provides a substrate for Fenton-like reactions, Mn 2+ Catalyzing H2O2 to generate highly toxic hydroxyl radicals (·OH) enables chemokinetic therapy, directly killing tumor cells; while chemotherapy drugs are released in large quantities after the shell disintegrates, directly inducing immunogenic death of tumor cells and releasing tumor-specific antigens and danger signals; at this time, the Mn loaded in S1... 2+Further leveraging the role of immune adjuvants, it activates the cGAS-STING innate immune pathway, promotes dendritic cell maturation and T cell activation, and transforms local tumor cell killing into a systemic anti-tumor immune response. The initiation of this cascade effect relies on the targeting guidance of S4 to enrich the nanosystem at the tumor site, the disintegration of the shell of S3 in the correct location to release active components, and the cross-linking agent of S2 to ensure the specificity of the shell's response. Ultimately, through the synergistic effect of each step, a deep integration of chemotherapy, chemokinetic therapy, and immunotherapy is achieved, forming a synergistic therapeutic effect of "local killing - immune activation - systemic protection," effectively inhibiting tumor recurrence and metastasis.

[0061] In some embodiments, in step S1, the mass ratio of the upconversion nanoparticles, the calcium peroxide, the chemotherapeutic drug (DOX), and the manganese chloride is 1:(0.5-2):(0.5-2):(0.1-0.5).

[0062] The upconversion nanoparticles are core-shell structured NaYF4:Yb,Er@NaYF4;

[0063] The chemotherapy drug is doxorubicin hydrochloride.

[0064] In some embodiments, in step S1, the stirring and mixing is carried out under light-protected conditions at a temperature of 20–25°C for 12–24 hours.

[0065] The parameter design in step S1 aims to achieve efficient and stable loading of each functional component: the core-shell structure of NaYF4:Yb,Er@NaYF4 provides a stable scaffold for the load and optimizes the upconversion imaging performance. Doxorubicin hydrochloride serves as a chemotherapy drug to ensure the tumor killing effect. The mass ratio of 1:(0.5~2):(0.5~2):(0.1~0.5) precisely matches the adsorption and coordination efficiency of upconversion nanoparticles, calcium peroxide, chemotherapy drugs and manganese chloride, achieving a balanced integration of imaging, self-oxygenation, chemotherapy, immune activation and chemokinetic therapy functions. The light-proof stirring environment of 20~25℃ and the reaction time of 12~24h provide mild and stable conditions for intermolecular physical adsorption and coordination, ensuring that each component is firmly bound to the core surface.

[0066] In some embodiments, in step S2, the molar ratio of cystamine to 3-aminophenylboronic acid is 1:(2-2.2);

[0067] The molar ratio of EDC to 3-aminophenylboronic acid is (1.2-1.5):1;

[0068] The molar ratio of NHS to EDC is 1:1.

[0069] In some embodiments, in step S2, the first amidation reaction is carried out under light-protected conditions, at a temperature of 20–25°C, and for a time of 6–12 h.

[0070] The parameters of step S2 are optimized for the efficient synthesis of the dual-response crosslinking agent: the molar ratio of cystamine to 3-aminophenylboronic acid 1:(2-2.2) ensures the complete introduction of borate groups at both ends of the crosslinking agent; the molar ratio of EDC to 3-aminophenylboronic acid (1.2-1.5):1 and NHS to EDC 1:1 precisely matches the stoichiometric requirements of the carboxyl activation and amidation reactions; the light-protected reaction environment of 20-25℃ and the reaction time of 6-12h ensure that the amidation reaction proceeds fully and efficiently generates a dual-response crosslinking agent containing both disulfide bonds and borate groups.

[0071] In some embodiments, in step S3, the weight-average molecular weight of the polyacrylic acid is 1800–2500 Da;

[0072] The mass ratio of the drug-loaded core to the polyacrylic acid is 1:(1-5);

[0073] The molar ratio of the carboxyl groups on the polyacrylic acid chain to the boric acid groups on the crosslinking agent is 1:(0.2-0.5).

[0074] In some embodiments, in step S3, the crosslinking reaction is carried out at a temperature of 20–25°C for 2–4 hours.

[0075] The parameters for step S3 focus on the optimal construction of the dual-response polymer shell: polyacrylic acid with a weight-average molecular weight of 1800–2500 Da possesses suitable reactivity and chain segment flexibility, easily forming a dense and responsive polymer network; the mass ratio of drug-loaded core to polyacrylic acid is 1:(1–5), and the molar ratio of polyacrylic acid carboxyl groups to crosslinking agent boric acid groups is 1:(0.2–0.5), ensuring uniform shell coating and moderate degree of crosslinking; the reaction temperature of 20–25°C and the reaction time of 2–4 h ensure the smooth progress of the crosslinking reaction, giving the shell both good sealing and protective properties and pH / GSH dual-response release characteristics.

[0076] In some embodiments, the activation method of the surface carboxyl groups in step S4 is as follows:

[0077] The intermediate product was dispersed in MES buffer solution with a pH of 5.5–6.5, and EDC and NHS were added. The mixture was then reacted at 20–25°C in the dark for 30–60 min to activate the surface carboxyl groups of the intermediate product.

[0078] The molar ratio of EDC to the carboxyl group to be activated is (1-2):1, and the molar ratio of NHS to EDC is 1:1.

[0079] In some embodiments, in step S4, the second amidation reaction is carried out in a PBS buffer with a pH of 7.5 to 8.5, at a reaction temperature of 20 to 25°C, and for a reaction time of 4 to 8 hours.

[0080] The mass ratio of the intermediate product to the cRGD-PEG-NH2 is 1:(0.5~2).

[0081] The parameters in step S4 provide support for the efficient and stable grafting of the targeting peptide: MES buffer at pH 5.5–6.5 is suitable for the carboxyl activation reaction, PBS buffer at pH 7.5–8.5 optimizes the amidation reaction environment, and the molar ratio of EDC to the carboxyl group to be activated (1–2):1 and NHS to EDC 1:1 ensures sufficient carboxyl activation and strong reaction specificity; the mass ratio of intermediate product to cRGD-PEG-NH2 1:(0.5–2) ensures the grafting density of the targeting molecule, and the reaction temperature of 20–25℃, the carboxyl activation time of 30–60 min, and the amidation reaction time of 4–8 h ensure that the targeting peptide is firmly grafted to the shell surface through amide bonds, significantly improving the tumor targeting enrichment ability of nanomaterials.

[0082] Figure 2 This is a schematic diagram of the structure of the anti-tumor targeted nanomaterial provided in the embodiments of this application.

[0083] Based on a general inventive concept, such as Figure 2 As shown, this application provides an antitumor-targeting nanomaterial prepared by the method described in any one of the above methods, wherein the nanomaterial has a core-shell targeting structure and comprises:

[0084] The drug-carrying core consists of upconversion nanoparticles, calcium peroxide nanoparticles adsorbed on the surface of the upconversion nanoparticles, chemotherapeutic drugs, and Mn. 2+ constitute;

[0085] A polymer shell encapsulating the drug-carrying core, the polymer shell being cross-linked from polyacrylic acid via a dual-responsive cross-linking agent containing disulfide bonds and phenylboronic acid groups;

[0086] cRGD-PEG targeting molecules are grafted onto the polymer shell via amide bonds.

[0087] The core-shell targeting structure of this anti-tumor targeted nanomaterial is gradually formed through an orderly process of stepwise assembly, precise cross-linking, and covalent grafting.

[0088] First, using upconversion nanoparticles as the core scaffold, and utilizing their abundant polar functional groups such as hydroxyl and carboxyl groups, a drug-carrying core is constructed through physical adsorption and coordination. When upconversion nanoparticles, calcium peroxide nanoparticles, chemotherapeutic drugs, and manganese chloride are mixed and stirred in an aqueous phase, calcium peroxide and chemotherapeutic drugs are adsorbed onto the surface of the upconversion nanoparticles by electrostatic attraction, van der Waals forces, or hydrogen bonds, while manganese chloride dissociates Mn... 2+ It then forms coordination bonds with the coordination sites in the above components, firmly anchoring itself on the core surface, and after purification, a structurally stable drug-loaded core is obtained.

[0089] Subsequently, a polymer shell was constructed through a crosslinking reaction: first, cystamine and 3-aminophenylboronic acid were used as raw materials to synthesize a dual-responsive crosslinking agent containing disulfide bonds and phenylboronic acid groups through an amidation reaction under EDC / NHS catalysis; then, the drug-loaded core was dispersed in a polyacrylic acid solution, and the dual-responsive crosslinking agent was added. The phenylboronic acid groups at both ends of the crosslinking agent formed borate ester bonds with the cis-diol structure (or cis-diol-like structure) on the polyacrylic acid chain. Through a large number of crosslinking sites, the linear polyacrylic acid chain was crosslinked into a dense three-dimensional polymer network, which finally wrapped around the drug-loaded core, forming a polymer shell with both pH responsiveness and glutathione responsiveness. The core-shell structured intermediate product was obtained after purification.

[0090] Finally, the targeting molecule is introduced through covalent grafting: Uncrosslinked carboxyl groups on the surface of the intermediate product are activated using EDC / NHS in a suitable buffer solution, converting them into highly active NHS esters. Then, cRGD-PEG-NH2 is added to the reaction system, where its terminal amino groups undergo amidation with the activated carboxyl groups, forming stable covalent bonds. This allows the cRGD-PEG targeting molecule to be firmly grafted onto the polymer shell surface. Through this step-by-step construction from the inside out, a core-shell targeting structure of "drug-loaded core - polymer shell - targeting molecule" is ultimately formed.

[0091] In summary, this application possesses significant technical advantages and application value, with its core advantages lying in the systematic design, functional synergy, and feasibility of implementation. In terms of structural design, the hierarchical construction of "drug-carrying core - polymer shell - targeting molecule" achieves precise integration of multifunctional modules, ensuring stable loading of each component while maintaining structural integrity through covalent bonding and cross-linking. Regarding the response mechanism, the polymer shell, relying on a dual-response cross-linking agent, achieves dual-sensitive release at pH and GSH levels, specifically disintegrating only within the tumor microenvironment and cells, significantly improving the accuracy of drug delivery and reducing systemic toxicity. In terms of therapeutic efficacy, it integrates chemotherapy, self-oxygenated enhanced chemokinetic therapy, and immunomodulatory therapy, forming a synergistic effect of "local killing - immune memory," which not only efficiently eliminates tumor cells but also inhibits metastasis and recurrence, overcoming the limitations of single-therapy approaches. Simultaneously, the introduction of upconversion nanoparticles enables imaging tracking, and cRGD targeting molecules significantly enhance tumor site enrichment efficiency. Furthermore, clearly controllable preparation parameters and mild reaction conditions ensure the reproducibility and scalability of the process.

[0092] The antitumor-targeting nanomaterials prepared in this application are primarily focused on the precision treatment and integrated diagnosis and treatment of malignant tumors. They can serve as a highly efficient nanodrug delivery platform, suitable for the treatment of various solid tumors expressing integrin αvβ3 receptors (such as liver cancer, lung cancer, breast cancer, and glioma). After administration via intravenous injection or other methods, targeted enrichment, precise drug release, and synergistic therapy enable specific killing of tumor tissues. Simultaneously, relying on upconversion imaging capabilities, they can be used for early tumor diagnosis, lesion localization, and efficacy monitoring during treatment, providing a basis for the development of individualized clinical treatment plans. Furthermore, the multi-therapeutic synergistic mechanism of this material can also be used to overcome tumor drug resistance and heterogeneity, providing new technical support for the combined treatment of advanced or metastatic tumors, and offering a referable design concept and preparation paradigm for the development of novel multifunctional antitumor nanomedicines.

[0093] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0094] Example 1

[0095] This embodiment provides a method for preparing anti-tumor targeted nanomaterials, the method comprising the following steps:

[0096] S1. Accurately weigh 10.0 mg of core-shell upconversion nanoparticles (NaYF4:Yb,Er@NaYF4, sourced from Xi'an Qiyue Biotechnology Co., Ltd.) and place them in a 50 mL round-bottom flask; add 20 mL of deionized water and sonicate for 30 min to ensure thorough dispersion; then add 10.0 mg of calcium peroxide nanoparticles (CAS No. 1305-79-9), 10.0 mg of doxorubicin hydrochloride (CAS No. 25316-40-9), and 2.0 mg of manganese chloride in sequence. At this point, the mass ratio of each component is 1:1:1:0.2. The flask is wrapped with aluminum foil to protect it from light and placed in a constant temperature water bath at 25°C. It is stirred at 300 rpm for 24 hours to allow the drug and ions to be fully adsorbed. The mixture is then transferred to a centrifuge tube and centrifuged at 10,000 rpm for 15 minutes. The supernatant is discarded. The precipitate is washed three times with deionized water to remove unadsorbed raw materials. The resulting precipitate is freeze-dried to obtain the drug-loaded core, which is then sealed and stored in the dark.

[0097] S2. Accurately weigh 152.3 mg (1.0 mmol) of cystamine (CAS No. 51-85-4) and 274.3 mg (2.0 mmol) of 3-aminophenylboronic acid (CAS No. 280563-63-5), and place them in a 100 mL reaction flask; add 50 mL of deionized water and stir until completely dissolved; add 382.9 mg (2.0 mmol) of EDC (chemical name 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 230.1 mg (2.0 mmol) of NHS (chemical name N-hydroxysuccinimide). At this point, the molar ratio of EDC to 3-APBA is 1.2:1, and the molar ratio of NHS to EDC is 1:1; react at 25 °C, protected from light, and at 400 rpm for 12 h to carry out the first amidation reaction; after the reaction is completed, a crosslinking agent solution with boric acid groups at both ends is obtained and stored at 4 °C for later use;

[0098] S3. Take 10.0 mg of the drug-loaded core prepared in step one and disperse it in 20 mL of aqueous solution containing 30.0 mg of polyacrylic acid (PAA, weight average molecular weight ~2000 Da, CAS number 9003-01-4). At this point, the mass ratio of the drug-loaded core to PAA is 1:3. Sonicate the mixture for 30 min to ensure thorough dispersion. Add 5 mL of the crosslinking agent solution prepared in step two. At this point, the molar ratio of carboxyl groups on the polyacrylic acid chain to boric acid groups on the crosslinking agent is 1:0.35. Stir the mixture at 300 rpm at 25 °C for 4 h to carry out the crosslinking reaction. After the reaction is complete, centrifuge the mixture at 12,000 rpm for 20 min. Wash the precipitate three times with deionized water and freeze-dry to obtain the intermediate product.

[0099] S4. Take 10.0 mg of the intermediate product obtained in step 3 and disperse it in 20 mL of MES buffer (pH 6.0, MES being 2-(N-morpholine)ethanesulfonic acid); add 19.1 mg (0.1 mmol) EDC and 11.5 mg (0.1 mmol) NHS, and activate at 25 °C in the dark for 45 min to activate the carboxyl groups on the surface of the intermediate product; centrifuge the activated product and redisperse it in 20 mL of PBS buffer (phosphate buffer) at pH 8.0; add 15.0 mg of cRGD-PEG-NH2 (targeting peptide, bound to amino groups and polyethylene glycol, with a terminal cyclic peptide cRGD, sourced from Xi'an Ruixi Biotechnology Co., Ltd.). At this point, the mass ratio of the intermediate product to cRGD-PEG-NH2 is 1:1.5; react at 25 °C for 8 h to carry out the second amidation reaction; after the reaction is complete, centrifuge the product at 12,000 rpm for 20 min and wash it three times with deionized water; after freeze-drying, the final antitumor targeting nanomaterial is obtained.

[0100] Transmission electron microscopy clearly reveals that the nanomaterials prepared in Example 1 exhibit a regular core-shell structure. The images show a distinct core-shell layer: the interior consists of a high-electron-density upconversion nanoparticle core, exhibiting a regular crystalline morphology that is approximately spherical or hexagonal; the exterior is encased in a polymer shell with relatively low electron density and uniform thickness. This core-shell structure is complete and continuous, with the shell completely enclosing the core, and the interface is clearly discernible.

[0101] From the particle size distribution, the nanoparticles obtained in Example 1 showed good dispersibility with no obvious agglomeration, and the overall size distribution was in the range of 150–200 nm. The core UCNPs had a particle size of approximately 20–30 nm, and the outer polymer shell had a relatively uniform thickness of approximately 20–30 nm. This regular core-shell structure demonstrates the success of the preparation method, with the drug-loaded core completely encapsulated in the polymer matrix, forming a stable nanocomposite.

[0102] Example 2

[0103] This embodiment provides a method for preparing anti-tumor targeted nanomaterials, the specific steps of which are as follows:

[0104] S1. Accurately weigh 10.0 mg of core-shell upconversion nanoparticles (NaYF4:Yb,Er@NaYF4) and place them in a 50 mL round-bottom flask. Add 20 mL of deionized water and sonicate for 30 min to ensure thorough dispersion. Add 15.0 mg of calcium peroxide nanoparticles, 15.0 mg of doxorubicin hydrochloride, and 1.0 mg of manganese chloride sequentially, with a mass ratio of 1:1.5:1.5:0.1. Wrap the flask in aluminum foil to protect it from light and place it in a 22°C constant temperature water bath. Stir at 350 rpm for 18 h. Centrifuge the mixture at 10,000 rpm for 15 min and discard the supernatant. Wash the precipitate three times with deionized water. Freeze-dry the precipitate to obtain the drug-loaded core and store it in a sealed container protected from light.

[0105] S2. Accurately weigh 152.3 mg (1.0 mmol) of cystamine and 301.7 mg (2.2 mmol) of 3-aminophenylboronic acid, and place them in a 100 mL reaction flask; add 50 mL of deionized water and stir to dissolve; add 478.6 mg (2.5 mmol) of EDC and 287.6 mg (2.5 mmol) of NHS, at which point the molar ratio of EDC to 3-APBA is 1.5:1, and the molar ratio of NHS to EDC is 1:1; react at 350 rpm for 6 h at 22 °C in the dark to complete the first amidation reaction; the resulting crosslinking agent solution is stored at 4 °C.

[0106] S3. Take 10.0 mg of drug-loaded core and disperse it in 20 mL of aqueous solution containing 50.0 mg of polyacrylic acid (weight average molecular weight ~2000 Da) at a mass ratio of 1:5; sonicate for 30 min to disperse it; add 8 mL of crosslinking agent solution; react at 22℃ and 350 rpm for 2 h to complete the crosslinking reaction; after the reaction, centrifuge at 12,000 rpm for 20 min, wash three times with deionized water, and freeze-dry to obtain the intermediate product;

[0107] S4. Take 10.0 mg of intermediate product and disperse it in 20 mL of MES buffer (pH 5.8); add 23.0 mg (0.12 mmol) EDC and 13.8 mg (0.12 mmol) NHS, and activate at 22 °C in the dark for 35 min; after centrifugation, redisperse in 20 mL of PBS buffer (pH 8.2); add 5.0 mg cRGD-PEG-NH2 at a mass ratio of 1:0.5; react at 22 °C for 6 h to complete the second amidation reaction; centrifuge at 12,000 rpm for 20 min, wash three times, and freeze-dry to obtain the final product.

[0108] Example 3

[0109] This embodiment provides a method for preparing anti-tumor targeted nanomaterials, the specific steps of which are as follows:

[0110] S1. Accurately weigh 10.0 mg of core-shell upconversion nanoparticles (NaYF4:Yb,Er@NaYF4) and place them in a 50 mL round-bottom flask; add 20 mL of deionized water and sonicate for 30 min; add 5.0 mg of calcium peroxide nanoparticles, 8.0 mg of doxorubicin hydrochloride and 4.0 mg of manganese chloride in sequence at a mass ratio of 1:0.5:0.8:0.4; stir at 250 rpm for 20 h at 20 °C under light-protected conditions; centrifuge at 10,000 rpm for 15 min and wash three times; freeze-dry to obtain the drug-loaded core and store in a sealed, light-protected container.

[0111] S2. Accurately weigh 152.3 mg (1.0 mmol) of cystamine and 288.1 mg (2.1 mmol) of 3-aminophenylboronic acid, and dissolve them in 50 mL of deionized water; add 414.8 mg (2.17 mmol) of EDC and 249.2 mg (2.17 mmol) of NHS, with a molar ratio of EDC to 3-APBA of 1.3:1 and a molar ratio of NHS to EDC of 1:1; react at 320 rpm for 9 h at 20 °C in the dark; the resulting crosslinking agent solution is stored at 4 °C.

[0112] S3. Take 10.0 mg of drug-loaded core and disperse it in 20 mL of aqueous solution containing 15.0 mg of polyacrylic acid (weight average molecular weight ~2000 Da) at a mass ratio of 1:1.5; sonicate for 30 min; add 3 mL of crosslinking agent solution; react at 20 °C and 320 rpm for 3 h; centrifuge at 12,000 rpm for 20 min, wash and freeze dry to obtain intermediate product;

[0113] S4. Take 10.0 mg of intermediate product and disperse it in 20 mL of MES buffer (pH 6.2); add 15.3 mg (0.08 mmol) EDC and 9.2 mg (0.08 mmol) NHS, and activate at 20 °C in the dark for 50 min; after centrifugation, disperse it in 20 mL of PBS buffer (pH 7.8); add 18.0 mg of cRGD-PEG-NH2 at a mass ratio of 1:1.8; react at 20 °C for 5 h to complete the reaction; centrifuge at 12,000 rpm for 20 min, wash three times, and freeze-dry to obtain the final nanomaterial.

[0114] The antitumor-targeting nanomaterials obtained in Examples 1-3 were subjected to physicochemical property testing.

[0115] (1) Size and particle size distribution: The core-shell morphology of the material was observed using a transmission electron microscope, and its hydration dynamics particle size and distribution (PDI) were measured by dynamic light scattering (DLS). The results are shown in Table 1.

[0116] Table 1 Size and particle size distribution of antitumor targeted nanomaterials

[0117]

[0118] (2) Surface potential (Zeta potential): The zeta potential was measured by a dynamic light scattering instrument to evaluate the colloidal stability. The results are shown in Table 2.

[0119] Table 2 Surface potentials of antitumor-targeting nanomaterials

[0120] Example Drug-loaded core potential (mV) Final product potential (mV) 1 -12.45±1.23 -18.56±1.05 2 -13.01±1.34 -20.12±1.28 3 -11.87±1.15 -16.89±0.97

[0121] (3) Drug loading performance: Ultraviolet-visible spectrophotometry was used. After the nanomaterials were completely destroyed in a buffer solution at pH 4.5, the absorbance of DOX at 480 nm was measured. The drug loading and encapsulation efficiency were calculated using a standard curve. The results are shown in Table 3.

[0122] Table 3 Drug loading performance of antitumor targeted nanomaterials

[0123] Example Drug loading (%) Encapsulation efficiency (%) 1 8.25±0.35 82.50±3.50 2 9.87±0.41 65.80±2.72 3 6.48±0.28 80.99±3.49

[0124] (4) In vitro drug release kinetics: The dialysis bag method was used. The nanomaterials were placed in different release media (pH 7.4, pH 6.5, pH 6.5 + 10 mM GSH), shaken at 37 °C, and samples were taken at predetermined time points. The DOX concentration was determined by HPLC or fluorescence spectroscopy. The results are shown in Table 4.

[0125] Table 4. In vitro drug release kinetics of antitumor targeted nanomaterials

[0126]

[0127] As shown in Tables 1 to 4, the antitumor-targeting nanomaterials prepared in Examples 1-3 of this invention have all successfully constructed well-defined and high-performance nanodelivery systems, exhibiting the following significant characteristics:

[0128] First, analysis of the structural characterization data (Table 1) showed that all examples formed regular core-shell structures. The core particle size remained within the range of 28-29 nm, confirming the stability of the upconversion nanoparticle substrate. The shell thickness was positively correlated with the amount of polyacrylic acid used, with Example 2 having the thickest shell (28.91 ± 2.89 nm) and Example 3 having the thinnest (21.05 ± 2.74 nm). Hydrated particle size and PDI data further indicated that all products exhibited good dispersibility (PDI less than 0.2), and the particle size distribution range (159-185 nm) was highly favorable for enrichment in tumor tissues via the EPR effect.

[0129] As can be seen from the surface potential changes (Table 2), after polymer encapsulation and targeted modification, the Zeta potential of the drug-loaded core significantly decreased from approximately -12 mV to approximately -19 mV. This change not only confirms the successful construction of the carboxyl-rich polyacrylic acid shell, but also indicates that the final product has better colloidal stability and a longer blood circulation time in the physiological environment.

[0130] The drug loading performance (Table 3) shows that different drug loading characteristics can be achieved by adjusting the proportions of each component. Example 2, using a higher drug feed ratio, achieved the highest drug loading (9.87±0.41%), but due to its thicker polymer shell, it suffered relatively more drug loss during washing, resulting in a lower encapsulation efficiency (65.80±2.72%). In contrast, Example 1 achieved the best balance between drug loading (8.25±0.35%) and encapsulation efficiency (82.50±3.50%).

[0131] Most importantly, the in vitro release data (Table 4) fully validates the dual-response characteristics of this nanosystem. In a pH 7.4 environment simulating normal tissue, the cumulative release rate after 48 hours was less than 23%, indicating good stability of the system in blood circulation. However, under pH 6.5 conditions simulating the tumor microenvironment, the release rate significantly increased to 40–50%, demonstrating the pH responsiveness of the phenylboronic ester bond. When both pH 6.5 and 10 mM GSH (simulating the intracellular environment of tumor cells) were present, all examples achieved burst releases of over 80%, fully demonstrating the synergistic effect of the dual-response mechanism of pH and GSH.

[0132] In summary, these data strongly demonstrate that the present invention has successfully constructed a targeted nanomaterial with a core-shell structure, good stability, efficient drug loading capacity, and intelligent release characteristics. In particular, its dual-response release behavior enables drug delivery with low leakage in normal tissues and specific release in tumor tissues, providing a reliable guarantee for reducing systemic toxicity and improving therapeutic efficacy.

[0133] The antitumor-targeting nanomaterials obtained in Example 1 were subjected to application performance testing.

[0134] (1) Cell uptake and targeting efficiency determination: Flow cytometry was used. U87-MG cells (human glioma cell line) that highly express integrin αvβ3 were used as the experimental model. The cells were co-incubated with nanomaterials loaded with DOX (with self-fluorescence) at 37°C for 4 h. The cells were then collected, and the average fluorescence intensity in the cells was detected by flow cytometry to quantify the cell uptake efficiency. The results are shown in Table 5.

[0135] Table 5. Cell uptake and targeting efficiency of antitumor-targeting nanomaterials in Example 1

[0136] experimental group Mean fluorescence intensity of U87-MG cells (highly expressing αvβ3) Free DOX 1250.45±105.67 Example 1 6850.67±450.23

[0137] (2) In vitro antitumor efficacy assay: The CCK-8 assay was used. U87-MG cells were co-incubated with samples of different concentrations for 48 h. The half-maximal inhibitory concentration (IC50) was calculated by detecting cell viability. 50 IC50, or the concentration of a drug required to kill half of the cells. 50 The lower the value, the stronger the toxicity. The test results are shown in Table 6.

[0138] Table 6. In vitro antitumor effects of antitumor-targeting nanomaterials in Example 1

[0139] experimental group <![CDATA[IC 50 (μg / mL)]]> Free DOX 2.45±0.25 Example 1 0.75±0.07

[0140] (3) In vivo imaging and tumor targeting assay: A U87-MG tumor-bearing mouse model was established. After tail vein injection of the material, the distribution of DOX fluorescence in vivo was observed using a small animal in vivo imaging system at 24 h. Mice were then sacrificed, and major organs and tumors were removed for in vivo imaging and fluorescence intensity quantification. The measured data are the fluorescence intensity of the ex vivo organs at 24 h, expressed in ×10⁻⁵ units. 8 p / s / cm 2 / sr, the measurement results are shown in Table 7.

[0141] Table 7. Tumor targeting results of the antitumor-targeting nanomaterials in Example 1

[0142] organs / tissues Free DOX Example 1 heart 1.89±0.23 1.35±0.15 liver 25.67±2.34 12.34±1.23 spleen 8.90±0.87 5.67±0.54 lung 4.56±0.45 3.45±0.32 kidney 12.34±1.20 7.82±0.76 tumor 5.67±0.55 15.78±1.50

[0143] (4) In vivo antitumor efficacy and safety determination: U87-MG tumor-bearing mice were randomly divided into groups and injected with the corresponding drug via the tail vein every 3 days for a total of 4 administrations. The tumor volume and mouse weight were measured regularly, and the tumor inhibition rate was calculated after the experiment. The results are shown in Table 8.

[0144] Table 8. In vivo antitumor efficacy and safety of the antitumor-targeting nanomaterials in Example 1

[0145] experimental group relative tumor volume Tumor inhibition rate Weight change rate PBS group 8.45±0.82 - 5.23％ Free DOX group 3.89±0.38 53.96％ -12.45％ Example 1 1.89±0.19 77.63％ -4.12％

[0146] As shown in Tables 5 to 8, the antitumor-targeting nanomaterials prepared in Example 1 exhibit significantly better overall performance than traditional free drugs at both the cellular and in vivo levels, specifically in the following three aspects:

[0147] First, regarding targeting and cellular uptake efficiency, as shown in Table 5, the average fluorescence intensity of the material from Example 1 in U87-MG cells reached 6850.67, which is 5.48 times that of free DOX (1250.45). This result strongly demonstrates that the active targeting mechanism mediated by the cRGD targeting peptide successfully achieved efficient recognition and enrichment of tumor cells that highly express integrin αvβ3.

[0148] Secondly, regarding therapeutic efficacy, this material exhibits a remarkable synergistic anti-tumor effect. As shown in Table 6, the IC of Example 1... 50 The concentration was as low as 0.75 μg / mL, significantly lower than the 2.45 μg / mL of free DOX, indicating that its in vitro cytotoxicity was approximately 3.27 times that of the latter. More importantly, in vivo efficacy data (Table 8) showed that the final tumor inhibition rate in the Example 1 group was as high as 77.63%, far superior to the 53.96% in the free DOX group. This significant improvement in therapeutic effect is not only due to the increased drug concentration at the tumor site caused by targeted delivery, but also benefits from the synergistic killing effect of chemokinetics and chemotherapy that may be triggered by the various components within the nanomaterial.

[0149] Finally, this nanosystem demonstrates significant advantages in terms of biosafety. As shown in Table 7, the in vivo distribution data shows that compared to free DOX, the fluorescence signal intensity of the material in Example 1 was significantly reduced in key normal tissues such as the heart, liver, and spleen, indicating that it can effectively reduce drug distribution in non-target tissues. This advantage directly translates into better in vivo safety. As shown in Table 8, free DOX treatment led to a 12.45% decrease in body weight in mice, showing significant systemic toxicity; while the body weight of the Example 1 group decreased by only 4.12%, with a significantly reduced toxic reaction, fully demonstrating the great value of nano-targeted delivery systems in reducing the toxic side effects of chemotherapy drugs.

[0150] In summary, the anti-tumor targeted nanomaterials obtained in Example 1 successfully achieved the design goals of "highly efficient targeting, synergistic therapy, and low toxicity and safety". Their comprehensive performance significantly surpasses that of traditional chemotherapy drugs, demonstrating great potential for clinical application.

[0151] The cGAS-STING pathway is one of the core pathways of innate immunity. When DNA released from tumor cell death is recognized by cGAS in the cytoplasm, it catalyzes the generation of the second messenger cGAMP, which in turn activates the STING protein, ultimately inducing the massive production of type I interferon (such as IFN-β) and initiating an anti-tumor immune response. Therefore, this application also measured the cGAS-STING pathway activation effect of the anti-tumor targeted nanomaterial of Example 1, and the results are shown in Table 9. The specific measurement method is as follows:

[0152] First, a mouse model bearing U87-MG human glioma was constructed until the tumor volume grew to approximately 100 mm. 3 Mice were randomly divided into three groups: a PBS negative control group, a free DOX conventional chemotherapy control group, and the nanomaterial group of Example 1, with five biological replicates in each group. The drug was administered via tail vein injection, once every three days for a total of two administrations. Mice were sacrificed 24 hours after the last administration, and the tumor tissue was completely removed for later use.

[0153] The obtained tumor tissue was flash-frozen in liquid nitrogen and then stored in an ultra-low temperature freezer at -80°C for subsequent protein and RNA extraction; a portion of fresh tumor tissue was also used to prepare a single-cell suspension for flow cytometry analysis.

[0154] Regarding specific detection indicators: Western blotting protein immunoblotting technology was used to analyze the phosphorylation level of STING protein. Total protein was extracted from tumor tissue, separated by electrophoresis, transferred to a membrane, and then incubated with rabbit anti-mouse p-STING antibody and STING antibody, respectively. After chemiluminescence development, the gray value ratio of p-STING to STING was calculated to accurately reflect the activation degree of the STING pathway.

[0155] The protein expression level of downstream effector molecule IFN-β was detected by enzyme-linked immunosorbent assay (ELISA). Tumor tissue homogenate supernatant was taken, and the operation was strictly carried out according to the kit instructions. The absorbance was measured using an ELISA reader, and the IFN-β concentration was calculated according to the standard curve.

[0156] The transcriptional levels of upstream key genes were analyzed by real-time quantitative PCR (qRT-PCR). Total RNA was extracted from tumor tissue and reverse transcribed into cDNA. The cGAS, STING and IFN-β genes were amplified using specific primers. GAPDH was used as an internal control. The relative mRNA expression levels of each gene were calculated using the 2^(-ΔΔCt) method.

[0157] Immune cell activation status was assessed by flow cytometry. Tumor tissue single-cell suspensions were prepared and stained with anti-CD3, anti-CD8 surface antibodies, and anti-granzyme B intracellular antibodies for analysis of CD8. + The proportion of granzyme B-positive cells in T cells was used to assess the degree of activation of cytotoxic T cells.

[0158] Table 9. Activation effect of cGAS-STING pathway on antitumor targeted nanomaterials in Example 1.

[0159]

[0160] As shown in Table 9, the cGAS-STING pathway activation data demonstrate that the anti-tumor targeted nanomaterials prepared in Example 1 exhibit significant advantages in activating innate immune pathways, forming a complete activation chain from the molecular to the cellular level.

[0161] First, at the level of activation of key proteins in the pathway, the phosphorylation level of STING protein in the Example 1 group reached 3.42±0.28, which was 3.42 times and 1.85 times that of the PBS group and the free DOX group, respectively. This data indicates that the nanomaterial can effectively activate the upstream switch of the cGAS-STING signaling pathway, laying a solid foundation for the cascade amplification of subsequent immune responses.

[0162] Secondly, at the gene transcription and protein expression levels, this material exhibits comprehensive activation effects. Not only did the expression level of STING mRNA increase to 3.89±0.31, but the mRNA expression of its upstream sensor cGAS also reached 4.35±0.35. More importantly, the downstream key effector molecule IFN-β achieved relative expression levels of 156.33±12.45 pg / mL at the protein level and 8.67±0.70 at the mRNA level, indicating that the entire signaling pathway from gene transcription to protein synthesis was effectively activated, generating a strong type I interferon response.

[0163] Ultimately, at the functional level of immune effector cells, this pathway activation successfully translated into a substantial anti-tumor immune response. Example 1: CD8+ in the tumor microenvironment. + The granzyme B positivity rate of T cells was as high as 38.91±3.05%, significantly higher than that in the free DOX group (15.67±1.23%) and the PBS group (5.23±0.45%). This indicates that after treatment with this nanomaterial, cytotoxic T cells were fully activated and possessed strong tumor-killing capabilities.

[0164] In summary, these data fully demonstrate that the nanomaterials of Example 1 can effectively activate the cGAS-STING signaling pathway, initiate a strong innate immune response, and ultimately drive the anti-tumor function of cytotoxic T cells, providing key molecular mechanism evidence for their synergistic immunotherapy.

[0165] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0166] Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0167] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing anti-tumor targeted nanomaterials, characterized in that, The method includes the following steps: S1. Upconversion nanoparticles, calcium peroxide nanoparticles, manganese chloride, and chemotherapeutic drugs are stirred and mixed in an aqueous phase to ensure that the calcium peroxide nanoparticles, the chemotherapeutic drugs, and Mn are mixed. 2+ Adsorbed onto the surface of the upconversion nanoparticles, the drug-loaded core is obtained after post-processing. S2. Cystamine and 3-aminophenylboronic acid are dissolved in water, and EDC and NHS are added. Then, the first amidation reaction is carried out to obtain a crosslinking agent with boric acid groups at both ends. S3. The drug-loaded core is dispersed in a polyacrylic acid solution, and the crosslinking agent is added. Then, a crosslinking reaction is carried out to form a dense polymer shell with both pH responsiveness and glutathione responsiveness on the outside of the drug-loaded core. After post-processing, an intermediate product is obtained. S4. The surface carboxyl groups of the intermediate product are activated, and then a second amidation reaction is carried out with cRGD-PEG-NH2 to graft the targeting peptide onto the polymer shell through amide bonds. After post-treatment, anti-tumor targeting nanomaterials are obtained.

2. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S1, the mass ratio of the upconversion nanoparticles, the calcium peroxide, the chemotherapeutic drug (DOX), and the manganese chloride is 1:(0.5-2):(0.5-2):(0.1-0.5). The upconversion nanoparticles are core-shell structured NaYF4:Yb,Er@NaYF4; The chemotherapy drug is doxorubicin hydrochloride.

3. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S1, the stirring and mixing are carried out under light-protected conditions at a temperature of 20–25°C for 12–24 hours.

4. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S2, the molar ratio of cystamine to 3-aminophenylboronic acid is 1:(2-2.2); The molar ratio of EDC to 3-aminophenylboronic acid is (1.2-1.5):1; The molar ratio of NHS to EDC is 1:

1.

5. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S2, the first amidation reaction is carried out under light-protected conditions, at a temperature of 20–25°C, for a time of 6–12 h.

6. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S3, the weight-average molecular weight of the polyacrylic acid is 1800-2500 Da; The mass ratio of the drug-loaded core to the polyacrylic acid is 1:(1-5); The molar ratio of the carboxyl groups on the polyacrylic acid chain to the boric acid groups on the crosslinking agent is 1:(0.2-0.5).

7. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S3, the crosslinking reaction is carried out at a temperature of 20–25°C for 2–4 hours.

8. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S4, the activation method of the surface carboxyl groups is as follows: The intermediate product was dispersed in MES buffer solution with a pH of 5.5–6.5, and EDC and NHS were added. The mixture was then reacted at 20–25°C in the dark for 30–60 min to activate the surface carboxyl groups of the intermediate product. The molar ratio of EDC to the carboxyl group to be activated is (1-2):1, and the molar ratio of NHS to EDC is 1:

1.

9. The method for preparing antitumor-targeting nanomaterials according to claim 1, characterized in that, In step S4, the second amidation reaction is carried out in PBS buffer with a pH of 7.5 to 8.5, at a reaction temperature of 20 to 25°C, and for a reaction time of 4 to 8 hours. The mass ratio of the intermediate product to the cRGD-PEG-NH2 is 1:(0.5~2).

10. An antitumor-targeting nanomaterial prepared by the method according to any one of claims 1 to 9, characterized in that, The nanomaterial has a core-shell targeted structure, including: The drug-carrying core consists of upconversion nanoparticles, calcium peroxide nanoparticles adsorbed on the surface of the upconversion nanoparticles, chemotherapeutic drugs, and Mn. 2+ constitute; A polymer shell encapsulating the drug-carrying core, the polymer shell being cross-linked from polyacrylic acid via a dual-responsive cross-linking agent containing disulfide bonds and phenylboronic acid groups; cRGD-PEG targeting molecules are grafted onto the polymer shell via amide bonds.

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