Two-stage targeting polymer prodrug and preparation method thereof

Abstract

The invention discloses a two-stage targeting high-molecular prodrug and a preparation method, and belongs to the technical field of biologication.According to the two-stage targeting high-molecular prodrug, an amphiphilic block copolymer framework serves as a basic structure for forming nano-micelles; the stage targeting ligand is connected to the skeleton and is used for targeting the prodrug to the liver tissue; the secondary targeting and dual-response drug release module is connected to the skeleton; the pH response shielding module is connected to the framework; the diagnosis and treatment body module is connected to the skeleton; and a core cross-linking agent is used for solving the problem of how to construct a two-stage targeted precise drug delivery system.

Description

Technical Field

[0001] The present invention relates to the field of dual-stage targeted drug preparation, and in particular to a dual-stage targeted polymer prodrug and a preparation method thereof. Background Art

[0002] Liver cancer is one of the common malignant tumors, and its treatment faces severe challenges. Targeted drug delivery systems aim to precisely deliver drugs to the tumor site to improve efficacy and reduce toxic side effects. They are current research hotspots. For the treatment of liver cancer, researchers have proposed a dual-stage targeting strategy, in which drug carriers are first enriched in the primary targeting of liver tissue, and then specifically recognize and enter the secondary targeting of liver cancer cells. This strategy can theoretically achieve precise attacks on liver cancer cells, but in practical applications, especially in the design of polymer prodrugs, it faces challenges arising from the complex physiological environment in the body. Existing technologies usually use the method of covalently linking primary and secondary targeting groups such as galactose for targeting liver cells and specific antibodies for targeting cancer cells to linear polymer backbones such as polyethylene glycol (PEG) to achieve dual-stage targeting. However, after entering the blood circulation, the preset targeting function of this type of design often decreases significantly due to the dynamic behavior of the molecules. On the one hand, Polymer chains undergo dynamic conformational changes in the blood, and their hydrophilic segments tend to wrap around the hydrophobic core, causing some of the targeting groups connected to the chain to be masked by their own segments, thereby losing the ability to bind to the target. Studies have shown that more than 50% of the targeting groups will fail as a result. On the other hand, when two different targeting groups are randomly distributed on the molecular chain and the spatial distance is too close, for example, less than 5 nanometers, steric hindrance and binding domain conflict will occur, causing the binding behavior of one group to interfere with the other group, resulting in a 40 to 60% reduction in the synergistic targeting efficiency. Therefore, the design of existing dual-stage targeted polymer prodrugs is mostly static molecular structure, which fails to fully consider its complex changes in the dynamic physiological environment of blood circulation. Problems such as the conformational collapse of the polymer chain, the mutual interference of the targeting groups, and the subsequent possible protein corona adsorption have jointly led to a significant reduction in the preset targeting function in the body, making it impossible to achieve the ideal therapeutic effect. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a dual-stage targeted polymer prodrug and a preparation method, which solves the problem of how to construct a dual-stage targeted precision drug delivery system.

[0004] In order to achieve the above-mentioned objectives, the present invention provides a dual-stage targeted polymer prodrug, which is a core-shell structured nanomicelle formed by self-assembly, comprising: an amphiphilic block copolymer skeleton as the basic structure for forming the nanomicelle; a primary targeting ligand connected to the skeleton, for targeting the prodrug to liver tissue; a secondary targeting and dual-responsive drug release module connected to the skeleton, for targeting liver cancer cells and responsively releasing drugs; a pH-responsive shielding module connected to the skeleton, for shielding the secondary targeting module under physiological conditions; a diagnostic and therapeutic module connected to the skeleton, for imaging and tracing the prodrug; and a core crosslinker, for cross-linking the core of the nanomicelle to enhance its structural stability.

[0005] Furthermore, the amphiphilic block copolymer backbone is poly(carboxybetaine methacrylate)-b-poly(ethylene glycol).

[0006] Furthermore, the secondary targeting ligand is a chemically modified galactosamine, which achieves secondary targeting to liver tissue by binding to the asialoglycoprotein receptor on the surface of hepatocytes.

[0007] Furthermore, the structure of the secondary targeting and dual-responsive drug release module includes: a GPC3 targeting peptide for specifically recognizing and binding to the GPC3 antigen on the surface of liver cancer cells; the chemotherapy drug doxorubicin; an active oxygen-sensitive bond connecting the doxorubicin to the GPC3 targeting peptide; and an MMP-2 cleavage site embedded within the GPC3 targeting peptide sequence.

[0008] Furthermore, the pH-responsive shielding module is a polyhistidine chain, which is in a folded state under the physiological pH conditions of blood circulation to shield the secondary targeting module, and stretches in the acidic microenvironment of tumor tissue to release the shielding.

[0009] Furthermore, the diagnostic and therapeutic module is a near-infrared fluorescent probe Cy7.5, and the core cross-linking agent is a disulfide bond cross-linking agent.

[0010] A method for preparing a dual-stage targeted polymer prodrug, the method comprising: preparing the required raw materials, including an amphiphilic block copolymer skeleton, a primary targeting ligand, a secondary targeting and dual-responsive drug release module, a pH-responsive shielding module, a diagnostic and therapeutic integration module, and a core crosslinking agent, wherein when preparing the secondary targeting and dual-responsive drug release module, a drug intermediate containing an active oxygen-sensitive bond is synthesized by reflux heating under anhydrous and oxygen-free conditions; subsequently, the skeleton is dissolved in a mixed solvent, and the secondary targeting and dual-responsive drug release module, the pH-responsive shielding module, and the diagnostic and therapeutic integration module are added, and a coupling reaction is carried out under inert gas protection using a gradient temperature program control method; then, the activated primary targeting ligand is added to the product after the coupling reaction, the pH value of the solution is adjusted to 7.0-7.5, and the polymer chains are spontaneously assembled into core-shell structured nanomicelles by constant stirring, and then the solution is allowed to stand for degassing; the core crosslinking agent is added to the formed micelle solution for core crosslinking, and the solution is purified by large-scale dialysis and ultrafiltration, and freeze-dried to obtain the prodrug.

[0011] Furthermore, the gradient temperature increase used in the coupling reaction is specifically: first react at 35°C for 10-14 hours, and then heat to 45°C for 10-14 hours.

[0012] Furthermore, the preparation of the pharmaceutical intermediate is completed by reflux heating doxorubicin and 4-formylphenylboronic acid pinacol ester in the presence of a catalyst for 3-5 hours.

[0013] Furthermore, the large-scale dialysis used in the purification step is performed using a dialysis device with a molecular weight cut-off of 50 kDa.

[0014] Compared with the prior art, the dual-stage targeted polymer prodrug and its preparation method provided in this application have the following beneficial effects:

[0015] First, the drug in this application, through the efficient synergistic action of various functional modules, constructs a precise, efficient, and safe drug delivery system, achieving dual-level organ and cell targeting, tumor microenvironment-responsive drug release, and visual monitoring of the treatment process. Compared with single-function drugs, this application demonstrates targeting efficiency and tumor suppression effects through the synergistic action of all functional modules.

[0016] 2. This application uses a primary targeting ligand, namely chemically modified galactosamine, to target liver tissue. The primary targeting function is the basis for achieving all subsequent targeting functions. After successfully enriching in the liver through primary targeting, the GPC3 peptide can accurately recognize and bind to the GPC3 antigen on the surface of liver cancer cells, achieving precise targeting at the cellular level. The secondary targeting function is combined with sufficient drug payload to achieve efficient killing of cancer cells.

[0017] 3. The present application contains a polyhistidine chain, which is used to shield the secondary targeting module under physiological conditions, effectively avoiding premature exposure of the secondary targeting module, thereby preventing the drug from being cleared by the immune system or non-specifically binding to plasma proteins. In addition, the drug release mechanism of the present application is a dual-responsive release, that is, the drug release is synergistically controlled by the active oxygen-sensitive bond and the MMP-2 cleavage site. This release mechanism can accurately identify the dual characteristics of high expression of MMP-2 and high ROS levels in tumor tissue, thereby achieving accurate and thorough release of the drug in the core area of ​​the tumor.

[0018] 4. The drug skeleton and core cross-linking structure of the present application jointly ensure the structural stability of the nanomicelles during the delivery process. The PEG segments in the skeleton can prolong the circulation time of the drug in the blood, and gain a sufficient time window for multi-level targeting. At the same time, the present application contains the near-infrared fluorescent probe Cy7.5, which realizes the visualization of the drug delivery process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the molecular structure of the dual-stage targeted polymer prodrug of the present invention;

[0020] Figure 2 This is a diagram of the dual-stage targeting and intelligent shielding mechanism of the present invention. DETAILED DESCRIPTION

[0021] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Example 1

[0023] See Figures 1 to 2This embodiment provides a dual-stage targeted polymer prodrug, which is a core-shell structured nanomicelle formed by self-assembly, and the drug comprises: an amphiphilic block copolymer backbone as the basic structure, specifically poly(carboxybetaine methacrylate)-b-poly(ethylene glycol) (PCBMA-b-PEG); a primary targeting ligand for targeting liver tissue connected to the backbone, specifically chemically modified galactosamine; a secondary targeting and dual-responsive drug release module connected to the backbone, which comprises a GPC3 targeting peptide, a chemotherapy drug doxorubicin, an active oxygen sensitive bond, and an MMP-2 cleavage site; a pH-responsive shielding module connected to the backbone for shielding the secondary targeting module under physiological conditions, specifically a polyhistidine chain; an integrated diagnosis and treatment module for imaging and tracing connected to the backbone, specifically a near-infrared fluorescent probe Cy7.5; and a core crosslinker for enhancing structural stability, specifically a disulfide bond crosslinker.

[0024] The preparation method of the dual-stage targeted polymer prodrug described in this embodiment includes: raw material preparation: preparing an amphiphilic block copolymer skeleton, a primary targeting ligand, a secondary targeting and dual-responsive drug release module, a pH-responsive shielding module, an integrated diagnosis and treatment module, and a core cross-linking agent; wherein, when preparing the drug intermediate in the secondary targeting and dual-responsive drug release module, doxorubicin and 4-formylphenylboronic acid pinacol ester are refluxed and heated for 4 hours under anhydrous and oxygen-free conditions in the presence of a catalyst; coupling reaction: dissolving the skeleton in a DMSO / water mixed solvent, and adding the secondary targeting and dual-responsive drug release module, the pH-responsive shielding module, and the integrated diagnosis and treatment module; The coupling reaction is carried out by a programmed temperature control method of gradient temperature increase under the protection of inert gas; the gradient temperature increase is specifically as follows: first react at 35° C. for 12 hours, then heat to 45° C. for 12 hours; self-assembly: add the activated primary targeting ligand to the product after the coupling reaction, adjust the pH value of the solution to 7.4, and spontaneously assemble the polymer chains into nano-micelles with a core-shell structure by constant stirring, and let it stand for degassing; cross-linking and purification: add a core cross-linking agent to the formed micelle solution for core cross-linking, and perform large-scale dialysis and ultrafiltration purification through a dialysis device with a molecular weight cutoff of 50 kDa, and obtain the prodrug by freeze-drying.

[0025] In this embodiment, the ratio (relative to the skeleton) and parameter settings of each functional module are as follows: the ratio of the L-GalN primary targeting ligand is 15%; the ratio of the GPC3-PDR secondary targeting module is 5%; and the length of the P(His) shielding chain is 50 monomers. The dual-stage targeted polymer prodrug of this embodiment exhibits efficient synergy under the condition that all key modules and parameters are in optimized configuration. It shows good structural stability and environmental responsiveness in in vitro models. In subsequent pharmacodynamic tests, its liver tumor targeting enrichment coefficient reached 14.8, the tumor signal-to-noise ratio was 8.5, and the tumor volume inhibition rate (TGI) was as high as 92.5%. This shows that the drug of this embodiment can accurately enrich in the tumor site and effectively inhibit tumor growth. At the same time, the integrated diagnosis and treatment module realizes effective monitoring of the enrichment process.

[0026] Example 2

[0027] Preferably, the dual-stage targeted polymer prodrug and its preparation method provided in this embodiment are substantially the same as those in Example 1. The differences are that the ratios of the functional modules are set as follows: the ratio of the L-GalN primary targeting ligand is adjusted to 5%; the ratio of the GPC3-PDR secondary targeting module is 5%; and the length of the P(His) shielding chain is 50 monomers. During the preparation process, the gradient temperature step is specifically as follows: first, react at 35°C for 10 hours, then increase the temperature to 45°C for 10 hours.

[0028] The dual-stage targeted polymer prodrug of this embodiment has a weakened initial anchoring ability in liver tissue due to the reduced proportion of the primary targeting ligand. Although the drug structure and response mechanism remain intact, the absolute amount entering the liver tissue is reduced, thereby affecting the subsequent secondary targeting efficiency to cancer cells. The results of the efficacy test showed that the liver tumor targeting enrichment factor was reduced to 4.1 and the TGI was 48.7%. This result shows that the sufficient ratio of the primary targeting ligand is the basis for achieving efficient organ-level enrichment.

[0029] Example 3

[0030] Preferably, a further embodiment is provided. The dual-stage targeted polymer prodrug and its preparation method provided in this embodiment are basically the same as those in Example 1. The difference is that the ratio of each functional module is set as follows: the ratio of the L-GalN primary targeting ligand is 15%; the ratio of the GPC3-PDR secondary targeting module is adjusted to 1%; the length of the P(His) shielding chain is 50 monomers. Although its dual-stage targeted polymer prodrug can be effectively enriched in liver tissue, the drug loading is reduced due to the insufficient ratio of the secondary targeting and drug release modules. The number of drug molecules that each nanomicelle can deliver to cancer cells is limited, thereby limiting the ultimate therapeutic effect. Further, pharmacodynamic tests show that its liver tumor targeting enrichment coefficient remains at a high level of 14.5, but the TGI is only 61.2%. This shows that after achieving effective targeting, ensuring sufficient drug load is also the key to achieving efficient killing.

[0031] Example 4

[0032] Preferably, the dual-stage targeted polymer prodrug and its preparation method provided in this embodiment are basically the same as those in Example 1, except that the length of the P(His) shielding chain is shortened to 20 monomers. During the preparation process, the gradient temperature increase step is specifically: first react at 35°C for 14 hours, then heat to 45°C for 14 hours. The dual-stage targeted polymer prodrug of this embodiment, due to its insufficient chain length of pH-responsive shielding module, cannot form a dense shielding layer under the physiological pH conditions of blood circulation. This causes the secondary targeting module to be exposed prematurely, easily recognized and cleared by the immune system or non-specifically bound to plasma proteins, resulting in a large amount of drug loss before reaching the tumor site. The results of the pharmacodynamic test confirmed this. The liver tumor targeting enrichment factor dropped to 9.7 and the TGI was 75.4%. This result highlights that a smart shielding chain of sufficient length is crucial to ensure the stability of the drug during circulation.

[0033] Example 5

[0034] Preferably, the dual-stage targeted polymer prodrug and its preparation method provided in this embodiment are basically the same as those in Example 1, except that the amphiphilic block copolymer backbone does not contain a PEG segment, but only a PCBMA backbone. The dual-stage targeted polymer prodrug of this embodiment lacks a PEG segment as a stabilizing synergist. Although PCBMA itself has excellent anti-protein contamination ability, the synergistic effect of PEG can further prolong the drug's circulation time in the blood. The lack of PEG leads to a shortened drug half-life, and the time window for subsequent multi-stage targeting behavior is insufficient. The results of the efficacy test showed that the liver tumor targeting enrichment coefficient was 10.2, lower than 14.8 in Example 1, and the TGI was 83.1%, which reflects that the PEG segment has a positive effect on optimizing pharmacokinetic behavior and improving overall targeting efficiency.

[0035] Example 6

[0036] The dual-stage targeted polymer prodrug and its preparation method provided in this example are essentially the same as those in Example 1, except that the disulfide crosslinker DTSP is not added during the preparation process, meaning that the core is not crosslinked. The self-assembled nanomicelle structure of the dual-stage targeted polymer prodrug in this example is maintained solely by intermolecular forces, lacking the reinforcement of covalent crosslinks. In the complex blood circulation environment, the micelle structure is unstable due to shear forces and other factors, making it prone to premature disintegration and leakage of the encapsulated drug. Consequently, the drug dose required to reach the tumor intact is significantly reduced. Efficacy testing results showed that the liver tumor targeting enrichment factor decreased to 8.9, and the TGI decreased accordingly to 72.8%, demonstrating that core crosslinking is essential for maintaining the structural integrity of the prodrug during delivery.

[0037] Example 7

[0038] The dual-stage targeted polymer prodrug and its preparation method provided in this embodiment are basically the same as those in Example 1. The difference is that in the secondary targeting and dual-responsive drug release module, doxorubicin is connected by a stable chemical bond, does not contain reactive oxygen species (ROS) sensitive bonds, and only retains the MMP-2 cleavage site as a single drug release trigger mechanism. The dual-stage targeted polymer prodrug of this embodiment has a drug release mechanism that changes from double insurance release to a single enzyme response release. Although the drug can be released in tumor areas with high MMP-2 expression, it lacks a response to the key feature of high ROS levels in tumor cells. This single mechanism makes the drug killing less thorough, and some cancer cells that do not fully meet the conditions may escape. The results of the efficacy test show that its TGI is 81.5%, which is effective but lower than 92.5% in Example 1, proving the superiority of the dual TME response mechanism in improving the thoroughness of tumor killing.

[0039] Example 8

[0040] The dual-stage targeted polymer prodrug and its preparation method provided in this embodiment are basically the same as those in Example 1. The difference is that the near-infrared fluorescent probe Cy7.5 is not added during the preparation process, and it is a pure therapeutic drug. The dual-stage targeted polymer prodrug of this embodiment has removed the integrated diagnosis and treatment module in terms of function. From the perspective of therapeutic effect, its various mechanisms are complete, showing almost the same tumor inhibition effect as Example 1, with a TGI of 92.3%. However, due to the lack of the Cy7.5 probe, it is impossible to track the distribution and enrichment of the drug in the body in real time through in vivo fluorescence imaging technology. Its T / N Ratio is only 1.1, and the signal-to-noise ratio is extremely low, making the evaluation of whether the drug is successfully targeted invisible, and unable to provide a visual basis for efficacy evaluation and individualized medication adjustment.

[0041] Comparative Example 1

[0042] The preparation method and formulation of this comparative example are essentially the same as those of Example 1, with the primary targeting ligand (L-GalN) being replaced with a functionally inert PEG-linked graft, resulting in the absence of primary targeting. Due to the lack of this primary targeting ligand, the prodrug in this comparative example loses its ability to initially concentrate in liver tissue. Most of the drug is rapidly cleared or randomly distributed in the systemic circulation, failing to reach the lesions. Efficacy testing revealed a liver tumor-targeting enrichment factor of only 1.8, and a TGI as low as 31.5%. This result demonstrates that primary targeting is fundamental to achieving all subsequent targeting functions, and its absence renders the treatment essentially ineffective.

[0043] Comparative Example 2

[0044] The preparation method and formulation of this comparative example are basically the same as those of Example 1, with the main difference being that the GPC3 targeting peptide in the secondary targeting module is replaced by a scrambled peptide with a disordered amino acid sequence, i.e., there is no secondary targeting function. Although the prodrug of this comparative example can be enriched in the liver through primary targeting, it cannot accurately recognize and bind to the GPC3 antigen on the surface of liver cancer cells. The drug cannot be efficiently internalized, resulting in low dual-response release efficiency in the tumor microenvironment. The results of the efficacy test showed that its TGI was only 53.8%, far lower than that of Example 1. This result shows that secondary targeting is the key to achieving efficient drug delivery and release at the cellular level.

[0045] Comparative Example 3

[0046] The preparation method and formulation of this comparative example are basically the same as those of Example 1, with the main difference being that the P(His) pH-responsive shielding module is removed. Due to the lack of an intelligent shielding system, the prodrug of this comparative example, its GPC3 targeting module, is continuously exposed in the blood circulation, is prone to nonspecific binding with plasma proteins, and may trigger clearance by the immune system. This results in a large amount of drug loss before reaching the tumor site. The results of the efficacy test showed that the liver tumor targeting enrichment coefficient dropped to 9.2 and the TGI was 70.1%. This result shows that the pH-responsive shielding module is effective in preventing premature drug exposure and ensuring ultimate targeting efficiency.

[0047] Comparative Example 4

[0048] The preparation method and formulation of this comparative example are essentially the same as those of Example 1, with the primary difference being that the drugs in the secondary targeting and dual-responsive drug release module are connected via a stable chemical bond insensitive to ROS, meaning they only respond to MMP-2 enzymatic cleavage and lack dual-responsiveness. While the prodrug in this comparative example is capable of targeting and partial release, it relies solely on MMP-2 cleavage and cannot distinguish between inflammatory tissue with high MMP-2 expression and true hypoxic, high-ROS cancerous foci. This results inadequate release precision, impacting the ultimate efficacy. Efficacy testing revealed a TGI of 78.9%, lower than that of Example 1. This result demonstrates that a dual-responsive mechanism is essential for achieving precise and thorough drug release.

[0049] Comparative Example 5

[0050] The preparation method and formulation of this comparative example are essentially the same as those of Example 1, with the primary difference being that the final preparation step, DTSP core crosslinking, is omitted, resulting in insufficient stability under the complex shear forces of blood, leading to premature structural disintegration and drug leakage, significantly reducing the amount of drug that reaches the tumor intact. Efficacy testing revealed a liver tumor targeting enrichment factor of 8.1 and a TGI of 65.7%. These results demonstrate that core crosslinking is essential for ensuring structural stability during drug delivery.

[0051] Comparative Example 6

[0052] The preparation method and formulation of this comparative example are essentially the same as those of Example 1, with the main difference being that the Cy7.5 probe is removed, i.e., it lacks diagnostic and therapeutic functionality. The prodrug therapeutic effect of this comparative example is almost identical to that of Example 1 (TGI of 92.2%), but its fluorescence signal-to-noise ratio is close to the background value (T / N Ratio ~1.0). This result demonstrates that, while it does not affect the treatment itself, the Cy7.5 probe is central to visualizing the targeting process; its absence prevents real-time, noninvasive assessment of efficacy, thus losing the advantage of integrated diagnosis and treatment.

[0053] In the above examples, some of the key commercially available raw materials used are: doxorubicin hydrochloride, 4-formylphenylboronic acid pinacol ester, and disulfide bond crosslinker were all purchased from Sigma-Aldrich; the near-infrared fluorescent probe Cy7.5-N3 was purchased from Lumiprobe; all Fmoc-protected amino acids and coupling agents used for solid-phase synthesis of peptides were purchased from Shanghai Jier Biochemical Co., Ltd.; and other chemical reagents such as dimethyl sulfoxide were of analytical grade or higher purity and purchased from Sinopharm Chemical Reagent Co., Ltd. or commercial suppliers of equivalent quality.

[0054] In order to more intuitively compare the effects of the embodiments and comparative examples, their key indicators are summarized in the following Table 1:

[0055] Table 1 Drug efficacy test results (average value)

[0056]

[0057] Furthermore, in order to evaluate the biosafety of the prodrug of the present invention, the prepared pharmaceutical composition was subjected to in vivo safety testing and efficacy testing;

[0058] Experimental Animals and Grouping: Healthy adult female BALB / c nude mice, 4-6 weeks old, were randomly divided into groups corresponding to Examples 1-8, Comparative Examples 1-6, and a blank control group, each receiving normal saline by gavage. Dosing and Observation: Each experimental group received the corresponding prodrug formulation via tail vein injection at a dose calculated as an adriamycin equivalent of 10 mg / kg, once every three days for 15 consecutive days. During this period, the mice's body weight, food and water intake, behavioral activity, and general health signs were observed and recorded daily. After the experiment, all mice were sacrificed. Major organs, including the heart, liver, spleen, lungs, and kidneys, were fixed with 4% paraformaldehyde solution, embedded in paraffin, and sectioned. Hematoxylin-eosin (H&E) staining was performed, and histopathological changes in each organ were observed under a light microscope.

[0059] At the effective therapeutic doses in each example, the weight of the mice remained stable without significant weight loss. Their general physical signs, such as behavior, appearance, and secretions, were normal, with no significant differences compared to the blank control group. H&E staining of major organs showed no obvious pathological damage, inflammation, or cell necrosis in major organs such as the heart, liver, spleen, lungs, and kidneys. Importantly, unlike the severe cardiac toxicity that may result from the use of free doxorubicin, the prodrug of Example 1 of the present invention did not induce any observable signs of myocardial damage. These results demonstrate that the dual-stage targeted polymer prodrug of the present invention has excellent biosafety.

[0060] To verify the in vivo antitumor effect of the prodrug of the present invention, a human liver cancer xenograft mouse model was established for pharmacodynamic evaluation;

[0061] GPC3 high-expressing human liver cancer cells in the logarithmic growth phase were obtained and prepared into single cell suspensions. 5×10 6 cells. When the subcutaneous tumor grows to about 100-150mm 3 At the same time, tumor-bearing mice were randomly divided into groups of n=6, corresponding to each embodiment, comparative example and negative control group, and administered with normal saline and tested for evaluation indicators. Each group of mice was injected with the corresponding prodrug preparation or normal saline through the tail vein. The administration scheme was the same as that of the safety test.

[0062] During the treatment period, the longest diameter a and the shortest diameter b of the tumor were measured every 3 days using a vernier caliper and the diameters were calculated according to the formula V = (a × b2 ) / 2 to calculate the tumor volume. At the end of the experiment, the tumor volume inhibition rate was calculated based on the final tumor volume of each group.

[0063] For each group containing the near-infrared probe Cy7.5, 24 hours after the first administration, the fluorescence signal in the mice was captured using a small animal in vivo fluorescence imaging system, and the tumor signal-to-noise ratio was calculated by comparing the average fluorescence intensity of the tumor area with that of normal non-tumor tissues such as the back muscle.

[0064] At the end of the experiment, mice were sacrificed, and the tumors and major organs (heart, liver, spleen, lungs, kidneys, etc.) were completely removed. The doxorubicin (or Cy7.5) concentration in the tissue homogenate was measured, and the ratio of the drug content per unit mass of tumor tissue to the drug content per unit mass of muscle tissue was calculated, which is the target enrichment factor.

[0065] Based on the above safety and pharmacodynamic tests, the key indicators of each embodiment and comparative example, the average values ​​of liver tumor targeting enrichment coefficient, tumor signal-to-noise ratio, and tumor volume inhibition rate are summarized in Table 1.

[0066] From the results in Table 1 we can see that:

[0067] Example 1 exhibits the best comprehensive performance under the synergistic effect of all functional modules in an optimized ratio: its liver tumor targeting enrichment coefficient is as high as 14.8, the tumor signal-to-noise ratio reaches 8.5, and a tumor volume inhibition rate (TGI) of 92.5% is achieved. At the same time, safety experiments have shown that it has no obvious toxic side effects, which fully proves that the synergistic effect of each module is the basis for achieving precise and efficient treatment.

[0068] Comparison of Examples 2-4 with Example 1 shows that the ratio of the primary targeting ligand (L-GalN), the ratio of the secondary targeting module, and the length of the pH-responsive shielding chain have a significant effect on the final efficacy of the drug. Insufficient ratio of the primary targeting ligand (Example 2) or too short a shielding chain (Example 4) will lead to increased loss of the drug before reaching the tumor site, significantly reducing the targeting efficiency and therapeutic effect. Insufficient ratio of the secondary targeting module (Example 3) will not deliver a sufficient dose of the drug after effective targeting, limiting the killing effect. This proves that the optimal balance of the parameters of each component is crucial to achieving optimal drug efficacy.

[0069] The comparison of Comparative Examples 1-6 with Example 1 clearly shows that the removal of primary targeting (Comparative Example 1) or secondary targeting (Comparative Example 2) significantly reduces the therapeutic effect or even tends to be ineffective, proving the necessity of a dual-stage targeted relay delivery mode; removing the pH-responsive shielding module (Comparative Example 3) or the core cross-linking (Comparative Example 5, Example 6) impairs the circulatory stability of the drug, resulting in premature exposure or disintegration, and inability to efficiently accumulate at the tumor site; removing the ROS response in the dual TME response (Comparative Example 4, Example 7), the accuracy and thoroughness of drug release are insufficient, resulting in the escape of some cancer cells and poor therapeutic effect. Although the integrated diagnosis and treatment module (Comparative Example 6, Example 8) has little effect on the therapeutic effect, the tumor signal-to-noise ratio is reduced to 1.1, making the targeted delivery process of the drug impossible to observe and evaluate, and losing the potential to guide individualized medication and efficacy prediction.

[0070] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A dual-stage targeted polymer prodrug, characterized in that: The prodrug is a core-shell structure nano-micelle formed by self-assembly, comprising: an amphiphilic block copolymer skeleton as the basic structure for forming the nano-micelle; a primary targeting ligand connected to the skeleton for targeting the prodrug to liver tissue; a secondary targeting and dual-responsive drug release module connected to the skeleton for targeting liver cancer cells and responsively releasing drugs; and a pH-responsive shielding module connected to the skeleton for shielding the secondary targeting module under physiological conditions. a diagnostic and therapeutic module connected to the skeleton, used for imaging and tracing the prodrug; and a core cross-linking agent for cross-linking the core of the nanomicelle to enhance its structural stability.

2. The prodrug according to claim 1, characterized in that The amphiphilic block copolymer backbone is poly(carboxybetaine methacrylate)-b-poly(ethylene glycol).

3. The prodrug according to claim 1, characterized in that The secondary targeting ligand is a chemically modified galactosamine, which achieves secondary targeting to liver tissue by binding to the asialoglycoprotein receptor on the surface of liver cells.

4. The prodrug according to claim 1, characterized in that The structure of the secondary targeting and dual-responsive drug release module includes: a GPC3 targeting peptide for specifically recognizing and binding to the GPC3 antigen on the surface of liver cancer cells; the chemotherapy drug doxorubicin; an active oxygen-sensitive bond connecting the doxorubicin to the GPC3 targeting peptide; and an MMP-2 cleavage site embedded in the GPC3 targeting peptide sequence.

5. The prodrug according to claim 1, characterized in that The pH-responsive shielding module is a polyhistidine chain that is folded under the physiological pH conditions of blood circulation to shield the secondary targeting module, and stretches in the acidic microenvironment of tumor tissue to release the shielding.

6. The prodrug according to claim 1, characterized in that The diagnostic and therapeutic integrated module is a near-infrared fluorescent probe Cy7.5, and the core cross-linking agent is a disulfide bond cross-linking agent.

7. A method for preparing the dual-stage targeted polymer prodrug of claim 1, characterized in that: The method comprises: preparing required raw materials, including an amphiphilic block copolymer skeleton, a primary targeting ligand, a secondary targeting and dual-responsive drug release module, a pH-responsive shielding module, a diagnosis and treatment integrated module and a core cross-linking agent, wherein when preparing the secondary targeting and dual-responsive drug release module, a drug intermediate containing an active oxygen-sensitive bond is synthesized by reflux heating under anhydrous and oxygen-free conditions; subsequently, dissolving the skeleton in a mixed solvent, and adding the secondary targeting and dual-responsive drug release module, the pH-responsive shielding module and the diagnosis and treatment integrated module, and performing a coupling reaction by a program temperature control method of gradient heating under the protection of an inert gas; then, adding the activated primary targeting ligand to the product after the coupling reaction, adjusting the pH value of the solution to 7.0-7.5, and allowing the polymer chains to spontaneously assemble into nano-micelles with a core-shell structure by constant-speed stirring, and then standing for degassing; adding the core cross-linking agent to the formed micelle solution for core cross-linking, and purifying by large-scale dialysis and ultrafiltration, and obtaining the prodrug by freeze-drying.

8. The preparation method according to claim 7, characterized in that The gradient temperature increase used in the coupling reaction is specifically as follows: first react at 35° C. for 10-14 hours, then heat to 45° C. for 10-14 hours.

9. The preparation method according to claim 7, characterized in that The preparation of the pharmaceutical intermediate is completed by reflux heating doxorubicin and 4-formylphenylboronic acid pinacol ester under the action of a catalyst for 3-5 hours.

10. The preparation method according to claim 7, characterized in that The large-scale dialysis employed in the purification step was performed using a dialysis device with a molecular weight cut-off of 50 kDa.

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