Anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability and applications thereof

Abstract

This invention relates to the field of biomedicine and discloses a composition for treating non-small cell lung cancer metastasis that targets replication stress vulnerability and its application. The composition includes a dual-responsive polymer prodrug, which consists of a block copolymer backbone, a matrix metalloproteinase cleavage peptide sequence coupled to the hydrophilic end, and an active molecular group coupled to the hydrophobic end based on an asymmetric grafting structure. The active molecular group comprises a replication stress inducer and an ATR kinase inhibitor. This invention maintains the amorphous phase of the micelle core through the asymmetric grafting structure, eliminating the permeation barrier caused by spontaneous crystallization of components. This amorphous core provides a proton permeation channel, ensuring the synchronous in-situ release of multiple target drugs, thereby blocking the DNA replication fork repair pathway and inducing mitotic catastrophe, maintaining the spatiotemporal overlap of multiple drug interventions in the pathological target area, and avoiding the risk of drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an anti-non-small cell lung cancer metastasis composition that targets replication stress vulnerability and its application. Background Technology

[0002] Currently, metastatic non-small cell lung cancer cells exhibit high levels of replication stress. They maintain genomic stability and evade apoptosis by upregulating ATR kinase-mediated response pathways, inducing replication stress, and blocking repair pathways to trigger mitotic catastrophe. This constitutes the basic pathway for intervention in metastatic clones in the field of biopharmaceutical manufacturing. Conventional drug administration methods often involve the physical mixing of active molecules. The distribution volume and metabolic cycle of active molecules after entering the physiological environment differ significantly, resulting in a spatiotemporal misalignment of peak blood drug concentrations for different target drugs in the lesion tissue. This misalignment prevents the induction of damage and the blocking of repair from coinciding on a molecular timescale, not only negating the synergistic effect but also providing tumor cells with an opportunity to initiate compensatory repair, thus inducing systemic drug resistance.

[0003] To ensure consistent distribution, polymer prodrug technology is employed. Industry research focuses on the stability of the carrier shell layer in response to the physiological environment, neglecting the decisive influence of the phase state of the micelle core surface on drug release kinetics. For example, Chinese invention patent application CN111956609A discloses an immune checkpoint blocking peptide prodrug nanomicelle, its preparation method, and its application. It utilizes matrix metalloproteinase-sensitive linker peptides to achieve shell layer removal and drug release. Under actual conditions, due to the strong aromatic ring stacking and intermolecular attraction between hydrophobic active molecules, the hydrophobic core of the micelle is prone to spontaneous local crystallization phase transition. The dense microcrystalline domain forms a physical barrier to proton permeation, blocking the approach of hydrated protons to acid-sensitive responsive bonds, resulting in release kinetic lag and non-uniformity, causing the preset synchronous release mechanism to fail at the surface physical level.

[0004] Therefore, the technical problem to be solved by this invention is how to suppress the crystallization of the core phase by regulating the spatial topological arrangement between components on the polymer backbone and ensuring the in-situ synchronous release of multi-target components in the lesion microenvironment. Summary of the Invention

[0005] This invention provides an anti-non-small cell lung cancer metastasis composition that targets replication stress vulnerability, comprising: A dual-responsive polymer prodrug, comprising a block copolymer backbone, a matrix metalloproteinase cleavage peptide sequence coupled to the hydrophilic end of the block copolymer backbone, and an active molecular group covalently grafted to the hydrophobic end of the block copolymer backbone; The active molecular group includes a replication stress inducer and an ATR kinase inhibitor; wherein, the active molecular group presents an asymmetric grafting structure at the hydrophobic end of the block copolymer backbone, so that the nanomicelles formed by the self-assembly of the dual-responsive polymer prodrug in the aqueous medium have an amorphous phase core. The matrix metalloproteinase cleavage peptide sequence and the hydrophilic end of the block copolymer backbone constitute the hydrophilic shielding segment of the nanomicelle. The amorphous core eliminates the spontaneously formed crystal permeation barrier of the active molecular group, causing the composition to undergo the following response process when it enters a tissue microenvironment characterized by weak acidity and matrix metalloproteinase overexpression: Step S11, the matrix metalloproteinase cleavage peptide sequence is broken in response to the matrix metalloproteinase in the tissue microenvironment, stripping off the hydrophilic shielding segment and exposing the amorphous core; Step S12, hydrated protons in the tissue microenvironment permeate into the core through the physical channel provided by the amorphous core and undergo chemical kinetic collisions with the acid-sensitive chemical bonds coupled to the active molecular group; Step S13, the replication stress inducer and the ATR kinase inhibitor are released in situ based on the breaking of the acid-sensitive chemical bonds, inducing replication stress collapse of the biological target.

[0006] Preferably, the active molecular group is grafted onto the hydrophobic end of the block copolymer backbone via a linker with a differentiated chain length. This differentiated chain length forms a steric hindrance gradient distribution at the hydrophobic end of the block copolymer backbone, inhibiting the recrystallization of the hydrophobic monomers in the active molecular group. The replication stress inducer includes gemcitabine derivatives or camptothecin derivatives. The ATR kinase inhibitor includes AZD6738 derivatives or VE-821 derivatives. The acid-sensitive chemical bond is selected from hydrazone bonds, acetal bonds, or orthoester bonds. After the composition enters the tissue microenvironment, the synchronous release time difference between the replication stress inducer and the ATR kinase inhibitor is less than 15 minutes.

[0007] Preferably, the matrix metalloproteinase cleavage peptide sequence is selected from the PVGLIG sequence, GPLGIAGQ sequence, or VPMSMRGG sequence; the linkage strength between the acid-sensitive chemical bond and the replication stress inducer is matched with the linkage strength between the acid-sensitive chemical bond and the ATR kinase inhibitor.

[0008] Preferably, the self-assembly logic of the dual-responsive polymer prodrug is determined by the following steps: Step S41: In the aqueous medium, the hydrophobic ends of the block copolymer backbone contract by hydrophobic forces to form the amorphous core; Step S42: The active molecular group utilizes the steric hindrance generated by the asymmetric grafting structure to block the planar stacking of aromatic rings between hydrophobic molecules; Step S43: The amorphous core is kept in a free state, providing a three-dimensional diffusion channel for the hydrated protons in the amorphous core.

[0009] Preferably, the main chain of the block copolymer is composed of hydrophilic segments and hydrophobic segments, wherein the hydrophilic segments include polyethylene glycol or polylactic acid-polyglycolic acid copolymers, and the hydrophobic segments include polyaspartic acid derivatives or polyglutamic acid derivatives; the replication stress inducer has a drug loading of 5% to 15% in the amorphous core, and the ATR kinase inhibitor has a drug loading of 8% to 20% in the amorphous core.

[0010] Preferably, the surface of the nanomicelles is modified with a targeting ligand, including integrin receptor-targeting peptide RGD or transferrin, to increase the enrichment ratio of the composition in the tissue microenvironment; the average particle size of the nanomicelles is 30 nm to 80 nm.

[0011] Preferably, the composition constructs a core-shell steric hindrance through the hydrophilic shielding segment in blood circulation, shielding the acid-sensitive chemical bond from attack by non-specific proteases; in the tissue microenvironment with a pH of 5.0 to 6.5, the breaking rate of the acid-sensitive chemical bond is increased by more than 10 times compared to the pH 7.4 environment.

[0012] Preferably, the molar ratio of the replication stress inducer and the ATR kinase inhibitor in the amorphous phase of the core is 1:3 to 3:1; the composition blocks the DNA replication fork repair pathway of the biological target by simultaneously releasing the active molecular group.

[0013] Preferably, the composition is used as a biopharmaceutical formulation to prepare a drug that blocks the clonal evolution of metastatic lesions in non-small cell lung cancer; the composition utilizes the amorphous phase core to maintain the diffusion rate of the hydrated proton in the core above a preset threshold, ensuring that the release kinetics of the active molecular group is dominated by the breaking frequency of the acid-sensitive chemical bond.

[0014] Application of an anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability; an anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability is used to prepare a drug that blocks the clonal evolution of metastatic lesions in non-small cell lung cancer.

[0015] Compared to existing technologies, the anti-non-small cell lung cancer metastasis composition of the present invention, which targets replication stress vulnerability, has the following advantages: 1. In the anti-non-small cell lung cancer metastasis composition, by defining a specific molar ratio of replication stress inducer molecules to ATR kinase inhibitor molecules on the polyamino acid backbone, a specific steric hindrance shielding field is generated by utilizing the larger spatial volume of the ATR kinase inhibitor molecules. This physically blocks the spontaneous crystallization tendency between adjacent replication stress inducer molecules due to aromatic ring stacking, and forces the hydrophobic graft segments to maintain an amorphous free phase in the assembled nanomicelle core. This avoids the water molecule permeation barrier formed by local crystallization of drug molecules in conventional co-loaded systems, thus providing a physical basis for the uniform response of subsequent acid-sensitive chemical bonds.

[0016] 2. Because the nanomicelle core is in a stable amorphous phase, hydrated protons in the environment can synchronously and indiscriminately penetrate to various spatial sites in the hydrophobic grafting region, so that the acid-sensitive chemical bonds of the coupled different active molecules are subjected to equal proton attack probabilities. Thus, based on the preset chemical kinetic equation, the two active components are released in situ synchronously, eliminating the release rate distortion caused by the difference in physical phase, solving the problem of the time misalignment of the peak concentration of active components in the lesion tissue, and ensuring a high degree of overlap in the spatiotemporal distribution of multi-target intervention.

[0017] 3. The composition forms a stable core-shell steric hindrance in the bloodstream through the cooperation of multiple responsive linkages and hydrophilic shielding segments, inhibiting the degradation of specific cleavage segments by non-specific proteases. After entering the microenvironment of non-small cell lung cancer metastatic lesions, the slightly acidic characteristics first trigger the disintegration of the hydrophilic shielding segments, and the matrix metalloproteinases highly expressed in the metastatic lesions precisely induce the disintegration of the micelle structure. This staged gating mechanism ensures that the drug efficacy molecules are released only in the target area, increases the enrichment ratio of active components in the lesion area, and reduces the toxic side effects on the normal hematopoietic system. Attached Figure Description

[0018] Figure 1 This is a diagram illustrating the structural construction of the nanomicelles of the present invention and their step-response release mechanism; Figure 2 This is a diagram showing the evolution and discrimination of the composition under different physiological environments and grafting ratios according to the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] It should be noted that all directional and positional terms used in this invention, such as: up, down, left, right, front, back, vertical, horizontal, inner, outer, top, bottom, transverse, longitudinal, center, etc., are only used to explain the relative positional relationship and connection between components in a specific state (as shown in the accompanying drawings). They are only for the convenience of describing this invention and do not require that this invention be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention. In addition, the descriptions of "first," "second," etc., in this invention are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0021] In the description of this invention, unless otherwise explicitly specified and limited, the terms installation, connection, and linking should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections; they can refer to direct connections or indirect connections through an intermediate medium; they can refer to the internal connection of two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0022] In the description of this specification, references to the terms "an embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0023] A composition for treating non-small cell lung cancer metastasis that targets replication stress vulnerability includes: A dual-responsive polymer prodrug, comprising a block copolymer backbone, a matrix metalloproteinase cleavage peptide sequence coupled to the hydrophilic end of the block copolymer backbone, and an active molecular group covalently grafted to the hydrophobic end of the block copolymer backbone; The active molecular group includes a replication stress inducer and an ATR kinase inhibitor; wherein, the active molecular group presents an asymmetric grafting structure at the hydrophobic end of the block copolymer backbone, so that the nanomicelles formed by the self-assembly of the dual-responsive polymer prodrug in the aqueous medium have an amorphous phase core. The matrix metalloproteinase cleavage peptide sequence and the hydrophilic end of the block copolymer backbone constitute the hydrophilic shielding segment of the nanomicelle. The amorphous core eliminates the spontaneously formed crystal permeation barrier of the active molecular group, causing the composition to undergo the following response process when it enters a tissue microenvironment characterized by weak acidity and matrix metalloproteinase overexpression: Step S11, the matrix metalloproteinase cleavage peptide sequence is broken in response to the matrix metalloproteinase in the tissue microenvironment, stripping off the hydrophilic shielding segment and exposing the amorphous core; Step S12, hydrated protons in the tissue microenvironment permeate into the core through the physical channel provided by the amorphous core and undergo chemical kinetic collisions with the acid-sensitive chemical bonds coupled to the active molecular group; Step S13, the replication stress inducer and the ATR kinase inhibitor are released in situ based on the breaking of the acid-sensitive chemical bonds, inducing replication stress collapse of the biological target.

[0024] Preferably, the active molecular group is grafted onto the hydrophobic end of the block copolymer backbone via a linker with a differentiated chain length. This differentiated chain length forms a steric hindrance gradient distribution at the hydrophobic end of the block copolymer backbone, inhibiting the recrystallization of the hydrophobic monomers in the active molecular group. The replication stress inducer includes gemcitabine derivatives or camptothecin derivatives. The ATR kinase inhibitor includes AZD6738 derivatives or VE-821 derivatives. The acid-sensitive chemical bond is selected from hydrazone bonds, acetal bonds, or orthoester bonds. After the composition enters the tissue microenvironment, the synchronous release time difference between the replication stress inducer and the ATR kinase inhibitor is less than 15 minutes.

[0025] Preferably, the matrix metalloproteinase cleavage peptide sequence is selected from the PVGLIG sequence, GPLGIAGQ sequence, or VPMSMRGG sequence; the linkage strength between the acid-sensitive chemical bond and the replication stress inducer is matched with the linkage strength between the acid-sensitive chemical bond and the ATR kinase inhibitor.

[0026] Preferably, the self-assembly logic of the dual-responsive polymer prodrug is determined by the following steps: Step S41: In the aqueous medium, the hydrophobic ends of the block copolymer backbone contract by hydrophobic forces to form the amorphous core; Step S42: The active molecular group utilizes the steric hindrance generated by the asymmetric grafting structure to block the planar stacking of aromatic rings between hydrophobic molecules; Step S43: The amorphous core is kept in a free state, providing a three-dimensional diffusion channel for the hydrated protons in the amorphous core.

[0027] Preferably, the main chain of the block copolymer is composed of hydrophilic segments and hydrophobic segments, wherein the hydrophilic segments include polyethylene glycol or polylactic acid-polyglycolic acid copolymers, and the hydrophobic segments include polyaspartic acid derivatives or polyglutamic acid derivatives; the replication stress inducer has a drug loading of 5% to 15% in the amorphous core, and the ATR kinase inhibitor has a drug loading of 8% to 20% in the amorphous core.

[0028] Preferably, the surface of the nanomicelles is modified with a targeting ligand, including integrin receptor-targeting peptide RGD or transferrin, to increase the enrichment ratio of the composition in the tissue microenvironment; the average particle size of the nanomicelles is 30 nm to 80 nm.

[0029] Preferably, the composition constructs a core-shell steric hindrance through the hydrophilic shielding segment in blood circulation, shielding the acid-sensitive chemical bond from attack by non-specific proteases; in the tissue microenvironment with a pH of 5.0 to 6.5, the breaking rate of the acid-sensitive chemical bond is increased by more than 10 times compared to the pH 7.4 environment.

[0030] Preferably, the molar ratio of the replication stress inducer and the ATR kinase inhibitor in the amorphous phase of the core is 1:3 to 3:1; the composition blocks the DNA replication fork repair pathway of the biological target by simultaneously releasing the active molecular group.

[0031] Preferably, the composition is used as a biopharmaceutical formulation to prepare a drug that blocks the clonal evolution of metastatic lesions in non-small cell lung cancer; the composition utilizes the amorphous phase core to maintain the diffusion rate of the hydrated proton in the core above a preset threshold, ensuring that the release kinetics of the active molecular group is dominated by the breaking frequency of the acid-sensitive chemical bond.

[0032] Application of an anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability; an anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability is used to prepare a drug that blocks the clonal evolution of metastatic lesions in non-small cell lung cancer.

[0033] Example 1: In the application scenario of intervening in non-small cell lung cancer metastases carrying replication stress burden and exhibiting micrometastatic characteristics, the metastatic cells carry oncogene mutations and are in a state of continuous replication stress. Conventional methods include using replication stress inducers and... When kinase inhibitors are administered via physical mixing, differences in apparent volume of distribution, plasma protein binding rate, and transmembrane rate among the active components after entering the physiological environment lead to a spatiotemporal misalignment of peak plasma concentrations of the two drugs in metastatic lesions. The replication stress inducer that arrives first triggers compensatory resistance in tumor cells, while the subsequent-arriving kinase inhibitor... Kinase inhibitors fail to match initial damage signals, leading to the failure of their co-lethal effect. Tumor cells utilize this time window to initiate repair mechanisms and develop systemic resistance. A composition targeting replication stress vulnerability in non-small cell lung cancer metastases comprises a dual-responsive polymer prodrug consisting of a block copolymer backbone, a matrix metalloproteinase cleavage peptide sequence coupled to a hydrophilic end, and a replication stress inducer grafted to a hydrophobic end. Kinase inhibitors constitute, replication stress inducers and Kinase inhibitors exhibit asymmetric grafting structures at the hydrophobic ends of the block copolymer backbone, with grafting molar ratios ranging from 1:2.5 to 1:3.2. Dual-responsive polymer prodrugs self-assemble in aqueous media to form core-shell structured nanomicelles within a grafting ratio range of 1:2.5 to 1:3.2. Kinase inhibitors utilize molecular volume to create a steric shielding effect, blocking the aromatic ring stacking between adjacent replication stress inducer molecules, thus maintaining the amorphous phase of the nanomicelle core.

[0034] The composition enters its In a blood circulation system with a pH of 7.4, nanomicelles, through a hydrophilic shielding segment, construct steric hindrance, inhibiting the non-specific degradation of matrix metalloproteinase (MMP) cleavage peptide sequences. Nanomicelles accumulate in the microenvironment of non-small cell lung cancer metastases, with a pH of 6.5 to 6.8. Environmentally responsive linkages break, stripping the hydrophilic shielding segment and exposing the MMP-cleaved peptide sequence to the MMP. The MMP catalyzes peptide sequence cleavage and triggers the disintegration of the nanomicelle structure. The hydrophobic graft segment, stripped of its outer shell, enters the lysosome via endocytosis. The lysosome has a pH of 5.0 to 5.5. Because the nanomicelle core maintains an amorphous phase and eliminates the osmotic barrier caused by crystallization, hydrated protons from the environment simultaneously permeate into the internal space of the hydrophobic graft segment. The acid-sensitive chemical bonds of the coupled active molecules experience an equal probability of proton collision, enabling the replication of stress inducers and... The in situ, proportional release of kinase inhibitors leads to synchronized peak concentrations of both types of active molecules within metastatic cells. This kinetic consistency blocks the buffer time of target cells in responding to DNA damage, locking the accumulation of damage signals and the blockage of repair pathways at the same time point, resulting in a synergistic enhancement index of metastatic tumor cell clones. Satisfy the following formula: ,in, For synergistic efficiency index, This refers to the dosage of the stress inducer in the composition at which a specific effect is produced. To replicate the same effect produced when the stress inducer is used alone, For the composition The dosage of kinase inhibitors at which a specific effect is produced. for The same dose of kinase inhibitors, when used alone, produces the same effect, and under the influence of the amorphous phase core, the composition is effective in the target region. When the value is less than 0.5, the composition achieves the elimination of non-small cell lung cancer metastatic cell clones through the synergistic lethal effect generated by this specific ratio window.

[0035] Example 2: The current experimental platform includes a resolution of 0.01 units. Temperature control accuracy is The dynamic light scattering instrument at 0.1℃ and the high-performance liquid chromatography (HPLC) instrument with a detection limit of 10 ng / mL were used. All data were obtained from the physical experimental platform. The stirring rate was set based on the balance between convective mass transfer efficiency and the mechanical strength of polymer micelles. When the dynamic viscosity of the reaction system was in the range of 1.0 mPa·s to 1.5 mPa·s, in order to ensure uniform distribution of the active pharmaceutical ingredient and prevent premature shear-induced core-shell structure stripping, the stirring rate was set at 200 rpm. Under this specific viscosity range and stirring rate, the local turbulent energy dissipation rate generated by the fluid in the reactor was controlled within a safe threshold. Although the overall fluid shear force itself cannot directly and physically tear the tiny micelle core at the nanoscale, an excessively high local velocity gradient will strip the dynamic hydration layer around the hydrophilic shielding section of the nanomicelles. The energy accumulation caused by high-frequency and intense intermolecular collisions is sufficient to... To overcome the hydrophobic interactions between polymer chains in the early stages of dynamic self-assembly, which lead to thermodynamic instability and exfoliation phase transition at the core-shell interface, the stirring rate was limited to maintain the hydrodynamic stability of the micelle mesostructure. The initial input data used a simulated bodily fluid environment containing 10% fetal bovine serum to introduce random interference from physiological electrolytes and protein adsorption. Multiple sample groups were set up, including the present invention sample group with a grafting molar ratio of 1:2.8, a lower limit control group with a grafting molar ratio of 1:2.0, an upper limit control group with a grafting molar ratio of 1:4.0, and a control group with a deletion feature (removal of matrix metalloproteinase cleavage peptide sequences). Transmission electron microscopy, by acquiring diffraction fingerprint data of the micelle core, showed that the present invention sample group exhibited diffuse ring characteristics, i.e., it was in an amorphous phase; while the 1:2.0 group showed obvious crystal plane reflection fringes, confirming the lower... The concentration of kinase inhibitors was insufficient to block the aromatic ring stacking interactions between replication stress inducer molecules.

[0036] exist In a release medium containing 20 nM matrix metalloproteinases and with a pH of 5.2, the cumulative release rate of the replication stress inducer in the sample group of this invention reached 85.3%. The cumulative release rate of the kinase inhibitor reached 86.7%, and the time difference between their simultaneous release was... The time was reduced to 1.4 min. Under this condition, the intermediate characteristic parameter, proton permeability coefficient, was measured by fluorescence quenching kinetics. for cm / s, confirming that the amorphous phase nucleus provides a low-resistance physical channel. When the grafting ratio is reduced to 1:2.0, drug molecules form a dense lattice, and the proton permeability coefficient... Descending to cm / s, resulting in release time lag Extended to 24.6 minutes, by changing the release medium. The value is used to establish the problem intensity gradient, simulating the acidity environment at different depths of tumor tissue. When the value decreases from 6.5 to 5.0, the synergistic effect index of the present invention sample group... The concentration decreased regularly from 0.48 to 0.35, showing a trend of acid-triggered synergistic enhancement. When the grafting ratio increased to 1:4.0, the micelle size increased from the initial 92.5 nm to 245.8 nm, accompanied by polymer precipitation. This indicates that steric overload disrupted the physical balance between hydrophobic interactions and hydrophilic repulsion. Experimental data confirmed that the grafting ratio range of 1:2.5 to 1:3.2 constitutes a working window that balances phase stability and kinetic synchronization. The final measured tumor cell inhibition rate was 42.6% higher than that of the physical mixing group. This composition achieves the elimination of non-small cell lung cancer metastatic cell clones through kinetic consistency.

[0037] In the application of cisplatin-resistant non-small cell lung cancer metastases, to verify the effect of different combinations of pharmacodynamic components on the maintenance of amorphous nuclei, the replication stress inducer was replaced with a camptothecin derivative, and... The kinase inhibitor was replaced with a VE-821 derivative, and the synthesis was completed under conditions of a grafting molar ratio of 1:3.1 and a water content of less than 0.01%. The activity of the two drugs was determined using high-performance liquid chromatography (HPLC). The value is 5.4, the release rate constant in the medium. ,in This is the first-order release rate constant, in units of... The camptothecin derivative was measured. It is 0.15 , Derivatives It is 0.14 The synergistic effect index of this component in a lysosomal simulated environment The value is 0.38, which confirms that after changing the type of active molecule, the steric hindrance gradient can still suppress the formation of hydrophobic monomers and maintain kinetic synchronicity.

[0038] Example 3: This example combines Figures 1 to 2 A description of a composition targeting replication stress vulnerability to non-small cell lung cancer metastasis and its application, such as... Figure 1As shown, the dual-responsive polymer prodrug consists of a block copolymer backbone that self-assembles into nanomicelles in an aqueous medium. The hydrophilic shielding segment is composed of a matrix metalloproteinase (MMP) cleavage peptide sequence coupled to the hydrophilic end and the block copolymer backbone. The amorphous core is composed of an asymmetric graft structure covalently grafted to the hydrophobic end. This structure contains a replication stress inducer, an ATR kinase inhibitor, and an acid-sensitive chemical bond. Upon entering the microenvironment, step S11, the MMP-responsive cleavage, is initiated, causing the MMP cleavage peptide sequence to break, thereby stripping away the hydrophilic shielding segment and exposing the amorphous core. The subsequent execution of step S12, namely physical channel penetration and kinetic collision, allows hydrated protons to penetrate through the physical channels provided by the amorphous phase core and undergo chemical kinetic collisions with the acid-sensitive chemical bonds of the coupled active molecular group. This ensures physical channel penetration while eliminating the crystallization penetration barrier, thereby triggering the release and executing step S13, namely acid-sensitive breakage and in-situ release. Through the breakage of acid-sensitive chemical bonds, the replication stress inducer and ATR kinase inhibitor are released in situ. This achieves biological targeting and evolution blocking through the synchronous in-situ action of the drugs, ultimately inducing the replication stress collapse of the biological target and blocking the clonal evolution of non-small cell lung cancer metastases.

[0039] like Figure 2As shown, the anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability utilizes a hydrophilic shielding segment to construct a core-shell steric hindrance in the bloodstream at pH 7.4 to shield against attacks from non-specific proteases on the acid-sensitive chemical bond. However, when the composition is in a weakly acidic tissue microenvironment with matrix metalloproteinase overexpression, the matrix metalloproteinase cleavage peptide sequence breaks, thereby stripping away the hydrophilic shielding segment and exposing the core. At this point, the grafting distribution state, i.e., the molar ratio of replication stress inducer and ATR kinase inhibitor at the hydrophobic end, exhibits different kinetic characteristics. In the lower limit control group with a grafting molar ratio of 1:2.0, a spontaneous local crystallization phase transition occurs, and a physical barrier for proton permeation is formed by dense microcrystalline domains, resulting in release kinetic lag and heterogeneity. Although the intrinsic diffusion rate of hydrated protons in conventional bulk media is extremely fast, far exceeding the normal breaking rate of acid-sensitive chemical bonds, this does not change the kinetic characteristics. However, the extremely strong hydrophobic repulsion formed within such dense microcrystalline domains reduces the absolute proton concentration of the micelles by several orders of magnitude. This sudden drop in concentration directly leads to an exponential decrease in the effective collision frequency of acid-catalyzed hydrolysis, transforming the originally unobstructed physical permeation process into a fundamental rate-limiting barrier that restricts the subsequent chemical bond breakage rate. If the grafting molar ratio is within the preset working window of 1:2.5 to 1:3.2, the spatial volume of the ATR kinase inhibitor is used to block the aromatic ring stacking between adjacent molecules and to forcibly maintain the amorphous free phase in the nanomicelle core, thereby achieving simultaneous permeation and in-situ release of hydrated protons to eliminate time misalignment. However, if the grafting molar ratio is within the upper limit control group of 1:4.0, steric hindrance overload disrupts the physical balance between hydrophobic interactions and hydrophilic repulsion, accompanied by polymer precipitation, causing the preset simultaneous release mechanism to fail at the surface physical level.

[0040] Example 4: In the preparation of a drug delivery system for non-small cell lung cancer metastases, due to the differences in solubility and reactivity mismatch of hydrophobic active molecules during the synthesis of polymer prodrugs, the final grafting ratio is prone to deviate from the preset window. When the molecular arrangement of the hydrophobic graft segments tends to be highly ordered, the dense microcrystalline domains generated by the micelle core bury the acid-sensitive linkage deep in the molecular array, causing the physical path of proton diffusion from the release medium to the interior to be blocked, thereby causing a spatiotemporal imbalance in the release concentration of the two active components and weakening the synchronous killing effect against replication stress vulnerability.

[0041] The composition achieves molecular structure construction by controlling a stepwise coupling procedure. The block copolymer backbone is dissolved in anhydrous dimethyl sulfoxide, and the water content of the system is controlled to be below 0.01%. An activated replication stress inducer is then added. Kinase inhibitors and replication stress inducers are coupled via an adipic acid linker with a length of 0.8 nm. The kinase inhibitor was coupled via a 2.4 nm long polyethylene glycol linker at a molar ratio of 1:3.5. The reaction was carried out at 35°C for 24 h. The molecular collision frequency was controlled by adjusting the dielectric constant of the solution to achieve a grafting molar ratio of 1:2.9. The preparation process used an anhydrous dimethyl sulfoxide and a low-polarity co-solvent dioxane to form a binary solvent system. Adjusting the volume ratio of the two solvents kept the overall dielectric constant of the reaction system between 38.5 and 42.0. The hydrodynamic radius change of the flexible chain at the ATR kinase inhibitor molecule's end under a specific dielectric constant controlled the steric hindrance in three dimensions, ensuring precise achievement of the target grafting ratio. The asymmetric occupancy gradient of the long and short chain linkers in three dimensions blocked the ordered arrangement path required for crystal nucleus growth, maintaining the amorphous phase of the nanomicelle core. High-performance liquid chromatography (HPLC) analysis confirmed that the monomer content was below 0.5%. Differential scanning calorimetry (DSC) was used to scan the thermodynamic behavior of the core within the range of -20°C to 150°C. No exothermic crystallization peak was observed in the sample group, and the glass transition temperature was [not specified]. The temperature of 42.5℃ indicates that the molecular chain segments are in an amorphous state with high free volume, while the control group, which eliminated the difference in the length of the connecting handles, showed a crystallization peak at 85.6℃, confirming that the steric gradient suppressed the phase ordering transition.

[0042] Proton diffusion coefficient of the composition in a simulated lysosomal environment Following the free volume diffusion law, its calculation formula is as follows: ,in, The proton diffusion coefficient is... A constant factor, The critical volume occupied by the motion of protons. The free volume of the kernel; in actual parameter extraction, the critical volume occupied by proton motion. It is obtained by directly converting the standard kinetic radius of the hydrated proton through the group contribution method constant, while the free volume of the nucleus is... This is achieved by performing offline positron annihilation lifetime spectroscopy tests on polymer solutions containing a preset grafting ratio, and calculating the result using the known quantum mechanical mapping constant between the measured average lifetime of positron-pair hybrids in the polymer amorphous region and the radius of free volume pores; because the asymmetric grafting structure maintains a high... Value, measured for This physical characteristic supports the synchronous hydrolysis of acid-sensitive linkages. The time difference between the peak plasma concentrations of the two drugs was measured to be less than 5.0 min, achieving the elimination of cell clones in metastatic lesions of non-small cell lung cancer. In the case of metastatic lesion microenvironment with different matrix metalloproteinase expression abundances, to confirm the supporting role of peptide sequences in the sensitivity of uncoating response, prodrug parallel sample groups containing PVGLIG, GPLGIAGQ, and VPMSMRGG sequences were prepared, and simulated blood circulation was monitored using fluorescence resonance energy transfer technology. The value was 7.4) and the tumor stroma ( The shielding stripping efficiency was calculated for each sample group under an environment containing 100 ng / mL matrix metalloproteinase 9) with a value of 6.8. ,in, The ratio of the actual number of cleaved peptide bonds to the initial total number of peptide bonds is measured in samples containing the VPMSMRGG sequence under pathological conditions. It reached 92.4%, while its performance in a simulated blood environment was... The value of less than 1.5% confirms that the peptide sequence has a definite response sensitivity to lesion-secreting enzymes while maintaining the stability of the circulatory system.

[0043] Example 5: In the scenario of producing polymer prodrugs from different batches of polyamino acid backbone raw materials, grafting kinetics calibration based on changes in solution conductivity was implemented to address the fluctuations in molecular weight distribution and polydispersity index of the block copolymer backbone. A series of dilute solutions containing backbones of different molar masses were prepared at 35°C. Active molecules coupled by adipic acid linkers were added dropwise to the reaction system. The critical point of molecular chain entanglement was determined by monitoring the dynamic viscosity evolution curve of the mixture, thus determining the grafting efficiency coefficient corresponding to the batch of backbone raw materials. ,in, The dimensionless process compensation factor is based on the measured grafting efficiency coefficient. The compensation volume of dimethylformamide was determined using empirical equations. When the coefficient decreased due to high molecular weight batches, dimethylformamide was dynamically added to the reaction system. Its strong hydrogen bond breaking ability stretched the polymer backbone, exposing deep reaction sites and bringing the grafting density back within the amorphous phase transition threshold. The steric hindrance energy variation caused by molecular weight fluctuations was compensated by adjusting the volume mixing ratio of dimethyl sulfoxide and dimethylformamide, thus ensuring the product... The grafting density of kinase inhibitors and replication stress inducers is within the amorphous phase transition threshold.

[0044] When the composition was applied to non-small cell lung cancer tissue environments with different matrix metalloproteinase expression gradients, the stability of the core-shell structure before reaching the lesion and the sensitivity of peeling triggering were established. Release parameters were established using in vitro enzymatic degradation simulation. Long-chain linkers with a pre-defined sequence were placed in a recombinant matrix metalloproteinase buffer containing a known concentration gradient. Dynamic light scattering was used to monitor the micelle hydrodynamic diameter from its initial state. Towards disintegration The time constant of the transition ,in, The initial hydrodynamic diameter. The diameter is the hydrodynamic diameter after disintegration. The time constant of diameter transition is used to establish a monotonic mapping relationship between enzyme concentration and hydrophilic shielding stripping rate. Covalent anchoring sites at the ends of peptide sequences are used to counteract interference noise caused by nonspecific protein adsorption. Within the matrix metalloproteinase concentration range of 50 ng / mL to 200 ng / mL, the response time variability is less than 8%. This calibration value serves as the physical basis for adjusting the grafting molar ratio to 1:2.8, maintaining the restricted conformation of prodrug molecules in blood circulation and achieving active exposure after entering the pathological target.

[0045] Example 6: In the production of polymer prodrugs from polyamino acid backbone raw materials with different reactivity, the functional group distribution density of different batches of raw materials varies. To ensure that the asymmetric grafting structure is in a predetermined distribution state, a block copolymer backbone solution is prepared at a constant temperature of 35°C. The content of terminal carboxyl groups is determined by potentiometric titration, and the activation coefficient is determined. ,in, The activation rate coefficient represents the ratio of the actual number of activated groups to the theoretical number of sites. Based on the activation rate coefficient... Determine the activating monomer of replication stress inducer and Corrected molar ratio of kinase inhibitor activating monomer ,in, To correct the molar ratio, it is used to determine the monomer feed ratio, and its calculation formula is as follows: Using this corrected molar ratio Instructions for feeding are given to adjust the grafting density, so that the replication stress inducer and... The grafting molar ratio of kinase inhibitors ranged from 1:2.5 to 1:3.2.

[0046] After the polymer prodrug forms nanomicelles in a polar aqueous medium, it is determined whether the order of the core is affected by the entanglement strength of the hydrophobic end segments. The micelle solution is extracted, and the scattering intensity curve is obtained using small-angle X-ray scattering (SAXS). The characteristic correlation length of the core molecules is calculated based on the scattering intensity distribution. Used to characterize the degree of order in molecular arrangement, if the characteristic correlation length is measured For phase transition thresholds greater than 2.0 nm, 2% ethanol (by mass) is added to the polar aqueous medium to adjust the polar environment and provide physical gaps. By adjusting the intersegmental forces, the core phase is restored to an amorphous state. The proton diffusion coefficient of the nanomicelle core in this amorphous state is then measured. Maintain at The above demonstrates the consistency of drug release kinetic curves under different operating conditions.

[0047] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A composition for treating non-small cell lung cancer metastasis that targets replication stress vulnerability, characterized in that, include: A dual-responsive polymer prodrug, comprising a block copolymer backbone, a matrix metalloproteinase cleavage peptide sequence coupled to the hydrophilic end of the block copolymer backbone, and an active molecular group covalently grafted to the hydrophobic end of the block copolymer backbone; The active molecular group includes a replication stress inducer and an ATR kinase inhibitor; wherein, the active molecular group presents an asymmetric grafting structure at the hydrophobic end of the block copolymer backbone, so that the nanomicelles formed by the self-assembly of the dual-responsive polymer prodrug in the aqueous medium have an amorphous phase core. The matrix metalloproteinase cleavage peptide sequence and the hydrophilic end of the block copolymer backbone constitute the hydrophilic shielding segment of the nanomicelle. The amorphous core eliminates the spontaneously formed crystal permeation barrier of the active molecular group, causing the composition to undergo the following response process when it enters a tissue microenvironment characterized by weak acidity and matrix metalloproteinase overexpression: Step S11, the matrix metalloproteinase cleavage peptide sequence is broken in response to the matrix metalloproteinase in the tissue microenvironment, stripping off the hydrophilic shielding segment and exposing the amorphous core; Step S12, hydrated protons in the tissue microenvironment permeate into the core through the physical channel provided by the amorphous core and undergo chemical kinetic collisions with the acid-sensitive chemical bonds coupled to the active molecular group; Step S13, the replication stress inducer and the ATR kinase inhibitor are released in situ based on the breaking of the acid-sensitive chemical bonds, inducing replication stress collapse of the biological target.

2. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, The active molecular group is grafted onto the hydrophobic end of the block copolymer backbone through a linker with a differential chain length. This differential chain length forms a steric hindrance gradient distribution at the hydrophobic end of the block copolymer backbone, inhibiting the recrystallization of the hydrophobic monomers in the active molecular group. The replication stress inducer includes gemcitabine derivatives or camptothecin derivatives. The ATR kinase inhibitor includes AZD6738 derivatives or VE-821 derivatives. The acid-sensitive chemical bond is selected from hydrazone bonds, acetal bonds, or orthoester bonds. After the composition enters the tissue microenvironment, the synchronous release time difference between the replication stress inducer and the ATR kinase inhibitor is less than 15 minutes.

3. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, The matrix metalloproteinase cleavage peptide sequence is selected from the PVGLIG sequence, GPLGIAGQ sequence, or VPMSMRGG sequence; the linkage strength between the acid-sensitive chemical bond and the replication stress inducer is matched with the linkage strength between the acid-sensitive chemical bond and the ATR kinase inhibitor.

4. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, The self-assembly logic of the dual-responsive polymer prodrug is determined by the following steps: Step S41: In the aqueous medium, the hydrophobic ends of the block copolymer backbone contract by hydrophobic forces to form the amorphous core; Step S42: The active molecular group utilizes the steric hindrance generated by the asymmetric grafting structure to block the planar stacking of aromatic rings between hydrophobic molecules; Step S43: The amorphous core is kept in a free state, providing a three-dimensional diffusion channel for the hydrated protons in the amorphous core.

5. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, The main chain of the block copolymer is composed of hydrophilic and hydrophobic segments. The hydrophilic segments include polyethylene glycol or polylactic acid-polyglycolic acid copolymers, and the hydrophobic segments include polyaspartic acid derivatives or polyglutamic acid derivatives. The replication stress inducer has a drug loading of 5% to 15% in the amorphous core, and the ATR kinase inhibitor has a drug loading of 8% to 20% in the amorphous core.

6. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, The surface of the nanomicelles is modified with targeting ligands, including integrin receptor-targeting peptide RGD or transferrin, to increase the enrichment ratio of the composition in the tissue microenvironment; the average particle size of the nanomicelles is 30 nm to 80 nm.

7. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, In the blood circulation, the composition constructs a core-shell steric hindrance through the hydrophilic shielding segment, shielding the acid-sensitive chemical bond from attack by non-specific proteases; in the tissue microenvironment with a pH of 5.0 to 6.5, the breaking rate of the acid-sensitive chemical bond is more than 10 times higher than that in the pH 7.4 environment.

8. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, The molar ratio of the replication stress inducer and the ATR kinase inhibitor in the amorphous phase of the core is 1:3 to 3:1; the composition blocks the DNA replication fork repair pathway of the biological target by simultaneously releasing the active molecular group.

9. The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability according to claim 1, characterized in that, This composition is used as a biopharmaceutical formulation to prepare a drug that blocks the clonal evolution of metastatic lesions in non-small cell lung cancer. The composition utilizes the amorphous phase core to maintain the diffusion rate of the hydrated proton in the core above a preset threshold, ensuring that the release kinetics of the active molecular group is dominated by the breaking frequency of the acid-sensitive chemical bond.

10. The application of a composition targeting replication stress vulnerability to non-small cell lung cancer metastasis, characterized in that, The anti-non-small cell lung cancer metastasis composition targeting replication stress vulnerability as described in claim 1 is used to prepare a drug that blocks the clonal evolution of metastatic lesions in non-small cell lung cancer.

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