pH / GSH spatiotemporal cascade-responsive antitumor nanomedicines, their preparation methods and applications

Abstract

This invention belongs to the field of biomedical polymer materials, and relates to pH / GSH spatiotemporal cascade-responsive antitumor nanomedicines, their preparation methods, and applications. The pH / GSH spatiotemporal cascade-responsive antitumor nanomedicines are formed by the self-assembly of polymer prodrugs with the structure shown in formula (1). The pH / GSH spatiotemporal cascade-responsive antitumor nanomedicines provided by this invention can highly sensitively identify and respond to the extremely narrow pH change window of the tumor microacid environment, achieving charge reversal on the nanomedicine surface and thus enhancing the cellular uptake efficiency of the nanomedicine. Subsequently, in response to the highly expressed GSH within the cells, it precisely releases the drug at specific points, killing tumor cells. Based on the dual significant characteristics of the acidic pH and GSH in the tumor microenvironment, this invention significantly improves the cellular uptake efficiency of the drug through a spatiotemporal cascade response mechanism of "pH-triggered charge reversal - GSH-induced controlled drug release," while simultaneously achieving precise and controllable drug release, effectively inhibiting cancer cell growth. This provides a general-purpose nanomedicine platform for solid tumor treatment that is structurally controllable, has excellent synergy, and possesses both basic research value and clinical translational potential. Formula (1)

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials, specifically relating to pH / GSH spatiotemporal cascade responsive antitumor nanomedicines, their preparation methods, and applications. Background Technology

[0002] Cancer is one of the major diseases that seriously threaten human health, with its global incidence and mortality rates continuing to rise. Currently, chemotherapy remains the primary first-line treatment, but traditional small-molecule chemotherapy drugs have significant drawbacks. On the one hand, due to their lack of targeting, small-molecule drugs exhibit severe toxic side effects on normal tissues; on the other hand, their penetration into solid tumors is extremely limited, meaning the drugs can only kill surface tumor cells, leading to frequent relapses and even metastasis to other sites, failing to achieve the desired therapeutic effect. Developing new drug delivery methods is one of the urgent challenges facing cancer treatment today.

[0003] Compared to normal tissues, tumor tissues produce large amounts of acidic substances such as lactic acid due to abnormal glycolytic metabolism, resulting in a lower pH value in their microenvironment compared to normal tissues (normal tissue pH is maintained at around 7.4, tumor cell extracellular microenvironment pH is maintained at around 6.5, and intracellular lysosomal / endosome pH is even lower at around 5.0). This creates a multi-level pH gradient from blood to the tumor microenvironment and then to intracellular organelles. Simultaneously, to cope with abnormal metabolism and oxidative stress, the expression levels of reducing substances such as glutathione (GSH) in tumor cells are significantly increased, forming a significant redox gradient. Based on the significant pH gradient and GSH concentration gradient of the tumor microenvironment, and other endogenous characteristics, novel stimulus-responsive nanomedicines can be constructed, providing a promising approach to addressing the challenges faced by small-molecule chemotherapy drugs. Nanocarriers of a certain size (typically 20–200 nm) can passively accumulate at tumor sites through the "enhanced permeability and retention" (EPR) effect, mitigating off-target effects of small molecule drugs to some extent. However, tumor tissue, due to its abnormal vascular system and uncontrolled growth, exhibits a dense extracellular matrix and high interstitial pressure, severely hindering the effective diffusion and deep penetration of nanomedicines from the perivascular area into the tumor interior. This results in a large amount of nanomedicine remaining in the peri-tumor region, significantly reducing its efficacy. Ensuring precise and controllable drug release at the lesion site while effectively penetrating deep into solid tumors to exert therapeutic effects remains an unresolved challenge in nanomedicine delivery.

[0004] Currently, the most effective method is to precisely design the carrier polymer structure and alter the surface properties of nanomedicines to induce a "charge flip"—a change in surface charge from negative to positive—under tumor-associated pH conditions. This allows for enhanced deep penetration and uptake of the nanomedicine by leveraging the electrostatic interactions between the positively charged nanomedicine and the negatively charged tumor cell membrane and extracellular matrix. This is considered one of the effective ways to overcome the bottleneck of deep drug delivery in solid tumors. The key to achieving this process lies not only in the simple introduction of pH-responsive groups, but also in the controllable design and systematic regulation of the carrier polymer's microstructure. Factors include the block sequence of pH-responsive fragments in the polymer, the type and proportion of tertiary amine monomers, and the length of hydrophilic / hydrophobic segments. These structural parameters collectively determine the aggregation state, protonation behavior, charge conversion efficiency, and subsequent penetration efficiency of nanomedicines in solid tumors under different pH environments. Currently, research on pH-responsive fragments containing different tertiary amine monomers, and on systematically comparing their pH sensitivity, surface charge conversion behavior, and controllability of drug release by controlling the block positions of these fragments in the polymer, remains limited. Existing research indicates that polymers containing protonable tertiary amines can be used to construct pH-responsive nanocarriers, but there are still significant shortcomings in the following aspects: First, the pH response sensitivity of some pH-responsive nanocarriers does not match the pH range of the tumor extracellular microenvironment (approximately pH 6.5–6.8), resulting in limited protonation of the tertiary amine, low charge-flipping efficiency, and difficulty in significantly improving deep penetration and cellular uptake of tumor tissues; Second, there is a lack of systematic structure-performance correlation studies on the synergistic effects of structural factors such as the block order of pH-responsive fragments in polymers (e.g., proximity to hydrophilic or hydrophobic segments, location in the shell or core), the type and ratio of tertiary amine monomers, and the length of hydrophilic / hydrophobic fragments on pH sensitivity, charge-flipping behavior, and the permeability of nanoparticles in tumors. In addition, although there are reports in the existing technology of using GSH-sensitive bonds (such as disulfide bonds) to achieve intracellular reduction response drug release, there is still a lack of polymer design strategies that are simple, controllable, have clear rules and can be universally applied to achieve the following simultaneously on the same carrier: (1) efficient pH-responsive charge flipping under weakly acidic tumor microenvironment conditions, thereby enhancing deep penetration of solid tumors; (2) rapid and specific drug release under high GSH levels in tumor cells, and to form a synergistic cascade amplification effect in time and space.

[0005] Based on the above overview, there is still an urgent need in this field to develop a pH / GSH dual-response antitumor nanomedicine system with a stable and controllable polymer structure, a pH response window that is highly matched with the tumor microenvironment, good synergistic pH / GSH dual response, and the ability to significantly improve deep tumor penetration and precise intracellular release while ensuring in vivo circulation stability. This aims to provide new technical approaches and nanomedicine platforms with potential clinical translation prospects for the treatment of solid tumors. Summary of the Invention

[0006] One of the objectives of this invention is to provide a novel pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine, which can achieve a high degree of matching between the pH response window and the tumor microenvironment and good synergy between pH / GSH dual responses, thereby significantly improving the release capacity of the nanomedicine.

[0007] The second objective of this invention is to provide a method for preparing the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicine.

[0008] The third objective of this invention is to provide the application of the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicine in the preparation of drugs for treating tumor diseases.

[0009] Existing antitumor nanodrug delivery systems suffer from the following problems: First, existing systems generally lack systematic regulation and research on the structure-activity relationship between polymer structural parameters (such as the block sequence of pH-responsive fragments, the type and ratio of tertiary amine monomers, and the length of hydrophilic / hydrophobic segments) and pH response, charge reversal, and drug release behavior, making it difficult to formulate universally applicable design principles. Second, the pH response window of existing pH or pH / GSH-stimulated responsive nanodrugs does not match the weakly acidic conditions of the tumor microenvironment well, and the charge reversal from negative to positive on the surface is insufficient, resulting in limited deep penetration and cellular uptake of nanodrugs in solid tumors. To address these problems in existing antitumor nanodrug delivery systems, this invention designs an antitumor nanodrug based on a spatiotemporal cascade response to pH / GSH in the tumor microenvironment. This nanodrug can sensitively respond to the acidic environment of the tumor microenvironment, achieving surface charge reversal to enhance the uptake efficiency by tumor cells and improve penetration into solid tumors. Subsequently, it responds to the highly expressed GSH within cells for precise drug release, killing tumor cells. The pH / GSH spatiotemporal cascade responsive antitumor nanomedicine provided by this invention is expected to break through the bottleneck of existing stimulus-responsive nanomedicines in deep delivery and precise drug release in solid tumors. It establishes a system structure-activity relationship of "polymer structure-pH response behavior-surface charge reversal-GSH-triggered drug release", providing a new technical approach and potential application nanomedicine platform for improving the efficacy and safety of chemotherapy.

[0010] The pH / GSH spatiotemporal cascade responsive antitumor nanomedicine provided by the present invention is formed by self-assembly of a polymer prodrug having the structure shown in formula (1); Equation (1) In formula (1), R1 and R2 are alkyl groups or cyclic molecular groups; R3 is a disulfide bond, diselenide bond or thioether bond; R4 is a group containing a dithioester group or a trithioester group; R is an anticancer chemotherapy drug source; m is an integer from 10 to 400, n is an integer from 5 to 200, and x is an integer from 1 to 200.

[0011] The method for preparing pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine provided by the present invention includes the following steps: under light-protected conditions, a polymer prodrug is dissolved in an organic solvent to obtain an organic phase; the organic phase is mixed with an aqueous phase using a microfluidic device; and the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine is assembled using a nanoprecipitation method based on microfluidic technology.

[0012] Based on the dual characteristics of acidic pH and GSH in the tumor microenvironment, this invention overcomes the limitations of existing nanomedicines, such as poor matching degree between pH response window and weakly acidic conditions in the tumor microenvironment, insufficient penetration ability of nanomedicines, and poor synergy of dual-response systems. It innovatively constructs a pH / GSH spatiotemporal cascade response type antitumor nanomedicine, which helps to achieve the following gains through the spatiotemporal cascade response mechanism of "pH-triggered charge reversal - GSH-induced drug release": (1) Enhanced penetration of solid tumors: The tertiary amine group of the pH response unit in the nanomedicine is protonated under the pH window of the tumor microacid environment. The corresponding blocks are driven to change from hydrophobic to hydrophilic, and the surface charge is flipped from negative to positive, which strengthens the interaction with the negatively charged tumor cell membrane. Through transmembrane transport, the dense cellular environment barrier of solid tumors is effectively broken through, which significantly improves the deep penetration efficiency and cell uptake rate of drugs in tumor tissues; (2) Precise and controllable drug release: After the above pH response, the nanomedicine is further exposed to the high concentration of GSH in the tumor cells. The hydrophobic core fragment is reduced and cleaved by GSH, realizing the targeted and continuous controllable release of chemotherapy drugs, which specifically inhibits the growth of cancer cells and greatly reduces the off-target toxicity of drugs. This invention establishes a system cascade pathway of "polymer structure - pH response charge flip - tumor penetration - GSH triggering drug release", which provides a general nanomedicine platform with controllable structure, excellent synergy, and both basic research value and clinical translation potential for the treatment of solid tumors. Attached Figure Description

[0013] Figure 1 The 1H NMR spectrum of the mPEG-CTA obtained in Preparation Example 1.

[0014] Figure 2 The 1H NMR spectrum of the DEAEMA obtained in Preparation Example 2.

[0015] Figure 3 The 1H NMR spectrum of MA-ss-OH obtained in Example 3.

[0016] Figure 4The 1H NMR spectrum of the MA-ss-PNP obtained in Preparation Example 3.

[0017] Figure 5 To prepare the mPEG- obtained in Example 4 b -P(DEAEMA- co The hydrogen NMR spectrum of (-MA-ss-PNP).

[0018] Figure 6 To prepare the mPEG- obtained in Example 4 b -P(DEAEMA- co The hydrogen NMR spectrum of (MA-ss-DOX).

[0019] Figure 7 To prepare the mPEG- obtained in Example 5 b -P(MA-ss-PNP) 1H NMR spectrum.

[0020] Figure 8 To prepare the mPEG- obtained in Example 5 b -P(MA-ss-PNP)- b - PDEAEMA's 1H NMR spectrum.

[0021] Figure 9 To prepare the mPEG- obtained in Example 5 b -P(MA-ss-DOX)- b - PDEAEMA's 1H NMR spectrum.

[0022] Figure 10 To prepare the mPEG- obtained in Example 6 b - PDEAEMA's 1H NMR spectrum.

[0023] Figure 11 To prepare the mPEG- obtained in Example 6 b -PDEAEMA- b -P(MA-ss-PNP) 1H NMR spectrum.

[0024] Figure 12 To prepare the mPEG- obtained in Example 6 b -PDEAEMA- b -P(MA-ss-DOX) 1H NMR spectrum.

[0025] Figure 13 The mPEG- prepared in Example 1 was used as a control. b -P(MA-ss-DOX) 1H NMR spectrum.

[0026] Figure 14The graph shows the zeta potential changes of nanomedicine PD-1 obtained in Example 1, nanomedicine PD-2 obtained in Example 2, nanomedicine PD-3 obtained in Example 3, and nanomedicine GD-1 obtained in Control Example 1 under different pH conditions.

[0027] Figure 15 The cumulative release curves of DOX for the nanomedicine PD-1 obtained in Example 1, the nanomedicine PD-2 obtained in Example 2, the nanomedicine PD-3 obtained in Example 3, and the nanomedicine GD-1 obtained in Control Example 1 are shown in the following conditions: pH 6.5 and 0 mM GSH.

[0028] Figure 16 The cumulative release curves of DOX for the nanomedicine PD-1 obtained in Example 1, the nanomedicine PD-2 obtained in Example 2, the nanomedicine PD-3 obtained in Example 3, and the nanomedicine GD-1 obtained in Control Example 1 are shown under the conditions of pH 6.5 and 10 mM GSH.

[0029] Figure 17 The graph shows the results of quantitative analysis of cellular uptake of nanomedicine PD-1 obtained in Example 1, nanomedicine PD-2 obtained in Example 2, nanomedicine PD-3 obtained in Example 3, and nanomedicine GD-1 obtained in Control Example 1 after incubation with A549 cells.

[0030] Figure 18 The graph shows the cell survival rate results after co-incubating A549 cells with different concentrations of nanomedicine PD-1 obtained in Example 1, nanomedicine PD-2 obtained in Example 2, nanomedicine PD-3 obtained in Example 3, and control nanomedicine GD-1 obtained in Example 1. Detailed Implementation

[0031] The pH / GSH spatiotemporal cascade responsive antitumor nanomedicine provided by the present invention is formed by self-assembly of a polymer prodrug having the structure shown in formula (1); Equation (1) In formula (1), R1 and R2 are alkyl groups or cyclic molecular groups; R3 is a disulfide bond, diselenide bond, or thioether bond; R4 is a group containing a dithioester group or a trithioester group; R is an anticancer chemotherapy drug source; m is an integer from 10 to 400, n is an integer from 5 to 200, and x is an integer from 1 to 200. Specifically, the alkyl group is preferably a C1 to C6 alkyl group, and examples include at least one of methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, and 4-methylpentyl. The cyclic molecular group can be a five-membered cyclic molecular group, a six-membered cyclic molecular group, or a seven-membered cyclic molecular group. R4 is preferably a group containing a trithioester. m can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 220, 250, 280, 300, 320, 350, 380, 400, etc. n can be 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, etc. x can be 1, 3, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, etc.

[0032] As described above, the polymer prodrug comprises a polyethylene glycol hydrophilic segment, a pH-responsive tertiary amine structural unit, and a reduced glutathione-responsive structural unit. It should be noted that the polymer prodrug with the structure shown in formula (1) is only used to represent the contained structural units and the number of repeating units in each structural unit, and is not used to limit the connection relationship between the structural units. The pH-responsive tertiary amine structural unit and the reduced glutathione-responsive structural unit can be linked to the polyethylene glycol hydrophilic segment through random copolymerization, and the pH-responsive tertiary amine structural unit and the reduced glutathione-responsive structural unit can also be linked to the polyethylene glycol hydrophilic segment through block copolymerization. That is, the polymer prodrug can be a random copolymer or a block copolymer. When the polymer prodrug is a block copolymer, the pH-responsive tertiary amine structural unit can be directly bonded to the hydrophilic segment of polyethylene glycol, while the reduced glutathione responsive structural unit is bonded to the pH-responsive tertiary amine structural unit. Alternatively, the reduced glutathione responsive structural unit can be directly bonded to the hydrophilic segment of polyethylene glycol, while the pH-responsive tertiary amine structural unit is bonded to the reduced glutathione responsive structural unit.

[0033] In the aforementioned pH / GSH spatiotemporal cascade-responsive antitumor nanomedicines, the anticancer chemotherapeutic agent corresponding to the anticancer chemotherapeutic drug source preferably contains hydroxyl (-OH) and / or amino (-NH2) groups. The anticancer chemotherapeutic agent is linked to the reduced glutathione-responsive monomer side chain via a carbamate or carbonate. Specifically, the anticancer chemotherapeutic agent may include at least one of the following: doxorubicin derivatives (DOX, EPB), epirubicin (EPB), paclitaxel (PTX), camptothecin derivatives (CPT, HCPT, SN38), and vinca alkaloids (VCR, VLB, NVB, VDS).

[0034] In the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicine, the particle size of the pH / GSH spatiotemporal cascade responsive antitumor nanomedicine is preferably 30 nm to 150 nm, such as 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, etc.

[0035] In the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicines, the zeta potential of the pH / GSH spatiotemporal cascade responsive antitumor nanomedicines is preferably -15 mV to 20 mV, such as -15 mV, -13 mV, -10 mV, -8 mV, -5 mV, -3 mV, 0 mV, 2 mV, 5 mV, 8 mV, 10 mV, 12 mV, 15 mV, 18 mV, 20 mV, etc.

[0036] In the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicine, the chemotherapeutic drug loading of the pH / GSH spatiotemporal cascade responsive antitumor nanomedicine is preferably 10% to 40%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, etc.

[0037] The method for preparing pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine provided by the present invention includes the following steps: under light-protected conditions, a polymer prodrug is dissolved in an organic solvent to obtain an organic phase; the organic phase is mixed with an aqueous phase using a microfluidic device; and the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine is assembled using a nanoprecipitation method based on microfluidic technology.

[0038] In a preferred embodiment, the polymer prodrug is prepared by the following method: using mPEG-R4 as shown in formula (2) as a macromolecular chain transfer agent, under the action of an initiator, the pH-responsive tertiary amine monomer shown in formula (3) and the reduced glutathione-responsive monomer shown in formula (4) are initiated to undergo RAFT polymerization, thereby linking the pH-responsive tertiary amine structural unit and the reduced glutathione-responsive structural unit to the hydrophilic segment of polyethylene glycol in a random copolymerization or block copolymerization manner. Then, the obtained polymer is subjected to ester exchange reaction with a hydroxyl-containing chemotherapeutic drug or to amino-containing chemotherapeutic drug to obtain a pH / GSH spatiotemporal cascade responsive polymer prodrug. Equation (2), Equation (3), Equation (4), In formula (2), m is an integer from 10 to 400; R4 is a group containing a dithioester group or a trithioester group; In formula (3), R1 and R2 are alkyl groups or cyclic molecular groups; In formula (4), R3 is a disulfide bond, a diselenide bond, or a thioether bond.

[0039] In the preparation of the above polymer prodrug, in formula (3), when R1 and R2 are cyclic molecular groups, the pH-responsive tertiary amine monomer can have the structure shown in formula (a), formula (b), or formula (c): Equation (a), Equation (b) Formula (c).

[0040] In the preparation of the aforementioned polymer prodrugs, mPEG-R4, as a macromolecular chain transfer agent, can simultaneously initiate the polymerization of pH-responsive tertiary amine monomers and reduced glutathione-responsive monomers, thus obtaining the polymer prodrug via random copolymerization; or mPEG-R4, as a macromolecular chain transfer agent, can first initiate the polymerization of pH-responsive tertiary amine monomers, and then initiate the polymerization of reduced glutathione-responsive monomers, thus obtaining the polymer prodrug via block copolymerization, where the pH-responsive tertiary amine structural unit is directly bonded to the macromolecular chain transfer agent, while the reduced glutathione-responsive structural unit is bonded to the pH-responsive tertiary amine structural unit; or mPEG-R4, as a macromolecular chain transfer agent, can first initiate the polymerization of reduced glutathione-responsive monomers, and then initiate the polymerization of pH-responsive tertiary amine monomers, thus obtaining the polymer prodrug via block copolymerization, where the reduced glutathione-responsive structural unit is directly bonded to the macromolecular chain transfer agent, while the pH-responsive tertiary amine structural unit is bonded to the reduced glutathione-responsive structural unit.

[0041] In the preparation process of the above-mentioned pH / GSH spatiotemporal cascade response antitumor nanomedicine, the molar ratio of the macromolecular chain transfer agent to the initiator is preferably 1:(0.2~0.5), such as 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, etc.

[0042] In the preparation of the aforementioned pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine, the macromolecular chain transfer agent can be commercially available or prepared using various existing methods. In one specific embodiment, the macromolecular chain transfer agent is generated by esterification of polyethylene glycol monomethyl ether with a RAFT chain transfer agent. The RAFT chain transfer agent contains a dithioester group or a trithioester group, specifically including at least one of the following: 4-cyano-4-[[(dodecylthio)thionylmethyl]thio]valerate (CTA-COOH), 4-cyano-4-(phenylthiocarbamoylthio)valerate (CPADB), 4-cyano-4-(propyltrithiocarbonate)valerate (CPPT), and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid (DMPA). The preferred molar ratio of polyethylene glycol monomethyl ether to RAFT chain transfer agent is 1:(4~8), such as 1:4, 1:5, 1:6, 1:7, 1:8, etc. The preferred conditions for the esterification reaction include a temperature of 25℃~30℃, such as 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, etc.; and a time of 24 h~48 h, such as 24 h, 28 h, 30 h, 32 h, 36 h, 40 h, 44 h, 48 h, etc. The esterification reaction is preferably carried out in the presence of a catalyst and a condensing agent. The catalyst is generally 4-dimethylaminopyridine (DMAP). The condensing agent is generally selected from at least one of N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and N,N'-diisopropylcarbodiimide (DIC).

[0043] In the preparation process of the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicine, the initiator can be any existing substance capable of generating free radicals to initiate monomer polymerization. Specifically, it can be selected from at least one of azo initiators, peroxide initiators, and redox initiators, preferably azo initiators, such as at least one of azobisisobutyronitrile (AIBN), azobisisoheptanenitrile (ABVN), azobisisovalerate (AMBN), and dimethyl azobisisobutyrate (AIBME).

[0044] In the preparation of the aforementioned pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine, the pH-responsive tertiary amine monomer is prepared by the following method: a substitution reaction is carried out by refluxing methacrylamide with ethanolamine containing different substituents in an organic solvent under the action of triethylamine. The organic solvent is preferably tetrahydrofuran (THF). The preferred conditions for the substitution reaction include a temperature of 70℃~80℃, such as 70℃, 72℃, 74℃, 76℃, 78℃, 80℃, etc.; and a time of 8 h~12 h, such as 8 h, 9 h, 10 h, 11 h, 12 h, etc.

[0045] In the preparation process of the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicine, the reduced glutathione responsive monomer can be prepared according to various existing methods. In a preferred embodiment, the reduced glutathione responsive monomer (MA-R3-PNP) is prepared by the following method: the diol shown in formula (5) is subjected to a primary nucleophilic substitution reaction with methacrylamide, and then the resulting diol methacrylate is subjected to a secondary substitution reaction with 4-nitrophenyl chloroformate to obtain the reduced glutathione responsive monomer. In a preferred embodiment, the reduced glutathione responsive monomer is prepared by the following method: S1. Methacrylamide chloride is subjected to a single-end nucleophilic substitution reaction with the diol shown in formula (5) under triethylamine catalysis to obtain the methacrylate monomer; S2. The methacrylate monomer is subjected to a nucleophilic substitution reaction with 4-nitrophenyl chloroformate (NPC) under triethylamine catalysis to obtain the reduced glutathione responsive monomer.

[0046] Equation (5) In formula (5), R3 is a disulfide bond, a diselenide bond, or a thioether bond.

[0047] In a preferred embodiment, the polymer prodrug is prepared according to the following chemical reaction process: (1) Preparation of macromolecular chain transfer agent mPEG-R4: Polyethylene glycol monomethyl ether was esterified with RAFT chain transfer agent to obtain macromolecular chain transfer agent mPEG-R4; (2) Preparation of pH-responsive tertiary amine monomers: Methacrylamide is subjected to a substitution reaction with ethanolamine containing different substituents to obtain pH-responsive tertiary amine monomers; (3) Preparation of reduced glutathione responsive monomer: Methacryl chloride and diol were subjected to a single-end nucleophilic substitution reaction under triethylamine catalysis, and the obtained methacrylate monomer was subjected to a nucleophilic substitution reaction with 4-nitrophenyl chloroformate under triethylamine catalysis to obtain reduced glutathione responsive monomer. (4) Preparation of polymer prodrug: mPEG-R4 is used as a macromolecular chain transfer agent. Under the action of an initiator, pH-responsive tertiary amine monomer and reduced glutathione-responsive monomer are initiated to undergo RAFT polymerization. Then, the obtained polymer is subjected to ester exchange reaction with hydroxyl-containing chemotherapeutic drugs or to amino-containing chemotherapeutic drugs to obtain pH / GSH spatiotemporal cascade responsive polymer prodrug.

[0048]

[0049] In the preparation process of the above-mentioned pH / GSH spatiotemporal cascade responsive antitumor nanomedicine, the preferred conditions for the RAFT polymerization reaction include a temperature of 65 °C to 75 °C, such as 65 °C, 68 °C, 70 °C, 72 °C, 75 °C, etc.; and a time of 20 h to 48 h, such as 20 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 48 h, etc.

[0050] In the preparation of the aforementioned pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine, the polymer obtained by RAFT polymerization of a pH-responsive tertiary amine monomer and a reduced glutathione-responsive monomer, initiated by mPEG-R4 as a macromolecular chain transfer agent, undergoes an ester exchange reaction with a hydroxyl-containing chemotherapeutic drug or an amino-containing chemotherapeutic drug to obtain a polymer prodrug. The preferred molar ratio of the reduced glutathione-responsive monomer to the chemotherapeutic drug in the polymer is 1:(2~10), such as 1:2, 1:3, 1:5, 1:8, 1:10, etc. The ester exchange reaction and the amino-amino-ester exchange reaction are preferably each independently including a temperature of 40℃~45℃, such as 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, etc.; and a time of 36 h~48 h, such as 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, 48 h, etc. The transesterification and amine transesterification reactions generally need to be carried out under light-protected conditions.

[0051] In the preparation process of the above-mentioned pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine, the aqueous phase is preferably a phosphate buffer solution. The pH range of the buffer solution can be 5.0~7.4, such as 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.4, etc. The mixing rate of the aqueous phase and the organic phase is preferably 10~50 μL / min, such as 10, 15, 20, 25, 30, 35, 40, 45, 50 μL / min, etc. The concentration of the polymer prodrug in the organic phase is preferably 0.1~10 mg / mL, such as 0.1, 0.5, 1, 2, 5, 8, 10 mg / mL, etc. The organic solvent is preferably dimethyl sulfoxide and / or N,N-dimethylformamide.

[0052] The present invention also provides the application of the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine in the preparation of drugs for treating tumor diseases.

[0053] The present invention will be further described in detail below through specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0054] Preparation Example 1: Preparation of macromolecular chain transfer agent mPEG-CTA

[0055] In a 250 mL round-bottom reaction flask with a side arm, accurately weighed polyethylene glycol monomethyl ether (3.0 g, 0.6 mmol, manufactured by Sigma-Aldrich, catalog number 81323-250G), 4-cyano-4-[[(dodecylthio)thiononemethyl]thio]valeric acid (CTA-COOH) (1.2 g, 3.0 mmol), 4-dimethylaminopyridine (DMAP) (0.18 g, 1.5 mmol), and 50 mL of ultra-dry anhydrous dichloromethane (CH2Cl2) were added and stirred to dissolve, yielding the reaction substrate. Separately, accurately measured N,N'-dicyclohexylcarbodiimide (DCC) (0.62 g, 3.0 mmol) was dissolved in 5 mL of anhydrous CH2Cl2 and placed in a clean, constant-pressure dropping funnel. This solution was slowly added dropwise to the round-bottom reaction flask containing the reaction substrate, while stirring continuously. The reaction was carried out overnight at 25 °C with stirring. After the reaction was complete, the reaction solution was filtered to remove insoluble matter, concentrated by rotary evaporation, and then precipitated in ice-cold anhydrous diethyl ether and centrifuged three times. The collected solid was placed in a vacuum drying oven and dried for 24 h. The resulting light yellow solid powder was the macromolecular chain transfer agent mPEG-CTA (2.3 g, yield 70%). The NMR spectrum of mPEG-CTA is shown below. Figure 1 As shown. From Figure 1 It can be seen that the macromolecular chain transfer agent mPEG-CTA was successfully prepared through esterification.

[0056] Preparation Example 2: Preparation of pH-responsive tertiary amine monomer (DEAEMA)

[0057] In a 250 mL round-bottom reaction flask with a side arm, precisely measured N,N-diethylethanolamine (11.7 g, 0.1 mol), hydroquinone (0.11 g, 0.001 mol) as a polymerization inhibitor, Et3N (10.1 g, 0.1 mol), and 100 mL of anhydrous tetrahydrofuran were added and stirred for 30 min to obtain the reaction substrate. Separately, methacryloyl chloride (10.4 g, 0.1 mol) was transferred to a clean constant-pressure dropping funnel and slowly added dropwise to the round-bottom reaction flask containing the reaction substrate. The reaction was continued with stirring for 2 h. After the reaction was complete, the reaction solution was filtered to remove triethylamine hydrochloride, followed by rotary evaporation to remove the tetrahydrofuran solvent. The residue was distilled under vacuum to finally obtain a colorless liquid product, which was the pH-responsive tertiary amine monomer DEAEMA (10.8 g, 58% yield). The NMR spectrum of DEAEMA is shown below. Figure 2 As shown. From Figure 2 It can be seen that the pH-responsive tertiary amine monomer DEAEMA was successfully prepared through a substitution reaction.

[0058] Preparation Example 3: Preparation of Reduced Glutathione Response Monomer (MA-ss-PNP)

[0059] S1. Take a clean, dry 250 mL round-bottom reaction flask with a side arm. Accurately weigh 10 g (65 mmol) of 2,2-dithiodiethanol, 4.8 g (47 mmol) of Et3N, and 70 mL of anhydrous THF into the reaction flask. Stir to dissolve for 30 min to obtain the reaction substrate. Separately, dissolve 3.34 g (32 mmol) of methacryloyl chloride in 40 mL of anhydrous THF and transfer the solution to a constant-pressure dropping funnel. Under ice-water bath conditions, slowly add the solution dropwise to the round-bottom reaction flask containing the reaction substrate, and then react at room temperature for 24 h. After the reaction was complete, insoluble matter was removed by filtration. The solution was concentrated by rotary evaporation and then redissolved in ethyl acetate. The solution was washed successively with deionized water (3 × 60 mL) and saturated brine (3 × 60 mL), dried over anhydrous Na₂SO₄, filtered, concentrated by rotary evaporation, and purified by silica gel column chromatography (ethyl acetate:petroleum ether = 1:1, V / V) to obtain a light white oily liquid (5.34 g, 75%). The NMR spectrum of this MA-ss-OH is shown below. Figure 3 As shown. From Figure 3 It can be seen that MA-ss-OH was successfully prepared from 2,2-dithiodiethanol and methacryloyl chloride via a single-end substitution reaction.

[0060] S2. Take a clean, dry 250 mL round-bottom reaction flask with a side arm. Under nitrogen protection, accurately weigh MA-ss-OH (4.92 g, 0.022 mol), Et3N (3.36 g, 0.033 mol), and 60 mL of anhydrous CH2Cl2 into the flask. Stir in an ice-water bath for 30 min until completely dissolved to obtain the reaction substrate. Separately, dissolve an appropriate amount of p-nitrophenyl chloroformate (NPC) (5.57 g, 0.028 mol) in 25 mL of anhydrous CH2Cl2 and transfer it to a constant-pressure dropping funnel. Slowly add the solution dropwise to the round-bottom reaction flask containing the reaction substrate under ice-water bath conditions. After the addition is complete, accurately weigh DMAP (0.54 g, 0.0044 mol) and add it to the reaction solution. Then, react at room temperature for 24 h. After the reaction was complete, insoluble matter was removed by filtration. The reaction solution was concentrated by rotary evaporation, redissolved in dichloromethane, washed with saturated brine (3 × 60 mL), dried over anhydrous Na₂SO₄, filtered, concentrated by rotary evaporation, and the crude product was purified by column chromatography (mobile phase: dichloromethane:petroleum ether = 4:1, V / V). The target product was a light yellow oily liquid (5.54 g, 65%). The NMR spectrum of this MA-ss-PNP is shown below. Figure 4 As shown. From Figure 4 It can be seen that MA-ss-PNP was successfully prepared by substitution reaction of MA-ss-OH and p-nitrophenyl chloroformate.

[0061] Preparation Example 4: This preparation example is used to illustrate the random copolymer mPEG- b -P(DEAEMA- co Preparation of -MA-ss-DOX

[0062] (1) Preparation of macromolecular chain transfer agent mPEG-CTA Similar to Preparation Example 1, the macromolecular chain transfer agent mPEG-CTA was obtained.

[0063] (2) Preparation of DEAEMA Similar to Preparation Example 2, DEAEMA was obtained.

[0064] (3) Preparation of MA-ss-PNP Similar to Preparation Example 3, MA-ss-PNP was obtained.

[0065] (4) mPEG- b -P(DEAEMA- co Preparation of -MA-ss-PNP) Take a clean, dry 10 mL Schlenk flask and evacuate and purge the reaction apparatus three times. Under nitrogen protection, add the weighed macromolecular chain transfer reagents mPEG-CTA (200 mg, 0.037 mmol), DEAEMA (172.2 mg, 0.92 mmol), MA-ss-PNP (724.9 mg, 1.8 mmol), AIBN (1.8 mg, 0.011 mmol), and 1.7 mL of ultra-dry anhydrous DMF in sequence. After stirring until dissolved, cycle the reaction system through freezing, evacuation, and nitrogen purging three times until no bubbles are generated. Then, place the reaction tube in a 70 °C oil bath for 40 h. After the reaction is complete, quench the reaction with liquid nitrogen. Slowly add the reaction solution dropwise to ice-cold ether and centrifuge to precipitate. Repeat the operation three times. Collect the solid powder and dry it in a vacuum oven for 24 h to obtain the target polymer as a light yellow solid (390 mg, yield 70.4%), which is the random copolymer mPEG- b -P(DEAEMA- co -MA-ss-PNP). The mPEG- b -P(DEAEMA- co The NMR spectrum of -MA-ss-PNP is as follows: Figure 5 As shown. From Figure 5 It can be seen that the macromolecular chain transfer agent mPEG-CTA successfully initiated the reversible addition-fragmentation chain transfer polymerization reaction of monomers DEAEMA and MA-ss-PNP to prepare the random copolymer mPEG- b -P(DEAEMA- co -MA-ss-PNP).

[0066] (5) mPEG- b -P(DEAEMA- co Preparation of -MA-ss-DOX Using a clean, dry 10 mL round-bottom reaction flask with a side arm, under nitrogen protection, accurately weigh DOX•HCl (292 mg, 0.50 mmol) and Et3N (50.9 mg, 0.50 mmol), and add 4.5 mL of ultra-dry anhydrous DMF. Stir at room temperature for 30 min to obtain the reaction substrate. Separately weigh mPEG- b -P(DEAEMA- co-MA-ss-PNP (300 mg, 0.02 mmol) was dissolved in 2.5 mL of anhydrous DMF and added to a round-bottom reaction flask containing the reaction substrate. The reaction was carried out in an oil bath at 40 °C for 40 h under light-protected conditions. After the reaction, the reaction solution was transferred to a MWCO1000 dialysis bag and dialyzed in DMF for two days until the dialysate was colorless. Then, it was transferred to a MWCO3500 dialysis bag and dialyzed in deionized water for three days. Freeze-drying yielded the target polymer as a dark red solid (250 mg, yield 59.7%), which is the random copolymer mPEG- b -P(DEAEMA- co -MA-ss-DOX). The mPEG- b -P(DEAEMA- co The NMR spectrum of (-MA-ss-DOX) is as follows: Figure 6 As shown. From Figure 6 It can be seen that mPEG- b -P(DEAEMA- c o-MA-ss-PNP) and the chemotherapy drug doxorubicin (DOX) successfully achieved an amino-ester exchange reaction.

[0067] Preparation Example 5: This preparation example illustrates the preparation of the block copolymer mPEG- b -P(MA-ss-DOX)- b -PDEAEMA preparation

[0068] (1) Preparation of macromolecular chain transfer agent mPEG-CTA Similar to Preparation Example 1, the macromolecular chain transfer agent mPEG-CTA was obtained.

[0069] (2) Preparation of MA-ss-PNP Similar to Preparation Example 3, MA-ss-PNP was obtained.

[0070] (3) mPEG- b Preparation of -P(MA-ss-PNP) A clean, dry 10 mL Schlenk flask was evacuated and purged with nitrogen three times. Under nitrogen protection, weighed macromolecular chain transfer reagents mPEG-CTA (200 mg, 0.037 mmol), AIBN (1.82 mg, 0.011 mmol), MA-ss-PNP (724.9 mg, 1.8 mmol), and 1.5 mL of ultra-dry anhydrous DMF were added sequentially. After dissolving at room temperature, the reaction system was cyclically frozen, evacuated, and purged with nitrogen three times until no bubbles were generated. Nitrogen was then introduced into the reaction system, and the reaction tube was placed in a 70 °C oil bath for 40 h. The reaction was quenched with liquid nitrogen, and the reaction solution was slowly added dropwise to ice-cold diethyl ether and centrifuged to precipitate. This process was repeated three times. The collected solid powder was dried in a vacuum oven for 24 h to obtain the target polymer as a light yellow solid powder (468 mg, yield 84%). Figure 8 It can be seen that the macromolecular chain transfer agent mPEG-CTA successfully initiated the reversible addition-fragmentation chain transfer polymerization of monomer MA-ss-PNP to prepare mPEG- b -P(MA-ss-PNP). The mPEG- b The NMR spectrum of -P(MA-ss-PNP) is as follows: Figure 7 As shown. From Figure 7 It can be seen that the macromolecular chain transfer agent mPEG-CTA successfully initiated the reversible addition-fragmentation chain transfer polymerization of monomer MA-ss-PNP to prepare mPEG- b -P(MA-ss-PNP).

[0071] (4) Preparation of DEAEMA Similar to Preparation Example 2, DEAEMA was obtained.

[0072] (5) mPEG- b -P(MA-ss-PNP)- b Preparation of PDEAEMA Take a dry and clean 10 mL Schlenk bottle, evacuate and purge with nitrogen three times, and under nitrogen protection, add the weighed macromolecular chain transfer reagent mPEG- b-P(MA-ss-PNP) (224 mg, 0.02 mmol), DEAEMA (74.1 mg, 0.4 mmol), AIBN (0.984 mg, 0.066 mmol), and 0.8 mL of ultra-dry anhydrous DMF were added. After stirring until dissolved, the reaction system was subjected to a three-cycle process of freezing, vacuuming, and nitrogen purging until no bubbles were generated. Then, the reaction tube was placed in a 70 °C oil bath for 40 h. After the reaction was completed, the reaction was quenched with liquid nitrogen. The reaction solution was slowly added dropwise to ice-cold diethyl ether and centrifuged to precipitate. This process was repeated twice. The solid powder was collected and dried in a vacuum oven for 24 h to obtain the target polymer as a light yellow solid (180 mg, yield 71%). This mPEG- b -P(MA-ss-PNP)- b -PDEAEMA's NMR spectrum is as follows Figure 8 As shown. From Figure 8 It can be seen that mPEG- b -P(MA-ss-PNP) successfully initiated a reversible addition-fragmentation chain transfer polymerization reaction of the monomer DEAEMA to prepare mPEG- b -P(MA-ss-PNP)- b -PDEAEMA.

[0073] (6) mPEG- b -P(MA-ss-DOX)- b Preparation of PDEAEMA Using a clean, dry 10 mL round-bottom reaction flask with a side arm, under nitrogen protection, accurately weigh out DOX•HCl (97.8 mg, 0.17 mmol), Et3N (17.07 mg, 0.17 mmol), and add 3.5 mL of ultra-dry anhydrous DMF. Stir at room temperature for 30 min to obtain the reaction substrate. Weigh out mPEG- b -P(MA-ss-PNP)- b -PDEAEMA (180 mg, 0.014 mmol) was dissolved in 2 mL of anhydrous DMF and added to a round-bottom reaction flask containing the reaction substrate. The flask was placed in an oil bath at 40 °C for 40 h under light-protected conditions. After the reaction, the reaction solution was transferred to a MWCO1000 regenerated cellulose dialysis bag and dialyzed against DMF for two days to remove excess DOX. Then, it was transferred to a MWCO3500 regenerated cellulose dialysis bag and dialyzed against deionized water for three days. Freeze-drying yielded the target polymer as a dark red solid (155 mg, yield 62.8%). This mPEG- b -P(MA-ss-DOX)- b -PDEAEMA's NMR spectrum is as follows Figure 9As shown. From Figure 9 It can be seen that mPEG- b -P(MA-ss-PNP)- b -PDEAEMA and the chemotherapy drug doxorubicin (DOX) successfully achieved an amino-ester exchange reaction.

[0074] Preparation Example 6: This preparation example illustrates the preparation of the block copolymer mPEG- b -PDEAEMA- b Preparation of -P(MA-ss-DOX)

[0075] (1) Preparation of macromolecular chain transfer agent mPEG-CTA Similar to Preparation Example 1, the macromolecular chain transfer agent mPEG-CTA was obtained.

[0076] (2) Preparation of DEAEMA Similar to Preparation Example 2, DEAEMA was obtained.

[0077] (3) mPEG- b Preparation of PDEAEMA Take a clean, dry 10 mL Schlenk flask and perform a vacuum-nitrogen purging process three times. Under nitrogen protection, add the weighed macromolecular chain transfer reagent mPEG-CTA (100 mg, 0.018 mmol), DEAEMA (171.3 mg, 0.92 mmol), AIBN (0.90 mg, 0.0055 mmol), and 0.5 mL of ultra-dry anhydrous DMF. Stir until dissolved. Perform a liquid nitrogen freezing-vacuuming-thawing cycle on the reaction solution three times until the reaction system is free of gas. Place the system in a 70 °C oil bath for at least 20 h. Immediately after the reaction stops, quench the reaction with liquid nitrogen. Transfer the product to a dialysis bag and dialyze it in ultrapure water for three days. Freeze-dry to obtain a white flocculent product (153 mg, yield 65%). This mPEG- b -PDEAEMA's NMR spectrum is as follows Figure 10 As shown. From Figure 10 It can be seen that mPEG- was successfully prepared via a reversible addition-fragmentation chain transfer polymerization reaction. b -PDEAEMA.

[0078] (4) Preparation of MA-ss-PNP Similar to Preparation Example 3, MA-ss-PNP was obtained.

[0079] (5) mPEG- b -PDEAEMA- bPreparation of -P(MA-ss-PNP) Take a dry and clean 10 mL Schlenk bottle, evacuate and purge with nitrogen three times, and under nitrogen protection, add the weighed macromolecular chain transfer reagent mPEG- b -PDEAEMA (180 mg, 0.026 mmol), MA-ss-PNP (525 mg, 1.34 mmol), AIBN (1.3 mg, 0.008 mmol), and 1.3 mL of ultra-dry anhydrous DMF were added. After stirring until dissolved, the reaction system was subjected to a three-cycle process of freezing, vacuuming, and nitrogen purging until no bubbles were generated. The reaction tube was then placed in a 70 °C oil bath for 40 h. After the reaction was completed, the reaction was quenched with liquid nitrogen. The reaction solution was slowly added dropwise to ice-cold ether and centrifuged to precipitate. This process was repeated twice. The collected solid powder was dried in a vacuum oven for 24 h to obtain the target polymer as a light yellow solid (280 mg, yield 85.9%). This mPEG- b -PDEAEMA- b The NMR spectrum of -P(MA-ss-PNP) is as follows: Figure 11 As shown. From Figure 11 It can be seen that mPEG- b -PDEAEMA successfully initiated a reversible addition-fragmentation chain transfer polymerization reaction of monomer MA-ss-PNP to prepare mPEG- b -PDEAEMA- b -P(MA-ss-PNP).

[0080] (6) mPEG- b -PDEAEMA- b Preparation of -P(MA-ss-DOX) Using a clean, dry 10 mL round-bottom reaction flask with a side arm, under nitrogen protection, accurately weigh out DOX•HCl (256 mg, 0.44 mmol), Et3N (44 mg, 0.44 mmol), and add 5.5 mL of ultra-dry anhydrous DMF. Stir at room temperature for 30 min to obtain the reaction substrate. Weigh out mPEG- b -P(MA-ss-PNP)- b-PDEAEMA (250 mg, 0.02 mmol) was dissolved in 2 mL of anhydrous DMF and added to a round-bottom reaction flask containing the reaction substrate. The flask was placed in an oil bath at 40 °C for 40 h under light-protected conditions. After the reaction, the reaction solution was transferred to a MWCO1000 regenerated cellulose dialysis bag and dialyzed against DMF for two days. Then, it was transferred to a MWCO3500 regenerated cellulose dialysis bag and dialyzed against deionized water for three days. Freeze-drying yielded the target polymer as a dark red solid (180 mg, yield 48.5%). This mPEG- b -PDEAEMA- b The NMR spectrum of -P(MA-ss-DOX) is as follows: Figure 12 As shown. From Figure 12 It can be seen that mPEG- b -PDEAEMA- b -P (MA-ss-PNP) and the chemotherapy drug doxorubicin (DOX) successfully achieved an amino-ester exchange reaction.

[0081] Comparative Preparation Example 1: This preparation example is used to illustrate the preparation of the GSH-single-responsive polymer mPEG- b Preparation of -P(MA-ss-DOX)

[0082] (1) Preparation of macromolecular chain transfer agent mPEG-CTA Similar to Preparation Example 1, the macromolecular chain transfer agent mPEG-CTA was obtained.

[0083] (2) Preparation of MA-ss-PNP Similar to Preparation Example 3, MA-ss-PNP was obtained.

[0084] (3) mPEG- b Preparation of -P(MA-ss-PNP) Similar to Preparation Example 5, MA-ss-PNP was obtained.

[0085] (4) mPEG- b Preparation of -P(MA-ss-DOX) Using a clean, dry 10 mL round-bottom reaction flask with a side arm, under nitrogen protection, accurately weigh out 124.7 mg (0.22 mmol) of doxorubicin hydrochloride (DOX•HCl), 43.52 mg (0.43 mmol) of Et3N, and add 5 mL of ultra-dry anhydrous DMF. Stir at room temperature for 30 min to obtain the reaction substrate. Separately weigh out mPEG- b-P(MA-ss-PNP) (200 mg, 0.022 mmol) was dissolved in 2 mL of ultra-dry DMF and added to a round-bottom reaction flask containing the reaction substrate. The reaction was carried out in an oil bath at 40 °C for 40 h, and the entire reaction system was protected from light. After the reaction, the reaction solution was transferred to a MWCO1000 regenerated cellulose dialysis bag and dialyzed against DMF for two days. Then it was transferred to a MWCO3500 regenerated cellulose dialysis bag and dialyzed against deionized water for three days. After freeze-drying, a red solid powder of the final target polymer (83.3 mg, yield 52%) was obtained. This mPEG- b The NMR spectrum of -P(MA-ss-DOX) is as follows: Figure 13 As shown. From Figure 13 It can be seen that mPEG- b -P (MA-ss-PNP) and the chemotherapy drug doxorubicin (DOX) successfully achieved an amino-ester exchange reaction.

[0086] Example 1: mPEG- b -P(DEAEMA- co Preparation of MA-ss-DOX nanomedicines.

[0087] The specific process for preparing nanomedicines using microfluidic technology is as follows: 20 mg of the random copolymer mPEG- synthesized in Preparation Example 4 was accurately weighed. b -P(DEAEMA- co The polymer solution (-MA-ss-DOX) was dispersed in 2 mL of DMF and stirred to form a homogeneous and clear solution. Using a "dropwise addition of the organic phase to the aqueous phase" method, the polymer solution was slowly added dropwise at a set flow rate (30 μL / min) to a single-necked flask containing 18 mL of phosphate buffer solution (pH 7.4, 0.01 M) using a constant-flow peristaltic pump, while the aqueous phase was kept under high-speed stirring. The mixture was stirred openly for 6 h until a significant Tyndall effect was observed in the solution. The reaction solution was then transferred to a dialysis bag with a molecular weight cutoff of 8000–14000, and phosphate buffer was used as the dialysis medium to remove the DMF organic solvent. Finally, the dialysis nanomedicine solution was concentrated using an ultrafiltration centrifuge tube to obtain the polymer self-assembled nanomedicine, named PD-1. This nanomedicine had a particle size of 89.14 nm, a potential of 5.31 mV, and an doxorubicin (DOX) loading of 17.3%.

[0088] Example 2: This example illustrates mPEG- b -P(MA-ss-DOX)- b -Preparation of PDEAEMA nanomedicines.

[0089] Nanomedicines were prepared according to the method of Example 1, except that the random copolymer mPEG- obtained in Preparation Example 4 was used. b -P(DEAEMA- co -MA-ss-DOX) uses the same weight parts of the block copolymer mPEG- obtained from Preparation Example 5. b -P(MA-ss-DOX)- b With PDEAEMA substituted and all other conditions remaining the same as in Example 1, a polymer self-assembled nanomedicine, named PD-2, was obtained. This nanomedicine had a particle size of 70.07 nm, a potential of 2.23 mV, and a doxorubicin (DOX) loading of 19.7%.

[0090] Example 3: This example illustrates mPEG- b -PDEAEMA- b Preparation of -P(MA-ss-DOX) nanomedicines.

[0091] Nanomedicines were prepared according to the method of Example 1, except that the random copolymer mPEG- obtained in Preparation Example 4 was used. b -P(DEAEMA- co -MA-ss-DOX) uses the same weight parts of the block copolymer mPEG- obtained from Preparation Example 6. b -PDEAEMA- b -P(MA-ss-DOX) was substituted, and the other conditions were the same as in Example 1, to obtain a polymer self-assembled nanomedicine, named PD-3. The nanomedicine had a particle size of 88.72 nm, a potential of 4.26 mV, and an adriamycin (DOX) loading of 15.8%.

[0092] Comparative Example 1: This example illustrates the use of GSH-single-response mPEG- b Preparation of -P(MA-ss-DOX) nanomedicines.

[0093] GSH-single-responsive nanomedicines were prepared according to the method of Example 1, except that the random copolymer mPEG- obtained in Preparation Example 4 was used instead. b -P(DEAEMA- co -MA-ss-DOX) used the same parts by weight of the polymer mPEG- synthesized from Control Preparation Example 1. b -P(MA-ss-DOX) was substituted, and all other conditions were the same as in Example 1, to obtain a polymer self-assembled nanomedicine, named GD-1. The nanomedicine had a particle size of 44.9 nm, a potential of -7.68 mV, and an doxorubicin (DOX) loading of 14.3%.

[0094] Test Example 1: This test example aims to illustrate the pH-responsive charge change properties of different nanomedicines. Accurately transfer 5 mL of GD-1, PD-1, PD-2, and PD-3 nanomedicine solutions into clean centrifuge tubes. Using a pipette, slowly and dropwise add 0.01 M dilute hydrochloric acid solution to each nanomedicine solution, monitoring the pH change in real time. By controlling the amount of dilute hydrochloric acid added, the pH of each nanomedicine solution was precisely adjusted to 7.4 and 6.5. The zeta potential of the nanomedicine solutions at each pH value was measured using a nanoparticle size and zeta potential analyzer. The results are shown below. Figure 14 As shown in the figure, at pH 7.4, the potential of the GSH-single-response nanodrug GD-1 is -7.68 mV, while the potentials of the pH / GSH spatiotemporal cascade response nanodrugs PD-1, PD-2, and PD-3 are 5.31 mV, 2.23 mV, and 4.26 mV, respectively. Furthermore, as the pH decreases from 7.4 to 6.5, the potential of nanodrug GD-1 remains negative at -7.76 mV, while the potentials of PD-1, PD-2, and PD-3 change to 6.22 mV, 4.03 mV, and 8.93 mV, respectively, showing a trend of gradually increasing potentials as the pH decreases.

[0095] Test Example 2: This test example aims to illustrate the drug release behavior of pH / GSH spatiotemporal cascade-responsive antitumor nanomedicines at different pH and GSH concentrations. (1) Plotting the DOX standard curve Under light-protected conditions, different masses of doxorubicin hydrochloride were accurately weighed and dissolved in 0.01 M phosphate buffer (pH 6.5). These solutions were then progressively diluted to prepare a series of standard solutions with concentration gradients of 40, 20, 10, 5, 2.5 μg / mL, and 1.25 μg / mL. Using pure PBS buffer as a blank reference, the UV absorption spectra of each concentration of standard solutions were scanned in the wavelength range of 300–700 nm, and the absorbance values ​​at the characteristic wavelength of 480 nm were recorded. An absorbance-concentration standard curve was plotted with the DOX concentration in the standard solution as the x-axis and the corresponding absorbance value at 480 nm as the y-axis.

[0096] (2) Investigation on the in vitro release characteristics of chemotherapy drugs The in vitro drug release behavior of pH / GSH spatiotemporal cascade-responsive antitumor nanomedicines was studied using dialysis at pH 6.5 and different GSH concentrations (0 mM and 10 mM). The release medium was a 0.01 M PBS buffer solution at pH 6.5 containing 0.5% Tween 80. The specific procedure was as follows: 2 mL of nanomedicine solution was packaged into a dialysis bag with a molecular weight cutoff of 8000–14000. The dialysis bag was then immersed in a 15 mL centrifuge tube containing the release medium. The tube was then incubated in a constant temperature shaker at 37 °C and 100 r / min, under light-protected conditions throughout. At predetermined time points, an appropriate amount of dialysis fluid was drawn from the centrifuge tube, and an equal volume of fresh buffer solution was added to maintain system volume stability. The DOX content in the samples was tested using a UV-Vis spectrophotometer. The cumulative release curve of chemotherapy drug DOX over time was plotted with release time as the x-axis and the total cumulative release of chemotherapy drugs as the y-axis. Three parallel samples were set up for each experiment.

[0097] The in vitro release curves of DOX for GD-1, PD-1, PD-2, and PD-3 nanomedicines at pH 6.5 and 0 mM GSH are shown below. Figure 15 As shown in the figure, the cumulative release of DOX from GD-1, PD-1, PD-2, and PD-3 nanomedicines within 72 hours was only about 13.8%, 17.5%, 15.1%, and 15.8%, respectively. This indicates that the leakage caused by drug-dependent simple diffusion is low. This characteristic can effectively reduce the toxic side effects of nanomedicines on normal tissues and cells when circulating in the blood, providing important support for their biosafety. The in vitro release curves of DOX for GD-1, PD-1, PD-2, and PD-3 nanomedicines at pH 6.5 and 10 mM GSH are shown below. Figure 16 As shown in the figure, the cumulative release of DOX by GD-1 nanomedicine within 72 hours is approximately 56.4%, while the cumulative release of DOX by PD-1, PD-2, and PD-3 nanomedicines within the same 72 hours reaches 78.1%, 75.0%, and 82.7%, respectively. This indicates that the nanomedicine can successfully release the chemotherapy drug DOX in the presence of GSH. Furthermore, the total cumulative release of PD-1, PD-2, and PD-3 nanomedicines is higher than that of GD-1 nanomedicine, and their release rate is also much faster than that of GD-1 nanomedicine in the initial period. This highlights the drug release advantage of pH / GSH spatiotemporal cascade responsive antitumor nanomedicines PD-1, PD-2, and PD-3 compared to GSH single-responsive antitumor nanomedicine GD-1 under the same drug release conditions.

[0098] Test Example 3: This test example aims to illustrate the quantitative analysis of cellular uptake of different nanomedicines using flow cytometry. A549 cells in the logarithmic growth phase were seeded at a density of 150,000 cells / well into 6-well plates and cultured in a constant temperature incubator at 37 ℃ and 5% CO2. After observing complete cell adhesion, the medium in each well was replaced with fresh medium containing different nanomedicines (DOX concentration set at 5 μg / mL), and the cells were incubated together for 4 h. The supernatant was collected into a centrifuge tube, washed three times with cold PBS, digested with trypsin, washed three times with PBS, and resuspended in PBS for flow cytometry analysis.

[0099] Flow cytometry was used to quantitatively analyze the uptake of different nanomedicines by A549 cells using cellular fluorescence uptake, such as... Figure 17 As shown in the figure, under pH 6.5 conditions and the same incubation time, the fluorescence intensities of PD-1, PD-2, and PD-3 nanomedicines were 1.8 times, 1.56 times, and 2.23 times that of the GD-1 nanomedicine group, respectively. The uptake of PD-1, PD-2, and PD-3 nanomedicines by A549 cells was significantly higher than that by GD-1 nanomedicine. This indicates that under the slightly acidic pH 6.5 conditions of tumor cells, the positive charge of PD-1, PD-2, and PD-3 can generate electrostatic interactions with the negatively charged cell membrane, thereby enhancing the efficient uptake of PD-1, PD-2, and PD-3 by tumor cells.

[0100] Test Example 4: This test example aims to illustrate the cytotoxicity experiments of different nanomedicines. Human non-small cell lung cancer cells (A549) in good growth condition were seeded into 96-well plates at an initial cell density of 5000 cells / well. The outer wells of the plate were discarded, and PBS was added instead. The plates were incubated at 37 °C with 5% CO2. Once the cells had adhered and grown to approximately 70% confluence with the culture dish, the original culture medium was aspirated, and fresh culture medium containing different groups of PD-1, PD-2, PD-3, and GD-1 nanomedicine solutions was added. The nanomedicine concentrations (calculated based on DOX equivalents) were 64, 32, 16, 8, 4, 2, 1, and 0.5 μM, with five replicates for each concentration. The plates were then quickly placed in the incubator. After a period of time, the 96-well plates were replaced with 10 μL of MTT solution in each well, and incubation continued for another 4 h. Next, 100 μL of Formazan dissolving solution was added to each well, and the cells were incubated in an incubator until complete dissolution of the formazan was observed under a microscope. The absorbance (OD) of each well at 570 nm was measured using a microplate reader. The culture medium used was adjusted to pH 6.5 with 0.01 M HCl solution. Cell viability was calculated using the following formula:

[0101] OD test OD blank and OD control These are the absorbance values ​​for the experimental group, the zeroing group, and the negative control group, respectively.

[0102] A study on cell survival rate of A549 cancer cells after 24 hours of treatment with different nanomedicine groups, as follows: Figure 18 As shown, the half-maximal inhibitory concentration (IC50) of the GD-1 group of nanomedicines is... 50 The half-maximal inhibitory concentration (IC50) was 19.32 μM, while the IC50 of the nanomedicines in the PD-1, PD-2, and PD-3 groups was... 50 The concentrations were 12.55 μM, 15.76 μM, and 11.11 μM, respectively, all lower than the IC50 of the GD-1 group. 50 The results indicate that the pH / GSH spatiotemporal cascade responsive antitumor nanomedicines PD-1, PD-2, and PD-3 are more cytotoxic than the GSH-single-responsive antitumor nanomedicine GD-1. This is because, compared to the single-GSH responsive GD-1 nanomedicine, which has no pH-responsive properties and a negative surface charge, resulting in limited cellular uptake, the PD-1, PD-2, and PD-3 nanomedicines first respond to low pH conditions, converting their surface charge to a positive charge, and are efficiently taken up by A549 cancer cells. After being taken up by the cells, the nanomedicines respond to the highly expressed GSH in the cells, thereby releasing the chemotherapeutic drug DOX, inhibiting cancer cell division, inducing apoptosis, and exhibiting a significant effect in inhibiting cancer cell growth.

[0103] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine, characterized in that, The pH / GSH spatiotemporal cascade responsive antitumor nanomedicine is formed by self-assembly of a polymer prodrug having the structure shown in formula (1); Equation (1) In formula (1), R1 and R2 are alkyl groups or cyclic molecular groups; R3 is a disulfide bond, diselenide bond or thioether bond; R4 is a group containing a dithioester group or a trithioester group; R is an anticancer chemotherapy drug source; m is an integer from 10 to 400, n is an integer from 5 to 200, and x is an integer from 1 to 200.

2. The pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to claim 1, characterized in that, The anticancer chemotherapy drug source corresponds to an anticancer chemotherapy drug containing hydroxyl and / or amino groups; Preferably, the anticancer chemotherapy drug is selected from at least one of doxorubicin, epirubicin, paclitaxel, camptothecin, and vincristine.

3. The pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to claim 1, characterized in that, The pH / GSH spatiotemporal cascade responsive antitumor nanomedicine has a particle size of 30 nm to 150 nm, a zeta potential of -15 mV to 20 mV, and a chemotherapy drug loading of 10% to 40%.

4. The method for preparing the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to any one of claims 1 to 3, characterized in that, The method includes the following steps: under light-protected conditions, a polymer prodrug is dissolved in an organic solvent to obtain an organic phase; the organic phase is mixed with an aqueous phase using a microfluidic device; and a pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine is assembled using a nanoprecipitation method based on microfluidic technology.

5. The method for preparing the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to claim 4, characterized in that, The polymer prodrug is prepared by the following method: using mPEG-R4 as shown in formula (2) as a macromolecular chain transfer agent, under the action of an initiator, the pH-responsive tertiary amine monomer shown in formula (3) and the reduced glutathione-responsive monomer shown in formula (4) are initiated to undergo RAFT polymerization, thereby linking the pH-responsive tertiary amine structural unit and the reduced glutathione-responsive structural unit to the hydrophilic segment of polyethylene glycol in a random copolymerization or block copolymerization manner. Then, the obtained polymer is subjected to ester exchange reaction with a hydroxyl-containing chemotherapeutic drug or to amino-containing chemotherapeutic drug to obtain a pH / GSH spatiotemporal cascade responsive polymer prodrug. Equation (2), Equation (3), Equation (4), In formula (2), m is an integer from 10 to 400; R4 is a group containing a dithioester group or a trithioester group; In formula (3), R1 and R2 are alkyl groups or cyclic molecular groups; In formula (4), R3 is a disulfide bond, a diselenide bond, or a thioether bond.

6. The method for preparing pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to claim 5, characterized in that, The molar ratio of the macromolecular chain transfer agent to the initiator is 1:(0.2~0.5). Preferably, the macromolecular chain transfer agent is generated by esterification of polyethylene glycol monomethyl ether with RAFT chain transfer agent; Preferably, the RAFT chain transfer agent contains a dithioester group or a trithioester group; Preferably, the RAFT chain transfer agent is selected from at least one of 4-cyano-4-[[(dodecylthio)thionylmethyl]thio]valeric acid, 4-cyano-4-(phenylthiocarbamoylthio)valeric acid, 4-cyano-4-(propyltrithiocarbonate)valeric acid, and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid; Preferably, the initiator is at least one of azo initiators, peroxide initiators, and redox initiators.

7. The method for preparing the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to claim 5, characterized in that, The reduced glutathione responsive monomer is prepared by the following method: the diol shown in formula (5) is subjected to a first nucleophilic substitution reaction with methacrylamide, and then the resulting diol methacrylate is subjected to a second nucleophilic substitution reaction with 4-nitrophenyl chloroformate to obtain the reduced glutathione responsive monomer. Equation (5) In formula (5), R3 is a disulfide bond, a diselenide bond, or a thioether bond.

8. The method for preparing the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to claim 4, characterized in that, The aqueous phase is a phosphate buffer solution; the organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide.

9. The method for preparing the pH / GSH spatiotemporal cascade-responsive antitumor nanomedicine according to claim 4, characterized in that, The mixing rate of the aqueous phase and the organic phase is 10~50 μL / min; the concentration of the polymer prodrug in the organic phase is 0.1~10 mg / mL.

10. The use of the pH / GSH spatiotemporal cascade responsive antitumor nanomedicine according to any one of claims 1 to 3 in the preparation of a drug for treating tumor diseases.

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