A multiple sensitive non-cationic nanogel, and a preparation method and application thereof

Abstract

The application relates to a multiple-sensitivity non-cationic nanogel as well as a preparation method and application thereof, and belongs to the technical field of medicines.The multiple-sensitivity non-cationic nanogel comprises the following components: a) DNA-miRNA hybrid; b) an amphiphilic monomer, the amphiphilic monomer is selected from one or more of polymethyl methacrylate diisopropyl amino ethyl ester-poly(3-[(3-acrylamide propyl) dimethyl ammonium] acetate-dimethyl acetyl-methacrylamide, acrylate-based polyethylene glycol-poly caprolactone, acrylate-based polyethylene glycol-polystyrene; c) gemcitabine-monomer conjugate; and d) a crosslinking agent, which can realize efficient targeted release of the DNA-miRNA and the gemcitabine in tumor tissues.

Description

Technical Field

[0001] This application relates to a multi-sensitivity non-cationic nanogel, its preparation method, and its application, belonging to the field of pharmaceutical technology. Specifically, it relates to a non-cationic nanogel delivery material with enzyme responsiveness, glutathione responsiveness, and pH responsiveness, and its preparation method, for synergistically encapsulating functional nucleic acid complexes with hydrophilic chemotherapeutic drugs. Background Technology

[0002] The combined application of gene therapy and chemotherapy holds promise for overcoming tumor drug resistance and improving cancer treatment outcomes. Among these approaches, utilizing functional nucleic acids (such as siRNA and miRNA) to regulate the expression of tumor-related genes to enhance the sensitivity of tumor cells to chemotherapeutic drugs has attracted significant attention. However, achieving efficient and safe co-delivery of nucleic acid drugs and small-molecule chemotherapeutic drugs remains a challenge, with the key lying in the design and optimization of the delivery vector.

[0003] Currently, the delivery of biomolecules mainly relies on viral and non-viral vectors. While viral vectors have high transfection efficiency, they suffer from potential immunogenicity, limited loading capacity, and complex preparation, thus restricting their clinical application. Therefore, non-viral delivery vectors, especially nanocarriers based on polymers or lipids, have become a research hotspot. However, existing non-viral delivery strategies, particularly in the co-delivery of nucleic acids and drugs, still face several challenges: 1) The inherent toxicity of cationic carriers and the biosafety issues related to inflammatory responses. Currently, most materials used for nucleic acid delivery are cationic (such as polyethyleneimine (PEI), cationic liposomes, etc.). These materials combine with nucleic acid molecules to form nanocomposites through electrostatic interactions of positive and negative charges. To ensure the stability of this complex in the bloodstream, it is usually prepared at a high nitrogen-to-phosphorus ratio (N / P), resulting in an excessive amount of positive charge remaining on the surface of the nanoparticles and a large amount of free cationic material in the system, which becomes the main source of cytotoxicity and potential inflammatory responses. These excess positive charges are also rapidly cleared by the reticuloendothelial system, such as the liver, affecting tumor targeting. Although existing studies have attempted to mitigate the toxic side effects of cationic compounds through strategies such as linking cationic groups with acid-sensitive bonds or introducing cross-links after compounding to neutralize charges, these methods are often complex and cannot fundamentally avoid the inherent biosafety risks of cationic components. Therefore, developing a novel nucleic acid loading material that is completely independent of cationic components and electrostatic interactions is crucial for improving therapeutic efficacy and safety. 2) Stability and reproducibility of the carrier complex. Nucleic acid loading based on electrostatic interactions is a self-assembly process involving complex kinetics and thermodynamics: there is both short-range electrostatic attraction between nucleic acids and cationic materials and long-range electrostatic repulsion between nucleic acid molecules, which compete with each other. This process is affected by various factors, such as the N / P ratio, the chemical properties of the material itself (charge density, hydrophobicity, molecular weight), mixing sequence, speed, and environment (such as pH, ionic strength). This complexity leads to the formed complex not being in the most stable thermodynamic state, and it is prone to secondary aggregation, phase separation, or drug leakage during storage or in vivo circulation, manifested as increased particle size, wider distribution, and decreased drug loading. This instability makes it difficult to precisely control and repeat the preparation process, directly affecting the consistency between different batches and the final therapeutic effect. 3) Problems with poor drug encapsulation synergy and difficulty in particle size control in co-delivery systems. Encapsulating nucleic acid molecules with different physicochemical properties alongside hydrophilic small-molecule chemotherapeutic drugs (such as gemcitabine and doxorubicin hydrochloride) in the same carrier is challenging. Traditional nanocarriers based on hydrophobic cores (such as micelles) are difficult to efficiently encapsulate hydrophilic drugs; while carriers based on electrostatic interactions are difficult to efficiently encapsulate both electrically neutral and weakly charged small-molecule drugs without affecting nucleic acid loading efficiency.Furthermore, the particle size and distribution of drug-loaded nanoparticles have a significant impact on their in vivo pharmacokinetic behavior (such as blood circulation time, tumor targeting and enrichment capacity, and tissue penetration depth): an appropriate particle size (typically 10-200 nm) is beneficial for passive targeting through the high permeability and retention effect (EPR effect) of solid tumors; while a uniform particle size distribution is key to ensuring consistent pharmacokinetic behavior and improving therapeutic efficacy. In existing technologies, whether self-assembled complexes or microparticles prepared using methods such as double emulsion, the particle size and uniformity are often difficult to control precisely, usually requiring cumbersome post-processing steps (such as sonication, homogenization, and extrusion), and the processing effect is limited. 4) Problems with existing nanogel technologies in nucleic acid encapsulation. Inverse microemulsion polymerization (IEP) is an effective method for preparing nanogels, which can carry out polymerization reactions in nanoscale "water cores," theoretically providing an ideal environment for encapsulating hydrophilic drugs (including nucleic acids). However, existing technologies using this method to prepare nanogels for nucleic acid delivery generally suffer from low encapsulation efficiency. The fundamental reason lies in the fact that in conventional reverse microemulsion polymerization, the polymerization reaction is initiated at multiple points within the aqueous phase of the emulsion droplet, lacking directional reinforcement of the oil-water interface. This results in a loose or heterogeneous cross-linked network structure, which easily "squeezes out" the bulky and structurally rigid nucleic acid molecules from the water core during gelation, thus failing to achieve efficient encapsulation. Currently, there is a lack of reverse microemulsion polymerization nanogel systems that can effectively solve this problem and achieve efficient synergistic encapsulation of nucleic acids and chemotherapeutic drugs.

[0004] Chinese patent application 202110853382.2 discloses a GSH-responsive gemcitabine nanoparticle, its preparation method, and its application. The provided GSH-responsive gemcitabine nanoparticles exhibit good blood compatibility, can inhibit the proliferation of B-cell lymphoma cells, and possess redox responsiveness, enabling controlled release of gemcitabine under stimulation from a high-concentration GSH tumor microenvironment, thereby improving the drug's efficacy against tumors and reducing toxic side effects. Chinese patent application 201910945589.5 discloses a gemcitabine-containing drug, its preparation method, pharmaceutical composition, and its application. It discloses a drug containing gemcitabine and nucleic acid nanoparticles. The nucleic acid domains of the gemcitabine-containing drug, after target modification, exhibit good targeting properties and can stably deliver gemcitabine with high reliability. However, this patent does not provide extensive research on the entire drug system, especially the dosage form, regarding its effect on drug release.

[0005] Based on the problems existing in the current technology, there is an urgent need to develop a new type of nanodelivery platform. This platform should be able to: 1) eliminate cationic components and solve the toxicity problem; 2) provide a new mechanism for efficient and stable nucleic acid loading that does not rely on electrostatic interactions; 3) achieve efficient synergistic encapsulation and controllable release of nucleic acids and hydrophilic small molecule chemotherapeutic drugs; and 4) have a simple and controllable preparation process that can produce nanoparticles with uniform particle size and stable performance. Summary of the Invention

[0006] The present invention aims to overcome the above-mentioned defects of the prior art and solve the following technical problems: 1) Overcoming cationic toxicity: Providing a non-cationic co-delivery carrier with high biosafety and low cytotoxicity to replace traditional cationic carriers.

[0007] 2) Achieve efficient co-encapsulation: Solve the problem that nucleic acids and hydrophilic small molecule chemotherapy drugs are difficult to be efficiently encapsulated by the same carrier due to their different physicochemical properties.

[0008] 3) Achieve intelligent targeted release: Construct a delivery system that can trigger drug release in response to specific stimuli of the tumor microenvironment (such as acidic pH, high concentration of glutathione, specific enzymes), thereby improving treatment specificity and reducing off-target toxicity.

[0009] 4) Simplified preparation process: A relatively simple and reproducible method for preparing nanogels is provided.

[0010] To address the aforementioned issues, a multi-sensitivity non-cationic nanogel, its preparation method, and its applications are provided. Through specific material design and polymerization methods, it achieves efficient co-encapsulation of nucleic acid drugs and small molecule chemotherapeutic drugs, responsive release in the tumor microenvironment, and synergistic anti-tumor effects, demonstrating significant technological advantages compared to existing technologies.

[0011] This application provides a multi-sensitivity non-cationic nanogel, comprising: a) DNA-miRNA hybrid; b) An amphiphilic monomer, wherein the amphiphilic monomer is selected from one or more of the following: poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methacrylamine, acrylate-based polyethylene glycol-polycaprolactone, and acrylate-based polyethylene glycol-polystyrene; c) Gemcitabine-monomer conjugate, wherein the structural formula of the gemcitabine-monomer conjugate is as follows: ; d) Crosslinking agent.

[0012] The amphiphilic monomers and gemcitabine monomer conjugates and asparagine endopeptidase-responsive crosslinking agents selected in this application are used to prepare nanogels loaded with gemcitabine and Tw / let-7c DNA-miRNA complexes via reverse emulsion polymerization, which have good drug-targeted release effects.

[0013] Optionally, the amphiphilic monomer is poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methacrylamine), with the following structural formula: .

[0014] The nanogel prepared from poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methacrylamine) exhibits better performance than the acrylate-based polyethylene glycol-polycaprolactone and acrylate-based polyethylene glycol-polystyrene nanogels. The latter two groups both possess moderately uniform particle sizes (66.4±3.6 nm and 64.9±5.2 nm, respectively), and their loading efficiency for Tw / let-7c is also more ideal (the measured encapsulation efficiencies are 77.6%). (±4.6% and 88.5±3.3%), but in the in vitro simulated release experiment, under pH 5.0 incubation conditions (simulating the physiological environment of tumor cell lysosomes), the Tw / let-7c release rates of the two groups of particles after 24 h were only 62.2±2.1% and 49.4±3.2%, respectively, which were lower than the release efficiency when using poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methylpropenamine as an amphiphilic monomer.

[0015] The hydrophobic segments of acrylate-based polyethylene glycol-polycaprolactone and acrylate-based polyethylene glycol-polystyrene do not have multi-level amine structures, while the hydrophobic segments of poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methacrylamine have tertiary amines, which can respond to acid stimulation and exhibit a proton sponge effect, thereby destroying the nanogel structure and making it more conducive to the release of Tw / let-7c.

[0016] Optionally, the m:n ratio in the amphiphilic monomer is 2:(1~4). Optionally, the m:n ratio in the amphiphilic monomer is 2:(2~4).

[0017] Optionally, the weight ratio of the gemcitabine-monomer coupling compound, the crosslinking agent, and the amphiphilic monomer is (3~5):(1~3):(1~3). Optionally, the weight ratio of the gemcitabine-monomer coupling agent, crosslinking agent, and amphiphilic monomer is 4:2:2.

[0018] It should be noted that those skilled in the art can, as needed, replace the linking bonds in the gemcitabine-monomer conjugate with other chemical bonds that can be broken in highly reactive chemical environments within tumor cells, such as acidic pH and reactive oxygen species (ROS). Those skilled in the art can also, as needed, replace gemcitabine with other drug options.

[0019] Optionally, the preparation method of the gemcitabine-monomer conjugate includes: S1. Dissolve triethylamine and 2,2'-dithiodiethanol in tetrahydrofuran, and add anhydrous THF solution of methacryloyl chloride dropwise at 1~10℃. After the addition is complete, restore the system to 20~35℃ and stir for 6~24h. After purification, ethyl 2-((2-hydroxyethyl)dimercapto)methacrylate is obtained. S2. Dissolve the obtained ethyl 2-((2-hydroxyethyl)dimercapto)methacrylate in chloroform, add 4-dimethylaminopyridine and triphosgene, and stir to react. S3. Dissolve the solid obtained from the reaction in N,N-dimethylformamide, add gemcitabine to react, and after purification, the gemcitabine-monomer conjugate is obtained.

[0020] Without using gemcitabine monomer conjugates, the encapsulation efficiencies of the prepared nanogels for Tw / let-7c and gemcitabine were 55.8±2.1% and 30.3±3.3%, respectively, which were far lower than those obtained using the gemcitabine-monomer conjugates in this application.

[0021] Optionally, the method for preparing the amphiphilic monomer includes: S1. 1-Dodecyl-(dimethylacetic acid)trithiocarbonate, diisopropylaminoethyl methacrylate, and azobisisobutyronitrile were dissolved in a DMF / water mixed solvent and reacted under nitrogen protection at 70±10℃ for 12~48h. Then, 3-[(3-acrylamidopropyl)dimethylammonium]acetate was added to continue the reaction. After the reaction was completed, the mixture was cooled to room temperature and azobisisobutyronitrile was added again. The reaction was continued under nitrogen protection at 70±10℃ for 12~48h. After purification, a white solid product was obtained. S2. Take the obtained white solid product and dissolve it in dimethyl sulfoxide with 3-methylacrylamide, N-hydroxysuccinimide and N,N-dicyclohexylcarbodiimide, and react for 12~48h. After purification, the amphiphilic monomer is obtained.

[0022] Optionally, the DNA-miRNA hybrid is a bifunctional complex with a tweezer-like structure formed by a single-stranded DNA that can bind to miR-21 and a tumor suppressor miRNA through complementary base pairing.

[0023] Optionally, the structure of the DNA-miRNA hybrid is as follows: .

[0024] Optionally, the crosslinking agent is an asparagine endopeptidase-sensitive crosslinking agent.

[0025] Optionally, the asparagine endopeptidase-sensitive cross-linking agent has the following structural formula: .

[0026] The proposed solution employs an asparagine endopeptidase-sensitive cross-linking agent, which achieves a good targeted release effect.

[0027] Optionally, the asparagine endopeptidase-sensitive crosslinking agent includes a polypeptide sequence of KGAANLGK or KGAAAGK.

[0028] Optionally, the asparagine endopeptidase-sensitive cross-linking agent comprises a polypeptide sequence of KGAANLGK.

[0029] Using a polypeptide with the sequence KGAAAGK as raw material, carbon-carbon double bonds were introduced through the reaction sites at both ends to prepare a cross-linking agent. The nanogel obtained by the scheme of this application and the nanogel prepared by the same method using KGAANLGK were compared in an in vitro simulated release experiment. It was found that at pH 5.0 and in the presence of GSH (glutathione) and Legumin (asparagine endopeptidase), the cumulative Tw / let-7c release rate of the KGAAAGK scheme in 24h was only 29.8±3.3%, which is much lower than the release efficiency when the KGAANLGK scheme is used as the cross-linking agent.

[0030] This application provides a method for preparing the above-mentioned multi-sensitivity non-cationic nanogel, the method comprising the following steps: 1) Dissolve the surfactant in the solvent n-hexane to obtain the organic phase; 2) After dissolving the amphiphilic monomer, gemcitabine-monomer conjugate, and cross-linking agent in a DMSO / DEPC aqueous solution, the DNA-miRNA hybrid was added to obtain the aqueous phase; 3) Under stirring, the aqueous phase is added to the organic phase, followed by the addition of initiator and accelerator to carry out the polymerization reaction; 4) After purification, the multi-sensitivity non-cationic nanogel is obtained.

[0031] Optionally, the initiator is selected from one or more of APS, TEMED, ammonium persulfate, sodium sulfite, and azobisisobutyrazoline hydrochloride; Optionally, the surfactant is selected from one or more of AOT, Span80, polyoxyethylene lauryl ether (Brij30), and potassium monododecyl phosphate (MAEPK); This application provides the application of the aforementioned multi-sensitivity non-cationic nanogels in the preparation of antitumor drugs.

[0032] The beneficial effects of this application include, but are not limited to: 1. The multi-sensitivity non-cationic nanogels of this application, their preparation methods and applications, are nanogels copolymerized from amphiphilic monomers, enzyme-sensitive crosslinking agents and drug-monomer conjugates. Using a non-cationic nanogel carrier system without cationic components, cationic toxicity can be fundamentally avoided.

[0033] 2. Based on the multi-sensitivity non-cationic nanogel, its preparation method, and its application, a co-encapsulation strategy of two drugs on one carrier is adopted. By combining physical encapsulation (nucleic acid) with covalent bonding (small molecule drug), two drugs with different physicochemical properties are simultaneously and efficiently loaded in a non-cationic nanogel to achieve synergistic therapeutic effects.

[0034] 3. Based on the multi-sensitivity non-cationic nanogel, its preparation method, and its application, the structure design of the nanogel enables it to respond sequentially or simultaneously to the acidic pH, specific enzyme (asparagine endopeptidase), and high concentration of reducing GSH in the tumor microenvironment, thereby achieving precise targeted release and synergistic and efficient release of drugs. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the structure of Tw / let-7c involved in Embodiment 1 of this application.

[0036] Figure 2 This is a schematic diagram illustrating the mechanism of action of the Tw / let-7c hybrid involved in Example 1 of this application.

[0037] Figure 3 The fluorescence spectra of the 200 nM Tw / let-7c hybrid involved in Example 1 of this application after incubation with different concentrations of miR-21 (Tw labeled BHQ1, let-7c labeled Cy3).

[0038] Figure 4The fluorescence spectra of the 200nM Tw / let-7c hybrid and 200nM miR-21 after incubation for different times in Example 1 of this application (Tw labeled BHQ1, let-7c labeled Cy3).

[0039] Figure 5 The fluorescence spectrum of the 200nM Tw / let-7c hybrid involved in Example 1 of this application after incubation with different miRNAs (200nM) for 60 min (Tw labeled BHQ1, let-7c labeled Cy3).

[0040] Figure 6 This is a synthetic route diagram of the CBPD involved in Embodiment 1 of this application.

[0041] Figure 7 The ¹H NMR spectrum of the CBPD monomer involved in Example 1 of this application is shown.

[0042] Figure 8 This is a synthetic route diagram of the gestigmine-containing reduction-sensitive monomer (GEMSS) involved in Example 1 of this application.

[0043] Figure 9 The ¹H NMR spectrum of the GEMSS monomer involved in Example 1 of this application is shown.

[0044] Figure 10 This is a synthetic route diagram of the asparagine endopeptidase-sensitive crosslinking agent (LEGCL) involved in Example 1 of this application.

[0045] Figure 11 The ¹H NMR spectrum of the LEGCL crosslinking agent involved in Example 1 of this application is shown.

[0046] Figure 12 The particle size distribution and zeta potential diagrams of nanogels prepared with different feed ratios according to Example 2 of this application are shown.

[0047] Figure 13 The diagram shows the encapsulation efficiency of Tw / let-7c by nanogels prepared with different feed ratios according to Example 2 of this application.

[0048] Figure 14 The diagram shows the encapsulation efficiency of gemcitabine in nanogels prepared with different feed ratios as described in Example 2 of this application.

[0049] Figure 15 Example 3 of this application shows the preparation (a) of the drug-loaded nanogel and its working schematic diagram (b).

[0050] Figure 16 The in vitro release curves of Tw / let-7c under different treatment conditions involved in Test Example 1 of this application are shown.

[0051] Figure 17 The in vitro release curves of gemcitabine under different treatment conditions involved in Test Example 1 of this application are shown.

[0052] Figure 18 The uptake effect of SK-Hep1 cells treated with NG@SCR, PEG-PEI@Tw / let-7c and NG@Tw / let-7c was detected by CLSM in Test Example 2 of this application.

[0053] Figure 19 This study examines the viability of SK-Hep1 cells after treatment with different groups of nanogels, as described in Test Example 3 of this application.

[0054] Figure 20 Tumor volume curves for different groups of mice involved in Test Example 4 of this application.

[0055] Figure 21 These are representative images of Bcl-2 immunohistochemical staining in different groups of tumor tissues involved in Test Example 4 of this application.

[0056] Figure 22 The viability of THLE-2, SK-Hep1, and Hep3B cells after treatment with different concentrations of blank nanogels, as described in Example 5 of this application, was tested.

[0057] Figure 23 HE-stained images of tissue sections of major organs treated with nanogel, as described in Test Example 5 of this application. Detailed Implementation

[0058] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0059] This invention provides a method for preparing a non-cationic nanogel for the synergistic delivery of DNA-miRNA hybrids and gemcitabine, hereinafter referred to as "co-delivery nanogel" or "NG@Tw / let-7c / Gem". This technology aims to achieve efficient co-encapsulation of nucleic acid drugs and small molecule chemotherapeutic drugs, tumor microenvironment-responsive release, and synergistic anti-tumor effects through specific material design and polymerization methods. The specific technical solution is as follows: Example 1 Key Components of Nanogels 1) Design of the intelligent DNA-miRNA hybrid (Tw / let-7c) This invention first designed and constructed a smart DNA-miRNA hybrid with dual gene regulation function, named Tw / let-7c, the structure of which is shown in the figure below. Figure 1 As shown.

[0060] This hybrid consists of a single-stranded DNA strand (called the "Tw" strand) and a tumor suppressor microRNA (miRNA, let-7c) that form a bifunctional complex with a "tweezers"-like structure through base partial complementary pairing. The Tw strand contains two functional regions: a binding region (loop region) designed to be completely complementary to the oncogenic miR-21 overexpressed in hepatocellular carcinoma (HCC); and a release region (stem region) designed to be partially complementary to the let-7c sequence, with several (e.g., 4) unpaired nucleotides intentionally reserved.

[0061] After entering tumor cells, the dynamic response mechanism of this hybrid is as follows: Figure 2 As shown, the working principle includes the following processes: 1) Target recognition: Overexpressed miR-21 in the cell binds to the binding region of the Tw chain through complete base complementarity pairing. 2) Competitive substitution and dissociation: Since the pairing of the Tw chain and let-7c is metastable, the fully complementary miR-21 has a stronger binding energy to the Tw chain, thus competitively displacing let-7c from the hybrid, leading to the dissociation of the Tw / let-7c hybrid. This process was verified by fluorescence resonance energy transfer (FRET) experiments. Figure 3 , Figure 4 and Figure 5 3) Dual gene regulation: After dissociation, the released let-7c exerts its tumor suppressor function; at the same time, the Tw chain binds stably to miR-21, inhibiting the activity of oncogenic miR-21.

[0062] This process achieves dual gene regulation by simultaneously inhibiting the oncogene (miR-21) and supplementing the tumor suppressor (let-7c). Those skilled in the art can refer to the protocol disclosed in Smartly responsive DNA-miRNA hybrids packaged inexosomes for synergistic enhancement of cancer cell apoptosis (DOI:10.1039 / d1nr08539e) for preparation or modification.

[0063] 2) Composition and structure of non-cationic nanogels To achieve efficient co-encapsulation and stimulus-responsive release of Tw / let-7c and gemcitabine, this invention designs and synthesizes functional polymeric monomers, and prepares nanogels via reverse microemulsion polymerization. The polymer network of these nanogels comprises the following key components: 2.1) Amphiphilic monomers (CBPD) The amphiphilic monomer is poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methylpropenylamine (CBPD), whose molecular structure contains both a hydrophilic betaine segment and a pH-responsive hydrophobic segment of poly(diisopropylaminoethyl methacrylate) (PDPA). In reverse microemulsion polymerization, CBPD tends to accumulate at the oil-water interface, and its functions are: ① to strengthen interfacial polymerization, forming a dense cross-linked interfacial layer, effectively preventing the internally encapsulated nucleic acid molecules (Tw / let-7c) from being "squeezed out" during polymerization, thereby significantly improving encapsulation efficiency; ② its PDPA segment protonates under acidic conditions (such as lysosome pH ~5.0), changing from hydrophobic to hydrophilic, producing a "proton sponge effect," promoting lysosomal escape of the nanogel. Its synthetic route is shown below. Figure 6 Characterization by ¹H-NMR (1H-NMR) Figure 7 As shown, the characteristic peaks confirm its structure.

[0064] The specific preparation method of the CBPD is as follows: 1-Dodecyl-(dimethylacetic acid)trithiocarbonate (DDAT, 0.75 g), diisopropylaminoethyl methacrylate (1.75 g), and azobisisobutyronitrile (AIBN, 33.74 mg) are dissolved in a 60 mL LDM / water mixed solvent. After nitrogen protection for 30 minutes, the system is transferred to a 70°C oil bath and stirred continuously for 24 hours. Subsequently, 3-[(3-acrylamidopropyl)dimethylammonium]acetate (0.98 g) is added, and the reaction continues overnight. The reaction solution is rapidly cooled to room temperature, and AIBN (6-8 g) is added again. After nitrogen protection for 45-60 minutes, the temperature is raised again to 70°C and the reaction continues for 24 hours. This desulfurized carbonate group is cycled three times to ensure complete conversion. The final product is purified by three precipitations with anhydrous diethyl ether, washed three times with acetone, and dried under vacuum to obtain 1.75 g of white solid. 1.5 g of the above white solid was dissolved together with 0.107 g of 3-methacrylamide, 0.216 g of N-hydroxysuccinimide (NHS), and 0.387 g of N,N-dicyclohexylcarbodiimide (DCC) in 30 mL of dimethyl sulfoxide (DMSO) and reacted at room temperature for 24 hours. The 1,3-dicyclohexylurea (DCU) byproduct generated during the reaction was removed by filtration through an oil-based membrane (0.22 μm). The filtrate was precipitated with anhydrous diethyl ether, centrifuged, washed three times, and then vacuum dried overnight to obtain a pale yellow solid (CBPD), the structural formula of which is shown below: .

[0065] Those skilled in the art can also refer to "A facile combined therapy of chemotherapeuticagent and microRNA for hepatocellular carcinoma using non-cationic nanogel" (DOI:10.1039 / d4tb02256d) for preparation or modification.

[0066] 2.2) Drug-monomer conjugates (GEMSS) To enable the hydrophilic small molecule chemotherapeutic drug gemcitabine to be stably and efficiently loaded into a gel network, this invention synthesizes a gemcitabine-monomer conjugate (GEMSS). Its synthetic route (…). Figure 8 Gemcitabine is linked to a polymerizable double methacrylate backbone via a glutathione (GSH)-responsive disulfide bond through a chemical reaction. The monomer is characterized by ¹H NMR spectroscopy as follows: Figure 9 As shown, the characteristic peaks confirm its structure. GEMSS participates in polymerization as a functional monomer, enabling gemcitabine to become a component of the gel network in a covalent form, effectively reducing premature drug leakage.

[0067] The specific preparation method of GEMSS is as follows: First, triethylamine (3.04 g) and 2,2'-dithiodiethanol (3.08 g) were weighed into a 250 mL reaction flask and dissolved in 100 mL of tetrahydrofuran (THF). Then, a 50 mL anhydrous THF solution of methacryloyl chloride (2.09 g) was prepared and added dropwise to the reaction mixture at 4 °C. After the addition was complete, the system was brought to room temperature and stirred for 12 hours. Then, insoluble substances were removed by filtration, and the filtrate was concentrated by rotary evaporation. The residue was redissolved in 100 mL of ethyl acetate, and the resulting solution was washed three times with saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3.9 g of a yellow liquid, namely ethyl 2-((2-hydroxyethyl)dimercapto)methacrylate (abbreviated as HDSEMA). Next, 0.45 g of HDSEMA was transferred to a 100 mL pear-shaped flask and dissolved in 25 mL of anhydrous chloroform under a nitrogen atmosphere. The flask was cooled in an ice bath, and 4-dimethylaminopyridine (DMAP, 0.49 g) and triphosgene (0.40 g) were added. The reaction mixture was then stirred at 25 °C for 8 hours. Afterward, the mixture was concentrated under reduced pressure to remove residual phosgene and solvent. The resulting solid was dissolved in 30 mL of anhydrous N,N-dimethylformamide (DMF). Gemcitabine (1.24 g) was added, followed by 5 mL of DMF, and the reaction was carried out overnight at room temperature. The crude product was precipitated by adding excess diethyl ether, washed three times with diethyl ether, and dried under vacuum to give a pale yellow solid (GEMSS), the structural formula of which is shown below:

[0068] 2.3) Enzyme-sensitive cross-linking agents (e.g., LEG-CL) To achieve rapid and specific drug release within tumor cells, this invention synthesizes a cross-linking agent (LEG-CL) that can be specifically cleaved by asparagine endopeptidase (Legumain, an enzyme highly expressed in tumor cell lysosomes). The synthetic route is described below. Figure 10 The cross-linking agent is characterized by a short peptide containing an asparagine endopeptidase recognition peptide sequence (KGAANLGK, SEQ ID NO.1), with the ε-amino groups of the lysine residues at both ends modified by methacrylamide groups. Its successful bifunctional modification was verified by ¹H-NMR (…). Figure 11 In the polymerization reaction, the double bonds of LEG-CL participate in the reaction, becoming cross-linking points that connect the polymer chains. In the presence of asparagine endopeptidase, this peptide is cleaved, leading to the disintegration of the entire nanogel network.

[0069] The specific preparation method of LEG-CL is as follows: A polypeptide (0.25 g) with the sequence KGAANLGK and methacryloyl chloride (0.33 g) were dissolved in 15 mL of DMSO and stirred overnight at room temperature. The reaction mixture was then dialyzed using a dialysis bag with a molecular weight cutoff (MWCO) of 300 Da. After purification by dialyzing (methanol, ultrapure water), the reaction solution was freeze-dried to obtain a white powder product (LEG-CL), the structural formula of which is shown below: .

[0070] The researchers also tested the peptide sequence of KGAAAGK (SEQ ID NO.2), but the results were slightly worse. KGAANLGK was then used in subsequent experiments.

[0071] Example 2: Formulation Optimization 1) Optimization of the ratio of hydrophobic to hydrophilic sections in CBPD CBPDs with repeating unit ratios of hydrophilic and hydrophobic blocks (betaine and diisopropylaminoethyl methacrylate) of 2:1, 2:2, 2:3, 2:4, and 2:5 (denoted as CBPD-1, CBPD-2, CBPD-3, CBPD-4, and CBPD-5, respectively) were prepared. The mass ratio of GEMSS, LEG-CL, and CBPD was fixed at 4:2:1. Nanogels loaded with Tw / let-7c and gemcitabine were prepared. The particle size and encapsulation efficiency of the nanogels for Tw / let-7c and gemcitabine were measured. The results are shown in Table 1 below.

[0072] Table 1. Particle size and drug encapsulation efficiency of drug-loaded nanogels prepared by CBPD with different ratios of repeating units in hydrophilic and hydrophobic segments.

[0073] Based on the results in Table 1, considering both particle size and drug encapsulation efficiency, it can be seen that the CBPD-3 group exhibits good particle size uniformity and high encapsulation efficiency for both Tw / let-7c and gemcitabine. Therefore, CBPD-3 was used for subsequent experiments.

[0074] For the CBPD-1 group, the amphiphilic monomer has a higher proportion of betaine segments (hydrophilic segments), which tend to distribute in the hydrophilic core during polymerization and crosslinking, causing the Tw / let-7c to be squeezed out. As the proportion of hydrophobic segments in CBPD increases, they tend to distribute at the oil-water interface, forming a crosslinked layer at the interface, thereby improving the drug loading efficiency. For the CBPD-4 and CBPD-5 groups, the proportion of hydrophobic segments further increases, and the molecular weight of the monomers also increases, resulting in unstable large particles after polymerization and crosslinking, which reduces the drug loading efficiency. In addition, the excessive hydrophobic components make the resulting samples difficult to disperse in water, which is not conducive to subsequent biological experiments.

[0075] 2) Optimization of the proportions of each component in the formulation The researchers also determined the optimal ratio of GEMSS, LEG-CL, and CBPD usage through system optimization.

[0076] according to Figure 12 , Figure 13 and Figure 14 The results show that when the mass ratio of GEMSS, LEG-CL and CBPD is 4:2:2, the prepared nanogel (NG@Tw / let-7c / Gem) has the best overall performance. At this time, the particle size is about 69.1 nm, the zeta potential is near neutral (-1.36 mV), and the encapsulation efficiency of Tw / let-7c and gemcitabine reaches about 90% and 85%, respectively.

[0077] 3) GEMSS is not used as a monomer. The researchers also compared the results using LEG-Cl as the crosslinking agent, CBPD as the monomer (feed ratio 2:2), n-hexane as the organic phase, Span80 and AOT as surfactants, and APS and TEMED as initiators, simultaneously loading Tw / let-7c complex (1 od) and gemcitabine monomer (4 mg). The encapsulation efficiencies of the obtained nanogels for Tw / let-7c and gemcitabine were measured to be 55.8 ± 2.1% and 30.3 ± 3.3%, respectively. Figure 13 and Figure 14 The results of this application show that the proposed method is significantly inferior to the nanogel obtained by using GEMSS as the monomer.

[0078] Example 3: Preparation method of nanogels The specific steps of the preparation method of the nanogel described in this invention are as follows: 1) Preparation of organic phase: Weigh the surfactant sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and sorbitan monooleate (Span80), dissolve them in the organic solvent n-hexane, and degas the resulting solution with nitrogen for 5 minutes to obtain an organic dispersion.

[0079] 2) Preparation of the aqueous phase mixture: The functional monomer GEMSS, the amphiphilic monomer CBPD, and the crosslinking agent LEG-CL were dissolved together in a mixed solvent of DMSO and DEPC water (volume ratio 1:1). Then, the Tw / let-7c hybrid dissolved in DEPC water was added to this mixture. This aqueous phase mixture was also degassed with nitrogen for 5 minutes.

[0080] 3) Formation and polymerization of the reverse microemulsion: Under stirring conditions, the degassed aqueous phase mixture from step (2) was added dropwise to the organic phase from step (1) to form a water-in-oil (W / O) reverse microemulsion. After stirring for about 10 minutes, the initiator ammonium persulfate (APS) and the accelerator N,N,N′,N′-tetramethylethylenediamine (TEMED) were added sequentially to initiate the free radical polymerization reaction. The reaction was carried out at 4°C for 2–4 hours.

[0081] 4) Precipitation and purification of the product: After polymerization, the reaction mixture was slowly added dropwise to an excess of pre-cooled acetone, and allowed to stand at -20°C to precipitate the nanogel. The precipitate was collected by centrifugation and dried under vacuum. The dried product was redispersed in phosphate-buffered saline (PBS, pH 7.4) and centrifuged (4500 rpm) to remove insoluble aggregates. The supernatant was further purified using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to finally obtain the pure co-delivered nanogel product NG@Tw / let-7c / Gem.

[0082] The preparation and working schematic diagram of drug-loaded nanogels are shown below. Figure 15 As shown.

[0083] Test Example 1: Stimulus-Responsive Release Study of Nanogels To investigate the stimulus-response release behavior under simulated tumor microenvironment conditions, the release characteristics of NG@Tw / let-7c / Gem under different conditions were analyzed. For example... Figure 16As shown, after 24 hours of incubation at physiological pH (pH 7.4), the cumulative release rate of Tw / let-7c was only 12.44%, indicating that the nanogel is structurally stable in a normal physiological environment. When the pH decreased to 5.0, the release rate increased slightly to 17.34%, which is attributed to the increased hydrophilicity of the gel due to the protonation of the PDPA block in CBPD under acidic conditions, but did not trigger significant structural disintegration. After adding 1 mM glutathione (GSH) at pH 5.0, the release rate increased to 25.58% after 24 hours, indicating that although the disulfide bonds in GEMSS can be cleaved by GSH, the cleavage mainly occurs at the chain ends, and the overall cross-linked network remains intact, thus limiting its promoting effect on drug release. However, when pH 5.0, 1 mM GSH, and 2 μM asparagine endopeptidase are present simultaneously, the release behavior of the nanogel changes significantly: the LEG-CL cross-linking agent is enzymatically cleaved, leading to rapid network disintegration and a sharp increase in Tw / let-7c release, with a cumulative release rate reaching 80.34% within 24 hours. Gemcitabine release shows a similar trend ( Figure 17 Under conditions of pH 5.0 + 1mM GSH + 2μM asparagine endopeptidase, the cumulative release rate reached 85.40% after 24 hours, significantly higher than the 6.24% in pH 7.4 PBS. However, under the enzyme-free but GSH-containing conditions at pH 5.0, the release rate only increased moderately to 31.29%. In summary, NG@Tw / let-7c / Gem remains stable under physiological conditions, but undergoes rapid disintegration under the combined stimulation of characteristic factors of the tumor microenvironment—acidic pH, high concentration of GSH, and overexpression of asparagine endopeptidase—thus achieving highly efficient simultaneous release of Tw / let-7c and gemcitabine.

[0084] Test Example 2: Study on Cellular Uptake and Intracellular Response of Nanogels The uptake and intracellular response of NG@Tw / let-7c in SK-Hep1 cells were investigated using confocal laser scanning microscopy (CLSM). To further compare the differences in intracellular response between NG@Tw / let-7c and the cationic carrier PEG-PEI@Tw / let-7c, FRET signal detection was performed using dual-labeled Tw / let-7c (Tw's 5' end labeled with BHQ1 quencher, and let-7c's 3' end labeled with Cy3 fluorophore). CLSM results showed that in the early stages of incubation with NG@Tw / let-7c, intracellular Cy3 fluorescence was weak, indicating a strong FRET signal and that the Tw / let-7c hybrid structure remained intact. With prolonged incubation, Cy3 fluorescence gradually increased, indicating that the hybrid dissociated in response to intracellular overexpression of miR-21, releasing Tw and let-7c. In contrast, the fluorescence enhancement in the PEG-PEI@Tw / let-7c group was significantly delayed, while the fluorescence intensity in the NG@SCR group, which served as the negative control, remained extremely weak throughout. Figure 18 The above results indicate that NG@Tw / let-7c can be effectively taken up by tumor cells and responds to intracellular miR-21 to achieve the dissociation and release of the hybrid, with a dissociation efficiency superior to that of the traditional cationic carrier PEG-PEI.

[0085] Test Example 3: In vitro cytotoxicity study of nanogels The in vitro killing effect of drug-loaded nanogels on SK-Hep1 liver cancer cells was evaluated using the CCK-8 assay. Cell viability was measured after incubating cells with different nanogel formulations for 48 hours. Figure 19 As shown, in the single-drug treatment groups, the cell viability after treatment with NG@Tw, NG@let-7c, and NG@Gem was 90.41%, 87.67%, and 61.67%, respectively, indicating that gemcitabine itself has a good antitumor effect, while the effect of nucleic acid drugs alone is relatively limited. In the combination therapy groups, the cell viability after treatment with NG@Tw / let-7c, NG@Tw / Gem, NG@let-7c / Gem, and NG@Tw / let-7c / Gem was 79.60%, 53.30%, 50.40%, and 40.12%, respectively. Among them, the NG@Tw / let-7c / Gem group, which simultaneously loaded with Tw / let-7c hybrid and gemcitabine, showed the most significant inhibitory effect on tumor cells, with cell viability decreasing to about 40%, which was significantly better than other groups. The above results indicate that co-delivery of Tw / let-7c hybrid and gemcitabine via nanogel can produce a synergistic effect, significantly improving the sensitivity of liver cancer cells to chemotherapeutic drugs.

[0086] Test Example 4: Study on the antitumor effect of drug-loaded nanogels in tumor-bearing mice. Based on in vitro experiments, the in vivo antitumor effect of NG@Tw / let-7c / Gem was further evaluated using a nude mouse model of SK-Hep1 subcutaneous tumors. Tumor-bearing mice were randomly divided into 8 groups, receiving either PBS control or different nanogel formulations. Tumor volume changes were measured periodically during treatment. Figure 20As shown, the single-drug treatment groups (NG@Tw, NG@let-7c, and NG@Gem) all exhibited some tumor-suppressive effects, but the efficacy was limited. In contrast, the combination therapy groups (NG@Tw / let-7c, NG@Tw / Gem, NG@let-7c / Gem, and NG@Tw / let-7c / Gem) showed stronger tumor-suppressive effects. Among them, the NG@Tw / let-7c / Gem group, which was simultaneously loaded with Tw / let-7c hybrid and gemcitabine, showed the most significant therapeutic effect, with tumor growth almost completely inhibited. At the treatment endpoint, the mean tumor volume in the NG@Tw / let-7c / Gem group was only 77.61±16.02 mm³, significantly smaller than that in the PBS control group (755.81±115.75 mm³), the NG@Tw / let-7c group (322.95±50.27 mm³), and the NG@Gem group (260.73±74.98 mm³). In addition, immunohistochemical staining analysis of tumor tissues showed that the expression level of the anti-apoptotic protein Bcl-2 in the NG@Tw / let-7c / Gem group was significantly reduced. Figure 21 This indicates that the combined treatment strategy effectively promoted tumor cell apoptosis. These results demonstrate that co-delivery of the Tw / let-7c hybrid and gemcitabine via nanogel can produce a significant synergistic anti-tumor effect in vivo, effectively inhibiting tumor growth.

[0087] Test Example 5: Biosafety Evaluation of Nanogels The biosafety of the nanogel was evaluated through in vitro cytotoxicity experiments and in vivo histological analysis.

[0088] 1) In vitro cytotoxicity evaluation: The cytotoxicity of blank nanogels against normal hepatocytes (THLE-2) and hepatocellular carcinoma cells (Hep3B, SK-Hep1) was detected using the CCK-8 assay. Figure 22 As shown, different concentrations of blank nanogels had no significant effect on the viability of the cells, indicating that the carrier material itself has good cell compatibility.

[0089] 2) In vivo tissue compatibility evaluation: H&E staining was performed on tissue sections of the major organs (heart, liver, spleen, lung, and kidney) of tumor-bearing mice. For example... Figure 23 As shown, compared with the control group, no obvious pathological changes were observed in the major organs of mice in each nanogel treatment group, indicating that the nanogel has good tissue compatibility when applied in vivo.

[0090] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A multi-sensitivity non-cationic nanogel, characterized in that, include: a) DNA-miRNA hybrid; b) An amphiphilic monomer, wherein the amphiphilic monomer is selected from one or more of the following: poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methacrylamine, acrylate-based polyethylene glycol-polycaprolactone, and acrylate-based polyethylene glycol-polystyrene; c) Gemcitabine-monomer conjugate, wherein the structural formula of the gemcitabine-monomer conjugate is as follows: ； d) Crosslinking agent.

2. The multi-sensitivity non-cationic nanogel according to claim 1, characterized in that, The amphiphilic monomer is poly(diisopropylaminoethyl methacrylate)-poly(3-[(3-acrylamidopropyl)dimethylammonium]acetate-dimethylacetyl-methacrylamine), and its structural formula is as follows: ； Optionally, the m:n ratio in the amphiphilic monomer is 2:(1~4). Optionally, the m:n ratio in the amphiphilic monomer is 2:(2~4).

3. The multi-sensitivity non-cationic nanogel according to claim 1, characterized in that, The weight ratio of the gemcitabine-monomer coupling compound, crosslinking agent, and amphiphilic monomer is (3~5):(1~3):(1~3). Optionally, the weight ratio of the gemcitabine-monomer coupling agent, crosslinking agent, and amphiphilic monomer is 4:2:

2.

4. The multi-sensitivity non-cationic nanogel according to claim 1, characterized in that, The method for preparing the gemcitabine-monomer conjugate includes: S1. Dissolve triethylamine and 2,2'-dithiodiethanol in tetrahydrofuran, and add anhydrous THF solution of methacryloyl chloride dropwise at 1~10℃. After the addition is complete, restore the system to 20~35℃ and stir for 6~24h. After purification, ethyl 2-((2-hydroxyethyl)dimercapto)methacrylate is obtained. S2. Dissolve the obtained ethyl 2-((2-hydroxyethyl)dimercapto)methacrylate in chloroform, add 4-dimethylaminopyridine and triphosgene, and stir to react. S3. Dissolve the solid obtained from the reaction in N,N-dimethylformamide, add gemcitabine to react, and after purification, the gemcitabine-monomer conjugate is obtained.

5. The multi-sensitivity non-cationic nanogel according to claim 1, characterized in that, The preparation method of the amphiphilic monomer includes: S1. 1-Dodecyl-(dimethylacetic acid)trithiocarbonate, diisopropylaminoethyl methacrylate and azobisisobutyronitrile were dissolved in a DMF / water mixed solvent and reacted under nitrogen protection at 70±10℃ for 12~48h. Then, 3-[(3-acrylamidopropyl)dimethylammonium]acetate was added to continue the reaction. After the reaction was completed, the mixture was cooled to room temperature and azobisisobutyronitrile was added again. The reaction was continued under nitrogen protection at 70±10℃ for 12~48h. After purification, a white solid product was obtained. S2. Take the obtained white solid product and dissolve it in dimethyl sulfoxide with 3-methylacrylamide, N-hydroxysuccinimide and N,N-dicyclohexylcarbodiimide, and react for 12~48h. After purification, the amphiphilic monomer is obtained.

6. The multi-sensitivity non-cationic nanogel according to claim 1, characterized in that, The DNA-miRNA hybrid is a bifunctional complex with a tweezer-like structure formed by a single-stranded DNA that can bind to miR-21 and a tumor suppressor miRNA through complementary base pairing.

7. The multi-sensitivity non-cationic nanogel according to claim 1, characterized in that, The crosslinking agent is an asparagine endopeptidase-sensitive crosslinking agent; Optionally, the asparagine endopeptidase-sensitive crosslinking agent includes a polypeptide sequence of KGAANLGK or KGAAAGK. Optionally, the asparagine endopeptidase-sensitive cross-linking agent comprises a polypeptide sequence of KGAANLGK.

8. The multi-sensitivity non-cationic nanogel according to claim 7, characterized in that, The structural formula of the asparagine endopeptidase-sensitive crosslinking agent is as follows: 。 9. The method for preparing the multi-sensitivity non-cationic nanogel according to any one of claims 1 to 8, characterized in that, The preparation method includes the following steps: 1) Dissolve the surfactant in the solvent n-hexane to obtain the organic phase; 2) After dissolving the amphiphilic monomer, gemcitabine-monomer conjugate, and cross-linking agent in a DMSO / DEPC aqueous solution, the DNA-miRNA hybrid was added to obtain the aqueous phase; 3) Under stirring, the aqueous phase is added to the organic phase, followed by the addition of initiator and accelerator to carry out the polymerization reaction; 4) After purification, the multi-sensitivity non-cationic nanogel is obtained.

10. The use of the multi-sensitivity non-cationic nanogel as described in any one of claims 1 to 8 in the preparation of antitumor drugs.

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