Enhanced penetration type double-response nanoparticles for co-delivery of chemotherapeutic drugs and gene drugs as well as preparation method and application of enhanced penetration type double-response nanoparticles

Abstract

The invention belongs to the technical field of medicines, and particularly relates to enhanced penetration type double-response nanoparticles for co-delivery of chemotherapeutic drugs and gene drugs as well as a preparation method and application thereof. According to the invention, albumin is used as a carrier of gemcitabine, an ROS responsive charge reversible cationic polymer is used as a delivery carrier of siRNA, and gemcitabine drug-loaded albumin and siRNA drug-loaded B-PDEA nanoparticles are coupled through MMP-2 responsive polypeptide to form the drug-loaded nanoparticles. After reaching a tumor area through the active targeting effect of albumin, the nanoparticles can be cut by MMP-2 in a microenvironment and release drug-loaded albumin, so that the size of a delivery system is reduced, and further tumor deep penetration and enhanced gemcitabine uptake are realized; meanwhile, after intracellular high-level ROS is recognized, the loaded siRNA can be selectively released and inhibit CD39 expression, tumor metabolism is adjusted, an immune microenvironment is remodeled and an anti-tumor immune response is activated to further realize chemotherapy sensitization while precise chemotherapy is performed, and the therapeutic efficacy on pancreatic cancer is expected to be enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a permeability-enhanced dual-response nanoparticle for co-delivering chemotherapeutic drugs and gene drugs, and a preparation method and application thereof. Background Art

[0002] The highly dense matrix of pancreatic cancer limits the delivery and penetration of chemotherapeutic drugs, leading to chemoresistance. This therapeutic challenge is closely related to the metabolic and immune crosstalk between multiple components in the tumor microenvironment (TME).

[0003] Apoptosis of tumor cells releases ATP as a danger-associated molecular pattern, leading to synergistic tumor suppression by stimulating immune responses. However, the upregulated expression of ectonucleotidase CD39 can catalyze ATP to generate AMP, and further hydrolyze AMP with the cooperation of CD73 to generate adenosine (ADO) with inhibitory function on immune effector cells and antigen-presenting cells; at the same time, the multiple KRAS mutations in pancreatic cancer reduce the expression of equilibrative nucleoside transporter (ENT), reduce the uptake of extracellular adenosine (eADO) by tumor cells, and aggravate the accumulation of eADO in the TME and the immunosuppressive tumor microenvironment. In addition, the reduced ENT also limits the endocytosis of the hydrophilic first-line chemotherapy drug gemcitabine, leading to intrinsic resistance. Therefore, based on the metabolic-chemotherapy-immune crosstalk characteristics of pancreatic cancer, targeting CD39, a key enzyme in the eATP-eADO axis, to design a drug delivery system that matches the metabolic reprogramming characteristics provides a comprehensive strategy for inhibiting tumor progression.

[0004] Although the reduction of ENT caused by KRAS mutations limits the uptake of gemcitabine, the enhanced macropinocytosis pathway provides hope for the use of albumin as a drug carrier to improve gemcitabine uptake. In addition, due to its ultra-small size, albumin exhibits a stronger ability to penetrate tumors. Therefore, the use of albumin as a drug delivery carrier can simultaneously enhance tumor penetration and cellular uptake of chemotherapeutic drugs. In order to regulate abnormal tumor metabolism and achieve chemotherapy sensitization, CD39 was selected as a research target. Due to the lack of specific inhibitors, small interfering RNA (siRNA) that can specifically degrade specific mRNA was selected to limit the ADO metabolic function of CD39, so it is necessary to design a matching delivery system to achieve targeted delivery and efficient transfection of siRNA. The structure of poly (2-acryloyl) ethyl (benzyl borate) diethylammonium bromide (B-PDEA) was reported to be positively charged, self-destructive and charge-reversing in response to high concentrations of ROS, and selectively release siRNA in tumor cells with high ROS levels, regulate abnormal metabolism, and synergistically promote anti-tumor immune response and chemotherapy sensitization. Summary of the invention

[0005] Aiming at the above problems encountered in the treatment of pancreatic cancer, the present invention designs and prepares a dual-responsive nanoparticle with enhanced penetration of tumor tissue based on the existing technical foundation, which is used for co-delivering chemotherapy drugs and adenosine metabolism regulating drugs. The nanoparticles are constructed by connecting the chemotherapy drug-loaded albumin and the polymer nanoparticles loaded with gene drugs through a responsive linker. The chemotherapy drug is gemcitabine, a first-line chemotherapy drug for pancreatic cancer, and the polymer is a cationic polymer B-PDEA obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization reaction, which has the structural characteristics of poly (2-acryloyl) ethyl (benzyl borate) diethylammonium bromide and has the charge flipping ability of high concentration ROS response. The gene drug is a small interfering RNA siCD39 that can interfere with the expression of tumor cell CD39, which is assembled with the cationic polymer through electrostatic interaction to form nanoparticles. The gemcitabine-loaded albumin and the nanoparticles loaded with siCD39 are connected by a responsive polypeptide that can respond to matrix metalloproteinase 2 (MMP-2) highly expressed in tumor tissue.

[0006] In the design of the present invention, albumin is used as a carrier of the chemotherapy drug gemcitabine to enhance tumor penetration and macropinocytosis-mediated endocytosis. Furthermore, in order to selectively release gene drugs, the study selected the ROS-responsive charge-reversible cationic polymer B-PDEA as a delivery carrier for siRNA. At the same time, considering the poor circulation stability and intratumoral accumulation of small-sized albumin, MMP-2-responsive polypeptides were used to couple gemcitabine-loaded albumin and siRNA-loaded B-PDEA nanoparticles to form larger-sized drug-loaded nanoparticles. When the nanoparticles reach the tumor area through the active targeting of albumin, they can be cut and released by MMP-2 in the microenvironment, achieving a reduction in the size of the delivery system, thereby achieving deep tumor penetration and enhanced gemcitabine uptake; at the same time, after recognizing high levels of intracellular ROS, the loaded siRNA can be selectively released and inhibit CD39 expression, regulating tumor metabolism, reshaping the immune microenvironment, and activating anti-tumor immune responses while performing precise chemotherapy to further achieve chemotherapy sensitization. The present invention obtains a chemotherapeutic drug-gene drug co-delivery system with ideal biocompatibility, enhanced circulation stability and tumor targeting ability through albumin modification and appropriate size design, which is expected to enhance the therapeutic efficacy of pancreatic cancer.

[0007] The above design integrates strategies such as promoting deep drug penetration, regulating tumor microenvironment, selectively releasing cytotoxic drugs, and activating anti-tumor immune response into a nanodelivery platform, providing a promising combined treatment strategy for pancreatic cancer.

[0008] In order to achieve the above object, the present invention provides the following technical solutions: The invention provides an enhanced penetration dual-responsive nanoparticle for co-delivering chemotherapeutic drugs and gene drugs, comprising gemcitabine-loaded albumin, a metabolism-regulating gene drug, a responsive polymer and cholesterol; the metabolism-regulating gene drug is a small interfering RNA siCD39 that inhibits CD39 expression; the responsive polymer at least comprises a dual-responsive polymer of MMP-2 and ROS; the metabolism-regulating gene drug is encapsulated in the responsive polymer and then encapsulated in the cholesterol; the gemcitabine-loaded albumin is coupled to the responsive polymer via an MMP-2 responsive polypeptide.

[0009] Preferably, the responsive polymer further comprises a ROS responsive polymer, and the molar ratio of the MMP-2 and ROS dual responsive polymer to the ROS responsive polymer is 1:4; and the ROS responsive polymer comprises a phenylboronic acid group.

[0010] The present invention also provides a method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs, comprising the following steps: (1) Gem and 6-maleimidocaproic acid are reacted at room temperature in the presence of an amidation catalyst and an acid-binding agent, and then separated and purified by silica gel chromatography to obtain the maleimidized gemcitabine prodrug Gem-Mal; (2) a. dissolving the MMP-2 responsive polypeptide (sequence: GPLGVRGC) and the acid-binding agent in a good solvent to obtain a MMP-2 responsive polypeptide solution; b. 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid and N-succinimide ester are dissolved in a good solvent, added to the MMP-2 response polypeptide solution, and reacted at room temperature to couple the MMP-2 response polypeptide with the chain transfer reagent to obtain reaction solution 1; c. The N-succinimidyl 6-maleimidocaproate is dissolved in a good solvent and added to the reaction solution in step b, and further reacted at room temperature to obtain a reaction solution II; d. The reaction solution 2 is dialyzed to remove impurities, and then freeze-dried and dehydrated to obtain a chain transfer reagent RAFT-pep-NHS coupled to a responsive polypeptide; (3) a. deoxygenating 4-cyano-4-(phenylcarbonylthio)pentanoic acid or the RAFT-pep-NHS described in step (2) and 2-(N,N-diethylamino)ethyl methacrylate and a catalyst, and subjecting the mixture to a polymerization reaction at 70° C. to obtain a reaction solution; b. after the reaction is completed, introducing air into the reaction solution described in step a to quench the reaction, concentrating under reduced pressure, and precipitating with a precipitant three times to obtain an enzyme-non-responsive polymer PDEA or an enzyme-responsive polymer PDEA-pep-NHS; c. Add 4-bromomethylphenylboronic acid and anhydrous K2CO3 to the PDEA or PDEA-pep-NHS obtained in step b, and stir the dissolved solution at room temperature under argon protection; d. After the reaction is completed, the solution obtained in step c is dialyzed with deionized water for 24 h, and lyophilized and dehydrated to obtain an enzyme-non-responsive cationic polymer B-PDEA or an enzyme-responsive cationic polymer B-PDEA-pep-NHS; (4) a. Dissolving the synthesized polymer materials B-PDEA and B-PDEA-pep-NHS in nuclease-free ultrapure water at different concentrations, and premixing them to obtain a working polymer solution; b. Dissolve the gene drug siCD39 (sequence: 5'-GGGCAGAUUCACUCAGGAA-3', 5'-UUCCUGAGUGAAUCUGCCC-3') in ultrapure water without nuclease at the required concentration, and add the siRNA solution to the working polymer solution obtained in step (4) a at the required N / P ratio, mix thoroughly and let stand at room temperature; (5) adding the cholesterol solution to the eggplant-shaped flask, removing the solvent by rotary evaporation to form a thin lipid film, adding the polymer solution obtained in step (4) to the eggplant-shaped flask containing the cholesterol film, ultrasonically treating the cholesterol film to rehydrate it, and stirring at room temperature overnight to obtain a cholesterol-coated polymer complex solution; (6) a. reacting human serum albumin (HSA) and 2-iminothiolane hydrochloride at room temperature, and obtaining HSA-SH having a free thiol group after desalting; b. adding the gemcitabine prodrug Gem-Mal obtained above to the HSA-SH solution obtained in step (6) in the required molar ratio, reacting at room temperature, desalting and freeze-drying to obtain the target product, gemcitabine drug-loaded albumin Gem-HSA; (7) Dissolve the gemcitabine-loaded albumin Gem-HSA obtained in step (6) b and add it to the cholesterol-coated polymer complex solution obtained in step (5), and mix thoroughly to obtain cascade-targeted nanoparticles siRNA@CPA.

[0011] Preferably, in step (1), the molar ratio of gemcitabine to 6-maleimidocaproic acid is 1:2; the catalyst is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); the acid-binding agent is N,N-diisopropylethylamine (DIPEA), and the molar ratio of the catalyst, the acid-binding agent and gemcitabine is (2-3):(2-3):1; further preferably, the reaction solvent is N,N-dimethylformamide (DMF), and the reaction time at room temperature is 48 h.

[0012] Preferably, the eluent for the chromatographic column separation in step (1) is methanol: dichloromethane 1:10, v:v.

[0013] Preferably, the good solvent for the MMP-2 responsive polypeptide in step (2) a is N,N-dimethylformamide (anhydrous DMF); the acid-binding agent is N,N-diisopropylethylamine (DIPEA), and the molar ratio of the acid-binding agent to the MMP-2 responsive polypeptide is (1-2):1.

[0014] Preferably, in step (2) b, the molar ratio of the MMP-2 responsive polypeptide to 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid N-succinimidyl ester is 1:(1-1.5), and the good solvent of the 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid N-succinimidyl ester is dimethyl sulfoxide (DMSO); further preferably, the solution of 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid N-succinimidyl ester should be dripped into the MMP-2 responsive polypeptide solution, and the dripping process is controlled to be completed within 15 minutes, and the activation reaction time at room temperature is 4 hours.

[0015] Preferably, in step (2) c, the molar ratio of the MMP-2 response polypeptide to N-succinimidyl 6-maleimidocaproate is 1:(1-1.5), and the good solvent for N-succinimidyl 6-maleimidocaproate is dimethyl sulfoxide (DMSO); further preferably, the solution of N-succinimidyl 6-maleimidocaproate should be dripped into the reaction solution 1 drop by drop, and the dripping process is controlled to be completed within 15 minutes, and the reaction time at room temperature is 4 hours.

[0016] Preferably, in step (2) d, a dialysis bag with a molecular weight cutoff of 1 kDa is used for the dialysis process; further preferably, the dialysis time in DMSO and deionized water is 24 h.

[0017] Preferably, in step (3) a, the molar ratio of 4-cyano-4-(phenylcarbonylthio)pentanoic acid or the RAFT-pep-NHS in step (2) to 2-(N,N-diethylamino)ethyl methacrylate is 1: (50-100); the catalyst is 2,2'-azobisisobutyronitrile (AIBN), and the molar ratio of AIBN to 4-cyano-4-(phenylcarbonylthio)pentanoic acid or the RAFT-pep-NHS is (0.5-1):1; further preferably, anhydrous methanol is used as the reaction solvent.

[0018] Preferably, the deoxygenation operation in step (3) a is performed in a Schlenk tube; specifically, after the materials in step (3) a are mixed and dissolved, the solution is added to a Schlenk tube, the solution in the tube is quickly frozen using liquid nitrogen, an oil pump is connected to suck and thaw the solution, high-purity nitrogen is introduced, and liquid nitrogen is used for quick freezing again, and the above liquid nitrogen repeated freeze-drying cycle is repeated 5-10 times to achieve deoxygenation; further preferably, the reaction time at 70° C. is 16 h.

[0019] Preferably, the precipitant in step (3) b is n-hexane precooled to -20°C. Specifically, the reaction solution in step a is concentrated under reduced pressure to 1 / 5 of the original volume, and then slowly dripped into precooled n-hexane. After sufficient precipitation, the n-hexane is removed by centrifugation. The precipitation is repeated 3 times, and then vacuum dried to obtain a precipitated product.

[0020] Preferably, in step (3) c, the molar ratio of PDEA, 4-bromomethylphenylboronic acid and anhydrous K2CO3 is 1:(100-150):(100-150), the solvent is N,N-dimethylformamide (DMF), and the reaction time at room temperature is 24 h.

[0021] Preferably, in step (3) d, a dialysis bag with a molecular weight cutoff of 3.5 kDa is used for the dialysis process.

[0022] Preferably, the molar ratio of B-PDEA to B-PDEA-pep-NHS in the working polymer solution in step (4) a is 4:1.

[0023] Preferably, the N / P ratio in step (4) b is 15:1; further preferably, the mixture is thoroughly mixed by pipetting 10 to 50 times, and the mixture is allowed to stand at room temperature for 30 minutes.

[0024] Preferably, the ultrasonic treatment process in step (5) is carried out in an ice bath, and the ultrasonic treatment time is 10 min.

[0025] Preferably, in step (6) a, the molar ratio of human serum albumin to 2-iminothiolane hydrochloride is 1:10; the reaction solvent is PBS buffer 8.0 containing 5 mM ethylenediaminetetraacetic acid (EDTA); further preferably, the reaction is carried out under nitrogen protection, and the reaction time at room temperature is 4 h.

[0026] Preferably, the molar ratio of Gem-Mal to HSA-SH in step (6) b is 1.2:1; the solvent is dimethyl sulfoxide (DMSO); further preferably, the final concentration of DMSO during the reaction is controlled to be 10%, v:v, and the reaction time at room temperature is 12-24h; Preferably, the desalting process in step (6) a and step (6) b is completed by a desalting column, the centrifugal force is 1000 g, the centrifugation time is 6 min, and the desalting is preferably repeated 3 times.

[0027] Preferably, the solvent of Gem-HSA in step (7) is ultrapure water that does not contain nuclease, and the mixture is thoroughly mixed by pipetting 10-50 times.

[0028] The enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs of the present invention are composed of chemotherapeutic drug-loaded albumin, metabolic regulation gene drugs, responsive linkers, ROS responsive polymers and cholesterol coatings. The chemotherapeutic drug is gemcitabine; the metabolic regulation gene drug is small interfering RNA siCD39 that inhibits CD39 expression; the triggering factor for the gemcitabine-loaded albumin to dissociate from the siCD39-loaded nanoparticles and achieve size reduction and increased penetration is the highly expressed MMP-2 in the tumor microenvironment, and the site of response stimulation is the MMP-2 response polypeptide; the stimulating release factor of the gene drug siCD39 is the high concentration of ROS in tumor cells, and the site of response stimulation is the phenylboronic acid group on the polymer side chain.

[0029] In the present invention, the gemcitabine drug-loaded albumin is obtained by reducing the surface thiol groups of human serum albumin with 2-iminothiolane hydrochloride, and then connecting the surface thiol groups of human serum albumin and the amino groups in the gemcitabine molecule with 6-maleimidocaproic acid under the action of a catalyst and an acid-binding agent.

[0030] In the present invention, the ROS responsive polymer skeleton is obtained by RAFT polymerization, and the ROS responsive element is phenylboronic acid, which is introduced by reacting 4-bromomethylphenylboronic acid with N,N-diethylamino on the polymer side chain under the action of a catalyst.

[0031] In the present invention, the siRNA-loaded nanoparticles are prepared by dissolving a polymer in ultrapure water without nuclease, adding an aqueous solution of siRNA and mixing, and then rehydrating the cholesterol forming the film.

[0032] In the present invention, the gemcitabine-loaded albumin is connected via an MMP-2 responsive peptide segment.

[0033] The room temperature in the present invention refers to 25°C.

[0034] The present invention also provides the use of the dual-responsive nanoparticles obtained by the above preparation method for co-delivering chemotherapeutic drugs and gene drugs in the preparation of drugs for treating pancreatic cancer.

[0035] Compared with the prior art, the present invention has the following advantages: (1) The nanoparticles of the present invention are easy to prepare, have strong compatibility and good stability, and can be used for the selective delivery of a variety of chemotherapeutic drugs and / or gene drugs containing reactive groups (such as amino groups, etc.).

[0036] (2) The nanoparticles of the present invention have a long circulation time in the body and do not show obvious systemic toxicity after multiple administrations.

[0037] (3) Compared with traditional intravenous administration, the nanoparticles of the present invention have obvious tumor accumulation and deep tumor penetration capabilities, and can effectively deliver drugs deep into the tumor, play a chemotherapy sensitization role, and reduce systemic toxicity.

[0038] (4) The nanoparticles of the present invention can effectively regulate abnormal adenosine metabolism in tumors, reduce the accumulation of immunosuppressive substances (such as adenosine) in the microenvironment, alleviate the immunosuppressive microenvironment, and activate anti-tumor immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the synthetic route of B-PDEA polymer.

[0040] Figure 2 The synthetic route of B-PDEA-pep-NHS polymer.

[0041] Figure 3 The microscopic morphology, particle size and distribution of siRNA@CPA nanoparticles in Example 1. a) Particle size and PDI data measured by DLS, b) TEM image of nanoparticles.

[0042] Figure 4 The loading of drugs in nanoparticles in Example 1 was investigated. a) Gel electrophoresis was used to verify the loading of siRNA at different N / P ratios; b) FRET effect of B-PDEA, nanoparticles, albumin, and nanoparticles + MMP-2.

[0043] Figure 5 The ROS response characteristics of the nanoparticles in Example 1. a) Molecular mechanism of polymer ROS responsiveness; b) Polymer molecules before and after response 1 H NMR change graph; c) HPLC detection result graph of the change of response product content during the response process.

[0044] Figure 6 The graphs show the results of investigating the ability of nanoparticles to release siRNA gene drugs in response to a high concentration of ROS in Example 1. a) siRNA release after treatment with different concentrations of H2O2; b) siRNA release differences between nanoparticles in tumor cells with high ROS levels and in stromal cells with low ROS levels (scale bar: 20 μm).

[0045] Figure 7 The graphs show the results of investigating the deep penetration ability of the nanoparticles in Example 1 into a) tumor tissues in vivo (scale bar: 200 μm) and b) tumor spheres in vitro (scale bar: 100 μm).

[0046] Figure 8 The figure shows the results of the investigation of the ability of free drugs or nanoparticles in Example 1 to regulate abnormal adenosine metabolism in tumor tissues. After administration, a) the protein expression of CD39 at the cell level in vitro was investigated; b) the concentration of extracellular adenosine (eADO) was investigated; c) the concentration of extracellular ATP (eATP) was investigated; d) the activation of immune cells and the polarization of pro-inflammatory phenotypes were investigated.

[0047] Fig. 9 The graph is a result of investigating the ability of free drugs or nanoparticles in Example 1 to promote the chemosensitization of tumor tissues. After administration, a) investigation of the apoptosis level of tumor cells in vitro; b) investigation of the proliferation and apoptosis level of tumor tissues in vivo. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0049] Example 1 A method for preparing enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs comprises the following steps: (1) A method for synthesizing a maleimidized gemcitabine prodrug: 100 mg of gemcitabine, 160.5 mg of 6-maleimidocaproic acid, 361.2 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 166 μL of N,N-diisopropylethylamine (DIPEA) were dissolved in 10 mL of anhydrous DMF and stirred at room temperature for 48 h. The reaction process is as shown in Formula I. The reaction was monitored by thin layer chromatography (methanol: dichloromethane = 1:10). After the solvent was removed by rotary evaporation, the crude product was purified by silica gel column (methanol: dichloromethane = 1:10) to obtain the maleimidized gemcitabine prodrug Gem-Mal in the form of a white solid.

[0050]

[0051] Formula I (2) Ⅰ. A method for synthesizing a polymer B-PDEA having ROS-responsive charge reversal, comprising the following steps: ① Dissolve 10.6 mg of 4-cyano-4-(phenylcarbonylthio)pentanoic acid, 645.2 mg of 2-(N,N-diethylamino)ethyl methacrylate and 4.92 mg of 2,2'-azobisisobutyronitrile (AIBN) in 2.5 mL of anhydrous methanol and add them to a Schlenk tube. The reaction solution was deoxygenated by repeated freeze-drying cycles with liquid nitrogen for five times. The polymerization reaction was carried out at 70°C for 16 h, and the reaction process was as shown in Formula II. After completion, the reaction was quenched by introducing oxygen by opening the Schlenk tube. The mixture solution was concentrated under reduced pressure to 1 / 5 of the original volume, and slowly dripped into n-hexane precooled to -20°C. The precipitation was repeated 3 times to obtain the enzyme-non-responsive polymer PDEA in the form of an orange-red viscous liquid.

[0052]

[0053] Formula II ② Dissolve 85 mg PDEA, 165.3 mg 4-bromomethylphenylboronic acid and 110.6 mg anhydrous K2CO3 in 5 mL anhydrous DMF. Stir at room temperature for 24 h under argon protection. The reaction process is as shown in Formula III. After completion, the reaction solution was dialyzed with deionized water (3.5k MWCO) for 24 hours. Freeze-dried and dehydrated to obtain flocculent gray-white solid poly (2-acryloyl) ethyl (benzyl borate) diethylammonium bromide B-PDEA. The overall synthetic route of B-PDEA is as follows: Figure 1 as shown in .

[0054]

[0055] Formula III II. A method for synthesizing a MMP-2 responsive and ROS responsive charge flipping polymer material B-PDEA-pep-NHS, comprising the following steps: ① 8 mg of MMP-2 responsive peptide (sequence: GPLGVRGC) and 3 μL of N,N-diisopropylethylamine (DIPEA) were dissolved in 1 mL of anhydrous DMF, and 5.3 mg of 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid N-succinimidyl ester was dissolved in 1 mL of anhydrous DMSO, and added dropwise to the DMF solution containing the peptide within 15 min, and the mixture was stirred at room temperature for 4 h. 4.6 mg of N-succinimidyl 6-maleimidocaproate was dissolved in 1 mL of anhydrous DMSO, and added dropwise to the reaction solution containing the peptide within 15 min, and the reaction was continued to stir at room temperature for 4 h. The reaction process is shown in Formula IV. After completion, the reaction solution was dialyzed with DMSO (1 k MWCO) for 24 h, and then dialyzed with deionized water (1 k MWCO) for 24 h. The chain transfer reagent RAFT-pep-NHS for coupling responsive peptides was obtained by freeze-drying and dehydration in the form of a flocculent dark pink solid.

[0056]

[0057] Formula IV ②26.3 mg RAFT-pep-NHS, 366.8 mg 2-(N,N-diethylamino)ethyl methacrylate and 3.7 mg 2,2'-azobisisobutyronitrile (AIBN) were dissolved in 2 mL of anhydrous methanol and added to a Schlenk tube. The reaction solution was deoxygenated by repeated freeze-drying cycles with liquid nitrogen for five times. The polymerization reaction was carried out at 70 ° C for 16 h, and the reaction process was as shown in Formula V. After completion, the reaction was quenched by introducing oxygen by opening the Schlenk tube. The mixture was concentrated under reduced pressure to 1 / 5 of the original volume, and slowly dripped into n-hexane precooled to -20 ° C. The precipitation was repeated 3 times to obtain the enzyme-responsive polymer PDEA-pep-NHS in the form of an orange-red viscous liquid.

[0058]

[0059] Formula V ③ Dissolve 50 mg PDEA-pep-NHS, 77.3 mg 4-bromomethylphenylboronic acid and 55.3 mg anhydrous K2CO3 in 3 mL anhydrous DMF. Stir at room temperature for 24 h under argon protection. The reaction process is as shown in Formula VI. After completion, the reaction solution was dialyzed with deionized water (3.5k MWCO) for 24 hours. Freeze-dried and dehydrated to obtain an enzyme-responsive cationic polymer B-PDEA-pep-NHS in the form of a flocculent gray-white solid. The overall synthetic route of B-PDEA-pep-NHS is as follows: Figure 2 as shown in .

[0060]

[0061] Formula VI (3) A method for preparing polymer nanoparticles siRNA@CPA for co-delivering siRNA and gemcitabine-loaded albumin, comprising the following steps: ① Dissolve B-PDEA and B-PDEA-pep-NHS at a concentration of 100 nmol / mL in ultrapure water without nuclease, and premix them at a molar ratio of 4:1 to obtain a working polymer solution.

[0062] ② Dissolve siCD39 at 10 nmol / mL in ultrapure water without nuclease, add it to the working polymer solution at a N / P ratio of 15:1, and pipette 10-50 times to mix thoroughly. Incubate the obtained polymer solution at room temperature for 30 min.

[0063] (4) Dissolve cholesterol in 1 mL of dichloromethane at a cholesterol:B-PDEA molar ratio of 5:1 and add to the eggplant-shaped flask. Remove the solvent by rotary evaporation to obtain a lipid film of cholesterol. Add 1 mL of the above polymer solution to the eggplant-shaped flask and treat with ultrasound for 10 min in an ice bath to rehydrate the cholesterol film. Stir at room temperature overnight.

[0064] (5) a. Dissolve 30 mg HSA in 2.5 mL PBS buffer 8.0 containing 5 mM EDTA, add 0.63 mg 2-iminothiolane hydrochloride, and react at room temperature under nitrogen for 4 h. After completion, the mixture is desalted by passing through a desalting column at a centrifugal force of 1000 g for 6 min. Repeat the desalting three times to collect the product HSA-SH.

[0065] b. Dissolve 10 mg of the above Gem-Mal in 50 μL DMSO, add it to the desalted product liquid HSA-SH at a molar ratio of 1.2:1, stir at room temperature overnight, desalt three times using the same method, collect the product and lyophilize and dehydrate to obtain Gem-HSA.

[0066] (6) Dissolve the gemcitabine-loaded albumin Gem-HSA obtained in step (5) b and add it to the cholesterol-coated polymer complex solution obtained in step (4), and mix thoroughly to obtain cascade-targeted nanoparticles siRNA@CPA.

[0067] The particle size, distribution and microscopic morphology of siRNA@CPA nanoparticles are shown in Figure 2. Figure 3 As shown: Under physiological conditions, the average particle size of the nanoparticles is 71.2 nm, with uniform distribution and round morphology (polydispersity coefficient is 0.321).

[0068] The siRNA loading capacity of B-PDEA nanoparticles was investigated: when the N / P ratio was greater than or equal to 10:1, the nanoparticles could load siRNA well. The coupling of B-PDEA nanoparticles with gemcitabine-loaded albumin was investigated. Figure 4 As shown in the figure, the fluorescence emission peak of B-PDEA in the nanoparticle group disappeared while the emission peak intensity of albumin increased significantly. The existence of the fluorescence resonance energy transfer (FRET) effect indicated that the two were successfully coupled and the distance was within 10 nm.

[0069] The MMP-2 responsiveness of siRNA@CPA nanoparticles was investigated. Figure 4 As shown in b: After adding MMP-2 treatment (nanoparticles + MMP-2), the observed FRET effect disappeared, and the independent emission peaks of B-PDEA nanoparticles and albumin reappeared, indicating that siRNA@CPA nanoparticles have MMP-2 responsiveness.

[0070] The ROS responsiveness of siRNA@CPA nanoparticles was investigated. Figure 5 a shows the ROS response mechanism of B-PDEA. Figure 5 As shown: b) 1 H NMR results showed that after H2O2 treatment, the characteristic peak of phenylboronic acid on the side chain of B-PDEA weakened, while the characteristic peak of the reaction product p-hydroxybenzyl alcohol (HMP) increased. c) HPLC results showed that with the extension of H2O2 treatment time, the peak area of ​​HMP increased and the response process was completed within 120 min.

[0071] The siRNA release behavior of siRNA@CPA nanoparticles in response to intracellular ROS was investigated. Figure 6 As shown: a) The loaded siRNA can be completely released in an environment with H2O2 above 1 mM; b) more release was observed in tumor cells (KPC) with higher intracellular ROS levels compared with stromal cells (NIH-3T3) (scale bar, 20 μm).

[0072] The tumor tissue penetration ability of siRNA@CPA nanoparticles was investigated. Figure 7 As shown: a) The MMP-2 response group (B-PDEA@Chole-pep-Alb) has a stronger deep tumor tissue accumulation signal than the non-response group (B-PDEA@Chole-Alb) (scale bar is 200 μm), and b) in the tumor sphere model, it was observed that MMP-2 treatment can significantly increase the ability of albumin signals to penetrate and accumulate in deep tumor tissues (scale bar is 100 μm). The above results show that the introduction of the design of MMP-2 responsive peptides is conducive to improving the tumor tissue penetration ability of nanoparticles and drug-loaded albumin.

[0073] The ability of siRNA@CPA nanoparticles to regulate tumor adenosine metabolism was investigated. Figure 8 As shown: Nanoparticles can significantly a) downregulate the expression level of CD39, thereby significantly b) reduce the extracellular adenosine concentration, c) increase the ATP concentration, leading to d) more local immune cell activation and pro-inflammatory phenotype polarization.

Claims

1. An enhanced penetration dual-responsive nanoparticle for co-delivering chemotherapeutic drugs and gene drugs, characterized in that: The invention comprises gemcitabine-loaded albumin, a metabolism-regulating gene drug, a responsive polymer and cholesterol; the metabolism-regulating gene drug is a small interfering RNA siCD39 that inhibits CD39 expression; the responsive polymer at least comprises a dual-responsive polymer of MMP-2 and ROS; the metabolism-regulating gene drug is encapsulated in the responsive polymer and then encapsulated in the cholesterol; the gemcitabine-loaded albumin is coupled to the responsive polymer via an MMP-2 responsive polypeptide.

2. The enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 1, characterized in that: The responsive polymer also includes a ROS responsive polymer, and the molar ratio of the MMP-2 and ROS dual responsive polymer to the ROS responsive polymer is 1:4; and the ROS responsive polymer contains a phenylboronic acid group.

3. The method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 2, characterized in that: The following steps are involved: (1) Gem and 6-maleimidocaproic acid are reacted at room temperature in the presence of an amidation catalyst and an acid-binding agent, and then separated and purified by silica gel chromatography to obtain the maleimidized gemcitabine prodrug Gem-Mal; (2) a. dissolving the MMP-2 responsive polypeptide (sequence: GPLGVRGC) and the acid-binding agent in a good solvent to obtain a MMP-2 responsive polypeptide solution; b. 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid and N-succinimide ester are dissolved in a good solvent, added to the MMP-2 response polypeptide solution, and reacted at room temperature to couple the MMP-2 response polypeptide with the chain transfer reagent to obtain reaction solution 1; c. The N-succinimidyl 6-maleimidocaproate is dissolved in a good solvent and added to the reaction solution in step b, and further reacted at room temperature to obtain a reaction solution II; d. The reaction solution 2 is dialyzed to remove impurities, and then freeze-dried and dehydrated to obtain a chain transfer reagent RAFT-pep-NHS coupled to a responsive polypeptide; (3) a. deoxygenating 4-cyano-4-(phenylcarbonylthio)pentanoic acid or the RAFT-pep-NHS described in step (2) with 2-(N,N-diethylamino)ethyl methacrylate and a catalyst, and then polymerizing them at 70° C. to obtain a reaction solution; b. After the reaction is completed, air is introduced into the reaction solution of step a to quench the reaction, and the reaction is concentrated under reduced pressure and precipitated three times with a precipitant to obtain an enzyme-non-responsive polymer PDEA or an enzyme-responsive polymer PDEA-pep-NHS; c. Add 4-bromomethylphenylboronic acid and anhydrous K2CO3 to the PDEA or PDEA-pep-NHS obtained in step b, and stir the dissolved solution at room temperature under argon protection; d. After the reaction is completed, the solution obtained in step c is dialyzed with deionized water for 24 h, and lyophilized and dehydrated to obtain an enzyme-non-responsive cationic polymer B-PDEA or an enzyme-responsive cationic polymer B-PDEA-pep-NHS; (4) a. Dissolving the synthesized polymer materials B-PDEA and B-PDEA-pep-NHS in nuclease-free ultrapure water at different concentrations, and premixing them to obtain a working polymer solution; b. Dissolve the gene drug siCD39 (sequence: 5'-GGGCAGAUUCACUCAGGAA-3', 5'-UUCCUGAGUGAAUCUGCCC-3') in ultrapure water without nuclease at the required concentration, and add the siRNA solution to the working polymer solution obtained in step (4) a at the required N / P ratio, mix thoroughly and let stand at room temperature; (5) adding the cholesterol solution to the eggplant-shaped flask, removing the solvent by rotary evaporation to form a thin lipid film, adding the polymer solution obtained in step (4) to the eggplant-shaped flask containing the cholesterol film, ultrasonically treating the cholesterol film to rehydrate it, and stirring at room temperature overnight to obtain a cholesterol-coated polymer complex solution; (6) a. reacting human serum albumin (HSA) and 2-iminothiolane hydrochloride at room temperature, and obtaining HSA-SH having a free thiol group after desalting; b. adding the gemcitabine prodrug Gem-Mal obtained above to the HSA-SH solution obtained in step (6) in the required molar ratio, reacting at room temperature, desalting and freeze-drying to obtain the target product, gemcitabine drug-loaded albumin Gem-HSA; (7) Dissolve the gemcitabine-loaded albumin Gem-HSA obtained in step (6) b and add it to the cholesterol-coated polymer complex solution obtained in step (5), and mix thoroughly to obtain cascade-targeted nanoparticles siRNA@CPA.

4. The method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 3, characterized in that: In step (1), the molar ratio of gemcitabine to 6-maleimidocaproic acid is 1:2; the catalyst is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); the acid-binding agent is N,N-diisopropylethylamine (DIPEA), and the molar ratio of the catalyst, the acid-binding agent and gemcitabine is (2-3):(2-3):1; preferably, the reaction solvent is N,N-dimethylformamide (DMF), and the reaction time at room temperature is 48 h; preferably, the eluent for the chromatographic column separation in step (1) is methanol: dichloromethane 1:10, v:v.

5. The method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 3, characterized in that: The good solvent of the MMP-2 responsive polypeptide described in step (2) a is N,N-dimethylformamide (anhydrous DMF); the acid-binding agent is N,N-diisopropylethylamine (DIPEA), and the molar ratio of the acid-binding agent to the MMP-2 responsive polypeptide is (1-2):1; Preferably, in step (2) b, the molar ratio of the MMP-2 responsive polypeptide to 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid N-succinimidyl ester is 1:(1-1.5), and the good solvent of the 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid N-succinimidyl ester is dimethyl sulfoxide (DMSO); further preferably, the solution of 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid N-succinimidyl ester should be dripped into the MMP-2 responsive polypeptide solution, and the dripping process is controlled to be completed within 15 minutes, and the activation reaction time at room temperature is 4 hours; Preferably, in step (2) c, the molar ratio of the MMP-2 response polypeptide to N-succinimidyl 6-maleimidocaproate is 1:(1-1.5), and the good solvent for N-succinimidyl 6-maleimidocaproate is dimethyl sulfoxide (DMSO); further preferably, the solution of N-succinimidyl 6-maleimidocaproate should be dripped into the reaction solution 1 drop by drop, and the dripping process is controlled to be completed within 15 minutes, and the reaction time at room temperature is 4 hours; Preferably, in step (2) d, a dialysis bag with a molecular weight cutoff of 1 kDa is used for the dialysis process; further preferably, the dialysis time in DMSO and deionized water is 24 h.

6. The method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 3, characterized in that: In step (3) a, the molar ratio of 4-cyano-4-(phenylcarbonylthio)pentanoic acid or the RAFT-pep-NHS in step (2) to 2-(N,N-diethylamino)ethyl methacrylate is 1: (50-100); the catalyst is 2,2'-azobisisobutyronitrile (AIBN), and the molar ratio of AIBN to 4-cyano-4-(phenylcarbonylthio)pentanoic acid or the RAFT-pep-NHS is (0.5-1):1; preferably, anhydrous methanol is used as the reaction solvent; Preferably, the deoxygenation operation in step (3) a is performed in a Schlenk tube; specifically, after the materials in step (3) a are mixed and dissolved, the solution is added to a Schlenk tube, the solution in the tube is quickly frozen using liquid nitrogen, an oil pump is connected to suck and thaw, high-purity nitrogen is introduced, and liquid nitrogen is used for quick freezing again, and the above liquid nitrogen repeated freeze-drying cycle is repeated 5-10 times to achieve deoxygenation; further preferably, the reaction time at 70° C. is 16 h; Preferably, the precipitant in step (3) b is n-hexane precooled to -20°C; specifically, the reaction solution in step a is concentrated under reduced pressure to 1 / 5 of the original volume, and then slowly dripped into precooled n-hexane. After sufficient precipitation, the n-hexane is removed by centrifugation, and the precipitation is repeated 3 times, and then vacuum dried to obtain a precipitated product; Preferably, in step (3) c, the molar ratio of PDEA, 4-bromomethylphenylboronic acid and anhydrous K2CO3 is 1:(100-150):(100-150), the solvent is N,N-dimethylformamide (DMF), and the reaction time at room temperature is 24 h; Preferably, in step (3) d, a dialysis bag with a molecular weight cutoff of 3.5 kDa is used for the dialysis process.

7. The method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 3, characterized in that: The molar ratio of B-PDEA to B-PDEA-pep-NHS in the working polymer solution in step (4) a is 4:1; Preferably, the N / P ratio in step (4) b is 15:1; further preferably, the mixture is fully mixed by pipetting 10 to 50 times, and the mixture is allowed to stand at room temperature for 30 minutes; Preferably, the ultrasonic treatment process in step (5) is carried out in an ice bath, and the ultrasonic treatment time is 10 min.

8. The method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 3, characterized in that: In step (6) a, the molar ratio of human serum albumin to 2-iminothiolane hydrochloride is 1:10; the reaction solvent is PBS buffer 8.0 containing 5 mM ethylenediaminetetraacetic acid (EDTA); Preferably, the reaction is carried out under nitrogen protection, and the reaction time is 4 h at room temperature; Preferably, in step (6) b, the molar ratio of Gem-Mal to HSA-SH is 1.2:1; the solvent is dimethyl sulfoxide (DMSO); further preferably, the final concentration of DMSO during the reaction is controlled to be 10%, v:v, and the reaction time at room temperature is 12-24 h; Preferably, the desalting process in step (6) a and step (6) b is completed by a desalting column, the centrifugal force is 1000 g, the centrifugation time is 6 min, and the desalting is preferably repeated 3 times.

9. The method for preparing the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 3, characterized in that: In step (7), the solvent of Gem-HSA is ultrapure water that does not contain nuclease, and the mixture is thoroughly mixed by pipetting 10-50 times.

10. Use of the enhanced penetration dual-responsive nanoparticles for co-delivering chemotherapeutic drugs and gene drugs according to claim 1 or 2 in the preparation of drugs for treating pancreatic cancer.

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