Glutamine metabolism regulation nano drug delivery system for enhancing targeting and penetration of pancreatic cancer as well as preparation method and application of glutamine metabolism regulation nano drug delivery system

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a glutamine metabolism regulation nano drug delivery system for enhancing pancreatic cancer targeting and penetration as well as a preparation method and application of the glutamine metabolism regulation nano drug delivery system. The metabolism regulation medicine is a glutamine transporter and a metabolic enzyme inhibitor; the nano drug delivery system is formed by self-assembly of a multifunctional block polymer and a drug; the polymer has collagen targeting peptide CBP modification, and can target high-abundance collagenous fibers to realize tumor enrichment; the MMP-7 response peptide can respond to high-concentration MMP-7 and trigger size reduction and deep penetration of the drug delivery system; the responsive drug release element can trigger drug release in a tumor intracellular reduction environment. The constructed nano drug delivery system is high in compatibility and suitable for selective delivery of various metabolic regulation drugs; pancreatic cancer drug accumulation and deep penetration can be remarkably enhanced, non-target tissue leakage is reduced, and accurate drug delivery and metabolism regulation strategy synergism and toxicity reduction are achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a glutamine metabolism regulation nano-drug delivery system for enhancing pancreatic cancer targeting and penetration, and a preparation method and application thereof. BACKGROUND

[0002] The rapid malignant proliferation ability of pancreatic cancer and the dense stroma and immunosuppressive tumor microenvironment (TME) hinder the infiltration and effect of drugs and immune cells, and limit the clinical prognosis of drug and immunotherapy programs. The above treatment difficulties are closely related to the extensive glutamine metabolic reprogramming in pancreatic cancer.

[0003] Specifically, pancreatic cancer cells upregulate the expression of glutamine transporter SLC1A5 and metabolic enzyme glutaminase (GLS), excessively uptake and metabolically utilize glutamine for material synthesis, energy supply and maintenance of intracellular redox homeostasis; at the same time, competitively limit the utilization of glutamine by other cell components in the TME, thereby inducing metabolic stress and tumor-promoting reprogramming, promoting tumor-associated fibroblasts to secrete extracellular matrix (ECM) and immune cell depletion, resulting in difficulty in infiltration and effect of drugs and immune cells. Therefore, glutamine addiction directly promotes the deterioration of pathological features in pancreatic cancer progression. Regulating glutamine addiction provides a promising new strategy for pancreatic cancer treatment.

[0004] However, the safety and effectiveness of glutamine metabolic regulation strategies are both challenged. Since normal cells (such as gastrointestinal cells) also depend on glutamine metabolism, metabolic regulation drugs often have intolerable gastrointestinal side effects; in contrast, tumor cells can trigger drug resistance by activating compensatory metabolic pathways under drug stimulation. In addition, ECM proliferation driven by glutamine addiction further hinders the perfusion and penetration of drugs, resulting in the inability of active ingredients of drugs to accumulate to an effective concentration in pancreatic cancer cells. The above factors jointly restrict the clinical translation of metabolic regulation therapy. Based on this, the inventors speculate that the use of multi-drug combination therapy and the assistance of tumor tissue targeting and ECM penetration delivery strategies can achieve specific enrichment of drugs in tumor cells rather than normal tissues or ECM, which is expected to improve the feasibility of glutamine metabolic regulation therapy.

[0005] In response to the challenge of treatment feasibility, prodrug strategies and delivery system design can provide corresponding solutions. 6-diazo-5-oxo-L-norleucine (DON) is the most classic GLS inhibitor. Extensive research has been devoted to the development of responsive prodrugs of DON to enhance its metabolic regulation efficacy and limit its cytotoxicity in normal tissues. At the same time, extensive studies have shown that nano-delivery systems matching the pathological characteristics can achieve the synergistic effect of drug therapy by changing the tissue distribution characteristics of the drug. Larger size nano-delivery systems have better circulation stability. According to the Stokes-Einstein equation, small size particles have stronger ECM penetration ability. Therefore, designing a responsive size-variable nano-delivery system is a feasible solution to simultaneously optimize the drug circulation stability and intratumoral accumulation behavior. In summary, the design of responsive prodrugs and nano-delivery systems matching the pathological characteristics of pancreatic cancer can improve the anti-tumor efficiency of glutamine metabolic regulation strategies while reducing off-target drug toxicity, addressing the clinical problems of metabolic regulation therapy. SUMMARY

[0006] The present application is aimed at the problems encountered in the above-mentioned glutamine metabolic regulation therapy for pancreatic cancer, based on the existing technical basis, a DON-responsive prodrug is synthesized, and a glutamine metabolic regulation nano-delivery system that enhances pancreatic cancer targeting and penetration is designed and prepared based on this. The system is constructed by loading metabolic regulation drugs in a multifunctional block polymer. The multifunctional block polymer is a polymer T-PPLN with pancreatic cancer tissue targeting, MMP-7 and reducing substance response ability, which is obtained by connecting polyethylene glycol (PEG) and polylysine as the backbone through matrix metalloproteinase-7 (MMP-7) responsive peptide, nitroimidazole modification, and finally coupling collagen targeting peptide CBP. The metabolic regulation drug is the GLS inhibitor NDY-J (DON prodrug) and the SLC1A5 inhibitor V-9302.

[0007] In the design of the present application, NDY-J acts as a DON prodrug and GLS inhibitor, with the ability to release active forms specifically in tumors, interfere with tumor cell glutamine metabolism. Further, in order to achieve synergistic effect of drugs and avoid compensatory pathway resistance, the glutamine transporter SLC1A5 inhibitor V-9302 is also used as a delivery drug.

[0008] In the design of the application, since nitroimidazole, NDY-J and V-9302 all have hydrophobicity and aromatic groups, the components in the prescription can self-assemble into a nanodelivery system through hydrophobic interaction and π-π stacking. After intravenous injection of the nanodelivery system, it can accumulate in the ECM of pancreatic cancer rich in collagen fibers through the collagen targeting ability of CBP. Further, under the action of high concentration MMP-7 in ECM, the nanodelivery system can realize responsive particle size reduction and enhanced penetration through de-PEGylation. Due to deep penetration and unshielding effect, the nanodelivery system is more easily taken up by tumor cells. When it reaches the tumor cells containing high concentration of reducing substances, nitroimidazole can be reduced and lead to the disintegration of the nanodelivery system and the synchronous release of NDY-J and V-9302, which synergistically inhibit the uptake and metabolism of glutamine by pancreatic cancer cells. The application inhibits the proliferation of pancreatic cancer supported by glutamine while restoring the availability of glutamine in the TME, reactivating anti-tumor immunity, and is expected to enhance the therapeutic efficacy for pancreatic cancer.

[0009] The above design integrates strategies such as promoting drug tumor accumulation and deep penetration, selectively releasing metabolic regulatory drugs, regulating tumor microenvironment, and activating anti-tumor immune response into one nanodelivery system, providing a promising combined treatment strategy for pancreatic cancer.

[0010] To achieve the above purpose, the application provides the following technical solutions: The application provides a preparation method of a glutamine metabolic regulation nanodelivery system for enhancing pancreatic cancer targeting and penetration, comprising the following steps: I. A synthesis method of DON prodrug NDY-J, comprising the following steps: (1) 5-oxopyrrolidine-2-carboxylic acid ethyl ester is reacted with lithium bis(trimethylsilyl)amide (LiHMDS) at low temperature, then fluorenylmethoxycarbonyl chloride is added. After a period of reaction, the temperature is raised to room temperature for reaction, and then the intermediate product NDY-I is obtained by silica gel column chromatography purification; (2) Trimethylsilyldiazomethane is reacted with n-butyllithium at low temperature for a period of time, then NDY-I is added. After a period of continuous reaction, the intermediate product NDY-J is obtained by silica gel column chromatography purification.

[0011] Preferably, the molar ratio of 5-oxopyrrolidine-2-carboxylic acid ethyl ester, LiHMDS and fluorenylmethoxycarbonyl chloride in step (1) is 1: (0.5-2): (2-5); further preferably, the reaction solvent is anhydrous tetrahydrofuran (THF), and the reaction temperature is-78 degrees Celsius, which is realized by dry ice bath.

[0012] Preferably, the fluorenylmethoxycarbonyl chloride in step (1) is slowly added to the reaction system, and the dropping time is controlled within 10-15 minutes; further preferably, the reaction time under dry ice bath is 2 hours, and the reaction time at room temperature is 16 hours.

[0013] Preferably, the eluent for column chromatography separation in step (1) is petroleum ether: ethyl acetate 3:1, v:v.

[0014] Preferably, the molar ratio of trimethylsilyl diazomethane, n-butyllithium and NDY-I in step (2) is 1:(1-1.2):(0.2-0.5); further preferably, the reaction solvent is anhydrous THF, the reaction temperature is-98~-116 degrees Celsius, and the reaction time is 30 minutes.

[0015] Preferably, NDY-I in step (2) is slowly added to the reaction system, and the dropping time is controlled within 10-15 minutes; further preferably, the reaction time after adding NDY-I is 30-60 minutes.

[0016] Preferably, the eluent for column chromatography separation in step (2) is chloroform: acetone 1:40, v:v.

[0017] II. A multifunctional block polymer T-PPLN with pancreatic cancer tissue targeting, MMP-7 and reducing substance response capability, comprising the following steps: (1) After stirring reaction of N(ε)-benzyloxycarbonyl-L-lysine and triphosgene under argon protection at 50 degrees Celsius, precipitating agent is used for precipitation, and then suction filtration is performed to obtain polymeric monomer Lys(Z)-NCA; (2) After stirring reaction of 1-methyl-2-nitroimidazole-5-methanol (NI) and p-nitrophenyl chloroformate (PNP-Cl) under argon protection at room temperature in the presence of a catalyst, silica gel column chromatography separation and purification are performed to obtain activated nitroimidazole PNP-NI; (3) After stirring reaction of azido group and N-hydroxy succinimide capped PEG with molecular weight of 5 kDa and N-1-(4,4-dimethyl-2,6-dioxohexyl) ethyl protected lysine capped MMP-7 responsive peptide (the sequence is shown as SEQ ID NO. 1) under room temperature in the presence of a catalyst, hydrazine hydrate is added to remove difluorovinyl protecting groups, dialysis and lyophilization are performed to obtain an intermediate product PEG-pep; (4) Lys(Z)-NCA obtained in step (1) is added to PEG-pep obtained in step (3), and stirring reaction is performed under argon protection at 50 degrees Celsius, and then dialysis and lyophilization are performed to obtain a block polymer PEG-pep-pLys(Z) protected by benzyloxycarbonyl; a strong acid is used to remove the benzyloxycarbonyl protecting group, and then dialysis and lyophilization are performed to obtain a block polymer PEG-pep-pLys. (5) The PNP-NI obtained in step (2) is added to the PEG-pep-pLys obtained in step (4), and the reaction is stirred under an argon atmosphere at room temperature under alkaline conditions. After dialysis and lyophilization, the multifunctional block polymer PEG-pep-pLys(NI), i.e., PPLN, is obtained; (6) The PPLN obtained in step (5) is dissolved, and the propargyl glycine-capped collagen-targeting peptide CBP (the sequence is shown in SEQ ID NO. 2) is added. In the presence of a catalyst, the reaction is stirred under an argon atmosphere at room temperature in the dark. After dialysis and lyophilization, the multifunctional block polymer T-PEG-pep-pLys(NI) with pancreatic cancer tissue targeting function, i.e., T-PPLN, is obtained.

[0018] Preferably, in step (1), the molar ratio of N(ε)-benzyloxycarbonyl-L-lysine to triphosgene is 1: (0.3-0.5); further preferably, anhydrous THF is used as the reaction solvent, and the reaction time is 3 hours.

[0019] Preferably, in step (1), the precipitant is n-hexane pre-cooled to -20°C. Specifically, the reaction solution is added dropwise to n-hexane stirred vigorously, and after sufficient precipitation, the n-hexane is removed by suction filtration, and the precipitate is washed with ice n-hexane 3 times, and then vacuum dried to obtain the precipitated product.

[0020] Preferably, in step (2), the catalyst is triethylamine and a catalytic amount of 4-(dimethylamino)pyridine (DMAP); further preferably, the molar ratio of NI, PNP-Cl, and triethylamine is 1: (1.5-2.0) : (1.5-2.0), DMAP is 1-5 mg, and anhydrous THF is used as the reaction solvent.

[0021] Preferably, in step (2), PNP-Cl should be added dropwise to the solution containing NI, and the dropwise addition process should be maintained for 30-60 minutes; further preferably, an ice water bath is used to control the reaction solution temperature to 0°C during the dropwise addition process.

[0022] Preferably, in step (2), the eluent for column chromatography separation is petroleum ether: ethyl acetate 4:1, v:v.

[0023] Preferably, in step (3), the catalyst is triethylamine and N,N-diisopropylethylamine (DIPEA); further preferably, the molar ratio of PEG, pep-Dde, and DIPEA is 1: (1.2-1.5) : (1.2-1.5), anhydrous DMSO is used as the reaction solvent, and the reaction time is 16 hours.

[0024] Preferably, in step (3), the concentration of hydrazine hydrate is 2-5%, and the reaction time is 2 hours.

[0025] Preferably, the dialysis process in step (3) is carried out in deionized water, and the dialysis external solution is replaced every 8 hours; further preferably, the dialysis process uses a dialysis bag with a molecular weight cut-off of 3.5 kDa.

[0026] Preferably, in step (4), the molar ratio of PEG-pep to Lys(Z)-NCA is 1: (25-35), anhydrous DMSO is used as the reaction solvent, and the reaction time is 72 hours.

[0027] Preferably, in step (4), the strong acid selected for the deprotection reaction is trifluoroacetic acid containing 5% hydrogen bromide; further preferably, the deprotection reaction is carried out at room temperature for 2-4 hours.

[0028] Preferably, the dialysis process in step (4) is carried out in deionized water, and the dialysis external solution is replaced every 8 hours; further preferably, the dialysis process uses a dialysis bag with a molecular weight cut-off of 10 kDa.

[0029] Preferably, in step (5), the alkaline conditions refer to the presence of triethylamine and DMAP in the reaction solution; further preferably, the molar ratio of PEG-pep-pLys, PNP-NI, and triethylamine is 1: (20-30) : (20-30), DMAP is 1-5 mg, anhydrous N,N-dimethylformamide (DMF) is used as the reaction solvent, and the reaction time is 24 hours.

[0030] Preferably, in step (5), the dialysis process is first carried out in anhydrous DMF for 24 hours, with the dialysis external solution being replaced every 8 hours; then carried out in deionized water for 48 hours, with the dialysis external solution being replaced every 12 hours; further preferably, the dialysis process uses a dialysis bag with a molecular weight cut-off of 10 kDa.

[0031] Preferably, in step (6), the catalyst is sodium ascorbate, cuprous iodide, and DIPEA, and the molar ratio of PEG-pep-pLys(NI), collagen-targeting peptide CBP, sodium ascorbate, cuprous iodide, and DIPEA is 1: (1.5-2) : (2-4) : (2-3) : (2-3), anhydrous DMF is used as the reaction solvent, and the reaction time is 24 hours.

[0032] Preferably, in step (6), the dialysis process is first carried out in 10 mM aqueous disodium ethylenediaminetetraacetate for 24 hours; then carried out in deionized water for 24 hours, with the dialysis external solution being replaced every 8 hours; further preferably, the dialysis process uses a dialysis bag with a molecular weight cut-off of 10 kDa.

[0033] III. The preparation method of the responsive nano-drug delivery system with pancreatic cancer tissue targeting and ECM enhanced penetration function, comprising the following steps: (1) The synthesized polymer materials PPLN and T-PPLN are dissolved in DMF respectively, and a working polymer solution is obtained by pre-mixing according to the proportion; (2) NDY-J and V-9302 are dissolved in DMF respectively, and a drug stock solution is obtained by pre-mixing according to the proportion; (3) The working polymer solution obtained in step (1) and the drug stock solution obtained in step (2) are mixed according to the proportion, and vortexed thoroughly to mix evenly; (4) The solution containing polymer and drug obtained in step (3) is slowly dropped into deionized water or buffer, and high-speed stirring is maintained during the dropping process to obtain a nano-drug delivery system dispersion; (5) The nano-drug delivery system dispersion obtained as described above is dialyzed in deionized water or buffer, and the supernatant is obtained after centrifugation of the dialyzed dispersion, and the supernatant is the multifunctional nano-drug delivery system.

[0034] Preferably, the molar ratio of PPLN and T-PPLN in the working polymer solution in step (1) is 4:1; further preferably, the total concentration of the polymer is 100 mg / mL.

[0035] Preferably, the molar ratio of NDY-J and V-9302 in the drug stock solution in step (2) is 2:1; further preferably, the concentration of NDY-J is 10 mg / mL.

[0036] Preferably, the volume ratio of the working polymer solution and the drug stock solution mixed in step (3) is 1:1; further preferably, the vortex mixing time is 5 minutes, and 3000 rpm centrifugation is performed for 5 minutes after vortexing to remove insoluble substances.

[0037] Preferably, the buffer in step (4) is phosphate buffer (pH 7.4) or physiological saline, and the dropping process should be carried out within 15-30 minutes; further preferably, the stirring speed is 1500 rpm.

[0038] Preferably, the dialysis buffer in step (5) is phosphate buffer (pH 7.4) or physiological saline, and a dialysis bag with a molecular weight cut-off of 10 kDa is selected; further preferably, 3000 rpm centrifugation is performed for 5 minutes after dialysis or filtration through a 0.22 μm microporous filter.

[0039] The application also provides the pancreatic cancer tissue-targeting, ECM-penetrating and response-releasing nanodelivery system for co-delivering metabolic regulatory drugs, which is composed of glutamine metabolic regulatory drugs, pancreatic cancer tissue-targeting elements, ECM enzyme environment response elements and tumor intracellular response release elements.

[0040] The multifunctional block polymer skeleton provided by the application comprises PEG, MMP-7 response peptides and nitroimidazole-modified polylysine blocks. The components are coupled by amide bonds. The polylysine is obtained by ring-opening polymerization of N-carboxylic anhydride; the collagen-targeting peptide CBP is modified at the end with an alkyne group, and the coupling of the targeting element is realized by the reaction of the azido group at the end of PEG through a Click reaction.

[0041] The multifunctional block polymer T-PPLN has amphiphilicity, wherein the collagen-targeting peptide-PEG end has hydrophilicity, and the polylysine end modified with a nitroimidazole has lipophilicity. Therefore, the T-PPLN and the PPLN can self-assemble to form nanoparticles in an aqueous system.

[0042] In the application, the nanodelivery system realizes drug loading through hydrophobic interaction and π-π stacking between the drug, the PPLN and the T-PPLN.

[0043] The room temperature referred to in the application is 25℃.

[0044] The application also provides the use of the responsive nanodelivery system with pancreatic cancer tissue targeting and ECM-enhanced penetration function in the preparation of a drug for treating pancreatic cancer.

[0045] Compared with the prior art, the application has the following advantages: (1) The nanodelivery system of the application is high in preparation convenience, strong in compatibility and good in stability, and can be applied to the selective delivery of various hydrophobic drugs and / or drug combinations.

[0046] (2) The nanodelivery system of the application has a relatively long circulation time in vivo, and does not show obvious systemic toxicity after multiple administrations.

[0047] (3) The nano drug delivery system of the present application has obvious tumor accumulation and deep tumor penetration ability compared with the traditional intravenous administration mode, can effectively deliver drugs to the deep part of the tumor, play the role of enhanced metabolic regulation, and reduce the systemic toxicity.

[0048] (4) The nano drug delivery system of the present application can specifically regulate the abnormal glutamine metabolism of tumor, reduce the uptake and utilization of glutamine in the microenvironment, restore the glutamine content in the microenvironment, relieve the immunosuppressive microenvironment, inhibit tumor proliferation, and activate anti-tumor immune response. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 It is a synthesis route map of DON prodrug NDY-J.

[0050] Figure 2 It is a synthesis route map of multifunctional block polymer T-PPLN.

[0051] Figure 3 It is a micro-morphology, particle size and distribution diagram of the nano drug delivery system J&V@T-PPLN NPs: a) particle size and polydispersity coefficient measured by DLS, b) TEM picture of the nano drug delivery system, the scale is 50 nm.

[0052] Figure 4 It is a drug loading condition investigation result diagram of the nano drug delivery system.

[0053] Figure 5 It is a drug release capacity investigation result diagram of the nano drug delivery system in the intracellular reducing environment: a) release curve of NDY-J in different environments; b) release curve of V-9302 in different environments.

[0054] Figure 6 It is a pancreatic cancer tissue in vivo targeting ability investigation result diagram of the nano drug delivery system; the scale is 100 mu m, and the tumor stroma marker is alpha-smooth muscle actin; wherein, the control drug delivery system is the nano drug delivery system J&V@PPLN NPs without collagen targeting peptide CBP and non-tumor tissue targeting.

[0055] Figure 7 It is a performance detection diagram of the nano drug delivery system: a) particle size change of the nano drug delivery system after being treated with different concentrations of MMP-7; b) in vitro tumor sphere penetration ability investigation and semi-quantitative results, the scale is 100 mu m; c) ECM penetration ability investigation and semi-quantitative results of the nano drug delivery system, the scale is 100 mu m, and the vascular endothelial marker is platelet endothelial cell adhesion molecule; wherein, the control drug delivery system is the nano drug delivery system J&V@T-PLN NPs without MMP-7 responsive peptide and with unchangeable size.

[0056] Figure 8 The results of investigating the ability of free drug or nanodelivery system to regulate pancreatic cancer glutamine metabolism; specific indicators include: a) glutamine consumption of KPC tumor cells; b) intracellular glutamate content of tumor cells; c) intracellular alpha-ketoglutarate content of tumor cells; d) intracellular glutathione content of tumor cells; e) intracellular NADPH / NADP + ratio of tumor cells and f) intracellular ATP content of tumor cells.

[0057] Figure 9 The results of investigating the anti-tumor function of nanodelivery system or control group in vivo, specific indicators include: a) survival of tumor-bearing model mice after administration; b) tumor volume change; c) body weight change of model mice during treatment; d) expression level of proliferation marker Ki67 in tumor tissue of model mice and e) H&E staining results of small intestine sections of model mice after treatment, scale is 200 μm. DETAILED DESCRIPTION

[0058] The advantages and characteristics of the present application will become more apparent with the description of specific embodiments. The embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0059] Drug loading refers to the percentage of the mass of the drug loaded in the nanodelivery system to the total mass of the nanodelivery system; encapsulation efficiency refers to the percentage of the mass of the drug loaded in the nanodelivery system to the actual mass of the drug added in the preparation process.

[0060] Example 1 A synthetic method of DON prodrug NDY-J, as shown in Figure 1 , includes the following steps: (1) Under argon protection, 314 mg of 5-oxopyrrolidine-2-carboxylic acid ethyl ester was dissolved in 10 mL of anhydrous THF, and the reaction system was cooled to -78 degrees Celsius by dry ice bath. 2 mL of 1 mol / L LiHMDS THF solution was slowly added dropwise within 5 minutes. The reaction was stirred at this temperature for 20 minutes. Another 2.58 g of fluorenylmethoxycarbonyl chloride was dissolved in 10 mL of anhydrous THF and cooled to -78 degrees Celsius. The fluorenylmethoxycarbonyl chloride solution was added dropwise to the reaction solution within 15 minutes. After stirring at this temperature for 2 hours, the reaction was gradually warmed to room temperature and continued to stir for 16 hours. The reaction process is as shown in formula I. The reaction was monitored by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). After the solvent was removed by rotary evaporation, the crude product was purified by silica gel column (petroleum ether: ethyl acetate = 3:1) to obtain the prodrug intermediate NDY-I in the form of a colorless solid.

[0061]

[0062] Formula I (2) Under argon protection, 0.5 mL of 2 mol / L trimethylsilyl diazomethane ethyl ether solution was dissolved in 6 mL of anhydrous THF, and the reaction system was cooled to -98 degrees Celsius by liquid nitrogen-ethanol bath. 0.55 mL of 2.5 mol / L n-butyllithium n-hexane solution was slowly added dropwise within 5 minutes. After stirring at this temperature for 30 minutes, it was cooled to -116 degrees Celsius. Another 82.7 mg of NDY-I was dissolved in 3 mL of anhydrous THF and cooled to -116 degrees Celsius. The NDY-I solution was slowly added dropwise to the reaction solution within 15 minutes. After stirring at this temperature for 30 minutes, it was slowly warmed to -78 degrees Celsius, and saturated ammonium chloride solution was added to quench the reaction. The reaction process is as shown in formula II. The reaction was monitored by thin layer chromatography (chloroform: acetone = 1:40). After the solvent was removed by rotary evaporation, the crude product was purified by silica gel column (chloroform: acetone = 1:40) to obtain the DON prodrug NDY-J in the form of a yellow solid.

[0063]

[0064] Formula II A method for synthesizing a multifunctional block polymer T-PPLN with pancreatic cancer tissue targeting, ECM enhanced penetration and response release, comprising the following steps: (1) Under argon protection, 1 g of N(ε)-benzyloxycarbonyl-L-lysine was dissolved in 30 mL of anhydrous THF with 441.8 mg of triphosgene. The reaction system was warmed to 50 degrees Celsius by oil bath, and stirred for 3 hours. The reaction process is as shown in formula III. After cooling to room temperature, it was precipitated in n-hexane pre-cooled to -20 degrees Celsius for 3 times, and then filtered to obtain the polymer monomer Lys(Z)-NCA in the form of a white solid.

[0065]

[0066] Formula III (2) Under argon protection, 252.2 mg of 1-methyl-2-nitroimidazole-5- methanol (NI), 430 μL of triethylamine and 1 mg of DMAP were dissolved in 15 mL of anhydrous THF. Separately, 594.1 mg of p-nitrophenyl chloroformate (PNP-Cl) was dissolved in 15 mL of anhydrous THF, and was added dropwise into the reaction solution in 60 minutes under ice bath. After the addition was completed, the temperature was slowly increased to room temperature, and the reaction was continued for 16 hours. The reaction process is shown in Formula IV. The reaction was monitored by thin layer chromatography (petroleum ether: ethyl acetate = 4: 1). After the solvent was removed by rotary evaporation, the crude product was purified by silica gel column (petroleum ether: ethyl acetate = 4: 1) to obtain activated nitroimidazole PNP-NI in the form of white solid.

[0067]

[0068] Formula IV (3) Under argon protection, 25.0 mg of MMP-7 responsive peptide, 100.0 mg of N3-PEG-NHS and 4.2 μL of DIPEA were dissolved in 1.5 mL of anhydrous DMSO, and the reaction was stirred at room temperature for 16 hours. After completion, the reaction solution was dialyzed (3.5k MWCO) with deionized water for 24 hours. After freeze-drying and dehydration, a flocculent white solid was obtained. The product was dissolved in 12 mL of 2% hydrazine hydrate in DMF solution, and the reaction was stirred at room temperature for 2 hours. The reaction process is shown in Formula V. After completion, the reaction solution was dialyzed (3.5k MWCO) with deionized water for 24 hours. After freeze-drying and dehydration, a flocculent white solid PEG-pep was obtained.

[0069]

[0070] Formula V (4) Under argon protection, 50.0 mg of PEG-pep and 74.3 mg of Lys(Z)-NCA were dissolved in 10 mL of anhydrous DMSO. The reaction system was heated to 50 degrees Celsius by oil bath, and the reaction was stirred for 72 hours. The reaction process is shown in Formula VI. After completion, the reaction solution was dialyzed (10k MWCO) with deionized water for 24 hours. After freeze-drying and dehydration, a flocculent white solid PEG-pep-pLys(Z) was obtained.

[0071]

[0072] Formula VI (4) 100.0 mg of PEG-pep-pLys(Z) was dissolved in 3 mL of trifluoroacetic acid, 90 μL of 33% hydrogen bromide acetic acid solution was added, and the reaction was stirred at room temperature for 3 hours. The reaction process is shown in formula VII. After completion, the reaction solution was dialyzed (7k MWCO) with deionized water for 24 hours. Freeze-drying dehydration obtained PEG-pep-pLys as a flocculent white solid.

[0073]

[0074] Formula VII (5) 50.0 mg of PEG-pep-pLys, 39.2 mg of PNP-NI, 18 μL of triethylamine and 1.2 mg of DMAP were dissolved in 4 mL of anhydrous DMF under argon protection, and the reaction was stirred at room temperature for 24 hours. The reaction process is shown in formula VIII. After completion, the reaction solution was first dialyzed (10k MWCO) with DMF for 24 hours, and then dialyzed (10k MWCO) with deionized water for 24 hours. Freeze-drying dehydration obtained PEG-pep-pLys(NI) as a flocculent white solid, namely PPLN.

[0075]

[0076] Formula VIII (6) 50.0 mg of PPLN, 10.0 mg of collagen-targeting peptide CBP, 2.1 mg of sodium ascorbate, 1.33 mg of cuprous iodide and 1.2 μL of DIPEA were dissolved in 5 mL of anhydrous DMF under argon protection. The reaction was stirred at room temperature for 24 hours in the dark. The reaction process is shown in formula IX. After completion, the reaction solution was first dialyzed (10k MWCO) with 10 mM aqueous disodium ethylenediaminetetraacetate for 24 hours, and then dialyzed (10k MWCO) with deionized water for 24 hours. Freeze-drying dehydration obtained Tar-PEG-pep-pLys(NI) as a flocculent white solid, namely T-PPLN.

[0077]

[0078] Formula IX The preparation method of the nano drug delivery system J&V@T-PPLN NPs for enhancing pancreatic cancer targeting and penetration, comprising the following steps: (1) PPLN and T-PPLN were dissolved in DMF at a concentration of 100 mg / mL respectively, and were pre-mixed at a molar ratio of 4:1 to obtain a working polymer solution.

[0079] (2) NDY-J and V-9302 were dissolved in DMF to obtain a drug stock solution with a concentration of 10 mg / mL and 5 mg / mL respectively.

[0080] (3) Mix 250 μL of the working polymer solution and 250 μL of the drug stock solution, and vortex thoroughly. Add 5 mL of the phosphate buffer (pH 7.4) into a 25 mL tomato flask, and stir rapidly at 1500 rpm. Under stirring, slowly drop the mixed polymer and drug solution using a microsyringe, and control the uniform dropping process for 20 minutes. After the dropping is completed, continue stirring for 5 minutes.

[0081] (4) After the stirring is completed, the dispersion is dialyzed (10 k MWCO) for 16 hours using the phosphate buffer (pH 7.4). After the dialysis is completed, filter the nano-drug delivery system J&V@T-PPLN NPs using a 0.22 μm microporous filter membrane.

[0082] The particle size and distribution and the micro-morphology of the J&V@T-PPLN NPs are as shown in FIG. 2A and FIG. 2B: under a physiological environment, the average particle size of the nano-drug delivery system is 83.47 nm, and the morphology is round (the polydispersity index is 0.206). Figure 3

[0083] The drug loading capacity of the J&V@T-PPLN NPs is investigated, and the results are as shown in FIG. 3A and FIG. 3B: the drug loading capacity of NDY-J is 7.49%, and the drug loading capacity of V-9302 is 3.72%, which can meet the in-vivo and in-vitro drug delivery requirements. Figure 4 The intracellular reduction environment responsive drug release capacity of the J&V@T-PPLN NPs is investigated, and the results are as shown in FIG. 4A and FIG. 4B: in a physiological environment (phosphate buffer pH 7.4) or an ECM environment (MMP-7), the drug release of the J&V@T-PPLN NPs is very little, while NDY-J and V-9302 can be rapidly released in the intracellular reduction environment of the tumor cells simulated by NADPH and microsomal enzymes, which indicates that the drug release of the nano-drug delivery system has a strong responsiveness and specificity.

[0084] Figure 5 The pancreatic cancer tissue targeting capacity of the J&V@T-PPLN NPs is investigated, and the results are as shown in FIG. 5A and FIG. 5B: after the tail vein injection of the pancreatic cancer tumor-bearing model mice, compared with the nano-drug delivery system J&V@PPLN NPs without the modification of the collagen targeting peptide CBP, the J&V@T-PPLN NPs have more accumulation in the tumors of the tumor-bearing mice (the scale is 200 μm). The above results indicate that the J&V@T-PPLN NPs have an ideal pancreatic cancer tissue targeting capacity.

[0085] The ECM penetration capacity of the J&V@T-PPLN NPs is investigated, and the results are as shown in FIG. 6A and FIG. 6B. Figure 6

[0086] Figure 7 ​​​​Results: a) The particle size of J&V@T-PPLN NPs was significantly reduced after MMP-7 treatment, and b) showed stronger deep penetration ability in the multicellular tumor spheroid model (scale bar, 200 μm). c) Compared with the non-MMP-7-responsive peptide-containing, size-invariant nanodelivery system J&V@T-PLN NPs, J&V@T-PPLN NPs showed a longer post-vascular migration distance in the tumor tissue of the pancreatic cancer-bearing model mice after tail vein injection (scale bar, 200 μm). The above results indicate that J&V@T-PPLN NPs have ideal ECM penetration ability.

[0087] The ability of J&V@T-PPLN NPs to regulate pancreatic cancer glutamine metabolism was investigated, as shown in Figure 8 Results: Compared with the free drug combination, J&V@T-PPLN NPs can significantly reduce the glutamine consumption of tumor cells (KPC) and reduce the intracellular content of glutamic acid and α-ketoglutaric acid, downstream metabolites of glutamine, and the production of reduced substances glutathione, NADPH, and ATP. Among them, G1 is the negative control group, G2 is the free DON treatment group, G3 is the free NDY-J treatment group, G4 is the free V-9302 treatment group, G5 is the free NDY-J and V-9302 combined treatment group, and G6 is the J&V@T-PPLN NPs treatment group. The dosages (DON, NDY-J 1 μM, V-9302 0.5 μM) and treatment times (24 hours) of each group are consistent.

[0088] The in vivo anti-tumor function of J&V@T-PPLN NPs was investigated, as shown in Figure 9 Results: Compared with the first-line clinical chemotherapy regimen AG, the nanodelivery system regulating glutamine metabolism has better in vivo anti-tumor efficacy. Among them, the pancreatic cancer-bearing model mice in the J&V@T-PPLN NPs administration group have the smallest tumor volume and the longest survival period. At the same time, the administration of the nanodelivery system does not cause obvious gastrointestinal damage, indicating that the drug delivery strategy design can improve the in vivo safety of glutamine metabolism regulation. Among them, T1 is the normal saline administration group, T2 is the clinical AG regimen administration group, T3 is the complete nanodelivery system J&V@T-PPLN NPs administration group, T4 is the non-MMP-7-responsive peptide-containing, size-invariant nanodelivery system J&V@T-PLN NPs administration group, and T5 is the non-tumor tissue-targeting nanodelivery system J&V@PPLN NPs administration group. The administration methods (tail vein injection) and administration frequencies (administration every 3 days) of each group are consistent. The dosages (NDY-J 1 mg / kg, V-9302 0.5 mg / kg) of T3 to T5 groups are consistent.

Claims

1. A glutamine metabolism-regulating nanodelivery system for enhancing the targeting and penetration of pancreatic cancer, characterized in that, The drug is composed of a polymer-encapsulated glutamine metabolism-regulating drug. The polymer is a multifunctional block polymer formed by linking PEG and polyamino acids modified with a controlled-release element through a responsive linker and modifying them with a tumor-targeting element. The assembly of the nano-drug delivery system and the loading process of the glutamine metabolism-regulating drug are achieved through hydrophobic interactions and π-π stacking. Preferably, the controlled-release element of the polymer is a nitroimidazole; the responsive linker is an MMP-7 responsive peptide; the tumor-targeting element is a collagen-targeting peptide CBP; and the glutamine metabolism-regulating drug is a GLS inhibitor NDY-J and an SLC1A5 inhibitor V-9302.

2. The preparation method of the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that, Includes the following steps: (1) a. After reacting ethyl 5-oxopyrrolidine-2-carboxylate with lithium bis(trimethylsilylamino) at -78 degrees Celsius, the intermediate product NDY-I was obtained by separation and purification by silica gel column chromatography; b. After reacting the intermediate product with trimethylsilyldiazomethane and n-butyllithium at -116 degrees Celsius, the glutaminase inhibitor prodrug NDY-J was obtained by separation and purification by silica gel column chromatography; (2) After reacting N(ε)-benzyloxycarbonyl-L-lysine with triphosgene at 50 degrees Celsius, the monomer Lys(Z)-NCA is obtained by precipitation with a precipitant; preferably, the molar ratio of N(ε)-benzyloxycarbonyl-L-lysine to triphosgene is 1: (0.3~0.5); the reaction solvent is anhydrous tetrahydrofuran; the reaction time at 50 degrees Celsius is 3 hours; the precipitant is n-hexane pre-cooled to -20 degrees Celsius; (3) After reacting 1-methyl-2-nitroimidazole-5-methanol, p-nitrophenyl chloroformate, triethylamine and 4-(dimethylamino)pyridine at room temperature, activated nitroimidazole PNP-NI is obtained by separation and purification by silica gel column chromatography; preferably, the molar ratio of 1-methyl-2-nitroimidazole-5-methanol, p-nitrophenyl chloroformate and triethylamine is 1: (1.5~2.0): (1.5~2.0), and the amount of 4-(dimethylamino)pyridine is 1~5 mg; the reaction solvent is anhydrous tetrahydrofuran; the p-nitrophenyl chloroformate should be added slowly dropwise, and the dropwise process should be maintained for 60 minutes; the reaction time at room temperature is 16 hours; the eluent for column chromatography separation is petroleum ether: ethyl acetate 4:1, v: v; (4) Polyethylene glycol N3-PEG-NHS with a molecular weight of 5 kDa, which is capped with azide and N-hydroxysuccinimide, and MMP-7 responsive peptide protected by difluorovinyl groups are reacted at room temperature in the presence of a catalyst. Then, hydrazine hydrate is added to remove the difluorovinyl groups. After dialysis and freeze-drying, PEG-peptide copolymer PEG-pep coupled with MMP-7 responsive peptide is obtained. (5) After the PEG-pep obtained in step (4) and the Lys(Z)-NCA obtained in step (2) are polymerized at 50 degrees Celsius, the PEG-amino acid copolymer PEG-pep-pLys(Z) protected by benzyloxycarbonyl is obtained by dialysis and freeze drying. (6) After removing the benzyloxycarbonyl protection of the PEG-pep-pLys(Z) obtained in step (5), the PEG-peptide-amino acid copolymer PEG-pep-pLys is obtained; (7) After reacting the PEG-pep-pLys obtained in step (6) and the PNP-NI obtained in step (3) in the presence of a catalyst, the mixture is then dialyzed and lyophilized to obtain a multifunctional block polymer PEG-pep-pLys(NI) without pancreatic cancer tissue targeting function, namely PPLN. (8) The PPLN obtained in step (7) was reacted with the collagen-targeting peptide CBP (sequence: LRELHLNNNC-Pra) capped with propargylglycine at room temperature, and then dialyzed and lyophilized to obtain the multifunctional block polymer Tar-PEG-pep-pLys(NI) with pancreatic cancer tissue targeting function, namely T-PPLN. (9) Dissolve the multifunctional block polymers PPLN and T-PPLN synthesized in steps (8) and (9) in N,N-dimethylformamide at different concentrations; dissolve the prodrug NDY-J and the glutamine transporter SLC1A5 inhibitor V-9302 synthesized in step (1) in DMF at different concentrations; mix the above four solutions thoroughly in the required proportions; (10) A solution containing polymer and drug is dropped into an aqueous system under stirring. After thorough stirring, the solution is then subjected to dialysis, centrifugation or filtration to obtain a glutamine metabolism-regulated nano-delivery system J&V@T-PPLNNPs that enhances the targeting and penetration of pancreatic cancer.

3. The method for preparing the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that: In step (1)a, the molar ratio of ethyl 5-oxopyrrolidine-2-carboxylate, lithium bis(trimethylsilylamino)amino, and fluorenemethyloxycarbonyl chloride is 1:1:5; the reaction solvent is anhydrous tetrahydrofuran; the eluent for column chromatography is petroleum ether:ethyl acetate 3:1, v:v; in step (1), the molar ratio of trimethylsilyldiazomethane, n-butyllithium, and NDY-I is 1:(1~1.2):(0.2~0.5); the reaction solvent is anhydrous tetrahydrofuran; the eluent for column chromatography is chloroform:acetone 1:40, v:v.

4. The method for preparing the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that: In step (4), the molar ratio of PEG to MMP-7 responsive peptide is 1:1.2; the catalyst is triethylamine and N,N-diisopropylethylamine; the reaction solvent is anhydrous dimethyl sulfoxide; the reaction time at room temperature is 16 hours; the concentration of hydrazine hydrate for deprotection is 2%, and the deprotection time is 2 hours; a dialysis bag with a molecular weight cutoff of 3.5 kDa is used in the dialysis process; and the dialysis time in deionized water is 24 hours.

5. The method for preparing the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that: In step (5), the molar ratio of PEG-pep to Lys(Z)-NCA is 1: (25~35); the reaction solvent is anhydrous dimethyl sulfoxide; the reaction time is 72 hours at 50 degrees Celsius; a dialysis bag with a molecular weight cutoff of 10 kDa is used for the dialysis process; and the dialysis time in deionized water is 24 hours.

6. The method for preparing the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that: In step (6), the deprotection reagent is trifluoroacetic acid containing 5% hydrogen bromide; the deprotection reaction time is 2-4 hours; a dialysis bag with a molecular weight cutoff of 7 kDa is used in the dialysis process; and the dialysis time in deionized water is 24 hours.

7. The method for preparing the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that: In step (7), the molar ratio of PEG-pep-pLys to PNP-NI is 1: (20~30); the catalyst is triethylamine and DMAP; the reaction solvent is anhydrous DMF; the reaction time at room temperature is 24 hours; a dialysis bag with a molecular weight cutoff of 10 kDa is used in the dialysis process; the dialysis time in DMF and deionized water is 24 hours and 48 hours, respectively.

8. The method for preparing the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that: In step (8), the molar ratio of PPLN to collagen-targeting peptide CBP is 1: (1.5~2); the catalyst for the Click reaction is sodium ascorbate, cuprous iodide and DIPEA; the reaction solvent is anhydrous DMF; the reaction process requires protection from light; the reaction time at room temperature is 24 hours; a dialysis bag with a molecular weight cutoff of 10 kDa is used in the dialysis process; the dialysis time in 10 mM disodium ethylenediaminetetraacetate aqueous solution and deionized water is 24 hours.

9. The method for preparing the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration according to claim 1, characterized in that: In step (9), the molar ratio of PPLN to T-PPLN is 4:1, and the total concentration of the polymer is 100 mg / mL; the concentrations of NDY-J and V-9302 are 10 mg / mL and 5 mg / mL, respectively; the volume ratio of the working polymer solution and the drug stock solution is 1:1; the aqueous phase system in step (10) is deionized water, phosphate buffer, or physiological saline; the droplet addition process should be carried out within 15 to 30 minutes; the stirring speed for thorough stirring is 1500 rpm; a dialysis bag with a molecular weight cutoff of 10 kDa is used for the dialysis process; after dialysis, centrifuge at 3000 rpm for 5 minutes or filter through a 0.22 μm microporous membrane.

10. The use of the glutamine metabolism-regulating nanodelivery system for enhancing pancreatic cancer targeting and penetration as described in claim 1 in the preparation of drugs for treating pancreatic cancer.

Images