Nanoparticle for treating pancreatic cancer by utilizing enamine N-oxide to trigger transendocytosis and cross-matrix barrier as well as preparation method and application of nanoparticle

Abstract

The invention belongs to the technical field of medicines, and particularly relates to nanoparticles for treating pancreatic cancer by utilizing enamine N-oxide to trigger transendocytosis and cross-matrix barrier as well as a preparation method and application of the nanoparticles. The nano particle is prepared by self-assembly of an amphiphilic block polymer and hydrophobic drugs GemC18 and Galunissertib, and the amphiphilic block polymer and the hydrophobic drugs GemC18 and Galunissertib are used as auxiliary materials. The chemotherapeutic drug is a hydrophobic prodrug GemC18 of a pancreatic cancer clinical first-line chemotherapeutic drug gemcitabine, the drug for regulating and controlling tumor matrix hyperplasia and immunosuppression is Galunissertib, and the polymer is an amphiphilic block polymer which is obtained through ring-opening polymerization reaction of N-carboxy anhydride (NCA), is modified by enamine N-oxide and has hypoxia response charge turnover capacity. And the GemC18 and the Galunissertib are assembled together with the pi-pi cationic polymer through a hydrophobic effect, so that the nanoparticles are formed. The nanoparticles can trigger an adsorption-mediated transendocytosis cross-matrix barrier, simultaneously respond to hypoxia, consume GSH (glutathione), up-regulate ROS (reactive oxygen species), correct charges, improve intra-tumor permeation of drugs, remodel a matrix and reverse immunosuppression.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across matrix barriers, as well as a preparation method and application thereof. Background Art

[0002] Pancreatic cancer has a desmoplastic stroma that impedes drug delivery and penetration, and the tumor is also immunosuppressive. The treatment of pancreatic cancer faces the challenges of stromal proliferation and immunosuppression.

[0003] Transforming growth factor-β (TGF-β) is highly expressed in pancreatic cancer. After being activated by TGF-β, TGF-β receptor phosphorylates SMAD2 / 3 to p SMAD2 / 3, then enter the cell nucleus with SMAD4 to initiate the expression of related genes. p The functions of SMAD2 / 3 include promoting the secretion of collagen by cancer-associated fibroblasts, upregulating the level of FOXP3+Treg cells, inducing the transformation of M1 macrophages to M2 macrophages, and inhibiting the anti-tumor function of CD8+T cells. p SMAD2 / 3 phosphorylation levels hold promise for remodeling matrix and reversing immune suppression.

[0004] Although matrix hyperplasia can limit the intratumoral penetration of gemcitabine, transcytosis as a mechanism across cell barriers offers hope for improving the intratumoral penetration of gemcitabine. Studies have reported that N-oxides have the ability to trigger adsorption-mediated transcytosis (AMT), so the use of amino acid polymers modified with enamine N-oxides as drug delivery vehicles can enhance tumor penetration. In order to remodel the matrix and reverse immunosuppression, we selected p SMAD2 / 3 is used as a research target. Galunisertib was selected to limit the phosphorylation of SMAD2 / 3, so it is necessary to design a matching delivery system to achieve targeted co-delivery of the hydrophilic drug gemcitabine and the hydrophobic drug Galunisertib. Gemcitabine was modified with octadecanoyl chloride to obtain hydrophobic GemC18. GemC18 and Galunisertib can be encapsulated in the hydrophobic core of the amphiphilic block polymers MPEG-pPLNO-pPhe and CBP-pPLNO-pPhe through hydrophobic interaction and π-π stacking. The amphiphilic block polymers MPEG-pPLNO-pPhe or CBP-pPLNO-pPhe structure have the ability to be electrically neutral, trigger transcytosis, undergo charge reversal in response to hypoxia, consume glutathione (GSH), and upregulate reactive oxygen species (ROS). They can selectively release drugs in hypoxic tumors and improve the intratumoral penetration of drugs. Summary of the invention

[0005] Aiming at the above problems encountered in the treatment of pancreatic cancer, the present invention designs and prepares a nanoparticle for the treatment of pancreatic cancer based on the existing technical foundation by using enamine N-oxide to trigger transcytosis across the matrix barrier. The nanoparticle is self-assembled by an amphiphilic block polymer and hydrophobic drugs GemC18 and Galunisertib. The chemotherapy drug is GemC18, a hydrophobic prodrug of gemcitabine, a first-line chemotherapy drug for pancreatic cancer, and the drug for regulating tumor matrix proliferation and immunosuppression is Galunisertib. The polymer is an amphiphilic block polymer with hypoxia-responsive charge reversal ability obtained by ring-opening polymerization of N-carboxyl cyclic anhydride (NCA) and modified by enamine N-oxide. GemC18 and Galunisertib are assembled together to form nanoparticles through hydrophobic interaction and π-π cationic polymers. The nanoparticles can trigger adsorption-mediated transcytosis across the matrix barrier, while responding to hypoxia, consuming GSH, upregulating ROS, and positively reversing the charge, improving the intratumoral penetration of the drug, and remodeling the matrix and reversing immunosuppression.

[0006] In the design of the present invention, the amino acid polymer modified with enamine N-oxide is used as a drug carrier to play the role of enhanced tumor penetration, hypoxia responsive charge reversibility, consumption of GSH, and upregulation of ROS. After the nanoparticles reach the tumor area through the active targeting of CBP, they can trigger transcytosis across the matrix barrier to achieve deep tumor penetration; at the same time, after the hypoxia response, the enamine N-oxide leaves as an unsaturated imine structure, consumes GSH by Michael addition, and at the same time, the amino acid polymer exposes the amine group, which can adsorb protons in the tumor and increase the positive charge density of the polymer, promoting the intratumoral penetration of the nanoparticles. While improving the intratumoral penetration of the drug, the matrix is ​​reshaped and the anti-tumor immune response is activated. The design of the present invention obtains ideal biocompatibility, enhanced circulation stability and tumor targeting ability through suitable size and CBP modification, and is expected to enhance the therapeutic efficacy of pancreatic cancer.

[0007] The above design integrates strategies such as promoting deep drug penetration, remodeling the matrix, and activating anti-tumor immune responses 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: Nanoparticles for the treatment of pancreatic cancer that utilize enamine N-oxide-triggered transcytosis across the matrix barrier are self-assembled from amino acid polymers modified with enamine N-oxide and the hydrophobic drugs GemC18 and Galunisertib.

[0009] Preferably, the amino acid polymer modified with enamine N-oxide includes polyethylene glycol polyamino acid polymer modified with enamine N-oxide, and amino acid polymer co-modified with enamine N-oxide and collagen targeting polypeptide CBP, and the sequence of CBP is LRELHLNNNC.

[0010] The method for preparing the nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier comprises the following steps: (1) Gem and octadecanoyl chloride were reacted at room temperature under the action of triethylamine, and then separated and purified by silica gel column chromatography to obtain octadecyl-modified gemcitabine prodrug GemC18; (2) CBP or MPEG500-NH2 and N6-benzyloxycarbonyl-L-lysine cyclic anhydride were polymerized at 50 °C; (3) After the reaction is completed, (S)-(-)-4-benzyloxyazacyclopentane-2,5-dione is added to the two reaction solutions obtained in step (2) respectively to continue the polymerization reaction; the reaction is terminated, the impurities are removed by dialysis, and the polymer CBP-pLys (Cbz) -pPhe modified with the targeted peptide CBP and the polymer MPEG-pLys (Cbz) -pPhe without the targeted peptide modification are obtained after freeze-drying and dehydration; (4) The two polymers obtained in step (3) are respectively dissolved in a trifluoroacetic acid solution containing 5% hydrobromic acid, and the dissolved solutions are stirred for reaction at room temperature for 2-3 hours. After the reaction, the obtained solution is dialyzed with deionized water for 24 hours, and freeze-dried for dehydration to obtain a polymer CBP-pLys-pPhe modified with a targeted peptide and a polymer MPEG-pLys-pPhe without a targeted peptide modification; (5) CBP-pLys-pPhe and MPEG-pLys-pPhe obtained in step (4) are dissolved in a good solvent, 4-nitrophenylprop-2-yn-1-yl carbonate and triethylamine are added, and the dissolved solutions are stirred and reacted under argon protection at room temperature; after the reaction, the reaction solutions are dialyzed with deionized water for 24 h, and freeze-dried to obtain CBP-pLP-pPhe and MPEG-pLP-pPhe; (6) CBP-pLP-pPhe or MPEG-pLP-pPhe and diethylhydroxylamine were dissolved in a chloroform solution containing 20% ​​trifluoroethanol and stirred at 50 °C for 24 h under argon protection. After completion, the reaction solution was precipitated with ether to obtain polymers CBP-pPLNO-pPhe and MPEG-pPLNO-pPhe that triggered transcytosis and hypoxia response; (7) Mix the synthesized polymer materials CBP-pPLNO-pPhe and MPEG-pPLNO-pPhe in the required molar ratio and dissolve them in DMSO to form a 30 mg / mL working polymer solution; (8) adding GemC18 and Galunisertib to the working polymer solution obtained in step (7) in the required molar ratio, mixing thoroughly and letting stand at room temperature; (9) The solution obtained in step (8) was slowly added dropwise into normal saline under stirring. After the addition was completed, it was dialyzed to remove impurities and filtered using a 0.22 μm filter membrane to obtain complete nanoparticles GemC18 / Gal@CPLNO.

[0011] Preferably, in step (1), the molar ratio of gemcitabine to octadecanoyl chloride is 1:1; the molar ratio of triethylamine to gemcitabine is 1:1.2; further preferably, the reaction solvent is dimethyl sulfoxide (DMSO), and the reaction time at room temperature is 24 h.

[0012] Further preferably, the eluent of the chromatographic column in step (1) is methanol: dichloromethane 1:20, v:v.

[0013] Preferably, in step (2), the molar ratio of CBP or MPEG500-NH2 to N6-benzyloxycarbonyl-L-lysine cyclic anhydride is 1:12. Further preferably, anhydrous N,N-dimethylformamide (DMF) is used as the reaction solvent; the reaction temperature is 50°C, and the reaction time is 48 h.

[0014] Preferably, the molar ratio of (S)-(-)-4-benzyloxazacyclopentane-2,5-dione to CBP or MPEG500-NH2 in step (3) is 7:1, the molecular weight cut-off of the dialysis bag is 3.5 kDa, the reaction temperature is 50 °C, and the reaction time is 48 h.

[0015] Preferably, the molecular weight cut-off of the dialysis bag in step (4) is 3.5 kDa.

[0016] Preferably, in step (5), the molar ratio of 4-nitrophenylprop-2-yn-1-yl carbonate to CBP-pLys-pPhe or MPEG-pLys-pPhe is 15.6:1; the molar ratio of triethylamine to 4-nitrophenylprop-2-yn-1-yl carbonate is 1.2:1; and the reaction time is 12 h.

[0017] Preferably, in step (6), the molar ratio of CBP-pLP-pPhe or MPEG-pLP-pPhe to diethylhydroxylamine is 1:60.

[0018] Preferably, the molar ratio of CBP-pPLNO-pPhe to MPEG-pPLNO-pPhe in the working polymer solution in step (7) is 1:4.

[0019] Preferably, in step (8), the molar ratio of GemC18 to Galunisertib is 2.13:1, and the mass ratio of MPEG-pLP-pPhe to GemC18 is 13.33:1; further preferably, the mixture is fully mixed by pipetting 10-50 times, and the mixture is allowed to stand at room temperature for 30 min.

[0020] Preferably, the dropwise addition process in step (9) is completed within 10-15 min, and the stirring speed is 1000 rpm.

[0021] The present invention also provides a method for synthesizing a polymer material MPEG-pPhe without transcytosis and hypoxia response functions, comprising the following steps: MPEG5000-NH2 and (S)-(-)-4-benzyloxyazacyclopentane-2,5-dione were dissolved in a good solvent and polymerized at 50°C. After the reaction was completed, the product was dialyzed to remove impurities and freeze-dried to obtain MPEG-pPhe.

[0022] Preferably, the good solvent is N,N-dimethylformamide (DMF), the reaction time is 48 h, and the molecular weight cutoff of the dialysis bag is 3.5 kDa.

[0023] The application of the above-mentioned nanoparticles that utilize enamine N-oxide to trigger transcytosis across the matrix barrier for the treatment of pancreatic cancer in drugs for the treatment of pancreatic cancer.

[0024] The nanoparticles for pancreatic cancer treatment using enamine N-oxide-triggered transcytosis across matrix barriers described in the present invention include a triggered transcytosis and hypoxia-responsive polymer, a chemotherapeutic drug GemC18, and a tumor matrix proliferation-regulating and immunosuppressive drug Galunisertib. The triggered transcytosis and hypoxia-responsive polymer penetrates tumor tissues through adsorption-mediated transcytosis, and its charge is positively converted in a hypoxic environment, consuming GSH and upregulating ROS; the chemotherapeutic drug is GemC18, a hydrophobic prodrug of gemcitabine; the tumor matrix proliferation-regulating and immunosuppressive drug is Galunisertib, which inhibits p SMAD2 / 3 levels, remodeling the matrix and activating anti-tumor immunity.

[0025] In the present invention, the triggered transcytosis and hypoxia responsive polymer is obtained by ring-opening polymerization of N-carboxyl cyclic anhydride (NCA), modifying the lysine amine group with 4-nitrophenylprop-2-yn-1-yl carbonate, and then reacting diethylhydroxylamine with the alkynyl group.

[0026] In the present invention, the nanoparticles are prepared by dissolving the polymer and the drug in DMSO, adding the mixture dropwise to physiological saline or deionized water for uniform mixing, and then dialyzing to remove impurities and filtering.

[0027] In the present invention, the GemC18 is obtained by reacting octadecanoyl chloride with the amine group of gemcitabine and separating and purifying it by silica gel chromatography column.

[0028] The room temperature in the present invention refers to 25°C.

[0029] 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 applied to the selective delivery of a variety of hydrophobic drugs.

[0030] (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.

[0031] (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 synergistic effect, and reduce systemic toxicity.

[0032] (4) The nanoparticles of the present invention can effectively reshape the tumor matrix, alleviate the immunosuppressive microenvironment, and activate the anti-tumor immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The synthetic route of the MPEG-pPLNO-pPhe polymer in the present invention is shown in FIG.

[0034] Figure 2 The synthetic route of the CBP-pPLNO-pPhe polymer in the present invention is shown in FIG.

[0035] Figure 3 The microscopic morphology, particle size and distribution of GemC18 / Gal@CPLNO nanoparticles in Example 1 of the present invention are shown in Figure 1. a) Particle size measured by DLS and TEM results, b) Stability data of nanoparticles.

[0036] Figure 4 The hypoxia responsiveness of the nanoparticles of the present invention. a) Molecular mechanism of the hypoxia responsiveness of the polymer; b) 1 H NMR was used to observe the changes of polymer molecules before and after the response; Figure 5The present invention investigates the ability of the nanoparticles to release drugs in response to hypoxic environments. a) The release of GemC18 after treatment with different concentrations of Fe2SO4 and Na2S2O4; b) The release of Galunisertib after treatment with different concentrations of Fe2SO4 and Na2S2O4.

[0037] Figure 6 The figures are as follows: a) investigation of the deep penetration ability of CPEG nanoparticles without transcytosis function in in vivo tumor tissue (scale bar is 2 mm); b) local magnified image of a (scale bar is 200 μm); c) investigation of the deep penetration ability of CPLNO nanoparticles with transcytosis function in in vivo tumor tissue (scale bar is 2 mm); d) local magnified image of c (scale bar is 200 μm); e) co-localization analysis data of b; f) co-localization analysis data of d; g) investigation of the deep penetration ability of in vitro tumor spheres (scale bar is 50 μm); h) co-localization analysis data of g.

[0038] Figure 7 This is an investigation of the effect of the nanoparticles of the present invention on regulating abnormal matrix proliferation in tumor tissue. After administration: a) Tumor tissue in vivo p a) Investigation of the expression of SMAD2 / 3; b) Investigation of the expression of type I collagen (Col1) in tumor tissue in vivo; c) Investigation of the expression of α-smooth muscle actin (αSMA) in tumor tissue in vivo; d) p Semi-quantitative results of SMAD2 / 3; e) semi-quantitative results of Col1; f) semi-quantitative results of αSMA.

[0039] Figure 8 This is an investigation of the nanoparticles of the present invention regulating the immunosuppression of tumor tissue. After administration: a) Tumor tissue in vivo p Investigation of SMAD2 / 3 protein expression; b) Flow cytometry data results of CD8+T cells in tumor tissue in vivo; c) Flow cytometry data results of M1 macrophages in tumor tissue in vivo; d) Flow cytometry data results of M2 macrophages in tumor tissue in vivo; e) M1 / M2 ratio of tumor tissue in vivo; f) Flow cytometry data results of Treg cells in tumor tissue in vivo.

[0040] Fig. 9 The present invention is used to investigate the ability of free drugs or nanoparticles of the present invention to enhance the chemotherapeutic effect of tumor tissues. After administration: a) investigate the proliferation level of tumor cells in vitro; b) investigate the proliferation and apoptosis level of tumor tissues in vivo. DETAILED DESCRIPTION

[0041] 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.

[0042] Example 1 Nanoparticles for pancreatic cancer treatment using enamine N-oxide to trigger transcytosis across matrix barriers and a preparation method thereof, comprising the following steps: (1) A method for synthesizing gemcitabine prodrug GEMC18, comprising the following steps: 1 g of gemcitabine, 1.27 g of octadecanoyl chloride and 633.74 μL of triethylamine (TEA) were dissolved in 15 mL of anhydrous DMF and stirred at room temperature for 24 h. The reaction process is as shown in Formula I. The reaction was monitored by thin layer chromatography (methanol: dichloromethane = 1:20). After the solvent was removed by rotary evaporation, the crude product was purified by silica gel column (methanol: dichloromethane = 1:20) to obtain maleimidized gemcitabine prodrug GemC18 in the form of a white solid.

[0043]

[0044] Formula I (2) Dissolve 400 mg MPEG500-NH2 and 2.62 g N6-benzyloxycarbonyl-L-lysine cyclic anhydride in 30 mL of anhydrous DMF and add them to a two-necked flask. The polymerization reaction is carried out at 50 °C for 48 h under nitrogen protection. The reaction process is shown in Formula II. After completion, the mixture is dialyzed to remove impurities and freeze-dried to obtain the polymer MPEG-pLys (Cbz).

[0045]

[0046] Formula II (3) 1.51 g MPEG-pLys (Cbz) and 478.23 mg (S)-(-)-4-benzyloxyazacyclopentane-2,5-dione were dissolved in 20 mL of anhydrous DMF, and the polymerization reaction was carried out at 50 °C for 48 h under nitrogen protection. The reaction process is shown in Formula III. After completion, the reaction solution was dialyzed with deionized water (3.5k MWCO) for 24 h. Freeze-dried and dehydrated to obtain a powdery white solid MPEG-pLys (Cbz) -pPhe.

[0047]

[0048] Formula III (4) Dissolve 500 mg of MPEG-pLys(Cbz)-pPhe in 20 mL of trifluoroacetic acid containing 5% hydrobromic acid and stir at room temperature for 2 h. The reaction process is as shown in Formula IV. Then dialyze with deionized water (3.5k MWCO) for 24 h. Freeze-dry and dehydrate to obtain MPEG-pLys-pPhe in the form of a flocculent white solid.

[0049]

[0050] Formula IV (5) Dissolve 379.5 mg of MPEG-pLys-pPhe, 304 mg of 4-nitrophenylprop-2-yn-1-yl carbonate and 334.5 μL of triethylamine in 10 mL of anhydrous DMSO and add them to a two-necked flask. The reaction is carried out at room temperature under nitrogen protection for 12 hours. The reaction process is as shown in Formula V. After completion, dialyze with deionized water (3.5 k MWCO) for 24 hours. Freeze-dry and dehydrate to obtain MPEG-pLP-pPhe in the form of a flocculent white solid.

[0051]

[0052] Formula V (6) Dissolve 207 mg MPEG-pLP-pPhe and 382.16 μL diethylhydroxylamine in 6 mL chloroform solution containing 20% ​​trifluoroethanol. Stir at 50 °C for 24 h under argon protection. The reaction process is as shown in Formula VI. After completion, the reaction solution is precipitated with ether to obtain a polymer MPEG-pPLNO-pPhe that triggers transcytosis and hypoxia response in the form of a flocculent orange-yellow solid. The overall synthetic route of MPEG-pPLNO-pPhe is as follows: Figure 1 as shown in .

[0053]

[0054] Formula VI The MPEG500-NH2 in step (2) is replaced by CBP, and the other reactants and reaction conditions remain unchanged. CBP-pPLNO-pPhe is synthesized by referring to the synthesis method of MPEG-pPLNO-pPhe in steps (2) to (6). The general synthesis route is as follows: Figure 2 as shown in .

[0055] (7) MPEG-pPLNO-pPhe and CBP-pPLNO-pPhe were premixed at a molar ratio of 4:1 and dissolved in DMSO to obtain a 30 mg / mL working polymer solution.

[0056] (8) Dissolve GemC18 in DMSO at a concentration of 4.5 mg / mL and dissolve Galunisertib in DMSO at a concentration of 33 mg / mL. Pipette the mixture 10 to 50 times to mix thoroughly according to the required ratio. Incubate the obtained polymer solution at room temperature for 30 min. Mix the DMSO solutions of GemC18 and Galunisertib in step (7) at the required ratio and pipette the mixture 10 to 50 times to mix thoroughly. Incubate the obtained mixed solution at room temperature for 30 min.

[0057] (9) The mixed solution obtained in step (8) is slowly added dropwise into physiological saline or deionized water while stirring. After the addition is completed, it is dialyzed to remove impurities (3.5k MWCO) and filtered to obtain the targeted nanoparticles GemC18 / Gal@CPLNO.

[0058] The particle size, distribution and micromorphology of the targeted nanoparticles GemC18 / Gal@CPLNO are shown in Figure 2. Figure 3 As shown: Under physiological conditions, the average particle size of the nanoparticles is 48.99 nm, the distribution is uniform and the morphology is round (the polydispersity coefficient is 0.275), the drug loading capacity of GemC18 is 9.58%, and the drug loading capacity of Galunisertib is 5.42%.

[0059] The hypoxia responsiveness of the targeted nanoparticles GemC18 / Gal@CPLNO was investigated. Figure 4 As shown: After adding FeSO4 and Na2S2O4 (nanoparticles + FeSO4 + Na2S2O4), Figure 4 The peaks at 6.45 ppm and 6.28 ppm observed in b disappeared, indicating that the enamine N-oxide was reduced and left, and the nanoparticles GemC18 / Gal@CPLNO had hypoxia response ability.

[0060] The drug release behavior of nanoparticles GemC18 / Gal@CPLNO in response to tumor hypoxia was investigated. Figure 5 As shown in the figure: a) Release of GemC18 after treatment with different concentrations of Fe2SO4 and Na2S2O4; b) Release of Galunisertib after treatment with different concentrations of Fe2SO4 and Na2S2O4. This shows that after hypoxia treatment, due to the gradual increase in positive electrical repulsion between polymers, the disintegration of nanoparticles is accelerated, resulting in faster release of drugs. The tumor tissue penetration ability of nanoparticles GemC18 / Gal@CPLNO was investigated, and the results are shown in the figure. Figure 6As shown: af) The nanoparticle group with transcytosis-triggering function (DID@CPLNO) has a deep tumor tissue accumulation signal farther away from the blood vessels (>400 μm) than the nanoparticle group without transcytosis function (DID@CPEG), and gh) in the tumor sphere model, it was observed that DID@CPLNO has a stronger ability to penetrate and accumulate in deep tumor tissues under normoxic and hypoxic acidic pH (scale bar is 50 μm). The above results show that the design of introducing enamine N-oxide transcytosis elements is conducive to improving the tumor tissue penetration ability of nanoparticles.

[0061] Different groups of formulations were prepared: G1: PBS; G2: Gemcitabine 5 mg / kg; G3: GemC18+Galunisertib; G4: GemC18 / Gal@PEG; G5: GemC18 / Gal@PLNO; G6: GemC18 / Gal@CPLNO, among which the dosage of GemC18 was 1.19 mg / kg and the dosage of Galunisertib was 0.674 mg / kg.

[0062] The ability of nanoparticles GemC18 / Gal@CPLNO to regulate tumor stroma was investigated. Figure 7 As shown: Nanoparticles can significantly a) downregulate p The expression levels of SMAD2 / 3 were significantly decreased, thereby a) reducing the expression level of Col1 and c) reducing the expression level of αSMA.

[0063] The ability of nanoparticles GemC18 / Gal@CPLNO to reverse immunosuppression was investigated. Figure 8 As shown: Nanoparticles can significantly a) downregulate p The expression levels of SMAD2 / 3 lead to more local immune cell activation and pro-inflammatory phenotype polarization (BF).

[0064] The tumor chemotherapy ability of nanoparticles GemC18 / Gal@CPLNO was investigated in vitro and in vivo. Fig. 9 As shown: a) Nanoparticles significantly enhanced the cytotoxicity of gemcitabine under hypoxic conditions at the cell level in vitro; b) At the tissue level in vivo, they induced more tumor cell apoptosis and led to a higher level of tumor proliferation inhibition.

Claims

1. Nanoparticles for pancreatic cancer treatment using enamine N-oxides to trigger transcytosis across matrix barriers, characterized in that: It is obtained by self-assembly of amino acid polymers modified with enamine N-oxide and hydrophobic drugs GemC18 and Galunisertib.

2. The nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 1, characterized in that: The amino acid polymer modified with enamine N-oxide includes polyethylene glycol polyamino acid polymer modified with enamine N-oxide, amino acid polymer co-modified with enamine N-oxide and collagen-targeting polypeptide CBP, and the sequence of CBP is LRELHLNNNC.

3. The method for preparing nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 2, characterized in that: The following steps are involved: (1) Gem and octadecanoyl chloride were reacted at room temperature under the action of triethylamine, and then separated and purified by silica gel column chromatography to obtain octadecyl-modified gemcitabine prodrug GemC18; (2) CBP or MPEG500-NH2 and N6-benzyloxycarbonyl-L-lysine cyclic anhydride were polymerized at 50 °C; (3) After the reaction is completed, (S)-(-)-4-benzyloxyazacyclopentane-2,5-dione is added to the two reaction solutions obtained in step (2) respectively to continue the polymerization reaction; the reaction is terminated, the impurities are removed by dialysis, and the polymer CBP-pLys (Cbz) -pPhe modified with the targeted peptide CBP and the polymer MPEG-pLys (Cbz) -pPhe without the targeted peptide modification are obtained after freeze-drying and dehydration; (4) The two polymers obtained in step (3) are respectively dissolved in a trifluoroacetic acid solution containing 5% hydrobromic acid, and the dissolved solutions are stirred for reaction at room temperature for 2-3 hours. After the reaction, the obtained solution is dialyzed with deionized water for 24 hours, and freeze-dried for dehydration to obtain a polymer CBP-pLys-pPhe modified with a targeted peptide and a polymer MPEG-pLys-pPhe without a targeted peptide modification; (5) CBP-pLys-pPhe and MPEG-pLys-pPhe obtained in step (4) are dissolved in a good solvent, 4-nitrophenylprop-2-yn-1-yl carbonate and triethylamine are added, and the dissolved solutions are stirred and reacted under argon protection at room temperature; after the reaction, the reaction solutions are dialyzed with deionized water for 24 h, and freeze-dried to obtain CBP-pLP-pPhe and MPEG-pLP-pPhe; (6) CBP-pLP-pPhe or MPEG-pLP-pPhe and diethylhydroxylamine were dissolved in a chloroform solution containing 20% ​​trifluoroethanol and stirred at 50 °C for 24 h under argon protection. After completion, the reaction solution was precipitated with ether to obtain polymers CBP-pPLNO-pPhe and MPEG-pPLNO-pPhe that trigger transcytosis and hypoxia response; (7) Mix the synthesized polymer materials CBP-pPLNO-pPhe and MPEG-pPLNO-pPhe in the required molar ratio and dissolve them in DMSO to form a 30 mg / mL working polymer solution; (8) adding GemC18 and Galunisertib to the working polymer solution obtained in step (7) in the required molar ratio, mixing thoroughly and letting stand at room temperature; (9) The solution obtained in step (8) was slowly added dropwise into normal saline under stirring. After the addition was completed, it was dialyzed to remove impurities and filtered using a 0.22 μm filter membrane to obtain complete nanoparticles GemC18 / Gal@CPLNO.

4. The method for preparing nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 3, characterized in that: In step (1), the molar ratio of gemcitabine to octadecanoyl chloride is 1:1; the molar ratio of triethylamine to gemcitabine is 1:1.2; preferably, the reaction solvent is dimethyl sulfoxide (DMSO), and the reaction time at room temperature is 24 h; preferably, the eluent of the chromatographic column in step (1) is methanol: dichloromethane 1:20, v:v.

5. The method for preparing nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 3, characterized in that: In step (2), the molar ratio of CBP or MPEG500-NH2 to N6-benzyloxycarbonyl-L-lysine cyclic anhydride is 1:

12. Preferably, anhydrous N,N-dimethylformamide (DMF) is used as the reaction solvent; the reaction temperature is 50°C and the reaction time is 48 h.

6. The method for preparing nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 3, characterized in that: The molar ratio of (S)-(-)-4-benzyloxyazacyclopentane-2,5-dione to CBP or MPEG500-NH2 in step (3) is 7:1, and the molecular weight cutoff of the dialysis bag is 3.5 kDa. The reaction temperature is 50 °C and the reaction time is 48 h; preferably, the molecular weight cutoff of the dialysis bag in step (4) is 3.5 kDa.

7. The method for preparing nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 3, characterized in that: In step (5), the molar ratio of 4-nitrophenylprop-2-yn-1-yl carbonate to CBP-pLys-pPhe or MPEG-pLys-pPhe is 15.6:1; the molar ratio of triethylamine to 4-nitrophenylprop-2-yn-1-yl carbonate is 1.2:1; and the reaction time is 12 hours.

8. The method for preparing nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 3, characterized in that: In step (6), the molar ratio of CBP-pLP-pPhe or MPEG-pLP-pPhe to diethylhydroxylamine is 1:60; Preferably, the molar ratio of CBP-pPLNO-pPhe to MPEG-pPLNO-pPhe in the working polymer solution in step (7) is 1:

4.

9. The method for preparing nanoparticles for treating pancreatic cancer by using enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 3, characterized in that: In step (8), the molar ratio of GemC18 to Galunisertib is 2.13:1, and the mass ratio of MPEG-pLP-pPhe to GemC18 is 13.33:1; preferably, the mixture is fully mixed by pipetting 10-50 times, and the mixture is allowed to stand at room temperature for 30 min; Preferably, the dropwise addition process in step (9) is completed within 10-15 min, and the stirring speed is 1000 rpm.

10. Use of the nanoparticles for treating pancreatic cancer by utilizing enamine N-oxide to trigger transcytosis across the matrix barrier according to claim 1 or 2 in drugs for treating pancreatic cancer.

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