Pancreatic cancer diagnosis and treatment integrated bionic nano material as well as preparation method and application thereof

Abstract

The invention provides a pancreatic cancer diagnosis and treatment integrated bionic nano material as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: preparing Fe-doped hollow mesoporous MnO2 spheres by taking mesoporous silica as a template, modifying the surfaces of the Fe-doped hollow mesoporous MnO2 spheres with polydopamine, stirring and adsorbing albumin paclitaxel / adriamycin and chloroquine, then mixing the Fe-doped hollow mesoporous MnO2 spheres with PANC02 cell membrane protein-loaded bionic liposome, and repeatedly extruding to prepare the pancreatic cancer diagnosis and treatment integrated bionic nano material. Aiming at the treatment requirement of improving PDAC prognosis, the bionic nanocomposite with ordered targeting and multi-dimensional TME remodeling effects is constructed, so that chemotherapy of PDAC is enhanced, related mechanisms are researched, theoretical and experimental foundations are expected to be laid for improving PDAC treatment resistance, and the bionic nanocomposite has important scientific significance and clinical application value for precise diagnosis and treatment of PDAC.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer, and a preparation method and application thereof. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors, with a five-year survival rate of only 11%. In recent years, although the diagnosis and treatment of this tumor have made certain progress, the high mortality rate is still a problem that plagues the world. PDAC patients lack specific clinical manifestations in the early stage and there is no effective screening method, so it is very easy to miss the diagnosis in the early stage, and most clinically confirmed cases have no indication for surgical resection. In terms of drug treatment, the PDAC tumor microenvironment (TME) is rich in a large number of fibrous matrix components, which makes the tumor tissue present with severe ischemia and interstitial hypertension, greatly limiting the delivery of drugs to the tumor site, resulting in poor treatment effect. In addition, the further remodeling of the TME during drug treatment is closely related to subsequent treatment resistance, which often leads to poor clinical prognosis due to tumor progression or recurrence. Therefore, it is crucial to find a multi-dimensional tumor microenvironment remodeling strategy to increase the delivery of drugs to tumor tissues and reduce the incidence of subsequent treatment resistance to improve the prognosis of PDAC patients.

[0003] In order to increase the delivery of drugs to the tumor site, it is urgent to find strategies for targeted loosening of the tumor extracellular matrix to achieve the initial remodeling of the TME. In terms of targeted drug delivery, cell membrane-wrapped nanocomplexes not only have good biocompatibility, but also have the advantages of long in vivo circulation time, targeting, immune escape, controlled drug release, and immune activation. In addition, biomimetic liposomes prepared by inserting tumor membrane proteins have the characteristics of strong membrane editability and are easier to meet different functional requirements. The strategy of sequentially targeting cancer-associated fibroblasts (CAFs) and tumor cells helps to increase the enrichment of drugs in tumor tissues and provides the necessary conditions for the initial remodeling of the tumor microenvironment.

[0004] In terms of matrix loosening in PDAC, the extracellular matrix of the tumor accounts for about 80% of the TME, forming a drug penetration barrier and seriously hindering the delivery of drugs into the tumor tissue. In order to break through this barrier, the research team from the University of Science and Technology of China, the Institute of Automation of the Chinese Academy of Sciences, and Zhejiang University reshaped the pancreatic cancer TME by degrading collagen and hyaluronic acid in the extracellular matrix, thereby promoting the penetration of drugs into tumor cells and achieving the synergistic effect of radiotherapy and chemotherapy. However, the extracellular matrix of tumor tissue is complex, and the degradation of a single component may not be enough to eliminate the effect of continuous extracellular matrix production by tumor-associated fibroblasts on the TME. CAFs, as the main component of stromal cells in the TME, can secrete a large amount of collagen and matrix proteins, and build a physical barrier to treatment resistance by promoting collagen deposition and excessive matrix fibrosis. Based on this, the research team from Zhejiang University, Harbin Institute of Technology, and the Institute of Chemistry of the Chinese Academy of Sciences used NO, photothermal therapy, metformin, etc. to directly kill CAFs or regulate the activity of pancreatic stellate cells (PSC) to inhibit the secretion of extracellular matrix as a whole, and obtained significant synergy of chemotherapy. It is worth noting that the first-line clinical chemotherapy drug albumin paclitaxel (Paclitaxel-albumin) has been found to target SPARC protein on the surface of fibroblasts and eliminate CAFs, which has the dual effects of remodeling TEM and killing tumor cells. Therefore, the strategy of albumin paclitaxel-loosening matrix combined with sequential targeting is expected to achieve initial remodeling of the tumor microenvironment and improve the efficacy of PDAC chemotherapy.

[0005] Tumor cells in PDAC only account for a minority of the total cells in the tumor, and the biological invasive behavior, heterogeneity, and treatment resistance of the disease are mainly driven by non-cancerous TME components. Although the short-term efficacy of albumin-paclitaxel to loosen the tumor stroma is acceptable, long-term clinical studies have found that the abundance of CAFs cells and the content of metallothionein in tumor tissues after chemotherapy are significantly increased, suggesting acquired chemotherapy resistance, and the effect of single-drug treatment is not obvious. Therefore, clarifying the key factors involved in CAFs activation and the effect of inhibiting its activation on tumor growth will help to achieve sustained benefits from chemotherapy. The activation of CAFs is closely related to the high autophagic activity of stromal fibroblasts and tumor cells. The high level of autophagy in stromal fibroblasts can downregulate RBPJ and p62 / SQSTM1 proteins and lead to CAFs activation. Tumor cells increase the level of lncRNA-CAF in stromal fibroblasts through exosomal lnc-CAF, block p62-dependent autophagic lysosomal degradation to upregulate IL-33 levels, and then upregulate the expression of CAFs markers (α-SMA, vimentin, and N-cadherin) in fibroblasts and activate CAFs. In addition, tumor cell autophagy can induce metabolic stress, inhibit chemotherapeutic drug-induced cell apoptosis and lead to tumor progression. CAFs autophagy promotes tumor growth by providing key metabolites or cytokines. Autophagy inhibitors can block the production of protective autophagy in target cells by blocking the binding of lysosomes to autophagosomes or inhibiting the activation of the autophagic pathway, thereby exerting a synergistic effect with chemotherapeutic drugs. In addition, when CAFs autophagy is defective, extracellular matrix production is reduced and TME is remodeled. Even if the autophagic ability of tumor cells remains normal, the growth of tumor tissue is still inhibited. Therefore, autophagy inhibitors further remodel the TME through the bidirectional inhibition of CAFs activation and protective autophagy, which is expected to reduce the incidence of acquired resistance and improve the efficacy of PDAC chemotherapy. Summary of the invention

[0006] The purpose of the present invention is to propose a bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer and its preparation method and application, to lay a theoretical and experimental foundation for improving the treatment resistance of PDAC, and to have important scientific significance and clinical application value for the precise diagnosis and treatment of PDAC.

[0007] The technical solution of the present invention is achieved in this way: The present invention provides a method for preparing an integrated bionic nanomaterial for the diagnosis and treatment of pancreatic cancer. The method comprises the following steps: using mesoporous silica as a template to prepare Fe-doped hollow mesoporous MnO2 spheres; after the surface is modified with polydopamine, albumin paclitaxel / doxorubicin and chloroquine are adsorbed by stirring; then the spheres are mixed with bionic liposomes loaded with PANC02 cell membrane proteins, and the liposomes are repeatedly extruded to obtain the integrated bionic nanomaterial for the diagnosis and treatment of pancreatic cancer.

[0008] As a further improvement of the present invention, Figure 1, including the following steps: S1. Preparation of mesoporous silica: dissolving alkyl orthosilicate in ethanol, adding porogen, water and ammonia, stirring to react, centrifuging, washing, drying and calcining to obtain mesoporous silica; S2. Synthesis of Fe-doped hollow mesoporous MnO2 spheres: prepare mesoporous silica into a suspension, dissolve potassium permanganate in water, dropwise add it into the mesoporous silica suspension, add iron salt, stir and react at room temperature, centrifuge, wash, resuspend, dropwise add alkali solution, heat and stir to react, centrifuge, wash, and dry to obtain Fe-doped hollow mesoporous MnO2 spheres; S3. Synthesis of modified Fe-doped hollow mesoporous MnO2 spheres: adding Fe-doped hollow mesoporous MnO2 spheres to water, adding dopamine hydrochloride and a catalyst, heating and stirring to react, centrifuging, washing, and drying to obtain modified Fe-doped hollow mesoporous MnO2 spheres; S4. Preparation of biomimetic liposomes: PANC02 cells in the logarithmic growth phase were digested and collected, membrane proteins were extracted using a membrane protein extraction kit to prepare a membrane protein solution; lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol were dissolved in chloroform, and the solvent was removed by rotary evaporation under reduced pressure to prepare liposomes, the membrane protein solution was added, mixed evenly, and repeatedly extruded using a liposome extruder with a polycarbonate membrane to prepare biomimetic liposomes; S5. Preparation of albumin paclitaxel / doxorubicin: Paclitaxel and doxorubicin were dissolved in ethanol, human serum albumin solution was added dropwise, stirred at room temperature, sonicated, dialyzed, and freeze-dried to obtain albumin paclitaxel / doxorubicin; S6 Preparation of integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer: Add albumin paclitaxel / doxorubicin, chloroquine and modified Fe-doped hollow mesoporous MnO2 spheres into water, stir to adsorb the drugs, centrifuge, wash, dry, mix the solid with bionic liposomes and add them into water, and extrude repeatedly with a liposome extruder with a polycarbonate membrane to obtain integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer.

[0009] As a further improvement of the present invention, the alkyl orthosilicate in step S1 is methyl orthosilicate or ethyl orthosilicate, the mass ratio of the alkyl orthosilicate, ethanol, porogen, water and ammonia water is 13-15:70-90:2-3:5-10:5-7, the porogen is hexadecyltrimethylammonium chloride, the stirring reaction time is 12-15h, the calcination temperature is 400-600°C, and the time is 2-3h.

[0010] As a further improvement of the present invention, the concentration of the suspension in step S2 is 10-15 g / L, the mass ratio of the potassium permanganate, the iron salt, and the mesoporous silica suspension is 4-6:1-2:100, the iron salt is ferric chloride or ferric nitrate, the time of the room temperature stirring reaction is 10-12 h, the alkali solution is 7-10wt% sodium carbonate solution, the temperature of the heated stirring reaction is 40-50°C, and the time is 7-10 h.

[0011] As a further improvement of the present invention, the mass ratio of the Fe-doped hollow mesoporous MnO2 spheres, dopamine hydrochloride and catalyst in step S3 is 10-15:4-6:1-2, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 2-4h.

[0012] As a further improvement of the present invention, the concentration of the membrane protein solution in step S4 is 5-10 mg / mL, the mass ratio of the lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol is 10-12:4-6:3-5:5-7, the number of repeated extrusions is 20-30 times, and the diameter of the liposome extruder of the polycarbonate membrane is 400 nm.

[0013] As a further improvement of the present invention, the concentration of the human serum albumin solution in step S5 is 10-15 mg / mL, the mass ratio of paclitaxel, doxorubicin and human serum albumin solution is 10-15:7-12:100, the stirring time at room temperature is 20-30 min, and the power of the ultrasound is 1000-1500 W for 5-10 min.

[0014] As a further improvement of the present invention, the mass ratio of albumin paclitaxel / doxorubicin, chloroquine and modified Fe-doped hollow mesoporous MnO2 spheres in step S6 is 3-4:1-2:10, the stirring and adsorption time of the drug is 1-2h, the mass ratio of the solid to the biomimetic liposome is 2-3:6-8, the number of repeated extrusions is 15-25 times, and the diameter of the polycarbonate membrane liposome extruder is 200nm.

[0015] The present invention further protects a bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer obtained by the above-mentioned preparation method.

[0016] The present invention further protects the use of the above-mentioned pancreatic cancer diagnosis and treatment integrated bionic nanomaterial in the preparation of drugs for diagnosing and treating pancreatic cancer and pancreatic ductal adenocarcinoma.

[0017] The present invention has the following beneficial effects: In response to the therapeutic needs for improving the prognosis of PDAC, the present invention constructs a bionic nanocomposite with sequential targeting and multi-dimensional TME remodeling effects to enhance the chemotherapy of PDAC and study the related mechanisms. The drug delivery to tumor tissues is increased through the ordered targeting mediated by the extracellular matrix targeting peptide CREKA and tumor cell membrane proteins; the drug delivery to tumor tissues is increased by using Fe-doped hollow mesoporous MnO2 as a drug carrier to achieve responsive drug release and MRI imaging in the acidic environment of tumors; the surface of Fe-doped hollow mesoporous MnO2 is modified by polydopamine, which improves the connection strength of bionic liposomes by forming hydrogen bonds and increasing the specific surface area, which helps to prepare bionic nanomaterials for integrated diagnosis and treatment of pancreatic cancer with higher mechanical properties, better stability, better safety and better biocompatibility, so as to better play a role in targeted diagnosis and treatment; albumin paclitaxel / doxorubicin is used to loosen the tumor matrix and initially reshape the TME, promoting the penetration of drugs deep into tumor tissues; chloroquine is used to inhibit CAFs activation and protective autophagy, further reshaping the TME, and the two drugs work synergistically to improve the chemotherapy effect and reduce the dosage, greatly reducing the adverse reactions of chemotherapy and the incidence of treatment resistance; finally, cytotoxic drugs and autophagy inhibitors are combined to achieve synergistic enhancement of chemotherapy. This invention is expected to lay a theoretical and experimental foundation for improving PDAC treatment resistance, and has important scientific significance and clinical application value for the precise diagnosis and treatment of PDAC.

[0018] In view of the major clinical problem of treatment resistance in PDAC, the present invention constructs a bionic cell membrane-modified pH-responsive nanocarrier loaded with albumin paclitaxel / doxorubicin and hydroxychloroquine. The bionic complex has the following features: 1) orderly targeting mediated by extracellular matrix targeting peptide CREKA and tumor cell membrane protein to increase the sequential delivery of drugs to tumor stroma and tumor cells; 2) with the help of Fe-doped hollow mesoporous MnO2 drug loading, responsive drug release and MRI imaging in the acidic environment of the tumor are achieved, and at the same time, it has the effect of reversing drug resistance. Usually, during chemotherapy, cells will develop drug resistance, thereby reducing the efficacy of the drug. By introducing the Fe element, this changes the The crystal structure of the hollow mesoporous MnO2 drug-loaded spheres will not cause drug resistance, has good effects, and reverses drug resistance; 3) Through albumin paclitaxel / doxorubicin to loosen the tumor matrix and initially reshape the TME, the drug penetration deep into the tumor tissue is promoted. The two drugs work synergistically to improve the chemotherapy effect and reduce the dosage, greatly reducing the adverse reactions of chemotherapy. Targeted drug delivery greatly improves the efficacy of the drug and can also avoid the impact on normal cells; 4) Chloroquine is used to inhibit CAFs activation and protective autophagy, further reshape the TME, and reduce the incidence of treatment resistance; 5) Combine cytotoxic drugs and autophagy inhibitors to achieve synergistic enhancement of chemotherapy.

[0019] (1) Designing a rational strategy to enhance drug accumulation in tumor tissues in response to the drug delivery difficulties in PDAC. The rich interstitial components in PDAC tumor tissues greatly limit the delivery of drugs to tumor cells. Therefore, designing a rational strategy to increase drug delivery is a key issue that needs to be urgently addressed in the treatment of PDAC. Studies have shown that the PDAC tumor microenvironment is rich in a large number of tumor-associated fibroblasts (CAFs), which can form the first line of defense for PDAC drug treatment by secreting collagen and matrix proteins. Therefore, designing a delivery strategy based on CAFs target molecules to enhance drug accumulation in the tumor stroma, and further loosening the tumor stroma by killing CAFs, can achieve TME remodeling and promote drug penetration into tumor tissues; thereafter, drugs enriched in tumor tissues can further enhance drug uptake in tumor cells with the help of homologous targeting of tumor cell membrane proteins. Solving this problem will lay a theoretical foundation for effective PDAC drug delivery.

[0020] (2) In view of the problem of drug resistance in PDAC treatment, design a reasonable strategy to enhance the efficacy of PDAC chemotherapy. PDAC tumors are highly heterogeneous and are very likely to develop drug resistance during treatment. Therefore, designing a reasonable strategy to reduce the occurrence of drug resistance is a key scientific issue facing PDAC. With the continuous development of tumor tissue pathology and molecular biology, researchers have found that there are a large number of activated CAFs in the TME after PDAC chemotherapy, which can protect tumor cells from drugs and immune cells. Based on this, selectively removing CAFs that promote tumorigenesis and regulating the activation and function of CAFs can effectively reshape the drug-resistant TME, reduce the incidence of acquired drug resistance, and ultimately achieve sustained benefits from PDAC chemotherapy. Solving this problem will provide a scientific basis for improving PDAC chemotherapy resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 This is a flow chart for preparing a bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer according to the present invention; Figure 2 This is a graph of the in vitro T1-weighted magnetic resonance imaging results of the bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Human serum albumin, CAS: 70024-90-7, purity>96, purchased from Merck Reagent; mouse pancreatic cancer cell PANC02 cells, purchased from Shanghai Yushao Biotechnology Co., Ltd.; membrane protein extraction kit, brand: Solarbio, product number EX1500. Example 1

[0025] like Figure 1 This embodiment provides a method for preparing a bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer, comprising the following steps: S1. Preparation of mesoporous silica: 13 g of methyl orthosilicate was dissolved in 70 g of ethanol, 2 g of hexadecyltrimethylammonium chloride, 5 g of water and 5 g of aqueous ammonia were added, the mixture was stirred for 12 h, centrifuged, washed, dried, and calcined at 400 ° C for 2 h to obtain mesoporous silica; S2. Synthesis of Fe-doped hollow mesoporous MnO2 spheres: mesoporous silica was prepared into a suspension with a concentration of 10 g / L, 4 g potassium permanganate was dissolved in 100 mL water, and the mixture was added dropwise to the 100 g mesoporous silica suspension, and 1 g ferric chloride was added. The mixture was stirred at room temperature for 10 h, centrifuged, washed, and resuspended in 100 mL water, and 30 g 7 wt% sodium carbonate solution was added dropwise. The mixture was heated to 40°C, stirred for 7 h, centrifuged, washed, and dried to obtain Fe-doped hollow mesoporous MnO2 spheres. S3. Synthesis of modified Fe-doped hollow mesoporous MnO2 spheres: 10 g of Fe-doped hollow mesoporous MnO2 spheres were added to 200 mL of water, 4 g of dopamine hydrochloride and 1 g of catalyst, heated to 40 ° C, stirred for 2 h, centrifuged, washed, and dried to obtain modified Fe-doped hollow mesoporous MnO2 spheres; The catalyst is a Tris-HCl solution with a pH of 8.5; S4. Preparation of biomimetic liposomes: PANC02 cells in the logarithmic growth phase were digested and collected, membrane proteins were extracted using a membrane protein extraction kit, and a membrane protein solution with a concentration of 5 mg / mL was prepared; 1 g of lecithin, 0.4 g of DSPE-PEG2000, 0.3 g of DSPE-PEG-CREKA, and 0.5 g of cholesterol were dissolved in 100 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to prepare liposomes, 100 mL of the membrane protein solution was added, mixed for 10 min, and repeatedly extruded using a liposome extruder with a polycarbonate membrane with a diameter of 400 nm to prepare biomimetic liposomes; S5. Preparation of albumin paclitaxel / doxorubicin: 1 g of paclitaxel and 0.7 g of doxorubicin were dissolved in 200 mL of ethanol, 10 g of a 10 mg / mL human serum albumin solution was added dropwise, stirred at room temperature for 20 min, ultrasonicated at 1000 W for 5 min, dialyzed, and freeze-dried to obtain albumin paclitaxel / doxorubicin; S6 Preparation of integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer: 3 g of albumin paclitaxel / doxorubicin, 1 g of chloroquine and 10 g of modified Fe-doped hollow mesoporous MnO2 spheres were added to 500 mL of water, stirred to adsorb the drugs for 1 h, centrifuged, washed and dried, 2 g of the obtained solid was mixed with 6 g of bionic liposomes and added to 200 mL of water, and repeatedly extruded 15 times using a liposome extruder with a polycarbonate membrane with a diameter of 200 nm to obtain integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer. Example 2

[0026] like Figure 1 This embodiment provides a method for preparing a bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer, comprising the following steps: S1. Preparation of mesoporous silica: 15 g of tetraethyl orthosilicate was dissolved in 90 g of ethanol, 3 g of hexadecyltrimethylammonium chloride, 10 g of water and 7 g of aqueous ammonia were added, the mixture was stirred for 15 h, centrifuged, washed, dried, and calcined at 600 ° C for 3 h to obtain mesoporous silica; S2. Synthesis of Fe-doped hollow mesoporous MnO2 spheres: mesoporous silica was prepared into a suspension with a concentration of 10 g / L, 6 g potassium permanganate was dissolved in 100 mL water, and the mixture was added dropwise to the 100 g mesoporous silica suspension, and 2 g ferric chloride was added. The mixture was stirred at room temperature for 12 h, centrifuged, washed, and resuspended in 100 mL water, and 40 g 10 wt% sodium carbonate solution was added dropwise. The mixture was heated to 50 ° C, stirred for 10 h, centrifuged, washed, and dried to obtain Fe-doped hollow mesoporous MnO2 spheres. S3. Synthesis of modified Fe-doped hollow mesoporous MnO2 spheres: 15 g of Fe-doped hollow mesoporous MnO2 spheres were added to 200 mL of water, 6 g of dopamine hydrochloride and 2 g of catalyst, heated to 50 ° C, stirred for 4 h, centrifuged, washed, and dried to obtain modified Fe-doped hollow mesoporous MnO2 spheres; The catalyst is a Tris-HCl solution with a pH of 9.5; S4. Preparation of biomimetic liposomes: PANC02 cells in the logarithmic growth phase were digested and collected, membrane proteins were extracted using a membrane protein extraction kit, and a membrane protein solution with a concentration of 10 mg / mL was prepared; 1.2 g of lecithin, 0.6 g of DSPE-PEG2000, 0.5 g of DSPE-PEG-CREKA, and 0.7 g of cholesterol were dissolved in 100 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes, 100 mL of the membrane protein solution was added, mixed for 10 min, and repeatedly extruded using a liposome extruder with a polycarbonate membrane with a diameter of 400 nm to obtain biomimetic liposomes; S5. Preparation of albumin paclitaxel / doxorubicin: 1.5 g of paclitaxel and 1.2 g of doxorubicin were dissolved in 200 mL of ethanol, 10 g of a 15 mg / mL human serum albumin solution was added dropwise, stirred at room temperature for 30 min, ultrasonicated at 1500 W for 10 min, dialyzed, and freeze-dried to obtain albumin paclitaxel / doxorubicin; S6 Preparation of integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer: 4 g of albumin paclitaxel / doxorubicin, 2 g of chloroquine and 10 g of modified Fe-doped hollow mesoporous MnO2 spheres were added to 500 mL of water, stirred to adsorb the drugs for 2 h, centrifuged, washed and dried, 3 g of the obtained solid was mixed with 8 g of bionic liposomes and added to 200 mL of water, and repeatedly extruded 25 times using a liposome extruder with a polycarbonate membrane with a diameter of 200 nm to obtain integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer. Example 3

[0027] like Figure 1 This embodiment provides a method for preparing a bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer, comprising the following steps: S1. Preparation of mesoporous silica: 14 g of tetraethyl orthosilicate was dissolved in 80 g of ethanol, 2.5 g of hexadecyltrimethylammonium chloride, 7 g of water and 6 g of aqueous ammonia were added, the mixture was stirred for 13 h, centrifuged, washed, dried, and calcined at 500 ° C for 2.5 h to obtain mesoporous silica; S2. Synthesis of Fe-doped hollow mesoporous MnO2 spheres: mesoporous silica was prepared into a suspension with a concentration of 12 g / L, 5 g potassium permanganate was dissolved in 100 mL water, and the mixture was added dropwise to the 100 g mesoporous silica suspension, and 1.5 g ferric nitrate was added. The mixture was stirred at room temperature for 11 h, centrifuged, washed, and resuspended in 100 mL water, and 35 g 8.5 wt% sodium carbonate solution was added dropwise. The mixture was heated to 45 °C, stirred for 8 h, centrifuged, washed, and dried to obtain Fe-doped hollow mesoporous MnO2 spheres. S3. Synthesis of modified Fe-doped hollow mesoporous MnO2 spheres: 12 g of Fe-doped hollow mesoporous MnO2 spheres were added to 200 mL of water, 5 g of dopamine hydrochloride and 1.5 g of catalyst, heated to 45 ° C, stirred for 3 h, centrifuged, washed, and dried to obtain modified Fe-doped hollow mesoporous MnO2 spheres; The catalyst is a Tris-HCl solution with a pH of 9; S4. Preparation of biomimetic liposomes: PANC02 cells in the logarithmic growth phase were digested and collected, membrane proteins were extracted using a membrane protein extraction kit, and a membrane protein solution with a concentration of 7 mg / mL was prepared; 1.1 g of lecithin, 0.5 g of DSPE-PEG2000, 0.4 g of DSPE-PEG-CREKA, and 0.6 g of cholesterol were dissolved in 100 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes, 100 mL of the membrane protein solution was added, mixed for 10 min, and repeatedly extruded using a liposome extruder with a polycarbonate membrane with a diameter of 400 nm to obtain biomimetic liposomes; S5. Preparation of albumin paclitaxel / doxorubicin: 1.2 g of paclitaxel and 1 g of doxorubicin were dissolved in 200 mL of ethanol, 10 g of a 12 mg / mL human serum albumin solution was added dropwise, stirred at room temperature for 25 min, ultrasonicated at 1200 W for 7 min, dialyzed, and freeze-dried to obtain albumin paclitaxel / doxorubicin; S6 Preparation of integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer: 3.5 g of albumin paclitaxel / doxorubicin, 1.5 g of chloroquine and 10 g of modified Fe-doped hollow mesoporous MnO2 spheres were added to 500 mL of water, stirred to adsorb the drugs for 1.5 h, centrifuged, washed and dried, 2.5 g of the obtained solid was mixed with 7 g of bionic liposomes and added to 200 mL of water, and repeatedly extruded 20 times using a liposome extruder with a polycarbonate membrane with a diameter of 200 nm to obtain integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer.

[0028] The prepared bionic nanomaterials for integrated diagnosis and treatment of pancreatic cancer were added to a H2O2 solution with a pH of 6.0 and glutathione was added to make the glutathione content 10 mg / mL. T1-weighted magnetic resonance imaging was investigated using a 0.5T magnetic resonance scanner. The results are shown in Figure 2 ,The results showed that as the concentration of the complex increased, the T1 signal gradually increased, indicating that the prepared bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer had good T1-weighted magnetic resonance imaging performance.

[0029] Comparative Example 1 Compared with Example 3, the difference is that step S3 is not performed.

[0030] The details are as follows: S1. Preparation of mesoporous silica: 14 g of tetraethyl orthosilicate was dissolved in 80 g of ethanol, 2.5 g of hexadecyltrimethylammonium chloride, 7 g of water and 6 g of aqueous ammonia were added, the mixture was stirred for 13 h, centrifuged, washed, dried, and calcined at 500 ° C for 2.5 h to obtain mesoporous silica; S2. Synthesis of Fe-doped hollow mesoporous MnO2 spheres: mesoporous silica was prepared into a suspension with a concentration of 12 g / L, 5 g potassium permanganate was dissolved in 100 mL water, and the mixture was added dropwise to the 100 g mesoporous silica suspension, and 1.5 g ferric nitrate was added. The mixture was stirred at room temperature for 11 h, centrifuged, washed, and resuspended in 100 mL water, and 35 g 8.5 wt% sodium carbonate solution was added dropwise. The mixture was heated to 45 °C, stirred for 8 h, centrifuged, washed, and dried to obtain Fe-doped hollow mesoporous MnO2 spheres. S3. Preparation of biomimetic liposomes: PANC02 cells in the logarithmic growth phase were digested and collected, membrane proteins were extracted using a membrane protein extraction kit, and a membrane protein solution with a concentration of 7 mg / mL was prepared; 1.1 g of lecithin, 0.5 g of DSPE-PEG2000, 0.4 g of DSPE-PEG-CREKA, and 0.6 g of cholesterol were dissolved in 100 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to prepare liposomes, 100 mL of the membrane protein solution was added, mixed for 10 min, and repeatedly extruded using a liposome extruder with a polycarbonate membrane with a diameter of 400 nm to prepare biomimetic liposomes; S4. Preparation of albumin paclitaxel / doxorubicin: 1.2 g of paclitaxel and 1 g of doxorubicin were dissolved in 200 mL of ethanol, 10 g of a 12 mg / mL human serum albumin solution was added dropwise, stirred at room temperature for 25 min, ultrasonicated at 1200 W for 7 min, dialyzed, and freeze-dried to obtain albumin paclitaxel / doxorubicin; S5 Preparation of integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer: 3.5 g of albumin paclitaxel / doxorubicin, 1.5 g of chloroquine and 10 g of Fe-doped hollow mesoporous MnO2 spheres were added to 500 mL of water, stirred to adsorb the drugs for 1.5 h, centrifuged, washed and dried, 2.5 g of the obtained solid was mixed with 7 g of bionic liposomes and added to 200 mL of water, and repeatedly extruded 20 times using a liposome extruder with a polycarbonate membrane with a diameter of 200 nm to obtain integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer.

[0031] Comparative Example 2 Compared with Example 3, the difference is that no biomimetic liposomes are added in step S6.

[0032] The details are as follows: S6 Preparation of integrated bionic nanomaterials for pancreatic cancer diagnosis and treatment: 3.5 g of albumin paclitaxel / doxorubicin, 1.5 g of chloroquine and 10 g of modified Fe-doped hollow mesoporous MnO2 spheres were added into 500 mL of water, stirred to adsorb the drugs for 1.5 h, centrifuged, washed and dried to obtain the material.

[0033] Comparative Example 3 Compared with Example 3, the difference is that chloroquine is not added in step S6.

[0034] The details are as follows: S6 Preparation of integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer: 5 g of albumin paclitaxel / doxorubicin and 10 g of modified Fe-doped hollow mesoporous MnO2 spheres were added to 500 mL of water, stirred to adsorb the drugs for 1.5 h, centrifuged, washed, and dried, 2.5 g of the obtained solid was mixed with 7 g of bionic liposomes and added to 200 mL of water, and repeatedly extruded 20 times using a liposome extruder with a polycarbonate membrane with a diameter of 200 nm to obtain integrated bionic nanomaterials for the diagnosis and treatment of pancreatic cancer.

[0035] Comparative Example 4 Compared with Example 3, the difference is that paclitaxel is not added in step S5.

[0036] The details are as follows: S5. Preparation of albumin doxorubicin: 2.2 g of doxorubicin was dissolved in 200 mL of ethanol, and 10 g of a 12 mg / mL human serum albumin solution was added dropwise. The mixture was stirred at room temperature for 25 min, ultrasonicated at 1200 W for 7 min, dialyzed, and freeze-dried to obtain albumin doxorubicin.

[0037] Comparative Example 5 Compared with Example 3, the difference is that doxorubicin is not added in step S5.

[0038] The details are as follows: S5. Preparation of albumin paclitaxel: 2.2 g of paclitaxel was dissolved in 200 mL of ethanol, and 10 g of a 12 mg / mL human serum albumin solution was added dropwise, stirred at room temperature for 25 min, ultrasonicated at 1200 W for 7 min, dialyzed, and freeze-dried to obtain albumin paclitaxel.

[0039] Comparative Example 6 Compared with Example 3, the difference is that no human serum albumin solution is added in step S5.

[0040] The details are as follows: S5. Preparation of paclitaxel / doxorubicin mixture: 1.2 g of paclitaxel and 1 g of doxorubicin were dissolved in 200 mL of ethanol and dried to prepare a paclitaxel / doxorubicin mixture.

[0041] Comparative Example 7 Compared with Example 3, the difference is that modified Fe-doped hollow mesoporous MnO2 balls are not added in step S6.

[0042] The details are as follows: S6 Preparation of integrated bionic nanomaterials for pancreatic cancer diagnosis and treatment: 1.75 g of albumin paclitaxel / doxorubicin and 0.75 g of chloroquine were added to 200 mL of water, stirred for 1.5 h, mixed with 7 g of bionic liposomes, and repeatedly extruded 20 times using a liposome extruder with a polycarbonate membrane of 200 nm in diameter to obtain the material.

[0043] Comparative Example 8 Compared with Example 3, the difference is that no ferric nitrate is added in step S2.

[0044] The details are as follows: S2. Synthesis of hollow mesoporous MnO2 spheres: mesoporous silica was prepared into a suspension with a concentration of 12 g / L. 5 g of potassium permanganate was dissolved in 100 mL of water and added dropwise to 100 g of the mesoporous silica suspension. The mixture was stirred at room temperature for 11 h, centrifuged, washed, and resuspended in 100 mL of water. 35 g of 8.5 wt% sodium carbonate solution was added dropwise. The mixture was heated to 45 °C, stirred for 8 h, centrifuged, washed, and dried to obtain hollow mesoporous MnO2 spheres.

[0045] Test Example 1 C166 cells (vascular endothelial cells) and THLE-3 cells (liver epithelial cells) were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin) under the following conditions: temperature 37°C, carbon dioxide concentration 5%. Subsequently, C166 cells and THLE-3 cells were inoculated in 96-well plates, and the cell density was controlled to be 4×10 5 After the cells were cultured overnight, the cells were treated with 100 μg / mL of the aqueous suspension of the product prepared in Example 1-3 or Comparative Example 1-8 for 24 hours. Deionized water was used as a blank control. Finally, the relative survival rate of the cells relative to the control group was determined according to the operating procedures of the MTT kit. The results are shown in Table 1.

[0046] Table 1

[0047] It can be seen from the above table that the products prepared in Examples 1-3 of the present invention have lower cytotoxicity and higher relative cell survival rate.

[0048] Test Example 2 The well-growing mouse pancreatic cancer tumor tissue was taken, cut into pieces, ground, and made into a tumor cell suspension (2g tumor tissue plus 12mL normal saline), and 0.2mL mouse pancreatic cancer Pan02 tumor cell suspension was inoculated into the subcutaneous left axillary back of male C57BL / 6 mice (body weight 18-20g), and the day of inoculation was recorded as D0. On the first day after tumor inoculation (D1), the experimental mice were randomly divided into PBS (solvent control) group, GEM (gemcitabine) 50.0mg / kg group and Example 1-3 or Comparative Example 1-8 (corresponding product) 10.0mg / kg group, with 10 mice in each group; at D1, the GEM group was intraperitoneally injected, once every 4d, for a total of 2 times; the PBS group and Example 1-3 or Comparative Example 1-7 10.0mg / kg group were subcutaneously administered once a day in the groin, for a total of 6 times.

[0049] At the end of the experiment (D7), the tumor volume was measured three times using a vernier caliper and the average value was taken. The volume calculation formula, including the long diameter and short diameter of the tumor, was: volume = 0.5 × long diameter × short diameter 2 Tumor growth inhibition rate (%) = [1-T (tumor volume of treatment group) / C (tumor volume of solvent control group)] × 100%. The results are shown in Table 2.

[0050] Table 2

[0051] It can be seen from the above table that the products prepared in Examples 1-3 of the present invention have a good inhibitory effect on pancreatic cancer tumors.

[0052] At the end of the experiment (D7), the spleen and thymus of the mice were removed, weighed and recorded, and the corresponding organ indexes were calculated.

[0053] Spleen index = spleen mass (mg) / body mass (g) × 10; thymus index = thymus mass (mg) / body mass (g) × 10. The results are shown in Table 3.

[0054] Table 3

[0055] Notes: Compared with the PBS group, P<0.05.

[0056] As can be seen from the above table, the products prepared in Examples 1-3 of the present invention have no significant effect on the thymus and spleen indexes of tumor-bearing mice, and therefore have no negative impact on the immunity of mice.

[0057] Test Example 3 Inhibitory effect on drug-resistant cells MiaPaCa-2 (CRL-1420™) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA); all cell lines were cultured in DMEM high glucose medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco).

[0058] The culture medium of MiaPaCa-2 / NPR (nab-Paclitaxel Resistance) cells was supplemented with 5 nM nab-paclitaxel. All cell lines were grown at 37°C in a humidified atmosphere with 5% CO2. Three thousand cells were seeded per well in a 96-well plate in a total volume of 100 µL of culture medium and cells were treated with different concentrations of drugs. IC 50 .

[0059] The drug-resistant cell line was established from MiaPaCa-2 cells. 5 1 mL of cells were seeded in 12-well plates and cultured overnight before treatment. 50 The cells were shocked for 24 h with 1% paclitaxel (200 μg / mL) of 1% paclitaxel. The pancreatic cancer cells were induced to develop resistance to paclitaxel. The culture medium was then changed twice a week, and the drug concentration was gradually increased when the cells grew well. The cells were transferred to a 75 cm 2 The cells were further cultured in tissue culture bottles, subcultured at 75-80% confluence, and directly seeded into a medium containing drugs, and divided into a control group, a positive drug group, Examples 1-3, and Comparative Example 8. Among them, the control group did not add drugs, and the positive drug group 1 (doxorubicin), the positive drug group 2 (paclitaxel), Examples 1-3, and the comparative example 8 group (integrated bionic nanomaterials for pancreatic cancer diagnosis and treatment) were added with different concentrations (0, 0.1, 1, 5, 10, 20, 40 μmol / mL) of the corresponding prepared product solutions. Each group had 6 replicate wells, and the experiment was repeated 3 times. The blank culture medium without cells was used as a blank group for control, and the absorbance (A) value was detected at a wavelength of 450nm using an enzyme marker.

[0060]

[0061] Inhibition rate (%) = (1-cell viability) × 100%. The IC of the drug was calculated using Graphpad Prism 8.0 software. 50 value.

[0062] The results are shown in Table 4.

[0063] Table 4

[0064] It can be seen from the above table that the integrated bionic nanomaterials for diagnosis and treatment of pancreatic cancer prepared in Examples 1-3 of the present invention have good in vitro inhibitory effects on drug-resistant cell lines.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing bionic nanomaterials for integrated diagnosis and treatment of pancreatic cancer. It is characterized in that Preparation of Fe-doped hollow mesoporous MnO using mesoporous silica as a template 2 The surface of the spheres was modified with polydopamine, and then albumin paclitaxel / doxorubicin and chloroquine were adsorbed by stirring. Then, they were mixed with bionic liposomes loaded with PANC02 cell membrane proteins and repeatedly extruded to obtain bionic nanomaterials for integrated diagnosis and treatment of pancreatic cancer.

2. The preparation method according to claim 1, It is characterized in that The following steps are involved: S1. Preparation of mesoporous silica: dissolving alkyl orthosilicate in ethanol, adding porogen, water and ammonia, stirring to react, centrifuging, washing, drying and calcining to obtain mesoporous silica; S2. Fe-doped hollow mesoporous MnO 2 Synthesis of the spheres: prepare mesoporous silica into a suspension, dissolve potassium permanganate in water, drop it into the mesoporous silica suspension, add iron salt, stir and react at room temperature, centrifuge, wash, resuspend, drop alkali solution, heat and stir to react, centrifuge, wash, and dry to obtain Fe-doped hollow mesoporous MnO 2 ball; S3. Modified Fe-doped hollow mesoporous MnO 2 Synthesis of spheres: Fe doping hollow mesoporous MnO 2 The balls were added into water, dopamine hydrochloride and catalyst were added, heated and stirred for reaction, centrifuged, washed and dried to obtain modified Fe-doped hollow mesoporous MnO 2 ball; S4. Preparation of biomimetic liposomes: PANC02 cells in the logarithmic growth phase were digested and collected, membrane proteins were extracted using a membrane protein extraction kit to prepare a membrane protein solution; lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol were dissolved in chloroform, and the solvent was removed by rotary evaporation under reduced pressure to prepare liposomes, the membrane protein solution was added, mixed evenly, and repeatedly extruded using a liposome extruder with a polycarbonate membrane to prepare biomimetic liposomes; S5. Preparation of albumin paclitaxel / doxorubicin: Paclitaxel and doxorubicin were dissolved in ethanol, human serum albumin solution was added dropwise, stirred at room temperature, sonicated, dialyzed, and freeze-dried to obtain albumin paclitaxel / doxorubicin; S6 Preparation of biomimetic nanomaterials for integrated diagnosis and treatment of pancreatic cancer: doping hollow mesoporous MnO with albumin paclitaxel / doxorubicin, chloroquine and modified Fe 2 The balls are added into water, stirred to absorb drugs, centrifuged, washed, dried, the solids are mixed with bionic liposomes and added into water, and repeatedly extruded by a liposome extruder with a polycarbonate membrane to obtain bionic nanomaterials for integrated diagnosis and treatment of pancreatic cancer.

3. The preparation method according to claim 2, It is characterized in that In step S1, the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate, the mass ratio of the alkyl orthosilicate, ethanol, porogen, water and ammonia water is 13-15:70-90:2-3:5-10:5-7, the porogen is hexadecyltrimethylammonium chloride, the stirring reaction time is 12-15h, the calcination temperature is 400-600°C, and the time is 2-3h.

4. The preparation method according to claim 2, It is characterized in that The concentration of the suspension in step S2 is 10-15 g / L, the mass ratio of the potassium permanganate, the iron salt, and the mesoporous silica suspension is 4-6:1-2:100, the iron salt is ferric chloride or ferric nitrate, the time of the room temperature stirring reaction is 10-12 h, the alkali solution is 7-10wt% sodium carbonate solution, the temperature of the heated stirring reaction is 40-50 ° C, and the time is 7-10 h.

5. The preparation method according to claim 2, It is characterized in that The Fe-doped hollow mesoporous MnO 2 The mass ratio of the balls, dopamine hydrochloride and the catalyst is 10-15:4-6:1-2, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50° C., and the time is 2-4 hours.

6. The preparation method according to claim 2, It is characterized in that The concentration of the membrane protein solution in step S4 is 5-10 mg / mL, the mass ratio of the lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol is 10-12:4-6:3-5:5-7, the number of repeated extrusions is 20-30 times, and the diameter of the liposome extruder of the polycarbonate membrane is 400 nm.

7. The preparation method according to claim 2, It is characterized in that The concentration of the human serum albumin solution in step S5 is 10-15 mg / mL, the mass ratio of paclitaxel, doxorubicin and human serum albumin solution is 10-15:7-12:100, the stirring time at room temperature is 20-30 min, the power of the ultrasound is 1000-1500 W, and the time is 5-10 min.

8. The preparation method according to claim 2, It is characterized in that The albumin paclitaxel / doxorubicin, chloroquine and modified Fe-doped hollow mesoporous MnO 2 The mass ratio of the spheres is 3-4:1-2:10, the stirring time for drug adsorption is 1-2h, the mass ratio of the solid to the biomimetic liposome is 2-3:6-8, the number of repeated extrusions is 15-25 times, and the diameter of the polycarbonate membrane liposome extruder is 200nm.

9. A bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the bionic nanomaterial for integrated diagnosis and treatment of pancreatic cancer as claimed in claim 9 in the preparation of drugs for diagnosing and treating pancreatic cancer and pancreatic ductal adenocarcinoma.

Images