Nanodrug for synergistically removing fibrosis and inhibiting pancreatic cancer metastasis and preparation method therefor

Abstract

The present invention belongs to the field of pharmaceutical formulations. Specifically disclosed are a nanodrug for synergistically removing fibrosis and inhibiting pancreatic cancer metastasis and a preparation method therefor. By means of the self-assembly of a small-molecule prodrug, excipient-related toxicity is avoided, and a high drug loading rate is achieved. By means of hybrid membrane encapsulation, homologous targeting of fibrotic adhesion interstitium and receptor protein-mediated active targeting are achieved. By means of the response of the prodrug molecule to ROS, massive, rapid, and traceless drug release is achieved in the fibrotic adhesion interstitium, removing the self-limiting characteristic of the release of a responsive small-molecule drug. The released drugs Cap and CA can simultaneously inhibit fibrosis signals / proteins, synergistically removing fibrosis. As the removal of fibrosis progresses, Cap and CA released rapidly and tracelessly in pancreatic cancer cells can simultaneously inhibit the metastasis / invasion signaling pathway of pancreatic cancer cells, synergistically inhibiting metastasis. The dynamic synergy between the process of removing fibrosis and the process of inhibiting metastasis provides a new approach for removing fibrosis in the treatment of pancreatic cancer.

Description

Nanomedicine for synergistic defibrosis and metastasis inhibition of pancreatic cancer and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical preparations, in particular to a nanomedicine for synergistic defibrosis and metastasis inhibition of pancreatic cancer and a preparation method thereof. BACKGROUND

[0002] Dense fibrotic adhesion stroma is one of the main reasons for the failure of pancreatic cancer treatment. As a typical histopathological feature of pancreatic cancer, it is a natural protective physical barrier for pancreatic cancer, which can block the effective delivery of chemotherapeutic drugs, and also protect cancer cells from the effects of conventional chemotherapeutic drugs, thereby leading to the development of chemotherapeutic drug resistance. Any effective chemotherapy must overcome the obstacles of fibrotic adhesion stroma, so how to efficiently defibrate has been a bottleneck problem in the treatment of pancreatic cancer. On the other hand, due to the strong invasiveness of pancreatic cancer cells, defibration will increase the risk of metastasis of pancreatic cancer, so how to effectively inhibit the metastasis of pancreatic cancer cells while efficiently defibrating becomes another difficult problem in the treatment of pancreatic cancer. Therefore, it is of great significance to construct a drug delivery system that can both efficiently defibrate and effectively inhibit the metastasis of cancer cells for the treatment of pancreatic cancer.

[0003] Nanomedicine opens up a new way to improve the therapeutic effect of cancer. Compared with small molecule drugs, nanomedicine has many advantages, such as longer blood circulation time in vivo, more efficient enrichment at tumor sites, and specific drug release in tumor microenvironment of functionalized nanomedicine. It can be seen that nanomedicine can effectively improve the targeting and bioavailability of drugs, thereby reducing the side effects of treatment. At present, nanomedicine mainly relies on excipients such as liposomes or polymers to form nanoparticles. Because the excipient occupies most of the mass of the nanomedicine, the drug loading capacity of the nanomedicine is usually low, which often leads to unsatisfactory treatment effect because the effective drug concentration is not reached, and if the drug dosage is increased, it is difficult to avoid the systemic toxicity caused by the excipient. Nanomedicine based on small molecule self-assembly perfectly solves the above problems. Without excipients, it has high drug loading rate, minimizes the toxic side effects caused by excipients, has more reliable safety in treatment, and the preparation method of small molecule self-assembled nanomedicine is often simpler and more environmentally friendly.

[0004] In recent years, in order to improve the treatment efficiency of pancreatic tumor, researchers have developed various nano drug delivery systems to regulate the fibrotic adhesion interstitium of pancreatic cancer. The strategies mainly include: (1) using polymers or liposomes as carriers to deliver TGF-β / Smad signal pathway inhibitors (jatrorrhizine, metformin, alpha-ambrettone, etc.), inhibit the activation pathway of pancreatic stellate cells, reduce the generation of cancer-associated fibroblasts, and reduce the formation of fibrotic adhesion interstitium; (2) using liposomes or inorganic nanoparticles as carriers to deliver pancreatic stellate cell activity inhibitors (pirfenidone, all-trans retinoic acid), and inhibit the activity of pancreatic stellate cells to slow down the formation of fibrotic adhesion interstitium; (3) using liposomes as carriers to deliver photothermal therapy agents to ablate the fibrotic adhesion interstitium of pancreatic cancer by photothermal effect. The above-mentioned defibrotic research greatly promotes the application of defibrotic nanomedicine, but there are still many shortcomings: first, it is difficult to achieve defibrosis and metastasis inhibition at the same time; second, with the help of a specific carrier, the drug loading rate is low, and it is difficult to achieve responsive release and poor selectivity; third, the signal pathway regulation is relatively single, and it is difficult to form a synergistic effect, and the defibrosis efficiency is low. SUMMARY

[0005] To solve the above technical problems, the present application provides a kind of nanomedicine of defibrosis and inhibition of pancreatic cancer metastasis synergy and preparation method thereof.

[0006] To achieve the above purpose, the present application is implemented according to the following technical solutions:

[0007] One of the purposes of the present application is to provide a preparation method of a nanomedicine for defibrosis and inhibition of pancreatic cancer metastasis synergy, comprising the following steps:

[0008] S1, trifluoroacetic acid, captopril Cap and cinnamyl aldehyde CA with a molar ratio of 1:1:1 are mixed, under the catalysis of trifluoroacetic acid, the mercapto group of captopril Cap and the aldehyde group of cinnamyl aldehyde CA occur condensation reaction, captopril Cap and cinnamyl aldehyde CA are connected through ROS sensitive tk bond, to obtain small molecule prodrug Cap-tkCA-Cap; and the small molecule prodrug Cap-tkCA-Cap is treated to obtain small molecule prodrug nanoparticles Cap-tkCA-Cap NPs;

[0009] S2, activated macrophage / fibroblast hybrid cell membrane is prepared;

[0010] S3, the obtained activated macrophage / fibroblast hybrid cell membrane is coated on the small molecule prodrug nanoparticles Cap-tkCA-Cap NPs to obtain the nanomedicine Cap-tkCA-Cap@CM.

[0011] Further, the step S1, the processing of the small molecule prodrug Cap-tkCA-Cap is specifically:

[0012] The small molecule prodrug Cap-tkCA-Cap is dissolved in dimethyl sulfoxide DMSO to obtain a mixed solution, the mixed solution is slowly dropped into deionized water under ultrasonic, and the dialysis bag with a molecular weight cut-off of 1000 Da is used for dialysis purification to obtain the small molecule prodrug nanoparticles Cap-tkCA-Cap NPs.

[0013] Specifically, the step S2 comprises:

[0014] S21, the macrophage RAW 264.7 is incubated with 10 ng / mL of LPS, and then the activated macrophage membrane is extracted by ultrasonic and centrifugation;

[0015] S22, the normal fibroblast NIH3T3 is incubated with 10 ng / mL of TGF-β1, and then the activated fibroblast membrane is extracted by ultrasonic and centrifugation;

[0016] S23, the activated macrophage membrane and the activated fibroblast membrane are mixed at a mass ratio of 1:1 and ultrasonic, and the membrane fusion is promoted by repeatedly extruding the polycarbonate porous membrane to obtain the hybrid cell membrane of activated macrophages / fibroblasts.

[0017] Specifically, the step S3 comprises:

[0018] The 1 mg / mL small molecule prodrug nanoparticles Cap-tkCA-Cap NPs are mixed with the hybrid membrane of 5 million activated macrophages and 5 million fibroblasts and then ultrasonic, and the nanodrug Cap-tkCA-Cap@CM is obtained by extruding the polycarbonate porous membrane.

[0019] Preferably, the pore size of the polycarbonate porous membrane is 100-400 nm.

[0020] The second object of the present application is to provide a nanodrug for defibrillation and inhibition of pancreatic cancer metastasis, which is prepared by the above method.

[0021] The mechanism of the nanodrug Cap-tkCA-Cap@CM prepared by the present application is:

[0022] In delivery, the hybrid film provides in vivo long circulation for the nanodrug, homologous targeting for fibroblasts in fibrotic adhesion interstitium of pancreatic cancer, and receptor protein-mediated targeting for HA and fibronectin in the interstitium, thereby efficiently accumulating in the fibrotic adhesion interstitium. In the fibrotic adhesion interstitium of pancreatic cancer, high levels of ROS cleave the tk bond of Cap-tkCA-Cap, rapidly, massively and tracelessly releasing Cap and CA; Cap and CA respectively inhibit the TGF-β / Smad signaling pathway, reduce the secretion of ECM proteins, and inhibit the formation of fibrotic adhesion interstitium, thereby synergistically de-fibrotizing. With the de-fibrotization, the biomimetic nanodrug is further rapidly released in pancreatic cancer cells, and Cap and CA respectively reduce the invasiveness of pancreatic cancer cells by inhibiting the TGF-β / Smad and PI3K / AKT signaling pathways, thereby synergistically inhibiting the occurrence of metastasis.

[0023] Compared with the prior art, the present application realizes no excipient-related toxicity and high drug loading rate by self-assembly of small molecule prodrugs, realizes homologous targeting and receptor protein-mediated active targeting of fibrotic adhesion interstitium by coating of the hybrid film, realizes massive, rapid and traceless drug release in the fibrotic adhesion interstitium by response of the prodrug molecules to ROS, and eliminates the self-limiting characteristics of responsive small molecule drug release; the released drugs Cap and CA can simultaneously inhibit fibrotic signals / proteins, thereby synergistically de-fibrotizing; with the de-fibrotization, Cap and CA which are rapidly and tracelessly released in pancreatic cancer cells can simultaneously inhibit the metastasis / invasion signaling pathways of pancreatic cancer cells, thereby synergistically inhibiting the occurrence of metastasis; the de-fibrotization process and the metastasis inhibition process are dynamically synergistic, thereby providing a new way for de-fibrotization of pancreatic cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a preparation diagram of the nanodrug synergistically de-fibrotizing and inhibiting pancreatic cancer metastasis according to the present application.

[0025] Fig. 2 is a structure of a small molecule prodrug Cap-tkCA-Cap by nuclear magnetic resonance hydrogen spectrum (H NMR). 1 H NMR).

[0026] Fig. 3 is a structure of a small molecule prodrug Cap-tkCA-Cap by nuclear magnetic resonance carbon spectrum (C NMR). 13 C NMR)

[0027] Fig. 4 is a high resolution mass spectrum (HR-MS) diagram of a small molecule prodrug Cap-tkCA-Cap.

[0028] Fig. 5 is a particle size change diagram of a small molecule prodrug nanoparticle Cap-tkCA-Cap NPs and a nanodrug Cap-tkCA-Cap@CM.

[0029] Figure 6 is a graph of the surface charge value of the nanodrug Cap-tkCA-Cap@CM.

[0030] Figure 7 is a transmission electron microscope (TEM) graph of Cap-tkCA-Cap NPs and Cap-tkCA-Cap@CM.

[0031] Figure 8 is a high resolution mass spectrometry (HR-MS) graph of the ROS-responsive Cap and CA drug release of the nanodrug Cap-tkCA-Cap@CM in a simulated physiological environment.

[0032] Figure 9 is a high performance liquid chromatography (HPLC) drug release graph of the ROS-responsive Cap and CA of the nanodrug Cap-tkCA-Cap@CM in a simulated physiological environment.

[0033] Figure 10 is the evaluation result of the defibrillation ability of Cap-tkCA-Cap@CM, the blank group, Cap, and CA groups.

[0034] Figure 11 is the expression of α-SMA, Fibronectin, and Col I in NIH3T3 treated with different concentrations of Cap-tkCA-Cap@CM.

[0035] Figure 12 is the result of the inhibition of pancreatic cancer cell metastasis without Cap-tkCA-Cap@CM and with the addition of Cap-tkCA-Cap@CM.

[0036] Figure 13 is the result of the inhibition of pancreatic cancer cell invasion without Cap-tkCA-Cap@CM and with the addition of Cap-tkCA-Cap@CM.

[0037] Figure 14 is the result of the inhibition of pancreatic cancer metastasis in mice without Cap-tkCA-Cap@CM and with the addition of the nanodrug Cap-tkCA-Cap@CM.

[0038] Figure 15 is the application effect of the nanodrug Cap-tkCA-Cap@CM in the treatment of pancreatic cancer in mice. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. The specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0040] The raw materials, reagents, equipment and tools used in the following embodiments are commercially available unless otherwise specified.

[0041] Captopril (Cap) was purchased from Sigma-Aldrich Company;

[0042] Cinnamaldehyde (CA), purchased from Sigma-Aldrich;

[0043] Macrophage RAW 264.7, purchased from Wuhan Pons Life Science Co., Ltd.;

[0044] Normal fibroblast NIH3T3, purchased from Wuhan Pons Life Science Co., Ltd.;

[0045] TGF-β1, purchased from Sigma-Aldrich.

[0046] Example 1, preparation of nanomedicine synergistic in defibering and inhibiting pancreatic cancer metastasis

[0047] As shown in FIG. 1, the present embodiment exemplarily demonstrates a preparation method of nanomedicine synergistic in defibering and inhibiting pancreatic cancer metastasis, and the specific steps are as follows:

[0048] S1, trifluoroacetic acid, captopril Cap and cinnamaldehyde CA with a molar ratio of 1:1:1 are mixed, under the catalysis of trifluoroacetic acid, the mercapto group of captopril Cap and the aldehyde group of cinnamaldehyde CA occur condensation reaction, captopril Cap and cinnamaldehyde CA are connected through ROS sensitive tk bond, to obtain small molecule prodrug Cap-tkCA-Cap; and the small molecule prodrug Cap-tkCA-Cap is treated to obtain small molecule prodrug nanoparticles Cap-tkCA-Cap NPs, the reaction formula is as follows:

[0049]

[0050] The structure of the small molecule prodrug Cap-tkCA-Cap is characterized by nuclear magnetic resonance hydrogen spectrum and carbon spectrum (1H NMR, 13C NMR) (see FIG. 2, FIG. 3) and high resolution mass spectrum (HR-MS) (FIG. 4); finally its structure is determined as the structure in the above reaction formula.

[0051] Then the small molecule prodrug Cap-tkCA-Cap is dissolved in dimethyl sulfoxide DMSO to obtain a mixed solution, the mixed solution is slowly dropped into deionized water under ultrasonic, and dialysis purification is carried out with a dialysis bag with a molecular weight cut-off of 1000 Da to obtain small molecule prodrug nanoparticles Cap-tkCA-Cap NPs.

[0052] S2, preparation of activated macrophage / fibroblast hybrid cell membrane;

[0053] S21, macrophage RAW 264.7 was incubated with 10 ng / mL of LPS at 37 °C for 12 hours, and the activated cell membrane surface integrin protein content was characterized using Western blotting (WB), and the protein ligands were vascular adhesion factor and fibrous tissue adhesion protein; then the activated macrophage membrane was extracted by ultrasonic (500 watts, 20 kilohertz, 5 minutes), centrifugation (30 minutes, 4 °C, 14000 g);

[0054] S22, normal fibroblast NIH3T3 was incubated with 10 ng / mL of TGF-β1 at 37 °C for 12 hours, and then the activated fibroblast membrane was extracted by ultrasonic (500 watts, 20 kilohertz, 5 minutes), centrifugation (30 minutes, 4 °C, 14000 g);

[0055] S23, the activated macrophage membrane and the activated fibroblast membrane were mixed at a mass ratio of 1:1 and ultrasonic (500 watts, 20 kilohertz, 5 minutes), and the membrane fusion was promoted by repeatedly extruding the polycarbonate porous membrane to obtain the hybrid cell membrane of activated macrophage / fibroblast.

[0056] S3, 1 mg / mL of small molecule prodrug nanoparticles Cap-tkCA-Cap NPs were mixed with 5 million hybrid membranes of activated macrophages and 5 million fibroblasts, and then ultrasonic (500 watts, 20 kilohertz, 5 minutes) was performed, and the nanomedicine Cap-tkCA-Cap@CM was obtained by extrusion through a polycarbonate porous membrane. The coating of the hybrid membrane was determined by gel electrophoresis, and the hydration particle size and surface charge value of the small molecule prodrug nanoparticles Cap-tkCA-Cap NPs and the nanomedicine Cap-tkCA-Cap@CM were determined by dynamic light scattering (DLS) and Zeta potential analyzer, and the particle size change was characterized by DLS at different times, and the results are shown in Figures 5 and 6. As shown in Figures 5 and 6, the small molecule prodrug can self-assemble into nanoparticles, and the nanomedicine Cap-tkCA-Cap@CM coated with the cell membrane has good stability. Transmission electron microscopy (TEM) was used to observe their morphological characteristics, and the TEM morphology of Cap-tkCA-Cap NPs and Cap-tkCA-Cap@CM is shown in Figure 7. As shown in Figure 7, the cell membrane is successfully coated on the surface of the nanoparticles.

[0057] Example 2, ROS-responsive drug release behavior of nanomedicine Cap-tkCA-Cap@CM in a simulated physiological environment

[0058] In the buffer solution, Cap-tkCA-Cap@CM was reacted with ROS (5 mM H2O2), and the drug release behavior of Cap and CA was characterized by HR-MS and high performance liquid chromatography (HPLC). The drug release kinetics data of the nano-drug Cap-tkCA-Cap@CM were obtained. First, Cap-tkCA-Cap@CM was dispersed in PBS at a concentration of 5 mg / mL, then H2O2 was added to make the concentration of H2O2 5 mM, then stirred at room temperature, and HR-MS test was carried out at 5 min, 15 min and 30 min, respectively. The results are shown in Figure 8. With the increase of time, the mass spectrometry peak intensity of Cap-tkCA-Cap gradually decreased, while the peak intensity of Cap and CA gradually increased, which fully proved that the nano-drug Cap-tkCA-Cap@CM released Cap and CA rapidly and without trace under the action of ROS. Second, Cap-tkCA-Cap@CM was dispersed in PBS at a concentration of 5 mg / mL, then H2O2 (5 mM) was added, and HPLC test was carried out at different time points (220 nm absorption wavelength to monitor the release of Cap, 291 nm absorption wavelength to monitor the release of CA). The results are shown in Figure 9. Cap-tkCA-Cap@CM has negligible release of Cap and CA without H2O2. When H2O2 is added, the release rate of Cap and CA is more than 70% within 10 min, and the release rate of Cap and CA is close to 95% at 30 min. This further proves that the nano-drug Cap-tkCA-Cap@CM has the performance of rapid response to release Cap and CA to ROS.

[0059] Example 3, Study on defibrillation and inhibition of metastasis and invasion of nano-drug Cap-tkCA-Cap@CM

[0060] (1) Evaluation of the defibrillation ability of Cap-tkCA-Cap@CM

[0061] The defibrillation ability of the biomimetic nanodrug was investigated by cell immunofluorescence staining: after the NIH3T3 cells were activated by incubating with 10 ng / mL of TGF-β1 for 24 hours, the cells were incubated with 5 mg / mL and 10 mg / mL of Cap-tkCA-Cap@CM, fixed with 4% formaldehyde for 15 minutes, then incubated with a-SMA, fibronectin (Fibronectin) primary antibody, and fluorescein-labeled secondary antibody, washed with PBS three times after removing the culture medium, incubated with cell core dye DAPI for 15 minutes, and subjected to fluorescence imaging. The results were compared with other different components (blank group, Cap, CA group), as shown in FIG. 10. After activation by TGF-β1, the fibrosis-related proteins a-SMA and Fibronectin were highly expressed. When Cap or CA was used for treatment, the cell fibrosis proteins decreased to a certain extent. When Cap-tkCA-Cap@CM was used for treatment at a concentration of 5 mg / mL, the cell fibrosis was significantly reduced. When the concentration of Cap-tkCA-Cap@CM was 10 mg / mL, the expression of a-SMA and Fibronectin in the cells was basically not observed, which proved that Cap-tkCA-Cap@CM had a significant defibrillation ability.

[0062] In addition, the expression of a-SMA, Fibronectin, and Col I in NIH3T3 treated with different concentrations of Cap-tkCA-Cap@CM (0.1 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL) was investigated by Western blotting (WB): the cells treated with the nanodrug were lysed, and the proteins were extracted from the cells. The proteins of each sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. After treating the membrane with T-TBS buffer containing 5% skim milk for 1 hour, the membrane was incubated with GAPDH (1:1000), a-SMA (1:5000), Col I (1:1000), and Fibronectin (1:1000) primary antibodies at 4°C overnight, and then incubated with secondary antibodies for 1 hour. Finally, the membrane was washed and imaged using a chemiluminescence detection kit. The results are shown in FIG. 11. As can be observed from FIG. 11, with the increase of the concentration of Cap-tkCA-Cap@CM, the expression of fibrosis proteins a-SMA, Fibronectin, and Col I decreased continuously. This further proved that the nanodrug Cap-tkCA-Cap@CM had an outstanding defibrillation ability.

[0063] (2) Investigation of the ability of Cap-tkCA-Cap@CM to inhibit the metastasis and invasion of pancreatic cancer cells

[0064] The ability of the biomimetic nanodrug to inhibit the metastasis and invasion of pancreatic cancer cells was evaluated by cell scratch test and Transwell invasion test.

[0065] Cell scratch test: After the pancreatic cancer cells (PANC-1) grew into a completely fused monolayer, a sterile pipette tip was used to make a linear scratch on the cell monolayer, and the cells were cultured with a culture medium containing Cap-tkCA-Cap@CM (10 mg / mL), and the component without the nanodrug was used as a control group. After 48 hours, the migration distance of the cells was observed and imaged, and the results are shown in Figure 12. From the results, it can be found that the scratch of the component without Cap-tkCA-Cap@CM has basically fused, while the growth of cells in the component incubated with the nanodrug Cap-tkCA-Cap@CM is basically not observed. This indicates that the nanodrug Cap-tkCA-Cap@CM can effectively inhibit the metastasis and invasion of PANC-1 cells.

[0066] Transwell invasion test: A chamber (8 μm) with Matrigel was used, and PANC-1 cells were added to the upper chamber, then serum-free medium containing Cap-tkCA-Cap@CM (10 mg / mL) was added, and complete medium containing 10% FBS was added to the lower chamber. After incubation for 24 hours, the cells on the surface of the membrane that were not infiltrated were removed by wiping, the infiltrated cells were fixed, stained with crystal violet, and imaged under a microscope by randomly selecting areas. The results are shown in Figure 13. From the figure, it can be found that when the nanodrug Cap-tkCA-Cap@CM is not used, PANC-1 cells rapidly metastasize and invade, while when the nanodrug Cap-tkCA-Cap@CM is used, very few PANC-1 cells can be observed, indicating that the nanodrug Cap-tkCA-Cap@CM has a significant inhibitory effect on the metastasis and invasion of PANC-1 cells.

[0067] Example 4, Application of Nanodrug Cap-tkCA-Cap@CM in Inhibiting Pancreatic Cancer Metastasis and in the Treatment of Pancreatic Cancer

[0068] (1) Study on the inhibition of pancreatic cancer metastasis by nanodrug Cap-tkCA-Cap@CM

[0069] Source of mice: Hangzhou Patsy Biological Technology Co., Ltd.; Variety: BALB / c mice, 20-25 g.

[0070] Select 6-8 week BALB / c mice, and inject PANC-1-luci cells (1 × 10 6 ) expressing luciferase and NIH3T3 cells (1 × 10 6) After mixing with Matrigel, it was injected into mice through the tail vein. One day later, the mice were administered Cap-tkCA-Cap@CM (10 mg / kg, once every two days, for a total of 5 times) through the tail vein. Ten minutes before imaging, the mice were injected with D-luciferin potassium salt (200 μL, 10 mg / mL). Bioluminescence imaging was performed to observe the metastasis of the tumor. PBS was injected as a control. The results are shown in FIG. 14. As shown in FIG. 14, the mice in the PBS group showed strong luminescence in the lungs, indicating that the pancreatic cancer cells had metastasized to the lungs. In sharp contrast to the PBS group, the mice in the nano-drug Cap-tkCA-Cap@CM group showed only very weak light, demonstrating that the nano-drug Cap-tkCA-Cap@CM can effectively inhibit the metastasis and invasion of pancreatic cancer cells in mice.

[0071] (3) Application of nano-drug Cap-tkCA-Cap@CM in the treatment of pancreatic cancer

[0072] On the basis of the aforementioned inhibition of pancreatic cancer defibering and metastasis, the nano-drug was explored for application in the treatment of pancreatic cancer by combining with the first-line chemotherapy drug for pancreatic cancer, gemcitabine (GEM). PANC-1 (1 × 10 6 ) and NIH3T3 cells (1 × 10 6 ) were mixed with Matrigel and inoculated subcutaneously into the right forelimb tissue of BALB / c nude mice to construct a subcutaneous tumor model. One week later, the mice were randomly divided into groups (PBS group, GEM group, Cap-tkCA-Cap@CM group, Cap-tkCA-Cap@CM + GEM group). The PBS group was injected intravenously with 200 μL of PBS, once every two days, from day 0 to day 12. The GEM group was injected intravenously with 15 mg / kg of GEM, once every two days, from day 6 to day 12. The Cap-tkCA-Cap@CM group was injected intravenously with 10 mg / kg of Cap-tkCA-Cap@CM, once every two days, from day 0 to day 12. The Cap-tkCA-Cap@CM + GEM group was injected intravenously with 10 mg / kg of Cap-tkCA-Cap@CM, once every two days, from day 0 to day 12, and 15 mg / kg of GEM, once every two days, from day 6 to day 12. The tumor volume was recorded every two days until day 24 to investigate the application effect of the nano-drug in the treatment of pancreatic cancer in mice. The results are shown in FIG. 15. The tumor of the mice was well inhibited after Cap-tkCA-Cap@CM administration for defibering and GEM administration for treatment, indicating that the nano-drug Cap-tkCA-Cap@CM has good effects in the treatment of pancreatic cancer.

[0073] To sum up, the present application realizes no excipient-related toxicity and high drug loading rate by self-assembly of small molecule prodrugs, realizes homologous targeting of fibrotic adhesion interstitial and receptor protein-mediated active targeting by coating of hybrid film, realizes massive, rapid and traceless drug release in fibrotic adhesion interstitial by response of prodrug molecules to ROS, and eliminates the self-limiting characteristics of responsive small molecule drug release; the released drugs Cap and CA can simultaneously inhibit fibrotic fibroin, and synergistically resolve fibrosis; with the progress of fibrosis resolution, Cap and CA released rapidly and tracelessly in the pancreatic cancer cells can simultaneously inhibit the metastasis and invasion of pancreatic cancer cells, and synergistically inhibit the occurrence of metastasis; the process of fibrosis resolution and the process of metastasis inhibition dynamically synergize, providing a new way for fibrosis resolution of pancreatic cancer treatment.

[0074] The technical scheme of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. A method for preparing a nanodrug that synergizes with the defibrosis and inhibits the metastasis of pancreatic cancer, characterized in that, The method comprises the following steps: S1, mixing trifluoroacetic acid, captopril Cap and cinnamaldehyde CA in a molar ratio of 1:1:1, under the catalysis of trifluoroacetic acid, the mercapto group of captopril Cap and the aldehyde group of cinnamaldehyde CA undergo condensation reaction, captopril Cap and cinnamaldehyde CA are connected through ROS-sensitive tk bond to obtain small molecule prodrug Cap-tkCA-Cap; and the small molecule prodrug Cap-tkCA-Cap is treated to obtain small molecule prodrug nanoparticles Cap-tkCA-Cap NPs; S2, preparing hybrid cell membranes of activated macrophages / fibroblasts; S3, using the obtained hybrid cell membranes of activated macrophages / fibroblasts to coat the small molecule prodrug nanoparticles Cap-tkCA-Cap NPs to obtain nano-drug Cap-tkCA-Cap@CM.

2. The method for preparing the nanomedicine synergistically defibrotic and pancreatic cancer metastasis inhibitor according to claim 1, characterized in that, In the step S1, the treatment of the small molecule prodrug Cap-tkCA-Cap is specifically: The small molecule prodrug Cap-tkCA-Cap is dissolved in dimethyl sulfoxide DMSO to obtain a mixed solution, the mixed solution is slowly dropped into deionized water under ultrasonic, and dialysis purification is carried out with a dialysis bag with a molecular weight cut-off of 1000 Da to obtain small molecule prodrug nanoparticles Cap-tkCA-Cap NPs.

3. The method for preparing the nanomedicine synergistically defibrotic and pancreatic cancer metastasis inhibitor according to claim 1, characterized in that, The step S2 comprises: S21, taking macrophage RAW 264.7 and incubating with 10 ng / mL of lipopolysaccharide LPS, then extracting the activated macrophage membrane by ultrasonic and centrifugation; S22, taking normal fibroblast NIH3T3 and incubating with 10 ng / mL of TGF-β1, then extracting the activated fibroblast membrane by ultrasonic and centrifugation; S23, mixing the activated macrophage membrane and the activated fibroblast membrane by ultrasonic at a mass ratio of 1:1, promoting membrane fusion by repeatedly extruding through a polycarbonate porous membrane to obtain a hybrid cell membrane of activated macrophages / fibroblasts.

4. The method of claim 1, wherein the preparation of the nanomedicine synergizing with the defiberization and the suppression of the pancreatic cancer metastasis is characterized by, The step S3 comprises: 1mg / mL of small molecule prodrug nanoparticles Cap-tkCA-Cap NPs is mixed with 5 million hybrid membranes of activated macrophages and 5 million fibroblasts after ultrasonic, and nano-drug Cap-tkCA-Cap@CM is obtained by extruding through a polycarbonate porous membrane.

5. The method for preparing the nanomedicine synergistically promoting defibrosis and inhibiting pancreatic cancer metastasis according to claim 3 or 4, characterized in that, The pore size of the polycarbonate porous membrane is 100-400 nm.

6. A nanodrug for synergistically resolving fibrosis and inhibiting pancreatic cancer metastasis, which is prepared by the method according to any one of claims 1-5.

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