Nano-drug co-delivery system for treating pancreatic cancer as well as preparation method and application of nano-drug co-delivery system

Abstract

The invention belongs to the field of biological medicine and nano-drugs, and particularly relates to a nano-drug co-delivery system for treating pancreatic cancer, a preparation method and application, the nano-drug co-delivery system is obtained by one-step self-assembly of poloxamer, 1, 2, 3, 4, 6-O-galloylglucose, NLG919 and docetaxel, and the poloxamer, 1, 2, 3, 4, 6-O-galloylglucose, NLG919 and docetaxel are in one-step self-assembly. The mass ratio of 6-O-galloylglucose to NLG919 to docetaxel is 5: 5: 3: 2, the NLG919 and docetaxel stably exist in a physiological environment and can be efficiently delivered to a tumor site, interaction between F-127 and PGG can be destroyed under a slightly acidic condition of a tumor microenvironment, release of NLG919 and DTX is triggered to play an anti-tumor function, potent chemical-immune synergistic treatment is achieved, and the NLG919 and the DTX can be used for treating tumors. The nano-drug delivery system provided by the invention is simple in preparation method and low in cost, and provides a new strategy for improving the treatment effect of pancreatic cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and nanomedicine, and specifically relates to a nanomedicine co-delivery system for treating pancreatic cancer, a preparation method and an application thereof. Background Art

[0002] Pancreatic cancer is a common malignant tumor of the digestive system. The main type is pancreatic ductal adenocarcinoma (PDAC). Its onset is insidious and its symptoms are atypical, which poses a challenge to early diagnosis. PDAC has a very poor prognosis, with a mortality rate ranking fourth among all malignant tumors and a 5-year survival rate of only 11%. It is expected to become the second leading cause of cancer death in the world by 2040. The causes of pancreatic cancer include smoking, poor eating habits, diabetes, chronic pancreatitis, and environmental factors such as long-term exposure to chemicals and long-term exposure to ionizing radiation may also be related to the occurrence of pancreatic cancer. Treatments for pancreatic cancer include surgical treatment, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. For patients with advanced or metastatic PDAC, chemotherapy is still the standard systemic treatment. Commonly used chemotherapy drugs for systemic chemotherapy include gemcitabine, albumin-bound paclitaxel, fluorouracil, etc., which are administered intravenously or orally. Although chemotherapy can reliably control tumors and stabilize clinical symptoms, its efficacy is limited and its toxicity is severe, often leading to adverse reactions such as nausea, vomiting, hair loss, and bone marrow suppression, thereby reducing patients' tolerance. Therefore, the development of new pancreatic cancer treatment strategies is an urgent clinical need.

[0003] Docetaxel (DTX) is a paclitaxel analogue and an important clinical treatment for pancreatic cancer. It is used in combination with gemcitabine and capecitabine to form a GTX neoadjuvant therapy regimen, which can achieve a remission rate of nearly 50% for solid pancreatic cancer tumors. As a cell mitotic microtubule inhibitor, docetaxel can cause G2 / M phase cell cycle arrest. In addition, DTX also has immunomodulatory functions, which can cause anti-tumor macrophage activation and promote immunogenic cell death (ICD) effects, expose tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), thereby activating dendritic cells (DCs), increasing T cell infiltration, and activating local tumor immunity. NLG919 is an indometamine-2,3-dioxygenase 1 (IDO1) inhibitor that can effectively reverse tumor immunosuppression caused by IDO1, help activate the host's immune system, and improve the efficacy of chemotherapy or other cancer therapies. Therefore, using DTX and NLG919 simultaneously to overcome the immunosuppressive TME of pancreatic cancer and achieve the synergistic effect of chemotherapy and immunotherapy is a two-pronged anti-tumor strategy.

[0004] The dense extracellular matrix of pancreatic cancer seriously hinders the distribution and perfusion of drugs, and the non-specific distribution of chemotherapy drugs can cause strong toxic side effects, which brings huge obstacles to the treatment of pancreatic cancer. The nano drug delivery system has the advantages of good biocompatibility and high targeting efficiency, and is a good choice for achieving targeted drug delivery and reducing toxic side effects.

[0005] The present invention uses a triblock copolymer of polyoxyethylene polyoxypropylene ether, poloxamer (Pluronic F-127, F-127) and PGG as building blocks, and constructs polyphenol-based nanoparticles - DTX + NLG919 @ F127-PGG (FPND) through a self-assembly strategy of bonding and hydrophobic interaction, thereby achieving co-delivery of NLG919 and DTX. The nanoparticles have a suitable size, uniform morphology and good stability, and show a potent pancreatic cancer therapeutic effect in the KPC pancreatic cancer mouse model. Summary of the invention

[0006] The primary purpose of the present invention is to provide a nano drug co-delivery system for treating pancreatic cancer, wherein the nano drug co-delivery system is obtained by one-step self-assembly of poloxamer, 1,2,3,4,6-O-galloyl glucose, NLG919 and docetaxel, and the mass ratio of the poloxamer, 1,2,3,4,6-O-galloyl glucose, NLG919 and docetaxel is 5:5:3:2.

[0007] Preferably, the poloxamer is poloxamer F127.

[0008] Preferably, the particle size of the nanoparticles in the nanodrug co-delivery system is 100-130 nm.

[0009] Preferably, the particle size of the nanoparticles in the nanodrug co-delivery system is 118 nm.

[0010] The second object of the present invention is to provide a method for preparing the nano drug co-delivery system, comprising the following steps:

[0011] (1) weighing poloxamer F-127, 1,2,3,4,6-O-galloyl glucose, NLG919 and docetaxel according to the mass ratio, and dissolving poloxamer F-127 and 1,2,3,4,6-O-galloyl glucose in an organic solvent to prepare a solution;

[0012] (2) mixing the poloxamer F-127 solution and 1,2,3,4,6-O-galloyl glucose solution prepared in step (1), stirring magnetically for 1 h, adding NLG919 and docetaxel powder, and stirring magnetically for 4 h;

[0013] (3) The mixed solution was transferred to a dialysis bag, dialyzed against deionized water to complete self-assembly, and freeze-dried to obtain DTX+NLG919@F127-PGG.

[0014] Preferably, the organic solvent in step (1) is dimethyl sulfoxide.

[0015] Preferably, the molecular weight cut-off of the dialysis bag in step (3) is 1000.

[0016] The third object of the present invention is to provide the use of the nano drug co-delivery system or the nano drug co-delivery system prepared according to the preparation method in the preparation of drugs or medical products for treating pancreatic cancer.

[0017] Preferably, the treatment method is chemo-immunotherapy.

[0018] Beneficial effects of the present invention: The nano drug co-delivery system prepared by the present invention self-assembles poloxamer and natural polyphenol 1,2,3,4,6-O-pentagalloylglucose through hydrogen bonding and hydrophobic interactions, and encapsulates NLG919 and docetaxel to form nanoparticles with a core-shell structure. The nano drug co-delivery system is stable under physiological conditions and can be efficiently delivered to the tumor site. Under the slightly acidic conditions of the tumor microenvironment, it will destroy the interaction between F-127 and PGG, triggering the release of NLG919 and DTX to exert anti-tumor function and achieve a strong chemical-immunotherapy synergistic treatment. The nano drug delivery system prepared by the scheme of the present invention has a simple preparation method and low cost, and provides a new strategy for improving the therapeutic effect of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below in conjunction with the accompanying drawings, in which:

[0020] Figure 1 Transmission electron microscopy image of the nano drug delivery system FPND

[0021] Figure 2 Hydrodynamic diameter of the nano drug delivery system FPND

[0022] Figure 3 H NMR spectrum of the nano drug delivery system FPND

[0023] Figure 4 Stability testing of nano drug delivery system FPND

[0024] Figure 5 Standard curves of DTX and NLG919 determined by HPLC

[0025] Figure 6 Cytotoxicity of the nano-drug delivery system FPND against mouse pancreatic cancer cells PANC02

[0026] Figure 7 Tumor volume changes in KPC homograft model after treatment with the nano drug delivery system FPND Note: Figure 7 A is the general picture of tumor tissue of mice in each group, B is the tumor growth of mice in each group, C is the weight of tumor tissue of mice in each group, and D is the weight change of mice in each group. DETAILED DESCRIPTION

[0027] The embodiments of the present invention will be described in detail below in conjunction with the examples to fully understand the purpose, features and effects of the present invention. The described embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Based on the embodiments of the present invention, other implementation benefits obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention.

[0028] In the following examples, unless otherwise specified, all reagents and consumables used can be obtained through commercial channels.

[0029] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods.

[0030] In the following examples, docetaxel (Docetaxel, DTX) is a paclitaxel analogue and an important clinical therapeutic drug for pancreatic cancer.

[0031] In the following embodiments, the 1,2,3,4,6-O-pentagalloylglucose (PGG) is a gallic tannin present in plants, has the characteristics of natural polyphenols and polyhydroxy groups, and can self-assemble with different materials or carriers through hydrogen bonds, hydrophilic and hydrophobic interactions, and metal coordination to form a nano-assembly with good biocompatibility.

[0032] In the following examples, the poloxamer is poloxamer F-127.

[0033] Example 1: A co-delivered nanodrug

[0034] The specific preparation process is:

[0035] (1) F-127, PGG, NLG919 and DTX were weighed according to the mass ratio (F-127: PGG: NLG919: DTX = 5:5:3:2), and F-127 and PGG were dissolved in 5 mL of DMSO to prepare solutions with a concentration of 10 mg / mL;

[0036] (2) The two solutions prepared in step (1) were mixed and magnetically stirred for 1 h, followed by addition of NLG919 and DTX powders and magnetic stirring for 4 h;

[0037] (3) The mixed solution of step (2) was transferred to a dialysis bag (molecular weight cutoff of 1000), dialyzed against deionized water for 72 h to complete self-assembly, the dialysate was collected, and freeze-dried for 48 h to obtain white FPND nanoparticles.

[0038] Example 2: Performance of FPND Nano-drug Delivery System

[0039] 1. Particle size and morphology

[0040] The morphology of the nano drug delivery system FPND was detected by transmission electron microscopy. The specific steps are as follows:

[0041] 10 μL of the prepared FPND nano drug delivery system dialysate was taken and dropped onto a copper grid for transmission electron microscopy and dried overnight. After the copper grid was dry, a transmission electron microscope was used to observe and take pictures.

[0042] Test results see Figure 1 It can be seen that F-127, PGG, NLG919 and DTX can self-assemble to obtain a core-shell structured nano drug delivery system at this feed ratio. The nanostructure is spherical, with uniform particle size and good dispersibility.

[0043] 2. Determination of hydrodynamic diameter

[0044] Dynamic light scattering was used to measure the hydrodynamic diameter of the FPND nano drug delivery system in water. Figure 2 The hydrodynamic diameter of the FPND nanodrug delivery system is approximately 118 nm, and this smaller diameter can achieve enrichment at the tumor site through the EPR effect.

[0045] 3. Structural identification by H NMR

[0046] The FPND nano drug delivery system was fully dissolved in deuterated DMSO and detected by nuclear magnetic resonance spectrometer. The results are shown in Figure 3 In the H NMR spectrum of FPND, the characteristic phenolic hydroxyl signal of PGG (δ=6.8-7.05 ppm), the proton signal of NLG919 (δ=7.42-7.46 ppm) and the proton signal of DTX (δ=4.5 ppm) were detected, indicating that PGG, NLG919 and DTX were successfully loaded into FPND.

[0047] 4. Stability test

[0048] (1) Prepare DMEM medium (pH=7.4) containing 10% fetal bovine serum (FBS) and acetate buffer solution at pH=5.0.

[0049] (2) 100 μL of the prepared FPND nano drug delivery system dialysate was dripped into 3 mL of DMEM medium (pH=7.4) containing 10% FBS and acetate buffer solution (pH=5.0), and then dispersed evenly by ultrasonication for 10 min.

[0050] (3) For the 10% FBS DMEM medium (pH = 7.4) system, the particle size distribution on days 0, 2, 4, 6, and 8 was measured using a dynamic light scattering instrument; for the pH = 5.0 acetate buffer solution system, the particle size distribution on days 0, 1, 2, 3, and 4 was measured using a dynamic light scattering instrument.

[0051] Depend on Figure 4The results show that after being placed in the 10% FBS DMEM medium (pH = 7.4) system for 8 days, the average particle size of the FPND nano drug delivery system only changed slightly, from 118nm to 167nm, indicating that the FPND nano drug delivery system has good stability in this system. After being placed in the pH = 5.0 acetate buffer solution for 4 days, the average particle size of the FPND nano drug delivery system increased from 118nm to 2186nm, indicating that FPND has significant acid sensitivity.

[0052] 5. Determination of drug content

[0053] (1) Accurately weigh 8 mg of DTX and NLG919, respectively, and dissolve them in DMSO to prepare DTX and NLG919 standard solutions of different concentrations (0.0625, 0.125, 0.25, 0.5, 1, 2 mg / mL).

[0054] (2) High performance liquid chromatography was used to detect the peak areas of different concentrations of DTX and NLG919 to prepare a standard curve. The detection wavelength was 254 nm (see Figure 5 ).

[0055] (3) Accurately weigh 2 mg of the FPND nano drug delivery system, dissolve it in DMSO, and prepare a 1 mg / mL solution. Use high performance liquid chromatography to detect the peak areas of DTX and NLG919, and calculate the content of the two drugs in the FPND nano drug delivery system through the standard curve. The drug content of DTX is 5.7%, and the drug content of NLG919 is 10.1%.

[0056] Example 3: Cytotoxicity detection of FPND nano-drug delivery system

[0057] The in vitro cytotoxicity of the nanodrug delivery system FPND on the pancreatic cancer cell line panc02 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) was evaluated by the CCK-8 method.

[0058] Panc02 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. 3 The cells were inoculated at a density of 100 μg / well in a 96-well plate and cultured for 24 h. When the cells adhered to the wall and grew well, different concentrations of FPND nano drug delivery system and free DTX (the equivalent concentration gradient of DTX was: 0, 0.1, 1, 5, 10, 50, 100, 500 ng / mL) were added. After 48 h of continuous culture, the drug effect was terminated, and the cell viability was detected using CCK-8, and the absorbance at 450 nm was measured using an enzyme marker.

[0059] Test results see Figure 6The cell viability of panc02 cells gradually decreased with the increase of DTX equivalent concentration. At the same time, when the DTX equivalent concentration was the same, FPND had a stronger cell killing ability than DTX alone. The IC50 value of FPND in panc02 cells was 2.8 times lower than that of free DTX.

[0060] Example 4: Evaluation of the in vivo anti-tumor activity of the FPND nano-drug delivery system

[0061] 1. Animal Experiments

[0062] (1) Animal preparation: 6-week-old C57BL / 6JGpt mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were adaptively housed in an SPF animal center for one week before the experiment.

[0063] (2) Preparation of spontaneous pancreatic cancer mouse tumor tissue: Fresh spontaneous pancreatic cancer mouse tumor tissue was obtained and washed twice in PBS containing a 1% penicillin-streptomycin mixture. The tissue was then placed in DMEM culture medium containing a 1% glutamine, 1% penicillin-streptomycin mixture and 10% FBS, cut into tissue blocks with a diameter of approximately 3 mm, and placed in a high-concentration matrix gel for mixing. All operations were completed in an ultra-clean bench.

[0064] (3) Construction of spontaneous pancreatic cancer allograft model in mice: C57BL / 6JGpt mice were captured and fully anesthetized with isoflurane. The axillary area was disinfected with 75% alcohol, and a surgical incision of approximately 0.5 cm was made with ophthalmic scissors. The tumor tissue wrapped with matrix gel was implanted subcutaneously in the mouse axilla, sutured with absorbable antibacterial sutures, and placed back in the mouse cage. After the mouse fully woke up, it was transferred to the cage rack.

[0065] (4) Grouping: Observe the mice every two days to measure tumor size and body weight. 3 At the time of treatment, the mice were randomly divided into 6 groups: control group, F127-PGG (FP) group, NLG919 group, DTX group, DTX+NLG919 (D+N) group, and FPND group, with 6 mice in each group;

[0066] (5) Dosage regimen: The equivalent dose of DTX in each treatment group containing DTX was 5 mg / kg, the dose of NLG919 was 8.9 mg / kg, and the dose of F127-PGG was 73.86 mg / kg. The frequency of administration was 3 times / week (once every two days), and the route of administration was tail vein injection.

[0067] (6) Tumor volume and body weight detection: The long and short diameters of the tumor were measured every two days, and the weight of the mice was recorded. The tumor volume was calculated by the following formula: Tumor volume (mm 3 )=(length×width 2 ) / 2.

[0068] (7) Specimen collection: The volume of subcutaneous tumor of mice reached 1500mm 3 The time was regarded as the merciful endpoint, and the mice were euthanized by overdose of anesthesia, and the tumors were carefully dissected out, weighed, and photographed for record.

[0069] like Figure 7 As shown, Figure 7 A is a general picture of the tumor tissue of mice in each group. It can be seen that the tumor volume of mice in the FPND group using the nano drug delivery system prepared in Example 1 is the smallest; Figure 7 The tumor volume growth curve in B shows that the growth rate of the tumor volume of mice in the FPND group using the nano drug delivery system prepared in Example 1 was significantly lower than that of the other groups, and the tumor volume was also smaller; Figure 7 C shows that the tumor weight of mice in the FPND group using the nano drug delivery system prepared in Example 1 was significantly lower than that in other groups. The statistical results of the weight changes of mice in each group are as follows Figure 7 As shown in D, the body weights of mice in the control group, FP group, NLG919 group and nanodrug delivery system FPND group were relatively stable without significant increase, while the body weights of mice in the DTX group and D+N group decreased after treatment. The above results indicate that nanodrug delivery system FPND treatment can significantly inhibit tumor progression in spontaneous pancreatic cancer mice and has significant in vivo antitumor activity.

[0070] In summary, the nano drug delivery system prepared by the scheme of the present invention forms a core-shell structure by self-assembly of poloxamer and natural polyphenol 1,2,3,4,6-O-pentagalloylglucose through hydrogen bonding and hydrophobic interaction to jointly load IDO1 inhibitor NLG919 and ICD inducer docetaxel to form a nano drug delivery system. The nano drug delivery system is stable under physiological conditions and can efficiently reach the tumor site. The slightly acidic environment of the tumor tissue will destroy the interaction between F127 and 1,2,3,4,6-O-pentagalloylglucose, triggering the release of NLG919 and DTX, achieving enhanced pancreatic cancer chemo-immunotherapy, effectively inhibiting the progression of pancreatic cancer, and exerting a strong anti-tumor activity. The nano drug delivery system prepared by the scheme of the present invention has a simple preparation method and low cost, and provides a new solution for improving the chemo-immunotherapy effect of pancreatic cancer.

[0071] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A nano drug co-delivery system for treating pancreatic cancer, characterized in that: The nano drug co-delivery system is obtained by one-step self-assembly of poloxamer, 1,2,3,4,6-O-galloyl glucose, NLG919 and docetaxel, and the mass ratio of poloxamer, 1,2,3,4,6-O-galloyl glucose, NLG919 and docetaxel is 5:5:3:

2.

2. The nano drug co-delivery system according to claim 1, characterized in that: The poloxamer is poloxamer F127.

3. The nano drug co-delivery system according to claim 1, characterized in that: The particle size of the nanoparticles in the nano-drug co-delivery system is 100-130 nm.

4. The nano drug co-delivery system according to claim 1, characterized in that: The particle size of the nanoparticles in the nano-drug co-delivery system is 118 nm.

5. The method for preparing the nano drug co-delivery system according to claim 1, characterized in that: The steps include: (1) weighing poloxamer F-127, 1,2,3,4,6-O-galloyl glucose, NLG919 and docetaxel according to the mass ratio, and dissolving poloxamer F-127 and 1,2,3,4,6-O-galloyl glucose in an organic solvent to prepare a solution; (2) mixing the poloxamer F-127 solution and 1,2,3,4,6-O-galloyl glucose solution prepared in step (1), stirring magnetically for 1 h, adding NLG919 and docetaxel powder, and stirring magnetically for 4 h; (3) The mixed solution was transferred to a dialysis bag, dialyzed against deionized water to complete self-assembly, and freeze-dried to obtain DTX+NLG919@F127-PGG.

6. The method for preparing the nano drug co-delivery system according to claim 5, characterized in that: The organic solvent in step (1) is dimethyl sulfoxide.

7. The method for preparing the nano drug co-delivery system according to claim 5, characterized in that: The molecular weight cut-off of the dialysis bag described in step (3) is 1000.

8. Use of the nano drug co-delivery system according to any one of claims 1 to 4 or the nano drug co-delivery system prepared by the preparation method according to any one of claims 5 to 7 in the preparation of drugs or medical products for treating pancreatic cancer.

9. The use according to claim 8, characterized in that The treatment method is combined chemotherapy.

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