CXCR4 inhibitory polymers for delivery of nucleic acid drugs and uses thereof

Abstract

The present invention provides CXCR4 inhibitory polymers for delivery of nucleic acid drugs and uses thereof. Specifically, the invention provides an application of an active component combination in preparation of a pharmaceutical composition or a kit for treating tumors or inhibiting tumor cells, and the active component combination comprises: (a) a first active component: a nucleic acid drug targeting RRM2; and (b) a second active ingredient: gemcitabine. The nucleic acid drug nanoparticles containing the CXCR4 inhibitory polymer can effectively inhibit the growth and metastasis of pancreatic cancer. In addition, when the nucleic acid drug nanoparticles provided by the invention are combined with gemcitabine, pancreatic cancer, especially drug-resistant or refractory pancreatic cancer, can be synergistically treated.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, in particular to a CXCR4 inhibitory polymer for delivering nucleic acid drugs and an application thereof. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor that is usually diagnosed at an advanced stage. It is characterized by a high metastatic rate and resistance to many therapeutic drugs. Due to the complex tumor microenvironment and the incompletely understood pathological mechanisms, traditional monotherapy strategies often fail to provide effective treatment options. Recently, immunotherapy and gene therapy have become cutting-edge therapies with broad prospects and great potential.

[0003] Therefore, there is an urgent need in the art to develop a nano-delivery system that can reduce chemotherapy resistance, inhibit tumor metastasis and growth, and thus effectively treat refractory pancreatic cancer. Summary of the invention

[0004] The purpose of the present invention is to provide a nano-delivery system that reduces chemotherapy resistance, inhibits tumor metastasis and growth, and thus effectively treats refractory pancreatic cancer.

[0005] In a first aspect of the present invention, there is provided a use of an active ingredient combination for preparing a pharmaceutical composition or a drug kit for treating tumors or inhibiting tumor cells, wherein the active ingredient combination comprises:

[0006] (a) a first active ingredient, which is a nucleic acid drug targeting RRM2; and (b) a second active ingredient, gemcitabine.

[0007] In another preferred embodiment, the first active ingredient is in the form of nucleic acid drug nanoparticles.

[0008] In another preferred embodiment, the nucleic acid drug targeting RRM2 is an antisense oligonucleotide (ASO) or siRNA drug targeting RRM2.

[0009] In another preferred embodiment, the nucleic acid drug includes siRNA.

[0010] In another preferred embodiment, the nucleic acid drug is siRNA targeting RRM2.

[0011] In another preferred embodiment, the siRNA contains a reverse complementary sense strand and an antisense strand, and the nucleotide sequence of the sense strand is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0012] In another preferred embodiment, the first active ingredient is a nucleic acid drug nanoparticle containing an antisense oligonucleic acid (ASO) targeting RRM2.

[0013] In another preferred embodiment, the molar ratio of the nucleic acid drug to gemcitabine is 1:20 to 20:1, preferably 1:10 to 10:1, more preferably 1:5 to 5:1, and most preferably 1:2 to 2:1.

[0014] In another preferred embodiment, the weight ratio of the nucleic acid drug to gemcitabine is 1:50 to 10:1, preferably 1:20 to 5:1, more preferably 1:15 to 2:1, and most preferably 1:10 to 1:1 or 1:5 to 1:1.

[0015] In another preferred embodiment, the tumor includes pancreatic cancer, preferably pancreatic ductal adenocarcinoma.

[0016] In another preferred embodiment, the tumor cells include pancreatic cancer cells, preferably pancreatic ductal adenocarcinoma cells.

[0017] In another preferred embodiment, the tumor cells include: PANC 02 cells and PANC-1 cells.

[0018] In another preferred embodiment, the pancreatic cancer is selected from the following group: drug-resistant pancreatic cancer, or refractory pancreatic cancer, or a combination thereof.

[0019] In another preferred embodiment, the pharmaceutical composition or drug kit is used to treat or is administered to a mammal, and more preferably the mammal is a rodent (such as a mouse, a rat) or a human.

[0020] In another preferred embodiment, the particle size (hydrodynamic diameter) of the nucleic acid drug nanoparticles is about 70-110 nm, preferably 80-100 nm, and more preferably 80-95 nm.

[0021] In another preferred embodiment, the surface potential (Zeta potential) of the nucleic acid drug nanoparticles is 6-10 mV, preferably 8-9.5 mV.

[0022] In another preferred example, the nucleic acid drug nanoparticles include a polymer and a nucleic acid drug.

[0023] In another preferred embodiment, the nucleic acid drug nanoparticles include a polymer, a nucleic acid drug and LNP (or a component of LNP).

[0024] In another preferred example, the nucleic acid drug nanoparticles include LNP and nucleic acid drugs.

[0025] In another preferred embodiment, the polymer has a polymerization unit represented by the following formula I:

[0026]

[0027] In another preferred embodiment, the polymer is a polymer obtained by reacting a first unit represented by formula II with a second unit represented by formula III.

[0028]

[0029]

[0030] In another preferred embodiment, the reaction is a Michael-type addition polymerization reaction.

[0031] In another preferred embodiment, the polymer is a CXCR4 inhibitory polymer.

[0032] In another preferred embodiment, the mass ratio of the polymer to the nucleic acid drug is 0.1-10:1, preferably 0.5-8:1, and more preferably 2-4:1.

[0033] In another preferred embodiment, the nucleic acid drug nanoparticles also include liposomes.

[0034] In another preferred embodiment, the liposomes include cationic liposomes (such as SM102), phospholipid DSPC, cholesterol and PEGylated lipids (such as cationic liposomes DMG-PEG2000).

[0035] In another preferred embodiment, the liposomes include or consist of: SM102, phospholipid DSPC, cholesterol and DMG-PEG2000 in a molar ratio of about 40:10-11:48-49:1-2.

[0036] In another preferred embodiment, in the nucleic acid drug nanoparticles, the weight ratio of the polymer to the liposome is 1:1500-1:2000, preferably 1:1500-1:1800, and more preferably 1:1600-1:1700.

[0037] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising a nucleic acid drug targeting RRM2, gemcitabine and a pharmaceutically acceptable carrier.

[0038] In another preferred embodiment, the nucleic acid drug targeting RRM2 is in the form of nucleic acid drug nanoparticles.

[0039] In another preferred embodiment, the nucleic acid drug nanoparticles include a polymer formed by mixing a polymer and a nucleic acid drug.

[0040] In another preferred embodiment, the polymer has a polymerization unit represented by the following formula I:

[0041]

[0042] In another preferred embodiment, the polymer is a CXCR4 inhibitory polymer.

[0043] In another preferred embodiment, the nucleic acid drug includes siRNA or ASO drug.

[0044] In another preferred embodiment, the nucleic acid drug is a siRNA or ASO drug targeting RRM2.

[0045] In another preferred embodiment, the siRNA contains a reverse complementary sense strand and an antisense strand, and the nucleotide sequence of the sense strand is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0046] In another preferred embodiment, the nucleic acid drug nanoparticles also include liposomes.

[0047] In another preferred embodiment, the pharmaceutical composition further comprises additional therapeutic drugs (such as anti-tumor agents).

[0048] In another preferred embodiment, the pharmaceutical composition further comprises a checkpoint inhibitor, CAR-T cells, or a combination thereof.

[0049] In another preferred embodiment, the pharmaceutical composition can significantly reduce the Ki67 index in pancreatic cancer cells.

[0050] In a third aspect of the present invention, there is provided an active ingredient combination, the active ingredient combination comprising:

[0051] (a) a first active ingredient, which is a nucleic acid drug targeting RRM2; and (b) a second active ingredient, gemcitabine.

[0052] In another preferred embodiment, the active ingredient combination consists of (a) a first active ingredient and (b) a second active ingredient.

[0053] In another preferred embodiment, the first active ingredient is in the form of nucleic acid drug nanoparticles.

[0054] In another preferred embodiment, the active ingredient combination consists of nucleic acid drug nanoparticles and gemcitabine.

[0055] In another preferred embodiment, the nucleic acid drug nanoparticles include a polymer having a polymer unit represented by the following formula I and a polymer formed by mixing a nucleic acid drug,

[0056]

[0057] In another preferred embodiment, the nucleic acid drug is a siRNA or ASO drug targeting RRM2.

[0058] In another preferred embodiment, the siRNA contains a reverse complementary sense strand and an antisense strand, and the nucleotide sequence of the sense strand is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0059] In another preferred embodiment, the active ingredient combination can significantly reduce the Ki67 index in pancreatic cancer cells.

[0060] In a fourth aspect of the present invention, there is provided a method for preparing the active ingredient combination according to the third aspect of the present invention, comprising the following steps:

[0061] (i) mixing a polymer having a polymer unit represented by the following formula I with a nucleic acid drug to obtain a first complex (or a first mixture):

[0062]

[0063] (ii) encapsulating the first complex to form a liposome loaded with the first complex.

[0064] In another preferred embodiment, the encapsulation treatment includes mixing the first complex with a raw material constituting liposomes, thereby forming liposomes loaded with the first complex.

[0065] In another preferred embodiment, the liposome loaded with the first complex is the nucleic acid drug nanoparticle.

[0066] In another preferred embodiment, in step (i), mixing is performed under the following conditions: temperature is 20° C.-30° C., and / or time is 10 min-30 min.

[0067] In another preferred embodiment, in step (i), the mass ratio of the polymer to the nucleic acid drug is 0.1-10:1, preferably 0.5-8:1, and more preferably 2-4:1.

[0068] In another preferred embodiment, the nucleic acid drug includes siRNA or ASO drug.

[0069] In another preferred embodiment, the nucleic acid drug is a siRNA or ASO drug targeting RRM2.

[0070] In another preferred embodiment, the siRNA contains a reverse complementary sense strand and an antisense strand, and the nucleotide sequence of the sense strand is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0071] In another preferred embodiment, the liposome raw materials include: cationic liposomes (such as SM102), phospholipid DSPC, cholesterol and PEGylated lipids (such as cationic liposomes DMG-PEG2000).

[0072] In another preferred embodiment, in step (ii), the liposome comprises SM102, phospholipid DSPC, cholesterol and DMG-PEG2000 in a molar ratio of 40:10-11:48-49:1-2.

[0073] In another preferred embodiment, step (ii) further comprises: the polymer is diluted with HEPES buffer to a concentration of 6-10 μg / ml, and the liposome is diluted with ethanol to a concentration of 3-5 mg / ml; the volume ratio of the polymer to the liposome is 1:2-5, preferably 1:2-4, and more preferably 1:3.

[0074] In another preferred embodiment, the conditions of the mixed reaction II include: temperature of 20°C-30°C, and time of 10min-30min.

[0075] In another preferred embodiment, in step (ii), the weight ratio of the polymer to the liposome is 1:1500-2000, preferably 1:1500-1800, and more preferably 1:1600-1700.

[0076] In another preferred embodiment, the polymer is prepared by a method comprising the following steps:

[0077] (a) mixing the compound represented by formula (II) and the compound represented by formula (III) in the presence of a reaction solvent I to carry out a Michael type addition polymerization reaction;

[0078]

[0079] In another preferred embodiment, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) is 1:0.8-1.2, preferably 1:0.9-1.1; more preferably 1:1.

[0080] In another preferred embodiment, the reaction solvent I is a methanol solution.

[0081] In another preferred embodiment, the compound represented by formula (II) is AMD3100, and its CAS number is 110078-46-1.

[0082] In another preferred embodiment, the compound represented by formula (III) is hexamethylenebisacrylamide, and its CAS number is 7150-41-6.

[0083] In another preferred embodiment, the reaction conditions of the polymerization reaction include: reacting at a temperature of 30-40° C. (preferably 37° C.) and a nitrogen environment for 1-4 days, preferably 2-3 days, and more preferably 2 days.

[0084] In another preferred embodiment, the method further comprises step (b): after 2 days of polymerization, adding additional AMD3100, and reacting for another 10-24 hours, preferably 10-18 hours, and more preferably 12 hours.

[0085] In another preferred embodiment, the method further comprises step (c): dialyzing the reaction solution obtained in (b) to obtain dialysate I, and freeze-drying the dialysate I to obtain the polymer.

[0086] In another preferred embodiment, the dialysis process comprises: dialysis with methanol for 1-3 days; and then dialysis with water at pH 4.0 for 1-3 days, preferably 1-2 days, and more preferably 2 days.

[0087] In a fifth aspect of the present invention, a medicine kit is provided, comprising:

[0088] (a) a first preparation, wherein the first preparation contains nucleic acid nanoparticles of a nucleic acid drug targeting RRM2 and a pharmaceutically acceptable carrier;

[0089] (b) a second formulation comprising gemcitabine and a pharmaceutically acceptable carrier;

[0090] (c) instructions describing a method for treating tumors by combining nucleic acid drug nanoparticles and gemcitabine.

[0091] In another preferred embodiment, the first preparation and the second preparation are independent of each other.

[0092] In another preferred embodiment, the first preparation and the second preparation are freeze-dried preparations or liquid preparations.

[0093] In another preferred embodiment, the first preparation and the second preparation are injections.

[0094] In another preferred embodiment, the first preparation is administered before, during or after the administration of the second preparation.

[0095] In another preferred embodiment, the nucleic acid drug nanoparticles include a polymer having a polymer unit represented by the following formula I and a polymer formed by mixing a nucleic acid drug,

[0096]

[0097] In another preferred embodiment, the nucleic acid drug is a siRNA or ASO drug targeting RRM2.

[0098] In another preferred embodiment, the siRNA contains a reverse complementary sense strand and an antisense strand, and the nucleotide sequence of the sense strand is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0099] In a sixth aspect of the present invention, there is provided an in vitro non-therapeutic method for inhibiting tumor cell growth, comprising the steps of:

[0100] Tumor cells are cultured in the presence of the active ingredient combination described in the third aspect of the present invention, thereby inhibiting the growth of tumor cells.

[0101] In another preferred embodiment, the tumor cells include pancreatic cancer cells.

[0102] In another preferred embodiment, the method comprises: mixing and culturing tumor cells with nucleic acid drug nanoparticles and gemcitabine.

[0103] In another preferred embodiment, the nucleic acid drug nanoparticles and gemcitabine can be added to the tumor cells simultaneously or sequentially.

[0104] In another preferred embodiment, the tumor cells include tumor cells in the logarithmic growth phase.

[0105] In another preferred embodiment, the number of tumor cells is 10 3 -10 8 Pieces / ml.

[0106] In another preferred embodiment, the culture time is 0.1-120 hours, preferably 1-96 hours.

[0107] In another preferred embodiment, the final molar concentration of the nucleic acid drug nanoparticles in the mixed culture medium is 50-1000 nM, preferably 80-500 nM, and more preferably about 100 nM.

[0108] In another preferred embodiment, the final molar concentration of gemcitabine in the mixed culture medium is 10-200 nM, preferably 25-150 nM, more preferably 50-100 nM, such as about 75 nM.

[0109] In a seventh aspect of the present invention, there is provided an in vitro non-therapeutic method for regulating immune cell function, comprising the steps of:

[0110] In the presence of the pharmaceutical composition of the second aspect of the present invention or the combination of active ingredients of the third aspect of the present invention, T cells are cultured to make CD8 + T cells and CD4 + The proportion of T cells increases, thereby regulating the function of immune cells.

[0111] In an eighth aspect of the present invention, a method for treating pancreatic cancer is provided, comprising the steps of:

[0112] A safe and effective amount of the pharmaceutical composition described in the second aspect of the present invention or the active ingredient combination described in the third aspect of the present invention is administered to a subject in need.

[0113] In another preferred embodiment, the method comprises administering the nucleic acid drug nanoparticles and gemcitabine in the pharmaceutical composition described in the second aspect of the present invention or the active ingredient combination described in the third aspect of the present invention to a subject in need simultaneously or selectively.

[0114] In another preferred embodiment, the administration includes oral administration or intravenous injection.

[0115] In another preferred embodiment, the subject is a human or non-human mammal.

[0116] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0117] In another preferred embodiment, the pancreatic cancer includes pancreatic ductal carcinoma and refractory pancreatic cancer.

[0118] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 Shown are the synthesis equation of PAMD in the embodiments of the present invention and its detection results, wherein Figure A is the synthesis equation of PAMD; Figure B is the NMR result of PAMD after synthesis; Figure C is the result of evaluating the ability of PAMD to form nanoparticles and encapsulate siRNA using agarose gel electrophoresis, wherein w / w represents the weight ratio of PAMD to siRNA, for example 0.2 represents a weight ratio of PAMD to siRNA of 0.2:1, and siRNA refers to siRNA alone, which refers to siRNA alone without PAMD.

[0120] Figure 2The figure shows the preparation and characterization results of nanoparticles. Among them, (A) is a schematic diagram of the structure of the nanomaterial LNP-PAMD / siRNA, PAMD / siRNA or LNP / siRNA, in which the yellow spherical structure represents LNP, the purple linear structure represents PAMD, and the blue and red linear structures represent siRNA; (B) characterizes the size and potential of the nanomaterial; (C) dynamic light scattering measurement results, proving that the nanoparticles are relatively uniformly distributed; (D) electron microscopy photos of LNP-PAMD / siRNA nanoparticles; (E) gel electrophoresis detection of single siRNA, nanoparticles (such as LNP-PAMD / siRNA, PAMD / siRNA or LNP / siRNA) with polymer (PAMD) and / or lipid (LNP) protecting siRNA in a simulated in vivo environment. The degradation rate or maintenance time results are shown.

[0121] Figure 3 The toxicity test results of the synthesized nanomaterials are shown in Figure 1. (A) The toxicity of PAMD / siRNA, LNP-PAMD / siRNA and gemcitabine (GEM) to PANC 02 and PANC-1 cells was tested using CCK8. The test results showed that the synthesized materials had low toxicity, lower than the toxicity of GEM; (B) IC50 results proved that both PANC 02 and PANC-1 cell lines had developed resistance to GEM.

[0122] Figure 4 The results show the inhibitory effect of compound PAMD on pancreatic cancer cells. (A) shows that the synthesized PAMD still retains the tumor metastasis inhibition property of AMD3100 inhibitor; (B) shows the results of Western Blot experiment, which shows that the synthesized nanoparticles (LNP-PAMD / siNC, PAMD / siNC) and the final product after assembly (LNP-PAMD / siRRM2) retain the property of AMD3100 to inhibit the Akt pathway.

[0123] Figure 5 The results show the absorption and internalization efficiency of nanoparticles by pancreatic cancer cell lines. (A) is the result of immunofluorescence detection, and (B) is the result of flow cytometry detection. The results show that compared with the individual materials (PAMD / siRNA, LNP / siRNA, siRNA Only), the assembled final product LNP-PAMD / siRNA can enter cells and show the same effect as the commercially available drug Lipofectamine 3000.

[0124] Figure 6The data show that the synthetic nanomaterial combined with GEM has the property of inhibiting cell growth at the cellular level. (A) The synthetic material LNP-PAMD / siRRM2 can effectively knock down the target molecule RRM2, and the knockdown efficiency is even much higher than the commercially available Lipo3000 (LIPO for short); (B) The CI is lower than 1, and the smaller the value, the better the synergistic effect between the drugs. The data prove that the nano drug LNP-PAMD / siRRM2 has a good synergistic effect with GEM; (C) CCK8 once again proves the high efficiency of the drug combination in killing PANC 02 and PANC-1 pancreatic cancer cells (D) Cell cloning also proves the inhibitory effect of the drug combination on the growth of PANC 02 and PANC-1 pancreatic cancer cells.

[0125] Figure 7 The figure shows the distribution of nanomedicine in vivo as proved by in vivo imaging. (A) In vivo imaging analyzes the distribution of nanomedicine in vivo, (B) is the statistical result of (A); compared with other organs, nanomedicine can better target tumors; (C) shows the changes in fluorescence intensity in different tissues after 4h and 24h for two nanomedicine administration methods (IP or IV); (D) shows the changes in fluorescence intensity in different tissues for different administration methods (IP or IV) of nanomedicine at two time points (4h or 24h); (E) is the distribution result of nanoparticles in tumor tissue; (F) In vivo imaging shows that the synthesized drug also has a good targeting effect at metastatic tumors; (G) Fluorescence co-localization experiment compares the effects of two different administration methods on drug enrichment at the tumor, including IP intraperitoneal injection and IV tail vein injection. The results show that the penetration depth of tail vein injection is not as good as that of intraperitoneal injection, and the fluorescence intensity (i.e., drug enrichment at the tumor) is also significantly lower than that of intraperitoneal injection.

[0126] Figure 8The results of the combination of nanomedicine LNP-PAMD / siRNA and GEM were verified in mice. (A) Schematic diagram of the dosing regimen of nanomedicine and GEM; (B) The results of monitoring the weight of mice during the administration period. The weight change was not large, indicating that the drug was relatively reliable and had little effect on the mice; (C) After the administration, the tumors of the mice were weighed, and the tumors in the targeted combined administration group were the smallest; (D) The size of the mouse tumor after administration was visually illustrated in the form of a picture; (E) The metastasis of the mice was counted, and the targeted combined administration group (G6 group) showed relatively good results; (F) q-PCR experiment detected the expression of the target gene RRM2 in tumor tissue; (G) WB experiment detected that the nanomaterial LNP-PAMD / siRNA in tumor tissue still retained AMD310 on the CXCR4 / CXCL12 axis 0 (AMD3100 is a CXCR4 / CXCL12 axis inhibitor), that is, LNP-PAMD / siRNA can inhibit the expression of CXCR4; (H) Immunohistochemical staining experiment detected the statistical results of the effect of nanoparticles LNP-PAMD / siRNA on the number of CD4, CD8, Ki67, CXCR4 and CXCR12 positive cells in the tumor immune microenvironment; among them, G1 is the PBS negative control group, G2 is the LNP-PAMD / siNC group, G3 is the LNP-PAMD / siRRM2 group, G4 is the GEM group, G5 is the LNP-PAMD / siNC+GEM group, and G6 is the LNP-PAMD / siRRM2+GEM group. DETAILED DESCRIPTION

[0127] After in-depth research, the inventors first discovered that the polymer PAMD synthesized by using CXCR4 inhibitor AMD3100 and hexamethylenebisacrylamide (HMBA) can be used as a siRNA nano delivery carrier and retain the inhibitory effect on the CXCR4 / CXCL12 axis; the nanoparticles LNP-PAMD / siRRM2 formed by LNP encapsulation of PAMD and siRRM2 can effectively reduce the expression of RRM2 in pancreatic cancer cells, effectively activate the immune system of mice, enhance the immune attack on tumor cells, and inhibit the metastasis of pancreatic cancer; in addition, the inventors unexpectedly discovered that the nanoparticles LNP-PAMD / siRRM2 have a synergistic effect after being used in combination with gemcitabine, reduce drug resistance, and effectively treat refractory pancreatic cancer. On this basis, the present invention was completed.

[0128] the term

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0130] As used herein, the term “including” or “comprising” includes “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0131] As used herein, the term "PAMD" and "PAMD polymer" are used interchangeably.

[0132] As used herein, the "PAMD / siRNA", "nanoparticles", "nucleic acid drug nanoparticles" and "siRNA nanoparticles" can be used interchangeably.

[0133] As used herein, the term "siRRM2" refers to siRNA that mediates RRM2 silencing, abbreviated as siRRM2.

[0134] Plerixafor (AMD 3100)

[0135] AMD3100 is an inhibitor of CXCR4 / CXCL12, with a CAS number of 110078-46-1 and a structural formula as shown below:

[0136]

[0137] The polymer PAMD in the present invention is a nanomolecule synthesized by AMD3100 and hexamethylenebisacrylamide (HMBA), and the polymerization unit of the polymer PAMD is as follows:

[0138]

[0139] Hexamethylenebisacrylamide (HMBA)

[0140] The CAS number of hexamethylenebisacrylamide (HMBA) is 7150-41-6 and the molecular formula is C 12 H 20 N2O2, its structural formula is as follows:

[0141]

[0142] Pancreatic ductal adenocarcinoma (PDAC)

[0143] Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor that is usually diagnosed at an advanced stage and is characterized by a high metastatic rate and resistance to many therapeutic drugs. CXCL12 is a chemokine that regulates integrin expression by binding to the CXCR4 receptor on T cells, thereby inhibiting T cell migration. This mechanism in the immunosuppressive tumor microenvironment of PDAC may limit T cell infiltration and attack on tumors. The CXCL12-CXCR4 axis plays a key role in tumor biology and affects multiple cell behaviors, including T cell migration, malignant cell proliferation, differentiation, migration, invasion, and metastasis. In PDAC cells, CXCL12 binding to CXCR4 activates Akt, ERK, and NF-κB signaling pathways, especially NF-κB activation, which promotes Shh gene expression, which in turn acts on pancreatic stellate cells (PSCs) through paracrine effects to induce more CXCL12 production, thus forming a positive feedback loop that promotes tumor development and immune evasion. This complex intercellular interaction reveals the importance of the CXCL12-CXCR4 axis in pancreatic cancer development and provides a potential therapeutic target.

[0144] Ribonucleotide reductase subunit M2 (RRM2)

[0145] RRM2 is key to DNA synthesis and can catalyze the biosynthesis of deoxyribonucleotides from ribonucleotide precursors, ensuring an important component of DNA replication. Its increased activity leads to an expansion of the dNTP pool, which in turn reduces the interaction between gemcitabine and DNA through molecular competition. Studies have shown that inhibiting RRM2 through different mechanisms can increase the sensitivity of pancreatic cancer cells to gemcitabine, while siRNA-mediated RRM2 silencing can inhibit the growth of bladder cancer cells even in the absence of gemcitabine. In addition, siRNA-mediated RRM2 silencing can enhance the anticancer activity of chemotherapeutic drugs, and combined use with chemotherapeutic drugs can produce a synergistic effect, thereby improving the effectiveness of chemotherapy.

[0146] In the present invention, siRRM2 is used to directly inhibit RRM2, thereby blocking the DNA repair mechanism and enhancing the response of pancreatic cancer to gemcitabine chemotherapy.

[0147] The technical solution of the present invention has the following main advantages:

[0148] 1. The active ingredient combination provided by the present invention can effectively inhibit the metastasis of pancreatic cancer.

[0149] 2. The nucleic acid drug nanoparticles provided by the present invention can effectively inhibit the growth of pancreatic cancer cells.

[0150] 3. The nucleic acid drug nanoparticles provided by the present invention have a specific synergistic effect with gemcitabine, reduce drug resistance, and effectively treat refractory pancreatic cancer.

[0151] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial channels unless otherwise specified.

[0152] Experimental methods:

[0153] 1. Synthesis and characterization of PAMD polymers

[0154] PAMD polymers are prepared by Michael-type addition polymerization. An equimolar mixture of AMD3100 (Mei Lun, catalog number MB5646, CAS: 110078-46-1) and HMBA (Absin, catalog number: abs42151273, CAS: 7150-41-6) was dissolved in a 70% methanol solution and reacted for 2 days at 37°C under nitrogen. Additional AMD3100 was then added and the reaction continued for 12 hours. After the reaction was completed, the mixture was dialyzed with methanol for 1 day and then with water (pH 4.0) for 2 days. The polymer solution was freeze-dried in a freeze dryer to obtain the final product (i.e., PAMD polymer). Nuclear magnetic resonance analysis was performed using a Varian INOVA 400MHz NMR instrument to verify the structure of the PAMD polymer.

[0155] 2. Preparation and Characterization of LNP-PAMD / siRNA Nanoparticles

[0156] The PAMD polymer synthesized in Experimental Method 1 and siRNA were mixed at a volume ratio of 1:1 in 20 mM HEPES buffer (pH 7.4) and incubated at room temperature for 20 minutes to prepare a PAMD / siRNA polymer.

[0157] When the siRNA is siNC (as shown in SEQ ID NO: 1 or SEQ ID NO: 3), a PAMD / siNC multimer is prepared;

[0158] When the siRNA is siRRM2 (as shown in SEQ ID NO: 2 or SEQ ID NO: 4), a PAMD / siRRM2 multimer is prepared;

[0159] The sense strand sequences of the relevant mouse or human siRNA are as follows:

[0160] Human siNC: 5'-AUGAACGUGAAUUGCUCAA-3' (SEQ ID NO: 1)

[0161] Human siRRM2: 5'-GCUAUAUUCUGGCUAAAGA-3; (SEQ ID NO: 2)

[0162] Mouse siNC:5'-UUCUCCGAACGUGUCACGUTT-3'(SEQ ID NO: 3)

[0163] Mouse siRRM2:5'-CCCAUCGAGUACCAUGAUATT-3' (SEQ ID NO: 4).

[0164] PANC 02 is a mouse cell and PANC-1 is a human cell. In subsequent experiments, if PANC 02 mouse cells are used, mouse siRNA should be selected; if PANC-1 human cells are used, human siRNA should be selected.

[0165] The ability of PAMD polymers to condense siRNA was evaluated by agarose gel electrophoresis analysis. PAMD / siRNA polymers at different weight ratios (including PAMD polymer:siRNA weight ratios of 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 3:1, and 4:1, and siRNA refers to control wells containing only siRNA and no PAMD) were loaded into 2% agarose gels, stained with 0.5 μg / mL ethidium bromide, and then run at 150 V for 15 minutes in 1×TAE buffer. The gel was then imaged using an E-Gel imager.

[0166] The results are as follows Figure 1 As shown in Figure C, when the weight ratio of PAMD polymer to siRNA is 1.5 or above, the PAMD / siRNA polymer can be fully concentrated. Therefore, the weight ratio of PAMD polymer to siRNA was selected to be 2:1 (ie, w / w=2) for the following LNP-PAMD / siRNA nanoparticle preparation.

[0167] The preparation of LNP-PAMD / siRNA nanoparticles is divided into two steps. First, siRNA and PAMD polymers are mixed in a weight ratio of 1:2 and reacted at room temperature for 20 minutes to form PAMD / siRNA polymers. Then, PAMD / siRNA polymers are fully mixed with lipid (LNP) solution in a volume ratio of 1:3, and after ultrafiltration through PBS, the final product LNP-PAMD / siRNA nanoparticles are obtained. The specific process is as follows:

[0168] The PAMD / siRNA polymer diluted in HEPES buffer at a concentration of 8.01 μg / ml was fully mixed with a lipid solution in ethanol at a concentration of 4.5 mg / ml in a volume ratio of 1:3, and reacted at room temperature for 20-30 minutes to prepare LNP / siRNA nanoparticles. The lipid solution was composed of SM102, phospholipid DSPC, cholesterol and DMG-PEG2000 in a molar ratio of 40:11:48.5:1.5. Then, after ultrafiltration with PBS, the final product LNP-PAMD / siRNA nanoparticles were obtained.

[0169] When the siRNA is siNC (as shown in SEQ ID NO: 1 or SEQ ID NO: 3), LNP-PAMD / siNC nanoplasmid is prepared;

[0170] When the siRNA is siRRM2 (as shown in SEQ ID NO: 2 or SEQ ID NO: 4), a LNP-PAMD / siRRM2 nanoplasmid is prepared;

[0171] The sense strand sequences of the relevant mouse or human siRNA are as follows:

[0172] Human siNC: 5'-AUGAACGUGAAUUGCUCAA-3' (SEQ ID NO: 1)

[0173] Human siRRM2: 5'-GCUAUAUUCUGGCUAAAGA-3; (SEQ ID NO: 2)

[0174] Mouse siNC:5'-UUCUCCGAACGUGUCACGUTT-3'(SEQ ID NO: 3)

[0175] Mouse siRRM2:5'-CCCAUCGAGUACCAUGAUATT-3' (SEQ ID NO: 4).

[0176] PANC 02 is a mouse cell and PANC-1 is a human cell. In subsequent experiments, if PANC 02 mouse cells are used, mouse siRNA should be selected; if PANC-1 human cells are used, human siRNA should be selected.

[0177] In the present invention, the concentration of siRRM2 used in the cell experiment is 100 μg / ml, wherein the concentration of Cy5.5 fluorescently labeled siRRM2 (referred to as siCy5.5 or Cy5.5-siRNA) is 11.8 μg / ml; the dose of siRRM2 used in the animal experiment is 2.5 mg / kg; during the experiment, the concentration of siRNA is pre-set, and the PAMD polymer: siRNA weight ratio is 2:1, and the volume ratio of PAMD and siRNA is 1:1 to prepare the PAMD / siRNA solution; and then the LNP-PAMD / siRNA solution is prepared at a volume ratio of (PAMD+siRNA): LNP of 3:1.

[0178] The hydrodynamic size and zeta potential of the nanoparticles were measured by dynamic light scattering (DLS) of NanoBrook Omni. The morphology of LNP-PAMD / siRNA nanoparticles was observed by transmission electron microscopy (TEM, using Tecnai G2 Spirit from FEI, USA).

[0179] 3. Stability of LNP-PAMD / siRNA Nanoparticles

[0180] The LNP-PAMD / siRNA nanoparticles prepared by the above method 2 were incubated with 10% fetal bovine serum at 37°C for different time periods. PAMD / siRNA, LNP / siRNA, and LNP-PAMD / siRNA nanoparticles were then destroyed with heparin, Trition X-100, heparin, and Trition X-100, respectively, to release the remaining siRNA. The amount of remaining siRNA was evaluated by agarose gel electrophoresis to examine the protective ability of siRNA. The samples were loaded into a 2% agarose gel containing 0.5 μg / mL ethidium bromide and electrophoresed at 150V for 15 minutes in 1×TAE buffer. The gel was then imaged using an E-Gel imager.

[0181] 4. Cell Culture

[0182] Human pancreatic cancer cells: PANC-1 cells were purchased from Wuhan Pronocell Life Sciences Co., Ltd. (Wuhan, China), and PANC02 cells were donated by Mr. Le Wenjun, a researcher at the South Campus of Oriental Hospital. Both cells were cultured in DMEM supplemented with 10% FBS, 100 units / ml penicillin, and 100 g / ml streptomycin, and grown in a medium temperature incubator at 37°C and 5% CO2.

[0183] 5. Western Blotting

[0184] Cells and tumor tissues were lysed on ice using RIPA lysis buffer, a mixture of phosphatase inhibitors and protease inhibitors. The lysate was then centrifuged at 12,000 rpm for 15 min and the supernatant was collected. The protein concentration was determined using a BCA protein assay kit (Thermo Scientific) and then normalized for uniform loading in subsequent experiments. A 10% polyacrylamide gel was prepared using an SDS-PAGE kit (Yamei, China) for protein separation and then the proteins were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for one hour and then incubated with primary antibodies - rabbit anti-CXCR4 (1:1000), rabbit anti-RRM2 (1:1000) and mouse anti-AKT (1:1000) at -4°C overnight. On the next day, the PVDF membrane was thoroughly washed three times with TBST and then incubated with the corresponding HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (dilution of 1:10000) at room temperature for one hour. After washing three more times with TBST, the membrane was developed using ECL reagents on a gel documentation system for chemiluminescence detection.

[0185] 6. Real-time polymerase chain reaction (RT-PCR)

[0186] 2×10 5 PANC 02 cells or 5 × 10 4 PANC-1 cells were seeded into six-well plates. After treating the cells with serum-free medium containing PAMD polymer and siRNA complexes (siNC or siRRM2) (at a concentration of 100 nM) for 4 hours, the cells were switched to medium containing gemcitabine (GEM) at a concentration of 25 nM and cultured for another 24 hours. After removing the serum-free medium, the cells were cultured in regular growth medium for another 24 hours. Total RNA was extracted according to the kit instructions provided by the manufacturer.

[0187] Complementary DNA (cDNA) was synthesized using 1 μg RNA and a reverse transcription kit. The relevant primer sequence information is as follows:

[0188] mRRM2 (forward: 5′-CCCATCGAGTACCATGATATT-3′ (SEQ ID NO: 5); reverse: 5′-ATCATGGTACTCGATGGGTT-3′ (SEQ ID NO: 6));

[0189] hRRM2 (Forward: 5′-GAAACGAGTTTCAGAGTATTT-3′ (SEQ ID NO: 7); Reverse: 5′-ATACTCTGAAACTCGTTTCTT-3′ (SEQ ID NO: 8)).

[0190] PCR conditions were as follows: 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, for a total of 30 cycles, and a final extension of 5 minutes at 72°C. The mRNA level relative to the internal control was calculated using the comparative threshold cycle (Ct) method.

[0191] 7. Cytotoxicity of PAMD polymers

[0192] PANC-1 cells (8000 cells / well) and PANC 02 cells (5000 cells / well) were seeded into 96-well plates and cultured overnight at 37°C. PAMD, LNP-PAMD / siNC, and LNP-PAMD / siRRM2 were then added to serum-free medium and cultured for 12 hours. The medium was then replaced with serum medium containing different concentrations of gemcitabine and cultured for another 24 hours. After the incubation period, CCK8 reagent was added according to the manufacturer's instructions and incubation was continued. After 2 more hours of culture, the absorbance of each well was measured at a wavelength of 450 nm using a multi-function microplate reader.

[0193] The relative cell viability was calculated as (sample / untreated) × 100. Dose-response analysis was performed using GraphPad Prism software to determine the IC 50 value.

[0194] 8. Inhibit cancer cell migration

[0195] PANC-1 cells and PANC 02 cells were digested with trypsin, resuspended in PBS, and counted to 1×10 5 and 9×10 4 . Cells were pretreated with ADM 3100 or PAMD polymer for 30 minutes and then resuspended in 300 μl serum-free medium and placed in the upper chamber coated with Matrigel. Serum-free medium with or without CXCL12 was added to the lower chamber. After 14 hours of culture in a 37°C incubator, cells were fixed with paraformaldehyde for 15 minutes and stained with crystal violet for 20 minutes. The crystal violet stain in the upper chamber was carefully removed with a cotton swab, and cell migration and invasion were observed under an optical microscope.

[0196] 9. Cell cloning experiment

[0197] 800 PANC-1 cells and 1000 PANC02 cells were inoculated in 6-well plates and cultured overnight. LNP-PAMD / siRRM2 and LNP-PAMD / siNC (100nm, w / w=2) were added in a 37°C incubator for 24 hours, and then gemcitabine was added. After 24 hours, the culture medium was replaced with a culture medium containing 10% fetal bovine serum and cultured. After 10 days of continued culture, the cells were stained with 0.1% crystal violet for 30 minutes, and then the excess crystal violet was rinsed off with PBS. Finally, the cell colony images were taken with a camera, and the number of colonies was calculated using image analysis software.

[0198] 10. Cellular Uptake

[0199] PANC-1 cells and PANC 02 cells were cultured in 6-well or 24-well plates at 37°C overnight. siRNA or siRNA in nanoparticles was labeled with Cy5.5, namely Cy5.5-siRNA or LNP-PAMD / Cy5.5-siRNA. 47.29 μl of LNP-PAMD / Cy5.5-siRNA nanoparticles were added to the well plate. After 4 hours, the cells were washed three times with PBS to remove the residual nanoparticles and then fixed with paraformaldehyde for 15 minutes. After washing with PBS three times again, they were incubated with DAPI at room temperature for 15 minutes, and the absorption of nanoparticles by cells was observed under a microscope. Similarly, after washing the cells with PBS, the cells were resuspended in PBS, and then the fluorescence intensity of the cells was measured for flow cytometry. The results of flow cytometry were analyzed using FlowJo software.

[0200] 11. In situ PDAC Model

[0201] Seven-week-old male C57 mice were anesthetized by intraperitoneal injection. A surgical incision of about 0.5 cm was then made in the left upper abdomen of the mouse. After confirming the location of the pancreas, 40 μl of Matrigel / PBS (1:1) and 6×10 4 After waiting for about 1 minute to ensure that the mixed solution was slightly solidified, the wound was sutured with absorbable surgical sutures.

[0202] 12. Drug biodistribution

[0203] Two weeks after the tumor cells were implanted in the above method 11, the animals were injected intravenously or intraperitoneally with 200 μl of Cy5.5 / siRNA or nanoparticles containing Cy5.5-siRNA. Four hours and 24 hours after administration, the mice were euthanized, and then the tumors and major organs were harvested and imaged using an in vivo imaging system. The fluorescence intensity of the tumors and major organs was quantified using the IVIS imaging system and the accompanying analysis software. The primary tumors were frozen and preserved by immersion in liquid nitrogen, and frozen sections were prepared for microscopic examination to observe the distribution of nanoparticles in the tumor.

[0204] 13. Antitumor Activity

[0205] Twelve days after pancreatic cancer PANC 02 cells were implanted into mice, the mice were randomly divided into six groups (n=6). Each group of mice received different treatments by intraperitoneal injection: PBS, LNP-PAMD / siNC, LNP-PAMD / siRRM2, gemcitabine (GEM), GEM+LNP-PAMD / siNC, and GEM+LNP-PAMD / siRRM2.

[0206] Starting from day 12, mice were intraperitoneally injected with PAMD polymer containing 2.5 mg / kg siRNA every 3 days until day 30. In addition, mice were intraperitoneally injected with gemcitabine at a dose of 25 mg / kg on days 14, 20, 26, and 32. Body weight was recorded every other day throughout the experiment.

[0207] On day 33, mice were euthanized and their primary tumor weights were measured. Macroscopic metastasis of organs in vivo was examined under a dissecting microscope. Major organs were collected and fixed with 4% paraformaldehyde and then embedded in paraffin. Tumor tissues were used for Western blot and reverse transcription polymerase chain reaction (RT-PCR) assays, while changes in the tumor immune microenvironment were evaluated by immunohistochemical staining.

[0208] Example 1: Preparation and characterization of LNP-PAMD / siRNA nanoparticles

[0209] AMD3100 is a CXCR4 antagonist based on a ring structure, which can specifically inhibit the signal transduction between CXCR4 and its ligand CXCL12. Based on the above experimental method, this example synthesized PAMD polymer ( Figure 1 A). PAMD polymers can both maintain the CXCR4 inhibitory activity of AMD3100 and condense nucleic acids into nanoparticles, preventing nucleic acid degradation and facilitating nucleic acid delivery. 1 The successfully synthesized PAMD was characterized by HNMR ( Figure 1 B).

[0210] Agarose gel electrophoresis was used to evaluate the ability of PAMD to condense siRNA. The results showed that when the weight ratio of PAMD polymer to siRNA was 1.5 or above, PAMD polymer could fully condense siRNA ( Figure 1 C). Then, the weight ratio of PAMD polymer to siRNA was selected to be 2:1 (ie, w / w=2) to prepare LNP-PAMD / siRNA nanoparticles, and the process was as follows:

[0211] (1) mixing siRNA and PAMD polymer in a weight ratio of 1:2 to form PAMD / siRNA polymer;

[0212] (2) PAMD / siRNA polymers diluted in HEPES buffer at a concentration of 8.01 μg / ml were fully mixed with a lipid solution in ethanol at a concentration of 4.5 mg / ml at a volume ratio of 1:3 to further encapsulate the PAMD / siRNA polymers and prepare LNP-PAMD / siRNA nanoparticles. The lipid solution consisted of SM102, phospholipid DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 40:10:48.5:1.5.

[0213] (3) After ultrafiltration with PBS, the final product, LNP-PAMD / siRNA nanoparticles, was obtained, with the structure shown in Figure 2 As shown in A, the polymer PAMD / siRNA is encapsulated inside the LNP.

[0214] Dynamic light scattering measurements showed that the average hydrodynamic size of LNP-PAMD / siRNA nanoparticles was 88.6 nm, the dispersion index was 0.12, and the zeta potential of the nanoparticles was 8.9 mV ( Figure 2 B-2C). Transmission electron microscopy (TEM) observations showed that the nanoparticles exhibited a uniform spherical morphology ( Figure 2 D).

[0215] To investigate the stability of the nanoparticles, the nanoparticles (LNP-PAMD / siRNA, LNP / siRNA or PAMD / siRNA) or naked siRNA (siRNA Only) were incubated with 10% FBS at 37°C for different time periods (0, 4, 8, 12, 16, 20 or 24 h). The remaining siRNA was released and detected by gel electrophoresis.

[0216] The results showed that there was no siRNA remaining in the naked siRNA group after 4 hours of incubation with 10% FBS, which means that unprotected siRNA is easily degraded in serum and quickly loses its activity. In contrast, the remaining siRNA in the other nanoparticle groups can withstand longer FBS incubation due to the protection of polymers (PAMD) or lipids (LNP). The siRNA in the PAMD / siRNA group and the LNP / siRNA group began to degrade within 24 hours, indicating that the protection ability of PAMD polymers or LNPs alone is limited. However, in the LNP-PAMD / siRNA group, siRNA remained stable after 24 hours, indicating that LNP-PAMD / siRNA nanoparticles have a good ability to protect siRNA under biological conditions ( Figure 2 E). This result highlights the dual protective effects of the PAMD polymer and lipid shell in the LNP-PAMD / siRNA group, which can condense siRNA and improve the stability of siRNA.

[0217] Example 2: LNP-PAMD reduces the high expression of CXCR4 in cells and simultaneously inhibits their migration activity

[0218] Based on the above experimental method, the cytotoxicity test of PANC 02 and PANC-1 pancreatic ductal adenocarcinoma cell lines in this example showed that PAMD / siRNA and LNP-PAMD / siRNA showed different cytotoxicity reactions ( Figure 3 A). The half maximal inhibitory concentration (IC) of PAMD on PANC 02 cells and PANC-1 cells 50 The results showed that the concentrations of LNP-PAMD in the PANC-02 cell line were 35.71ug / mL and 55.3ug / mL, respectively, which were slightly lower than the half maximal inhibitory concentration (IC 50 80.51ug / mL and 113ug / mL respectively)( Figure 3 B) IC 50 The values ​​were affected by the amount of LNP added, which may be due to the ability of nanocarriers to regulate the release rate and bioavailability of drugs. However, LNPs can enhance the selective accumulation of drugs in cancer cells through specific uptake mechanisms such as receptor-mediated endocytosis, thereby improving efficacy without increasing toxicity.

[0219] The CXCR4 / CXCL12 signaling pathway plays a central role in the occurrence and development of cancer. AMD3100, as a specific inhibitor of the CXCR4 / CXCL12 axis, can significantly inhibit the migration and invasion of tumor cells by blocking this signaling pathway. To evaluate the effect of PAMD on pancreatic cancer cell migration, migration experiments were performed in PANC 02 and PANC-1 cell lines using 20nM CXCL12 as a chemoattractant. In a comparative study of compounds PAMD and AMD3100, it was found that at the optimal safety concentration, 1μg / ml compound PAMD had a better inhibitory effect on PANC 02 and PANC-1 pancreatic cancer cell lines than 300nM AMD3100 ( Figure 4 A).

[0220] In the present invention, 300 nM AMD3100 was used as a control group, and cells were treated with 1 ug / mL of PAMD, LNP-PAMD / siNC and LNP-PAMD / siRRM2, and then a Western Blot experiment was performed.

[0221] The experimental results showed that PAMD, LNP-PAMD and LNP-PAMD / siRRM2 could effectively inhibit the activation of AKT signaling pathway ( Figure 4 B). The AKT signaling pathway has been shown to play an important role in cell migration and invasion, cell survival and proliferation, and metabolic regulation of tumor cells. This finding provides important experimental evidence for the further study and potential clinical application of the compound LNP-PAMD.

[0222] Example 3: Stability and cellular uptake of nanoparticles

[0223] Based on the above experimental method, this embodiment uses an optical microscope to observe the absorption behavior of nanoparticles by PANC 02 and PANC-1 pancreatic cancer cell lines. The absorption of nanoparticles in cells was quantitatively evaluated using flow cytometry technology. The internalization efficiency of Cy5.5-labeled siRNA nanoparticles was evaluated using PANC 02 and PANC-1 pancreatic cancer cell lines. The nanoparticles were incubated with cells in a 37°C incubator for 4 to 5 hours to simulate in vivo conditions. After the culture was completed, cells treated with LNP-PAMD / siCy5.5 were observed using a fluorescence microscope, wherein siCy5.5 refers to siRRM2 fluorescently labeled with Cy5.5, and the concentration of Lipofectamine 3000 was used according to the instructions to ensure that the volume of siRNA was the same, the concentration of siRNA was 100 μg / ml, and the concentrations of LNP / siRNA, PAMD / siRNA and LNP-PAMD / siRNA were all 1 μg / ml.

[0224] The experimental results showed that the Cy5.5 fluorescence signal was almost undetectable in cells containing only siRNA, while the fluorescence intensity of cells containing only PAMD / siRNA was 60%. After the addition of LNP / siRNA, the fluorescence intensity increased significantly to about 90%, which is comparable to the fluorescence intensity of the group using the Lipofectamine 3000 transfection reagent. The addition of LNP significantly improved the intracellular delivery efficiency of siRNA, thereby enhancing the detection of the fluorescence signal. This phenomenon may be due to the fact that LNP enhances the cell penetration ability of PAMD / siRNA nanoparticles and promotes the effective internalization of siRNA, thereby improving the detection sensitivity of fluorescently labeled siRNA. This improvement in delivery efficiency may be due to the fact that the special structure or composition of LNP promotes the interaction between PAMD / siRNA nanoparticles and cell membranes, thereby improving the absorption of PAMD / siRNA nanoparticles by cells ( Figure 5 A).

[0225] Flow cytometry analysis of cells treated with the same samples as above yielded consistent results ( Figure 5 B). Specifically, in the PANC-1 pancreatic cancer cell line, cells treated with LNP-PAMD / siRNA complexes showed higher fluorescence intensity than cells treated with PAMD / siRNA complexes, indicating that LNP-PAMD / siRNA complexes have higher labeling efficiency in cells. In addition, similar experiments conducted in the PANC 02 pancreatic cancer cell line also reproduced this phenomenon, further confirming the advantages of LNP-PAMD / siRNA complexes in cell labeling.

[0226] Example 4: LNP-PAMD effectively knocks out the expression of RRM2 in cells

[0227] Based on the above experimental methods, quantitative polymerase chain reaction (qPCR) technology was used in this study to evaluate the intracellular transfection efficiency of LNP-PAMD nanoparticles. Among them, the concentration of Lipofectamine 3000 was used according to the instructions to ensure the same volume of siRNA, the concentration of siRRM2 was 100μg / ml, and the concentrations of LNP / siRRM2, PAMD / siRRM2 and LNP-PAMD / siRRM2 were all 1μg / ml.

[0228] The results showed that LNP-PAMD / siRRM2 nanoparticles could significantly inhibit the expression of RRM2 protein in two pancreatic cancer cell lines. Specifically, in the PANC 02 cell line, the expression level of RRM2 decreased by about 60% after treatment with LNP-PAMD / siRRM2 nanoparticles. Similarly, in the PANC-1 cell line, the expression of RRM2 was also reduced by about 60% ( Figure 6 A).

[0229] In addition, the half inhibitory concentration (IC50) of gemcitabine (GEM) in PANC-02 and PANC-1 pancreatic cancer cell lines was determined, and the IC 50 The values ​​were 11.47 μg / mL and 17.13 μg / mL ( Figure 3 B). These data showed that PANC 02 and PANC-1 cell lines were less sensitive to gemcitabine, suggesting that PANC 02 and PANC-1 cell lines may have developed resistance to this commonly used chemotherapeutic drug, gemcitabine, which may be one of the key factors leading to clinical treatment failure.

[0230] The CCK8 assay is widely used as a method to assess cell proliferation and viability. The inventors quantified the cytotoxicity of RRM2 knockout combined with gemcitabine (GEM) treatment on PANC 02 and PANC-1 cell lines using CCK8 assay. In the absence of GEM, 100 nM LNP-PAMD / siRRM2 had 10% and 20% cytotoxicity against PANC 02 and PANC-1 cell lines, respectively ( Figure 6 B) (wherein, the molar concentration of LNP-PAMD / siRRM2 is 100 nM, which is equivalent to a concentration of 1.33 μg / ml. Therefore, its cytotoxicity at 100 nM is ( Figure 6 B) with Figure 3 In this study, the inventors set the maximum concentration of GEM to 100 nM, and used the concentration gradient reduction method to evaluate the synergistic cytotoxic effect of GEM and LNP-PAMD / siRRM2 complex on cells.

[0231] In the PANC 02 cell line, the cell killing rate was about 10% when 25nM GEM was used alone, and the cell killing rate was about 10% when 100nM PAMD / siRRM2 was used alone. Unexpectedly, when the two were used in combination, the cell killing rate was significantly increased to 40% ( Figure 6 B). This result indicates that combining CXCR4 inhibitors with gene-targeted therapy can significantly enhance the cytotoxic effect of GEMs on PANC-02 cells.

[0232] In the PANC-1 cell line, the cell killing rate was about 50% when 100nM GEM was used alone, and the cell killing rate was about 40% when 100nM PAMD / siRRM2 was used alone. Also unexpectedly, when LNP-PAMD / siRRM2 and GEM (i.e., siRRM2+GEM) were used in combination (LNP-PAMD / siRRM2+GERM group), the cell killing rate was synergistically increased to about 85% ( Figure 6 B).

[0233] After confirming the effect of RRM2 knockout on cells, the inventors further studied the inhibitory effect of RRM2 knockout combined with gemcitabine (GEM) on cell proliferation. In order to quantitatively evaluate the synergistic effect of LNP-PAMD / siRRM2 and GEM in pancreatic cancer cell lines, the inventors calculated the combination index (CI).

[0234] The results showed that in the PANC 02 cell line, the combined treatment with 100 nM LNP-PAMD / siRRM2 and 25 nM GEM had a combined index (CI) value of 0.23, while in the PANC-1 cell line, the combined treatment with 100 nM LNP-PAMD / siRRM2 and 25 nM GEM had a combined index (CI) value of 0.57 ( Figure 6 C). CI values ​​less than 1 are generally considered to be indicative of strong synergistic activity, and these values ​​were significantly lower than 0.5, indicating that LNP-PAMD / siRRM2 had a significant synergistic cytotoxic effect when used in combination with GEM.

[0235] The results indicate that LNP-PAMD / siRRM2+GEM nanoparticles (ie, siRRM2+GEM) have synergistic anticancer activity against pancreatic cancer cells.

[0236] Example 5: Distribution of Nanoparticles in Tumor-bearing Mice

[0237] In the orthotopic PANC 02 pancreatic tumor model, the inventors evaluated the accumulation ability of nanoparticles. PANC 02 pancreatic tumors were implanted in mice for 4 weeks. When the tumor volume was approximately 200 cubic millimeters, Cy5.5-labeled LNP-PAMD / siRNA nanoparticles were administered by tail vein injection (IV) and intraperitoneal injection (IP), and the weight and tumor volume of the mice were monitored. Subsequently, the distribution of nanoparticles in tumors and major organs was analyzed 4 hours and 24 hours after administration. PAMD / siRNA and LNP / siRNA were used as controls to verify the importance of lipids to the ability of LNP-PAMD / siRNA polyplexes to penetrate tumors in vivo. Among them, the doses of LNP-PAMD / siRNA, PAMD / siRNA and LNP / siRNA were 6.25 mg / kg, respectively.

[0238] Using in vivo imaging technology, the siRNA signal intensity of different nanoparticles in animals was demonstrated. The nanoparticles were injected into mice via intraperitoneal injection and circulated in the body for 4 hours. Using small animal in vivo imaging technology, the inventors successfully detected the fluorescent signal of siRNA in the liver and tumor sites. It is worth noting that the accumulation of LNP-PAMD / siRNA composite nanoparticles containing PAMD / siRNA and LNP / siRNA components in orthotopic pancreatic tumors was significantly higher than that of control nanoparticles containing only PAMD or LNP components. This result shows that the accumulation effect of LNP-PAMD / siRNA composite nanoparticles in in situ pancreatic tumors is significantly enhanced ( Figure 7 A).

[0239] After the nanoparticles circulated in the body for 24 hours, the inventors conducted a quantitative analysis of the fluorescent signals in the tumors and major organs. The results showed that the LNP-PAMD / siRNA particles increased the accumulation of siRNA in primary tumors by 1.73 times compared with the PAMD / siRNA particles. The accumulation of siRNA in primary tumors by LNP-PAMD particles was significantly higher than that of LNP particles, reaching an accumulation effect of 2.12 times ( Figure 7 B).

[0240] Although the fluorescence intensity in the tumor area also decreased, at 24 hours, more drugs were effectively targeted and delivered to the tumor site through the blood, resulting in accumulation. This distribution pattern may be related to the characteristics of the drug carrier, including its size, surface modification, and passive targeting ability, which together promote the penetration and retention of drugs in the tumor microenvironment.

[0241] In the present invention, the inventors explored the effects of different administration routes on the in vivo distribution of nanoparticles. Specifically, tail vein injection and intraperitoneal injection were compared, and the dynamic distribution of nanoparticles in the body was observed at 4 hours and 24 hours.

[0242] The results showed that LNP-PAMD / siRNA nanoparticles accumulated significantly in the tumor and liver within 4 hours, whether injected intravenously (IV) or intraperitoneally (IP). However, as the circulation time was extended to 24 hours, the accumulation of these nanoparticles in the tumor site was significantly reduced ( Figure 7 C), which may be related to the in vivo clearance of nanoparticles, specific interactions with the tumor microenvironment, or other biological factors.

[0243] Further data analysis showed that although the distribution patterns of these nanoparticles in tumors and organs were roughly similar, the fluorescence intensity of siRNA in tumor tissues at both time points after intraperitoneal injection was significantly higher than that after tail vein injection, about three times that of tail vein injection ( Figure 7 D). This suggests that intraperitoneal injection may be a more effective method of drug delivery because it can improve the efficiency of drug absorption by tumor tissue. This difference may be due to the fact that intraperitoneal injection brings the drug closer to the primary tumor site, thereby taking advantage of the local biological properties of the tumor, such as the enhanced permeability and retention effect (EPR), to promote the accumulation of drugs in the tumor.

[0244] As we all know, pancreatic cancer has a high metastatic potential, which poses a huge challenge to treatment. The inventors used in vivo imaging technology to find that LNP-PAMD / siRNA nanoparticles showed a high affinity for both orthotopic pancreatic tumors and distant metastatic lesions ( Figure 7 F). This finding highlights the potential targeting ability of LNP-PAMD / siRNA nanoparticles in tumor therapy. Normally, the surface of normal organs is covered with a continuous layer of mesothelial cells, which hinders the penetration and accumulation of nanoparticles. In contrast, tumor tissues, especially peritoneal tumors and metastases, often have an incomplete or interrupted mesothelial cell layer. This structural disruption may promote the penetration and accumulation of nanoparticles in tumor tissues.

[0245] In this study, in order to gain a deeper understanding of the distribution characteristics of nanoparticles in tumor tissue, the inventors used microscopic imaging technology to conduct a detailed analysis of cryogenic tumor tissue sections. Among them, the dose of the siRNA only group was 2.5 mg / kg, and the doses of LNP-PAMD / siRNA, PAMD / siRNA, and LNP / siRNA were 6.25 mg / kg, respectively. The inventors observed that in the PAMD / siRNA group and the siRNA group, these components mainly accumulated in the peripheral areas of the tumor. In contrast, in the LNP-PAMD / siRNA group, these components appeared not only in the peripheral areas of the tumor, but also in the central areas of the tumor, indicating that LNP-PAMD nanoparticles have better tumor penetration ability. ( Figure 7 E).

[0246] In addition, the obvious co-localization of siRNA and LNP-PAMD / siRNA component fluorescence further confirmed that the nanoparticles were present in a more intact form within the tumor tissue ( Figure 7 G). These findings are of great significance for optimizing drug delivery strategies and injection timing to improve the penetration and uniform distribution of nanoparticles in tumor tissues.

[0247] Example 6: In vivo antitumor activity

[0248] C57 mice with intact immune function were selected as experimental mice. By transplanting the PANC-02 cell line into C57 mice, a stable tumor model was successfully established to simulate the role of CXCR4 in the tumor immune microenvironment of pancreatic ductal adenocarcinoma (PDAC). On this basis, the efficacy of different treatment options was evaluated.

[0249] Based on the above experimental method, this example selects mice with similar weight and age between 6 and 8 weeks to establish an orthotopic tumor model. When the tumor grows to a predetermined size, the mice are treated with seven consecutive doses of LNP-PAMD / siRRM2 or LNP-PAMD / siNC (siRNA is 2.5 mg / kg, LNP-PAMD is 6.25 mg / kg) by intraperitoneal injection. In this study, mice received adjuvant combination therapy with gemcitabine (GEM) by intraperitoneal injection of nanoparticles, once a week from the start of treatment ( Figure 8 A). This study recorded in detail the changes in the weight of mice during the treatment process, and the results showed that there was no significant change in the weight of mice in all treatment groups ( Figure 8 B), indicating that the treatment was well tolerated. After 4 weeks of treatment, the mice were euthanized, and tumor and metastasis samples from different treatment groups were collected for observation and weighing.

[0250] The results showed that the combination of LNP-PAMD / siRRM2 and GEM showed the best anti-tumor activity, and the mice treated with the combination of LNP-PAMD / siRRM2 and GEM had the lightest tumor weight, highlighting the great potential of this combination therapy for the treatment of pancreatic cancer ( Figure 8 C and 8D).

[0251] To evaluate the safety of the treatment, hematoxylin-eosin (HE) staining was performed on the main organs of the mice in this study to observe any toxic pathological changes caused by the treatment.

[0252] The results showed that no obvious pathological changes were observed in the major organs of mice in all treatment groups, further confirming the safety of the treatment.

[0253] In addition, this study also observed a significant reduction in tumor metastasis in mice treated with PAMD. Among them, the tumor metastasis rate in the LNP-PAMD / siRRM2 group combined with the GEM group was the lowest, and even 2 mice had no obvious signs of tumor metastasis in the spleen. This result shows that the combined treatment has a significant anti-tumor effect ( Figure 8 E).

[0254] The expression of target gene RRM2 in tumor tissue was detected by qPCR experiment. The results showed that compared with the PBS control group (G1 group), GEM alone (G4 group) did not affect the change of target gene RRM2 level; nanoparticles LNP-PAMD / siRRM2 (G3 group) could significantly reduce the relative level of target gene RRM2, and it was found that when combined with GEM (G6 group), it could further reduce the relative level of target gene RRM2 ( Figure 8 F).

[0255] The WB experiment was used to detect the effect of nanomaterial LNP-PAMD / siRNA on the CXCR4 / CXCL12 axis in tumor tissues. The results showed that the nanomaterial LNP-PAMD / siRNA (G2 group or G3 group) still retained the CXCR4 / CXCL12 axis inhibitor properties of AMD3100, that is, LNP-PAMD / siRNA could inhibit the expression of CXCR4 ( Figure 8 G).

[0256] This example aims to further explore the effect of nanoparticles on the tumor immune microenvironment. To this end, after collecting tumor samples, immunohistochemical staining experiments were performed. The experimental results showed that LNP-PAMD / siRRM2 treatment can significantly reduce the Ki67 index in mouse tumor tissue ( Figure 8 H), which indicates that the treatment can effectively inhibit the proliferation of tumor cells. Ki67 is a biomarker of cell proliferation, and its reduced level directly reflects the slowing of tumor cell proliferation. In addition, the reduction of Ki67 index also means the weakening of tumor invasion ability, which can reduce the risk of tumor recurrence and metastasis, thereby improving the prognosis of patients. Further immunohistochemical staining analysis showed that after LNP-PAMD / siRRM2 treatment, the number of CD4 and CD8 positive cells in tumor tissue increased significantly ( Figure 8 H). This finding suggests that the treatment may have effectively activated the immune system of mice and enhanced the immune attack on tumor cells. At the same time, this also means that the tumor tissue may have undergone T cell infiltration and the immune microenvironment of the tumor may have changed. This change in the immune microenvironment may have broken the original immunosuppressive state of the tumor microenvironment and promoted the infiltration of more immune-active cells. Changes in the distribution and interaction of these immune cells in the tumor microenvironment may be closely related to the improved efficacy brought about by LNP-PAMD / siRRM2 combined with GEM treatment. This experiment aims to evaluate the effects of nanoparticles on the infiltration and activation state of immune cells in tumor tissues by detecting specific immune markers, so as to intuitively observe the changes in immune cells in the tumor microenvironment after nanoparticle treatment. In this way, we can have a more comprehensive understanding of the regulatory effects of nanoparticle therapy on the tumor immune microenvironment and provide a scientific basis for future tumor treatment strategies.

[0257] discuss

[0258] CXCR4 and its ligand CXCL12 play a crucial role in regulating tumor growth and metastasis, so inhibitors targeting this signaling pathway have become a hot topic of research. Although the development of CXCR4 / CXCL12 inhibitors has made progress in recent years, monotherapy alone cannot provide a definitive and effective treatment for pancreatic cancer, a malignant tumor that is usually diagnosed at an advanced stage and metastasizes rapidly. Previous research results have shown that AMD3100, as a CXCR4 inhibitor, does not directly induce cell death when used alone. Instead, its clinical value is mainly reflected in its ability to mobilize chronic myeloid leukemia cells from protective microenvironments such as the bone marrow. This mechanism helps to increase the sensitivity of these cells to chemotherapy, thereby enhancing the therapeutic effect. The treatment of pancreatic cancer is particularly complex, in part because tumor cells show significant resistance to commonly used first-line chemotherapy drugs. This resistance greatly limits the effectiveness of chemotherapy, resulting in limited treatment options, and has therefore become one of the key obstacles that need to be overcome in the treatment of pancreatic cancer. Although traditional pancreatic cancer treatments face many challenges, siRNA-based treatments have become a research hotspot due to their unique advantages. The high specificity of siRNA enables it to precisely target specific oncogenes, while its customizability allows researchers to design siRNA to inhibit different molecular targets as needed. This flexibility provides the possibility of developing personalized treatment plans and helps overcome the heterogeneity and drug resistance of pancreatic cancer.

[0259] In this study, the inventors designed and synthesized an innovative nanoparticle carrier based on the CXCR4 inhibitor AMD3100, which can effectively encapsulate small interfering RNA (siRNA) molecules. By adopting a strategy of combined treatment with the first-line chemotherapy drug gemcitabine (GEM), the aim is to improve the efficacy of metastatic pancreatic ductal adenocarcinoma (PDAC). These nanoparticles are designed to take advantage of the targeting ability of AMD3100 and the gene silencing effect of siRNA, combined with the cytotoxicity of GEM, to work synergistically and strive to achieve a breakthrough in the treatment of metastatic PDAC. The results showed that the newly developed CXCR4 inhibitor nanoparticle PAMD / siRNA, when administered alone, was similar to AMD3100 in its ability to reduce CXCR4 expression and effectively block the activation of the AKT signaling pathway. In addition, other experiments have also found that when PAMD / siRNA nanoparticles are used in combination with gemcitabine (GEM), they can significantly improve the high resistance of pancreatic cancer to chemotherapy, which may be closely related to the drug controlled release and targeted delivery properties of PAMD / siRNA nanoparticles. Through systematic in vitro cell experiments and in vivo animal model experiments, it was verified that PAMD nanoparticles have low cytotoxicity, providing a safe basis for their clinical application. Lipid nanoparticle (LNP) modification has been shown to significantly improve the cellular delivery efficiency of nanomedicines. The experimental results of this application showed that LNP-modified nanomedicines had higher cellular absorption rates in various cell lines compared with unmodified controls. This finding emphasizes the importance of LNP modification in improving the delivery efficiency of nanomedicines and provides valuable inspiration for the design of nanomedicines.

[0260] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of an active ingredient combination, characterized in that: A pharmaceutical composition or a drug kit for preparing a tumor treatment or tumor cell inhibition, wherein the active ingredient combination comprises: (a) a first active ingredient, which is a nucleic acid drug targeting RRM2; and (b) a second active ingredient, gemcitabine.

2. The use according to claim 1, characterized in that The first active ingredient is in the form of nucleic acid drug nanoparticles.

3. The use according to claim 1, characterized in that The weight ratio of the nucleic acid drug to gemcitabine is 1:50 to 10:1, preferably 1:20 to 5:1, more preferably 1:15 to 2:1, and most preferably 1:10 to 1:1 or 1:5 to 1:

1.

4. The use according to claim 1, characterized in that The tumor includes pancreatic cancer, preferably pancreatic ductal adenocarcinoma.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a nucleic acid drug targeting RRM2, gemcitabine and a pharmaceutically acceptable carrier.

6. An active ingredient combination, characterized in that The active ingredient combination includes: (a) a first active ingredient, which is a nucleic acid drug targeting RRM2; and (b) a second active ingredient, gemcitabine.

7. A method for preparing the active ingredient combination according to claim 6, characterized in that: The following steps are involved: (i) mixing a polymer having a polymer unit represented by the following formula I with a nucleic acid drug to obtain a first complex (or a first mixture): (ii) encapsulating the first complex to form a liposome loaded with the first complex.

8. A medicine box, characterized in that: The medicine kit comprises: (a) a first preparation, wherein the first preparation contains nucleic acid nanoparticles of a nucleic acid drug targeting RRM2 and a pharmaceutically acceptable carrier; (b) a second formulation comprising gemcitabine and a pharmaceutically acceptable carrier; (c) instructions describing a method for treating tumors by combining nucleic acid drug nanoparticles and gemcitabine.

9. An in vitro non-therapeutic method for inhibiting tumor cell growth, comprising the steps of: Tumor cells are cultured in the presence of the active ingredient combination of claim 6, thereby inhibiting tumor cell growth.

10. The method according to claim 9, characterized in that The tumor cells include pancreatic ductal carcinoma and refractory pancreatic cancer.

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