Preparation method and application of biodegradable PLGA-TK-PEG nano drug carrier

Abstract

The invention discloses a method for preparing a biodegradable PLGA-TK-PEG nano drug carrier, including the synthesis of PLGA-TK-PEG high molecular polymer and the preparation of PLGA nanoparticles, and obtaining PLGA nanoparticles with good performance by centrifugation. The invention also discloses a method for preparing a smart response PLGA nano drug modified by a targeted molecule loaded with a chemotherapeutic drug, a Chinese medicine monomer or a gene drug, and the use of the aforementioned PLGA nano drug in the preparation of a drug for treating liver cancer, lung cancer, brain cancer or pancreatic cancer. The nano drug can enter tumor cells through targeting and endocytosis, and can controllably release the anticancer drug carried under the condition of high content of ROS in tumor cells, thereby achieving the purpose of inhibiting tumor cells more efficiently and with low toxicity. The diameter of the nano drug is about 50-300nm, and the blank nanoparticles have excellent biocompatibility. The targeted modification can serve the purpose of specifically delivering the nano drug to tumor cells, and can controllably release the anticancer drug carried under the condition of 0.1-100mM H2O2.

Description

Technical Field

[0001] The invention belongs to the research field of anticancer nanomedicines, and specifically relates to a preparation method and application of an anticancer PLGA nanomedicine modified with a targeting molecule based on ROS release. Background Art

[0002] Liver cancer is one of the malignant tumors with the highest morbidity and mortality in my country, which seriously endangers human health and life safety. Although there have been many new advances in early diagnosis, surgery, radiotherapy, chemotherapy and targeted therapy of liver cancer, the current 5-year survival rate of liver cancer is still around 20%. Once metastasis occurs, the 5-year survival rate will be less than 5% (CA Cancer J.Clin.2018,68:394-424). Therefore, the development of highly effective and low-toxic anti-liver cancer drugs is a major problem that needs to be solved urgently around the world.

[0003] Neddylation modification is a new type of anti-tumor molecular target protein. Neddylation modification is a newly discovered protein post-translational modification pathway, which is the process of binding the ubiquitin-like small molecule NEDD8 to the substrate molecule. Its main function is to regulate protein activity. NEDD8, as the most studied ubiquitin-like (Ub) molecule, has a homology of up to 80% with the ubiquitin molecule. Neddylation modification is an energy-consuming cascade reaction process, which is catalyzed by NEDD8 activating enzyme E1 (NAE), NEDD8 conjugating enzyme E2 and NEDD8 ligase E3 successively. Finally, NEDD8 forms an isopeptide with the lysine residue of the substrate molecule through its C-terminal 76 glycine residue and connects to the target protein to enable the substrate to function. So far, studies have found that the NEDD8 cascade reaction includes 1 E1 (heterodimer formed by NAE1 and UBA3), 2 E2s (UBC12 and UBE2F), and some E3s (J.Natl.Cancer Inst.2014,106:dju083). It has been reported that highly activated Neddylation and highly expressed NAE1, UBA3 and UBC12 were detected in the liver cancer tissues of patients. If the activating enzyme E1 (NAE), conjugating enzyme E2 and ligase E3 of NEDD8 are knocked down, the activity of Neddylation in liver cancer tissues will inevitably be inhibited, thereby inhibiting the occurrence and development of liver cancer (Mol. Cell Proteomics. 2015, 14: 499-509).

[0004] RNA interference (RNAi) refers to the phenomenon that endogenous or exogenous short-chain RNA induces the degradation of homologous mRNA, leading to gene silencing. Two Americans, Andrew Fell and Craig Mello, won the 2006 Nobel Prize in Physiology for this. Why has RNAi technology been so successful in the field of scientific research, but few drugs have entered Phase II / III clinical trials? This is mainly because of the instability of siRNA, which requires a suitable carrier to transport siRNA to the site of action. Therefore, it is urgent to develop new gene nanomedicines to achieve the purpose of gene therapy (Nat. Nanotechnol. 2019, 14: 388-397).

[0005] Nano drugs can embed gene drugs (siRNA or Crispr-cas9 plasmid, etc.) due to their small size effect, and can also modify targeting molecules and intelligent response groups, etc. Therefore, they have the advantages of EPR (high permeability and retention of solid tumors), extended elimination half-life of drugs, targeted delivery, controlled release, and penetration of body barriers (blood-brain barrier), and have made great progress in cancer treatment. Representatives of nano drugs are liposome doxorubicin (Doxil) and albumin paclitaxel (Abraxane), the former reduces the cardiotoxicity of doxorubicin, while the latter avoids the allergic reaction of the solvent castor oil. Inorganic nanoparticles such as inert metals (gold, silver, platinum), magnetic Fe3O4, mesoporous silica nanoparticles, quantum dots, etc. are used as nano drug carriers. Ultra-small nanoparticles can be metabolized by the kidneys, but this is at the expense of impaired renal function (Med.Chem.2014,21:3837-3853). Therefore, biodegradable nanomaterials are increasingly used in the biopharmaceutical field, such as PLGA (polylactic-co-glycolic acid), PLA (polylactic acid), cyclodextrin, chitosan, etc., which have been approved by the FDA.

[0006] The new generation of nanomedicines should not only be able to selectively deliver anticancer drugs to tumor tissues, but also release anticancer drugs in a controlled or intelligent response manner once they reach the tumor tissues. Researchers have explored many intelligent response nanomedicines, such as external stimuli (light, magnetic field, ultrasound, etc.) and internal stimuli (pH, temperature, enzymes, redox potential, etc.) (ACS Nano 2013, 7: 8455-8468). Among these reported intelligent response systems, the unique heterogeneous redox potential gradient in the tumor site has attracted widespread attention. Human cells are inevitably exposed to reactive oxygen species (ROS) from external factors (such as UV) and intracellular aerobic metabolism. A small amount of ROS is an important signal molecule that regulates the normal physiological activities of cells. High levels of ROS will act on biological macromolecules including DNA, causing damage to them and destroying their functions, becoming an important driving force for the occurrence of diseases such as tumors. A large number of studies have shown that the level of ROS in tumors is generally higher than that of normal controls from the same tissue source. Therefore, nanomedicines based on ROS smart response can be designed to selectively release anticancer drugs in ROS-rich tumor cells. Although there have been some reports on inorganic nanomedicines based on ROS smart response, there are few studies on biodegradable organic nanomedicines, which seriously restricts the development prospects of nanomedicines. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, according to an embodiment of the present invention, it is hoped to provide a method for preparing a nano-delivery system of anti-cancer chemotherapy drugs with biodegradability, intelligent response and targeted delivery, and propose a method for preparing a PLGA nano-drug modified with a targeted molecule loaded with an anti-cancer drug, and further verify the good anti-cancer effect and low toxic side effects of the nano-drug in in vivo cell experiments and in vitro animal experiments.

[0008] According to the embodiment, the present invention provides a method for preparing a biodegradable PLGA-TK-PEG nano drug carrier, comprising the following steps:

[0009] Using ethanolamine as an initiator, ring-opening polymerization of GA (glycolide) and LA (lactide) was carried out under the catalysis of 2-ethylhexanoate tin (II). Equal amounts of LA and GA were added to a Schlenk flask at 100-200°C, and then 2-hydroxyethyl disulfide and 2-ethylhexanoate tin (II) were added under nitrogen protection and stirring, and the reaction was carried out for 12-72 hours. The resulting viscous solution was diluted with chloroform, precipitated in ether, and dried in an oven to obtain PLGA-NH2.

[0010] In a Schlenk flask, TK (thioketal) was dissolved in DMF (N,N-dimethylformamide), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and HOBT (1-hydroxybenzotriazole) were added, and then PLGA-NH2 was added. The reaction was stirred at 30°C for 48 hours. The product was dialyzed (molecular weight cutoff (MWCO)) and freeze-dried to obtain PLGA-TK-COOH.

[0011] In a Schlenk flask, NH2-PEG-NH2 was dissolved in DMF, and then PLGA-TK-COOH, EDCI and HOBT were added. The reaction was stirred at 30°C for 48 hours. The product was dialyzed (molecular weight cutoff (MWCO)) and freeze-dried to obtain PLGA-TK-PEG-NH2.

[0012] In a Schlenk flask, FA was dissolved in DMF, EDCI and HOBT were added, and then PLGA-TK-PEG-NH2 was added. The reaction was stirred at 30°C for 72 h. The product was dialyzed (molecular weight cutoff (MWCO)) and freeze-dried to obtain PLGA-TK-PEG-FA.

[0013] Preferably, in the method for preparing the aforementioned biodegradable PLGA-TK-PEG nano drug carrier of the present invention, the molecular weight of PLGA is 2k-100k, and the molecular weight of PEG is 2k-20k.

[0014] According to the embodiments, the nano drug carrier prepared by the preparation method of the aforementioned biodegradable PLGA-TK-PEG nano drug carrier of the present invention is used to load chemotherapy drugs, anticancer Chinese medicine monomers or gene drugs to prepare targeted molecular modified smart response PLGA nano drug.

[0015] Preferably, the nano drug carrier prepared by the preparation method of the aforementioned biodegradable PLGA-TK-PEG nano drug carrier of the present invention is used to load chemotherapeutic drugs or anticancer Chinese medicine monomers to prepare a targeted molecule-modified smart response PLGA nano drug, comprising the following steps:

[0016] 5-1000 mg of PLGA-TK-PEG-FA polymer and 0.1-500 mg of chemotherapy drugs or anticancer Chinese medicine monomers are dissolved in 0.1-10 ml of chloroform or dichloromethane, and 0.1-100 mg of CD-Chol is dissolved in 0.1-10 ml of chloroform or dichloromethane. Ultrasonic emulsification is performed for 1-20 minutes under ice bath conditions of 20-500 W. Stirring or rotary evaporation is performed in a fume hood overnight to completely evaporate the chloroform or dichloromethane. PLGA nanoparticles with good performance are obtained by centrifugation, and the particle size is 50-300 nm.

[0017] Preferably, the nano drug carrier prepared by the preparation method of the aforementioned biodegradable PLGA-TK-PEG nano drug carrier of the present invention is used to load gene drugs to prepare the targeted molecular modified smart response PLGA nano drug, comprising the following steps:

[0018] 0.1-500 mg of gene drug is dissolved in 25 ml of DEPC water or double distilled water, and then PLGA-TK-PEG-FA and CD-Chol solution are added, and ultrasonic emulsification is performed for 1-20 min in an ice bath at 20-500 W. Stir or rotary evaporate in a fume hood overnight to completely evaporate the chloroform or dichloromethane, and PLGA nanoparticles with good performance are obtained by centrifugation, and the particle size is 50-300 nm.

[0019] The present invention has developed a PLGA nano drug loaded with gene drugs and modified with targeting molecule FA, which not only has a good targeting effect on liver cancer cells with high expression of folate receptor (FR), but also contains ketalthiolate groups, which can be controllably released by the high ROS level in liver cancer cells. And the application of the PLGA gene nano drug in disease treatment, especially in cancer treatment, such as common liver cancer, lung cancer, pancreatic cancer and brain cancer, mainly includes in vitro cell experiments, in vivo animal experiments and clinical experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the synthesis and application of FA-modified PLGA nanomedicine loaded with siRNA for ROS intelligent response;

[0021] Figure 2 The structural formula and NMR spectrum of the synthesized PLGA-TK-PEG polymer are shown below;

[0022] Figure 3 (A) TEM image and (B) particle size distribution diagram of PLGA nanoparticles, scale bar 100 nm;

[0023] Figure 4 The biocompatibility of PLGA-TK-PEG nanoparticles to (A) normal liver cells QSG-7701, (B) liver cancer cells HepG2 and (C) liver cancer cells Huh7;

[0024] Figure 5The intelligent response of PLGA nanomedicine loaded with Nile red (NR), the content of ROS in normal liver cells QSG-7701 and liver cancer cells HepG2 and Huh7, (A) fluorescence intensity of DCFH-DA observed by fluorescence microscope, (B) fluorescence value of DCFH-DA analyzed by flow cytometry, scale bar 200μm, (C) intelligent response of NR-loaded PLGA nanomedicine under different concentrations of H2O2;

[0025] Figure 6 The targeting effect of FA-modified NR-loaded PLGA nanomedicine, (A) flow cytometry analysis of the fluorescence value of NR in normal liver cells QSG-7701, liver cancer cells HepG2 and Huh7, (B) fluorescence microscopy analysis of the fluorescence intensity of NR, scale bar 25μm;

[0026] Figure 7 The knockdown efficiency of oncogene UBC12 in liver cancer cells HepG2 and Huh7 was verified by transfection efficiency of siRNA nanomedicine. (A) Fluorescence microscopy of Cy3-modified siRNA, scale 10 μm, (B) Flow cytometry analysis, (C) Western blot detection of knockdown efficiency of oncogene UBC12 in liver cancer cells HepG2 and Huh7 by PLGA nanocarrier and lipo2000 transfection reagent, β-actin was used as internal reference;

[0027] Figure 8 Knocking down the oncogene UBC12 can inhibit the proliferation of liver cancer cells HepG2 and promote their apoptosis. (A) The inhibitory effect of the PLGA gene drug on the proliferation of liver cancer cells HepG2, (B) AnnexinV-FITC cell flow cytometry apoptosis graph, (C) apoptosis statistics graph, *p<0.05, **p<0.01;

[0028] Fig. 9 Knocking down the oncogene UBC12 can inhibit the proliferation of liver cancer cell Huh7 and promote its apoptosis. (A) The inhibitory effect of the PLGA gene drug on the proliferation of liver cancer cell HepG2, (B) AnnexinV-FITC cell flow cytometry apoptosis graph, (C) apoptosis statistics graph, *p<0.05, **p<0.01;

[0029] Fig.10 The inhibitory effects of the PLGA gene drug, the PLGA gene drug control group (random NT sequence), free siRNA, and PLGA nanoparticles on Huh7 nude mouse subcutaneous tumors, (A) changes in tumor volume, (B) changes in mouse body weight during drug administration, (C) tumor size after taking tumor tissue, (D) tumor weight, (E) WB results of UBC12 protein knockdown in tumor tissues of each group, **p<0.01, ***p<0.001;

[0030] Fig.11 (A) Immunohistochemistry images of cleaved caspase-3, Ki-67 and UBC12, scale bar 50 μm, (B) immunohistochemistry statistical graph, *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. These embodiments should be understood to be only used to illustrate the present invention and not to limit the scope of protection of the present invention. After reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0032] The present invention has developed a PLGA nano-drug loaded with siRNA and modified with FA. The FA on the surface of the nano-drug can target the FR highly expressed on the membrane of liver cancer cells and enter the tumor cells through endocytosis. Under the condition of high ROS content in tumor cells, the loaded gene drug siRNA can be controllably released, thereby achieving the purpose of inhibiting liver cancer cells. The schematic diagram is shown in FIG. Figure 1 shown.

[0033] First, PLGA-TK-PEG polymer was synthesized and characterized by nuclear magnetic resonance, and its purity was above 90%. Figure 2 Then, PLGA nanoparticles were prepared by solvent evaporation method, and transmission electron microscopy (TEM) and dynamic light scattering (particle size distribution) were used to characterize the PLGA nanoparticles, as shown in FIG. Figure 3 As shown, the PLGA nanoparticles have a spherical morphology and their average diameter is approximately 90 nm.

[0034] In the United States, PLGA has passed FDA certification and has been officially included in the United States Pharmacopoeia as a pharmaceutical excipient. PEG modification can not only increase the water solubility of anticancer drugs, but also increase their half-life in the body and reduce immunogenicity. Therefore, the PLGA-TK-PEG nanoparticles theoretically have excellent biocompatibility. Figure 4 As shown, blank PLGA nanoparticles have no obvious cytotoxicity to normal liver cells QSG-7701, liver cancer cells HepG2 and Huh7, proving that the blank PLGA nanoparticles do have excellent biocompatibility.

[0035] The present invention is based on the fact that high levels of ROS in tumor cells can controllably open the thioketal group of nanomedicines. First, the content of ROS in normal liver cells QSG-7701 and liver cancer cells HepG2 and Huh7 was detected. Figure 5As shown in Figures A and 5B, the ROS content in liver cancer cells HepG2 and Huh7 is higher than that in normal liver cells, so the high ROS content in tumor cells can be used to controllably release PLGA nanomedicines with thioketal groups. Then, under the condition of 1×PBS buffer, the intelligent response of different concentrations of H2O2 to PLGA nanomedicines containing thioketal groups was verified. Figure 5 As shown in Figure C, under different concentrations of H2O2, NR-loaded PLGA nanodrugs can be degraded and released by H2O2.

[0036] In order to verify the excellent targeting of the PLGA gene drug, the anticancer drug was released only in tumor cells. The PLGA nanodrug loaded with NR (50 μg / ml) modified with the targeting molecule FA was incubated with normal liver cells QSG-7701 and liver cancer cells HepG2 and Huh7 for 4 hours. Figure 6 As shown in A, the normal liver cell QSG-7701 has a slight rightward shift, which is due to the nonspecific adsorption of QSG-7701. Compared with the normal cell QSG-7701, the PLGA nanoparticles are more targeted on the surface of liver cancer cells HepG2 and Huh7, which is due to the specific adsorption of FR highly expressed on the surface of these two liver cancer cells, thereby achieving the purpose of targeted delivery of anticancer drugs. After fixation with 4% paraformaldehyde and staining of the cell nucleus with 0.5μg / ml Hoechst, Figure 6 The fluorescence microscopy analysis results of B showed that liver cancer cells can endocytose more PLGA nanodrugs, so the targeting molecule FA can play the purpose of specifically delivering anticancer gene drugs.

[0037] In order to test the knockdown efficiency of the PLGA gene nanodrug on liver cancer cells HepG2 and Huh7 in in vitro cell experiments, the transfection efficiency of the gene nanodrug in liver cancer cells was first observed, and then the gene nanodrug and lipo2000 transfection reagent were used to transfect and knock down the cancer gene UBC12, and their knockdown efficiency was observed by Western blot. The sequence of the siRNA-loaded FA-modified PLGA gene nanodrug, whose Cy3-modified siRNA knockdown cancer gene UBC12 is: 5'-ACUCCAUAAUUUAUGGCCUGCAGUATT-3'. Among them, the loading amount of siRNA in the PLGA gene nanodrug can be firstly made into a standard curve based on the ultraviolet absorption value of Cy3 at 550nm using Cy3-labeled siRNA, and then the PLGA gene nanodrug is dissolved in 0.5M NaOH solution, the amount of Cy3-siRNA is measured, and then the loading amount of siRNA in the PLGA nanodrug is calculated. Then, the PLGA gene nanomedicine was incubated with liver cancer cells HepG2 and Huh7 for 24 hours, and the transfection efficiency of the gene nanomedicine was analyzed by fluorescence microscopy and flow cytometry. Figure 7 As shown in A, the transfection efficiency of the gene nanomedicine in liver cancer cells is relatively high, at over 95%. Figure 7 The flow cytometry results of B also confirmed that the gene nanodrug could be well transfected into liver cancer cells. Finally, Western blot was used to detect the knockdown efficiency of the oncogene UBC12 by the PLGA gene nanodrug and lipo2000 transfection reagent. 6 HepG2 and Huh7 cells were inoculated in 6-well plates and cultured for 24 hours. HepG2 and Huh7 liver cancer cells were co-incubated with PLGA gene nanomedicine (containing 1 μg / ml siRNA) or gene nanomedicine containing random NT sequences for 72 hours, and then the knockdown efficiency was detected by Western blot. Figure 7 As shown in Figure C, in liver cancer cells HepG2 and Huh7, the PLGA can knock down UBC12 well, while its random NT sequence cannot effectively knock down the oncogene UBC12. Although the knockdown efficiency is slightly weaker than that of the transfection reagent lipo2000, PLGA gene nanomedicine can be tried in animal models and clinical applications, so it still has great clinical exploration value.

[0038] Then, we analyzed the proliferation and apoptosis of liver cancer cells after knocking down the UBC12 gene. 3HepG2 and Huh7 cells were seeded in 96-well plates and cultured for 24 hours. PLGA gene nanomedicine (containing 1 μg / ml siRNA) or gene medicine containing NT sequence was co-incubated with HepG2 and Huh7 cells for 72 hours, and then CCK-8 was used to detect cell proliferation. Figure 8 As shown in A, knocking down UBC12 can effectively inhibit the proliferation of liver cancer cells HepG2, while random NT sequences have little effect on the proliferation of HepG2. Subsequently, the apoptosis of liver cancer cells HepG2 after knocking down UBC12 was verified. Figure 8 As shown in AnnexinV-FITC cell flow cytometry apoptosis graphs and apoptosis statistical analysis in B and 8C, after the oncogene UBC12 was knocked down by the PLGA nanomedicine, the apoptosis of liver cancer cells HepG2 increased significantly, while the NT sequence had little effect on the apoptosis of HepG2. The above results show that the PLGA gene nanomedicine can inhibit the proliferation of liver cancer cells HepG2 and promote their apoptosis by knocking down the UBC12 oncogene.

[0039] Fig. 9 The proliferation and apoptosis experiments of liver cancer cells Huh7 in A, 9B and 9C showed that the PLGA gene nanomedicine knocked down the oncogene UBC12, inhibited the proliferation of liver cancer cells Huh7, and promoted its apoptosis, while the random NT sequence could not affect the proliferation and apoptosis of liver cancer cells Huh7. In summary, the PLGA nanomedicine can effectively knock down the oncogene UBC12, and after UBC12 is knocked down, it will inhibit the proliferation of liver cancer cells and promote their apoptosis. The random NT sequence has little effect on the proliferation and apoptosis of liver cancer cells.

[0040] 3×10 6 Huh7 cells, after 7 days, formed a cell mass of about 100 mm 3 The mice with subcutaneous tumors were divided into 5 groups (n=6 in each group) according to the average tumor volume. They were given 100μl of PBS, PLGA nanoparticles (about 0.1mg), free siRNA (6μg), random siRNA-loaded PLGA nanoparticles, and siRNA-loaded PLGA nanoparticles (containing 6μg siRNA) respectively, and the drugs were injected into the tail vein every three days. Before drug administration, the tumor volume was measured with a vernier caliper and the mice were weighed. The tumor volume was calculated by (length × width 2 ) / 2. After three weeks of treatment, all mice in the five groups were sacrificed, and tumor tissues were collected for detection of tumor burden, cleaved caspase-3 and Ki-67 immunohistochemistry, and UBC12 knockdown efficiency (Western blot or immunohistochemistry).

[0041] like Fig.10 As shown in A, only siRNA-loaded PLGA nanodrugs have a significant inhibitory effect on Huh7 subcutaneous tumors, while free siRNA has no significant inhibitory effect on Huh7 subcutaneous tumors. This may be because free siRNA is unstable in body fluids and cannot enter cells. Even if a small amount of free siRNA enters cells, it is easily affected by its degradation enzymes in lysosomes or cytoplasm. NT sequence siRNA-loaded PLGA nanodrugs also have no significant inhibitory effect on Huh7 subcutaneous tumors, which is consistent with the knockdown efficiency results in in vitro cell experiments. This shows that NT sequence siRNA cannot knock down UBC12, and also proves the excellent selectivity of the PLGA gene nanodrug. Fig.10 As shown in Figure B, the five groups of mice did not lose a lot of weight, indicating that the toxic side effects of both PLGA nanoparticles and siRNA-loaded PLGA nanodrugs were not very obvious, and the PLGA gene nanodrug had excellent biocompatibility. Fig.10 C and Fig.10 As shown in Figure D, after the mice were killed and the tumor tissues were collected, the weighing results of the tumor tissues also showed that the siRNA-loaded PLGA nanodrugs were more effective in inhibiting the occurrence and development of tumors than the NT sequence siRNA nanodrugs or free siRNA. Fig.10 As shown in E, the WB results of UBC12 in tumor tissues showed that only the PLGA gene nanomedicine group had the phenomenon of knocking down UBC12, which proved the effect of the siRNA nanomedicine in inhibiting liver cancer cells at the protein level.

[0042] Tumor specimens were subjected to gravimetric analysis, formalin fixation, paraffin sectioning, and immunohistochemical analysis. Fig.10 A and Fig.10 As shown in B, the results of immunohistochemical cleaved caspase-3 and Ki-67 analysis showed that the PLGA gene nanomedicine had less tumor proliferation factor (Ki-67) and more apoptosis factor (cleaved caspase-3) compared with other groups, and therefore was more able to inhibit the growth of liver cancer cells Huh7. Among them, the immunohistochemical results of UBC12 showed that the siRNA nanomedicine had a significant effect of knocking down UBC12, thereby further inhibiting the Neddylation pathway and inhibiting the occurrence and development of liver cancer.

[0043] In summary, the present invention has developed a PLGA nanodrug with FA modified on the surface and loaded with UBC12 siRNA. Through the targeting effect of FA, the nanodrug can specifically deliver UBC12 siRNA only to tumor cells through endocytosis, and the high ROS level in tumor cells can break the ketal thiol group of the nanodrug, achieving the purpose of controlled release. In vitro cell experiments and in vivo animal experiments show that the PLGA gene nanodrug can effectively knock down the UBC12 protein of tumor cells, inhibit the activity of Neddylation, and promote the apoptosis of tumor cells, thereby achieving the purpose of inhibiting the occurrence and development of liver cancer. The ROS intelligent responsive, FA-targeted PLGA nanodrug has certain practical guiding significance in the research and development of anti-cancer gene nanodrugs, especially in the process of siRNA gene drugs going to the clinic.

Claims

1. A biodegradable targeted molecule-modified PLGA nanomedicine, characterized in that: The nano drug comprises a PLGA-TK-PEG-FA nano drug carrier and a loaded gene drug; Wherein, the targeting molecule is folic acid; Wherein, the nano drug is prepared by a method comprising the following steps: 0.1-500 mg of gene medicine is dissolved in 25 ml of DEPC water or double distilled water, and then PLGA-TK-PEG-FA nano drug carrier and DC-Chol solution are added, wherein the DC-Chol solution is obtained by dissolving DC-Chol in dichloromethane or chloroform; ultrasonic emulsification is performed for 1-20 minutes under ice bath conditions of 20-500 W, stirring or rotary evaporation is performed in a fume hood overnight to completely volatilize chloroform or dichloromethane, and PLGA nano medicine with good performance is obtained by centrifugation; The nano drug can controllably release the loaded gene drug in the presence of 0.1-100 mM H2O2; the gene drug is siRNA, and the sequence of the siRNA is 5'-ACUCCAUAAUUUAUGGCCUGCAGUATT-3'; Wherein, the PLGA nanomedicine is used to target liver cancer cells.

2. The nano drug according to claim 1, characterized in that The PLGA-TK-PEG-FA nano drug carrier is prepared by a preparation method comprising the following steps: Using ethanolamine as an initiator, GA and LA are ring-opening polymerized under the catalysis of 2-ethylhexanoate tin (II); adding equal amounts of LA and GA into a Schlenk flask at 100-200° C., then adding 2-hydroxyethyl disulfide and 2-ethylhexanoate tin (II) under nitrogen protection and stirring, and reacting for 12-72 hours; diluting the obtained viscous solution with chloroform, precipitating in ether, and drying in an oven to obtain PLGA-NH2; In a Schlenk flask, TK was dissolved in DMF, EDCI and HOBT were added, and then PLGA-NH2 was added. The reaction was stirred at 10-60°C for 12-72h. The product was dialyzed and freeze-dried to obtain PLGA-TK-COOH; In a Schlenk flask, NH2-PEG-NH2 was dissolved in DMF, and then PLGA-TK-COOH, EDCI and HOBT were added. The reaction was stirred at 10-60°C for 12-72h. The product was dialyzed and freeze-dried to obtain PLGA-TK-PEG-NH2. In a Schlenk flask, FA was dissolved in DMF, EDCI and HOBT were added, and then PLGA-TK-PEG-NH2 was added. The reaction was stirred at 10-60°C for 24-96h. The product was dialyzed and freeze-dried to obtain PLGA-TK-PEG-FA nanodrug carrier.

3. The nano drug according to claim 2, characterized in that: The molecular weight of PLGA is between 2k-100k, and the molecular weight of PEG is between 2k-20k.

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