Nano-drug taking FABP5 as target and capable of improving liver cancer radiofrequency ablation curative effect as well as preparation method and application of nano-drug

Abstract

The invention relates to the field of anti-tumor drugs, and discloses a nano medical drug taking FABP5 as a target as well as a preparation method and application of the nano medical drug. The nano medical drug is prepared by integrating a lipid nano shell with pH response, cycloarginine-glycine-aspartic acid and an erythrocyte membrane, and encapsulating nano particles of superparamagnetic iron oxide and sgFABP5. The nano medical medicine has the application potential of synergistically enhancing the tumor treatment effect after the liver cancer radiofrequency ablation operation.

Description

Technical Field

[0001] This application relates to the field of anti-tumor drugs. More specifically, it relates to a nano-drug targeting FABP5 for enhancing the efficacy of radiofrequency ablation in liver cancer, its preparation method and applications. Background Art

[0002] Hepatocellular carcinoma is the fourth leading cause of cancer death globally. Radiofrequency ablation (RFA) is an important means of local treatment for hepatocellular carcinoma. Its principle is that the electrode needle provides an alternating current to generate a high-frequency electromagnetic field, causing the molecules in the tumor tissue to collide and rub against each other to generate heat. The local temperature of the tumor tissue and its surrounding area can reach 60-120°C, so that the tumor cells undergo coagulative necrosis. However, RFA still has limitations. The "heat sink effect" leads to a reduction in the ablation area, incomplete tumor ablation, an increased local recurrence rate after ablation of tumors near major blood vessels, and poor patient prognosis, which is a key limiting factor leading to the recurrence and progression of residual cancer.

[0003] Fatty acid-binding protein 5 (FABP5) is a chaperone protein for intracellular fatty acids and plays a key role in lipid metabolism and cell proliferation. FABP5 is highly expressed in various cancers and is closely related to the growth, development and metastasis of tumors. It also affects the survival and function of immune cells in the tumor microenvironment (TME), making it an attractive target for novel therapeutic strategies. FABP5 is also a biomarker for ferroptosis. Inhibiting FABP5 will lead to fatty acid accumulation, excessive generation of ferrous ions and lipid peroxidation, ultimately triggering ferroptosis. In addition, our research found that the expression of FABP5 increases after RFA treatment. Reducing the level of FABP5 can significantly improve the therapeutic effect on HCC. FABP5 is a potential target affecting the RFA effect in liver cancer. Knockdown of FABP5 not only inhibits the growth and metastasis of liver cancer, but also enhances the efficacy of radiofrequency ablation in liver cancer. It can also further inhibit the progression of liver cancer by promoting RFA-induced immune cell death (ICD), enhancing the antigen presentation ability of dendritic cells (DCs) and promoting T cell infiltration.

[0004] When knocking down FABP5 in combination with radiofrequency ablation (RFA) of liver cancer, the inhibition of FABP5 expanded and maintained the anti-tumor immune response induced by RFA, in which the activation of the STING / TBK1 pathway played a key role. In addition, nanocarrier-mediated FABP5 deficiency promoted RFA-induced immunogenic cell death (ICD), enhanced the antigen presentation ability of dendritic cells (DCs) and promoted T cell infiltration, increasing the efficacy of RFA for liver cancer. Developing novel drugs and intervention measures targeting FABP5 mRNA is a new research direction to improve the treatment effect of liver cancer. Using small guide RNA (sgRNA) to target FABP5 mRNA is the main method to inhibit FABP5 expression. However, naked sgRNAs have poor stability and are easily degraded, making it difficult for them to reach the tumor site and exert their effects. Lipid nanoparticles (LNPs) are lipid-formed nanoparticles commonly used to deliver sgRNAs and can be enriched in tumor tissues through the retention effect and active targeting.

[0005] Therefore, it is necessary to design a feasible therapeutic nanomedicine to improve the stability of sgRNAs, effectively avoid their degradation, and enable them to reach the tumor site smoothly and exert their effects. Summary of the Invention

[0006] To solve the technical problem that naked sgRNAs have poor stability and are easily degraded, making it difficult for them to reach the tumor site and exert their effects, the present invention provides the preparation and application of a nanomedicine targeting FABP5. Specifically, pH-responsive lipid nanoshells (phLips) are integrated with cyclic arginine-glycine-aspartic acid (cRGD) and red blood cell membranes (RBCM), encapsulating superparamagnetic iron oxide (SPIO) and sgFABP5, endowing it with higher tumor targeting and longer efficacy, and having better effects when combined with RFA.

[0007] To achieve the above invention purpose, the following technical solutions are adopted in this application:

[0008] In the first aspect, this application provides a nanomedicine targeting FABP5 (named: FS@RBCM / cRGD-phLip), which is a nanoparticle obtained by integrating pH-responsive lipid nanoshells with cyclic arginine-glycine-aspartic acid and red blood cell membranes, and encapsulating superparamagnetic iron oxide and sgFABP5.

[0009] In the second aspect, this application provides a preparation method of the nanomedicine described in the first aspect, including the following steps:

[0010] Step 1, synthesizing nanoparticles by the thin-film dispersion method: Dissolve Chol, DPPC, DOTAP, DSPE-PEOz, DSPE-PEG2K-cRGD, and Cy5.5 in an organic solvent and dissolve them by ultrasonic oscillation;

[0011] Step 2: Rotary evaporation. After evaporation, a thin film is formed on the inner wall of the flask, and it is left to dry overnight.

[0012] Step 3: Filtration through a membrane. After rotary evaporation to dryness, add nuclease-free water to dissolve it, ultrasonically disperse the mixture evenly, filter it through a 200 nm - 100 nm filter membrane, and store it in the dark at 4°C.

[0013] Step 4: Dilute liposomes.

[0014] Step 5: Loading plasmid into liposomes. Dissolve the pSpCas9-sgRRM2 plasmid with DEPC water, mix it according to the mass ratio of plasmid:liposome of 1:(1 - 8), vortex it, and incubate it at room temperature to obtain the product.

[0015] In a third aspect, the present application provides the use of the nanomedicine drug described in the first aspect as an enhancer in the radiofrequency ablation treatment of liver cancer.

[0016] Furthermore, the above use refers to administering the nanomedicine drug for treatment after radiofrequency ablation of liver cancer.

[0017] In a fourth aspect, the present application provides the use of the nanomedicine drug described in the first aspect in combination therapy with radiofrequency ablation treatment of liver cancer and the ferroptosis inhibitor Lipro1.

[0018] In summary, the present application has the following beneficial effects:

[0019] The nanomedicine drug FS@RBCM / cRGD-phLip provided by the present application has the potential for synergistically enhancing the tumor treatment effect after radiofrequency ablation of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :(A) Schematic diagram of the construction of FS@RBCM / cRGD-phLip; (B - C) Representative transmission electron microscope (TEM) images showing the morphology of the modified lipid nanoparticles; (D) Dynamic light scattering (DLS) analysis showing the size distribution of the nanoparticles; (E) Elemental analysis of the nanoparticles using TEM; (F) Statistical histogram showing the size measurement results of five different LNP formulations; (G) Zeta potential analysis comparing the surface charges of the formulations; (H) Agarose gel electrophoresis evaluating the binding efficiency of LNP-sgRNA complexes with different mass ratios.

[0021] Figure 2 :(A) Immunofluorescence micrographs and time-course analysis of the mean fluorescence intensity (MFI) in LO2 and HCC-LM3 cells.

[0022] Figure 3: (A) Coomassie blue staining was used to compare the protein compositions in RBCM, RBCM-cRGD conjugate, and dual-targeted RBCM / cRGD-phLips nanoparticles; (B) Western blot analysis of the expression of CD47 in RBCM, RBCM-cRGD, and RBCM / cRGD-phLips formulations with GAPDH as an internal control and CD47 as an erythrocyte marker; (C) Western blot results showed the expression of FABP5 in HCC-LM3 and SK-HEP-1 cells treated with or without LNP-encapsulated sgFABP5; (D) Cell viability assay was performed to compare the effects of @phLips, @RBCM / cRGD-phLips, and SPIO@RBCM / cRGD-phLips on SK-HEP-1 and HCC-LM3 cells; (E) Representative nuclear magnetic resonance imaging scans were used to illustrate the progression of HCC under different treatment methods, as well as the quantitative analysis of the relative T2-weighted signal intensities of SPIO@phLips and SPIO@RBCM / cRGD-phLips; (F-G) Fluorescence imaging of the distribution of nanoparticles inside and outside organs and tumors.

[0023] Figure 4 : (A) Flowchart of the mouse model protocol after RFA; (B) Comparison of tumor sizes under different treatment conditions after RFA; (C-D) Statistical histograms of tumor weights and volumes in each group; (E) Line graph of tumor growth kinetics in each treatment group; (F) Line graph representing the individual growth trajectories of mice; (G) Statistical histogram showing the body weight changes of mice in each treatment group.

[0024] Figure 5 : (A) Immunofluorescence images showing CD8+ cells in HCC tumor tissues; (B) Representative images of CD8+ and GZMA+ cells in HCC tumor tissues, as well as the statistical percentage of GZMA+ cells among CD8+ cells; (C-F) Quantification levels of TNF-α (C), IL-2 (D), IL-12 (E), and INF-γ (F) in HCC tumor tissues under different treatment conditions.

[0025] Figure 6 : (A) Schematic flowchart of the animal model for treating HCC cells with the ferroptosis inhibitor Lipro1, RFA, and the nanoparticle combination delivery system; (B) Comparison of tumor sizes in each treatment group; (C) Statistical histogram of tumor weights in each group; (D) Violin plot describing the tumor volume distribution in each treatment group; (E) Line graph showing the individual growth trajectories of mice; (F) Tumor growth curves in each treatment group; (G) Histogram showing the body weight changes of mice in different treatment groups; (H) Representative H&E images of the tumor tissue morphology in each treatment group. Detailed implementation

[0026] The technical solutions and effects of this application will be further described in detail below in conjunction with embodiments and accompanying drawings. It can be understood that the specific embodiments described herein are only used to explain the invention-creation and are not intended to limit the invention-creation.

[0027] In the FS@RBCM / cRGD-phLips of the present invention, we encapsulated LNPs with red blood cell membranes, encapsulated sgRNA conjugated with FABP5 Cy5.5 in LNPs with a diameter of 100 nm, and connected RGD polypeptides to the LNPs. Cyanine dye 5.5 (Cy5.5) and superparamagnetic iron oxide (SPIO) were sandwiched between the lipid layers to form part of the LNP core. The mass ratio of LNPs to sgRNA was 1:4. LNPs are lipid-formed nanoparticles used to deliver sgRNA and can be enriched in tumor tissues through the retention effect and active targeting. Adding RGD peptides to traditional LNPs can make LNPs have stronger targeting. In addition, RGD peptides can also promote the penetration of drugs and improve their bioavailability, thereby enhancing the therapeutic effect. Superparamagnetic iron oxide (SPIO) endows LNPs with good biocompatibility and high sensitivity of magnetic resonance imaging signals, enabling precise detection and tracking.

[0028] In addition, to simulate the RFA conditions, we performed in vitro heat treatment of cells at 45 °C to construct high-metastasis human hepatocellular carcinoma cell lines HCC-LM3, human hepatocellular carcinoma cell line SK-HEP1, and murine hepatocarcinoma cell line Hepa1-6. And we further established a mouse subcutaneous tumor-bearing model, injecting 1×10 6 cells / mL of Hepa1-6 cells subcutaneously into the inner side of the right lower limb of the mouse. When the tumor volume reached approximately 100 mm 3 , an 18G bipolar ablation needle was inserted into the tumor tissue. The radiofrequency tail needle was connected to the radiofrequency transmitter, and ablation was performed at a temperature of 60 °C for two minutes. A mouse liver cancer radiofrequency ablation postoperative model was constructed and treated with FS@RBCM / cRGD-phLip.

[0029] Example 1: Preparation of nanoparticles

[0030] Grouping of nanoparticles:

[0031] (1) @RBCM / cRGD-phLips: Without SPIO, not loaded with pSpCas9-sgFABP5;

[0032] (2) SPIO@RBCM / cRGD-phLips: Containing SPIO, not loaded with pSpCas9-sgFABP5;

[0033] (3) sgFABP5@RBCM / cRGD-phLips: Without SPIO, loaded with pSpCas9-sgFABP5;

[0034] (4) FS@RBCM / cRGD-phLips: Containing SPIO, loaded with pSpCas9-sgFABP5.

[0035] Step 1, synthesis of nanoparticles by thin film dispersion method:

[0036] Calculate the corresponding masses according to the molecular weights of Chol, DPPC, DOTAP, DSPE-PEOz, DSPE-PEG2K-cRGD, and Cy5.5 and accurately weigh them. Dissolve them in an organic solvent (chloroform: methanol volume ratio 3:1 (1.5 ml: 0.5 ml)), and dissolve them by ultrasonic oscillation in an eggplant-shaped flask.

[0037] First, synthesize the groups without loading pSpCas9-sgFABP5, with and without SPIO, and preliminarily quantify the reagents: DPPC 14.66 mg; DOTAP 6.98 mg; CHOL 3.86 mg; Cy5.5 0.5 mg; DSPE-PEG2K-cRGD 6.70 mg; DSPE-PEOz 10.99 mg; SPIO 1.09 mg.

[0038] Step 2, rotary evaporation:

[0039] Use a rotary evaporator at 45 - 50 °C, 100 rpm, for 15 - 20 min. After evaporation, a thin film is formed on the bottle wall and dried overnight.

[0040] Step 3, membrane filtration:

[0041] After rotary evaporation, add 1 ml of nuclease-free water to dissolve it. Disperse the mixture evenly by an ultrasonic cleaner, and filter it using a liposome extruder (200 nm - 100 nm filter membrane), extrude about 9 times, and store it in the dark at 4 °C.

[0042] Step 4, dilution of liposomes:

[0043] The DOTAP in the liposomes is 0.0068 g, and the concentration is 6.8 μg / μl. Take 50 μl of liposomes and add 290 μl of DEPC water to dilute the DOTAP concentration to 1 μg / μl.

[0044] Secondly, synthesize the groups loaded with pSpCas9-sgFABP5, with or without SPIO.

[0045] Step 5, liposome loading of plasmid:

[0046] (1) Plasmid: pSpCas9-sgFABP5 plasmid;

[0047] (2) Centrifuge the sample at 12,000 r / min for 1 minute before opening to ensure that the lyophilized plasmid DNA is at the bottom of the collection tube;

[0048] (3) Initially determine the mass ratios: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8. Mix plasmid (pSpCas9-sgFABP5) and liposome (LP) according to different mass ratios, gently vortex for 30 s, and incubate at room temperature for 15 min.

[0049] As Figure 1 shown, FS@RBCM / cRGD-phLip was successfully prepared. Figure 1 B and Figure 1 C show the @RBCM / cRGD-phLips nanoparticles under transmission electron microscopy (TEM). These nanoparticles exhibit a spherical structure and uniform morphology. The hydrodynamic diameter of the nanoparticles is 136 ± 28 nm ( Figure 1 D), within which the nanoparticles can effectively penetrate cell membranes and have good biodistribution characteristics, suitable for tumor-targeted drug delivery. Elemental analysis confirmed the presence of iron (Fe) element on its surface ( Figure 1 E). The particle size distribution range of the nanoparticles is 150 - 200 nm ( Figure 1 F), and the Zeta potential range is 20 - 30 mV. Gel electrophoresis analysis showed that when sgRNA was mixed with lipid nanoparticles (LNPs) at a mass ratio of 1:4, sgRNA could be successfully encapsulated into LNPs ( Figure 1 H).

[0050] Example 2: Construction of disease model

[0051] (1) To study the synergistic anti-tumor effect of FABP5-targeted nanoparticles and RFA in vivo, we established a subcutaneous tumor-bearing model by injecting 1×10 6 cells / mL of Hepa1-6 cells subcutaneously into the inner side of the right lower limb of mice.

[0052] The mice were evenly divided into five groups:

[0053] 1) At the end of the 21-day experiment on the tumors of the control group mice, the mice will be sacrificed to harvest tumor tissues, and during this period, the tumor weight, volume and growth status of the mice will be recorded.

[0054] 2) RFA+RP treatment group. On the 8th, 11th, 14th, 17th, and 21st days after tumor inoculation, RP was directly injected into the mice for treatment. On the 11th day, an 18G bipolar ablation needle was placed into the tumor tissue, the radiofrequency tail needle was connected to the radiofrequency transmitter, the radiofrequency temperature was set at 60°C, and radiofrequency ablation was repeated 2 times per minute. During this period, the tumor weight, volume, and growth status of the mice were recorded, and the mice were sacrificed on the 21st day to harvest the tumor tissue.

[0055] 3) RFA+SRP treatment group. On the 8th, 11th, 14th, 17th, and 21st days after tumor inoculation, SRP was directly injected into the mice for treatment. On the 11th day, an 18G bipolar ablation needle was placed into the tumor tissue, the radiofrequency tail needle was connected to the radiofrequency transmitter, the radiofrequency temperature was set at 60°C, and radiofrequency ablation was repeated 2 times per minute. During this period, the tumor weight, volume, and growth status of the mice were recorded, and the mice were sacrificed on the 21st day to harvest the tumor tissue.

[0056] 4) RFA+FRP treatment group. On the 8th, 11th, 14th, 17th, and 21st days after tumor inoculation, FRP was directly injected into the mice for treatment. On the 11th day, an 18G bipolar ablation needle was placed into the tumor tissue, the radiofrequency tail needle was connected to the radiofrequency transmitter, the radiofrequency temperature was set at 60°C, and radiofrequency ablation was repeated 2 times per minute. During this period, the tumor weight, volume, and growth status of the mice were recorded, and the mice were sacrificed on the 21st day to harvest the tumor tissue.

[0057] 5) RFA+FSRP treatment group. On the 8th, 11th, 14th, 17th, and 21st days after tumor inoculation, FSRP was directly injected into the mice for treatment. On the 11th day, an 18G bipolar ablation needle was placed into the tumor tissue, the radiofrequency tail needle was connected to the radiofrequency transmitter, the radiofrequency temperature was set at 60°C, and radiofrequency ablation was repeated 2 times per minute. During this period, the tumor weight, volume, and growth status of the mice were recorded, and the mice were sacrificed on the 21st day to harvest the tumor tissue.

[0058] It can be seen from Figure 4 that in the in vivo experiment of mice, RP was used as the control group, and SRP, FRP, and FSRP were used as the treatment groups for experimental research ( Figure 4 A). The experimental results showed that compared with the control group, the tumor weight and volume of each treatment group were significantly reduced, and the tumor growth rate was significantly slowed down. Among them, the nanoparticles loaded with superparamagnetic iron oxide (SPIO) or single-stranded guide FABP5 gene editing system (sgFABP5) alone showed comparable anti-tumor effects, while the dual-loaded nanoparticles (SPIO / sgFABP5) showed the strongest tumor suppression effect ( Figure 4 B-F). In addition, there were no significant changes in the body weights of all experimental group mice, and no obvious cytotoxicity of the nanoparticles was observed during the entire experimental period ( Figure 4 G).

[0059] (2) Previous evidence indicated that the anti-tumor effect of FABP5-targeted nanoparticles combined with RFA was associated with the induction of ferritin deposition in HCC. To verify this mechanism, we used the ferritin deposition inhibitor Lipro1 and established a subcutaneous tumor-bearing model. Hepa1-6 cells at a density of 1×10 6 cells / mL were injected subcutaneously into the medial side of the right lower limb of mice.

[0060] The mice were evenly divided into 4 groups:

[0061] 1) In the control group, the mice were sacrificed at the end of the 20-day experiment to harvest tumor tissues. During this period, the tumor weight, volume, and growth status of the mice were recorded.

[0062] 2) In the Lipro1 treatment group, Lipro1 was directly injected into the mice on days 7, 10, 13, 16, and 20 after tumor inoculation. During this period, the tumor weight, volume, and growth status of the mice were recorded, and the mice were sacrificed on day 20 to harvest tumor tissues.

[0063] 3) In the RFA+FSRP treatment group, FSRP was directly injected into the mice on days 7, 10, 13, 16, and 20 after tumor inoculation. On day 10, an 18G bipolar ablation needle was placed into the tumor tissue, the radiofrequency tail needle was connected to the radiofrequency transmitter, the radiofrequency temperature was set at 60°C, and radiofrequency ablation was repeated for 2 minutes. During this period, the tumor weight, volume, and growth status of the mice were recorded, and the mice were sacrificed on day 20 to harvest tumor tissues.

[0064] 4) In the Lipro1+RFA+FSRP treatment group, FSRP and Lipro1 were directly injected into the mice on days 7, 10, 13, 16, and 20 after tumor inoculation. On day 10, an 18G bipolar ablation needle was placed into the tumor tissue, the radiofrequency tail needle was connected to the radiofrequency transmitter, the radiofrequency temperature was set at 60°C, and radiofrequency ablation was repeated for 2 minutes. During this period, the tumor weight, volume, and growth status of the mice were recorded, and the mice were sacrificed on day 20 to harvest tumor tissues.

[0065] As Figure 6 known, to clarify the mechanism of action of dual-loaded nanoparticles in exerting anti-hepatocellular carcinoma (HCC) effects by inducing ferroptosis, the experimental group combined the application of the ferroptosis inhibitor Lipro1 on the basis of dual-loaded nanoparticle treatment ( Figure 6 A). The results showed that compared with the control group, the dual-loaded nanoparticles exhibited a significant inhibitory effect on the weight, volume, and growth rate of tumor tissues, while the combined administration of Lipro1 could reverse the above effects ( Figure 6 B-F). Lipro1 did not show significant biotoxicity ( Figure 6 G).Figure 6 H further confirmed that Lipro1 could inhibit the anti-tumor effect mediated by sgFABP5.

[0066] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A nanomedicine drug targeting FABP5, characterized in that, The nano-medical drug is a nanoparticle that integrates a pH-responsive lipid nano-shell with cyclo-arginine-glycine-aspartic acid and red blood cell membrane, encapsulating superparamagnetic iron oxide and sgFABP5.

2. The preparation method of the nanomedicine drug according to claim 1, characterized in that, It includes the following steps: Step 1, synthesizing nanoparticles by thin-film dispersion method: Dissolve Chol, DPPC, DOTAP, DSPE-PEOz, DSPE-PEG2K-cRGD, and Cy5.5 in an organic solvent and dissolve them by ultrasonic oscillation; Step 2, rotary evaporation. After evaporation, a thin film is formed on the bottle wall and dried overnight; Step 3, membrane filtration: After rotary evaporation to dryness, add nuclease-free water to dissolve it, ultrasonically disperse the mixture evenly, filter it through a 200nm - 100nm filter membrane, and store it in the dark at 4°C; Step 4, diluting liposomes; Step 5, liposome loading plasmid: Dissolve the pSpCas9-sgRRM2 plasmid with DEPC water, mix it according to the mass ratio of plasmid:liposome of 1:(1 - 8), vortex, and incubate at room temperature to obtain it.

3. Use of the nano-medical drug according to claim 1 as an enhancer in the radiofrequency ablation treatment of liver cancer.

4. The use according to claim 3, characterized in that, The said use refers to administering the nano-medical drug for treatment after radiofrequency ablation of liver cancer.

5. Use of the nano-medical drug according to claim 1 in combination therapy with radiofrequency ablation treatment of liver cancer and ferroptosis inhibitor Lipro1.

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