Drug-loaded biomimetic nanodecoys based on genetic engineering, their preparation methods and applications

Abstract

This invention belongs to the field of medicine, specifically disclosing a drug-loaded biomimetic nanodecoy based on genetic engineering, its preparation method, and its application. The drug-loaded biomimetic nanodecoy of this invention comprises CCR2-overexpressing nanovesicles, and the hydrophobic regions of the nanovesicles are loaded with curcumin. This invention also provides a method for preparing the drug-loaded biomimetic nanodecoy based on genetic engineering and its application. The overexpressed CCR2 is used to adsorb excess CCL2 to inhibit the binding of macrophages to CCL2, preventing macrophage chemotaxis and subsequent TGF-β production. This inhibits the activation of hepatic stellate cells by removing pro-fibrotic mediators upstream; simultaneously, curcumin is released to block the downstream TGF-β / Smad signaling pathway, thereby inhibiting the activation of hepatic stellate cells.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and in particular relates to a drug-loaded biomimetic nanodecoy based on genetic engineering, its preparation method and application. Background Technology

[0002] Liver fibrosis is a chronic disease caused by factors such as viral infections or metabolic disorders. If the process of liver fibrosis is not effectively controlled, it may eventually develop into cirrhosis, and even liver failure and hepatocellular carcinoma. However, there are currently no approved drugs for the treatment of liver fibrosis, making the development of novel anti-fibrotic therapies that can inhibit this pathological process highly promising.

[0003] The process of liver fibrosis is accompanied by a decrease in the degradation of extracellular matrix (ECM) proteins and an increase in their deposition, gradually forming fibrous scars to replace damaged liver tissue. Hepatic stellate cells (HSCs) are the main precursor cells of myofibroblasts that produce ECM and are key to the occurrence and development of liver fibrosis. In various types of chronic liver disease (CLD), approximately 82-96% of myofibroblasts are formed by the activation of hepatic stellate cells. Therefore, inhibiting hepatic stellate cell activation to alleviate liver fibrosis is a feasible therapeutic approach. Furthermore, the activation of hepatic stellate cells is not an isolated process; it also depends on interactions with other cells. Macrophages are one of the important cells involved in the progression of liver fibrosis. Peripheral monocytes can bind to chemokines, particularly CC motif chemokine ligand 2 (CCL2), via the chemokine receptor CCR2, chemokines, and chemotactic molecules, attracting damaged liver tissue and polarizing into pro-fibrotic macrophages. These macrophages release pro-fibrotic cytokines such as transforming growth factor β (TGF-β), promoting the activation of hepatic stellate cells. Theoretically, blocking the CCL2 / CCR2 axis could weaken the bridge between liver inflammation and hepatic stellate cell activation, offering new possibilities for treating liver fibrosis. The CCR2 inhibitor cenicriviroc, a dual CCR2 / CCR5 antagonist initially developed as an anti-HIV drug, has shown anti-fibrotic effects in animal models of liver fibrosis. It blocks the CCL2 / CCR2 signaling pathway by binding to the CCR2 and CCR5 receptors, respectively. However, this inhibitor failed to achieve its primary outcome in a phase II clinical trial. Therefore, precisely and effectively blocking the CCL2 / CCR2 signaling pathway in liver fibrosis remains a significant challenge. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned problems by providing a drug-loaded biomimetic nanodecoy based on genetic engineering, its preparation method, and its applications. The invention proposes the preparation of CCR2-overexpressing nanovesicles loaded with the hydrophobic drug curcumin. Through the overexpression of CCR2 on their surface, these nanovesicles specifically adsorb CCL2, thereby significantly inhibiting macrophage infiltration and reducing the production of pro-fibrotic mediators such as transforming growth factor β (TGF-β). Simultaneously, CNV-C improves the delivery of curcumin in the liver, enabling precise delivery and inhibiting hepatic stellate cell activation by blocking the downstream TGF-β / Smad signaling pathway.

[0005] A drug-loaded biomimetic nanodecoy based on genetic engineering is characterized by comprising CCR2 overexpressing nanovesicles, wherein the hydrophobic regions of the nanovesicles are loaded with curcumin.

[0006] In the aforementioned genetically engineered drug-loaded biomimetic nanodecoys, the nanovesicles are obtained by transfecting human embryonic kidney cell lines with a lentiviral vector carrying the CCR2 gene.

[0007] A liver fibrosis treatment drug comprising the aforementioned genetically engineered drug-loaded biomimetic nanodecoy.

[0008] Among the aforementioned drugs for treating liver fibrosis, the mechanism of action of these drugs in the treatment of liver fibrosis is as follows:

[0009] The drug accumulates in the fibrotic liver; overexpression of CCR2 and nanosize effect promote drug accumulation in the liver.

[0010] In fibrotic liver, the drug reduces CCL2 levels and inhibits macrophage recruitment by overexpressing CCR2, mimicking macrophage binding to CCL2.

[0011] The drug releases its internally loaded curcumin in the liver;

[0012] Curcumin blocks the TGF-β / Smad signaling pathway, directly inhibiting the activation of hepatic stellate cells.

[0013] Overexpression of CCR2 promotes the accumulation of nanovesicles in the liver and, through binding to CCL2, absorbs excess CCL2 in the liver, thereby reducing the infiltration of monocyte-derived macrophages and consequently reducing the production of the pro-fibrotic cytokine TGF-β, thus inhibiting hepatic stellate cell activation upstream. Curcumin mainly inhibits hepatic stellate cell activation through its direct pharmacological action, while CCR2-overexpressing nanovesicles indirectly inhibit hepatic stellate cell activation by improving drug delivery and regulating immune cell behavior. The combined use of both, acting simultaneously upstream and downstream, can effectively improve therapeutic efficacy and achieve more effective treatment for liver fibrosis.

[0014] A method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering, comprising:

[0015] Prepare a lentiviral vector carrying the CCR2 gene and prepare a cell line;

[0016] The cell line was transfected with the lentiviral vector described above to construct CCR2-cells overexpressing CCR2.

[0017] The cell membranes of CCR2- cells were obtained through freeze-thaw treatment and centrifugation.

[0018] The obtained cell membranes were extruded using a liposome extruder containing a porous polycarbonate filter membrane to form vesicles of uniform size, thereby obtaining CCR2 overexpressing nanovesicles (CNVs).

[0019] Curcumin was loaded into the hydrophobic region of the nanovesicles to form a yellow suspension, thus obtaining CNV-C.

[0020] In the above-mentioned method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering, the method for preparing nanovesicles (CNVs) using CCR2-overexpressing CCR2-cells specifically includes:

[0021] Resuspend CCR2- cells in a cell membrane extraction reagent containing 1 mM PMSF and incubate on ice for several minutes;

[0022] The cell suspension was subjected to two freeze-thaw cycles, and then centrifuged at 1000±200g for several minutes at 4℃.

[0023] Collect the supernatant and centrifuge at 16,000±6000g at 4°C for several minutes to separate cell membrane fragments;

[0024] The cell membrane was redispersed in PBS phosphate buffer, and the protein content was measured.

[0025] Subsequently, the cell membrane was extruded sequentially through high-pore and low-pore porous polycarbonate filter membranes using an extruder to obtain CCR2-overexpressing nanovesicles (CNVs).

[0026] In the above-mentioned method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering, an ice bath is used for 10-20 minutes, such as 15 minutes.

[0027] Centrifuge at 1000±200g at 4℃ for 5-30 minutes, such as 10 minutes;

[0028] Centrifuge at 16,000±6000g at 4℃ for 20-50 minutes, or 30 minutes, to separate cell membrane fragments;

[0029] The high pore size is 300nm-500nm, such as 400nm, and the low pore size is 100nm-300nm, such as 200nm.

[0030] In the above-mentioned method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering, loading curcumin into the hydrophobic region of the nanovesicles to form a yellow suspension to obtain CNV-C specifically includes:

[0031] The prepared nanovesicles (CNVs) were mixed with curcumin in a buffer solution at a ratio of 1:(1±0.2), preferably at a ratio of 1:1, and stirred for 30 minutes at room temperature.

[0032] The mixture was centrifuged at 16,000±6000g at 4°C to obtain CNV-C.

[0033] In the above-mentioned method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering, the cell line is the human embryonic kidney cell line.

[0034] In the above-mentioned method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering, the cell line is HEK293T, HEK293A, or HEK293E.

[0035] The advantages of this invention are:

[0036] By constructing a cell line that overexpresses CCR2 to obtain cell membrane-derived nanovesicles, on the one hand, we can obtain nanovesicles that express CCR2 on the surface, and on the other hand, we can load hydrophobic drugs into the lipid bilayer of the cell membrane structure to realize a genetically engineered CCR2 overexpression nanovesicle CNV-C loaded with curcumin.

[0037] In CNV-C, overexpressed CCR2 is used to adsorb excess CCL2 to inhibit the binding of macrophages to CCL2, thereby preventing macrophage chemotaxis and the subsequent production of TGF-β. This inhibits the activation of hepatic stellate cells by removing pro-fibrotic mediators upstream. At the same time, curcumin is released to block the downstream TGF-β / Smad signaling pathway, thereby inhibiting the activation of hepatic stellate cells.

[0038] Overexpression of CCR2 promotes the accumulation of nanovesicles in the liver and, by binding to CCL2, absorbs excess CCL2 in the liver, thereby reducing the infiltration of monocyte-derived macrophages and thus reducing the production of the profibrotic cytokine TGF-β. Attached Figure Description

[0039] Figure 1 This is a schematic diagram illustrating the preparation process of the drug-loaded biomimetic nanodecoy based on genetic engineering and the treatment mechanism of liver fibrosis according to the present invention.

[0040] Figure 2 These are the preparation and physicochemical characterization diagrams of CNV-C of this invention;

[0041] Figure 3 This is a graph showing the anti-fibrotic efficacy of CNV-C in vivo.

[0042] Figure 4 This is an analysis and comparison of inflammation and fibrosis-related biomarkers in mice after drug treatment in different groups;

[0043] Figure 5 This validates the molecular mechanism by which CNV-C alleviates liver fibrosis.

[0044] Figure 6 It refers to the pharmacokinetics and biodistribution of CNV-C in vivo;

[0045] Figure 7 This is a diagram showing the biosafety assessment results of CNV-C;

[0046] Figure 8 This refers to the serum CCL2 level in mice 8 hours after administration of 300 mg / kg APAP.

[0047] Figure 9 This refers to the inhibitory effect of CNV-C on macrophage migration and hepatic stellate cell activation in vitro;

[0048] In the figure: *, p<0.05; **, p<0.01; ***, p<0.001; ns, no significance (p>0.05). Detailed Implementation

[0049] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0050] This invention provides a drug-loaded biomimetic nanodecoy based on genetic engineering, its preparation method, and its application in liver fibrosis. Specifically, cell membranes are extracted from a cell line overexpressing CCR2 to prepare CCR2-overexpressing nanovesicles (CNVs). Curcumin (CUR) is then loaded into the CNVs to obtain the drug-loaded biomimetic nanodecoy, CNV-C. Finally, this drug-loaded biomimetic nanodecoy, CNV-C, is used for the treatment of liver fibrosis.

[0051] like Figure 1 As shown, in order to verify the efficacy and mechanism of action of the above-mentioned drug-loaded biomimetic nanodecoy, this embodiment uses mice as experimental subjects, prepares corresponding drug-loaded biomimetic nanodecoys, and applies the drug-loaded biomimetic nanodecoys to mice with liver fibrosis induced by carbon tetrachloride.

[0052] Firstly, as Figure 1A. HEK293T cells were transfected with CCR2-mScarlet lentivirus to construct CCR2-overexpressing cells. Subsequently, CNVs were prepared by extracting and extruding the cell membranes of these CCR2-HEK293T cells. Finally, curcumin was loaded into the lipid bilayer of the CNV to form CNV-C. Figure 2 As shown, the drug-loaded biomimetic nanodecoy CNV-C is prepared in the following manner:

[0053] (1) A stable CCR2-overexpressing transfected cell line (CCR2-HEK293T) was constructed by transfecting wild-type human embryonic kidney cells (HEK293T) with CCR2-mScarlet lentivirus:

[0054] The mouse CCR2 gene was cloned into the lentiviral vector plasmid PGMLV-CMV-Mouse_CCR2-mScarlet-PGK-Puro. Lipofectamine was used. TM HEK 293T cells were transfected with the viral packaging vectors psPAX2 and PMD2G plasmid, as well as a lentiviral vector plasmid, using transfection reagent 2000 for 72 hours. Lentiviral supernatant containing mouse CCR2 and the mScarlet tag (mScarlet is a red fluorescent protein) was collected by ultracentrifugation (24000g, 4℃, 4 hours). HEK293T cells were then co-cultured with lentivirus and polyethyleneimine (10 μg / mL) for 48 hours, followed by puromycin selection (2 μg / mL) to obtain cell lines stably expressing CCR2-mScarlet. To confirm the expression of CCR2-mScarlet in the engineered HEK 293T cell lines, staining was performed for observation in this example. Figure 2 Image A shows confocal laser scanning microscopy (CLSM) images of wild-type HEK293T cells and CCR2-HEK293T cells. The location of the cell nucleus can be observed through Hoechst-stained blue fluorescence, the distribution of the cell membrane can be observed through DiO-stained green fluorescence, and the localization of the target protein CCR2 can be observed through the red fluorescence of the mScarlet-labeled fusion protein. "Merged" indicates co-localization analysis. The co-localization fluorescence signal between the mScarlet reporter protein (red) and the DiO-stained cell membrane (green) observed by CLSM verifies the successful expression of CCR2-mScarlet on the cell membrane.

[0055] (2) Obtaining CCR2-overexpressing nanovesicles (CNVs):

[0056] CCR2-HEK293T cells (5 × 10^7) were resuspended in cell membrane extraction reagent containing 1 mM PMSF and incubated on ice for 15 minutes. The cell suspension was subjected to two freeze-thaw cycles and then centrifuged at 1000 g for 10 minutes at 4 °C. The supernatant was collected and centrifuged at 16,000 g for 30 minutes at 4 °C to separate cell membrane fragments. The cell membranes were redispersed in PBS (phosphate-buffered saline) and protein content was determined using a BCA assay kit. Subsequently, the cell membranes were extruded sequentially through 400 nm and 200 nm porous polycarbonate filters to obtain CNVs. Figure 2 B is the immunoblotting analysis of NV and CNV. Compared with nanovesicles (referred to as NV) prepared from wild-type HEK293T cells, the immunoblotting analysis showed CCR2 expression on CNV. Figure 2 C is the transmission electron microscope (TEM) image of CNV, scale bar = 200 nm. Figure 2 F is the hydration diameter of CNV and CNV-C in PBS (n=3). Transmission electron microscopy (TEM) confirmed that CNV has a vesicular structure and its hydration diameter is 223.0±2.2nm.

[0057] (3) Prepare CNV-C and load it into the lipid bilayer of CNV using the lipophilicity of CUR to form a yellow suspension:

[0058] The prepared CNV and CUR were mixed in PBS and stirred at room temperature for 30 minutes. The mass ratio of CNV to CUR was 1:1. The mixture was then centrifuged at 16,000 g at 4 °C to obtain CNV-C. Figure 2 E represents the UV-Vis absorption spectra of CUR in DMSO organic solvent and CNV and CNV-C in PBS. As can be seen, the UV-Vis spectrum of the prepared CNV-C shows a characteristic absorption peak of CUR at 436 nm, indicating successful CUR loading. Furthermore, it can be observed that its absorbance has a linear relationship with concentration; based on its absorbance, the concentration of CUR can be further quantified. In the CNV-C obtained by the above method, the optimal drug loading capacity of CUR is 45.3±0.7%, and the corresponding encapsulation efficiency is 82.8±2.2%.

[0059] also, Figure 2 D is a TEM image of CNV-C, scale bar = 200nm. It can be seen that CNV-C maintains the complete vesicle structure after loading CUR. Figure 2 F is the hydration diameter of CNV and CNV-C in PBS (n=3). Figure 2G represents the zeta potential of CNV and CNV-C in PBS (n=3). It can be seen that the hydration diameter and zeta potential of CNV-C are 224.9±11.6nm and -13.6±1.7mV, respectively, and they did not change significantly after CUR loading. Figure 2 H represents the cumulative release rate of curcumin in CNV-C (n=3). Notably, CNV-C exhibited a controllable drug release capability, releasing 64.1±3.0% of CUR within 72 hours, indicating that CNV-C has the potential to serve as a sustained-release carrier for CUR.

[0060] Subsequently, as Figure 1 B. In this embodiment, carbon tetrachloride (CCl4)-induced mice were used to verify the efficacy of CNV-C in treating liver fibrosis. Following tail vein injection, CNV-C accumulated in the fibrotic liver and reduced macrophage infiltration and TGF-β production by adsorbing CCL2. Simultaneously, curcumin released from CNV-C amplified the therapeutic effect by enhancing its effectiveness in inhibiting hepatic stellate cell activation, thereby alleviating liver fibrosis, as detailed below:

[0061] (1) Figure 3A is the treatment schedule for CCl4-induced liver fibrosis in mice. Mice were injected with CCl4, CUR, CNV, NV-C (curcumin-loaded nanovesicles that do not express CCR2), and CNV-C at specified time points and sacrificed for analysis at week 7. Specifically, C57BL / 6 mice were randomly assigned to six different groups, each containing 5 mice: control, CCl4 model group, CUR group (8 mg / kg), CNV group (10 mg / kg), NV-C group (18 mg / kg, equivalent to 8 mg / kg CUR content), and CNV-C group (18 mg / kg, equivalent to 8 mg / kg CUR content). For the CUR group, CUR was dissolved in PBS containing 10% DMSO and 10% SBE-β-CD, while the drug formulations for the other groups were prepared in PBS. Except for the control group, the mice in the other five groups were intraperitoneally injected twice weekly with 1 mL / kg CCl4 (1:4 diluted in olive oil) for 6 consecutive weeks to induce liver fibrosis. The control group received an equal volume of PBS via intraperitoneal injection. Starting from week 3, the CUR, CNV, NV-C, and CNV-C groups received intravenous injections of the corresponding doses of the drug twice weekly, while the control and CCl4 model groups received an equal volume of PBS solution. At week 7, mice were euthanized by isoflurane anesthesia, and liver tissue and serum samples were collected. The degree of liver fibrosis was assessed using H&E staining, Sirius Red staining, and immunohistochemical staining targeting α-SMA (α-smooth muscle actin) and COL1A1 (collagen 1A1). Macrophage infiltration was assessed using F4 / 80 immunostaining. The expression of α-SMA, COL1A1, TGF-β (transforming growth factor β), Smad3, p-Smad3 (phosphorylated Smad3), and actin in the liver was detected by Western blotting. The mRNA expression changes of α-SMA, COL1A1, IL-1β (interleukin 1β), and TNF-α (tumor necrosis factor α) were determined by real-time quantitative PCR (qPCR). The level of CCL2 (CC motif chemokine ligand 2) in the liver was detected using a CCL2 ELISA kit.

[0062] Figure 3 B shows representative images of H&E, Sirius Red, α-SMA, and COL1A1 staining from livers of different groups of mice, scale bar = 100 μm. Extensive hepatocyte degeneration and necrosis, accompanied by inflammatory cell infiltration and disruption of hepatic cord structure, were observed in the CCl4 group. Furthermore, abundant collagen fiber deposition was observed in the portal vein region, leading to the formation of fibrous septa and pseudolobules. Conversely, the CNV-C group showed relatively intact liver structure with reduced inflammatory cell infiltration and collagen fiber proliferation. Only mild fibrosis remission was observed in the CUR, CNV, and NV-C groups. Figure 3 C represents a quantitative analysis of Sirius Red positive regions in different groups (n=5). The quantitative analysis of Sirius Red staining also showed that the percentage of positive staining regions in the CNV-C group was significantly lower than in other groups, indicating that CNV-C achieved a better anti-fibrotic effect. Furthermore, this embodiment also used immunohistochemical staining to estimate the expression levels of α-SMA and COL1A1 in mouse liver to reflect hepatic stellate cell activation. Figure 3 As shown in Figure B, CCl4-induced mouse liver showed increased expression of α-SMA and COL1A1 in the portal vein region and liver parenchyma, which was significantly reduced by CNV-C treatment.

[0063] Figure 3 D represents a quantitative analysis of α-SMA positive regions in different groups (n=5). Figure 3 E is a quantitative analysis of the COL1A1 positive region (n=5). Figure 4 A represents the immunoblotting analysis of α-SMA and COL1A1 expression in the livers of mice in each group. The aforementioned findings were obtained through... Figure 3 Quantitative analysis of α-SMA and COL1A1 positive regions in D and 3E, and Figure 4 Immunoblot analysis of A can provide further confirmation. Furthermore, Figure 4 B, C, D, and E represent the relative gene expression of α-SMA, COL1A1, IL-1β, and TNF-α in the livers of mice in each group. It was found that in the CNV-C group, the gene expression of α-SMA and COL1A1, as well as the expression of inflammatory markers including IL-β and TNF-α, were significantly downregulated. These results suggest that CNV-C can effectively alleviate liver fibrosis in vivo by combining its CCL2 adsorption capacity with its loaded curcumin.

[0064] To elucidate the therapeutic mechanism behind CNV-C treatment, this embodiment also examined macrophage infiltration in mouse livers. Figure 5 Image A shows representative images of F4 / 80 immunohistochemical staining in the livers of mice from different groups, along with corresponding quantitative analyses (n=5). Scale bar = 50 μm. Immunohistochemistry showed a significant reduction in F4 / 80-positive macrophage infiltration in the CNV-C group compared to other treatment groups. Furthermore, this example also used enzyme-linked immunosorbent assay (ELISA) to quantify the level of CCL2 in mouse livers. Figure 5B represents the CCL2 levels in the livers of different mouse groups (n=3). As expected, the CNV-C group exhibited the lowest CCL2 levels among all treatment groups. CCL2 levels were also reduced in the CUR and NV-C groups, which may be attributed to CUR's inhibition of CCL2 expression in the liver. These results indicate that CNV-C reduces hepatic macrophage infiltration by simultaneously adsorbing CCL2 released from liver tissue and inhibiting its production. Furthermore, the reduction in macrophage infiltration further leads to a decrease in the release of pro-fibrotic cytokines such as TGF-β, which activate the TGF-β signaling pathway and promote hepatic stellate cell activation and ECM generation. Therefore, this study also investigated whether CNV-C treatment inhibited the TGF-β / Smad signaling pathway in fibrotic livers. Figure 5 C represents the immunoblotting analysis of TGF-β, Smad3, and p-Smad3 expression in the livers of different mouse groups. It can be seen that the level of TGF-β was significantly reduced in the CNV-C group. Furthermore, phosphorylation of the downstream molecule Smad3 was also inhibited after CNV-C treatment. These experiments confirm that CUR delivery combined with the removal of the upstream fibrotic mediator CCL2 significantly enhances the inhibitory effect of the TGF-β / Smad signaling pathway, thereby effectively improving the therapeutic effect.

[0065] Ensuring that drug molecules circulate in the body for an extended period and accumulate effectively at the site of the lesion is crucial for maximizing their therapeutic efficacy. Therefore, such as Figure 6 As shown, this embodiment further investigated the pharmacokinetics and biodistribution of CNV-C in C57BL / 6 mice. By analyzing the changes in serum CUR concentration after intravenous injection of CUR or CNV-C, the experiment found that the blood half-life of CNV-C was 2.3 times longer than that of free CUR. Figure 6 A) indicates that using cell membrane nanovesicles as a carrier can prolong the in vivo circulation time of CUR. To further visualize the biodistribution of CNV-C, CNV loaded with IR780 (CNV-IR780) was intravenously injected into C57BL / 6 mice for in vivo fluorescence imaging. IR780 was chosen because it is a near-infrared dye with similar hydrophobicity to CUR. In vivo fluorescence imaging clearly showed that CNV-IR780 tended to accumulate in the liver compared to free IR780. Figure 6 B). Furthermore, to better quantify the accumulation of IR780 in major organs, organs were collected 24 hours post-injection for ex vivo fluorescence imaging. It can be seen that CNV-IR780 accumulation in the liver was significantly increased compared to free IR780. Figure 6(C and D). In addition, the biosafety of CNV-C was evaluated in C57BL / 6 mice after administration of CNV-C (8 mg / kg CUR) twice a week for 3 consecutive weeks. Blood samples and major organs were collected for biochemical and histopathological analysis. Figure 7 A shows representative images of H&E stained organ sections from each group of mice, with a scale bar of 100 μm. As can be seen, no abnormal changes or pathological damage were observed in any of the examined tissue samples. Figure 7 B represents the serum biochemical parameters (serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, urea, creatinine, total bilirubin, platelet count, hemoglobin, red blood cell count, monocyte count, neutrophil count, and white blood cell count) in mice treated with CNV-C (n=5). These parameters showed no significant changes compared to normal mice after repeated administration. These results indicate that CNV-C can prolong the in vivo circulation time of CUR, improve its accumulation in the liver, and exhibit good biocompatibility.

[0066] Furthermore, in order to study the drug mechanism of CNV-C, this embodiment also conducted in vitro experiments on it:

[0067] Assessing the adsorption capacity of CNV-C for CCL2: Serum samples were collected from mice with an acetaminophen (APAP)-induced acute liver injury model. A significant increase in serum CCL2 levels was observed 8 hours after APAP administration. Figure 8 ).

[0068] Serum samples were mixed with NV, CNV, or CNV-C, and the supernatant was collected after centrifugation for testing. Figure 9 A is a schematic diagram of the CCL2 adsorption assay and the transwell migration assay. CCL2-rich serum was collected 8 hours after mouse APAP modeling, while low-CCL2 serum was obtained by incubating CCL2-rich serum with CNV-C and then removing CNV-C by centrifugation before the transwell assay. Figure 9 B represents the adsorption capacity of NV, CNV, and CNV-C for CCL2 as detected by ELISA. The ordinate represents the residual CCL2 concentration in serum samples after incubation with NV, CNV, and CNV-C (n=3). The CCL2 levels in the CNV and CNV-C groups showed a concentration-dependent decrease, decreasing from 8.9±0.8 ng / mL to 4.4±0.2 ng / mL and 4.3±0.1 ng / mL, respectively. The NV group, lacking CCR2 expression, did not show a significant change in CCL2 adsorption. These results indicate that CNV-C exhibits a CCR2-dependent adsorption effect on CCL2.

[0069] Macrophage infiltration into the liver is regulated by the CCL2 / CCR2 signaling axis after injury and mediates liver inflammation and fibrosis. To verify the effect of CNV-C on macrophage migration in vitro, RAW264.7 cells were cultured in serum pretreated with CNV-C and evaluated using a transweld assay. The migration ability of RAW264.7 cells was enhanced when cultured with CCL2-rich serum. Figure 9 C represents the crystal violet staining and corresponding quantitative analysis of migrating RAW264.7 cells (n=5), scale bar=20 μm. Macrophage migration was significantly inhibited in the CNV and CNV-C groups, while the NV group had no effect on macrophage migration. These results indicate that CNV-C, as a nanodecoy, can effectively inhibit CCL2-mediated macrophage migration.

[0070] Furthermore, this embodiment investigated the inhibitory effect of CNV-C on TGF-β-induced activation of hepatic stellate cells. Human hepatic stellate cells (LX-2) were exposed to TGF-β (10 ng / mL) and co-incubated with CUR, NV-C, CNV, or CNV-C for 24 hours (30 μM CUR). Figure 9 D is a representative CLSM image of α-SMA expression in LX-2 cells after different drug treatments, with α-SMA (red) and DAPI-labeled nuclei (blue), scale bar = 20 μm. Immunofluorescence staining showed a significant decrease in the fluorescence intensity of α-SMA (a marker of hepatic stellate cell activation) in the CUR, NV-C, and CNV-C groups, indicating the inhibitory effect of CUR on hepatic stellate cell activation. Furthermore, Figure 9 E is an immunoblotting analysis of α-SMA and COL1A1 expression in LX-2 cells after different drug treatments, confirming the decrease in α-SMA and COL1A1 expression levels after CNV-C treatment. These results collectively demonstrate that CNV-C can inhibit hepatic stellate cell activation as an antifibrotic drug by loading CUR.

[0071] In summary, this study developed a biomimetic nanodecoy, CNV-C, loaded with CUR (crystal urea nitrogen) through genetically engineered cell membrane nanovesicles. CNV-C specifically adsorbs CCL2 via its overexpressed CCR2, significantly inhibiting macrophage infiltration, thereby reducing the production of pro-fibrotic mediators and suppressing the fibrotic process. Furthermore, CNV-C improves CUR delivery in fibrotic liver, effectively inhibiting hepatic stellate cell activation by blocking the downstream TGF-β / Smad signaling pathway. Animal experiments demonstrated that CNV-C protects liver structure and reduces collagen fiber proliferation in a CCl4-induced liver fibrosis model. As the first research achievement using genetically engineered nanodecoys to treat liver fibrosis, this study provides a new paradigm for developing biomimetic nanomedicine for liver fibrosis-related diseases.

[0072] The specific embodiments described in this example are merely illustrative of the present invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A drug-loaded biomimetic nanodecoy based on genetic engineering, characterized in that, This includes extracting cell membranes from a cell line that overexpresses CCR2 and preparing CCR2-overexpressing nanovesicles (CNVs), wherein the hydrophobic regions of the nanovesicles are loaded with curcumin to obtain the drug-loaded biomimetic nanodecoy CNV-C. Drug-loaded biomimetic nanodecoy CNV-C significantly inhibits macrophage infiltration by specifically adsorbing CCL2 through its overexpressed CCR2, thereby reducing the production of profibrotic mediators.

2. The drug-loaded biomimetic nanodecoy based on genetic engineering according to claim 1, characterized in that, The nanovesicles were obtained by transfecting human embryonic kidney cell lines with a lentiviral vector carrying the CCR2 gene.

3. A method for preparing a drug-loaded biomimetic nanodecoy based on genetic engineering as described in claim 1 or 2, characterized in that, include: Prepare a lentiviral vector carrying the CCR2 gene and prepare a cell line; The cell line was transfected with the lentiviral vector described above to construct CCR2-cells overexpressing CCR2. The cell membranes of CCR2- cells were obtained through freeze-thaw treatment and centrifugation. The obtained cell membranes were extruded using a liposome extruder containing a porous polycarbonate filter membrane to form vesicles of uniform size, thereby obtaining CCR2 overexpressing nanovesicles (CNVs). Curcumin was loaded into the hydrophobic region of the nanovesicles to form a yellow suspension, thus obtaining CNV-C.

4. The method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering according to claim 3, characterized in that, The specific methods for preparing nanovesicles (CNVs) using CCR2-overexpressing CCR2-cells include: Resuspend CCR2- cells in a cell membrane extraction reagent containing 1 mM PMSF and incubate on ice for 10-20 minutes; The cell suspension was subjected to two freeze-thaw cycles, and then centrifuged at 1000±200 g for 5-30 minutes at 4°C. Collect the supernatant and centrifuge at 16,000±6000 g for 20-50 minutes at 4°C to precipitate cell membrane fragments; The cell membrane was redispersed in a buffer solution, and its protein content was measured. Subsequently, the cell membrane was extruded sequentially through high-pore and low-pore porous polycarbonate filter membranes using an extruder to obtain CCR2-overexpressing nanovesicles (CNVs). The high pore size is 300nm-500nm, and the low pore size is 100nm-300nm.

5. The method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering according to claim 4, characterized in that, The process of loading curcumin into the hydrophobic regions of the nanovesicles to form a yellow suspension to obtain CNV-C specifically includes: The prepared nanovesicles (CNVs) were mixed with curcumin in a buffer solution at a ratio of 1:(1±0.2). The mixture was centrifuged at 16,000 ± 6,000 g at 4 °C to obtain CNV-C.

6. The method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering according to claim 5, characterized in that, The cell line mentioned is the human embryonic kidney cell line.

7. The method for preparing drug-loaded biomimetic nanodecoys based on genetic engineering according to claim 6, characterized in that, The cell lines mentioned are HEK293T, HEK293A, or HEK293E.

8. A drug for treating liver fibrosis, characterized in that, Including the drug-loaded biomimetic nanodecoy based on genetic engineering as described in claim 1 or 2; The mechanism of action of drugs in the treatment of liver fibrosis is as follows: Drugs accumulate in fibrotic livers; In fibrotic liver, the drug reduces CCL2 levels and inhibits macrophage recruitment by overexpressing CCR2, mimicking macrophage binding to CCL2. The drug releases its internally loaded curcumin in the liver; Curcumin directly inhibits the activation of hepatic stellate cells by blocking the TGF-β / Smad signaling pathway.

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