Preparation method and application of LRG1-targeted enzyme-responsive nano-drug

Abstract

The invention provides an enzyme-responsive nano-drug targeting LRG1 protein, and belongs to the technical field of biological medicines, and the nano-drug is formed by co-assembling an amphiphilic drug conjugate and a carried hydrophobic drug. Wherein the drug conjugate comprises a hydrophilic end of ET peptide targeting LRG1 and a hydrophobic end of the cytotoxic chemotherapeutic drug, the hydrophilic end is covalently linked with the hydrophobic segment through an enzyme-responsive linker, and the hydrophobic drug is entrapped in the amphiphilic drug conjugate. The preparation method comprises the following steps: dissolving the amphipathic drug conjugate and the hydrophobic drug in an organic phase, uniformly mixing the amphipathic drug conjugate and the hydrophobic drug under an ultrasonic condition, dispersing the mixture into a water phase, and standing at room temperature to obtain the targeted response type nano-drug which can prolong the in-vivo circulation time of the drug, realize efficient targeting of a colorectal tumor part, remarkably inhibit the growth of the colorectal tumor of a mouse, and has a good application prospect. DNA damage of tumor cells is promoted, tumor cell apoptosis is induced, and an effective and accurate novel method is provided for enhancing the colorectal cancer treatment effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of an LRG1-targeted enzyme-responsive nanomedicine. Background Art

[0002] Colorectal cancer is the third most common digestive malignancy in the world. Colorectal cancer patients have a poor prognosis due to local invasion and distant metastasis. The 5-year survival rate of colorectal cancer patients with distant metastasis is only 14%. In recent years, even though early detection and treatment methods have improved, colorectal cancer is still one of the leading causes of cancer death worldwide, causing more than 900,000 deaths each year. Chemotherapy for colon cancer is an important part of clinical treatment and is widely used in adjuvant therapy, postoperative metastasis, and treatment of advanced patients. In the chemotherapy regimen for colorectal cancer, camptothecin and its derivatives, as potent topoisomerase I inhibitors, can bind to the DNA-topoisomerase I complex during DNA replication, inhibit the reconnection of topoisomerase I after cutting DNA, thereby causing DNA breakage. Camptothecin intercalates into DNA and inhibits topoisomerase I, leading to DNA loss and cell death, which in turn triggers the activation of multiple DNA repair pathways, including poly(ADP-ribose) polymerase (PARP)-dependent. Therefore, the combination of drugs to overcome the DNA self-repair mechanism after treatment with the chemotherapy drug camptothecin is an effective dual-drug combination therapy strategy. Olaparib is a PARP inhibitor that inhibits the PARP-mediated DNA damage repair process. Olaparib can inhibit the DNA damage repair pathway induced by camptothecin, causing DNA damage to accumulate, thereby promoting tumor cell apoptosis and enhancing the toxic effect of camptothecin on tumor cells. The combined use of anti-tumor drugs will help overcome drug resistance, improve the toxic effect on tumor cells after medication, reduce the dose-limited toxicity of single drugs and expand the treatment range. However, due to the poor water solubility of camptothecin, strong toxic side effects, and easy loss of metabolic activity, its clinical application is often limited by severe side effects, including bone marrow suppression, cholinergic syndrome, and delayed diarrhea. In order to expand the therapeutic effect and reduce the adverse reactions associated with camptothecin and its derivatives, it is key to deliver these therapeutic drugs specifically to the tumor site.

[0003] In recent years, with the clinical application of Doxil, Abraxane, Onivyde and Genexol-PM, nanomedicines have shown significant advantages in achieving dual drug delivery, improving therapeutic effects and reducing systemic toxicity. The targeting methods of nanomedicines are divided into active targeting and passive targeting. Passive targeting is the process of using the physiological characteristics of tumor tissue to achieve the natural accumulation of nanomedicines in tumor tissues in the body. However, due to its dependence on the physiological characteristics of tumor tissues, the accuracy of passive targeting is limited, resulting in only some drugs being able to reach the tumor site, which may increase the risk of systemic side effects. In addition to passive targeting, the active targeting technology of nanomedicines has also shown significant advantages in tumor treatment, which can significantly improve the tumor tissue targeting of drugs, reduce systemic side effects, and prolong the circulation time of drugs in the body. In order to improve the delivery accuracy of CPT, researchers have explored a variety of receptors, including albumin, sweepers, folic acid and integrin receptors, to enhance the targeting of CPT to colorectal cancer tissues. Although these targeted nanomedicines have shown positive effects, the heterogeneity of colorectal tumors means that many patients may not be able to fully benefit from these treatments due to insufficient expression of target molecules. Therefore, there is an urgent need to find new molecular markers and develop corresponding nanomedicines to meet this demand.

[0004] Leucine-rich alpha-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that plays a vital role in a variety of pathological processes, including tissue fibrosis, cell proliferation, epithelial-mesenchymal transition, and neovascularization. LRG1 is expressed at significantly elevated levels in a variety of cancers, including pancreatic cancer, breast cancer, bladder cancer, and colorectal cancer, and plays a role in promoting the development and metastasis of these cancers, suggesting that LRG1 has the potential to become a diagnostic biomarker. Although LRG1 is considered a potential therapeutic target, the development of nanomedicines targeting LRG1 has been slow due to the lack of specific ligands. Recently, our research team identified a 12-amino acid peptide, ET (ESYSAKHRIMLT), through phage display technology, which has high affinity to LRG1 and has been shown to have therapeutic effects on breast cancer and renal fibrosis with high LRG1 expression in vivo. These studies provide a scientific basis for the application of LRG1 in the treatment of colorectal cancer and point out the direction of future research. Summary of the invention

[0005] In view of the above situation, the present invention provides a preparation method and application of an LRG1-targeted enzyme-responsive nanomedicine, which provides an effective and precise new method for enhancing the therapeutic effect of colorectal cancer.

[0006] The present invention aims to precisely deliver camptothecin and olaparib to the site of colorectal tumors by targeting LRG1 to achieve efficient anti-tumor effects. After intravenous administration, the nanodrug is mainly accumulated in the colorectal tumor tissue due to the overexpression of LRG1 in colorectal tumor tissues and the high affinity of ET peptides for LRG1. After colorectal cancer cells internalize the nanodrug, the elevated cathepsin B cleaves the glycine bond in the amphiphilic drug conjugate, triggering the release of camptothecin. At the same time, the nanodrug decomposes, rapidly releases olaparib, blocks the DNA damage repair mechanism, and synergistically induces DNA damage with camptothecin, increasing the mortality rate of tumor cells. This synergistic mechanism greatly enhances the anti-tumor efficacy of subcutaneous and in situ models of colorectal cancer in mice, highlighting the potential of the strategy of targeting LRG1 and the synergistic effect of camptothecin and olaparib in the combined treatment of colorectal cancer. Accordingly, the present invention provides an enzyme-responsive nanodrug targeted by LRG1, which is formed by co-assembly of an amphiphilic drug conjugate and a loaded hydrophobic drug. The drug conjugate contains the hydrophilic end of the ET peptide targeting LRG1 and the hydrophobic end of the cytotoxic chemotherapy drug. The hydrophilic end is covalently linked to the hydrophobic segment through an enzyme-responsive linker, and the hydrophobic drug is encapsulated in the interior of the amphiphilic drug conjugate.

[0007] The ET peptide sequence targeting LRG1 is ESYSAKHRIMLT, as shown in SEQ ID NO.1; the cytotoxic chemotherapy drug is camptothecin; the enzyme-responsive linker is a glycine bond cleavable by cathepsin B; and the encapsulated hydrophobic drug is olaparib.

[0008] Based on the same inventive concept, the present invention provides a method for preparing an LRG1-targeted enzyme-responsive nanomedicine, the steps of which are:

[0009] (1) dissolving an amphiphilic drug conjugate and a hydrophobic drug in an organic phase, wherein the organic phase is dimethyl sulfoxide;

[0010] (2) The organic phase containing the amphiphilic drug conjugate and the hydrophobic drug is uniformly mixed under ultrasonic conditions and then dispersed into the aqueous phase, and allowed to stand at room temperature to obtain an enzyme-responsive nanodrug, wherein the aqueous phase is deionized water or a phosphate buffer with a pH of 7.4.

[0011] Application of the enzyme-responsive nanomedicine prepared by the above method in the preparation of drugs for treating colorectal cancer.

[0012] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0013] The method of the present invention is simple and easy to operate. The prepared enzyme-responsive nanomedicine based on LRG1 targeting can achieve efficient enrichment and long-term retention in colorectal tumor tissues through active targeting of LRG1 targeting peptides and passive targeting of nanomedicines. The glycine bond in the amphiphilic drug conjugate is cleaved by the elevated cathepsin B in the colorectal tumor, triggering the release of camptothecin. At the same time, the nanomedicine decomposes and rapidly releases olaparib, blocking the DNA damage repair mechanism, and synergistically induces DNA damage with camptothecin, increasing the mortality rate of tumor cells. This synergistic mechanism greatly enhances the anti-tumor efficacy of subcutaneous and in situ models of mouse colorectal cancer, highlighting the potential of the strategy of targeting LRG1 and the synergistic effect of the combined treatment of colorectal cancer with camptothecin and olaparib. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a combination index diagram of different molar ratios of camptothecin and olaparib in colorectal cancer cell MC38 in the present invention.

[0015] Figure 2 This is a transmission electron microscope image of the nanomedicine prepared in the present invention.

[0016] Figure 3 This is the mass spectrum of the nano drug prepared by the present invention after being treated with cathepsin B.

[0017] Figure 4 This is a graph verifying the affinity of the nanomedicine prepared in the present invention to LRG1 in colorectal tumor tissue.

[0018] Figure 5 The nano drug prepared by the present invention inhibits proliferation, induces DNA damage, and induces apoptosis of colorectal cancer cells MC38

[0019] Figure 6 These are representative images of tumors and statistical graphs of tumor mass after the nanomedicine prepared in the present invention was used to treat mice.

[0020] Figure 7 This is a statistical diagram of the changes in the expression levels of apoptosis-related proteins in mice after the treatment with the nanomedicine prepared by the present invention.

[0021] Figure 8 The results of the blood routine and blood biochemical indexes of mice after the treatment with the nano drug prepared by the present invention

[0022] Fig. 9 The results are the toxic effects of the nano drug prepared by the present invention on the main organs of mice after the treatment.

[0023] Fig.10 This is a structural diagram of the amphiphilic molecule CPT-ESYSAKHRIMLT in the nanomedicine prepared in the present invention.

[0024] Fig.11 The structural formula diagram of LRG1 targeting peptide SEQ ID NO.1 in the nanomedicine prepared by the present invention. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described in detail below in conjunction with the embodiments.

[0026] The present invention is specifically implemented by the following embodiments:

[0027] Example 1

[0028] An enzyme-responsive nanomedicine targeting LRG1 protein, characterized in that the nanomedicine is co-assembled by an amphiphilic drug conjugate and a carried hydrophobic drug. The drug conjugate comprises the hydrophilic end of an ET peptide targeting LRG1 and the hydrophobic end of a cytotoxic chemotherapy drug, the hydrophilic end is covalently linked to the hydrophobic segment through an enzyme-responsive linker, and the hydrophobic drug is encapsulated in the amphiphilic drug conjugate.

[0029] Example 2

[0030] A method for preparing an enzyme-responsive nanomedicine based on LRG1 targeting, the steps of which are:

[0031] (1) Weigh 2 mg of the amphiphilic drug conjugate as a carrier and 1 mg of olaparib as a hydrophobic drug and dissolve them in 10 μL of dimethyl sulfoxide. The mass ratio of the carrier to the drug is 2:1.

[0032] (2) The solvent obtained in step (1) was dispersed in a phosphate buffer solution at pH 7.4, the power was 120 W, the ultrasonic time was 25 min, and the mixture was allowed to stand at room temperature for 1 h to obtain an enzyme-responsive nanomedicine.

[0033] Example 3

[0034] A method for preparing an enzyme-responsive nanomedicine based on LRG1 targeting, the steps of which are:

[0035] (1) Weigh 2 mg of the amphiphilic drug conjugate as a carrier and 1 mg of olaparib as a hydrophobic drug and dissolve them in 10 μL of dimethyl sulfoxide. The mass ratio of the carrier to the drug is 2:1.

[0036] (2) The solvent obtained in step (1) was dispersed in ultrapure water, the power was 120 W, the ultrasonic time was 25 min, and the mixture was allowed to stand at room temperature for 1 h to obtain enzyme-responsive nanomedicine.

[0037] The enzyme-responsive nanomedicine prepared by the above method is used in the preparation of drugs for treating colorectal cancer. Tumor tissue highly expresses cathepsin B.

[0038] The above are only embodiments, which are used to illustrate the specific facts of the present invention, and are not intended to limit the protection scope of the present invention. Any technical solutions that are essentially the same as those of the present application and are made by using equivalent replacement means or transformations shall fall within the protection scope of the present application.

[0039] The preparation method of the present invention is simple and easy to operate. The prepared enzyme-responsive nanomedicine can highly express cathepsin B in the colorectal tumor site, and can be effectively used in the preparation of drugs for treating colorectal cancer. Experiments have shown that the effect is very good. The relevant information is as follows:

[0040] 1. Verify the optimal synergistic ratio of camptothecin and olaparib in colorectal cancer cells MC38

[0041] MC38 cells with good growth status were inoculated in 96-well plates at 3000 cells / well, placed in an incubator for 24 hours, and then a solution with a camptothecin concentration of 0.5 μM and an olaparib concentration of 0.25 μM (camptothecin concentration: olaparib concentration = 1:0.5) was prepared using complete medium, and four concentrations were gradiently diluted and added to the 96-well plate for further incubation for 48 hours. The absorbance value at 450 nm was measured using an ELISA reader, and the cell survival rate was calculated. The Combination Index (CI) of camptothecin and olaparib in MC38 cells at a concentration of 1:0.5 was calculated using CompuSyn software to evaluate the synergistic effect of the two drugs at this concentration. The CI values ​​of camptothecin and olaparib at concentrations of 1:1, 1:2, and 1:4 (maximum camptothecin concentration of 0.5 μM) were calculated using the same method as above to evaluate the synergistic effect of the two drugs at different concentrations. As shown in the results, CPT and OLA have a significant synergistic effect at 1:0.5 and 1:1 (CI < 0.5), however, the synergistic effect is not obvious at lower concentrations (0.5 < CI < 1), and at 1:0.5, the two drugs show antagonistic effects at the lowest concentration (CI > 1). In contrast, when the molar ratio of the two drugs was 1:2 and 1:4, all treatment groups showed strong synergistic effects. In order to further optimize the ratio of the two drugs, we chose a CPT and OLA molar ratio of 1:2 for subsequent studies, while ensuring the synergistic killing effect, to optimize the dose of OLA and minimize potential toxic side effects.

[0042] 2. Morphology Characterization

[0043] The obtained nano drug (named OCE NM) was used for morphological characterization. The prepared nanomedicine was spherical and had a relatively uniform particle size. The particle size distribution of the obtained nanomedicine was 175-182 nm, and the average particle size was about 179.0 nm. The structure of the amphiphilic molecule was: CPT-ESYSAKHRLMLT, and the structure of the LRG1 targeting peptide SEQ ID NO.1 was: ESYSAKHRLMLT. For details, see Fig.10 , 11 .

[0044] 3. Carrier-drug ratio experiment

[0045] By adding a fixed mass of amphiphilic carrier (0.5 mg) and different masses of olaparib, the mass of olaparib carried in the nanodrug was analyzed, and the encapsulation rate and drug loading rate of the nanodrug were calculated. With the increase of the amount of olaparib input, the encapsulation rate of the nanodrug showed a trend of gradual decrease as a whole. In particular, when the ratio of carrier to OLA was 1:4, the encapsulation rate showed a significant decrease. In addition, the drug loading rate was positively correlated with the mass of OLA input. The more OLA mass was input, the higher the drug loading rate. According to previous research results, the synergistic killing effect of camptothecin and olaparib was best when the molar ratio of the two was 1:2. It was calculated that when the mass ratio of amphiphilic carrier to olaparib was 1:0.5, the molar ratio of camptothecin and olaparib in the obtained nanodrug was exactly 1:2. Finally, the condition of carrier: drug of 1:0.5 was selected to prepare the nanodrug, and the ratio of carrier to drug was controlled in subsequent experiments to achieve the maximum killing effect.

[0046]

[0047] 4. Experiment to determine the enzyme response of nanomedicine in tissue protease B solution.

[0048] The changes in morphology and molecular weight after enzyme treatment reflect the enzyme responsiveness. The nanomedicine obtained in Example 3 was added to 1 μg / mL of cathepsin B buffer, and the incubation conditions were: 25 mM 2-(N-morpholino)ethanesulfonic acid and 5 mM dithiothreitol. It was placed in a 37°C constant temperature shaker and incubated for more than two hours. The incubation solution was mixed with an equal volume of dichloromethane, and the mass spectra before and after the enzyme reaction were detected by MALDI-TOF MS, as shown in FIG. Figure 2 As shown, characteristic peaks of functional peptides appeared after enzyme response, indicating that after enzyme incubation, the amphiphilic molecule was broken to release the LRG1 targeting peptide ESYSAKHRIMLT (ET) and camptothecin.

[0049] 5. Verify the affinity experiment between the drug and LRG1 secreted by colorectal cancer cell MC38 cells.

[0050] MC38 cells were cultured at a rate of 5×10 5Cells / well were seeded in a 6-well plate and placed in an incubator for 24 h. OCM was diluted with serum-free medium at a concentration of 3.5 μg / mL for DSPE-PEG-Cy5. NM -Cy5 and OCE NM -Cy5 was added to the cells (protected from light throughout the process). The cells were digested at 1h and 4h after drug addition, and the precipitate was washed once with PBS to completely remove the Cy5 dye that was not taken up by the cells. The cell precipitate was resuspended in 300μL PBS to prepare a single cell suspension, and the fluorescence intensity of Cy5 in each group was analyzed by flow cytometry. The results were further analyzed using FlowJo software, and statistical analysis was performed using GraphPad Prism software. As shown in the figure, the PBS group served as a negative control, and its Cy5 fluorescence intensity remained unchanged at 1h and 4h. After the nanodrug and cells were incubated for 1h, OCE NM -Cy5 compared to OCM NM -Cy5 showed a higher uptake efficiency, with the fluorescence intensity increased by 21.3%. As the incubation time was extended to 4 h, the uptake difference between the two nanomedicines increased further. NM -Cy5-treated cells have a higher Cy5 fluorescence intensity than OCM NM -Cy5 increased by 22.9%. This observation was further confirmed by laser confocal fluorescence microscopy (Figure), which showed that OCE NM The accumulation in cells is higher than that of OCM NM The above results confirm that OCE NM The in vitro targeting ability of colorectal cancer cells MC38 was enhanced, which was mainly attributed to the high expression of LRG1 in colorectal cancer cells and the high affinity of the ET peptide modified on the nanoparticles to LRG1.

[0051] VI. Verify the effect of drugs on inhibiting proliferation, inducing DNA damage, and inducing apoptosis in colorectal cancer cells MC38

[0052] MC38 cells were seeded in a 96-well plate at 3000 cells / well and placed in an incubator for 24 hours. Different drug dilutions with the same amount of camptothecin were added and incubated in the incubator for another 48 hours. CCK-8 reagent was added, the absorbance at 450nm was measured, and the cell survival rate of each group was calculated. The results are shown in the figure. Compared with the non-targeted control, OCE NM The IC50 of OCE decreased by 48.4%, from 1.90 μM to 0.98 μM, indicating that NM By targeting the LRG1 receptor on the surface of colorectal cancer cells, the cytotoxic effect of the drug was significantly enhanced.

[0053] MC38 cells were seeded in a 6-well plate at 300 cells / well and placed in an incubator for 24 hours. Different drugs were used for treatment for 48 hours (the corresponding CPT amount was 20nM). The cells were incubated for 7 days, and the culture medium was replaced every two days. When the cell clones grew to an appropriate size and were not fused, the original culture medium was discarded, the cells were washed three times with PBS, and 1mL of 4% paraformaldehyde was added to fix the cells for 20 minutes. The fixative was discarded, the cells were washed three times with PBS, 1mL of 0.1% crystal violet stain was added, and the cells were stained in the dark for 20 minutes. After that, the cells were washed with tap water until the excess stain was completely washed away, dried, and photographed. The results are shown in the figure, compared with the non-targeted control of 81±10.2, OCE NM The number of cell clones decreased by 69.5%, indicating that OCE NM Significantly increased the cytotoxic effect of the drug, thereby effectively inhibiting cell proliferation

[0054] MC38 cells and NIH3T3 cells were digested, centrifuged, and counted. Each droplet volume was 16 μL, containing 2×10 4 Cells (MC38∶NIH3T3=3∶1) were resuspended in drug-containing medium at a concentration of 1.25×10 6 cells / mL. Add 2μL of 5% methylcellulose solution to each droplet to make a final concentration of 0.5%. Add 1mL PBS to a six-well plate, add several drops on the lid, incubate inverted in an incubator for 48h, and take photos. The results are shown in the figure. OCE NM The cell sphere area of ​​the group was (3.0±0.1)×10 5 μm 2 , compared with the non-targeted control (4.8±0.2)×10 5 μm 2 The above conclusions prove that OCE NM It can inhibit the proliferation of colorectal cancer cells MC38.

[0055] MC38 cells were cultured at 2×10 5Cells / well were inoculated in a 6-well plate and placed in an incubator for 24 hours. The cells were treated with different drugs for 48 hours (the corresponding CPT amount was 0.1 μM). Agarose gel and cells were added to a clean glass slide and placed in a freshly prepared pre-cooled working solution and lysed at 4°C for 2 hours. After washing the surface lysis solution with PBS, place it in a horizontal electrophoresis tank, pour in freshly prepared alkaline electrophoresis solution, and place it at room temperature for 40 minutes to unwind the DNA. Turn on the electrophoresis instrument, set the voltage to 25V, and the electrophoresis time to 30 minutes. Wash the surface electrophoresis solution with PBS, add Tris-HCl (pH7.5) neutralizing solution, and neutralize at 4°C for 3×10 minutes. Wash the surface neutralizing solution with PBS, add 20 μL PI staining solution, cover with a coverslip, stain in the dark for 10 minutes, observe under a fluorescent inverted microscope, and take pictures and record. The results are shown in the figure. OCE NM The comet tail length was 110.8±9.3μm, which was 0.64 times longer than the non-targeted control group (67.6±8.2μm), indicating more significant DNA damage. In addition, the expression level of the DNA damage marker γH2AX protein showed consistent results, confirming that OCE NM Can effectively induce DNA damage.

[0056] MC38 cells were cultured at 2×10 5 Cells / well were seeded in a 6-well plate and placed in an incubator for 24 hours. The cells were treated with different drugs for 48 hours (the corresponding CPT amount was 0.1 μM). The apoptotic cells in the culture medium were collected and digested with EDTA-free trypsin. The cell pellet was resuspended in 100 μL Bindingbuffer working solution, 5 μL Annexin V-FITC was added and blown evenly, and stained for 5 minutes in the dark. 5 μL PI staining solution and 400 μL PBS were added and blown evenly, and then immediately analyzed by flow cytometry. The results showed that OCE NM The percentage of apoptotic cells in OCE was 21.5±3.9%, which was 0.64 times higher than that of the non-targeted control (11.2±1.4%). NM It induces DNA strand breaks and promotes cell apoptosis through the Bcl-2 / Bax / caspase-3 signaling pathway.

[0057] 7. Experiment on the inhibitory effect of nanomedicine on colorectal tumors

[0058] All animals were inoculated subcutaneously on the left side to induce colorectal subcutaneous tumor models. The specific steps were as follows: MC38-Luc cells with good growth status were inoculated at a rate of 2×10 7The concentration of cells / ml was diluted into a cell suspension and placed on ice for later use. The mice were anesthetized with isoflurane, 100 μL of the cell suspension was taken and inoculated subcutaneously into the mice. After the cell inoculation was completed, the mice were returned to the cage for further observation. When the tumor volume reached 100 mm 3 At 14:00, the in vivo anti-tumor study of different drugs was started, and the equivalent dose of CPT in the experimental group was 2.5 mg / kg; the drug was administered once every two days through the tail vein and all mice were weighed and recorded. After seven consecutive administrations, blood samples were collected from the mice after anesthesia. Some of them were collected into anticoagulant tubes for blood routine testing, and the rest were collected into centrifuge tubes and placed at room temperature for 2 hours. After centrifugation at 8000 rpm for 5 minutes, serum was collected for blood biochemical indicators. After collecting blood samples, the mice were killed by cervical dislocation, and the heart, liver, spleen, lung, and kidney were fixed in 4% paraformaldehyde for paraffin sectioning and H&E staining for pathological analysis; tumor tissues were photographed and weighed, and the tumor inhibition rate was calculated. Subsequently, part of the tumor tissue was frozen at -80°C for Western blot experiments after protein extraction to detect protein expression levels, and the remaining tissues were fixed with 4% paraformaldehyde for paraffin sectioning and H&E staining or immunohistochemical staining analysis.

[0059] The results showed that compared with non-targeted controls, OCE NM The tumor volume was reduced by 75.8% and the tumor inhibition rate was 90.6%. Western blot results showed that compared with the non-targeted control, OCE NM The expression of DNA damage protein γH2AX increased by 76%, the expression level of anti-apoptotic protein Bcl-2 decreased by 46%, the expression level of pro-apoptotic protein Bax increased by 58%, and the expression level of cleaved-caspase-3 increased by 82.5%. The results of blood routine, blood biochemistry and H&E staining of major organs showed that OCE NM The common side effects of camptothecin were significantly reduced. NM It has significant anti-tumor effects in vivo and inhibits tumor growth by promoting DNA damage to induce tumor cell apoptosis. NM It showed low toxic side effects in the safety evaluation, providing strong experimental evidence for its use as a potential anti-tumor drug.

[0060] It should also be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0061] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts, all of which fall within the scope of protection of the present invention.

[0062] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

[0063] It can be clearly shown from the above that the present invention provides an enzyme-responsive nanomedicine based on LRG1 targeting, which can simultaneously deliver camptothecin and olaparib to colorectal cancer cells by specifically targeting LRG1. After effective internalization by tumor cells, due to the action of elevated cathepsin B, OCE NM The OCE is destroyed, resulting in the rapid release of both drugs. The released camptothecin induces significant DNA strand breaks, while olaparib inhibits the DNA damage repair mechanism, which together leads to increased DNA damage and promotes cell apoptosis. NM It showed excellent anti-tumor efficacy in both subcutaneous and orthotopic tumor models, and significantly reduced the side effects associated with camptothecin. The strategy of co-delivering camptothecin and olaparib to colorectal cancer by targeting LRG1 provides an effective and precise new method to enhance the therapeutic effect of colorectal cancer.

Claims

1. An enzyme-responsive nanodrug targeting LRG1 protein, characterized in that: The nano drug is formed by the co-assembly of an amphiphilic drug conjugate and a carried hydrophobic drug. The drug conjugate contains the hydrophilic end of the ET peptide targeting LRG1 and the hydrophobic end of the cytotoxic chemotherapy drug. The hydrophilic end is covalently connected to the hydrophobic segment through an enzyme-responsive linker, and the hydrophobic drug is encapsulated in the amphiphilic drug conjugate. The sequence of the targeting peptide ET having affinity to LRG1 is ESYSAKHRIMLT, as shown in SEQ ID NO.1; the cytotoxic chemotherapy drug is camptothecin; the enzyme-responsive linker is a glycine bond cleavable by cathepsin B; and the encapsulated hydrophobic drug is olaparib.

2. The method for preparing the enzyme-responsive nanomedicine targeting LRG1 protein according to claim 1, characterized in that: The following steps are involved: (1) dissolving an amphiphilic drug conjugate and a hydrophobic drug in an organic phase, wherein the organic phase is dimethyl sulfoxide; (2) Under ultrasonic conditions, the organic phase containing the amphiphilic drug conjugate and the hydrophobic drug is uniformly mixed and then dispersed into the aqueous phase, and allowed to stand at room temperature to obtain the enzyme-responsive nanomedicine, wherein the aqueous phase is deionized water or a phosphate buffer with a pH of 7.

4.

3. The method for preparing the enzyme-responsive nanomedicine targeting LRG1 protein according to claim 2, characterized in that: (1) Weigh 2 mg of the amphiphilic drug conjugate as a carrier and 1 mg of olaparib as a hydrophobic drug and dissolve them in 10 μL of dimethyl sulfoxide. The mass ratio of the carrier to the drug is 2:

1. (2) The solvent obtained in step (1) was dispersed in a phosphate buffer solution at pH 7.4, the power was 120 W, the ultrasonic time was 25 min, and the mixture was allowed to stand at room temperature for 1 h to obtain an enzyme-responsive nanomedicine.

4. The method for preparing the enzyme-responsive nanomedicine targeting LRG1 protein according to claim 2, characterized in that: (1) Weigh 2 mg of the amphiphilic drug conjugate as a carrier and 1 mg of olaparib as a hydrophobic drug and dissolve them in 10 μL of dimethyl sulfoxide. The mass ratio of the carrier to the drug is 2:

1. (2) The solvent obtained in step (1) was dispersed in deionized water, the power was 120 W, the ultrasonic time was 25 min, and the mixture was allowed to stand at room temperature for 1 h to obtain an enzyme-responsive nanomedicine.

5. Use of the nanomedicine prepared according to the method according to any one of claims 2, 3 or 4 in the treatment of colorectal cancer.

6. Use of the nanomedicine prepared according to the method of any one of claims 2, 3 or 4 in treating diseases with high expression of cathepsin B.

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